Laser Capture Microdissection (lcm) Expressed Genes In Human And Rat At Different Stages Of Spermatogenic Cells Differences

Spermatogenesis is a series of highly orderly processes, involving multiple gene expression under precise temporal and spatial regulation. The cloning and identification of these genes are of great value in delineating the mechanism of spermatogenesis including spermiogenesis. To elucidate the molecular mechanisms involved in the rhythmic cycling of germinal epithelial cells located in the seminiferous tubules, the method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) were adopted to identify and to isolate the differentially expressed genes during germ cell differentiation. In the present study, over 11,000 each of human and rat primary spermatocytes and round spermatids were isolated by LCM. Total RNAs were extracted from the two species of cells and used as templates to reverse transcribe the first-strand cDNA. Subsequently, the reversed double-stranded cDNA was synthesized by long distance PCR (LD PCR). The SSH was performed by utilizing two hybridizations followed by two suppressive PCR amplifications. The subtracted cDNAs obtained were cloned into pGEM?-T Easy vector and transformed in E. coli DH5a. The average insert size of the cDNA isolated from 150 randomly picked white clones was 500 bp, ranging from of 250 bp to 1.7 kb.The screening was performed by identify the human primary spermatocyte-specific cDNAs against those of round spermatid. Using the dot-blotting method, a total of 421 clones were spotted onto Hybond-N+ nylon membranes and hybridized with tester cDNA and driver cDNA probe. Strong signals were obtained with 390 clones. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. A search for sequence homology in the GenBank DNA and EST database by BLAST revealed that the distribution of EST clusters for high homology genes, high homology ESTs and novel ESTs were 58.7%, 1.77% and 39.5% , respectively, in primary spermatocyte-specific cDNAs. Using the dot-blot method, a total of 918 rat cDNA clones (603 primary spermatocyte-specific, 315 round spermatid-specific)...