The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed d...The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.展开更多
A cDNA, designated as rtSH3p13, was isolated from a rat testis cDNA library. It consists of 1463 bp nuclear acids,which encodes a protein of 312 amino acids and was assigned the GenBank accession number AF227439. The ...A cDNA, designated as rtSH3p13, was isolated from a rat testis cDNA library. It consists of 1463 bp nuclear acids,which encodes a protein of 312 amino acids and was assigned the GenBank accession number AF227439. The deduced rtSH3p13 protein is a truncated isoform of SH3p13 as a result of mRNA alternative splicing. It is mainly expressed in the rat testis, detected in spermatids at the steps 8-19 of spermiogenesis, and found around the acrosome. During postnatal development, rtSH3p13 appears on day 18 and reaches maximum on day 60. Further experimental results suggested that rtSH3p13 forms a complex with activated epidermal growth factor receptor (EGFR) and interacts with synaptojanin I. Surprisingly, similar to SH3 domain, the V region of rtSH3p13 also inhibits endocytosis in CHO cells.Our results reveal a link between an rtSH3p13-synaptojanin-clathrin complex-mediated formation of pits and the process of spermiogenesis.展开更多
EGF was localized in human fetal, adult and cryptorchid testis and seminoma using an immunohistochemical method. Leydig cells of adult testes stained intensely for EGF while those of fetal testes showed weak staining ...EGF was localized in human fetal, adult and cryptorchid testis and seminoma using an immunohistochemical method. Leydig cells of adult testes stained intensely for EGF while those of fetal testes showed weak staining reaction. In addition, some spermatogonia and occasional peritubular myoid cells of adult testis stained positively. In cryptorchid testis, there were clusters of Leydig cells interspersed between seminiferous tubules with varying staining intensities. The number of positively stained cells in the cryptorchid testis were fewer than that in adult testes. The decrease in the number of Leydig cells producing EGF may account for the spermatogenesis arrest and infertility associated with this disorder.Intense staining of the cytoplasm of seminoma cells was observed, suggesting that the high production of EGF may be related to their invasive property. In conclusion, EGF is produced by human testis and Leydig cells are the principal source of this cytokine.展开更多
文摘The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.
基金This study was supported by grants from National Natural Science Foundation of China(No.30240019,30300060)National High Technology Research and Development Program of China(No.2001 AA221I31)+1 种基金Major State Basic Research Project(No.Gl 999055901)State Ministry of Science and Technology Program(No.2002BA7llA0l).
文摘A cDNA, designated as rtSH3p13, was isolated from a rat testis cDNA library. It consists of 1463 bp nuclear acids,which encodes a protein of 312 amino acids and was assigned the GenBank accession number AF227439. The deduced rtSH3p13 protein is a truncated isoform of SH3p13 as a result of mRNA alternative splicing. It is mainly expressed in the rat testis, detected in spermatids at the steps 8-19 of spermiogenesis, and found around the acrosome. During postnatal development, rtSH3p13 appears on day 18 and reaches maximum on day 60. Further experimental results suggested that rtSH3p13 forms a complex with activated epidermal growth factor receptor (EGFR) and interacts with synaptojanin I. Surprisingly, similar to SH3 domain, the V region of rtSH3p13 also inhibits endocytosis in CHO cells.Our results reveal a link between an rtSH3p13-synaptojanin-clathrin complex-mediated formation of pits and the process of spermiogenesis.
文摘EGF was localized in human fetal, adult and cryptorchid testis and seminoma using an immunohistochemical method. Leydig cells of adult testes stained intensely for EGF while those of fetal testes showed weak staining reaction. In addition, some spermatogonia and occasional peritubular myoid cells of adult testis stained positively. In cryptorchid testis, there were clusters of Leydig cells interspersed between seminiferous tubules with varying staining intensities. The number of positively stained cells in the cryptorchid testis were fewer than that in adult testes. The decrease in the number of Leydig cells producing EGF may account for the spermatogenesis arrest and infertility associated with this disorder.Intense staining of the cytoplasm of seminoma cells was observed, suggesting that the high production of EGF may be related to their invasive property. In conclusion, EGF is produced by human testis and Leydig cells are the principal source of this cytokine.
