Cloning of cDNA for newt WT1 and the differential expression during spermatogenesis of the Japanese newt, Cynops pyrrhogaster

To investigate the function of Wilms' tumor 1 (WT1) during spermatogenesis, cDNA for newt WT1 homolog was cloned and the expression of WT1 in newt testes was examined. The cDNA is 2089 bp in length and encodes 426 amino acid (aa) residues. The deduced aa sequence shares 76 and 79% homology with human and Xenopus WT1, respectively. Northern blot analysis shows that WT1 mRNA, 3.2 and 4.5kb in length, are expressed in the testis and kidney. Both WT1 mRNA species are detected in various stages of spermatogenesis, but the 3.2kb mRNA is highly expressed in spermatogonia and mature sperm stages, while the amount of 4.5kb mRNA is almost constant throughout spermatogenesis. In situ hybridization reveals that WT1 mRNA is localized in Sertoli cells. Moreover, immunohistochemical analysis shows that WT1 protein is highly expressed in the nuclei of Sertoli cells in early spermatogonia and mature sperm stages, but not in pericystic cells or germ cells. These results suggest that WT1 is involved in the regulation of gene expression in Sertoli cells, depending on the spermatogenic stage.     

Cloning and characterization of a testis-specific thymosin beta 10 cDNA. Expression in post-meiotic male germ cells.

Thymosin beta 10 is one of a small family of proteins closely related in sequence to thymosin beta 4, recently identified as an actin-sequestering protein. A single molecular weight species of thymosin beta 10 mRNA is present in a number of rat tissues. In adult rat testis, an additional thymosin beta 10 mRNA of higher molecular weight was identified. Nucleotide sequencing of cDNA clones complementary to the testis-specific thymosin mRNA indicated that this mRNA differed from the ubiquitous thymosin beta 10 mRNA only in its 5'-untranslated region, beginning 14 nucleotides upstream of the translation initiation codon. These results, together with primer extension experiments, suggest that the two thymosin beta 10 mRNAs are transcribed from the same gene through a combination of differential promoter utilization and alternative splicing. The novel thymosin beta 10 mRNA could be detected only in RNA isolated from sexually mature rat testis. Both mRNAs were present in pachytene spermatocytes; only the testis-specific mRNA was detected in postmeiotic haploid spermatids. Immunoblot analysis using specific antibodies showed that the thymosin beta 10 protein synthesized in adult testis was identical in size to that synthesized in brain. Immunohistochemical analysis showed that the protein was present in differentiating spermatids, suggesting that the testis-specific thymosin beta 10 mRNA is translated in haploid male germ cells.     

Molecular cloning and characterization of SRG-L, a novel mouse gene developmentally expressed in spermatogenic cells

Full-length cDNA of a novel mouse gene upregulated in late stages of spermatogenic cells was cloned from mouse testis using overlapping RT-PCR and RACE. The mRNA of the gene was expressed mainly in diplotene/pachytene spermatocytes, round and elongating spermatids. We named this gene as SRG-L (Spermatogenesis Related Gene expressed in late stages of spermatogenic cells, GenBank Accession No. AY352586). The tissue-specific analysis showed a higher expression level in testis and spleen. The gene is mapped on chromosome 8q33.1 and contains 18 exons. The full-length of cDNA is 2,843 bp with an open reading frame (ORF) of 2,625 bp that encodes a 104 kDa protein (874 amino acids) with a putative transmembrane region. The bioinformatics analysis revealed that the SRG-L has two conserved regions, transglutaminase-like homologues domain and D-serine dehydratase domain, rich phosphorylation sites and methylation sites. The SRG-L protein was detected in diplotene/pachytene spermatocytes and spermatids by immunohistochemical staining and Western blot. The results suggest that SRG-L may play definite roles regulating differentiation of germ cells during spermatogenesis, particularly during meiosis and spermiogenesis.     

A new species of enkephalin precursor mRNA with a distinct 5'-untranslated region in haploid germ cells

To elucidate the primary structure of preproenkephalin (A) mRNA expressed by haploid germ cells (round spermatids) in rat testis, we have screened a lambda gt11 cDNA library for preproenkephalin cDNA inserts. The largest cDNA insert contained a protein-coding sequence encoding 269 amino acid residues as well as 327 and 309 bases of the 5'- and 3'-untranslated regions, respectively. The protein-coding region plus 3'-untranslated region of the mRNA was over 99% homologous to that of brain preproenkephalin mRNA, whereas the 5'-untranslated region contained a distinct sequence including a partial sequence of intron A of the preproenkephalin gene [(1984) J. Biol. Chem. 259, 14301-14308; (1984) J. Biol. Chem. 259, 14309-14313]. Northern blot analysis using a 5'-end-specific probe showed that this type of preproenkephalin mRNA exists exclusively in the germ cells.     

The nucleotide sequence of boar transition protein 2 (TNP2) cDNA and haploid expression of the gene during spermatogenesis

A cDNA clone coding for boar transition protein 2 (TNP 2) was isolated from a randomly primed cDNA library of boar testis. Sequence analysis revealed an open reading frame of 414 bp (corresponding to 138 amino acids), 33 bp of the 5' untranslated and about 300 bp of the 3' untranslated region. As compared to TNP 2 of mouse and rat, similarity with TNP 2 of the boar is approximately 70% at the nucleotide level and only about 40% on the basis of amino acid sequence. The similarity between boar and bull TNP2 is 77% and 64%, respectively. Northern blot experiments with RNA of different boar tissues and in situ hybridization on mature boar testis sections revealed testis-specific expression of the TNP 2 gene which is restricted to haploid germ cells. Hybridization experiments of boar TNP2 cDNA with testicular RNA of boar, bull, rat and mouse revealed decreasing intensities of the hybridization signals. With human testicular RNA no hybridization could be obtained.     

Identification and characterization of a haploid germ cell-specific nuclear protein kinase (Haspin) in spermatid nuclei and its effects on somatic cells.

We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I-III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (for haploid germ cell-specific nuclear protein kinase). Western blot analysis showed that specific antibodies recognized a protein of Mr 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells.