Reproduction. Mammalian Ovary. Amitabh Krishna; Raj Kamal Srivastava Arnab Banerjee

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1 Reproduction Mammalian Ovary Amitabh Krishna; Raj Kamal Srivastava Arnab Banerjee Department of Zoology, Banaras Hindu University, Varanasi , 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 Conclusions a. Regulation of folliculogenesis b. Regulation of steroidogenesis Corresponding author: Prof. Amitabh Krishna, Department of Zoology, Banaras Hindu University, Varanasi

2 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.

3 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.

4 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.

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6 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.

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8 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.

9 Table 1 S.No. Type of Follicle Characteristics 1 Primordial Follicle Primary oocyte surrounded by a single layer of spindle shaped (pre) granulosa cells and a basement membrane. There are pools of resting follicles. 2 Primary Follicle Primary oocyte surrounded by a single layer of cuboidal granulosa cells and a basement membrane. They are the first stage of follicle growth 3 Secondary Follicle (Pre-antral Follicle) Primary oocyte surrounded with zona pellucida and several (2-8) layers of granulosa cells. The surrounding ovarian stroma cells form theca layers, which are separated by granulosa cells by basement membrane. 4 Antral Follicle 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. 5 Graafian Follicle (Pre-ovulatory Follicle) 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. 6 Atretic Follicle Morphologically, it is characterized by necrosis of both the oocyte and the granulose cells. Their nuclei become pyknotic and the cell degenerate.

10 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 10

11 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). 11

12 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. 12

13 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 13

14 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). 14

15 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 T 15

16 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 p34 cdc2 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 16

17 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 (C- 19 steroid), ovarian steroidogenesis proceeds primarily along the 4 pathways (Fig. 7). Unlike 17

18 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 α- hydroxypregnenolone. The conversion to progesterone requires the action of 3βhydroxysteroid dehydrogenase, which shifts the double bond from the 5 to 4 position. 17 α- hydroxypregnenolone 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. 18

19 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 19

20 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. 20

21 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 21

22 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 22

23 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 23

24 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

25 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 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