| Egg production performance,an essential economic trait in poultry production,depends mainly on follicular development,selection,and ovulation.Many factors influence follicular development,and the follicular granular and theca layers play an important role in regulating follicular development.Reproductive hormones and related genes precisely regulate this complex biological process.Unlike poultry,pigeons lay only two eggs during the laying cycle.In this study,we determined the laying interval of pigeons,measured the diameter and number of follicles on days 1,3,5,and 7 of the laying interval(LI1,LI3,LI5 and LI7),and mapped follicular development in pigeons,we used ELISA to determine the concentrations of GnRH,FSH,LH,PRL,P4,T,and E2 in the serum of pigeons at LI1,LI3,LI5 and LI7,P4,T,and E2 concentrations in the serum of pigeons at LI1,LI3,LI5,and LI7,RT-qPCR was used to detect the expression of CYP17A1,CYP19A1,HSD3B1,and HSD17B1,critical genes for steroid hormone synthesis in the granulosa layer,StAR,CYP11A1,and HSD3B1 in the granular layer,and gonadal axis reproductive hormone-related genes and their receptor expression(GnRH1,GnRHR,FSH,LH,FSHR,LHR,AR,ER,PR)and expression of critical genes for yolk deposition(APO-B,APO-VLDL II,VTG2,ER).The concentrations of FSH,LH,P4,T,and E2 in the yolk of different follicle grades were measured 6 h before ovulation,the expression of critical genes for steroid hormone synthesis CYP17A1,HSD3B1,and HSD17B1 in the inner follicular layer,the expression of the critical gene for steroid synthesis CYP19A1 in the outer follicular layer,and the expression of StAR,StAR,HSD3B1,and HSD17B1 in the granulosa layer were measured using RT-qPCR 6 h before ovulation,CYP11A1,and HSD3B1 expression in the granulosa layer.The main findings are as follows:(1)Female pigeons develop follicles in pairs,with two preovulatory follicles(F1 and F2)on the ovary and small follicles(SF1)developing in pairs on the ovary divided into SF1,SF2,SF3,and SF4,with F1 present at the LI1 stage and F2 developing at the LI3,with F1 and F2 follicles proliferating in volume during the LI3-LI5.GnRH expression in the serum of hens during the laying interval was highest in the LI1,and the concentrations of LI3,LI5,and LI7 decreased(P<0.01),FSH and LH hormone levels in the serum were higher in the pre-laying interval than in the later phase,and also showed a decreasing trend in the late egg-laying interval,P4 showed an increasing trend in the LI1-LI5 phase and decreased in the LI5-LI7 phase,E2 and T showed fluctuating changes during the egg-laying interval,but there was no significant difference in E2 during the egg-laying interval(P>0.05).(2)The expression trend of CYP19A1,an essential gene for steroid hormone synthesis,gradually decreased in F1 follicular membrane cells.The expression of CYP17A1 in F1 membrane cells in the LI3 stage was significantly higher than that in LI5 and LI7 stages(P<0.01),and the expression of HSD3B1 and HSD17B1 in the LI7 stage was the highest.The expression of CYP19A1 and HSD17B1 were significantly higher in the SF1 membrane cells of pigeons in the LI7 stage than in the LI5 stage(P<0.05),and the expression of HSD3B1 was significantly higher in the LI3 stage than in the other stages(P<0.01).The expression pattern of essential genes in steroid hormone synthesis pathway in F1 follicular granulosa cells,StAR expression was the lowest in LI1 and the highest in LI7,significantly higher than other periods(P<0.05).CYP11A1 and HSD3B1 showed the same expression trend as StAR,the lowest expression in LI1,and the highest in LI7,with an increasing trend in the egg-laying interval.StAR and HSD3B1 in F2 follicular granulosa cells gradually increased during the egg-laying interval,and the expression of CYP11A1 in the LI5 stage was significantly higher than in the LI7(P<0.01).The expression of StAR,CYP11A1,and HSD3B1 in SF1 follicular granulosa cells There was no significant difference in the expression of StAR,and CYP11A1 in SF1 follicular granulosa cells(P>0.05).The gene expression levels of gonadal axis-related genes differed in different egg-laying intervals.There was no significant difference in the expression of GnRH 1 in the hypothalamus of hens in different egg-laying intervals(P>0.05),and the expression of GnRHR was the lowest in the LI3 stage and significantly lower than that in the LI1 and LI5 stages(P<0.05).The expression trends of FSH and LH in the pituitary gland were the same,with the highest expression of FSHR at LI7 and the highest expression of LHR at LI1,which was significantly higher than the other periods(P<0.05).There was no significant difference in ovarian PR expression levels at the egg-laying interval(P>0.05),AR and ER expression was higher at LI3 than at other periods(P<0.05),and the difference was not significant at other periods(P>0.05).The expression of yolk synthesis genes APO-B and APO-VLDL II decreased in LI7 during the egg-laying interval and showed a decreasing trend.The expression of VTG2 and ER gradually increased in the liver and was significantly higher in LI7 than in LI1,LI3,and LI5(P<0.01).(3)Reproductive hormones were differentially expressed in the yolk of different follicle grades in preovulatory hens,FSH,LH,P4,T,and E2 contents were higher in SF1 yolk than in F1 and F2,and F1 concentration was higher than that of F2(P<0.05).StAR expression was highly significantly higher in F1 granulosa cells of preovulatory follicles than in F2 and SF1 granulosa cells(P<0.01),CYP11A1 was not significantly different in F1 and F2 granulosa cells(P>0.05).The expression of CYP17A1 was significantly higher in SF1 than F1 in preovulatory follicular endometrial cells(P<0.05),HSD3B1 was significantly higher in SF1 theca cells than F1(P<0.05),HSD17B1 was not significantly different in different grades of follicles(P>0.05).In summary,the pigeon reproductive hormones FSH,LH,PRL and P4 play a major role in the laying interval.The expression of P4 synthesis-related genes in the granulosa layer of graded follicles gradually increased as follicles continued to mature,and the expression of undifferentiated P4 related genes in the granular layer of SF1 was low,but the SF1 membrane layer could synthesize T and E2.The results of the study provide a theoretical basis for elucidating the effects of reproductive hormones on follicle development. |
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