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Pachytene Spermatocytes (pachytene + spermatocyte)
Selected AbstractsMolecular cloning and characterization of SRG-L, a novel mouse gene developmentally expressed in spermatogenic cellsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2006Quanhong Ma Abstract Full-length cDNA of a novel mouse gene upregulated in late stages of spermatogenic cells was cloned from mouse testis using overlapping RT-PCR and RACE. The mRNA of the gene was expressed mainly in diplotene/pachytene spermatocytes, round and elongating spermatids. We named this gene as SRG-L (Spermatogenesis Related Gene expressed in late stages of spermatogenic cells, GenBank Accession No. AY352586). The tissue-specific analysis showed a higher expression level in testis and spleen. The gene is mapped on chromosome 8q33.1 and contains 18 exons. The full-length of cDNA is 2,843 bp with an open reading frame (ORF) of 2,625 bp that encodes a 104 kDa protein (874 amino acids) with a putative transmembrane region. The bioinformatics analysis revealed that the SRG-L has two conserved regions, transglutaminase-like homologues domain and D -serine dehydratase domain, rich phosphorylation sites and methylation sites. The SRG-L protein was detected in diplotene/pachytene spermatocytes and spermatids by immunohistochemical staining and Western blot. The results suggest that SRG-L may play definite roles regulating differentiation of germ cells during spermatogenesis, particularly during meiosis and spermiogenesis. Mol. Reprod. Dev. 1075,1083, 2006. © 2006 Wiley-Liss, Inc. [source] Lipoprotein lipase and endothelial lipase in human testis and in germ cell neoplasmsINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 1 2010J. E. Nielsen Summary The aim of this study was to investigate endothelial lipase (EL, LIPG) and lipoprotein lipase (LPL) mRNA and protein expression in normal human testis and testicular germ cell tumours (GCT). Both EL and LPL were expressed in normal seminiferous tubules and in the interstitial compartment. EL mRNA and protein were found in all germ cells as well as in Sertoli and Leydig cells. EL mRNA was abundant in pre-invasive carcinoma in situ (CIS) cells and GCTs, and EL protein was present in the cytoplasm of these cells. LPL mRNA was also relatively abundant in germ cells, Sertoli cells, CIS cells and GCTs. The LPL protein, however, was restricted to the cell membranes of pachytene spermatocytes and spermatids in normal tubules, absent from CIS cells and scarcely represented in tumours. The distribution of LPL protein in non-seminomas resembled the distribution of OCT3/4, a marker of embryonal carcinoma. The results suggest that both EL and LPL participate in the supply of nutrients and steroidogenesis in the testes, and that especially EL may be important for the supply of cholesterol for testosterone production in the Leydig cells. The partial cellular separation of the expression of the two lipases in normal testis suggests the existence of distinct biological roles, perhaps developmentally regulated, as indicated by the LPL expression in GCTs with embryonic features. A high expression of EL and abundance of lipid in tubules with CIS may have a diagnostic value. [source] Molecular cloning of several rat ABC transporters including a new ABC transporter, Abcb8, and their expression in rat testisINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2006Nathalie Melaine Summary Several members of the ABC transporter superfamily play an important role in testicular physiology and defence against anticancer drugs. Using a reverse transcription-polymerase chain reaction strategy with degenerate primers and rat testis RNA as template, we have looked for the presence of other members of this superfamily. Of the six partial cDNA found, five corresponded to ABC transporters already known ,Mdr1b, Mrp1, Tapl/Abcb9, Umat/Abcb6 and Sur2/Abcc9, and one presented a strong homology with mouse and human ABCB8. Using a 5, and 3, RACE approach, we cloned the full-length cDNA and found that the predicted protein presented 92% and 80% homology with the mouse and human proteins respectively. Strong expression of rat abcb8 was found in heart, brain and testis when compared with liver, lung and spleen. In the testis, rat abcb8 was expressed both in the somatic Sertoli cells and peritubular cells and in the germline (spermatogonia and pachytene spermatocytes). Furthermore, Umat/Abcb6 was very highly expressed in the testis (high amounts in meiotic pachytene spermatocytes and low amount in post-meiotic early spermatids). In conclusion, we confirm the presence of several ABC transporters in the testis and also provide evidence of the presence of Abcb8 in the organ. [source] Expression of Mina53, a product of a Myc target gene in mouse testisINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2006MAKOTO TSUNEOKA Summary Recently we have identified a novel gene mina53 (mina), which is a direct transcriptional target of oncoprotein Myc. Mina53 protein was shown to be highly expressed in tumour cells and to play a role in cell proliferation. Here we report the expression of Mina53 in mouse testis, which contains proliferating cells and expresses many cancer-related genes. Immunohistochemical studies by using newly produced monoclonal antibody to Mina53 showed that Mina53 was expressed in the nuclei of spermatogonia. Mina53 was also expressed in meiotic prophase cells such as preleptotene, leptotene and zygotene, and weakly in early pachytene spermatocytes, but was absent in late pachytene spermatocytes, spermatids and mature sperm. The expression pattern of Mina53 was quite similar to that of proliferation cell nuclear antigen (PCNA). Using experimental cryptorchid testis, it was found that Mina53 was highly expressed in undifferentiated spermatogonia, which were PCNA-positive. These results suggest that Mina53 is prominently expressed in proliferating, undifferentiated spermatogonia, and plays a role in cell proliferation from the spermatogonial stage to the meiotic prophase in spermatogenesis, but not in meiotic divisions per se. [source] Aberrant distribution of junctional complex components in retinoic acid receptor alpha-deficient miceMICROSCOPY RESEARCH AND TECHNIQUE, Issue 6 2010Sanny S.W. Chung Abstract Retinoic acid receptor alpha (RAR,)-deficient mice are sterile, with abnormalities in the progression of spermatogenesis and spermiogenesis. In this study, we investigated whether defective retinoid signaling involved at least in part, disrupted cell,cell interactions. Hypertonic fixation approaches revealed defects in the integrity of the Sertoli-cell barrier in the tubules of RAR,-deficient testes. Dye transfer experiments further revealed that coupling between cells from the basal to adluminal compartments was aberrant. There were also differences in the expression of several known retinoic acid (RA)-responsive genes encoding structural components of tight junctions and gap junctions. Immunostaining demonstrated a delay in the incorporation of zonula occludens (ZO-1), a peripheral component protein of tight junctions, into the Sertoli cell tight junctions. Markedly reduced expression of connexin-40 in mutant pachytene spermatocytes and round spermatids was found by in situ hybridization. An ectopic distribution of vimentin and disrupted cyclic expression of vimentin, which is usually tightly regulated during spermiogenesis, was found in RAR,-deficient testes at all ages examined. Thus, the specific defects in spermiogenesis in RAR,-deficient testes may correlate with a disrupted cyclic expression of RA-responsive structural components, including vimentin, a downregulation of connexin-40 in spermatogenic cells, and delayed assembly of ZO-1 into Sertoli cell tight junctions. Interestingly, bioinformatic analysis revealed that many genes that are components of tight junctions and gap junctions contained potential retinoic acid response element binding sites. Microsc. Res. Tech., 2010. © 2009 Wiley-Liss, Inc. [source] Mammalian sperm quality and aromatase expressionMICROSCOPY RESEARCH AND TECHNIQUE, Issue 8 2009Serge Carreau Abstract In most mammalian species the aromatase is encoded by a single gene (cyp19), which contains 18 exons, 9 of them being translated. In adult rats, together with Leydig cells germ cells represent an additional source of estrogens. The amount of P450arom transcript is threefold higher in pachytene spermatocytes compared to younger cells (spermatogonia-preleptotene spermatocyte) or round spermatids; conversely, aromatase activity is more intense in haploid cells. In man besides Leydig cells, we have shown the presence of a biologically active aromatase and of estrogen receptors (ER, and ERß) in immature germ cells and ejaculated spermatozoa. Concerning aromatase, a 30% decrease of the amount of mRNA is observed in immotile compared to motile sperm fraction from the same sample; moreover, the aromatase activity is diminished. We have amplified aromatase mRNA by RT-real time PCR in spermatozoa from asthenospermic, teratospermic, and asthenoteratospermic men and recorded respectively 44, 52, and 67% decreases of the amount of transcripts as compared to controls. Statistical analyses between the sperm morphology and the aromatase/GAPDH ratio have revealed a high degree of correlation (r = ,0.64) with the percentage of abnormal spermatozoa (especially microcephaly and acrosome malformations). Alterations of sperm number and motility have been described in men genetically deficient in aromatase, which together with our data, suggest a likely role for aromatase/estrogens in the acquisition of sperm motility. Therefore besides gonadotrophins and testosterone, estrogens produced locally should be considered as a physiologically relevant hormone involved in the regulation of mammalian spermatogenesis. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc. [source] Expression of zinc finger protein 105 in the testis and its role in male fertilityMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2010Huaxin Zhou Using an in silico approach, we identified a putative zinc finger domain-containing transcription factor (zinc finger protein 105, ZFP105) enriched in the adult mouse testis. RT-PCR analyses showed that Zfp105 was indeed highly expressed in adult mouse testis and that its expression was regulated during postnatal development. To further characterize Zfp105 expression, we generated a Zfp105:,-galactosidase (LacZ) knock-in reporter mouse line (Zfp105LacZ/+) in which a Zfp105:LacZ fusion gene was expressed. Whole-mount LacZ analyses of adult Zfp105LacZ/+ tissues showed robust LacZ staining in the testis, very weak staining in the ovary, and no staining in the spleen, liver, kidney, heart, lung, thymus, adrenal gland, uterus, or oviduct. Sectional LacZ staining showed that ZFP105 was highly expressed in pachytene spermatocytes. ZNF35, the human ortholog of Zfp105, was also highly expressed in human testis. Immunofluorescence analysis showed that ZNF35 was located primarily in the cytoplasm of male germ cells. More importantly, reduced male fertility was observed in adult Zfp105LacZ/LacZ mice. Histological studies showed the presence of undifferentiated spermatogenic cells in the lumen of seminiferous tubules at stage VII and in the epididymal lumen of adult Zfp105LacZ/LacZ mice. Taken together, our results suggest that ZFP105 is a male germ-cell factor and plays a role in male reproduction. Mol. Reprod. Dev. 77: 511,520, 2010. © 2010 Wiley-Liss, Inc. [source] A shared promoter region suggests a common ancestor for the human VCX/Y, SPANX, and CSAG gene families and the murine CYPT familyMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2008Martin A. Hansen Abstract Many testis-specific genes from the sex chromosomes are subject to rapid evolution, which can make it difficult to identify murine genes in the human genome. The murine CYPT gene family includes 15 members, but orthologs were undetectable in the human genome. However, using refined homology search, sequences corresponding to the shared promoter region of the CYPT family were identified at 39 loci. Most loci were located immediately upstream of genes belonging to the VCX/Y, SPANX, or CSAG gene families. Sequence comparison of the loci revealed a conserved CYPT promoter-like (CPL) element featuring TATA and CCAAT boxes. The expression of members of the three families harboring the CPL resembled the murine expression of the CYPT family, with weak expression in late pachytene spermatocytes and predominant expression in spermatids, but some genes were also weakly expressed in somatic cells and in other germ cell types. The genomic regions harboring the gene families were rich in direct and inverted segmental duplications (SD), which may facilitate gene conversion and rapid evolution. The conserved CPL and the common expression profiles suggest that the human VCX/Y, SPANX, and CSAG2 gene families together with the murine SPANX gene and the CYPT family may share a common ancestor. Finally, we present evidence that VCX/Y and SPANX may be paralogs with a similar protein structure consisting of C terminal acidic repeats of variable lengths. Mol. Reprod. Dev. 75: 219,229, 2008. © 2007 Wiley-Liss, Inc. [source] Expression of a novel T-complex testis expressed 5 (Tctex5) in mouse testis, epididymis, and spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2007Y.B. Han Abstract Expression of T-complex testis expressed 5 (Tctex5), an orthologue of protein phosphatase-1 inhibitor-3 (PPP1R11), was enhanced in mouse testis and was also expressed in epididymis and spermatozoa. There were three transcripts of Tctex5 including one brain specific and two common transcripts dominant in mouse testis. Tctex5 protein isoforms (75, 52, 32, 25, and 14.3 kDa) were identified. Isoforms of 75 and 52 kDa were spermatogenic-specific and were found in protein fraction containing nuclei, mitochondria, and flagellum accessory, and also in protein fraction containing mainly membranes. Tctex5 was localized in nuclei of pachytene spermatocytes, round spermatocytes, cytoplasm of Sertoli cells in testis; cilia, secretion bodies and nuclei of epithelial cells and interstitium smooth muscle cells in epididymis; and head and principal piece of tail in epididymal spermatozoa. The results suggested that Tctex5 might be a specific protein phosphatase-1 inhibitor in sperm; various Tctex5 transcripts and isoforms and cellular locations imply its different roles in spermatogenesis. Nuclei-type isoforms (75 and 52 kDa) might take part in nucleus remodeling during spermatogenesis whilst membrane-type isoform (52 kDa) might be responsible for dephosphorylation of proteins during capacitation. The other isoforms might play general roles for all kinds of cell types. Mol. Reprod. Dev. 74: 1132,1140, 2007. © 2007 Wiley-Liss, Inc. [source] p19Ink4d and p18Ink4c cyclin-dependent kinase inhibitors in the male reproductive axisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2007Gregory M. Buchold Abstract The loss of the cyclin-dependent kinase inhibitors (CKIs) p18Ink4c and p19Ink4d leads to male reproductive defects (Franklin et al., 1998. Genes Dev 12: 2899,2911; Zindy et al., 2000. Mol Cell Biol 20: 372,378; Zindy et al., 2001. Mol Cell Biol 21: 3244,3255). In order to assess whether these inhibitors directly or indirectly affect male germ cell differentiation, we examined the expression of p18Ink4c and p19Ink4d in spermatogenic and supporting cells in the testis and in pituitary gonadotropes. Both p18Ink4c and p19Ink4d are most abundant in the testis after 18 days of age and are expressed in purified populations of spermatogenic and testicular somatic cells. Different p18Ink4c mRNAs are expressed in isolated spermatogenic and Leydig cells. Spermatogenic cells also express a novel p19Ink4d transcript that is distinct from the smaller transcript expressed in Sertoli cells, Leydig cells and in other tissues. Immunohistochemistry detected significant levels of p19Ink4d in preleptotene spermatocytes, pachytene spermatocytes, condensing spermatids, and Sertoli cells. Immunoprecipitation-Western analysis detected both CKI proteins in isolated pachytene spermatocytes and round spermatids. CDK4/6-CKI complexes were detected in germ cells by co-immunoprecipitation, although the composition differed by cell type. p19Ink4d was also identified in FSH+ gonadotrophs, suggesting that this CKI may be independently required in the pituitary. Possible cell autonomous and paracrine mechanisms for the spermatogenic defects in mice lacking p18Ink4c or p19Ink4d are supported by expression of these CKIs in spermatogenic cells and in somatic cells of the testis and pituitary. Mol. Reprod. Dev. 74: 997,1007, 2007. © 2007 Wiley-Liss, Inc. [source] Analysis of the mRNA expression of the TGF-Beta family in testicular cells and localization of the splice variant TGF-,2B in testisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 10 2006Lutz Konrad Abstract The transforming growth factors (TGF)-,, TGF-,1, TGF-,2, and TGF-,3, and their receptors [T,RI, T,RII, T,RIII (betaglycan)] elicit many functions in the testis, for example, they perturb the blood testis barrier (BTB). Although expression of the ligands and receptors have been investigated, the alternative splice variants are incompletely examined. We therefore have analyzed all ligands, the receptors, and the splice variants T,RIB, T,RIIB, and TGF-,2B in testicular cells from rat and mouse. In mouse, the novel transcript variant TGF-,2B was identified and was found in Leydig cells, spermatogonia, pachytene spermatocytes, and in the apical regions of the Sertoli cells in adult testis. Even though expression of the splice variant T,RIB could be shown in mouse and rat, we never found the isoform T,RIIB in the rat cell lines studied. Whereas in all testicular cells expression of all TGF-, ligands could be shown, receptor mRNA expression was slightly more diverse. Furthermore, expression pattern of the splice variants was more heterogeneous, for example, T,RIB was not detectable in adult Sertoli cells, primary peritubular cells, and immortalized peritubular cells. The heterogeneous expression of the receptors and especially of the splice variants might provide possible clues for the different functions of the TGF-, ligands in testicular cells. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Stage-dependent Dishevelled-1 expression during mouse spermatogenesis suggests a role in regulating spermatid morphological changesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2006Pengpeng Ma Abstract Dishevelled (Dsh in Drosophila or DVL in mice) is a member of the highly conserved Wg/Wnt signaling pathway, which regulates important processes such as cell proliferation, polarity, and specification of cell fate. Three orthologous genes of Dishevelled (Dvl-1, Dvl-2, and Dvl-3) have been found in both humans and mice. They play pivotal roles in regulating cell morphology and a variety of changes in cell behaviors. In the present study, we show that the expression of Dvl-1 is stage-dependent during mouse spermatogenesis, although Dvl-2 and Dvl-3 show relative consistent expression. The expression of Dvl-1 mRNA first appears in pachytene spermatocytes, increases in round and elongating spermatids, and then turns to an undetectable level in mature sperm cells. Analyses of immunohistochemistry and immunofluorescence staining show that DVL-1 is present diffusely in the cytoplasm of pachytene spermatocytes and exhibits mainly a vesicular pattern and perinuclear distribution and a weak diffusely cytoplasmic signal in round and elongating spermatids. The vesicular pattern of DVL-1 has been observed by previous studies in somatic cells, and suggested to play roles in signal transduction. Immunoprecipitation experiments show that DVL-1 coimmunprecipitates with spermatogenic cells ,-actin rather than ,-tubulin. These results indicate that DVL-1 may be involved in spermatid morphological changes during mouse spermiogenesis through mediating signal transduction and/or regulating actin cytoskeleton organization. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Increased recombination frequency showing evidence of loss of interference is associated with abnormal testicular histopathologyMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003Susannah Varmuza Abstract Nondisjunction leading to aneuploid gametes has been linked genetically to both increases and decreases in recombination frequency on the aneuploid chromosome. In the present study, we present physical evidence of increased frequency of recombination nodules as measured by Mut-S-like homologue-1 (MLH1) foci on pachytene chromosomes from sterile male mice homozygous for a mutation in the protein phosphatase 1c, (PP1c,) gene. The pattern of elevated recombination frequency in PP1c, mutant spermatocytes is consistent with a loss of interference. Previous studies demonstrated: (1) spermiogenesis is impaired starting at step 8 with a severe reduction in elongating and condensed spermatids; (2) spermatids and sperm exhibit elevated rates of DNA fragmentation; and (3) haploid gametes exhibit elevated levels of aneuploidy. Morphometric analysis of developing testes revealed that the first wave of meiosis proceeds at a normal rate in mutant testes, a surprising result given that the PP1 inhibitor okadaic acid has been shown to accelerate progression of spermatocytes from pachytene to the first meiotic division (MI). Evidence of abnormal testicular histopathology is apparent at 3 weeks, before the appearance of haploid gametes, eliminating the possibility that the mutant phenotype is caused by the presence of abnormal spermatids, but coincident with the appearance of the first set of mid to late pachytene spermatocytes. These observations lead us to conclude that the PP1c, mutation causes a complex phenotype, including subtle adverse effects on meiosis, possibly mediated by defective signaling between germ cells and Sertoli cells. Mol. Reprod. Dev. 64: 499,506, 2003. © 2003 Wiley-Liss, Inc. [source] Testicular protein Spag5 has similarity to mitotic spindle protein Deepest and binds outer dense fiber protein Odf1MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2001Xueping Shao Abstract Outer dense fibers (ODF) and the fibrous sheath (FS) are major cytoskeletal structures in the mammalian sperm tail. The molecular mechanisms underlying their morphogenesis along the axoneme or their function are poorly understood. Recently, we reported the cloning and characterization of Odf2, a major ODF protein, and Spag4, an axoneme-binding protein, by virtue of their strong interaction with Odf1, the 27 kDa major ODF protein. We proposed a crucial role for leucine zippers in molecular interactions during sperm tail morphogenesis. Here we report the cloning and characterization of a novel gene, Spag5, which encodes a 200 kDa testicular protein that interacts strongly with Odf1. Spag5 is transcribed and translated in pachytene spermatocytes and spermatids. It bears 73% similarity with the mitotic spindle protein Deepest of unknown function. We identified two putative leucine zippers in the C-terminal part of the Spag5 protein, the downstream one of which is involved in interaction with Odf1. Interestingly, these motifs are present in Deepest. These results highlight the importance of the leucine zipper in sperm tail protein interactions. Mol. Reprod. Dev. 59: 410,416, 2001. © 2001 Wiley-Liss, Inc. [source] |