Endothelial and steroidogenic cell migration are regulated by WNT4 in the developing mammalian gonad

The signalling molecule WNT4 has been associated with sex reversal phenotypes in mammals. Here we show that the role of WNT4 in gonad development is to pattern the sex-specific vasculature and to regulate steroidogenic cell recruitment. Vascular formation and steroid production in the mammalian gonad occur in a sex-specific manner. During testis development, endothelial cells migrate from the mesonephros into the gonad to form a coelomic blood vessel. Leydig cells differentiate and produce steroid hormones a day later. Neither of these events occurs in the XX gonad. We show that WNT4 represses mesonephric endothelial and steroidogenic cell migration in the XX gonad, preventing the formation of a male-specific coelomic blood vessel and the production of steroids. In the XY gonad, Wnt4 expression is downregulated after sex determination. Transgenic misexpression of Wnt4 in the embryonic testis did not inhibit coelomic vessel formation but vascular pattern was affected. Leydig cell differentiation was not affected in these transgenic animals and our data implies that Wnt4 does not regulate steroidogenic cell differentiation but represses the migration of steroidogenic adrenal precursors into the gonad. These studies provide a model for understanding how the same signalling molecule can act on two different cell types to coordinate sex development.     

Sexually dimorphic gene expression in the developing mouse gonad

Over the course of a few days, the bipotential embryonic mouse gonad differentiates into either a testis or an ovary. Though a few gene expression differences that underlie gonadal sex differentiation have been identified, additional components of the testicular and ovarian developmental pathways must be identified to understand this process. Here we report the use of a PCR-based cDNA subtraction to investigate expression differences that arise during gonadal sex differentiation. Subtraction of embryonic day 12.5 (E12.5) XY gonadal cDNA with E12.5 XX gonadal cDNA yielded 19 genes that are expressed at significantly higher levels in XY gonads. These genes display a variety of expression patterns within the embryonic testis and encode a broad range of proteins. A reciprocal subtraction (of E12.5 XX gonadal cDNA with E12.5 XY gonadal cDNA) yielded two genes, follistatin and Adamts19, that are expressed at higher levels in XX gonads. Follistatin is a well-known antagonist of TGFbeta family members while Adamts19 encodes a new member of the ADAMTS family of secreted metalloproteases.     

Constitutive connective tissue growth factor expression in scleroderma fibroblasts is dependent on Sp1

Fibrotic diseases such as scleroderma (systemic sclerosis, SSc) are characterized by an excessive production of extracellular matrix and profibrotic proteins such as connective tissue growth factor (CTGF). In normal dermal fibroblasts, CTGF is not expressed unless induced by proteins such as tumor growth factor-beta (TGFbeta). Conversely, in fibroblasts cultured from fibrotic lesions CTGF mRNA and protein are constitutively expressed, even in the absence of exogenously added TGFbeta. Thus, studying the mechanism underlying CTGF overexpression in SSc fibroblasts is likely to yield valuable insights into the basis of the fibrotic phenotype of SSc and possibly other scarring disease. CTGF overexpression is mediated primarily by sequences in the CTGF promoter. In this report, we identify the minimal promoter element involved with the overexpression of CTGF in SSc fibroblasts. This element is distinct from the element necessary and sufficient for the induction of CTGF expression by TGFbeta in normal fibroblasts. Within this region is a functional Sp1 binding site. Blocking Sp1 activity reduces the elevated, constitutive levels of CTGF promoter activity and protein expression observed in SSc fibroblasts. Relative to those prepared from normal dermal fibroblasts, nuclear extracts prepared from SSc fibroblasts possess increased Sp1 binding activity. Removal of phosphate groups from nuclear extracts enhanced Sp1 binding activity, suggesting that phosphorylation of Sp1 normally reduces Sp1 binding to DNA. Thus, the constitutive overexpression of CTGF in SSc fibroblasts seems to be independent of TGFbeta signaling but dependent at least in part on Sp1.     

Evidence for activation of Amh gene expression by steroidogenic factor 1

The anti-Müllerian hormone gene (Amh) is responsible for regression in males of the Müllerian ducts. The molecular mechanism of regulation of chicken Amh expression is poorly understood. To investigate the regulation of chicken Amh expression, we have cloned Amh cDNAs from quail and duck as well as the promoter regions of the gene from chicken, quail, and duck. The expression patterns of Amh during embryonic development in these three species were found to be similar, suggesting that the regulatory mechanisms of Amh expression are conserved. The sequence of the proximal promoter of Amh contains a putative binding site for steroidogenic factor 1 (SF1), the protein product of which can up-regulate Amh in mammals. We showed here that SF1 is able to activate the chicken Amh promoter and binds to its putative SF1 binding site. These results suggest that SF1 plays a role in regulation of Amh expression in avian species.     

A FTZ-F1-containing yeast artificial chromosome recapitulates expression of steroidogenic factor 1 in vivo

Steroidogenic factor 1 (SF-1/Nr5a1) is an orphan nuclear receptor encoded by the Ftz-F1 gene and is required for gonad and adrenal development and regulation of hormone production within the reproductive and adrenal axes. To extend our understanding of Ftz-F1 and its role in SF-1 expression, we identified and characterized a yeast artificial chromosome (YAC) containing Ftz-F1. Within this YAC, Ftz-F1 is centrally located and flanked by genes encoding a second orphan nuclear receptor, germ cell nuclear factor, and proteasome (prosome, macropain) subunit beta type 7. Three lines of transgenic mice carrying the YAC were generated and in two lines (lines 7 and 14), RT-PCR and ribonuclease protection analysis showed that expression of transgenic SF-1 mimicked that of endogenous SF-1, both spatially and quantitatively. In the third line (line 15), pituitary and hypothalamic expression were absent. Comparison of the integrated transgenes revealed that line 15 was truncated at the end of intron 4 and revealed a region within the locus that is responsible for SF-1 expression in the pituitary and hypothalamus. The line 14 transgene was introduced into a mouse strain lacking functional SF-1. Examination of SF-1-deficient, transgene-positive mice revealed that the YAC was able to rescue adrenal and gonad development, which normally arrests in the SF-1-null embryos and showed that the 153-kb transgene integrated in line 14 is sufficient to properly direct SF-1 expression and support its biological activity. Thus, the study defines a region of Ftz-F1 that contains the requisite set of regulatory elements to direct SF-1 cell-specific expression and all temporal and quantitative changes need for its biological activity.     

Dax1 suppresses P450arom expression in medaka ovarian follicles

Dax1 is a member of an unusual orphan nuclear receptor family, and is known to regulate P450arom in mammals and is involved in sex differentiation in some vertebrates. To investigate whether Dax1 is involved in the regulation of the steroidogenic pathway for estrogen biosynthesis in medaka ovarian follicles, we isolated Dax1 cDNA from adult medaka ovaries and analyzed its expression pattern in medaka gonads. In adult ovaries, Dax1 mRNA was detected only in postvitellogenic follicles and was not detected in previtellogenic and vitellogenic follicles. In adult testis, Dax1 mRNA was not detected. We compared the expression pattern of Dax1 with that of Foxl2, Ad4BP/Sf-1, P450c17, and P450arom by in situ hybridization using adjacent sections. In contrast to Dax1 expression, these genes were co-expressed in vitellogenic follicles but were not detected in postvitellogenic follicles. Thus, in medaka ovarian follicles, Dax1 did not show any overlapping expression patterns against Foxl2, Ad4BP/Sf-1, P450c17, and P450arom. Moreover, co-transfection experiments demonstrated that Dax1 inhibits Ad4BP/Sf-1- and Foxl2-mediated P450arom expression. On the other hand, during early sex differentiation, Dax1 mRNA was not detected in both males and females. Our results suggest that Dax1 down-regulates Ad4BP/Sf-1- and Foxl2-mediated P450arom expression in medaka ovarian follicles.     

Leydig cell-specific expression of DAX1 improves fertility of the Dax1-deficient mouse

Dax1 is an orphan nuclear receptor expressed in both Leydig and Sertoli cells of the testis. Mutation of DAX1 in humans causes adrenal failure and hypogonadotropic hypogonadism. Targeted mutagenesis of Dax1 in mice reveals a primary gonadal defect characterized by overexpression of aromatase and cellular obstruction of the seminiferous tubules and efferent ductules, leading to germ cell death and infertility. Transgenic expression of DAX1 under the control of the müllerian-inhibiting substance promoter, which is selectively expressed in Sertoli cells, improves fertility but does not fully correct the histological abnormalities in the testes of Dax1 knockout (Dax1KO) mice. We therefore hypothesized that Dax1 may also play a crucial role in other somatic cells of the testis, namely the Leydig cells. A 2.1-kilobase fragment of the murine LH receptor 5'-promoter (LHR-DAX1) was used to generate transgenic mice that selectively express DAX1 in Leydig cells. Expression of the LHR-DAX1 transgene caused no observable phenotype in wild-type mice but improved fertility when expressed in Dax1KO males (rescue [RS]). Although testicular size was not increased in LHR-DAX1 RS animals, aromatase expression was restored to normal levels, and sperm production was increased. Testicular pathology was only slightly improved in RS mice compared to Dax1KO animals. Taken together with the result of previous studies of DAX1 expression in Sertoli cells, we conclude that the testis phenotype of Dax1KO mice reflects the combined effects of Dax1 deficiency in both Sertoli and Leydig cells.     

Two regions within the proximal steroidogenic factor 1 promoter drive somatic cell-specific activity in developing gonads of the female mouse

Targets of steroidogenic factor 1 (SF1; also known as NR5A1 and AD4BP) have been identified within cells at every level of the hypothalamic-pituitary-gonadal and -adrenal axes, revealing SF1 to be a master regulator of major endocrine systems. Mouse embryos express SF1 in the genital ridge until Embryonic Day 13.5 (E13.5). Thereafter, expression persists in the male and is substantially lower in the female gonad until birth. We hypothesize that the sexually dimorphic expression of Sf1 during gonadogenesis is mediated by sex-specific regulation of its promoter. To investigate dimorphic regulation within the fetal gonad, we developed an experimental strategy using transient transfection of E13.5 gonad explant cultures and evaluated various Sf1 promoter constructs for sexually dimorphic DNA elements. The proximal Sf1 promoter correctly targeted reporter activity to SF1-expressing cells in both XY and XX gonads. Stepwise deletion of sequences from the Sf1 promoter revealed two regions that affected regulation within female gonads. Mutation of both sequences together did not cause further disruption of reporter activity, suggesting the two sites might work in concert to promote activity in female somatic cells. Results from gel mobility shift assays and fetal gonad-chromatin immunoprecipitation showed that TCFAP2 binds to one of the two female-specific sites within the proximal promoter of Sf1. Together, we show that transient transfection experiments performed within developing testes and ovaries are a powerful tool to uncover elements within the Sf1 promoter that contribute to sex-specific expression.     

Kallmann syndrome 1 gene is expressed in the marsupial gonad

Kallmann syndrome is characterized by hypogonadotrophic hypogonadism and anosmia. The syndrome can be caused by mutations in several genes, but the X-linked form is caused by mutation in the Kallmann syndrome 1 (KAL1). KAL1 plays a critical role in gonadotropin-releasing hormone (GnRH) neuronal migration that is essential for the normal development of the hypothalamic-pituitary-gonadal axis. Interestingly, KAL1 appears to be missing from the rodent X, and no orthologue has been detected as yet. We investigated KAL1 during development and in adults of an Australian marsupial, the tammar wallaby, Macropus eugenii. Marsupial KAL1 maps to an autosome within a group of genes that was added as a block to the X chromosome in eutherian evolution. KAL1 expression was widespread in embryonic and adult tissues. In the adult testis, tammar KAL1 mRNA and protein were detected in the germ cells at specific stages of differentiation. In the adult testis, the protein encoded by KAL1, anosmin-1, was restricted to the round spermatids and elongated spermatids. In the adult ovary, anosmin-1 was not only detected in the oocytes but was also localized in the granulosa cells throughout folliculogenesis. This is the first examination of KAL1 mRNA and protein localization in adult mammalian gonads. The protein localization suggests that KAL1 participates in gametogenesis not only through the development of the hypothalamic-pituitary-gonadal axis by activation of GnRH neuronal migration, but also directly within the gonads themselves. Because KAL1 is autosomal in marsupials but is X-linked in eutherians, its conserved involvement in gametogenesis supports the hypothesis that reproduction-related genes were actively recruited to the eutherian X chromosome.     

Expression of vascular endothelial growth factor receptor 1 in bone marrow-derived mesenchymal cells is dependent on hypoxia-inducible factor 1

Bone marrow-derived cells are recruited to sites of ischemia, where they promote tissue vascularization. This response is dependent upon the expression of vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1), which mediates cell migration in response to VEGF or placental growth factor (PLGF). In this study, we found that exposure of cultured mouse bone marrow-derived mesenchymal stromal cells (MSC) to hypoxia or an adenovirus encoding a constitutively active form of hypoxia-inducible factor 1 (HIF-1) induced VEGFR1 mRNA and protein expression and promoted ex vivo migration in response to VEGF or PLGF. MSC in which HIF-1 activity was inhibited by a dominant negative or RNA interference approach expressed markedly reduced levels of VEGFR1 and failed to migrate or activate AKT in response to VEGF or PLGF. Thus, loss-of-function and gain-of-function approaches demonstrated that HIF-1 activity is necessary and sufficient for basal and hypoxia-induced VEGFR1 expression in bone marrow-derived MSC.     

Male-specific cell migration into the developing gonad

Background: The gene Sry acts as a developmental switch, initiating a pathway of gene activity that leads to the differentiation of testis rather than ovary from the indifferent gonad (genital ridge) in mammalian embryos. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. To investigate the contribution of mesonephric cells to this process, gonads from wild-type mice (CD1), and mesonephroi from a transgenic strain ubiquitously expressing β-galactosidase (ROSA26), were grafted together in vitro. After culture, organs were fixed and stained for β-galactosidase activity to identify cells contributed from the mesonephros to the male or female gonad.Results: Migration of mesonephric cells occurred into XY but not XX gonads from 11.5–16.5 days post coitum (dpc). Somatic cells contributed from the mesonephros were distinguished by their histological location and by available cell-specific markers. Some of the migrating cells were endothelial; a second population occupied positions circumscribing areas of condensing Sertoli cells; and a third population lay in close apposition to endothelial cells.Conclusions: Migration from the mesonephros to the gonad is male specific at this stage of development and depends on an active signal that requires the presence of a Y chromosome in the gonad. The signals that trigger migration operate over considerable distances and behave as chemoattractants. We suggest that migration of cells into the bipotential gonad may have a critical role in initiating the divergence of development towards the testis pathway.     

Skeletal muscle HIF-1alpha expression is dependent on muscle fiber type

Oxygen homeostasis is an essential regulation system for cell energy production and survival. The oxygen-sensitive subunit alpha of the hypoxia inducible factor-1 (HIF-1) complex is a key protein of this system. In this work, we analyzed mouse and rat HIF-1alpha protein and mRNA expression in parallel to energetic metabolism variations within skeletal muscle. Two physiological situations were studied using HIF-1alpha-specific Western blotting and semiquantitative RT-PCR. First, we compared HIF-1alpha expression between the predominantly oxidative soleus muscle and three predominantly glycolytic muscles. Second, HIF-1alpha expression was assessed in an energy metabolism switch model that was based on muscle disuse. These two in vivo situations were compared with the in vitro HIF-1alpha induction by CoCl(2) treatment on C(2)C(12) mouse muscle cells. HIF-1alpha mRNA and protein levels were found to be constitutively higher in the more glycolytic muscles compared with the more oxidative muscles. Our results gave rise to the hypothesis that the oxygen homeostasis regulation system depends on the fiber type.     

Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression,which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells

Stimulation of human colon cancer cells with insulin-like growth factor 1 (IGF-1) induces expression of the VEGF gene, encoding vascular endothelial growth factor. In this article we demonstrate that exposure of HCT116 human colon carcinoma cells to IGF-1 induces the expression of HIF-1 alpha, the regulated subunit of hypoxia-inducible factor 1, a known transactivator of the VEGF gene. In contrast to hypoxia, which induces HIF-1 alpha expression by inhibiting its ubiquitination and degradation, IGF-1 did not inhibit these processes, indicating an effect on HIF-1 alpha protein synthesis. IGF-1 stimulation of HIF-1 alpha protein and VEGF mRNA expression was inhibited by treating cells with inhibitors of phosphatidylinositol 3-kinase and MAP kinase signaling pathways. These inhibitors also blocked the IGF-1-induced phosphorylation of the translational regulatory proteins 4E-BP1, p70 S6 kinase, and eIF-4E, thus providing a mechanism for the modulation of HIF-1 alpha protein synthesis. Forced expression of a constitutively active form of the MAP kinase kinase, MEK2, was sufficient to induce HIF-1 alpha protein and VEGF mRNA expression. Involvement of the MAP kinase pathway represents a novel mechanism for the induction of HIF-1 alpha protein expression in human cancer cells.