Characterization of a new TSPY gene family member in Yq (TSPYq1)

We investigated subinterval 6E on the human Y chromosome, a region frequently deleted in infertile males. YAC yOX17, mapped within subinterval 6E by STS-PCR, was analyzed for the presence of new genes. TSPYq1, a member of the TSPY multi-copy gene family, was isolated and characterized from a yOX17 cosmid subclone. PCR and FISH analysis performed on normal subjects and on patients with microdeletions of Yq suggested the presence of multiple copies of TSPY in Yq.     

Generation and characterization of a transgenic mouse with a functional human TSPY

To generate an animal model that is suitable for the analysis of regulation and expression of human testis-specific protein, Y-encoded TSPY, a transgenic mouse line, TgTSPY9, harboring a complete structural human TSPY gene was generated. Fluorescence in situ hybridization and Southern analyses show that approximately 50 copies of the human TSPY transgene are integrated at a single chromosomal site that maps to the distal long arm of the Y chromosome. The transgene is correctly transcribed and spliced according to the human pattern and is mainly expressed in testicular tissue, with spermatogonia and early primary spermatocytes (leptotene and zygotene) as expressing germ cells. TSPY transgenic mice are phenotypically normal, and spermatogenesis is neither impaired nor enhanced by the human transgene. The present study shows that a human TSPY gene integrated into the mouse genome follows the human expression pattern although murine tspy had lost its function in rodent evolution millions of years ago.     

Molecular docking uncovers TSPY binds more efficiently with eEF1A2 compared to eEF1A1

Testis-specific protein, Y-encoded (TSPY) binds to eukaryotic translation elongation factor 1 alpha (eEF1A) at its SET/NAP domain that is essential for the elongation during protein synthesis implicated with normal spermatogenesis. The eEF1A exists in two forms, eEF1A1 (alpha 1) and eEF1A2 (alpha 2), encoded by separate loci. Despite critical interplay of the TSPY and eEF1A proteins, literature remained silent on the residues playing significant roles during such interactions. We deduced 3D structures of TSPY and eEF1A variants by comparative modeling (Modeller 9.13) and assessed protein–protein interactions employing HADDOCK docking. Pairwise alignment using EMBOSS Needle for eEF1A1 and eEF1A2 proteins revealed high degree (~92%) of homology. Efficient binding of TSPY with eEF1A2 as compared to eEF1A1 was observed, in spite of the occurrence of significant structural similarities between the two variants. We also detected strong interactions of domain III followed by domains II and I of both eEF1A variants with TSPY. In the process, seven interacting residues of TSPY’s NAP domain namely, Asp 175, Glu 176, Asp 179, Tyr 183, Asp 240, Glu 244, and Tyr 246 common to both eEF1A variants were detected. Additionally, six lysine residues observed in eEF1A2 suggest their possible role in TSPY–eEF1A2 complex formation essential for germ cell development and spermatogenesis. Thus, more efficient binding of TSPY with eEF1A2 as compared to that of eEF1A1 established autonomous functioning of these two variants. Studies on mutated protein following similar approach would uncover the causative obstruction, between the interacting partners leading to deeper understanding on the structure–function relationship.     

Expression of a candidate gene for the gonadoblastoma locus in gonadoblastoma and testicular seminoma

The gonadoblastoma locus on the Y chromosome (GBY) predisposes the dysgenetic gonads of XY females to develop in situ tumors. It has been mapped to a critical interval on the short arm and adjacent centromeric region on the Y chromosome. Currently there are five functional genes identified on the GBY critical region, thereby providing likely candidates for this cancer predisposition locus. To evaluate the candidacy of one of these five genes, testis-specific protein Y-encoded (TSPY), as the gene for GBY, expression patterns of TSPY in four gonadoblastoma from three patients were analyzed by immunohistochemistry using a TSPY specific antibody. Results from this study showed that TSPY was preferentially expressed in tumor germ cells of all gonadoblastoma specimens. Additional study on two cases of testicular seminoma demonstrated that TSPY was also abundantly expressed in all stages of these germ cell tumors. The present observations suggest that TSPY may either be involved in the oncogenesis of or be a useful marker for both types of germ cell tumors.     

CK2-dependent C-terminal phosphorylation at T300 directs the nuclear transport of TSPY protein

TSPY (testis-specific protein, Y-encoded) is a member of the greater SET/NAP family of molecules with various functions, e.g., in chromatin remodeling, regulation of gene expression, and has been implicated to play a role in the malignant development of gonadoblastoma, testicular and prostate cancer. Here we demonstrate that the C-terminus has a functional role for the nucleo-cytoplasmatic shuttling of the TSPY protein. Using various combinations of in vitro mutagenesis and enhanced green fluorescent protein reporter gene-expression experiments we were able to show that while the deletion of C-terminus leads to a decreased stability and enhanced degradation of the protein, the selective mutation of a C-terminal CK2 phosphorylation site (T300) prevents the TSPY protein from entering the nucleus. We conclude that phosphorylation of the (T300) residue is a necessary and functional prerequisite for TSPY's transport into the nucleus reminding of comparable data from a related Drosophila molecule, NAP1.     

Sexing single bovine blastomeres using TSPY gene amplification

The testis-specific protein Y-encoded gene (TSPY) is a Y-specific gene present in variable copy number in many mammalian species, including cattle. We tested the applicability of the TSPY gene as a Y-specific marker to predict preimplantation embryo sex in Nelore (Bos indicus) cattle. Two blastomeres were removed from each embryo. A total of 36 single blastomeres and the remaining cells of their 18 matched in vitro conceived embryos were screened for TSPY amplification by nested-PCR. The results obtained from a single blastomere and the remaining cells of the same embryo were concordant in all cases. All blastomeres (16/16) from eight embryos produced with sexed sperm (specific for production of male embryos) were TSPY-positive. We conclude that TSPY is a good male-specific marker, the usefulness of which is probably enhanced by the high copy number. Other methods that are less time-consuming, such as real-time PCR, could be improved with the use of the TSPY gene sequences to generate primers and/or probes. This is the first report to demonstrate the applicability of the TSPY gene for sexing single cells in cattle.     

Organization and concerted evolution of the ampliconic Y-chromosomal TSPY genes from cattle

The Y-chromosomal gene TSPY (testis-specific protein Y-encoded) is probably involved in early spermatogenesis and has a variable copy number in different mammalian species. Analysis of bovine BAC clones leads to an estimate of 90 TSPY loci on the bovine Y chromosome. Half of these loci (TSPY-M1 and TSPY-M2) contain a single copy, while the other loci (TSPY-C) contain a cluster of three, possibly four, truncated pseudogenes. Fluorescence in situ hybridization indicated that the TSPY loci are located mainly on the short arm (Yp). The TSPY genes appear to account for about 2.5% of the Y chromosome and contain several published bovine Y-chromosomal microsatellites. The homology of TSPY and the major Y-chromosomal repetitive elements BRY.2 from cattle and OY.1 from sheep (80-85% similarity) further illustrates how the Y chromosome is shaped by rearrangements and horizontal spreading of the most abundant sequences. A comparison of TSPY-M1 sequences from different BAC clones and from related bovine species suggests concerted evolution as one of the mechanisms of the rapid evolution of the mammalian Y chromosome.     

Discovery of gene families and alternatively spliced variants by RecA-mediated cloning

Probing the functional complexity of the human genome will require new gene cloning techniques, not only to discover intraspecies gene homologs and interspecies gene orthologs, but also to identify alternatively spliced gene variants. We report homologous cDNA cloning methods that allow cloning of gene family members, genes from different species, and alternatively spliced gene variants. We cloned human 14-3-3 gene family members using DNA probes with as much as 35% sequence divergence, cloned alternatively spliced gene forms of Rad51D, and cloned a novel splice form of the human 14-3-3 theta gene with a unique expression pattern. Interspecies gene cloning was demonstrated for the mouse Rad51C and mouse beta-actin genes using human gene probes. The gene family cloning method is fast, efficient, and free from PCR errors; moreover, it exploits the abilities of RecA protein to pair homologous or partially homologous DNA sequences stably in kinetically trapped, multistranded DNA hybrids that can be used for subsequent gene clone enrichment.     

A comparison of TSPY genes from Y-chromosomal DNA of the great apes and humans: Sequence,evolution, and phylogeny

The genes for testis-specific protein Y (TSPY) were sequenced from chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and baboon (Papio hamadryas). The sequences were compared with each other and with the published human sequence. Substitutions were detected at 144 of the 755 nucleotide positions compared. In overviewing five sequences, one deletion in human, four successive nucleotide insertions in orangutan, and seven deletions/insertions in baboon sequence were noted. The present sequences differed from that of human by 1.9% (chimpanzee), 4.0% (gorilla), 8.2% (orangutan), and 16.8% (baboon), respectively. The phylogenetic tree constructed by the neighbor-joining method suggests that human and chimpanzee are more closely related to each other than either of them is to gorilla, and this result is also supported by maximum likelihood and strict consensus maximum parsimony trees. The number of nucleotide substitutions per site between human and chimpanzee, gorilla, and orangutan for TSPY intron were 0.024, 0.048, and 0.094, respectively. The rates of nucleotide substitutions per site per year were higher in the TSPY intron than in the TSPY exon, and higher in the TSPY intron than in the ZFY (Zinc Finger Y) intron in human and apes. © 1996 Wiley-Liss, Inc.