C H A P T E R

4
Development of the Mammalian
Ovary and Follicles
Katja Hummitzsch, Helen F. Irving-Rodgers, Jeff Schwartz,
Raymond J. Rodgers

Abbreviations mammals include the ovulation of oocytes and produc-

tion of hormones that act to coordinate reproductive
Bmp2 bone morphogenetic protein 2
Ctnnb1 beta-catenin activity among organs. The endocrinology of the adult
DAX1 dosage-sensitive sex reversal, adrenal hypoplasia crit- ovary is relatively unique as the regulation of hormone
ical region, on chromosome X, gene 1 secretion by the ovary is determined by formation and
DAZL deleted in azoospermia-like regression of the follicles and corpora lutea. The diversity
dpc day post coitum of cell types in the ovary serves a breadth of endocrine,
E embryonic day (mouse development)
Emx2 empty spiracles homeobox 2 oogenic, and other functions. The multiplicity of cell
Fog2 friend of GATA types dictates the necessity of precise cell-cell communi-
FOXL2 forkhead box L2 cation within the ovary for proper function. Much recent
FST/ fst follistatin research in the ovary has focused on how cells are regu-
Gata4 GATA binding protein 4
lated and how they communicate with each other. It is
GREL gonadal-ridge epithelial-like
LGR5 leucine-rich repeat-containing G-protein coupled
therefore of increasing importance to understand the cell
receptor 5; also known as GPR49 or GPR67 lineages and cell-fate decisions that occur cyclically and
Lhx9 Lim homeobox protein 9 linearly during the functional life of the ovary.
Lypd6 LY6/PLAUR domain containing 6 Since much of the research effort in this area has been
Magi2 membrane-associated guanylate kinase, WW and PDZ
directed toward humans and agricultural and laboratory
domain containing 2
OCT3/4 octamer-binding transcription factor 4; also known as species, we will mainly reference this literature. Much of
POU5F1 the published research on fetal development of ovaries in
PD postnatal day primates and agricultural species relies extensively on
PGC primordial germ cell timed histological observations of the developing ovary.
Rspo1 R-spondin 1
Using this approach, recent research has been directed
Sf1 steroidogenic factor 1
SYPC3 synaptonemal complex protein 3 toward the identification and study of the epithelial
VASA mouse vasa homologue, MVH or DEAD and the stromal compartments and has documented
(Asp-Glu-Ala-Asp) box polypeptide 4, DDX4 the important role of mass migration of stromal cells dur-
WNT4/ Wnt4 Wingless-Type MMTV Integration Site Family, ing formation of the ovary. Studies of mice, in particular,
Member 4
have relied heavily on lineage tracing techniques and on
Wt1 Wilm’s tumor suppressor gene
manipulation of gene expression during development.
Additionally, many studies of rodents have focused on
Introduction comparisons between development of the female and
male gonads. Although this is important, less is known
The reproductive strategies that have evolved in dif- about the systematic development of the ovary. Thus,
ferent species have been the result of strong natural selec- the understanding of development of the ovary across
tion pressures. Thus, it is no surprise that a wide variation species is somewhat disjointed.
exists between species in the development and structure The origin of granulosa cells was always believed to be
and function of female gonads. The key roles of ovaries in an epithelial cell, but over the years, three separate

The Ovary 71 © 2019 Elsevier Inc. All rights reserved.

https://doi.org/10.1016/B978-0-12-813209-8.00004-2
72 4. DEVELOPMENT OF THE MAMMALIAN OVARY AND FOLLICLES

Granulosa cells of rete ovarii origin

(From regressing nephrons of the mesonephros) Granulosa cells of surface epithelial origin

(A) (B)

Surface epithelium Mesonephric tubules Basal lamina Fibre

Granulosa cell Oogonia, oocyte Stromal cell Capillary
FIG. 1 Illustrations of earlier theories on key aspects of ovary formation. (A) The somatic epithelial granulosa cells were considered to arise from
other epithelial cells of rete ovarii of the medulla [1], which in turn arose from the nephrons of the mesonephros. (B) The surface epithelial cells con-
tribute somatic cells to the ovigerous cords, which in turn give rise to the somatic epithelial granulosa cells [2,3].

pathways were hypothesized. Initially, they were pro-
posed to be derived from the nephrons of the mesoneph-
ros (Fig. 1; reviewed recently in Refs. [4, 5]). The
mesonephros acts as a temporary kidney early in gesta-
tion and contributes cells to the developing gonads, dif-
ferently between the male and female ([6–12]; for
review see Refs. [13, 14]). In ovaries, mesonephric tubules
are found in the hilum and medulla where they are
referred to as the rete ovarii. They persist into adulthood.
The hypothesis that rete ovarii give rise to granulosa cells
was based on the close association of rete ovarii with
oocytes [1,15]. This was further strengthened by the
demonstration that the presence of rete ovarii correlated
with onset of meiosis [16] and follicle formation [17].
Subsequently, it was suggested that cells derived from
the ovarian surface epithelium give rise to the granulosa
cells [2,3]. However, a more recent examination of
bovine ovarian development suggested that granulosa
cells are not derived from differentiated ovarian surface
epithelial cells; rather, both the apical ovarian surface epi-
thelium and the granulosa cells arise from a common pre-
cursor population of gonadal ridge epithelial-like (GREL)
cells [18]. GREL cells, in turn, are derived from cells of the
surface of the mesonephros [18], which replicated to
form the gonadal ridge/ovarian primordium. This is
the model that we present in this chapter. The origins
and development of germ cells and the cumulus cells are
covered in other chapters. This chapter will focus on the
origins of somatic cells and the formation of structures of
the ovary.
GONADAL (OR GENITAL) RIDGE
FORMATION
The sexually undifferentiated gonadal ridge forms as a
thickening of the coelomic or surface epithelium in an
anterior/posterior direction on the ventral side of the
mesonephros. The mesonephros acts as transient fetal
kidney in mammals and in females regresses around
55 days post coitum (dpc) in cow (term 283 d) [19]
and 75 dpc in sheep (term 147 d) [3]. Gonadal ridge for-
mation occurs 33 dpc in humans (term 266 d) [20],
between 27 and 31 dpc in the cow [21], 23 dpc in sheep
[22], 25 dpc in goat (term 150 d) [23], and embryonic
day (E)10.3–10.4 in mice (term 20 d) [24] (Table 1).
The initial thickening and full development of the
gonadal ridge requires the expression of the transcription
factor Gata4 (GATA-binding protein 4; [24]) in coopera-
tion with Fog2 (Friend of GATA; [51]), Sf1 (steroidogenic
factor 1), Wt1 (Wilm’s tumor suppressor gene), Lhx9 (Lim
homeobox protein 9; reviewed in Ref. [52]), and Emx2
(empty spiracles homeobox 2; [53]). Conditional knock-
out of Gata4 in mice decreased proliferation of coelomic
or surface epithelial cells before the thickening process
of the gonadal ridges would normally occur and to
reduced Sf1 and Lhx9 expression [24]. Homozygous null
mutations in any of the other genes resulted in regression
of the developing gonadal ridge in mouse embryos.
The coelomic or surface epithelium of the mesonephros
proliferates and undergoes phenotypical changes
(Fig. 2A–C), giving rise to GREL cells in bovine [18],

I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS

 GONADAL (OR GENITAL) RIDGE FORMATION 73
TABLE 1 Comparison of Key Events During Ovarian Development Between Rodents, Primates, and Ruminants
Rodents Primatesa Ruminants

Gestational length Mouse: 18–22 days Human: 40 weeks Cow: 279–287 days
Rat: 21–23 days Sheep: 144–152 days
Goat: 148–152 days
Genital ridge development
Formation Mouse: E10.3–10.4 [24] Human: week 5 [20] Cow: 27–38 dpc [19,21]
Rat: E12 [25] Sheep: 23 dpc [22]
Goat: 25 dpc [23]

Thickening of coelomic epithelium Mouse [24] Human [26,27] Cow [19]

Rat [25]
Basement membrane dissolution (below Mouse [24]
coelomic epithelium/mesonephric surface
epithelium)
Sexual differentiation Mouse: E10.5–11.5 dpc [29]
Rat: E14.5 [30]
Oogenesis
PGC migration Mouse: E9.5–11.5 [32]
PGC arrival at genital ridge Mouse: E10.5 [34]
Germ cell differentiation Synchronized in ovigerous cords;
from anterior to posterior of
ovary [34]
Human [26–28]
Human: week 6–7 [20]
Rhesus monkey: week 7–8
[31]
Human: week 5 [20,33]
Human: week 6 [33]
Zonal; medullar (mature) to
proximal (immature) [18,31]
Cow [18,19]
Cow: 40 dpc [21]
Sheep: 28 dpc [22]
Goat: 34 dpc [23]
Cow: 23–31 dpc [21]
Cow: 32 dpc [21]
Sheep: 23 dpc [3]
Zonal; medullar (mature)
to proximal
(immature) [35]

Mitosis Rat: E14.5–18.5 [36] Human: 2–7 months [36] Cow: 50–150 dpc [37]
Rhesus monkey: 7–19 weeks
[31]
Germ cell nests/cysts Mouse [34] Human [38] Cow [19]
Entry into meiosis Mouse: E13.5 [39] Human: week 11 [35] Cow: 70–80 dpc [37]
Rat: E17–18 [40] Rhesus monkey: 8 weeks [31] Sheep: day 55 [2]
Goat: 55 dpc [23]
Waves of germ cell apoptosis Mouse: E11.5–13.5 and E17.5–D9 Human: around week 22 [41] Cow: 130–170 dpc [37]
[39] Sheep: 75–100 dpc [36]
Rat: E17–20 [40]
Meiosis arrest Mouse: E17.5-PD5 [34] Cow: 120–150 dpc [37]
Ovigerous cords
Formation Mouse: E13.5 [42] Human: week 9–12 [28] Cow: around 60 dpc [43]
Rat: E13 [25] Sheep: 38–75 dpc [2]
Goat: 36 dpc [23]

Connection with surface/no. separation Mouse [42] Human [28] Cow [18,19]
from ovarian surface by basement Sheep [4]
membrane (early stage)
Breakdown Mouse: E17.5 [39] Human: week 20 [39] Cow: 90–130 dpc [18,44]
Follicle formation and activation

Primordial follicle Mouse: after birth [PD1–7, [45]] Human: week 18–20 [28,47] Cow: around 90 dpc [44]
Rat: week 1 after birth [46] Rhesus monkey: 20–23 [31] Sheep: around 100
dpc [2]
Goat: 90 dpc [23]
Continued

I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS

74 4. DEVELOPMENT OF THE MAMMALIAN OVARY AND FOLLICLES

TABLE 1 Comparison of Key Events During Ovarian Development Between Rodents, Primates, and Ruminants—cont’d
Rodents Primates Ruminants

Region of primordial follicle Formation Mouse: medulla (first wave), Inner cortex, close to Inner cortex, close to
cortex (second wave) [48] medulla [28] medulla [2,18]
Rat: inner zone [46]
Primary follicle Rat: PD5 [49] Human: at 40 weeks all stages Cow: 140 dpc [44]
of folliculogenesis observed Sheep: 90 dpc [3]
[50]b
Preantral follicle Cow: 210 dpc [44]
Sheep: 120 dpc [3]
Antral follicle Rat: PD15 [49] Cow: 240–285 dpc [19]
Sheep: 135 dpc [3]
a
Literature about nonhuman primates with specific time points for developmental stages is rare. It mostly focuses on physiological aspects during pregnancy or on generational effects in
disease animal models such as PCOS.
b
Human fetal ovary samples from the third trimester are rare, and therefore the exact timing of the different follicle types after follicle activation is not available.

(A)

(B)

(C) (D) (E)

(F) (G) (H)

Surface epithelium
Germ cells:
GREL cell Fibre Oogonia
Primordial Degenerative
Granulosa cell Capillary Primary oocyte (arrested at
Mitotic Meiotic
Stromal cell Basal lamina prophase l)

FIG. 2 Schematic diagram of ovarian development. The development of the ovary commences at the mesonephric surface (A) in the location of the
future gonadal ridge. Some mesonephric surface epithelial cells change phenotype into GREL cells (B). (C) The GREL cells proliferate and the basal
lamina underlying the mesonephric surface epithelium breaks down. GREL cells continue to proliferate and PGCs (gray) migrate into the ridge
between the GREL cells (D). Stroma from the mesonephros, including blood vessels, continues to expand and penetrates the ovary toward the ovar-
ian surface. As it does so it branches and thus corrals proliferating oogonia and GREL cells into forming the ovigerous cords (E). The cords are sur-
rounded by a basal lamina at their interface with stroma, but are open to the ovarian surface, and compartmentalization into cortex and medulla
becomes obvious. Once the stroma has expanded to just below the surface the outermost GREL cells become partitioned on the surface separated
from the underlying stroma by a basal lamina and then the GREL cells differentiate into the ovarian surface epithelial cells (F). Ovigerous cords are
partitioned into smaller cords, commencing first at the interface with the medulla. Eventually, the cords are partitioned into follicles containing GREL
cells that differentiate into granulosa cells and oogonia that become oocytes. The first primordial follicles appear in the inner cortex-medulla region,
surrounded by a basal lamina (G). (H) Finally, the ovarian surface is covered by a simple epithelium. A tunica albuginea, densely packed with fibers,
develops from the stroma below the surface epithelial basal lamina. Some primordial follicles become activated and commence development into
primary and preantral follicles. Modified from Hummitzsch et al. [18] and incorporating information from Smith et al. [54].

I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS

 OVIGEROUS CORD FORMATION
which start to express the cytoskeletal protein vimentin,
as occurs with the subepithelial mesenchyme in the rat
[25]. GREL cells express cytokeratins 18 and 19,
plakophilin-2, and desmoglein-2 as do mesonephric sur-
face epithelial cells [18]. Expression of cytokeratins 8, 18,
and 19 has been found in the undifferentiated gonad in
the mouse [42], whereas no cytokeratin 18 expression
was detected in the rat [55].
Also during development, the basal lamina underly-
ing the mesonephric surface epithelium where the
gonadal ridge forms undergoes remodeling in a process
controlled by Gata4 [24] as shown in mice. A similar
incomplete basal lamina has been also observed in bovine
ovarian development [19]. Presumably, this allows the
primordial germ cells (PGCs) and mesenchymal or
stroma cells from the mesonephros to penetrate between
the proliferating surface epithelial cells of the developing
gonadal ridge [18,26]. Mesonephric cell migration has
been observed in the ovaries of human around 46 dpc
[26] and sheep as early as 30 dpc (reviewed in Ref. [54]).
This penetrating mesonephric stroma contains a vascular
capillary bed, providing blood supply to the developing
ovary. Thus, the early ovarian primordium contains two
population of somatic cells; cells similar in appearance to
the coelomic or surface epithelium, now termed GREL
cells, and cells similar to blastemal cells of the mesoneph-
ros [26]. Based upon the description of their penetrating
behavior, these blastemal cells of the mesonephros are
the stromal cells from the mesonephros (Fig. 2D).
PGCs, the carrier of genetic information of the next
generation and the precursors for oogonia and oocytes,
arise from the yolk sac (for review see [33,56–59]) and
migrate through the primitive gut into dorsal mesentery
and then laterally to the gonadal ridges. This process is
regulated by the expression of stem cell factor and
stromal-derived factor-1 by the gonadal ridge and sur-
rounding mesenchyme (reviewed in Ref. [33]). In mice,
PGCs start to migrate at E7.5 and enter the gonadal ridge
at E10.5 ( [60], Table 1). In humans, the migration occurs
in gestational week 5 and the PGCs arrive at week 6
(reviewed in Ref. [33]). PGCs begin to proliferate during
the migration process.
The colonization of the gonadal ridges by PGCs is fol-
lowed by sex determination (reviewed in Ref. [61]) and
subsequent differentiation into oogonia in the developing
ovary (reviewed in Ref. [33]). The sex determination
occurs in the human at week 6–7 [20], around 40 dpc in
the cow [21], at 28 dpc in the sheep [22], after 25 dpc
in the goat [62], at 23 dpc in the pig (term 114 d) [63],
and around E10.5–11.5 in mice [29] (Table 1). The differ-
entiation into an ovary is characterized by increased
expression of Wnt4 (wingless-type MMTV integration
site family, Member 4), Ctnnb1 (beta-catenin), Rspo1
(R-spondin 1), Foxl2 [Forkhead box 2, [64]], FST (follistatin),
DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
75
critical region, on chromosome X, gene 1), and meiosis-
marker SYCP3 (synaptonemal complex protein 3) [65]. Loss
of expression of some of these genes, such as Foxl2 and
Wnt4, results in female-to-male sex reversal (reviewed in
Ref. [52]).
PGCs migrate by amoeboid movement to the gonadal
ridge during which they begin to proliferate. Once in the
gonadal ridge, they cease migration and are classified as
oogonia. Oogonia have a higher mitotic rate than PGCs
and form germ cell nests. These oogonial nests undergo
mitosis synchronously, and the oogonia are connected
by intercellular bridges caused by cytokinesis that is
incomplete at this stage [19,34,38]. Recently, it has been
shown that the proliferation of germ cells is dependent
upon the expression of Wt1 in somatic cells of the gonadal
ridge [60]. Functional point mutation of Wt1 in mice
resulted not only in a decrease of cell number from
mitotic arrest and morphological change from an epithe-
lial to mesenchymal phenotype in somatic cells but also in
a decrease in germ cell numbers early in oogenesis
(E10.5–12.5).
OVIGEROUS CORD FORMATION
After formation of the gonadal ridge and sex differen-
tiation, the stroma from the mesonephros penetrates
further into the gonadal ridge/ovarian primordium,
which at this stage is composed of GREL cells, PGCs,
and oogonial nests [18]. This process creates areas of
stroma, characterized by expression of COUP-TfiI [(also
known as nuclear receptor subfamily 2 Group F Member
2 (NR2F2)] in stromal cells, alternating with areas of
GREL cells/germ cells. This in effect produces the oviger-
ous cords composed of GREL cells and germ cells, and the
cords are therefore initially “open” to the surface of
the ovary as no distinct surface epithelium underlain
by a basal lamina has been established at this stage
[2,18,19,28] (Fig. 2E). The ovigerous cord formation
occurs gradually from inside of the primordium toward
the outer zone in line with the branching of the stroma
as it penetrates toward the surface of the ovary. The
penetrating stroma has been observed previously in
human [50] and described as “cell streams” in sheep [3].
Importantly, the ovigerous cords are separated from the
stromal compartment by a continuous basal lamina
[3,18,19,28], composed of subunits of laminin 111 and
collagens type IV and type XVIII, perlecan, and nidogens
1 and 2. The somatic GREL cells inside the ovigerous
cords strongly express FOXL2, a marker of granulosa
cells (the role of FOXL2 in the ovary is discussed in
Ref. [66]). Furthermore, the GREL cells still express
cytokeratins 18 and 19 [18,67], plakophilin-2, and
desmoglein-2 [18]. Expression of cytokeratins 8 and
16–19 has also been identified in the somatic cells of the
76 4. DEVELOPMENT OF THE MAMMALIAN OVARY AND FOLLICLES
ovigerous cords of the fetal rat ovary [25]. The entry of
oogonia into meiosis is first initiated in oogonia at the
base of the ovigerous cords closest to the medulla and
is triggered by retinoic acid, which is synthesized by alde-
hyde dehydrogenases in the mesonephros (Aldh1a2/3)
and in the ovary (Aldh1a1) [68].
REGIONALIZATION—CORTEX AND
MEDULLA
The formation of ovigerous cords results in regionali-
zation of the developing ovary into cortex and medulla in
human [69], bovine [18], sheep [2], and goat [23]. In mice,
a morphological regionalization can be observed close to
birth at day E18.5 [70], but at a molecular level, the cortex
and medulla can be distinguished as early as E12.5 by
expression of Bmp2 (bone morphogenetic protein 2,
[70]), Lypd6 (LY6/PLAUR Domain Containing 6), and
Magi2 (membrane-associated guanylate kinase, WW
and PDZ domain containing 2, [71]) in the cortex and
Wnt4 and Fst [70] in the medulla. In general, the medulla
is relatively much smaller in mice than humans and other
species studied, and in the adult mouse ovary, it is even
less obvious (reviewed in Ref. [72]). The cortex contains
the ovigerous cords early in development and a vascular-
ized stroma. Proliferation of oogonia continues to occur
in the outer cortex, and maturation into oocytes and for-
mation of first follicles happen at the interface of inner
cortex and medulla (Fig. 2G). The medulla is character-
ized by lymphatic and large blood vessels, connective tis-
sue, and remnants of mesonephric tubules (rete ovarii)
[19,50]. The greatest number of Ki67-positive stromal
cells occurs mainly in the medulla between 120 and 180
dpc in bovine [19].
OVIGEROUS CORD BREAKDOWN AND
FOLLICLE FORMATION
Later in gestation, the stroma continues to penetrate
the cortical region and the ovigerous cords, partitioning
them into smaller groups of germ cells and GREL cells
(Fig. 2F). The gradual breakdown of ovigerous cords
proceeds from the interface between the medulla and
the cortex and extends to the periphery of the cortex
as the stroma expands toward the surface, as has been
detected in fetal human [35,50,73], cattle [18,74,75],
sheep [2,36,76], mouse [77], and postnatal rat ovaries
[78,79]. It reflects the same gradual pattern reported in
the maturation process of oogonia/oocytes as observed
by expression of germ cell markers temporally from
OCT3/4 (octamer-binding transcription factor 4; also
known as POU5F1), DAZL (deleted in azoospermia-
like), and then to VASA (also known as mouse vasa
homologue, MVH or DEAD (Asp-Glu-Ala-Asp) box
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
polypeptide 4 (DDX4) [18,35]). Since there is a basal lam-
ina at the interface of the penetrating stroma and the ovi-
gerous cords, the GREL cells are the only somatic cells
inside the cords, rendering them to be the precursor of
the granulosa cells of follicles [18]. Less clear is whether
GREL cells are identical to pregranulosa cells of primor-
dial follicles or if GREL cells differentiate into pregranu-
losa cells upon follicle formation. The factors that initiate
any transition are also unknown.
As development progresses, significant proportions of
oogonia undergo apoptosis, with the oogonial nests
being reduced to individual oocytes arrested at the
pachytene stage of meiosis I. These are surrounded by
a finite number of GREL cells to form the primordial fol-
licles (Fig. 2F). The basal lamina that thus far separated
the ovigerous cords from the surrounding stroma now
surrounds individual follicles. The number and potential
quality of follicles with which the ovary is endowed, are
ultimately determined by the complex interactions
between the germ cell, GREL cells, and the stroma. Folli-
cle formation occurs in human, ruminants (cow, sheep,
goat), and pig before birth, whereas in the less precocious
mice and other rodents, follicle formation starts shortly
after birth (Table 1). Reports of the first primordial folli-
cles in the human vary from as early as week 11.5 [80]
to as late as between 16 and 20 weeks of gestation
[47,81]. In cattle primordial follicles appear at 90 dpc
[44], around 100 dpc in sheep [2], between 60 and
90 dpc in goat [82], at 70–90 dpc in pig [83], and in mice
between postnatal days (PD)1–7 [45]. The large disparity
in the emergence of primordial follicles observed, even
in the mouse, suggests that this is a real biological varia-
tion, and not just due to experimental methodological
differences.
ESTABLISHMENT OF PRIMORDIAL
FOLLICLE RESERVE
At the early stages of ovarian development, oogonia
are highly proliferative that results in a peak in germ cell
numbers with 6,800,000 between gestational weeks 16–20
in human [84], 2,700,000 at 110 dpc in cow [37], 850,000 at
75 dpc in sheep [36], approximately 15,000 at E15–20 in
mice [41], in the fifth gestational month in rhesus monkey
[31], and at E18.5 in rat [84]. These numbers then reduce
drastically because of declining germ cell proliferation
rates and increasing cell death. The total number of germ
cells decreases by 80%–90% between 5 months and birth
in human [84], 75–100 dpc in sheep [36], and 130–170 dpc
in cattle [37], and by 60% between E18.5 and PD2 in rat
[84]. It has been shown for rat fetal and postnatal ovaries
and human fetal ovaries that there are three waves of
germ cell degeneration [84]. The first wave includes oogo-
nia undergoing mitosis (shortly before meiotic entry),
 FORMATION OF THE OVARIAN SURFACE EPITHELIUM
the second waves affects oocytes at pachytene stage,
and the third concerns oocytes at diplotene stage [84].
Two waves of germ cell death have been reported in mice.
The first occurs during the period of meiosis at E13.5–15.5
and the second wave during follicle formation at
E17.5–PD 1 [85]. The main mechanism for germ cell
death is apoptosis mediated by gene products of the
BCL2 family members (reviewed in Ref. [54]). Recent
studies in mice suggest also an involvement of autophagy
in germ cell death as a response to nutritional stress
around birth. In addition, some oocytes on the surface
of the ovary are lost by germ cell extrusion [86]. Reasons
for the elimination of germ cells are chromosomal
abnormalities (failure of mitosis and meiosis), defective
mitochondrial genomes, insufficient pregranulosa cells,
and degeneration of oocytes during restructuring of
ovigerous cords and cysts into follicles (for review see
Ref. [34, 41]).
FOLLICLE ACTIVATION
Activation of primordial follicles and subsequent
differentiation into primary, preantral, and antral follicles
can be observed late in gestation in the cow [19] and
sheep [3] (Table 1). Follicles in the mature stages undergo
atresia before birth [19], such that only primordial folli-
cles remain in the ovary. Similar events occur in mice
where two classes of primordial follicles have been iden-
tified—medullary primordial follicles which will be acti-
vated shortly after birth and go through folliculogenesis
fast before dying by apoptosis/follicular atresia in the
first 3 months after birth and cortical follicles which will
be activated through adulthood (reviewed in Ref. [48]).
The medullary follicles contain granulosa cells arising
from precursor cells that expressed Foxl2 in the fetal
ovary, whereas granulosa cells from cortical follicles
are derived from Lgr5 (Leucine-rich repeat-containing
G-protein coupled receptor 5; also known as GPR49 or
GPR67)-positive cells, which were located in the cortex
and at the ovarian surface. Mork et al. [87] concluded
that the two distinct granulosa cell population might
still descend from one progenitor line that would be
compatible with the GREL cell model described in the
cow [18].
FORMATION OF THE OVARIAN
SURFACE EPITHELIUM
Previous literature implied that the mature ovarian
surface epithelium originates from the mesoderm-
derived epithelial layer lining the intraembryonic coelom
and the area where the gonad is formed. The gonadal
blastema or ovarian primordium is partly formed by pro-
liferation of this surface layer (reviewed in Ref. [88]).
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
77
Interestingly, when the ovarian primordium first forms,
it lacks a subepithelial basal lamina, except at the base
of the ovary where it arises and protrudes from the meso-
nephros (Fig. 2D) (cow [18,19], human [28], and mouse
[42]). Thus, the base or hilum of the ovary is mesonephros
and its epithelium remains intact while the remainder of
the primordium develops. All cells on the surface of fetal
mice ovaries, except those at the base, express Lgr5 at
E16.5 [89]. This may contribute to the observation that
the hilum or base of postnatal mice ovaries is richer in
stem cells with greater oncogenic potential than other
locations on the ovarian surface [90]. The absence of a
mature surface epithelium during early ovarian develop-
ment is further supported by the observation that sur-
face epithelial cell expression of estrogen receptor 1
(also known as ERα), a characteristic of the adult ovarian
surface epithelial layer, does not occur until at least ges-
tational day 55 in sheep [91] and day 75 in cattle [43].
The early ovarian primordium is composed of GREL
cells that differentiate from the mesonephric surface epi-
thelium, together with germ cells, and stroma penetrating
from the mesonephros. GREL cells lining the outside of
the developing ovary are tightly connected by adherens
junctions to form a protective cover [18]. At the stage of
ovigerous cords, the GREL cells nearest the surface
appear to express cytokeratin 19 [19] and the desmosomal
proteins plakophilin-2 and desmoglein-2 more strongly
than the GREL cells closer to the medulla [18]. Similar
zonal expression has been observed in mouse ovaries,
with only cells in the outer cortical zone being positive
for cytokeratin 19 [42]. Through the branching and
expansion of stroma into the cortical area and lateral
extension below the ovarian surface, a basal lamina forms
under the GREL cell layers at the surface. Thus, the GREL
cells on the surface begin to differentiate into surface epi-
thelial cells. A well-defined basement membrane can be
observed around the fifth month of human gestation
(reviewed in Ref. [88]) and around 150 dpc in bovine
[18]. At this point, the surface epithelium is still multilay-
ered and becomes a single layer between 180 and 200 dpc
in bovine [18,19] as a result of increased apoptosis from
165 dpc onward [19].
The current model of formation of the ovarian surface
epithelium and granulosa cells of follicles has both cell
types derived from precursor GREL cells. Support for
the concept that both, granulosa and surface epithelial
cell types, might arise from one precursor cell [18] comes
from the common morphology described in bovine fetal
ovaries of elongated columnar cells with elongated cen-
tral nuclei and dark eosinophilic cytoplasm [19]. Another
important consideration is that there are two develop-
mental origins of ovarian surface epithelial cells. The ini-
tial surface epithelial cells covering the major apical
portion of the developing ovary are derived from the
GREL cells, which in turn were originally derived from
the mesonephric surface epithelial cells. However, the
78 4. DEVELOPMENT OF THE MAMMALIAN OVARY AND FOLLICLES
hilum or base of the ovary is directly derived from meso-
nephros and its epithelium is a classic epithelium that
remains as such during development of the ovary. The
implications of two apparently distinct developmental
pathways are not known but are possibly related to the
higher onogenicity and stemness of epithelial cells at
the hilum versus the remainder of the apically situated
surface epithelial cells [92].
FORMATION OF THE OVARIAN TUNICA
ALBUGINEA
The tunica albuginea is a nonvascularized [93], thick
fibrous-rich layer, composed mainly of structural colla-
gens and other extracellular proteins (collagen type I,
decorin, versican), located below the ovarian surface
[18,94,95]. The ovarian tunica albuginea is not as thick
as that of the testis and forms late during development,
after the ovarian surface epithelium has been established.
In cattle, this occurs between 240 and 285 dpc [19] and in
rat around E17 [25]. The tunica albuginea appears to have
some degree of zonation and is variable in thickness from
one location to another in the ovary [95].
The tunica albuginea originates from the mesonephric
stroma that penetrates the ovary in “cell streams” that
extend to just below the surface as described previously
(Fig. 2) [18]. Unlike the other stromal compartments
within the ovary, the tunica albuginea and the outer cor-
tex containing primordial follicles are avascular. The fac-
tors initiating the changes in the cortical stroma resulting
in formation of the tunica albuginea are not known.
Given the proximity to the surface epithelium, it is possi-
ble that factors from the surface epithelial cells influence
the adjacent stromal cells to develop into the fibrous
tunica. Aberrant changes to the tunica albuginea have
been observed in human conditions such as polycystic
ovary syndrome (PCOS), where ovaries exhibit a sub-
stantially thicker tunica albuginea containing more colla-
gen and also have increased thicknesses of cortical and
subcortical stroma [96]. This might negatively influence
folliculogenesis and ovulation.
FORMATION OF THE THECA INTERNA
AND EXTERNA
The first thecal cells can be identified at the follicular
preantral stage and therefore during late gestation in
human [50], bovine [19], and sheep [3], but only after
birth in mouse and rat [97]. Thecal layers form close to
the basal lamina surrounding the membrana granulosa
of the follicle and they start to differentiate into a theca
interna and theca externa during follicle growth and
antrum formation. The outer layer, the theca externa is
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
composed of fibroblasts, collagen fibers, larger venules,
lymphatic vessels, nerve fibers, and cells with contractile
filaments, whereas the theca interna contains specialized
steroidogenic cells, fibroblasts, immune cells, and capil-
laries. These steroidogenic thecal cells synthesize andro-
gen precursors required by granulosa cells for estradiol
production. A role of the theca externa has not been iden-
tified, but it may play a role in follicular fluid expulsion
during ovulation (reviewed in Ref. [98]).
Steroidogenic thecal cells most likely arise from stem
cells [99] within the stroma and indeed a potential stem
cell niche in the theca has previously been identified
[100]. Certain chondroitin/dermatan sulfate epitopes
(antibodies 7D4, 3C5, and 4C3), which marked stem cell
niches in other tissues, have been detected in the ovary. A
study in pigs identified an alkaline phosphate-positive
cell population in cultured thecal cells from small antral
follicles [101]. In addition to the mesenchymal surface
markers CD29, CD44, and CD90, the cells also expressed
the pluripotency marker SOX2. Furthermore, these cells
showed multipotency potential by differentiating into
osteocytes, adipocytes, and oocyte-like cells. Recently in
mouse, it was shown that Indian hedgehog (Ihh) and
Desert hedgehog (Dhh) expression in granulosa cells is
required for the formation of thecal cells, which express
the downstream target Gli [102]. Among other effects,
double-knockout of Ihh and Dhh results in disruption
of folliculogenesis at the preantral stage and fewer
CYP17A1-expressing cells in the ovarian mesenchyme.
Furthermore, during gestation, Gli-positive cells are
only found in the mesonephros, and it appears that
Gli-positive cells from the mesonephros are the origin
of stem cells for thecal lineage.
EARLY FORMATION OF THE
VASCULATURE
Vascularization of the developing ovary is less pro-
nounced and occurs later than in the developing testis,
making the degree of vascularization one of the earliest
distinguishing morphological features between the
female and male gonads [103]. When the stroma pene-
trates into the gonadal ridge/ovarian primordium, it con-
tains endothelial cells assembled into mature capillaries
surrounded by a subendothelial basal lamina as observed
in human [28] and bovine fetal ovaries [18]. Blood vessels
in human fetal ovaries express prokineticin receptor 1,
which is involved in angiogenesis later in gestation
(14–19 weeks; [104]). A complex vascular network,
expressing PECAM1 and calveolin-1, has been observed
early (E13.5) in the developing mouse ovary [105].
Disruption of calveolin-1 in mice resulted in decreased
angiogenesis [106]. Furthermore, cells associated with
vasculature are in the stromal compartment between
 REFERENCES
germ cell nests between E14.5 and 18.5 and they express
MafB (MAF BZIP Transcription Factor B) [107], which has
been recently shown to be a regulator of endothelial cell
sprouting [108]. Thus, the initial capillary network of the
ovarian cortex is not likely formed by vasculogenesis but
rather by the sprouting or splitting forms of angiogenesis
[109], allowing expansion of the existing capillary net-
work derived directly from vasculature in the mesoneph-
ros. This appears to also happen in mouse where is was
observed that “few endothelial cells crossed the border
between the mesonephros and the XX gonad” [110] and
“the XX gonad recruits vasculature by a typical angiogenic
process” [103].
Lymphatic vessels are visible in the hilum area/
medulla of the developing human ovary at 15 weeks
[111], but in the mouse they only appear around PD10,
the time when the first wave of growing follicles become
estrogenic [112]. As the follicles continue to grow, highly
branched lymphatic vessels are recruited to the theca and
stromal layers around each follicle, resulting in the estab-
lishment of the ovarian lymphatic network [113,114].
Subsequently, the lymphatic network is remodeled to
accommodate the growth of each new follicle wave
throughout the reproductive life span [113,114]. Neolym-
phangiogenesis around developing follicles is prevented
by blockade of VEGFR3 (vascular endothelial growth fac-
tor receptor 3) signaling, causing a reduction in follicle
viability and hormone secretion [115].
CONCLUSIONS
The latest model of ovarian development presented,
while elegant in its simplicity, has been developed sub-
stantially only from timed histological observations of
developing ovaries. Further study is indicated of how
GREL cells develop into either surface epithelial cells or
granulosa cells, and perhaps any influence of the oogo-
nia/oocytes in this process. The developmental origins
of surface epithelial cells at the base or hilum of the ovary
differ from those on the rest of the ovary. Cells from these
regions have been found to differ in stemness and onco-
genicity. Given the recent new hypotheses on the cellular
origins of ovarian cancer, this discovery warrants further
investigations. The mesonephros-derived stroma pene-
trates into the developing ovary, branching as it does
so. It is likely that the movement of the stroma is respon-
sible for the physical formation of ovigerous cords,
surface epithelium, and follicles. The penetrating stroma
also brings with it the initial vascular supply of the ovary.
Additional research is now needed to interrogate and
refine these concepts further to enable a fuller under-
standing of the development of the ovary and its follicles.
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
79
References
[1] Byskov AG, Lintern-Moore S. Follicle formation in the immature
mouse ovary: the role of the rete ovarii. J Anat 1973;116(Pt 2):
207–17.
[2] Sawyer HR, Smith P, Heath DA, Juengel JL, Wakefield SJ,
McNatty KP. Formation of ovarian follicles during fetal develop-
ment in sheep. Biol Reprod 2002;66(4):1134–50.
[3] Juengel JL, Sawyer HR, Smith PR, Quirke LD, Heath DA, Lun S,
et al. Origins of follicular cells and ontogeny of steroidogenesis in
ovine fetal ovaries. Mol Cell Endocrinol 2002;191(1):1–10.
[4] Maheshwari A, Fowler PA. Primordial follicular assembly in
humans—revisited. Zygote 2008;16(4):285–96.
[5] Wilhelm D, Palmer S, Koopman P. Sex determination and gonadal
development in mammals. Physiol Rev 2007;87(1):1–28.
[6] Inomata T, Eguchi Y, Nakamura T. Origin of mullerian duct and
its later development in relation to Wolffian duct and anogenital
distance in the rat. Nihon Juigaku Zasshi 1989;51(4):693–701.
[7] Inomata T, Eguchi Y, Nakamura T. Origin of mullerian duct and
its later developmental changes in relation to wolffian duct in
bovine fetuses. Zentralbl Veterinarmed A 1989;36(3):166–74.
[8] Inomata T, Inoue S, Sugawara H, Kajihara H, Shinomiya T,
Wagai I, et al. Developmental changes in paramesonephric and
mesonephric ducts and the external genitalia in swine fetuses dur-
ing sexual differentiation. J Vet Med Sci 1993;55(3):371–8.
[9] Inomata T, Ninomiya H, Sakita K, Kashiwazaki N, Ito J, Ariga M,
et al. Developmental changes of Mullerian and Wolffian ducts in
domestic cat fetuses. Exp Anim 2009;58(1):41–5.
[10] Paranko J, Foidart JM, Pelliniemi LJ. Basement membrane in dif-
ferentiating mesonephric and paramesonephric ducts of male and
female rat fetuses. Differentiation 1985;29(1):39–49.
[11] Vazquez MD, Bouchet P, Foliguet B, Gerard H, Mallet JL,
Leheup B. Differentiated aspect of female and male mouse meso-
nephroi. Int J Dev Biol 1998;42(4):621–4.
[12] Vazquez MD, Bouchet P, Mallet JL, Foliguet B, Gerard H,
LeHeup B. 3D reconstruction of the mouse’s mesonephros. Anat
Histol Embryol 1998;27(5):283–7.
[13] Mullen RD, Behringer RR. Molecular genetics of Mullerian duct for-
mation, regression and differentiation. Sex Dev 2014;8(5):281–96.
[14] Shaw G, Renfree MB. Wolffian duct development. Sex Dev
2014;8(5):273–80.
[15] Loffler S, Horn LC, Weber W, Spanel-Borowski K. The transient
disappearance of cytokeratin in human fetal and adult ovaries.
Anat Embryol (Berl) 2000;201(3):207–15.
[16] Byskov AG. Does the rete ovarii act as a trigger for the onset of
meiosis? Nature 1974;252(5482):396–7.
[17] Byskov AG, Skakkebaek NE, Stafanger G, Peters H. Influence of
ovarian surface epithelium and rete ovarii on follicle formation.
J Anat 1977;123(Pt 1):77–86.
[18] Hummitzsch K, Irving-Rodgers HF, Hatzirodos N, Bonner W,
Sabatier L, Reinhardt DP, et al. A new model of development of
the mammalian ovary and follicles. PLoS One 2013;8(2)e55578.
[19] Kenngott RA, Vermehren M, Ebach K, Sinowatz F. The role of
ovarian surface epithelium in folliculogenesis during fetal devel-
opment of the bovine ovary: a histological and immunohisto-
chemical study. Sex Dev 2013;7(4):180–95.
[20] Hanley NA, Hagan DM, Clement-Jones M, Ball SG, Strachan T,
Salas-Cortes L, et al. SRY, SOX9, and DAX1 expression patterns
during human sex determination and gonadal development.
Mech Dev 2000;91(1–2):403–7.
[21] Wrobel KH, Suss F. Identification and temporospatial distribution
of bovine primordial germ cells prior to gonadal sexual differen-
tiation. Anat Embryol (Berl) 1998;197(6):451–67.
[22] Payen E, Pailhoux E, Abou Merhi R, Gianquinto L,
Kirszenbaum M, Locatelli A, et al. Characterization of ovine
80 4. DEVELOPMENT OF THE MAMMALIAN OVARY AND FOLLICLES
SRY transcript and developmental expression of genes involved
in sexual differentiation. Int J Dev Biol 1996;40(3):567–75.
[23] Pailhoux E, Vigier B, Vaiman D, Servel N, Chaffaux S, Cribiu EP,
et al. Ontogenesis of female-to-male sex-reversal in XX polled
goats. Dev Dyn 2002;224(1):39–50.
[24] Hu YC, Okumura LM, Page DC. Gata4 is required for formation of
the genital ridge in mice. PLoS Genet 2013;9(7)e1003629.
[25] Frojdman K, Paranko J, Virtanen I, Pelliniemi LJ. Intermediate fil-
ament proteins and epithelial differentiation in the embryonic
ovary of the rat. Differentiation 1993;55(1):47–55.
[26] Wartenberg H. Development of the early human ovary and role of
the mesonephros in the differentiation of the cortex. Anat Embryol
(Berl) 1982;165(2):253–80.
[27] Gruenwald P. The development of the sex cords in the gonads of
man and mammals. Am J Anat 1942;70(3):359–97.
[28] Heeren AM, van Iperen L, Klootwijk DB, de Melo Bernardo A,
Roost MS, Gomes Fernandes MM, et al. Development of the fol-
licular basement membrane during human gametogenesis and
early folliculogenesis. BMC Dev Biol 2015;15:4.
[29] Hacker A, Capel B, Goodfellow P, Lovell-Badge R. Expression of
Sry, the mouse sex determining gene. Development 1995;121(6):
1603–14.
[30] Mittwoch U, Delhanty JDA, Beck F. Growth of differentiating tes-
tes and ovaries. Nature 1969;224:1323.
[31] Baker TG. A quantitative and cytological study of oogenesis in the
rhesus monkey. J Anat 1966;100(Pt 4):761–76.
[32] Anderson R, Copeland TK, Sch€ oler H, Heasman J, Wylie C. The
onset of germ cell migration in the mouse embryo. Mech Dev
2000;91(1):61–8.
[33] De Felici M. Origin, migration, and proliferation of human pri-
mordial germ cells. In: Coticchio G, editor. Oogenesis XII.
London: Springer-Verlag; 2013. p. 364.
[34] Pepling ME. From primordial germ cell to primordial follicle:
mammalian female germ cell development. Genesis 2006;44
(12):622–32.
[35] Anderson RA, Fulton N, Cowan G, Coutts S, Saunders PT. Con-
served and divergent patterns of expression of DAZL, VASA and
OCT4 in the germ cells of the human fetal ovary and testis. BMC
Dev Biol 2007;7:136.
[36] McNatty KP, Smith P, Hudson NL, Heath DA, Tisdall DJ, WS O,
et al. Development of the sheep ovary during fetal and early neo-
natal life and the effect of fecundity genes. J Reprod Fertil Suppl
1995;49:123–35.
[37] Erickson BH. Development and radio-response of the prenatal
bovine ovary. J Reprod Fert 1966;10:97–105.
[38] Motta PM, Makabe S, Nottola SA. The ultrastructure of human
reproduction. I. The natural history of the female germ cell: origin,
migration and differentiation inside the developing ovary. Hum
Reprod Update 1997;3(3):281–95.
[39] Rosario R, Adams IR, Anderson RA. Is there a role for DAZL
in human female fertility? Mol Hum Reprod 2016;22
(6):377–83.
[40] Wartenberg H, Ihmer A, Schwarz S, Miething A, Viebahn C.
Mitotic arrest of female germ cells during prenatal oogenesis. A
colcemid-like, non-apoptotic cell death. Anat Embryol (Berl)
2001;204(5):421–35.
[41] Kerr JB, Myers M, Anderson RA. The dynamics of the primordial
follicle reserve. Reproduction 2013;146(6):R205–15.
[42] Appert A, Fridmacher V, Locquet O, Magre S. Patterns of keratins
8, 18 and 19 during gonadal differentiation in the mouse: sex- and
time-dependent expression of keratin 19. Differentiation 1998;
63(5):273–84.
[43] Garverick HA, Juengel JL, Smith P, Heath DA, Burkhart MN,
Perry GA, et al. Development of the ovary and ontongeny of
mRNA and protein for P450 aromatase (arom) and estrogen
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
receptors (ER) alpha and beta during early fetal life in cattle. Anim
Reprod Sci 2010;117(1–2):24–33.
[44] Yang MY, Fortune JE. The capacity of primordial follicles in fetal
bovine ovaries to initiate growth in vitro develops during mid-
gestation and is associated with meiotic arrest of oocytes. Biol
Reprod 2008;78(6):1153–61.
[45] Kerr JB, Duckett R, Myers M, Britt KL, Mladenovska T, Findlay JK.
Quantification of healthy follicles in the neonatal and adult mouse
ovary: evidence for maintenance of primordial follicle supply.
Reproduction 2006;132(1):95–109.
[46] Frojdman K, Malmi R, Pelliniemi LJ. Lectin-binding carbohy-
drates in sexual differentiation of rat male and female gonads.
Histochemistry 1992;97(6):469–77.
[47] Fowler PA, Flannigan S, Mathers A, Gillanders K, Lea RG,
Wood MJ, et al. Gene expression analysis of human fetal ovarian
primordial follicle formation. J Clin Endocrinol Metab 2009;94(4):
1427–35.
[48] Zheng W, Zhang H, Liu K. The two classes of primordial follicles
in the mouse ovary: their development, physiological functions
and implications for future research. Mol Hum Reprod 2014;20(4):
286–92.
[49] Mangoushi MA. The fate of grafted fetal ovaries in the rat. J Anat
1974;118(Pt 3):601–10.
[50] Konishi I, Fujii S, Okamura H, Parmley T, Mori T. Development
of interstitial cells and ovigerous cords in the human fetal ovary:
an ultrastructural study. J Anat 1986;148:121–35.
[51] Efimenko E, Padua MB, Manuylov NL, Fox SC, Morse DA,
Tevosian SG. The transcription factor GATA4 is required for fol-
licular development and normal ovarian function. Dev Biol 2013;
381(1):144–58.
[52] Wilhelm D, Yang JX, Thomas P. Mammalian sex determination
and gonad development. Curr Top Dev Biol 2013;106:89–121.
[53] Miyamoto N, Yoshida M, Kuratani S, Matsuo I, Aizawa S. Defects
of urogenital development in mice lacking Emx2. Development
1997;124(9):1653–64.
[54] Smith P, Wilhelm D, Rodgers RJ. Development of mammalian
ovary. J Endocrinol 2014;221(3):R145–61.
[55] Fridmacher V, Locquet O, Magre S. Differential expression of
acidic cytokeratins 18 and 19 during sexual differentiation of
the rat gonad. Development 1992;115(2):503–17.
[56] Bendel-Stenzel M, Anderson R, Heasman J, Wylie C. The origin
and migration of primordial germ cells in the mouse. Semin Cell
Dev Biol 1998;9(4):393–400.
[57] Chuva de Sousa Lopes SM, Roelen BA. On the formation of germ
cells: the good, the bad and the ugly. Differentiation 2010;
79(3):131–40.
[58] Matsui Y. The molecular mechanisms regulating germ cell devel-
opment and potential. J Androl 2010;31(1):61–5.
[59] Saitou M, Yamaji M. Germ cell specification in mice: signaling,
transcription regulation, and epigenetic consequences. Reproduc-
tion 2010;139(6):931–42.
[60] Chen SR, Zheng QS, Zhang Y, Gao F, Liu YX. Disruption of genital
ridge development causes aberrant primordial germ cell prolifer-
ation but does not affect their directional migration. BMC Biol
2013;11:22.
[61] Eggers S, Sinclair A. Mammalian sex determination-insights from
humans and mice. Chromosome Res 2012;20(1):215–38.
[62] Pannetier M, Tilly G, Kocer A, Hudrisier M, Renault L,
Chesnais N, et al. Goat SRY induces testis development in XX
transgenic mice. FEBS Lett 2006;580(15):3715–20.
[63] Parma P, Pailhoux E, Cotinot C. Reverse transcription-polymerase
chain reaction analysis of genes involved in gonadal differentia-
tion in pigs. Biol Reprod 1999;61(3):741–8.
[64] Gustin SE, Hogg K, Stringer JM, Rastetter RH, Pelosi E, Miles DC,
et al. WNT/beta-catenin and p27/FOXL2 differentially regulate
 REFERENCES
supporting cell proliferation in the developing ovary. Dev Biol
2016;412(2):250–60.
[65] Ross DG, Bowles J, Hope M, Lehnert S, Koopman P. Profiles of
gonadal gene expression in the developing bovine embryo. Sex
Dev 2009;3(5):273–83.
[66] Pannetier M, Chassot AA, Chaboissier MC, Pailhoux E. Involve-
ment of FOXL2 and RSPO1 in ovarian determination, develop-
ment, and maintenance in mammals. Sex Dev 2016;10(4):167–84.
[67] Kenngott RA, Sauer U, Vermehren M, Sinowatz F. Expression of
intermediate filaments and germ cell markers in the developing
bovine ovary: an Immunohistochemical and laser-assisted micro-
dissection study. Cells Tissues Organs 2014;200(2):153–70.
[68] Bowles J, Feng CW, Miles K, Ineson J, Spiller C, Koopman P.
ALDH1A1 provides a source of meiosis-inducing retinoic acid
in mouse fetal ovaries. Nat Commun 2016;7:10845.
[69] Stoop H, Honecker F, Cools M, de Krijger R, Bokemeyer C,
Looijenga LH. Differentiation and development of human female
germ cells during prenatal gonadogenesis: an immunohistochem-
ical study. Hum Reprod 2005;20(6):1466–76.
[70] Yao HH, Matzuk MM, Jorgez CJ, Menke DB, Page DC, Swain A,
et al. Follistatin operates downstream of Wnt4 in mammalian
ovary organogenesis. Dev Dyn 2004;230(2):210–5.
[71] Chen H, Palmer JS, Thiagarajan RD, Dinger ME, Lesieur E,
Chiu H, et al. Identification of novel markers of mouse fetal ovary
development. PLoS One 2012;7(7)e41683.
[72] Jimenez R. Ovarian organogenesis in mammals: mice cannot tell
us everything. Sex Dev 2009;3(6):291–301.
[73] Kerr CL, Hill CM, Blumenthal PD, Gearhart JD. Expression of plu-
ripotent stem cell markers in the human fetal ovary. Hum Reprod
2008;23(3):589–99.
[74] Burkhart MN, Juengel JL, Smith PR, Heath DA, Perry GA,
Smith MF, et al. Morphological development and characterization
of aromatase and estrogen receptors alpha and beta in fetal ova-
ries of cattle from days 110 to 250. Anim Reprod Sci 2010;117(1–2):
43–54.
[75] van den Hurk R, Dijkstra G, van Mil FN, Hulshof SC, van den
Ingh TS. Distribution of the intermediate filament proteins vimen-
tin, keratin, and desmin in the bovine ovary. Mol Reprod Dev
1995;41(4):459–67.
[76] McNatty KP, Fidler AE, Juengel JL, Quirke LD, Smith PR,
Heath DA, et al. Growth and paracrine factors regulating follicu-
lar formation and cellular function. Mol Cell Endocrinol 2000;
163(1–2):11–20.
[77] Bullejos M, Koopman P. Germ cells enter meiosis in a rostro-
caudal wave during development of the mouse ovary. Mol
Reprod Dev 2004;68(4):422–8.
[78] Rajah R, Glaser EM, Hirshfield AN. The changing architecture
of the neonatal rat ovary during histogenesis. Dev Dyn 1992;
194(3):177–92.
[79] Hirshfield AN. Heterogeneity of cell populations that contribute
to the formation of primordial follicles in rats. Biol Reprod
1992;47(3):466–72.
[80] Kurilo LF. Oogenesis in antenatal development in man. Hum
Genet 1981;57(1):86–92.
[81] Gondos B. Cellular interrelationships in the human fetal ovary
and testis. Prog Clin Biol Res 1981;59B:373–81.
[82] Zhou Z, Wan Y, Zhang Y, Wang Z, Jia R, Fan Y, et al. Follicular
development and expression of nuclear respiratory factor-1 and
peroxisome proliferator-activated receptor gamma coactivator-1
alpha in ovaries of fetal and neonatal doelings. J Anim Sci
2012;90(11):3752–61.
[83] Ding W, Wang W, Zhou B, Zhang W, Huang P, Shi F, et al. For-
mation of primordial follicles and immunolocalization of PTEN,
PKB and FOXO3A proteins in the ovaries of fetal and neonatal
pigs. J Reprod Dev 2010;56(1):162–8.
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
81
[84] Baker TG. A quantitative and cytological study of germ cells in
human ovaries. Proc R Soc Lond B Biol Sci 1963;158:417–33.
[85] Sarraj MA, Drummond AE. Mammalian foetal ovarian develop-
ment: consequences for health and disease. Reproduction 2012;
143(2):151–63.
[86] Rodrigues P, Limback D, McGinnis LK, Plancha CE, Albertini DF.
Multiple mechanisms of germ cell loss in the perinatal mouse
ovary. Reproduction 2009;137(4):709–20.
[87] Mork L, Maatouk DM, McMahon JA, Guo JJ, Zhang P,
McMahon AP, et al. Temporal differences in Granulosa cell spec-
ification in the ovary reflect distinct follicle fates in mice. Biol
Reprod 2011;86(37):1–9.
[88] Auersperg N, Wong AS, Choi KC, Kang SK, Leung PC. Ovarian
surface epithelium: biology, endocrinology, and pathology.
Endocr Rev 2001;22(2):255–88.
[89] Rastetter RH, Bernard P, Palmer JS, Chassot AA, Chen H,
Western PS, et al. Marker genes identify three somatic cell types
in the fetal mouse ovary. Dev Biol 2014;394(2):242–52.
[90] Flesken-Nikitin A, Hwang CI, Cheng CY, Michurina TV,
Enikolopov G, Nikitin AY. Ovarian surface epithelium at the junc-
tion area contains a cancer-prone stem cell niche. Nature 2013;
495(7440):241–5.
[91] Juengel JL, Heath DA, Quirke LD, McNatty KP. Oestrogen receptor
alpha and beta, androgen receptor and progesterone receptor
mRNA and protein localisation within the developing ovary and
in small growing follicles of sheep. Reproduction 2006;131(1):81–92.
[92] Flesken-Nikitin A, Odai-Afotey AA, Nikitin AY. Role of the stem
cell niche in the pathogenesis of epithelial ovarian cancers. Mol
Cell Oncol 2014;1(3)e963435.
[93] van Wezel IL, Rodgers RJ. Morphological characterization of
bovine primordial follicles and their environment in vivo. Biol
Reprod 1996;55(5):1003–11.
[94] Hatzirodos N, Bayne RA, Irving-Rodgers HF, Hummitzsch K,
Sabatier L, Lee S, et al. Linkage of regulators of TGF-beta activity
in the fetal ovary to polycystic ovary syndrome. FASEB J 2011;
25(7):2256–65.
[95] Prodoehl MJ, Irving-Rodgers HF, Bonner W, Sullivan TM,
Micke GC, Gibson MA, et al. Fibrillins and latent TGFβ binding
proteins in bovine ovaries of offspring following high or low pro-
tein diets during pregnancy of dams. Mol Cell Endocrinol
2009;307:133–44.
[96] Hughesdon PE. Morphology and morphogenesis of the Stein-
Leventhal ovary and of so-called “hyperthecosis” Obstet Gynecol
Surv 1982;37(2):59–77.
[97] Guraya SS, Uppal J. Morphological and histochemical studies on
the foetal and postnatal ovaries of the field rat (Millardia meltada).
Acta Morphol Neerl Scand 1978;16(4):287–304.
[98] Young JM, McNeilly AS. Theca: the forgotten cell of the ovarian
follicle. Reproduction 2010;140(4):489–504.
[99] Honda A, Hirose M, Hara K, Matoba S, Inoue K, Miki H, et al. Iso-
lation, characterization, and in vitro and in vivo differentiation of
putative thecal stem cells. Proc Natl Acad Sci U S A 2007;104(30):
12389–94.
[100] Hatzirodos N, Nigro J, Irving-Rodgers HF, Vashi AV,
Hummitzsch K, Caterson B, et al. Glycomic analyses of ovarian fol-
licles during development and atresia. Matrix Biol 2012;31:45–56.
[101] Lee YM, Kumar BM, Lee JH, Lee WJ, Kim TH, Lee SL, et al. Char-
acterisation and differentiation of porcine ovarian theca-derived
multipotent stem cells. Vet J 2013;.
[102] Liu C, Peng J, Matzuk MM, Yao HH. Lineage specification of ovar-
ian theca cells requires multicellular interactions via oocyte and
granulosa cells. Nat Commun 2015;6:6934.
[103] Coveney D, Cool J, Oliver T, Capel B. Four-dimensional analysis
of vascularization during primary development of an organ, the
gonad. Proc Natl Acad Sci U S A 2008;105(20):7212–7.
82 4. DEVELOPMENT OF THE MAMMALIAN OVARY AND FOLLICLES
[104] Eddie SL, Childs AJ, Kinnell HL, Brown P, Jabbour HN,
Anderson RA. Prokineticin ligands and receptors are expressed
in the human fetal ovary and regulate germ cell expression of
COX2. J Clin Endocrinol Metab 2015;100(9):E1197–205.
[105] Bullejos M, Bowles J, Koopman P. Extensive vascularization of
developing mouse ovaries revealed by caveolin-1 expression.
Dev Dyn 2002;225(1):95–9.
[106] Morais C, Ebrahem Q, Anand-Apte B, Parat MO. Altered angio-
genesis in caveolin-1 gene-deficient mice is restored by ablation
of endothelial nitric oxide synthase. Am J Pathol 2012;180(4):
1702–14.
[107] Maatouk DM, Mork L, Hinson A, Kobayashi A, McMahon AP,
Capel B. Germ cells are not required to establish the female path-
way in mouse fetal gonads. PLoS One 2012;7(10)e47238.
[108] Jeong H-W, Hernández-Rodríguez B, Kim J, Kim K-P, Enriquez-
Gasca R, Yoon J, et al. Transcriptional regulation of endothelial
cell behavior during sprouting angiogenesis. Nat Commun
2017;8(1):726.
[109] Kovacic JC, Moore J, Herbert A, Ma D, Boehm M, Graham RM.
Endothelial progenitor cells, angioblasts, and angiogenesis—old
terms reconsidered from a current perspective. Trends Cardiovasc
Med 2008;18(2):45–51.
I. THE OVARIAN FOLLICULAR APPARATUS: OPERATIONAL CHARACTERISTICS
[110] Brennan J, Karl J, Capel B. Divergent vascular mechanisms down-
stream of Sry establish the arterial system in the XY gonad. Dev
Biol 2002;244(2):418–28.
[111] Kleppe M, Kraima AC, Kruitwagen RFPM, Van Gorp T, Smit NN,
van Munsteren JC, et al. Understanding lymphatic drainage path-
ways of the ovaries to predict sites for sentinel nodes in ovarian
cancer. Int J Gynecol Cancer 2015;25(8):1405–14.
[112] Brown HM, Dunning KR, Robker RL, Pritchard M, Russell DL.
Requirement for ADAMTS-1 in extracellular matrix remodeling
during ovarian folliculogenesis and lymphangiogenesis. Dev Biol
2006;300(2):699–709.
[113] Brown HM, Robker RL, Russell DL. Development and hormonal
regulation of the ovarian lymphatic vasculature. Endocrinology
2010;151(11):5446–55.
[114] Svingen T, Francois M, Wilhelm D, Koopman P. Three-
dimensional imaging of Prox1-EGFP transgenic mouse gonads
reveals divergent modes of lymphangiogenesis in the testis and
ovary. PLoS One 2012;7(12)e52620.
[115] Rutkowski JM, Ihm JE, Lee ST, Kilarski WW, Greenwood VI,
Pasquier MC, et al. VEGFR-3 neutralization inhibits ovarian lym-
phangiogenesis, follicle maturation, and murine pregnancy. Am J
Pathol 2013;183(5):1596–607.