For the purpose of marine environmental radioactivity monitoring, stable Cs and 137Cs concentrations were obtained from 214 marbled flounder (Pleuronectes yokohamae) samples collected from off the coast of Ibaraki prefecture in Japan in 2005 and 2006. Prior to the investigation, an analytical method for the determination of Cs in muscle of marbled flounder samples by inductively coupled plasma mass spectrometry (ICP-MS) was seriously considered from the view point of sample preparation (wet way) to a Cs measurement. Since major problems in determining Cs by the ICP-MS is the interference of matrix materials and molecular ions on Cs peak area (m/z 133), a known amount of In (m/z 115) was added to the sample solutions as an internal standard. To check the accuracy of the determination, duplication analysis and standard reference materials were used. Analytical results of Cs in muscles samples by ICP-MS agreed well with duplicated samples, and standard reference materials. The standard length and body weight were obtained from a total of 214 flounder samples. The stable Cs, stable K and 137Cs concentrations in muscle were determined by ICP-MS, flame photometry and radiochemical analysis, respectively. The mean standard length correlates closely with the mean body weight. The stable Cs concentration was in proportion to the body weight, also, the 137Cs concentration correlated closely with the stable Cs concentration in muscle. On the other hand, a stable K concentration was found to be uniform, and unrelated with 137Cs and stable Cs concentrations. The specific activities, the ratio of 137Cs/stable Cs in muscle, was found to be 5.5∼6.7 with an average of 6.2±0.35 (SD) among five different size groups of the standard length of marbled flounder samples. From these results, ICP-MS provided useful tools for the determination of a trace amount of Cs in muscle samples, combined with wet way pretreatment techniques. In addition, marbled flounder proved to be a useful species for comparing the radioactivity of fishes in coastal seas around Japan with each other in the environmental radioactivity monitoring program.

Background: Ovarian follicular development is primarily dependent on pituitary gonadotropins. Identification of gonadotropin- inducible genes in the ovary is one of the effective approaches for the study of follicular development. In this study we identify rat homologue of p120, a nuclear transcription co-activator, as one of the FSH inducible genes in the rat granulosa cells.Methods: A full-length cDNA encoding rat p120 was cloned, and expression of the gene in the ovary was examined by Northern blotting. Tissue localization of p120 was examined by in situ hybridization. Cellular functions of p120 were studied by co-transfection of rat p120 gene together with estrogen receptor (ER)-alpha, ER-beta, androgen receptor (AR), or progesterone receptor (PR) genes.Results: A full-length cDNA encoding rat p120 was characterized as a protein with 957 amino acid residues. Rat p120 was expressed ubiquitously, but strongly in the ovary and the testis. Expression of p120 mRNA was also induced in vivo by PMSG or PMSG/hCG treatment. Strong expression of p120 mRNA was observed in the granulosa cells of pre-ovulatory large antral follicles. Progesterone receptor was co-localized with p120 in the large antral follicles. Co-transfection experiments revealed that rat p120 activated AR, ER-alpha, ER-beta, and PR in the presence of their respective ligands.Conclusion: These observations suggest that rat p120 is strongly induced in the ovarian granulosa cells, and may work together with PR in the granulosa cells of ovulatory follicles to promote the ovulation process.

Adult stem cells from bone marrow, referred to as mesenchymal stem cells or marrow stromal cells (MSCs), are defined as pluripotent cells and have the ability to differentiate into multiple mesodermal cells. In this study, we investigated whether MSCs from rat, mouse, and human are able to differentiate into steroidogenic cells. When transplanted into immature rat testes, adherent marrow-derived cells (including MSCs) were found to be engrafted and differentiate into steroidogenic cells that were indistinguishable from Leydig cells. Isolated murine MSCs transfected with green fluorescence protein driven by the promoter of P450 side-chain cleaving enzyme gene (CYP11A), a steroidogenic cell-specific gene, were used to detect steroidogenic cell production in vitro. During in vitro differentiation, green fluorescence protein-positive cells, which had characteristics similar to those of Leydig cells, were found. Stable transfection of murine MSCs with a transcription factor, steroidogenic factor-1, followed by treatment with cAMP almost recapitulated the properties of Leydig cells, including the production of testosterone. Transfection of human MSCs with steroidogenic factor-1 also led to their conversion to steroidogenic cells, but they appeared to be glucocorticoid- rather than testosterone-producing cells. These results indicate that MSCs represent a useful source of stem cells for producing steroidogenic cells that may provide basis for their use in cell and gene therapy.

Ovarian follicular development is primarily depen‑ dent on pituitary gonadotropins. We report here the identification of two genes a nuclear transcription co- activator p120 and a novel serine protease-like pro‑ tein as FSH inducible genes in the rat granulosa cells. Characterization of the genes revealed that rat p120 may work together with PR in the granulosa cells of large antral follicles to promote the ovulation events and that the novel serine protease-like protein may be involved in the proteolytic process during the follicular development and ovulation process.

Abstract Basic helix-loop-helix (bHLH) proteins regulate transcription from the E box sequence (5′-CANNTG-3′) 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 SHARP-2 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 3′,5′-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.

Exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of toxic manifestations, including fetal death. In order to evaluate the effects of low dose TCDD on placental function, pregnant Holtzman rats were given a single oral dose of 1600 ng TCDD/kg body wt or an equivalent volume of vehicle (control) on gestation day 15 (GD15), and changes in the gene expression in the placenta on GD20 were analyzed by two comprehensive methods, representational difference analysis (RDA) and DNA microarray technology. Candidates of TCDD-inducible and -suppressive genes were selected. Quantitative real-time PCR analysis was then performed to verify the induction or suppression levels of the candidate genes. Finally, we identified 81 TCDD-inducible and 21 TCDD-suppressive genes from the placenta of TCDD-treated Holtzman rats on GD20. One of the remarkable profiles of the gene expression was that glucose transporters were strongly up-regulated by the TCDD treatment. Furthermore, many interferon-inducible genes were also up-regulated by the treatment. They included several cytokines such as IP-10 known as a potent angiogenesis inhibitor. In addition, interferon molecules are known to suppress angiogenesis. The above observations suggest that activation of the interferon signaling pathway and the induction of anti-angiogenic factors by TCDD might have a role in causing the inhibition of neovascularization, resulting in the hypoxic state of placenta and increased incidence of fetal death.

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.