Effects Of Recombinant Adiponectin On Hormone Secretion In Huoyan Geese Ovarian Granulosa Cells

As a new type of adipocytokines,adiponectin（AdipoQ）plays an important role not only in regulating lipid metabolism,but also in regulating animal reproductive system.At present,AdipoQ has been researched in human,rat and pig,and there has been some gratifying progress.But there are not many studies in poultry,and there are little reports about the effect of AdipoQ on ovarian hormone secretion.As a local breed,Huoyan goose is known for its excellent production performance.But in recent years,production performance of Huoyan goose has been degraded due to the irregular hybridization.This study expressed and purified recombinant AdipoQ after expression vector pET-28a-AdipoQ was constructed.And then this study examined the effect of recombinant AdipoQ on P₄ and E₂ production in Huoyan geese ovarian granulose cells to determine whether recombinant AdipoQ could be involved in the steroidogenesis process.This study also detected the expression levels of StAR,CYP11A1 and CYP19A1 mRNA and protein.This study investigate the possible role of AdipoQ in hormone secretion in Huoyan geese ovarian grannulosa cells,which establishes foundation to research the production performance and the regulation mechanism by AdipoQ in Huoyan geese.In this study,six laying period（10 months）Huoyan geese were selected as test object.The first,ovaries of the six laying period Huoyan geese were collected,respectively.And then the study designed specific primers according to AdipoQ gene sequence of anser cygnoides in GenBank.The CDS sequence of AdipoQ was amplified and connected with the pMD18-T vector.After that,pMD18-T-AdipoQ and pET-28a were respectively restriction enzyme digested by EcoRⅠand XhoⅠto contruct pET-28a-AdipoQ.After expression and purification of pET-28a-AdipoQ,the study obtained the His-tag recombinant AdipoQ and did Huoyan geese ovarian granulose cells with it（1μg/mL,2.5μg/mL and 5μg/mL）.This study measured P₄ and E₂ production by ELISA and detected the expression levels of StAR,CYP11A1 and CYP19A1 by qRT-PCR,IF and Western blot techniques in Huoyan geese ovarian granulose cells.This study had shown that recombinant AdipoQ was obtained under the condition of 0.4 mM IPTG and induced expression of 6 h at 30℃and purified using Ni-Agarose Resin.This study also showed that 24 h recombinant AdipoQ treatment affected P₄ and E₂ production in Huoyan geese ovarian granulose cells.P₄production increased significantly（P<0.01）and E₂ production decreased significantly（P<0.05）.The expression level of StAR mRNA and protein increased significantly（P<0.01）.Besides,the expression level of CYP11A1 mRNA and protein also increased significantly（P<0.05）.In contrast,the expression level of CYP19A1 mRNA decreased significantly（P<0.05）and the expression level of CYP19A1 protein was decreasing（P>0.05）.This study also suggested that the fluorescence of StAR and CYP11A1 protein enhanced（P<0.05）,whereas the fluorescence of CYP19A1 protein had the tendency to weaken（P>0.05）.This study demonstrated that there are some effects of AdipoQ on hormone secretion in Huoyan geese ovarian grannulosa cells,the mechanism of which may through that AdipoQ can promote the synthesis and secretion of P₄ by up regulation of the expression of StAR and CYP11A1 and also inhibit the synthesis and secretion of E₂ by down regulating the expression of CYP19A1.