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Chapter 1 - The Ovary: A General Overview of Follicle Formation and Development

Published online by Cambridge University Press:  09 November 2022

Rachel Cutting
Affiliation:
Human Fertilisation and Embryology Authority, London
Mostafa Metwally
Affiliation:
Sheffield Teaching Hospitals and the University of Sheffield

Summary

This chapter provides a summary of human ovarian physiology. The reader is introduced to basic concepts of sex determination, in particular how primordial germ cells and somatic cells differentiate and multiply to establish the reserve of primordial follicles. Key molecular and cellular events associated with primordial and preantral stages of follicle development are described. The chapter then provides an overview of antral follicle development, which is subdivided to consider the morphological changes and the role of granulosa and theca cells in steroidogenesis, along with development of the oocyte. The changes that occur as the follicle matures towards ovulation are also considered, in particular the fundamental role of hormones, cumulus expansion and resumption of meiosis. Finally, establishment and maintenance of the corpus luteum is briefly discussed. Diagrams of steroidogenesis and an overview of follicle formation and development are provided to supplement the text.

Type
Chapter
Information
Publisher: Cambridge University Press
Print publication year: 2022

1.1 Introduction

In the context of reproduction, the key functions of the ovary are to provide an environment that supports the maturation of oocytes for ovulation alongside adequate production of sex steroids to prepare for and sustain a potential pregnancy. These functions are not mutually exclusive, as each depends on follicle and oocyte growth and maturation progressing in a coordinated and timely manner. Our current understanding of how the ovary is formed, with its limited supply of follicles, and how individual follicles develop and achieve these functions of oocyte support and steroidogenesis are summarised briefly this chapter.

1.2 Sex Determination and Early Gonadal Development

In humans, male and female embryos are morphologically similar until around week 6 of development when activation of the molecular programme from sex chromosomes allocated at fertilisation sets in motion the formation of a sex-specific gonadal phenotype. Prior to this, a small cluster of pluripotent primordial germ cells (PGCs), first identified in the posterior yolk sac endoderm, begins to proliferate and migrate along the hindgut, through the dorsal mesentery, to eventually settle on either side of the developing aorta in the emerging gonads, or ‘gonadal ridges’. This migration of PGCs, driven by chemotactic signals (e.g. KIT/KITLG), occurs alongside proliferation and inward migration of somatic cells to create a bulge on the ventromedial aspect of each mesonephros called the gonadal ridge. The gonadal ridges, covered by coelomic epithelium, develop alongside the two adjacent embryonic ducts, the Wolffian (also known as the mesonephric) and Müllerian (also known as the paramesonephric) ducts, which all together constitute a bipotential reproductive system.

Subsequent differentiation of the gonads is generally dependent on the genetic constitution of the embryo. In males, brief and timely expression of the sex-determining region on the Y chromosome (SRY gene) in somatic cells, together with other transcription factors (e.g. SF1 and WT1), drives expression of SRY-box transcription factor 9 (SOX9). This transcription factor in turn activates the expression of an array of genes that drives a pre-Sertoli cell phenotype, including cues that amplify and propagate the molecular programme along adjacent somatic and germ cells (e.g. FGF9 and PTGDS), leading to the differentiation of the fetal testis. PGCs, now called spermatogonia, undergo further proliferation before arresting in mitosis. The production of testosterone from newly differentiated Leydig cell precursors stimulates the Wolffian ducts to develop into components of the male reproductive tract – the vas deferens, epididymis and seminal vesicles – while the production of anti-Müllerian hormone (AMH), driven by SOX9 in pre-Sertoli cells, causes regression of the Müllerian ducts.

Female XX embryos lack SRY, and therefore SOX9 expression remains low in somatic cells within the developing gonad. These cells, thought to be derived from the mesenchyme or coelomic epithelium overlying the gonadal ridge, additionally express factors that actively repress SOX9 and its targets. Such factors, including RSPO1, WNT4, CTNNB, FST and FOXL2, are necessary to promote and maintain the differentiation of these cells into ‘pre-granulosa’ cells. Adequate expression of these proteins is fundamentally important for maintaining the phenotype of this lineage, even into adulthood, with experimental examples of mutations shown to precipitate a partial gonadal sex reversal or the development of an ovotestis phenotype. Secreted signals from the pre-granulosa cells are important for (1) stimulating PGCs (now called oogonia) to arrest in meiosis (see Section 1.3), and (2) promoting differentiation of nearby somatic cells, from an alternative lineage, into ‘pre-theca’ cells. Thus, the pre-granulosa cells initiate a series of developmental events to define the tissue that will eventually become the ovary. In comparison with the fetal testis, the relatively low levels of testosterone and AMH cause the Wolffian ducts to regress and the Müllerian ducts to persist, the latter of which will form the precursor structures to the oviduct, uterus and upper vagina in the female [Reference She and Yang1].

1.3 Follicle Formation: Establishment of the Ovarian Reserve

As the fetal ovary is being established, the germ cells – now called oogonia – continue to proliferate by mitosis to form dense oogonial cell clusters; however, cell division is incomplete, and many of the oogonia remain connected to each other with a shared cytoplasm. Retinoic acid, which is produced by the somatic cells of the ovary and the adjacent mesonephros, binds to stimulated by retinoic acid 8 (STRA8) receptors on oogonia to initiate the process of meiosis, while at the same time inhibiting further proliferation of these cells. Oogonia proceed relatively slowly through the early stages of meiosis to arrest in the diplotene stage of prophase I, at which point they are called oocytes. In humans, arrested oocytes are identifiable in fetal ovaries between 10 and 24 weeks of gestation. It is during this time that primordial follicle formation occurs, a process whereby pre-granulosa cells invade and interpose between the germ cells in their clusters to envelop individual oocytes. Germ cells that fail to enter meiosis or become completely encapsulated in pre-granulosa cells degenerate by apoptosis [Reference Wang, Zhou and Xia2].

It is generally believed that the net effect of these mitotic and apoptotic processes throughout this period leads to the final establishment of the ovarian reserve. Based on a relatively small number of histological studies, the average number of follicles in the human ovarian reserve (i.e. in both ovaries) is estimated to be 500,000–1,000,000 at the time of birth [Reference Wallace and Kelsey3, Reference Hansen, Knowlton and Thyer4]. Evidence for de novo synthesis of oocytes after this time is still a matter of debate, although recent studies have identified a small population of ‘oogonial stem cells’ that persist into adulthood, and these can be isolated and differentiated into oocyte-like cells in vitro [Reference White, Woods and Takai5]. More recent studies in mice have shown that adult somatic cells of non-ovarian lineages can be ‘reprogrammed’ in vitro to generate new oocytes capable of fertilisation and generation of offspring [Reference Tian, Liu and Ye6, Reference Hikabe, Hamazaki and Nagamatsu7]. Thus, research efforts are being directed towards developing novel fertility preservation strategies through artificial amplification of the ovarian reserve, although the medical potential of these findings is still far from being realised.

1.4 Follicle Development

1.4.1 Primordial Follicles

Primordial follicles, situated in the cortex of the ovary, constitute the most numerous population of follicles at any one time. Each primordial follicle consists of a small, primary oocyte (~20 μm in diameter) arrested in prophase I of meiosis, surrounded by a layer of squamous pre-granulosa cells and enveloped by a basement membrane. Within the oocyte, the nucleus, also known as the germinal vesicle, contains chromosomes in the dictyate state, a configuration conducive for active gene transcription. Both the oocyte and pre-granulosa cells are tightly connected by junctional complexes to allow for bidirectional communication and preservation of a viable, yet relatively quiescent phenotype. Thus, although primordial follicles are often referred to as ‘dormant’, it is important to note that they are still intrinsically active, with the oocyte and surrounding pre-granulosa cells undergoing normal cellular metabolism and homeostasis.

From the time the reserve is established within the fetal ovary, the number of primordial follicles progressively decreases; in other words, the ovarian reserve begins a steady trajectory of decline. For most females, the main reason why the ovary eventually runs out of follicles is because they are continuously recruited to grow. In adults, this continuous activation eventually leads to the menopause, a natural event that occurs in women at an average age of 51 years, when fewer than 1,000 viable primordial follicles remain. Interestingly, it has been proposed that the initial size of the ovarian reserve formed during fetal development is a predictor of the timing of menopause, as there is a strong association between the rate of follicle loss and the advancement of chronological age [Reference Wallace and Kelsey3]. Genetic variation, which can influence the initial size of the ovarian reserve but also the rate of primordial follicle activation, can also impact the timing of menopause, which, if it occurs before the age of 40, is termed premature ovarian insufficiency. Regardless of genetic influence, there is a continuous departure of primordial follicles from the ovarian reserve as they activate and enter a trajectory of irreversible growth. The remaining non-growing primordial follicles may be retained in a relatively quiescent but potentially vulnerable state for up to 40–50 years before being activated. However, this protracted period of suspended animation makes them particularly susceptible to chronic and acute exposures to environmental toxicants. Products of cigarette smoking, diet and alcohol consumption are all lifestyle factors reported to affect the viability or rate of decline of the ovarian reserve. Therefore, it is not only the quantity of primordial follicles that becomes diminished with age but also the quality. This is important in the context of female fertility, especially now that the age of first-time parents has steadily increased over the past 40 years [Reference Nelson, Telfer and Anderson8].

The question of why some primordial follicles are activated to grow while others stay arrested is a major area of interest in reproductive science and medicine. Numerous molecular signals have been implicated in a range of models; however, an intricate balance of stimulatory and inhibitory factors (e.g. KL, BMP4, BMP7, bFGF, LIF and KGF) along with spatial access to these factors are likely to be important. Transgenic mouse models have established that adequate expression of key transcription factors (e.g. Nobox, Sohlh1, Sohlh2, Foxo3a and Lhx8) are essential for oocyte activation. Studies have also found that AMH, produced by developing preantral follicles, exerts an inhibitory influence on the primordial pool, while factors that activate the PI3K/AKT/mTOR signalling pathways in pre-granulosa cells and oocytes have a stimulatory effect on growth. Identifying how these pathways are regulated in this context is now the subject of many research groups. In most mammalian species, an increase in the rate of pre-/granulosa cell division, accompanied by morphological changes in shape of these cells from a squamous to a cuboidal form, as well as a relatively abrupt increase in oocyte growth, are all morphological features characteristic of follicle activation [Reference Kallen, Polotsky and Johnson9, Reference Ford, Beckett, Roman, McLaughlin and Sutherland10].

1.4.2 Preantral Follicles

Follicles committed to activate and grow eventually establish a tightly packed single layer of cuboidal granulosa cells. Once this layer is complete, these primary-stage follicles require adequate expression of oocyte-derived factors for further development, specifically growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), members of the transforming growth factor β (TGFβ) family. These molecular signals act on surrounding somatic cells, which in turn signal back to the oocyte – possibly by KIT/KITL – to ensure both cell types develop in synchrony. The oocyte also develops a glycoprotein-rich zona pellucida (ZP) coat, which remains throughout the life of the oocyte. The ZP is important for fertilisation and pre-implantation development and is only shed just prior to implantation. Despite the presence of this relatively thick barrier, granulosa cells develop long thread-like processes, called transzonal projections, which extend through the ZP to the surface of the oocyte where they connect to gap junctions. The existence of gap junctions between the oocyte and granulosa cells, and also between adjacent granulosa cells, is vital for allowing bidirectional molecular communication.

It is also during the primary stage when stromal stem cells begin to differentiate into a layer of theca cells and associate with the basement membrane. As preantral follicle development progresses, granulosa and theca cells continue to proliferate under the influence of local growth factors – principally originating from the oocyte (e.g. GDF9, BMP15) but also from the surrounding cells and tissues. Several signalling pathways play key roles at this stage, including the TGFβ (e.g. activin), insulin-like growth factor (IGF) and epidermal growth factor (EGF) pathways; however, many others are also implicated. The combination of these mitogenic signals causes multilayering of the somatic cells and further growth of the oocyte, leading to overall expansion of the follicle into a secondary-stage or multilayered preantral follicle [Reference Edson, Nagaraja and Matzuk11].

Importantly, these early stages of preantral follicle development occur independently of gonadotrophins and steroid hormones. As such, early follicle development occurs throughout pre-pubertal life – even in the fetal ovary – although fetal preantral/small antral follicles will never develop much further due to insufficient levels of follicle-stimulating hormone (FSH). In the post-pubertal ovary, FSH from the pituitary binds to functional FSH receptors expressed on granulosa cells of preantral follicles. FSH augments the actions of local growth factors to mainly stimulate the rate of follicle growth by promoting cell proliferation and, with further development, stimulates the differentiation of follicle cells to become steroidogenic (see Section 1.4.3.2). Most of the large preantral/small antral follicles that develop when FSH levels are low –for example, before puberty (Figure 1.1), or during the luteal phase of the menstrual cycle – will perish, a process known as atresia. However, in the post-pubertal ovary, if the timing is right, around 7–10 follicles per ovary per cycle will be selected for further development to the antral stage.

Figure 1.1 The human neonatal ovary. Numerous primordial follicles are embedded in the stroma, and a small antral follicle measuring approximately 400 μm in diameter is evident (centre). Although the antral follicle appears well developed, it is destined to undergo atresia due to insufficient levels of gonadotrophins in early life. Scale bar = 200 μm.

Image kindly provided by Dr Suzannah Williams and Briet Bjarkadottir (University of Oxford).

1.4.3 Antral Follicles

In the human ovary, the developmental journey from a small primordial (0.03 mm/30 μm diameter) to a large preantral follicle (0.2 mm/200 μm) occurs over several months. By comparison, antral follicle development (>200 μm) occurs rapidly over a 6-week time frame, with significant expansion (from 2 to 25 mm) during the final 2 weeks corresponding to the follicular phase of the menstrual cycle. During antral follicle growth, a series of developmental events ensures that the ovary communicates effectively with the hypothalamus and pituitary to set in motion a feedback mechanism with the gonad that regulates the production of sex steroids to prepare the reproductive tract and coordinate this with ovulation. The following sections provide a brief summary of the developmental changes that occur in the oocyte and somatic compartments as gonadotrophin-dependent follicles develop towards the ovulatory stage.

1.4.3.1 Morphological Changes

As large preantral follicles become increasingly responsive to gonadotrophins, fluid rich in proteins, mostly derived from granulosa cell secretions and serum transudate, begins to appear. These discrete pockets of fluid eventually coalesce into a single, large antrum, which allows unimpeded diffusion of growth factors and molecules. This is important because, while the theca layer is highly vascularised, blood vessels never penetrate the basement membrane of the follicle until ovulation occurs; therefore, granulosa cells and the oocyte are sustained in a nutrient-rich environment despite their relative distance from the vasculature. Granulosa and theca cell proliferation occurs at an increased rate due to the powerful actions of steroid and peptide hormones and local growth factors, although the rapid expansion of the antral follicle throughout the follicular phase of the menstrual cycle occurs mostly as a consequence of the increased antral volume. Within the antral follicle, the granulosa cells differentiate into two specialised types: (1) the cumulus cells surrounding and in direct contact with the fully grown oocyte, and (2) the mural granulosa cells at the periphery of the follicle. Likewise, the theca cells differentiate into two specialised layers: (1) the vascularised theca interna, which is in direct contact with the basement membrane, and (2) the more peripheral theca externa, containing fibroblasts and smooth muscle-like cells, providing structural integrity to the expanding follicle.

1.4.3.2 Steroidogenesis and Atresia

The mural granulosa cells, along with the theca interna cells, are dependent on gonadotrophins for survival and steroidogenesis. The ‘two-cell, two-gonadotrophin hypothesis’ proposed in the 1970s refers to luteinising hormone (LH) binding and activating the LH receptors (LHRs) expressed on theca interna cells, stimulating production of androgen, while FSH signals via the FSH receptors (FSHRs) on mural granulosa cells to convert androgen to oestrogen [Reference Hillier, Whitelaw and Smyth12]. FSHRs and LHRs are G protein-coupled cell-surface receptors that activate second messengers – cyclic adenosine monophosphate (cAMP), which drives protein kinase A (PKA), as well as other kinase pathways – which each signal to the nucleus to drive transcription of target genes. Such targets are varied but include inhibitors of apoptosis, such as the BCL-2 family, which are important for follicle maintenance and survival. Inadequate gonadotrophin signalling in the antral follicle leads to somatic cell death and loss of basement membrane integrity, allowing leukocyte infiltration, collapse of the antrum and eventual oocyte demise. This degenerative process – otherwise known as atresia – is a discrete mechanism resulting in rapid clearance of follicles that are no longer destined for further development. Although atresia can occur at any stage of follicle development, it is predominantly a feature of antral follicles and is more easily recognisable microscopically in larger follicles due the relatively high number of cells. It is estimated that, in humans, atresia accounts for the loss of over 99.9% of the follicles from the original reserve at birth. Therefore, in order for antral follicles to survive, they need to express functional gonadotrophin receptors, particularly for FSH, during the early stages, and LH in the later stages to coincide with the cyclical rise in systemic gonadotrophins.

Of the small proportion of follicles that remain responsive to adequate levels of gonadotrophins (i.e. during the follicular phase of the menstrual cycle), cAMP/PKA signalling also drives the expression of key enzymes involved in growth and steroidogenesis. In theca cells, key steroidogenic enzymes include cholesterol side change cleavage/cytochrome P450 (encoded by the CYP11A1 gene), which converts cholesterol, derived from the circulation, to pregnenolone within the mitochondria (Figure 1.2). 3-β-Hydroxysteroid dehydrogenase (HSD3B1) then converts pregnenolone to progesterone. Under the action of 17α-hydroxylase/17,20-lyase enzyme (CYP17A1), both pregnenolone and progesterone can be modified to the 17α-hydroxy forms, which are the precursor substrates for the androgens. These are the first rate-limiting steps involved in steroidogenesis.

Figure 1.2 Steroid synthesis in antral follicles. During antral follicle development, luteinising hormone (LH) binds to the LH receptor on theca cells, which regulates the expression and activity of key steroidogenic enzymes, allowing the modification of cholesterol into androgens. Testosterone and androstenedione diffuse across the basement membrane where they are converted to oestrogens through the activity of aromatase (CYP19A1), which is regulated by follicle-stimulating hormone (FSH) signalling in granulosa cells. In pre-ovulatory follicles, LH receptor is also expressed in granulosa cells to further amplify progesterone synthesis. Note: steroidogenic enzymes are presented as protein symbols (refer to main text for protein name) with corresponding coloured arrows indicating enzyme activity.

Progestagens, often referred as the grandparental steroids, are converted to the androgens dehydroepiandrosterone (DHEA) and androstenedione in the presence of CYP17A1. Importantly, 17β-hydroxysteroid dehydrogenase 1 (HSD17B1) converts these androgens into androstenediol and testosterone, respectively. Dihydrotestosterone (DHT), a potent, non-aromatisable androgen, is also produced from the activity of 5α-reductase 1 (SRD5A1) on testosterone. Additionally, both DHEA and androstenediol can be further converted by HSD3B1 to androstenedione and testosterone, respectively, both of which are the precursor substrates for the oestrogens. These substrates, being lipophilic steroids, are able to readily diffuse through the basement membrane into the adjacent granulosa cells.

Although theca cells are capable of producing small amounts of oestrogens, granulosa cells fundamentally lack the enzyme (i.e. CYP17A1) required to produce the androgenic substrates. Instead, FSH/FSHR signalling in granulosa cells drives aromatase (CYP19A1) activity, which enables conversion of the theca-derived, aromatisable steroids androstenedione and testosterone to oestrogens. Here, oestradiol-17β is derived from aromatisation of testosterone, or through conversion of oestrone by HSD17B1. Oestradiol is the major form of oestrogen secreted back across the basement membrane into the theca interna where it enters the circulation through the large blood vessels [Reference Duffy, Ko, Jo, Brannstrom and Curry13, Reference Palermo14].

Although steroid hormones are fundamentally important for endocrine signalling throughout the body, both steroid and peptide hormones have important roles in further enhancing antral follicle development. Androgens from the theca, along with FSH, as well as oestrogens from the granulosa cells, all stimulate the further proliferation of granulosa cells. Androgens also promote aromatase activity, together creating a positive-feedback mechanism to significantly increase oestrogen output during the follicular phase of the cycle [Reference Sánchez and Smitz15]. Other growth factors, such as IGF-1 and other TGFβ members (e.g. activins, inhibins and follistatin), are produced in response to gonadotrophin signalling and are also implicated in increasing the rate of granulosa and theca cell proliferation. FSH also causes elevated expression of inhibin B (inhibin α and βB heterodimer), while both FSH and LH promote the expression of inhibin A (inhibin α and βA heterodimer); thus, as follicle development proceeds, the elevated ratio of inhibin A:inhibin B along with rising oestrogen in the blood can be used as markers of follicle expansion. Inhibins and oestrogens are also important for supressing FSH, a feedback mechanism that causes a shift in hormonal support during the latter half of the follicular phase. In the human menstrual cycle, this results in regression of subsidiary follicles, allowing a single follicle to go on to ovulation (see Section 1.4.4).

1.4.3.3 Oocyte Development

Although the oocyte is arrested in the first prophase of meiosis throughout preantral and antral follicle development, it is far from inactive. Oocyte growth continues throughout the preantral stage to reach a maximum size of around 120 μm in small antral follicles. During this time, the oocyte synthesises and accumulates a vast amount of RNA – approximately 200× more relative to a somatic cell. Much of the mRNA is actively transcribed and polyadenylated for translation in a stage-dependent manner as the oocyte develops and becomes competent to resume meiosis; however, many mRNAs are also transcribed and deadenylated for degradation or storage towards the end of follicle development, a process indispensable for development into a viable embryo. During antral follicle development, the oocyte also begins to synthesise the machinery required for further meiotic progression. Maturation-promoting factor (MPF), a protein complex involving cyclin-dependent kinase (CDK) and cyclin B, is produced to enable meiotic progression but is held in abeyance by high levels of cAMP. The relationship between the oocyte and the adjacent granulosa cells is crucial for the maintenance of cAMP. This is because granulosa cells provide a source of cyclic guanosine monophosphate (cGMP) via gap junctions to inhibit the activity of phosphodiesterase (PDE3A), an enzyme that acts to degrade cAMP (see Section 1.4.4.2) [Reference Jaffe and Egbert16].

1.4.4 The Ovulatory Follicle

In mono-ovulatory species such as humans, usually only one antral follicle will continue to develop to the pre-ovulatory stage. Although the mechanism for this selection is not yet fully understood, it is thought that follicles that produce comparatively high levels of IGF and the receptor IGFR1, in response to both FSH activity and oocyte signals, exhibit augmented steroidogenesis, which in turn supports a rapid advancement of follicle growth. The elevated levels of oestrogen produced by the pre-ovulatory follicle eventually promote LHR expression in the mural granulosa cells. Oestrogens entering the circulation also alter the activity of gonadotropin-releasing hormone-expressing neurons in the hypothalamus, resulting in a surge of LH secretion from the pituitary. These acutely high levels of circulating LH bind to LHR in the pre-ovulatory follicle to initiate a host of events in the lead up to ovulation 24–36 hours later. The following sections summarise the key changes that occur in the follicle and oocyte during that time.

1.4.4.1 Cumulus Expansion

Throughout antral follicle development, cumulus cells surrounding the oocyte are fairly closely packed, forming a unit known as the cumulus–oocyte complex (COC). Within a few hours of the LH surge, the cumulus cells surrounding the oocyte loosen and the COC undergoes rapid expansion. This process is precipitated by the elevation in LH signalling in mural granulosa cells, which drives expression of EGF-like factors (amphiregulin, epiregulin), which are secreted into the antrum to bind EGF receptors located on cumulus cells. These EGF-like signals, along with growth factors secreted from the oocyte (e.g. GDF9, BMP15 and others) provide a powerful cocktail of stimulants that promotes the upregulation of genes necessary for rapid proliferation and differentiation (e.g. HAS2, PTGS2, PTX3 and TNFAIP6) of cumulus cells, which, in turn, secrete a gel-like matrix of hyaluronan and stabilisation factors causing expansion. This expanded cumulus is important for nourishment of the oocyte and the embryo during the peri-conception period, and also aids in oocyte pick-up by the Fallopian tube [Reference Sánchez and Smitz15, Reference Robker, Hennebold and Russell17].

1.4.4.2 Resumption of Meiosis

As the cumulus expands, the gap junctions/transzonal projections between the oocyte and surrounding cells close. This loss of cGMP source from the granulosa cells allows PDE3A activation and cAMP degradation in the oocyte. The consequential reduction in cAMP causes dephosphorylation of CDK1 and activation of MPF to enable meiosis to resume. Microscopically, the changes can be visualised by the breakdown of the germinal vesicle envelope (also known as germinal vesicle breakdown), formation and assembly of the meiotic spindle (enabling chromosomal segregation), formation of cortical granules towards the periphery of the oocyte and the subsequent extrusion of the first polar body following completion of the first meiotic division. A cytostatic factor then blocks MPF causing the oocyte to arrest again, this time in metaphase II. New proteins are then synthesised to prepare for the completion of meiosis to produce a haploid gamete for fertilisation [Reference Sánchez and Smitz15, Reference Jaffe and Egbert16].

1.4.4.3 Ovulation

In the mural granulosa cells, as well as production of EGF-like factors, LHR signalling drives expression of steroidogenic enzymes (CYP11A1, HSD3B1) to enable the production of progesterone. The mural granulosa cells lose the ability to respond to oestrogens and instead the LH and progesterone stimulate mitogenesis and further progesterone synthesis. Although the elevation in progesterone is essential for ovulation to occur, it is also believed to depress the growth of less mature follicles, thus enabling dominance of a pre-ovulatory follicle. The actions of progesterone lead to considerable expansion of the pre-ovulatory follicle and cause it to bulge from the ovarian surface with a relatively thin layer of granulosa and theca cells. This thin layer, called the stigma, is avascular and is subject to the progesterone-stimulated activities of proteolytic enzymes such as matrix metalloproteinases (MMPs), tissue inhibitors of MMPs, plasminogen activator and cyclo-oxygenase/prostaglandins. The internal pressure of the follicle along with the contractile activity of the theca externa results in follicle rupture, leading to expulsion of follicular fluid and the COC around 24–36 hours after the LH surge [Reference Duffy, Ko, Jo, Brannstrom and Curry13].

1.5 Corpus Luteum

Following ovulation, the basement membrane of the follicle collapses and is breached by invading blood vessels. The loss of oocyte-derived factors along with the LH-induced promotion of steroidogenesis causes the remaining granulosa and theca cells to differentiate into large and small lutein cells, respectively. Both cells continue to produce progesterone, but androgens, oestrogens, oxytocin and inhibin A are also produced by the corpus luteum. The initial action of LH along with the paracrine activity of progesterone provides luteotrophic support to allow the structure to persist until pregnancy occurs, at which point human chorionic gondadotrophin from the developing conceptus replaces LH. If no pregnancy occurs, the loss of peptide hormone signalling and the progressive reduction in progesterone is associated with luteal regression towards the end of the menstrual cycle, with reabsorption of the corpus luteum [Reference Stouffer, Bishop, Bogan, Xu and Hennebold18].

1.6 Summary

The ovary is an incredibly dynamic organ: from the time it is formed in the fetus and right throughout adult reproductive life, there is constant remodelling (Figure 1.3). The activity of specific transcription factors drives the differentiation of somatic and germ cells, which become arranged into the basic germ cell units – the primordial follicles. From this precious reserve, a proportion of follicles are constantly being activated to grow, a process driven by local cues and involving timely activity of cell-specific signals. Further development throughout the preantral stages depends on synchronous growth of the oocyte and somatic cells, which progressively become responsive and eventually dependent on the timely rise in gonadotrophins from the pituitary. Throughout the process, the developmental and maturational changes in the oocyte, granulosa and theca cells are critical determinants in ensuring the production of sex steroids and eventually the release of a mature metaphase II oocyte at the time of ovulation. The surrounding cells and structures, which have not been discussed here but include the vasculature, lymphatics and nerves, all set within stroma and encased by a surface epithelium, must also cope with the ongoing demands of internal restructuring. The complexity of these interactions in time and space is fundamental for ovarian function and female fertility.

Figure 1.3 Summary of germ cell development from follicle formation to ovulation. Key stage-specific events are indicated in the text boxes. CL, corpus luteum; FSH, follicle-stimulating hormone; LH, luteinising hormone; MII, metaphase II.

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Figure 0

Figure 1.1 The human neonatal ovary. Numerous primordial follicles are embedded in the stroma, and a small antral follicle measuring approximately 400 μm in diameter is evident (centre). Although the antral follicle appears well developed, it is destined to undergo atresia due to insufficient levels of gonadotrophins in early life. Scale bar = 200 μm.

Image kindly provided by Dr Suzannah Williams and Briet Bjarkadottir (University of Oxford).
Figure 1

Figure 1.2 Steroid synthesis in antral follicles. During antral follicle development, luteinising hormone (LH) binds to the LH receptor on theca cells, which regulates the expression and activity of key steroidogenic enzymes, allowing the modification of cholesterol into androgens. Testosterone and androstenedione diffuse across the basement membrane where they are converted to oestrogens through the activity of aromatase (CYP19A1), which is regulated by follicle-stimulating hormone (FSH) signalling in granulosa cells. In pre-ovulatory follicles, LH receptor is also expressed in granulosa cells to further amplify progesterone synthesis. Note: steroidogenic enzymes are presented as protein symbols (refer to main text for protein name) with corresponding coloured arrows indicating enzyme activity.

Figure 2

Figure 1.3 Summary of germ cell development from follicle formation to ovulation. Key stage-specific events are indicated in the text boxes. CL, corpus luteum; FSH, follicle-stimulating hormone; LH, luteinising hormone; MII, metaphase II.

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