Male Sex DeterminationdPhenotypic

Gabriel Livera, CEA – INSERM – Universities Paris 7 and Paris 11, Fontenay-aux-Roses, France
© 2018 Elsevier Inc. All rights reserved.

The male sex determination is a sequential process that starts during fetal life in mammals. The building of the testis from the undif-
ferentiated bipotential gonad is the process central to establish the male characteristics in the embryo and later. The somatic cells of
the male gonad will impose the spermatogenic fate to the neighboring germ cells and act on distant tissues through hormone
production to virilise the reproductive tract and other organs. The formation of testes in the embryo is thus key for the establishment
of maleness. During fetal life, the proper differentiation of the testis is not only mandatory for allowing the sperm production later,
in adulthood, but also to permit the masculinization of the fetus with the development of appropriate reproductive organs both
intern and extern.

Testicular Differentiation

Before the testes acquire their adult oval-shape, side by side with the epididymides, in a scrotal position they undergo a developmental
process starting with two long fine threads that occupy most of the abdominal cavity, the undifferentiated embryonic gonads. The
many cell types of the testis are all formed early during development with a fast and sequential sequence of differentiation (Svingen
and Koopman, 2013). In mammals, this sequence is usually initiated during the first third of the gestation. The testis comprises
various somatic cells and germ cells and all have to acquire a male fate during development. However, it is only the testicular somatic
cells, more specifically the Sertoli and Leydig cells, that are required to properly masculinize the embryo (Fig. 1).
The gonad appears first as an anlage on the ventral face of the mesonephros, a primitive kidney present during embryonic devel-
opment in mammals. The thickening of it due to cell proliferation and migration of various cell types, including the primordial germ

(A)
peritubular cell

testicular cord
Sertoli cell

gonocyte
interstitium

Leydig cell
mesenchymal cell
microcapillary

(B) (C) (D)

(E) (F)

Fig. 1 Testicular differentiation. (A) Various testicular cell types appear during fetal life and form the testicular cords and the interstitium.(B) Histo-
logical section from a 15.5 dpc mouse fetal testis. (C) Sertoli cells stained with AMH from a 8 wpf human testis. (D) Gonocytes stained with DDX4 in
a 13.5 dpc mouse testis. (E) Peritubular myoid cells stained with a-SMA in a 15.5 dpc mouse testis. (F) Leydig cells stained with 3-bHSD in a 17.5
dpc mouse testis. Scale bars: 10 mm, B, E and F; 30 mm, C; 100 mm, D. AMH, anti-Müllerian Hormone; DDX4, Dead box polypeptide 4/VASA
homolog; a-SMA, alpha smooth muscle actin; 3-bHSD, 3-beta hydroxy steroid deshydrogenase.

88 Encyclopedia of Reproduction, 2nd edition, Volume 1 https://doi.org/10.1016/B978-0-12-801238-3.64363-5

 Development j Male Sex DeterminationdPhenotypic 89

cells, will result in an undifferentiated bipotential gonad. Such can be observed at 11.5 days post-conception (dpc) in the mouse and
5 weeks post fertilization (wpf) in humans. The gonads then contain the bipotential precursors of somatic and germ cells.
Shortly after, in XY embryo, testicular cords form (Nel-Themaat et al., 2009). This is the first morphological event that allows
distinguishing a testis from an ovary. It relies on the differentiation of the Sertoli cells. The first Sertoli cells can be observed as early
as 12.0 dpc in the mouse testis and about 6 wpf in humans. Embryonic Sertoli cells are the conductor of the male sex determination.
These cells will provoke the rapid appearance of the steroidogenic testicular cells, the Leydig cells, as soon as 12.5 dpc. They also
trigger the differentiation of the peritubular myoid cells surrounding the cords and impose the male fate to the germline.

Testicular Cords
The gonadal anlage results from a thickening of the coelomic epithelium due to cell proliferation and migration. These cells are
believed to be the precursors of the somatic lineages in the genital crest. Interestingly the XY embryonic gonad grows slightly thicker
that its XX counterpart from then on.
Sertoli cells are the first to differentiate within the future testis (Magre and Jost, 1991). These cells provide support and protection
to the male germline and are thus termed supporting cell lineage. In the mouse embryo, around 12.5 dpc, Sertoli cells that have
differentiated polarize and aggregate to surround groups of germ cells. The tube-like structures then created are the testicular cords,
the rudiments of the seminiferous tubules that will be observed in the adult testis. The simultaneous formation of several cords has
been demonstrated to be a two step process: first, a large bundle of Sertoli and germ cell formed, second it is partitioned in cord-
forming units due to its fractionation by cells migrating from the mesonephros toward the coelomic epithelium (Cool et al., 2012).
Sertoli cells do proliferate actively during the fetal life and cease progressively multiplying during post-natal life. In the mouse,
Sertoli cell proliferation is almost null around 15 days postpartum (dpp) (Vergouwen et al., 1991). Thus the fetal and post-natal
multiplication of Sertoli cell is key to establish the testicular size. Indeed it is accepted that each Sertoli cell can only support a given
number of germ cells in the adult testis.
Myoid cells surround the testicular cords, forming an additional outer layer. These peritubular cells appear at 13.5 dpc in the
mouse and aggregate around Sertoli cells. Then, they rapidly elongate and flatten. At 14.5 dpc, peritubular cells start expressing
markers of smooth muscular cells such as alpha smooth muscle actin (a-SMA). In the human, peritubular cells are morphologically
recognizable at 12 weeks of gestation. Together with Sertoli cells they will produce a basement membrane or basal lamina
(Merchant-Larios and Taketo, 1991). In the adult peritubular myoid cells display a contractile function needed to allow sperm
progression in the tubules.
The Sertoli cells develop tight junctions between adjacent cells. This is part of what is called the blood testis barrier (BTB). Such
BTB is a structure required for the proper progression of spermatogenesis. In the mouse the BTB is set up between 15 and 20 dpp.
This coincides with the period when a lumen appears in the testis cords; then these are not anymore cords but tubules. This trans-
formation of testis cords in seminiferous tubules is a mark of their terminal differentiation, albeit these do not produce yet sperm
cells. The tubular diameter will progressively increase to accommodate the developing seminiferous epithelium until adulthood.

Vascularization
The embryonic testis displays very early a specific vasculature. The embryonic gonads contain numerous microcapillaries and in the
case of the embryonic testis a blood vessel becomes visible very early on the ventral part of the gonad just beneath the epithelium
(Brennan et al., 2002). This male-specific coelomic vessel will be the future spermatic artery. It likely originates from the cells that
migrated from the mesonephros as these bear endothelial cell markers and are thus believed to form the vasculature of the gonadal
primordium. The distinctive coelomic vessel can be observed in the male fetal gonad by 13.5 dpc. The specific arterial system set up
in the male embryonic is believed to facilitate the proper exportation of male hormones.

Leydig Cells
The interstitial tissue, in-between the cords, contains the Leydig cells that are responsible for the production of androgens. Fetal
Leydig cells form a population distinct from the adult ones. Fetal Leydig cells appear just after initial sex differentiation and induce
masculinization of the fetus by producing androgens (Habert et al., 2001). Leydig cell number increases throughout fetal life from
about 200 at 12.5 dpc up to more than 10,000 in the new-born mouse. At the same time, those cells progressively increase in size
and aggregate forming clumps often near or around blood vessels. Leydig cells produce testosterone as early as they differentiate and
testosterone production peaks about 17.5 dpc in the mouse, and 14 wpf in humans (Livera et al., 2006). These androgens will be
mandatory for the masculinization of the fetus. Of interest, fetal Leydig cells differ from the Leydig cell population observed in the
adult testis. Indeed, at puberty a new population of adult Leydig cells will replace earlier populations. These later have small and
relatively fewer cytoplasmic lipid droplets in comparison to fetal Leydig cells (Haider et al., 1995). Fetal Leydig cells in rodents can
produce testosterone independently of luteinizing hormone stimulation (LH), while adult Leydig cells heavily depend upon LH
stimulation. This appears untrue in the case of human fetal Leydig cells that are literally bathed in chorio-gonadotropin (LH
analog). Perhaps the best criterion differentiating fetal Leydig cells from their adult counterpart is their ability to respond to multiple
LH-stimulation without desensitization. This feature likely accounts for their formidable androgen production. Of note, fetal Leydig
cells also produce another hormone required for proper masculinization (INSL-3).
90 Development j Male Sex DeterminationdPhenotypic

Germ Cells
During fetal life, the testis contains exclusively mitotic germ cells, the meiotic and post-meiotic cells will only be formed after birth
with meiotic onset at puberty. The germ cells are not formed within the embryonic gonad but originate from outside. The primor-
dial germ cells (PGC) migrate from a region quite distant from the gonads, being at the base of the allantois at 6.5 dpc. After a long
journey, germ cells finally reach the genital crests around 10 dpc. At that time they are termed post-migratory PGCs, they actively
proliferate and are often observed with patches of condensed chromatin in their nuclei. In the rodents, once included in testis cords,
germ cells will progressively stop dividing. Then they are often referred as “gonocytes” (Culty, 2009). The exact reason for this
mitotic arrest is unknown though it has been hypothesized that it may facilitate epigenetic events occurring then. The quiescence
phase of gonocytes starts from 14.5 or 15.5 dpc and lasts up to birth in the mouse. At that time gonocytes are easily recognized due
to their large spherical nuclei containing fine chromatin granules and often two globular nucleoli. In the human testis no quiescence
phase has been reported possibly due to the lack of synchronicity of the human germline during development. During the begin-
ning of postnatal life, in rodents, the gonocytes relocate progressively from the center of the cords to the basement membrane. This
will also be the place homing the future spermatogonia that derived from the gonocytes. The transition from gonocyte to a presper-
matogonial type in contact with the basement membrane occurs before birth in humans.

Masculinisation of the Reproductive Tract

Internal and external genitalia develop after the testicular differentiation. The internal reproductive organs aredapart the testesdthe
epididymis, the vas deferens and the accessory glands (seminal vesicles, prostate and bulbourethral glands). The epididymis is
a long duct connected to the seminiferous tubules in its anterior part (head) and in which sperm will mature. The vas deferens
is the portion of tube connected to the posterior end of epididymis (cauda). The accessory glands provide fluids to lubricate the
duct system and sustain the sperm. This forms the reproductive tract that passes the sperm from testes up to the ejaculation. The
external genital organs are the penis and the scrotum, a pouch-like structure holding the testes below the body wall.

Internal Genitalia
Unlike the gonads, the male and female reproductive tracts originate from different primordia. Prior to sex determination, embryos
contain two complete sets of ductal structures. The male genital tract arises from the Wolffian ducts and the female one from Mülle-
rian ducts (Rey et al., 2000). Those ducts are also termed respectively mesonephric and paramesonephric ducts due to their position
in the mesonephros bordering the gonad. In the male embryo, the primordium of the female tract, the Müllerian ducts, will disap-
pear while the primordium for male tract will develop. The regression of the Müllerian duct is due to the anti-Müllerian hormone
(AMH) production by Sertoli cells during development. Androgens produced by fetal Leydig cells actively maintain Wolffian ducts,
which give rise to the epididymis, vas deferens and seminal vesicles. The external genitalia differentiate from the urogenital sinus.
External genitalia define the sex for the civil status in the human species.
The two pairs of ducts co-exist in the mesonephroi in both sexes at the beginning. The Wolffian duct appears as a continuous
tube that runs the length of the urogenital ridge. Thus the Wolffian duct is present all along the mesonephros; it is nearby the Mülle-
rian duct. Wolffian ducts open in the urogenital sinus at the most posterior end of the embryo.
The first event evidencing a male differentiation of the reproductive tract is the regression of the Müllerian ducts. This regression
is observable at 14 dpc in the mouse male embryo and at 8 wpf in human ones (Dyche, 1979). These have almost completely
disappeared by 9 wpf. Second, shortly after, due to the development of the definitive kidney (metanephros) and under the action
of androgens the Wolffian ducts will give rise to the male reproductive tract. This witnesses a functional change from urinary to
reproductive function. Testosterone allows the stabilization and the differentiation of Wolffian ducts (Welsh et al., 2007). The
main organ resulting from this differentiation is the epididymis. The formation of the epididymis involves a complex program
to achieve the formation of a long tube with a complex pattern of coiling, formation of septae, and regional functionalization.
The cranial part of the duct become connected to the testis cords forming the rete testis and the portion of the duct just below
tremendously elongate and becomes convoluted to form the epididymis. The caudal part of the duct will develop a muscular enve-
lope and form the vas deferens. In the most distal part of each duct, just prior the junction with the sinus, a lateral diverticulum will
grow and produce the seminal vesicle. Thus the main intern accessory reproductive organs are all derived from the Wolffian ducts.
The prostate and bulbourethral gland originate from the differentiation of the uro-genital sinus (Table 1).
Pioneer studies by Alfred Jost evidenced that in utero castration of rabbit fetuses prior to sexual differentiation systematically
triggered a female-type development of the genitalia (Jost, 1953). This demonstrated that only testicular hormones are key for
both the regression of the Mullerian ducts (AMH) and the development of Wolffian ducts (testosterone).

External Genitalia
The external genital differentiation is also profoundly stroke by the presence of testicular androgens (mostly dihydro-testosterone in
this case). The external genitalia differentiate from a bipotential anlage composed of the genital tubercle, folds and outer swellings
that remain sexually undifferentiated until 9 wpf in human embryos (Yamada et al., 2003). Then male specific change will appear
 Development j Male Sex DeterminationdPhenotypic 91

Table 1 Chronology of the main events during male differentiation

Mouse Human

Formation of the gonadal primordium 10 dpc 4 wpf

Colonization by the primordial germ cells 10.5–11.5 dpc 4–5 wpf
Formation of testicular cords 12.5 dpc 7 wpf
Differentiation of Leydig cells 12.5 dpc 8 wpf
Regression of Müllerian ducts 14 dpc 8 wpf
Masculinization of external genitalia 16 dpc 10 wpf
Appearance of seminal vesicle 16 dpc 10 wpf
Appearance of prostatic buds 17 dpc 10 wpf
Increased anogenital distance 16 dpc 11–13 wpf
Fully descended testes 25 dpp 30 wpf

dpc, days post-conception; dpp, days post-partum; wpf, weeks post-fertilization.

Modified from Saint-Dizier M. & Chastand-Maillard S: La reproduction animale et humaine. (2014) Chapter 1: p. 19–39. Quae,
Versailles, France.

with a near-complete male phenotype observable at the beginning of the second trimester (14 wpf). The genital tubercle elongates
and grows to form the penis. The folds located below, on each side of the urethral groove, will close over the groove. The swellings
on the outer sides of the folds, also referred as labio scrotal ridges, will form a pouch to later accommodate the testes, the scrotum.
As the scrotum forms, testes descend down the posterior abdominal wall, through the inguinal canal. This descent is a long process
starting around 10 wpf and usually completed around 30 wpf. Testicular descent also depends on androgen and on another Leydig
cell secretion the insulin-like 3 (INSL-3). Testicular descent is decomposed in a two phases process with INSL-3 mediating the first
part, trans-abdominal, and androgens mediating the second part, inguino-scrotal (Klonisch et al., 2004). In mice, testicular descent
is mostly post-natal and completed by 25 dpp. This extra-abdominal position of the testes provides them with a temperature lower
than the body temperature, a difference mandatory for proper spermatogenesis. However, one should keep in mind that not all
mammals have descended testes.

Male Determination of Other Tissues

The development of male-specific features is obviously not only restricted to the gonads and reproductive tract, albeit studies and
consensus remain scarce in other tissues. The perineal growth and the digit ratio are indicators that have been promoted as markers
of virilisation. The brain evidently is also primed differently in males and females. Though this is far from exhaustive other param-
eters have not yet received detailed attention.
The development of the genital tubercle and genital swellings into, respectively, the penis and scrotum is accompanied by an
increase in perineal growth and thus a longer anogenital distance (distance from the anus to the posterior base of the genital
tubercle). This distance is longer in males than in females in most mammals (twice as long in rodents) (Welsh et al., 2008). As
this feature is easy to observe at birth, it is often used as a marker of the proper exposure to androgens and proper masculinisation
during fetal life.
The ratio of the lengths of the index and ring finger (2D,4D digit ratio) has been proposed as another marker of androgen action
during development in mice and humans (McIntyre, 2006). Males have generally a longer ring finger (digit 4) relative to the length
of index finger (digit 2) than females. This parameter is quite attractive as it is easy to measure though it remains debated.
Sexual dimorphism of brain structures has been reported in various species. This sexual differentiation of the brain induces
neuroanatomical changes that are believed to impact behavior. However many environmental factors also shape the brain and
this complexity has limited a clear view of what is due to a direct hormonal effect. Maybe one of the best examples that male
and female brains differ at the anatomical level is the existence of a nucleus in the rat brain with a different size according to
the sex, the “sexually dimorphic nucleus of the preoptic area” of the hypothalamus (Gorski et al., 1978). In the male rat the volume
of this structure is about three to eight times larger than in females and this sexual difference is mostly established during post-natal
development. This structure is implicated in sexual behavior in animals. The volume or neuron number has since been reported to
differ in numerous other areas of the brain albeit many variations in species exist. Lastly, early differences observed between the XX
and XY embryonic brains (prior testicular steroidogenesis) also point toward likely cell-autonomous events possibly related to the
chromosomal constitution. This possibility that some male features might rely on cell-autonomous events (not being the conse-
quence of having testes) is similar to what has been reported in marsupials, where the formation of a scrotum, or a pouch, is a conse-
quence of the absence or presence of two X chromosomes.
Anecdotally, the mammary gland development differs according to the sex in some mammals but not in others. Though in
humans, at birth, roughly similar mammary structures are observed in boys and girls, in rats and mice, the post-natal mammary
structures appear strikingly different. In the rat fetus, the mammary anlage shows a male-specific atrophy of the rudimentary
buds. This suppresses nipple formation during post-natal life, another parameter often used to monitor proper masculinisation,
albeit exclusively in rodents.
92 Development j Male Sex DeterminationdPhenotypic

In conclusion, the testicular development is key to maleness acquisition. The hormonal secretions of the developing testis ensure
that the development of the gonad is properly coordinated with that of the rest of the organism to ensure the complete establish-
ment of the male reproductive potential once adult. Testicular androgens are the main endocrine factors promoting masculine
phenotypes. A period of high testosterone production in males during the fetal life drives the differentiation of primary reproductive
tissues. Nevertheless, the particular pattern of testosterone production varies among vertebrate species and differences in potential
target tissues are widely debated.

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