Testis-specific Protein Y-encoded Gene Research Articles

PCR amplification, sequencing, and in silico analysis of holandric genes TSPYand SRY in Nili Ravi buffalo

The present work has been designed to study the evolutionary perspectives of two important holandric genes, namely, testis-specific protein, Y-encoded (TSPY), and sex determining region, Y-encoded (SRY) in Nili Ravi buffalo. These Y-chromosomal genes are responsible for male sex development. The evolutionary aspects of these genes have not been studied in the Nili Ravi buffaloes from India. A study was carried out on the peripheral blood samples of three adult Nili Ravi bulls belonging to university farm. DNA was extracted using the phenol-chloroform isoamyl alcohol (PCI) technique and reported primer sequences were used to amplify the genes (partially) using a thermocycler. Three biological samples were analyzed using Sanger sequencing and were aligned using Clustal Omega. Separate phylogenetic trees for the genes were constructed using MEGA7 software. The selection pressure analysis and evolutionary divergence were individually studied for each of the genes. The current study reports the partial TSPY and SRY gene sequences of Indian Nili Ravi buffalo. Besides, the molecular evolution analysis indicates that the genes have experienced some purifying selection during the evolutionary process. Molecular characterization of these holandric genes (TSPY and SRY) in Nili Ravi will find applications in sex determination in Nili Ravi and other buffaloes.

Identification of Genetic Variants in Y-Chromosome Specific Genes Associated with Seminal Production in Murrah Buffaloes

Background: Marker assisted selection based strategies are essential to identify superior quality bulls at an early age for frozen semen production. The present study aims to determine the genetic variants in the Y-chromosome specific genes, sex determining region on Y-chromosome (SRY) and testis specific protein Y-encoded (TSPY) in association with semen production traits in Murrah buffalo bulls. Methods: The genomic DNA extracted from the blood samples of 70 Murrah buffalo bulls maintained under semen production in the frozen semen station were utilized for PCR amplification. The sequences of SRY and TSPY genes were screened for single nucleotide polymorphism using the seqman module of DNASTAR LASERGENE software. The single nucleotide variations in comparison with reference sequences and phylogenetic tree construct were obtained using Clustal W and MegAlign module. Result: The analysis revealed that the sequences of SRY and TSPY genes were monomorphic in nature, devoid of variations specifying that the sequences are highly conserved among the Murrah buffalo bulls studied. Hence, association studies with seminal traits could not be performed. However, the exon sequence of SRY gene of Murrah buffalo showed a single nucleotide variation at position 209 (G less than A) compared to the sequences of Nili-Ravi and Kundi buffaloes. Similarly, the TSPY gene exhibited a significant number of single nucleotide variations compared with Bos taurus sequences. The phylogenetic tree analysis evinced a close genetic relationship with less divergence between Murrah buffalo and other buffalo breeds and species in the bovinae family. Further extensive studies on SRY, TSPY and other Y-chromosome specific genes in a large, diverse population of Murrah buffalo bulls could help to discover a considerable number of SNP markers related to semen production for the early selection of breeding bulls.

Malignant ovarian germ cell tumours in gonadal Y chromosome mosaicism

AimsPrevious studies have shown that two partially overlapping mechanisms are responsible for the development of malignant ovarian germ cell tumours (MOGCT): either spontaneous mutations, mostly in KIT gene, or the...

Expression of the human TSPY gene in the brains of transgenic mice suggests a potential role of this Y chromosome gene in neural functions

The testis specific protein Y-encoded (TSPY) is a member of TSPY/SET/NAP1 superfamily, encoded within the gonadoblastoma locus on the Y chromosome. TSPY shares a highly conserved SET/NAP-domain responsible for protein--protein interaction among TSPY/SET/NAP1 proteins. Accumulating data, so far, support the role of TSPY as the gonadoblastoma gene, involved in germ cell tumorigenesis. The X-chromosome homolog of TSPY, TSPX is expressed in various tissues at both fetal and adult stages, including the brain, and is capable of interacting with the multi-domain adapter protein CASK, thereby influencing the synaptic and transcriptional functions and developmental regulation of CASK in the brain and other neural tissues. Similar to TSPX, we demonstrated that TSPY could interact with CASK at its SET/NAP-domain in cultured cells. Transgenic mice harboring a human TSPY gene and flanking sequences showed specific expression of the human TSPY transgene in both testis and brain. The neural expression pattern of the human TSPY gene overlapped with those of the endogenous mouse Cask and Tspx gene. Similarly with TSPX, TSPY was co-localized with CASK in neuronal axon fibers in the brain, suggesting a potential role(s) of TSPY in development and/or physiology of the nervous system.

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.

TSPY Gene Copy Number and Levels of mRNA for TSPY and Cell Cycle, Meiosis and Early Male Germ Cell Marker Genes in Holstein Bulls.

Testis-specific protein, Y-encoded (TSPY) is present in varying gene copy number in both human and cattle. Copy number variation of TSPY has been linked to spermatogenesis and is hypothesized to be a potential indicator of male fertility although there is limited data on TSPY expression in testicular tissue. Mice have only a single, non-functional TSPY gene whereas cattle have multiple functional copies and since slaughterhouse testicular material is readily available, cattle are a better model species for TSPY analyses. This study aims to compare TSPY copy number and mRNA transcript levels with that of various genes related to cell cycle and meiosis in Holstein bulls. To do this we measured TSPY copy number in DNA extracted from blood (n=51) and TSPY expression in total mRNA extracted from testicular tissue (n=26) with real time polymerase chain reaction (PCR). Our results show that TSPY copy number is negatively correlated to TSPY mRNA expression in the testis (r=-0.694, p<0.0001). We also found negative correlations of TSPY copy number and positive correlations of TSPY mRNA expression with various cell cycle genes (CCNB1, CCNB2, CDK1), meiotic genes (RAD51, SYCP3 and MLH1) and markers of early germ cells (UCHL1, TRPC2). Based on our results, it appears that TSPY expression may represent a good marker of early male germ cells. The negative correlation that we found between TSPY copy number and TSPY expression may have useful applications; TSPY copy number could be measured in blood to predict the amount of early germ cells in individual bulls. Supported by National Sciences Engineering and Research Council grant 364747-08; Canadian Research Chair program; and L'Alliance Boviteq Inc. (poster)

Transgenic Mouse Studies to Understand the Regulation, Expression and Function of the Testis-Specific Protein Y-Encoded (TSPY) Gene

The TSPY gene, which encodes the testis-specific protein, Y-encoded, was first discovered and characterized in humans, but orthologous genes were subsequently identified on the Y chromosome of many other placental mammals. TSPY is expressed in the testis and to a much lesser extent in the prostate gland, and it is assumed that TSPY serves function in spermatogonial proliferation and/or differentiation. It is further supposed that TSPY is involved in male infertility and exerts oncogenic effects in gonadal and prostate tumor formation. As a member of the TSPY/SET/NAP protein family, TSPY is able to bind cyclin B types, and stimulates the cyclin B1-CDK1 kinase activity, thereby accelerating the G2/M phase transition of the cell cycle of target cells. Because the laboratory mouse carries only a nonfunctional Y-chromosomal Tspy-ps pseudogene, a knockout mouse model for functional research analyses is not a feasible approach. In the last decade, three classical transgenic mouse models have been developed to contribute to our understanding of TSPY regulation, expression and function. The different transgenic mouse approaches and their relevance for studying TSPY regulation, expression and function are discussed in this review.

Mutations in the TSPYL1 gene associated with 46,XY disorder of sex development and male infertility

We screened 100 individuals with anomalies of testicular development or function for mutations in the TSPYL1 gene. A 46,XY female with complete gonadal dysgenesis carried a p.K320R mutation in the highly conserved NAP domain, and a 46,XY male with idiopathic azoospermia harbored a p.R89H mutation, and this data supports the hypothesis that mutations in TSPYL1 may contribute to anomalies of testicular development/function.

Gonadoblastoma locus and the TSPY gene on the human Y chromosome

The gonadoblastoma (GBY) locus is the only oncogenic locus on the human Y chromosome. It is postulated to serve a normal function in the testis, but could exert oncogenic effects in dysgenetic gonads of individuals with intersex and/or dysfunctional testicular phenotypes. Recent studies establish the testis-specific protein Y-encoded (TSPY) gene to be the putative gene for GBY. TSPY serves normal functions in male stem germ cell proliferation and differentiation, but is ectopically expressed in early and late stages of gonadoblastomas, testicular carcinoma in situ (the premalignant precursor for all testicular germ cell tumors), seminomas, and selected nonseminomas. Aberrant TSPY expression stimulates protein synthetic activities, accelerates cell proliferation, and promotes tumorigenicity in athymic mice. TSPY binds to type B cyclins, enhances an activated cyclin B-CDK1 kinase activity, and propels a rapid G(2)/M transition in the cell cycle. TSPY also counteracts the normal functions of its X-homologue, TSPX, which also binds to cyclin B and modulates the cyclin B-CDK1 activity to insure a proper G(2)/M transition in the cell cycle. Hence, ectopic expression and actions of the Y-located TSPY gene in incompatible germ cells, such as those in dysgenetic or ovarian environments and dysfunctional testis, disrupt the normal cell cycle regulation and predispose the host cells to tumorigenesis. The contrasting properties of TSPY and TSPX suggest that somatic cancers, such as intracranial germ cell tumors, melanoma, and hepatocellular carcinoma, with detectable TSPY expression could exhibit sexual dimorphisms in the initiation and/or progression of the respective oncogenesis.

TSPY Expression Is Variably Altered in Transgenic Mice with Testicular Feminization1

TSPY (testis-specific protein, Y-encoded) genes are expressed in premeiotic germ cells and round spermatids. The topology and timing of TSPY expression, and also its homology to members of the TTSN-family, suggest that TSPY is a proliferation factor for germ cells. There is also evidence for a role of TSPY in the aetiology of testis cancer. TSPY is a candidate for GBY, the elusive gonadoblastoma locus on the human Y chromosome, which is thought to predispose dysgenetic gonads of 46, XY sex-reversed females to develop gonadoblastoma. We have previously generated a TSPY transgenic mouse line (Tg(TSPY)9Jshm) that carries approximately 50 copies of the human TSPY gene on the mouse Y chromosome. In order to elucidate TSPY expression under complete androgen insensitivity and to investigate a possible role of TSPY in gonadal tumorigenesis, we have now generated sex-reversed TSPY transgenic Ar(Tfm) mice hemizygous for the X-linked testicular feminization mutation (Ar(Tfm)). We can show that the TSPY transcript is aberrantly spliced in the testes of TSPY-Ar(Tfm) mice, and that TSPY expression is upregulated by androgen insensitivity in some but not all animals. TSPY transgenic mice showed significantly increased testes weights. In one TSPY transgenic Ar(Tfm) animal, spermatogenesis proceeded beyond meiotic prophase. No tumors of germ cell origin were found in the testes of TSPY-Ar(Tfm) mice. Five out of 46 TSPY transgenic Ar(Tfm) mice, and 3 out of 31 age-related NMRI-Ar(Tfm) controls developed Leydig cell tumors, whereas none of the age-matched Ar(Tfm) mice (n=44) on a wild type background were affected by Leydig cell tumorigenesis.

Testis-specific protein Y-encoded gene is expressed in early and late stages of gonadoblastoma and testicular carcinoma in situ

The testis-specific protein Y-encoded gene (TSPY) is a tandem repeat gene located at the critical region for the gonadoblastoma locus on Y chromosome that predisposes the dysgenetic gonads of intersex individuals to oncogenesis. The expression and molecular properties of TSPY suggest that it is the putative gene for the gonadoblastoma locus on Y chromosome. In this study, we examined the expression of TSPY and other germ cell tumor markers in 4 cases of gonadoblastoma using immunostaining techniques. Our results showed that TSPY expression was closely associated with initiation and various stages of gonadoblastoma development. TSPY protein localized with established germ cell tumor markers, such as the placental alkaline phosphatase, c-KIT, and OCT3/4, in the same tumor cells of both gonadoblastoma and adjacent carcinoma in situ, the precursor for germ cell tumors. These findings support the candidacy of TSPY as the gene for the gonadoblastoma locus on Y chromosome and suggest that TSPY could be a significant marker for these types of germ cell tumors.

Finding of Hybrid Individuals between Native Japanese Macaques and Introduced Rhesus Macaques in the Bousou Peninsula, Chiba, Japan

In order to assess hybridization of rhesus macaques (Macaca mulatta) from an introduced population with native Japanese macaques (M. fuscata) in the Bousou Peninsula, we examined nine individuals for diagnostic genetic markers of blood proteins (adenosine deaminase and NADH-dependent diaphorase), TSPY (testis-specific protein, Y-encoded) gene and mitochondrial DNA. Two hybrid individuals were confirmed in an introduced group of rhesus macaques inhabiting the southernmost area of the peninsula. Close examination of genotypes revealed that one of the hybrids resulted from crossbreeding in the group for more than one generation. An adult male hybrid was also captured at Sugeno, Ichihara, located in the central hilly zone of the peninsula. Judging from the mtDNA profile, his natal group was not a rhesus but Japanese macaque group. These results suggest reciprocal hybridization between rhesus and Japanese macaques in the peninsula.

Gonadoblastoma, Testicular and Prostate Cancers, and the TSPY Gene

The role of the Y chromosome in oncogenesis of human cancers has been somewhat controversial. Both gain and loss of the Y chromosome in different leukemia, lymphoma, and solid tumors have been reported (Abeliovich et al. 1994; Brothman 1997; Dave et al. 1996; Geburek et al. 1997; Jackson-Cook et al. 1996; Kirk et al. 1994; Konig et al. 1994; Mertens et al. 1997; Riske et al. 1994; Watanabe et al. 1996). Conceivably, both oncogenes and tumor-suppressor genes exist on this chromosome, and they may act at different points during tumorigenesis, particularly for cancers of male-specific organs, such as the testis and the prostate.

Supernumerary marker chromosomes (SMCs) in Turner syndrome are mostly derived from the Y chromosome.

DNA and FISH (fluorescence in situ hybridization) analysis were carried out in 12 patients with stigmata of Turner syndrome to determine whether the Supernumerary Marker Chromosome (SMC) found cytogenetically in each of these patients was derived from the Y chromosome. The presence of a Y chromosome in these patients may predispose them to develop gonadoblastoma. PCR-Southern blot analysis, followed by FISH, was used to detect the presence of Y chromosome material. The Sex determining Region Y (SRY), Testis Specific Protein Y-encoded (TSPY) and Y-chromosome RNA Recognition Motif (YRRM) genes, which map at Yp11.31, Yp11.1-11.2 and Yp11.2/Yq11.21-11.23, respectively, were selected as markers, because they span the whole Y chromosome, and more importantly, they are considered to be involved in the development of gonadoblastoma. It was shown that in 12 patients, all of whom had an SMC, the SMC of 11 was derived from the Y chromosome. Furthermore, the presence of the SRY, TSPY and YRRM gene sequences was determined and FISH analysis confirmed the Y origin of the SMCs. The methodology described in this report is a rapid, reliable and sensitive approach which may be easily applied to determine the Y origin of an SMC carried in Turner syndrome. The identification of an SMC is important for the clinical management and prognostic counseling of these patients with Turner syndrome.

Molecular isolation and characterization of an expressed gene from the human Y chromosome.

Using a positional cloning approach, we have isolated an expressed gene from a flow-sorted Y chromosome cosmid library. The isolation of this gene was based on the identification of the Y-231 cosmid that contains CpG rich sequences (HTF islands) in its human insert. The Y-231 cosmid was capable of detecting a 1.3 kb transcript in poly (A)+ RNA samples from human testis. Several cDNA clones were isolated from a human testis cDNA library constructed in lambda gt10. In addition, DNA-mediated gene transfer and restriction enzyme mapping experiments demonstrated that two functional transcriptional units are present within the Y-231 cosmid. DNA sequencing analysis showed that the largest cDNA clone contains 1075 bp of unique sequence and a poly (A) track at the 3' end of the corresponding mRNA. An open reading frame of 762 bp that encodes a predicted protein of 253 amino acids with a calculated molecular weight of 28.9 kD was identified. The Y-231 structural gene encompasses approximately 2.7 kb of genomic sequence and contains six exons that are interrupted by five introns. The Y-231 gene shares very high (97%) identity at the DNA level to a previously described Y-specific gene, testis specific protein Y-encoded (TSPY) gene, suggesting the possibility that these two genes are related, if not identical. However, the TSPY gene has been postulated to be intronless. Further PCR and RT-PCR analyses of these two genes and their transcripts have provided evidence supporting the hypothesis that they are the same gene and are members of a Y-specific repeated gene family containing intronic sequences. The Y-231 (TSPY) gene is conserved in the male genome and expressed in the testis of the chimpanzee, suggesting that it may play an important role in the physiology of this organ in man and the great ape.