Effects Of AdipoQ On Reproductive Hormone Expression And Potential Signaling Pathways In Hypothalamic Neurons Of Laying Hens

The HPG axis plays an important role in the reproductive development and functional maintenance of poultry.After receiving all kinds of information analyzed and integrated by the central nervous system,the hypothalamus at the top of the HPG axis secretes GnRH to stimulate the secretion of GTH in the anterior pituitary and promote gonadal development and steroid hormone secretion.In recent years,it has been found that adipose tissue is not only an important energy storage tissue of animal body,but also an active endocrine organ,which secretes a variety of fat factors,one of which is that the biological function of AdipoQ,AdipoQ is mediated by AdipoRs.AdipoQ and its receptors are expressed in hypothalamus,pituitary,gonad,uterus,embryo and other gonads and reproductive tissues of different animals,indicating that AdipoQ and AdipoRs play an important role in regulating animal reproduction and embryonic growth and development.At present,the effect of AdipoQ and AdipoRs on mammalian HPG axis has been confirmed,but there are few reports in poultry field,and the research focuses on the expression and role of gonadal tissue,but the expression and regulation of central system(hypothalamus-pituitary)has not been reported.Therefore,in this experiment,Hyland brown laying hens were used as experimental materials to culture primary hypothalamic neurons to study the effects of AdipoQ and AdipoRs on reproductive hormone secretion and related receptor expression of hypothalamic neurons in laying hens,and to clarify their action targets and regulatory mechanisms,so as to lay a foundation for improving the theory of poultry reproductive regulation.The main results of this study are as follows:1.The expression and distribution of AdipoQ and AdipoRs in hypothalamus of laying hens and their morphological relationship with GnRH neuronsThe localization of adiponectin and its receptor in hypothalamus of Hyland brown laying hens was studied by immunohistochemical method.The results showed that AdipoQ and AdipoR2 were located in the main hypothalamic nuclei that regulate food intake and energy balance,namely lateral hypothalamic area(LHA),funnel nucleus(IN),dorsomedial nucleus(DMN)and paraventricular nucleus(PVN).The morphological relationship between adiponectin receptor and gonadotropin-releasing hormone(GnRH)neurons was analyzed by immunofluorescence double labeling.The results showed that AdipoR1 and AdipoR2 were located in GnRH neurons of IN and DMN.The results suggest that adiponectin and its receptors may play an important role in the endocrine network that integrates energy balance and reproduction.2.Effects of overexpression or interference of AdipoQ on the expression of reproductive hormones and related receptors in hypothalamic neurons of laying hensIn this experiment,we successfully cultured and identified the primary hypothalamic neurons of laying hens,and verified the successful overexpression and interference of AdipoQ gene in hypothalamic neurons by qRT-PCR and WB techniques.The expression of reproduction-related genes and their receptors in hypothalamic neurons of laying hens were detected by qRT-PCR technique.the results showed that GnRH m RNA decreased significantly after overexpression of AdipoQ,while AdipoR1,AdipoR2,ER,PR,LHR and FSHR m RNA increased significantly,GnRH m RNA increased significantly after interfering with AdipoQ,and AdipoR1,AdipoR2,ER,PR,LHR and FSHR m RNA decreased significantly.Through qRT-PCR,ELISA and WB techniques,the results show that overexpression of AdipoQ can inhibit the production of GnRH gene,interfere with AdipoQ will promote the production of GnRH gene,and the levels of hormone and protein are consistent with the trend of genetics.3.Effects of overexpression or interference of AdipoQ on potential signal pathways of hypothalamic neurons in laying hensIn this experiment,primary hypothalamic neurons of laying hens were cultured and Compound C(AMPK inhibitor)was added alone.AMPK,AdipoQ,AdipoR1,AdipoR2,ER,PR,LHR and FSHR m RNA decreased and GnRH mRNA increased significantly by qRT-PCR technique.Overexpression or interference with AdipoQ and Compound C(AMPK inhibitor)co-treatment,through the study of qRT-PCR and ELISA techniques,the results showed that AdipoQ inhibited the production of GnRH through AMPK signal pathway,and had a certain effect on AdipoQ,AdipoR1,AdipoR2,ER,PR,LHR and FSHR m RNA.When LY294002(PI3K inhibitor)was added alone,PI3 K,AdipoQ,AdipoR1,AdipoR2,ER,PR,LHR,FSHR m RNA decreased and GnRH m RNA increased significantly by qRT-PCR technique.Overexpression or interference with AdipoQ and LY294002(PI3K inhibitor)cotreatment,through qRT-PCR and ELISA techniques,the results showed that AdipoQ inhibited the production of GnRH through PI3 K signal pathway,and had certain effects on AdipoQ,AdipoR1,AdipoR2,ER,PR,LHR and FSHR m RNA.4.Effects of interfering AdipoRs and AdipoRon co-treatment on the expression of reproductive hormones and related receptors in hypothalamic neurons of laying hensIn this experiment,the safe concentration range of AdipoRon was determined by CCK-8,qRT-PCR and WB techniques,and the successful interference of AdipoR1 and AdipoR2 genes in hypothalamic neurons was verified.The cells were co-treated with interfering AdipoR1 or AdipoR2 at the concentration of 5 μ g / ml of AdipoRon for 72 hours.The expression of related genes and receptors were detected by qRTPCR,ELISA and WB.The results showed that AdipoRon could inhibit the interference of AdipoR1 and AdipoR2 to a certain extent.Adding AdipoRon alone,interfering with AdipoR1 or AdipoR2 alone and co-processing had certain effects on AdipoQ,AdipoR1,AdipoR2,GnRH,ER,PR,LHR and FSHR m RNA.Through the study of qRT-PCR,ELISA and WB techniques,the results showed that adding AdipoRon alone,interfering with AdipoR1/AdipoR2 alone and co-treatment could inhibit the production of GnRH,and the levels of hormone and protein were consistent with the gene trend.These results suggest that adiponectin and its receptors may play an important role in the integration of energy balance and reproductive endocrine network.Overexpression of AdipoQ can inhibit the secretion of GnRH,interfere with AdipoQ and promote the secretion of GnRH.AdipoQ affects the secretion of GnRH through AMPK and PI3 K signaling pathways,and AdipoRon inhibits the interference of AdipoRs.The addition of AdipoRon,interference with AdipoRs and co-treatment have certain effects on the secretion of related reproductive genes and GnRH.