Sex specific retinoic acid signaling is required for the initiation of urogenital sinus bud development

The mammalian urogenital sinus (UGS) develops in a sex specific manner, giving rise to the prostate in the male and the sinus vagina in the embryonic female. Androgens, produced by the embryonic testis, have been shown to be crucial to this process. In this study we show that retinoic acid signaling is required for the initial stages of bud development from the male UGS. Enzymes involved in retinoic acid synthesis are expressed in the UGS mesenchyme in a sex specific manner and addition of ligand to female tissue is able to induce prostate-like bud formation in the absence of androgens, albeit at reduced potency. Functional studies in mouse organ cultures that faithfully reproduce the initiation of prostate development indicate that one of the roles of retinoic acid signaling in the male is to inhibit the expression of Inhba, which encodes the βA subunit of Activin, in the UGS mesenchyme. Through in vivo genetic analysis and culture studies we show that inhibition of Activin signaling in the female UGS leads to a similar phenotype to that of retinoic acid treatment, namely bud formation in the absence of androgens. Our data also reveals that both androgens and retinoic acid have extra independent roles to that of repressing Activin signaling in the development of the prostate during fetal stages. This study identifies a novel role for retinoic acid as a mesenchymal factor that acts together with androgens to determine the position and initiation of bud development in the male UGS epithelia.     

Characterization of fetal urogenital sinus-induced prostatic hyperplasia in the mouse: time course, hormonal requirement, age dependency, and responsiveness of various adult organs to growth induction by fetal urogenital sinus tissues

Mouse prostatic hyperplasia can be induced experimentally by the direct implantation of fetal urogenital sinus (UGS) or its mesenchyme (UGM) tissue in situ. This study characterized the time course, the requirement of sex steroids, and the optimal ages of donor and host tissues necessary to induce the maximal overgrowth of the adult mouse prostate gland in this model system. To test the potential uses of these fetal inductors as general growth-promoting substances for other adult organs, we have also tested directly the activity of fetal UGS in several non-UGS-derived adult organs. These results were compared with the growth-promoting effect achieved by fetal UGM in order to gain further insight into the relative contribution of UGS/UGM in the overall growth responses. Peak DNA synthesis in the implanted prostate occurred at three time periods-Days 4, 7-16, and 35. At Day 4, DNA synthesis may have reflected tissue repair following surgical trauma, but the DNA synthesis on Days 7-16 and 35 is attributable to growth of the chimeric (enlarged) prostate gland. Initiation and maintenance of hyperplasia required testicular androgens. Exogenous testosterone propionate (175 micrograms/day) did not induce additional prostatic overgrowth in intact, sexually mature hosts, but promoted additional overgrowth in immature and pubertal hosts. Exogenous estrogen (17 beta-estradiol dipropionate, 20 micrograms/day) inhibited fetal UGS-induced prostatic overgrowth by inhibiting the hypothalamic-pituitary-testicular axis. UGS derived from fetuses of Days 14, 16, or 18 of gestation had similar growth-inductive capability in intact adult hosts, but this capability was restricted soon after birth.(ABSTRACT TRUNCATED AT 250 WORDS)     

WNT5A selectively inhibits mouse ventral prostate development

The establishment of prostatic budding patterns occurs early in prostate development but mechanisms responsible for this event are poorly understood. We investigated the role of WNT5A in patterning prostatic buds as they emerge from the fetal mouse urogenital sinus (UGS). Wnt5a mRNA was expressed in UGS mesenchyme during budding and was focally up-regulated as buds emerged from the anterior, dorsolateral, and ventral UGS regions. We observed abnormal UGS morphology and prostatic bud patterns in Wnt5a null male fetuses, demonstrated that prostatic bud number was decreased by recombinant mouse WNT5A protein during wild type UGS morphogenesis in vitro, and showed that ventral prostate development was selectively impaired when these WNT5A-treated UGSs were grafted under under kidney capsules of immunodeficient mice and grown for 28 d. Moreover, a WNT5A inhibitory antibody, added to UGS organ culture media, rescued prostatic budding from inhibition by a ventral prostatic bud inhibitor, 2,3,8,7-tetrachlorodibenzo-p-dioxin, and restored ventral prostate morphogenesis when these tissues were grafted under immunodeficient mouse kidney capsules and grown for 28 d. These results suggest that WNT5A participates in prostatic bud patterning by restricting mouse ventral prostate development.     

Mouse urogenital development: a practical approach

A detailed knowledge of the developmental anatomy of the embryonic mouse urogenital tract is required to recognize mutant urogenital phenotypes in transgenic and knock-out mice. Accordingly, the purpose of this article is to review urogenital development in the mouse embryo and to give an illustrated methodological protocol for the dissection of urogenital organ rudiments at 12-13 days of gestation (E12-13) to isolate the urogenital ridge and at E16 to isolate the seminal vesicle, Müllerian duct, Wolffian duct, and prostatic rudiment, the urogenital sinus (UGS). The UGS can be cultured and, in the presence of testosterone, prostatic buds form in vitro. Because of the importance of mesenchymal-epithelial interactions in urogenital development, methods for the isolation of epithelium and mesenchyme from the embryonic urogenital sinus are also described. Urogenital sinus mesenchyme (UGM) and urogenital sinus epithelium (UGE) can be used to construct tissue recombinants that can either be grown in vitro or grafted in vivo for the study of epithelial-mesenchymal interactions in prostatic development.     

Catalog of mRNA expression patterns for DNA methylating and demethylating genes in developing mouse lower urinary tract

The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltrasferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease.     

Catalog of mRNA expression patterns for DNA methylating and demethylating genes in developing mouse lower urinary tract

The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltrasferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease.     

The role of smooth muscle in regulating prostatic induction

We have examined the role that smooth muscle plays during prostatic organogenesis and propose that differentiation of a smooth muscle layer regulates prostatic induction by controlling mesenchymal/epithelial interactions. During development of the rat reproductive tract, an area of condensed mesenchyme involved in prostatic organogenesis is formed. This mesenchyme (the ventral mesenchymal pad, VMP) is found in both males and females, yet only males develop a prostate. We demonstrate that a layer of smooth muscle differentiates between the VMP and the urethral epithelium, and that there is a sexually dimorphic difference in the development of this layer. Serial section reconstruction showed that the layer formed at approximately embryonic day 20.5 in females, but did not form in males. In cultures of female reproductive tracts, testosterone was able to regulate the thickness of this layer resulting in a 2.4-fold reduction in thickness. We observed that prostatic buds were present in some female reproductive tracts, and determined that testosterone was able to stimulate prostatic organogenesis, depending upon the bud position relative to the smooth muscle layer. In vitro recombination experiments demonstrated that direct contact with the VMP led to the induction of very few epithelial buds, and that androgens dramatically increased bud development. Taken together, our data suggest that differentiation of a smooth muscle layer regulates signalling between mesenchyme and epithelium, and comprises part of the mechanism regulating prostatic induction.     

Estrogenic environmental chemicals and drugs: mechanisms for effects on the developing male urogenital system

Development and differentiation of the prostate from the fetal urogenital sinus (UGS) is dependent on androgen action via androgen receptors (AR) in the UGS mesenchyme. Estrogens are not required for prostate differentiation but do act to modulate androgen action. In mice exposure to exogenous estrogen during development results in permanent effects on adult prostate size and function, which is mediated through mesenchymal estrogen receptor (ER) alpha. For many years estrogens were thought to inhibit prostate growth because estrogenic drugs studied were administered at very high concentrations that interfered with normal prostate development. There is now extensive evidence that exposure to estrogen at very low concentrations during the early stages of prostate differentiation can stimulate fetal/neonatal prostate growth and lead to prostate disease in adulthood. Bisphenol A (BPA) is an environmental endocrine disrupting chemical that binds to both ER receptor subtypes as well as to AR. Interest in BPA has increased because of its prevalence in the environment and its detection in over 90% of people in the USA. In tissue culture of fetal mouse UGS mesenchymal cells, BPA and estradiol stimulated changes in the expression of several genes. We discuss here the potential involvement of estrogen in regulating signaling pathways affecting cellular functions relevant to steroid hormone signaling and metabolism and to inter- and intra-cellular communications that promote cell growth. The findings presented here provide additional evidence that BPA and the estrogenic drug ethinylestradiol disrupt prostate development in male mice at administered doses relevant to human exposures.     

Proprotein convertases modulate budding and branching morphogenesis of rat ventral prostate

The onset of prostate morphogenesis is involved in the interaction between mesenchyme and epithelium. Proprotein convertases (PCs) activate a variety of growth and differentiation factors including mesenchymal and epithelial factors, such as insulin-like growth factor (IGF) and transforming growth factor-beta (TGF-beta), which induce ductal budding and branching. In this study, we provide evidence that PCs play a critical role in prostatic budding from the urogenital sinus (UGS) and ductal branching morphogenesis of the neonatal rat ventral prostate. PCs were expressed only in the epithelial cells of neonatal rat prostate. PC activity in the ventral prostate was modulated by endogenous androgen. PC inhibition suppressed prostatic budding and branching. Taken together, our data indicates that androgen-induced PCs initiate the development of the prostate.     

Sonic hedgehog regulates prostatic growth and epithelial differentiation

The Sonic hedgehog (SHH)-signalling pathway mediates epithelial-mesenchymal interactions in several tissues during development and disease, and we have investigated its role in rat ventral prostate (VP) development. We have demonstrated that Shh and Ptc expression correlates with growth and development of the prostate and that their expression is not regulated by androgens in the VP. Prostatic budding was induced in response to testosterone in Shh null mouse urogenital sinus (UGS) explants grown in vitro and in rat UGS explants cultured with cyclopamine, suggesting that SHH-signalling is not critical for prostatic induction. SHH-signalling was disrupted at later stages of VP development (in vitro), resulting in a reduction in organ size, an increase in ductal tip number, and reduced proliferation of ductal tip epithelia. The addition of recombinant SHH to VPs grown in vitro caused a decrease in ductal tip number and expansion of the mesenchyme. In the presence of testosterone, inhibition of SHH-signalling accelerated the canalisation of prostatic epithelial ducts and resulted in ducts that showed morphological similarities to cribiform prostatic intraepithelial neoplasia (PIN). The epithelia of these ducts also demonstrated precocious and aberrant differentiation, when examined by immunohistochemistry for p63 and cytokeratin 14. In conclusion, we show that SHH-signalling is not essential for prostatic induction, but is important for prostatic growth, branching, and proliferation, and that androgen-stimulated growth in the absence of signalling from the SHH pathway results in aberrant epithelial differentiation.     

Genistein represses the induction of prostatic buds by testosterone]

Genistein, a phytoestrogen and a kind of endocrine disrupters, inhibits tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor. It is also effective both in the suppression of the prostatic cell proliferation and the prostate carcinogenesis. We have recently demonstrated that several growth factors, like EGF, transforming growth factor-alpha (TGF-alpha), or keratinocyte growth factor (KGF), can induce prostatic bud formation in the absence of androgen. The present study was performed to investigate whether genistein can suppress testosterone-induced prostatic bud formation. Urogenital sinuses of 16.5-day male rat fetuses were cultured organotypically for 5 days in a serum-free medium containing 10 or 100 ng/ml genistein and 50 ng/ml testosterone. The number and total volume of prostatic buds were analyzed by laser scanning microscopy and computerized. We found that genistein inhibits significantly testosterone-induced prostatic bud formation. In the presence of genistein, cell proliferation of the sinus epithelium was suppressed and the number of prostatic buds and total volume of the buds were reduced as compared with those in the sinuses cultured with testosterone alone. Genistein did not appear to cause necrosis of the sinus. These results support our hypothesis that growth factors like EGF secreted from the sinus mesenchyme activated by testosterone are involved in the induction and stimulation of growth of the prostatic buds.     

Tissue interactions and prostatic growth. I. Induction of adult mouse prostatic hyperplasia by fetal urogenital sinus implants

A homologous chimeric prostate was produced by implantation of intact fetal urogenital sinus(es) (UGS) into the ventral prostate gland (VP) of an adult athymic mouse. A 10- to 20-fold overgrowth of the chimeric lobe of ventral prostate gland, as measured by glandular wet weight and by DNA content, was observed 4 to 9 wk following UGS implantation. The overgrowth was prostate-like as indicated by histologic composition and by responses to endogenous androgen, and was composed of both host and donor cells in about equal proportions as shown by glucose phosphate isomerase isozymic profiles. Unlike the canine model for prostatic hyperplasia, the mouse prostatic overgrowth occurred in the complete absence of exogenous sex steroids. The histoarchitecture of the chimeric VP and the isozymic detection of the contribution to the overgrowth by host cells have provided strong evidence that adult prostatic cells have been recruited to respond proliferatively by cellular interactions with fetal UGS. The demonstration of cellular interactions followed by reactivation of the fetal growth potential provides direct experimental evidence in support of McNeal's hypothesis that the reactivation of fetal growth potential may account for the development of human benign prostatic hyperplasia (BPH).     

Role of p63 and basal cells in the prostate

The prostate contains two major epithelial cell types - luminal and basal cells - both of which develop from urogenital sinus epithelium. The cell linage relationship between these two epithelial types is not clear. Here we demonstrate that luminal cells can develop independently of basal cells, but that basal cells are essential for maintaining ductal integrity and the proper differentiation of luminal cells. Urogenital sinus (UGS) isolated from p63(+/+) and p63(-/-) embryos developed into prostate when grafted into adult male nude mice. Prostatic tissue that developed in p63(-/-) UGS grafts contained neuroendocrine and luminal cells, but basal cells were absent. Therefore, p63 is essential for differentiation of basal cells, but p63 and thus basal cells are not required for differentiation of prostatic neuroendocrine and luminal epithelial cells. p63(-/-) prostatic grafts also contained atypical mucinous cells, which appeared to differentiate from luminal cells via activation of Src. In the response to castration, regression of p63(-/-) prostate was inordinately severe with almost complete loss of ducts, resulting in the formation of residual cystic structures devoid of epithelium. Therefore, basal cells play critical roles in maintaining ductal integrity and survival of luminal cells. However, regressed p63(-/-) prostate did regenerate in response to androgen administration, indicating that basal cells were not essential for prostatic regeneration.