| Mammalian spermatogenesis is a complex process in which spermatogenic cells undergo differentiating and giving rise to spermatozoa. A variety of biology events occur during spermatogenesis, such as self-renewal of spermatogonia, meiotic division of spermatocyte and the spermiogenesis of spermatid. Spermatogenesis is the major function of adult testis. Many genes specifically and/or highly expressed in this tissue are involved in this process.Two strategies were used to identify the genes related to spermatogenesis: 1. Differential display RT-PCR Two populations of cells (DNA contains are 2n and 4n) in seminiferous tubule were sorted, the fragments specifically expressed in 4n cells were obtained and sequenced. 2. Differential hybridization of human testis cDNA micorarrays Human testis cDNA microarrays were constructed by spotted the PCR product amplified from human testis library. Then membranes were hybridized using the probes of embryonic testis (6 months) and adult testis.In first strategy, six ESTs were obtained from the 4n cells, one of them, R5, has high similarity with the Mus musculus cytosolic sialic acid 9-O-acetylesterase. The full-length gene of sialic acid-specific acetylesterase was isolated from human testis cDNA library, two transcripts of full length were obtained. They were named as homo sapiens sialic acid-specific acetylesterase I (HSEI) and homo sapiens sialic acid-specific acetylesterase II (HSEII). HSEI cDNA consists of 1990bp and contains an open reading frame (from 410bp to 1876bp), encoding a protein of 488 amino acids. The sequence of HSEI was depositedin the GenBank. Accession number is AF300796. The nucleotides length of HSEII is 2750bp, an open reading frame is between 13bp and 1584bp, encoding a protein of 523 amino acids. Accession number is AF303378.The main sequences of amino acids of HSEI and HSEII are same, but HSEII protein has an extra 35aa in its N terminal. Blasting the nucleotides sequences of HSEI and HSEII with data of GenBank, we locate them in 11q24. Nine exons of them are same, but two exons of HSEI and one exon of HSEII are different. It hints that HSEI and HSEII are two transcripts of HSE gene transcribed from the different transcription start sites. Curiously the exon (225-374bp) of HSEI is same with one of exons (1370-1519bp) of SP17, but they use different transcription chain. High resolution Stanford TNG radiation hybrid panel was used to localize HSEI and HSEII in human chromosome. They were localized in chromosome 11, coincident with the result of the genome sequence blast. The transcription patterns of HSEI and HSEII in various human tissues were investigated by northern blot, the results show that there are three bands, the sizes are about 2.7kb, 6.0kb and 7.5kb. The transcript of ~2.7kb is HSEII, it is expressed ubiquitously, but highly expressed in testis, .colon and prostate. The bands of ~6.0kb and ~7.5kb are the novel isofoms that haven't been cloned. The cellular localization of HSE was done by using in situ hybridization, the results showed that primary spermtatocytes and spermatids in seminiferous tubular have positive signals. The subcellular localizations of HSEI and HSEII were identified by GFP fusion protein. HSEI-GFP fusion protein was evenly distributing in cytoplasm, while HSEII-GFP was not evenly distributing in cytoplasm and there were many bright spots in cytosol. HSEI-GFP and HSEII-GFP had no signal in the region of their nucleus.In the second strategy, 592 clones were found, whose signals of hybridization in adult testis are 3 times higherthan that in embryonic testis. In the 317 unigenes of 592 clones, 42 novel full-length genes and 90 novel testis-specific transcripts were obtained. They were deposited in the GenBank. One of testis-specific transcripts, testis-specific calpastatin (tCAST) and two novel full-length genes, NYD-SP27 and human ubiquitin-like fusion protein (hUFP), were selected for study.In tCAST, signal intensity of hybridization in adult testis and embryo testis were 137.51 and 38.02 respective...
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