Perturbations of genes essential for Müllerian duct and Wölffian duct development in Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.

Development of the human bladder and ureterovesical junction.

The urinary bladder collects urine from the kidneys and stores it until the appropriate moment for voiding. The trigone and ureterovesical junctions are key to bladder function, by allowing one-way passage of urine into the bladder without obstruction. Embryological development of these structures has been studied in multiple animal models as well as humans. In this report we review the existing literature on bladder development and cellular signalling with particular focus on bladder development in humans. The bladder and ureterovesical junction form primarily during the fourth to eighth weeks of gestation, and arise from the primitive urogenital sinus following subdivision of the cloaca. The bladder develops through mesenchymal-epithelial interactions between the endoderm of the urogenital sinus and mesodermal mesenchyme. Key signalling factors in bladder development include shh, TGF-ß, Bmp4, and Fgfr2. A concentration gradient of shh is particularly important in development of bladder musculature, which is vital to bladder function. The ureterovesical junction forms from the interaction between the Wolffian duct and the bladder. The ureteric bud arises from the Wolffian duct and is incorporated into the developing bladder at the trigone. It was previously thought that the trigonal musculature developed primarily from the Wolffian duct, but it has been shown to develop primarily from bladder mesenchyme. Following emergence of the ureters from the Wolffian ducts, extensive epithelial remodelling brings the ureters to their final trigonal positions via vitamin A-induced apoptosis. Perturbation of this process is implicated in clinical obstruction or urine reflux. Congenital malformations include ureteric duplication and bladder exstrophy.

Development of urogenital system in the Spix cavy: A model for studies on sexual differentiation.

This study documented, for the first time, the morphological patterns of differentiation of male and female genital organs of Spix cavy (Galea spixii) using histological and ultrastructural analyses, with immuno-localization of steroidogenic enzymes, cytochromes P450 aromatase (P450arom) and 17α-hydroxylase/17, 20-lyase (P450c17), involved in the synthesis of estrogens and androgens respectively throughout fetal sexual development. Undifferentiated gonads of Spix cavy develop into ovaries in females after 25 days of gestation (DG), exhibiting P450arom immunoreactivity. After 25 DG, paramesonephric ducts develop and form oviducts, uterine horns and cranial portion of the vagina. The caudal portion of the vagina originates from the urogenital sinus, and a vaginal closure membrane is present at the end of gestation. Partial channeling of the urethra into the clitoris occurs after 40 DG, but complete channeling never occurs. A preputial meatus emerges near the tip of organ. In males, undifferentiated gonads develop into testes at 25 DG and develop immunoreactivity for P450c17, which is required for androgens synthesis and likely maintenance of mesonephric ducts. Mesonephric ducts develop subsequently, forming the epididymis and ductus deferens. The pelvic urethra develops after 25 DG with channeling into the penis occurring around 30 DG. This is the first morphological study describing the process of sexual differentiation during gestation in a hystricomorph rodent and one of the most comprehensive analyses conducted in any mammal. Male genital organ development follows the general pattern described in other domestic mammals, but does not include formation of the baculum as occurs in mice and rats. In females, clitoral development includes partial canalization by the urethra and development of a preputial meatus. Further studies are required to clarify the mechanisms involved in the differentiative processes described.

Non-canonical Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension.

Congenital anomalies of the kidney and urinary tract (CAKUT) affect about 1 in 500 births and are a major cause of morbidity in infants. Duplex collecting systems rank among the most common abnormalities of CAKUT, but the molecular basis for this defect is poorly understood. In mice, conditional deletion of Wnt5a in mesoderm results in bilateral duplex kidney and ureter formation. The ureteric buds (UBs) in mutants emerge as doublets from the intermediate mesoderm (IM)-derived nephric duct (ND) without anterior expansion of the glial cell line-derived neurotrophic factor (Gdnf) expression domain in the surrounding mesenchyme. Wnt5a is normally expressed in a graded manner at the posterior end of the IM, but its expression is down-regulated prior to UB outgrowth at E10.5. Furthermore, ablation of Wnt5a in the mesoderm with an inducible Cre at E7.5 results in duplex UBs, whereas ablation at E8.5 yields normal UB outgrowth, demonstrating that Wnt5a functions in IM development well before the formation of the metanephros. In mutants, the posterior ND is duplicated and surrounding Pax2-positive mesenchymal cells persist in the nephric cord, suggesting that disruption of normal ND patterning prompts the formation of duplex ureters and kidneys. Ror2 homozygous mutants, which infrequently yield duplex collecting systems, show a dramatic increase in incidence with the additional deletion of one copy of Wnt5a, implicating this receptor in non-canonical Wnt5a signaling during IM development. This work provides the first evidence of a role of Wnt5a/Ror2 signaling in IM extension and offers new insights into the etiology of CAKUT and possible involvement of Wnt5a/Ror2 mutations.

Fibroblast growth factor receptor signaling in kidney and lower urinary tract development.

Fibroblast growth factor receptors (FGFRs) and FGF ligands are highly expressed in the developing kidney and lower urinary tract. Several classic studies showed many effects of exogenous FGF ligands on embryonic renal tissues in vitro and in vivo. Another older landmark publication showed that mice with a dominant negative Fgfr fragment had severe renal dysplasia. Together, these studies revealed the importance of FGFR signaling in kidney and lower urinary tract development. With the advent of modern gene targeting techniques, including conditional knockout approaches, several publications have revealed critical roles for FGFR signaling in many lineages of the kidney and lower urinary tract at different stages of development. FGFR signaling has been shown to be critical for early metanephric mesenchymal patterning, Wolffian duct patterning including induction of the ureteric bud, ureteric bud branching morphogenesis, nephron progenitor survival and nephrogenesis, and bladder mesenchyme patterning. FGFRs pattern these tissues by interacting with many other growth factor signaling pathways. Moreover, the many genetic Fgfr and Fgf animal models have structural defects mimicking numerous congenital anomalies of the kidney and urinary tract seen in humans. Finally, many studies have shown how FGFR signaling is critical for kidney and lower urinary tract patterning in humans.

Lgr4 controls specialization of female gonads in mice.

Leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is a type of membrane receptor with a seven-transmembrane structure. LGR4 is homologous to gonadotropin receptors, such as follicle-stimulating hormone receptor (Fshr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr). Recently, it has been reported that Lgr4 is a membrane receptor for R-spondin ligands, which mediate Wnt/beta-catenin signaling. Defects of R-spondin homolog (Rspo1) and wingless-type MMTV integration site family, member 4 (Wnt4) cause masculinization of female gonads. We observed that Lgr4(-/-) female mice show abnormal development of the Wolffian ducts and somatic cells similar to that in the male gonads. Lgr4(-/-) female mice exhibited masculinization similar to that observed in Rspo1-deficient mice. In Lgr4(-/-) ovarian somatic cells, the expression levels of lymphoid enhancer-binding factor 1 (Lefl) and Axin2 (Axin2), which are target genes of Wnt/beta-catenin signaling, were lower than they were in wild-type mice. This study suggests that Lgr4 is critical for ovarian somatic cell specialization via the cooperative signaling of Rspo1 and Wnt/beta-catenin.

FGF8 is essential for formation of the ductal system in the male reproductive tract.

During development of the urogenital tract, fibroblast growth factor 8 (Fgf8) is expressed in mesonephric tubules, but its role in this tissue remains undefined. An evaluation of previously generated T-Cre-mediated Fgf8-deficient mice (T-Cre; Fgf8(flox/Δ2,3) mice), which lack Fgf8 expression in the mesoderm, revealed that the cranial region of the Wolffian duct degenerated prematurely and the cranial mesonephric tubules were missing. As a result, the epididymis, vas deferens and efferent ductules were largely absent in mutant mice. Rarb2-Cre was used to eliminate FGF8 from the mesonephric tubules but to allow expression in the adjacent somites. These mutants retained the cranial end of the Wolffian duct and formed the epididymis and vas deferens, but failed to elaborate the efferent ductules, indicating that Fgf8 expression by the mesonephric tubules is required specifically for the formation of the ductules. Ret knockout mice do not form the ureteric bud, a caudal outgrowth of the Wolffian duct and progenitor for the collecting duct network in the kidney, but they do develop the cranial end normally. This indicates that Fgf8, but not Ret, expression is essential to the outgrowth of the cranial mesonephric tubules from the Wolffian duct and to the development of major portions of the sex accessory tissues in the male reproductive tract. Mechanistically, FGF8 functions upstream of Lhx1 expression in forming the nephron, and analysis of Fgf8 mutants similarly shows deficient Lhx1 expression in the mesonephric tubules. These results demonstrate a multifocal requirement for FGF8 in establishing the male reproductive tract ducts and implicate Lhx1 signaling in tubule elongation.

Crucial Roles of the Mesenchymal Androgen Receptor in Wolffian Duct Development.

Wolffian duct (WD) maintenance and differentiation is predominantly driven by the androgen action, which is mediated by the androgen receptor (AR). It is well established that the mesenchyme indicates the fate and differentiation of epithelial cells. However, in vivo developmental requirement of mesenchymal AR in WD development is still undefined. By designing a mesenchyme-specific Ar knockout (ARcKO), we discovered that the loss of mesenchymal Ar led to the bilateral or unilateral degeneration of caudal WDs and cystic formation at the cranial WDs. Ex vivo culture of ARcKO WDs invariably resulted in bilateral defects, suggesting that some factor(s) originating from surrounding tissues in vivo might promote WD survival and growth even in the absence of mesenchymal Ar. Mechanistically, we found cell proliferation was significantly reduced in both epithelial and mesenchymal compartments; but cell apoptosis was not affected. Transcriptomic analysis by RNA sequencing of E14.5 mesonephroi revealed 131 differentially expressed genes. Multiple downregulated genes (Top2a, Wnt9b, Lama2, and Lamc2) were associated with morphological and cellular changes in ARcKO male embryos (ie, reduced cell proliferation and decreased number of epithelial cells). Mesenchymal differentiation into smooth muscle cells that are critical for morphogenesis was also impaired in ARcKO male embryos. Taken together, our results demonstrate the crucial roles of the mesenchymal AR in WD maintenance and morphogenesis in mice.

Gata3 acts downstream of beta-catenin signaling to prevent ectopic metanephric kidney induction.

Metanephric kidney induction critically depends on mesenchymal-epithelial interactions in the caudal region of the nephric (or Wolffian) duct. Central to this process, GDNF secreted from the metanephric mesenchyme induces ureter budding by activating the Ret receptor expressed in the nephric duct epithelium. A failure to regulate this pathway is believed to be responsible for a large proportion of the developmental anomalies affecting the urogenital system. Here, we show that the nephric duct-specific inactivation of the transcription factor gene Gata3 leads to massive ectopic ureter budding. This results in a spectrum of urogenital malformations including kidney adysplasia, duplex systems, and hydroureter, as well as vas deferens hyperplasia and uterine agenesis. The variability of developmental defects is reminiscent of the congenital anomalies of the kidney and urinary tract (CAKUT) observed in human. We show that Gata3 inactivation causes premature nephric duct cell differentiation and loss of Ret receptor gene expression. These changes ultimately affect nephric duct epithelium homeostasis, leading to ectopic budding of interspersed cells still expressing the Ret receptor. Importantly, the formation of these ectopic buds requires both GDNF/Ret and Fgf signaling activities. We further identify Gata3 as a central mediator of beta-catenin function in the nephric duct and demonstrate that the beta-catenin/Gata3 pathway prevents premature cell differentiation independently of its role in regulating Ret expression. Together, these results establish a genetic cascade in which Gata3 acts downstream of beta-catenin, but upstream of Ret, to prevent ectopic ureter budding and premature cell differentiation in the nephric duct.

Wolffian duct differentiation by physiological concentrations of androgen delivered systemically.

In developing mammalian males, conversion of the Wolffian ducts into the epididymides and vasa deferentia depends on androgen secretion by the testes, whereas in females these ducts remain in a vestigial form or regress. However, there is continuing uncertainty whether the androgen needs to be delivered locally, either by diffusion from the adjacent testis or, by secretion into the lumen of the duct, or whether circulating androgens maintain and virilize the Wolffian ducts. To resolve this uncertainty, we transplanted either day 0-2 or day 8-9 post-partum testes beneath the flank skin of three groups of neonatal (days 0-1) female tammar wallabies, where they developed and secreted physiological levels of hormones. The Wolffian ducts of all these females were retained and had formed extensive epididymides when examined at days 25, 34 and 87 after birth. In the two older groups of females, sampled after the time of prostatic bud formation, the urogenital sinus was virilized and there was extensive prostatic development similar to that of normal males of the same age, showing that androgen secretion had occurred. Virilization of the Wolffian ducts occurred during an early but short-lived window of sensitivity. This study provides the first clear evidence that under physiological conditions virilization can be mediated by circulating androgen.

Ift25 is not a cystic kidney disease gene but is required for early steps of kidney development.

Eukaryotic cilia are assembled by intraflagellar transport (IFT) where large protein complexes called IFT particles move ciliary components from the cell body to the cilium. Defects in most IFT particle proteins disrupt ciliary assembly and cause mid gestational lethality in the mouse. IFT25 and IFT27 are unusual components of IFT-B in that they are not required for ciliary assembly and mutant mice survive to term. The mutants die shortly after birth with numerous organ defects including duplex kidneys. Completely duplex kidneys result from defects in ureteric bud formation at the earliest steps of metanephric kidney development. Ureteric bud initiation is a highly regulated process involving reciprocal signaling between the ureteric epithelium and the overlying metanephric mesenchyme with regulation by the peri-Wolffian duct stroma. The finding of duplex kidney in Ift25 and Ift27 mutants suggests functions for these genes in regulation of ureteric bud initiation. Typically the deletion of IFT genes in the kidney causes rapid cyst growth in the early postnatal period. In contrast, the loss of Ift25 results in smaller kidneys, which show only mild tubule dilations that become apparent in adulthood. The smaller kidneys appear to result from reduced branching in the developing metanephric kidney. This work indicates that IFT25 and IFT27 are important players in the early development of the kidney and suggest that duplex kidney is part of the ciliopathy spectrum.

The critical time window for androgen-dependent development of the Wolffian duct in the rat.

Androgens are thought to separately regulate stabilization and differentiation of the Wolffian duct (WD), but the time windows for these effects are unclear. To address this, fetal rats were exposed to flutamide within either an early window (EW) [embryonic day 15.5 (E15.5) to E17.5], when the WD degenerates in the female, or a later window (LW) (E19.5-E21.5), when the WD morphologically differentiates in the male, or during the full window of WD development (FW) (E15.5-21.5). WDs were examined for abnormalities during fetal (E21.5) or postnatal life, and anogenital distance and prostate presence/absence were recorded. Exposure to FW- or EW-flutamide, but not to LW-flutamide, induced comparable abnormalities in the fetal WD at E21.5, namely reduced WD coiling, reduced cell proliferation, reduced epithelial cell height, altered epithelial vimentin expression, and reduced expression of smooth muscle actin in the WD inner stroma. Exposure to EW- or FW-flutamide, but not to LW-flutamide, resulted in incomplete/absent WDs in more than 50% of males by adulthood, although such abnormalities were infrequent in fetal life. These findings suggest that androgen action during the EW is sufficient to promote WD morphological differentiation several days later. Because the androgen receptor is expressed in the WD stroma but not in the epithelium during this EW, WD differentiation is likely to be dependent on androgen-mediated signaling from the stroma to the epithelium. In conclusion, the critical window for androgen action in regulating WD development in the rat is between E15.5 and E17.5. This window is also important for prostate formation and anogenital distance masculinization.

MSX2 promotes vaginal epithelial differentiation and wolffian duct regression and dampens the vaginal response to diethylstilbestrol.

In utero exposure to diethylstilbestrol (DES) leads to patterning defects in the female reproductive tract (FRT) and a propensity to the development of vaginal adenocarcinomas in humans. In the mouse, DES treatment similarly induces a plethora of FRT developmental defects, including stratification of uterine epithelium and presence of glandular tissue in cervix and vagina. Uterine abnormalities are associated with repression of the homeobox gene Msx2, and DES leads to an altered uterine response in Msx2 mutants including a dilated uterine lumen. Here we investigate the role of Msx2 in normal vaginal development and in FRT response to DES. During vaginal development, Msx2 is required for Tgfbeta2 and Tgfbeta3 expression and for proper vaginal epithelial differentiation. Moreover, Msx2 is involved in caudal Wolffian duct regression by promoting apoptosis. Consistently, neonatal DES exposure represses Msx2 expression in the Wolffian duct epithelium and inhibits its apoptosis and subsequent regression. Intriguingly, although DES treatment also represses Msx2 expression in the vaginal epithelium, a much more severe DES-induced vaginal phenotype was observed in Msx2 mutant mice, including a complete failure of Müllerian vaginal epithelial stratification and a severely dilated vaginal lumen, accompanied by loss of p63 and water channel protein expression. These results demonstrate a critical role for Msx2 in counteracting the effect of DES on FRT patterning and suggest that the response to DES may be highly variable depending on the genotype of an individual.

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

Androgen-dependent mechanisms of Wolffian duct development and their perturbation by flutamide.

Androgens play a vital role in Wolffian duct (WD) development, but the mechanisms that underlie this are unknown. The present study used in utero exposure of pregnant rats to the androgen receptor antagonist flutamide (50 or 100 mg/kg) to explore possible mechanisms. Pregnant rats were treated from embryonic d 15.5 (E15.5), and WDs were isolated from fetuses from E17.5-E21.5 and from adults. WD morphology was evaluated, and total length of the duct lumen was determined in fetal samples. Fetal WDs were immunostained for androgen receptor and stromal (inner and outer) and/or epithelial-cell-specific markers and analyzed for cell proliferation and apoptosis. In adulthood, most flutamide-exposed males lacked proximal WD-derived tissues, whereas at E18.5-E19.5, a time when the WD has completely regressed in females, a complete normal WD was present in all flutamide-exposed animals. This suggests that flutamide, at doses of 50 or 100 mg/kg, interferes with WD differentiation, not stabilization. Consistent with this, WD elongation/coiling increased in controls by 204% between E19.5 and E21.5 but increased less significantly (103%) in flutamide-exposed animals. This was associated with reduced cell proliferation, but there was no increase in apoptosis or change in expression of androgen receptor mRNA or protein. Flutamide treatment impaired differentiation of inner stromal cells, shown by decreased expression of smooth muscle actin, before effects were noted in the epithelium, consistent with androgens driving WD development via stromal-epithelial interactions. In conclusion, WD differentiation is far more susceptible to blockade of androgen action than is its initial stabilization, and these effects may be mediated by disruption of stromal-epithelial interactions.

Novel function of LHFPL2 in female and male distal reproductive tract development.

Congenital reproductive tract anomalies could impair fertility. Female and male reproductive tracts are developed from Müllerian ducts and Wolffian ducts, respectively, involving initiation, elongation and differentiation. Genetic basis solely for distal reproductive tract development is largely unknown. Lhfpl2 (lipoma HMGIC fusion partner-like 2) encodes a tetra-transmembrane protein with unknown functions. It is expressed in follicle cells of ovary and epithelial cells of reproductive tracts. A spontaneous point mutation of Lhfpl2 (LHFPL2(G102E)) leads to infertility in 100% female mice, which have normal ovarian development, ovulation, uterine development, and uterine response to exogenous estrogen stimulation, but abnormal upper longitudinal vaginal septum and lower vaginal agenesis. Infertility is also observed in ~70% mutant males, which have normal mating behavior and sperm counts, but abnormal distal vas deferens convolution resulting in complete and incomplete blockage of reproductive tract in infertile and fertile males, respectively. On embryonic day 15.5, mutant Müllerian ducts and Wolffian ducts have elongated but their duct tips are enlarged and fail to merge with the urogenital sinus. These findings provide a novel function of LHFPL2 and a novel genetic basis for distal reproductive tract development; they also emphasize the importance of an additional merging phase for proper reproductive tract development.

Androgen receptor expression in developing male reproductive organs.

The distribution of androgen receptors (AR) in developing male BALB/c mouse reproductive organs was determined by 3H-dihydrotestosterone steroid autoradiography. The efferent ductules, urogenital sinus (UGS) and Wolffian ducts, and their derivatives, the epididymis, ductus deferens, seminal vesicles, coagulating glands, prostate and bulbouretheral glands, were examined in mice from 13-days fetal (gestation = 19-20 days) to 10 days postnatal. All organs contained AR in their mesenchymal/stromal cells at all times examined. The Wolffian ducts and UGS did not contain epithelial AR on days 13-14 or 16 of gestation. The efferent ductule was the first site of epithelial AR expression in the male tract during development; this organ had epithelial AR on day 16 and at all subsequent times. The epididymis and ductus deferens contained epithelial AR beginning on day 19 of gestation. Seminal vesicle and coagulating gland epithelium was AR- at birth, became weakly AR+ on day 1, and was strongly AR+ on day 2 and subsequently. Prostatic epithelium was AR- up to day 4, when some positive epithelial cells were seen; the prostatic epithelium was strongly AR+ on day 6 and subsequently. The last organ to begin expressing epithelial AR was the bulbouretheral gland; this epithelium did not become clearly AR+ until day 8 postnatally. In summary, these results indicate that initial epithelial AR expression in the male reproductive tract occurs in a clear temporal sequence and proceeds in a cranial-caudal direction. Epithelial AR first appear in the efferent ductules, followed by initial epithelial AR expression in Wolffian-derived organs and finally in the UGS-derived organs. The factors controlling initial epithelial AR expression are unclear, but mesenchyme may be involved.

In vitro androgen-induced growth and morphogenesis of the Wolffian duct within urogenital ridge.

Male mouse urogenital ridges (URs) at 15.5 days of gestation (vaginal plug = day 0) containing Wolffian (WDs) and Mullerian ducts were cultured for 4 days with or without gonads in serum-free medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 supplemented with insulin, transferrin, cholera toxin, epidermal growth factor, and BSA). URs without gonads were grown in serum-free medium with testosterone (T, 10(-7) M), 5 alpha-dihydrotestosterone (DHT, 10(-8) M), T (10(-7) M) plus cyproterone acetate (antiandrogen, 10(-5) M), or T (10(-8) M) plus 390 MSD (17 beta-N, N-diisopropylcarbamoyl-4-aza-5 alpha- androstan-3-one, an inhibitor of 5 alpha-reductase, 10(-5) M). After 4 days of culture the number of epididymal curvatures that appeared in the upper portion of WDs were quantified. DNA content of URs grown in serum-free medium was also measured. Both T and DHT increased DNA contents in a dose- (T = 10(-8) to 10(-10) M, DHT = 10(-9) to 10(-11) M) and time-dependent manner. DHT was approximately 10-fold more effective than T in eliciting epididymal coiling and increasing DNA content of URs. The effects of T or DHT were mimicked by coculture with fetal testes. Epididymal coiling and an increase in DNA content occurred in URs grown in the presence of T plus 390 MSD. By contrast, URs cultured without androgens or with T plus cyproterone acetate failed to undergo epididymal coiling and to increase DNA content. The conversion rate per mg protein of [1 beta, 2 beta-3H]T into [3H]DHT was 0.30-fold lower in 15.5 day URs cultured over a 4-day period in comparison to urogenital sinuses whose development is known to be dependent upon DHT. These data suggest that T is an important hormone in the development of the upper portion of WDs, although it is not possible to exclude a role for DHT in the development of the epididymis.

Residual activity of mutant androgen receptors explains wolffian duct development in the complete androgen insensitivity syndrome.

Development of the Wolffian ducts (WD) into epididymides and vasa deferentia is dependent on testosterone. Patients with the complete androgen insensitivity syndrome (CAIS) are therefore not expected to develop these structures. However, WD derivatives have been described in cases of CAIS. It is thought that these may be remnants. This study assesses the degree of WD development in 33 patients with CAIS and investigates whether this development was androgen dependent. Epididymides and vasa deferentia were identified in 70% of patients with substitution mutations in the androgen receptor ligand-binding domain. They were more developed than epididymides and vasa deferentia from 16- to 20-wk-old male fetuses, suggesting that the WD had been stimulated to grow, rather than failed to regress. Receptors with substitutions in the ligand-binding domain were normally expressed and showed residual response to androgens in transactivation assays. Patients with premature stop codons or frameshift mutations, which prevented androgen receptor expression, or DNA-binding domain mutations that abolished transcriptional activity did not have epididymides or vasa deferentia. We hypothesize that mutant receptors with residual activity in vitro respond to high local testosterone concentrations in vivo, thereby stimulating WD development. The classification of androgen insensitivity in such patients should be considered severe rather than complete.

Luteinizing hormone-dependent activity and luteinizing hormone-independent differentiation of rat fetal Leydig cells.

Addition of 5x10(-2) U/ml recombinant luteinizing hormone (LH) to testes from fetuses at 16.5 day post conception (dpc) cultured for 5 days increased the number of Leydig cells by 34% and the acute LH-stimulated testosterone production by 600%. To determine whether these positive effects of LH in vitro are physiologically relevant in vivo, fetuses were decapitated on days 16.5 pc (before the onset of LH expression in the hypophysis) or 18.5 pc (before the surge of LH in the fetal plasma) and removed at 21.5 dpc. The number of fetal Leydig cells per testis and the acute LH-stimulated testosterone production by the testes ex vivo were unaltered by decapitation. Since, in all groups, the number of Leydig cells doubled between 16.5 and 18.5 dpc and between 18.5 and 21.5 dpc, these results suggest that neither the appearance of new fully differentiated fetal Leydig cells nor the maintenance of differentiated functions in existing fetal Leydig cells depend on LH during late fetal life, although this hormone is present in the plasma. Decapitation reduced the testosterone concentrations in the plasma (-56%) and in the testis in vivo (-67%) and the basal testosterone secretion of the testis ex vivo (-70%). This suggests that LH is required to maintain the physiological activity of the Leydig cell during late fetal life. However, the decrease of the in vivo testosterone production after decapitation was not sufficient to impair the growth of the Wolffian ducts and the lengthening of the anogenital distance. In conclusion, during late fetal life in the rat, Leydig cells are LH-independent for their functional differentiation and LH-dependent for their activity.