Steroid hormones and the development of reproductive organs.

It has been more than 150 years since the physiological function of androgen was reported for the first time in fowl. This finding has served as a basis for many studies focusing on steroid hormones from various aspects. These studies have significantly enhanced our knowledge about the structures of steroid hormones, their synthetic pathways, enzymes involved in the synthetic pathways, steroid hormone-specific receptors, actions of steroid hormones through receptor binding, and the differentiation of steroidogenic cells. However, there are still many attractive and important issues in these areas, some of which are currently being addressed. In this review, we trace the history and findings of the previous studies on steroid hormones, summarize our present understanding in this area, and discuss issues that remain to be elucidated.

Disruption of aryl hydrocarbon receptor (AhR) induces regression of the seminal vesicle in aged male mice.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates diverse dioxin toxicities. Despite mediating the adverse effects, the AhR gene is conserved among animal species, suggesting important physiological functions for AhR. In fact, a recent study revealed that AhR has an intrinsic function in female reproduction, though its role in male reproduction is largely unknown. In this study, we show age-dependent regression of the seminal vesicles, probably together with the coagulating gland, in AhR(-/-) male mice. Knockout mice had abnormal vaginal plugs, low sperm counts in the epididymis, and low fertility. Moreover, serum testosterone concentrations and expression of steroidogenic 3betahydroxysteroiddehydrogenase (3betaHsd) and steroidogenic acute regulatory protein (StAR) in testicular Leydig cells were decreased in AhR(-/-) males. Taken together, our results suggest that impaired testosterone synthesis in aged mice induces regression of seminal vesicles and the coagulating glands. Such tissue disappearance likely resulted in abnormal vaginal plug formation, and eventually in low fertility. Together with previous findings demonstrating AhR function in female reproduction, AhR has essential functions in animal reproduction in both sexes.

A steroidogenic cell line with differentiation potential from mouse granulosa cells, transfected with Ad4BP and SV40 large T antigen genes.

Several steroidogenic cell lines of granulosa cells (GC) have been used to elucidate differentiation mechanisms of GC during folliculogenesis. These cell lines, however, are of limited usefulness since they have lost some of their differentiation potential. The transcription factor adrenal-4 binding protein (Ad4BP), also known as steroidogenic factor-1 or NR5A1, is essential for the expression of all P-450 steroidogenic enzymes. By transfection with the Ad4BP gene together with SV40 DNA, we have generated several steroidogenic cell lines. One selective clone, named 4B2, retained its steroidogenic potential and was therefore analyzed in depth. This cell line responded to 8-Br-cAMP by displaying differentiation characteristics similar to those occurring in the differentiation process of primary cultured GC, including enhanced progesterone secretion, a cell shape change from a fibroblastic to epithelioid conformation, elongated mitochondria, increased gap junction formation and inhibition of cell proliferation. Prostaglandin E2 (PGE2), an intraovarian regulator of GC, stimulated cAMP production, and this eicosanoid, like 8-Br-cAMP, induced differentiation properties with the exception of cell conformation in 4B2 cells. These results suggest that expression of Ad4BP may provide the basis for a repertoire of cAMP-sensitive differentiation properties, including morphological alterations and growth inhibition. Thus, the 4B2 cell line may serve as a tool for elucidation of differentiation mechanisms that are under the control of Ad4BP.

Association of single nucleotide polymorphisms in the IL-18 gene with sarcoidosis in a Japanese population.

Interleukin-18 (IL-18) and interleukin-12 (IL-12) synergistically stimulate interferon-gamma (IFN-gamma) production from Th1 cells. The levels of serum IL-18 and IFN-gamma and bronchoalveolar lavage fluid IL-18 were elevated in patients with sarcoidosis. The polymorphisms of the IL-18 gene may play a possible role in expression regulation of the gene. We investigated the roles of the polymorphisms in the development of sarcoidosis. We examined two single nucleotide polymorphisms of the IL-18 gene in 119 patients with sarcoidosis and 130 healthy control subjects. Our results showed that the frequency of sarcoidosis patients with the CA genotype at position -607 was significantly higher than that with the AA genotype (OR = 2.200) and a significantly higher proportion of patients had the C allele at -607 compared with that of the controls (OR = 2.123). No significant differences were seen in the distribution of the genotypes or phenotype frequencies at position -137. There was no specific organ involvement associated with a certain genotype or phenotype. In IL-18 gene polymorphisms, the C allele at position -607 might be a genetic risk factor for sarcoidosis in this Japanese population.

Expression profiles of COUP-TF, DAX-1, and SF-1 in the human adrenal gland and adrenocortical tumors: possible implications in steroidogenesis.

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF), DAX-1, and steroidogenic factor-1 (SF-1) are orphan members of the nuclear hormone receptor superfamily. COUP-TF and DAX-1 have been shown to negatively regulate the transcriptional activity of SF-1, a steroidogenic cell-specific activator of various steroidogenic cytochrome P450 genes. We therefore examined the expression levels and immunolocalization of COUP-TF, DAX-1, and SF-1 in human adrenal gland (NL) and adrenocortical adenomas, and compared the results with CYP17 expression levels and its enzyme activities to study their potential correlation with adrenocortical steroidogenesis. In NL (n = 10), expressions of COUP-TF, DAX-1, and SF-1 were detected in the nuclei of adrenocortical cells, but not in the medulla. In cortisol-producing adenomas causing Cushing syndrome (CS, n = 20), CYP17 expression was upregulated (298 +/- 2% vs NL 98 +/- 4%), whereas expression levels of both COUP-TFs (COUP-TFI, 52 +/- 5% vs NL 98 +/- 4%; COUP-TFII, 18 +/- 4% vs NL 98 +/- 4%) and DAX-1 (42 +/- 4% vs NL 100 +/- 4%) were reduced. In deoxycorticosterone-producing adenomas (DOC, n = 2), on the other hand, CYP17 expression was extremely reduced (8 and 12% vs NL 98 +/- 4%), whereas DAX-1 expression increased markedly (350 and 360% vs NL 100 +/- 4%). Expression levels of SF-1 did not differ between NL (100 +/- 8%) and CS (106 +/- 10%), but its expression appeared to be decreased in DOC (25 and 20%). These results showed CYP17 expression to be upregulated and downregulated in CS and DOC, respectively, in a manner reciprocal to that of its repressors, COUP-TF and/or DAX-1. In summary, the results indicate that co-localization of COUP-TF, DAX-1, and SF-1 in NL was lost in adrenocortical tumors and that these orphan receptors play an important role in the regulation of steroidogenesis in human adrenals.

Comparative localization of Dax-1 and Ad4BP/SF-1 during development of the hypothalamic-pituitary-gonadal axis suggests their closely related and distinct functions.

Two nuclear receptors, Ad4BP/SF-1 and Dax-1, are essential regulators for development and function of the mammalian reproductive system. Similarity in expression sites, such as adrenal glands, gonads, pituitary, and hypothalamus, suggests a functional interaction, and the phenotype similarities were manifested in Ad4BP/SF-1-deficient mice and in cases of natural human mutations of Dax-1. In this study, quantitative reverse transcriptase polymerase chain reaction analyses revealed that expression profiles of Dax-1 in embryonic gonads are different between the two sexes and also from those of Ad4BP/SF-1. Immunohistochemical analyses clarified the spatial and temporal expressions of the Dax-1 protein during development of tissues composing the hypothalamic-pituitary-gonadal axis. During gonadal development, Dax-1 occurred after Ad4BP/SF-1 exhibiting a sexually dimorphic expression pattern at indifferent stages, indicating a possibility of Dax-1 involvement in earliest sex differentiation. When cord formation begins in the testis at embryonic day 12.5 (E12.5), Dax-1 was expressed strongly in Sertoli cells, but its expression level markedly decreased in Sertoli cells and increased in interstitial cells between E13.5 and E17.5. In the female, Dax-1 was strongly expressed in the entire ovarian primordium from E12.5 until E14.5, and then its expression level was decreased and limited to cells near the surface epithelium between E17.5 and postnatal day 0 (P0). During postnatal development of the testis, the variable staining of Dax-1 in Sertoli cells was detected as early as P7 and Dax-1-expressing Leydig cells became rare. In the postnatal ovary, Dax-1 expression was detected in granulosa cells with variable staining intensity, and occasionally in interstitial cells. During pituitary organogenesis, Dax-1 but not Ad4BP/SF-1 was expressed in the dorsal part of Rathke's pouch from E9.5. Later in development after E14.5, the distribution of Dax-1 overlapped with that of Ad4BP/SF-1, being restricted to gonadotropic cells in the anterior pituitary. In the ventromedial hypothalamus (VMH), Dax-1 and Ad4BP/SF-1 were mostly colocalized throughout the embryonic and postnatal development. Thus, the coexpression of Dax-1 and Ad4BP/SF-1 indicates their closely related functions in the development of the reproductive system. Furthermore, we noticed the presence of cells that express Dax-1 but not Ad4BP/SF-1, further indicating additional functions of Dax-1 in an Ad4BP/SF-1-independent molecular mechanism.

Identification of novel first exons in Ad4BP/SF-1 (NR5A1) gene and their tissue- and species-specific usage.

It has been demonstrated that the mammalian Ad4BP/SF-1 (NR5A1) gene is regulated precisely in sex, tissue, and developmental stage specific manners. To clarify the complex transcriptional regulation, we investigated in the present study whether the gene transcription is regulated by multiple promoters accompanied by noncoding first exons. Novel first exons (Io and Ig) were identified downstream of the already identified exon Ia. Nucleotide sequences revealed that Ia and Ig exons were well conserved, whereas Io exon was less conserved among the mouse, rat, and human genes. Interestingly, the splice donor of the mouse and human Io and human Ig exons do not satisfy the consensus sequence. Transcripts containing Ia, Io, and Ig were detected in all rat tissues examined, while the transcript containing Io was undetectable in the corresponding tissues of mice. The lack of exon Io usage in the mouse was confirmed by transient transfection assays with cultured cells. Quantitative RT-PCR analysis revealed that the transcript containing Ig exon was the main product in the pituitary but significantly less in the spleen, suggesting that the regulation of Ad4BP/SF-1 gene transcription in the pituitary and spleen is distinct from that of other tissues. The above findings, together with the structural abnormality at the splice donor site, suggest that acquisition of the multiple first exons enables the Ad4BP/SF-1 gene to be regulated differentially in different animal species and in different tissues in the same animal.

Orphan receptors COUP-TF and DAX-1 as targets in disordered CYP17 expression in adrenocortical tumors.

CYP17 gene transcription is activated by SF-1 binding to a cyclic AMP-responsive sequence within the promoter region of the gene, and its transcription is inhibited by COUP-TF binding to the sequence. Another orphan receptor, DAX-1, is shown to act as a suppressor of SF-1-mediated transcription. We examined the expression level of these orphan receptors in adrenocortical tumors and compared the results with CYP17 expression. CYP17 was highly expressed in cortisol-producing adenomas, whereas COUP-TF and DAX-1 expression levels were very low. In deoxycorticosterone-producing adenomas, on the other hand, CYP17 expression was extremely low, whereas DAX-1 was highly expressed and SF-1 expression was slightly decreased. In conclusion, the reciprocal expression of CYP17 and the transcriptional repressors COUP-TF and DAX-1 indicates that these orphan receptors have a pathophysiologic role in the excessive hormone production in cortisol- and deoxycorticosterone-producing adrenocortical tumors.

Sex reversal caused by Mus musculus domesticus Y chromosomes linked to variant expression of the testis-determining gene Sry.

When the Y chromosomes from certain populations of Mus musculus domesticus are introduced into the mouse strain C57BL/6 (B6), testis determination can fail, resulting in gonads developing either as ovotestes (with both ovarian and testicular components) or as ovaries. Not all Y(DOM) chromosomes cause sex reversal. Y(DOM) chromosomes are divided into three classes based upon their ability to induce testes in B6. The molecular basis underlying the three Y(DOM) classes is an enigma. The simplest explanation is that they harbor different alleles of the testis-determining gene, Sry. Sequencing of Sry(DOM) genes has indeed identified polymorphisms. However, none were unequivocally linked to the sex-reversal trait. It was concluded that all SRY(DOM) proteins are functionally equivalent. Using a semiquantitative RT-PCR assay, we now show that representatives of the three Y(DOM) classes have variant Sry expression patterns, that severity of sex reversal correlates with Sry mRNA titers, and that genetic correction of the sex reversal results in the upregulation of Sry expression. We propose that the variant Sry expression patterns result from polymorphisms at the site of a putative Sry enhancer.

Saccharomyces cerevisiae cultured under aerobic and anaerobic conditions: air-level oxygen stress and protection against stress.

Cells of Saccharomyces cerevisiae were grown aerobically and anaerobically, and levels of the protective compounds, cysteine and glutathione, and activities of defensive enzymes, catalase and superoxide dismutase, against an oxygen stress were determined and compared in both cells. Aerobiosis increased both the compounds and enzyme activities. The elevated synthesis of glutathione could be associated with the increased levels of cysteine which in its turn was found to be controlled by the oxygen-dependent activation of cystathionine beta-synthase.

The role of human MBF1 as a transcriptional coactivator.

Multiprotein bridging factor 1 (MBF1) is a coactivator which mediates transcriptional activation by interconnecting the general transcription factor TATA element-binding protein and gene-specific activators such as the Drosophila nuclear receptor FTZ-F1 or the yeast basic leucine zipper protein GCN4. The human homolog of MBF1 (hMBF1) has been identified but its function, especially in transcription, remains unclear. Here we report the cDNA cloning and functional analysis of hMBF1. Two isoforms, which we term hMBF1alpha and hMBF1beta, have been identified. hMBF1alpha mRNA was detected in a number of tissues, whereas hMBF1beta exhibited tissue-specific expression. Both isoforms bound to TBP and Ad4BP/SF-1, a mammalian counterpart of FTZ-F1, and mediated Ad4BP/SF-1-dependent transcriptional activation. While hMBF1 was detected in the cytoplasm by immunostaining, coexpression of the nuclear protein Ad4BP/SF-1 with hMBF1 induced accumulation of hMBF1 in the nucleus, suggesting that hMBF1 is localized in the nucleus through its binding to Ad4BP/SF-1. hMBF1 also bound to ATF1, a member of the basic leucine zipper protein family, and mediated its activity as a transcriptional activator. These data establish that the coactivator MBF1 is functionally conserved in eukaryotes.

Dax-1 as one of the target genes of Ad4BP/SF-1.

The DAX-1 (also known as AHC) gene encodes an unusual member of the nuclear hormone receptor superfamily. DAX-1 plays a critical role during gonadal and adrenal differentiation since mutations of the human DAX-1 gene cause X-linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. In recent studies, DAX-1 was reported to function as a transcriptional suppressor of Ad4BP/SF-1, a critical transcription factor in gonadal and adrenal differentiation. With respect to implication of Ad4BP/SF-1 in the transcriptional regulation of the DAX-1 gene, inconsistent findings have been previously reported. We investigated the upstream region of the mouse Dax-1 (also known as Ahch) gene and identified a novel Ad4/SF-1 site by transient transfection and electrophoretic mobility shift assays. In addition, immunohistochemical analyses with a specific antibody to Dax-1 indicated the presence of immunoreactive cells in steroidogenic tissues, pituitary gland, and hypothalamus. Although the distributions of Dax-1 and Ad4BP/SF-1 were very similar, they were not completely identical. The expression of Dax-1 was significantly impaired in knock-out mice of the Ftz-f1 gene, which encodes Ad4BP/ SF-1. Taken together, our findings indicate that Ad4BP/SF-1 controls the transcription of the Dax-1 gene.

Structural and functional abnormalities in the spleen of an mFtz-F1 gene-disrupted mouse.

The spleen has two main functions. The first is to provide a proper microenvironment to lymphoid and myeloid cells, whereas the second involves clearance of abnormal erythrocytes. Ad4BP/SF-1, a product of the mammalian FTZ-F1 gene (mFTZ-F1), was originally identified as a steroidogenic, tissue-specific transcription factor. Immunohistochemical examination of the mammalian spleens confirmed the expression of Ad4BP/SF-1 in endothelial cells of the splenic venous sinuses and pulp vein. In mFtz-F1 gene-disrupted (KO) mice, several structural abnormalities were detected in the spleen, including underdevelopment and nonuniform distribution of erythrocytes. Examination of the spleen of KO fetuses showed failure of development of certain tubular structures during embryogenesis. These structures are normally assembled by Ad4BP/SF-1 immunoreactive cells, and most likely form the vascular system during later stages of development. Other structural abnormalities in the spleen of the KO mice included defects in the tissue distribution of type-IV collagen, laminin, c-kit, and vimentin. These morphologic defects in the vascular system were associated with a decrease in the proportion of hematopoietic cells, although differentiation of these cells was not affected significantly. A high number of abnormal red blood cells containing Howell-Jolly bodies were noted in the KO mice, indicating impaired clearance by the splenic vascular system. We also detected the presence of an mRNA-encoding cholesterol side-chain cleavage P450 in the spleen, resembling the findings in steroidogenic tissues such as the gonads and adrenal cortex. The mRNA transcript was not involved in splenic structural defects as it was detected in the spleens of both normal and KO mice, indicating that the regulatory mechanism of the P450 gene in the spleen is different from that in steroidogenic tissues. Our results indicate that a lack of the mFtz-F1 gene in mice is associated with structural and functional abnormalities of the splenic vascular system.

Molecular cloning and expression of the SF-1/Ad4BP gene in the frog, Rana rugosa.

SF-1/Ad4BP is a transcriptional factor that was originally found to be a mammalian homologue of the Drosophila Ftz-F1 (fushi tarazu factor 1) (), and transcribed from a gene designated the Ftz-F1 gene (). Ftz-F1 gene-deficient mice lack adrenal glands and gonads. Besides mammals, however, the SF-1/Ad4BP cDNA has only been isolated to date in fish and birds. To understand its role(s) for adrenal and gonadal development in vertebrates, cloning of this gene in animals other than mammals is required. In this study, we succeeded to isolate frog (Rana rugosa) SF-1/Ad4BP cDNA from a testis lambdagt10 cDNA library. It encoded a protein of 468 amino acids, and its open reading frame (ORF) shared 70% similarity with that of chicken OR2.1 (a SF-1/Ad4BP homologue) and 62% with bovine SF-1/Ad4BP. SF-1/Ad4BP mRNA was expressed in the testes, brains, adrenals/kidneys and spleens, but not ovaries, of adult frogs. In addition, we also cloned the 5'-untranslated region (4.6kb) of the SF-1/Ad4BP gene with exons I and II. Genomic structure analysis revealed that frog SF-1/Ad4BP was also transcribed from the same gene as that of mammals. However, many Ftz-F1-related proteins have been reported so far. The Ftz-F1 gene does not encode all of those Ftz-F1-related proteins. Thus, the name of Ftz-F1 is not adequate for the gene coding SF-1/Ad4BP. Here, we propose the use of SF-1/Ad4BP instead of Ftz-F1 for the gene that encodes SF-1/Ad4BP in vertebrates.