RESUMO
Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.
Assuntos
AMP Cíclico/metabolismo , Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos , Oócitos/crescimento & desenvolvimento , 1-Metil-3-Isobutilxantina/administração & dosagem , Animais , Bovinos , Colforsina/administração & dosagem , Criopreservação , Feminino , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Vitrificação/efeitos dos fármacosRESUMO
Aqueous extracts of Rhizopus oryzae (Aq-ROU) have a broad range of physiological activity. Here we identified a new physiological effect of Aq-ROU in rat hepatocyte cell line RLN-10. Aq-ROU induced the accumulation of nitrite, a stable metabolite nitric oxide (NO), in cell culture medium and induced potent diaminofluorescein-FM diacetate staining in the cells. Real-time reverse transcriptase (RT)-PCR analysis showed marked inducible NO synthase gene expression. Additionally, markedly enhanced expression of p22(phox) and temporally increased expression of NADPH oxidase1 indicated that superoxide was produced. Nuclear translocation of nuclear factor-kappa (NF-κ) B p65 increased remarkably following Aq-ROU and following lipopolysaccharide treatment, a potent activator of NF-κB. Ammonium pyrrolidine-1-carbodithioate, an inhibitor of NF-κB, inhibited NO production following Aq-ROU treatment. Our data indicate that Aq-ROU induces NO production and potentially the production of superoxide, which may contribute to the broad range of physiological effects observed for Aq-ROU ingested by animals.
Assuntos
Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Óxido Nítrico/biossíntese , Rhizopus/química , Água/química , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NADH NADPH Oxirredutases/genética , NADPH Oxidase 1 , NADPH Oxidases/genética , Óxido Nítrico Sintase Tipo II/genética , Pirrolidinas/farmacologia , Ratos , Superóxidos/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
We investigated the role of nitric oxide (NO) in vascular endothelial growth factor (VEGF) expression in the rat placenta. A nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME), was constantly infused into pregnant rats 6-24 h before sacrifice on gestational day (GD) 15.5. NO production declined to about 15% of the control level as monitored by NO trapping and electron paramagnetic resonance spectroscopy. VEGF mRNA expression was temporally decreased by L-NAME, but recovered to normal levels after 24 h of treatment, whereas hypoxia inducible factor (HIF)-1α and induced NOS (iNOS) expression increased. VEGF expression decreased significantly in placental explants after 6 h of co-treatment with L-NAME and lipopolysaccharide, an iNOS inducer. Our data indicate that NO induce VEGF expression in vivo and in vitro in the rat placenta, suggesting that peaked NO production was maintained by a reciprocal relationship between NO and VEGF via HIF-1α.