Ovarian Development

The ovaries’ two major functions are (1) release of mature ova at the time of ovulation and (2) the secretion of steroid hormones. Cyclic release of pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control both functions. In turn, gonadotropin-releasing hormone (GnRH), which the hypothalamus secretes into the pituitary portal circulation, regulates the release of the two hormones. The ovarian steroid hormones stimulate the growth of the reproductive organs, foster the development of secondary sexual characteristics, and help maintain the implanted blastocyst. The production of these hormones depends upon the development and maturation of the follicles and the formation of the corpus luteum.

Folliculogenesis

The primordial germ cells arise from the yolk sac endoderm and migrate via the hindgut mesentery to the gonadal ridge, the migration commencing 3 weeks after fertilization.⁵ At this stage the proliferating germ cells constitute the dominant growth, forming a finite complement of about six million ova by the sixth month of fetal life, to which there is no later addition. This proliferation is evident by the 8th week and is marked by the 13th week when the ovary consists largely of oogonial and oocytic clusters that taper proximally, with ‘cords’ only in the rete. Pregranulosa cells enclose individual germ cells to form primordial follicles (Figure 23.3A). At the primary oocyte stage, the cells are in the prophase of the first meiotic division and DNA synthesis resumes only in the event of fertilization. At birth, the ovary consists largely of scanty, loose stroma, enclosing primordial and maturing follicles and folded around stems of vascular connective tissue. The characteristic ovarian stroma develops during the first year. The total number of germ cells at birth exhibits a considerable variation around an approximate average of 500,000, already a significant loss from the second trimester maximum.

Follicular Maturation

Follicular maturation begins in the third trimester of intrauterine life, becoming more frequent later. The development of a follicle at this stage of life is followed by its atresia. The number of maturing follicles becomes considerable in the first 2 years of postnatal life, falls to a low level at 4 years, and then increases again. At puberty individual follicles start to rupture and form corpora lutea, which then in turn regress.

Typically, a single dominant follicle develops, resulting in a single ovulation during each cycle. During the early follicular phase selection of a dominant follicle takes place. This complex process involves insulin-like growth factors and locally produced substances.6–8 The early rise of FSH levels helps recruit the dominant follicle. The transient decrease in FSH during the midfollicular phase prevents development of subdominant follicles. The largest follicle has more granulosa cells and therefore more FSH receptors, enabling its continued growth despite the low FSH levels.⁹ Moreover, the dominant follicle’s granulosa cells are not only sensitized to FSH but at this stage express LH receptors and therefore respond directly to LH as well as FSH.^10,11

Follicular maturation comprises enlargement of the ovum, formation of the zona pellucida around it, proliferation of the granulosa cells, and formation of a cavity among the latter. The enlarging follicle expands gradually. Several months are required for a new growing follicle to reach the preantral stage (0.15 mm), and an additional 70 days to reach a 2 mm size. It is likely that several hundred primordial follicles initiate growth each month, but only 20 or so become ‘precursor’ (preovulatory) follicles. From the time they enter the selectable stage during the late luteal phase, preovulatory follicles become sensitive to cyclic changes of FSH in terms of granulosa cell proliferation. From the midfollicular phase, the preovulatory follicle synthesizes high quantities of estradiol, and, after the midcycle gonadotropin surge, very large amounts of progesterone. At this stage, the follicle is maximally responsive to gonadotropins, and especially to LH, which triggers granulosa wall dissociation and cumulus expansion as well as oocyte nuclear maturation. At this phase, the follicle is about 2–5 mm in diameter, with a fluid-filled antrum, more than one million granulosa cells, and a single oocyte. The other follicles undergo atresia at an early stage of development. Thus, as the follicle develops, its responsiveness to gonadotropins progressively increases under the control of local factors acting in an autocrine/paracrine fashion.¹² In addition to the critical endocrine signaling pathways between the hypothalamus, pituitary, and ovary, it is now evident that the proteins of the TGF-β superfamily secreted by the oocyte itself are important in influencing the microenvironment of the developing follicle.¹³

Granulosa cell proliferation begins when the ovum starts to enlarge. The cells change from flat to cuboidal and then form several layers around the ovum (Figure 23.3B). Small periodic acid–Schiff (PAS)-positive droplets form between the granulosa cells and condense around the ovum to form small spaces. Enlargement or fusion of these spaces produces first an arcuate crevice and then a reniform cavity, the antrum (Figure 23.3C). The granulosa cells form a palisade at the periphery of the follicle and in turn are surrounded by a pale layer of specialized ovarian stroma, the theca interna. A basal lamina separates the granulosa cells from the cells of the theca interna. The theca externa is a poorly defined zone formed by the merging of the theca interna with the surrounding ovarian stroma (Figure 23.3D). Theca cells are fundamental for follicular growth, synthesizing all androgens required by the developing follicles for conversion into estrogens by the granulosa cells, and providing structural support of the growing follicle as it progresses through the developmental stages. Theca cells are likely recruited from surrounding stromal tissue by factors secreted from an activated primary follicle.¹⁴ During maturation, the cytoplasm of the cells comprising the theca interna accumulates lipids, increases in amount, and becomes foamy or granular. In the dominant preovulatory follicle, the theca cell layer is well supplied with blood vessels but the granulosa cell layer remains avascular.

The follicular fluid is a modified transudate from the theca and adjacent vessels, which are believed to become more permeable because of locally released histamine. The same transudative process leads to the development of a loose-textured vascular shell around the outside of the follicle, the theca externa. As the antrum enlarges, the granulosa cell layer generally thins, but persists as a mound, the cumulus oophorus, around the ovum (Figure 23.3C). Granulosa cells aspirated from the developing follicle at this stage tend to be single or in small clusters with scanty amphophilic cytoplasm (Figure 23.4). Outside the cumulus the theca interna shows a corresponding thickening, the theca cone, which has a role in loosening the coherence of the tissues. The mature, preovulatory follicle is known as a graafian follicle. The final phase in follicular maturation is abrupt and largely mediated by the LH surge and a complex interaction of local cytokines.¹⁵ It includes rapid accumulation of follicular fluid, loosening of the theca, and discharge of the fluid contents and ovum with the few attendant granulosa cells that form the corona radiata. The follicle then collapses, taking on a corrugated outline that becomes exaggerated as transformation into a corpus luteum advances. Bleeding into the thecal tissues may occur at about the time of ovulation but, as the granulosa is as yet avascular, there is normally no bleeding into the follicular cavity and peritoneum.

Formation of the Corpus Luteum

The formation of the corpus luteum comprises two processes: (1) blood vessels grow into the collapsed granulosa cell layer from the theca, carrying theca cells with them (some bleeding now takes place into the cavity and, because of this, the corpus luteum of the current cycle appears bluish when seen through the surface of the ovary); and (2) the cells comprising the granulosa cell layer luteinize (Figure 23.5), the cells becoming strikingly large and pale, and readily distinguishable from the smaller and somewhat less pale luteinized theca cells (‘theca–lutein cells’). Ultrastructural studies of the luteinized cells show abundant smooth endoplasmic reticulum with vesicular dilatations, mitochondria with tubular cristae, and numerous small lipid droplets, which are the familiar general features of the steroid-secreting cell.

When pregnancy occurs, the corpus luteum remains unchanged for about 50 days. It then doubles in size during the following 10 days and remains thus enlarged until about day 80, when it begins to shrink as the placenta takes over progesterone production. Hyaline bodies, about 10–20 µm in size, are found sparingly among the luteinized cells only in the corpus luteum of pregnancy (Figure 23.6A). Another histologic feature that, when present, distinguishes the corpus luteum of pregnancy from that of menstruation is the organization of the central coagulum to produce mature, avascular, collagenous connective tissue over a time period not available to the corpus luteum of menstruation prior to its programmed involution (Figure 23.6B).

Involution of the Corpus Luteum

Unless stimulated by β-human chorionic gonadotropin (β-hCG), produced by the trophoblast of the fertilized zygote, the corpus luteum begins its programmed involution at about day 9, i.e., about day 23 of a normal 28 day cycle. The biologic mechanisms responsible for this physiologic destructive process of the corpus luteum (structural luteolysis) are complex and centered on apoptosis.16,17 Hyaline and fatty changes appear to a variable extent in the luteinized granulosa cells (Figure 23.7A), together with an increase of acid phosphatase and PAS-positive intracellular material. This apoptotic change may occur acutely, with frank necrosis (Figure 23.7B). The average corpus luteum reaches about 1.5 cm in maximum size. It has shrunk only slightly by the end of the cycle, but then rapidly gets smaller. As it does so, its wavy lemon-yellow form becomes thinner and the color deepens to a darker yellow or orange. Eventually, the luteal pigment and the cells disappear. After several months, the corpus luteum completes its transformation into a much folded, collapsed shell of white, hyaline collagen, the corpus albicans (Figure 23.7C). Corpora albicantia persist in the medulla and are often conspicuous on the cut surface, particularly in later life. Regression of the corpus luteum of pregnancy is occasionally accompanied by residual microcalcispherule formation (Figure 23.7D).

Follicular Atresia

During each cycle many follicles start to mature but usually only one ruptures to form a corpus luteum. Occasionally, two or three synchronous corpora lutea are found (one mechanism of multiple pregnancies). The follicles that do not complete maturation become atretic. In atretic antral follicles, granulosa cells stop proliferating and become apoptotic. Atresia may start at any stage during maturation and proceeds at rates varying from follicle to follicle and in different parts of the same follicle.18,19 Atresia is triggered by a lack of some essential factors supporting follicular development. Specifically, terminal follicular development is strictly dependent upon gonadotropin supply (FSH, then LH in the final preovulatory stage). In addition, paracrine factors (growth factors, CKs, steroids, constituents of extracellular matrix) also play important roles in amplifying gonadotropin action in follicular cells.²⁰ The features of atresia include degeneration and shrinkage of the ovum; apoptosis of the granulosa cells or their transformation into connective tissue cells (Figure 23.8A); reversion of the cells of the theca interna to their original stromal form; invasion and obliteration of the shrinking cavity by stromal cells; and the formation of a hyalinized, collagenous membrane at the margin of the follicle. Eventually, the hyaline membrane alone persists to mark the site of the former follicle, which now appears as a compact white or gray nodule with a wavy outline, the corpus fibrosum (Figure 23.8B).

Macrophages laden with lipofuscin may accumulate around corpora albicantia and corpora fibrosa and may remain there for long periods. Care should be taken not to confuse degenerating luteinized cells containing lipofuscin and old hemorrhage with endometriosis.

During pregnancy, follicle maturation and atresia diminish in the first 10 weeks. They then become more active and are associated with marked theca-cell luteinization (Figure 23.8C).The cellular stroma left after follicular atresia contributes to production of the cellular stroma that typifies the mature ovary and is absent at birth. Stroma formation depends largely on follicular activity and is depressed if follicles are lacking or inactive and increased if maturation and atresia are excessive, as in polycystic ovary syndrome. The medullary and juxtahilar stroma thus has a direct ‘follicular’ ancestry, which makes it easier to understand why the tumors that may rarely arise from it are similar to those that develop in the functioning cortical stroma, i.e., tumors of sex cord–stromal type.

The Hormonal Background of the Ovarian Cycle

The normal menstrual (ovarian) cycle is depicted in Figure 23.9. Increasing levels of FSH stimulation are required for follicular growth.²¹ Follicular antrum formation and antral expansion are absolutely dependent on pituitary FSH to which the larger follicles are more responsive. Early antral follicles (≤5 mm) develop normally even in infant ovaries but preovulatory (≥20 mm) growth depends on the adult FSH levels reached in ovulatory menstrual cycles and on the follicle capacity for estrogen synthesis. Follicular estrogens feeding back to the hypothalamic–pituitary axis control gonadotropin secretion, the secretion of FSH showing peaks on about days 4 and 14. Local interaction between estrogens and gonadotropins helps to coordinate preovulatory follicular maturation. Signaling by the gonadal paracrine activin and inhibin, which are members of the TGF-β superfamily, is crucial for follicular and oocyte development.22,23 Immature antral follicles produce activin that enhances granulosa cell sensitivity and responsiveness to FSH, thus promoting follicular development, and simultaneously suppresses thecal androgen synthesis. Appropriate stimulation by FSH diverts the follicle to formation of inhibin, which suppresses the secretion of FSH and promotes androgen synthesis by LH-stimulated theca cells. Androgens in turn synergize with FSH to augment inhibin synthesis.

LH acts on specific receptors on theca cells to stimulate androgen production, with positive correlations between androgen receptor expression and follicular cell proliferation.¹⁵ Furthermore, androgens are active through a conversion to estrogens in granulosa cells. The acute follicular enlargement leading to ovulation is associated with the peak of LH secretion on day 14 of the normal 28 day cycle, triggered by estrogen secreted by the dominant follicle. If pregnancy does not ensue, the corpus luteum’s life span is maximally about 14 days. As mentioned previously, its degeneration begins by day 23 (day 9 postovulation). Its persistence during that time depends on the continuous low level of LH secretion, but on no other known luteotrophic stimulus. When pregnancy occurs, chorionic gonadotropin fosters maintenance of the corpus luteum.

All known steroidogenic sites in the ovary produce each of the main ovarian hormones from acetate by way of cholesterol. These include progesterone, androstenedione, testosterone, dihydroandrosterone, estradiol, and estrone 1, which are the main successive stages in cholesterol transformation. The exact roles of the granulosa cells and theca cells in the preovulatory follicle are difficult to study; the complex inter-relationships of the cells to each other are virtually impossible to reproduce in vitro. However, clearly the granulosa cells can synthesize pregnenolone and progesterone and convert androgens to estrogens by aromatization. Thecal tissue produces mainly androstenedione, with small amounts of estradiol. As the granulosa cell layer in the preovulatory follicle is avascular, the hormonal environment of these cells (and also that of the oocyte) is different from that of the blood. It is dependent, in part, upon the activity of the adjacent theca cells. Thus, the products of the granulosa cells must diffuse through at least some of the theca cell layer to reach the ovarian venous blood. While follicular fluid analysis gives only an indirect indication of the activity of both types of follicular cells, the dominant and therefore the most active preovulatory follicle in an ovary has the highest levels of estradiol in the fluid. This dominant follicle has the highest estrogen : androgen ratio in its secreted hormones of all the follicles and its granulosa cells have the highest capacity for androgen aromatization in vitro. Aromatase activity in the granulosa cells falls off shortly before ovulation, and synthesis of progesterone commences just prior to ovulation in response to the LH surge.

The levels of LH, FSH, and estradiol fall after ovulation, but there is sufficient LH to maintain the sensitive luteinized granulosa cells of the corpus luteum. Together the luteinized theca cells and luteinized granulosa cells of the corpus luteum produce both estradiol and its major product progesterone. Normal corpus luteum function depends on numerous regulatory factors, such as prostaglandins, oxytocin, steroids, growth factors, cytokines, and so forth.²⁴ The theca cells produce primarily estrogen and the luteinized granulosa cells produce mainly progesterone, which is reflected in the high plasma progesterone level during this phase of the cycle and the high pregnanediol excretion from the time of ovulation until the day or two preceding menstruation. Both hormones reach their peak in the midsecretory phase of the cycle, when estradiol levels are similar to those of the mid to late follicular phase. The stromal tissue produces mainly androstenedione, which has about one-tenth the androgenic activity of testosterone and is partly converted into the latter peripherally. The effects of these hormones on the pituitary are largely mediated through the hypothalamus. Secretion of FSH is inhibited by sufficient amounts of either estrogens or progesterone; the effect of estrogens on LH secretion is thought to be biphasic, and is stimulatory over a short length of time and inhibitory in the long term.

The ovaries are the primary source of androgens in the postmenopausal period when testosterone production in particular is maintained or even boosted, presumably due to increased stimulation of the ovarian stroma by LH.

The Ovarian Hilum and Its Vicinity

The rete ovarii consists of multiple intercommunicating channels lined by deeply staining, cuboidal to flattened epithelial cells exhibiting a characteristically angular, jagged-looking cross section (Figure 23.10). Nearer to the ovary the rete tubules are more rounded in outline and in this situation they often show traces of a muscle and connective tissue covering. The origin of the rete ovarii is disputed. It is generally considered as homologous with the straight seminiferous tubules of the testis. The relevant immunoprofile of the rete tubules includes strong reactivity for CD10²⁵ and nonreactivity for calretinin.³

Small groups of lutein-like cells are found in the mesovarium and in the ovarian hilum, often adjacent to nerves. These are the so-called ‘ovarian hilar’ (hilum or hilus) cells (Figure 23.11). They resemble the interstitial cells of the testis (Leydig cells) and contain lipofuscin pigment and Reinke crystals. They produce both androgens and estrogens and express α-inhibin and calretinin.³

Afollicular Ovarian Failure (Primary Ovarian Insufficiency, Premature Follicular Depletion)