| Prolactin(PRL)is a polypeptide hormone encoded by a single gene.PRL can not only initiate and maintain lactation in mammals,but also affect ovarian development and ovulation in female animals.However,there are few studies about the effect of PRL on follicular development,and the mechanism of PRL in ewes follicular development has not been reported.Granular cells are an important structure of follicles,in addition to supporting follicular growth,the hormone secreted by granular cells can also maintain oocyte maturation.Therefore,the biological function of PRL in follicular development and atrettage can be elucidated by exploring the influence of PRL on the function of ewes granulosa cells.In this test,the ovary granular cells of the small-tailed Han sheep around 1 year old were used as the experimental material.Granular cells were cultured by adding PRL at different concentrations in culture medium,CCK-8 method was used to measure granular cells proliferation,Annexin V-FITC/PI stained granule cells,flowing cytometry was used to detect granulocyte apoptosis,ELISA kit to detect the content of estrogen(E2)and progesterone(P4)in the supernatant of granular cells,RT-qPCR detection of granular cells apoptosis and relative expression of hormone secretion-related genes,in order to study the effect of PRL on the proliferation,apoptosis and hormone secretion of sheep ovarian granulosa cells,and clarify the mechanism of PRL regulating sheep follicle development.The following results were mainly obtained:(1)Set up vitro culture system of sheep ovarian granulosa cells,added different concentrations of PRL(0,0.05,0.5,5 ?g/mL),and the granulosa cells were cultured continuously for 7d.Cell activity(absorbance of 450nm)was measured by cck-8 method every 24 hours,and the growth curve of granulosa cells was plotted.The results showed that PRL promoted the proliferation of sheep granulosa cells,and the concentration of PRL promoted significantly was 0.5 g/mL(P<0.05).(2)Flowing cytometry was used to detect the apoptosis of ewes granulosa cells at different concentrations of PRL(0,0.05,0.5,5?g/mL)after 24h and 48h treatment.The results showed that low concentration(0.05?g/mL)of PRL significantly promoted granular cell apoptosis after 24h treatment with PRL(P<0.05),while medium concentration(0.5 ?g/mL)and high concentration(5 ?g/mL)of PRL significantly inhibited granulosa cell apoptosis(P<0.05).After 48h treatment with PRL,granulosa cells apoptosis rates of 0.05,0.5 and 5?g/mL PRL were significantly lower than those of the control group(P<0.05).This indicated that the effect of PRL on the apoptosis of granulosa cells in sheep was related to the concentration and duration of PRL,and the short time of low concentration of PRL was a promoting effect on the apoptosis of granulosa cells.(3)To explore the regulatory mechanism of PRL on granulosa cell apoptosis,RT-qPCR was used to detect the mRNA expression levels of pro-apoptotic factors(Bax,p53 and Caspase3)and anti-apoptotic factors(Bcl-2)after sheep granulosa cells were treated with different concentrations of PRL(0,0.05,0.5,5 ?g/mL)for 24h.The results showed that adding PRL significantly up-regulated the expression of Bcl-2 mRNA(P<0.05),and increased with the increase of PRL concentration.Added low concentration(0.05 ?g/mL)PRL significantly up-regulated the expression levels of Bax and p53 mRNA(P<0.05).There was no significant difference in the effect of adding medium concentration(0.5?g/mL)and high concentration(5?g/mL)PRL on the mRNA expression of Bax,p53 and Caspase3(P>0.05).This indicates that the low concentration of PRL leads to granulosa cell apoptosis by promoting of proapoptotic factor gene expression.(4)Different concentrations of PRL(0,0.05,0.5,5 ?g/mL)were used to treat ewes granular cells for 24h,and detected cells that E2 and P4 secreted in the supernatant fluid by ELISA.The results showed that adding of PRL inhibited the secretion of E2 in granular cell.The E2 secretion in the medium concentration(0.5 ?g/mL)and high concentration(5 ?g/mL)groups was significantly lower than that in the control group and low concentration(0.05 ?g/mL)group(P<0.05).The addition of PRL significantly promoted the secretion of granular cell P4(P<0.05).P4 secretion increased with the increase of PRL concentration,but there was no significant difference in P4 secretion between the test groups(P>0.05).(5)Different concentrations of PRL(0,0.5 ?g/mL)were used to treat ewes granular cells for 24 h,48 h and 72 h.ELISA was used to detect the secretion levels of E2 and P4 in the superscript of the cells.The results showed that 0.5 ?g/mL PRL significantly inhibited E2 secretion in sheep ovarian granulosa cells after treatment for 24h,48h and 72h(P<0.05),E2 secretion after 72 hours of PRL treatment was higher than that after 48 hours,but the difference was not significant(P>0.05).0.5?g/mL PRL significantly promoted P4 secretion in sheep ovarian granulosa cells after 24h,48h and 72h treatment(P<0.05),and the P4 concentration gradient increased with time,indicating that the inhibition of E2 secretion and the promotion of P4 secretion by PRL on granulosa cells did not decrease with time.(6)RT-qPCR was used to detect the relative expression levels of FSHR,PRLR,LHR,CYP19,CYP11,3?-HSD and StAR genes after sheep granulosa cells were treated with different concentrations of PRL(0,0.05,0.5,5?g/mL)for 24h.The results showed that PRL up-regulated the mRNA expression levels of FSHR,PRLR,LHR,CYP11,3?-HSD and StAR,and down-regulated the mRNA expression levels of CYP19.This indicated that PRL stimulated FSH and LH signaling,inhibited E2 secretion of sheep granulosa cells by down-regulating CYP19,and up-regulated the expression levels of CYP11,3?-HSD and StAR to promote the P4 secretion of granulosa cells.In summary,PRL promoted the proliferation of sheep ovarian granulosa cells,and the apoptosis effect on granulosa cells was different depending on the concentration of PRL.The low concentration of PRL promoted the apoptosis though stimulated the Bax and p53 signals.In addition,PRL expanded FSH signaling by up-regulating the expression levels of PRLR,FSHR,and LHR mRNA,down-regulating CYP19 to inhibit E2 secretion from sheep granulosa cells,up-regulating the expression levels of CYP11,3?-HSD,and StAR to promote P4 secretion,and the effect of granulocyte hormone secretion does not diminish within a certain period of time.The results of this experiment provide a new theoretical basis for the study of the mechanism of follicle development by PRL. |
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