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.

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.

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.

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.

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.

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.

Impact of stress on oocyte quality and reproductive outcome.

Stress is an important factor that affects physical and mental status of a healthy person disturbing homeostasis of the body. Changes in the lifestyle are one of the major causes that lead to psychological stress. Psychological stress could impact the biology of female reproduction by targeting at the level of ovary, follicle and oocyte. The increased level of stress hormone such as cortisol reduces estradiol production possibly by affecting the granulosa cell functions within the follicle, which results deterioration in oocyte quality. Adaptation of lifestyle behaviours may generate reactive oxygen species (ROS) in the ovary, which further affects female reproduction. Balance between level of ROS and antioxidants within the ovary are important for maintenance of female reproductive health. Physiological level of ROS modulates oocyte functions, while its accumulation leads to oxidative stress (OS). OS triggers apoptosis in majority of germ cells within the ovary and even in ovulated oocytes. Although both mitochondria- as well as death-receptor pathways are involved in oocyte apoptosis, OS-induced mitochondria-mediated pathway plays a major role in the elimination of majority of germ cells from ovary. OS in the follicular fluid deteriorates oocyte quality and reduces reproductive outcome. On the other hand, antioxidants reduce ROS levels and protect against OS-mediated germ cell apoptosis and thereby depletion of germ cells from the ovary. Indeed, OS is one of the major factors that has a direct negative impact on oocyte quality and limits female reproductive outcome in several mammalian species including human.

Apoptosis in mammalian oocytes: a review.

Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

Changes in signal molecules and maturation promoting factor levels associate with spontaneous resumption of meiosis in rat oocytes.

The present study was aimed to find out changes in signal molecules and maturation promoting factor (MPF) levels during meiotic cell cycle progression from diplotene and metaphase-II (M-II) arrest, a period during which oocyte achieves meiotic competency. Data suggest that high levels of adenosine 3'-5'-cyclic monophosphate (cAMP), guanosine 3'-5'-cyclic monophosphate (cGMP), and nitric oxide (NO) are associated with diplotene arrest, while reduction in their levels correlates with reduced MPF level and meiotic resumption from diplotene arrest. On the other hand, increased intracellular NO, calcium (Ca(2+) ) as well as hydrogen peroxide (H2 O2 ) levels correlate with decreased cAMP, Thr-161 phosphorylated cyclin-dependent kinase-1 (Cdk1) as well as cyclin B1 levels. The decreased Thr-161 phosphorylated Cdk1 and cyclin B1 level reduce MPF level leading to exit from M-II arrest in oocytes cultured in vitro. These data suggest that the decrease of cAMP level and increase of cytosolic free Ca(2+) as well as H2 O2 levels associate with the reduced MPF level and meiotic resumption from diplotene arrest. On the other hand, increase of NO, cGMP, Ca(2+) as well as H2 O2 levels are associated with reduced MPF and spontaneous exit from M-II arrest in rat oocytes cultured in vitro.

Reduction of nitric oxide level leads to spontaneous resumption of meiosis in diplotene-arrested rat oocytes cultured in vitro.

The present study was aimed to investigate whether a decrease of nitric oxide (NO) level is beneficial for sponateous resumptiom of meiosis in diplotene-arrested oocytes cultured in vitro. For this purpose, diplotene-arrested oocytes were collected from ovary of immature female rats after a single subcutaneous injection of 20 IU pregnant mare's serum gonadotropins (PMSG) for 48 h. In vitro effects of S-nitroso-l-acetyl penicillamine (SNAP; an NO donor) and aminoguanidine (AG; an inducible NOS [iNOS] inhibitor), intracellular NO, cyclic guanosine monophosphate (cGMP), Cdc25B, Thr-14/Tyr-15 and Thr-161 phosphorylated cyclin-dependent kinase-1 (CDK1), and cyclin B1 levels were analyzed. The SNAP inhibited spontaneous meiotic resumption form diplotene arrest in a concentration-dependent manner, while AG-induced meiotic resumption form diplotene in 0.1 mmol/L 3-isobutyl-1-methylxanthine (IBMX)-treated oocytes in a concentration-dependent manner. The intracellular NO as well as cGMP levels were decreased significantly during spontaneous meiotic resumption from diplotene arrest. The reduction of Cdc25B expression level was associated with the accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. However, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels were reduced significantly during meiotic resumption from diplotene arrest. Taken together, these data suggest that the inhibition of iNOS expression leads to a decrease of NO and cGMP levels thereby decreasing Cdc25B level. The reduced CDC25 B level leads to accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. As a result, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels are decreased leading to maturation-promoting factor (MPF) inactivation. The inactive MPF finally induced meiotic resumption from diplotene stage in rat oocytes cultured in vitro.

A moderate increase of hydrogen peroxide level is beneficial for spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

Hydrogen peroxide (H2O2) acts as a signaling molecule and modulates various aspects of cell functions in a wide variety of cells including mammalian germ cells. We examined whether a decreased level of intra-oocyte cyclic 3',5'-adenosine monophosphate (cAMP) leads to accumulation of H2O2, and if so, whether a moderate increase of H2O2 inactivates maturation promoting factor (MPF) during spontaneous resumption of meiosis in rat oocytes cultured in vitro. Removal of cumulus cells and culture of denuded oocytes in vitro significantly decreased oocyte cAMP level and led to spontaneous meiotic resumption from diplotene arrest. The reduced oocyte cAMP level was associated with an increased oocyte H2O2 level and reduced catalase activity. Exogenous supplementation of H2O2 induced meiotic resumption from diplotene arrest in a concentration- and time-dependent manner in oocytes treated with 0.1 mM of 3-isobutyl-1-methylxanthine, while dibutyryl-cAMP and 3-t-butyl-4-hydroxyanisole inhibited the stimulatory effect of exogenous H2O2. The increased intra-oocyte H2O2 level induced Thr-14/Tyr-15 phosphorylation of CDK1, while Thr-161 phosphorylated CDK1 and cyclin B1 levels were reduced significantly. These results suggest that a decreased level of intra-oocyte cAMP is associated with an increased level of H2O2. The increased level of H2O2 was associated with high phosphorylation of Thr-14/Tyr-15 and dephosphorylation of the Thr-161 residue of CDK1 and reduced the cyclin B1 level, which eventually inactivated MPF. The MPF inactivation triggered spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

Melatonin protects against clomiphene citrate-induced generation of hydrogen peroxide and morphological apoptotic changes in rat eggs.

The present study was aimed to determine whether clomiphene citrate-induces generation of hydrogen peroxide in ovary, if so, whether melatonin could scavenge hydrogen peroxide and protect against clomiphene citrate-induced morphological apoptotic changes in rat eggs. For this purpose, forty five sexually immature female rats were given single intramuscular injection of 10 IU pregnant mare's serum gonadotropin for 48 h followed by single injections of 10 IU human chorionic gonadotropin and clomiphene citrate (10 mg/kg bw) with or without melatonin (20 mg/kg bw) for 16 h. The histology of ovary, ovulation rate, hydrogen peroxide concentration and catalase activity in ovary and morphological changes in ovulated eggs were analyzed. Co-administration of clomiphene citrate along with human chorionic gonadotropin significantly increased hydrogen peroxide concentration and inhibited catalase activity in ovary, inhibited ovulation rate and induced egg apoptosis. Supplementation of melatonin reduced hydrogen peroxide concentration and increased catalase activity in the ovary, delayed meiotic cell cycle progression in follicular oocytes as well as in ovulated eggs since extrusion of first polar body was still in progress even after ovulation and protected against clomiphene citrate-induced egg apoptosis. These results clearly suggest that the melatonin reduces oxidative stress by scavenging hydrogen peroxide produced in the ovary after clomiphene citrate treatment, slows down meiotic cell cycle progression in eggs and protects against clomiphene citrate-induced apoptosis in rat eggs.

Reactive oxygen and nitrogen species during meiotic resumption from diplotene arrest in mammalian oocytes.

Mammalian ovary is metabolically active organ and generates by-products such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) on an extraordinary scale. Both follicular somatic cells as well as oocyte generate ROS and RNS synchronously and their effects are neutralized by intricate array of antioxidants. ROS such as hydrogen peroxide (H(2)O(2)) and RNS such as nitric oxide (NO) act as signaling molecules and modulate various aspects of oocyte physiology including meiotic cell cycle arrest and resumption. Generation of intraoocyte H(2)O(2) can induce meiotic resumption from diplotene arrest probably by the activation of adenosine monophosphate (AMP)-activated protein kinase A (PRKA)-or Ca(2+)-mediated pathway. However, reduced intraoocyte NO level may inactivate guanylyl cyclase-mediated pathway that results in the reduced production of cyclic 3',5'-guanosine monophosphate (cGMP). The reduced level of cGMP results in the activation of cyclic 3',5'-adenosine monophosphate (cAMP)-phosphodiesterase 3A (PDE3A), which hydrolyses cAMP. The reduced intraoocyte cAMP results in the activation of maturation promoting factor (MPF) that finally induces meiotic resumption. Thus, a transient increase of intraoocyte H(2)O(2) level and decrease of NO level may signal meiotic resumption from diplotene arrest in mammalian oocytes.