Differential Expression Of LRH-1 In Follicles Of Laying And Broody Chicken And Its Effect On Proliferation And Apoptosis Of Granulosa Cells

Ovary is the initial environment of animal life cycle,in which continuous automatic repair takes place.The stability of this dynamic feedback system stems from the cycle of cell proliferation and apoptosis in follicles.The endocrine signaling pathways involved hormones,hormone receptors and cytokines.In mammalian ovary,LRH-1 is specifically expressed in granulosa cells and luteal cells,which can promote follicular development and maintain ovulation.However,the effects of LRH-1 on reproductive function of poultry,especially on nest individuals,have not been reported.Therefore,it is of great significance to study the regulation of LRH-1 on reproductive performance of poultry.Sichuan Mountainous Black-bone Chicken?yellow feather line?has strong broody habits,with low egg production and low egg laying frequency,which results in low reproduction rate.Broody habit affects the development of breeding poultry production.In this experiment,eighty hens with broody behavior were selected from 30-week-old Sichuan Mountainous Black-bone Chicken?yellow feather line?,and 40 laying individuals were used as controls to study the characteristics of nesting period in the population,and to analyze the expression difference of LRH-1 in the follicles of broody and laying individuals.Then ten 30-week-old Rohmann layers were selected to study the effects of LRH-1 on the development of granulosa cells by isolating and culturing granulosa cells of follicles,and to explore the key role of LRH-1 in the selection of follicles and the maturation of follicles,in order to elucidate the regulation mechanism of LRH-1 on the proliferation and apoptosis of granulosa cells.The main results of this study are as follows:?1?The results of nest phenotype data showed that compared with laying hens'body weight and feed intake?2.4 kg and 113 g/d,respectively?,those of laying hens continued to decreased with the increase of nesting time(2.0 kg and 67 g/d for the 14^thday of nesting,respectively),while the body temperature of laying hens decreased significantly on the 7^thday of nesting?from 42?to 41.7??,but then recovered.?2?The expression level of LRH-1 in follicles at different stages of Sichuan Mountainous Black-bone Chicken was analyzed by quantitative fluorescence PCR.We found that LRH-1was expressed in follicles of different grades and small yellow follicle,and the expression level of LRH-1 in follicles of F1 and F6 was significantly higher than those in other follicles?P<0.05?.The expression pattern of cytochrome P450 family 11 subfamily A polypeptide 1?CYP11A1?regulating progesterone synthesis is contrary to that of cytochrome P450 family19 subfamily A polypeptide 1?CYP19A1?regulating estrogen synthesis.CYP11A1 has the highest expression level in F1 follicles,but none expression in small yolk follicles at laying and broody period.CYP19A1 expression was not detected in F1,but expressed in small yolk follicles at laying stage.Meanwhile,it was highly expressed in F6 follicles.?3?In the process of culturing granulosa cells of Rohmann layer follicles in vitro,flow cytometry was used to detect the apoptotic rate of granulosa cells.It was found that 36 h after interfering with LRH-1,inhibition of LRH-1 could significantly increase the early and late apoptotic rate of granulosa cells compared with negative control group?control group:5.2%and 5.0%;transfection group:14.1%and 13.5%,P<0.05?;and quantitative fluorescence PCR detected the relative expression of apoptotic gene Bcl-2 and p53 decreased significantly?P<0.05?.After 24 hours of interference,the concentration of reproductive hormones in cell culture medium was detected by ELISA.The results showed that the concentration of estrogen and progesterone decreased significantly?P<0.05?.?4?The granulosa cells were transfected with LRH-1 overexpression vector,and the proliferation of granulosa cells was detected by CCK-8 method.The results showed that over-expression of LRH-1 could significantly promote the proliferation of granulosa cells at24 h after transfection?OD=1.8 in the negative control group,OD=1.2 in the transfection group,P<0.01?,and the effect increased gradually with the duration of transfection.In this experiment,after 24 h of transfection,the gene of PCNA and Bcl-2 increased significantly compared with the control group?P<0.05?.In conclusion,LRH-1 is mainly expressed in the granular layer of follicles F1 and F6 of chickens,and plays a role in promoting cell proliferation during follicular development.In addition,LRH-1 inhibits follicular granulosa cell apoptosis by promoting the synthesis of estradiol and progesterone.