AB1294 Sigma-AldrichAnti-Cytochrome P450 Side Chain Cleavage Enzyme Antibody, a.a. 509-526

NFAT (the nuclear factor of activated T cells) upregulation has been linked to cellular transformation intrinsically, but it is unclear whether and how tissue cells with NFAT activation change the local environment for tumor initiation and progression. Direct evidence showing NFAT activation initiates primary tumor formation in vivo is also lacking. Using inducible transgenic mouse systems, we show that tumors form in a subset of, but not all, tissues with NFATc1 activation, indicating that NFAT oncogenic effects depend on cell types and tissue contexts. In NFATc1-induced skin and ovarian tumors, both cells with NFATc1 activation and neighboring cells without NFATc1 activation have significant upregulation of c-Myc and activation of Stat3. Besides known and suspected NFATc1 targets, such as Spp1 and Osm, we have revealed the early upregulation of a number of cytokines and cytokine receptors, as key molecular components of an inflammatory microenvironment that promotes both NFATc1(+) and NFATc1(-) cells to participate in tumor formation. Cultured cells derived from NFATc1-induced tumors were able to establish a tumorigenic microenvironment, similar to that of the primary tumors, in an NFATc1-dependent manner in nude mice with T-cell deficiency, revealing an addiction of these tumors to NFATc1 activation and downplaying a role for T cells in the NFATc1-induced tumorigenic microenvironment. These findings collectively suggest that beyond the cell autonomous effects on the upregulation of oncogenic proteins, NFATc1 activation has non-cell autonomous effects through the establishment of a promitogenic microenvironment for tumor growth. This study provides direct evidence for the ability of NFATc1 in inducing primary tumor formation in vivo and supports targeting NFAT signaling in anti-tumor therapy.

Endothelin-2 (EDN2), a potent vasoconstrictive peptide, is transiently produced by periovulatory follicles at the time of ovulation when corpus luteum (CL) formation begins. EDN2 induces contraction of ovarian smooth muscles ex vivo via an endothelin receptor A-mediated pathway. In this study, we aimed to determine if EDN2 is required for normal ovulation and subsequent CL formation in?vivo. In the ovaries of a mouse model that globally lacks the Edn2 gene (Edn2 knockout mouse; Edn2KO), histology showed that post-pubertal Edn2KO mice possess follicles of all developmental stages, but no corpora lutea. When exogenous gonadotropins were injected to induce super-ovulation, Edn2KO mice exhibited significantly impaired ovulation and CL formation compared to control littermates. Edn2KO ovaries that did ovulate in response to gonadotropins did not contain histologically and functionally identifiable CL. Intra-ovarian injection of EDN2 peptide results suggest partial induction of ovulation in Edn2KO mice. Endothelin receptor antagonism in wild type mice similarly disrupted ovulation, CL formation, and progesterone secretion. Overall, this study suggests that EDN2 is necessary for normal ovulation and CL formation.

Fetal rat phthalate exposure produces a spectrum of male reproductive tract malformations downstream of reduced Leydig cell testosterone production, but the molecular mechanism of phthalate perturbation of Leydig cell function is not well understood. By bioinformatically examining fetal testis expression microarray data sets from susceptible (rat) and resistant (mouse) species after dibutyl phthalate (DBP) exposure, we identified decreased expression of several metabolic pathways in both species. However, lipid metabolism pathways transcriptionally regulated by sterol regulatory element-binding protein (SREBP) were inhibited in the rat but induced in the mouse, and this differential species response corresponded with repression of the steroidogenic pathway. In rats exposed to 100 or 500 mg/kg DBP from gestational days (GD) 16 to 20, a correlation was observed between GD20 testis steroidogenic inhibition and reductions of testis cholesterol synthesis endpoints including testis total cholesterol levels, Srebf2 gene expression, and cholesterol synthesis pathway gene expression. SREBP2 expression was detected in all fetal rat testis cells but was highest in Leydig cells. Quantification of SREBP2 immunostaining showed that 500 mg/kg DBP exposure significantly reduced SREBP2 expression in rat fetal Leydig cells but not in seminiferous cords. By Western analysis, total rat testis SREBP2 levels were not altered by DBP exposure. Together, these data suggest that phthalate-induced inhibition of fetal testis steroidogenesis is closely associated with reduced activity of several lipid metabolism pathways and SREBP2-dependent cholesterologenesis in Leydig cells.

The platelet-derived growth factor (PDGF) signaling pathway regulates numerous lineages of mesenchymal cell origin during development and in the adult. The transcriptional targets of this pathway have been shown to be required in several PDGF-dependent processes, but the roles of these targets in specific tissues is just beginning to be identified. In this study, we show that five different PDGF target genes are essential for male and/or female fertility. Mutations in each of these five different genes lead to defects in the steroid-producing cells in the testis and/or ovary and altered hormone production, suggesting that the PDGF pathway controls steroidogenesis through these genes in both sexes. Furthermore, conditional mutations of both PDGF receptors revealed a requirement in steroid-producing cells in multiple organs, including the testis, ovary, and adrenal cortex. Therefore, PDGF signaling may constitute a common mechanism in the control of multiple steroidogenic lineages.

Hepatocyte growth factor (HGF) regulates many cellular functions acting through c-Met, its specific tyrosine kinase receptor. We previously reported that in prepuberal rats HGF is secreted by the peritubular myoid cells during the entire postnatal testicular development and by the Sertoli cells only at puberty. We have also demonstrated that germ cells at different stages of development express c-Met and that HGF modulates germ cell proliferation and apoptosis. In the present article, we extend our study to the interstitial compartment of the testis and demonstrate that the c-Met protein is present on Leydig cells. The receptor is functionally active as demonstrated by the detected effects of HGF. We report in this article that HGF significantly increases the amount of testosterone secreted by the Leydig cells and decreases the number of Leydig cells undergoing apoptosis. The antiapoptotic effect of HGF is mediated by caspase-3 activity because the amount of the active fragment of the enzyme is decreased in Leydig cells cultured in the presence of HGF. However, treatment with the growth factor does not modify the expression levels of caspase-3 mRNA. These data indicate that HGF regulates the functional activities of Leydig cells. Interestingly, the steroidogenetic activity of the cells is increased by HGF in cultured explants of testicular tissues as well as the antiapoptotic effect of HGF. Therefore, our data indicate that HGF has a crucial role in the regulation of male fertility.

Phthalates are chemical plasticizers used in a variety of consumer products; in rodents, they alter testicular development, leading to decreased testosterone synthesis and maldevelopment of the reproductive tract. Here, our goals were to discover a set of biomarker genes that respond early after relatively low-dose-level dibutyl phthalate (DBP) exposure and map the responding testicular cell types. To identify testicular phthalate biomarker genes, 34 candidate genes were examined by quantitative PCR at 1, 2, 3, or 6 h after exposure of Gestational Day 19 rats to DBP dose levels ranging from 0.1 to 500 mg/kg body weight. Twelve genes (Ctgf, Cxcl10, Dusp6, Edn1, Egr1, Fos, Ier3, Junb, Nr4a1, Stc1, Thbs1, and Tnfrsf12a) were identified with increased expression by 1-3 h at 100 or 500 mg/kg DBP, and 7 of these 12 genes had increased expression by 6 h at 10 mg/kg DBP. Using in situ hybridization of fetal testis cryosections from DBP-exposed rats, the temporal cellular expression of 10 biomarker genes was determined. Genes with a robust response at 1 h (Dusp6, Egr1, Fos, and Thbs1) were induced in peritubular myoid cells. For Egr1 and Fos, the interstitial compartment also showed increased expression at 1 h. Cxcl10 and Nr4a1 were induced by 1-3 h in both sparsely located interstitial cells and peritubular myoid cells. By 3 h, Stc1 was induced in Leydig cells, and Edn1, Ier3, and Tnfrsf12a were increased in Sertoli cells. These data reveal a complex early cascade of phthalate-induced cellular responses in the fetal testis, and for the first time suggest that peritubular myoid cells are an important proximal phthalate target cell.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant and causes adverse effects on female reproduction when administered to rats. Our aims were to study effects of gestational and lactational exposure to TCDD on ovarian steroidogenesis and steroidogenic enzyme expression of offspring on postnatal day (PND) 14 in the rat and sensitivity of enzymatically isolated ovarian follicles to TCDD in vitro. Synthetic estrogen diethylstilbestrol (DES) was used as a treatment control. Serum progesterone (P4) level in offspring increased significantly on PND 14 in the TCDD (1 microg/kg)-exposed group while body weight, FSH and E2 levels were not changed. In ovarian follicles of offspring on PND 14 in the TCDD-exposed groups, protein expression of cytochrome P-450 aromatase, cytochrome P-450 cholesterol side-chain cleavage, steroidogenic acute regulatory protein, 3beta-hydroxy-steroid-dehydrogenase/Delta(5)-Delta(4) isomerase type 1, or P4 receptor was not affected. TCDD decreased E2 and P4 production in ex vivo follicle culture. DES at a dose level of 0.1mg/kg was dystocic while a dose 0.02 mg/kg increased ovarian ex vivo E2 and testosterone production without affecting P450arom activity indicating stimulation of early steps of steroidogenic pathway. Data suggests that TCDD has multiple targets in ovarian steroidogenesis, but the inhibitory action represented as decreased follicular steroid hormone production ex vivo is not apparent at the ovarian protein expression. Furthermore, TCDD had no direct effect on immature rat ovarian steroidogenesis in vitro suggesting that the follicle culture method is not a sensitive method to study the mechanisms of TCDD action.

The wealth of knowledge about the function and regulation of adult Leydig cells, the cells within the mammalian testis that produce testosterone, make these cells ideal for studying principles and mechanisms of aging. A hallmark of mammalian aging is decreased serum testosterone concentration. In the Brown Norway rat, this has been shown to be associated with the reduced ability of aged Leydig cells to produce testosterone in response to LH. Herein, we demonstrate that culturing the aged cells with dibutyryl cAMP, a membrane-permeable cAMP agonist that bypasses the LH receptor-adenlyly cyclase cascade, restores testosterone production to levels comparable to those of young cells and also restores steroidogenic acute regulatory protein and P450scc, the proteins involved in the rate-limiting steps of steroidogenesis. These results strongly suggest that signal transduction deficits are responsible for reduced steroidogenesis by aged Leydig cells and that bypassing signal transduction reverses the steroidogenic decline by the aged cells.

In mammals, the concentration of testosterone in blood serum typically becomes reduced with aging. We have shown that, in rats, this results from reduced testosterone production by individual Leydig cells. To gain an understanding of the mechanisms by which Leydig cell steroidogenic function changes with aging, we compared the gene expression profiles in Leydig cells isolated from young and old Brown Norway rats, focusing on possible changes in genes involved in, or associated with, steroidogenesis. We also explored the effects of caloric restriction, an intervention shown to delay or inhibit age-associated pathologic and biologic changes in a number of systems and organisms, on the expression of these genes. Of the total of 1176 genes arrayed on each of the five membranes per group that we used, about 500 (45%) were detectable in Leydig cells isolated from young and old rat testes, with the same genes detectable in cells from both ages. An ANOVA model was applied to statistically analyze the 500 detectable genes in the four treatment groups. The primary criterion by which interesting genes were selected was a P value of /=0.05. With aging, a large number of genes were seen with fold changes of >1.5. However, when P/=0.05 was used as the selection criterion, only 45 genes were seen to change significantly with age. Most, but not all, of these genes also had fold changes of >1.5. Among the 500 genes, 17 were found to decrease in expression with aging, the most prominent among which were genes involved in steroidogenesis and stress response/free radical scavenging. Long-term caloric restriction had little effect on the 45 genes altered by age. In contrast to the effects of caloric restriction as seen in many other cell types and systems, the vast majority (44/45) of the Leydig cell genes that changed with age were not 'rescued' by this intervention.