Human Spermatogenesis Gene Tsmcak And The Of Nyd-sp16 Cloning And Functional Analysis

Spermatogenesis, the fundamental function of adult testis, is a continuum of cellular differentiation in which three principal phases can be discerned: spermatogonia renewal and proliferation, meiosis and spermiogenesis. It is a complex process involving cell division, differentiation and interaction between cells in the microenvironment of the seminiferous tubule. Many genes specifically or highly expressed in testis tissue are involved in the process of Spermatogenesis.Cloning of Spermatogenesis related genes and further functional study are very important to the elucidation of molecular mechanism involving in meiosis and spermiogenesis process. It is also important to clinical diagnosis and treatment of male infertility and male molecular contraception.In order to isolate novel genes expressed in tissues, many approaches based on different display measurement, such as suppressed subtractive hybridization (SSH), differential display reversed transcripted PCR (DDRT-PCR) and cDNA microarray technology have been developed and improved. In our lab, a human testis cDNA microarray was used to compare the genes differentially expressed between adult and fetal testes and 1522 differentially expressed clones were found. Among these clones, 40 were novel testis development and Spermatogenesis associated genes.In present study, two genes (tsMCAK and NYD-SP16) were selected for primary functional study.Part 1. Study on tsMCAK geneChromosome segregation during mitosis is a critical transport event and is accomplished through the dynamics of the mitotic spindle which iscomposed of many microtubules and kinesin-related proteins (KRPs). Among these KRPs, one family contains a central motor domain. The members of this family have been found in many species, such as human somatic cell, mouse, hamster, Xenopus and diatom. Previous study indicated that MCAK-like proteins were required for chromosome separation and cell division, proliferation. But the relationship between MCAK-like protein and testis function has not been reported.In this study, by using human testis cDNA microarray and differentially hybridization technique, a novel splice variant ofhuman mitotic centromere-associated kinesin (HsMCAK) gene was cloned. It was a new member of KRPs and named as testis-specific MCAK (tsMCAK) for its specific expression in testis tissue.The hybridization intensity of tsMCAK gene in adult and fetal testes was 45.84 and 12.65, respectively. This result indicated that tsMCAK was expressed 2.62 folds more in adult testis than fetal testis and it is a gene expressed highly in adult testis. The full length of tsMCAK cDNA is 2401 bp with a complete ORF of 2013 bp encoding a 671 amino acids protein. The nucleotide sequence of tsMCAK was submitted to GenBank under the accession number AY026505. Sequence analysis showed that it is highly homologous with HsMCAK gene; its peptide sequence is completely homologous with that of HsMCAK and just 54 amino acids shorter than HsMCAK; both proteins have a central kinesin motordomain and sequence homology within the motor domain between two proteins is 100% identity. Splicing comparison of tsMCAK with HsMCAK showed that tsMCAK gene is a splice variant of HsMCAK in testis tissue.tsMCAK gene is mapped to chromosome 1P34. Human multiple tissue cDNA PCR showed that tsMCAK-specific mRNA was particularly and strongly expressed in human testis tissue and no expression in othertissues. Expression study of tsMCAK gene in the testes of patients with spermatogenic obstacle showed that expression lose of tsMCAK gene is correlated with male infertility and confirmed location of tsMCAK gene in spermatogenic cells. The complete protein homology and identical conserved domain suggested that the function of tsMCAK in testis should be similar to that of HsMCAK. tsMCAK may be important for the chromosome segregation and proliferation and division of spermatogenic cells during spermatogenesis.In conclusion, tsMCAK' is a candidate gene for the testis-specific KRPs. Its specifically high ex...