Differential expression of the zinc finger gene Zfp105 during spermatogenesis

We report the isolation of Zfp105, the mouse homolog of the human ZNF35 zinc finger gene. Zfp105 and ZNF35 are highly conserved at the protein and nucleotide level, and Zfp105 maps to a region of mouse Chromosome (Chr) 9 that is homologous to the human region containing ZNF35. Zpf105 is highly expressed in the testis, especially in pachytene spermatocytes and round spermatids. The possible role of this gene product in maintaining an ordered germ cell differentiation process is discussed. Received: 11 February 1998 / Accepted: 1 May 1998     

Expression and function of the testis-predominant protein LYAR in mice

Mammalian spermatogenesis is a complex process involving an intrinsic genetic program of germ cell-specific and -predominant genes. In the present study, we analyzed the Ly-1 reactive clone (Lyar) gene in the mouse. Lyar, which is known to be expressed abundantly in the testis, encodes a nucleolar protein that contains a LYAR-type C2HC zinc finger motif and three nuclear localization signals. We herein confirmed that Lyar is expressed predominantly in the testis, and further showed that this expression is specific to germ cells. Protein analyses with an anti-LYAR antibody demonstrated that the LYAR protein is present in spermatocytes and spermatids, but not in sperm. To assess the functional role of LYAR in vivo, we used a genetrap mutagenesis approach to establish a LYAR-null mouse model. Lyar mutant mice were born live and developed normally. Male mutant mice lacking LYAR were fully fertile and showed intact spermatogenesis. Taken together, our results demonstrate that LYAR is strongly preferred in male germ cells, but has a dispensable role in spermatogenesis and fertility.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.     

Ablation of Ggnbp2 impairs meiotic DNA double‐strand break repair during spermatogenesis in mice

Gametogenetin (GGN) binding protein 2 (GGNBP2) is a zinc finger protein expressed abundantly in spermatocytes and spermatids. We previously discovered that Ggnbp2 resection caused metamorphotic defects during spermatid differentiation and resulted in an absence of mature spermatozoa in mice. However, whether GGNBP2 affects meiotic progression of spermatocytes remains to be established. In this study, flow cytometric analyses showed a decrease in haploid, while an increase in tetraploid spermatogenic cells in both 30‐ and 60‐day‐old Ggnbp2 knockout testes. In spread spermatocyte nuclei, Ggnbp2 loss increased DNA double‐strand breaks (DSB), compromised DSB repair and reduced crossovers. Further investigations demonstrated that GGNBP2 co‐immunoprecipitated with a testis‐enriched protein GGN1. Immunofluorescent staining revealed that both GGNBP2 and GGN1 had the same subcellular localizations in spermatocyte, spermatid and spermatozoa. Ggnbp2 loss suppressed Ggn expression and nuclear accumulation. Furthermore, deletion of either Ggnbp2 or Ggn in GC‐2spd cells inhibited their differentiation into haploid cells in vitro. Overexpression of Ggnbp2 in Ggnbp2 null but not in Ggn null GC‐2spd cells partially rescued the defect coinciding with a restoration of Ggn expression. Together, these data suggest that GGNBP2, likely mediated by its interaction with GGN1, plays a role in DSB repair during meiotic progression of spermatocytes.     

The identification and characterization of a testis-specific cDNA during spermatogenesis

Using bioinformatics and experimental validation, we obtained a cDNA (named srsf) which was exclusively expressed in the mouse testes. RT-PCR analysis showed that srsf mRNA was not expressed in the gonad during the sex determination period or during embryogenesis. In developing mouse tests, srsf expression was first detected on post-natal day 10, reached its highest level on day 23, and then reduced to and remained at a moderate level throughout adulthood. In situ hybridization analysis demonstrated that srsf mRNA was expressed in pachytene spermatocytes and round spermatids in the testes. The predicted protein contains one RNA-binding domain (RBD) and a serine-arginine rich domain (RS), which are characterized by some splicing factors of SR family members. These findings indicate that srsf may play a role during spermatogenesis.     

A Novel Testis-Enriched Gene Spata33 Is Expressed during Spermatogenesis

With an increasing incidence of male idiopathic infertility, identification of novel genes involved in spermatogenesis is an important aspect for the understanding of human testicular failure. In the present study, we have identified a novel gene Spata33, also called as 4732415M23Rik or C16orf55, which is conserved in mammalian species. Spata33 was predominantly expressed in the postpartum and adult mouse testes at mRNA and protein levels. Its expression was increased during the first wave of the spermatogenesis, indicating that Spata33 may be associated with the meiotic process. Further immunohistochemistry analysis revealed that Spata33 was mainly expressed in the spermatocytes, spermatogonia and round spermatids. Its expression was uniformly distributed in the nucleus and cytosol in these germ cells, which was further confirmed by Spata33-tagged with GFP staining in the GC-1 and TM4 cells. These results indicated that Spata33 was predominantly expressed in the mouse testis and associated with spermatogenesis. Identification and characterization of the novel testis-enriched gene Spata33 may provide a new route for understanding of spermatogenesis failure.     

Loss of TSLC1 causes male infertility due to a defect at the spermatid stage of spermatogenesis

Tumor suppressor of lung cancer 1 (TSLC1), also known as SgIGSF, IGSF4, and SynCAM, is strongly expressed in spermatogenic cells undergoing the early and late phases of spermatogenesis (spermatogonia to zygotene spermatocytes and elongating spermatids to spermiation). Using embryonic stem cell technology to generate a null mutation of Tslc1 in mice, we found that Tslc1 null male mice were infertile. Tslc1 null adult testes showed that spermatogenesis had arrested at the spermatid stage, with degenerating and apoptotic spermatids sloughing off into the lumen. In adult mice, Tslc1 null round spermatids showed evidence of normal differentiation (an acrosomal cap and F-actin polarization indistinguishable from that of wild-type spermatids); however, the surviving spermatozoa were immature, malformed, found at very low levels in the epididymis, and rarely motile. Analysis of the first wave of spermatogenesis in Tslc1 null mice showed a delay in maturation by day 22 and degeneration of round spermatids by day 28. Expression profiling of the testes revealed that Tslc1 null mice showed increases in the expression levels of genes involved in apoptosis, adhesion, and the cytoskeleton. Taken together, these data show that Tslc1 is essential for normal spermatogenesis in mice.     

Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea). V. The temporal germ cell development strategy of the testis

The germ cell development strategy during spermatogenesis was investigated in the black swamp snake (Seminatrix pygaea). Testicular tissues were collected, embedded in plastic, sectioned by ultramicrotome, and stained with methylene blue and basic fuchsin. Black swamp snakes have a postnuptial pattern of development, where spermatogenesis occurs from May to July and spermiation is completed by October. Though spatial relationships are seen between germ cells within the seminiferous epithelium during specific months, accumulation of spermatogonia and spermatocytes early in spermatogenesis and the depletion of spermatocytes and accumulation of spermatids late in spermatogenesis prevent consistent cellular associations. This temporal germ cell development within an amniotic testis is consistent with that seen in other recently studied temperate reptiles (slider turtle and wall lizard). These reptiles’ temporal development is more similar to the developmental strategy found in anamniotes than the spatial germ cell development that characterizes birds and mammals. Our findings also imply that a third germ cell development strategy may exist in temperate breeding reptiles. Because of the phylogenetic position of reptiles between anamniotes and other terrestrial amniotes, this common germ cell development strategy shared by temperate reptiles representing different orders may have significant implications as far as the evolution of sperm development within vertebrates.     

Abnormal sperm development in pcd 3J-/- mice: the importance of Agtpbp1 in spermatogenesis

Homozygous Purkinje cell degeneration (pcd) mutant males exhibit abnormal sperm development. Microscopic examination of the testes from pcd(3J)-/- mice at postnatal days 12, 15, 18 and 60 revealed histological differences, in comparison to wild-type mice, which were evident by day 18. Greatly reduced numbers of spermatocytes and spermatids were found in the adult testes, and apoptotic cells were identified among the differentiating germ cells after day 15. Our immunohistological analysis using an antihuman AGTPBP1 antibody showed that AGTPBP1 was expressed in spermatogenic cells between late stage primary spermatocytes and round spermatids. A global gene expression analysis from the testes of pcd(3J)-/- mice showed that expression of cyclin B3 and de-ubiquitinating enzymes USP2 and USP9y was altered by >1.5-fold compared to the expression levels in the wild-type. Our results suggest that the pcd mutant mice have defects in spermatogenesis that begin with the pachytene spermatocyte stage and continue through subsequent stages. Thus, Agtpbp1, the gene responsible for the pcd phenotype, plays an important role in spermatogenesis and is important for survival of germ cells at spermatocytes stage onward.