A mouse protein that localizes to acrosome and sperm tail is regulated by Y-chromosome

BACKGROUND: Acrosomal proteins play crucial roles in the physiology of fertilization. Identification of proteins localizing to the acrosome is fundamental to the understanding of its contribution to fertilization. Novel proteins are still being reported from acrosome. In order to capture yet unreported proteins localizing to acrosome in particular and sperm in general, 2D-PAGE and mass spectrometry analysis of mouse sperm proteins was done. RESULTS: One of the protein spots identified in the above study was reported in the NCBI database as a hypothetical protein from Riken cDNA 1700026L06 that localizes to chromosome number 2. Immunofluorescence studies using the antibody raised in rabbit against the recombinant protein showed that it localized to mouse acrosome and sperm tail. Based on the localization of this protein, it has been named mouse acrosome and sperm tail protein (MAST, [Q7TPM5 (http://www.ncbi.nlm.nih.gov/protein/Q7TPM5)]). This protein shows 96% identity to the rat spermatid specific protein RSB66. Western blotting showed that MAST is expressed testis-specifically. Co-immunoprecipitation studies using the MAST antibody identified two calcium-binding proteins, caldendrin and calreticulin as interacting partners of MAST. Caldendrin and calreticulin genes localize to mouse chromosomes 5 and 8 respectively. In a Yq-deletion mutant mouse, that is subfertile and has a deletion of 2/3rd of the long arm of the Y chromosome, MAST failed to localize to the acrosome. Western blot analysis however, revealed equal expression of MAST in the testes of wild type and mutant mice. The acrosomal calcium-binding proteins present in the MAST IP-complex were upregulated in sperms of Yq-del mice. CONCLUSIONS: We have identified a mouse acrosomal protein, MAST, that is expressed testis specifically. MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome. The misexpression of all the proteins identified in a complex in the Yq-del mice invokes the hypothesis of a putative pathway regulated by the Y chromosome. The role of Y chromosome in the regulation of this complex is however not clear from the current study

In vivo identification, survival, and functional efficacy of transplanted hepatocytes in acute liver failure mice model by FISH using Y-chromosome probe

Hepatocyte transplantation has excited much interest in lending temporary metabolic support to a failing liver following acute liver injury. The exact site from which they act and the clinical, biochemical, and histological changes in the recipient body following hepatocyte transplantation is yet to be worked out. The present study is an attempt to delineate location and function of transplanted hepatocytes and also the overall survival of these cells with a fluorescent in situ hybridization (FISH) technique using a Y-chromosome-specific probe in a carbon tetrachloride (CCl4)-induced mice model of fulminant hepatic failure. Fifty-five syngenic adult Swiss female mice of approximately the same age and body weight were divided into three groups. Group-1 (n=15), which received mineral oil, served as a negative control. Group-II (n=15) received CCl4 (3 mL/kg) 40% vol/vol in mineral oil, by gavage served as positive control for hepatic failure. Group-III (n=25) received intrasplenic transplantation of syngenic single cell suspension of hepatocytes in Hanks medium, after 30 h of CCl4 administration. Male Swiss adult mice (n=15) served as donors of hepatocytes. The overall survival of animals in groups I to III was 100, 0, and 70%, respectively, by 2 wk of the study period. Transplanted hepatocytes were identified by Periodic Acid Schiff (PAS) staining and confirmed with a FISH technique using the Y-chromosome probe. The majority of exogenously transplanted hepatocytes were found in the liver and spleen sections even after 1 wk of hepatocyte transplantation. Transplanted cells were mostly found to be translocated into the sinusoids of the liver. Transplanted hepatocytes were found to be beneficial as a temporary liver support in a failing liver, significantly improving the survival of the animals. In the present study, the FISH technique was used to unequivocally distinguish the transplanted cells from the host, and thus describes a model for studying the distribution and survival of the transplanted cells

Novel noncoding RNA from human Y distal heterochromatic block (Yq12) generates testis-specific chimeric CDC2L2

The human Y chromosome, because it is enriched in repetitive DNA, has been very intractable to genetic and molecular analyses. There is no previous evidence for developmental stage- and testis-specific transcription from the male-specific region of the Y (MSY). Here, we present evidence for the first time for a developmental stage- and testis-specific transcription from MSY distal heterochromatic block. We isolated two novel RNAs, which localize to Yq12 in multiple copies, show testis-specific expression, and lack active X-homologs. Experimental evidence shows that one of the above Yq12 noncoding RNAs (ncRNAs) trans-splices with CDC2L2 mRNA from chromosome 1p36.3 locus to generate a testis-specific chimeric β sv13 isoform. This 67-nt 5′UTR provided by the Yq12 transcript contains within it a Y box protein-binding CCAAT motif, indicating translational regulation of the β sv13 isoform in testis. This is also the first report of trans-splicing between a Y chromosomal and an autosomal transcript

Cytogenetic and molecular study of 370 infertile men in South India highlighting the importance of copy number variations by multiplex ligation-dependent probe amplification

Male infertility is a common and severe problem affecting 7% of population. The main objective of this study is to identify the chromosomal abnormalities, Y microdeletions in infertile men and also to access the frequency of abnormal sperm count. Based on the sperm count and viability, the infertile men were grouped as Azoospermia, Asthenospermia, Oligospermia and the remaining as Idiopathic infertility. A total of 370 infertile men and 60 normal control men were recruited. Chromosomal abnormalities were identified in 3 men (3/370). The prevalence of Y microdeletions in the infertile group is 8/370 in the Azoospermia factor (AZF) region with four AZFc deletion/duplication, two AZFa deletion, one AZF b & AZFc deletion and one case of total AZF a, AZFb & AZFc deletion. However, only five cases of Y microdeletions were identified by Multiplex PCR but an additional three cases by MLPA (Multiplex ligation-dependent probe amplification). Fluorescencein situhybridisation also confirmed the deletions. Here, we performed MLPA post-multiplex PCR, and our study revealed good yield of the Y microdeletion identification. The partial duplications which are difficult to be identified can now be easily identified by MLPA, and hence, we recommend MLPA as the choice of investigation compared to multiplex PCR for infertile men

Mice With Partial Deletion of Y-Heterochromatin Exhibits Stress Vulnerability

The role of Y chromosome in sex determination and male fertility is well established. It is also known that infertile men are prone to psychological disturbances. Earlier studies in the laboratory identified genes expressed in testes that are putatively regulated by Y chromosome in man and mouse. With the availability of a Y-deleted mouse model, that is subfertile, we studied the effect of a partial deletion of Y-chromosomal heterochromatin on mouse behavior when compared to its wild type. The partial Y-deleted mice exhibited anxiety like phenotype under stress when different anxiety (open field test and elevated plus maze, EPM test) and depression related tests (tail suspension and force swim) were performed. The mutant mice also showed reduction in hippocampal neurogenesis and altered expression of neurogenesis markers such as Nestin, Sox2, Gfap, NeuroD1 and Dcx using quantitative real time PCR (qPCR) analysis. The genes with altered expression contained short stretches of homology to Y-derived transcripts only in their Untranslated Regions (UTRs). Our study suggests putative regulation of these genes by the Y chromosome in mouse brain altering stress related behavior

Y chromosomal noncoding RNAs regulate autosomal gene expression via piRNAs in mouse testis.

Abstract Background Deciphering the functions of Y chromosome in mammals has been slow owing to the presence of repeats. Some of these repeats transcribe coding RNAs, the roles of which have been studied. Functions of the noncoding transcripts from Y chromosomal repeats however, remain unclear. While a majority of the genes expressed during spermatogenesis are autosomal, mice with different deletions of the long arm of the Y chromosome (Yq) were previously also shown to be characterized by subfertility, sterility and sperm abnormalities, suggesting the presence of effectors of spermatogenesis at this location. Here we report a set of novel noncoding RNAs from mouse Yq and explore their connection to some of the autosomal genes expressed in testis. Results We describe a set of novel mouse male-specific Y long arm (MSYq)-derived long noncoding (lnc) transcripts, named Pirmy and Pirmy-like RNAs. Pirmy shows a large number of splice variants in testis. We also identified Pirmy-like RNAs present in multiple copies at different loci on mouse Y chromosome. Further, we identified eight differentially expressed autosome-encoded sperm proteins in a mutant mouse strain, XYRIIIqdel (2/3 Yq-deleted). Pirmy and Pirmy-like RNAs have homology to 5′/3′UTRs of these deregulated autosomal genes. Several lines of experiments show that these short homologous stretches correspond to piRNAs. Thus, Pirmy and Pirmy-like RNAs act as templates for several piRNAs. In vitro functional assays reveal putative roles for these piRNAs in regulating autosomal genes. Conclusions Our study elucidates a set of autosomal genes that are potentially regulated by MSYq-derived piRNAs in mouse testis. Sperm phenotypes from the Yq-deleted mice seem to be similar to that reported in inter-specific male-sterile hybrids. Taken together, this study provides novel insights into possible role of MSYq-derived ncRNAs in male sterility and speciation