517 research outputs found
Mouse versus Rat: Profound Differences in Meiotic Regulation at the Level of the Isolated Oocyte
Cumulus cell-enclosed oocytes (CEO), denuded oocytes (DO), or dissected follicles were obtained 44–48 hr after priming immature mice (20–23 days old) with 5 IU or immature rats (25–27 days old) with 12.5 IU of equine chorionic gonadotropin, and exposed to a variety of culture conditions. Mouse oocytes were more effectively maintained in meiotic arrest by hypoxanthine, dbcAMP, IBMX, milrinone, and 8-Br-cGMP. Atrial natriuretic peptide, a guanylate cyclase activator, suppressed maturation in CEO from both species, but mycophenolic acid reversed IBMX-maintained meiotic arrest in mouse CEO with little activity in rat CEO. IBMX-arrested mouse, but not rat, CEO were induced to undergo germinal vesicle breakdown (GVB) by follicle-stimulating hormone (FSH) and amphiregulin, while human chorionic gonadotropin (hCG) was ineffective in both species. Nevertheless, FSH and amphiregulin stimulated cumulus expansion in both species. FSH and hCG were both effective inducers of GVB in cultured mouse and rat follicles while amphiregulin was stimulatory only in mouse follicles. Changing the culture medium or altering macromolecular supplementation had no effect on FSH-induced maturation in rat CEO. The AMP-activated protein kinase (AMPK) activator, AICAR, was a potent stimulator of maturation in mouse CEO and DO, but only marginally stimulatory in rat CEO and ineffective in rat DO. The AMPK inhibitor, compound C, blocked meiotic induction more effectively in hCG-treated mouse follicles and heat-treated mouse CEO. Both agents produced contrasting results on polar body formation in cultured CEO in the two species. Active AMPK was detected in germinal vesicles of immature mouse, but not rat, oocytes prior to hCG-induced maturation in vivo; it colocalized with chromatin after GVB in rat and mouse oocytes, but did not appear at the spindle poles in rat oocytes as it did in mouse oocytes. Finally, cultured mouse and rat CEO displayed disparate maturation responses to energy substrate manipulation. These data highlight significant differences in meiotic regulation between the two species, and demonstrate a greater potential in mice for control at the level of the cumulus CEO
Genetic Dissection of Epidermal Growth Factor Receptor Signaling during Luteinizing Hormone-Induced Oocyte Maturation
Recent evidence that luteinizing hormone (LH) stimulation of ovulatory follicles causes transactivation of the epidermal growth factor receptor (EGFR) has provided insights into the mechanisms of ovulation. However, the complete array of signals that promote oocyte reentry into the meiotic cell cycle in the follicle are still incompletely understood. To elucidate the signaling downstream of EGFR involved in oocyte maturation, we have investigated the LH responses in granulosa cells with targeted ablation of EGFR. Oocyte maturation and ovulation is disrupted when EGFR expression is progressively reduced. In granulosa cells from mice with either global or granulosa cell-specific disruption of EGFR signaling, LH-induced phosphorylation of MAPK3/1, p38MAPK, and connexin-43 is impaired. Although the LH-induced decrease in cGMP is EGFR-dependent in wild type follicles, LH still induces a decrease in cGMP in Egfrdelta/f Cyp19-Cre follicles. Thus compensatory mechanisms appear activated in the mutant. Spatial propagation of the LH signal in the follicle also is dependent on the EGF network, and likely is important for the control of signaling to the oocyte. Thus, multiple signals and redundant pathways contribute to regulating oocyte reentry into the cell cycle
Molecular Regulation of Noradrenaline in Bovine Corpus Luteum
Noradrenergic stimulation increases progesterone, oxytocin and prostaglandins in the bovine luteal tissue. Better understanding of noradrenaline (NA) role in bovine the corpus luteum (CL) can advance our current knowledge on the regulatory mechanism of CL function. The present study was conducted to explore the source of noradrenaline and further to investigate whether nerve growth factor (NGF), insulin like growth factor 1 (IGF1) and transforming growth factor b1 (TGFb1) influence the expression of dopamine-b-hydroxylase (DBH), biosynthetic enzyme of NA in cultured bovine luteal cells. Corpora lutea were collected and classified into stages of early, developing, mid, late, and regressed. Messenger RNA (mRNA) and protein expression of DBH were studied throughout the estrous cycle. Additionally, DBH protein expression was examined in cultured mid luteal cells after tumour necrosis factor alpha/interferon gamma (TNFa/IFNg)-induced apoptosis or after treatment with NGF, IGF1, and TGFb1. DBH mRNA and protein expressions were detected throughout the cycle without significant changes in the protein level while mRNA showed a decrease at the developing stage (P < 0.05). Interestingly, NGF, IGF1, and TGFb1 increased DBH expression in cultured luteal cells (P < 0.05). The overall findings suggest non-neural source of noradrenaline in the bovine CL which appears to be regulated by NGF, IGF1, and TGFb1 indicating intraluteal molecules play an important and unrecognized role in the CL function
Regulation of Asymmetrical Cytokinesis by cAMP during Meiosis I in Mouse Oocytes
Mammalian oocytes undergo an asymmetrical first meiotic division, extruding half of their chromosomes in a small polar body to preserve maternal resources for embryonic development. To divide asymmetrically, mammalian oocytes relocate chromosomes from the center of the cell to the cortex, but little is known about the underlying mechanisms. Here, we show that upon the elevation of intracellular cAMP level, mouse oocytes produced two daughter cells with similar sizes. This symmetrical cell division could be rescued by the inhibition of PKA, a cAMP-dependent protein kinase. Live cell imaging revealed that a symmetrically localized cleavage furrow resulted in symmetrical cell division. Detailed analyses demonstrated that symmetrically localized cleavage furrows were caused by the inappropriate central positioning of chromosome clusters at anaphase onset, indicating that chromosome cluster migration was impaired. Notably, high intracellular cAMP reduced myosin II activity, and the microinjection of phospho-myosin II antibody into the oocytes impeded chromosome migration and promoted symmetrical cell division. Our results support the hypothesis that cAMP plays a role in regulating asymmetrical cell division by modulating myosin II activity during mouse oocyte meiosis I, providing a novel insight into the regulation of female gamete formation in mammals
The Role of Cilostazol, a Phosphodiesterase 3 Inhibitor, on Oocyte Maturation and Subsequent Pregnancy in Mice
It is important to identify effective contraceptive drugs that cause minimal disruption to physiological processes. Phosphodiesterase 3 (PDE3) inhibitors suppress meiosis in oocytes by decreasing the level of cAMP and blocking the extrusion of the first polar body. In this study, we tested the PDE3 inhibitor, cilostazol, as a potential contraceptive agent. The effects of cilostazol treatment in vitro and in vivo on the suppression of oocyte maturation in a mouse model were investigated. The results indicated that treatment with increasing concentrations of cilostazol led to a dose-dependent arrest in meiosis progression. The effective in vitro concentration was 1 µM and was 300 mg/kg in vivo. The effect of cilostazol was reversible. After removal of the drug, meiosis resumed and mouse oocytes matured in vitro, and showed normal chromosome alignment and spindle organization. After fertilization using an ICSI method, the oocytes showed normal morphology, fertilization rate, embryo cleavage, blastocyst formation, and number of viable pups when compared with controls. The offspring showed similar body weight and fertility. In vivo, the mice became infertile if the drug was injected sequentially, and became pregnant following discontinuation of cilostazol. More importantly, no side effects of cilostazol were observed in treated female mice as demonstrated by blood pressure and heart rate monitoring. It is concluded that cilostazol, a drug routinely used for intermittent claudication, can effectively inhibit oocyte maturation in vitro and in vivo, does not affect the developmental potential of oocytes following drug removal and has few side effects in female mice treated with this drug. These findings suggest that cilostazol may be a potential new contraceptive agent that may facilitate an efficacy and safety study of this drug
Influence of mycotoxin zearalenone and its derivatives (alpha and beta zearalenol) on apoptosis and proliferation of cultured granulosa cells from equine ovaries
BACKGROUND: The mycotoxin zearalenone (ZEA) and its derivatives, alpha and beta-zearalenol (alpha and beta-ZOL), synthesized by genera Fusarium, often occur as contaminants in cereal grains and animal feeds. The importance of ZEA on reproductive disorders is well known in domestic animals species, particularly in swine and cattle. In the horse, limited data are available to date on the influence of dietary exposure to ZEA on reproductive health and on its in vitro effects on reproductive cells. The aim of this study was to evaluate the effects of ZEA and its derivatives, alpha and beta-ZOL, on granulosa cells (GCs) from the ovaries of cycling mares. METHODS: The cell proliferation was evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test after 3 days exposure at different concentrations of ZEA and its derivatives (from 1 × 10-7 to 0.1 microM). The apoptosis induction was evaluated after 1 day exposure, by DNA analysis using flow cytometry. RESULTS: An increase in cell proliferation with respect to the control was observed in the presence of ZEA at 1 × 10-3 and 1 × 10-4 microM and apoptosis was induced by all mycotoxins at different concentrations. CONCLUSION: The simultaneous presence of apoptosis and proliferation in GC cultures treated with zearalenones could indicate that these mycotoxins could be effective in inducing follicular atresia. These effects of zearalenones may result from both direct interaction with oestrogen-receptors as well as interaction with the enzymes 3alpha (beta)-hydroxysteroid dehydrogenase (HSD), involved in the synthesis and metabolism of endogenous steroid hormones. These cellular disturbances, described for the first time in equine GCs cultured in vitro, could be hypothesized as referred to reproductive failures of unknown ethiology in the mare
Pharmacological Analyses of Protein Kinases Regulating Egg Maturation in Marine Nemertean Worms: A Review and Comparison with Mammalian Eggs
For development to proceed normally, animal eggs must undergo a maturation process that ultimately depends on phosphorylations of key regulatory proteins. To analyze the kinases that mediate these phosphorylations, eggs of marine nemertean worms have been treated with pharmacological modulators of intracellular signaling pathways and subsequently probed with immunoblots employing phospho-specific antibodies. This article both reviews such analyses and compares them with those conducted on mammals, while focusing on how egg maturation in nemerteans is affected by signaling pathways involving cAMP, mitogen-activated protein kinases, Src-family kinases, protein kinase C isotypes, AMP-activated kinase, and the Cdc2 kinase of maturation-promoting factor
EGF increases expression and activity of PAs in preimplantation rat embryos and their implantation rate
BACKGROUND: Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs) have been implicated in mammalian fertilization, early stages of development and embryo implantation. As in-vitro developing embryos resulted in lower implantation rate than those developed in-vivo we assume that a reduced PAs activity may be involved. In the present work we studied the effect of EGF on PAs activity, quantity and embryo implantation. METHODS: Zygotes were flushed from rat oviducts on day one of pregnancy and grown in-vitro in R1ECM supplemented with EGF (10 ng/ml) and were grown up to the blastocyst stage. The control groups were grown in the same medium without EGF. The distribution and quantity of the PAs were examined using fluorescence immunohistochemistry followed by measurement of PAs activity using the chromogenic assay. Implantation rate was studied using the embryo donation model. RESULTS: PAs distribution in the embryos was the same in EGF treated and untreated embryos. Both PAs were localized in the blastocysts' trophectoderm, supporting the assumption that PAs play a role in the implantation process in rats. EGF increased the quantity of uPA at all stages studied but the 8-cell stage as compared with controls. The tissue type PA (tPA) content was unaffected except the 8-cell stage, which was increased. The activity of uPA increased gradually towards the blastocyst stage and more so due to the presence of EGF. The activity of tPA did not vary with the advancing developmental stages although it was also increased by EGF. The presence of EGF during the preimplantation development doubled the rate of implantation of the treated group as compared with controls
Extra- and intra-ovarian factors in polycystic ovary syndrome: impact on oocyte maturation and embryo developmental competence
background: Polycystic ovary syndrome (PCOS) is a common metabolic dysfunction and heterogeneous endocrine disorder in women of reproductive age. Although patients with PCOS are typically characterized by increased numbers of oocytes retrieved during IVF, they are often of poor quality, leading to lower fertilization, cleavage and implantation rates, and a higher miscarriage rate. methods: For this review, we searched the database MEDLINE (1950 to January 2010) and Google for all full texts and/or abstract articles published in English with content related to oocyte maturation and embryo developmental competence. results: The search showed that alteration of many factors may directly or indirectly impair the competence of maturating oocytes through endocrine and local paracrine/autocrine actions, resulting in a lower pregnancy rate in patients with PCOS. The extra-ovarian factors identified included gonadotrophins, hyperandrogenemia and hyperinsulinemia, although intra-ovarian factors included members of the epidermal, fibroblast, insulin-like and neurotrophin families of growth factors, as well as the cytokines. conclusions: Any abnormality in the extra- and/or intra-ovarian factors may negatively affect the granulosa cell-oocyte interaction, oocyte maturation and potential embryonic developmental competence, contributing to unsuccessful outcomes for patients with PCOS who are undergoing assisted reproduction.Obstetrics & GynecologyReproductive BiologySCI(E)PubMed49REVIEW117-331
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