Spermatogenesis Stage-specific Expression Of The Gene Isolation And Expression

Spermatogenesis is a complex course during which many genes are transcripted and translated under stringent temporal and spatial regulation. The identification and study of these differentially expressed genes is of great value to delineate the mechanism of spermatogenesis.Attributed to the extremely array rhythm of rat germinal cells in seminiferous tubule, the method of DDRT-PCR combined with segmentation technique of the seminiferous tubule was adopted to identify and isolate the differentially expressed genes in spermatogenesis.Two segments i.e. II VI stages and VII VIII stages were isolated under the anatomic microscope depending on the differential dioptres. 11 VI stages contain spermatogonia dividing into types A and B of spermatogonia, transition of primary spermatocytes to pachytene spermatocytes,and spennatids of 2 6 steps and 15 18 steps. VII VIII stages contain A type of spermatogonia,resting spennatocytes,pachytene spermatocytes, and 7 8 and 19 steps of spermatids, and the releasing of the matured spemiotozoa. This segment is without cell-division events.The total RNA of these two segments were extracted and reverse transcripted to cDNA, and were amplified with 3 single base-anchored polyT₁₂N(N is C,G or T) primers matched with the upstream arbitrary primers. The products were electrophoresly segregated and auto-radiophotographed. 112 differential cDNA bands were picked out and 76 were confirmed to be of difference in the two segments by reverse Northern dot-blot hybridyzation. 36 cDNA bands, in which 17 bands with a ratio of more than 10, were selected to be cloned and sequenced ,and 22 ESTs were obtained by means of submitting the new sequences to GenBank. The GenBank Accession Numbers are : AF094604, AF094605, AF094606,AF094607, AF095318, AF095320, AF095321,...