REVIEW

Testis Development
Juho-Antti Mäkelä,1 Jaakko J. Koskenniemi,1,2 Helena E. Virtanen,1 and Jorma Toppari1,2

1
Research Centre for Integrative Physiology and Pharmacology, Institute of Biomedicine, University of Turku,

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20520 Turku, Finland; and 2Department of Pediatrics, Turku University Hospital, 20520 Turku, Finland
ORCiD numbers: 0000-0001-8489-8724 (J.-A. Mäkelä); 0000-0003-2228-334X (J. Toppari).

ABSTRACT Production of sperm and androgens is the main function of the testis. This depends on normal development of both testicular
somatic cells and germ cells. A genetic program initiated from the Y chromosome gene sex-determining region Y (SRY) directs somatic cell
specification to Sertoli cells that orchestrate further development. They first guide fetal germ cell differentiation toward spermatogenic
destiny and then take care of the full service to spermatogenic cells during spermatogenesis. The number of Sertoli cells sets the limits of
sperm production. Leydig cells secrete androgens that determine masculine development. Testis development does not depend on germ
cells; that is, testicular somatic cells also develop in the absence of germ cells, and the testis can produce testosterone normally to induce full
masculinization in these men. In contrast, spermatogenic cell development is totally dependent on somatic cells. We herein review germ cell
differentiation from primordial germ cells to spermatogonia and development of the supporting somatic cells. Testicular descent to scrota is
necessary for normal spermatogenesis, and cryptorchidism is the most common male birth defect. This is a mild form of a disorder of sex
differentiation. Multiple genetic reasons for more severe forms of disorders of sex differentiation have been revealed during the last decades,
and these are described along with the description of molecular regulation of testis development. (Endocrine Reviews 40: 857 – 905, 2019)

M ale reproductive health is largely determined

already in early development of the testis.
Differentiation of somatic cells to Sertoli cells in the
early bipotential gonad starts the male-specific devel-
opment and guides the germ cells to the spermatogenic
lineage. Sertoli cells coordinate differentiation of other
somatic cells in the developing testis, including Leydig
cells that produce testosterone required to masculinize
the fetus. Normal differentiation and maintenance of
Sertoli cell identity is decisive for the future reproductive
health, and the number of Sertoli cells determines
sperm production capacity. In this review we describe
germ cell specification, differentiation of testicular so-
matic cells, and disorders that arise from defects in testis
development.
For this narrative review we have followed the
literature with the search terms primordial germ cell,
testis and stem cell, or spermatogenesis.

Primordial Germ Cell Specification
Specification of the germline precursors during early
embryonic development is essential for perpetuation of
the species, as these cells will eventually give rise to
mature gametes, sperm, and eggs, the vectors of
transgenerational genetic information. There are two
distinct modes of germline specification in metazoans:
preformation and epigenesis (). The former refers to
inheritance of maternal determinants, whereas in the
latter mode germ cells are specified by inductive signals.
Preformation is based on synthesis, transportation, and
consequential unequal distribution and accumulation
of specific RNAs and proteins to a certain area within
the oocyte cytoplasm, called the germ plasm. When
embryogenesis starts, the cells that inherit the germ
plasm are recruited into the germline. In the epigenesis
mode no maternally deposited germ plasm has been
observed, and the germ lineage is specified from a rather ISSN Print: 0163-769X
homogeneous population of embryonic cells by in- ISSN Online: 1945-7189
ductive signals from embryonic and extraembryonic Printed: in USA
Copyright © 2019
sources. In mice, humans, and probably most other
Endocrine Society
mammals, the germline is determined by the epigenesis Received: 14 May 2018
mode, and the first cells of the germ lineage are called Accepted: 17 July 2018
primordial germ cells (PGCs). PGCs are specified before First Published Online:
gastrulation during early embryonic development both 26 December 2019

doi: 10.1210/er.2018-00140 https://academic.oup.com/edrv 857

REVIEW

ESSENTIAL POINTS
· Cells of the germ lineage are specified at an extraembryonic location and migrate to the presumptive gonad during early
fetal development
· Induction of sex-determining region Y (SRY) expression in the bipotential gonad defines the onset of testicular
differentiation and determines the sex of the developing individual
· Specification of somatic gonadal cells to Sertoli cells determines testicular differentiation, including germ cell
development to spermatogenic cells
·· Sertoli cell identity requires constant maintenance
Defects in the genetic machinery regulating testis development cause disorders of sex differentiation
· Testicular hormones regulate development of reproductive tract and external genitalia

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in humans (likely during the second week after fertil-
ization) and mice [embryonic day (E)  to ] (). In the
XY embryo, they form the first distinctive sub-
population of embryonic cells that can be considered
the predecessors of spermatogonial stem cells (SSCs)—
the cells responsible for lifelong sperm production in
men and perpetuation of the species.
Research on early PGCs or their immediate pre-
cursors has been hampered by their low number and
embedded location among the somatic cells of the
perigastrulation embryo. However, the mechanisms
and the molecular cues responsible for specification of
mouse and human PGCs have been elucidated during
recent years. Adoption of PGC fate embarks these cells
on a developmental trajectory that clearly separates
them from the surrounding somatic cells, and their
early differentiation is characterized by three different
but interconnected events: repression of the somatic
mesodermal program, reacquisition of pluripotent
gene expression, and global reprogramming of the
epigenome. The first two are discussed below, and the
last one is discussed later in this article. In this section
we describe the current understanding about PGC
specification in mice and humans.
PGC specification in the mouse
Signaling pathways upstream of mouse
PGC specification
In mice, PGCs are induced from the posterior
proximal epiblast cells of the egg cylinder at ~E..
Extraembryonic ectoderm (ExE)–derived bone mor-
phogenetic protein (BMP) and BMPb, as well as
BMP originating from the posterior visceral endo-
derm, act on a subset of epiblast cells (Fig. a), and
subsequently a founder population of  to  mouse
PGCs (mPGCs) is observed at the base of the de-
veloping allantois in extraembryonic mesoderm of
an E. embryo (–). BMPs act via SMAD sig-
nal transducers, and phosphorylation of SMAD,
SMAD, and SMAD triggers complex formation
with SMAD and its nuclear translocation. Loss of any
of the involved BMPs, SMADs ,  and , but not that
of SMAD, leads to a severe reduction in the total
mPGC number or their complete absence, but gene
dosage also plays a role (–).
Germ cell competence is initially induced by
BMP in as few as six cells of the proximal epiblast on
E., just preceding the onset of gastrulation ().
These founder cells are in direct contact with the
overlying ExE and they differ from their somatic
neighbors by the expression of B lymphocyte–
induced maturation protein- [BLIMP, officially
PR/SET domain (PRDM)] (, ). The ability to
activate BLIMP expression is not restricted to a
specific subset of epiblast cells, but it is the local high
BMP that specifies the mPGC precursors in a
WNT-dependent manner (, , ). Additionally,
anterior visceral endoderm secretes BMP signaling
antagonists [including cerberus  (CER) and dick-
kopf  (DKK)] to inhibit mPGC specification of
anterior epiblast cells (Fig. a) (, ). It is, however,
a very strictly temporally restricted process (E.~E.),
and competence of the epiblast cells to give rise
to mPGCs is markedly reduced after E. ().
Additionally, mPGC precursors need to express
BLIMP at a sufficiently high level to commit to the
germ lineage ().
Wnt is expressed by epiblast cells and in the
posterior visceral endoderm (Fig. a). Its expression is
directly or indirectly activated by BMP, which has a
crucial role (i) in priming epiblast cells to respond to
WNT signaling activation in a proper manner, and (ii)
in the induction of Blimp and PR/SET domain 
(Prdm), another mPGC specification gene ().
BMP/SMAD and WNT/b-catenin signaling path-
ways thus work in parallel to ensure sustained ex-
pression of BLIMP and its downstream targets, and
PRDM, whose expression is greatly enhanced by T
(Brachyury, officially TBXT), an effector and target of
WNT signaling (Fig. b) (). Importantly, mPGC
formation fails in Wnt and catenin b (Ctnnb)
mutants (Ctnnb encodes the WNT signaling trans-
ducer b-catenin) regardless of BMP signaling (, ).
These data indicate that BMPs alone cannot induce or
maintain mPGC specification to a sufficient extent but

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Figure 1. (a) PGC specification in the mouse. Mouse PGC precursors are induced in the E6.5 embryo from the posterior proximal
epiblast cells of the egg cylinder. BMP4 (green) and BMP8b (light green) from ExE, and BMP2 (khaki) from the posterior visceral
endoderm (PVE), plus WNT3 (orange) from the epiblast and PVE act on a subset of epiblast cells, which are recruited into the germline
as PGC precursors. Anterior visceral endoderm (AVE) secretes BMP signaling antagonists to inhibit ectopic mPGC specification in the
anterior epiblast. (b) Pathways involved in formation of mPGC precursors. ExE-derived BMP4 and BMP2 from the posterior visceral
endoderm (PVE) act via SMAD signal transducers to inhibit the expression of somatic genes and induce Blimp1 and Prdm14 in mPGC
precursors. WNT3 is directly or indirectly activated by BMP4. The concerted action of BMP/SMAD and WNT3/b-catenin signaling
pathways is needed to maintain the high expression of Blimp1 and Prdm14, and specification of mPGCs. (c) PGC specification in
humans. The epiblast of an E12 human embryo takes the shape of a disc and is surrounded by the amnion on the dorsal side and
hypoblast on the ventral side. The exact origin of hPGC precursors is not known, but they likely arise in either the posterior epiblast or
dorsal amnion in response to signals (BMPs, WNTs) derived from the epiblast, amnion, or hypoblast. Inhibitory signals are probably also
at play to prevent ectopic hPGC formation. (d) Synthesis of putative interactions involved in hPGC(LC) specification. WNT signaling
activates the expression of EOMES in hPGCLC precursors. EOMES then induces the expression of SOX17, which is also a target of BMP
signaling. TFAP2C and BLIMP1 are consequently upregulated, resulting in adoption of germ cell fate and repression of the somatic
program.

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depend on WNT/b-catenin/T signaling for suste-
nance of BLIMP and PRDM expression.
BLIMP1 as the master regulator of early mPGCs
BLIMP is a DNA-binding protein that is known
mainly to repress but also to activate gene expression
(–). BLIMP-positive cells of an E. embryo
are a subset of cells that are known to express
interferon-induced transmembrane protein 
(IFITM, also known as mil- or fragilis) at E. ().
There is a progressive increase in their number, and at
the midstreak stage (E.) ~ BLIMP-expressing

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REVIEW
cells are observed in a tight cluster. At the early bud
stage (E.) their number has risen to ~ cells.
Genetic lineage tracing experiments have revealed
that all BLIMP-positive cells of the E. embryo
are committed to the germ lineage and are thus the
earliest identified definitive precursors of adult
mouse germline stem cells (). Blimp mutant cells
fail to specify as mPGCs, highlighting the impor-
tance of BLIMP for germline development (, ).
BLIMP not only initiates mPGC specification but
it also works as a major coordinator responsible for
adoption of mPGC-specific gene expression via direct
repression of the somatic program and promoting
induction of other mPGC specification genes, in-
cluding Prdm, transcription factor (TF) AP-g
(Tfapc), DND miRNA-mediated repression inhibitor
 (Dnd), Kit, and Nanos (, ). The first two of
these (PRDM and TFAPC) form a tripartite
mutually interdependent transcriptional complex with
BLIMP, whereas the remaining three (DND, KIT,
and NANOS) are critical for the early embryonic
germ cell development and survival (–). Besides
BLIMP, these cells also express high levels of tissue-
nonspecific alkaline phosphatase (TNAP, officially
ALPL), IFITM (fragilis), and developmental
pluripotency-associated protein (DPPA) (also known
as stella and PGC), and—in stark contrast to their
mesodermal neighbors—display repression of Hox
(Homeobox) genes, especially Hoxa and Hoxb, two
key genes for mesodermal commitment (, –).
BLIMP also mediates repression of the incipient
somatic program in mPGC precursors and promotes
acquisition of germ cell characteristics, including
reactivation of pluripotency-associated gene expres-
sion (, , ). One mode of BLIMP action is
modification of histone methylation in a complex with
protein arginine methyltransferase  (PRMT), and
the BLIMP/PRMT complex has been implicated in
maintenance of mPGC lineage during migration ().
Blimp-deficient mPGC-like cells fail to repress the
somatic program (including Hox cluster activity,
epithelial–mesenchymal transition-associated genes,
cell cycle progression genes, and DNA methylation
machinery), and they inconsistently activate the
mPGC transcriptional signature (e.g., Dppa, Nanos)
().
Subsequent to BLIMP, Prdm is induced in the
mPGC precursors at E. in a BMP-dependent
manner, and its expression is maintained by
BLIMP (, , , ). In Prdm-null mice, mPGCs
are formed at lower numbers and there is a nearly
complete loss of mPGCs by E. (). BLIMP di-
rectly induces Tfapc, which is expressed in mPGCs
from E. onward. TFAPC exerts its effect via
BLIMP and is indispensable for germline develop-
ment and maintenance (, ). Tfapc-deficient
mPGC-like cells do not migrate and are lost (prob-
ably via somatic differentiation) at ~E., resulting in
860 Mäkelä et al Testis Development
gonads devoid of germ cells and consequent sterility in
Tfapc-null mice of both sexes (). In vitro data show
that Tfapc-deficient mPGCs fail to repress the so-
matic program and do not upregulate germ cell
markers Nanos and deleted in azoospermia-like
(Dazl), a situation reminiscent of and partially phe-
nocopying loss of Blimp (, ).
BLIMP, PRDM, and TFAPC collectively
constitute a transcription regulatory network essential
for the adoption of germ cell identity in the mouse (,
, ). BLIMP-dependent induction of Tfapc,
maintenance of it by PRDM, and the mutually
interdependent expression of PRDM and BLIMP
create a functional triad that is needed to ensure
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proper and timely specification and commitment of
mPGCs (, , ). PRDM and BLIMP are likely
the master regulators of mPGC development, whereas
TFAPC appears to have an auxiliary role in mPGC
formation and maintenance, and it probably modu-
lates the response of a subset of BLIMP and PRDM
targets (, ).
mPGC-like cell induction in vitro
mPGC-like cells (mPGCLCs) with germline compe-
tence and spermatogenic differentiation ability can be
derived in vitro from pluripotent stem cells (PSCs)
either via inducible expression of TFs or by treating the
cultured cells with cytokines (, , ). Notably,
there prevails a narrow window of action for mPGC-
inducing signals in vitro, too, and the transient pre-
gastrulating epiblast-like cell (EpiLC) population that
arises from pluripotent cells rather quickly loses its
competence to give rise to mPGCLCs. Interestingly,
when the mPGCLC state is induced via introduction
of TFs, the nascent mPGCLCs do not display a
transient upregulation of mesodermal genes (Hoxa,
Hoxb, and T), as is the case in vivo and when the
mPGC-like state is induced with cytokines (, , ,
). These data suggest that upon specification nascent
mPGCs not only activate an mPGC program but also a
somatic mesodermal program, and that the former
subsequently extinguishes the latter.
Similar to their precursor cells, EpiLCs, mPGCLCs
have a limited lifespan, and they cannot be maintained
for more than  days in vitro (). Culture conditions
that would enable long-term maintenance of these
cells are yet to be defined, although the requirements
for their robust expansion have been recently char-
acterized (). Comparative analyses have shown that
mPGCLCs derived from embryonic stem cells (ESCs)
are very similar to migratory (until ~E.) mPGCs
(). In vitro mPGC specification is therefore
considered a reliable reconstitution of the in vivo
events and it is considered to provide a relevant model
to analyze the mechanisms responsible for formation,
development, and maintenance of mPGCs. Among
other things, this has enabled researchers to look into
the reprogramming of mPGC epigenome (discussed
Endocrine Reviews, August 2019, 40(4):857–905

later in this review) that would not be feasible in vivo
(). However, there are some obvious differences
between mPGCLCs and in vivo mPGCs. For instance,
day  mPGCLCs express at best only low levels of
genes—such as Dazl and DEAD-box helicase 
(Ddx)—that are upregulated in mPGCs upon arrival
in the primordial gonad and considered to play a role
in silencing of retrotransposons in the male and in
onset of meiosis in the female (–). Their low
expression in mPGCLCs is attributed to accumula-
tion of repressive and lack of active histone marks at
these genes, and it may also indicate that mPGCLCs
are more equivalent to migratory mPGCs (). A recent
report by Hill et al. () suggests that proper activation
of these genes depends on a concerted action of
multiple factors, which provides an explanation for
why it has been difficult to recapitulate it in vitro.
Reactivation of pluripotent gene expression in
the germline
An interesting feature in mPGC development is the
reactivation of pluripotent gene expression. NANOG,
octamer-binding TF  (OCT, officially POUF), and
sex-determining region Y (SRY) box (SOX) form a
core gene regulatory network (GRN) and promote the
pluripotent state in the inner cell mass of a blastocyst.
NANOG is re-expressed in the posterior epiblast at
E., whereas SOX is detected in the anterior epiblast,
and OCT is ubiquitously expressed in the prestreak,
early streak, and midstreak embryos (, ). In Sox-
deficient embryos, mPGC specification is compro-
mised, and SOX also plays a role in promoting
mPGC proliferation (). NANOG is strictly not
needed for mPGC specification, but proliferation and
survival of migrating mPGCs and germline develop-
ment depend on NANOG to a certain extent (–).
In vitro, BMP induces mPGCLCs in EpiLCs in lower
numbers in Nanog mutant cells, suggesting that
NANOG is required for robust mPGC(LC) formation
(). Interestingly, recent research has demonstrated
that a single NANOG target gene, estrogen-related
receptor b (Esrrb), can substitute for Nanog loss in
germline development (). Surprisingly, however,
overexpression of NANOG in EpiLCs is able to induce
mPGCLC independently of BMP and WNT signaling.
Moreover, SOX represses mPGCLC state induction
by NANOG in EpiLCs, whereas it has no effect on
BMP-mediated mPGCLC formation ().
These data provide an important example of
context-dependent action of pluripotency-associated
factors during development: while working in an in-
terdependent and synergistic fashion to form the self-
sustaining ESC core GRN, SOX is a NANOG
antagonist during mPGC(LC) induction. The sequence
and dynamics of Blimp, Prdm, and Tfapc activation
differ somewhat whether mPGCLC formation is in-
duced using BMP or overexpression of NANOG, but
the derived cells are transcriptionally almost identical
doi: 10.1210/er.2018-00140
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(). In summary, these data show that BMP/SMAD
and NANOG work independently and synergistically
during mPGCLC formation. These data suggest that
there might be a number of interacting and at least
partially redundant mechanisms and pathways in
function to ensure germline commitment of a sufficient
subset of epiblast cells.
PGC specification in humans
That the germline is established in humans between E
and E makes research on human PGC (hPGC)
specification impracticable. Hence, knowledge con-
cerning the molecular events in pregastrulating hu-
man embryo, where hPGCs are specified, is lacking.
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The disc-shaped epiblast of a human post-
implantation embryo is sandwiched by the amnion on
the dorsal side and cells of the yolk sac–contributing
hypoblast on the ventral side. This structural orga-
nization separates the putative hPGC precursors in the
epiblast from trophectoderm, the functional coun-
terpart of the mouse ExE. It is therefore likely that the
inducing agent originates from an alternative source,
such as the hypoblast, or is secreted by the epiblast cells
themselves, as is the case in the rabbit, another
mammalian species with disc-shaped epiblasts ()
(Fig. c). An intriguing new insight into the discussion
was introduced by Sasaki et al. (), who studied the
specification of PGCs in the cynomolgus monkey. “Strikingly, SOX17, a critical TF
They demonstrated that cynomolgus monkey PGCs for endoderm lineages, is
are induced before gastrulation in the dorsal amnion essential for hPGC(LC)
in response to signals derived from the amnion itself specification.”
and the extraembryonic mesenchyme. Given the
similarity of early embryonic development between
humans and the cynomolgus monkey, these data
suggest that hPGCs may arise mainly in the nascent
amnion instead of the posterior epiblast. Thus, it
seems that PGCs of different mammalian species arise
in distinct parts of the perigastrulation embryo in a
manner that reflects diverse strategies for embryonic
patterning in a particular species.
Although the exact embryonic origin of hPGCs
awaits clarification, subsequent human germline de-
velopment is rather well characterized. As reviewed by
De Felici (), microscopic studies by Politzer (,
, ) and Witschi () identified a group of
 to  large, spherical cells in the endoderm of the
yolk sac in a -week-old human embryo (). During
the following days, a substantial increase in their
number was also recorded, and the size of the pre-
migratory hPGC population rose to ~ cells.
Markers of early [ to  weeks post coitum (wpc)]
hPGCs include TNAP, NANOG, OCT, stage-specific
embryonic antigen  (SSEA), and KIT (–). The
expression of DAZL, DDX, and fucosyltransferase 
(FUT) are subsequently activated or enhanced in
maturing hPGCs/gonocytes ( to  wpc) (, ).
Notably (and in contrast to mPGCs), hPGCs do not
express SOX (). Morphologically, early hPGCs do
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not differ much from neighboring somatic cells, but as
they mature, they grow in size and become round or
oval and have a clear cytoplasm and a large nucleus
with one or two prominent nucleoli (, , ).
However, maturing hPGCs are not a morphologically
homogeneous population of cells ().
Although experimental and definitive data are
lacking, it has been proposed that a subset of posterior
epiblast cells (or cells of the dorsal amnion) are
specified as hPGC precursors between E and E in
response to autocrine and/or paracrine signals (BMP
and WNT) (Fig. c). During the following days these
cells move out of the embryo proper and are tran-
siently incorporated into the extraembryonic meso-
derm. At E they are found near the base of allantois
in the wall of the definitive yolk sac, and upon ini-
tiation of TNAP expression are considered true
hPGCs ().
Induction of human PGCLCs
The basic mechanisms of hPGC specification have
been elucidated in vitro. Although EpiLCs are readily
induced from murine ESCs by Activin A and basic
fibroblast growth factor (bFGF), the propensity of
human ESCs (hESCs) for human PGCLC (hPGCLC)
formation in the presence of bFGF is poor, and the
approach used for mouse cells fails to render hu-
man PSCs competent for the germline (, –).
Nonetheless, hPGCLCs can be induced from hESCs
and human-induced PSC (hiPSCs), and robust culture
systems for the derivation of hPGCLCs have been
available since  (–). Either hESCs cultured
in a medium containing inhibitors against glycogen
synthase kinase b (GSKB), c-Jun N-terminal kinase
(JNK), MAPK kinase (MEK), and p (5 i culture)
or hiPSCs stimulated by Activin A and a WNT sig-
naling agonist (ACTA plus CHIR) display high
competency for germline fate and, in the presence of
BMPs, stem cell factor (SCF), epidermal growth factor,
and leukemia inhibitory factor (LIF), they robustly give
rise to hPGCLCs (, ). The transcriptional profiles
of hPGCLCs are highly similar independent of the
induction method or the starting material (hESCs or
hiPSCs) ().
A shared feature in PGC(LC) specification between
humans and mice seems to be a dependency on a
signaling network that is orchestrated by three key
proteins. However, as described in more detail below,
the identity of the proteins involved and, for the
proteins that are shared, their significance for robust
PGC specification depends on the species. Strikingly,
SOX, a critical TF for endoderm lineages, is essential
for hPGC(LC) specification (, , ). It is the
earliest known marker for hPGCLCs and works up-
stream of BLIMP () (Fig. d). Sox is also induced
in the mouse when the germ lineage is established, but
it is dispensable for mPGC specification (, ). Data
concerning the induction of TFAPC in hPGCLC
862 Mäkelä et al Testis Development
precursors is contradictory. Recently it was shown that
TFAPC is induced in nascent hPGCLCs by BMP
signaling, that is, independent of SOX, and it was
postulated that TFAPC not only drives a distinctive
developmental program but is also needed for sus-
tained expression of SOX, BLIMP, NANOG, OCT,
and NANOS (). This is in stark contrast to its
somewhat minor role during mPGC specification ().
Other reports imply that SOX and BLIMP act
upstream of TFAPC for hPGCLC specification (,
). Both SOX and BLIMP were shown to be
necessary for hPGCLC formation, but SOX alone
was able to induce germ cell genes independent of
cytokines in i-cultured germ cell–competent hESCs
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(). A novel regulatory network composed of OCT,
paired box  (PAX), and BLIMP was also recently
implicated in hPGC induction (). How all of these
TFs interact and to what extent they display re-
dundancy is not well understood at present. BMP
and BMP (but not BMP or BMPa) work in-
terchangeably in induction of hPGCLCs, whereas SCF,
epidermal growth factor, and LIF have an auxiliary role
in the process and they support the maintenance of
nascent hPGCLCs () (Fig. d).
When hiPSCs are used as a source of hPGCLCs, the
cultured cells first give rise to incipient mesoderm/
primitive streak-like cells (iMeLCs), which modestly
and transiently upregulate genes characteristic of me-
sodermal lineages [including T and eomesodermin
(EOMES)] (). In the presence of BMP, iMeLCs then
robustly generate hPGCLCs. Recently it was shown that
the germline competence in iMeLCs depends on ap-
propriate dosage and duration of WNT signaling (, ).
WNT signaling activates EOMES, which in turn induces
the hPGCLC specification program by upregulating the
expression of key genes, most notably SOX.
At the moment, there is no consensus as to what is
the hierarchy of different TFs during hPGC(LC)
specification, which TFs are absolutely required for
the process, and what is the relative importance of the
TFs involved. There are at least two plausible expla-
nations for the discrepancies in the available data: first,
the cells of origin (i-cultured hESCs vs hiPSCs cul-
tured under a conventional condition) probably bear
distinct states of primed pluripotency that might affect
their route to germ cell fate; and second, both ablation
and excessive overexpression of TFs might blur the
physiological significance of the findings. Future
studies that precisely define the function of key TFs
in a physiologically meaningful way are warranted.
PGC specification in humans vs mice
Many aspects of mammalian PGC specification seem
to have been conserved in evolution, but there are also
obvious differences in the mechanisms responsible for
PGC specification in humans and mice. What then
might explain the observed dissimilarities? First, the
early postimplantation development of mouse and
Endocrine Reviews, August 2019, 40(4):857–905

human embryos at the time when PGCs are formed
may explain such differences: the human embryo is a
bilaminar flat disc, whereas the mouse embryo forms a
cup-shaped egg cylinder (). Second, in the early
postimplanation embryo of the mouse, the PGC-
inducing ExE is situated on top of the epiblast, but
an equivalent structure does not exist in humans ().
Third, mouse and human PSCs, which are the in vitro
source of PGCLCs, differ in their regulation, growth
requirements, and cell morphology (). Human PSCs
depend on bFGF for long-term maintenance, whereas
mouse PSCs rely on LIF to support the pluripotent
state. Human ESCs actually resemble more (in terms
of cytokine dependence, epigenetics, and tran-
scriptomics) mouse epiblast stem cells (EpiSCs), an
epiblast cell-like cell line that is refractory to PGC
induction, than mouse ESCs (, –).
Human PGCLCs are transcriptionally close to
hPGCs of -week-old male human embryos (corre-
sponds to E. to E. in the mouse) but, similar to
mPGCLCs, express a significantly lower level of germ
cell and meiosis-related markers, such as DDX and
DAZL (, , ). In addition to the critical role of
SOX, the expression level of SOX is another ob-
vious difference between human and mouse PGC(LC)s.
SOX is rapidly downregulated in human PSCs
destined to germ cell fate and it is undetectable in
hPGCLCs and hPGCs but has an important role in
mPGC specification and proliferation (, , ). In-
terestingly, BLIMP, working downstream of SOX,
may not be as important in humans as in mice ().
Moreover, the role of PRDM in hPGC(LC) speci-
fication is unclear, as PRDM expression is very low in
nascent hPGCLCs and it is only weakly induced during
the following few days (, ). PRDM is repressed in
late hPGCs, and its overexpression is associated with
delayed differentiation of hPGC(LC)s and germ cell
tumor (GCT) formation (). The interdependent
BLIMP-PRDM-TFAPC GRN, which is needed for
robust specification of mPGCs, thus does not seem to
exist in human. Moreover, Krüppel-like factor 
(KLF), a gene associated with naive pluripotency,
is sharply upregulated upon hPGCLC, but not
mPGC(LC), specification (, , , ).
Available pieces of data suggest that hPGCs are
induced from a transient peri-gastrulating mesoderm-
like state (, ). The molecular bases of human and
mouse PGCLC induction and in vitro maintenance are
similar, but the transcriptional programs downstream
of induction display considerable divergence be-
tween the two species and depend partially on dif-
ferent signaling cascades. Most strikingly, a clearly
noticeable transient induction and subsequent re-
pression of the somatic mesodermal program is
observed only during mPGC(LC) induction, and it is
probably needed for proper activation of the tri-
partite transcriptional network that does not exist in
humans (, ).
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In summary, in vitro–specified PGCs faithfully
mimic most of the key events that are associated with
PGC formation in vivo: repression of somatic genes,
reactivation of pluripotency gene expression, and global
reprogramming of the epigenome. Owing to epigenetic
regulation, implementation of the germ cell–specific
transcriptional program is incomplete (as in early PGCs
in vivo), which might reflect lack of an appropriate
environment, that is, similar to the one that PGCs are
exposed to upon and after integration into the gonadal
primordium (, , , ). It is also possible that
hPGCLCs correspond to early postspecification hPGCs,
and similar to mPGCLCs do not express germ cell–
specific genes DDX and DAZL, and display just some
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but not all aspects of epigenetic reprogramming (, ).
Further maturation of hPGCLCs probably depends on
identifying the proper cytokines that enable hPGCLCs
to proceed on their developmental pathway.
PGC homing
As described in the previous section, PGC precursors
are likely induced in the human embryo within the
epiblast or dorsal amnion during the second embryonic
week. In human, PGCs are initially observed at an
extraembryonic location on E and the first PGCs
colonize the gonadal ridges (GRs) during the fifth
week postconception (, ). Hence, PGCs need to
“…the fate of mismigrated
translocate a considerable distance to contribute to the
PGCs depends not only
developing bipotential gonad (Fig. a). In mammalians on prosurvival factors but
they reach gonads by both active and passive means, also on environmental
that is, migration and displacement caused by em- permissiveness.”
bryonic growth movements (, ). A four-step ap-
proach can be used to describe PGC homing: (i)
transepithelial, (ii) dorsal, and (iii) lateral migration
followed by (iv) coalescence with somatic cells to form
the complete embryonic gonads (Fig. b). To what
extent the displacement of PGCs is due to active
(migration) and passive (folding, growth, and devel-
opment of embryonic structures) mechanisms is a
matter of debate, and the whole concept of germ cell
migration during embryonic development has been
questioned (). However, many lines of evidence
suggest that PGCs are migratory cells: PGCs are motile
cells both in vivo and in vitro, mouse GR explants attract
isolated early PGCs over long distances in vitro, and
ectopic expression of a putative PGC attractant results
in aberrant germ cell migration in vivo (–). The
conclusion from these studies and numerous other
reports is that PGCs actively migrate in the developing
embryo, at least over relatively short distances ().
Route of PGC gonad-oriented displacement
At the time of PGC specification, the prospective
gonads near the developing root of the mesentery exist
as slightly condensed structures of the coelomic epi-
thelium (CE). To build a functional gonad, PGCs need
to be translocated from the yolk sac endoderm to
the embryo proper, and the somatic precursors of
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REVIEW

Figure 2. (a) Site of nascent hPGCs. hPGCs are observed near the base of the developing allantois in the wall of the definitive yolk sac
that is outside the embryo proper, soon after their specification. As the embryo folds, the part of extraembryonic endoderm that is
inhabited by the nascent PGCs is incorporated into the embryo and forms the midgut and the hindgut. (b) PGC displacement. The
route of PGC migration/displacement during early fetal development is shown. A four-step approach can be used to describe PGC
homing: (i) transepithelial in the endodermal epithelium, (ii) dorsal through the mesenchyme of the gut mesentery, and (iii) lateral in
the mesoderm, and finally (iv) coalescence with somatic cells of the GRs to contribute to the embryonic gonads.

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864 Mäkelä et al Testis Development Endocrine Reviews, August 2019, 40(4):857–905


the gonad need to proliferate and reorganize to form
the GRs, the primordia of the future testis or ovary.
Roughly  week after specification at an extraem-
bryonic location, PGCs get reincorporated into the
embryo proper as the embryonic disc folds and the
portion of the yolk sac that hosts PGCs becomes
the hindgut and the midgut. Consequently, PGCs can
be found as single cells near the aorta among the
endodermal cells of the primitive hindgut and
midgut epithelium. These events take place during
the fourth embryonic week and precede the for-
mation of GRs.
If indeed PGCs were to actively migrate from the
yolk sac to the GR, they would first need to push
endodermal epithelial cells out of their way within the
yolk sac (stalk) and in the primitive gut (i), then
penetrate into to surrounding mesenchyme and move
dorsally through the gut mesentery (ii), and, upon
arriving at the preaortic region in the midline of the
abdominal dorsal body wall, move a little farther in the
lateral direction (iii) to make it to the GR (iv) (Fig. b).
Recent and previous research also question the axiom
of long-range PGC migration and provide data to
support passive displacement of PGCs from the
endoderm to the GR (, ). However, these data
support the notion of short-range migration of PGCs
at particular steps of homing, for instance, when
PGCs cross the basal lamina of the endodermal
epithelium to enter the mesenchyme. This particular
step might involve epithelial-to-mesenchymal
transition, a process that converts well-organized
and compact epithelial cells into migratory mes-
enchymal cells.
Physical guidance of PGC migration
It has been suggested that nerve fibers serve to guide
the movement of PGCs from the dorsal hindgut
mesentery via the preaortic region to the gonadal
anlage (). Indeed, both (first) hPGCs and nerve
fibers arrive at GRs during the fifth embryonic week
(). There is, however, a wide spatial distribution of
hPGCs during the th to th wpc, and during de-
velopment some hPGCs are still located in the gut
epithelium, whereas others are already enclosed within
the nascent testis cords (, , ). These observations
bring up many questions that have been insufficiently
addressed to date. What is the reason for the wide
spatial distribution of hPGCs within the developing
embryo/fetus? What is the latest time point of gonadal
entry? What is the mechanism that drives gonad-
oriented movement of hPGCs at later time points?
What is the destiny of hPGCs that fail to reach the
gonad and what is the initial cause for improper
displacement?
If ectopic PGCs are not cleared by apoptosis, they
might give rise to GCTs (). In humans, extragonadal
GCTs are most commonly found along the midline
and within the central nervous system, which supports
doi: 10.1210/er.2018-00140
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the notion of nerve fibers playing an important role
in directional guidance and promotion of PGC
survival (, , ). Research done in mice suggests
that PGC survival can only be supported within the
migratory path, and if PGCs fall outside this tra-
jectory, cell survival–promoting signals are too weak
to maintain these ectopic PGCs and they undergo
apoptosis (, , ). Interestingly, not all stray
PGCs perish, and mPGCs that end up in the adrenals
produce oocytes and enter meiosis independent of
the sex (). It has also been speculated that PGCs
might contribute to the pool of adult-type hema-
topoietic stem cells that emerge in the aorta–
gonad–mesonephros region during embryogenesis
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(). Thus the fate of mis-migrated PGCs depends
not only on prosurvival factors but also on envi-
ronmental permissiveness. Owing to a distinctively
different epigenetic status, it is unlikely that the stray
PGCs could contribute to somatic tissues, with he-
matopoietic stem cells probably being the remarkable
exception.
In the mouse, migratory PGCs are mostly found
in tissues as single cells but they occasionally extend
long processes to other PGCs and might move
within a tissue as a group of cells (). The molecular
control of PGC migration is another much-debated
topic. To what extent is the proper guidance based on
attraction, and what is the role of repulsive cues? It
has been suggested that mediators such SCF
and fibronectin, which are abundantly expressed
along the migratory route, and adhesion molecules
expressed by PGCs themselves are responsible for
early (before the fifth week of embryonic develop-
ment) guidance of PGCs prior to establishment of the
connections of the nervous system (, , , ). If
this hypothesis holds true, once the nerve connec-
tions are established, the chance of going astray
would be considerably diminished. Besides providing
a physical lifeline for PGCs, the nervous system
might also use other means to aid PGC homing, and
Schwann cells have been suggested to secrete
lysophosphatidic acid, which attracts and guides
PGCs ().
The route of PGC homing is virtually identical
in humans and mice. Migrating PGCs move in an
amoeboid fashion and have a dynamic cell mor-
phology, broad leading edge, and low adhesiveness and
is therefore reminiscent of immune cell migration ().
Surprisingly, migratory PGCs proliferate and have
been suggested to depend on anaerobic energy pro-
duction (). However, many of the genes that are
enriched in migratory hPGCs, when compared with
later fetal germ cells, are associated with aerobic
respiration (). PGC proliferation and survival/
apoptosis are controlled by a number of genes, as
recently reviewed by Hamer and de Rooij (). The
molecular control of PGC migration has been char-
acterized only in the mouse.
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Molecular mechanisms of PGC migration
Although the molecular mechanisms responsible for
attracting, guiding, and, finally, stopping hPGCs are
largely unknown, there is a handful of molecular
players that have been shown to regulate mPGC
trafficking. Molecular regulation of embryonic germ
cell migration in different species has recently been
reviewed by Barton et al. (). Although numerous
mechanisms—chemotactic movement based on tissue-
wide gradient of an attractant, dynamic adhesion to
close-by PGCs and extracellular matrix (ECM)
components along the migration path, lipid
modification-mediated PGC repulsion, other repulsive
cues, and neuronal guidance—have been suggested to
be involved in PGC homing, the significance of dif-
ferent mechanisms in mammalian species is not well
understood (, ). In mice, the data supporting
involvement of C-X-C motif chemokine ligand 
(CXCL)/C-X-C motif chemokine receptor 
(CXCR), SCF/KIT, cadherin  (CDH), and ITGB
(integrin subunit b) in PGC migration is, however,
relatively solid.
CXCL12/CXCR4. CXCL expressed in the
body wall mesenchyme and GR and its cognate
receptor CXCR on mPGCs form the best charac-
terized system taking part in guidance of PGCs (,
, ). Although being dispensable for the trans-
epithelial migration of PGCs, it is needed for proper
homing of PGCs in the mesenchyme. Lack of either
Cxcl or Cxcr results in very few intragonadal
PGCs, whereas ectopic expression of CXCL leads
to reorientation and aberrant migration of mPGCs
(, ). Despite that the CXCL/CXCR system
has been suggested to protect PGCs from cell death,
the main role for survival promotion of PGCs
has been assigned to SCF/KIT signaling (, ,
, ).
SCF/KIT. The SCF/KIT system has also been
shown to be important for continued motility of
mPGCs (). Despite being expressed along the
migratory pathway both in humans and mice and
forming a gradient with the highest level of ex-
pression in the GR, the role of SCF as a directional
cue has been questioned (, , , ). WNTA/
receptor tyrosine kinase–like orphan receptor 
(ROR) signaling has been suggested to work in
synergy with SCF/KIT system and potentiate the
mPGC response to SCF (). Lack of either Wnta
or Ror results in fewer mPGCs colonizing the GRs
(). ROR is expressed by postmigratory human
germ cells in an -week male fetus, but it is not
known whether it plays a role in the migration of
hPGCs ().
Cell adhesion. Two adhesion molecules, CDH
and ITGB, also contribute to mPGC homing and
are needed specifically to aid in exit from the hindgut
(–). CDH is a cell–cell adhesion molecule,
whereas ITGB interacts with the ECM. Whereas
866 Mäkelä et al Testis Development
loss of Cdh results in mis-migration and ectopic
PGCs, Itgb-deficient mPGCs colonize the GRs
with a poor efficiency (, ). A role for ITGB in
the mPGC migratory process is also supported by
the presence of fibronectin, an ECM molecule that
interacts with ITGB, along the route of mPGC
migration. Fibronectin is also required for mPGC
displacement in vitro (). Additionally, TGFb and
FGF signaling have been implicated in mPGC mi-
gration (–).
For successful homing of PGCs, an equally im-
portant question as to what induces and guides PGC
migration is what eventually stops it. It has been
presumed that PGCs become nonmotile upon co-
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alescing with somatic gonadal cells. However, de-
scendants of PGCs display motile properties later in
life; that is, in the fetal/perinatal period as gonocytes
migrate to the basal lamina of the testis cords, and in
the adult as SSCs constantly move within the basal
compartment of the seminiferous epithelium
(–).
By E. postmigratory mPGCs/early gonocytes
become nonmotile and fail to migrate even when
transferred into a permissive environment (). The
mechanism responsible for stopping PGC migration
once they have reached the gonad is yet to be char-
acterized. It is, however, well documented that upon
arriving to the GRs, the transcriptome of PGCs is
altered: for instance, markers of an adult germline cell,
Ddx, Dazl, and germ cell nuclear acidic protein
(Gcna), are induced, whereas Tnap, Ssea, Blimp, and
Kit are downregulated (–). A similar process
takes place in humans, and hundreds of genes are
differentially expressed between migrating hPGCs and
mitotic germ cells that have settled in the gonads (,
, ). These data challenge the notion of gonocytes
being equivalent to PGCs and representing just a
nonmotile version of them (). A dozen master
regulators of gene expression networks, including SIX
homeobox (SIX), lysine demethylase A (KDMA),
and SOX, have been implicated in adoption of
gonadal hPGC/early gonocyte transcriptional signa-
ture ().
If indeed a chemotactic gradient is the main
determinant of PGC gonad-oriented movement,
then reaching the highest concentration of an at-
tractant (i.e., the GR) would provide a natural stop
sign for PGCs. However, cessation of direc-
tional movement is one thing and loss of motility is
another. To contribute to a new organ, the inherent
motile behavior of PGCs has to be repressed. How
it mechanistically takes place is not known, but it
has been suggested that cell–cell adhesion be-
tween PGCs, as well as PGCs and gonadal somatic
cells, has a key role in suppression of PGC motility
(). The molecular events in charge of gonadal
somatic cell specification are the topic of the fol-
lowing section.
Endocrine Reviews, August 2019, 40(4):857–905

Testis Determination: Genetics
and Embryology
Gonads of an adult individual, testes or ovaries, have
their origin in GRs, two long and thin embryonic
structures that run craniocaudally on the dorsal sur-
face of the coelomic cavity, overlying the mesonephri.
Onset of CE cell proliferation in E. mice or  to 
wpc humans is the first sign of presumptive gonad
formation. Subsequently, the underlying basal lamina
partially disintegrates to allow migration of the mitotic
progeny into the dorsal inner mesenchyme to form
areas of cellular condensation, later observed as the
GRs. All of the cells in GRs (supporting cells, ste-
roidogenic cells, germ cells) are thought to be capable
of differentiating into either testicular or ovarian
lineages. This bipotentiality of cell fates makes GRs
unique primordia in organogenesis.
The bipotential gonad and adrenal gland both arise
from a specific region of intermediate mesoderm in the
early embryo, the adrenogonadal primordium (AGP).
The primordial adrenal gland can be seen as a distinct
structure at ~ days postconception (dpc) in humans.
In the ,XY embryo the developing gonad remains
bipotential until  dpc when the expression of Y
chromosome gene SRY is transiently induced in a
subset of somatic cells of the gonadal primordium
(). This process, called sex determination, precedes
by a couple of weeks the onset of sex differentiation
that depends on induction of steroidogenic gene ex-
pression and production of testosterone in the de-
veloping male. Development of external genitalia and
phallic growth are among the earliest signs of an-
drogen action. Testosterone synthesized by fetal
Leydig cells is also needed to stabilize Wolffian
structures, whereas Sertoli cell–derived anti-Müllerian
hormone (AMH) acts to regress the Müllerian ducts,
the source of the fallopian tubes, uterus, and upper
vagina in females (). Timelines of prenatal testis
development in mice and humans are provided in Fig.
a and b.
The inability of a ,XY embryo to activate the
expression of SRY in a temporally, spatially, or
quantitatively proper manner results in a disorder of
sex development (DSD). DSDs are relatively rare
congenital conditions that are characterized by atypical
development of chromosomal, gonadal, and ana-
tomical sex. DSDs have their origin in complete or
partial gonadal dysgenesis, or in undervirilization
or undermasculinization due to defective androgen
synthesis or action. ,XY complete gonadal dysgen-
esis is defined by female external genitalia, well-
developed Müllerian duct–derived structures, and
streak gonads. ,XY partial gonadal dysgenesis is
characterized by varying degrees of masculinization of
the external genitalia, coexistence of Müllerian and
Wolffian structures, and incomplete testis formation.
The etiology of ,XY DSDs is variable, and mutations
doi: 10.1210/er.2018-00140
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involving SRY account for only % to % of all cases
of ,XY complete gonadal dysgenesis (). As dis-
cussed later in this section, more than a dozen other
genes have been associated with ,XY gonadal dys-
genesis (). Spontaneous mutations, deletions,
duplications, or translocations in humans and in-
troduction of transgenes into mice have shown that
either SRY or SOX alone is sufficient to induce testis
development in an XX embryo (–). Among
the SOX family genes, SOX and SOX have also
been associated with human testicular or ovotes-
ticular ,XX DSDs (, ).
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Formation of the bipotential gonad
Located on the medial surface of the urogenital ridge,
the GR becomes morphologically distinct in human
embryo during the fifth developmental week. GRs
arise within the intermediate mesoderm as a result of
CE proliferation and ingression. Proliferation of CE
cells starts at ~E. in the mouse and these cells ul-
timately give rise to most of the somatic cell types in
the presumptive testis or ovary (, ). CE can thus
be regarded as a source of multipotent progenitor cells
(). Mitotic activity of CE cells and their ingression
into the interior of the tissue result in formation of
thickened, elongated, and thin structures, the GRs, by
E. in the mouse—the time of Sry activation and
arrival of PGCs (Fig. a). Cells of the GR contribute to “…SRY is merely a trigger and
the future gonads, but cell migration from elsewhere in does very little per se to
the embryo—at least vascular endothelial cells (VECs) promote entry into the male
from the neighboring mesonephros, macrophages pathway….”
from the yolk sac or fetal liver, and germ cells from the
base of allantois—is also needed for complete testicular
organogenesis.
TFs essential for GR formation
Wilms tumor  (Wt) is among the earliest genes to be
expressed in the mouse urogenital ridge at E ().
WT-positive cells of the gonadal primordium sub-
sequently contribute to Sertoli cells and at least two
different interstitial cell lineages (). E. can be
considered the earliest stage of organogenesis of the
mouse gonads or GRs. A number of genes have been
implicated in GR formation. However, the phenotype
of Wt, GATA-binding protein (Gata), empty spi-
racles homeobox  (Emx), LIM homeobox  (Lhx),
and nuclear receptor subfamily  group A member 
(Nra)–mutant mice is most severe, suggesting that
these factors are absolutely crucial for the earliest steps
of gonadal development (–). Nra [also
known as steroidogenic factor  (Sf)] expression is
induced coincidentally with the onset of CE cell
proliferation in a WT/LHX-dependent manner
(). Similar to the mouse, WT and NRA are
expressed very early in the human presumptive gonad
and their expression precedes that of SRY by ~ days
(, ). WT is indispensable for the activation of
Nra and Dax (officially Nrb) expression, and its
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Figure 3. (a) Formation of the testis and timeline of mouse prenatal testis development. CE cells start to proliferate at ~E9.5 when
Nr5a1 is also induced. Many signaling pathways converge on the level of NR5A1. The underlying basement membrane disintegrates and
the cells ingress into the interior of the tissue, resulting in formation of the GRs by E10.5 when Sry is induced. The first PGCs have already
arrived in the GRs at this stage. Pre-Sertoli cells are subsequently specified and testiculogenesis starts. Sertoli cells orchestrate the
process. Other somatic populations are either recruited from the undifferentiated precursors or they migrate from elsewhere, such as
VECs from the mesonephros (MN). Sertoli cells encase PGCs into protocords. Fully developed testis cords are first observed at E13.5. (b)
Timeline of first and early second trimester testis development in humans. (c) Regulation of SRY expression in (i) the mouse and (ii)
humans. (i) In mice, a modular approach can be used to gain an insight into the complex interplay of factors involved in induction of Sry.
Modules 1 through 3 converge on a key TF that directly takes part in regulation of Sry expression. Epigenetic regulatory mechanisms
constitute module 4 that controls the access of TFs to the Sry gene and its regulatory elements and enhancers. (ii) In humans, the same
key TFs are responsible for the activation of SRY expression as in the mouse but, although many similarities can be observed,
considerable species-dependent differences likely exist.

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loss results in an early block in gonadal development, steroidogenic tissues and die soon after birth due to
lack of reproductive organs, and late embryonic le- adrenal insufficiency (). GRs initially develop in
thality (). Nra-null mice do not develop these mice but they subsequently regress due to

868 Mäkelä et al Testis Development Endocrine Reviews, August 2019, 40(4):857–905


Figure 3. (Continued)
apoptosis (). These data suggest that NRA is not
needed for the onset of gonadal development but
rather for the maintenance and differentiation of
somatic cells within the GRs. Many signaling pathways
converge on the level of NRA (Fig. a), and ac-
cordingly misregulation of Nra is associated with
most mouse mutant embryos that display impaired
gonadal development.
GATA expression in the CE precedes the onset of
epithelial proliferation (). In conditional Gata
knockout mice, CE cells proliferate less and integrity of
the basement membrane is maintained. The cellular
changes driving the thickening of CE do not thus
occur, and the CE of these mice remains as a mor-
phologically undifferentiated monolayer. This is re-
lated to Emx deficiency, which results in early failure
of GR development due to inability of CE cells to
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migrate through the basement membrane (). In the
absence of GATA, Nra and Lhx are not induced
and the CE shows no features of gonadal differenti-
ation (). Chromobox homolog  (CBX) also
works upstream of Nra during early gonadal de-
velopment, and Cbx-null mice display early gonadal
growth defects, although normal CE proliferation
(, ). The expression of Wt and Emx is not
affected in Cbx-null gonads at E., but that of
Gata, Nra, and Lhx is dramatically decreased,
suggesting that CBX exerts its effect on gonadal
development via GATA and NRA.
Nra expression is also regulated by SIX and
SIX, and Six/ double-mutant mice have small
gonads and complete loss of NRA-expressing cells.
Gonadal expression of Wt, Gata, and Lhx, however,
is not affected, implying that loss of Six/ specifically
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impinges on Nra (). PBX homeobox  (PBX)
and insulin and insulin-like growth factor signaling
have also been implicated in Nra regulation at the
time of GR formation (, ). In conclusion, many
factors participate in the timely and proper activation
of Nra in the CE (Fig. a), and formation of
NRA-expressing progenitor cells represents the
initial stage of gonadal growth and development in
both sexes.
According to current knowledge, gonadal pri-
mordia of XY and XX embryos are indistinguishable
until induction of SRY at  dpc and E. in humans
and mice, respectively, and comprise an unorganized
mixture of somatic and germ cells (Fig. a). Although
the somatic cells of embryonic gonads actively pro-
liferate in both sexes, activation of SRY expression is
accompanied by a proliferative burst and accelerated
growth of the testis when compared with the age-
matched ovary ().
SRY activation and entry into the male
developmental pathway
SRY expression is induced in human embryonic gonad
at ~ dpc and in the mouse at E. (, –).
There are a few aspects of SRY that deserve special
attention. First, both in humans and mice, SRY is only
transiently expressed. Whereas in the mouse Sry ex-
pression returns to the preinduction level by E., in
the human embryo SRY expression is downregulated
at ~ dpc and it reaches the baseline transcript level
by  dpc (, , ). Second, there is a specific
window of induction for SRY expression probably in
both species, but experimental data show that if Sry is
not upregulated by E., the bipotential gonad will
assume the ovarian fate (). Third, SRY is merely a
trigger and does very little per se to promote entry into
the male pathway and depends on its downstream
factors (most notably SOX) for initiation of the
genetic program that ultimately leads to testis de-
termination and male sex differentiation. Fourth, it is
not just the timing but also the intensity of expression,
and SRY abundance needs to cross a specific threshold
level for proper induction of the downstream ma-
chinery ().
There are at least a dozen factors that take part in
regulation of Sry expression in mice in a partially
interdependent, synergistic, or redundant fashion (Fig.
c). Larney et al. () have proposed a modular
approach to provide a coherent model that would
explain how these factors are functionally organized
upstream of Sry. According to the model, each module
is centered upon a key TF (GATA, NRA, and
WT) that interacts with other proteins. The inter-
acting factors have complex functions and they might
act as coactivators, posttranslational modifiers, or
upstream regulators of primary factors. A fourth
module, which is somewhat different in nature, is
provided by epigenetic regulatory mechanisms that
870 Mäkelä et al Testis Development
either permit or inhibit access to the promoter and
regulatory elements of Sry.
Next, we use a similar approach and immerse into
the function of these modules in detail and aim at
providing an organized description of SRY regulation
(Fig. c). Despite obvious differences between humans
and mice in the structure of the SRY gene itself or in
the regulatory regions that surround it, as pointed out
by Larney et al. (), owing to scarcity of research
material and at the risk of irrelevant findings, we still
have to resort to studies conducted with mice to
understand how mammalian male development is
initially induced.
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Module 1
GATA works at the heart of module . At least three
different factors or signaling cascades control the
action of GATA. The most intimate of these is the
zinc finger cofactor friend of GATA (FOG, officially
ZFPM), and loss of either Fog or elimination of
direct interaction between the two results in reduced
expression of Sry, loss of Sox induction, and a con-
sequent XY sex-reversal phenotype (). The role of
GATA action in sex determination is highlighted by
the fact that loss of Gata induced at E. (the time of
Sry induction), but not at later time points, causes
an early block in Sertoli cell differentiation and con-
current male-to-female sex reversal (). GATA
binding to Sry promoter does not depend solely on
interaction with FOG but also on a complex MAPK
(mitogen-activated protein kinase) signaling cascade
that results in phosphorylation of GATA (, ).
GATA is directly phosphorylated by p, and si-
multaneous inactivation of pa and pb MAPKs
leads to reduced Sry expression and gonadal sex re-
versal (). GATA phosphorylation and testis de-
velopment is also impaired in MAPK kinase kinase 
(Mapk)– and growth arrest and DNA damage-
inducible g (Gaddg)–mutant embryos (–).
MAPK and GADDg are thought to function up-
stream of p MAPKs, and reduced phosphorylation of
GATA and low expression of Sry are associated with
reduced levels of phosphorylated p (, ). In-
terestingly, Gaddg deficiency can be compensated for
by overexpression of MAPK, suggesting that these two
factors interact in the same cellular pathway during
gonadal sex determination. The role for GADDg in
the activation of GATA via the MAPK cascade is
supported by the fact that Gaddg expression shares
two key features of Sry expression, that is, narrow
temporal window and center-to-pole spatial expansion,
and, importantly, it is detected earlier than Sry ().
There are no known cases of human XY DSD due
to GADDg or MAPK mutations, but single
nucleotide changes in a related protein, MAPK,
are associated with testis dysgenesis and persistence
of Müllerian derivatives (). Interestingly, mice
lacking the cognate gene display overtly normal
Endocrine Reviews, August 2019, 40(4):857–905

testis development, implying species-specific dif-
ferences in involvement of MAPK family members
in SRY regulation ().
GADDg also provides a putative link to insulin/
IGF signaling and their role in sex determination.
Constitutive ablation of either via loss of insulin or the
IGF receptor are associated with growth and differ-
entiation defects of the AGP, adrenal gland agenesis,
and male-to-female sex reversal (, ). Although it
is not clear whether the sex differentiation defect in
these mice is due to a direct (insulin/IGF signaling
being integrated into one or more of the modules) or
an indirect (general proliferation defect with sub-
sequent reduction in the number of pre-Sertoli cells)
mechanism, implicit data suggest that insulin/IGF
signaling might be integrated into module  via
GADDg (). Homeoprotein TFs SIX and SIX
are also involved in Sry regulation via modules  and .
They directly regulate the expression of Fog and
Nra (the key TF of module ). SIX and SIX display
at least partial functional redundancy, as loss of Six
or Six alone does not affect embryonic gonadal
development, but loss of both leads to reduced Sry
expression and impaired testicular differentiation
().
The role of GATA in SRY regulation in humans is
unclear. Whereas GATA alone poorly activates the
SRY promoter, another well-known SRY regulator,
WT, potentiates its effect, as discussed later ().
Importantly, however, note that the complexity of
GATA regulation (phosphorylation and interaction
with FOG) might explain a lack of an effect in in vitro
promoter transactivation assays. Although GATA
mutations have been associated with ,XY DSDs,
cardiac defects are the most common clinical feature of
GATA mutation carriers (). It has been specu-
lated, however, that the mutations in these patients do
not interfere with the ability of GATA to interact and
synergize with other proteins (WT, NRA, FOG)
upon SRY regulation. GATA and FOG are expressed
in the  wpc human embryonic gonad, suggesting a role
in early gonad development ().
Module 2
The second group of factors implicated in regulation
of SRY converge on NRA, which directly binds to
the SRY promoter (, ). NRA (more com-
monly known as SF) is one of the most essential
factors of gonadal and adrenal development, and
NRA mutations account for one fifth of the cases of
,XY gonadal dysgenesis (). NRA is a master
regulator of gene expression throughout gonadal and
adrenal development, in steroidogenic tissues, and
during gametogenesis (). NRA mutations are
associated with a spectrum of phenotypes: from ,XY
gonadal dysgenesis and female external genitalia to
normal male sexual development with distal hypo-
spadias (, ). NRA is highly expressed in all
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derivatives of the AGP: testis, ovary, and the adrenal
gland (). Nra is induced very early in the mouse
gonadal primordium (~E.), but upon sex de-
termination its expression becomes sexually di-
morphic (). In the testis, Sertoli cells and Leydig
cells continue to express high levels of NRA,
whereas in the ovary it is transiently downregulated
and reactivation of its expression foreshadows the
onset of steroid hormone production ().
CBX, a core subunit of multiprotein polycomb
repressive complex  (PRC), is a chromatin modu-
lator that is associated with male-to-female sex reversal
in both humans and mice (, ). CBX is
expressed in the human embryonic testis at the time of
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SRY induction, implicating a role in early testis de-
velopment (). PRC takes part in posttranslational
modification of histones and is classically associated
with a transcriptionally repressive state of many genes,
but in the embryonic gonad it (or at least its CBX
component) is also implicated in activation of gene
expression (). Research performed on a human
Sertoli-like cell line suggests that CBX directly
regulates a number of genes involved in testis de-
termination or associated with DSD, including NRA
and SOX (). Notably, SRY, GATA (as has been
shown in the mouse), or WT do not appear on the list
of CBX-upregulated genes (, ). The conclusion
from the existing data is that CBX exerts its effect on
“…WT1 is the most important
regulation of SRY via stimulation of NRA expression regulator of SRY expression in
(, , ). This is a common theme for other humans.”
factors of module , too. These are SIX/SIX, LHX,
and CREB-binding protein/p-interacting trans-
activator, with Glu/Asp-rich carboxyl-terminal do-
main (CITED).
As discussed above, Nra is a direct target of SIX/
SIX (). It is therefore likely that both CBX and
SIX/SIX work in many at least partially redundant
ways to ensure sufficient activation of Sry transcription.
LHX is another sex-independent early marker of the
presumptive gonadal region of E. mouse urogenital
ridges (). Expression of Nra is dramatically re-
duced in Lhx-null mice, and they consequently display
complete gonadal agenesis (). Interestingly, Lhx is a
putative target of CBX in mice but not in humans (,
). CITED is a context-dependent transcriptional
coactivator or repressor that is expressed early on (E)
in the mouse AGP (). During adrenal development
CITED and WT cooperatively upregulate Nra in
the primordial adrenal gland (). Cited-deficient
male mice display delayed testis determination and
distorted testis histology (). Although it is likely
that a CITED/WT/NRA-dependent mechanism
operates in the gonadal part of AGP, too, CITED is at
best a relatively weak indirect inducer of Sry (, ).
There are no known cases of human DSD associated
with SIX, SIX, LHX, or CITED loss of function
(). A recent report speculates that a related gene,
CITED, might be more relevant than CITED in
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humans (). CITED is upregulated after sex de-
termination in the fetal testis, whereas CITED is
ubiquitously expressed in  to  wpc human testis
(). However, there are no data available from gonads
at the bipotential stage, and therefore the involvement
of CITED/ in human testis determination remains to
be shown.
Module 3
At the center of module  is WT, a TF that regulates
not only the expression of Sry but also that of Nra,
Dax, and Amh. Owing to its direct (SRY is a direct
target of WT) and indirect (via upregulating other
SRY expression-stimulating factors) effects, WT is
the most important regulator of SRY expression in
humans (, ).
There are many isoforms of WT that display
differential transcription regulatory functions, but the
main difference between the major isoforms is the
presence or absence of a three–amino acid sequence
[lysine-threonine-serine (KTS)]. In mice, an isoform
that lacks the KTS sequence, that is, WT(2KTS), but
not the longer isoform, that is, WT(1KTS), has been
shown to regulate the expression of Nra together
with LHX, making WT(2KTS) part of module 
(). This is an example of at least somewhat dif-
ferential functions that the isoforms have in devel-
opment and tissue maintenance as also illustrated by
phenotypic dissimilarities between Wt(2KTS) and
Wt(1KTS) knockout mice (). Wt(2KTS)-null
mice have streak gonads in both sexes and abnormal
Müllerian or Wolffian duct development, whereas
Wt(1KTS)-deficient mice display XY sex reversal
(). These data suggest that WT(2KTS) plays the
primary role in GR development, and once the
bipotential gonad is formed, WT(1KTS) induces a
testis differentiation cascade in XY individuals. Ad-
ditional pieces of data imply that the situation might
be more complicated in practice, and WT(1KTS)
has been implicated in testis determination via post-
transcriptional regulation of Sry mRNA ().
WT(2KTS) strongly activates the SRY promoter in
humans and generally displays stronger control on other
promoters [AMH and androgen receptor (AR)], as well
(, ). In vitro data show that human WT directly
binds to the SRY promoter and induces its transcription.
Interestingly, WT and SRY then synergistically regulate
gene expression in the nascent testis (). Both major
isoforms of WT have also been suggested to cooperate
with GATA to induce SRY in humans (). Moreover,
WT interacts with NRA to regulate SRY, but also
AMH and DAX (). Involvement of DAX brings
another layer of complexity, as DAX has been shown
to impinge on WT/NRA function by physically
opposing their interaction. Dosage of DAX is appar-
ently also important because DAX overexpression has
been associated with XY sex reversal both in humans
and mice (–). ,XY patients who carry a
872 Mäkelä et al Testis Development
duplication of the genomic area harboring the human
DAX gene (Xp) display a range of phenotypes from
partial to complete gonadal dysgenesis, emphasizing the
DAX dosage sensitivity of male sex determination (,
).
Male-to-female sex reversal in Dax-overexpressing
mice is likely due to functional antagonism between
SRY/NRA and DAX (). Excess DAX in-
terferes with activation of SRY downstream targets,
most notably Sox, by inhibiting the binding of Sox
stimulatory factors (SRY and NRA) to the Sox
promoter region (, ). Although there are no
known definitive reported cases of XY sex reversal
due to DAX loss of function, Dax-deficient mice
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display a variety of gonadal phenotypes from
complete male-to-female sex reversal to slightly
distorted prenatal testis development depending on
their genetic background (–). Similar to
overexpressing mice, disorder of male sex de-
termination stems from reduced expression/action
of SOX (, ). Interestingly, DAX expression
is induced in the human ,XY indifferent gonadal
primordium very early and roughly  week before
SRY, whereas in mice Dax is detected after Sry, at
E. (). Whereas DAX expression is main-
tained in both sexes through sex determination and
in the fetal gonads in humans, in mice the expression
in post–sex determination gonads is sexually di-
morphic with increased expression in the ovaries
(, , ). These data suggest that DAX might
play a somewhat different role in sex determination
and the maintenance of gonadal function in humans
and mice. Although it is unlikely that DAX would be
crucial for Sry regulation in mice owing to temporal
disconnect of its expression, it might be involved in
the same process in humans. Nonetheless, for male
sex determination to succeed its dosage needs to be
balanced in both species. Moreover, the action of
DAX during gonadal development might vary
depending on the availability of corepressors or other
interacting proteins ().
Module 4
Whereas the three previous modules centered on
individual TFs, their upstream regulatory mechanisms,
and multilayered interaction between the modules,
module  is different in nature, as it is needed to make
DNA accessible for transcriptional activators and thus
enable the function of modules  to .
DNA methylation status of a gene promoter
region is considered an indication of its transcrip-
tional activity. Whereas hypermethylated promoters
are associated with silent genes, actively tran-
scribed genes are usually characterized by promoter
hypomethylation. The Sry promoter region is hyper-
methylated in the mouse embryo at E., but this
genomic region undergoes spatially gonad-restricted
demethylation in the XY embryo within the
Endocrine Reviews, August 2019, 40(4):857–905

subsequent days and becomes hypomethylated by
E. (). Paralleling Sry downregulation, the sites
are remethylated by E.. These stage-specific
demethylation/remethylation events at the Sry pro-
moter are undoubtedly critical for proper expression
of Sry, but the underlying mechanisms are yet to be
identified. An obvious candidate with demethyl-
ase activity and association with Sry regulation,
GADDg might not play a role, as the Sry promoter
is hypomethylated in Gaddg-null mice at E.
(). DNA methylation has been shown to control
SRY in human cell lines, and SRY expression is
stimulated by a DNA-demethylating agent ().
Histone modifications are also an elemental part
of a genome’s epigenetic regulation. Some histone
marks are associated with repressed transcriptional
status, whereas others are found at loci of active
transcription. Methylation of histone  at lysine 
(HK) belongs to the former. HK demethylase,
lysine demethylase A (KDMA), has been implicated
in Sry regulation in mice, and Kdma-mutant mice
exhibit reduced levels of Sry and male-to-female sex
reversal with a variable phenotypic penetrance
depending on their genetic background. KDMA
probably acts independently from other modules, as
the expression of Gata, Nra, Wt, and others is not
altered in Kdma-deficient mice at E. ().
According to a model proposed by Kuroki et al. (),
KDMA accumulates at the Sry locus in the XY
bipotential gonad prior to induction of Sry. This re-
sults in HK demethylation, deheterochromatiniza-
tion, and opening up of the chromatin structure,
allowing access of TFs. However, based on a recent
study, removal of repressive marks is not sufficient but
H lysine  acetylation, a positive histone mark, is
also required for the proper activation of Sry ex-
pression (). What is responsible for recruitment of
histone-modifying enzymes to the Sry promoter at the
correct time is not known.
Over the threshold
According to the presented model (Fig. c), these four
modules constitute the molecular machinery that gov-
erns the regulation of mouse Sry. Species-dependent
differences in the function, significance, regulation, and
even the identity of the modules are likely, and the
current knowledge does not allow any comprehensive
conclusions about SRY regulation in humans to be
drawn. However, gene knockout studies in mice and
naturally occurring mutations in human have indicated
that induction of SRY depends on flawless concerted
action of different modules, and failure of any of them
might result in subthreshold expression of SRY and
impaired male sex determination and differentiation.
Once the threshold level of SRY protein is reached,
downstream events are triggered, and SRY alone or in
synergy with other TFs (such as WT) consequently
stimulates the expression of male-specific genes. Direct
doi: 10.1210/er.2018-00140
REVIEW
and indirect data imply that all of the modules are
activated to a similar extent in XX and XY embryos
prior to E., which is consistent with the notion that
all of the cells within the gonadal primordium are
poised between two developmental pathways. The
decision as to which path to follow depends on the
presence (endogenous or introduced) or absence of
the Sry gene that then dictates the fate of the gonad by
inducing differentiation of pre-Sertoli cells ().
Consistently, the gene expression patterns in pre-
sumptive testes and ovaries bifurcate soon after the
onset of SRY expression. The ovarian pathway and
differentiation into granulosa cells, the female somatic
counterpart, is concomitantly suppressed through
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downregulation of Wnt by FGF, an indirect target of
SRY (, ).
Sertoli cell specification
Sertoli cells are the first somatic cell type to be specified
in the presumptive testis, and a sufficient number of
them are required to initiate and maintain testicular
differentiation and block the ovarian pathway. Given
their critical role at the onset of male development, it is
important to understand what determines the number
of pre-Sertoli cells, as initially defined by expression of
SRY and subsequently by that of SOX. Whether the
Sertoli cells of an adult individual are the descendants
of the cells that activated Sry during its narrow window “Sertoli cells coordinate the
of embryonic expression, or whether it is possible to differentiation of all other cell
recruit new cells into Sertoli cell fate later, has been types within the testis,
debated (, ). Using a lineage-tracing approach, including germ cells.”
Sekido et al. () concluded that SRY acts uniquely
within the supporting cell lineage to direct the cell fate
decision, and under normal conditions cells that have
not initially expressed SRY are not recruited into this
lineage. According to this hypothesis, new Sertoli cells
cannot be recruited from Sry-negative somatic cells of
the gonadal primordium or the surrounding mesen-
chyme. Svingen and Koopman (), however, pro-
vided many lines of evidence supporting the notion
that mesonephric, CE, or GR cells that express Sox
at a low or undetectable level can be persuaded to
become SOX-positive pre-Sertoli cells. Furthermore,
research performed on chimeric mice shows that XX
somatic cells, which by definition are Sry-negative, can
adopt Sertoli cell identity (). These data strongly
support the idea of cell-nonautonomous entry into
the Sertoli cell fate. There is also a dramatic FGF-
stimulated increase in the number of pre-Sertoli cells
between E. and E. as SRY/SOX-expressing
cells proliferate, resulting in an overall increase in
the XY gonad size (–). In Wt(1KTS)-deficient
and Six/-mutant mice, cell proliferation within this
period is reduced, resulting in too few Sertoli cells to
maintain testicular differentiation and consequent
male-to-female sex reversal (, ). Notably, it is
not just the level of Sry expression per cell, but also the
number of Sry-expressing pre-Sertoli cells that is
https://academic.oup.com/edrv 873
REVIEW
critical for entry into the testis development pathway
().
The specification of Sertoli cells is a key event
during testis formation (Fig. a). Sertoli cells co-
ordinate the differentiation of all other cell types
within the testis, including the germ cells. Having a
single cell type as the orchestrator of testis differen-
tiation allows the fate of several cell lineages to be
coordinately specified.
SOX9
Commencing at ~E. in mice, upregulation of Sox
by SRY is a critical step for the development of a
fertile male (Figs. a and a). Similar to Sry, Sox is
upregulated in the GR following a center-to-pole
pattern (). Intriguingly, the successive waves of
Sry and Sox expression are not induced by a GR
central domain–derived diffusible factor, as they still
occur in isolated and cultured anterior and posterior
pole GR segments (). In ,XY human embryo,
SOX is upregulated following SRY, and it reaches
the highest expression at  dpc (). SOX protein
is not detected in a -wpc human XY embryo, but a
high level of SOX is observed in -wpc fetal human
testis (). SOX is indispensable and sufficient for
testicular organogenesis, and SOX can functionally
substitute for the lack of SRY and induce testis de-
velopment in an XX embryo both in humans and
mice (–, –).
The action of SOX during human sex de-
termination is strictly dose sensitive, and a critical
threshold for SOX expression has to be reached to
maintain testicular differentiation. Notably, hetero-
zygous mutations of SOX are associated with com-
plete ,XY gonadal dysgenesis, whereas duplication of
the region in chromosome  that houses the human
SOX gene results in ,XX testicular DSD (, ,
, ). SOX duplication is, however, a relatively
rare cause of ,XX testicular DSD, but there are a
number of reports of similar cases with an identified
mutation in the putative SOX regulatory region,
suggesting that ectopic and aberrant activation of
SOX might be the causative factor there (–).
Similarly, mutations at the SOX upstream enhancer
regions can result in partial or complete loss of SOX
transcription in the ,XY embryo and development
of an ovary instead of a testis (). The critical
thresholds for gene activation seem to be species-
dependent, as Sox heterozygous mutant males are
normal and a homozygous deletion is required to
reverse the sex of the affected XY mice ().
These findings make SOX the most important
direct target of SRY. Once induced above a certain
threshold level, SOX then orchestrates a cascade of
gene interactions and maintains its own expression via
an autoregulatory mechanism (Fig. a) (). SOX
coordinates the function of a male-specific tran-
scriptional network to achieve the following outcomes:
874 Mäkelä et al Testis Development
maintenance of Sertoli cell differentiation and identity,
suppression of the ovarian pathway, maintenance
of the Wolffian structures, and repression of the
Müllerian ones.
The expression of mouse Sox is controlled by
factors that bind to a .-kb enhancer region, called
testis-specific enhancer of Sox (TES) (). Prior to
SRY, NRA binds to and activates TES, then from
E. on SRY and NRA synergistically upregulate
Sox expression via TES, and after downregulation of
SRY, Sox expression is maintained by FGF, pros-
taglandin D synthase (PTGDS), doublesex and mab-
–related TF (DMRT), and, as described above, by
SOX itself (Fig. a) (, ). The expression of Fgf
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is directly or indirectly stimulated by SOX, and these
two thus seem to form a positive feedback loop that
maintains the high expression of both the genes and
represses that of Wnt, a key gene in ovarian devel-
opment ().
Interestingly, an FGF signal radiating outward
from the center of GR is needed for the sustained
expression of SOX at the anterior and posterior poles
(). Both FGF and prostaglandin D (PGD, a
product of PTGDS) have been implicated in re-
cruitment of SOX-low/negative cells into Sertoli cell
fate as a means of increasing the population size of pre-
Sertoli cells, which is critical for testis formation
(–). Emphasizing the importance of FGF
signaling, Sertoli cell differentiation is impaired in Fgf
and FGF receptor (Fgfr) (FGF receptor)–null mice,
and they consequently exhibit partial or complete
male-to-female sex reversal (, , ). The sig-
nificance of FGF/FGFR signaling in the nascent
human testis has not been characterized, but a het-
erozygous point mutation in FGFR has recently been
associated with ,XY male-to-female sex reversal
(). Additionally, FGF is expressed very early
( wpc) in the human embryonic testis, suggesting a
role during human testis differentiation, too ().
SOX also forms another feed-forward loop: PTGDS
is a direct target of SOX, and PTGDS in turn pro-
motes SOX transcriptional activity by promoting its
nuclear translocation (, , ). The significance
of PTGDS for maintenance of SOX expression is
probably less important than that of FGF signaling, as
testicular organogenesis is only mildly affected in
Ptgds-deficient mice and no known cases of DSD
with a mutated PTGDS gene in human patients have
been reported (, ). Working as an autocrine and
paracrine factor, PGD is, however, a potent agent,
and it can partially masculinize female embryos in its
own right (, ). DMRT, a gene needed for the
maintenance of Sertoli cell or male identity, supports
the expression of Sox and represses the female
pathway by suppressing forkhead box L (Foxl) ().
The significance of TES for human SOX expression
has been justifiably questioned, as there are no known
cases of DSD associated with TES mutation in
Endocrine Reviews, August 2019, 40(4):857–905
 REVIEW

humans. It has rather been shown that human SOX is
regulated by a distal enhancer element, termed RevSex
().
AMH
One of the most critical factors secreted by nascent
Sertoli cells is AMH. It belongs to the TGFb family of
growth factors and its production is maintained in the
testis until puberty (). For this reason it is
considered a specific marker for Sertoli cell functional
immaturity. AMH has a pivotal role in driving the
regression of Müllerian ducts, whereas androgens
derived from the fetal Leydig cells are responsible for
the maintenance of Wolffian ducts and their sub-
XY double-knockout gonads SRY was shown to ac-
tivate the expression of  genes, including desert
hedgehog (Dhh), Sox, and cytochrome P family
 subfamily B member  (Cypb) (). DHH is
crucial for fetal Leydig cell specification, SOX is a
close homolog of SOX and might compensate for its
loss, and retinoic acid (RA)–degrading enzyme
CYPB is specifically expressed in the testis to
prevent premature meiotic entry of germ cells (,
, ). These data provide a more multifaceted

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sequent development into male accessory sex glands
and urogenital structures. Recently, AMH has been
proposed to control gonocyte entry into a mitotically
inactive state during fetal development, and in the
neonatal mouse AMH has been implicated to promote
germ cell transition from gonocytes to spermatogonia
(, , ). When AMH or its action is missing,
Müllerian derivatives persist, emphasizing the signif-
icance of AMH for male urogenital tract development
().
AMH is upregulated in the human embryonic
testis during the eighth developmental week, and a
high protein expression of AMH is detected in the
human fetal testis from  wpc on (, ). Similar to
Sry, Amh expression is regulated by a number of
essential testicular factors: SOX, NRA, WT, and
GATA all have a stimulatory effect on Amh ex-
pression, whereas DAX inhibits its transcription via
an antagonistic function (–). Amh is a direct
target of NRA, but NRA also regulates Amh
expression synergistically with WT(2KTS) and
SOX// (, ). A similar synergism has been
observed between GATA and WT(2KTS) ().
These are just a few examples of the complex network
of factors and interactions behind the regulation of
Amh transcription. Although SOX is needed for the
induction of Amh expression, it remains to be elu-
cidated how different factors converge on Amh reg-
ulation and how they collaborate, cooperate, and
interact to control the sex-dependent expression of
AMH ().

Other SOX family genes

For good reason, SOX has been regarded as a de facto
inducer of male development that is needed for both
activation of a testis-specific transcriptional program
and repression of the ovarian pathway (Fig. a).
However, Nicol and Yao () asked a very important
question: What is to become of the bipotential gonad
if both SOX and b-catenin, SOX’s functional
counterpart in the female, are absent? Interestingly,
both XY and XX gonads progressively lean toward the
testis fate, but XY double-knockout gonads are more
masculinized than are their XX counterparts. In the
Figure 4. (a) Regulation of SOX9 and its role in testiculogenesis. Sox9 is initially induced by
the synergistic action of SRY and NR5A1. Later, SOX9 maintains its own expression either directly
or by feed-forward loops with PTGDS, FGF9/FGFR2, and DMRT1. SOX9 orchestrates testicular
development by maintaining Sertoli cell differentiation/identity and repression of the ovarian
pathway either directly or via DMRT1 and FGF9 signaling. DHH and AMH are among the key SOX9
downstream genes needed for the successful development of the male urogenital system. (b)
Maintenance of the testicular phenotype. DMRT1 maintains male sex postnatally together with
SOX9. Besides maintaining the expression of testicular genes, they repress those that are active in
ovarian development (Wnt4, b-catenin). DMRT1 also silences RA action at sex-sensitive loci (Foxl2,
Esr2). When DMRT1 is absent, RA is able to activate feminizing genes, which also repress Dmrt1 and
Sox9 that are needed to maintain Sertoli cell identity. AR signaling impinges on Foxl2 transcription
in mouse Sertoli cells. RAR, RA receptor; T, testosterone.

doi: 10.1210/er.2018-00140 https://academic.oup.com/edrv 875

REVIEW
role for SRY in testis determination than previously
thought.
In general, the role of SOX family genes in testis
determination is intriguing. Besides the two indis-
pensable genes (SRY and SOX), other members of
the family have been implicated in transformation of
the bipotential gonad into a testis. Following SOX,
SOX and SOX are upregulated in the mouse
XY gonad at ~E (, ). Sox-null mice display
progressive degeneration of the seminiferous epi-
thelium and infertility by  month of age (, ).
Use of inducible knockout mouse models has shown
that the initial testis determination is dependent on
SOX, but SOX and SOX act synergistically dur-
ing differentiation and maturation of testicular
structures (, ). SOX and SOX have both
been associated with female-to-male sex reversal in
overexpressing transgenic mouse models and in
,XX patients with a duplication in the genetic
regions that encompasses either of these genes (,
, , ). The ability of SOX and SOX to
direct male development can be explained by two
different mechanisms: either they substitute for lack
of SRY, or they activate the same genes as SOX.
There is evidence for both (, ). A lack of
gonadal phenotype in prenatal Sox- and Sox-
deficient mice suggests that neither is essential for
testis determination or differentiation. Recently, SOX
C group proteins SOX, SOX, and SOX have
also been implicated in testicular organogenesis
(). Sox deficiency results in elongated gonads in
both sexes at E. and in more testis cords in XY
embryos (). In conclusion, SOX family genes are
absolutely crucial for testicular organogenesis. Their
functional redundancy due to shared transcriptional
targets calls for their levels of expression to be tightly
regulated in both sexes, as misregulation might result
in impaired gonadal development and partial or
complete gonadal dysgenesis.
Formation of the fetal testis
Testis cord formation
Soon after the emergence of SOX-expressing pre-
Sertoli cells, cells within the long and thin GRs start
to reorganize. Aggregation of pre-Sertoli cells initi-
ates testis cord formation, and germ cells are sub-
sequently encased within the nascent cords whereas
non-Sertoli somatic cells are left on the outside (Fig.
a). The gonadal length in mice is reduced until
E., whereas its width quadruples from E. to
E. (). Around  protocords are initially
formed but their number is reduced to ~ by E.
when these structures form sharp-edged loop-like
structures that are surrounded by the testicular
interstitium, and are now referred to as testis cords
(). As a result of Sertoli cell proliferation, the testis
cords subsequently become longer and wider and are
876 Mäkelä et al Testis Development
connected at their proximal regions to a single cord-
like structure, the presumptive rete testis that runs
parallel to the mesonephros. The individual cords
further elongate and start to coil by E., which is
probably an indication that the intratesticular space
becomes limiting due to emergence of the tunica
albuginea, a stiff fibrous capsule surrounding the
testis (, ).
SOX has been shown to be indispensable for testis
cord formation, but during subsequent testicular
differentiation its function can be substituted for by
SOX and SOX (, ). As reviewed by Svingen
and Koopman (), in vitro and in vivo data imply
that neurotrophic tyrosine kinase receptors and their
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ligands are involved in the aggregation and cord
formation process of pre-Sertoli cells. FGF has also
been implicated in testis cord formation but its effect
might be indirect (stimulation of pre-Sertoli cell
proliferation and maintenance of their identity) (,
). Inhibition of TGFb, Activin, and Nodal signaling
also impinges on early testiculogenesis, suggesting that
ligands and receptors of these pathways are needed for
normal male gonadal development (). Notably,
germ cell–derived molecules are not essential for cord
formation, as testis cords do develop in germ cell–
deficient mice (, ). However, according to a
recent report, cord formation is severely affected in
We/We (Kit mutant) mice that have very few intra-
gonadal germ cells due to a PGC migratory defect
(). The authors take it as a proof of importance of
bidirectional (Sertoli–germ cell) signaling during early
testiculogenesis, but further independent approaches
are needed to confirm that the phenotype is not due to
indirect effects of abnormal SCF/KIT signaling.
Origin of other testicular cell types
As discussed above, hPGCs arrive at the developing
gonad over a wide temporal window, and many
hPGCs are found outside the presumptive testis at
the time of SOX induction during the seventh week
of development (, , , ). It is not known
when cord formation and tubularization have pro-
ceeded far enough in the human fetal testis to im-
pede incorporation of late migrating PGCs into
the testis cords. It is, however, conceivable that the
formation of the tunica albuginea makes testicular
entrance for PGCs, or any other cell type, very
difficult if not impossible (). Formation of the
testis cords brings changes to the nomenclature and
the properties of the cell types involved: pre-Sertoli
cells become fetal or immature Sertoli cells that
undergo a transition from a mesenchymal cell-like
state to a polarized epithelial cell, and PGCs that
are engulfed by testis cords become gonocytes (or
prespermatogonia/prospermatogonia in rodents)
whose fetal and prepubertal development is the
topic of one of the following sections. PGC-to-
gonocyte transition encompasses a significant shift
Endocrine Reviews, August 2019, 40(4):857–905

in germ cell transcriptome toward what could be
called a gonadal-stage germline marker expression
profile.
Not only the Sertoli and germ cells but also all the
other cell types present in the adult testis originate
from precursors that are specified or recruited into
testis parenchyma during fetal development. VECs and
peritubular myoid cells (PMCs) are intimately involved
in the formation of testis cords. VECs are derived from
the mesonephros. Vascular endothelial growth factor
(VEGF) and platelet-derived growth factor (PDGF)
signaling mediate the migration of VECs to the XY
gonad from E., and formation of testicular arteries
is subsequently observed (–) (Fig. a). For-
mation of testicular vasculature and lymphangio-
genesis has been recently reviewed elsewhere ().
Blocking the migration of these cells is detrimental
for testicular morphogenesis, and endothelial cells
have a crucial role in establishing tissue architecture
during testiculogenesis (–). Platelet and en-
dothelial cell adhesion molecule  (PECAM)–positive
putative VECs are first observed in the human testis at
 wpc, but vascularization proceeds slowly and large
vessels with PECAM-stained endothelia are first seen
in  wpc testicular interstitium, but they might begin
to form as early as  wpc ().
Despite decades of research, the origin of PMCs
remains profoundly mysterious. Similar to VECs, they
were previously thought to derive from the meso-
nephros, but more recent research has elucidated that
the immigrant cells are exclusively endothelial and do
not contribute to PMCs or other interstitial lineages
(, –). PMCs at the periphery of mouse testis
cords become morphologically distinct by E. ().
Clearly identifiable testis cords or protocords in  wpc
human fetal testis seem to lack PMCs, but smooth
muscle actin–positive PMCs enclose the testis cords by
 wpc ().
One remarkable difference in the histology of
rodent and human testicular tissue is the organization
of peritubular cells. Whereas only one layer of PMCs
encapsulates rodent seminiferous tubules, the sheet of
peritubular cells in humans is thick and consists of five
to seven cellular layers and intertwined ECM, and is
called the lamina propria. Besides PMCs, fibroblasts
are also incorporated into the human lamina propria.
Impaired spermatogenesis is often associated with
thickening of the lamina propria due to accumulation
of ECM between the cellular layers (). Both Sertoli
cells and PMCs contribute to the ECM and the basal
lamina of the seminiferous epithelium ().
An intriguing population of testicular somatic cells
that is often neglected is the macrophages. Although it
has been known already for quite some time that
interstitial macrophages intimately interact with
Leydig cells and play a role in androgen production, a
new subset of macrophages was recently discov-
ered intermingled with PMCs in the mouse testis
doi: 10.1210/er.2018-00140
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(–). Although being implicated in control of
SSC maintenance and differentiation, the ultimate
function for these cells is yet to be defined. Recently, it
was shown that the ontogeny of testicular macrophage
subsets differs, and although the interstitial macro-
phages are derived from the yolk sac, the origin of
peritubular macrophages is distinct and they emerge
postnatally from bone marrow–derived progenitors
(). As the mouse ages, the interstitial compartment
is also replenished by bone marrow–derived
progenitors.
Development of Wolffian derivatives and external
genitalia depends on production and secretion of
androgens by fetal Leydig cells relatively early during
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fetal development. Upregulation of genes encoding
the key known components of testicular steroido-
genesis [LHCGR, STAR, HSDB, hydroxysteroid
b-dehydrogenase  (HSDB), CYPA, and
CYPA] is observed already roughly a week after
SRY, during the eighth wpc, and they generally
reach a plateau of high expression by the end of the
following week (). Similar to PMCs, the embry-
onic origin of (fetal) Leydig cells is a topic of heated
debate. Disruption of embryonic Hedgehog (HH)
signaling results in abnormalities in both the cell
populations, suggesting that HH signaling might act
on a common precursor. The testis-specific member
of the HH family of secreted proteins, DHH, pro-
“…SOX family genes are
duced by (pre)Sertoli cells, is one of the first genes to absolutely crucial for testicular
be expressed in the mouse XY gonad after Sry (). organogenesis.”
In the mouse testis, HH signaling plays a crucial role
in specification and differentiation of fetal Leydig
cells and PMCs (, –). Whereas Dhh-mu-
tant mice are azoospermic and display abnormal
testicular histology, DHH mutations in humans have
been associated with complete or partial ,XY go-
nadal dysgenesis (, –). In conclusion, both
Leydig cells and PMCs are likely specified by Sertoli
cell–dependent mechanisms. This signifies the need
to maximize the population size of pre-Sertoli cells
by direct (high endogenous SRY) or indirect (re-
cruitment into Sertoli cell fate via secreted factors)
means. Specification of the interstitial cell lines, in-
cluding fetal Leydig cell population and the origin of
adult-type Leydig cells, have been recently reviewed
elsewhere ().
Testicular Descent
Testicular descent from intra-abdominal position into
the scrotum is a continuum that has often been de-
scribed to have two main phases: the transabdominal
phase and the inguinoscrotal phase. In the trans-
abdominal phase the testes are anchored close to the
inner entrance of the future inguinal canal, and in the
inguinoscrotal phase the testes pass through the in-
guinal canal into the scrotum. The two phases have
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REVIEW
been described to occur in humans between the th
and th gestational weeks (GWs) and between the
th and th GWs, respectively (, ). In rodents,
the inguinoscrotal phase occurs postnatally around
puberty (, ).
Gubernaculum is a mesenchymal structure be-
tween testis and inguinal area and it has an important
role in testicular descent. According to mouse models,
the transabdominal phase of testicular descent is de-
pendent on Leydig cell hormone insulin-like peptide 
(INSL) inducing male-like development of the
gubernaculums (the shortening of the cord and en-
largement of the bulb) (–), which anchor the
testes into the inguinal area and prevent them from
ascending when the fetus grows (). Signaling of
INSL and its receptor [relaxin family peptide receptor
 (RXFP)] affects myogenic differentiation of the
gubernacular mesenchymal cells possibly via WNT/
b-catenin pathways (, ). If the male-like de-
velopment of the gubernaculum is disrupted, the testes
are freely moving and they locate high in the abdomen
(–).
In humans, both male and female fetuses have been
described to show in early pregnancy an inner descent
of gonads that is dependent on descending anlage of
the diaphragm (). Later on, after regression of the
Müllerian ducts and the involution of the cranial
mesonephric ligament in male fetuses, the testes glide
across the genital ducts (Wolffian ducts) and dive into
swollen gubernaculums (). However, it has also
been suggested that gubernaculum is attached only to
ductus deferens and epididymis but not directly to
testis (, ). INSL is likely to play a role in
testicular descent also in humans, because it has been
detected only in amniotic fluid of male fetuses (,
). Congenital cryptorchidism has been associated
with reduced INSL levels in cord blood (, ).
However, mutations in the genes of INSL or its
receptor are rare in cryptorchid patients (, ).
Boys with persistent Müllerian duct syndrome due to
mutations in the genes of AMH or its receptor
have abnormally long gubernaculums and unilateral
cryptorchidism or bilateral intra-abdominal testes
(, ). Thus, also AMH is likely to have a role in
the transabdominal phase of testicular descent in
humans, in the shortening of the gubernacular cord.
In the mouse, AMH is not needed for the descent of
the testes (). However, it has been suggested that
AMH may still affect the length of the gubernacular
cord ().
In rodents, in the inguinoscrotal phase, the bulb of
the gubernaculum remodels and everts into a hollow
cone, and this eversion creates the processus vaginalis
(a diverticulum of the peritoneal membrane) ().
Thereafter, the gubernaculum elongates to the
scrotum similar to a limb bud and becomes sec-
ondarily attached to the scrotal skin (, ). The
processus vaginalis remains open in rodents and the
878 Mäkelä et al Testis Development
inguinal canal is plugged by a fat pad on the epi-
didymis ().
The inguinal canal differentiates around the
gubernaculum during early fetal development in
humans (). Prior to the transinguinal passage of the
testes, swelling of the gubernaculums has been ob-
served and this widens the inguinal canals (, ).
However, no eversion of the gubernaculum occurs in
the inguinoscrotal phase of testicular descent in
humans (). After the testes, epididymides, and
gubernaculums have passed through the inguinal
canal as a unit, possibly with the help of intra-
abdominal pressure, the gubernaculums start to
shrink (, ). The testes and epididymis and
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gubernaculum migrate from the external inguinal ring
into the scrotum following the genital branch of the
genitofemoral nerve (). The transinguinal descent
of testes starts around GW  to , and usually testes
have descended into the scrotum by the th GW
(–). The scrotal ligament remains as a remnant
of gubernaculum and the processus vaginalis closes
().
Androgens are important for the inguinoscrotal
phase of testicular descent, and this phase is usually
disrupted both in mice and boys with androgen in-
sensitivity (testicular feminization syndrome) (,
). However, mutations of the AR are rare in pa-
tients with isolated undescended testis (). In the
mouse, androgens also cause the regression of the
cranial gonadal ligament, the cranial suspensory
ligament, during the transabdominal phase of tes-
ticular descent (). However, androgens are
needed already before the inguinoscrotal phase, and
only exposure to antiandrogens during the out-
growth of the gubernaculum or earlier disrupts the
postnatal inguinoscrotal descent of testes in rodents
(, ). The target of androgens in testicular
descent is unknown, but animal studies suggest that
androgens affect migration of the gubernaculum in
the inguinoscrotal phase indirectly via genito-
femoral nerve and its neurotransmitter calcitonin
gene–related peptide (CGRP) and inguinoscrotal fat
pad (, ). In addition to androgens, the INSL/
RXFP-mediated pathway has also been suggested
to have a role in the inguinoscrotal descent of the
testis ().
The prevalence of undescended testis has been
reported to be % to % among full-term boys in
prospective cohort studies. Higher prevalence has
been observed among premature babies and in boys
born small for gestational age (). Usually the
inguinoscrotal phase of testicular descent is dis-
rupted (). Spontaneous descent of undescended
testis is often noticed during the first few months of
life, likely due to temporary postnatal activation of the
hypothalamus–pituitary–gonadal (HPG) axis and as-
sociated increase in reproductive hormone levels (,
–).
Endocrine Reviews, August 2019, 40(4):857–905

Disorders of Testicular Development
Most DSDs have an unknown etiology, although our
knowledge on the molecular genetic basis of gonadal
development has rapidly increased. Disorders of tes-
ticular development can originate on different levels:
development of the bipotential gonad, differentiation
of the testis, or function of the testis. Germ cells are not
necessary for gonadal differentiation that occurs under
endocrine, paracrine, and autocrine regulation. A
bipotential gonad develops from the gonadal pri-
mordium in the CE on the ventral side of the me-
sonephros. Several genes are active at this early phase
on GW  to , including GATA, WT, and the
nuclear receptor SF (NRA) (). Some mutations
in WT lead to ,XY gonadal dysgenesis (),
whereas some NRA mutations can cause agenesis of
both the adrenal gland and gonads (). However, the
phenotypic variation is large and adrenal agenesis is
not as frequent as gonadal dysgenesis ().
Differentiation of the supporting cells in the
bipotential gonad to testicular Sertoli cells is the de-
cisive step in sex-specific development. This is initiated
by the SRY gene that is activated on  dpc (, ).
Factors that are active in the early phase of devel-
opment (GATA, SF, WT) and MAPK pathway are
controlling SRY, as well as epigenetic modulation by
DNA methylation, histone modification, and chro-
matin modeling (). Gene variants of CREB-binding
protein and its related paralog p that operate in
histone modifications have been associated with DSDs
in some cases (). Mutations in SRY are causing
,XY gonadal dysgenesis, whereas most ,XX tes-
ticular DSD is caused by a translocation of SRY-
containing piece of chromosome Y ().
Specification of Sertoli cell lineage is secured by the
SRY target gene SOX (). Mutations in SOX cause
,XY gonadal dysgenesis and severe campomelic
dysplasia that are often lethal early in life ().
Mutations in the distal enhancer of SOX, RevSex,
have also been associated with DSDs (). SOX acts
in concert with DMRT, GATA, SF, and WT to
control the target genes necessary for testicular de-
velopment. Once SOX gene is activated, it can
upregulate its own expression via FGF and PGD
(, ) (Fig. a). Ovarian differentiation is promoted
by WNT, R-spondin  (RSPO), and b-catenin, which
are suppressed by SOX and FGF tilting the devel-
opment into the male direction. When SOX activity is
compromised, the central part of the gonad may de-
velop into testis, whereas the poles differentiate to the
ovarian direction, causing a rare ovotesticular dysgen-
esis. The SOX gene family includes other members in
addition to SRY and SOX, such as SOX and SOX,
that may also contribute to gonadal development, be-
cause duplications of the SOX-containing gene region
in chromosome q is associated with ,XX gonadal
dysgenesis (). Deletions in chromosome p
doi: 10.1210/er.2018-00140
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including DMRT can cause ,XY gonadal dysgenesis
(). Sertoli cells are rapidly proliferating first until
GW , then after birth during the minipuberty for the
first  months of life, and finally at early puberty before
the onset of spermatogenesis ().
Sertoli cells form the seminiferous cords that also
encompass the germ cells that have migrated from the
yolk sac via hindgut to the gonad. Sertoli cell–derived
AMH induces regression of paramesonephric struc-
tures that otherwise develop to oviducts, uterus, and
the upper third of vagina. Mutations in AMH or its
receptors cause the persistent Müllerian duct syn-
drome where a boy has both male and female internal
sex organs.
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In the interstitial space some of the progenitor cells
differentiate to Leydig cells. These cells activate Notch
signaling, downregulate WT, and upregulate SF and
steroidogenic enzymes, leading to active steroid syn-
thesis. A balance between proliferating progenitor cells
and differentiating steroidogenic cells has to be
maintained. Aristaless-related homeobox (ARX) and
chicken ovalbumin upstream promoter TFII (COUP-
TFII, officially NRF) are hallmarks of progenitor-
like cells, and their expression declines whereas that of
SF and steroidogenic enzymes increases. Mutations
in ARX can lead to poor masculinization of the ,XY
fetus due to a small number of Leydig cells ().
Notch signaling controls the population of the pro-
“Differentiation of…testicular
genitor cells, and the loss of action leads to an in- Sertoli cells is the decisive step
creased mass of fetal Leydig cells and a declined in sex-specific development.”
progenitor pool (). Mutations in the mastermind-
like domain containing  (MAMLD) gene, a gene
required in Notch signaling, can lead to complete
,XY gonadal dysgenesis (). Sertoli cells play an
important role in proliferation and differentiation of
Leydig cells by secreting DHH and PDGF (, ).
Mutations leading to compromised DHH signaling
can also cause ,XY gonadal dysgenesis (). Fetal
Leydig cells involute after birth and adult Leydig cells
develop from an own progenitor pool ().
In rodents, fetal Leydig cells are unable to syn-
thesize testosterone, and they secrete androstenedione
and other androgens that are considerably less potent
than testosterone. Production of testosterone from
these precursors then takes place in Sertoli cells that
express HSDB (, ). In the human fetus,
Leydig cells differentiate right after Sertoli cells at  to
 wpc, and the testis starts to produce increasing
amounts of testosterone. Androgens are needed for
masculinization of internal and external genital tract.
Testosterone is further converted to DHT in the target
tissues by a-reductase, which is necessary for effective
androgen action, because DHT is much more potent
than testosterone. Development of the prostate and
external genitalia is critically dependent on DHT. Loss
of function of a-reductase and defects in steroido-
genic machinery in Leydig cells result in various de-
grees of ,XY DSD where undermasculinization can
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REVIEW
present with undescended testes, hypospadias, micro-
phallus, and a short anogenital distance up to completely
female external genitalia. Hypogonadotropic hypo-
gonadism can cause a similar phenotype, albeit usually
less severe, because the Leydig cells need pituitary go-
nadotropin stimulation, particularly during the latter
half of pregnancy when placental chorion gonadotropin
levels decline. Inactivating luteinizing hormone (LH)
receptor mutations lead to ,XY DSD with a female
phenotype ().
The most common reason for ,XY DSD is in-
sensitivity of the AR, which may lead to completely
female external phenotype in the case of complete
androgen insensitivity or undermasculinization when
the defect is partial. Mutations are found usually in the
exomes of AR, but intronic variants have also been
identified ().
Maintenance of Testicular Phenotype
The genetic sex of an individual is determined at the
moment of fertilization. For the first  weeks the
development of XY and XX embryos is synchronized,
but then induction of SRY sets the ball rolling and the
future males embark on a developmental trajectory
that ultimately gives rise to all the characteristics that
are collectively referred to as “maleness.” As discussed
above, determination of the gonadal phenotype de-
pends on a concerted action of a number of genes, very
few of which are critical, and the process is strictly
regulated in time and space. Surprisingly, the sex
identity of gonadal supporting cells is not fixed once
specified but needs to be constantly maintained.
Failure to do so might result in cellular trans-
differentiation and compromise the reproductive
function (, ). Emerging data suggest that there
are two TFs that are responsible for the maintenance
of the sex identities of gonadal supporting cells by
promoting a specific sex-dependent transcriptional
circuitry and repressing the opposite one (Fig. b).
These two mutually antagonistic factors are DMRT
and FOXL.
DMRT1 in testis development
DMRT transcripts are detected alongside with SRY in
 to  wpc male human embryos but not in female
embryos (). During the first half of pregnancy,
DMRT is principally expressed by Sertoli cells, but
thereafter the expression in germ cells becomes
dominant. However, prenatal germ cells, from -wpc
PGCs to mitotically quiescent gonocytes, express
DMRT (). Importantly, no DMRT expression is
observed in the somatic compartment of the ovary,
and it is lost from oogonia upon meiotic entry (,
). A similar expression pattern is found in the
mouse, and DMRT expression becomes sexually
dimorphic at the onset of gonadal differentiation with
880 Mäkelä et al Testis Development
decreasing levels in the ovary and increasing expres-
sion in the testis ().
Mutations in the human DMRT gene have been
associated with ,XY gonadal dysgenesis and male-
to-female sex reversal (–). Interestingly, Dmrt-
null XY mice display overtly normal embryonic
testicular development, but the Sertoli cells of these
mice fail to mature and do not cease to proliferate
(). Germ cell maintenance in these mice is severely
disrupted, and they are totally depleted by  weeks of
age (, ). Adult Dmrt-null mice are charac-
terized by dysgenic testes with few poorly organized
tubules lacking the lumen, as well as testicular fibrosis
and/or Leydig cell hyperplasia (). Cell type–specific
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ablation studies of Dmrt further demonstrate that
Dmrt expression in germ cells is needed for their
homing from the tubular lumen to the basement
membrane of the seminiferous epithelium, a critical
step for the onset of spermatogenesis and formation
of germline stem cell pool (). Germ cell survival,
maintenance, and differentiation also depend on
Dmrt expression in Sertoli cells, suggesting that
DMRT is a master regulator of Sertoli cell function.
According to these data, there might be a discrepancy
in the role of DMRT in testis development in hu-
mans and mice, and DMRT is probably needed at
an earlier stage in humans to maintain the gonadal
somatic cell identity.
FOXL2 in ovarian development
FOXL is a critical gene in ovarian development and
maintenance of the ovarian function. It is expressed in
both granulosa and theca cells. However, the signifi-
cance of FOXL varies considerably depending on the
species. Although it is indispensable for female de-
velopment in the goat, and XX goat embryos lacking
Foxl develop testes, in humans and mice loss of its
function is associated with ovarian failure and in-
fertility (–). A conditional deletion of Foxl in
the adult mouse ovary leads to reprogramming of the
ovarian granulosa and theca cells to the testicular
counterparts, Sertoli and Leydig cells, respectively
(). This transdifferentiation process is driven by
induction and activation of Dmrt and Sox in the
ovary. Conversely, loss of Dmrt in the adult testis
leads to de-repression of Foxl, downregulation of
Sox, and transformation of testicular cell types into
ovarian equivalents (, ). These data provided
the first definitive proof that somatic cell identities of
gonadal-supporting and steroidogenic cells are not
irreversible, and active measures need to be taken to
maintain their characteristics and functionality. Such
plasticity of cell fates is indeed surprising because one
might expect that gonadal somatic cells would
employ a set of mechanisms to permanently silence
the genes that are involved in sex determination
and differentiation of the opposite sex, such as
chromatin and histone modifications. Self-evidently,
Endocrine Reviews, August 2019, 40(4):857–905

however, this is not the case and the gonadal phenotype
must be considered a relatively labile property.
The maintenance mechanism of gonadal somatic
cell sex identity
How does the loss of Dmrt then mechanistically con-
tribute to the activation of female-specific transcrip-
tional circuitry and adoption of ovarian supporting cell
identity? It has been demonstrated that fetal sex de-
termination, transdifferentiation, and maintenance of
the sex postnatally are mechanistically related to each
other (). DMRT maintains male sex postnatally
together with SOX, and the genes that they repress
include those that are active in the female sex de-
termination network (Fig. b). Additionally, DMRT
silences RA action at sex-sensitive loci [e.g., Foxl, es-
trogen receptor  (Esr)] (). When DMRT is absent,
RA is able to activate feminizing genes, which, besides
promoting the granulosa cell fate, repress the male
program that is needed to maintain Sertoli cell identity.
Interestingly, AR signaling also impinges on Foxl
transcription in mouse Sertoli cells, suggesting that
testosterone is actively involved in the maintenance of
Sertoli cell identity (). Thus, two highly potent
hormones, testosterone and RA, work in opposing di-
rections with respect to maintenance of testicular go-
nadal phenotype, and DMRT is required to restrict the
action of RA to permit its beneficial spermatogenesis-
promoting action and prevent the harmful induction
of the feminizing genes (Fig. b). Interestingly,
enforced expression of Dmrt alone in XX mouse
fetal gonads is sufficient to drive testicular differ-
entiation and development of male secondary sex
characteristics (, ).
As discussed above, DMRT is expressed in the
GRs at the time of sex determination in both
humans and mice (, ). If the bipotential
gonad assumes ovarian fate, Dmrt is promptly
downregulated, whereas in the testis its expression is
enhanced (). When does the DMRT function
then become essential for the maintenance of tes-
ticular gonadal identity? Based on the available data,
it is hard to judge. Both global Dmrt-null mice, as
well as mice where Dmrt has been ablated in a
Sertoli cell–specific manner from E. on, have
seemingly normal prenatal testis development (,
, ). However, in both, the maintenance of
testicular structures is severely compromised soon
after birth. Whereas global Dmrt deficiency results
in germ cell depletion and Sertoli cell hyperplasia by
postnatal day , the Sertoli cell–specific ablation of
Dmrt from E. on does not seem to have any
immediate effect on testiculogenesis until  weeks of
age, when the first signs of gradual loss of Sertoli cell
identity become evident (, ). Subsequently,
the number of SOX-expressing gonadal-supporting
cells is dramatically reduced, the gonadal architecture is
rearranged, and Foxl is strongly induced by  month
doi: 10.1210/er.2018-00140
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of age (). These cases of testicular dysgenesis
are phenocopied by conditional inactivation of Sox
on a Sox-knockout background resulting in down-
regulation of Dmrt (, ). In conclusion, these
data indicate that SOX/ are needed for the main-
tained expression of Dmrt. The opposite is equally
true, and the data suggest that SOX is involved yet in
another feed-forward loop to maintain its high ex-
pression (Fig. ) ().
Roughly a -month period is needed for the effects
of Dmrt loss to become visible independent of the
time of the genomic rearrangement (, ). This
indicates that there are redundant mechanisms that act
together with DMRT to maintain testicular identity
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and buffer against detrimental effects of stochastic
changes in DMRT expression. The factors that are
involved act either to maintain SOX expression or
repress the induction of feminizing genes. Such a
function has been assigned at least to FGF, that,
besides stabilizing SOX and stimulating its expres-
sion, is primarily responsible for repression of female
genes, including Wnt (, , ). Importantly,
note that gonadal SOX expression is repressed by
WNT and its downstream effector b-catenin, and
WNT also indirectly impinges on Sry (–).
Furthermore, it is also possible that estrogen synthesis
may have to reach a specific threshold for the proper
onset of the feminizing program and repression of the “In humans, fetal and
testicular one. Estrogen dependency and existence of prepubertal differentiation of
an FGF-like pro-maleness mechanism might together the germline is characterized
provide an explanation for the -month schedule of by asynchronicity….”
testis-to-ovary transformation.
Based on association of human chromosome p
deletions removing DMRT with ,XY male-to-
female sex reversal, it is reasonable to assume that
DMRT function in humans and mice is conserved.
However, Dmrt-null mice are born male, whereas
,XY patients, who carry a mutation at chromosome
, may have fully feminized external genitalia and
display complete gonadal dysgenesis (, ). The
difference may be ostensible and can be explained by
considerably longer human gestation that may permit
reprogramming from testis to ovary already during
fetal development. Nonetheless, species-dependent
differences in molecular control of the process can-
not be ruled out.
Regulation of Gonocyte Proliferation,
Quiescence, and Differentiation
A theory prevails that the sex identity of PGCs is
undefined prior to determination of the somatic sex.
The sex of PGCs and their entry into the male or
female gametogenic pathway is dictated by strong
inductive signals from the gonadal somatic cells. In
humans, embryonic/fetal germ cells can be subdivided
into three categories (in chronological order): PGCs
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(migratory), gonocytes (proliferatively active), and
prespermatogonia (displaying variable mitotic activ-
ity). This is confirmed by recent RNA sequencing
analysis performed on germ cells isolated from - to
-wpc human male embryos, indicating that there are
three transcriptionally distinct populations of germ
cells in the male fetus and transition from one state to
the following involves dramatic changes in tran-
scriptome (). In rodents, the developmental dynamics
are somewhat different, as gonocytes first divide actively,
then enter a phase of mitotic quiescence, and re-
sumption into the cell cycle coincides with differenti-
ation into spermatogonia. Gonocytes-to-spermatogonia
transition involves remarkable changes in cell shape,
size, gene/protein expression, and intratubular location.
In humans, fetal and prepubertal differentiation of the
germline is characterized by asynchronicity, as gonads
of both sexes simultaneously contain at least two de-
velopmentally distinct subpopulations of germ cells
(). In this section, we first concentrate on rodent fetal
and early postnatal germ cell development because it
helps to understand the dynamics and the molecular
regulation of the same events in humans. In the
latter part, human prespermatogenic germ cells are in
focus.
Rodent prespermatogenesis
In mice, the first PGCs arrive at the presumptive gonad
at ~E, and most of the PGCs have colonized the GRs
by E. (, ). When Sry is induced, gonadal PGCs
are incorporated into testis cords starting at E and
from then on the germ cells that are present in the
testis cords are referred to as gonocytes or prosper-
matogonia (, , ). The term prespermato-
genesis is sometimes used to denote the phase of
testicular development involving gonocytes (, ).
Furthermore, to dissect the gonocyte population into
functional categories, appendixes M-, T-, and T- are
occasionally used. The term M-gonocytes (M for
multiplying; M-prospermatogonium) refers to mi-
totically active early gonocytes, T-gonocytes (T for
transitional; T-prospermatogonia) are mitotically
quiescent but undergo genome-wide epigenetic
reprogramming (the topic of the following section),
and T-gonocytes (T-prospermatogonia) are the
proliferating and migratory immediate predecessors of
spermatogonia (Fig. a).
This nomenclature is somewhat confusing for at
least two reasons: first, gonocytes differentiate in an
asynchronous manner, resulting in the coexistence of
two subsets of gonocytes at most time points of
prespermatogenesis; and second, recent research has
elucidated that there is no clear demarcation between
T-gonocytes and spermatogonia, as proliferatively
quiescent gonocytes gain characteristics of sper-
matogonia (spermatogonia-specific molecular sig-
nature, physical contact with the basal lamina of testis
cords, and mitotic reactivation) in an unconcerted
882 Mäkelä et al Testis Development
fashion and over a relatively long period of time
(–). Owing to the absence of a marker that is
exclusively expressed in the gonocytes, it is also very
hard to pinpoint a specific time point when gonocyte
proliferation becomes spermatogonial (self-)renewal.
This is especially true in those rodents (including
the mouse and rat) where spermatogenesis proceeds
almost without a delay once the germ cells have
settled on the basal lamina of testis cords. Spermatogenic
differentiation of gonocytes/early spermatogonia
can, however, be unambiguously determined by the
onset of stimulated by retinoic acid gene  (Stra)
and Kit expression ().
Mouse gonocytes remain proliferatively active in
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the nascent testis and their numbers increase expo-
nentially at least until E. (M-gonocytes) ().
However, markers that are associated with cell cycle
arrest are enriched at E., and gonocytes enter
mitotic quiescence by E. to E. (T-gonocytes) in
an asynchronous (and strain-dependent) fashion and
do not resume cell cycle before birth (, , ,
). Mitotic reactivation (T-gonocytes) takes place
in a strain-dependent manner ~ day after birth and
according to recent data concurs with gonocytes-to-
spermatogonia transition (GST) (Fig. a) (, ).
GST involves dramatic changes in germ cell shape,
intracordal position, and transcriptome/translatome.
The gonocyte is a large, spherical cell with a
prominent nucleus and it is located at the center of
the testis cord, whereas spermatogonia are semi-
circular cells at the periphery of seminiferous tubules.
Upon GST, and concomitantly with the onset of
relocation from the center to the periphery of testis
cords, perinatal germ cells start to express markers
that are associated with SSCs in the adult mouse:
GDNF family receptor a (GFRa), rearranged
during transfection (RET), and promyelocytic leu-
kemia zinc finger (PLZF, officially ZBTB) ().
The antimeiotic gene Nanos, which is expressed
already by early mouse gonocytes (E.), is also
upregulated at the same stage (, ). NANOS is
an RNA-binding protein that directly interacts with
the carbon catabolite repressor –negative on TATA
(CCR-NOT) deadenylase complex, resulting in deg-
radation of specific RNAs. Recently it was suggested
that NANOS would act as a gatekeeper of GST and it
would suppress the expression of spermatogonial
markers in a context-dependent fashion (). During
human fetal testis development induction of NANOS
coincides specifically with entry into mitotic quiescence
(). Notably, GST strictly does not take place syn-
chronously in the mouse testis, and whereas the first
gonocytes gain contact with the basal lamina before
birth or just after it, most gonocytes have translocated
and transited into spermatogonia by postnatal day
(PND) . (, ).
Undergoing GST is crucial for germ cell differ-
entiation, as the gonocytes that fail to migrate to the
Endocrine Reviews, August 2019, 40(4):857–905

periphery of seminiferous tubules are cleared by ap-
optosis (). Germ cell apoptosis is a common event
during early postnatal testis development in both mice
and rats, and despite becoming mitotically active, the
total number of germ cells is roughly halved from
PND . to . and PND  to , respectively (, ).
In general, the perinatal testis development is similar
in the mouse and rat but the events occur  to  days
later in the rat.
Note that the spermatogonia of prespermatogenic
testis represent a heterogeneous population of cells in
terms of protein expression, mitotic activity, and cell
fate (). While a proportion of nascent spermato-
gonia is set aside to form the pool of self-renewing
SSCs, the remainder give rise to the differentiating
cells of the first round of spermatogenesis, a dis-
tinctive developmental program that apparently lacks
the differentiation-priming step (, ). To our
knowledge, it is not known what regulates the bi-
furcation of cell fates in the early postnatal testis, but
RA signaling—a sign of spermatogenic commitment
and onset of male germ cell differentiation—is acti-
vated in a subset of mouse spermatogonia in a patch-
like fashion as early as PND  (, ). It is likely
that either (i) germ cells developmentally carry de-
terminants that inherently predispose or preordain
them into either of the two destinies, or (ii) the so-
matic environment (primarily Sertoli and myoid
cells) dictates the cellular fate of nascent spermato-
gonia by spatiotemporally regulating the availability
of differentiation-inducing (RA) or SSC-maintaining
agents. There is compelling evidence to support
both the suggested mechanisms, and heterogeneous
expression of receptors for differentiation/stemness-
promoting factors that are asymmetrically distrib-
uted within the seminiferous epithelium might
explain the differential propensities of nascent
spermatogonia into either of the developmental
pathways (, ).
Molecular control of rodent prespermatogenesis
Escape from premature meiosis. Intracordal
localization protects gonocytes from meiosis-inducing
RA, and the PGCs that fail to get incorporated into the
testis cords will enter meiosis, undergo malignant
transformation (GCT), or perish (, , , ). There
are at least two mechanisms that prevent the premature
meiotic entry of gonocytes: first, somatic cells of the testis
(Sertoli, myoid, and Leydig cells) express CYP en-
zymes that degrade the abundant mesonephros-derived
RA; and second, Sertoli cell–derived FGF upregulates
Nanos and Cripto, and thus maintains the expression of
pluripotency-associated genes (Fig. b) (–). At
least in humans, the gonadal somatic cells in the male
also express lower level of enzymes needed in RA
synthesis than do the female equivalents, which probably
also affects the local concentration of bioavailable RA
(). Similar to the mouse, CYPB is also expressed by
doi: 10.1210/er.2018-00140
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human fetal testicular somatic cells, but preferentially by
Leydig cells and to a lesser extent by Sertoli and peri-
tubular cells ().
RNA-binding protein NANOS acts cell autono-
mously to repress meiosis in gonocytes, and Nanos
deficiency results in male sterility due to loss of germ
cells via premature meiosis (, ). Interestingly, RA
works as an FGF antagonist and downregulates
Nanos in both fetal and postnatal germ cells ().
CRIPTO is an obligate coreceptor of the autocrine and
self-enhancing Nodal signaling pathway that is tran-
siently active in mouse gonocytes (). Nodal sig-
naling extends the period of pluripotency-related gene
expression in XY germ cells, and thus indirectly delays
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the onset of differentiation. Additionally, ex vivo ex-
periments have demonstrated that Nodal signaling
is a potent inhibitor of meiotic entry (, ). In-
terestingly, components of the Nodal pathway are
dynamically expressed in human PGCs/gonocytes, too
().
Exit from the cell cycle. Besides avoiding entry
into meiosis, early gonocytes also have to stop pro-
liferating. TGFb signaling and Activin have been
shown to exert an antiproliferatory effect on PGCs,
and they seem to work together to induce the mitotic
arrest of gonocytes, too (, –). Cyclin in-
hibitor p-mediated dephosphorylation of retino-
blastoma protein (pRB) might be one of the “The gonocytes-to-
downstream mechanisms that TGFb signaling em- prespermatogonia transition
ploys to impinge on gonocyte cycling (, , ). (GPT) in humans is a gradual
Recently, the multifunction prostanoid PGD process.”
[reviewed by Rossitto et al. ()] has also been im-
plicated in the same process through promoting ex-
pression and nuclear localization of CDK inhibitor
p (). PGD might also indirectly block meiotic
entry and support mitotic arrest by inducing Nanos
(, ).
Loss of phosphatase and tensin homolog (Pten),
Dmrt, and Dnd have been associated with formation
of testicular teratoma due to a failure to arrest gon-
ocyte mitosis (, –). DND promotes mitotic
arrest by posttranscriptional mechanisms that likely
involve stabilization of p and p mRNAs (, ).
The role of DND in fetal germ cells is intriguing, for
in addition to contributing to the onset of mitotic
quiescence, DND has also been suggested to protect
these cells from apoptosis and malignant trans-
formation, maintain their identity, and suppress
meiosis in collaboration with NANOS (–).
Lastly, androgen action in vivo and in vitro has been
noted to inhibit gonocyte proliferation, and RA
degradation might be important to arrest gonocyte
mitosis (Fig. b) (, ). TGFb signaling and FGF
also promote the survival of quiescent gonocytes and,
unlike M- or T-gonocytes, T-gonocytes undergo
apoptosis highly infrequently (, , ).
Downregulation of pluripotency genes.
Among the numerous cell lineages of a developing
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Figure 5. Developmental regulation of gonocytes. (a) Germ cell populations of the developing testis in the mouse. Insets shown (B–D)
refer to panels (b), (c), and (d). (b) M-gonocytes actively proliferate in the nascent testis cords that have not yet fully developed. They also
retain the expression of pluripotency-associated genes, such as Nanog, Oct4, and Sox2, due to activity of the Nodal/Cripto signaling. M-
gonocytes are protected from premature entry into meiosis by expression of antimeiosis gene NANOS2 and CYP26 activity in Sertoli cells
resulting in degradation of extragonadal RA. Nanos2 and Cripto are both FGF9 signaling targets. As a consequence of p21 and p27
upregulation and stabilization, the cell cycle of M-gonocytes is subsequently slowed down. TGFb signaling and PGD2 have been shown to
control this process extrinsically, whereas DND1 is cell-autonomously involved. AMH and testosterone (T) potentially also contribute. (c)
Gonocytes enter mitotic quiescence between E13.5 and E15.5 and become T1-gonocytes. This is accompanied by selective downregulation
of pluripotency-related genes as a result of DMRT1 and SOX4 action. Cell-extrinsically, PGD2 also contributes to this process. On the
contrary, Nanos2 and Dnmt3l are upregulated. At this stage testis cord formation has been completed and peritubular cells encase the
testis cords. (d) Perinatally, gonocytes start to display signs of GST. GST encompasses translocation of germ cells from the lumen to
the basement membrane of the testis cord, acquisition of a transcriptomic signature typical of spermatogonia (PLZF, GFRa1, RET), and cell
cycle resumption. Many factors have been implicated in control of GST, but the data supporting a specific role for SCF/KIT and PDGF
signaling in control of the gonocyte migration is rather solid. Gonocytes probably use actin-based (small spheres organized into filaments),
pseudopod-like protrusions to gain contact with the basement membrane. CDH1-mediated cell adhesion may also play a significant role in

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gonocyte translocation. A subset of nascent spermatogonia is forced to enter spermatogenesis soon after GST by Sertoli cell–derived RA.
E2, estradiol. (e) Germ cell populations of the developing human testis. The developing testis houses two developmentally distinct
populations of fetal germ cells for almost 1 y. (f) Genome-wide reprogramming of the fetal germ cell epigenome. In the preimplantation
embryo the epigenome is demethylated and the lowest average CpG (5mC) methylation is observed at E3.5. The DNA is subsequently
remethylated and PGCs are specified in the posterior epiblast that exhibits relatively high global DNA methylation (~70%). Following
specification, PGC DNA is redemethylated in two stages (global and locus-specific demethylation), and at ~E13.5, 95% of CpG sequences
are in an unmethylated state. This period is accompanied by dynamic changes in chromatin modifications also. During the following few
days the gonocyte epigenome undergoes genome-wide de novo DNA methylation and acquires the paternal epigenetic signature.
PIWI–piRNA-mediated mechanisms involved in silencing of TEs are first implemented around this time. Late gonocytes and nascent
spermatogonia exhibit relatively high levels of global DNA methylation. Although DNA methyltransferases are active during
spermatogenesis, changes in DNA methylome during spermatogenesis are rather subtle. Dotted line indicates the postulated 5-
methylcytosine dynamics in humans. Spheres stand for available data at specific time points during human fetal development. gPGC,
gonadal PGC; M, M-gonocytes; T1, T1-gonocytes; T2, T2-gonocytes.

884 Mäkelä et al Testis Development Endocrine Reviews, August 2019, 40(4):857–905

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fetus, the germline is characterized by prolonged ex- puberty or during early adulthood they break free
pression of pluripotency-associated genes (). In from dormancy to form CIS, which then gives rise to a
mouse gonocytes, the Nodal/Cripto signaling pathway seminomatous or nonseminomatous testicular tumor.
is thought to maintain their expression in an FGF-
dependent manner a couple of days longer than in XX
germ cells (, ). Pluripotency genes are, however, Figure 5. (Continued)
selectively downregulated as gonocytes enter the mi-
totically quiescent phase between E. and E. (Fig,
c) (). Most notably, the expression of core net-
work genes of pluripotent state maintenance, that is,
Nanog, Sox, and Oct, are repressed. These three
genes have been implicated in embryonic germ cell
differentiation, proliferation, and survival, respectively
(, , ). Similar changes (with the exception of

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SOX, whose expression is lacking altogether) are
observed in human fetal germ cells, as well, as they stop
proliferating (). Note that in addition to these genes,
numerous other pluripotency-associated genes are
intimately linked with male germline development
and maintenance, including spalt-like  (SALL) and
undifferentiated embryonic cell TF (UTFI), for in-
stance (, ).
Silencing of Nanog and Sox is at least partially due
to methylation of their promoters, whereas OCT is
downregulated by posttranscriptional mechanisms
(). By E., E., and E. the protein ex-
pressions of SOX, OCT, and NANOG, respectively,
become substantially reduced or undetectable in male
germ cells (, ). DMRT has been suggested to
repress the expression of Sox, Nanog, and Oct cell
autonomously, and using both direct and indirect
means (). Furthermore, PGD has been implicated
in downregulation of these genes by a DMRT-
independent mechanism (). Very recently, SOX
was shown to be needed for the proper differentiation
of gonocytes, and via an unknown mechanism Sox
deficiency resulted in prolonged expression of CRIPTO
and NANOG and inability of the gonocytes to induce
Nanos and DNA methyltransferase (Dnmt)l—two
markers for gonocyte quiescence ().
The inability of Sox-null mice to repress plurip-
otent gene expression might also originate from a
failure to downregulate the mechanisms that are re-
sponsible for their prolonged expression, such as
Nodal/Cripto or FGF signaling, as has been suggested
to explain the prolonged expression of these genes in a
conditional Emx-knockdown mouse model (,
). Notably, the inability to repress the expression of
these and other cellular pluripotency genes (such as
Dppa, Dppa, and Cdh) has been associated with
testicular tumorigenesis, and testicular GCTs are the
most frequent cause of cancer in men between  and
 years of age (, , ). Based on morpho-
logical and gene expression similarities, it has been
suggested that testicular GCTs are derived from
gonocytes that fail to undergo correct fetal differen-
tiation and are maintained within the testicular tissue
as pre–carcinoma in situ (CIS) cells (, ). In

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Extrinsic control of GST. Given the signifi-
cance of GST for formation of the SSC pool and
avoidance of a pre-CIS lesion, its molecular control is
surprisingly poorly characterized. Fortunately some
conclusions can be drawn, nonetheless. Recently, the
FGF signaling dependency of GST was demonstrated
(Fig. d) (). Culture of testicular fragments from
the E. mouse in the presence of an FGF signaling
inhibitor had an adverse effect on GST, and after a
-day culture germ cells failed to translocate to the
periphery of testis cords, remained mitotically qui-
escent, and did not display a transcriptomic signature
typical of nascent spermatogonia (). Contrary to
earlier research, it was demonstrated that RA action is
dispensable for GST, and FGF signaling actually
works upstream of the RA pathway and may thus
control spermatogonial differentiation (, ).
Moreover, WNT/b-catenin has recently been im-
plicated in control of GST, as gonocytes, in which the
pathway is constitutively activated, are hyper-
proliferative and strongly express cyclin-D, a post-
GST, spermatogonia-associated marker (, ).
Interestingly, a subset of miRNAs involved in the
regulation of WNT/b-catenin and PTEN signaling
pathways is differentially expressed in gonocytes and
spermatogonia isolated from -day-old and - to
-day-old mice, respectively (). This is an in-
teresting observation considering the emerging role
of WNT signaling in promoting undifferentiated
spermatogonial differentiation and proliferation, as
well as the suggested role for PTEN in SSC main-
tenance (, , ).
Both SCF/KIT and PDGF signaling have been
implicated in gonocyte migration in rodents, and Sertoli
cells secrete the ligands of the pathways, whereas the
receptors are located on gonocytes (–). PDGF(R)
signaling also stimulates cell cycle resumption in germ
cells upon and after GST, and might potentiate or prime
gonocytes to respond to RA (Fig. d) (, –). In
vitro effects of a number of cytokines, hormones, and
growth factors on rodent PGC/gonocyte proliferation
and apoptosis have been studied in the past, but lack of
in vivo research greatly lessens the significance of these
findings (). Moreover, robust actin turnover in both
gonocytes and Sertoli cells is important for proper
migration, and gonocytes probably employ actin-based
pseudopod-like protrusions to gain contact with the
basal lamina. Actin dynamics are probably also required
for CDH-mediated cell adhesion that might play a
significant role in gonocyte repositioning (, ). A
disintegrin and metalloproteinase (ADAM)/integrin/
tetraspanin complexes have also been implicated in
translocation of the gonocytes and their subsequent
anchoring to the basal lamina ().
Interestingly, most of the above-mentioned
mechanisms are dysregulated in the mutant testes
that lack reproductive homeobox  (Rhox). As a
result, gonocyte migration and differentiation into
886 Mäkelä et al Testis Development
SSCs is compromised and Rhox-deficient mice
display gradual and progressive spermatogenic decline
due to impaired SSC maintenance (). Notch sig-
naling in Sertoli cells is probably also involved in GST
and, when excessive or improperly timed, it can
jeopardize gonocyte development (). Recently, it
was proposed that gonocyte-derived ligands [Delta-
like ligand  (DLL) and Jagged- (JAG)] might
actually control the activity of Notch signaling in
testicular somatic cells, providing a piece of evidence
for reciprocal two-way signaling between the germline
and the soma in the developing testis (). GST or
maintenance of nascent spermatogonia is also severely
impaired in inducible Pelota (Pelo) knockout mice
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via a thus-far-unknown mechanism (Fig. d) ().
Prespermatogenic germ cell differentiation
in humans
As discussed above, the molecular basis of regulation
of gonocyte differentiation in mice and humans are
remarkably similar. Molecular control responsible for
the phasic development of human fetal germ cells was
recently characterized by Li et al. () who identi-
fied . TFs that might play crucial roles as the
master regulators of gene expression networks in
human PGCs/gonocytes as they progress from mi-
gratory to nonmotile mitotic and proliferatively qui-
escent phases. Despite obvious similarities at the
molecular level, the dynamics of the process are
somewhat different, as human fetal germ cells do not
collectively enter a phase of mitotic quiescence (similar
to ~E. to E. in mouse) but a subset (likely a
constantly changing one) is proliferating throughout
fetal development (Fig. e) ().
The first gonocytes migrate to the basal lamina of
the testis cords at ~ wpc to give rise to germ cells that
are referred to as prespermatogonia in humans (not to
be confused with mouse prospermatogonia, an al-
ternative term for gonocytes). This coincides with the
downregulation of pluripotency factors and although
practically all germ cells of the first trimester embryo
express OCT, the number of OCT-expressing cells
decreases rapidly during the second trimester ().
The gonocytes-to-prespermatogonia transition (GPT)
in humans is a gradual process, and by midgestation
most of the germ cells in the fetal testis have un-
dergone GPT (). There is a simultaneous increase
in NANOS expression suggesting a conserved anti-
meiotic role for this factor in both human and mouse
prespermatogenesis (, , ). As testis devel-
opment proceeds from the earliest phases toward
midgestation, DDX expression is enhanced (, ).
OCT and DDX start to display a mutually exclusive
pattern of expression, and although OCT-positive
cells are heterogeneously distributed within the testis
cords, DDX-expressing cells are preferentially located
at the periphery (, , ). DDX thus becomes a
marker for prespermatogonia, whereas OCT is
Endocrine Reviews, August 2019, 40(4):857–905

preferentially expressed by the gonocytes. Notably,
% of DDX-positive cells in an -wpc fetal testis do
not express the proliferation marker Ki-, which
makes DDX a good marker for mitotically inactive
germ cells at this stage and suggests that early pre-
spermatogonia are mitotically quiescent (). In-
terestingly, prespermatogonia exist as syncytia of two
or more cells connected by cytoplasmic bridges, a
characteristic that is shared with spermatogenic cells
(, ).
Until  wpc Ki- is predominantly expressed by
the OCT-positive gonocytes. Subsequently, both
gonocytes and prespermatogonia display a compara-
ble mitotic activity, but with advancing age pre-
spermatogonia become proliferatively inactive and the
few germ cells that proliferate are almost without
exception centrally located OCT-expressing gono-
cytes (). Interpretations of these data are that
prespermatogonia upregulate DDX and down-
regulate OCT, enter short-term mitotic quiescence,
resume cell cycle for a short period at the end of the
second trimester and the beginning of the third tri-
mester, and then enter another proliferatively quies-
cent period before birth. OCT expression persists
into early postnatal life ( to  months) and is almost
exclusively seen in centrally located gonocytes, making
fetal germ cell differentiation in humans a pro-
nouncedly asynchronized process (, ). This is in
strict contrast to rodent prespermatogenesis where
OCT expression is lost in quiescent gonocytes by
E. and E. in mice and rats, respectively (,
).
As discussed above, prespermatogenic germ cell
development does not proceed in synchrony in ro-
dents either. However, the scale of asynchronicity
between humans and mice or rats is dramatically
different, and a considerably longer timespan (up to
 year) is required for it to conclude in humans.
NANOG expression in the human fetal and early
postnatal testis follows a similar pattern to that of
OCT, and practically all germ cells of the early second
trimester fetus express it (). Minipuberty, charac-
terized by a surge in gonadotropin and reproductive
hormonal activity, takes place soon after birth (at ~ to
/ months of age) and is considered the final stage of
gonocyte differentiation into infantile spermatogonia
(–).
Prespermatogenic germ cell expansion
In humans, migrating PGCs are proliferatively active
but their cell cycle is accelerated once they have
reached the GR and been enclosed within the testis
cords as gonocytes. The number of germ cells in the
male embryo has been suggested to increase from
~ at the beginning of the fifth embryonic week to
, in the -week-old embryo (). Until midg-
estation the increase in germ cell number in the
human fetus follows a curvilinear pattern (). The
doi: 10.1210/er.2018-00140
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total germ cell number in the male roughly doubles
in a week during  to  wpc, but the growth rate is
subsequently reduced and it takes twice the time to
double the number of germ cells during  to  wpc
(). Finally,  weeks are needed to give rise to a
twofold increase in germ cell count during  to
 wpc. The number of Ki-–positive germ cells then
steadily decreases toward birth, but a stage that would
be characterized by very low germ cell mitotic activity
or total quiescence cannot be discerned. This might be
masked by the asynchronous nature of the process and
coexistence of gonocytes and prespermatogonia dur-
ing an extended period of time. A subset of human
fetal germ cells at a specific developmental phase
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(having undergone GPT, for instance) might enter a
mitotically quiescent phase to undergo epigenetic
reprogramming.
The number of somatic cells in the male embryo
increases at a comparable rate to that of germ cells
until  wpc, but then the growth slows down (). In
their data set, Li et al. () noted that somatic cells
of the fetal gonads generally expressed fewer cell
cycle–related genes than did the neighboring germ
cells, and only ~% of somatic cells actively pro-
liferated. Prepubertal ( to  years) development of
germ cells is characterized by variable mitotic activity
and isolated phases of spermatogonial proliferation
“Resetting of the germ cell
and involution that may be accompanied by emer-
epigenome during embryonic
gence of scarce meiotic or postmeiotic germ cells and fetal development is
(–). The number of spermatogonia is slightly critical for perpetuation of the
reduced during the first  years of life, but it is then species.”
increased to reach a plateau by  years of age ().
The early reduction in spermatogonial numbers might
be due to apoptosis of gonocytes (mistaken as sper-
matogonia) after minipuberty, whereas the increase
that is seen during  to  years of age reflects the
proliferation of type A spermatogonia and their dif-
ferentiation into type B spermatogonia (). In total,
the number of germ cells is increased during infancy,
and the testicular volume of -year-old boys is
twofold to threefold higher than in newborns (,
). The onset of puberty, however, is characterized
by exponential growth of the spermatogonial pop-
ulation and testis volume (, ).
Genome-Wide Reprogramming of
the Epigenome
Epigenetic marks and factors collectively contribute to
the molecular memory of a cell. They ensure that a cell
faithfully executes the transcriptional program needed
for the fulfillment of its task and maintenance of its
function. The epigenetic marks of a differentiated
somatic cell are relatively stable, but the epigenome
undergoes extensive reprogramming in the mam-
malian preimplantation embryo and the germline
(, ). To generate a totipotent state, epigenetic
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REVIEW
marks need to be removed during early development.
Gametogenesis also calls for erasure of parental epi-
genetic memories and removal of potential epi-
mutations from the germline (). There are also
~ imprinted genes in mammals and most of them
are found in clusters (). The proper imprinted
expression of these genes is controlled by imprinting
control regions. In the male germline, maternal and
paternal imprints are erased in PGCs and early
gonocytes and re-established during fetal development
(, ). Resetting of the germ cell epigenome
during embryonic and fetal development is critical for
perpetuation of the species.
DNA methylation typically acts to repress gene
transcription. Many genes (such as meiotic genes)
in the mammalian genome exhibit a gonad-specific
pattern of expression and only take part in gameto-
genesis. Other tissues and physiological processes must
therefore employ mechanisms (such as DNA meth-
ylation) to repress the ectopic expression of these
genes. Failure to demethylate the DNA that hosts these
genes or their regulatory regions (promoters and
enhancers) in fetal germ cells might impede their
expression during gametogenesis and result in failure
to produce sperm or eggs. Fetal germ cells undergo
genome-wide reprogramming of the epigenome that
erases the epigenetic memory of the previous gener-
ation and is postulated to create a blank slate upon
which sex-specific mechanisms then work to establish
an androgenetic or gynogenetic (in males and females,
respectively) epigenetic signature.
Activation of germline genes in nascent gonocytes
is partially mediated by DNA demethylation. Despite
global hypomethylation of the genome, transcription
is not promiscuously activated in PGCs (, ).
PGCs must therefore recruit other mechanisms to
avoid uncontrolled transcription of DNA, including
activation of transposable elements (TEs). However,
not everything gets demethylated, and there are ge-
nomic regions that at least partially escape the resetting
of the epigenome, potentially contributing to trans-
generational epigenetic inheritance (TEI).
Global demethylation of PGC genome
The epigenetic marks on the zygotic DNA need to be
extensively erased to reset the epigenome of cells of the
early embryos for naive pluripotency (Fig. f). In the
preimplantation embryo the epigenome undergoes
early demethylation and the lowest average CpG
methylation of all preimplantation embryonic stages is
observed in the inner cell mass of an E. mouse
blastocyst (). This is, however, a transient state and
the DNA is subsequently and rapidly remethylated.
mPGCs are then derived from the proximal epiblast of
the early postimplantation embryo and they exhibit
global DNA methylation levels similar to their somatic
neighbors that are primed for lineage differentiation
(, , ).
888 Mäkelä et al Testis Development
Soon following specification, the DNA of nascent
mPGCs is redemethylated to around half of the levels
that are observed in the epiblast cells. This is called
stage I (or global) DNA demethylation and it is fol-
lowed by stage II, encompassing locus-specific DNA
demethylation commencing at ~E. and concluding
at E. when DNA methylation during fetal germ cell
development reaches its lowest point (, ). At
E. ~% of cytosines at CpG sequences in genomic
DNA are in an unmethylated state, whereas in E.
epiblast CG dinucleotides carry a methyl residue in ~
out of  cases (, ). Consequently, almost all
genomic features become hypomethylated. PGC
epigenetic reprogramming entails not only global
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DNA demethylation but also erasure of genomic
imprints, reactivation of the X chromosome, and
genome-wide reorganization of histone modifications
(, , –).
In the process of PGC epigenetic reprogramming the
genome becomes demethylated by both replication-
independent and replication-dependent mechanisms
(). The former refers to active conversion of
-methylcytosines (mCs) to -hydroxymethylcytosines
(hmCs) by ten-eleven translocation methylcytosine
dioxygenase enzymes (TET), whereas the latter seem-
ingly passive mechanism involves repression of DNA
methyltransferase action: DNA methylation in pro-
liferating PGCs is not maintained owing to suppression
of enzymes that carry out maintenance or de novo DNA
methylation (, –). As DNA gets replicated,
CpGs of the daughter strand are not methylated, and
consequently every round of DNA replication halves the
total number of methylated CG dinucleotides per unit of
DNA, a phenomenon that is also called replication-
coupled dilution.
Demethylation in mPGCs
In the mouse, replication-coupled dilution relies on
BLIMP and PRDM, two key genes in mPGC
specification, which repress the expression of de novo
DNMTs Dnmta and Dnmtb, and ubiquitin-like with
PHD and ring finger domains  (Uhrf), a factor
needed to recruit DNMT, the maintainer of cellular
DNA methylation signature (, , ). This
mechanism is likely to be activated in mPGCs upon
specification (stage I), whereas mC-to-hmC con-
version that accounts for erasing imprints and
demethylation of meiotic gene promoters does not
become significant before mPGCs settle in the gonadal
primordia (stage II) (, , , ). DNA
methylation at imprinting control regions and meiotic
gene promoters is preserved by DNMT, an action
that is needed to prevent precocious differentiation of
gonocytes ().
Three TET enzymes (TET to TET) have been
implicated in hydroxylation of mC, but TET and
TET have been considered to play the primary role in
the process, whereas TET is probably dispensable for
Endocrine Reviews, August 2019, 40(4):857–905

mC-to-hmC conversion in the germline (, ).
Upregulation of TET and TET in mPGCs at E. is
followed by an increase of hmC levels that reach their
peak at ~E (, ). Cells can replace hmC (or its
derivatives) enzymatically by unmethylated cytosines,
but data suggest that the decline of hmC after E. is
mainly due to replication-coupled dilution (, ,
). The significance of TET-mediated demethyla-
tion was recently severely questioned and it was
suggested that the canonical demethylating activity of
TET is restricted to removal of aberrant residual
DNA methylation and protection of newly deme-
thylated DNA from reacquiring this mark. Moreover,
TET was implicated in a crucial role in germline
development through activation of germline genes in
gonocytes via a DNA demethylation-independent
mechanism ().
Demethylation in hPGCs
hPGCs follow essentially similar dynamics in terms of
DNA demethylation as described above for mPGCs,
but some differences do exist and not all observations
in mice can be faithfully extrapolated to humans (,
, ). First, although repression of DNMTA,
DNMTB, and UHRF are associated with replication-
coupled loss of CG dinucleotide methylation in
hPGCs, too, partially or completely different factors
must be at play, as PRDM is likely indispensable for
the process (, , , ). Second, imprinted
genes undergo TET-mediated hydroxymethylation
already in migratory hPGCs, which is in line with the
data showing TET and TET induction already in
early hPGCLCs (, , ). Thus DNA demethy-
lation in hPGCs cannot be subdivided into equally
distinct stages as has been previously considered to
exist in mice. At around the time of GR colonization,
hPGCs of the sixth embryonic week already exhibit
substantial demethylation, and global methylation of
CG dinucleotides is at %. Epigenetic ground state is
achieved in germ cells isolated from -week-old male
fetuses (). At this point .% of CpGs in the
genomic DNA are found unmethylated ().
Other aspects of PGC epigenome reprogramming
In PGCs that undergo epigenetic reprogramming,
DNA methylation and transcription need to be
uncoupled, and therefore a mechanism independent
of DNA methylation is required to protect PGCs from
the harmful effects of aberrant gene expression and
activation of TEs [reviewed by Tang et al. ()]. This
task has been assigned to chromatin modifications,
and accordingly, there is a dynamic turnover of histone
marks in PGCs at and around the time of low global
mC levels (Fig. f). Species-to-species variation in the
exact means that are used, however, is typical. In-
terestingly, the factors (BLIMP/PRDM) whose
action is needed to lower DNA methylation in a
genome-wide fashion also drive the dynamic changes
doi: 10.1210/er.2018-00140
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in chromatin modifications during the course of male
germ cell epigenetic reprogramming ().
The levels of a repressive histone mark trimethy-
lation of lysine  on histone  (HKme) persis-
tently increase in migratory mPGCs, are maintained in
nascent gonocytes, and are downregulated as gono-
cytes enter mitotic quiescence (Fig. f) (, , ).
Concomitantly with enhancement of HKme,
there is a loss of dimethylation of lysine  on histone
H (HKme; a repressive mark) in mPGCs (,
). Whereas HKme behaves similarly in hPGCs,
HKme is only transiently enriched in migratory
germ cells and depleted in hPGCs upon GR coloni-
zation. The significance of this difference at the mo-
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lecular level is not understood. Dimethylations of
arginine  on HA and H (HA/HRme) are also
increased in migrating mPGCs but downregulated
upon arrival at the embryonic gonad (Fig. f) ().
Notably, HA/HRme levels do not fluctuate
during hPGC development (). HA/HRmes
have been suggested to be critical for the repression
of TEs in early PGCs that have lost HKme and
do not yet have an active Piwi-interacting RNA
(piRNA)–mediated mechanism for TE silencing
(discussed below) (). Notably, the PIWI–piRNA
pathway might be active in hPGCs relatively early, as
the key components of the pathway are upregulated in
- to -wpc hPGCs and the expression is maintained “Epigenetic ground state is
in both gonocytes and prespermatogonia (, ). achieved in germ cells isolated
Despite nearly complete erasure of cytosine from 11-week-old male
methylation in mPGCs and hPGCs, some DNA se- fetuses.”
quences are protected and they retain relatively high
levels of DNA methylation (, , , ). Es-
pecially, evolutionarily young and potentially haz-
ardous TEs, pericentric heterochromatin, some loci
associated with TEI, but also some single copy genes
remain methylated. DNA methylation at these escapee
loci might be maintained by HKme- or tripartite
motif containing  (TRIM)–mediated and UHRF-
independent recruitment of DNMT (). DNA
methylation-mediated TEI through the germline has
been under heated debate recently. However, these
marks would have to tolerate two waves of epigenetic
reprogramming in every generation and be susceptible
to environmental agents to qualify as carriers of trans-
generational nongenetic information to subsequent
generations with phenotypic consequences, and
therefore the significance of DNA methylation in TEI
has justifiably been questioned ().
Acquisition of paternal epigenetic signature
Knowledge regarding the mechanisms of remethyla-
tion during gonocyte/fetal germ cell quiescence is still
in its infancy. This is especially true for human fetal
germ cells. De novo DNA methylation is a genome-
wide process and it also involves the three known
paternally methylated germline differentially methyl-
ated regions, but not much is known about how
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paternal-specific DNA methylation imprints are mech-
anistically established in the male germline.
In the male mouse germline, prenatal de novo
methylation of DNA starts at ~E. and is completed
by E. (, , ). Although there is considerable
variation, by E. gonocytes display on average %
methylation of CG dinucleotides. During subsequent
differentiation the DNA of male germ cells gets further
methylated and spermatozoa display % to %
methylation at CpG sites (, ). Interestingly, DNA
methylome does not markedly change between
adult human spermatogonial stem cells and sperm,
suggesting that the methylation pattern of germ cells is
largely established prior to onset of spermatogenesis
().
As discussed earlier in this text, fetal germ cell entry
into mitotic quiescence is accompanied with dramatic
changes in transcription in both humans and mice. In
the mouse, DNMTs (Dnmta, Dnmtb and Dnmtl) are
among the genes whose expression is strongly induced in
gonocytes between E. and E. (, , ). This
coincides with methylation of Nanog and Sox pro-
moters that is associated with their transcriptional re-
pression in the male germline (). These data suggest
that repression of pluripotency-associated genes is due to
global remethylation of the gonocyte genome. In-

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terestingly, DNMTA, but not DNMTB, has been
shown to be essential for de novo DNA methylation of
the imprinted loci in germ cells of both sexes (). In the
human fetal testis, the highest levels of DNMTA and
DNMT are recorded during the nd developmental
week but the levels are relatively high throughout weeks
 to  (). Owing to the paucity of knowledge we can
only presume that this is the period when human
prespermatogonial DNA is remethylated and the pa-
ternal imprints are established. Low methylation
levels are maintained in male PGCs at least until 
wpc (). Although the germ cell DNA is extensively
remethylated already during prenatal development,
the spermatogenic cell genome also undergoes dy-
namic changes in methylation in mitotic and early
meiotic cells (). Interestingly, spermatogenic
differentiation commitment within the mouse
germline is associated with strong upregulation of
DNMTA and DNMTB (). Their increased
expression probably contributes to the irreversibility
of the process, as differentiating spermatogonia
cannot revert back to the self-renewing state.
Restoration of DNA methylation is important not
only to establish the paternal epigenetic signature but
also to reinstate the control on gene expression. Gon-
ocytes and their developmental successors also employ
other means to repress transcription. A male germline-
specific mechanism of TE silencing is recruited in the
mouse embryo at E. when the expression of MILI, a
PIWI family protein, is first observed in gonocytes (Fig.
f) (). MILI interacts with piRNAs to carry out its role
in transposon mRNA and gene silencing (, ).
Another PIWI family member, MIWI, follows a couple
of days later. Although MILI expression is maintained in
Figure 6. The number of Sertoli cells during childhood and puberty in primates based on studies
by Rey et al., 1993 (500), Simorangkir et al., 2012 (504), and Cortes et al., 1987 (499). The number of the male germline until postmeiotic cells, MIWI is
adult subjects (all .25 years of age) are shown at age 18 in the lowest panel. The data were transiently expressed and is depleted from most male
extracted from original publications using WebPlot Digitizer v4.18. [Data from Cortes D, Muller J, mouse germ cells by  days after birth and displays a
Skakkebaek NE. Proliferation of sertoli cells during development of the human testis assessed by restricted pattern of expression within the differentiation-
stereological methods. Int J Androl 1987;10(4):589–596; from Rey RA, Campo SM, Bedecarras P, et al. primed subset of undifferentiated spermatogonia (,
Is infancy a quiescent period of testicular development? Histological, morphometric, and functional
). Notably, the effect of piRNAs on TE silencing
study of the seminiferous tubules of the cebus monkey from birth to the end of puberty. J Clin
Endocrinol Metab 1993;76(5):1325–1331; and from Simorangkir DR, Ramaswamy S, Marshall GR,
is through de novo DNA methylation, and the
et al. Sertoli cell differentiation in rhesus monkey (macaca mulatta) is an early event in puberty and PIWI–piRNA pathway is therefore tethered into
precedes attainment of the adult complement of undifferentiated spermatogonia. Reproduction transcriptional control via DNA methyltransferase
2012;143(4):513–522.] action ().

890 Mäkelä et al Testis Development Endocrine Reviews, August 2019, 40(4):857–905

 REVIEW

Postnatal Testicular Growth
Sertoli cell proliferation and maturation
In adulthood, the number of Sertoli cells correlates
both with testicular volume and sperm output both in
rodents and in humans (, ). Sertoli cells provide
the physical framework for adult testicular micro-
architecture, habitats, and regulation of germ cells, and
their intercellular junctions facilitate and form the bulk
of the blood–testis barrier. First, the overall events that
take place in Sertoli cell proliferation and maturation as
well as the circulating markers of Sertoli cell function are
briefly described. Then, the changes during the three
developmental periods, that is, the neonatal period,
cells before puberty (). Thus, before the onset of
puberty testicular volume provides an estimate of the
Sertoli cell numbers, assuming that the interindividual
differences in interstitial volume are small. Previously,
testicular volume has been measured in autopsy studies
by weighing or by water displacement (, , ).
In more recent studies ultrasound has been used
(–), which allows longitudinal follow-up.
Inhibin B. Inhibins are testicular peptides that
are generally thought to inhibit the activity of the
hypothalamus (). Inhibin B is the predominant
inhibin in primates and it is expressed predominantly
in the Sertoli cells before and as a joint product of
Sertoli cells and spermatocytes after the onset of

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juvenile period, and puberty, are reviewed in more detail. spermatogenesis (, ).
The circulating inhibin B concentration correlates
Pattern of Sertoli cell proliferation in with the Sertoli cell number in rodents, and the
autopsy studies
Counting of Sertoli cells in histological sections under
stereomicroscopy is the gold standard for their quan-
tification. However, such data are scarce as, in humans,
they require autopsy studies. A cross-sectional autopsy
study suggests that the increase in Sertoli cell number
seems to take place mostly during the first neonatal
months (i.e., minipuberty) and during puberty ().
However, the results of this study are limited by the
small number of subjects, large interindividual differ-
ence in the number of Sertoli cells, and the inevitable
cross-sectional nature of the study.
The importance of puberty and minipuberty in
Sertoli cell proliferation is supported by experimental
studies in nonhuman primates. In Cebus monkeys and
marmosets, the number of Sertoli cells increases sharply
during minipuberty, and a slight increase was found in
early puberty (, ). In contrast, the increase in
Sertoli cell number in rhesus monkeys during puberty is
rapid, whereas a little increase in their number takes
place during the neonatal period (–). The
numbers of Sertoli cells during primate lifetimes based
on autopsy and experimental studies are shown in Fig.
. Thus, the relative importance between either of the
two periods for the final Sertoli cell number in humans
remains uncertain, especially as the experimental in-
hibition of Sertoli cell proliferation during the mini-
pubertal period in marmosets does not seem to translate
to reduced Sertoli cell number in adulthood ().

Biomarkers of Sertoli cell number and maturation

Substantial interindividual variability has been observed
in the number of Sertoli cells in adulthood. Less invasive
biomarkers of Sertoli cells provide a good picture on
Sertoli cell development, as they allow longitudinal
follow-up. The longitudinal pattern of testicular growth
by ultrasonography, circulating inhibin B, and AMH as
well as their temporal relationship with Sertoli cell
number in humans are shown in Fig. .
Testicular volume. In Cebus monkeys, ~% of Figure 7. The longitudinal pattern of biomarkers of Sertoli cell proliferation and maturation in
the testicular cells in the seminiferous tubules are Sertoli humans and their temporal relationship with the number of Sertoli cells.

doi: 10.1210/er.2018-00140 https://academic.oup.com/edrv 891

REVIEW
hemicastration halves the circulating concentration in
rhesus monkeys () and humans (). In humans,
the inhibin B concentration correlates with testicular
volume during minipuberty ().
AMH. AMH is an interesting circulating
marker in terms of functional maturation. The ex-
pression of AMH is switched on early during the fetal
Sertoli cell development, and it causes regression of the
Müllerian ducts (). During the fetal, neonatal, and
juvenile periods, AMH is secreted exclusively by the
Sertoli cells (). The levels of AMH peak during
minipuberty, and later very early during puberty at  to
 years (, ), consistent with the observed
testicular growth and the increase in Sertoli cell
numbers. The loss of AMH secretion occurs early
during puberty (, ), as a result of the increase in
intratesticular testosterone concentration and the
entry of germ cells into meiosis already at the age when
circulating testosterone concentrations are low and/or
indistinguishable (, ).
GnRH, FSH, and LH. Hypothalamic GnRH
and gonadotropic FSH and LH are not direct biomarkers
of Sertoli cell function. However, the peak in activity of
the HPG axis during minipuberty and puberty clearly
coincides with the periods of Sertoli cell proliferation.
Furthermore, a compensatory increase in FSH levels
often indicates a Sertoli cell dysfunction or clearly low
numbers of Sertoli cells. During the juvenile presumed
hiatus between minipuberty and puberty, the nocturnal
peaks in GnRH in circulation seem to be of importance
for the functional maturation of Sertoli cells.
Figure 8. Testicular growth during minipuberty based on autopsy studies by Berensztein et al.,
2002 (506) and Bidlingmaier et al., 1983 (505). The data were extracted from original publications
using WebPlot Digitizer v4.18. [Data from Bidlingmaier F, Dorr HG, Eisenmenger W, et al.
Testosterone and androstenedione concentrations in human testis and epididymis during the first
two years of life. J Clin Endocrinol Metab 1983;57(2):311–315; and from Berensztein EB, Sciara MI,
Rivarola MA, Belgorosky A. Apoptosis and proliferation of human testicular somatic and germ cells
during prepuberty: High rate of testicular growth in newborns mediated by decreased apoptosis. J
Clin Endocrinol Metab 2002;87(11):5113–5118.]
892 Mäkelä et al Testis Development
Sertoli cell proliferation and maturation
during minipuberty
During the first postnatal months, there is an ap-
proximately fourfold increase in the number of Sertoli
cells in humans and rhesus monkeys, although the
increase is even greater in Cebus monkeys (, ).
This growth is caused by a similar increase in the length
of seminiferous tubules, whereas the diameter of the
seminiferous tubules remains unaltered (, , ).
The increase in the number of Sertoli cells during
minipuberty seems to be driven both by proliferation
and protection from apoptosis, and it seems to take
place mostly during the first postnatal month in
humans (). This results in a marked increase in the
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size of the testes. Based on autopsy studies (shown in
Fig. ) and studies utilizing ultrasonography to assess
testicular volume, testicular growth takes place until
the age of  months (–). A cross-sectional
study utilizing ultrasonography reported that tes-
ticular volume may even decrease after the age of
 months ().
There are several candidates for factors that drive
Sertoli cell development during minipuberty. The
obvious first candidates are the gonadotropins FSH
and LH. Experimental silencing of the HPG axis by
administration of a GnRH antagonist is associated
with a % reduction in Sertoli cell number by the end
of minipuberty (). Furthermore, FSH/LH supple-
mentation of baby boys with hypogonadotropic
hypogonadism markedly increases the testicular vol-
ume and circulating concentrations of inhibin B and
AMH according to several case reports (–).
The FSH receptor is expressed already starting from
the fetal period (). Men with an inactivating FSH
receptor mutation have reduced semen quality and
testicular volume in adulthood, compatible with the
diminished minipubertal proliferation of Sertoli cells
(), although this effect may also be caused later
during testicular development. It remains still unknown
whether the LH/androgen secretion contributes to
the proliferation of Sertoli cells during minipuberty.
Circulating AMH levels are higher during mini-
puberty among subjects with androgen insensitivity,
suggesting that the androgens might affect the ex-
pression of AMH. Neonatal Sertoli cells do not
express AR before the onset of puberty in humans
and nonhuman primates (, , ). Thus, the
potential effect of LH/androgens has to be relayed
either by Leydig cells or PMCs, which do express
the AR.
Sertoli cell proliferation and maturation during
the juvenile period
Although the period between minipuberty and true
puberty is characterized by silence of the HPG axis
and, for example, lack of circulating testosterone, it has
been suggested that during this period the testis might
remain “silently active” (). Especially toward the
Endocrine Reviews, August 2019, 40(4):857–905
 REVIEW

end of the juvenile period, the GnRH axis starts to
exhibit pulsatile activity nocturnally.
During this hiatus in activity of the HPG axis,
testosterone is undetectable and inhibin B and AMH
levels first decline from the peak levels during mini-
puberty, and then remain stable (, ). A small
proportion of Sertoli cells seems to proliferate in
rhesus monkeys during the juvenile period between
minipuberty and true puberty (). Owing to the
relatively small number of subjects in autopsy studies,
it remains unknown whether a small increase in Sertoli
cell number takes place in humans as well ().
However, relative to minipuberty or true puberty, the
contribution of the juvenile period does not appear
Interestingly, when comparing the studies, the peak
and loss of AMH appears to begin already before the
testicular volume of $ mL by orchidometer, which is
often considered a marker of pubertal onset, as it is the
first reliable marker of the imminent peak in testicular
growth. Our longitudinal study suggests that clear
testicular growth can be shown by ultrasonography long
before the attainment of testicular volume $ mL.
Thus, such testicular growth might be a marker of
Sertoli cell proliferation (). Studies in XXSxrb mice
(a mouse line displaying premeiotic failure in sper-
matogenesis due to presence of two X chromosomes)
suggest that the downregulation in AMH seems to be
complemented by the entry into meiosis, as AMH levels

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that significant. Alternatively, this period seems im-
portant for the maturation of Sertoli cells, which in-
cludes changes in morphology and expression of
various enzymes.
Puberty. This period coincides with the reac-
tivation of the hypothalamic–pituitary axis, which
initially occurs nocturnally. The small number of
subjects in studies precludes careful analysis of when
the peak in Sertoli cell proliferation occurs. However,
Sertoli cell proliferation takes place early during the
pubertal transition in rhesus monkeys, when the
circulating nocturnal testosterone concentrations had
not exhibited a robust pubertal increase ().
Among men with hypogonadotropic hypogonadism,
the gonadotropin substitution with FSH alone in-
creases the testicular volume and the circulating
levels of inhibin B and AMH (–). FSH in-
creases the proliferation of Sertoli cells in humans,
although both LH and FSH are able to stimulate the
proliferation of Sertoli cells in late juvenile rhesus
monkeys ().
After the possible peak or plateau in AMH, the
levels decline rapidly while a concomitant increase in
inhibin B levels takes place (, , ). The levels
of AMH inversely correlate with circulating an-
drogen levels and remain persistently elevated in
mice or boys who lack a functional AR (, ).
Additionally, the treatment of men with hypo-
gonadotropic hypogonadism with the combination
of FSH and LH/human chorionic gonadotropin de-
creases AMH levels (). In contrast, treatment with
FSH alone increases the circulating levels of AMH in
men with hypogonadotropic hypogonadism ().
remained elevated despite the typical pubertal mor-
phological changes ().
Adulthood. Until recently, there has been a
general agreement that Sertoli cells cease to proliferate
during puberty, and thus they might not proliferate
during adulthood. A recent study suggested that
Sertoli cells of the seminiferous tubules in the tran-
sitional zone near to rete testis may elongate also in
adulthood in rats (). However, it is not known if
this is true in humans. In young adults, sperm pro-
duction does not increase between  and  years of
age, which reflects the cessation of Sertoli cell pro-
liferation at puberty and implicates that sperm pro-
duction cannot be augmented in adult men (). An
exception to this rule is hypogonadotropic hypo-
gonadism that prevents puberty, and gonadotropin
treatment is needed for the initiation of puberty and
induction of spermatogenesis in adulthood.
Concluding Remarks
Rapid development of research technologies has ad-
vanced our knowledge on testis development and
function. Molecular mechanisms of DSDs are now
known for a large number of syndromes. Emerging
new data on epigenetics will be important for further
understanding of disease mechanisms. Such data may
also explain complex interactions between genes and
the environment. Comprehensive research on genes,
epigenetic mechanisms, and environmental factors,
including lifestyle and endocrine disruptors, is needed
to solve many open questions.

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Acknowledgments
We acknowledge Emmi Rotgers, Anne Jørgensen, and
Humphrey Yao for sharing their review manuscript titled “At
the Crossroads of Fate—Somatic Cell Lineage Specification in
the Fetal Gonad” during finalization of our review. We also
doi: 10.1210/er.2018-00140
thank Lise Akslglaede for sharing the data that were used to
produce Figure 7.
Financial Support: This work was supported by grants
from the Academy of Finland, the Sigrid Jusélius Foundation,
the Novo Nordisk Foundation, Turku University Hospital,
and by the Emil Aaltonen Foundation.
Correspondence and Reprint Requests: Jorma Toppari,
MD, PhD, Institute of Biomedicine, University of Turku, Kii-
namyllynkatu 10, 20520 Turku, Finland. E-mail: jortop@utu.fi.
Disclosure Summary: The authors have nothing to
disclose.
Abbreviations
5hmC, 5-hydroxymethylcytosine; 5mC, 5-methylcytosine;
AGP, adrenogonadal primordium; AMH, anti-Mul̈ lerian
hormone; AR, androgen receptor; bFGF, basic FGF; BLIMP1,
B lymphocyte-induced maturation protein-1; BMP, bone
morphogenetic protein; CBX2, chromobox homolog 2; CDH1,
cadherin 1; CE, coelomic epithelial/epithelia/epithelium; CIS,
carcinoma in situ; CITED, CREB-binding protein/p300-
interacting transactivator, with Glu/Asp-rich carboxyl-
terminal domain; CXCL12, C-X-C motif chemokine ligand
12; CXCR4, C-X-C motif chemokine receptor 4; CYP26B1,
cytochrome P450 family 26 subfamily B member 1; DAZL,
depleted in azoospermia-like; DDX4, DEAD-box helicase 4;
DHH, desert hedgehog; DMRT1, doublesex and mab-3–related
TF1; DND1, DND miRNA-mediated repression inhibitor 1;
DNMT, DNA methyltransferase; dpc, days postconception;
DPPA, developmental pluripotency-associated protein; DSD,
disorder of sex development; E, embryonic day; ECM, extra-
cellular matrix; EMX2, empty spiracles homeobox 2; EOMES,
eomesodermin; EpiLC, epiblast-like cell; ESC, embryonic stem
cell; ExE, extraembryonic ectoderm; FGF, fibroblast growth
factor; FGFR, FGF receptor; FOG2, friend of GATA2; FOXL2,
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REVIEW
forkhead box L2; GADD45g, growth arrest and DNA damage-
inducible 45g; GATA, GATA-binding protein; GCT, germ cell
tumor; GPT, gonocytes-to-prespermatogonia transition; GR,
gonadal ridge; GRN, gene regulatory network; GST, gonocytes-
to-spermatogonia transition; GW, gestational week; H2A/
H4R3me2, dimethylations of arginine 3 on hsitones H2A and
H4; H3K9, histone 3 at lysine 9; H3K27me3, trimethylation of
lysine 27 on histone 3; H3K9me2, dimethylation of lysine
9 on histone H3; hESC, human ESC; HH, hedgehog; hiPSC,
human-induced pluripotent stem cell; HOX, Homeobox;
HPG, hypothalamus–pituitary–gonadal; hPGC, human PGC;
hPGCLC, human PGC-like cell; iMeLC, incipient mesoderm/
primitive streak-like cell; INSL3, insulin-like peptide 3; ITGB1,
integrin subunit b1; KDM3A, lysine demethylase 3A; KTS,
lysine-threonine-serine; LH, luteinizing hormone; LHX9, LIM
homeobox 9; LIF, leukemia inhibitory factor; MAP3K4, MAPK
kinase kinase 4; mPGC, mouse PGC; mPGCLC, mPGC-like cell;
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NR5A1, nuclear receptor subfamily 5 group A member 1;
OCT4, octamer-binding TF4; PDGF, platelet-derived growth
factor; PDRM14, PR/SET domain 14; PGC, primordial germ cell;
PGD2, prostaglandin D2; piRNA, Piwi-interacting RNA; PMC,
peritubular myoid cell; PND, postnatal day; PRDM, PR/SET
domain; PSC, pluripotent stem cell; PTEN, phosphatase and
tensin homolog; PTGDS, prostaglandin D2 synthase; RA, reti-
noic acid; ROR2, receptor tyrosine kinase–like orphan receptor
2; SCF, stem cell factor; SF1, steroidogenic factor 1; SIX, SIX
homeobox; SOX, SRY-box; SRY, sex-determining region Y; SSC,
spermatogonial stem cell; TE, transposable element; TEI,
transgenerational epigenetic inheritance; TES, testis-specific
enhancer of Sox9; TET, ten-eleven translocation; TF, tran-
scription factor; TFAP2C, TF AP-2g; TNAP, tissue-nonspecific
alkaline phosphatase; UHRF1, ubiquitin-like with PHD and ring
finger domains 1; VEC, vascular endothelial cell; wpc, weeks post
coitum; WT1, Wilms tumor 1..
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