| Melatonin(MT)is an indoleamine hormone mainly secreted by the pineal gland,which regulates the reproductive activity of seasonal breeding animals.Sheep are short-day animals.With the increase of light exposure and MT secretion,ovarian follicles develop,mature and ovulate,and enter the breeding season.Granulosa cells are the main secretory cells in the follicle.The hormones,cytokines and proteins secreted by granulosa cells can not only maintain the growth and development of follicles,but also promote the maturation of oocytes and ovulation of follicles.Therefore,by exploring the effect of MT on the function of ovarian granulosa cells,this study provides a theoretical basis for exploring the mechamism of MT on reproduction of sheep.effect of MT on the regulation mechanism of seasonal reproduction in sheep.In this study,2-3 mm follicles of sheep in a slaughterhouse were collected as experimental materials,and different concentrations of MT(0,10-9,10-8,10-7,10-6 M)were added to the granulosa cells during the culture process.The proliferation of granulosa cells was detected by CCK-8 method.Annexin V-FITC/PI staining and flow cytometry were used to detect the apoptosis of sheep granulosa cells.According to the results of proliferation and anti-apoptosis of different concentrations of MT,the concentration of 10-7 MT was selected for subsequent experiments to detect the effects of MT on steroid hormone secretion and the expression of pituitary hormone receptor genes and steroid synthesis pathway genes and proteins.The secretion levels of progesterone(P4),estradiol(E2),cyclic adenosine monophosphate(cAMP),antioxidant dismutase(SOD)and reactive oxygen species(ROS)in the supernatant of granulosa cell culture medium were detected by ELISA.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression levels of related genes.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression levels of related genes.Western blot(WB)was used to detect the protein expression of MT receptor genes,pituitary gonadotropin receptor genes and steroid hormone synthesis related genes.The specific research results were as follows:(1)The addition of MT 10-7 M in the culture system of ovarian granulosa cells has a better proliferation effect.Compared with the control group,all MT supplemented groups significantly promoted the proliferation activity of OVine granulosa cells after treatment with different concentrations of MT for 24 h(P<0.05),and the cell proliferation activity of 10-8 M and 10-7 M groups was significantly higher than that of 10-9 M group(P<0.05),and the cell proliferation activity of 10-7 M group was the highest.However,there was no significant difference between 10-8 M and 10-6 M groups(P>0.05),and there was no significant difference between 10-6 M and 10-9 M groups(P>0.05).MT treatment after 48 h and 72 h,the MT group add cell proliferation activity were significantly higher than the control group(P<0.05),and the cell proliferation activity of the 10-6 M group was significantly higher than that of the 10-9 M group(P<0.05),and the cell proliferation activity was highest at the concentration of 10-7 M MT.However,there was no significant difference between 10-8 M and 10-6 M(P>0.05).(2)The addition of MT 10-7 M in the culture system of ovarian granulosa cells had a better anti-apoptosis effect.Compared with the control group,the granulosa cell apoptosis rate of 10-7 and 10-6 M MT treatment for 24 h was significantly decreased(P<0.01),and that of 10-8 M MT treatment for 24 h was significantly decreased(P<0.05),but there was no significant difference in the apoptosis rate of granulosa cells treated with 10-9 M MT(P>0.05).Compared with the 10-9 M group,the 10-8,10-7 and 10-6 M MT treatment significantly reduced the apoptosis rate(P<0.01),and the 10-7M MT treatment group had the lowest apoptosis rate,but there was no significant difference among the 10-8,10-7 and 10-6 M MT treatment groups(P>0.05).Compared with the 10-9 M group and the control group,the apoptosis rates of cells treated with 10-8,10-7 and 10-6 M MT were significantly decreased(P<0.01),and the apoptosis rate of cells treated with 10-7 M MT was still the lowest.However,there was no significant difference between the 10-8,10-7 and 10-6 M MT groups(P>0.05).This indicates that the anti-apoptotic effect of MT does not increase with the increase of the concentration when the concentration reaches a certain concentration,while the low concentration of MT has no effect on the apoptosis of granulosa cells.Further study showed that 10-7 M MT treatment for 48 h significantly up-regulated the mRNA expression of Bcl-2(P<0.05),and significantly down-regulated the mRNA expression of Bax and Caspase-3 and the ratio of Bax/Bcl-2(P<0.05).It was proved that MT could inhibit the apoptosis of granulosa cells by up-regulating the expression of anti-apoptotic genes and down-regulating the expression of pro-apoptotic genes.(3)The addition of MT to the ovarian granulosa cell culture system promoted the antioxidant capacity of the cells.After 48 h treatment with 10-7 M MT,the addition of MT significantly inhibited the secretion of ROS(P<0.05),promoted the secretion of SOD(P<0.05),and protected the proliferation and apoptosis of granulosa cells.(4)The addition of MT to the ovarian granulosa cell culture system downregulated P4 levels.After granulosa cells were treated with 10-7 M MT for 48h,the secretion levels of P4,E2,and cAMP in the supernatant of granulosa cell culture medium were measured.The results showed that 10-7 M MT significantly inhibited P4 secretion from granulosa cells(P<0.05),but had no significant effect on E2 and cAMP secretion(P>0.05).Further study showed that 10-7 M MT treatment for 48 h significantly up-regulated the mRNA and protein expression of MT1 in granulosa cells(P<0.01),and significantly down-regulated the mRNA and protein expression of FSHR(P<0.05).There was no significant difference in the mRNA and protein expression of MT2,LHR and CYP19A1(P>0.05).It was demonstrated that MT supplementation upregulated MT receptor genes and downregulated gonadotropin receptor genes and genes related to P4 synthesis,confirming the inhibitory effect of MT on P4 secretion.In conclusion,MT can promote the proliferation and inhibit the apoptosis of sheep granulosa cells by up-regulating the expression of anti-apoptotic genes,down-regulating the expression of pro-apoptotic genes and enhancing the antioxidant capacity.P4 secretion from granulosa cells is inhibited by inhibiting the mRNA and protein expression of FSHR,STAR,3β-HSD and CYP11A1,and the reduction of P4 secretion can alleviate the negative feedback regulation of GnRH in the hypothalamus,thereby helping to initiate the activity of the reproductive axis. |
PDF Full Text Request