Testis-specific Protein Research Articles

Putative molecular mechanism underlying sperm chromatin remodelling is regulated by reproductive hormones

BackgroundThe putative regulatory role of the male reproductive hormones in the molecular mechanism underlying chromatin condensation remains poorly understood. In the past decade, we developed two adult male rat models wherein functional deficits of testosterone or FSH, produced after treatments with 20 mg/Kg/d of cyproterone acetate (CPA) per os, for a period of 15 days or 3 mg/Kg/d of fluphenazine decanoate (FD) subcutaneously, for a period of 60 days, respectively, affected the rate of sperm chromatin decondensation in vitro. These rat models have been used in the current study in order to delineate the putative roles of testosterone and FSH in the molecular mechanism underlying remodelling of sperm chromatin.ResultsWe report that deficits of both testosterone and FSH affected the turnover of polyubiquitylated histones and led to their accumulation in the testis. Functional deficits of testosterone reduced expression of MIWI, the 5-methyl cap binding RNA-binding protein (PIWIlike murine homologue of the Drosophila protein PIWI/P-element induced wimpy testis) containing a PAZ/Piwi-Argonaut-Zwille domain and levels of histone deacetylase1 (HDAC1), ubiquitin ligating enzyme (URE-B1/E3), 20S proteasome α1 concomitant with reduced expression of ubiquitin activating enzyme (ube1), conjugating enzyme (ube2d2), chromodomain Y like protein (cdyl), bromodomain testis specific protein (brdt), hdac6 (histone deacetylase6), androgen-dependent homeobox placentae embryonic protein (pem/RhoX5), histones h2b and th3 (testis-specific h3). Functional deficits of FSH reduced the expression of cdyl and brdt genes in the testis, affected turnover of ubiquitylated histones, stalled the physiological DNA repair mechanism and culminated in spermiation of DNA damaged sperm.ConclusionsWe aver that deficits of both testosterone and FSH differentially affected the process of sperm chromatin remodelling through subtle changes in the ‘chromatin condensation transcriptome and proteome’, thereby stalling the replacement of ‘dynamic’ histones with ‘inert’ protamines, and altering the epigenetic state of condensed sperm chromatin. The inappropriately condensed chromatin affected the sperm chromatin cytoarchitecture, evident from subtle ultrastructural changes in the nuclei of immature caput epididymal sperm of CPA- or FD-treated rats, incubated in vitro with dithiothreitol.

Testis-Specific Protein Y-Encoded Copy Number Is Correlated to Its Expression and the Field Fertility of Canadian Holstein Bulls

Testis-specific protein Y-encoded (TSPY) is present in varying copy number in both human (20–76 copies) and cattle (37–200 copies), and some studies have linked this variation to semen quality in men. The purpose of this study was to determine if TSPY copy number is associated with fertility in bulls by using adjusted non-return rates, a commonly used measure of field fertility in Canada. In addition, we investigated the associations between TSPY copy number and its expression as well as specific semen parameters, such as average sperm concentration, sperm count, ejaculate volume, and motility. In 2 independent trials, TSPY copy number was shown to be positively correlated to adjusted non-return rates (trial #1: Spearman r = 0.34, p < 0.05; trial #2: Spearman r = 0.77, p < 0.01). Furthermore, TSPY copy number was inversely correlated to TSPY mRNA expression in the testis (Pearson r = –0.71, p < 0.0001). There were no correlations of TSPY copy number or expression with the semen parameters measured. Therefore, TSPY copy number might represent a potential marker of bull fertility, but its mechanism does not appear to be directly related to the semen characteristics analyzed as part of this study.

The bromodomain-containing protein tBRD-1 is specifically expressed in spermatocytes and is essential for male fertility

SummaryBy a conserved cellular differentiation process, spermatogenesis leads to formation of haploid sperm for successful reproduction. In Drosophila and in mammals, post-meiotic spermatid differentiation depends on several translationally repressed and stored mRNAs that are often expressed exclusively in the testis through a cell type specific transcriptional program. In Drosophila, the mRNAs of proteins required for post-meiotic chromatin reorganisation, like ProtB and Mst77F, are transcribed in meiotic spermatocytes and subjected to translational repression for days. Transcription of many of these translationally repressed mRNAs depends on testis-specific homologs of TATA box binding protein-associated factors (tTAFs). Here, we identified the testis-specific bromodomain protein, tBRD-1, that is only expressed in primary spermatocytes. Bromodomain proteins are able to recognise and bind acetylated histones and non-histone proteins. We generated tbrd-1 mutant flies and observed that function of tBRD-1 is required for male fertility. tBRD-1 partially colocalised with tTAFs, TAF1 and Polycomb to a Fibrillarin-deficient region within the spermatocyte nucleolus. The nucleolar localisation of tBRD-1 depended on tTAF function but not the other way round. Further, we could show that ectopically expressed tBRD-1-eGFP is able to bind to the interbands of polytene chromosomes. By inhibitor treatment of cultured testis we observed that sub-cellular localisation of tBRD-1 may depend on the acetylation status of primary spermatocytes.

The testis-specific double bromodomain-containing protein BRDT forms a complex with multiple spliceosome components and is required for mRNA splicing and 3'-UTR truncation in round spermatids.

Members of the BET (bromodomain and extra terminal motif) family of proteins have been shown to be chromatin-interacting regulators of transcription. We previously generated a mutation in the testis-specific mammalian BET gene Brdt (bromodomain, testis-specific) that yields protein lacking the first bromodomain (BRDTΔBD1) and observed disrupted spermiogenesis and male sterility. To determine whether BRDTΔBD1 protein results in altered transcription, we analyzed the transcriptomes of control versus BrdtΔBD1/ΔBD1 round spermatids. Over 400 genes showed statistically significant differential expression, and among the up-regulated genes, there was an enrichment of RNA splicing genes. Over 60% of these splicing genes had transcripts that lacked truncation of their 3′-untranslated region (UTR) typical of round spermatids. We selected four of these genes to characterize: Srsf2, Ddx5, Hnrnpk and Tardbp. The 3′-UTRs of Srsf2, Ddx5 and Hnrnpk mRNAs were longer in mutant round spermatids and resulted in reduced protein levels. Tardbp was transcriptionally up-regulated and a splicing shift toward the longer variant was observed. All four splicing proteins were found to complex with BRDT in control and mutant testes. We thus suggest that, along with modulating transcription, BRDT modulates gene expression as part of the splicing machinery. These modulations alter 3′-UTR processing in round spermatids; importantly, the BD1 is essential for these functions.

Ischemic cardiac tissue conditioned media induced differentiation of human mesenchymal stem cells into early stage cardiomyocytes

Mesenchymal stem cells (MSCs) are multipotent, can be easily expanded in culture and hence are an attractive therapeutic tool for cardiac repair. MSCs have tremendous potential to transdifferentiate to cardiac lineage both in vitro and in vivo. The present study examined the differentiation capacity of conditioned media derived from ischemic cardiac tissue on human MSCs. Human Bone marrow-derived MSCs after due characterization by immunocytochemistry and flow cytometry for MSC specific markers were induced by culture media derived from ischemic (n=13) and non-ischemic (n=18) human cardiac tissue. Parallel cultures were treated with 5-azacytidine (5-azaC), a potent cardiomyogen. MSCs induced with ischemic conditioned media formed myotube like structures, expressed sarcomeric Troponin I, alpha myosin heavy chain proteins and were positive for cardiac specific markers (Nkx2.5, human atrial natriuretic peptide, myosin light chain-2a, GATA-4) as was observed in 5-azaC treated cells. However, uninduced MSCs as well as those induced with non-ischemic cardiac conditioned media still maintained the fibroblast morphology even after 3weeks post-induction. Transmission electron microscopic studies of cardiomyocyte-like cells derived from MSCs revealed presence of sarcomeric bands but failed to show gap junctions and intercalated discs as of adult cardiomyocytes. These findings demonstrate that ischemic cardiac conditioned media induces morphological and molecular changes in MSCs with cardiac features, but at a primitive stage. Proteomics analysis of the ischemic conditioned media revealed differential expression of three relevant proteins (C-type lectin superfamily member 13, Testis-specific chromodomain protein Y2 and ADP/ATP translocase 1), whose exact role in cardiac regeneration needs further analysis.

Protein disulfide isomerase homolog PDILT is required for quality control of sperm membrane protein ADAM3 and male fertility

A disintegrin and metalloproteinase 3 (ADAM3) is a sperm membrane protein critical for both sperm migration from the uterus into the oviduct and sperm primary binding to the zona pellucida (ZP). Here we show that the testis-specific protein disulfide isomerase homolog (PDILT) cooperates with the testis-specific calreticulin-like chaperone, calsperin (CALR3), in the endoplasmic reticulum and plays an indispensable role in the disulfide-bond formation and folding of ADAM3. Pdilt(-/-) mice were male infertile because ADAM3 could not be folded properly and transported to the sperm surface without the PDILT/CALR3 complex. Peculiarly we find that not only Pdilt(-/-), but also Adam3(-/-), spermatozoa effectively fertilize eggs when the eggs are surrounded in cumulus oophorus. These findings reveal that ADAM3 requires testis-specific private chaperones to be folded properly and that the principle role of ADAM3 is for sperm migration into the oviduct but not for the fertilization event. Moreover, the importance of primary sperm ZP binding, which has been thought to be a critical step in mammalian fertilization, should be reconsidered.

Mice overexpression of &lt;i&gt;human augmenter of liver regeneration&lt;/i&gt; (hALR) in male germ cells shows abnormal spermatogenesis and reduced fertility

Human augmenter of liver regeneration (hALR) is a sulfhydryl oxidase that is highly expressed in spermatogonia and early spermatocytes. To investigate the physiological effects of hALR in spermatogenesis, we generated a hALR transgenic mouse model driven by the human TSPY (testis-specific protein, Y-encoded) promoter that allows the transgene to be specifically activated in the testes. hALR content was found to be increased in both germ cells. The histological and TUNEL analysis of transgenic testes revealed a number of spermatogenetic defects including primary spermatocyte overpopulation followed by depletion through apoptosis, degenerating and detached nucleated germ cells, haploid cell loss and intraepithelial vacuoles of varying sizes. In line with these features, adult transgenic male mice also displayed a reduction in fertility. Our data suggest that regulated spatial and temporal expression of hALR is required for normal testicular development and spermatogenesis, and overexpression of hALR results in influencing the sperm morphology and quantity and the eventual reduction in male fertility. Present findings in the mouse may be of interest to human male fertility.

Induction of Fetal Hemoglobin by Transcriptional Co-Activators MTF-1 and TSPYL1

Abstract 353Sickle cell disease (SCD) impacts one of 400 African-Americans born each year. Augmentation of fetal hemoglobin (HbF) levels is widely accepted as the most effective method for treating SCD, but hydroxyurea (HU) is currently the only approved drug that increases HbF. Thus, there is a need for the development of new therapies for this disease, including the identification of transcriptional activators that specifically up-regulate γ-globin (HbF). Developmental regulation of human β-like globin gene switching is controlled by several parameters, including cis- and trans-acting transcriptional determinants. Understanding the mechanisms underlying control of globin gene expression, particularly those involved in activation of γ-globin expression (HbF) is important for developing new treatments for SCD.Metal-responsive transcription factor-1 (MTF-1) is a key regulator of zinc metabolism in higher eukaryotes that controls the metal-inducible expression of metallothioneins and a number of other genes directly involved in the intracellular sequestration and efflux transport of zinc. Previous studies demonstrated that MTF-1 plays an essential role in liver development and that MTF-1-deficient mice display an anemic phenotype, suggesting a role for MTF-1 in hematopoiesis. In our study, when murine MTF-1 was expression was enforced, we observed a 5-fold increase in γ-globin expression in K562 cells. We also demonstrated increased γ-globin expression in adult blood from MTF-1 human β-globin locus yeast artificial chromosome (β-YAC) bi-transgenic (bigenic) mouse lines at the mRNA level by quantitative real-time RT-PCR (qPCR) and at the protein level by FACS analysis. Lastly, γ-globin gene expression was induced 12-fold in bone marrow cells (BMCs) derived from these bigenic mice compared to BMCs derived from β-YAC-only mice, and 3-fold after 6 hours of zinc treatment in β-YAC-only BMCs. Corroborative studies including zinc-deficient and zinc replete diets in β-YAC mice and erythroid-specific MTF-1 loss-of-function in loxP-flanked-MTF-1 LCR-β-globin promoter-Cre β-YAC mice further support a role for MTF-1 in g-globin gene expression. Chromatin immunoprecipitation (ChIP) analysis did not show recruitment of MTF-1 to any γ-globin gene-proximal metal response elements (MREs), the DNA motif that MTF-1 binds to control zinc metabolism genes. However, GATA-2 co-immunoprecipitated with MTF-1 in MTF-1 β-YAC BMCs, but not in β-YAC-only BMCs, suggesting that reactivation of γ-globin expression by MTF-1 might be mediated by a MTF-1-GATA-2 protein complex. ChIP experiments indicated that MTF-1 and GATA-2 co-occupy the same sites in the γ-globin promoter. Two of the stronger co-recruitment regions contain not only GATA sites, but also non-canonical MREs that vary by 1 or 2 bp from the canonical 7 bp MRE core. Interestingly, GATA-2 was induced 2-fold in adult blood of MTF-1 β-YAC mice, and also 3.5-fold in MTF-1 β-YAC BMCs treated with zinc for 6 hours. Our data suggest that activation of γ-globin by MTF-1 is mediated by protein-protein interaction with GATA-2 and that this multi-protein complex is targeted to GATA sites located in the γ-globin gene-promoters via binding of the GATA-2 protein.In a previous study we identified testis-specific protein Y-like 1 (TSPYL1) as a candidate gene involved in activation of γ-globin (de Andrade et al., 2006, Blood Cells, Mol. & Dis. 37:82). TSPYL1 mRNA level was increased 2–5 fold in deletional hereditary persistence of fetal hemoglobin (HPFH-2) subjects and decreased in a carrier of the Sicilian δβ-thalassemia trait. TSPYL1 is a transcription factor that is a member of the nucleosome assembly protein (NAP) family. TSPYL1 is not a DNA-binding protein; thus it exerts its effect through protein-protein interactions. When we enforced expression of human TSPYL1 in K562 cells an 11-fold induction of γ-globin expression was obtained. A reduction of γ-globin expression was observed following TSPYL1 knockdown in K562 cells. qPCR analysis of blood from TSPYL1 β-YAC bigenic mice showed that γ-globin expression was increased 4–12-fold. Taken together, our data strongly support the evidence that MTF-1 and TSPYL1 reactivate γ-globin expression in adult erythropoiesis. These two proteins represent potential new targets in strategies to reactivate γ-globin in hemoglobinopathies where higher levels of HbF would have beneficial effects. Disclosures:No relevant conflicts of interest to declare.

The first bromodomain of the testis-specific double bromodomain protein Brdt is required for chromocenter organization that is modulated by genetic background

Mice homozygous for a mutation ( Brdt ∆BD1/∆BD1 ) lacking the first bromodomain of Brdt, a testis-specific member of the BET family of double-bromodomain containing proteins, are sterile and exhibit profound defects in chromatin remodeling during spermiogenesis. We have now observed that a prominent feature of the aberrant spermatid nuclei is a fragmented chromocenter, a structure comprised of peri-centromeric heterochromatin. There was a concomitant increase in the levels of heterochromatin protein 1 alpha (Hp1α), suggesting that the presence of multiple chromocenters was correlated with a spread of heterochromatin beyond the normal centromeric region. Brdt protein was normally present throughout the nucleus but was excluded from the chromocenter. A more densely staining region of Brdt protein appeared to separate sirtuin 1 (Sirt1) protein from contact with the chromocenter. Although still nuclear, this unique localization of Brdt protein was lost in Brdt ∆BD1/∆BD1 mutant spermatids and Brdt and Sirt1 overlapped around the chromocenters. There was also ectopic localization of the H1 histone family, member N, testis-specific (H1fnt) protein in Brdt ∆BD1/∆BD1 round spermatids, which may be linked to the previously reported loss of polarized localization of peri-nuclear heterochromatin foci. The extent of chromocenter fragmentation was more severe and penetrant in mutant testes on a pure 129Sv/Ev as compared to a pure C57Bl/6 background. Indeed, all aspects of the mutant phenotype were more severe on the 129 Sv/Ev background. Contrary to previous studies in genetic models where fragmented chromocenters were observed in spermatids, the Brdt ∆BD1/∆BD1 mutant spermatids do not undergo apoptosis (on either background). These observations suggest that the first bromodomain of Brdt is critical in the formation and/or maintenance of an intact chromocenter and implicate this structure in proper remodeling of the chromatin architecture of the sperm head.

Verification of Male Infertility Biomarkers in Seminal Plasma by Multiplex Selected Reaction Monitoring Assay

Seminal plasma is a promising biological fluid to use for noninvasive clinical diagnostics of male reproductive system disorders. To verify a list of prospective male infertility biomarkers, we developed a multiplex selected reaction monitoring assay and measured the relative abundance of 31 proteins in 30 seminal plasma samples from normal, nonobstructive azoospermia and post-vasectomy individuals. Median levels of some proteins were decreased by more than 100-fold in nonobstructive azoospermia or post-vasectomy samples, in comparison with normal samples. To follow up the most promising candidates and measure their concentrations in seminal plasma, heavy isotope-labeled internal standards were synthesized and used to reanalyze 20 proteins in the same set of samples. Concentrations of candidate proteins in normal seminal plasma were found in the range 0.1-1000 μg/ml but were significantly decreased in nonobstructive azoospermia and post-vasectomy. These data allowed us to select, for the first time, biomarkers to discriminate between normal, nonobstructive azoospermia, and post-vasectomy (simulated obstructive azoospermia) seminal plasma samples. Some testis-specific proteins (LDHC, TEX101, and SPAG11B) performed with absolute or nearly absolute specificities and sensitivities. Cell-specific classification of protein expression indicated that Sertoli or germ cell dysfunction, but not Leydig cell dysfunction, was observed in nonobstructive azoospermia seminal plasma. The proposed panel of biomarkers, pending further validation, could lead to a clinical assay that can eliminate the need for testicular biopsy to diagnose the category of male infertility, thus providing significant benefits to patients as well as decreased costs associated with the differential diagnosis of azoospermia.

OCT4 immunohistochemistry may be necessary to identify the real risk of gonadal tumors in patients with Turner syndrome and Y chromosome sequences

The aim of this study was to investigate the frequency of gonadal tumors among patients with Turner syndrome (TS) carrying Y-derivative sequences in their chromosomal constitution. Six out of 260 patients with TS were selected based on mosaicism of the entire Y chromosome; 10 were included because Y-derivative sequences have been detected by PCR with specific oligonucleotides (sex-determining region on the Y, testis specific-protein, Y and DYZ3) and further confirmed by FISH. The 16 patients were subjected to bilateral gonadectomy at ages varying from 8.7 to 18.2 years. Both histopathological investigation with hematoxylin and eosin (H&E) and immunohistochemical analysis with anti-octamer-binding transcription factor 4 (OCT4) antibody were performed. Gonadal neoplasia was not detected in any of the 32 gonads evaluated by H&E; however, four gonads (12%) from three patients (19%) had positive OCT4 staining in 50-80% of nuclei, suggesting the existence of germ cell tumors (gonadoblastoma or in situ carcinoma). Evaluation of the real risk of development of gonadal tumors in TS patients with Y-derivative sequences in their chromosomal constitution may require a specific histopathological study, such as immunohistochemistry with OCT4.

SEQUENCE RECOMBINATION IN EXON 1 OF THE TSPY GENE IN MEN WITH IMPAIRED FERTILITY

The aim of this study was to evaluate TSPY (testis specific protein on the Y chromosome) gene and 5'UTR (UnTranslated Region) polymorphisms in men with impaired fertility compared to fertile controls. We analyzed 72 infertile men and 31 fertile controls usingconventional sequencing analysis to find crucial SNPs (single nucleotide polymorphism) and other changes. The most remarkable changes were found in the 1(st) exon only. In one half of the both infertile men and fertile controls, the most frequent finding was 26 SNPs with a similar pattern. In the other half we found highly relevant changes, generating a stop codon in the first third of exon 1. Early termination cut down the protein by 78.5%. This kind of change was not found in the fertile controls. No correlation was found between the spermiogram and the changes leading to the stop codon. The distribution of men with deletions, insertion and higher gene copy number was not statistically different. The changes found in exon 1 in infertile men could fundamentally affect the process of spermatogenesis. These findings could significantly enhance our understanding of the molecular-genetic causes of male infertility.

The THO complex is required for nucleolar integrity in Drosophila spermatocytes

The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. Here, we isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, our study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes.

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...

Transplantation Monitoring by Plasma DNA Sequencing

Over the past 15 years, there has been increasing interest in the use of cell-free nucleic acids in plasma for molecular diagnostics, especially for cancer detection and prenatal diagnosis. In 1998, Lo et al. showed that donor-derived DNA sequences could be detected in the plasma of transplant recipients (1). This group further showed that different transplanted organs—e.g., the heart, liver, and kidney—appeared to release different amounts of DNA into the plasma, probably relating to the size of the organ (2). Furthermore, details in the design of the molecular assay have been shown to have important implications for detecting circulating donor-derived DNA. For example, the use of a detection target, DYS14 (1,3), which is a member of a multicopy Y chromosome–specific gene, TSPY1 3 (testis specific protein, Y-linked 1), has produced a higher sensitivity in detecting circulating donor-derived DNA than when the single-copy gene SRY (sex-determining region Y) is used (2). As cell death is generally accepted to be an important reason for the release of DNA into the plasma, Lo et al. further hypothesized that the measurement of donor-derived DNA in the plasma of transplant recipients might be used for monitoring graft rejection (1). This hypothesis has subsequently been shown to be correct by a number of independent workers (4),(5). The usefulness of this strategy depends, however, on the ability to design molecular assays that allow the detection of donor-specific DNA sequences. Most workers in this field have thus far used …

Cancer Testis Antigen, Ropporin, Is a Potential Target for Multiple Myeloma Immunotherapy

Despite recent improvements in standard pharmacologic treatments of multiple myeloma (MM), immunotherapy may prove to be more effective due to its higher specificity and lower toxicity. A novel cancer/testis antigen, ropporin, is a testis-specific protein localized in the sperm flagella. Comparing ropporin expression in healthy and MM samples, we did not detect ropporin expression in the normal tissues, but positive signals were found in 44% of the MM primary samples. The immunogenicity of ropporin was confirmed by the presence of specific antibodies detected by enzyme-linked immunosorbent assay in patients' serum. Our results show that ropporin is a novel cancer/testis antigen for MM. Except for in the testis, an immune privileged site, ropporin was not expressed in normal tissues, but was present in MM cell lines and patients' samples. Noteworthy, we show for the first time that ropporin was present at the cell surface of MM plasma cells. We suggest that ropporin is a promising target for MM immunotherapy, as we were able to generate human leukocyte antigen class I-restricted cytotoxic lymphocytes able to kill autologous MM cells.

TSPYL2 Is Important for G1 Checkpoint Maintenance upon DNA Damage

Nucleosome assembly proteins play important roles in chromatin remodeling, which determines gene expression, cell proliferation and terminal differentiation. Testis specific protein, Y-encoded-like 2 (TSPYL2) is a nucleosome assembly protein expressed in neuronal precursors and mature neurons. Previous studies have shown that TSPYL2 binds cyclin B and inhibits cell proliferation in cultured cells suggesting a role in cell cycle regulation. To investigate the physiological significance of TSPYL2 in the control of cell cycle, we generated mice with targeted disruption of Tspyl2. These mutant mice appear grossly normal, have normal life span and do not exhibit increased tumor incidence. To define the role of TSPYL2 in DNA repair, checkpoint arrest and apoptosis, primary embryonic fibroblasts and thymocytes from Tspyl2 deficient mice were isolated and examined under unperturbed and stressed conditions. We show that mutant fibroblasts are impaired in G1 arrest under the situation of DNA damage induced by gamma irradiation. This is mainly attributed to the defective activation of p21 transcription despite proper p53 protein accumulation, suggesting that TSPYL2 is additionally required for p21 induction. TSPYL2 serves a biological role in maintaining the G1 checkpoint under stress condition.

Testis-specific glyceraldehyde-3-phosphate dehydrogenase: origin and evolution

BackgroundGlyceraldehyde-3-phosphate dehydrogenase (GAPD) catalyses one of the glycolytic reactions and is also involved in a number of non-glycolytic processes, such as endocytosis, DNA excision repair, and induction of apoptosis. Mammals are known to possess two homologous GAPD isoenzymes: GAPD-1, a well-studied protein found in all somatic cells, and GAPD-2, which is expressed solely in testis. GAPD-2 supplies energy required for the movement of spermatozoa and is tightly bound to the sperm tail cytoskeleton by the additional N-terminal proline-rich domain absent in GAPD-1. In this study we investigate the evolutionary history of GAPD and gain some insights into specialization of GAPD-2 as a testis-specific protein.ResultsA dataset of GAPD sequences was assembled from public databases and used for phylogeny reconstruction by means of the Bayesian method. Since resolution in some clades of the obtained tree was too low, syntenic analysis was carried out to define the evolutionary history of GAPD more precisely. The performed selection tests showed that selective pressure varies across lineages and isoenzymes, as well as across different regions of the same sequences.ConclusionsThe obtained results suggest that GAPD-1 and GAPD-2 emerged after duplication during the early evolution of chordates. GAPD-2 was subsequently lost by most lineages except lizards, mammals, as well as cartilaginous and bony fishes. In reptilians and mammals, GAPD-2 specialized to a testis-specific protein and acquired the novel N-terminal proline-rich domain anchoring the protein in the sperm tail cytoskeleton. This domain is likely to have originated by exonization of a microsatellite genomic region. Recognition of the proline-rich domain by cytoskeletal proteins seems to be unspecific. Besides testis, GAPD-2 of lizards was also found in some regenerating tissues, but it lacks the proline-rich domain due to tissue-specific alternative splicing.

Testes-specific protease 50 (TSP50) promotes cell proliferation through the activation of the nuclear factor κB (NF-κB) signalling pathway

TSP50 (testes-specific protease 50) is a testis-specific expression protein, which is expressed abnormally at high levels in breast cancer tissues. This makes it an attractive molecular marker and a potential target for diagnosis and therapy; however, the biological function of TSP50 is still unclear. In the present study, we show that overexpression of TSP50 in CHO (Chinese-hamster ovary) cells markedly increased cell proliferation and colony formation. Mechanistic studies have revealed that TSP50 can enhance the level of TNFα (tumour necrosis factor α)- and PMA-induced NF-κB (nuclear factor κB)-responsive reporter activity, IκB (inhibitor of NF-κB) α degradation and p65 nuclear translocation. In addition, the knockdown of endogenous TSP50 in MDA-MB-231 cells greatly inhibited NF-κB activity. Co-immunoprecipitation studies demonstrated an interaction of TSP50 with the NF-κB-IκBα complex, but not with the IKK (IκB kinase) α/β-IKKγ complex, which suggested that TSP50, as a novel type of protease, promoted the degradation of IκBα proteins by binding to the NF-κB-IκBα complex. Our results also revealed that TSP50 can enhance the expression of NF-κB target genes involved in cell proliferation. Furthermore, overexpression of a dominant-negative IκB mutant that is resistant to proteasome-mediated degradation significantly reversed TSP50-induced cell proliferation, colony formation and tumour formation in nude mice. Taken together, the results of the present study suggest that TSP50 promotes cell proliferation, at least partially, through activation of the NF-κB signalling pathway.

The Prognostic Significance of Multiple Molecular Markers in Blood of Patients With Multimodal Treatment of Esophageal Cancer

Introduction: Human CABYR is a testis specific polymorphic calcium binding tyrosine phosphorylation regulative protein isolated from spermatozoa. This protein is localized to the sperm flagellum in association with the fibrous sheath; it can bind calcium after being phosphorylated during sperm capacitation. Six transcript variants of CABYR encoding five protein isoforms have been identified. Normally, CABYR is expressed in the human germ line but not in adult human tissues, thus, it is considered a cancer testis (CT) protein. A high frequency of expression in tumor tissues and restricted expression in normal tissues make CT proteins attractive cancer vaccine targets. Increased expression of CABYR a/b and c isoforms in lung cancers and isoforms c and d in brain tumors have been reported. The expression patterns of CABYR genes in colorectal cancer (CRC) are not well characterized. The aim of this study is to evaluate the expression of CABYR a/b and c in CRC tumors and to determine if they hold promise as vaccine targets. Method: Consenting CRC patients (pts) who underwent elective resection for whom tumor samples were available were included in the study. Demographic, clinical, and pathologic data were collected prospectively. Tissues were OCT embedded and stored at -80°C until analysis. Total purified RNA was isolated from tissue samples and cDNA synthesized. CABYR a/b and c expression was analyzed by quantitative PCR (QPCR) using the SYBR Green platform. Tumor and normal tissue expression levels were determined and compared as were tumor and testis CABYR expression levels. Tumors with expression levels 0.1% or more than the testis were considered positive. Expression of CABYR a/b and c in a set of normal human tissues was analyzed by RT-PCR. Results: A total of 47 paired CRC and normal tissue specimens (18 M/ 29 F, age 65+/-16.8) were studied (85% colon,15% rectal). Stage breakdown was as follows: Stage 2, 26; Stage 3, 19; and Stage 4, 2 pts. The percent of pts with a relative expression ratio of malignant to normal tissues (MN ratio) over 1 was 70% for CABYR a/b and 72% for CABYR-c. The percent with both a MN ratio over 1 and expression levels over 0.1% of testis was 23.4% for CABYR-a/b and 25.5% for CABYR-c. Except for very low expression of CABYR a/b in the brain, CABYR a/b or c was not expressed in normal tissues. Conclusion: RT-PCR analysis confirmed the frequent overexpression of CABYR a/b and c in most CRC tumors compared to adjacent normal tissues. In 23-26% of tumors expression was more than 0.1% of the expression level of testis. A larger and more diverse group of tumors (Stage 1-4) needs to be assessed to determine if CABYR expression correlates with T, N, or final tumor stage. Evaluation of blood for anti-CABYR antibodies is also needed. These results support CABYR as a potential therapeutic vaccine candidate in CRC.