| Nitric oxide(NO)is a gas free radical.It presents in many cell of many tissues, participates in many physiological and pathophysiological processes of many systems including recirculating system,nervous system,digestive system,respiratory system, apparatus system and immune system.NO is also closely involved in female mammalian reproductive system,such as follicle and oocyte development,ovulation,corepus luteum regress,ferlility,blastocyst implantation,pregnant sustaining,delivery adjustment, menstrual cycle control.Research about NO's effects on granulosa cell accumulates on mouse which includes also human being mainly.There was seldom report in other kinds of animal.This study was to investigate the effects of NO on cell apoptosis,cell cycle,on porcine oocyte maturation and embryo later and on hormone secretion.Exogenous NO was provided with sodium mitroprusside(SNP)(0mM(control),0.01mM,0.1mM and 1mM)to the medium of granulosa cell and COCs.Aider culture,Cell morphology was observed,apoptosis rate,cell cycle of porcine granulosa and cumulus and hormone secretion were deected,porcine oocyte maturation rate and embryo development were examined.Result is shown as below.There were no difference in the granulosa cell morphology in 0.01mM and 0mM.The adhered granulosa cell contract,became abnormal and the digested cell surface is not smooth,appearanced bubbles in 0.1 mM and 1mM.0.01 mM SNP depressed granulosa cell apoptosis rate of follicles 2 mm and 4 mm in diameter(P＜0.05),but had no effects in cumulus apoptosis.In contrast,0.1 and 1mM SNP promoted the apoptosis of granulosa cell and cumulus.Different cell type had different apoptosis rate:the apoptosis rate of cumulus was under granulosa cell of follicle 2mm,2mm under 4mm(P＜0.05).1mM SNP had the most obviously effects on apoptosis, the apoptosis rate achieved 55%in follicular granulosa cell in 2mm,depressed 47% compared to control,in 4mm,depressed 42%.9%in cumulus cell,depressed 8%.SNP can decrease dose-dependly G0/G1 stage cell rate of cumulus and granulosa of follicle 2mm and 4mm in diameter(P＜0.05),The G0/G1 stage cell rate may drop to 32%in 1mM from 84%in control in 2mm follicular granulosa,drop to 31%from 76%in 4mm. there was no significant difference in 0.01mM follicular granulosa in 4mm and cumulus compared with control.SNP may increase S stage and G2/M stage cell rate,especially in 1mM(P＜0.05).0.1mM and 1mM SNP can inhibit estradiol secrete of granulosa cell of follicle 2mm and 4mm(P＜0.05),and 1mM inhibit progesterone secrete.0.01mM SNP promoted oocyte maturation which was stimulated by cool(P＜0.05),the maturation rate of oocyte raised to 18%from 15%in control,but it had no effects on parthenogenetic embryo development later.0.1mM and 1 mM suppressed oocyte maturation rate, cleavage rate.There was no blastocyst in 0.1mM and 1mM.The results up show that NO can influence granulosa cell morphology,apoptosis,cell cycle and steroid hormone secretion,when granulosa cell and cumulus are cultured in vitro,furthermore effects of NO are more difference in different NO concentration,the high concentration NO promoted abnormal morphology of granulosa.The low concentration NO did not;The high concentration NO promoted apoptosis of cumulus and granulosa in 2mm and 4mm.The low concentration NO depressed apoptosis; Meanwhile NO can also dose-dependent decreased G0/G1 stage rate,increased S stage and G2/M stage rate;high concentration NO can inhibit estradiol and progesterone secrete of granulosa of follicle 2mm and 4mm(P＜0.05);High concentration harmed oocyte maturation and embryo development later. |
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