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Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system, similar to that of mammals. However, under high temperature conditions, XX medaka is masculinised by elevation of cortisol, the major teleost glucocorticoid. In this study, to identify novel factors in the gonads acting downstream from cortisol during sexual differentiation, we performed RNA sequencing (RNA-seq) analysis using the gonadal regions of larvae reared at normal temperature with and without cortisol, and at high temperature. The RNA-seq and real-time PCR analyses showed that expression of some peroxisome proliferator-activated receptor α (PPARα) signalling-targeted genes was increased by cortisol. PPARα agonist treatment induced masculinisation of XX medaka in some cases, and co-treatment of the agonist with cortisol further induced masculinisation, whereas treatment of pparaa knockout medaka with cortisol or the agonist did not induce masculinisation. This study provides the first evidence that PPARα is involved in environmental sex determination in vertebrates.

Cyclooxygenase 2 (COX-2) is an inducible rate-limiting enzyme for prostanoid production. Because COX-2 represents one of the inducible genes in mouse mesenchymal stem cells upon differentiation into Leydig cells, we investigated COX-2 expression and production of prostaglandin (PG) in Leydig cells. Although COX-2 was undetectable in mouse testis, it was transiently induced in Leydig cells by human chorionic gonadotropin (hCG) administration. Consistent with the finding that Leydig cells expressed aldo-keto reductase 1B7 (PGF synthase) and PGE synthase 2, induction of COX-2 by hCG caused a marked increase in testicular PGF 2α and PGE 2 levels. Using mouse Leydig cell tumor-derived MA-10 cells as a model, it was indicated by reporter assays and electron mobility shift assays that transcription of the COX-2 gene was activated by CCAAT/enhancer-binding protein β (C/EBPβ) with cAMP-stimulation. C/EBPβ expression was induced by cAMP-stimulation, whereas expression of C/EBP homolog protein (CHOP) was robustly downregulated. Transfection of CHOP expression plasmid inhibited cAMP-induced COX-2 promoter activity. In addition, CHOP reduced constitutive COX-2 expression in other mouse Leydig cell tumor-derived TM3 cells. These results indicate that COX-2 is induced in Leydig cells by activation of C/EBPβ via reduction of CHOP expression upon gonadotropin-stimulation to produce PGF 2α and PGE 2 .

Ovaries represent one of the primary steroidogenic organs, producing estrogen and progesterone under the regulation of gonadotropins during the estrous cycle. Gonadotropins fluctuate the expression of various steroidogenesis-related genes, such as those encoding steroidogenic enzymes, cholesterol deliverer, and electronic transporter. Steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP)/NR5A1 and liver receptor homolog-1 (LRH-1) play important roles in these phenomena via transcriptional regulation. With the aid of cAMP, SF-1/Ad4BP and LRH-1 can induce the differentiation of stem cells into steroidogenic cells. This model is a useful tool for studying the molecular mechanisms of steroidogenesis. In this article, we will provide insight into the transcriptional regulation of steroidogenesis-related genes in ovaries that are revealed from stem cell-derived steroidogenic cells. Using the cells derived from the model, novel SF-1/Ad4BP- and LRH-1-regulated genes were identified by combined DNA microarray and promoter tiling array analyses. The interaction of SF-1/Ad4BP and LRH-1 with transcriptional regulators in the regulation of ovarian steroidogenesis was also revealed.

Although 11-ketotestosterone (11KT) and testosterone (T) are major androgens in both teleosts and humans, their 5α-reduced derivatives produced by steroid 5α-reductase (SRD5A/srd5a), i.e., 11-ketodihydrotestosterone (11KDHT) and 5α-dihydrotestosterone (DHT), remains poorly characterized, especially in teleosts. In this study, we compared the presence and production of DHT and 11KDHT in Japanese eels and humans. Plasma 11KT concentrations were similar in both male and female eels, whereas T levels were much higher in females. In accordance with the levels of their precursors, 11KDHT levels did not show sexual dimorphism, whereas DHT levels were much higher in females. It is noteworthy that plasma DHT levels in female eels were higher than those in men. In addition, plasma 11KDHT was undetectable in both sexes in humans, despite the presence of 11KT. Three srd5a genes (srd5a1, srd5a2a and srd5a2b) were cloned from eel gonads. All three srd5a genes were expressed in the ovary, whereas only both srd5a2 genes were expressed in the testis. Human SRD5A1 was expressed in testis, ovary and adrenal, whereas SRD5A2 was expressed only in testis. Human SRD5A1, SRD5A2 and both eel srd5a2 isoforms catalyzed the conversion of T and 11KT into DHT and 11KDHT, respectively, whereas only eel srd5a1 converted T into DHT. DHT and 11KDHT activated eel androgen receptor (ar)α-mediated transactivation as similar fashion to T and 11KT. In contrast, human AR and eel arβ were activated by DHT and11KDHT more strongly than T and 11KT. These results indicate that in teleosts, DHT and 11KDHT may be important 5α-reduced androgens produced in the gonads. In contrast, DHT is the only major 5α-reduced androgens in healthy humans.

In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)—a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor β superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells.

Medaka (Oryzias latipes) are teleost fish with a XX/XY sex determination system. Recently, it was reported that high temperature (HT) induced the masculinization of XX medaka by increasing the levels of cortisol, a major glucocorticoid produced by interrenal cells in teleosts. Cortisol secretion is regulated by adrenocorticotropic hormone (ACTH) secreted from the pituitary gland, which is partly regulated by corticotropin-releasing hormone (CRH) secreted from the hypothalamus. In teleosts, two crh paralogs, named crha and crhb, have been identified. Recently, the expression of crhb but not crha was upregulated by HT during gonadal sex differentiation period in medaka and loss-of-functions of its receptors under HT suppressed masculinization of XX medaka and increase of cortisol levels, suggesting that crhb is involved in masculinization induced by HT. However, the transcriptional regulation of crhb under HT has not been elucidated. We analyzed the gene expression pattern in the hypothalamus of medaka embryos incubated under HT using DNA microarray. The expressions of heat shock protein (hsp) genes, such as hsp70.1 and hsp30, were increased. Overexpression of hsp70.1 or hsp30 in cultured rat hypothalamic 4B cells significantly induced crh gene expression. Moreover, hypothalamic hsp70.1-overexpressing transgenic medaka also showed increased crhb gene expression that increased cortisol levels compared with fish incubated at a normal temperature. These results provide the first evidence that HSPs induce cortisol levels by elevating crhb gene expression in the hypothalamus.

Context: 11-ketotestosterone (11-KT) is a novel class of active androgen. However, the detail of its synthesis remains unknown for humans. Objective: The objective of this study was to clarify the production and properties of 11-KT in human. Design, Participants, and Methods: Expression of cytochrome P450 and 11β-hydroxysteroid dehydrogenase types 1 and 2 (key enzymes involved in the synthesis of 11-KT) were investigated in human gonads. The production of 11-KT was investigated in Leydig cells. Plasma concentrations of testosterone and 11-KT were measured in 10 women and 10 men of reproductive age. Investigation of its properties was performed using breast cancer-derived MCF-7 cells. Results: Cytochrome P450 and 11β-hydroxysteroid dehydrogenase types 1 and 2 were detected in Leydig cells and theca cells. Leydig cells produced 11-KT, and relatively high levels of plasma 11-KT were measured in both men and women. There was no sexual dimorphism in the plasma levels of 11-KT, even though testosterone levels were more than 20 times higher in men than in women. It is noteworthy that the levels of testosterone and 11-KT were similar in women. In a luciferase reporter system, 11-KT activated human androgen receptor-mediated transactivation. Conversely, 11-KT did not activate estrogen receptor-mediated transactivation in aromatase-expressed MCF-7 cells, whereas testosterone did following conversion to estrogen. 11-KT did not affect the estrogen/estrogen receptor -mediated cell proliferation of MCF-7 cells. Furthermore, it significantly inhibited cell proliferation when androgen receptor was transfected into MCF-7 cells. Conclusions: The current study indicates that 11-KT is produced in the gonads and represents a major androgen in human. It can potentially serve as a nonaromatizable androgen.

Porcine steroid hormone profiles have some unique characteristics. We previously studied human and murine steroidogenesis using steroidogenic cells-derived from mesenchymal stem cells (MSCs). To investigate porcine steroidogenesis, we induced steroidogenic cells from porcine subcutaneous preadipocytes (PSPA cells), which originate from MSCs. Using cAMP, adenovirus-mediated introduction of steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP) induced the differentiation of PSPA cells into sex steroid-producing cells. Introducing SF-1/Ad4BP also induced the aldo-keto reductase 1C1 (AKR1C1) gene. Porcine AKR1C1 had 17β-hydroxysteroid dehydrogenase activity, which converts androstenedione and 11-ketoandrostenedione into testosterone (T) and 11-ketotestosteorne (11KT). Furthermore, differentiated cells expressed hydroxysteroid 11β-dehydrogenase 2 (HSD11B2) and produced 11KT. HSD11B2 was expressed in testicular Leydig cells and the adrenal cortex. 11KT was present in the plasma of both immature male and female pigs, with slightly higher levels in the male pigs. T levels were much higher in the male pigs. It is noteworthy that in the female pigs, the 11KT levels were >10-fold higher than the T levels. However, castration altered the 11KT and T plasma profiles in the male pigs to near those of the females. 11KT induced endothelial nitric oxide synthase (eNOS) in porcine vascular endothelial cells. These results indicate that 11KT is produced in porcine adrenal glands and testes, and may regulate cardiovascular functions through eNOS expression.

Polycyclic aromatic hydrocarbons (PAHs) are pollutants that exert harmful effects on marine invertebrates, however, the molecular mechanism underlying PAH action remains unclear. We investigated the effect of PAHs on the ascidian Ciona intestinalis type A (Ciona robusta). First, the influence of PAHs on early Ciona development was evaluated. PAHs such as dibenzothiophene, fluorene, and phenanthrene resulted in formation of abnormal larvae. PAH treatment of swimming larva induced malformation in the form of tail regression. Additionally, we observed the Ciona aryl hydrocarbon receptor (Ci-AhR) mRNA expression in swimming larva, mid body axis rotation, and early juvenile stages. The time correlation between PAH action and AhR mRNA expression suggested that Ci-AhR could be associated with PAH metabolism. Lastly, we analyzed Ci-AhR mRNA localization in Ciona juveniles. Ci-AhR mRNA was localized in the digestive tract, dorsal tubercle, ganglion, and papillae of the branchial sac, suggesting that Ci-AhR is a candidate for an environmental pollutant sensor and performs a neural function. Our results provide basic knowledge on the biological function of Ci-AhR and PAH activity in marine invertebrates.

Steroid hormones are mainly produced in adrenal glands and gonads. Because steroid hormones play vital roles in various physiological processes, replacement of deficient steroid hormones by hormone replacement therapy (HRT) is necessary for patients with adrenal and gonadal failure. In addition to HRT, tissue regeneration using stem cells is predicted to provide novel therapy. Among various stem cell types, mesenchymal stem cells can be differentiated into steroidogenic cells following ectopic expression of nuclear receptor (NR) 5A subfamily proteins, steroidogenic factor-1 (also known as adrenal 4 binding protein) and liver receptor homolog-1, with the aid of cAMP signaling. Conversely, these approaches cannot be applied to pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, because of poor survival following cytotoxic expression of NR5A subfamily proteins. However, if pluripotent stem cells are first differentiated through mesenchymal lineage, they can also be differentiated into steroidogenic cells via NR5A subfamily protein expression. This approach offers a potential suitable cells for future regenerative medicine and gene therapy for diseases caused by steroidogenesis deficiencies. It represents a powerful tool to investigate the molecular mechanisms involved in steroidogenesis. This article highlights our own and current research on the induction of steroidogenic cells from various stem cells. We also discuss the future direction of their clinical application.

Open Access CC BY-NC-ND, Regulation of intestinal secretion is important for body fluid homeostasis. We investigated the role of three MAP kinases (MAPKs) as negative regulators in muscarinic cholinoceptor (mAChR)-mediated intestinal secretion in mice. Electrophysiological analyses revealed that mAChR stimulation enhanced intestinal chloride secretion, which was further augmented by the inhibition of JNK but not by that of ERK or p38 with specific inhibitors SP600125, U0126 or SB203580, respectively. Immunoblot analyses in colonic mucosa showed that mAChR stimulation increased MAPKs phosphorylation that was suppressed by the specific inhibitor for each MAPK. This suggests that JNK is a major negative regulator in mAChR-induced intestinal secretion.

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

Pluripotent stem cells maintain their pluripotency and undifferentiated status through a network of transcription factors. Liver receptor homolog-1 (Lrh-1) is one of these, and regulates the expression of other important transcription factors such as Oct-3/4 and Nanog. In early embryo and embryonic stem (ES) cells, Lrh-1 is transcribed using a unique promoter. In this study, we investigated the transcriptional regulation of embryonic Lrh-1 using ES and embryonal carcinoma F9 cells. Reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays demonstrated that Sox2 and Gabp proteins bind to the promoter region of embryonic Lrh-1, and are necessary for its activation. The Sox2 site showed strong promoter activity and affinity for protein binding. Upon differentiation into the parietal endoderm by retinoic acid and cAMP, Lrh-1 promoter activity and transcripts were markedly reduced within 24 h. At the same time, Sox2 and Gabp binding to the promoter region of Lrh-1 were decreased, followed by a reduction of their expression. These results indicate that embryonic Lrh-1 expression is regulated by both Sox2 and Gabp. Our study presents new insights into the transcription factor network of pluripotent stem cells.

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified.In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.

Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gαq/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca2+-dependent early phase p38 MAPK phosphorylation and extracellular Ca2+-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or ionomycin also phosphorylate p38 MAPK by Ca2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression.

Ferredoxin reductase (FDXR, also known as adrenodoxin reductase) is a mitochondrial flavoprotein that transfers electrons from NADPH to mitochondrial cytochrome P450 enzymes, mediating the function of an iron–sulfur cluster protein, ferredoxin. FDXR functions in various metabolic processes including steroidogenesis . It is well known that multiple steroidogenic enzymes are regulated by a transcription factor steroidogenic factor-1 (SF-1, also known as Ad4BP). Previously, we have shown that SF-1 transduction causes human mesenchymal stem cell differentiation into steroidogenic cells. Genome-wide analysis of differentiated cells, using a combination of DNA microarray and promoter tiling array analyses, showed that FDXR is a novel SF-1 target gene. In this study, the transcriptional regulatory mechanism of FDXR was examined in steroidogenic cells. A chromatin immunoprecipitation assay revealed that a novel SF-1 binding region was located within intron 2 of the human FDXR gene. Luciferase reporter assays showed that FDXR transcription was activated through the novel SF-1 binding site within intron 2. Endogenous SF-1 knockdown in human adrenocortical H295R and KGN cells decreased FDXR expression. In H295R cells, strong binding of two histone markers of active enhancers, histones H3K27ac and H3K4me2, were detected near the SF-1 binding site within intron 2. Furthermore, the binding of these histone markers was decreased concurrent with SF-1 knockdown in H295R cells. These results indicated that abundant FDXR expression in these steroidogenic cells was maintained through SF-1 binding to the intronic enhancer of the FDXR gene.

Steroidogenic factor 1 (SF-1) is a master regulator for steroidogenesis. In this study, we identified novel SF-1 target genes using a genome-wide promoter tiling array and a DNA microarray. SF-1 was found to regulate human glutathione S-transferase A (GSTA) family genes (hGSTA1-hGSTA4), a superfamily of detoxification enzymes clustered on chromosome 6p12. All hGSTA genes were up-regulated by transduction of SF-1 into human mesenchymal stem cells, while knockdown of endogenous SF-1 in H295R cells down-regulated all hGSTA genes. Chromatin immunoprecipitation assays, however, revealed that SF-1 bound directly to the promoters of hGSTA3 and weakly of hGSTA4. Chromosome conformation capture assays revealed that the coordinated expression of the genes was based on changes in higher-order chromatin structure triggered by SF-1, which enables the formation of long-range interactions, at least between hGSTA1 and hGSTA3 gene promoters. In steroidogenesis, dehydrogenation of the 3-hydroxy group and subsequent Δ(5)-Δ(4) isomerization are thought to be enzymatic properties of 3β-hydroxysteroid dehydrogenase (3β-HSD). Here, we demonstrated that, in steroidogenic cells, the hGSTA1 and hGSTA3 gene products catalyze Δ(5)-Δ(4) isomerization in a coordinated fashion with 3β-HSD II to produce progesterone or Δ(4)-androstenedione from their Δ(5)-precursors. Thus, hGSTA1 and hGSTA3 gene products are new members of steroidogenesis working as Δ(5)-Δ(4) isomerases.

Liver receptor homolog-1 (LRH-1) is a member of the nuclear receptor 5A (NR5A) subfamily. It is expressed in granulosa cells of the ovary and is involved in steroidogenesis and ovulation. To reveal the transcriptional regulatory mechanism of LRH-1, we determined its transcription start site in the ovary using KGN cells, a human granulosa cell tumor cell line. 5′-rapid amplification of cDNA ends PCR revealed that human ovarian LRH-1 was transcribed from a novel transcription start site, termed exon 2o, located 41 bp upstream of the reported exon 2. The novel LRH-1 isoform was expressed in the human ovary but not the liver. Promoter analysis and an EMSA indicated that a steroidogenic factor-1 (SF-1) binding site and a GC box upstream of exon 2o were required for promoter activity, and that SF-1 and specificity protein (Sp)-1/3 bind to the respective regions in ovarian granulosa cells. In KGN cells, transfection of SF-1 increased ovarian LRH-1 promoter activity and SF-1-dependent reporter activity was further enhanced when peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was cotransfected. In Drosophila SL2 cells, Sp1 was more effective than Sp3 in enhancing promoter activity, and co-transfection of the NR5A-family synergistically increased activity. Infection with adenoviruses expressing SF-1 or PGC-1α induced LRH-1 expression in KGN cells. These results indicate that the expression of human LRH-1 is regulated in a tissue-specific manner, and that the novel promoter region is controlled by the Sp-family, NR5A-family and PGC-1α in ovarian granulosa cells in a coordinated fashion.

It is well known that the androgen/androgen receptor (AR) pathway is involved in both male and female fertility in mammals. AR knockout female mice are reported to exhibit various abnormalities in follicle development, and a subfertile phenotype. In exogenous gonadotropin-induced superovulation, serum androgen levels were robustly elevated in female mice at the periovulatory stage after human chorionic gonadotropin (hCG) treatment. At this stage, ovarian AR proteins were strongly expressed in cumulus cells. Because these results suggested that the androgen/AR pathway is involved in ovulation, we investigated the expression of ovulation-related genes in the mouse ovary treated with the nonaromatizable androgen, 5α-dihydrotestosterone (DHT). DHT treatment induced the expression of the genes for cyclooxyganase-2 (Cox-2 or prostaglandin endoperoxidase synthase 2) and the epidermal growth factor-like factor, amphiregulin (Areg), in the ovary, whereas their hCG-induced expression was suppressed by the AR antagonist flutamide. These genes were also induced by DHT in AR-expressing primary granulosa and granulosa tumor-derived cells. Reporter assays, electrophoretic shift mobility assays and chromatin immunoprecipitation assays demonstrated that androgen response sequence(s) existing upstream of each gene were responsible for androgen responsiveness and were occupied by the AR in periovulatory granulosa cells. Our results suggest that the androgen/AR pathway is involved in the ovulatory process via expression of the Cox-2 and Areg genes in periovulatory granulosa cells.

CC-BY-NC-ND, Intestinal epithelial cells form a tight barrier to act as selective physical barriers, repelling hostile substances. Tumor necrosis factor-α (TNF-α) is a well characterized pro-inflammatory cytokine which can compromise intestinal barrier function and the suppression of TNF-α function is important for treatment of inflammatory bowel disease (IBD). In this study, we investigated the contribution of G-protein-coupled receptor (GPCR)-induced signalling pathways to the maintenance of epithelial barrier function. We first demonstrated the existence of functional muscarinic M3 and histamine H1 receptors in colonic epithelial cell HT-29/B6. As we previously reported, muscarinic M3 receptor prevented TNF-α-induced barrier disruption through acceleration of TNF receptor (TNFR) shedding which is carried out by TNF-α converting enzyme (TACE). M3 receptor-mediated suppression of TNF-α function depends on Gαq/11 protein, however, histamine H1 receptor could not ameliorate TNF-α function, while which could induce Gαq/11 dependent intracellular Ca2+ mobilization. We found that p38 MAPK was predominantly phosphorylated by M3 receptor through Gαq/11 protein, whereas H1 receptor barely upregulated the phosphorylation. Inhibition of p38 MAPK abolished M3 receptor-mediated TNFR shedding and suppression of TNF-α-induced NF-κB signalling. The p38 MAPK was also involved in TACE- mediated EGFR transactivation followed by ERK1/2 phosphorylation. These results indicate that not H1 but M3 receptor-induced activation of p38 MAPK might contribute to the maintenance of epithelial barrier function through down-regulation of TNF-α signalling and activation of EGFR.

Steroidogenic factor-1 (SF-1, also known as Ad4BP) has been demonstrated to be a primary transcriptional regulator of steroidogenic-related genes. However, mRNA for liver receptor homolog-1 (LRH-1), which together with SF-1, belongs to the NR5A nuclear receptor family, is expressed at much higher levels than SF-1 mRNA in the human gonad. In our previous studies, we demonstrated that SF-1 induced the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into steroidogenic cells such as Leydig or adrenocortical cells. The introduction of LRH-1 into human MSCs (hMSCs) with the aid of cAMP also induced the expression of steroidogenic enzymes, including CYP17, and their differentiation into steroid hormone-producing cells. Promoter analysis, EMSA, and chromatin immunoprecipitation assay using LRH-1-transduced hMSCs indicated that three LRH-1 binding sites were responsible for CYP17 transactivation. Immunohistochemical studies showed that LRH-1 protein was expressed in human Leydig cells. The CYP17 promoter region was highly methylated in hMSCs, whereas it was demethylated by the introduction of LRH-1 and cAMP treatment. These results indicate that LRH-1 could represent another key regulator of the steroidogenic lineage in MSCs and play a vital role in steroid hormone production in human Leydig cells.

Basic helix-loop-helix (bHLH) proteins regulate transcription from the E box sequence (59-CANNTG-39) located in the regulatory region of most gene promoters. The rat enhancer of split- and hairy-related protein 2 (SHARP-2) is a member of the bHLH protein family. To analyze the possible role of SHARP-2 in the rat ovary, the regulation of the expression of the SHARP2 gene was examined, and the SHARP-2 protein was characterized. Northern blot analysis revealed that the level of SHARP-2 mRNA abruptly and temporarily increases as the result of the action of LH, i.e., eCG or hCG treatment alone or hCG after eCG treatment, in the rat ovary, as indicated by the treatment of primary cultured rat granulosa cells with hCG after FSH treatment or of mouse Leydig MA-10 cells with hCG or 8-bromoadenosine 39,59-cyclic monophosphate. An in situ hybridization analysis showed that eCG treatment increases the level of the SHARP-2 transcript in theca interna cells and that hCG treatment, after the administration of eCG, increases the level of the SHARP-2 transcript in granulosa cells. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into primary cultured granulosa cells and MA-10 cells revealed that the entire coding sequence of SHARP-2 fused to the GFP is localized in the nucleus. The transcriptional activity of SHARP-2 also was examined using transient DNA transfection experiments. When an expression vector encoding the full length of SHARP-2 was cotransfected with thymidine kinase promoter-luciferase reporter plasmids, with or without E box sequences, into MA-10 cells, the luciferase activity was decreased in an E box-dependent manner. We conclude that the level of SHARP-2 mRNA is regulated by gonadotropins and that SHARP-2 functions as a transcriptional repressor localized in the nucleus. cyclic adenosine monophosphate, gene regulation, granulosa cells, ovary, theca cells

Zinc-fingers and homeoboxes (ZHX) 1 is a transcription factor that interacts with the activation domain of the A subunit of nuclear factor-Y (NF-YA). Using a yeast two-hybrid system, a novel ubiquitous transcription factor ZHX2 as a ZHX1-interacting protein was cloned. ZHX2 consists of 837 amino acid residues and contains two zinc-finger motifs and five homeodomains (HDs) as well as ZHX1. The mRNA is expressed among various tissues. ZHX2 not only forms a heterodimer with ZHX1, but also forms a homodimer. Moreover, ZHX2 interacts with the activation domain of NF-YA. Further analysis revealed that ZHX2 is a transcriptional repressor that is localized in the nuclei. Since ZHX2 shares a number of properties in common with ZHX1, we conclude that all these come under the ZHX family. The minimal functional domains of ZHX2 were then characterized. The dimerization domain with both ZHX1 and ZHX2 is the region containing HD1, the domain that interacts with NF-YA is the HD1 to HD2 region, the repressor domain is the HD1 to a proline-rich region. Lastly, using an immunoprecipitation assay, we showed that ZHX2 intrinsically interacts with NF-YA in HEK-293 cells and that ZHX2 represses the promoter activity of the cdc25C gene stimulated by NF-Y in Drosophila Schneider line 2 cells. Thus the ZHX family of proteins may participate in the expression of a number of NF-Y-regulated genes via a more organized transcription network.

In newt testis, spermatocytes never appear during winter, because secondary spermatogonia die by apoptosis just before meiosis. In the current study, we examined the effect of low temperatures on spermatogenesis. Incubation of newts at low temperatures (8, 12, 15 degrees C) induced defects in spermatogenesis in a temperature-dependent manner. At 8 degrees C, multinucleated giant cells (MGCs) were observed in spermatocytes and spermatogenesis never proceeded beyond meiosis. Although spermatocytes completed meiotic divisions at 12 degrees C, severe cell death was observed in the spermatids. At 15 degrees C both normal and abnormal spermiogenesis were observed. Under these conditions, impaired meiotic synapsis/recombination and down-regulation of the expression of the DMC1 protein, which play pivotal roles in meiotic pairing in eukaryotes, were also observed. Furthermore, to examine the quality of the sperm produced at low temperature for supporting development, artificial insemination was performed. The eggs inseminated with spermatozoa derived from newts kept at 15 degrees C demonstrated a restricted developmental capacity, even though these spermatozoa had an equal capacity for carrying out fertilization to those kept at 22 degrees C. These results suggest that meiosis at low temperatures cause the production of abnormal spermatozoa. Conservation and the significance of this phenomenon in poikilothermic vertebrates living in the temperate zones are also discussed.

The mouse zinc-fingers and homeoboxes 1 (ZHX1 ) gene was cloned and its transcriptional regulatory mechanism analysed. The mouse ZHX1 gene spans approximately 29 kb and consists of five exons. Exons 1 ‐3 contain the nucleotide sequence of the 5 0 -noncoding region of mouse ZHX1 cDNA, exon 4 contains a part of the 5 0 -noncoding region, an entire coding sequence, and a part of the 3 0 -noncoding sequence, and exon 5 contains the resulting 3 0 -noncoding sequence. The ZHX1 gene exists as one copy in the haploid mouse genome. Two species of ZHX1 mRNA with or without the nucleotide sequence of the third exon are produced by an alternative splicing. To investigate the regulatory elements involved in the transcription of the ZHX1 gene, transient DNA transfection experiments with ZHX1/firefly luciferase reporter genes were performed using a lipofection method. Functional analyses of a series of 5 0 - and 3 0 -deletion constructs of the reporter genes revealed that the nucleotide sequence between 2 59 and þ50 is required for full promoter activity in mouse embryonal carcinoma F9 cells. Two positive regulatory cis-acting elements in the region were identified. These elements, designated as Box A and Box B, are located between nucleotides 2 47 and 242 and þ 22 and þ27, respectively, and synergistically stimulate transcription of the mouse ZHX1 gene. Electrophoretic mobility shift assays with specific competitors and antibodies show that PEA3 and Yin and Yang 1 (YY1) bind to Box A and Box B, respectively. Thus, we conclude that PEA3 and YY1 synergistically stimulate the transcription of the ZHX1 gene. q 2002 Elsevier Science B.V. All rights reserved.

Ovarian follicular development is initiated by FSH secreted from the pituitary gland. The FSH-induced follicular development involves granulosa cell proliferation and differentiation. We demonstrated that a growth factor of epidermal growth factor (EGF) family epiregulin was rapidly induced in the primary culture of rat ovarian granulosa cells by FSH within 1 h. Epiregulin gene expression was also observed in granulosa cells of antral ovarian follicles from pregnant mare’s serum gonadotropin-primed rats in vivo. To analyze the regulation of gene expression of epiregulin, we isolated and characterized the rat epiregulin gene of 22.1 kb, including 3.8 kb of 5′-upstream region as well as all five exons and four introns. We determined the transcriptional start site of rat epiregulin gene by primer extension analysis and then characterized the upstream promoter region of the gene. By using a luciferase reporter system, deletion and mutation analyses of rat epiregulin gene promoter region revealed that 125 bp upstream of transcriptional start site was essential, and that two CT boxes and one GT box within this region were important for the gene expression. We also demonstrated by EMSAs that Sp1/Sp3 proteins were involved in the epiregulin gene expression via the upstream sequence. Involvement of Sp1/Sp3 was also demonstrated that transfection of Sp1 or Sp3 expression plasmids dramatically increased the epiregulin gene promoter activities about 90- or 7.9-fold, respectively, in Drosophila SL2 cells that lack endogenous Sp family proteins. Such an increase in the promoter activity was also observed in mammalian cells when NIH-3T3 cells were used. In conclusion, we demonstrated here for the first time that EGF-type growth factor epiregulin is rapidly and strongly induced in the ovarian granulosa cells by FSH stimulation, and that two CT boxes and one GT box present in the upstream region are essential for the promoter activity of rat epiregulin. We also demonstrated that Sp family members play crucial roles in the epiregulin promoter activity through the CT boxes. The restricted and hormonally regulated expression of epiregulin in the rat ovarian granulosa cells may correspond to the physiological relevance of this peptide growth factor to the FSH-induced ovarian follicular growth and maturation.

LH receptor gene expression is primarily regulated via specific interactions of trans-acting proteins and cis-acting DNA sequences in the upstream region of the gene. In this study, we report, using luciferase assays, that the region between -171 and -137 base pairs (bp) is essential for basal expression of the rat LH receptor gene. To identify factors that interact with the region between -171 and -137 bp and regulate expression of the gene, a rat granulosa cell cDNA library was screened using a yeast one-hybrid system. A positive clone, isolated by the screening, encodes a transcription factor early growth response gene-1 (Egr-1). To determine the sequence to which Egr-1 protein binds, electrophoretic mobility shift assay (EMSA) was employed. The Egr-1 protein was produced by an in vitro transcription/translation system using a full-length rat Egr-1 cDNA. The upstream region between -171 and -137 bp contains 2 overlapping Egr-1 consensus sequences. The EMSA revealed that Egr-1 binds independently to both sites. The overexpression of Egr-1 in MA-10 cells caused an approximately 2-fold increase in reporter luciferase activity. However, no induction of the luciferase activity was observed when luciferase constructs that lacked or had mutations in either or both of the Egr-1 sites were used, indicating that Egr-1 positively regulates LH receptor gene expression. In differentiated granulosa cells that had been pretreated with FSH for 48 h, the levels of both mRNA and Egr-1 protein were induced by hCG or cAMP, reaching maximal levels approximately 1.5 h after treatment and then returning to basal levels 8 h thereafter. No Egr-1 mRNA or protein was detected in undifferentiated granulosa cells, even after stimulation with 8-bromoadenosine-cAMP. These results suggest that Egr-1 functions only in luteinized granulosa cells after stimulation with hCG or cAMP. In conclusion, the findings demonstrate that Egr-1 actually binds to the regulatory upstream region of the LH receptor gene and positively regulates receptor gene expression. In addition, Egr-1 expression was observed only in luteinized granulosa cells after stimulation with hCG or cAMP. The present study provides further support to the hypothesis that Egr-1 plays important roles in the pituitary-gonadal axis.

Zinc-fingers and homeoboxes 1 (ZHX1) is a protein which interacts with the activation domain of the A subunit of nuclear factor-Y. To analyze the physiological role(s) of ZHX1, we searched ZHX1-interacting protein(s) using a yeast two-hybrid system. The rat counterpart of ZHX1 cDNAs was cloned from an ovarian granulosa cell complementary DNA (cDNA) library, indicating that ZHX1 is able to form a homodimer. An analysis of the nucleotide sequence and its deduced amino acid sequence show that rat ZHX1 consists of 873 amino acid residues. Northern blot analysis shows that ZHX1 messenger RNA is expressed ubiquitously and that the level in the ovary are not regulated by gonadotropins. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into human embryonic kidney HEK293 cells reveal that full-length ZHX1 fused to the GFP is localized in the nuclei. Thus, we report on the molecular cloning, expression and characterization of full-length rat ZHX1 cDNA.

We previously reported that mammalian FSH induced differentiation of secondary spermatogonia into primary spermatocytes in organ culture of newt testicular fragments, whereas in medium lacking FSH primary spermatocytes never appeared. Here, we investigated why spermatogonia fail to form primary spermatocytes in the absence of FSH. Spermatogonia maintained proliferative activity and viability at about half the level of those cultured in the presence of FSH, progressed into the seventh generation, but became moribund during the G2/M phase. Thus, the eighth generation of spermatogonia never appeared, suggesting that cell death is the chief reason why primary spermatocytes fail to form in the absence of FSH. The presence of Dmc1, a molecular marker for the spermatocyte stage, confirmed our microscopic observations that spermatogonia differentiated into primary spermatocytes in the presence of FSH. Thus, FSH is indispensable for the completion of the last spermatogonial mitosis, a prerequisite for the conversion of germ cells from mitosis to meiosis. Because prolactin induced apoptosis in spermatogonia during the seventh generation, we propose that a checkpoint exists for the initiation of meiosis in the seventh generation whereby spermatogonia enter meiosis when the concentration ratio of FSH to prolactin is high but fail to do so when the ratio is low.

Hormone replacement therapy is necessary for patients with adrenal and gonadal failure. Steroid hormone treatment is also employed in aging people for sex hormone deficiency. These patients undergo such therapies, which have associated risks, for their entire life. Stem cells represent an innovative tool for tissue regeneration and the possibility of solving these problems. Among various stem cell types, mesenchymal stem cells have the potential to differentiate into steroidogenic cells both in vivo and in vitro. In particular, they can effectively be differentiated into steroidogenic cells by expressing nuclear receptor 5A subfamily proteins (steroidogenic factor-1 and liver receptor homolog-1) with the aid of cAMP. This approach will provide a source of cells for future regenerative medicine for the treatment of diseases caused by steroidogenesis deficiencies. It can also represent a useful tool for studying the molecular mechanisms of steroidogenesis and its related diseases.

The transcription factor SF-1 (steroidogenic factor-1) is a master regulator of steroidogenesis. Previously, we have found that SF-1 induces the differentiation of mesenchymal stem cells into steroidogenic cells. To elucidate the molecular mechanisms of SF-1-mediated functions, we attempted to identify protein components of the SF-1 nuclear protein complex in differentiated cells. SF-1 immunoaffinity chromatography followed by MS/MS analysis was performed, and 24 proteins were identified. Among these proteins, we focused on C/EBPβ (CCAAT/enhancer-binding protein β), which is an essential transcription factor for ovulation and luteinization, as the transcriptional mechanisms of C/EBPβ working together with SF-1 are poorly understood. C/EBPβ knockdown attenuated cAMP-induced progesterone production in granulosa tumour-derived KGN cells by altering STAR (steroidogenic acute regulatory protein), CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and HSD3B2 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2) expression. EMSA and ChIP assays revealed novel C/EBPβ-binding sites in the upstream regions of the HSD3B2 and CYP11A1 genes. These interactions were enhanced by cAMP stimulation. Luciferase assays showed that C/EBPβ-responsive regions were found in each promoter and C/EBPβ is involved in the cAMP-induced transcriptional activity of these genes together with SF-1. These results indicate that C/EBPβ is an important mediator of progesterone production by working together with SF-1, especially under tropic hormone-stimulated conditions.

Gonadotropins are essential for ovarian follicular development and differentiation. To identify genes that are rapidly induced by gonadotropin in the immature rat ovary, ovarian genes were screened by a subtraction cloning procedure. cDNA clones encoding novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family GIOT1 and -2 (gonadotropin-inducible transcription factor 1 and 2), were identified. Two isoforms of GIOT2 (GIOT2 alpha and 2 beta), which are probably produced by alternative splicing, also exist. Nucleotide sequence analysis revealed that GIOT1, but not GIOT2, contains the krüppel-associated box-A domain at the NH(2) terminus. RNA analyses revealed that these mRNAs were rapidly and temporarily induced by gonadotropins in the rat testis as well as in the ovary. In situ hybridization study revealed that expression of GIOT1 was induced in theca interna cells in the ovary and Leydig cells in the testis. Interestingly, the gene expression of GIOT1 is restricted to the pituitary, adrenal, testis, and ovary, while GIOT2 gene is expressed ubiquitously. A functional analysis of GIOT1 and -2 by a GAL4-based mammalian one-hybrid system revealed that GIOT1, but not GIOT2, is a transcriptional repressor and that the krüppel-associated box-A domain of GIOT1 is responsible for the transcriptional repressor activity. A GAL4-based yeast two-hybrid system was also used to identify proteins that interact with the rat GIOT1. We cloned genes encoding rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta, both of which are transcription-regulatory proteins. Interaction of these proteins with GIOT1 was directly demonstrated by GST pull-down assay. Our data strongly suggest that GIOT1 may function as a novel transcriptional repressor by working with rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta proteins and may play a significant role at the transcription level in the folliculogenesis.

A comparative study of natriuretic peptide receptor (NPR) was performed by cloning the NPR-A receptor subtype from the bullfrog (Rana catesbeiana) brain and analyzing its functional expression. Like other mammalian NPR-A receptors, the bullfrog NPR-A receptor consists of an extracellular ligand binding domain, a hydrophobic transmembrane domain, a kinase-like domain and a guanylate cyclase domain. Sequence comparison among the bullfrog and mammalian receptors revealed a relatively low ( approximately 45%) similarity in the extracellular domain compared to a very high similarity ( approximately 92%) in the cytoplasmic regulatory and catalytic domains. Expression of NPR-A mRNA was detected in various bullfrog tissues including the brain, heart, lung, kidney and liver; highest levels were observed in lung. Functional expression of the receptor in COS-7 cells revealed that frog atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) elicited cyclic guanosine 3'5'-monophosphate production by stimulating the receptor in a dose-dependent manner from 10(-10) M concentrations. Rat ANP was also effective in stimulating the frog receptor whereas rat BNP and porcine BNP were less responsive to the receptor. On the other hand, frog C-type natriuretic peptide (CNP) as well as porcine CNP stimulated the receptor only at high concentrations (10(-7) M). This clearly indicates that the bullfrog receptor is a counterpart of mammalian NPR-A, and is specific for ANP or BNP but not for CNP.

In newts elevated titers of plasma prolactin (PRL), induced by low temperature, cause apoptosis in the penultimatemitotic stage of spermatogonia, and this cell death is suppressed by antiserum against newt PRL, but only during the initial 3 days of exposure (Yazawa et al., 1999). Thus, factors other than PRL must be involved in spermatogonial death. Follicle-stimulating hormone (FSH) may be a plausible candidate. Accordingly, the current study examined the activityof FSH on the proliferation and survival of spermatogonia at low temperatures in vivo and in vitro. Porcine FSH (pFSH) administration in vivo inhibited spermatogonial death induced at 12 degrees C, but failed to do so at8 degrees C. Also pFSH promoted in vitro the proliferation of spermatogonia at 12 degrees C, but not at 8 degrees C. Furthermore,dibutyryl cyclic AMP stimulated in vitro DNA synthesis of secondary spermatogonia at 12 degrees C, but not at 8 degrees C. These different responses to temperatures were not caused by different levels of mRNA for the receptor of follicle-stimulating hormone, the numberof FSH binding sites, or FSH binding affinity to its receptors in the testicular cells. Thus, the results indicate that a temperature-sensitive period exists duringthe postreceptor process and is responsible for thelack of response of newt testis to FSH at 8 degrees C.

We previously showed in vivo and in vitro, that among the spermatogenic stages of the newt, prolactin (PRL) induces apoptosis specifically in the penultimate stage of secondary spermatogonia. In the current report, we demonstrate in vitro that cycloheximide (CHX), an inhibitor of protein synthesis, induces morphological apoptotic changes similar to those caused by PRL, such as chromatin condensation and apoptotic body formation. Next, we found that Z-VAD-fmk, an inhibitor of various caspases, suppressed the apoptosis induced by PRL and CHX, but ICE inhibitor Ac-YVAD-CHO or caspase-3 inhibitor Ac-DEVD-CHO did not. As high caspase activity was present in extracts of testes treated with CHX, we suggest that an unidentified caspase induces the morphological changes of apoptosis in newt spermatogonia.

Ovarian granulosa cells undergo cell growth and cytodifferentiation during follicular maturation. In a number of tissues, the gene expression that is responsible for the cytodifferentiation is largely dependent on E box(es) located upstream of the responsible genes. In this study, we report on the cloning of cDNA(s) encoding E box (59-CACGTG-39)-binding protein from a rat granulosa cell cDNA library using a yeast one-hybrid system. When multiple E box sequences were used as target, we obtained a positive clone that encodes the rat homologue of upstream stimulatory factor 2 (USF2). An analysis of the nucleotide sequence and its deduced amino acid sequence reveals that rat USF2 protein consists of 346 amino acid residues and belongs to the basic helix-loop-helix/leucine zipper protein family. Northern blot analysis shows that rat USF2 mRNA exists as multiple forms between 1.6 and 2.2 kilobases. The size of the cloned insert was identical to that of the transcript of maximal length. Electrophoretic mobility shift assays showed that in vitrotranslated rat USF2 specifically binds to the E box. In addition, cotransfection experiments with luciferase-reporter constructs in HepG2 cells reveal that the overexpression of rat USF2 leads to an increase of luciferase activity in the E box sequence-dependent manner. Thus, we report molecular cloning, expression, and functional characterization of full-length rat USF2 cDNA. gene regulation, granulosa cells, ovary

Cell death is a common feature during spermatogenesis and, in some seasonal breeding animals, is often observed at the transition stage from spermatogonia to spermatocytes. In the Japanese red-bellied newt, we have previously shown that this cell death is caused by the elevated titer of plasma PRL that occurs after animals are transferred to low temperature, suggesting that cell death causes the cessation of spermatocytogenesis from late autumn to early spring. In the present report, first we show that the injection of PRL into newts causes apoptosis in spermatogonia after the sixth mitotic division, the penultimate one before spermatogonia normally enter meiosis. Second, we demonstrate in organ cultures of testes fragments that PRL acts directly on the testes. Third, we show that the action by PRL is inhibited by FSH dose dependently.

We examined the effect of low temperatures on annexin V expression in newt testis. When newts were transferred to a low temperature (12 degrees C), up-regulation of annexin V protein was observed in secondary spermatogonia. In primary spermatocytes, high levels of annexin V expression were observed at both 12 degrees C and 22 degrees C, but at 12 degrees C the protein was localized in part of the cytoplasm of primary spermatocytes. These results indicate that in newt testis annexin V is a cold-sensitive protein, suggesting the possibility that annexin V might have a cold stress-related function in newt germ cells.

Ferredoxin 1 (FDX1; adrenodoxin) is an iron-sulfur protein that is involved in various metabolic processes, including steroid hormone synthesis in mammalian tissues. We investigated the transcriptional regulation of FDX1 in ovarian granulosa cells. Previously, we reported that the NR5A family, including steroidogenic factor-1 (SF-1) and liver receptor homolog-1 could induce differentiation of human mesenchymal stem cells (hMSCs) into steroidogenic cells. A ChIP assay showed that SF-1 could bind to the FDX1 promoter in differentiated hMSCs. Luciferase reporter assays showed that transcription of FDX1 was synergistically activated by the NR5A family and 8Br-cAMP treatment through two SF-1 binding sites and a CRE-like sequence in a human ovarian granulosa cell line, KGN. Knockdown of FDX1 attenuated progesterone production in KGN cells. These results indicate transcription of FDX1 is regulated by the NR5A family and cAMP signaling, and participates in steroid hormone production in ovarian granulosa cells.

5-Aminolevulinic acid synthase 1 (ALAS1) is a rate-limiting enzyme for heme biosynthesis in mammals. Heme is essential for the catalytic activities of P450 enzymes including steroid metabolic enzymes. Nuclear receptor 5A (NR5A) family proteins, steroidogenic factor-1 (SF-1), and liver receptor homolog-1 (LRH-1) play pivotal roles in regulation of steroidogenic enzymes. Recently, we showed that expression of SF-1/LRH-1 induces differentiation of mesenchymal stem cells into steroidogenic cells. In this study, genome-wide analysis revealed that ALAS1 was a novel SF-1-target gene in differentiated mesenchymal stem cells. Chromatin immunoprecipitation and reporter assays revealed that SF-1/LRH-1 up-regulated ALAS1 gene transcription in steroidogenic cells via binding to a 3.5-kb upstream region of ALAS1. The ALAS1 gene was up-regulated by overexpression of SF-1/LRH-1 in steroidogenic cells and down-regulated by knockdown of SF-1 in these cells. Peroxisome proliferator-activated receptor-γ coactivator-1α, a coactivator of nuclear receptors, also strongly coactivated expression of NR5A-target genes. Reporter analysis revealed that peroxisome proliferator-activated receptor-γ coactivator-1α strongly augmented ALAS1 gene transcription caused by SF-1 binding to the 3.5-kb upstream region. Finally knockdown of ALAS1 resulted in reduced progesterone production by steroidogenic cells. These results indicate that ALAS1 is a novel NR5A-target gene and participates in steroid hormone production.

Replacement of deficient steroid hormones by hormone replacement therapy is necessary for patients with adrenal and gonadal failure. However, patients undergoing these therapies have associated risks during their entire life. Stem cells represent an innovative tool for tissue regeneration and gene therapy, and the possibility of solving these problems. Among various stem cell types, mesenchymal stem cells can be differentiated into steroidogenic cells by stable expression of steroidogenic factor-1 (SF-1) and cAMP treatment. The same approach cannot be applied to other undifferentiated cells such as embryonic stem (ES) cells because of poor survival following expression of SF-1. However, ES cells can also be differentiated into steroidogenic cells with SF-1 via the mesenchymal cell lineage. These approaches will provide a source of cells for future regenerative medicine and gene therapy of diseases caused by steroidogenesis deficiencies.

POR (cytochrome P450 oxidoreductase) is a ubiquitously expressed gene encoding an electron donor to all microsomal P450 enzymes and several non-P450 enzymes. POR mutations cause an autosomal recessive disorder characterized by skeletal dysplasia, adrenal dysfunction, and disorders of sex development. Although recent studies have indicated the presence of a CpG-rich region characteristic of housekeeping genes around the untranslated exon 1 (exon 1U) and a tropic effect of thyroid hormone on POR expression via thyroid hormone receptor-β, detailed regulatory mechanisms for the POR expression remain to be clarified.Our objective was to report a pivotal element of the proximal promoter of POR.We first studied three patients (cases 1-3) with POR deficiency due to compound heterozygosity with an p.R457H mutation and transcription failure of an apparently normal allele, by oligoarray comparative genomic hybridization and serial direct sequencing of the deletion fusion points. Consequently, a 2,487-bp microdeletion involving exon 1U was identified in case 1 and an identical 49,604-bp deletion involving exon 1U and exon 1 was found in cases 2 and 3. We next analyzed the 2,487-bp region commonly deleted in cases 1-3 by in silico analysis, DNA binding analysis, luciferase assays, and methylation analysis. The results showed a critical function of the evolutionally conserved SP1 binding sites just upstream of exon 1U, especially the binding site at the position -26/-17, in the transcription of POR.The results suggest that the SP1 binding sites constitute an essential element of the POR proximal promoter.

Transformants of mesenchymal stem cells (MSCs) stably expressing steroidogenic factor-1 (SF-1) undergo differentiation into steroidogenic cell-lineages by stimulation with cyclic-adenosine mono-phosphate (cAMP). Another member of NR5A nuclear orphan receptors, Liver-specific receptor homologue-1 (LRH-1), was also able to differentiate MSCs. On the other hand, we found that embryonic stem (ES) cells were hardly induced to differentiate into steroidogenic cell-lineage by the similar treatment. In this study, we developed a novel method to differentiate ES cells into steroidogenic cells. We introduced SF-1 into mouse ES cells at ROSA26 locus under regulation of Tetracycline-off (Tet-off) in order to express SF-1 in the cells at desired period. When SF-1 was induced to express after the ES cells had been differentiated into mesenchymal cell-lineage, steroid hormones were produced from the SF-1 expressing cells. This provides a safer method for supplying sufficient amount of differentiated cells toward future regenerative medicine.

Previously, we have demonstrated that mesenchymal stem cells could be differentiated into steroidogenic cells through steroidogenic factor-1 and 8bromo-cAMP treatment. Use of liver receptor homolog-1, another of the nuclear receptor 5A family nuclear receptors, with 8bromo-cAMP also resulted in the differentiation of human mesenchymal stem cells into steroid hormone-producing cells. The same approaches could not be applied to other undifferentiated cells such as embryonic stem cells or embryonal carcinoma cells, because the over-expression of the nuclear receptor 5A family is cytotoxic to these cells. We established embryonic stem cells carrying tetracycline-regulated steroidogenic factor-1 gene at the ROSA26 locus. The embryonic stem cells were first differentiated into a mesenchymal cell lineage by culturing on collagen IV-coated dishes and treating with pulse exposures of retinoic acid before expression of steroidogenic factor-1. Although the untreated embryonic stem cells could not be converted into steroidogenic cells by expression of steroidogenic factor-1 in the absence of leukemia inhibitory factor due to inability of the cells to survive, the differentiated cells could be successfully converted into steroidogenic cells when expression of steroidogenic factor-1 was induced. They exhibited characteristics of adrenocortical-like cells and produced a large amount of corticosterone. These results indicated that pluripotent stem cells could be differentiated into steroidogenic cells by the nuclear receptor 5A family of protein via the mesenchymal cell lineage. This approach may provide a source of cells for future gene therapy for diseases caused by steroidogenesis deficiencies.

In poikilothermic vertebrates, sex determination is sometimes influenced by environmental factors such as temperature. However, little is known about the molecular mechanisms underlying environmental sex determination. The medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Recently, it was reported that XX medaka can be sex-reversed into phenotypic males by high water temperature (HT; 32-34 degrees C) treatment during the sex differentiation period. Here we report that cortisol caused female-to-male sex reversal and that metyrapone (an inhibitor of cortisol synthesis) inhibited HT-induced masculinization of XX medaka. HT treatment caused elevation of whole-body levels of cortisol, while metyrapone suppressed the elevation by HT treatment during sexual differentiation. Moreover, cortisol and 33 degrees C treatments inhibited female-type proliferation of germ cells as well as expression of follicle-stimulating hormone receptor (fshr) mRNA in XX medaka during sexual differentiation. These results strongly suggest that HT induces masculinization of XX medaka by elevation of cortisol level, which, in turn, causes suppression of germ cell proliferation and of fshr mRNA expression.

In vertebrates, sex is normally determined by genotype. However, in poikilothermal vertebrates, including reptiles, amphibians, and fishes, sex determination is greatly influenced by environmental factors, such as temperature. Little is known about the molecular mechanisms underlying environmental sex determination in these species. The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. However, XX flounder can be induced to develop into predominantly either phenotypic females or males, by rearing at 18 or 27 C, respectively, during the sex differentiation period. Therefore, the flounder provides an excellent model to study the molecular mechanisms underlying temperature-dependent sex determination. We previously showed that an aromatase inhibitor, an antiestrogen, and 27 C treatments cause masculinization of XX flounder, as well as suppression of mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, estrogen administration completely inhibits masculinization by these treatments, suggesting suppression of cyp19a1 mRNA expression, and the resultant estrogen biosynthesis may trigger masculinization of the XX flounder induced by high water temperature. Here, we demonstrated that cortisol causes female-to-male sex reversal by directly suppressing cyp19a1 mRNA expression via interference with cAMP-mediated activation and that metyrapone (an inhibitor of cortisol synthesis) inhibits 27 C-induced masculinization of XX flounder. Moreover, cortisol concentrations in 27 C-reared juveniles were significantly higher than in 18 C-reared fishes during sexual differentiation. These results strongly suggest that masculinization by high water temperature is ascribable to elevation of cortisol concentration during gonadal sex differentiation in the flounder.

NR4A1, also called NGFI-B in the rat, Nur77 in the mouse and TR3 in humans, belongs to the orphan nuclear steroid hormone receptor superfamily and is one of the immediate-early genes. In the endocrine organs, including the gonads, NGFI-B/Nur77 gene expression is rapidly induced by pituitary hormones. NGFI-B/Nur77 expression was found to be rapidly reduced by an estrogenic endocrine disrupter, diethylstilbestrol (DES) in theca interna cells of immature rat ovaries. DES treatment also triggered a rapid decrease of serum luteinizing hormone (LH) levels, suggesting that DES acts on the hypothalamo-pituitary axis to suppress LH secretion from the pituitary. The transcriptional regulation of NGFI-B/Nur77 by LH/human chorionic gonadotropin (hCG) or 8-bromoadenosine 3'-5'-cyclic monophosphate (8 Br-cAMP) was examined in mouse Leydig tumor cells MA-10. Luciferase assays using NGFI-B/Nur77 promoter constructs and electric mobility shift assays (EMSA) showed that NGFI-B/Nur77 gene expression was mediated through three of the four activator protein-1 (AP-1)-like sites, namely the -233 AP-1, -213 AP-1 and -69 AP-1 sites adjacent to the transcription start site of the NGFI-B/Nur77 promoter. We also demonstrated here that both the Jun family and cAMP-responsive element binding (CREB) proteins bind to the -233 AP-1 site, whereas the main binding protein to the -213 AP-1 site was CREB, and Jun family protein to the -69 AP-1 site, respectively. The rapid induction of NGFI-B/Nur77 gene expression by LH/hCG in MA-10 cells appears to be mediated by both CREB and Jun family proteins through the cAMP-protein kinase A (PKA) pathway.

We have shown previously that Cyp11b1, an 11β-hydroxylase responsible for glucocorticoid biosynthesis in the adrenal gland, was induced by cAMP in androgen-producing Leydig-like cells derived from mesenchymal stem cells. We found that Cyp11b1 was induced in male Leydig cells, or female theca cells, when human chorionic gonadotropin was administered in immature mice. Expression of Cyp11b1 in rodent gonads caused the production of 11-ketotestosterone (11-KT), a major fish androgen, which induces male differentiation or spermatogenesis in fish. As in teleosts, plasma concentrations of 11-KT were elevated in human chorionic gonadotropin-treated mice. In contrast to teleosts, however, plasma concentrations of 11-KT were similar in both sexes, despite levels of testosterone, a precursor substrate, being about 20 times higher in male mice. Because expression of 11β-hydroxysteroid dehydrogenase type 2, was much higher in the mouse ovary than in the testis, conversion of testosterone into 11-KT may occur more efficiently in the ovary. In a luciferase reporter system that was responsive to and activated by androgens, 11-KT efficiently activated mammalian androgen receptor-mediated transactivation. Our results suggest that the androgen metabolic pathway is conserved between teleosts and mammals, despite sexual dominance and reproductive functions of 11-KT being altered during evolution.

Steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1) belong to the nuclear receptor superfamily and are categorized as orphan receptors. In addition to other nuclear receptors, these play roles in various physiological phenomena by regulating the transcription of target genes. Both factors share very similar structures and exhibit common functions. Of these, the roles of SF-1 and LRH-1 in steroidogenesis are the most important, especially that of SF-1, which was originally discovered and named to reflect such roles. SF-1 and LRH-1 are essential for steroid hormone production in gonads and adrenal glands through the regulation of various steroidogenesis-related genes. As SF-1 is also necessary for the development of gonads and adrenal glands, it is also considered a master regulator of steroidogenesis. Recent studies have clearly demonstrated that LRH-1 also represents another master regulator of steroidogenesis, which similarly to SF-1, can induce differentiation of non-steroidogenic stem cells into steroidogenic cells. Here, we review the functions of both factors in these steroidogenesis-related phenomena.