Shail K. Chaube

Meiotic cell cycle arrest in mammalian oocytes.

Meiotic cell cycle in mammalian oocytes is a dynamic process that involves several stop/go channels. The cell cycle arrest in oocyte occurs at various stages such as diplotene, metaphase‐I (M‐I), metaphase‐II (M‐II), and so called metaphase‐like arrest (M‐III). Leutinizing hormone surge induces meiotic resumption from diplotene arrest in follicular microenvironment by overriding several factors responsible for the maintenance of meiotic arrest. The inhibitory factors are synthesized in oocyte or in the associated follicular somatic cells and transferred to the oocyte. The major factors include hypoxanthine, cyclic adenosine 3′, 5′‐monophosphate, cyclic guanosine 3′, 5′‐monophosphate, reactive oxygen species, protein kinase A, and protein kinase C. In the presence of active protein kinases, epidermal‐like growth factors are produced that activate mitogen‐activated protein kinase in cumulus granulosa cells. The maturation promoting factor, cytostatic factors, and spindle assembly checkpoint proteins are also involved in that maintenance of arrest at various stages of meiotic cell cycle in mammalian oocytes. In this review, we briefly summarize the role of these factors in the maintenance of meiotic cell cycle arrest in mammalian oocytes. J. Cell. Physiol. 223:592–600, 2010.

Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro

Hydrogen peroxide (H2O2) is known to induce cell cycle arrest and apoptosis in various somatic cell types cultured in vitro. We hypothesize that this reactive oxygen species (ROS) could modulate cell cycle and induce morphological features characteristics of apoptosis in oocytes cultured in vitro. To test this hypothesis, immature and mature oocytes were cultured in medium containing various doses of H2O2 with or without caspase-3 inhibitor for various times. The treatment of H2O2 induced germinal vesicle break down (GVBD) in all immature oocytes followed by initiation of shrinkage. Some of immature oocytes (but not mature oocytes) also showed membrane blebbing. On the other hand, H2O2 treatment inhibited first polar body emission in mature oocytes just prior to initiation of shrinkage. The cytoplasmic granulation and fragmentation into apoptotic bodies were observed in mature oocytes during later stages of H2O2 treatment. The shrinkage was induced by H2O2 in a dose- and time-dependent manner in both immature and mature oocytes. Although, H2O2-induced degeneration was observed in both immature and mature oocytes after 2.0 hrs of treatment, immature oocytes were more susceptible to undergo quick shrinkage, membrane blebbing and degeneration. Co-addition of caspase-3 inhibitor prevented shrinkage and degeneration of both immature and mature oocytes except membrane blebbing that was observed at higher doses of H2O2 after 1.0 hr of culture. Treatment of H2O2 induced bax protein expression (3 times), DNA fragmentation and caspase-3 activity (2.5 times) in oocytes undergoing morphological apoptotic changes. These findings clearly suggest that H2O2 induced GVBD in immature oocytes, inhibited first polar body extrusion in mature oocytes prior to initiation of morphological changes characteristic of apoptosis such as shrinkage, membrane blebbing and cytoplasmic fragmentation prior to degeneration.

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 (H2O2) 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 H2O2 can induce meiotic resumption from diplotene arrest probably by the activation of adenosine monophosphate (AMP)‐activated protein kinase A (PRKA)—or Ca2+‐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 H2O2 level and decrease of NO level may signal meiotic resumption from diplotene arrest in mammalian oocytes. J. Cell. Biochem. 111: 521–528, 2010.

Intracellular levels of hydrogen peroxide and nitric oxide in oocytes at various stages of meiotic cell cycle and apoptosis

The objective was to find out the functional roles of hydrogen peroxide (H2O2) and nitric oxide (NO) during various stages of meiotic cell cycle and apoptosis in rat oocytes. For this purpose, 30 oocytes from each stage such as diplotene, metaphase-I (M-I), metaphase-II (M-II) and apoptosis were collected and intracellular H2O2, total nitrite level and inducible nitric oxide synthase (iNOS) expression were analysed. This study demonstrated that generation of a tonic level of H2O2 induces meiotic resumption in diplotene-arrested oocytes and further increase may lead to apoptosis. Conversely, reduction in iNOS expression and total nitrite level are associated with meiotic resumption in diplotene-arrested oocytes, but induce apoptosis in aged oocytes. These results suggest that generation of a tonic level of H2O2, reduced iNOS expression and total nitrite level are associated with meiotic resumption, while more generation of H2O2 and sustained reduced total nitrite level are linked with oocyte apoptosis in rat.

Subchronic supplementation of lithium carbonate induces reproductive system toxicity in male rat.

Lithium is frequently used as an effective drug for the treatment of several psychiatric disorders in human. This alkali element and its salt, at its higher doses, may lead to various side effects or has several toxic effects after prolonged therapeutic use. To test this hypothesis, the present study was designed to investigate the adverse effect of subchronic exposure of lithium carbonate on reproductive organs of male rat. Rats were exposed to lithium carbonate at doses of 500, 800, 1100 mg/kg of diet for 90 days. The weight of reproductive organs, histology of testis, epididymis, seminal vesicle, prostate, testicular interstitial fluid volume (IFV), testosterone level, sperm morphology and fertility index were analyzed. Treatment with higher doses of lithium carbonate (i.e. 800, 1100 mg/kg diet) significantly reduced testes, epididymis and accessory sex organs weights, whereas, lower dose (500 mg/kg diet) did not show any untoward effect. Similarly, the sperm number from cauda epididymis and daily sperm production was significantly decreased with higher doses of lithium carbonate. The serum testosterone levels and IFVs were also reduced significantly. Seminal vesicle and prostate secretions were completely blocked and spermatozoa were not seen in the lumen of epididymis and vas deference. Histological studies have revealed that lithium carbonate (1100 mg/kg) caused degeneration of spermatogenic cells and vacuolization of sertoli cells cytoplasm in the testis. The sperm transit rate and production of abnormal spermatozoa were significantly (P<0.01) increased. When the lithium carbonate-treated males were mated with normal cyclic females, the fertility index declined to 50% even after 30 days of withdrawal of lithium carbonate treatment. These results clearly suggest that subchronic exposure of lithium carbonate promote reproductive system toxicity and reduces fertility of male rats.

Effect of exogenous melatonin on X-ray induced cellular toxicity in lymphatic tissue of Indian tropical male squirrel, Funambulus pennanti.

Purpose: The radioprotective ability of melatonin was investigated in the Indian tropical rodent, Funambulus pennanti during its reproductively inactive phase (RIP) when peripheral melatonin is high and the animal is under the influence of environmental stresses. Materials and methods: Fifty male squirrels were divided into two sets of 25 each; with set 1 receiving 0.9% saline and set 2 receiving 25 μg melatonin/100 g body weight/day for four weeks during evening hours. Five saline-treated and five-melatonin treated squirrels were sacrificed at times of 4, 24, 48 and 72 hours following 2.06 Gy X-ray radiation. Remaining squirrels that were not exposed to X-ray were used as control and melatonin treated, respectively. Total leukocyte count (TLC) and lymphocyte count (LC) in peripheral blood and lipid peroxidation (LPO) status, superoxide dismutase (SOD) activity, total antioxidant status (TAS), apoptotic percentage on the basis of morphological changes and DNA fragmentation and caspase-3 activity were measured in the spleens of squirrels. Results: Peripheral blood and spleens with higher intrinsic levels of environmental and X-radiation induced free radical generation had inhibited TLC, LC, SOD activity and TAS and increased LPO and apoptosis. Melatonin pre-treatment significantly reduced and altered the changes brought on by X-radiation. Conclusion: Exogenous melatonin with its anti-apoptotic and antioxidant properties additively increased the immunity of the squirrels, by protecting their hematopoietic system and lymphoid organs against X-ray radiation induced cellular toxicity.

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.

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 (Ca2+) as well as hydrogen peroxide (H2O2) 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 Ca2+ as well as H2O2 levels associate with the reduced MPF level and meiotic resumption from diplotene arrest. On the other hand, increase of NO, cGMP, Ca2+ as well as H2O2 levels are associated with reduced MPF and spontaneous exit from M‐II arrest in rat oocytes cultured in vitro.

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H2O2) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca2+/Mg2+-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca2+/Mg2+-supplemented medium. In Ca2+/Mg2+-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 µm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H2O2 (84.40±0.50 ng/egg) when compared to control eggs (80.46±1.34 ng/egg). The higher concentration of calcium ionophore (1.6 µm) induced apoptosis and pronounced generation of intracellular H2O2 (92.43±0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H2O2 level (81.20±1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H2O2 in rat eggs.

Neem (Azadirachta indica L.) leaf extract deteriorates oocyte quality by inducing ROS-mediated apoptosis in mammals

Neem (Azadirachta indica L.) leaf has been widely used in ayurvedic system of medicine for fertility regulation for a long time. The molecular mechanism by which neem leaf regulates female fertility remains poorly understood. Animal studies suggest that aqueous neem leaf extract (NLE) induces reactive oxygen species (ROS) - mediated granulosa cell apoptosis. Granulosa cell apoptosis deprives oocytes from nutrients, survival factors and cell cycle proteins required for the achievement of meiotic competency of follicular oocytes prior to ovulation. Under this situation, follicular oocyte becomes more susceptible towards apoptosis after ovulation. The increased level of hydrogen peroxide (H2O2) inside the follicular fluid results in the transfer of H2O2 from follicular fluid to the oocyte. The increased level of H2O2 induces p53 activation and over expression of Bax protein that modulates mitochondrial membrane potential and trigger cytochrome c release. The increased cytosolic cytochrome c level induces caspase-9 and caspase-3 activities that trigger destruction of structural and specific proteins leading to DNA fragmentation and thereby oocyte apoptosis. Based on these animal studies, we propose that NLE induces generation of ROS and mitochondria-mediated apoptosis both in granulosa cells as well as in follicular oocyte. The induction of apoptosis deteriorates oocyte quality and thereby limits reproductive outcome in mammals.