Construcktion Of RNA-SEQ CDNA Library Of Round Spermatid And Matural Spermatozoa Of Male Mice

Spermatogenesis is a complex proliferation process essential for all species undergoing sexual reproduction. It consists of three stages:mitosis in spermatogonia, meiosis in spermatocytes, and spermiogenesis. During spermiogenesis, round spermatids undergo complex morphological, biochemical, and physiological modifications resulting in the formation of mature spermatozoa under precise temporal and spatial regulation. Spermatogenesis in the rat consists of several unique morphologic cellular associations between Sertoli cells and developing germ cells within the seminiferous epithelium. To elucidate the molecular mechanisms involved in the rhythmic cycling of germinal epithelial cells located in the seminiferous tubules,the complexity of the cellular associations leads to difficulty in the isolation of individual cells at a defined stage of development for the study of their unique patterns of gene expression. Thus,the first important thing is to get a method of isolating the puritied cells.We successfully isolated about3×104round spermatids from freezing temperature-embedding testicular biopsies. Addationally, many of mature spermatozoas were isolated from caput epididymis. High quanlity total RNA were isolated from both round spermatid and mature spermatozo. RNA plays an important role in spermiogenesis, spermatozo living. Transcriptome analysis by RNA-Seq can provide a comprehensive understanding of molecular mechanisms involved in specific biological processes from the information on gene structure and function. While in round spermatids histones and non-histone proteins are replaced by transition proteins, in elongating spermatids, transition proteins are removed from the condensing chromatin and are replaced by protamines, which are the principal basic nuclear proteins of maturespermatozoa. Addiontionally,cells isolated by LCM were limitided partly. So the challenge is that total RNA were not enough for constructing libraries of RNA-Seq.The problem was soloved via linear amplification of mRNA.Firstly, we isolated puritied round spermatids by LCM and matural spermatozos from caput epididymis. Then the total RNA were amplified via mRNA linear ampification and successfully constructed the libraried for RNA-Seq.So the downstream sequencing can be started.