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Human TSPY is a candidate oncogene and is supposed to function as a proliferation factor during spermatogenesis. It is the only mammalian protein-coding gene known to be organized as a tandem repeat gene family. It is expressed at highest level in spermatogonia and to a lower amount in primary spermatocytes. To characterize the human TSPY promoter we used the luciferase reporter system in a mouse spermatogonia derived cell line (GC-1spg) and in a GC-4spc cell line, that harbour prophase spermatocytes of the preleptotene and early pachytene stage. We isolated a 1303 bp fragment of the 5′-flanking region of exon 1 that shows significant promoter activity in GC-1spg and reduced activity in GC-4spc cells. In order to gain further insight into the organization of the TSPY-promoter, stepwise truncations of the putative promoter sequence were performed. The resulting fragments were cloned into the pGL3-vector and analysed for reporter gene activity in the murine germ cell lines GC-1spg and GC-4spc, leading to the characterization of a core promoter (−159 to −1), an enhancing region (−673 to −364) and a silencing region (−1262 to −669). Database research for cis-active elements yielded two putative SOX-like binding sites in the enhancing region and reporter gene activity was drastically reduced when three nucleotides of the AACAAT SOX core sequence were mutated. Our findings strongly suggest that testis-specific expression of human TSPY is mediated by Sox proteins. (Mol Cell Biochem 276: 159–167, 2005)

We have studied the process of tspy gene silencing in murine evolution. We have isolated functional tspy sequences from Apodemus agrarius, A. sylvaticus, A. flavicollis, and Mus platythrix (subgenus Pyromys) and nonfunctional tspy sequences from species of the subgenus Mus. We present two alternative models as to how tspy may have lost its function in the murine lineage.

Molecular aspects of murine evolution were studied by sequencing, and subsequently comparing, introns of the Y-chromosomal tspy genes from Apodemus agrarius, A. sylvaticus, A. flavicollis, Mus platythrix (subgenus Pyromys), M. booduga (subgenus Leggada), and from species of the subgenus Mus, including M. cervicolor, M. macedonicus and M.spretus. Estimates of nucleotide substitution rates in these lineages were in perfect agreement with phylogenetic data previously published by She et al. (1990), Catzeflis et al. (1992; 1993), and Lyon et al. (1996). The only exception was provided by a comparatively late divergence of M. spretus and M. macedonicus. Our data also suggest that M.booduga diverged from the subgenus Mus about 3 Myr ago.

Sequences homologous to human and bovine TSPY were isolated from M. musculus testicular cDNA, and a nearly full-length gene was polymerase chain reaction (PCR) amplified from mouse genomic DNA. This gene is apparently non-functional. Contrary to the situation encountered in species along the primate and artiodactyl lineages, in which TSPY is moderately repetitive, murine Tspy appears to be single copy. Murine Tspy is located on Yp, i.e. in the same syntenic group as in man. Sequence comparisons of murine, human and bovine TSPY exons suggest that TSPY became non-functional during rodent evolution.

We have isolated both a full-length rat Tspy cDNA from testicular mRNA by rapid amplification of cDNA ends (RACE) and RT-PCR and a full-length rat Tspy gene from genomic DNA by PCR. In contrast to the mouse, where Tspy is present in a single copy and is apparently functionless, and to man and cattle, where TSPY is organized in a moderately repetitive cluster, the rat Tspy locus apparently consists of one complete functional and one truncated, probably nonfunctional, copy, coherently localized on Yp, as revealed by FISH analysis.

Using expressed sequence tag (EST) sequence information, novel genes related to the Y chromosome gene family TSPY were isolated and characterized. Because of a significant homology to TSPY, the novel genes were designated TSPY-Like (TSPYL) and were assigned as new members of the TSPY-SET-NAP1L1 family. A human TSPYL gene was localized on chromosome 6 and a murine Tspyl gene was localized on chromosome 10. Expression of TSPYL was observed in all tissues investigated, as well as early onset during development. Both the human and murine Tspyl homologs lack introns over the entire region so far investigated and are thought to have arisen by an ancient retroposition event. Retroposition of Tspyl genes is supported by the isolation of a murine Tspyl pseudogene on chromosome 12 which also lacks intronic sequences, and by its observed proximity to an R element, a family of dispersed repetitive DNA.

We have isolated genomic sequences as well as transcripts from the bovine homolog of the human testis-specific protein, Y-encoded, TSPY which—in both species—is located on the Y Chromosome (Chr), organized as a gene family with a variable number of members, and expressed exclusively in the testis. 1266 bp of bovine TSPY specific sequence have been isolated from a testis cDNA library, by RT-PCR analyses and by Rapid Amplification of cDNA Ends (RACE). A bovine TSPY gene 4 is organized in seven exons, and transcripts are polyadenylated at various 3′ ends. Consensus polyadenylation signals AA UUAAA are missing. Microheterogeneous sequence variation is found between TSPY family members. In addition, homologies to other Y-located repeated sequence families, BRY, have been discovered; these sequences are presumably derived from ancient members of the TSPY cluster, now forming a separate, probably nonfunctional subfamily. Bovine TSPY is subject to differential splicing. In the adult, it is expressed in early germ-cell stages, and expression could also be detected in fetal testis. Comparison with the human homolog shows the highest degree of similarity in the coding regions of exons 2, 3, and 4, which are also precisely conserved regarding their length.