Ashutosh N. Pandey

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.

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.

Cellular internalization and stress response of ingested amorphous silica nanoparticles in the midgut of Drosophila melanogaster

BACKGROUND Amorphous silica nanoparticles (aSNPs) are used for various applications including food industry. However, limited in vivo studies are available on absorption/internalization of ingested aSNPs in the midgut cells of an organism. The study aims to examine cellular uptake of aSNPs (<30nm) in the midgut of Drosophila melanogaster (Oregon R(+)) owing to similarities between the midgut tissue of this organism and human and subsequently cellular stress response generated by these nanoparticles. METHODS Third instar larvae of D. melanogaster were exposed orally to 1-100μg/mL of aSNPs for 12-36h and oxidative stress (OS), heat shock genes (hsgs), membrane destabilization (Acridine orange/Ethidium Bromide staining), cellular internalization (TEM) and apoptosis endpoints. RESULTS A significant increase was observed in OS endpoints in the midgut cells of exposed Drosophila in a concentration- and time-dependent manner. Significantly increased expression of hsp70 and hsp22 along with caspases activation, membrane destabilization and mitochondrial membrane potential loss was also observed. TEM analysis showed aSNPs-uptake in the midgut cells of exposed Drosophila via endocytic vesicles and by direct membrane penetration. CONCLUSION aSNPs after their internalization in the midgut cells of exposed Drosophila larvae show membrane destabilization along with increased cellular stress and cell death. GENERAL SIGNIFICANCE Ingested aSNPs show adverse effects on the cells of GI tract of the exposed organism thus their industrial use as a food-additive may raise concern to human health.

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 mares 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.

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

Abstract 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.

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.

miRNA profiling provides insights on adverse effects of Cr(VI) in the midgut tissues of Drosophila melanogaster

Cr(VI), a well-known environmental chemical, is reported to cause various adverse effects on exposed organisms including genomic instability and carcinogenesis. Despite available information on the underlying mechanism of Cr(VI) induced toxicity, studies regarding toxicity modulation by epigenetic mechanisms are limited. It was therefore, hypothesized that the global miRNA profiling in Cr(VI) exposed Drosophila, a genetically tractable model organism, will provide information about mis-regulated miRNAs along with their targeted genes and relevant processes. Third instar larvae of Drosophila melanogaster (Oregon R(+)) were exposed to 5.0-20.0 μg/ml of Cr(VI) for 24 and 48 h. Following miRNA profile analysis on an Agilent platform, 28 of the 36 differentially expressed miRNAs were found to be significantly mis-regulated targeting major biological processes viz., DNA damage repair, oxidation-reduction processes, development and differentiation. Down-regulation of mus309 and mus312 under DNA repair, acon to oxidation-reduction and pyd to stress activated MAPK cascade respectively belonging to these gene ontology classes concurrent with up-regulation of dme-miR-314-3p, dme-miR-79-3p and dme-miR-12-5p confirm their functional involvement against Cr(VI) exposure. These findings assume significance since majority of the target genes in Drosophila have functional homologues in humans. The study further recommends Drosophila as a model to explore the role of miRNAs in xenobiotic induced toxicity.

Synthesis and Characterization of the Heterometallic Aggregate Pb2Al5(μ3-O)(μ4-O)(μ-OiPr)9(OiPr)3(μ-OAc)3

A novel heterometallic aggregate Pb2Al5(μ3-O)(μ4-O)(μ-OiPr)9(OiPr)3(μ-OAc)3 obtained from the interaction of Pb(OAc)2 and Al(O–iPr)3 is the first structurally characterized example based on lead and aluminum. This compound has been isolated in high yield and examined by 1H-, 13C-, and 27Al NMR, and in the solid state by X-ray crystallography.