The Self-secretory Function Of KISS1/GPR54 In Goat Leying Cells

KISS1(Kisspeptin as its protein)and G protein coupled receptor 54(G Protin-coupled Receptor 54,GPR54)play an important role in regulating animal reproduction through hypothalamic-pituitary-gonadal axis.Our previou studies have shown that Kisspeptin can be used as a new target to control the sexual desire and reproduction of animals.However,the mechanism of immune castration with Kisspeptin is not clear.Hormone immunity is mainly mediated by antibody neutralization,which affects the central axis of the hypothalamus-pituitary.The expression of KISS1 in testicular tissue is higher than that of other tissues,so it remains to be seen whether KISS1 gene immunity can achieve the goal of castration by autocrine of testicular tissue.Dazu black goatwas used as the test animal,the expression of KISS1/GPR54 in Leydig cells were analyzed by the immunohistochemical and quantitative PCR and enzyme-linked immunosorbent assay.To elucidate the mechanism of KISS gene immune castration through the autocrine,the autocrine and function of Kisspeptinafter overexpression processing were further analyzed in leydig cells.This study is expected to further enhance the immunocastration effect of KISS1 gene vaccine and provide theoretical basis for the wide application of hormone immunoregulation technology in animals.The specific methods and results of this study are as follows:(1)Expression distributions of KISS1 and GPR54 in goat tissues.The m RNA content of KISS1 and GPR54 genes were detected by q PCR in the hypothalamus,pituitary,testis,epididymis,pancreas,heart,liver,spleen,lung,stomach,kidney,intestine,and lymph nodes of goat.This result showed that expression of KISS1 and GPR54 was expressed in various tissues and higher expression in the hypothalamus,pituitary,and testis.(2)The expression of Kisspeptin and GPR54 in Leydig cells of goat.The expressions of Kisspeptin and GPR54 in testicular tissue were detected by immunohistochemistry,and the results showed that the expressions of Kisspeptin and GPR54 protein were found in Leydig cells and spermatogenic cells.Western Blotting and RT-PCR were used to detect the expressions of KISS1 and GPR54 in cultured Leydig cells.The results showed that both KISS1 and GPR54 genes and proteins were positively expressed in Leydig cells.(3)The effect of Kisspeptin overexpression on the autocrine of Kisspeptin in goat Leydig cells.The Leydig cells were treated with 100 nmol Kisspeptin,100 nmol Kisspeptin +100nmol peptin234 and physiological saline.The expressions of KISS1 and GPR54 were detected by q PCR method.The results showed that the m RNA levels of KISS1 and GPR54 treated by Kisspeptin was significantly higher than control group.(4)The effect of Kisspeptinby overexpressionprocessing on the autocrine function(or testosterone secretion)of Leydig cells in goat.The Leydig cells were treated with 100 nmol Kisspeptin,100 nmol Kisspeptin +100 nmol peptin234 and physiological saline.The secretion of testosterone was detected by ELISA.The results showed that Kisspeptin over expression could significantly increase the secretion of testosterone,and testosterone secretion didn't decresed significantly after treatment of antagonist.These results indicate that Kisspeptin can promote the testosterone secretion of Leydig cells through autocrine mode,and KISS1 gene immunity can exert an immunocastration effect by autocrine mode.