cDNA cloning and localization of Sp3111 (also called Ms4a14) in the rat testis.

With tetraspanning topology, members of the membrane-spanning four-domain subfamily A (MS4A) may facilitate signaling or ion channel functions in many tissues. In this study, we report the cloning of a full-length cDNA from rat testis, designated Ms4a14 (Sp3111), which encodes the MS4A protein with 1139 amino acid residues. In situ hybridization and immunohistochemical analyses indicate that Ms4a14 is predominantly expressed from round spermatids to spermatozoa at specific stages in the rat testis at both the mRNA and protein level. Immunofluorescence analysis revealed that MS4A14 (SP3111) is located in the acrosome and the midpiece of the flagellum in mature sperm. Previously, we explored and reported the involvement of MS4A14 in reproductive functions, using antibody blockage during IVF and a transgenic RNA interference method in a mouse model. Our results suggested that MS4A14 is involved in fertilization and zygote division. As MS4A14 protein exists in mammals, such as humans, cows, dogs, and rodents, MS4A14 may play a ubiquitous role in mammalian reproduction.

Gonadotropins, via cAMP, negatively regulate GATA-1 gene expression in testicular cells.

We and others demonstrated that the mRNAs encoding GATA-binding proteins, GATA-1 and GATA-4, were detected in mouse and rat testis, and in isolated rat Sertoli cells and testicular tumor cell lines derived from Leydig and Sertoli cells. In this study, we investigated the possible effects of gonadotropins and cAMP on the expression of GATA-binding protein genes in testicular cells. Unexpectedly, FSH negatively regulated GATA-1 (but not GATA-4) mRNA in a dose-dependent manner in primary cultures of rat Sertoli cells isolated from 21-d-old animals. GATA-1 mRNA was also negatively regulated by cAMP in a dose- and time-dependent manner in MA-10, a mouse Leydig tumor cell line. When 0.3 mM cAMP was administered to MA-10 cell cultures for 4 h, more than 95% of the GATA-1 mRNA and protein was abolished. The reduction of GATA-1 mRNA by cAMP can be mimicked by treatment with forskolin, which elevates intracellular cAMP levels. The inhibitory effect of cAMP was specific to the GATA-1 gene, given that GATA-4 and alpha-tubulin mRNA levels were not changed by any of the cAMP treatments. Inhibin alpha-subunit mRNA, on the other hand, was evidently increased by cAMP treatment in both MA-10 and Sertoli cells. However, inhibin alpha-subunit mRNA levels were elevated at 60-90 min before the suppression of GATA-1 mRNA detected. The inhibitory effect of cAMP on GATA-1 mRNA and protein was shown to be specific to testicular cells. The GATA-1 mRNA expressed in MEL, a mouse erythroid leukemia cell line, was not affected by cAMP. The reduction of GATA-1 mRNA by cAMP can be prevented when a translational inhibitor, cycloheximide, is added. In summary, we demonstrated that gonadotropins via cAMP negatively regulate the mRNA and protein levels of GATA-1, but not GATA-4, in testicular cells. The inhibitory effect on GATA-1 gene expression was specific to testicular cells and was not observed in erythroid cells.

Cloning and characterization of rat spermatid protein SSP411: a thioredoxin-like protein.

In an attempt to identify new sperm-specific genes that are involved in sperm maturation, fertilization, and embryo development, such as the mammalian ortholog of the sperm-supplied protein gene, spe-11, in Caenorhabditis elegans, we cloned and characterized a new spermatid-specific protein gene, ssp411, from adult rat testes. The ssp411 cDNA shared >85% sequence identity with an unnamed human protein, FLJ21347, and an uncharacterized mouse testicular protein called transcript increased in spermiogenesis 78 (TISP78). A 2.8-kb ssp411 mRNA was expressed in a testis-specific and age-dependent manner; the mRNA was evident at 28 days and remained at high levels throughout adulthood. An SSP411 protein of molecular weight 88 000 was detected in testicular extracts by Western blot analysis. Ssp411 mRNA and SSP411 protein, as analyzed by in situ hybridization and immunohistochemistry, were both expressed in a stage-dependent fashion during the cycle of the seminiferous epithelium. The ssp411 mRNA was predominantly localized to round and elongated spermatids, with maximal expression at stages VII-XII. The SSP411 protein was mainly observed in elongated spermatids and reached its highest levels during stages V-VI. A conserved thioredoxin-like domain was detected in the N-terminal region of SSP411 and its orthologs. An analysis of the predicted 3-dimensional structural modeling and folding pattern further suggested that SSP411 is identifiable as a member of thioredoxin family. In summary, we have identified a new rat spermatid protein gene, ssp411, and its orthologs in human and mouse and demonstrated that SSP411 might belong to a testis-specific thioredoxin family. This suggests that SSP411 may play a role in sperm maturation, fertilization, and/or embryo development, as has been shown in thioredoxin family.