A Preliminnary Study On The Isolation And In Vitro Culture Of Buffalo Leydig Cells

This study was undertaken to research the testis of guangxi swamp buffalo. Based on the integration of culture methods for Leydig cells from many other species, we firstly cultured the buffalo Leydig cells in vitro. And we do something others about cryopreservation and revive, then a preliminary selection of important proteins associated with androgen production was made by studying expression growth characteristics and protein profiles of Leydig cells; this should provide important information on molecular mechanisms of male reproductive regulation. This research was divided into four sections.Chapter one:This describes the preliminarily selection of a suitable culture system for buffalo Leydig cells in vitro, and was made after careful consideration of culture methods used for in vitro culture of Leydig cells of other species. These showed that selection of a Petri dish, the time of digestion, the use of Percoll affect Leydig cell growth. At result, by combination with collagenase IV digestion for30-40min, and density with Percoll (gradient1.090、1.080、1.065、1.045), replace nutrient solution after6h, it was possible to culture successfully buffalo Leydig cells. Chapter two:This chapter describes the purification and identification of viability of Leydig cells obtained from Chapter one, and the conduct freeze-thaw cycles of cells growing in a viable state. The results showed that①cell growth three days, need digestion for3min; growth more than seven days will digestion30-40min; and②compared to primary Leydig cells, reproduction and proliferation of cells decreased significantly after subculture;③The optimal cryoprotectant was found to be70%DMEM/F12culture medium (without serum)+20%FBS (fetal bovine serum)+10%DMSO.Chapter three:The production of testosterone by Leydig cells stimulated by human chorionic gonadotropin (HCG) is described. By investigating treatments using different concentrations of HCG (0IU,25IU,50IU, and100IU), androgen levels were found to be sensitive to the HCG with the maximum concentration at50IU.Chapter four:By using high performance liquid chromatography and gas chromatography combined with mass spectrometry (LCMS, GCMS),3062proteins were separated from buffalo Leydig cells. In terms of metabolic pathways, Leydig cells have many metabolic and physiological functions, including the action of steroids in male hormones, and four types of proteins that are involved in steroid synthesis were identified:P450(cytochrome P450)、 lanosterol synthase、sterol o-acyltransferase1、NAD(P)dependent steroid dehydrogenase-like. In conclusion, this research has established a culture system that is suitable for buffalo Leydig cells. Identification of cell purity was conducted successfully, and research was also performed on cell subculturing and cryopreservation. Finally, by separating proteins related to the secretion of androgen by Leydig cells, a foundation has been established for understanding the mechanisms of reproduction in male buffalo.