Masamichi Doiguchi

Bisphenol A-induced apoptosis of cultured rat Sertoli cells

Bisphenol A (BPA) was examined for its effects on cultured Sertoli cells established from 18-day-old rat testes. We demonstrated that exposure of cultured Sertoli cells to BPA decreased the cell viability in a dose- and a time-dependent manner and that exposure to BPA brought about morphologic changes of the cells, such as membrane blebs, cell rounding, cytoskeletal collapse, and chromatin condensation or fragmentation, all of which conform to the morphologic criteria for apoptosis. Immunocytochemistry showed that active caspase-3, a major execution caspase, was expressed in round Sertoli cells positively labeled by the TUNEL method. Co-localization of active caspase-3 and aggregated actin fragments was also observed in the round Sertoli cells. Theses results suggest that BPA induces cell death of Sertoli cells by promoting apoptosis. Apoptosis-inducing cell death was observed in cells exposed to 150-200 microM BPA, while BPA at <100 microM had only slight cytotoxic effects on the cells.

Complementary DNA Cloning and Characterization of Rat spergen-1, a Spermatogenic Cell-Specific Gene-1, Containing a Mitochondria-Targeting Signal

Abstract To elucidate the molecular mechanisms involved with spermiogenesis in testis, we performed differential display screening to isolate genes that are developmentally up-regulated during rat testis development. One of the cDNAs isolated by differential display was highly expressed in testis. Both reverse transcription-polymerase chain reaction and Northern blot analysis showed that the expression level of the gene developmentally increased. By screening the rat testis cDNA library, we successfully isolated rat cDNA clones encoding the entire open-reading frame of 462 base pairs coding a small protein of 154 amino acids. Because in situ hybridization revealed that the gene was specifically expressed in haploid spermatids in the rat seminiferous tubules, it was designated as spergen-1 (spermatogenic cell-specific gene-1). The recently opened database of the full-length mouse cDNA collection contains a mouse gene that is homologous to rat spergen-1. Subcellular fractionation followed by immunoblot analysis revealed that spergen-1 protein was associated with mitochondria. The transfection experiments performed in COS-7 cells suggested that spergen-1 has a N-terminal mitochondria-targeting signal. We suggest that spergen-1 might be involved in spermiogenesis by transiently associating with spermatid mitochondria.

Spermatid-Specific Expression of Iba1, an Ionized Calcium Binding Adapter Molecule-1, in Rat Testis

Abstract Postnatal development of mammalian seminiferous tubules can be divided into three phases: spermatogonial mitosis, spermatocyte meiosis, and a postmeiotic phase in which drastic morphological changes occur in spermatids (spermiogenesis). In an attempt to elucidate the molecular mechanisms involved in spermiogenesis, we have applied a differential display method to identify genes that are developmentally up-regulated during rat testis development. One of the cDNA fragments isolated by differential display turned out to be iba1, an ionized calcium binding adapter molecule-1, that contains two EF hand-like motifs. Expression of iba1 mRNA in the rat testis was detected first at 4 wk in postnatal development and then increased up to adulthood. Using the antibody against a synthetic peptide corresponding to the N-terminal Iba1 protein, we discovered that Iba1 protein was not detectable by immunohistochemistry in spermatogonia, spermatocytes, and round spermatids in adult rat testis but was specifically expressed in the cytoplasm of elongate spermatids (steps 10–19) as well as in residual bodies that are ultimately engulfed by Sertoli cells. In situ hybridization, on the other hand, revealed that iba1 mRNA is present in round spermatids as well as early elongate spermatids (steps 1–12) but not in late spermatids, suggesting that iba1 mRNA undergoes post-transcriptional regulation. Because Iba1 protein is specifically expressed in the cytoplasm of elongate spermatids, which is finally engulfed as residual bodies into Sertoli cells, we suggest that Iba1 may be involved in the final stage of spermiogenesis (i.e., in elimination of the residual cytoplasm from spermatids).

Complementary DNA Cloning and Characterization of Rat spergen-2, a Spermatogenic Cell-Specific Gene 2 Encoding a 56-Kilodalton Nuclear Protein Bearing Ankyrin Repeat Motifs

Abstract Differential display in combination with a cDNA cloning approach were used to isolate a novel gene, spergen-2, which has an open reading frame of 1500 nucleotides and encodes a protein of 500 amino acids that contains ankyrin repeat motifs and a putative nuclear localization signal. Expression of spergen-2 is developmentally upregulated in testis. In situ hybridization revealed that spergen-2 mRNA is expressed in spermatocytes and round spermatids (steps 1–6). Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spergen-2 protein is predominantly expressed in nuclei of late spermatocytes (stages IX–XIV) and spermatids (steps 1–11), indicating the restricted expression of spergen-2 during spermatogenesis. In nucleoplasm of spermatogenic cell nuclei, spergen-2 tends to localize in the interchromosome space with relatively low DNA density. These findings indicate a potential role of spergen-2 in spermatogenesis, especially in cell differentiation from late pachytene spermatocytes to spermatids or in early spermatid differentiation.