Yukiko Ogino

Regulation of outgrowth and apoptosis for the terminal appendage: External genitalia: Development by concerted actions of BMP signaling

Extra-corporal fertilization depends on the formation of copulatory organs: the external genitalia. Coordinated growth and differentiation of the genital tubercle (GT), an embryonic anlage of external genitalia, generates a proximodistally elongated structure suitable for copulation, erection, uresis and ejaculation. Despite recent progress in molecular embryology, few attempts have been made to elucidate the molecular developmental processes of external genitalia formation. Bone morphogenetic protein genes (Bmp genes) and their antagonists were spatiotemporally expressed during GT development. Exogenously applied BMP increased apoptosis of GT and inhibited its outgrowth. It has been shown that the distal urethral epithelium (DUE), distal epithelia marked by the Fgf8 expression, may control the initial GT outgrowth. Exogenously applied BMP4 downregulated the expression of Fgf8 and Wnt5a, concomitant with increased apoptosis and decreased cell proliferation of the GT mesenchyme. Furthermore, noggin mutants and Bmpr1a conditional mutant mice displayed hypoplasia and hyperplasia of the external genitalia respectively. noggin mutant mice exhibited downregulation of Wnt5a and Fgf8 expression with decreased cell proliferation. Consistent with such findings, Wnt5a mutant mice displayed GT agenesis with decreased cell proliferation. By contrast, Bmpr1a mutant mice displayed decreased apoptosis and augmented Fgf8 expression in the DUE associated with GT hyperplasia. These results suggest that some of the Bmp genes could negatively affect proximodistally oriented outgrowth of GT with regulatory functions on cell proliferation and apoptosis. The DUE region can be marked only until 14.0 dpc (days post coitum) in mouse development, while GT outgrowth continues thereafter. Possible signaling crosstalk among the whole distal GT regions were also investigated.

Androgen dependent development of a modified anal fin, gonopodium, as a model to understand the mechanism of secondary sexual character expression in vertebrates

Male external genitalia show structural variations among species. Androgenic hormones are essential for the morphological specification of male type copulatory organs, while little is known about the developmental mechanisms of such secondary sexual characters. Western mosquitofish Gambusia affinis may offer a clue to the sexual differentiation researches, because they show a prominent masculine sexual character for appendage development, anal fin to gonopodium (GP) transition, and its formation could be induced in early juvenile fry by exogenously supplied androgens. We show that GP development is promoted by androgen dependent augmentation of sonic hedgehog (Shh) expression. Two AR cDNAs were cloned and identified as ARα and ARβ from western mosquitofish. Both ARs were predominantly expressed in the distal region of outgrowing anal fin rays. Exposure of fry to androgen caused anal fin outgrowth concomitant with the Shh induction in the distal anal fin ray epithelium. When AR signaling was inhibited by its antagonist flutamide in fry, the initial induction of the Shh was suppressed accompanying retarded anal fin outgrowth. Similar suppression of anal fin outgrowth was induced by treatment with cyclopamine, an inhibitor of Shh signaling. These observations indicate that androgen dependent Shh expression is required for anal fin outgrowth leading to the formation of a genital appendage, the GP in teleost fishes. Androgen‐induced GP formation may provide insights into the expression mechanism regulating the specification of sexual features in vertebrates.

Comparative responsiveness to natural and synthetic estrogens of fish species commonly used in the laboratory and field monitoring

Exposure to estrogenic chemicals discharged into the aquatic environment has been shown to induce feminization in wild freshwater fish and although fish species have been reported to differ in their susceptibility for these effects, empirical studies that directly address this hypothesis are lacking. In this study, in vitro ERα activation assays were applied in a range of fish species used widely in chemical testing (including, zebrafish, fathead minnow, medaka) and/or as environmental monitoring species (including, roach, stickleback, carp) to assess their comparative responsiveness to natural (estrone, estradiol, estriol) and synthetic (17α-ethinylestradiol (EE2), diethylstilbestrol (DES)) estrogens. In vivo exposures to EE2 via the water (nominal 2 and 10 ng/L for 7 days) were also conducted for seven fish species to compare their responsiveness for hepatic vitellogenin (VTG) mRNA induction (an ER mediated response). Of the fish species tested, zebrafish ERα was found to be the most responsive and carp and stickleback ERα the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ERα-mediated response sensitivity order of zebrafish > medaka > roach > fathead minnow > carp > stickleback. For VTG mRNA induction in vivo, the order of species responsiveness was: rainbow trout (not tested in the ERα activation assays) > zebrafish > fathead minnow > medaka > roach > stickleback > carp. Overall, the responses to steroid estrogens in vitro via ERα compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ERα-mediated responses indicative of estrogenic responses (VTG induction) in vivo.

A mutation in the receptor Methoprene-tolerant alters juvenile hormone response in insects and crustaceans

Juvenile hormone is an essential regulator of major developmental and life history events in arthropods. Most of the insects use juvenile hormone III as the innate juvenile hormone ligand. By contrast, crustaceans use methyl farnesoate. Despite this difference that is tied to their deep evolutionary divergence, the process of this ligand transition is unknown. Here we show that a single amino-acid substitution in the receptor Methoprene-tolerant has an important role during evolution of the arthropod juvenile hormone pathway. Microcrustacea Daphnia pulex and D. magna share a juvenile hormone signal transduction pathway with insects, involving Methoprene-tolerant and steroid receptor coactivator proteins that form a heterodimer in response to various juvenoids. Juvenile hormone-binding pockets of the orthologous genes differ by only two amino acids, yet a single substitution within Daphnia Met enhances the receptors responsiveness to juvenile hormone III. These results indicate that this mutation within an ancestral insect lineage contributed to the evolution of a juvenile hormone III receptor system.

Evolutionary History and Functional Characterization of Androgen Receptor Genes in Jawed Vertebrates

Vertebrates show diverse sexual characters in sexually attractive and reproductive organs, which are regulated by steroid hormones, particularly androgens. However, the evolutionary history of androgen receptor (AR) gene remains largely unknown on the basis of phylogenic and functional analyses. To elucidate the evolutionary history and functional diversification of AR genes in vertebrates, we cloned the AR cDNAs from a shark, basal ray-finned fishes (Actinopterygii), namely bichir and sturgeon (Acipenseriformes), and teleosts including a basal teleost, arowana (Osteoglossiformes). Molecular phylogenetic analysis revealed that the gene duplication event that gave rise to two different teleost ARs (alpha and beta) likely occurred in the actinopterygian lineage leading to teleosts after the divergence of Acipenseriformes but before the split of Osteoglossiformes, which is compatible with the phylogenetic timing of teleost-specific genome duplication. Searching for AR genes in the medaka genome indicated that the teleost AR gene duplication has been associated with the duplication between chromosomes 10 and 14. Our functional analysis revealed that the shark AR activates the target gene via androgen response element by classical androgens. The teleost ARalpha showed the unique intracellular localization with a significantly higher transactivating capacity than that by teleost ARbeta. These findings indicate that the most ancient type of AR, as activated by the classical androgens as ligands, emerged before the Chondrichthyes-Osteichthyes split, and the AR gene was duplicated during the teleost-specific genome duplication event. We report here for the first time the accurate evolutionary history of AR gene and functional characterization of AR duplicates in teleost lineage.

Embryonic development of mouse external genitalia: insights into a unique mode of organogenesis

SUMMARY The mammalian external genitalia are specialized appendages for efficient copulation, internal fertilization and display marked morphological variation among species. In this paper, we described the embryonic development of mouse genital tubercle (GT), an anlage of the external genitalia utilizing the scanning electron microscope (SEM) analysis. It has been shown that the Distal Urethral Epithelium (DUE) may fulfill an essential role in the outgrowth control of the GT. Our present SEM analysis revealed a small distal protrusion at the tip of the GT of normal embryos as well as some morphological differences between male and female embryonic external genitalia. Previous analysis shows that the teratogenic dose of Retinoic Acid (RA) induces a drastic marformation of the urethral plate, but not gross abnormalities for GT outgrowth. Interestingly, a small distal protrusion at the tip of GT was clearly observed also after RA treatement. Furthermore, we showed that treatment with anti‐androgen flutamide resulted in the demasculinization of the GT in males. The unique character of GT development and the sexual dimorphism are discussed.

Differing species responsiveness of estrogenic contaminants in fish is conferred by the ligand binding domain of the estrogen receptor.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) induces a range of adverse effects, notably on reproduction and reproductive development. These responses are mediated via estrogen receptors (ERs). Different species of fish may show differences in their responsiveness to environmental estrogens but there is very limited understanding on the underlying mechanisms accounting for these differences. We used custom developed in vitro ERα reporter gene assays for nine fish species to analyze the ligand- and species-specificity for 12 environmental estrogens. Transcriptonal activities mediated by estradiol-17β (E2) were similar to only a 3-fold difference in ERα sensitivity between species. Diethylstilbestrol was the most potent estrogen (∼ 10-fold that of E2) in transactivating the fish ERαs, whereas equilin was about 1 order of magnitude less potent in all species compared to E2. Responses of the different fish ERαs to weaker environmental estrogens varied, and for some considerably. Medaka, stickleback, bluegill and guppy showed higher sensitivities to nonylphenol, octylphenol, bisphenol A and the DDT-metabolites compared with cyprinid ERαs. Triclosan had little or no transactivation of the fish ERαs. By constructing ERα chimeras in which the AF-containing domains were swapped between various fish species with contrasting responsiveness and subsequent exposure to different environmental estrogens. Our in vitro data indicate that the LBD plays a significant role in accounting for ligand sensitivity of ERα in different species. The differences seen in responsiveness to different estrogenic chemicals between species indicate environmental risk assessment for estrogens cannot necessarily be predicted for all fish by simply examining receptor activation for a few model fish species.

External genitalia formation: role of fibroblast growth factor, retinoic acid signaling, and distal urethral epithelium.

Abstract: The process of fetal external genitalia development might be divided into two processes. The first process accomplishes the initial outgrowth of the anlage, genital tubercle (GT). Previous analysis suggests that the distal urethral epithelium (DUE) of the GT, the Fgf8‐expressing region, regulates the outgrowth of the GT. The second process eventually generates the sexually dimorphic development of the external genitalia, which is dependent on the action of steroid hormones. Several key genes, for example, RARs, RXRs, RALDH2, and CYP26, were dynamically expressed during GT development. The teratogenic dose of RA at 9.0 d.p.c. induced a drastic malformation of the urethral plate during GT formation, but did not show gross abnormalities in its outgrowth. In RA‐treated embryos, Fgf8 expression was still detected in the distal GT regions. Possible regulatory roles of the FGF and RA signaling systems in external genitalia formation are discussed.

Essential Roles of Androgen Signaling in Wolffian Duct Stabilization and Epididymal Cell Differentiation

The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2α-Cre;AR(flox/Y) mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation.

Positive regulation of steroidogenic acute regulatory protein gene expression through the interaction between Dlx and GATA-4 for testicular steroidogenesis

Split hand/foot malformation (SHFM) is syndromic ectrodactyly often associated with mental retardation and/or craniofacial defects. Several clinical reports previously described urogenital dysplasia such as micropenis, hypospadias, and small testis in SHFM patients. Genetic lesions in the Dlx5 and Dlx6 (Dlx5/6) locus are associated with the human genetic disorder SHFM type 1. Although Dlx5/6 are expressed in the testis, their possible function of Dlx5/6 during testis differentiation has not been described. In this study, we show that Dlx5/6 are expressed in the fetal Leydig cells during testis development. We examined the effect of Dlx5 expression on the promoter activation of the steroidogenic acute regulatory protein (StAR) gene, which is essential for gonadal and adrenal steroidogenesis, in a Leydig cell line. Dlx5 efficiently activates the StAR promoter when GATA-4, another transcription factor essential for testicular steroidogenesis, was coexpressed. The transcriptional activation required the GATA-4-recognition element in the StAR promoter region and Dlx5 can physically interact with GATA-4. Furthermore, we herein show that the double inactivation of Dlx5 and Dlx6 in the mouse leads to decreased testosterone level and abnormal masculinization phenotype. These results suggest that Dlx5 and Dlx6 participate in the control of steroidogenesis during testis development. The findings of this study may open the way to analyze human congenital birth defects.