DnaJC18, a Novel Type III DnaJ Family Protein, is Expressed Specifically in Rat Male Germ Cells.

Mammalian spermatogenesis occurs in a precise and coordinated manner in the seminiferous tubules. One of the attempts to understand the detailed biological process during mammalian spermatogenesis at the molecular level has been to identify the testis specific genes followed by study of the testicular expression pattern of the genes. From the subtracted cDNA library of rat testis prepared using representational difference analysis (RDA) method, a complimentary DNA clone encoding type III member of a DnaJ family protein, DnaJC18, was cloned (GenBank Accession No. DQ158861). The full-length DnaJC18 cDNA has the longest open reading frame of 357 amino acids. Tissue and developmental Northern blot analysis revealed that the DnaJC18 gene was expressed specifically in testis and began to express from postnatal week 4 testis, respectively. In situ hybridization studies showed that DnaJC18 mRNA was expressed only during the maturation stages of late pachy- tene, round and elongated spermatids of adult rat testis. Western blot analysis with DnaJC18 antibody revealed that 41.2 kDa DnaJC18 protein was detected only in adult testis. Immunohistochemistry study further confirmed that DnaJC18 protein, was expressed in developing germ cells and the result was in concert with the in situ hybridization result. Confocal microscopy with GFP tagged DnaJC18 protein revealed that it was localized in the cytoplasm of cells. Taken together, these results suggested that testis specific DnaJC18, a member of the type III DnaJ protein family, might play a role during germ cell maturation in adult rat testis.

Cadmium up-regulates transcription of the steroidogenic acute regulatory protein (StAR) gene through phosphorylated CREB rather than SF-1 in K28 cells.

Cadmium is a widely used heavy metal in industry and affects the male reproductive system of animals, including humans, as a result of occupational and environmental exposures. However, the molecular mechanism underlying its effect on steroidogenesis in gonads remains unclear. In this study, we demonstrated that exposure of K28 mouse testicular Leydig tumor cells to cadmium led to a significant increase in the mRNA level, promoter activity and protein level of the steroidogenic acute regulatory protein (StAR), an essential factor for steroid biosynthesis. It has been well documented that StAR gene transcription is regulated by multiple transcription factors, including cAMP-responsive element binding protein (CREB) family members and SF-1. Cadmium treatment caused an increase in CREB phosphorylation but did not alter the CREB protein level in the nucleus. EMSA studies revealed that cadmium-induced phosphorylated CREB formed specific complexes with the proximal region of the StAR gene promoter. Furthermore, co-transfection with a CREB expression plasmid significantly increased cadmium-induced StAR promoter activity. However, the nuclear level and the affinity of SF-1 protein for the StAR proximal promoter were dramatically decreased upon exposure to cadmium. Taken together, these results suggest that cadmium up-regulates StAR gene expression through phosphorylated CREB rather than through SF-1 in mouse testicular Leydig cells.

The novel, actin-like protein Tact3 is expressed in rodent testicular haploid germ cells.

Mouse testis actin-like proteins 1 and 2 (mTact1 and mTact2), which are expressed in murine haploid germ cells, have been described previously. Here, we report the cloning and characterization of a third actin-like protein from rat, rat testis actin-like protein 3 (rTact3). The complete cDNA of the rTact3 gene was approximately 3.7 kb in length, and its corresponding amino acid sequence consisted of 1219 amino acids. The rTact3 gene lacks introns, similar to mTact1 and mTact2. The 356 C-terminal amino acids of rTact3 showed 43% homology with mTact1, whereas the 863 N-terminal amino acids did not show any significant homology. Northern blot analysis revealed that rTact3 mRNA was expressed only in adult rat testes and not during the prepubescent stage. In situ hybridization revealed that rTact3 was expressed exclusively during round and elongated spermatids maturation stages in rat testes. Immunohistochemical experiments using antibodies raised against a synthetic peptide showed that the expression of the rTact3 protein was also restricted in round and elongated spermatids, specifically in the head and acrosome of mature rat sperm. The 5'-flanking region of the mTact3 gene was found to contain a TATA-box motif as well as two putative CREB/c-Jun and five C/EBP motifs. mTact3 promoter activity was enhanced in a dose-dependent manner by the transfection of CREB, c-Jun, or C/EBP in NIH3T3 cells. These results suggest that Tact3 proteins might play an important role in rodent germ-cell development.

The changed immunoreactivity of StarD6 after pilocarpine-induced epilepsy.

This study was designed whether StarD6 is also affected by neurodegenerative stimuli, as other START proteins may be one of the delivery proteins in cholesterol metabolism of neurosteroid synthesis. Increased immunoreactivity of StarD6 was observed in all zones of the hippocampus by the administration of pilocarpine (350 mg/kg, intraperitoneally). More intense StarD6 immunolocalization was seen in the strata radiatum and lacunosum-moleculare, particularly in CA1-2 areas until 12 h after pilocarpine-induced epilepsy. StarD6 protein level was transiently increased at 3 h after pilocarpine-treated rat hippocampus comparing with normal rat. This raises the possibility that StarD6 might have nuclear roles and contribute to subsequent formation of neuroprotective steroids after excitotoxic brain injury.

Cadmium-induced apoptosis is mediated by the translocation of AIF to the nucleus in rat testes.

Cadmium (Cd) is a highly toxic metal that affects a variety of cellular events, such as cell proliferation, differentiation and survival. Cd generates reactive oxygen species (ROS) that induce apoptosis. We previously demonstrated that Cd induces apoptosis in testicular germ cells and that apoptosis was prevented by the administration of ascorbic acid (AA), an ROS scavenger. However, little is known about the signaling pathways underlying Cd-induced apoptosis in rat testes. Here, we report that Cd-induced apoptosis in rat testes was associated with the translocation of apoptosis inducing factor (AIF) from mitochondria to the nucleus, and that this was prevented by treatment with AA. Cd-induced cleavage of poly ADP-ribose polymerase-1 (PARP-1), and this was also inhibited by treatment with AA. Taken together, these results suggest that Cd-induced ROS was responsible for the upregulation of PARP-1, the translocation of AIF to the nucleus, and apoptosis of testicular cells in rat testes.

Molecular cloning and expression of steroidogenic acute regulatory protein from bullfrog (Rana catesbeiana).

Steroidogenic acute regulatory protein (StAR) transfers cholesterol from the outer mitochondrial membrane to the inner membrane where the cytochrome P450 side chain cleavage enzyme (P450scc) resides. This process is the rate-limiting step in steroidogenesis. StAR cDNAs have been cloned and characterized from a range of different species. To investigate the role of StAR in the amphibian system, we cloned a full-length StAR cDNA from bullfrog (Rana catesbeiana) using reverse transcription polymerase chain reaction (RT-PCR) in conjunction with rapid amplification of cDNA ends (RACE). The putative full-length bullfrog StAR (bfStAR) cDNA was 1862 base pairs (bp) in length, and the longest open reading frame (ORF) encoded a protein of 284 amino acids. Amino acid sequence comparison showed that amphibian StAR has a high degree of sequence identity, ranging from 62% to 98%, with StAR proteins of other species. Similar to other species, bfStAR contained two conserved domains, the mitochondrial targeting domain and cholesterol-binding domain, in the N-terminus and C-terminus of the protein, respectively. Northern blot analysis and RT-PCR indicated that StAR mRNA is expressed in the gonads and adrenal gland. Transfection of green monkey kidney (COS-1) cells with an expression construct for bfStAR revealed that it encoded 34 and 27kDa proteins that were recognized by antiserum raised against the human StAR-related lipid transfer (START) domain.

Expression of ectodermal neural cortex 1 and its association with actin during the ovulatory process in the rat.

Ectodermal neural cortex (ENC) 1, a member of the kelch family of genes, is an actin-binding protein and plays a pivotal role in neuronal and adipocyte differentiation. The present study was designed to examine the gonadotropin regulation and action of ENC1 during the ovulatory process in immature rats. The levels of ENC1 mRNA and protein were stimulated by LH/human chorionic gonadotropin (hCG) within 3 h both in vivo and in vitro. In situ hybridization analysis revealed that ENC1 mRNA was localized not only in theca/interstitial cells but also in granulosa cells of preovulatory follicles but not of growing follicles in pregnant mare's serum gonadotropin/hCG-treated ovaries. LH-induced ENC1 expression was suppressed by a high dose of protein kinase C inhibitor RO 31-8220 (10 microM) but not by low doses of RO 31-8220 (0.1-1.0 microM), suggesting the involvement of atypical protein kinase C. ENC1 was detected in both nucleus and cytoplasm that was increased by LH/hCG treatment. Both biochemical and morphological analysis revealed that LH/hCG treatment increased actin polymerization within 3 h in granulosa cells. Interestingly, ENC1 physically associated with actin and treatment with cytochalasin D, an actin-depolymerizing agent, abolished this association. Confocal microscopy further demonstrated the colocalization of ENC1 with filamentous actin (F-actin). The present study demonstrates that LH/hCG stimulates ENC1 expression and increases F-actin formation in granulosa cells. The present study further shows the physical association of ENC1 and F-actin, implicating the role of ENC1 in cytoskeletal reorganization during the differentiation of granulosa cells.

Expression of the putative sterol binding protein Stard6 gene is male germ cell specific.

Mammalian spermatogenesis is orchestrated by many specific molecular and cellular events. To understand the detailed mechanism by which spermatogenesis is controlled, the specific genes involved in this process must be identified and studied. From the subtracted cDNA library of rat testis prepared using the representational difference analysis (RDA) method, we isolated the cDNA clone of steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) protein 6 (Stard6). Stard6 cDNA consists of 1146 base pairs of nucleotides and has the longest open reading frame, of 227 amino acids. Northern blot analysis revealed Stard6 mRNA to be testis-specific. The mRNA transcript appeared from the third week of postnatal development, and the expression level increased up to adulthood. Moreover, in situ hybridization showed Stard6 mRNA expression to be germ cell-specific and expressed only during the maturation stages of round and elongated spermatids of adult rat testis. Western blot analysis with Stard6 antibody revealed a 28-kDa Stard6 protein only in testis. Immunohistochemistry further confirmed localization of Stard6 protein expressed in mature germ cells, in concert with the in situ hybridization result. Taken together, these results suggest that Stard6, a member of the START protein family, may play a role during germ cell maturation in adult rat testis.

Effect of ascorbic acid supplementation on testicular steroidogenesis and germ cell death in cadmium-treated male rats.

Cadmium (Cd) is one of the environmental pollutants affecting various tissues and organs including testis. Harmful effect of Cd in testis is known to be germ cell degeneration and impairment of testicular steroidogenesis. Animals treated with high doses of Cd (0.2 and 0.3 mg/100g BW) showed a significant decrease in serum testosterone (T) level, but a significant induction of testicular lipid peroxidation levels. TUNEL assay showed that low doses of Cd (0.13 and 0.15 mg/100g BW) exhibited typical characteristics of apoptosis while high doses of Cd caused more necrosis than apoptosis. In contrast, supplementation with ascorbic acid reduced testicular lipid peroxidation levels. Ascorbic acid supplementation restored testicular 3beta-hydroxysteroiddehydrogenase (HSD) and 17beta-HSD enzyme activities, 3beta-HSD and cytochrome P450 side chain cleavage (P450(scc)) mRNA levels and serum T concentration to normal in Cd-administered rats. Moreover, administration of ascorbic acid prevented germ cell apoptosis as demonstrated by the reduced number of TUNEL-positive cells in germinal epithelium and inhibited Cd-induced necrosis. These results indicate that ascorbic acid have protective roles in vivo on the Cd-induced overall testicular damage including impaired steroidogenesis and germ cell death possibly through scavenging the reactive oxygen species generated by Cd administration.

Molecular mechanism of suppression of testicular steroidogenesis by proinflammatory cytokine tumor necrosis factor alpha.

Tumor necrosis factor alpha (TNF-alpha) has been demonstrated to inhibit steroidogenesis in Leydig cells at the transcriptional level of steroidogenic enzymes. However, the molecular mechanism of this observed gene repression is not well understood. We now demonstrate that nuclear factor kappaB (NF-kappaB) activated by TNF-alpha inhibits the transactivation of orphan nuclear receptors, which regulate the expression of steroidogenic-enzyme genes. TNF-alpha treatment suppressed the luteinizing-hormone-induced or Nur77/SF-1-stimulated promoter activity of steroidogenic-enzyme genes in Leydig cells. The TNF-alpha-mediated gene suppression was blocked by treatment with an inhibitor of NF-kappaB. In addition, overexpression of the p65 (RelA) subunit of NF-kappaB showed the same effect as TNF-alpha and inhibited Nur77 transactivation, suggesting the involvement of NF-kappaB activation in the observed gene repression. Physical association of Nur77 with p65 was revealed by mammalian two-hybrid, GST pull-down, and coimmunoprecipitation analyses. The NF-kappaB inhibition of Nur77 transactivation was likely due to the competition of p65 for Nur77 binding with coactivators. Finally, chromatin immunoprecipitation assays revealed that TNF-alpha treatment caused the recruitment of NF-kappaB to the promoter of the steroidogenic-enzyme p450c17 gene, supporting the hypothesis that the TNF-alpha-mediated gene repression involves NF-kappaB inhibition of the transcriptional activity of Nur77 and other orphan nuclear receptors. These findings provide a molecular mechanism underlying the inhibition of testicular steroidogenesis by proinflammatory cytokines.