Showing papers by "Shail K. Chaube published in 2016"

Impact of stress on oocyte quality and reproductive outcome.

Abstract: 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.

Moderate increase of reactive oxygen species triggers meiotic resumption in rat follicular oocytes.

Abstract: Aim The mammalian ovary generates reactive oxygen species (ROS) on an extraordinary scale; however, the role of ROS during meiotic cell cycle progression in follicular oocytes remains poorly understood. The present study was aimed to determine whether a moderate increase of ROS level in the ovary is beneficial for meiotic resumption from diplotene arrest in follicular oocytes. Methods Cumulus oocyte complexes were collected from the ovaries of female rats that had been treated with either: (i) pregnant mare's serum gonadotrophin; or (ii) pregnant mare's serum gonadotrophin + human chorionic gonadotrophin. We analyzed morphological changes, ROS and hydrogen peroxide levels, catalase activity, 3′,5′-cyclic adenosine monophosphate and 3′,5′-cyclic guanosine monophosphate levels, Thr14/Tyr15, Th-161, total cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels. Results Human chorionic gonadotrophin treatment induced meiotic resumption from diplotene arrest and extrusion of first polar body in cumulus oocyte complexes collected from ovaries and cultured for 3 h in vitro. Meiotic resumption from diplotene arrest was associated with increased ROS and hydrogen peroxide levels but decreased 3′,5′-cyclic adenosine monophosphate as well as 3′,5′-cyclic guanosine monophosphate levels. The reduced cyclic nucleotide levels were associated with decreased Thr161 phosphorylated Cdk1 and cyclin B1 level but increased Thr14/Tyr15 phosphorylated Cdk1 level leading to maturation promoting factor destabilization. Destabilized maturation-promoting factor triggered meiotic resumption from diplotene arrest and progression to metaphase-I as well as metaphase-II stage in follicular oocytes. Conclusion Our findings suggest that a moderate increase of ROS in the ovary is beneficial for meiotic resumption from diplotene arrest and extrusion of first polar body in follicular oocytes.

Involvement of Reactive Oxygen Species in Meiotic Cell Cycle Regulation and Apoptosis in Mammalian Oocytes

Abstract: The mammalian ovary is a metabolically active organ and generates a large amount of reactive oxygen species (ROS) during final stages of folliculogenesis. ROS modulate physiological arrest (i.e., diplotene arrest) in follicular oocytes as well as metaphase-II (M-II) arrest in ovulated oocytes in most of the mammalian species. A moderate increase in the level of ROS could be beneficial for meiotic resumption from diplotene and M-II arrest in oocytes. The increased production of ROS, decreased antioxidant system, drug treatment, pathological conditions, stress, and several other factors may lead to accumulation of ROS in the ovary. Increased levels of ROS may generate oxidative stress (OS), which could induce meiotic cell cycle arrest in oocytes. OS triggers granulosa cell apoptosis and thereby reduces the transfer of nutrients and survival factors to the oocytes, leading to apoptosis. In vitro culture conditions, reduced survival factors, and destabilized maturation promoting factor (MPF) may generate ROS and thereby OS in follicular and ovulated oocytes. OS induces apoptosis in diplotene- and M-II-arrested oocytes through mitochondria-mediated pathway. The deterioration in oocyte quality resulting from ROS-mediated apoptosis may negatively impact the outcome of assisted reproductive technology (ART) in several mammalian species, including humans.

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

Abstract: 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.

Maturation promoting factor destabilization facilitates postovulatory aging-mediated abortive spontaneous egg activation in rat.

Abstract: The present study was designed to investigate whether destabilization of maturation promoting factor (MPF) leads to postovulatory aging-mediated abortive spontaneous egg activation (SEA). If so, we wished to determine whether changes in Wee-1 as well as Emi2 levels are associated with MPF destabilization during postovulatory aging-mediated abortive SEA in rats eggs aged in vivo. For this purpose, sexually immature female rats were given a single injection (20 IU IM) of pregnant mare serum gonadotropin for 48 h followed by single injection of human chorionic gonadotropin (20 IU). Ovulated eggs were collected after 14, 17, 19 and 21 h post-hCG surge to induce postovulatory aging in vivo. The morphological changes, Wee1, phosphorylation status of cyclin dependent kinase 1(Cdk1), early mitotic inhibitor 2 (Emi2), anaphase promoting complex/cyclosome (APC/C), cyclin B1, mitotic arrest deficient protein (MAD2) levels and Cdk1 activity were analyzed. The increased Wee 1 level triggered phosphorylation of Thr-14/Tyr-15 and dephosphorylation of Thr-161 residues of Cdk1. The decrease of Emi2 level was associated with increased APC/C level and decreased cyclin B1 level. Changes in phosphorylation status of Cdk1 and reduced cyclin B1 level resulted in destabilization of MPF. The destabilized MPF finally led to postovulatory aging-mediated abortive SEA in rat eggs. It was concluded that the increase of Wee 1 but decrease of Emi2 level triggers MPF destabilization and thereby postovulatory aging-mediated abortive SEA in rat eggs.

Rat: An Interesting Model to Study Oocyte Meiosis in Mammals

Abstract: In most of the mammalian species, oocyte meiosis takes very long time and passes through several stop/go channels to generate female gamete [1-3]. Within the ovarian follicles, oocytes are normally arrested at diplotene stage of first meiotic prophase for a long time. Meiotic resumption from diplotene stage in follicular oocytes takes place after pituitary gonadotropins surge. Oocytes then progress through metaphase-I (M-I stage) to metaphase-II (M-II stage) just prior to ovulation in several mammalian species. Meiotic resumption from diplotene arrest to M-II arrest is a very important period because follicular oocyte achieves meiotic competency during this period. The M-II arrest is a physiological arrest in preovulatory oocytes and exit from M-II arrest normally occurs due to sperm triggering at the time of fertilization in most of the mammalian species including human. Studies carried out in last two decades suggest that the rat is an interesting and peculiar animal model among mammals to analyse meiotic cell cycle regulation in oocytes within the ovarian follicle and even after ovulation [3,4].

Involvement of Cyclin-Dependent Kinase 1 during Postovulatory Aging-Mediated Abortive Spontaneous Egg Activation in Rat Eggs Cultured In Vitro.

Abstract: Freshly ovulated rat eggs do not remain arrested at metaphase II (MII) and undergo exit from MII arrest with initiation of extrusion of the second polar body (PBII), a characteristic feature of abortive spontaneous egg activation (SEA). The biochemical and molecular changes during postovulatory aging-mediated abortive SEA remain poorly understood. We investigated the morphological, cellular, and molecular changes during postovulatory aging-mediated abortive SEA in eggs cultured in vitro. Our results suggest that postovulatory egg aging in vitro induced initiation of PBII extrusion in a time-dependent manner. Postovulatory aging increased Wee1 kinase and Thr-14/Tyr-15 phosphorylated cyclin-dependent kinase 1 (Cdk1) levels, whereas Thr-161 phosphorylated Cdk1 and cyclin B1 levels were significantly decreased in eggs cultured in vitro. The early mitotic inhibitor 2 (Emi2) level was significantly reduced, but anaphase promoting complex/cyclosome (APC/C) and mitotic arrest deficient protein (MAD2) levels were increased initially and then reduced during a later period of in vitro culture. These results suggest that an increased Wee1 kinase level modulated the specific phosphorylation status of Cdk1, increased Cdk1 activity, and decreased the cyclin B1 level. Furthermore, the decreased Emi2 level was associated with an increased level of APC/C and decreased level of cyclin B1, which resulted in maturation promoting factor (MPF) destabilization and finally led to postovulatory aging-mediated abortive SEA in rat eggs cultured in vitro.

Abortive Spontaneous Egg Activation: A Limiting Factor For Reproductive Outcome in Mammals

Abstract: Most of the mammalian species possess asynchronous ovary that exhibit different stages of follicles. During final stages of folliculogenesis, mammalian ovary ensures the generation of competent oocytes required for fertilization and embryonic development. To achieve this goal, large number of incompetent or defective germ cells (>99%) are eliminated from ovary through follicular atresia via apoptosis. At the time of puberty, limited number of germ cells are available in the ovary (<1%), which are culminated into oogonia and then primary oocytes [1]. These primary oocytes are arrested at diplotene stage until pituitary gonadotropin surge at the time of puberty [2]. Gonadotropins trigger meiotic resumption from diplotene arrest and these oocytes progresses to metaphase-II (M-II) stage just prior to ovulation [2,3].

A 12-Crown-4 Ether Containing Dipeptide Boc-12-Crown-4-l-DOPA-Gly-OMe Induces Cell Cycle Arrest and Apoptosis in Rat Eggs Cultured In Vitro

Abstract: The study was designed to investigate whether crown ether containing dipeptide Boc-12-crown-4-l-DOPA-Gly-OMe has potential to induce meiotic cell cycle arrest and apoptosis in rat eggs cultured in vitro. The immature female rats were subjected to superovulation induction protocol and ovulated eggs were collected from ampulla of the fallopian tube. Ovulated eggs arrested at metaphase-II (M-II) stage of meiotic cell cycle were cultured in media-199 with or without various concentrations (0.0, 0.025, 0.050, 0.10, and 0.20 mM) of dipeptide for 3 h in vitro. Morphological apoptotic changes, hydrogen peroxide (H2O2) concentration, cytochrome c level, caspase-3 level as well as activity and DNA fragmentation were analysed in eggs cultured in vitro. Culture of M-II arrested eggs in plain medium for 3 h in vitro induced meiotic exit from M-II arrest in majority of eggs as evidenced by initiation of extrusion of second polar body (II PB). The dipeptide induced maintenance of M-II arrest and morphological apoptotic features in a concentration-dependent manner prior to degeneration. The dipeptide-induced morphological features were associated with increased H2O2 and cytochrome c levels in treated eggs. The increased cytochrome c induced caspase-3 level and activity and thereby DNA fragmentation as evidenced by DAB positive staining in treated eggs. Our results suggest that dipeptide Boc-12-C-4-l-DOPA-Gly-OMe induces cell cycle arrest at M-II stage and apoptosis in rat eggs cultured in vitro.