| With the development of research and methodology,it is found that adiponectin and its receptor exist in the gonads and reproductive tissues of many animals,which indicates that adiponectin and its receptor play an important role in regulating animal reproduction and embryo growth and development.Most of these studies are focused on mammals,but few on the reproductive function of poultry.Granulosa cells are good experimental models for the study of follicles development in poultry.In this experiment,Hy-line Brown Laying Hen was used as the experimental material,qRT-PCR and Western blot(WB)were used to study the biological function and regulatory mechanism of AdipoQ and AdiooRs in chicken ovarian granulosa cells in vitro,which laid a foundation for improving the theory of reproductive regulation in poultry.1.Expression and distribution of AdipoQ and AdiooRs genes in chicken tissues and ovaries(1)The expression of AdipoQ and adipors in chicken tissues and follicles was studied.The results showed that the mRNA of AdipoQ and adipors genes were widely distributed in various tissues and follicles of chicken.The expression of AdipoQ was the highest in the hypothalamus and fat(P<0.05).AdipoR1 was mainly expressed in leg muscles and hypothalamus,and AdipoR2 was mainly expressed in hypothalamus and pituitary.The expression of AdipoR1 was the lowest in F3 and the highest in SWF,SYF and LWF.The expression of AdipoQ was the highest in F1 and SYF follicles.The expression of AdipoR2 was the highest in F1 and F2 follicles.(2)The results of qRT PCR showed that AdipoQ and its receptor existed in granulosa cells and theca cells.The results of qRT PCR showed that the expression level of AdipoQ mRNA in theca cells was about 40 times higher than that in granulosa cells(P<0.001).The expression level of AdipoR1 mRNA in theca cells was about 2 times lower than that in granulosa cells(P<0.05).(3)The distribution and expression of AdipoQ and AdiooRs in ovary and follicle were analyzed by qRT-PCR and immunohistochemistry.The results of immunohistochemistry showed that the positive signals of AdipoQ,AdipoR1 and AdipoR2 were found in ovary.In addition,AdipoQ showed immunopositive signal in ovarian granulosa cells,but AdipoR1 and AdipoR2 were not detected in granulosa cells.2.The effect of AdipoRon on the proliferation,apoptosis and secretory function of primary ovarian granulosa cells(1)There was no effect on the viability of granulosa cells treated with AdipoRon(5ug/ml,10ug/ml)for 24 hours.At 48h and 72h,the activity of granulosa cells decreased significantly(P<0.05).The results showed that the apoptotic rate of granulosa cells was not affected by the treatment of AdipoRon(5ug/ml,10ug/ml)for 24 hours.The results showed that AdipoRon had no effect on the proliferation and apoptosis of ovarian granules in a certain concentration and time range.(2)In this experiment,the primary ovarian granulosa cells were isolated by enzyme digestion method.After being attached to the wall for 12 hours,the primary ovarian granulosa cells were treated with 0(control group),5,10ug/ml AdipoRon for 24 hours.The amount of ovarian hormone secretion in cell culture medium was measured by ELISA.The results showed that AdipoRon affected the secretion of ovarian reproductive hormone.AdipoRon(5ug/ml,10ug/ml)significantly inhibited the secretion of E2(P<0.05),and significantly stimulated the secretion of LH,FSH,PROG,AdipoR1,AdipoR2,GnRH(P<0.05).3.Analysis of the effect of adipoRon combined with interference or overexpression of AdipoRs on primary ovarian granulosa cellsOverexpression or interference of AdipoRs and AdipoRon(5ug/ml,10ug/ml)on the expression of reproductive hormone and receptor related genes in chicken ovarian granulosa cells for 24 hours.The results show that:(1)The level of ACC,p450scc,STAR and CYP19A1 mRNA in granulosa cells treated by AdipoRon(5ug/ml,10ug/ml)alone increased significantly(P<0.05)。AdipoRon(5ug/ml,10ug/ml)significantly inhibited the secretion of E2 and stimulated the secretion of LH,FSH,prog.AdipoR1,AdipoR2 and GnRH(P<0.05).We speculate that in the granulosa cells of chicken ovary,AdipoQ can promote the binding of LH and LHR receptor,thus stimulating the development of ovarian follicles and ovulation.(2)Interference with the combination of AdipoR1 or AdipoR2 and adiporon(5ug/ml,10ug/ml)can significantly increase the expression of FSHR and LHR,and promote the secretion of FSH and LH.Therefore,it can promote the development of small follicles and stimulate the secretion of steroid hormones.Over expression of AdipoRs had no effect on the levels of FSHB,STAR and GnRH mRNA,and the expression of LHR gene decreased significantly(P<0.05).Over expression of AdipoRs significantly stimulated the secretion of LH,decreased the expression of LHR,and affected its reproductive function.AdipoRon(5ug/ml,10ug/ml)treatment alone or overexpression of AdipoRs and adiporon(5ug/ml,10ug/ml)increased the expression of GnRH,promoted the secretion of GnRH,and promoted the binding of GnRH and GnRH receptor.AdipoRon(5ug/ml,10ug/ml)alone or overexpressing AdipoRs and AdipoRon jointly promoted p38MAPK mRNA level in granulosa cells.It can be inferred that AdipoQ is involved in p38 signaling pathway of chicken granulosa cells and plays a physiological role in early mammalian development.(3)Interference with AdipoRs significantly inhibited the secretion of AdipoRs,and overexpression of AdipoRs significantly promoted the secretion of AdipoRs.Overexpression or interference of AdipoRs combined with AdipoRon(5ug/ml,10ug/ml)significantly inhibited the secretion of E2,and promoted the secretion of LH,FSH,PROG.AdipoR1,AdipoR2,GnRH in granulosa cells(P<0.05).In addition,overexpression of AdipoR1orAdipoR2 significantly promoted the secretion of AdipoR1 and AdipoR2,and inhibited the secretion of AdipoQ(P<0.05).The secretion of adiponectin was negatively correlated with the abundance of AdipoR1 and AdipoR2 mRNA.The overexpression or interference of adipors combined with adiporon,respectively,promoted the secretion of progesterone and ovulation of follicles.In conclusion,according to our current research results,AdipoRon and its receptor can coordinate with a variety of factors to regulate the ovarian function of chicken,and play an important role in the initiation of sexual maturity and ovarian follicle development.The specific regulatory mechanism needs to be further studied. |
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