The Ovarian Life Cycle

Oocyte Ultrastructure

The oocyte is a large cell with a large, highly transcriptionally active nucleus, also known as the germinal vesicle, and an abundant cytoplasm rich in organelles and proteins. The growing oocyte generates all the components required for ovulation, fertilization, and the early stages of preimplantation embryo development before transcription begins from the newly formed embryonic genome. Essential cytoplasmic components include ribosomes, mitochondria, and maternal mRNAs that are stored for later translation into proteins required for maturation and embryo development. Ribosomes and maternal mRNAs are found in the cytoplasm associated with fibrillar cytoskeletal structures known as cytoplasmic lattices, which are composed of keratins, tubulin, and other highly abundant oocyte proteins. ^, After transcription ceases in the fully grown oocyte, the nucleus also serves as a storage depot for RNAs and RNA-binding proteins.

Oocytes contain far more mitochondria than somatic cells. ^, Oocyte mitochondria are also unique in their structure and DNA content relative to somatic cells in that they are spherical, have few cristae, and contain only one to two copies of mitochondrial DNA each. Primordial germ cells begin with about 10 mitochondria, and the numbers of mitochondria increase rapidly throughout germ cell migration to the gonad, entry into meiosis, and primordial follicle formation when the oocytes each contain ∼6000 mitochondria. With the onset of oocyte growth, mitochondrial replication continues, with an estimated 300,000 to 400,000 mitochondria in fully grown human oocytes. At this stage, the mitochondria form aggregates that are tightly associated with smooth endoplasmic reticulum membranes. The substantial number of oocyte mitochondria generate the adenosine triphosphate (ATP) needed to support oocyte development, maturation, fertilization, and early embryo development. Diminished mitochondrial function, in some cases due to mitochondrial DNA mutations, is thought to be a major contributor to reproductive aging and premature ovarian insufficiency.

Critical Genes Expressed During Oocyte Growth

The growing oocyte expresses a number of genes that are essential for successful follicular development, fertilization, and preimplantation development. Mouse knockout studies have identified several oocyte-specific transcription factors essential for folliculogenesis. Figla, Sohlh1 (spermatogenesis- and oogenesis-specific basic helix-loop-helix 1), and Lhx8 (LIM homeobox 8) appear to be required for the formation of primordial follicles from naked primordial oocytes, whereas Nobox (newborn ovary homeobox) is involved in the transition of primordial to primary follicles (see Fig. 8.4 ). Dazl , Cpeb1 (cytoplasmic polyadenylation element binding protein 1), and Ybx2 (previously known as Msy2 ) encode DNA- or RNA-binding proteins involved in regulating mRNA translation within the oocyte.

Oocyte growth is accompanied by formation of the zona pellucida (ZP), an extracellular matrix surrounding the oocyte. The ZP protects the developing germ cell in the follicle, the ovulated egg in the oviduct, and the preimplantation embryo. It also serves as the initial site of contact with sperm, and after fertilization, becomes a barrier that discourages polyspermy. Three genes have been characterized that encode ZP1 (ZPA), ZP2 (ZPB), and ZP3 (ZPC), which are the main sulfated glycoproteins of the ZP. A polymer of ZP2 and ZP3 proteins forms filaments that are interconnected by ZP1, a minor component of the ZP. FIGLA regulates the coordinated expression of these genes during the oocyte growth phase. Humans and rats have a fourth ZP-1-like subunit (ZP4) that is not expressed in mice.

Mice lacking ZP1 form structurally abnormal ZP and have reduced fecundity. In mice lacking ZP2, a thin ZP forms that is not retained in preovulatory follicles. The antral-stage follicle number is substantially reduced, few eggs are ovulated, and no two-cell-stage embryos can be found in mated animals. Moreover, blastocysts derived from in vitro fertilized oocytes from females lacking ZP2 do not undergo normal development. Mice lacking ZP3 form no ZP despite the expression of the other zona proteins. Few eggs are ovulated, and the females are sterile. As in the ZP2 mutant, in vitro fertilized eggs from ZP3-deficient mice do not develop beyond the blastocyst stage.

Oocytes express growth factors in the TGF-β superfamily that are secreted and regulate the development of the follicle. These factors are described in detail below.

Several oocyte-specific “maternal effect” genes required during preimplantation embryo development have been identified in mouse oocytes. Four of these genes encode proteins that form a “subcortical maternal complex” in oocytes, including Nlrp5 (NACHT; leucine-rich repeat; and PYD-containing 5, also known as MATER ), Khdc3 (KH domain containing 3, also known as Filia ), Ooep (oocyte-expressed protein, also known as Floped ), and Tle6 (transducin-like enhancer of split 6). ^, Peptidylarginine disulfide isomerase, type VI (PADI6) may also function in this complex based on its similar localization patterns in the oocyte and cleavage stage embryo. Mouse oocytes lacking NLRP5, PADI6, or OOEP do not have cytoplasmic lattices, are defective in their ability to synthesize proteins, and fail to develop following fertilization. ^, KHDC3 is required in oocytes and early embryos to regulate spindle function; embryos of Khdc3 -null female mice develop poorly because of a high incidence of aneuploidy. Nlrp2 , a gene related to Nlrp5 , is expressed in both oocytes and granulosa cells during folliculogenesis. Maternal NLRP2 is required for successful development in early embryos.

Paralogues of the mouse subcortical maternal complex genes are expressed in human oocytes and likely function similarly. Indeed, women homozygous for a point mutation at a phosphorylation site in TLE6 are sterile due to a failure of embryo cleavage following fertilization. Similarly, human oocytes lacking PADI6 undergo developmental arrest following fertilization.

Zar1 (zygote arrest 1) encodes a cytoplasmic protein required for the transition from fertilized egg to cleaving embryo by an unknown mechanism. Gclm (glutamate-cysteine ligase, modifier subunit) encodes a protein that regulates the synthesis of glutathione, which is critical for controlling cellular redox status. Mouse embryos deficient in maternal GCLM have deficits in their ability to develop to the blastocyst stage. Npm2 (nucleoplasmin 2) encodes a nuclear protein generated before oocyte maturation that affects heterochromatin organization and histone deacetylation. In the absence of NPM2 , ovulation and fertilization occur normally, but there is a complete failure of preimplantation embryo development. Dppa3 ( Stella ), in addition to its role in primordial germ cell specification, is a maternal effect gene required for normal preimplantation embryo development.

Oocyte-Derived Factors and the Control of Follicular Growth and Maturation

The concept that the oocyte plays an important role in follicular function and is far more than a passive inhabitant of the follicle emerged from two observations: (1) that follicular survival depends on the presence of viable germ cells and (2) that removal of the oocyte from an antral follicle is followed by luteinization, indicating that the oocyte produces factors that restrain the terminal differentiation of granulosa cells. ^, Additional support for this concept was derived from experiments in which midsized oocytes from mouse secondary follicles were transferred by a grafting procedure into primordial follicles. This transfer resulted in a doubling of the rate of development of the primordial follicular cells in the presence of the secondary follicle oocyte.

The effects of the oocyte on follicular growth are mediated in part by closely related members of the TGF-β superfamily that are selectively or specifically produced by the oocyte. These factors, growth differentiation factor (GDF)-9 and BMP15, influence granulosa and theca cell function ( Fig. 8.7 ) (see Chapter 6 ). The importance of GDF9 and BMP15 has been established through genetic manipulation of mice and the discovery of spontaneous mutations in the genes encoding GDF9 and BMP15, which affect follicular dynamics in sheep. ^, In humans, there is a correlation between higher GDF9 and BMP15 expression and the overall quality of the oocyte and subsequent embryo, and some genetic variants have been proposed to cause primary ovarian dysfunction. ^,

Growth Differentiation Factor-9

GDF9, encoded by a gene on chromosome 5q31.1, is highly expressed by oocytes and, to a lesser extent, by primate granulosa cells. It forms homodimers but also heterodimers with BMP15. The follicles of GDF9-deficient mice arrest in growth at the primary stage, yet the oocytes continue to grow at a faster rate than wild-type oocytes, progressing to advanced stages of differentiation seen in the antral follicles of normal mice. However, there are ultrastructural abnormalities in the interconnections between granulosa cells and oocytes; the oocytes ultimately die, leaving a ribbon of ZP behind. The theca also does not form around the follicles, implicating GDF9 in the organization or proliferation of this follicular component. Studies in the rat also indicate that GDF9 stimulates the growth of primary follicles, consistent with the block to progression at the primary stage in GDF9-deficient mice. ^,

GDF9 has a variety of actions on granulosa cells and theca cells that are species-specific, acting at least in part through interaction with the activin-like receptor (ALK)-5 (TGF-βRI) and BMP receptor type 2 (BMPR2) receptor complex. ^, In rodents, GDF9 stimulates granulosa cell differentiation, including induction of luteinizing hormone (LH) receptors and steroidogenesis. In cumulus cells, GDF9 promotes the expression of hyaluronan synthase 2, pentraxin 3, and tumor necrosis factor inducible gene 6 (TSG6), the latter being proteins that are incorporated into the proteoglycan extracellular matrix of the cumulus oophorus complex. It also suppresses urokinase expression while stimulating prostaglandin-endoperoxide synthase-2 (PTGS2, also known as COX2), prostaglandin synthesis, and progesterone formation. ^, LH receptor expression is suppressed by GDF9, which would discourage luteinization of the cumulus cells. These actions of GDF9 promote unique phenotypes in the granulosa cells surrounding the oocyte, which are exposed to the highest GDF9 concentrations. GDF9 inhibits human theca cell steroidogenesis in vitro . It also stimulates theca cell proliferation, a finding consonant with the apparent role of GDF9 in the murine ovary in controlling thecal development.

Bone Morphogenetic Protein-15

BMP15, also known as GDF9b, encoded by a gene on the X chromosome, is another member of the TGF-β superfamily produced by oocytes. It is related structurally to GDF9 and shares a similar pattern of expression. Targeted deletion of the murine Bmp15 gene causes a modest ovarian phenotype in nullizygous animals of subfertility with diminished ovulation and fertilization rates. However, mice nullizygous for Bmp15 and heterozygous for a Gdf9 mutation have severely impaired fertility, with abnormalities in folliculogenesis and cumulus cell function. Spontaneous point mutations in the ovine Bmp15 gene (e.g., Inverdale and Hanna sheep) result in phenotypes that differ from those in Bmp15 knockout mice. In the heterozygous state, the number of follicles ovulating is increased, and thus there is an increase in fecundity. However, primary ovarian failure, with a phenotype resembling the murine Gdf9 knockout, is observed in ewes homozygous for the mutations. In vitro , BMP15 stimulates granulosa cell mitosis. Thus, its absence in vivo would be predicted to impair follicular growth, which is consistent with the ovarian abnormalities in homozygous mutant sheep.

BMP15 binds to a receptor complex consisting of BMPR1B (ALK6) and BMPRII. A point mutation in BMPR1B in Booroola sheep is associated with an additive increase in ovulation rate based on the copy number of mutant alleles. Targeted deletion of the Bmpr2 gene in mice does not affect follicular development, but it does yield an infertility phenotype because of defects in cumulus cell expansion that prevent in vivo fertilization.

Both BMP15 and KIT-ligand participate in a negative feedback loop: BMP15 stimulates KIT-ligand expression by granulosa cells, whereas KIT-ligand inhibits BMP15 expression in oocytes. In the presence of an oocyte, both BMP15 and KIT-ligand stimulate granulosa cell mitosis. The observations that only the oocyte expresses KIT, and that KIT-ligand suppresses expression of BMP15, a granulosa cell mitogen, suggest that the oocyte must be involved in producing another granulosa cell mitogen.

GDF9 and BMP15 are both synthesized as proproteins that form dimers and are then proteolytically processed to yield the bioactive molecules. Notably, the Inverdale mutation that inactivates BMP15 dramatically impairs the proteolytic processing of both the mutant BMP15 and wild-type GDF9 in coexpressing cells. This observation suggests that the phenotype of the Inverdale sheep may be the result, at least in part, of GDF9 deficiency due to interference by mutant BMP15 with wild-type GDF9 processing. Similarly, cells coexpressing mutant human forms of BMP15 and GDF9 associated with premature ovarian failure have decreased production of the mature proteins, likely because of impaired posttranslational processing. Although both GDF9 and BMP15 homodimers have biological activity, heterodimers of the two proteins are far more bioactive as indicated by their potency in regulating granulosa cell survival, granulosa cell functions that support oocyte metabolism, and cumulus cell expansion during maturation. These findings suggest that GDF9:BMP15 heterodimers, rather than homodimers of the individual proteins, are the essential functional ligands secreted by the oocyte.

Intercellular Communication Between Granulosa Cells and the Oocyte

Granulosa cells are interconnected by an extensive network of gap junctions, which are important for metabolic exchange and transport of small molecules between neighboring cells. The number of gap junctions per granulosa cell increases as follicles develop, coupling them into a functional syncytium. Moreover, granulosa cells extend cytoplasmic processes through the ZP to form gap junctions with the plasma membrane of the oocyte.

Gap junctions are comprised of hexameric arrays of proteins called connexins. Connexin-37 and connexin-43 are two of the most important follicular connexins, and they form gap junctions with different permeability properties. Connexin-37 is the predominant connexin in the oocyte, whereas connexin-43 predominates in granulosa cells. However, at least the first layer of granulosa cells surrounding the oocyte also expresses connexin-37. Communication between granulosa cells and the oocyte occurs through homotypic connexin-37 gap junctions, whereas gap junctional communication between granulosa cells is via homotypic connexin-43 complexes. Follicle-stimulating hormone (FSH) increases gap junction communication by raising levels of mRNA encoding the main connexins in granulosa cells and oocytes. The number of granulosa cell projections to the oocyte through the ZP is also increased. In addition, TGF-β1 and all-trans retinoic acid synthesized locally by cumulus cells induce connexin-43 expression in granulosa cells.

In antral follicles, oocytes are restrained from resuming meiosis via diffusion of cyclic GMP (cGMP) through gap junctions. The ovulatory surge of LH suppresses connexin-43 mRNA expression and causes mitogen-activated kinase-mediated phosphorylation of connexin-43 protein that ultimately results in gap junction closure and disruption of cell-to-cell metabolic coupling.

The importance of connexins to follicular function was shown in the ovarian phenotype of connexin-37 and connexin-43 knockout mice. ^, In connexin-37-deficient mice, created by targeting the Gja4 gene that encodes this protein, follicle growth is arrested at the preantral stage; oocyte growth, although it commences, is also subsequently arrested before meiotic competence is achieved, resulting in loss of oocytes and formation of luteinized structures. Connexin-43-deficient mice, created by targeting the Gja1 gene, have an ovarian phenotype characterized by diminished germ cell number and impaired growth of follicles beyond the primary stage. Other connexins are expressed in the ovary, but their specific functions are unknown.

Granulosa Cells and the Convergence of Multiple Signaling Pathways

Granulosa cells express receptors for, and respond to, a large number of factors that are either generated locally within the follicle or that enter the follicular compartment from the blood. These include oocyte-derived factors, autocrine/paracrine factors produced by the granulosa cells themselves, products of theca cells, and circulating factors derived from the pituitary and other tissues such as fat. The repertoire of signaling molecules that affect primates as well as other animal granulosa cells in vitro and in vivo beyond FSH and LH is extensive, encompassing hypothalamic factors (e.g., kisspeptin, and gonadotropin-releasing hormone [GnRH]), other pituitary hormones (e.g., growth hormone and prolactin), a large array of growth factors (e.g., epidermal growth factor [EGF] family members, TGFβ family members, and insulin-like growth factors [IGFs]), hormones controlling metabolism (e.g., insulin), vascular tone and angiogenesis (e.g., endothelins and apelin), cytokines (e.g., tumor necrosis factor-α [TNF-α]), and adipokines (e.g., leptin, adiponectin, resistin). Some of these factors have a critical role as documented from the phenotypes associated with mutations in humans or spontaneous or induced gene mutations in animals such as previously described for GDF9 and BMP15. The hierarchical and temporal dominance of the actions of the plethora of factors that can act on granulosa cells has yet to be established.

In addition to the multiple factors that act on the array of receptors expressed on granulosa cells, microvesicles and exosomes participate in communication within the follicle. MicroRNAs are one of the better characterized cargoes of these shed components, which act as rheostats to fine-tune gene expression. ^,

Granulosa Cell Endocrine Activity

The key steroid hormone produced by the preovulatory granulosa cells is estradiol. The synthesis of estrogens requires a collaborative relationship with adjacent theca cells, which produce the immediate precursors (androgens) for the aromatization reaction (see Chapter 4 ). The control of this process is under the direction of LH, acting on thecal elements, and FSH, acting on the granulosa compartment through multiple signaling pathways including protein kinase A and AKT (also known as protein kinase B) ( Fig. 8.8 ; see Chapter 2 ). The actions of LH and FSH are modulated by local factors produced by the somatic cells of the follicle and the oocyte, as noted above, including BMPs.

The two-cell–two-gonadotropin model is a formidable example of the integrated function of the different cellular components of the follicle ( Fig. 8.8 ). Studies of isolated granulosa cells have shown that FSH, but not LH, stimulates estrogen production when the cells are provided with an aromatizable substrate. In contrast, isolated human theca cells do not produce substantial amounts of estrogens but instead secrete dehydroepiandrosterone, androstenedione, and smaller amounts of testosterone when adenylate cyclase activity is stimulated. The aromatase activity of granulosa cells is estimated to be at least 700 times greater in the granulosa cells of large preovulatory follicles than in theca cells, arguing strongly for the cellular compartmentalization of estrogen synthesis outlined in the two-cell–two-gonadotropin model.

Estradiol undergoes metabolism in the ovary, generating a number of hydroxylated molecules (e.g., 2-hydroxyestradiol, 4-hydroxyestradiol), some undergoing further metabolism to methylated steroids by catechol-O-methyl transferase (e.g., 2-methoxyestradiol). These estrogen metabolites display pro- or antiangiogenic activity and influence oocyte maturation and the development of oocyte competence (see Chapter 4 ).

Inhibin, a member of the TGF-β protein superfamily, is a heterodimeric 32-kDa glycoprotein composed of two subunits—α and β—linked by disulfide bonds. There is a single common α subunit, but there are different β subunits, denoted β _A and β _B (see Chapter 6 ). The αβ _A and αβ _B heterodimers are named inhibin A and B, respectively. In the ovary, the primary source of inhibin is granulosa cells. The main endocrine role of inhibin, for which it was discovered and named, is to suppress pituitary FSH production. In vitro , inhibin augments LH- and IGF-stimulated androgen production by theca cells, and elevated inhibin production by granulosa cells in polycystic ovary syndrome (PCOS) may contribute to the hyperandrogenemia associated with this condition.

Although both isoforms of inhibin have similar biological properties, their synthesis is regulated differently during the follicular and luteal phases. Inhibin B is secreted mainly during the early follicular phase, with levels decreasing in the midfollicular phase and subsequently declining further after the LH surge. Concentrations of inhibin A are low during the first half of the follicular phase but increase during the midfollicular phase and peak during the luteal phase. The differential production of inhibin A and inhibin B is supported by measurements made on follicles of different sizes that showed that inhibin A levels rise with increasing follicular size while inhibin B levels reach a peak in follicles of 9 to 10 mm. Activins are related TGF-β superfamily members formed of dimers of inhibin subunits (e.g., activin A is a dimer of inhibin A subunits) (see Chapter 6 ). They are produced by granulosa cells and oocytes and appear to have mainly autocrine or paracrine roles in follicular development and corpus luteum function. A binding protein, follistatin, blocks activin activity.

Granulosa cells produce other members of the TGF-β superfamily that have local as well as endocrine roles, including anti-müllerian hormone (AMH, also known as müllerian inhibiting substance), which plays important roles in follicular dynamics by restraining the entry of primordial follicles into the growing pool (see Chapter 6 ). AMH is expressed by granulosa cells at the time primordial follicles are recruited to grow into the preantral stages. It is produced as a preprohormone that is activated by proteolytic processing at the N-terminus. However, the cleaved pro-region remains associated with the mature AMH. The highest AMH expression is observed in preantral and small antral follicles. Consequently, AMH levels in serum reflect the number of growing follicles in the size range of 2 to 9 mm and are used as a biomarker of ovarian reserve. In the mouse ovary, the absence of AMH causes primordial follicles to be recruited more rapidly, resulting in the early exhaustion of the follicular endowment. AMH also reduces the sensitivity of follicles to FSH and the number of LH receptors on granulosa cells. Based on its activities, AMH has been proposed to be involved in the pathophysiology of PCOS (see Chapter 22 ). Although some adult extraovarian tissues express AMH receptors (AMHR2), including the human endometrium and adrenal cortex, the roles of AMH in female reproduction outside of the ovary have yet to be elucidated.

Granulosa Cell Phenotypic Heterogeneity

Granulosa cells display different phenotypes within the follicle, depending on their location. The mural granulosa cells located near the basal lamina, granulosa cells lining the antrum, and cumulus granulosa cells each have distinguishing features that are determined, in part, by their relative proximity to the oocyte and theca cells and, consequently the paracrine substances released by the oocyte and theca cells. The mural granulosa cells in the antral follicle express the greatest steroidogenic activity ( Fig. 8.9 ). In addition, mural granulosa cells in the preovulatory follicle have the highest level of LH receptors, but there is heterogeneity in LH receptor expression, with receptors being absent from inner mural cells, and present in 13% to 48% of the outer mural granulosa cells. In contrast, natriuretic peptide receptor 2 (NPR2), which produces the cGMP that inhibits resumption of meiosis, is present throughout the follicle but more concentrated in the cumulus cells.

The cumulus cells encapsulating mature mouse oocytes are more transcriptionally synchronized when compared with cumulus cells surrounding immature oocytes. They have a distinctive molecular signature that includes expression of Slc38a3 , which encodes a sodium-coupled neutral amino acid transporter, and higher expression of AMH and secreted frizzled-related protein-4. Cumulus cells and granulosa cells close to the antrum do not express DNA damage-inducible transcript 4-like (DDIT4L), a suppressor of mechanistic target of rapamycin (mTOR) signaling, whereas mural granulosa cells express high levels of this protein. As a result, the cellular metabolism regulator, mTOR, is activated in the cells closest to the oocyte, which improves the transfer of nutrients to the oocyte and increases oocyte developmental competence.

Cumulus cells proliferate after the LH surge and are active in producing an extracellular matrix consisting of hyaluronan, proteoglycans, and proteoglycan-binding proteins when stimulated by the prostaglandins generated in response to the ovulatory stimulus. The elaboration of this matrix leads to the preovulatory expansion of the cumulus-oocyte complex, which is essential for ovulation. Differential patterns of expression of prostaglandin E (EP) receptors allow granulosa cell subpopulations to respond uniquely to PGE ₂ during ovulation (see Chapter 4 ).

Luteinized granulosa cells of the ovulated follicle undergo terminal differentiation to give rise to the large luteal cell population of the corpus luteum. These cells have an increased capacity to synthesize progesterone, resulting from upregulation of the machinery required to acquire cholesterol from the circulating lipoproteins, which now have access to the granulosa-lutein cells because of the neovascularization of the developing corpus luteum. The granulosa-lutein cells also retain the capacity to synthesize estrogens from androgen precursors produced by theca lutein cells.