The stages of cell division leading to the production of the female gamete begin early in fetal life. The process is halted until puberty when ovulation begins, and then halted again until fertilization. The developing egg is called an oocyte; it differentiates into a mature haploid ovum (or egg). Although dramatic progress in the development of an oocyte takes place during the follicular phase, the development of the follicle begins about 3 months prior to the menstrual cycle in which it is released at ovulation (Fig. 4.2). Folliculogenesis begins with the recruitment of primordial follicles from the ovarian pool; the signals which initiate recruitment are unknown. At the beginning of the cycle, a number of primary follicles are recruited to undergo initial development, stimulated by luteinizing hormone (LH). These secondary follicles express follicle-stimulating hormone (FSH) receptors. One follicle is selected to continue development as the dominant follicle. It is this follicle that releases the ovum at ovulation. The remnants of the follicle then become the corpus luteum.

In the female fetus, the primordial germ cells migrate to the ovary at 21 days postfertilization and become oogonia. They proliferate by mitotic division and then differentiate into primary oocytes; by 20 weeks of gestation, in each ovary, there are about 10 million primary oocytes ready to enter meiotic division (see Chapter 5). The oocytes degenerate throughout fetal life so the female neonate is born with a finite number of 250000–500000 oocytes; this is all the oocytes she will ever have. Degeneration of the oocytes continues throughout postnatal life; there are fewer than 200000 oocytes left at puberty and numbers continue to fall. However, if one assumes a reproductive life of about 35–40 years, the maximum number of ova released from the ovaries will be no more than a few hundred. Although it was thought that the oocytes number was established during fetal development, it is now suggested that some ovarian oogonia still unassociated with follicular cells persist from the fetal period and can undergo mitosis thus acting as a stem cell population of oocytes in postnatal life (Johnson et al., 2004).

Meiosis is a specialized cell division involved in the production of gametes (see Chapter 7, p. 149, for a detailed description). In it, the number of chromosomes is reduced from 46 (known as the diploid number) to 23 (the haploid number). In the first part of meiosis, the DNA replicates, so each chromosome is composed of two chromatids. The homologous chromosomes pair up along their long axes and crossing over occurs so the chromosomes exchange genetic material. In humans, two or three exchanges of DNA occur per chromosome pair ensuring that the combination of genes will be unique (Box 4.2). This is achieved by areas of fusion (called chiasmata) forming between adjacent chromosomes (see Chapter 7). The oocyte remains arrested in the prophase stage of division I of meiosis for a number of years until sexual maturity (Fig. 4.3). During this time, the primary oocytes synthesize the extracellular matrix and the secretory vesicles. Arrested meiosis provides a specialized mechanism to allow the oocyte to grow while it contains duplicate copies of chromosomes and, therefore, twice as much DNA is available to direct synthesis of RNA as in a somatic cell. The hormones of puberty release the oocyte from the first meiotic block; this process is called oocyte maturation. The oocyte resumes division I of meiosis. The chromosomes re-condense, the nuclear envelope breaks down and the meiotic spindle forms. The chromosomes then segregate into the two daughter nuclei. The cytoplasm divides asymmetrically to form a large secondary oocyte and a small polar body, each of which contains 23 chromosomes as pairs of chromatids. Meiotic division then arrests for a second time, so the oocyte released at ovulation has still not completed meiosis. Activation of the oocyte and release from the second meiotic block occurs at fertilization. In the second meiotic division, the sister chromatids finally segregate into two nuclei and the cytoplasm again divides asymmetrically, this forming the mature ovum and a second polar body, each with 23 chromosomes. Because both divisions of the cytoplasm are asymmetrical, the ovum is large. Effectively, the polar bodies are small packages of cytoplasm which contain discarded chromosomes. During in vitro fertilization (IVF), observing the presence of the second polar body indicates that fertilization has occurred (see Chapter 6).

In the female, mitosis of the gametes stops in the fetal period, whereas in the male, mitotic division of gametogenesis begins at puberty and continues until senescence (see Box 4.3). The termination of mitosis in the female, and entry into meiosis, is under the control of protein complexes, maturation-promoting factor (MPF) and cytostatic factor (CSF), which regulate the oocyte’s progression through meiosis (Dale et al., 1999). MPF phosphorylates multiple proteins involved in chromosome condensation and the breakdown of the nuclear envelope. The hormones of puberty increase MPF activity at the exit of the first meiotic block. It is hypothesized that the second meiotic block occurs because the maturing oocyte accumulates CSF which inhibits cell division. Oocyte activation and the release from the second meiotic block are thought to be triggered by a component of the sperm that enters the oocyte at fertilization and causes CSF degradation.

Both premature arrest at the end of oocyte maturation and parthenogenic release from meiotic arrest are thought to cause infertility. As women age, the extended duration of arrested meiosis, which may last for over 50 years, probably results in the meiotic spindles (see Fig. 7.11, p. 153) becoming increasingly fragile; this leads to an increased rate of abnormalities such as Down’s syndrome and failed implantation.

Development of a mature female gamete depends on complex interactions between the developing gamete and the surrounding cells forming the outer layers of the follicle. Mitosis is completed during fetal development. During the first meiotic prophase, the primordial germ cells stimulate organization of the surrounding cells to form the granulosa cells (flattened cuboidal epithelial cells) which condense and encircle them forming the primordial follicles. The follicular cells secrete a basement membrane around the outside forming a cellular unit (Fig. 4.4). These granulosa cells are connected to the oocyte by gap junctions through which nutrients can be transported. The primitive oocyte therefore has two layers and is about 18 μm in diameter. A few follicles may resume development spontaneously and incompletely throughout fetal and neonatal life. However, regular recruitment of the primordial follicles into the pool of growing follicles begins at puberty when levels of FSH increase, so the primordial follicles containing oocytes may stay arrested in meiosis for decades.

From puberty, a few primordial follicles spontaneously restart their development each day, forming a continuous stream of growing preantral or primary follicles. Most of these early follicles fail to develop fully and undergo atresia (fail to develop any further); less than 0.1% of follicles will ovulate and develop into a corpus luteum. The granulosa cells of follicles undergoing atresia accumulate lipid droplets and reduce protein synthesis. Both the granulosa cells and the oocyte become apoptotic (undergo ‘programmed cell death’). White blood cells invade the dying follicular cell mass and scar tissue forms.

As the majority of the follicles regress rather than progress through development, the ovary has a dense population of atretic follicles resulting in an irregular corrugated outer surface of the ovary. The development of primordial follicles into primary or preantral follicles takes about 85 days; the preantral phase is the longest phase of development of the oocyte. Initiation and progress through this early follicular development is independent of pituitary hormones but there may be paracrine regulation by cytokines (see Chapter 3), such as epidermal growth factor (EGF; Box 4.4). These developing follicles do not secrete significant amounts of steroid hormones. Further follicular development requires pituitary support (secretion of FSH and LH).

Early in the cycle, the concentration of FSH is sufficient to support the further development of some preantral follicles. The preantral follicles that are optimal for further development are of the appropriate size and maturity to respond and have adequate FSH receptors. Recruitment of the follicles is related to the interaction between the FSH concentration and the number of FSH receptors on the developing follicles. Therefore, the number of follicles surviving is related to the amount of FSH present. The antral development phase, ovulation and luteal phase comprise one cycle in the humans. In other mammalian species, antral expansion occurs in the luteal phase of the previous cycle, thus shortening non-fertile cycles.

Usually about 15–20 preantral follicles are rescued from atresia each month and undergo initial stages of development and marked enlargement in response to the increasing FSH concentration at the beginning of each cycle. Several components contribute to the growth of the follicle: the oocyte enlarges, the follicular cells divide and further stromal cells are recruited to form the expanded outer layers of the follicle. The oocyte itself increases in diameter to 60–120 μm. It synthesizes large amounts of ribosomal RNA (rRNA) and messenger RNA (mRNA) to increase its protein stores ready for the maturation of the oocyte and fertilization, but does not resume meiosis. The follicular cells divide into several layers of granulosa cells, which secrete an amorphous and acellular translucent jelly, the zona pellucida. The zona pellucida is formed from condensation of glycoprotein and accumulates between the granulosa cells and the oocyte, acting as an extracellular coat of the oocyte. It has an important role in sperm binding and penetration during fertilization (see Chapter 6). Although the zona pellucida acts to separate the oocyte from the avascular granulosa cells, cytoplasmic processes penetrate the zona pellucida forming gap junctions at the oocyte surface. These allow delivery of low molecular weight substrates, such as nucleotides and amino acids, and cellular signalling molecules into the oocyte. Gap junctions also exist between granulosa cells.

The third component of follicular growth is condensation of ovarian stromal cells on the basement membrane (membrana propria) of the follicle. These recruited cells form a loose matrix of spindle-shaped cells around the follicle, known as the thecal layer. The cells differentiate into two layers: the theca interna, an inner layer of highly vascular glandular cells, and the theca externa, a poorly vascularized fibrous capsule.

There is critical bidirectional paracrine and juxtacrine communication between the oocyte and the surrounding somatic or follicular cells of the ovary, the granulosa and thecal cells. FSH is required both for granulosa cell differentiation and division (Richards and Pangas, 2010). The granulosa cells acquire receptors for FSH and oestrogen and the theca interna cells acquire receptors for LH. Synthesis of steroid hormones by the follicle requires cell cooperation (Fig. 4.5; Hillier et al., 1994). Interstitial glands lie within the stroma and between the developing follicles. They are formed of steroidogenic cells and produce androgens for secretion and aromatization to oestrogen in follicles. LH stimulates the theca interna cells to synthesize androgens (testosterone and androstenedione) from acetate and cholesterol but these cells initially have limited capacity to synthesize oestrogens. Androgens from the theca interna cells diffuse to the avascular granulosa cells. The granulosa cells are unable to synthesize androgens but can aromatize androgens to oestrogens (oestradiol-17β and oestrone). The enzyme aromatase (CYP19) is involved in the steroid biosynthesis pathway leading to increased oestrogen production. FSH stimulates production of insulin-like growth factor I (IGF-I), which stimulates aromatase activity and hence oestrogen production. Activins and oestradiol enhance the actions of FSH (Richards and Pangas, 2010). Small amounts of LH are required to amplify follicular oestrogen production. The steroids are secreted into the bloodstream, where they have a systemic effect, and into the follicular fluid, where they may have a paracrine role. Androgens also stimulate aromatase. Oestrogens stimulate granulosa cells to proliferate and express further oestrogen receptors. Therefore, oestrogen further stimulates oestrogen output, an example of positive feedback. This increases the amount of circulating oestrogen from the most advanced or ‘dominant’ follicle (therefore, monitoring oestrogen output is a guide to the maturity of the most mature follicles). The dominant follicle, which undergoes the most growth, will enlarge from 20 to 200–400 μm diameter. The dominant follicle produces oestradiol, which inhibits FSH secretion. However, the dominant follicle develops exquisite sensitivity to FSH and can continue to respond to the decreasing concentration of FSH. The smaller follicles, destined to become atretic, lose their responsiveness to FSH and do not develop LH receptors (Scheele and Schoemaker, 1996).

Early in the antral phase under the influence of FSH, the granulosa cells produce inhibin B and activin which suppresses androgen output and increases the aromatizing capability of the granulosa cells, thus promoting oestrogen synthesis. Later in the antral phase under the influence of both FSH and LH, the granulosa cells switch to producing inhibin A which stimulates androgen output and attenuates the aromatizing activity. Thus, androgen output is regulated by activin and inhibins and the ratio of inhibin A:inhibin B acts as a marker of follicular growth

The effect of the LH surge is twofold. First, it stimulates the terminal growth phase of the preovulatory follicle and the meiotic and cytoplasmic maturation of the oocyte, culminating in expulsion of the oocyte from the ovary. These effects include re-initiation of oocyte meiosis and expansion of the cumulus cell–oocyte complex (Richards and Pangas, 2010). Second, it causes luteinization, a series of endocrine changes within the follicular cells that result in a different hormone secretory profile in the second half of the cycle.

Within a few hours of the LH surge, there are dramatic changes in the oocyte, which resumes meiotic division. There may also be a positive signal from the granulosa cells or a reduction of gap junction communication, which decreases the flow of meiosis-arresting substances to the oocyte. Progression through the remainder of the first meiotic division results in half the chromosomes (as paired chromatids) and almost all the cytoplasm being enclosed in the secondary oocyte, which is destined to become the ovum. The remaining chromosomes and very little cytoplasm are enclosed in a membrane forming a very small cell, known as the first polar body (see Fig. 4.3). Thus, the secondary oocyte keeps the bulk of the materials that were synthesized earlier in follicular development; these are conserved for the zygote. The chromosomes of the secondary oocyte enter the second meiotic division and go on to the next stage of division, called metaphase (see Fig. 7.11), where they align on the spindle. However, meiosis is then immediately arrested for a second time; this is regulated by CSFs. Meiosis resulting in the production of a mature female pronucleus will not resume until successful fertilization following ovulation. By this time, the oocyte will already contain the sperm nucleus. Thus, there is actually no time when the oocyte is a true gamete in the sense of being a cell with only 23 chromosomes, as is the case of a spermatozoon.

Concurrently, the LH surge promotes maturation of the cytoplasmic compartment of the oocyte. The cytoplasmic processes between the oocyte and the granulosa cells withdraw and contact is lost. The Golgi apparatus (see Table 1.1) synthesizes lysosome-like cortical granules, which align under the surface of the oocyte. Protein synthesis continues but the profile of the proteins synthesized changes as the oocyte prepares for fertilization. The gonadotrophin surge stimulates the cumulus cells surrounding the oocyte (see Fig. 4.4) to secrete hyaluronic acid, which disperses the cumulus cells embedding them in a mucus-like matrix.

Ovulation is triggered by the mid-cycle surge of LH, which occurs in response to sustained high levels of oestrogen released from the developing dominant follicle. The single mature preovulatory follicle has a diameter of 2–2.5 cm in an ovary that is approximately 3 cm long. It was this structure that de Graaf identified and named in 1672. The increased size and changed position of the follicle mean that it protrudes from the surface of the ovary (see Fig. 4.2). This results in the thinning of the layer of epithelial cells between the wall of the follicle and the peritoneal cavity. As expansion continues, the wall becomes thinner and avascular, and the cells appear to dissociate.

About 36 h after the LH surge, ovulation occurs and the oocyte is expelled from the ovary (see Fig. 4.2). The LH surge stimulates the production of a cascade of proteolytic enzymes, including renin and other trypsin-like enzymes from thecal cells, which digest the follicle wall. The biochemical changes, including generation of oxygen free radicals, that precede ovulation are similar to those seen in inflammation. Plasminogen activator, which converts procollagenase to collagenase, is produced by granulosa cells resulting in the breakdown of the connective tissue. Progesterone production rises immediately after the LH surge and the preovulatory increase in progesterone may be important in follicular rupture as it decreases formation of collagen. Prostaglandins increase vascular permeability, which maintains the intrafollicular pressure as fluid begins to leak through the eroded follicular wall. Small contractile waves also ripple across the ovary increasing the intrafollicular pressure. The force is cushioned by the follicular fluid, so the pressure generated is targeted at the weakened ovarian surface, causing it to rupture. Some women experience abdominal pain around the time of ovulation on the same side of the ovary producing the ovum. This is referred to as Mittelschmerz pain (derived from German ‘middle pain’). As the follicle ruptures and the ovarian surface is breached, the fluid washes out the oocyte, which is surrounded by the granulosa (cumulus) cells, from the ovary to the exterior. The oocyte is swept into the uterine tube by the fimbria. It is then propelled towards the uterus by peristaltic muscular activity and cilia movements of the epithelial cells lining the tube. After taking years (12–50 years) to complete maturation, the oocyte is then viable and fertilizable for only about a day.

After ovulation, the residual parts of the follicle remaining in the ovary collapse into the space and form the corpus luteum (‘yellow body’; see Fig. 4.4). There is some bleeding and fibrotic activity in the cavity, which allows formation of a fibrin core around which the remaining granulosa cells congregate. The structure is enclosed by a capsule of fibrous thecal cells. The basement membrane between the granulosa cells and thecal cells breaks down allowing vascularization of the interior. This allows increased transport of cholesterol precursor to the luteinizing granulosa cells to maintain a high rate of progesterone secretion. A few of the thecal cells disperse to the stroma tissue. The granulosa cells first luteinize, then stop dividing and hypertrophy into large luteal cells. The luteal cells are rich in mitochondria, endoplasmic reticulum and Golgi bodies and have numerous lipid droplets and lutein, a yellow carotenoid pigment.

Luteinization is associated with a progressive increment in progesterone secretion from the corpus luteum. The outer thecal cells form a stem cell population of smaller luteal cells which have numerous LH receptors and produce progesterone and androgens. Levels of progesterone rise until the middle of the luteal phase (see Fig. 4.6). The corpus luteum produces oestrogen and inhibin as well as progesterone. All three hormones inhibit secretion of FSH from the anterior pituitary gland and therefore prevent further development of follicles.

It has been suggested that a cause of fertility problems may be inadequate production of progesterone at the time of ovulation and during the subsequent luteal phase. However, exogenous administration of progesterone or human gonadotrophin (hCG) has had limited success in clinical practice. It appears that most women have a proportion of their cycles with a low progesterone output without their overall fertility being affected (Hamilton-Fairley and Johnson, 1998). A shortened luteal phase can lead to intermenstrual bleeding, premenstrual ‘spotting’ and short cycles.

The corpus luteum also synthesizes relaxin, secretion of which peaks in the middle of the luteal cycle (Johnson et al., 1993), probably regulated by LH. Relaxin may be involved in promoting the growth of the myometrium and cervix and growth and secretory activity of the endometrium (Huang et al., 1991).

Effectively, the corpus luteum is an endocrine gland producing oestrogen and progesterone. The LH surge stimulates its growth and activity. Unless fertilization occurs, the life of the corpus luteum is very short, and it undergoes spontaneous luteolysis (degeneration and regression) after about 6 days. The corpus luteum appears to have an age-related decrease in responsiveness to LH (Zeleznik and Hillier, 1996) and so requires progressively more LH for survival. Following the LH surge, LH concentration in the luteal phase is low, so luteolysis will occur. Blood flow to the corpus luteum falls and the follicular tissue becomes ischaemic. The concentrations of oestrogen and progesterone begin to fall as the degenerating corpus luteum stops hormone production. Thus, luteolysis terminates a non-fertile cycle. As the level of oestrogen falls, the inhibition on the hypothalamus will be abrogated and FSH secretion will resume, ready for the next cycle. The atrophying corpus luteum loses its yellow pigment, so it becomes known as a corpus albicans (‘white body’). It gradually contracts over a period of months, leaving a white scar tissue which is absorbed into the stromal tissue of the ovary.

If fertilization occurs, then hCG, which has structural similarities to LH, rescues the corpus luteum from luteolysis, stimulating its further growth and production of steroid hormone up to the 10th week when placental endocrine function becomes established. Human chorionic gonadotrophin has a longer half-life than LH, so it provides a sustained and more intense stimulus. If fertilization occurs, then the concentration of relaxin also continues to rise until the end of the first trimester.

Organs that respond to hormonal changes have receptors for the hormones. Responses can change because hormone levels fluctuate or because receptor density on the target organs alters. The principal actions of oestrogen and progesterone during the monthly cycle are on the endometrium which is one of the tissues most sensitive to ovarian steroid hormones. The endometrium undergoes cyclical changes: the growth of the uterine wall in expectation of an embryo, and its degeneration if fertilization does not take place. In the first half of the cycle, the uterus goes through a proliferative phase. Oestrogen stimulates the epithelial cells of the basal layer of the endometrium to divide and proliferate, forming a thick mucosal wall with numerous endometrial glands (Fig. 4.8). Oestrogen also stimulates proliferation of the stoma and glands and angiogenesis (growth of new blood vessels): extensive vascular tissue, spiral arteries and veins develop within the endometrium. Within the space of a few days, the effect of oestrogen is to increase the height of the wall from 0.5 to 5 mm, a remarkable 10-fold increase. The thickness of the endometrium is monitored by ultrasound in assisted conception (see Chapter 6) to assess whether it is optimal for implantation; the insertion of embryos into a uterine cavity with an endometrium less than 5 mm thick is unlikely to be successful. The myometrium does not grow extensively during the menstrual cycle. During the proliferative phase, oestrogen primes the endometrial cells by inducing the synthesis of progesterone receptors.