Rsd-9 Function In Vivo Spermatogenic Remodeling Model And The Process Of Spermatogenesis Related Genes

Spermatogenesis is a complex developmental process that includes the mitotic proliferation of spermatogonial stem cells,meiotic prophase,division of spermatocytes, and morphological changes of haploid spermatids to highly specialized spermatozoa.The developmental program of spermatogenesis is dependent upon precise,developmental stage and germ cell type-specific gene expression.Therefore,the cloning and identification of these differentially expressed genes is of great value to delineate the mechanism of spermatogenesis.In the present study,we've developed a novel in vivo system to further elucidate the mechanism of spermatogenesis based on the technique of microinjection into seminiferous tubules of mouse.Then,we determined the functions of the RSD-9 gene that is a germ cell type-specific gene.In the past,we established the technique of microinjection into seminiferous tubules of mouse and the germ cell transplantation system.Based on it,The GC1 cells stably expressed GFP were transplanted into mouse seminiferous tubules.After three spermatogenic cycles,the frozen sections from testis tissues were observed by microscopy.In the seminiferous tubules of mouse testis of the experimental group, donor-derived cells developed into elongated spermatids identified by the staining of the acrosomes with TRITC-PNA.The green fluorescent-labeled cells stained with TRITC-PNA showed that donor GC1 spg cells did undergo differentiation into elongated spermatids.However,no green fluorescence from the spermatogenic clone was observed within the control side of the testicular seminiferous tubules,or within the epididymis.Based on the above findings,with each recipient mouse,one testis was used to transplant GC1 spg cells that stably expressed pRNAT/H1-RSD-9-RNAi-negative and the other one was transplanted GC1 spg cells that stably expressed pRNAT/H1-RSD-9-RNAi.After two to three spermatogenic cycles,the transplanted GC1 spg cells that stably expressed pRNAT/H1-RSD-9-RNAi-negative can differentiate and form donor-derived spermatogenic cell colonies and sperms in the recipient testes.But, the transplanted GC1 spg cells that stably expressed pRNAT/H1-RSD-9-RNAi can't differentiate and form donor-derived spermatogenic cell colonies in the recipient testes. Knock-down of the RSD-9 gene led to the stagnation of the process of spermatogenesis and the absence of the acrosome. RSD-9 was isolated from the mouse testis cDNA library using the fragment coding the coiled-coil region and the variable region of rtSH3P13 as bait in yeast two-hybrid system.RSD-9 protein has a structure similarity with GrpE,which serves as an ADP/ATP exchange factor in the Hsp70 chaperone machinery.Northern blot results revealed that RSD-9 gene was specifically expressed in brain and testis.Furthermove, immunohistochemistry has been performed to detect the localization of RSD-9 protein in testis tissues a significant staining pattern with anti-RSD-9 was detected from round and elongated spermatids to mature sperms,whereas no staining was detected in spermatogonia,spermatocytes closing to basal membrane as well as in Sertoli cells and Leydig cells.Then,we demonstrated that the RSD-9 protein interacted with rtSH3P13 in vitro and in vivo by GST-pulldown and Co-IP.Based on this background,we began our functional research of RSD-9 gene.By Blot overlay assay,we found RSD-9 protein bound with both ATP and GTP through its P-loop motif.Because rtSH3P13 is known to be involved in clathrin-dependent endocytosis,we examined the effect of overexpressing RSD-9 on this process.And we found that expression of RSD-9 andâ–³RSD-9 could regulate the endocytosis of transferrin.The clathrin-mediated endocytosis is very crucial for the process of architecture of acrosome,Knock-down of endogenous RSD-9 interfered the architecture of the acrosome,which confirms that RSD-9 is needed for this acrosome biogenesis process.