Reactive oxygen species signalling in the deterioration of quality of mammalian oocytes cultured in vitro: Protective effect of antioxidants.

The in vitro fertilization (IVF) is the first choice of infertile couples worldwide to plan for conception. Besides having a significant advancement in IVF procedure, the success rate is still poor. Although several approaches have been tested to improve IVF protocol, minor changes in culture conditions, physical factors and/or drug treatment generate reactive oxygen species (ROS) in oocytes. Due to large size and huge number of mitochondria, oocyte is more susceptible towards ROS-mediated signalling under in vitro culture conditions. Elevation of ROS levels destabilize maturation promoting factor (MPF) that results in meiotic exit from diplotene as well as metaphase-II (M-II) arrest in vitro. Once meiotic exit occurs, these oocytes get further arrested at metaphase-I (M-I) stage or metaphase-III (M-III)-like stage under in vitro culture conditions. The M-I as well as M-III arrested oocytes are not fit for fertilization and limits IVF outcome. Further, the generation of excess levels of ROS cause oxidative stress (OS) that initiate downstream signalling to initiate various death pathways such as apoptosis, autophagy, necroptosis and deteriorates oocyte quality under in vitro culture conditions. The increase of cellular enzymatic antioxidants and/or supplementation of exogenous antioxidants in culture medium protect ROS-induced deterioration of oocyte quality in vitro. Although a growing body of evidence suggests the ROS and OS-mediated deterioration of oocyte quality in vitro, their downstream signalling and related mechanisms remain poorly understood. Hence, this review article summarizes the existing evidences concerning ROS and OS-mediated downstream signalling during deterioration of oocyte quality in vitro. The use of various antioxidants against ROS and OS-mediated impairment of oocyte quality in vitro has also been explored in order to increase the success rate of IVF during assisted reproductive health management.

Meiotic Instability Generates a Pathological Condition in Mammalian Ovum.

Maintenance of metaphase-II (M-II) arrest in ovum is required to present itself as a right gamete for successful fertilization in mammals. Surprisingly, instability of meiotic cell cycle results in spontaneous exit from M-II arrest, chromosomal scattering and incomplete extrusion of second polar body (PB-II) without forming pronuclei so called abortive spontaneous ovum activation (SOA). It remains unclear what causes meiotic instability in freshly ovulated ovum that results in abortive SOA. We propose the involvement of various signal molecules such as reactive oxygen species (ROS), cyclic 3',5' adenosine monophosphate (cAMP) and calcium (Ca2+) in the induction of meiotic instability and thereby abortive SOA. These signal molecules through their downstream pathways modulate phosphorylation status and activity of cyclin dependent kinase (cdk1) as well as cyclin B1 level. Changes in phosphorylation status of cdk1 and its activity, dissociation and degradation of cyclin B1 destabilize maturation promoting factor (MPF). The premature MPF destabilization and defects in other cell cycle regulators possibly cause meiotic instability in ovum soon after ovulation. The meiotic instability results in a pathological condition of abortive SOA and deteriorates ovum quality. These ova are unfit for fertilization and limit reproductive outcome in several mammalian species including human. Therefore, global attention is required to identify the underlying causes in greater details in order to address the problem of meiotic instability in ova of several mammalian species icluding human. Moreover, these activated ova may be used to create parthenogenetic embryonic stem cell lines in vitro for the use in regenerative medicine.Graphical abstract.

Cyclic nucleotide phosphodiesterase inhibitors: possible therapeutic drugs for female fertility regulation.

Cyclic nucleotide phosphodiesterases (PDEs) are group of enzymes responsible for the hydrolysis of cyclic adenosine 3', 5' monophosphate (cAMP) and cyclic guanosine 3', 5' monophosphate (cGMP) levels in wide variety of cell types. These PDEs are detected in encircling granulosa cells or in oocyte with in follicular microenvironment and responsible for the decrease of cAMP and cGMP levels in mammalian oocytes. A transient decrease of cAMP level initiates downstream pathways to cause spontaneous meiotic resumption from diplotene arrest and induces oocyte maturation. The nonspecific PDE inhibitors (caffeine, pentoxifylline, theophylline, IBMX etc.) as well as specific PDE inhibitors (cilostamide, milrinone, org 9935, cilostazol etc.) have been used to elevate cAMP level and inhibit meiotic resumption from diplotene arrest and oocyte maturation, ovulation, fertilization and pregnancy rates both in vivo as well as under in vitro culture conditions. The PDEs inhibitors are used as powerful experimental tools to demonstrate cyclic nucleotide mediated changes in ovarian functions and thereby fertility. Indeed, non-hormonal nature and reversible effects of nonspecific as well as specific PDE inhibitors hold promise for the development of novel therapeutic drugs for female fertility regulation.

Cilostamide and rolipram prevent spontaneous meiotic resumption from diplotene arrest in rat oocytes cultured in vitro.

The involvement of specific phosphodiesterases (PDEs) in the modulation of cAMP and thereby spontaneous meiotic resumption remains poorly understood. This work aims to evaluate the effects of cilostamide and rolipram (PDE 3A and PDE 4D inhibitors) on spontaneous meiotic resumption from diplotene arrest in rat oocytes cultured in vitro. For this purpose, diplotene-arrested cumulus oocyte complexes (COCs) were collected from rat ovary. The COCs and denuded oocytes were exposed to various concentrations of cilostamide (0.0, 2.5, 5.0 and 10 µM) and rolipram (0, 10, 50 and 100 µM) for various times (0, 3, 5, 7, 14, 16, 18, 20, 22 and 24 h). Cilostamide inhibited spontaneous meiotic resumption in a concentration- and time-dependent manner in COCs and denuded oocytes. Although rolipram showed inhibition of spontaneous meiotic resumption up to some extent, cilostamide was more potent to prevent spontaneous meiotic resumption in both COCs and denuded oocytes. Cilostamide significantly reduced PDE 3A expression, increased cAMP level and prevented spontaneous meiotic resumption in COCs and denuded oocytes. Although rolipram inhibited PDE 4D expression in cumulus cells, increased cAMP level but was not sufficient to prevent spontaneous meiotic resumption. We conclude that both drugs prevent spontaneous resumption from diplotene-arrest through PDE 3A/PDE 4D-cAMP mediated pathway. However, as compare to rolipram, cilostamide was more potent in preventing spontaneous resumption from diplotene-arrest in rat oocytes cultured in vitro. Thus, cilostamide could be used as a potential candidate for the development of female contraceptive drug in future.

Autophagy in hypoxic ovary.

Oxygen deprivation affects human health by modulating system as well as cellular physiology. Hypoxia generates reactive oxygen species (ROS), causes oxidative stress and affects female reproductive health by altering ovarian as well as oocyte physiology in mammals. Hypoxic conditions lead to several degenerative changes by inducing various cell death pathways like autophagy, apoptosis and necrosis in the follicle of mammalian ovary. The encircling somatic cell death interrupts supply of nutrients to the oocyte and nutrient deprivation may result in the generation of ROS. Increased level of ROS could induce granulosa cells as well as oocyte autophagy. Although autophagy removes damaged proteins and subcellular organelles to maintain the cell survival, irreparable damages could induce cell death within intra-follicular microenvironment. Hypoxia-induced autophagy is operated through 5' AMP activated protein kinase-mammalian target of rapamycin, endoplasmic reticulum stress/unfolded protein response and protein kinase C delta-c-junN terminal kinase 1 pathways in a wide variety of somatic cell types. Similar to somatic cells, we propose that hypoxia may induce granulosa cell as well as oocyte autophagy and it could be responsible at least in part for germ cell elimination from mammalian ovary. Hypoxia-mediated germ cell depletion may cause several reproductive impairments including early menopause in mammals.

Necroptosis in stressed ovary.

Stress is deeply rooted in the modern society due to limited resources and large competition to achieve the desired goal. Women are more frequently exposed to several stressors during their reproductive age that trigger generation of reactive oxygen species (ROS). Accumulation of ROS in the body causes oxidative stress (OS) and adversely affects ovarian functions. The increased OS triggers various cell death pathways in the ovary. Beside apoptosis and autophagy, OS trigger necroptosis in granulosa cell as well as in follicular oocyte. The OS could activate receptor interacting protein kinase-1(RIPK1), receptor interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) to trigger necroptosis in mammalian ovary. The granulosa cell necroptosis may deprive follicular oocyte from nutrients, growth factors and survival factors. Under these conditions, oocyte becomes more susceptible towards OS-mediated necroptosis in the follicular oocytes. Induction of necroptosis in encircling granulosa cell and oocyte may lead to follicular atresia. Indeed, follicular atresia is one of the major events responsible for the elimination of majority of germ cells from cohort of ovary. Thus, the inhibition of necroptosis could prevent precautious germ cell depletion from ovary that may cause reproductive senescence and early menopause in several mammalian species including human.

Necrosis and necroptosis in germ cell depletion from mammalian ovary.

The maximum number of germ cells is present during the fetal life in mammals. Follicular atresia results in rapid depletion of germ cells from the cohort of the ovary. At the time of puberty, only a few hundred (<1%) germ cells are either culminated into oocytes or further get eliminated during the reproductive life. Although apoptosis plays a major role, necrosis as well as necroptosis, might also be involved in germ cell elimination from the mammalian ovary. Both necrosis and necroptosis show similar morphological features and are characterized by an increase in cell volume, cell membrane permeabilization, and rupture that lead to cellular demise. Necroptosis is initiated by tumor necrosis factor and operated through receptor interacting protein kinase as well as mixed lineage kinase domain-like protein. The acetylcholinesterase, cytokines, starvation, and oxidative stress play important roles in necroptosis-mediated granulosa cell death. The granulosa cell necroptosis directly or indirectly induces susceptibility toward necroptotic or apoptotic cell death in oocytes. Indeed, prevention of necrosis and necroptosis pathways using their specific inhibitors could enhance growth/differentiation factor-9 expression, improve survivability as well as the meiotic competency of oocytes, and prevent decline of reproductive potential in several mammalian species and early onset of menopause in women. This study updates the information and focuses on the possible involvement of necrosis and necroptosis in germ cell depletion from the mammalian ovary.

Role of granulosa cell mitogen-activated protein kinase 3/1 in gonadotropin-mediated meiotic resumption from diplotene arrest of mammalian oocytes.

In mammals, preovulatory oocytes are encircled by several layers of granulosa cells (GCs) in follicular microenvironment. These follicular oocytes are arrested at diplotene arrest due to high level of cyclic nucleotides from encircling GCs. Pituitary gonadotropin acts at the level of encircling GCs and increases adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) and activates mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. The MAPK3/1 disrupts the gap junctions between encircling GCs and oocyte. The disruption of gap junctions interrupts the transfer of cyclic nucleotides to the oocyte that results a drop in intraoocyte cAMP level. A transient decrease in oocyte cAMP level triggers maturation promoting factor (MPF) destabilization. The destabilized MPF finally triggers meiotic resumption from diplotene arrest in follicular oocyte. Thus, MAPK3/1 from GCs origin plays important role in gonadotropin-mediated meiotic resumption from diplotene arrest in follicular oocyte of mammals.

Impact of stress on female reproductive health disorders: Possible beneficial effects of shatavari (Asparagus racemosus).

Stress is deeply rooted in the society and women are frequently exposed to psychological, physical and physiological stressors. Psychological stress disturbs reproductive health by inducing generation of reactive oxygen species (ROS) and thereby oxidative stress (OS). The increased OS may affect physiology of ovary, oocyte quality and cause female reproductive health disorders. To overcome stress-mediated reproductive health disorders in women, shatavari (Asparagus racemosus) is frequently recommended in Ayurvedic system of medicine. Although shatavari is one of the major health tonics and most popular rasayana drugs to treat reproductive ailments of women, underlying mechanism of shatavari action at the level of ovary remains poorly understood. Based on the existing studies, we propose that shatavari may improve female reproductive health complications including hormonal imbalance, polycystic ovarian syndrome (PCOS), follicular growth and development, oocyte quality and infertility possibly by reducing OS level and increasing antioxidants level in the body. Further studies are required to elucidate the mechanism of shatavari actions at the level of ovary and oocyte that directly impacts the reproductive health of women.

Germ cell depletion from mammalian ovary: possible involvement of apoptosis and autophagy.

Mammalian ovary contains millions of germ cells during embryonic life but only few of them are culminated into oocytes that achieve meiotic competency just prior to ovulation. The majority of germ cells are depleted from ovary through several pathways. Follicular atresia is one of the major events that eliminate germ cells from ovary by engaging apoptotic as well as non-apoptotic pathways of programmed cell death. Apoptosis is characterized by several morphological changes that include cell shrinkage, nuclear condensation, membrane blebbing and cytoplasmic fragmentation by both mitochondria- as well as death receptor-mediated pathways in encircling granulosa cells and oocyte. Although necroapoptosis have been implicated in germ cell depletion, autophagy seems to play an active role in the life and death decisions of ovarian follicles. Autophagy is morphologically characterized by intracellular reorganization of membranes and increased number of autophagic vesicles that engulf bulk cytoplasm as well as organelles. Autophagy begins with the encapsulation of cytoplasmic constituents in a membrane sac known as autophagosomes. The autophagic vesicles are then destroyed by the lysosomal enzymes such as hydrolases that results in follicular atresia. It seems that apoptosis as well as autophagy could play active roles in germ cells depletion from ovary. Hence, it is important to prevent these two pathways in order to retain the germ cells in ovary of several mammalian species that are either threatened or at the verge of extinction. The involvement of apoptosis and autophagy in germ cell depletion from mammalian ovary is reviewed and possible pathways have been proposed.

Journey of oocyte from metaphase-I to metaphase-II stage in mammals.

In mammals, journey from metaphase-I (M-I) to metaphase-II (M-II) is important since oocyte extrude first polar body (PB-I) and gets converted into haploid gamete. The molecular and cellular changes associated with meiotic cell cycle progression from M-I to M-II stage and extrusion of PB-I remain ill understood. Several factors drive oocyte meiosis from M-I to M-II stage. The mitogen-activated protein kinase3/1 (MAPK3/1), signal molecules and Rho family GTPases act through various pathways to drive cell cycle progression from M-I to M-II stage. The down regulation of MOS/MEK/MAPK3/1 pathway results in the activation of anaphase-promoting complex/cyclosome (APC/C). The active APC/C destabilizes maturation promoting factor (MPF) and induces meiotic resumption. Several signal molecules such as, c-Jun N-terminal kinase (JNK2), SENP3, mitotic kinesin-like protein 2 (MKlp2), regulator of G-protein signaling (RGS2), Epsin2, polo-like kinase 1 (Plk1) are directly or indirectly involved in chromosomal segregation. Rho family GTPase is another enzyme that along with cell division cycle (Cdc42) to form actomyosin contractile ring required for chromosomal segregation. In the presence of origin recognition complex (ORC4), eccentrically localized haploid set of chromosomes trigger cortex differentiation and determine the division site for polar body formation. The actomyosin contractile activity at the site of division plane helps to form cytokinetic furrow that results in the formation and extrusion of PB-I. Indeed, oocyte journey from M-I to M-II stage is coordinated by several factors and pathways that enable oocyte to extrude PB-I. Quality of oocyte directly impact fertilization rate, early embryonic development, and reproductive outcome in mammals.

Role of Mitogen Activated Protein Kinase and Maturation Promoting Factor During the Achievement of Meiotic Competency in Mammalian Oocytes.

The oocyte quality remains as one of the major problems associated with poor in vitro fertilization (IVF) rate and assisted reproductive technology (ART) failure worldwide. The oocyte quality is dependent on its meiotic maturation that begins inside the follicular microenvironment and gets completed at the time of ovulation in most of the mammalian species. Follicular oocytes are arrested at diplotene stage of first meiotic prophase. The resumption of meiosis from diplotene arrest, progression through metaphase-I (M-I) and further arrest at metaphase-II (M-II) are important physiological requirements for the achievement of meiotic competency in mammalian oocytes. The achievement of meiotic competency is dependent upon cyclic stabilization/destabilization of maturation promoting factor (MPF). The mitogen-activated protein kinase3/1 (MAPK3/1) modulates stabilization/destabilization of MPF in oocyte by interacting either with signal molecules, transcription and post-transcription factors in cumulus cells or cytostatic factors (CSFs) in oocyte. MPF regulates meiotic cell cycle progression from diplotene arrest to M-II arrest and directly impacts oocyte quality. The MAPK3/1 activity is not reported during spontaneous meiotic resumption but its activity in cumulus cells is required for gonadotropin-induced oocyte meiotic resumption. Although high MAPK3/1 activity is required for the maintenance of M-II arrest in several mammalian species, its cross-talk with MPF remains to be elucidated. Further studies are required to find out the MAPK3/1 activity and its impact on MPF destabilization/stabilization during achievement of meiotic competency, an important period that decides oocyte quality and directly impacts ARTs outcome in several mammalian species including human. J. Cell. Biochem. 119: 123-129, 2018. © 2017 Wiley Periodicals, Inc.

Human Chorionic Gonadotropin Mediated Generation of Reactive Oxygen Species Is Sufficient to Induce Meiotic Exit but Not Apoptosis in Rat Oocytes.

Generation of reactive oxygen species (ROS) is associated with final stages of follicular development and ovulation in mammals. The human chorionic gonadotropin (hCG) mimics the action of luteinizing hormone and triggers follicular development and ovulation. However, it remains unclear whether hCG induces generation of ROS, if yes, whether hCG-mediated increased level of ROS could induce meiotic exit and/or apoptosis in rat oocytes. For this purpose, cumulus-oocyte complexes (COCs) were collected from ovary of experimental rats injected with 20 IU pregnant mare's serum gonadotropin for 48 h followed by 20 IU hCG for 0, 7, 14, and 21 h. The morphological changes in COCs, meiotic status of oocyte, total ROS, hydrogen peroxide (H2O2), inducible nitric oxide synthase (iNOS), nitric oxide (NO), Bax, Bcl-2, cytochrome c, telomerase reverse transcriptase (TERT) expression levels, and DNA fragmentation were analyzed in COCs. Our data suggest that hCG surge increased total ROS as well as H2O2 levels but decreased iNOS expression and total NO level in oocytes. The hCG-mediated increased level of ROS was sufficient to induce meiotic cell cycle resumption in majority of oocytes as evidenced by meiotic exit from diplotene as well as metaphase-II (M-II) arrest and their meiotic status. However, increase of ROS level due to hCG surge was not sufficient to trigger Bax and cytochrome c expression levels and DNA fragmentation in COCs. In addition, increased TERT activity was observed in oocytes collected 21 h post-hCG surge showing onset of oocyte aging. Taken together, these results suggest that hCG induces generation of ROS sufficient to trigger meiotic exit from diplotene, as well as M-II arrest, but not good enough to induce apoptosis in rat oocytes.

Reduction of nitric oxide level results in maturation promoting factor destabilization during spontaneous meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro.

Nitric oxides (NO) act as one of the major signal molecules and modulate various cell functions including oocyte meiosis in mammals. The present study was designed to investigate the mechanism of NO action during spontaneous meiotic exit from diplotene arrest (EDA) in rat cumulus oocytes complexes (COCs) cultured in vitro. Diplotene-arrested COCs collected from ovary of immature female rats after 20 IU pregnant mare's serum gonadotropins (PMSG) for 48 h were exposed to various concentrations of NO donor, S-nitroso-N-acetyl penicillamine (SNAP) and inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG) for 3 h in vitro and downstream factors were analyzed. Our results suggest that SNAP inhibited, while AG induced EDA in a concentration-dependent manner. The iNOS-mediated total NO, cyclic nucleotides and cell division cycle 25B (Cdc25B) levels were reduced significantly. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated cyclin-dependent kinase 1 (Cdk1) level and decreased Thr161 phosphorylated Cdk1 as well as cyclin B1 levels leading to maturation promoting factor (MPF) destabilization. The destabilized MPF finally induced spontaneous EDA. Taken together, these results suggest that reduction of iNOS-mediated NO level destabilizes MPF during spontaneous EDA in rat COCs cultured in vitro.

Maturation promoting factor destabilization mediates human chorionic gonadotropin induced meiotic resumption in rat oocytes.

Human chorionic gonadotropin (hCG) mimics the action of luteinizing hormone (LH) and triggers meiotic maturation and ovulation in mammals. The mechanism by which hCG triggers meiotic resumption in mammalian oocytes remains poorly understood. We aimed to find out the impact of hCG surge on morphological changes, adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), Wee1, early mitotic inhibitor 2 (Emi2), anaphase-promoting complex/cyclosome (APC/C), meiotic arrest deficient protein 2 (MAD2), phosphorylation status of cyclin-dependent kinase 1 (Cdk1), its activity and cyclin B1 expression levels during meiotic resumption from diplotene as well as metaphase-II (M-II) arrest in cumulus oocyte complexes (COCs). Our data suggest that hCG surge increased cyclic nucleotides level in encircling granulosa cells but decreased their level in oocyte. The reduced intraoocyte cyclic nucleotides level is associated with the decrease of Cdc25B, Thr161 phosphorylated Cdk1 and Emi2 expression levels. On the other hand, hCG surge increased Wee1, Thr14/Tyr15 phosphorylated Cdk1, APC/C as well as MAD2 expression levels. The elevated APC/C activity reduced cyclin B1 level. The changes in phosphorylation status of Cdk1 and reduced cyclin B1 level might have resulted in maturation promoting factor (MPF) destabilization. The destabilized MPF finally triggered resumption of meiosis from diplotene as well as M-II arrest in rat oocytes.

Carbenoxolone reduces cyclic nucleotides level, destabilizes maturation promoting factor and induces meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro.

BACKGROUND: Disruption of gap junction and transfer of cyclic nucleotides to the oocyte lead to meiotic exit from diplotene arrest (EDA) in mammals. In the present study, we examined whether a gap junction blocker, carbenoxolone (CBX) could induce EDA by reducing cyclic nucleotides level and destabilizing maturation promoting factor (MPF) in rat oocytes cultured in vitro. METHODS: Diplotene-arrested cumulus oocyte complexes (COCs) were collected from ovary of immature female rats after 20 IU pregnant mare's serum gonadotropins (PMSG) for 48h. These diplotene-arrested COCs were incubated with various concentration of CBX for 3h in vitro. The morphological changes, meiotic status of oocyte, inducible nitric oxide synthase (iNOS), total nitric oxide (NO), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), changes in specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels were analyzed. RESULTS: CBX induced EDA in a concentration-dependent manner. The iNOS expression, total NO and cyclic nucleotides level were significantly decreased. The reduced cyclic nucleotides level resulted in the decrease of Cdc25B expression level. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated Cdk1 level. However, Thr161 phosphorylated Cdk1 as well as cyclin B1 levels were significantly reduced leading to MPF destabilization. The destabilized MPF finally induced EDA in rat COCs cultured in vitro. CONCLUSIONS: Our results suggest that CBX blocked gap junctions interrupted the transfer of cyclic nucleotides to the oocyte. Reduction of cyclic nucleotides level destabilized MPF and induced EDA in vitro. Thus, CBX could be used to induce meiotic maturation under in vitro culture conditions during assisted reproductive technology (ART) programs.

Nitric oxide signaling during meiotic cell cycle regulation in mammalian oocytes.

Nitric oxide (NO) acts as a major signal molecules and modulate physiology of mammalian oocytes. Ovarian follicles generate large amount of NO through nitric oxide synthase (NOS) pathway to maintain diplotene arrest in preovulatory oocytes. Removal of oocytes from follicular microenvironment or follicular rupture during ovulation disrupt the flow of NO from granulosa cells to the oocyte that results a transient decrease of oocyte cytoplasmic NO level. Decreased NO level reduces cyclic nucleotides level by inactivating guanylyl cyclases directly or indirectly. The reduced cyclic nucleotides level modulate specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and triggers cyclin B1 degradation. These changes result in maturation promoting factor (MPF) destabilization that finally triggers meiotic resumption from diplotene as well as metaphase-II (M-II) arrest in most of the mammalian species.

Abortive Spontaneous Egg Activation: An Emerging Biological Threat for the Existence of Mammals.

Mammals are important for balancing the natural ecosystem, but in the past few decades, several species have rapidly been entered under threatened category worldwide. The environmental changes, loss of natural habitats, human activities, and thereby stress are responsible for a gradual decline in reproductive outcome. Stress induces generation of reactive oxygen species (ROS). High physiological level of ROS drives abortive spontaneous egg activation (SEA), while beyond the physiological level causes oxidative stress (OS). The OS induces apoptosis and deteriorates egg quality that limits reproductive outcome. The reduced reproductive outcome is one of the major causes for gradual decline in population size of several mammalian species. Despite having several conservation programs, a gradual decline in species reproductive outcome and their population size is the serious concern for the existence of threatened mammalian species. Thus, it is important to identify and prevent the underlying causes responsible for abortive SEA, which could be an emerging problem for several mammalian species that are threatened or at the verge of extinction.

Role of Cyclic Nucleotide Phosphodiesterases During Meiotic Resumption From Diplotene Arrest in Mammalian Oocytes.

Cyclic nucleotide phosphodiesterases (PDEs) are group of enzymes that hydrolyze cyclic nucleotides in wide variety of cell types including encircling granulosa cells as well as associated oocytes. One group of PDEs are located in encircling granulosa cells and another group get expressed in the oocyte, while few other PDEs are expressed in both compartments. The PDE1A, PDE4D, PDE5A, PDE8A, and PDE8B are granulosa cell specific PDEs that hydrolyze adenosine 3',5'-cyclic monophosphate (cAMP) as well as guanosine 3',5'-cyclic monophosphate (cGMP) with different affinities. PDE3A, PDE8A as well as PDE9A are expressed in oocyte and specifically responsible for the cyclic nucleotide hydrolysis in the oocyte itself. Few other PDEs such as PDE7B, PDE10A, and PDE11A are either detected in granulosa cells or oocytes. Activation of these PDEs either in encircling granulosa cells or in oocyte directly or indirectly reduces intraoocyte cAMP level. Reduction of intraoocyte cAMP level modulates phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and triggers cyclin B1 degradation that destabilizes maturation promoting factor (MPF) and/or increases Cdk1 activity. The destabilized MPF and/or increased Cdk1 activity leads to resumption of meiosis, which initiates the achievement of meiotic competency in preovulatory follicles of several mammalian species. Use of specific PDEs inhibitors block cyclic nucleotides hydrolysis that results in increase of intraoocyte cyclic nucleotides level, which leads to maintenance of meiotic arrest at diplotene stage in vivo as well as in vitro. Thus, cyclic nucleotide PDEs play important role in the achievement of meiotic competency by reducing intraoocyte cyclic nucleotides level in mammalian oocytes. J. Cell. Biochem. 118: 446-452, 2017. © 2016 Wiley Periodicals, Inc.

Presence of encircling granulosa cells protects against oxidative stress-induced apoptosis in rat eggs cultured in vitro.

Increased oxidative stress (OS) due to in vitro culture conditions can affect the quality of denuded eggs during various assisted reproductive technologies (ARTs). Presence of intact granulosa cells may protect eggs from OS damage under in vitro culture conditions. The present study was aimed to investigate whether encircling granulosa cells could protect against hydrogen peroxide (H2O2)-induced egg apoptosis in ovulated cumulus oocyte complexes (COCs) cultured in vitro. The OS was induced by exposing COCs as well as denuded eggs with various concentrations of H2O2 for 3 h in vitro. The morphological changes, total reactive oxygen species (ROS) as well as catalase expression, Bax/Bcl-2, cytochrome c levels and DNA fragmentation were analysed in COCs as well as denuded eggs. Our results suggest that H2O2 treatment induced morphological apoptotic features in a concentration-dependent manner in denuded eggs cultured in vitro. The 20 µM of H2O2 treatment induced OS by elevating total ROS level, reduced catalase and Bcl-2 expression levels with overexpression of Bax and cytochrome c and induced DNA fragmentation in denuded eggs cultured in vitro. The presence of encircling granulosa cells protected H2O2-induced morphological apoptotic features by preventing the increase of Bax, cytochrome c expression levels and DNA fragmentation in associated egg. However, 20 µM of H2O2 was sufficient to induce peripheral granulosa cell apoptosis in COCs and degeneration in few denuded eggs cultured in vitro. Taken together our data suggest that the presence of encircling granulosa cells could be beneficial to protect ovulated eggs from OS damage under in vitro culture conditions during various ART programs.