Studies On The Effects Of Nitric Oxide On The Growth And Development Of Porcine Preantral Follicles

Preantral follicle was the early development of mammalian ovarian follicle,but only a few of them can be fully grown to maturation and ovulation because most of them became atresic or degenerated before formation of antrum folliculin.It is a waste of genetic resources.It would be of theoretical and practical importance to study and utilize these resources.Recently,many studies demonstrated that nhric oxide,as a signal molecular,also has its key role in the control of mammalian reproductive system,such as its involvement in the control of growth and development of oocyte.But there had been little reports about the effects of nitric oxide on the growth and development of preantral follicles.In order to observe the follicular survival,basal membrane integrity,follicular morphological normality,antrum formation,follicular diameter changes,and at the end of the culture,the development of follicle oocyte and the release of 17β-estrodiol and progesterone were also studied by using the way of culturing porcine preantral follicles, the treatments were:NO donor of SNP(sodium nitroprusside)(0,0.001,0.01,0.1 and 1mmol/L),NOS substrate of L-Arg(600μmol/L)and NO-S-inhibitor of L-NNA(1 mmol/L) and L-NAME(1 mmol/L).The results were as follows:①There were no significant differences on the growth during cultural period in follicle diameter among SNP treatments(P＞0.05),and all the treatments showed increase in diameter;the diameter of L-ARG+L-NNA treatment on day 4 were significantly higher than that of L-NNA,L-NAME and L-ARG+L-NAME(275μm vs 257μm,254μm,258μm,P＜0.05).The change on day 6 is similar to that on day 4,and the L-ARG+L-NNA treatment was significantly higher than the two inhibitors(P＜0.05);②The follicular survival rate of 1mmol/L SNP was significantly lower than that of 0.001mmol/L SNP(61.6%vs81.5%,P＜0.05).On day 2,the follicle survival rate of control, L-ARG and L-ARG+L-NNA treatments had significantly higher than that of L-NNA, L-NAME and L-ARG+L-NAME(P＜0.05);the treatments of L-ARC,L-NAME and L-ARG+L-NNA on day 4 had significantly higher than that of L-NNA and L-ARG+L-NAME(P＜0.05);the treatments of Control,L-ARG and L-ARG+L-NNA on day 6 were significantly higher than that of L-NNA and L-NAME treatments(P＜0.05),and the survival rate of L-ARG+L-NAME had significantly higher than that of L-NAME(66.7 %vs51.9%,P＜0.05);③The basal membrane integrity rate of 1mmol/L SNP was significantly lower than that of 0.001 mmol/L SNP and Control during the later cultural period(Day 4:61.8%vs90.9%,85.5%,P＜0.05;Day 6:55.2%vs87.6%,77.1%,P＜0.05); the basal membrane integrity rate of L-NAME treatment on day 4 was significantly lower than that of Control,L-ARG,L-ARG+L-NNA and L-ARG+L-NAME treatments(68.7% vs82.3%,81.1%,84.9%,81.8%,P＜0.05);the treatment of L-ARG on day 6 was significantly higher than that of L-NNA,L-NAME,L-ARG+LNNA and L-ARG+L-NAME(P＜0.05);④The conditions of morphological normality rate was the same like the basal membrane integrity,1mmol/L SNP was significantly lower than the Control and 0.001mmol/L SNP treatment(Day 4:54.9%vs,73.8%,71.8%,P＜0.05;Day 6: 45.2%vs60.6%,68.8%,P＜0.05);in addition,the treatment of 0.1mmol/L SNP on day 6 was significantly lower than that of 0.001mmol/L SNP(P＜0.05);The morphonorgical normality rate of L-NAME treatment for 4 day's culture was significantly lower than that of Control,L-ARG and L-ARG+L-NAME(P＜0.05),the treatments of two inhibitors on day 6 was significantly lower than the other treatments(P＜0:05);Compare to the Control, the treatment of L-ARG could significantly reverse the inhibitory effect of L-NNA and L-NAME(P＜0.05);⑤The antrum formation rate of treatments of SNP showed significant difference on day 4,with highest antrum formation rate of 0.001 mmol/L SNP reach to 50 %;in addition,the antrum formation rate of 0.001mmol/L SNP was significantly higer than that of 0.1 and 1mmol/L SNP treatments(73.1%vsS0.0%,47.6%,P＜0.05);The antrum formation rate of L-ARG and Control on day 4 were significantly higher than that of L-NNA,L-NAME and L-ARG+L-NAME(27.5%,28.5%vs12.6%,13.2%,16.8%, P＜0.05);the treatments of L-ARG and Control were significantly higher than that of inhibitors regardless of with or without L-Arg(P＜0.05);⑥At the end of the culture,from the oocyte normality rate,apart from 1 mmol/L SNP significantly lower than that of 0.001mmol/L SNP(P＜0.05),there were no significant difference among other treatments(P＞0.05);compare to treatments of L-ARG,L-NNA and L-NAME were significantly decreased in terms of the oocyte normality rate(58.4%vs33.5%,35.6%, P＜0.05);in addition,L-Arg could significantly reverse the inhibitory effect of L-NNA(P＜0.05),but could partially reverse the inhibitory effect of L-NAME(P＜0.05);the COC recovery rate of treatments of SNP were 22.9%,37.3%,25.6%,20.7%and 19.4%, respectively,and the COC recovery rate of 0.001mmol/L SNP was significantly higher than other treatments including the Control(P＜0.05);compare to Control,the COC recovery rate of treatment of L-Arg could significantly reverse the inhibitory effect of L-NNA and L-NAME(21.7%vs11.9%,8.9%,P＜0.05);⑦The levels of 17β-estrodiol of the Control on day 4 was significantly higher than those of the other SNP treatments (P＜0.05);apart from there were no significant differences on day 6 between 0.001mmol/L SNP and Control,the treatment of Control was significantly higher than that of other treatments(P＜0.05),and with the increase of SNP concentrations,the concentrations of 17β-estrodiol decrease;also,the release of progesterone of 1mmol/L SNP treatment on day 4 were significantly higher than that of Control,0.001 and 0.01mmol/L SNP(P＜0.05);the progesterone concentrations of treatment of Control on day 6 was significantly lower than those of the other SNP treatments(P＜0.05),and for the levels of progesterone there had doseage dependent manner with the increasement of SNP concentrations.The results indicated that during the growth and development of porcine preantral follicle,nitric oxide would involved in follicle growth,maintaining basal membrane integrity and morphological normality,antrum formation,oocyte normal development and controling of gonadal steroid hormone secretion.