Reproduction
Mammalian Ovary
Amitabh Krishna;
Raj Kamal Srivastava
Arnab Banerjee
Department of Zoology,
Banaras Hindu University,
Varanasi-221005, India
Contents
Introduction
Development of the ovary
Anatomy of the ovary
a. Follicular types and structure
b. Corpus luteum formation and demise
c. Interstitial cells
Functions of the ovary
a. Generation of female gametes
b. Mechanism of ovulation
c. Production of hormones
Regulation of ovarian functions
a. Regulation of folliculogenesis
b. Regulation of steroidogenesis
Conclusions
Corresponding author: Prof. Amitabh Krishna, Department of Zoology, Banaras Hindu University,
Varanasi 221 005 E-Mail: [email protected]
INTRODUCTION:
The ovary is a multi-compartmental female gonad with broad range of distinct biological
properties. The primary function of the female gonad is the differentiation and release of the
mature oocyte or egg during each reproductive cycle that is fully competent for fertilization and
successful propagation of the species. Additionally, the ovary produces steroids that allow the
development of female secondary sexual characteristics and support pregnancy. The ovarian
hormones also regulate puberty development, ovulation and reproductive cycle. The functions of
ovary are controlled by various cell types. In addition to germ cells, ovary consists of support
cells, hormone producing cells, blood vessels and nerve supply. In the ovary, each germ cell is in
contact with multiple supporting cells, known as granulosa cells and thecal cells, forming an
ovarian follicle. There are two main functional units within the ovary: the follicle and the
corpus luteum. They perform different functions, are transient, and appear at different phases of
the reproductive cycle. The follicle contains the female germ cells, the oocyte; following
maturation (the process of folliculogenesis), the oocyte is released from the ovary (ovulation).
The corpus luteum is formed from the mature follicle, after it has ovulated; one of its main
functions is to secrete hormone, progesterone, which is essential for preparation of uterus for the
initial stages of pregnancy in a fertile cycle. In most mammals the reproductive process in the
female occurs in a predetermined sequence of events, which includes three stages: i. The
follicular phase: the final stages of follicular growth and maturation; ii. Ovulation: the release
of the oocyte from the mature follicle; and iii. The luteal phase: the formation of a corpus
luteum and progesterone synthesis.
DEVELOPMENT OF THE OVARY:
Sexual differentiatiation is a sequential process beginning with the establishment of chromosomal
sex at fertilization, followed by the development of gonadal sex, and culminating in the
development of secondary sexual characteristics. Alfred Jost formulated this paradigm in the late
1940s, which has become the central dogma of sexual differentiation (Fig. 1). The testis-determining
gene(s), SRY, located on the y-chromosome is necessary for development of the mammalian testis.
In the absence of y-chromosome or testis determining gene(s), testis fail to develop and ovaries
form. Two x-chromosomes appear to be essential for the development of normal ovaries as
individuals with a single x chromosome develop gonads that are only partially differentiated. The
ovary-determining gene has not yet been identified.
In humans the primordial gonad, called as genital ridges, are formed during the third and fourth
weeks of embryogenesis by the proliferation of the coelomic epithelium and condensation of the
underlying mesenchyme on each side of the midline between the primitive kidney (mesonephros)
and the dorsal mesentry. Initially the primordial gonads do not contain germ cells; the germ cells are
formed in the endoderm of the yolk sac near the allantoic evagination. During the fifth week of
gestation the germ cells begin to leave the yolk sac (primitive gut) where they may be easily
recognized histologically by strong positive staining for alkaline phosphatase and migrate through
the mesentry to the genital ridge. The migration is thought to follow a chemotactic substance
elaborated by the genital ridge. The undifferentiated gonad has generally been considered to be
composed of peripheral cortical and central medullary regions. In the male, sex differentiation of the
gonads involves development of the medullary primordium and suppression of the cortex. In the
female, on the other hand, the cortical region develops, whereas medullary differentiation is
suppressed.
Development of ovary in the vertebrate can be classified into four stages (Fig. 2). During the first
stage, primordial germ cells from the yolk sac endoderm migrate to the genital ridge through the
dorsal mesentry by ameboid movement. Prior to migration, the germ cells divide mitotically. Once
migration starts, mitosis is inhibited until the germ cells reach the genital ridge. The second stage
starts with the arrival of the primordial germ cells to the genital ridge. It consists of the proliferation
of primordial germ cells and the coelomic epithelium on the genital ridges. The surface epithelium of
the genital ridges infiltrate the mesenchymal loose connective tissue and form the primary sex
cords. At this stage the gonad is known as Indifferent gonads that are identical in both sexes. The
Indifferent gonads are composed of three distinct cell types: (1) germ cells, (2) supporting cells that
are derived from the coelomic epithelium of the genital ridge and that will differentiate either into
the Sertoli cells of the testis or the granulosa cells of the ovary, and (3) stromal (interstitial) cells
derived from the mesenchyme of the genital ridge. During the third stage, initial primary sex cords
degenerate and a new cortical sex cords develop near the cortical region. The fourth and final stage
of ovarian formation is characterized by development of the cortex and involution of the medulla of
the indifferent gonad.
The mechanisms that control differentiation of the indifferent gonad into an ovary or a testis are
poorly understood. At approximately 11th week of development in human the germ cells in the
ovary are referred to as oogonia. From this point on, the oogonial endowment is subject to three
simultaneous ongoing processes: mitosis, meiosis, and atresia (degeneration). The oogonia, which
enter the prophase of the first meiotic division, known as primary oocytes. From around sixteen
weeks of gestation in human, these oocytes become surrounded by a single layer of spindle-shaped
(non-cuboidal) primordial (pre)-granulosa cells, giving rise to primordial follicles. The oocytes,
which failed to get surrounded by pre-granulosa cells undergo atresia.
The number of germ cells peaks at six to seven millions by twenty weeks of gestation, at that time
two-thirds of the total germ cells are intra-meiotic dictyate primary oocytes, while the remaining
one-third are still oogonia. Many of the primordial follicles start to mature but development is
arrested at an early stage and atresia follows. This process is very rapid during fetal life and at birth
the number of germ cells is reduced to one or two million from seven million. Early follicular
development leading to atresia continues at a lower rate during childhood and reproductive life,
leaving 400,000 follicles at puberty and a few hundred by the menopause. Only four to five
hundred follicles will ovulate in the course of a reproductive life span.
ANATOMY OF OVARY:
Ovaries lies on either side of the upper pelvic cavity between the external and internal iliac arteries.
Unlike the testes, which descend into the scrotum, the ovaries remain in the abdominal cavity and do
not require cooler temperature for normal function. The ovary is an intraperitoneal structure attached
to the sidewall of the pelvis by a fold of peritoneum called mesovarium. The upper portion of this
fold carries the ovarian artery and pampiniform plexus of ovarian veins and is called the
infundibulo-pelvic ligament. The lower portion is continuous with the broad ligament. The fallopian
tube curls up the anterior surface of the ovary and projects over its upper pole, whilst the inferior
pole is connected to the cornu of the uterus by the ovarian ligament. The ovarian artery, a branch of
the aorta, anastomoses with the terminal branch of the uterine artery and become a common vessel
called the ramus ovaricus artery before spiraling into the medulla of the ovary. In the human the
ovarian contribution is greater than that of the uterine artery.
The ovary is organized into an outer cortex and an inner medulla (Fig. 3). The germ cells are
located within the cortex. An epithelial layer of cubiodal cells resting on a basement membrane
covers the surface of the ovary. This layer, termed the germinal or serous epithelium, is continuous
with the peritoneum. Underlying the germinal epithelium is a layer of dense connective tissue
termed the tunica albuginea. Embedded in the connective tissue of the cortex are the follicles
containing the, female gamete or germ cell, oocyte. The number and the size of the follicles vary
depending on the age and reproductive state of the female. Prior to the attainment of sexual
maturation, numerous spindle shaped cells accumulate in the ovary and make up the ovarian stroma.
These are thought to arise from the dormant genital ridge mesenchyme and are the source of ovarian
androgens. The stroma cells also participate in the formation of the thecal cells of secondary
follicles. Numerous primordial and a few primary follicles are scattered throughout the stroma with
the greatest concentration at the periphery of the cortex. A number of secondary follicles are seen in
deeper layers; prior to ovulation one or more of these will grow towards the surface. The medulla
contains connective and interstitial tissues. Blood vessels, lymphatcs, and nerves enter the medulla
via the hilus. The hilum contains numerous hilar cells, which are of mesenchymal origin and
equivalent to the Leydig cells of the testis. Other cells in the cortex are steriodogenic cells termed
interstitial cells. These cells are derived from the thecal cells of atretic follicles and are found in
nests cord throughout the life of female. An adult ovary reveals a mixture of structures of different
histological appearance scattered throughout the ovary. This includes follicles of varying sizes,
atretic (degenerating) follicles, corpora lutea and interstitial cells.
Follicular Types and Structure: The follicles (follicle is Latin for “little bag”) are structurally
the most conspicuous and functionally the most important units in the ovarian cortex. The existence
of follicles of different sizes (primordial, primary, secondary, tertiary (early antral), preovulatory
(Graafian) and atretic follicles) (Table 1 & Fig. 4) reflects specific changes associated with their
growth, development, and fate. At the end of the follicular phase of the reproductive cycle, the
Graafian follicles that reach maturity release its ovum by the process known as ovulation. After
ovulation, the ovulated follicle develops into the corpus luteum.
A follicle consists of an oocyte; surrounding granulosa cells and follicular wall or thecal layer. The
granulosa cells are separated from the thecal cells by a basement membrane. Between the oocyte and
the surrounding granulosa cells is present a thin transparent membrane, the zona pellucida. In
mature follicles, the thecal layer can be further divided in to the theca interna, containing
differentiated steroid producing cells, and the theca externa, consisting of mainly connective tissue.
The boundary between the theca interna and theca externa is not clear; neither is the boundary
between the theca externa and the ovarian stroma. The blood and nerve supply terminate in the theca
interna. There are no blood vessels in the granulosa layer during any stage of follicular growth. It is
the avascular nature of the granulosa cell compartment that necessitates inter-cellular contact
between neighboring cells. Thus the granulosa cells are inter-connected by extensive intercellular
gap junctions. The gap junctions are composed of proteins called connexins-37. It is generally
presumed that these specialized cell junctions may be important in metabolic exchange and in the
transport of small molecules between neighboring granulosa cells. Moreover, the granulosa cells
extend cytoplasmic process to form gap junction like unions with the plasma membrane of the
oocytes. Follicular granulosa cells are heterogeneous in nature and their level of differentiation is not
uniform. Granulosa cells show at least two populations: mural or membrana granulosa cells and
cumulus oophorus. Accordingly, the mural or membrane granulosa cells, (the cells adjacent with
basement membrane) are steroidogenically more active than cumulus cells. For example, mural or
membrane granulosa cells generally have a higher intracellular level of 3β-hydroxysteroid
dehydrogenase, Glucose-6-phosphatase and cytochrome P450 enzymes. The mural granulosa cells
also possess a generous luteinizing hormone (LH) receptors complement. The absence of
cytochrome P450 activity in cumulus cells suggests the absence of steroidogenic activity. The
overall LH receptor content and level of LH-responsiveness appears substantially diminished in
cumulus granulosa cells relative to mural granulose cells. These observations have given rise to the
suggestion that cumulus granulosa cells may perhaps function in a stem cell capacity. According to
this view, cumulus oophorus may act like a feeder layer engaged in active multiplication.
Table 1
S.No.
Type of Follicle
1
Primordial Follicle
2
Primary Follicle
3
Secondary Follicle
(Pre-antral Follicle)
4
Antral Follicle
5
Graafian Follicle
(Pre-ovulatory
Follicle)
6
Atretic Follicle
Characteristics
Primary oocyte surrounded by a single layer of spindle shaped
(pre) granulosa cells and a basement membrane. There are
pools of resting follicles.
Primary oocyte surrounded by a single layer of cuboidal
granulosa cells and a basement membrane. They are the first
stage of follicle growth
Primary oocyte surrounded with zona pellucida and several (28) layers of granulosa cells. The surrounding ovarian stroma
cells form theca layers, which are separated by granulosa cells
by basement membrane.
Primary oocyte surrounded with zona pellucida and several
layers of granulosa cells layers contain many smaller cavities
(antral) with their surrounding rosette of granulose cells are
called “Call-Exner bodies”. The fluid, liquor folliculi, is
formed by secretion from surrounding cells. Theca layers can
be divided into inner steroidogenic compact layer, theca
interna and outer loose layer of stroma, theca externa.
Characterized by the formation of a large fluid filled cavity,
antrum. The granulose cells project into the antrum in the area
of the primary oocyte forming a mound known as the cumulus
oophorus. Oocyte attains its maximum size.
Morphologically, it is characterized by necrosis of both the
oocyte and the granulose cells. Their nuclei become pyknotic
and the cell degenerate.
Very few follicles reach the developmental stage capable of being ovulated. Most follicles
degenerate (become atretic). Follicular atresia can take place during any stage of the follicular
development. Atresia is initiated very early in life (as soon as the first primordial follicles develop
in the fetal ovary) and occurs at any stage of follicular maturation. It takes place throughout
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prepubertal development and at every menstrual cycle. Morphologically, it is characterized by
necrosis of both the oocyte and the granulosa cells. The nuclei of granulosa cells become pyknotic
and the cells degenerate. In other follicles, death of the oocyte is one of the first events to occur.
Interestingly, some oocytes are stimulated to resume meiosis during the initial phases of atresia and
they extrude the first polar body before dying. In marked contrast to the granulosa cells, thecal cells
lose their differentiated condition, and instead of dying, return to the pool of interstitial cells not
associated with follicles. The atretic follicle, on the other hand, is invaded by fibroblasts and
become an avascular, nonfunctional scar.
The primary oocyte enlarges in diameter early in follicular development and undergoes no
subsequent enlargement. Reduction division, which began with the formation of the oocyte is
resumed by preovulatory gonadotropin surge about twelve to thirty-six hours before ovulation.
The secondary oocyte thus formed immediately enters the second meiotic division, but the meiotic
division again arrested at metaphase until fertilization. The meiotic division is completed at the
time of fertilization, when the second polar body is extruded, and the female pronucleus is formed.
The very prolonged meiosis of the primary oocyte is due to an inhibitory effect of the granulosa
cells (Meiosis Inhibitory Substance or MIS) through their cytoplasmic extensions; and these are
withdrawn before meiosis is resumed.
Ovulation consists of rapid follicular enlargement followed by protrusion of the follicle from the
surface of the ovarian cortex. Rupture of the follicle results in the extrusion of an oocyte-cumulus
complex into the bursa of the ovary and its transport into the fallopian tubes. Endoscopic
visualization of the ovary around the time of ovulation reveals that elevation of a conical “stigma”
on the surface of the protruding follicle precedes rupture.
Corpus Luteum formation and demise: The corpus luteum is an endocrine gland that develops
rapidly from the ovulated follicle and performs vital functions in the reproductive process, namely,
the secretion of progesterone, which is necessary for the implantation of the blastocyst and
maintenance of pregnancy. The process by which the post-ovulatary follicle differentiates to
become the corpus luteum is known as luteinization. Both luteinization and ovulation has a
common stimulus, the preovulatory LH surge. Profound and radical changes occur within a
relatively short period of the process of luteinization and the formation of the corpus luteum (Fig.
5).
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Granulosa cells along with thecal cells infiltrate the collapsed follicle together with a rich supply of
blood vessels. The filtrating cells undergo hypertrophy and hyperplasia. Granulosa cells
particularly undergo massive hypertrophy, with many fold volume increases relative to their
preovulatory size. In most mammalian species, the cells derived from granulosa cells have been
designated as Large Luteal Cells or granulosa- lutein cells and those from thecal cells as Small
Luteal Cells or Thecal-lutein Cells. Cyclin D2 is a gonadotropin responsive gene involved in
granulosa cell proliferation, as its targeted deletion impairs both normal and gonadotropin induced
granulosa cell mitosis. The Cip/Kip family of kinase inhibitors regulates cyclin D complexes.
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Cyclin D2 expression is down regulated within 4 hours in granulosa cells undergoing luteinization,
which suggests that the LH surge arrests mitosis by concurrent inhibition of cyclin D2 and upregulation of p27 kip 1 and p21 cip1. The cellular hypertrophy involves not only a multifold increase
in the cytoplasmic volume of the cells, but also remarkable changes in the intracellular organelles
such as the mitochondria, smooth endoplasmic reticulum, and lipid droplets. The rapid cell
transformation that occurs early in the life of the corpus luteum is also characterized by the
disappearance of key proteins and by either the reappearance of some or at least a marked
enhancement of others. The expression of follicle stimulating hormone (FSH)-receptor, a protein
expressed only in granulosa cells of the follicles, becomes undetectable with luteal formation. The
expression of enzymes such as P450 17α hydroxylase and P450 aromatase, involved in the
synthesis of androgens and estradiol, is reduced to low or undetectable levels. The inhibition of
the synthesis of these enzymes is only transient in species such as rat and humans, whereas it is
sustained throughout the life span of the corpus luteum in other species such as bovine, ovine and
rabbit. The disappearance of P450 17α hydroxylase and P450 aromatase, the rate limiting enzymes
in androgen and estrogen synthesis, in the sheep and cows has been employed as a marker for
luteinization. In contrast to the down regulation of the FSH-receptor, P450 aromatase, and P450
17α hydroxylase, the expression of other proteins, such as prolactin-receptors and steroidogenic
enzymes, 3β-hydroxysteroid dehydrogenase (HSD) and P450 side chain cleavage (SCC),
increases remarkably and remain elevated until the end of pregnancy. The expression of P450 SCC
enzyme increases within 7 hours of the ovulatory stimulus in the rat corpus luteum. Expression of
Steroidogenic acute regulatory protein (StAR) has been shown to undergo luteinizationdependent up-regulation. StAR imports cholesterol into mitochondria, and is essential for
steroidogenesis. Its expression pattern renders it an important marker of the luteinization process.
Luteinization triggers up-regulation of the cholesterol-trafficking pathways (lipoprotein receptor,
cholesterol transport proteins, and the enzymes that catalyze cholesterol synthesis, cholesterol ester
lytic enzymes) to meet a dramatically elevated substrate requirement. A prominent increase in
expression of low-density lipoprotein (LDL)-receptor was demonstrated in the follicle, beginning
soon after the ovulatary stimulus and persisting through the luteal phase correlating with the
progesterone level. Circulating high-density lipoprotein (HDL) contributes cholesterol to luteal
steroid synthesis, and is the principal cholesterol supply in murine rodents. The cellular uptake of
HDL occurs via a scavanger receptor type 1, class B (SR-B1) has been elucidated. The
abundance of its expression correlates with luteinization of granulosa cells, and SR-B1 content is
directly correlated with the acquisition of cholesterol by granulosa cells. Expression of SR-B1
increases several folds during luteinization. The conversion of a follicle to corpus luteum requires
that high surge level of LH to provoke ovulation. This gonadotropin is then also required, albeit at
much lower levels, for the maintenance of the corpus luteum. However, in some species, prolactin
is also an important component of the so called “luteotrophic complex”. Unless pregnancy occurs,
the functional life of the corpus luteum is short and limited to luteal phase of the cycle. Luteolysis
begins with shunting of blood vessel going to the corpus luteum, following which lysosomes
initiate a process of lipolysis. Withdrawal of LH support under a variety of experimental
circumstances virtually invariably results in luteal demise. A specific luteolytic factor has not been
isolated in primates, but prostaglandin F2 alpha from the endometrium may fulfill this function in
other species. During luteolysis the luteal cells become necrotic, progesterone secretion ceases, and
the corpus luteum is invaded by macrophages and then by fibroblasts. Endocrine function is rapidly
lost and the corpus luteum is replaced by a scar-like tissue called the corpus albicans.
Interstitium- The interstitial cells are located in the loose connective tissue of both the cortex
and the medulla, arising from mesenchymal cells of the stromal compartment. They are
androgen-producing cells. The interstitial cells lie within the stroma and between the
developing follicles. They are composed of aggregates of steroidogenic-like cells, which
contain extensive smooth endoplasmic reticulum and lipid droplets. In the rabbit, in which these
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cells are well developed, they synthesize progesterone and 20α-hydroxyprogesterone, and are
sensitive to the LH activity observed after coitus. The interstitial cells are also have been
implicated in the synthesis of androgens as in human, rat and rabbit ovaries, and it is possible
that this tissue serves as an additional source of androgens for both secretion and
aromatization in the follicles.
The interstitium of the ovary also contains extravascular macrophages, lymphocytes and
polymorphonuclear granulocytes at various stages of the reproductive cycle. The resident
ovarian representatives of the white blood cell may constitute potential in situ modulators of
ovarian function, acting through the local secretion of regulatory cytokines.
FUNCTIONS OF THE OVARY
The major functions of the ovary are the differentiation (oogenesis and folliculogenesis) and
release of the female gamete or mature oocyte (ovulation) for fertilization and the production
of hormones (steroidogenesis) for regulation of female reproductive organs and their functions.
Generation of female gamete:The production of functional female gametes requires two inter-connected processes:
Oogenesis and Folliculogenesis.
Oogenesis- Oogenesis is the process of formation and maturation of female gametes or oocyte
for fertilization. The oocytes, provide the maternal genetic material and nutrients for early
development of the embryo. The ovary nurtures thousands of oocytes and functions as an
incubator for their development. Oogenesis begins during fetal development with formation of
primordial germ cells from a small number of stem cells at an extragonadal site and ends years
later in the sexually mature adult with activation of ovulated eggs (Fig 6).
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T
h
e primordial germ cells divide mitotically producing species-specific numbers of oogonia.
Oogonia become meiotic oocytes that progress through meiosis to a haploid state at the time of
ovulation. Meiosis is the reduction division unique for germ cells. It consists of two divisions,
which result in the production of the haploid gametes. The oogonia undergo the first meiosis
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early in life, often during fetal life. It is now called primary oocyte. However, the meiosis in the
primary oocytes are arrested at the diplotene stage until shortly after ovulatory surge of
gonadotropin. The termination of mitosis and early entry into meiosis is evidently evoked by a
meiosis initiation factor derived from cells of the mesonephric tissue, as the removal of this
tissue prevents meiosis. The consequence of this early termination of mitosis is that, by the time
of birth, a female has all the oocytes within her ovary that she will ever have. If these oocytes
are lost, for example by exposure to x-irradiation, they cannot be replaced from stem cells and
the (woman) female will be infertile. This situation is distinctly different from that in the male
in which the mitotic proliferation of spermatogonial stem cells continues throughout adult
reproductive life. The mechanisms, which control meiotic arrest of the oocyte in the diplotene
stage are not fully known. The meiotic arrest is needed as an important checkpoint to ensure
that the oocyte has time to grow big enough before fertilization in order to sustain the following
embryogenesis. As soon as the oocyte reaches diplotene stage, it must be enclosed by the
granulosa cells and a basement membrane to form a primordial follicle. Early follicular growth
is recognized by multiplication of the granulosa cells and simultaneous enlargement of the
oocyte. The first meiosis is reinitiated prior to ovulation resulting in the germinal vesicle
(oocyte nucleus) breakdown and produces a large haploid secondry oocyte and a tiny first polar
body. The meiosis is regulated by the activity of p34cdc2 kinase and cyclin B. These are
components of a functional activity generally called maturation promoting factor (MPF).
MPF activity is triggered by preovulatory LH-surge. Fully-grown oocytes undergo meiotic
maturation and become suitable for fertilization at the time of ovulation. This occurs as a result
of changes in intercellular communication between follicular components, as well as changes in
levels of various factors, including cyclic AMP, calcium, and steroids. Only fraction of original
germ cell population survives and fewer still successfully progress to ovulation in adult life, the
great majority is destined to undergo apoptosis or atresia. In human ovaries, for example, germ
cell number peaks around mid gestation at approx 7 millions, decreases to 1 or 2 millions by
birth, and declines to approx 250,000 by puberty; of these survivors, only 400 or 500 follicle
will ovulate during the reproductive life span.
Folliculogenesis:- Folliculogenesis is the process by which follicles develop and mature.
Maturation of oocytes (oogenesis) is closely associated with the development of follicle.
Folliculogenesis always begins in the innermost part of the ovarian cortex in mammals.
Primordial follicle consists of primary oocyte surrounded by a single layer of flattened
granulosa cells, the membrane granulosa. As primordial follicles develop in to primary follicles,
the membrana garnulosa gradually transform from a flat into a cuboidal shaped cell. Follicles
develop through primordial, primary and secondary stages before acquiring an antral cavity. At
the antral stage most follicles undergo atresia, however, under optimal gonadotropin stimulation
that occurs after puberty, a few of these follicles rescued (selected) to reach the preovulatory
stage called dominant follicle. As a primary follicle continues to grow, granulosa cells divide
mitotically and acquire the thecal layer that encloses granulosa layers. Secondary follicles have
membrana granulosa with two to six layers of granulosa cells. The theca layers form around the
secondary follicles. During formation of tertiary follicles, granulosa cells secrete fluid that
accumulates between granulosa cells. Large amount of additional fluid diffuses out of thecal
blood vessels and are added to the secretion of granulosa cells. This fluid-filled space is called
as the antrum or antral cavity, and the fluid is called follicular fluid. Follicular fluid contain
steroid and protein hormones, anti-coagulants, enzymes, and electrolytes and is similar to blood
serum in appearance and contents. Tertiary follicles have a membrana granulosa of more than
four cell layers, and the theca layer is now differentiated into an inner theca intrna and outer
theca externa. Oocytes in tertiary follicles are suspended in follicular fluid by a stalk of
granulosa cells, the cumulus oophorus. Immediately surrounding oocytes is a thin ring of
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granulosa cells, the corona radiata. At this stage, the follicle is called as Graafian follicle and
appears as a transparent vesicle that bulges from the surface of the ovary. Even though one of
the functions of the ovary is to produce oocytes, the majority of oocytes never ovulate. The
number of the oocytes reaches its maximum soon after the ovaries are formed. At birth a female
has all the follicles she will have in her life, no new follicles are made after birth. The majority
of the follicles (70-99%) are eliminated by a process termed atresia. Recent studies have
demonstrated that apoptosis is the molecular mechanism underlying follicular atresia.
Mechanism of Ovulation:Ovulation is a direct result of the LH surge and occurs some (12-36) hours after the LH peak.
The LH surge induces multiple changes in the dominant follicle, which occur within a relatively
short time. These changes include oocyte maturation, granulosa cells luteinization, activation
of proteolytic enzymes, and other local factors. One of the earliest responses of the ovary to a
rise in LH is increased blood flow, resulting from an LH-mediated release of vasodilator
substances such as Vascular Endothelial Growth Factor (VEGF), prostaglandins,
histamine and bradykinin. The preovulatory follicle switches from estrogen producing to a
progestin-producing structure. There is also an increased production of follicular fluid,
disaggregation of granulosa cells, and detachment of the oocyte-cumulus complex from the
follicular wall. As ovulation approaches, the follicle enlarges and protrudes from the surface of
the ovary. In response to the surge, plasminogen activator is produced by thecal and granulosa
cells of the dominant follicle and converts plasminogen to plasmin. Plasmin is a proteolytic
enzyme that acts directly on the follicular wall and stimulates the production of collagenase
enzymes, which digest the connective tissue matrix. The thinning and increased distensibility of
the wall facilitates the rupture of the follicle. The extrusion of the oocyte-cumulus cell mass is
aided by smooth muscle contractions.
Production of Hormones:The ovary produces both steroid and non-steroid hormones. Steroid hormones are derived from
cholesterol; they bind to sex-binding proteins and are metabolized in the liver and kidney.
Non-steroidal hormones are protein or polypeptides. The ovarian hormones act on the
hypothalamus and pituitary to regulate the secretion of hormones by these two tissues, thus
establishing the hypothalamus-pituitary-ovary axis. The ovarian hormones also affect
functions of the reproductive tract. This action of ovarian hormones is important because the
success of follicle development, ovulation, fertility and eventually embryonic development
depends on correct functioning of hypothalamus, pituitary and reproductive tract.
a. Steroid hormones of the ovary:They are non-polar, fat soluble hormones generally derived from cholesterol and having a
cyclopentane-perhydro-phenanthrene ring core. They have intracellular receptors, which are
not readily soluble in blood and are transported by carrier proteins or sex hormone binding
proteins. The synthetic form can often be administered orally.
Ovarian steroidogenesis proceeds along the biosynthetic pathway as outlined in Fig. 7. The
main product of the follicle is estradiol, while progesterone is produced by the corpus luteum.
Small amounts of androgens are produced by both follicles and corpus luteum as well as by
ovarian stroma cells. Ovarian steroidogenesis depends on the availability of cholesterol, which
is produced locally from acetate or taken up from the circulation via low-density lipoprotein
(LDL) receptor. Conversion of cholesterol to pregnenolone by P450 side chain cleavage
enzyme is a rate-limiting step regulated by gonadotropin. From pregnenolone to androgen (C19 steroid), ovarian steroidogenesis proceeds primarily along the ∆4 pathways (Fig. 7). Unlike
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testes, the ovary has an active cytochrome P450 aromatase enzyme and the major products of
follicle are estrogen. Thus the three major steroid hormones produced by the ovary are:
progesterone, androgen and estrogen.
Progesterone: Progestins (Progesterone and 17α-hydroxyprogesterone) are predominantly
produced by luteal cells. Luteal cells have the LH receptors and primarily secretes progesterone
and in some mammals it also secretes estrogen in response to LH stimulation. Granulosa cells
of preovulatory follicle prior to ovulation also secretes small quantity of progesterone.
Progesterone also serves as a precursor for androgen and estrogen synthesis. Pregnenolone
formed from the cholesterol may be converted either to progesterone or 17 αhydroxypregnenolone. The conversion to progesterone requires the action of 3βhydroxysteroid dehydrogenase, which shifts the double bond from the ∆5 to ∆4 position. 17 αhydroxypregnenolone is converted by the P450 17α hydroxylase enzyme to
dehydroepiandrostenedione (DHEA). The DHEA can then be converted to androstenedione.
Androstenedione is the major androgen secreted by the ovary, although a small amount of
DHEA and testosterone are also released. In circulation progesterone binds primarily to
transcortin and albumin. The serum levels of progesterone in cycling woman ranges from
undetectable to 10 ng/ml, with the peak at Day eight after ovulation. Progestins are degraded in
the liver and kidney as sulfate or glucuronide conjugates and excreted in the urine. The major
metabolite of progesterone is pregnanediol, which conjugated with glucuronide gets excreted
in the urine.
Androgen: Androgen is principally secreted by testis in male, but also by adrenal cortex. In
females, the ovary also secretes androgen. In the ovary, androgen is produced primarily by the
theca interna (thecal cells) in the preovulatory follicle and interstitial cells. These cells are
amply endowed with P450c 17α-hydroxylase enzyme activity, capable of converting
pregnenolone and progesterone, respectively to, DHEA and androstenedione. These are further
converted to testosterone in the steroidogenic pathway (Fig. 7). The major ovarian androgen are
androstenedione and testosterone. The thecal cells have LH-receptors, and LH acts to stimulate
the production of androstenedione and testosterone (both are estrogen precursors), which are
converted to estrogen. When androgen is secreted in abnormally high quantities, they interfere
with the proper functioning of the ovaries leading to the development of polycystic ovary and
hirsutism.
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Estrogen: Estrogen are produced predominantly by granulosa cells, utilizing androstenedione
as a precursor produced by the thecal cells, granulosa cells have FSH receptors, and FSH
stimulates in granulosa cells aromatization of thecal androgen to produce estrogen. Early in the
follicular phase, the granulosa cells contain only FSH-receptors. As the follicle grows in
response to the action of FSH and estrogen production increases as a result of the action of LH
on theca cells and FSH on granulosa cells. This is known as two cells two gonadotropin
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theory of estrogen synthesis (Fig. 8). As the serum estrogen level rises, it enhances further
actions of FSH and consequently inducing the development of LH receptors on the granulosa
cells. Once LH–receptors develop, granulosa cells begin to secrete progesterone, which helps in
ovulatory process. After ovulation, granulosa cells change to luteal cells. The LH stimulates
luteal cells to secrete both progesterone and estrogen. Both LH and FSH bind to their specific
receptors and trigger a cAMP mediated estrogen production. Reciprocally, estrogen feeds
positively and negatively back to stimulates and inhibit LH and FSH synthesis and secretion at
the hypothalamus and pituitary levels, respectively. Approx 60% of the estrogen secreted is
transported bound to steroid hormone binding globulin (SHBG), 20% is bound to albumin,
and the remaining 20% are in free form. The serum level of estrogen in a cycling woman range
from undetectable to 700 pg/ml. Estrogens is degraded in the liver and kidney. It stimulates the
growth and development of the uterus, fallopian tubes, cervix, vagina, labia and breasts at
puberty and controls reproductive cycle.
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b. Non-steroidal hormones of the ovary:
Relaxin: One of the peptide hormones to be recognized as a product of the ovary was relaxin. It
is mainly produced by the corpus luteum during pregnancy but has also been found in decidual
tissue, placenta and human seminal plasma. The secretion of relaxin from the corpus luteum is
stimulated by human chorionic gonadotropin (hCG). Relaxin does not appear to be single
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polypeptide since three closely related peptides of a molecular weight of about 9000 have been
shown to have relaxin-like activity. It is a dimer peptide; consist of an alpha and a beta chain
connected by two disulphide bridges (Fig. 9). Structurally, it is similar to insulin and insulinlike growth factor (IGF) suggesting that they derive from the duplication of a common
ancestral gene. The relaxin genes were cloned by early 1970s.
Two forms of relaxins have been discovered, namely, relaxin H1 and relaxin H2. Corpora
lutea of menstrual cycle and pregnancy are the main sites for relaxin H2 production. Relaxin H1
expression is identified in the deciduas and placenta but not in the ovary. The serum relaxin
levels consistently rise after the LH surge during the menstrual cycle. Although the absolute
level of relaxin is low, relaxin reaches its maximum concentration during the first trimester of
normal pregnancy and then gradually declines to term. LH stimulates the production of relaxin
during the menstrual cycle and hCG during pregnancy. The main effect of relaxin is to induce
relaxation of the pelvic bones and ligaments, inhibit myometrial motility, and soften the cervix.
In addition, relaxin has been shown to induce uterine growth. It is clear, therefore, that the
hormone plays an important role in both maintaining uterine quiescence and favoring the
growth and softening of the reproductive tract during pregnancy.
Inhibin Family: For many years it was suspected that the ovary produces a peptide hormone
that exerts selective inhibitory control over the secretion of follicle-stimulating hormone (FSH).
This protein, originally described in testicular extracts and termed inhibin, has later been
isolated also from follicular fluid. Subsequently two other polypeptides were isolated and
characterized, activin and follistatin, based on their ability to effect on the production of FSH
by the pituitary in mammals. Inhibins are characterized as heterodimeric glycoproteins
consisting of a common α -subunit combined with one of two β-subunits, βA or βB (Fig. 10).
The two subunits (α and βA or βB) are held together by disulfide bonds, producing two
different inhibins termed inhibin-A (α-βA) and inhibin-B (α-βB). Activins are characterized as
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homodimeric glycoproteins consisting of two β-subunits. Three forms of activin, activin-A
(βA-βA), activin-B (βB-βB) and activin-AB (βA-βB) have been isolated and shown to have
similar biological functions. Follistatin is a single-chain polypeptide originally isolated from
ovarian fluid as pituitary FSH secretion inhibitor.
The major sites for inhibins synthesis are granulosa cells in preovulatory follicles in the ovary.
Inhibin has also been synthesized in the corpus luteum. The granulosa cell, but not the corpus
luteum is the major site for activin production. The major sites for follistatin synthesis are
granulosa cells, luteal cells and thecal cells. Originally, inhibin was detected in serum
throughout the estrous cycle. Inhibin rises in the late follicular phase and during mid cycle and
its level is even higher during the luteal phase. The level of inhibin parallels the serum
progesterone level. Levels of activin, unlike inhibin, do not seem to fluctuate during the estrous
cycle. The production of inhibin is primarily controlled by FSH and is associated with
development of follicles. LH and androgen have also been reported to stimulate the production
of inhibin. Inhibin selectively suppresses both basal and GnRH-stimulated FSH synthesis and
secretion without influencing LH secretion. Follistatin protein is only detected in tertiary
follicles and newly formed corpus luteum. It was later found that follistatin is a high affinity
binding protein and exerts its effects primarily through binding and neutralization of activin.
Inhibin has paracrine and autocrine functions in the ovary. Although inhibin has been found to
bind to granulosa cells, the receptor for inhibin has not been isolated. Inhibin stimulates the
proliferation of luteinized granulose cells and suppresses FSH-mediated estrogen production by
granulose cells. It also stimulates androgen production and synergises with LH and insulin-like
growth factor (IGF)-I to increase androgen production by thecal cells. Current data suggest that
inhibin plays a role in the regulation of follicular development. Injection of inhibin into the
ovary increases follicular diameter. Inhibin has been also suggested as a tumor suppressor gene,
as the inhibin α-subunit gene knock-out led to the development of granulosa cell tumors in
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mice. Activin often acts as a functional antagonist of inhibin in pituitary as well as in many
other tissues. The neutralization of activin activity by follistatin has been observed in many
biological systems. It must be noted that not all activin functions were neutralized by follistatin.
The interaction between activin and follistatin is probably important to the local regulation of
activin’s function.
Intraovarian Growth factors: Several growth factor families [EGF, TGF, FGF, IGF and
Cytokines] have been shown to be produced and have autocrine and paracrine functions in
the ovary. Among these, IGF has been the best studied. Two IGFs, IGF-I and IGF-II, have been
described. Both are dimeric peptides consisting of one A chain and one B chain linked by
disulfide bonds. The IGF elicit their biological effects by binding to the specific cell surface
receptors (Type I and Type II) on the target. In addition, IGFs also bind to six serum IGFbinding proteins (IGFBPs) with comparable high affinity to IGF receptor. IGFBPs can either
inhibit or potentiate IGF action at the level of target cells by sequesteration or releasing of IGF
through the change of IGF-binding affinity. Stimulating IGFBP-protease may eliminate the
inhibitory effect of IGFBP on IGF. In ovary FSH stimulates IGFBP-protease.
IGF-I is mainly expressed in the theca-interstitial cells, whereas IGF-II expression is localized
to the granulosa cells in human. IGFs promote granulosa cell proliferation and differentiation as
well as production of androgen by theca cells. The expression of IGFs is stimulated by
gonadotropin and estrogen. FSH action has become increasingly apparent during the past
decade. FSH also stimulates the expression of IGF receptors and increases binding of IGF to
granulosa cells. IGF concentrations in follicular fluid are slightly increased during late follicular
growth and decreased in atresia. However, the concentrations of IGFBPs are dramatically
changed at the same time. The major function of IGFs seems to be to potentiate the action of
gonadotropins during follicular development. IGFs synergize with FSH to increase estrogen,
progesterone and inhibin production. IGFs also induce LH receptor expression and increase
ovarian androgen production. Both growth and steroidogenesis of granulosa cells are stimulated
by IGFs.
REGULATION OF OVARIAN FUNCTION
A. Regulation of Folliculogenesis: Folliculogenesis is the process by which follicles develop
and mature. At any given time, follicles are found under four conditions: resting, growing,
degenerating, or ready to ovulate. Some of these processes occur without hormonal
intervention, while others are controlled by an intricate relationship between the gonadotropins,
steroids, and local intra-ovarian growth factors.
Progression from primordial to primary follicles occurs at a relatively constant rate throughout
fetal, juvenile, prepubertal, and adult life. Once primary follicles leave the resting reservoir,
they are committed for further development, and typically only one will ovulate in the human
female. The conversion from primordial to primary follicles is believed to be independent of
gonadotropins. The exact signal that recruits a follicle from a resting to a growing pool is
unknown; it could be programmed by the cell genome or influenced by local ovarian factors.
Early follicular development is gonadotropin independent, whereas the follicular development
beyond early antral follicle is gonadotropin-dependent. Development beyond early antral
follicle begins at puberty and continues in a cyclic manner throughout the reproductive years.
Maturation of primary follicles to the preantral stage takes several weeks in human. The
potential role of the oocyte in early follicle development is provided by studies of growth
differentiation factor-9 (GDF-9), a homodimeric protein of the transforming growth factorβ (TGF-β)/activin family. GDF-9 is produced by growing oocytes of primary and larger
24
follicles but is absent in primordial follicles. In mutant mice, disruption of the GDF-9 gene
prevents follicle development beyond the primary stage. These studies demonstrated the
importance of oocyte-granulosa cell interactions during early stages of follicle development.
Besides GDF-9, kit ligands and BMP-15 are highly expressed in secondary follicles, they are
likely to play important role in preantral follicle development. Granulosa-oocyte
communication is essential for normal oocyte growth in early follicles. Immature oocytes
separated from granulosa cells do not grow. In mice, a gap junction protein, connexin-37, is
expressed at the oocyte-granulosa cell junction by the time follicles have developed to the
secondary stage, whereas follicles of mice that lack connexin 37 do not progress normally.
The critical hormone responsible for progration from preantral to antral stage is FSH. However,
pure preparation of FSH is less effective than those containing some LH, indicating that both
hormones, at a certain ratio, are required. The granulosa cells of early antral follicles acquire
receptors for FSH and start producing estrogen. Mitosis of the granulosa cells are stimulated by
FSH and estradiol. FSH has been shown to stimulate the expression of cyclin D2, a cell cycle
protein important in the G1 phase of cell division. Mice lacking cyclin D2 are infertile, and
granulosa cell replication is impaired as early as the secondary follicle stage. As the number of
granulosa cells increases, production of estrogens, binding capacity for FSH, size of follicle,
and volume of the follicular fluid all increase markedly. FSH and LH are important trophic
factors for the proliferation and survival of follicular somatic cells and the cyclic recruitment of
antral follicles. In rats, estrogens are potent antiapoptotic hormones in early antral follicles.
Follicle estrogen production is dependent upon both FSH stimulation of aromatase in the
granulosa cells and LH stimulation of androstenedione production by theca cells. Moreover,
FSH induces granulosa cell sensitivity to LH by increasing LH receptors in granulosa cells and
prepares for the luteinization of granulosa cells in response to the ovulatory LH-surge in
mammals. In contrast, only LH stimulates theca cells, and LH receptors are present from the
beginning of the formation of the theca layer.
In addition to blood-borne hormones, antral follicles are exposed to a unique
microenvironment. The follicular fluid contains different concentrations of pituitary hormones,
steroids, peptides and growth factors. Some are present in follicular fluid at a concentration
100-1000 times higher than in the circulation. The follicular fluid contains other substances,
including inhibin, activin, GnRH-like peptide, growth factors, opioid peptides, oxytocin, and
plasminogen activator.
Recent rodent studies indicate that preantral follicles in serum-free cultures undergo
apoptosis despite exposure to gonadotropins, suggesting that gonadotropins are probably not
survival factors at early stages of folliculogenesis. An elaborate intra follicular control
mechanism ensures the survival of preovulatory follicles. The onset of apoptosis in
preovulatory follicles in a serum-free culture is prevented by treatment with FSH and LH. In
addition, treatment with growth hormone or local factors including IGF-I, EGF, TGF-α, and
Fibroblast growth factor-2, likewise suppresses follicle cell apoptosis. Interleukin-1 β is also
a survival factor for preovulatory follicles. Although gonadotropins are the most important
survival factors for preovulatory follicles, this array of extracellular signals acting through
endocrine, paracrine, autocrine, or juxtacrine mechanisms, ensures their survival for ovulation.
B. Regulation of Steroidogenesis: The main steroid hormone of the follicle is estradiol, while
progesterone is produced by the corpus luteum. Small amounts of androgens are produced by
both structures and by ovarian interstitial cells. Ovarian steroidogenesis depends on the
availability of cholesterol, which is produced locally from acetate or taken up from the
circulation via low-density lipoprotein (LDL) receptors. Conversion of cholesterol to
pregnenolone by P450 SCC enzyme is a rate-limiting step regulated by gonadotropins. From
pregnenolone to androgens (C-19 steroids), ovarian steroidogenesis proceeds primarily along
25
the delta 4 pathway (Fig. 7). Unlike the testes, the ovary has an active cytochrome P450
aromatase; and so the main products of the follicle are estrogens, rather than androgens.
The estradiol synthesis of the follicle requires cooperation between granulosa and theca cells as
well as coordination between FSH and LH. This is commonly known as two-cell and two
gonadotropin theory (Fig. 8) of follicular estradiol synthesis. This requires unique partnership
in steroid synthesis between theca and granulosa cells. The principal site of estrogen synthesis
in the ovary is granulosa cells under the control of FSH. FSH stimulates not only estrogen
production of granulosa cells at all stages of follicular development but also progesterone
synthesis by mature follicles before ovulation. Androgen production appears to be the primary
steroidogenic function of theca cells in response to LH. The expression of LH receptors is timedependent. Theca cells acquire LH receptors at a relatively early stage, whereas LH receptors
on the granulosa cells are induced by a combined action of FSH and estradiol only in the
maturing follicles. Androgens from theca cells provide substrates for granulosa cells to
synthesize estrogens. The action of LH on theca androgen production, together with the action
of FSH in granulosa cell estrogen synthesis, forms the basis of the “Two-cell, two-hormone”
theory for the control of steroidogenesis in the ovary.
CONCLUSION
Our knowledge of ovary has greatly increased during the last decade. Whereas the role of
gonadotropin as primary regulator of ovarian functions remains indisputable, the role played by
local intraovarian factors has become increasingly apparent during the past decade. The most
ovarian cell types including oocyte, produce a variety of peptides that act locally to influence
gonadotropin actions, either positively or negatively. In this respect, it is fascinating to note the
large number of follicular functions that are controlled by a balance between activating and
inhibitory factors. Despite the advancements in our understanding of various ovarian functions,
many processes remain poorly understood. Virtually all of our knowledge on biological actions
of intraovarian factors on regulation of ovarian functions are based on in vitro studies, therefore
the extent to which results from these studies can be extrapolated to the in vivo level remains
uncertain. Further advancement in our understanding of ovarian functions depend on the
development of in vivo model to study the potential role of these intraovarian factors in normal
and abnormal ovarian functions.
Suggested Reading:
1. Brinster RL. Germ line stem cell transplantation and transgenesis. Science 2002; 296:
2174-2176.
2. Matzuk MM, Burns KH, Viveiros MM and Eppig JJ. Intercellular communication in the
mammalian ovary: oocytes carry the conservation. Science 2002; 296: 2178-2180.
3. Swain A and Lovell-Badge R. Mammalian sex determination: a molecular drama.
Genes and Development 1999; 13: 755-767.
4. Murphy BD. Models of luteinization. Biology of Reproduction 2000; 63: 2-11.
5. Armstrong DG and Webb R. Ovarian follicular dominance: the role of intraovarian
growth factors and novel proteins. Biology of Reproduction 1997; 2: 139-146.
6. Gougeon A. Regulation of ovarian follicular development in primates: facts and
hypotheses. Endocrine Reviews 1996; 17: 121-155.
7. McGee EA and Hsueh AJW. Initial and cyclic recruitment of ovarian follicles.
Endocrine Reviews 2000; 21: 200-214.
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8. Erickson GF. Ovarian anatomy and physiology. In: Menopause: Biology and
Pathobiology 2000; Academic Press
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