The period encompassing the weeks before a birth, the birth, and the few weeks thereafter is called:

Establishment of Primordial Follicle Reserve

At the early stages of ovarian development, oogonia are highly proliferative that results in a peak in germ cell numbers with 6,800,000 between gestational weeks 16–20 in human [84], 2,700,000 at 110 dpc in cow [37], 850,000 at 75 dpc in sheep [36], approximately 15,000 at E15–20 in mice [41], in the fifth gestational month in rhesus monkey [31], and at E18.5 in rat [84]. These numbers then reduce drastically because of declining germ cell proliferation rates and increasing cell death. The total number of germ cells decreases by 80%–90% between 5 months and birth in human [84], 75–100 dpc in sheep [36], and 130–170 dpc in cattle [37], and by 60% between E18.5 and PD2 in rat [84]. It has been shown for rat fetal and postnatal ovaries and human fetal ovaries that there are three waves of germ cell degeneration [84]. The first wave includes oogonia undergoing mitosis (shortly before meiotic entry), the second waves affects oocytes at pachytene stage, and the third concerns oocytes at diplotene stage [84]. Two waves of germ cell death have been reported in mice. The first occurs during the period of meiosis at E13.5–15.5 and the second wave during follicle formation at E17.5–PD 1 [85]. The main mechanism for germ cell death is apoptosis mediated by gene products of the BCL2 family members (reviewed in Ref. [54]). Recent studies in mice suggest also an involvement of autophagy in germ cell death as a response to nutritional stress around birth. In addition, some oocytes on the surface of the ovary are lost by germ cell extrusion [86]. Reasons for the elimination of germ cells are chromosomal abnormalities (failure of mitosis and meiosis), defective mitochondrial genomes, insufficient pregranulosa cells, and degeneration of oocytes during restructuring of ovigerous cords and cysts into follicles (for review see Ref. [34, 41]).

17.3.8 Stem Cells

Zygote that is formed with the fertilization of an oogonium by sperm is the first form of the organism. While, on the one hand, it ensures the numerical increase with the cell divisions which the zygote passes through, on the other hand, these cells differentiate bring about specialized cell types. Nevertheless, need of the cells to reproduce and differentiate lasts not only just during the embryonic development but for a lifetime of an adult organism. With the aim of being able to continue its vitality, the organism constantly replaces new ones instead of the cells that are lost for different reasons such as cellular aging, damage, or injury. Stem cells can be defined as the cells that embody the characteristics of renewing itself and differentiation and that can be colonized. Here with the characteristic of self-renewal, it is stated that at least one of the cells that are brought into being after the stem cell has been divided carry the characteristics of the stem cell. Self-renewal feature explains both as a result of mitosis the ability to reproduce in a certain number and the ability to protect stem cell pool with asymmetrical division. Differentiation is the ability to form different cell types of the stem cells and is defined with the concept of potency. For example, except placental structures, embryonic stem cells (Fig. 17.2C) can differentiate to all the cells that form the organism and therefore are called as pluripotent. Stem cells that are settled in the organs and differentiate to only one or two cell types are named as unipotent and multipotent stem cells, respectively. Colony features are defined as the capacity of stem cells to form new stem cells. Features that can form a colony are the cells that can be cultured successfully (Lanza et al., 2009; Yilmaz et al., 2016).

In mammals, the fertilized oocyte, zygote, 2-cell, 4-cell, 8-cell, and morula resulting from cleavage of the early embryo are examples of totipotent cells (ability to form a complete organism). The inner cell mass (ICM) of the 5- to 6-day-old human blastocyst (Fig. 17.2D) is the source of pluripotent human embryonic stem cells. During embryonic development, the ICM develops into two distinct cell layers, the epiblast and the hypoblast. The hypoblast forms the yolk sac that later becomes redundant in the human, and the epiblast differentiates into the three primordial germ layers (ectoderm, mesoderm, and endoderm). Pluripotent embryonic stem cells can give rise to many cell types in vitro, including cells specific to endodermal tissues. Advances in the understanding as to how ES cells differentiate should provide answers for reprogramming of stem cells from adult tissues.

Classification of stem cells according to different features is possible. But none of these can provide a complete classification. On the other hand, generally, it is an appropriate approach to classify stem cells according to the source they are taken as being embryo-derived stem cells and adult stem cells and according to the potency features as pluripotent, multipotent, and unipotent. Just as embryo-derived stem cells can be pluripotent stem cells that are derived from ICM of blastocyst (Fig. 17.2D) or epiblast, they can be multipotent stem cells that are trophoblast or neural crest sourced. While recently the only known source of pluripotent stem cells is trophoblast, ICM and somatic cells that have been fully differentiated recently have been reprogrammed with the influence of some of the transcription factors and produced pluripotent cells called as induced pluripotent stem cells. Pluripotent stem cells have great importance in terms of regenerative medicine with high differentiation capacity they have. Stem cells that are in different organ and tissues of an organism that has completed its development at a certain level are named as adult stem cells, and mostly these cells have multipotent or unipotent character with lessened potency. Among these come especially mesenchymal stem cells. Nearly all tissues such as epithelial tissue, digestive system, musculoskeletal system, nerve system, reproductive system, and heart and vascular system have stem cells liable to ensure their renewal. Whereas the feature of a cell’s high potency also brings the possibility of transforming into a cancer cell, mesenchymal stem cells do not carry this risk (Bongso and Lee, 2011; Przyborski, 2016).

Nowadays the most frequently used cells in the culture of the specialized cells are the stem cells. Stem cell having very special settlements in the organism reveals the necessity of these cells’ culture conditions being very special as well because stem cells are found inside of a rather special microenvironment called niche mostly in a silent manner and the signals they receive from here are quite important in terms of determining the fate of the cells. Getting out of the silent state with signals that come from the microenvironment, a stem cell can go to the cell division again; these cells that are divided as a result of the interaction with this microenvironment can differentiate. In this respect, to know well the conditions that must be provided for the cell that is cultured in the stem cell culture is quite significant. For instance, in mouse embryonic stem cell culture, to prevent these cells from differentiating, to plant/cultivate these cells on mouse embryonic fibroblasts, and to add Leukemia Inhibitory Factor (LIF) into the culture environment are required. However, in a suspension culture where this is not provided and grasping feature is prevented, mouse embryonic stem cells will differentiate in a short time and form embryoid body structures that are seen equal to a 6-day-old embryo (Freshney et al., 2007; Fauza and Bani, 2016).

Ovarian gametogenesis (oogenesis)

Oogenesis begins in fetal life when the primordial germ cells, or oogonia, migrate to the genital ridge. The number of oogonia increases dramatically from about 600,000 by the second month of fetal life to a maximum of about 7 million by the sixth to seventh month. The oogonia then begin meiotic division (they are now referred to as primary oocytes) until they reach the diplotene stage of the prophase (the germinal vesicular stage), in which they will remain until stimulation by gonadotropins in adulthood during the menstrual cycle (discussed later). However, by a process of apoptosis and atresia of the enveloping follicle, which starts prenatally and persists throughout childhood, the number of primary oocytes declines drastically from about 2 to 4 million at birth to become 90% depleted by puberty. Further depletion of the pool occurs throughout adulthood, so that by age 37 only about 25,000 and by age 50 only about 1000 oocytes remain.

In recent years, the traditional dogma that mammals have fixed, nonrenewable oocyte stores established before birth has been challenged. Some studies suggest that adult mammalian ovaries possess pluripotent germline stem cells (GSCs) that can differentiate into oocytes, as well as other cell types. Nonmammalian organisms, such as Drosophila, do possess ovarian GSCs. Whereas the existence of spermatogonial cells in the adult human testis that give rise to pluripotent GSCs is well accepted, there is considerable evidence that disputes the existence of mammalian adult ovarian GSCs. At this point, there is not sufficient evidence to prove that mammalian oogenesis occurs after birth (Hanna, 2014).

Oogenesis

During fetal life the developing ovaries become populated with primordial germ cells (oogonia), which continue to divide by mitosis until a few weeks before birth. After this time, no new oocytes are produced, and the female is born with all the oocytes she will ever have (approximately 1 000 000), which are not replaced. From early in gestation, fetal oogonia enter meiosis, reaching the first prophase stage, whereupon they become arrested and remain so for up to 50 years until just before ovulation. During this arrest, the oocyte with the surrounding layer of flattened granulosa cells is known as a primordial follicle. These primordial follicles are scattered throughout the cortex of the ovaries, surrounded by interstitial connective tissue. The majority of ovarian oocytes become atretic by puberty, leaving only about 250 000 available in the reproductive phase of life. Of these, only about 400 will be ovulated.

In the ovary there is continual recruitment of small numbers of primordial follicles to start folliculogenesis, which is a lengthy process taking 6 months or longer. This recruitment continues until the supply of primordial follicles is exhausted, around the time of the menopause. Folliculogenesis encompasses recruitment of a cohort of primordial follicles from the resting pool, initiation of follicle and oocyte growth; this is followed by final selection and maturation of a single preovulatory follicle, with the remaining follicles being eliminated by atresia. During this time, the oocyte grows from 35 µm to 120 µm in diameter, undergoes meiosis to produce a haploid gamete, produces large amounts of stable RNA to support early embryonic development and acquires the nuclear and cytoplasmic maturity to undergo fertilization and embryogenesis.

Following recruitment, the granulosa cells of the primordial follicle become cuboidal in shape and undergo cell division. When the follicle reaches the secondary stage, with two layers of granulosa cells, a layer of theca cells develops around the follicle. The theca and granulosa cells of the follicle, which are epithelial in nature, create a specialized microenvironment for the developing oocyte. At the same time, the granulosa cells secrete a glycoprotein coat around the oocyte, known as the zona pellucida. Later on, this will provide species-specific sperm receptors at fertilization, and protect the embryo before implantation. Microvilli extend from the granulosa cells through the zona pellucida to the plasma membrane of the oocyte and are intimately involved in the transfer of nutrients and signalling molecules between the two.

When there are several layers of granulosa cells and the oocyte is fully grown, a fluid-filled cavity (the antrum) appears, and starts expanding. The oocyte itself is pushed to one side and is surrounded by two or three layers of tightly knit granulosa cells, the corona radiata. From now until ovulation, follicular development is subject to endocrine control, predominantly by follicle stimulating hormone (FSH). At the beginning of each menstrual cycle, there is a group of about 20 small antral follicles, only one of which will ovulate 2 weeks later. The rest of the group undergo atresia, and die by apoptosis.

After antrum formation, the rate of cell division in the granulosa cell population slows down, and these cells differentiate and become steroidogenic, utilizing theca-derived androgen to produce increasing amounts of oestradiol. In the mid-follicular phase, a dominant follicle emerges and its secretion is responsible for about 95% of circulating oestradiol levels in the late follicular phase. During the final maturation of the follicle, the corona cells become columnar and less tightly packed. The primary oocyte resumes meiotic maturation in response to the onset of the mid-cycle surge of luteinizing hormone (LH). The germinal vesicle breaks down and the first polar body, containing one of each pair of homologous chromosomes (23 in total) and a minute amount of cytoplasm, is extruded. The oocyte (now termed a secondary oocyte) is ovulated while proceeding through the second meiotic division, where it arrests again at metaphase II, and is only stimulated to complete meiosis at fertilization. Each of the 23 chromosomes consists of two chromatids. At fertilization the pairs of chromatids separate, with 23 being retained in the oocyte and 23 being expelled in the second polar body. With the entry of the sperm containing its complement of 23 chromosomes, diploidy is restored.

Development of the Female Gamete (See Chapter 184)

If the gonad develops into an ovary, the primordial germ cells become oogonia and continue mitotic division.9 Mitotic division of these cells has been observed in humans up to the seventh fetal month10 but ceases sometime shortly before birth. No oogonia form subsequent to the birth of the infant after a normal full-term pregnancy.

In both males and females, the germ cells form a syncytium while dividing.11,12 These intercellular connections permit communication and facilitate the high degree of synchrony that has been observed during both mitotic and meiotic divisions.13-15

By the eighth or ninth week after fertilization, some oogonia enter prophase of meiosis I and become primary oocytes.9-16 Meiosis begins first deep to the surface of the human ovary and then expands toward the surface. Thus, at an appropriate fetal stage, oogonia are found superficially, oocytes deep to the surface, and small follicles at the inner part of the ovarian cortex.9 It has been suggested that a diffusible meiosis-activating substance is secreted by rete cells (derived from the mesonephros), which lie in the center of the ovary, and good experimental evidence is available to support this hypothesis.17-20

The oocyte goes through the leptotene, zygotene, and pachytene stages of meiosis I, and it then arrests at the diplotene stage. At this point the oocyte becomes surrounded by a single, incomplete layer of flat follicular cells9; this unit is called a primordial follicle. The follicle’s large central nucleus is known as the germinal vesicle. A crescent-shaped assembly of cellular organelles containing mitochondria, endoplasmic reticulum, the Golgi complex, lysosomes, and annulate lamellae (stacked parallel membrane arrays with pores) remain clustered adjacent to the nucleus.15,21 Once it has been incorporated into a primordial follicle, the oocyte enters a long period of quiescence, beginning before birth in humans and ending in either atresia or ovulation.

Once sexual maturity is attained, a small number of oocytes begins the process of folliculogenesis or follicle maturation during each menstrual cycle.22 The oocyte grows and eventually becomes one of the largest cells in the human body.23 The organelles disperse throughout the cytoplasm and the germinal vesicle (nucleus) enlarges. It increases its complement of nuclear pores, facilitating transport of molecules between nucleoplasm and cytoplasm. The follicular cells resume mitosis and increase markedly in size, changing in shape from squamous to cuboidal, and the follicle becomes surrounded by a basement membrane. Those follicles containing an oocyte surrounded by a single layer of cuboidal follicular cells are known as unilaminar primary follicles, to distinguish them from earlier or later stages.3

During further growth of the primary follicle, a thick, acellular coat, the zona pellucida, begins to form between the oocyte and the follicular cells. Mitotic activity increases the number of follicle cell layers, and the follicle is now called a multilaminar primary follicle. The expanding follicle compresses the surrounding ovarian stoma, which organizes into a compact layer adjacent to the basement membrane of the follicle. This layer of stromal cells is called the theca interna, and its cells have the capacity to produce androgens when stimulated by luteinizing hormone activity (Figure 4-2). The theca interna is vascularized, but the epithelial layers of follicle cells remain avascular.

The zona pellucida is important in the process of fertilization because it contains sperm receptors, takes part in induction of the acrosome reaction, and becomes a block to polyspermy. It also may act after fertilization as a smooth, slippery envelope to contain the sticky ball of cells of the morula-stage embryo; these are free to adhere to the uterine endothelium when the zona breaks down, just before implantation.

The zona pellucida is made up of three separate filamentous glycoproteins, ZP1 through ZP3, which differ in molecular weight and isoelectric point and account for virtually all protein in the zona pellucida. ZP1 cross-links these filaments, resulting in a three-dimensional matrix that is permeable to large macromolecules. ZP3 serves as a species-specific sperm receptor and also induces the acrosome reaction in sperm at contact. At or shortly after fertilization, these two characteristics are lost, reducing the likelihood of polyspermy.24-26 The ZP3 gene has been cloned; it is expressed only in oocytes, and then only during the growth phase of oogenesis.27 The human genes for ZP2 and ZP3 are located on chromosomes 16 and 7, respectively,28 but the location of ZP1 remains unknown. The interesting story of these zona pellucida proteins has been the subject of a popularized account29 as well as several reviews.28,30,31 Radiolabeling studies in mice indicate that all three glycoproteins are synthesized by the oocyte itself, rather than by the follicular cells.32 Furthermore, immunofluorescence studies show that zona pellucida antigens are present within human oocytes but not in follicular cells.33 Studies in species other than the mouse, however, suggest that the granulosa cells that surround the oocyte also may play a role in the synthesis of zona pellucida components.30

Numerous cytoplasmic projections of the follicular cells penetrate the zona pellucida to contact the cell membrane of the oocyte. In humans, these filopodial extensions of the follicular cell may actually lie deeply buried in the oocyte, in straight invaginations or pits.34 These pits are lined by the oocyte cell membrane; however, no cytoplasmic continuity exists between the two cell types. Animal studies have demonstrated the presence of gap junctions along the association of these two cell membranes, permitting transfer of small molecules (molecular weight of approximately 1000) between them.22

As the primary follicle enlarges, the follicular cells begin to produce follicular fluid, which collects within the intercellular spaces between follicle cells. These spaces coalesce to form a large fluid-filled cavity called the antrum, which is characteristic of the secondary (vesicular) follicle. The antrum expands, and the oocyte becomes located on one side of the follicle, where it is embedded within a mound of follicle cells known as the cumulus oophorus. The layers of follicular cells immediately surrounding the oocyte are termed the corona radiata. Because of its increased size, the follicle further compresses the surrounding ovarian stroma. A looser, less organized layer of flattened stromal cells encircles the follicle superficial to the theca interna. This is called the theca externa, and its cells have no steroid-secreting activity (see Figure 4-2). A few days before ovulation, one secondary follicle becomes dominant and inhibits the growth of the remaining secondary follicles. The dominant follicle, now called a graafian follicle, can reach several centimeters in diameter. The oocyte is approximately 100 μm in diameter at this stage. Approximately a day before ovulation, its nuclear membrane breaks down, the nucleolus disappears, and the first polar body forms, containing one of the two sets of chromosomes. Meiosis I is completed, and the oocyte proceeds to meiosis II, but it again arrests on reaching metaphase. In most mammalian species including humans, meiosis II resumes only after the oocyte is penetrated by a sperm.35,36 Completion of meiosis in the fertilized oocyte results in production of the second polar body.

Follicles of any stage can undergo atresia. Atresia begins in the fetus and continues into menopause until all follicles have disappeared. At birth, approximately 2 million primordial follicles are present within the two ovaries. It has been estimated that half of the 2 million follicles present at birth are atretic at that time.10 In humans, follicular growth starts before birth, and the newborn ovary contains multilaminar primary follicles as well as primordial follicles. Follicular growth and subsequent atresia are continuous during human childhood, and it has been clearly stated that “quiescent ovaries in which follicular growth is absent do not occur in normal children.”37

Little is known about control of atresia. For example, it is not known whether atresia is initiated by action of the follicular cells, by that of the oocyte, or by both.22 The process of atresia, however, can be manipulated experimentally.37 Approximately 40,000 follicles are present in the two ovaries of a young adult woman, indicating a reduction to 2% of the pool originally present at birth.38

These stages, up to and including the newly fertilized mature ovum, are summarized in Table 4-1. Most or all of the RNA and protein found in a mature oocyte are synthesized during oocyte growth. Those macromolecules present in the oocyte of an atretic follicle are degraded and the degradation products subsequently used for new synthesis.23

As estimated from an assumed fertility span of 30 years, approximately 400 eggs are shed during a woman’s lifetime.35 Thus, approximately 1 in every 100 of the eggs present in a young adult completes maturation and is ovulated; the rest degenerate. A human female has her full complement of eggs, albeit immature, on the day she is born. This is not the case for sperm development in males.

Development of the Female Gamete (See Chapter 150)

If the gonad develops into an ovary, the primordial germ cells become oogonia, and mitotic division continues.9 Mitotic division of these cells has been observed in humans up to the seventh fetal month10 but ceases sometime shortly before birth. No oogonia form after the birth of the infant after a normal full-term pregnancy.

In both males and females the germ cells form a syncytium while dividing.11,12 These intercellular connections permit communication and facilitate the high degree of synchrony that has been observed during both mitotic division and meiotic division.13-15

By the eighth or ninth week after fertilization, some oogonia enter prophase of meiosis I and become primary oocytes.9-16 Meiosis begins first deep to the surface of the human ovary and then expands toward the surface. Thus, at an appropriate fetal stage, oogonia are found superficially, oocytes deep to the surface, and small follicles at the inner part of the ovarian cortex.9 It has been suggested that a diffusible meiosis-activating substance is secreted by rete cells (derived from the mesonephros), which lie in the center of the ovary, and good experimental evidence is available to support this hypothesis.17-20

The oocyte goes through the leptotene, zygotene, and pachytene stages of meiosis I, and it then stops at the diplotene stage. At this point the oocyte becomes surrounded by a single, incomplete layer of flat follicular cells9; this unit is called a primordial follicle. The follicle's large central nucleus is known as the germinal vesicle. A crescent-shaped assembly of cellular organelles containing mitochondria, endoplasmic reticulum, the Golgi complex, lysosomes, and annulate lamellae (stacked parallel membrane arrays with pores) remains clustered adjacent to the nucleus.15,21 Once it has been incorporated into a primordial follicle, the oocyte enters a long period of quiescence, beginning before birth in humans and ending in either atresia or ovulation.

Once sexual maturity is attained, a small number of oocytes begin the process of folliculogenesis, or follicle maturation, during each menstrual cycle.22 The oocyte grows and eventually becomes one of the largest cells in the human body.23 The organelles disperse throughout the cytoplasm, and the germinal vesicle (nucleus) enlarges. It increases its complement of nuclear pores, facilitating transport of molecules between nucleoplasm and cytoplasm. The follicular cells resume mitosis and increase markedly in size, changing in shape from squamous to cuboidal, and the follicle becomes surrounded by a basement membrane. Those follicles containing an oocyte surrounded by a single layer of cuboidal follicular cells are known as unilaminar primary follicles, to distinguish them from cells of earlier or later stages.3

During further growth of the primary follicle, a thick, acellular coat, the zona pellucida, begins to form between the oocyte and the follicular cells. Mitotic activity increases the number of follicular cell layers, and the follicle is now called a multilaminar primary follicle. The expanding follicle compresses the surrounding ovarian stoma, which organizes into a compact layer adjacent to the basement membrane of the follicle. This layer of stromal cells is called the theca interna, and its cells have the capacity to produce androgens when stimulated by luteinizing hormone activity (Figure 3-2). The theca interna is vascularized, but the epithelial layers of follicular cells remain avascular.

The zona pellucida is important in the process of fertilization because it contains sperm receptors, takes part in induction of the acrosome reaction, and becomes a block to polyspermy. It may also act after fertilization as a smooth, slippery envelope to contain the sticky ball of cells of the morula-stage embryo; these cells are free to adhere to the uterine endothelium when the zona breaks down, just before implantation.

The zona pellucida is made up of three separate filamentous glycoproteins, zona pellucida glycoprotein 1 (ZP1) ZP1 through ZP4, which differ in molecular weight and isoelectric point and account for virtually all protein in the zona pellucida. ZP1 crosslinks these filaments, resulting in a three-dimensional matrix that is permeable to large macromolecules. ZP3 serves as a species-specific sperm receptor and also induces the acrosome reaction in sperm on contact. At or shortly after fertilization, these two characteristics are lost, reducing the likelihood of polyspermy.24-26 The ZP3 gene has been cloned; it is expressed only in oocytes, and then only during the growth phase of oogenesis.27 ZP1, ZP2, and ZP3 are located on chromosomes 19, 7, and 5, respectively, while ZP4 is located on chromosomes 11, 16, 7, and 1.27a The interesting story of these zona pellucida proteins has been the subject of a popularized account29 and several reviews.28,30,31 Radiolabeling studies in mice indicate that all three glycoproteins are synthesized by the oocyte itself, rather than by the follicular cells.32 Furthermore, immunofluorescence studies show that zona pellucida antigens are present within human oocytes but not in follicular cells.33 Studies in species other than the mouse, however, suggest that the granulosa cells that surround the oocyte also may play a role in the synthesis of zona pellucida components.30

Numerous cytoplasmic projections of the follicular cells penetrate the zona pellucida to contact the cell membrane of the oocyte. In humans these filopodial extensions of the follicular cell may actually lie deeply buried in the oocyte, in straight invaginations or pits.34 These pits are lined by the oocyte cell membrane; however, no cytoplasmic continuity exists between the two cell types. Animal studies have demonstrated the presence of gap junctions along the association of these two cell membranes, permitting transfer of small molecules (molecular weight of approximately 1000) between them.22

As the primary follicle enlarges, the follicular cells begin to produce follicular fluid, which collects within the intercellular spaces between follicular cells. These spaces coalesce to form a large fluid-filled cavity called the antrum, which is characteristic of the secondary (vesicular) follicle. The antrum expands, and the oocyte becomes located on one side of the follicle, where it is embedded within a mound of follicular cells known as the cumulus oophorus. The layers of follicular cells immediately surrounding the oocyte are termed the corona radiata. Because of its increased size, the follicle further compresses the surrounding ovarian stroma. A looser, less organized layer of flattened stromal cells encircles the follicle superficial to the theca interna. This is called the theca externa, and its cells have no steroid-secreting activity (see Figure 3-2). A few days before ovulation, one secondary follicle becomes dominant and inhibits the growth of the remaining secondary follicles. The dominant follicle, now called a graafian follicle, can reach several centimeters in diameter. The oocyte is approximately 100 µm in diameter at this stage. Approximately 1 day before ovulation, its nuclear membrane breaks down, the nucleolus disappears, and the first polar body forms, containing one of the two sets of chromosomes. Meiosis I is completed, and the oocyte proceeds to meiosis II, but it again stops on reaching metaphase. In most mammalian species, including humans, meiosis II resumes only after the oocyte has been penetrated by a sperm.35,36 Completion of meiosis in the fertilized oocyte results in production of the second polar body.

Follicles of any stage can undergo atresia. Atresia begins in the fetus and continues into menopause until all follicles have disappeared. At birth approximately 2 million primordial follicles are present within the two ovaries. It has been estimated that half of the 2 million follicles present at birth are atretic at that time.10 In humans, follicular growth starts before birth, and the newborn ovary contains multilaminar primary follicles and primordial follicles. Follicular growth and subsequent atresia are continuous during human childhood, and it has been clearly stated that “quiescent ovaries in which follicular growth is absent do not occur in normal children.”37

Little is known about control of atresia. For example, it is not known whether atresia is initiated by action of the follicular cells, by that of the oocyte, or by both.22 The process of atresia, however, can be manipulated experimentally.37 Approximately 40,000 follicles are present in the two ovaries of a young adult woman, indicating a reduction to 2% of the pool originally present at birth.38

These stages, up to and including the newly fertilized mature ovum, are summarized in Table 3-1. Most or all of the RNA and protein found in a mature oocyte are synthesized during oocyte growth. Those macromolecules present in the oocyte of an atretic follicle are degraded, and the degradation products are subsequently used for new synthesis.23

As estimated from an assumed fertility span of 30 years, approximately 400 eggs are shed during a woman's lifetime.35 Thus approximately 1 in every 100 of the eggs present in a young woman completes maturation and is ovulated; the rest degenerate. A human female has her full complement of eggs, albeit immature, on the day she is born. This is not the case for sperm development in males.

1. The Fixed “Pool” and “Resting Reserve” of Primordial Follicles

Three sequential partially overlapping processes—oogonial proliferation, initiation of meiosis I with conversion of oogonia to primary oocytes, encapsulation of the oocyte by pregranulosa cells forming the primordial follicle—establish the reservoir of ovarian primordial follicles.[6]

In humans, primordial germ cells arise in the yolk sac endoderm and migrate to the gonadal ridge (they do not survive elsewhere) by the seventh week of gestation. Oogonia proliferate by mitosis during migration and at a greatly accelerated pace once in the gonadal ridge resulting in a peak germ cell content in excess of 6–7 million at 20 weeks of fetal life. Thereafter oogonial division dramatically declines, in effect ending at midgestation thereby creating a “fixed endowment” of germ cells which cannot be replenished.[7, 8] Transformation of individual oogonia into primary oocytes (entry into the first stages of meiosis) is initiated at 11–12 weeks and continues over the remainder of fetal life. Also around midgestation primordial follicle formation begins as a single layer of pregranulosa cells envelopes each oocyte in a process that continues until just after birth (Fig. 2A.2). After oocytes are enclosed within the primordial follicles they remain arrested for variable time intervals in the dictyate state of meiosis I and remain in this condition in a “resting pool.” Oocytes not surrounded by pregranulosa cells are lost presumably by extrusion beyond the ovarian capsule or more likely by programmed cell death (apoptosis).[9]

The variable duration of the period of arrest and the factors which modify it are not understood. However, from within minutes or even decades after formation, a primordial follicle may leave the resting pool and initiate oocyte enlargement and granulosa cell differentiation and proliferation to a variety of preantral or antral stages (Fig. 2A.3). At any point during this process atresia may take place. As a result, from a peak oocyte number of 6–7 million at 20 weeks, only 1–2 million exist at birth and at puberty an average of 200,000 follicles remain in the ovary.[7] During reproductive life, in addition to the monthly ovulation of a single dominant follicle, continuing reactivation, growth, and atresia of follicles at the rate of about 1000 per month results in an unrelenting decrease in the original follicle reserve pool. Approximately 8–10 years before menopause, concomitant with rising levels of follicle stimulating hormone (FSH) and diminished inhibins, increasing percentages of follicles are lost from the resting pool.[10] This terminal acceleration in follicle depletion rate “serves as a ticking clock to time the onset of menopause.” Eventually as a result of ovarian follicle exhaustion menopause occurs at about 51 years of age, “a time point that has remained constant for centuries.”[11]

Gene Expression During PGCs’ Migration and Proliferation

Human PGCs are precursors of oocytes and are crucial for maintenance of the species. In female gonads, PGCs differentiate into oogonia by mitosis [15,16], which undergo massive proliferation by mitotic divisions from 6 to 8 weeks gestation [17] and then become primary oocytes that arrest at the prophase of the first meiotic division. During this phase, PGCs migrate to the gonadal ridges from the hindgut by crossing the dorsal mesentery. A series of factors, including the transforming growth factor beta (TGFβ) family members and germ cell-derived transcription factors [18,19], are involved in this process. The expression levels of BMP2 and BMP4 increase in mice PGCs, while the expression level of Activin increases in human PGCs. The transcriptional factors that play important roles in survival, migration, and proliferation of PGCs include SOX17, BLIMP1, PRDM14, OCT4, NANOG, FIGα, NANOS3, and DND1 [20].

Through incomplete division of the cytoplasm (incomplete cytokinesis) during mitosis, oogonia form germ cell cysts that will develop into ovarian follicles [21]. The growth phase of oogenesis is initiated when oogonia finish their mitotic division, together with their enlargement to form primary oocytes and meiosis initiation. During 11–20 weeks of embryonic life before birth, the first meiotic division in primary oocytes is initiated in humans. During this process, several RNA binding proteins, exemplified by DAZL and BOLL, are involved in different stages of human meiotic division [22]. PGCs react upon signals secreted by neighboring somatic cells through receptor/ligand interactions to facilitate their migration through the hindgut and into the developing gonads. Released by granulosa cells, KITL recognizes its receptor KIT on the oocyte, inducing gonad development. Using single-cell RNA-sequencing techniques (RNA-seq), a recent study revealed that several signaling pathways are coordinately and reciprocally enriched between PGCs and their gonadal niche cells [23]. For example, the NOTCH signaling pathway is specifically activated in gonadal somatic cells, while the BMP signaling pathway is activated in PGCs by BMP2 secreted from the neighboring granulosa cells [23].

Oogenesis and oocytes

Oogenesis begins in utero and continues throughout a female’s reproductive years (Fig. 2.7).

In utero phase: Primordial germ cells arrive at the genital ridge by the 5th week of the development, when they are labeled as oogonia. These multiply by mitosis without atresia and by 6–7 weeks, about 10,000 oogonia exist. By 8 weeks, 600,000 oogonia are present, which then undergo a simultaneous process of mitosis, meiosis, and atresia, to peak at 20 weeks to 6–7 million cells, of which about 60% are primary oocytes and the rest are oogonia. Oogonial mitosis ends by approximately 7th month and atresia peaks by 5th month, after which, by 7th month, oogonial atresia is complete while follicular atresia sets in, to continue throughout reproductive years.18 At birth, 1–2 million germ cells persist, which are primary oocytes in prophase of the first meiotic division.19

Prepuberty and puberty: Germ cell mass reduces further to 300,000–400,000 at the pubertal onset, out of which only about 400 are taken on for ovulation throughout a female’s life span. After the first LH surge at puberty, the primary oocytes complete the 1st meiotic division with conversion to secondary oocyte and the formation of 1st polar body.

B. Embryologic Development and Maturation of Germ Cells in Women

The germ cells incorporated in the developing ovary multiply at high rate, and, by 24 weeks of gestation, there are 7 million oogonia in the primitive ovaries. They continue to multiply, but most die by apoptosis, so that only about 1 million primary oocytes are left at birth [4]. These decrease to about 400,000 by puberty. The surviving oogonia are arrested at the prophase of meiosis. Completion of the first division of meiosis does not occur until the time of ovulation. Only about 400 of these oocytes actually mature and are released by ovulation in a woman's lifetime; the others undergo apoptosis and die at various stages of development. In contrast to the situation in the male, female germ cells stop dividing by mitosis during prenatal life. As mentioned previously that may be the reason for the low rate of development of ovarian GCTs.