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.

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.

Increased level of reactive oxygen species persuades postovulatory aging-mediated spontaneous egg activation in rat eggs cultured in vitro.

The present study was aimed to find out whether increased level of reactive oxygen species (ROS) particularity hydrogen peroxide (H2O2) could persuade postovulatory aging-mediated abortive spontaneous egg activation (SEA) in rat eggs cultured in vitro. For this purpose, ROS and H2O2 levels, mitochondria distribution and its membrane potential, p286-CaMK-II, Emi2, Thr-161 phophorylated cyclin-dependent protein kinase1 (Cdk1) as well as cyclin B1 levels, in vitro effects of 3-tert-butyl-4 hydroxy anisole (BHA), pentoxifylline and dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) were analyzed during postovulatory aging-induced abortive SEA in vitro. Data of the present study suggest that postovulatory aging increased H2O2 levels, disturbed mitochondrial distribution pattern and mitochondrial membrane potential (MMP) in eggs. There was an significant increase of p286-CaMK-II level, while Emi2 level reduced significantly during egg aging in vitro. The reduced Emi2 level was associated with decreased Thr-161 phosphorylated cyclin-dependent kinase-1 (Cdk1) as well as cyclin B1 level in aged eggs that underwent abortive SEA. Further, supplementation of pentoxifylline, db-cAMP, and BHA protected postovulatory aging-mediated abortive SEA in concentration-dependent manner. These data suggest that postovulatory aging increased H2O2 levels, reduced MMP, and increased p286-CaMK-II. The increased p286-CaMK-II was associated with reduced Emi2 level and maturation-promoting factor levels during postovulatory aging-mediated abortive SEA. Drugs that elevate cAMP directly or indirectly and BHA protected postovulatory aging-mediated abortive SEA possibly by reducing ROS level in rat eggs cultured in vitro.

Nitric oxide signals postovulatory aging-induced abortive spontaneous egg activation in rats.

OBJECTIVE: The aim of this study was to determine whether an increase of intracellular nitric oxide (NO) level signals postovulatory aging-induced abortive spontaneous egg activation (SEA) in rats. METHODS: Freshly ovulated eggs (arrested at metaphase-II stage; M-II) were cultured in vitro for 3 hours to induce postovulatory egg aging. The morphological changes, inducible nitric oxide synthase (iNOS) expression, NO, cytosolic free Ca(2+), 3',5' cyclic guanosine monophosphate (cGMP), cell division cycle 25B (Cdc25B) and Wee1 levels, specific phosphorylation (pThr-14/Tyr-15) as well as total cyclin-dependent kinases-1 (Cdk1) (PSTAIRE) levels were analyzed. RESULTS: Postovulatory aging induced generation of NO possibly through an iNOS-mediated pathway. The increase in NO level was associated with augmented cytosolic free Ca(2+) as well as cGMP levels in aged eggs. A significant increase in Wee1 level and decrease of Cdc25B level were observed in aged eggs. An accumulation of phosphorylated Cdk1 (pThr-14/Tyr-15) level was observed in aged eggs, while total Cdk1 (PSTAIR) level remained unchanged. CONCLUSION: Our study demonstrates that generation of NO through an iNOS-mediated pathway increases cytosolic free Ca2+and cGMP levels. High levels of these signal molecules trigger the accumulation of phosphorylated Cdk1 in aged eggs. Thus, NO signals the accumulation of phosphorylated Cdk1 and induces postovulatory aging-induced abortive SEA in the rat.

RyR channel-mediated increase of cytosolic free calcium level signals cyclin B1 degradation during abortive spontaneous egg activation in rat.

In few mammalian species including rat, post-ovulatory aging induces abortive spontaneous egg activation (SEA), which is morphologically characterized by exit from metaphase-II (M-II) arrest. A possibility exists that the RyR channel-mediated insufficient increase of cytosolic free Ca(2+) level could be one of the causes for post-ovulatory aging-induced abortive SEA. To test this possibility, eggs collected after 17 h post-hCG surge were cultured with or without various concentrations of nifedipine (NF), ruthenium red (RR), and KN-93 for 3 h in vitro. Morphological changes characteristic of abortive SEA, cytosolic free Ca(2+) level, cyclin B1 level, and meiotic status were analyzed. Data of the present study indicate that NF and RR inhibited post-ovulatory aging-induced abortive SEA in a concentration-dependent manner. Further, RR protected against RyR channel as well as caffeine-mediated increase of cytosolic free Ca(2+) level. In addition, KN-93 inhibited post-ovulatory aging-induced abortive SEA in a concentration-dependent manner. An increase of cytosolic free Ca(2+) level was associated with a reduction of cyclin B1 level during post-ovulatory aging-induced abortive SEA. These data indirectly suggest the involvement of RyR channels in the increase of cytosolic free Ca(2+) level. The increased cytosolic free Ca(2+) level triggers cyclin B1 degradation possibly through CaMK-II activity during post-ovulatory aging-induced abortive SEA in rat eggs cultured in vitro.

An insufficient increase of cytosolic free calcium level results postovulatory aging-induced abortive spontaneous egg activation in rat.

PURPOSE: The present study was aimed to find out whether postovulatory aging-induced abortive spontaneous egg activation (SEA) is due to insufficient increase of cytosolic free Ca(2+) level. METHODS: Immature female rats (22-24 days old) were subjected to superovulation induction protocol. Eggs were collected 14, 17 and 19 h post-hCG surge to induce in vivo egg aging. The eggs were collected 14 h post-hCG surge and cultured in vitro for 3, 5 and 7 h to induce in vitro egg aging. The morphological changes, rate of abortive SEA, chromosomal status and cytosolic free Ca(2+) levels were analyzed. RESULTS: Postovulatory aging induced morphological features characteristics of abortive SEA in a time-dependent manner in vivo as well as in vitro. The extracellular Ca(2+) increased rate of abortive SEA during initial period of culture, while co-addition of a nifedipine (L-type Ca(2+) channel blocker) protected against postovulatory aging-induced abortive SEA. However, CI induced morphological features characteristics of egg activation (EA) in a dose-dependent manner. As compare to control, an increase of cytosolic free Ca(2+) level (1.42 times) induced abortive SEA, while further increase of cytosolic free Ca(2+) level (2.55 times) induced EA. CONCLUSION: Our results show that an insufficient cytosolic free Ca(2+) level is associated with postovulatory aging -induced abortive SEA, while furthermore increase is required to induce EA in rat.

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.