Effects Of AdipoQ And AdipoRs On Hormone Secretion And Expression Of Related Genes In Primary Pituitary Cells Of Laying Hens

Pituitary gland is one of the most important endocrine organs of animals,which consists of pituitary gland and neurohypophysis.Many hormones secreted by pituitary gland act on many target organs and tissues of the body through systemic circulation,and play an important role in regulating the reproduction,growth,development and metabolism of animals.A large number of studies have shown that adiponectin(AdipoQ)and its receptors(AdipoRs)are expressed and play pleiotropic roles in many tissues and cells of different species,including chicken adenohypophysis,but its biological role and mechanism in chicken adenohypophysis have not been reported.In this study,Hailan brown laying hens were used as experimental materials,and the expression and distribution of AdipoQ and its receptor in chicken pituitary tissue,their physiological effects on chicken pituitary cells and their potential mechanisms were explored at individual level and cellular level,so as to provide a basis for perfecting the physiological regulation theory of poultry.The main research contents and results are as follows:1 Distribution and localization of AdipoQ and AdipoRs in pituitary of laying hensThe pituitary tissues of 12 45-week-old healthy Hailan brown laying hens with similar weight were collected,and the expression of AdipoQ and its receptor m RNA and protein were detected by q RT-PCR,Western Blotting and immunohistochemistry.The results showed that AdipoQ and AdipoRs were widely expressed in hypothalamus,pituitary and ovary,and their m RNA and protein expression trends were similar.AdipoQ expression in pituitary was lower than that in hypothalamus but higher than that in ovary,and AdipoR1 and AdipoR2 were the highest in pituitary.2 Effects of over-expression/interference Adiopo Q on proliferation and apoptosis of chicken embryo primary adenohypophysis cellsHyland brown eggs with 15-18 embryo ages were selected to collect adenohypophysis under aseptic conditions,and the primary adenohypophysis cells of chicken embryos were isolated and cultured.CCK-8 method and flow cytometry were used to evaluate the effects of Adiopo Q on the viability,proliferation and apoptosis of adenohypophysis cells.Overexpressing Adiopo Q plasmid for 36 h,the activity of pituitary cells increased significantly(P<0.05).After 36 h of transfection,the activity of pituitary cells decreased significantly(P<0.05).After the expression of Adiopo Q gene for 36 h,the number of cells in S phase(P<0.01)and G2/M phase(P<0.05)increased significantly,and the number of cells in G0/G1 phase decreased significantly(P<0.05).The cell proliferation promoting genes CDK1(P<0.01)and CCND1(P<0.05)The expression levels of apoptosis-promoting genes Fas and Fas L were significantly down-regulated(P<0.01),Caspase3 was significantly down-regulated(P<0.05),and Bcl2 was significantly up-regulated(P<0.05).The effect of interfering Adiopo Q gene for 36 h on cell number and apoptosis in each cycle was contrary to the over-expression results,with the proliferation-promoting genes CDK1 and PCNA decreasing significantly(P<0.01)and CCND1 decreasing significantly(P<0.05).The expression levels of pro-apoptotic genes Casepase3 and Fas were significantly up-regulated(P<0.05),Fas L was significantly up-regulated(P<0.01)and Bcl2 was significantly down-regulated(P<0.01).3 Effects of over-expression/interference of AdipoQ on hormone s Secretion,related gene e Expression and AMPK signal pathway in chicken embryo primary pituitary cellsELISA and q RT-PCR were used to detect the effects of overexpression/interference of AdipoQ 36 h on the expression of hormones and related genes.The results showed that after overexpression of AdipoQ,the hormone secretion and the expression of corresponding genes of LH,TSH,PRL and ACTH were significantly down-regulated,and the m RNA expression of GH and Gn RHR and the hormone content of FSH were significantly reduced.The levels of GH,PRL and ACTH hormones and the expression of LHβ and Gn RHR m RNA were significantly increased after AdipoQ interference.AMPK inhibitor(Compound C)and overexpression/interference AdipoQ gene were used to co-treat adenohypophysis cells to explore the AMPK signaling pathway.WB method was used to detect the effect of overexpression/interference of AdipoQ for 36 h on AMPK phosphorylation in cells.Over-expression of AdipoQ significantly increased AMPK phosphorylation level(P<0.01).The phosphorylation level of AMPK decreased significantly after interfering with AdipoQ(P<0.01).When Compound C was used to reduce AMPK phosphorylation level in adenohypophysis cells,the hormone levels and corresponding gene expressions of FSH,LH and TSH,and the m RNA level of GH were significantly up-regulated(P<0.05).4 Effects of co-treatment with AdipoRs and AdipoRon on hormone secretion and related gene expression in chicken pituitary cellsELISA and q RT-PCR were used to study the effect of transfection of AdipoR1 or AdipoR2 interfering fragments and co-treatment with AdipoRon with a safe concentration of 5 μg/m L on adenohypophysis cells.The results showed that compared with NC control group,the contents of hormones GH and PRL and the expressions of genes LHβ,PRL and ACTH in si-AdipoR1 group were significantly increased(P<0.05),and the expressions of genes FSHβ and LHβ in si-Adipor1+AdipoRon group were significantly increased(P<0.05).Compared with NC control group,the levels of LH and GH hormones and the expressions of Gn RHR,TSHβ and GH in si-AdipoR2 group were significantly increased(P<0.05),while the levels of LHβ and GH in si-Adipor2+AdipoRon group were significantly increased compared with AdipoRon group(P<0.05).To sum up,AdipoQ and its receptors are expressed and distributed in chicken pituitary,and AdipoQ can promote the proliferation of chicken pituitary cells,inhibit their apoptosis and hormone secretion.Adipoq can inhibit the synthesis and secretion of chicken pituitary hormones FSH,LH,TSH and GH through AMPK signaling pathway.AdipoQ substitute(AdipoRon)inhibits the synthesis of LH,Gn RHR,TSH and GH by chicken adenohypophysis cells by acting with AdipoR1,and inhibits the secretion of FSH,LH,GH,PRL and ACTH by chicken adenohypophysis cells by acting with AdipoR2.