Fate of the germ cells in mammalian ovary: A review

INTRODUCTION

Fetal ovary of the mammals contains millions of germ cells and majority of germ cells (>99%) are depleted from cohort of ovary at the time of birth.^[1-4] Elimination of germ cells takes place following various apoptotic as well as non-apoptotic cell death pathways.^[4-6] Existing studies suggest that germ cells are depleted from the cohort of ovary following apoptosis, autophagy, necroptosis, and necrosis-mediated pathways.^[4,7,8] Elimination of poor quality germ cells leads to a quorum sensing mechanism that relies on oocyte health in shaping the germ cells pool in adult ovary. Indeed, rapid depletion of germ cells could be a part of selection, to retain only oocytes of highest quality that has ability to develop into right female gametes.^[9] However, the biological significance of rapid germ cell elimination from mammalian ovary remains poorly understood. Although recent evidences suggest the presence of oocyte like stem cell in adult ovary and opens a new source of oocyte, its quality and fate remain controversial.^[1,3,10]

Ovary is able to retain only <1% of germ cells that are culminated into oogonia.^[11,12] During folliculogenesis, oogonia enters into meiosis and gets converted into primary oocytes. Majority of primary oocytes are arrested at diplotene stage of first meiotic prophase from birth to puberty.^[4,10,12,13] Diplotene arrest in primary oocytes is the longest period of meiosis and may last for several months to years depending on the mammalian species.^[13] At puberty, resumption from diplotene arrest is triggered by pituitary gonadotropins surge.^[14] Once resumed from diplotene arrest, oocyte passes through metaphase-I (M-I) to metaphase-II (M-II) stage by extruding first polar body (PB-I). After ovulation, oocyte remained arrested at M-II stage until fertilization in several mammalian species.^[1,14,15]

Freshly ovulated oocytes in most of the mammalian species are encircled with dispersed cumulus granulosa cells, show clear cytoplasm, absence of germinal vesicle, and possess PBI. These morphological features are normally used for the selection of the right female gamete for successful fertilization in majority of mammalian species including human. However, freshly ovulated oocytes of few mammalian species do not wait for fertilizing spermatozoa and quickly undergo spontaneous exit from M-II arrest by initiating the extrusion of second polar body (PB-II). Although these oocytes initiate extrusion of PB-II, but it never gets completed due to scattered chromosomes in the cytoplasm. The large amount of cytoplasm moves toward PB-II area and generates a pathological condition and limits reproductive outcome in several mammalian species including human. The present review article focuses on the fate of germ cells including its depletion from ovary, conversion into fertilizable oocyte, and generation of pathological condition of spontaneous oocyte activation (SOA) in mammals.

FATE OF GERM CELLS

Human females are born with a fixed number (5–7 millions) of germ cells in the ovary that decreases throughout the postnatal life.^[13] Germ cell numbers decline rapidly and only 400,000 follicles are reported at puberty out of which a few hundred are present at the time of menopause.^[16] Almost 10–20 primordial follicles are selected to get converted into growing follicles and one follicle ovulates in each reproductive cycle, remaining undergo follicular atresia in human.^[17] Therefore, a large number of germ cells are eliminated from the cohort of ovary through follicular atresia.^[17] The follicular atresia may occur through various cell death pathways including apoptosis, necrosis, necroptosis, and autophagy.^[4,18-20]

Apoptosis eliminates the majority of germ cells at all stages of reproductive cycle.^[14,16,21] Both extrinsic and intrinsic pathways are involved during oocyte apoptosis in mammals.^[18,20] A cross-talk between encircling granulosa cells as well as oocyte is important for the survival of both cell types.^[22] Granulosa cell apoptosis enhances the susceptibility of oocyte toward programmed cell death (PCD).^[23] Granulosa cell death interrupts the supply of various nutrients, growth factors, and cyclic nucleotides which enhances the generation of ROS in diplotene-arrested follicular oocyte.^[24,25] The diplotene-arrested immature oocyte is more vulnerable to H₂O₂-induced apoptosis as compare to mature oocyte arrested at M-II.^[26,27] The high level of ROS modulates mitochondria membrane potential (MMP), which triggers the release of cytochrome c from mitochondria that activate upstream and downstream caspases in oocytes. Studies suggest that the reduced phosphorylation of cyclin-dependent kinase-1 at Thr-161 destabilizes maturation promoting factor (MPF) and allows oocyte apoptosis through FasL-mediated pathway.^[28]

Growing bodies of evidences suggest the involvement of necrosis during follicular atresia^[19] and ATP depletion triggers necrosis, while its adequate level promotes apoptosis.^[19,20] Abnormally high level of intracellular calcium ([Ca⁺⁺]i) as well as oxidative stress can cause necrotic death in mouse^[29] and human oocyte.^[30] Growing body of evidences suggests the involvement of necroptosis in germ cells elimination from mammalian ovary.^{[19,22,31,32]} The high levels of ROS inhibit caspases and shift the death pathway toward necroptosis.^[33] Oxidative stress increases the expression of RIPK1 and RIPK3 in granulosa cells of the human ovary that drives cell death pathway toward necroptosis. Acetylcholinesterase causes necroptosis in granulosa cells of the human ovary under stressful conditions.^[18] The necrostatin-1 inhibits necroptosis in mouse oocyte and enhances the expression of growth differentiation factor-9 (GDF-9) as well as Bcl-2.^[19,34]

The germ cell loss from ovary may also occur through nonapoptotic pathways of PCD.^[4,6] For instance, autophagy could be involved in the elimination of germ cells from the ovary.^[4,7] Autophagy is initiated by enclosing cytoplasmic constituent of a cell in a membrane sac autophagosomes.^[35,36] The lysosomal hydrolases degrade autophagic vesicles and their components in a dying cell.^[36] Autophagy, which maintains cellular homeostasis in normal conditions, may be induced by nutrient starvation,^[37] amino acid deprivation,^[38] pathogen infection,^[39] damaged organelles^[40] such as endoplasmic reticulum,^[41] hypoxia,^[42] and under oxidative stress condition.^[43]

By triggering the accumulation of autophagosomes, autophagy can also induce apoptosis in granulosa as well as luteal cells.^[44] This is supported by the observation that the activation of Akt and mTOR inhibits, while downregulation of Akt and mTOR induces granulosa cell autophagy during folliculogenesis.^[45] Follicle-stimulating hormone (FSH)-mediated increase of mTOR activity is suppressed by Akt inhibitor, suggesting that the Akt controls mTOR activity as well as autophagy in rat granulosa cells.^[46] Based on these observations, we propose that autophagy could be involved in follicular atresia and germ cell elimination from the mammalian ovary.

More than 40% of oocyte shows biochemical and morphological features of apoptosis and autophagy, suggesting the elimination of germ cells through a new form of PCD.^[36] Further, inhibition of autophagy increases apoptosis, while inhibition of apoptosis resulted in autophagy.^[47,48] In some cases, both apoptosis and autophagy are reported simultaneously.^[48] Hence, process of apoptosis and autophagy functions together in the elimination of germ cells from prepubertal rat ovary.^[47] In addition, oocyte expresses one or more than 1 markers of autophagy suggesting that autophagy plays an important role during germ cell elimination from rat ovary.^[48]

The nutrient deprivation, temperature, stress, and hypoxia are known inducers of autophagy.^[49] However, hypoxia is a potent stimulus among all to induce autophagy^[50] which is operated through AMPK-mTOR, UPR, and PKC-JNK pathways in variety of somatic cells.^[51,52] Hypoxia may be generated at high altitude and exposure to polluted air.^[53] A follicular oocyte is encircled by several layers of granulosa cells that separate the oocyte from blood supply. Hence, these layers of somatic cells generate barrier for O₂ as well as nutrients supply to the follicular oocyte and thus pO₂ is compromised within the follicular microenvironment. Growing body of evidences suggests that hypoxia affects the function of ovary in several ways. For instance, it may deplete follicular reserve of spiny mouse,^[54] retards luteal growth in sheep ovary^[55] and follicular development in hamster,^[56] and promotes ROS generation and follicular aging in human granulosa cells.^[57,58] Based on these observations, we proposed the involvement of hypoxia-mediated autophagy in the mammalian ovary.^[20,59] Thus, conversion of germ cells into oocytes and finally ovum and their loss during the process of conversion are very important in mammals including humans.^[20,59] The germ cell reserve in the ovary and production of the healthy oocyte during reproductive life is the primary requirement for mammals including human.

FATE OF MEIOTIC EXIT FROM DIPLOTENE ARREST IN FOLLICULAR OOCYTES

High intracellular levels of cyclic nucleotides (cAMP and cGMP) are mainly responsible for the maintenance of diplotene arrest and levels of these cyclic nucleotides are regulated by cyclic nucleotide phosphodiesterases in mammalian follicular oocytes.^[60] The PDEs reduce cAMP and cGMP levels by hydrolyzing the phosphodiester bond in oocyte and granulosa cells. Based on the distribution, characteristics, and substrate specificity, there are several families of PDEs which are reported in mammals. The PDE 3A, one of the most important enzymes among all, regulates meiotic cell cycle in mouse,^[61] rat,^[62] bovine,^[63] and human oocyte.^[64]

Gap junction disruption affects the transfer of cAMP and cGMP from encircling granulosa cells to oocyte. Gap junction disruption is initiated by cyclic nucleotide signaling and operated by MOS/MEK/MAP3/1 pathways. Mitogen-activated protein kinase3/1 (MAPK3/1), a serine threonine (Ser/Thr) kinase, plays important roles in follicular growth, development, and maturation of mammalian ovary.^[65] Activated MAPK3/1 phosphorylates Ser/Thr residues of many substrates that are implicated in stress responses, growth inhibition and apoptosis in mammalian ovary.^[66] MAPK3/1 modulates transcription and post-transcriptional factors, phosphorylation of connexin-43 in the granulosa cells and thus disrupts gap junctions of encircling granulosa cells of pre-ovulatory follicles.^[67]

Gap junction disruptions through MAPK3/1-mediated pathway interrupt the transfer of cAMP and cGMP from granulosa cells to oocyte that results in the decrease of intraoocyte cyclic nucleotides level and initiates downstream pathways to induce MPF destabilization.^[60,68-70] The MPF destabilization results in meiotic resumption from diplotene arrest.^[71,72]

FATE OF MEIOTIC EXIT FROM M-I ARREST IN FOLLICULAR OOCYTES

The oocyte reaches to M-I stage both in vivo and in vitro culture conditions once the meiosis is resumed from diplotene arrest. However, oocyte does not progress beyond the M-I stage under in vitro culture conditions in rat oocytes.^{[10,12,73,74]} It is still obscure that why diplotene arrested follicular oocytes do not advanced beyond M-I under in vitro culture conditions. The human chorionic gonadotropin surge activates meiotic cell cycle progression from M-I stage and then oocyte achieves physiological arrest at M-II stage and extrudes PB-I.^[10,73,74] Polar body formation is an important step to form haploid set of chromosomes in a female gamete. Mammalian oocytes undergo several crucial processes, including meiotic spindle formation and migration, chromosomal segregation, and polar body extrusion. Various signal molecules such as c-Jun N-terminal kinase (JNK2), sentrin-specific protease 3, mitotic kinesin-like protein 2, a regulator of G-protein signaling (RGS2), Epsin2, and polo-like kinase 1 regulate meiotic spindle organization and chromosomal segregation.^[21]

The functional and active spindle assembly checkpoint proteins such as Bub 1, Bub 2, and Mad2 kinases are present to ensure the correct segregation of homologous chromosomes and trigger a cell cycle arrest in metaphase if there is any incorrect chromosomal attachment. For instance, overexpression of Mad2 leads to meiotic cell cycle arrest at M-I with abnormal spindle formation.^[75] On the other hand, RanGTP gradient induces cortical differentiation and regulates actin cytoskeleton during M-I arrest in oocyte.

Both Rho family GTPase and Cdc-42 play important roles in the formation and extrusion of PB-I.^[76] Growing body of evidences suggests that origin recognition complex (ORC4) helps in the removal of chromosomes during PB-I formation. Thus, meiotic cell cycle progression from M-I to M-II is critical since the oocyte prepares itself to release PB-I and get converted into the right female gamete which is the right choice for fertilization.^[14,75,77]

FATE OF MEIOTIC EXIT FROM M-II ARREST AT THE TIME OF OVULATION

Oocytes at the time of ovulation are normally arrested at M-II stage in mammals. They are morphologically characterized by the complete extrusion of PB-I. These ovulated oocytes wait for fertilization in the ampulla of the fallopian tube for several hours. However, in the absence of fertilization within the required window period, they undergo postovulatory aging-mediated spontaneous exit from M-II arrest.^[78] These oocytes can be morphologically characterized by incomplete extrusion of PB-II so-called abortive SOA.^[78-80] Studies suggest that several molecular changes are involved including cytosolic Ca⁺⁺ and ROS levels.^[79,80] Moderate increase of cytosolic free Ca⁺⁺ is one of the important participants that lead to postovulatory aging-mediated abortive exit from M-II arrest in rat oocytes.^[79,80] In aging oocytes under in vitro conditions, it has been observed that the increase of cytosolic free Ca⁺⁺ level elevates ROS level.^[79] Further, the elevated ROS levels disturb MMP in rat oocytes.^[79] Increased levels of both Ca⁺⁺ and ROS modulate cytostatic factors and result in MPF destabilization.^[81] The increase in cytosolic free Ca⁺⁺ level also reduces Emi2 level but increases p268-CaMK-II level to destabilize MPF. The destabilized MPF finally results in postovulatory aging-mediated abortive SOA with the initiation of extrusion of PB-II.^[81] Indeed, postovulatory aging-induced abortive SOA is a pathological condition that makes oocytes unfit for fertilization and limits assisted reproductive technologies outcome in mammals.

CONCLUSION

Ovary contains millions of germ cells and their fate decisions start at the time of birth itself. The poor quality and defective germ cells are eliminated rapidly through apoptosis, autophagy, necrosis, and necroptosis cell death pathways. Only few germ cells are culminated into oocytes that are arrested at diplotene stage until puberty. The fate of diplotene arrest starts with the gonadotropin surge during puberty. Oocyte passes through M-I to M-II and extrudes PB-I at the time of ovulation. In few species, oocytes do not wait for fertilizing spermatozoa and quickly undergo abortive SOA, leading to deterioration in oocyte quality. Thus, only few oocytes are selectively recruited having fertilization ability, while majority of poor quality germ cells as well as oocyte are either eliminated or become useless for mammals including human.