Wolffian Duct Differentiation Research Articles

Postnatal differentiation and regional histological variations in the ductus epididymidis of the Congjiang Xiang pig

The Congjiang Xiang pig is a rare Chinese miniature breed whose epididymal histologic features through the postnatal development are poorly understood. To clarify the histomorphological differences between each region of epididymis during postnatal development, 24 male Congjiang Xiang pigs aged from neonatal (15 d), peri-puberty (30 d), puberty (60 d) to adult (180 d) stages, were examined. Postnatal differentiation of the different regions (I–V) of the epididymis started from birth and continued until maturity that showed regional variations. Developmental progression was disto-proximal. At the neonatal stage, Wolffian duct differentiation starts in the distal region, then ascends to the middle region which forms regions V, IV and III, respectively. A simple lined cuboidal in the epidydimal epithelial, which gradually differentiated into a pseudostratified columnar with stereocilia from neonatal to post-pubertal. After puberty cell rearrangement occurred in the epithelium, differentiation accelerated, and spermatozoon seen in the lumen, especially the lumen of region II. In region III, both halo and apical cells were frequently observed. At the post-pubertal stage, clear cells were frequently observed in Region IV-V, and the epididymal duct was markedly increased in size and fully packed with spermatozoa. The information presented in this study will be helpful for future evaluations of Congjiang Xiang pig fertility. After puberty cell rearrangement occurred in the epithelium, differentiation accelerated, and spermatozoon seen in the lumen, especially the lumen of region II. In region III, both halo and apical cells were frequently observed. At the post-pubertal stage, clear cells were frequently observed in Region IV–V, and the epididymal duct was markedly increased in size and fully packed with spermatozoa. The information presented in this study will be helpful for future evaluations of Congjiang Xiang pig fertility.

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.

New Insights into the Role of Androgens in Wolffian Duct Stabilization in Male and Female Rodents

Androgen-mediated wolffian duct (WD) development is programmed between embryonic d 15.5 (e15.5) and 17.5 in male rats, and WD differentiation has been shown to be more susceptible to reduced androgen action than is its initial stabilization. We investigated regulation of these events by comparing fetal WD development at e15.5-postnatal d0 in male and female androgen receptor knockout mice, and in rats treated from e14.5 with flutamide (100 mg/kg/d) plus di-n(butyl) phthalate (500 mg/kg/d) to block both androgen action and production, testosterone propionate (20 mg/kg/d) to masculinize females, or vehicle control. In normal females, WD regression occurred by e15.5 in mice and e18.5 in rats, associated with a lack of epithelial cell proliferation and increased apoptosis, disintegration of the basement membrane, and reduced epithelial cell height. Exposure to testosterone masculinized female rats including stabilization and partial differentiation of WDs. Genetic or chemical ablation of androgen action in males prevented masculinization and induced WD regression via similar processes to those in normal females, except this occurred 2-3 d later than in females. These findings provide the first evidence that androgens may not be the only factor involved in determining WD fate. Other factors may promote survival of the WD in males or actively promote WD regression in females, suggesting sexually dimorphic differences in the preprogrammed setup of the WD.

Virilization and 5α-reduced Androgens: Insights from Marsupials.

In eutherian mammals gonadal differentiation takes place in the early fetus, and the androgen mediated events of virilization occur long before birth. In contrast, in marsupial mammals such as the tammar wallaby, the young is born at a relatively poor state of differentiation, and almost all of gonadal and sexual differentiation occurs after birth. We have therefore used the tammar wallaby as a model species to investigate the endocrinology of virilization, since the young is accessible for experimental manipulation during the period of virilization. In addition, the onset of virilization of the Wolffian duct (day 10), prostate (day 25) and phallus (day 60) are widely separated allowing us to investigate the possibly different mechanisms underlying the virilisation of each. We have previously established that the main circulating androgen driving virilization is not testosterone or DHT, but 5α-androstane-3α,17β-diol (adiol), which is converted to DHT in target tissues. We hypothesised that the relatively early timing of Wolffian duct (WD) differentiation may be due to high concentrations of androgens secreted down the WD from the testes. However, we found that WD were retained and differentiated in neonatal females with neonatal testis grafts under the skin, showing that local transport of androgens down the WD is not needed for WD virilization. To further investigate the formation of 5α-reduced androgens in tammars we obtained partial sequences of the tammar homologues of steroid-5α-reductases type 1 and 2 (SRD1 and SRD2), which had about 80% amino acid homology with man and mouse isoforms. Both isoforms were expressed in the testes and ovaries, urogenital sinus/prostate and urogenital tubercle/phallus of both male and female pouch young collected between the day of birth and day 150 post partum, and dynamic changes in expression were detected by real-time rtPCR that may be important for the timing of virilization. Further work is ongoing to determine expression patterns for other relevant steroid metabolising enzymes. Marsupials, because of the accessibility of the young at the critical stages, provide excellent models for the investigation of the endocrine control of virilization. This research received support from the Australian NHMRC.

230. Megalin, RAP and Nkx3.1 expression in the developing reproductive tract of a marsupial, the tammar wallaby

Androgens induce the differentiation of the urogenital sinus (UGS) to form a prostate. An early marker of this response is upregulation of the transcription factor Nkx3.1 in the urogenital epithelium in the precursors of prostatic buds. In tammars, prostate differentiation begins ~3 weeks after birth and after the time the testis starts to secrete androgens, and 2 weeks after androgen stimulated Wolffian duct differentiation. The reason for this delay in prostate differentiation is unexplained. Androgen receptors are present in the UGS, and the potent androgen, androstanediol, induces prostatic development in females. Whilst androgens may diffuse into cells by across the cell membrane, there is increasing evidence that steroids are also internalised actively via the cell-surface transport molecule Megalin. We are exploring the possibility that the delay may be related to the establishment of a Megalin-mediated pathway. Megalin is a cell surface receptor expressed on epithelia and mediates the endocytosis of a wide range of ligands, including SHBG-bound sex steroids. Megalin action is regulated by Receptor Associated Protein (RAP), which acts as an antagonist to Megalin action. This study cloned partial sequences of Megalin, RAP and Nkx3.1 and examined their expression in the developing urogenital sinus of the tammar wallaby using RT–PCR. The cellular distribution of Megalin protein in the developing UGS was examined using immunohistochemistry. Megalin, RAP and Nkx3.1 in the tammar were all highly conserved with eutherian orthologueues. Megalin and Nkx3.1 transcripts were detected in the liver, kidney, ovary, testis and developing urogenital sinus of male and female tammars. In the developing UGS of the tammar, there was strong staining for Megalin protein in the urogenital epithelium with some diffuse staining in the surrounding mesenchyme. Together, these results suggest that Megalin could be a key gene in the mediation of androgen action in prostatic development in the tammar wallaby.

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.

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.

Altered Gene Expression During Rat Wolffian Duct Development following Di(n-Butyl) Phthalate Exposure

Di(n-butyl) phthalate (DBP) is a common plasticizer and solvent that disrupts androgen-dependent male reproductive development in rats. In utero exposure to 500 mg/kg/day DBP on gestation days (GD) 12 to 21 decreases androgen biosynthetic enzymes, resulting in decreased fetal testicular testosterone levels. One consequence of prenatal DBP exposure is malformed epididymides in adult rats. Reduced fetal testosterone levels may be responsible for the malformation, since testosterone is required for Wolffian duct stabilization and their development into epididymides. Currently, little is understood about the molecular mechanisms of Wolffian duct differentiation. The objective of this study was to identify changes in gene expression associated with altered morphology of the proximal Wolffian duct following in utero exposure to DBP. Pregnant Crl:CD(R) (SD) rats were gavaged with corn oil vehicle or 500 mg/kg/day DBP from GD 12 to GD 19 or 21. There were only small morphological differences between control and DBP-exposed Wolffian ducts on GD 19. On GD 21, 89% of male fetuses in the DBP dose group showed marked underdevelopment of Wolffian ducts, characterized by decreased coiling. RNA was isolated from Wolffian ducts on GD 19 and 21. Together with empirical information, cDNA microarrays were used to help identify candidate genes that could be associated with the morphological changes observed on GD 21. These candidate genes were analyzed by real-time RT-PCR. Changes in mRNA expression were observed in genes within the insulin-like growth factor (IGF) pathway, the matrix metalloproteinase (MMP) family, the extracellular matrix, and in other developmentally conserved signaling pathways. On GD 19, immunolocalization of IGF-1 receptor protein demonstrated an increase in cytoplasmic expression in the mesenchymal and epithelial cells. There was also a variable decrease in androgen receptor protein in ductal epithelial cells on GD 19. This study provides insight into the effects of antiandrogens on the molecular mechanisms involved in Wolffian duct development. The altered morphology and changes in gene expression following DBP exposure are suggestive of altered paracrine interactions between ductal epithelial cells and the surrounding mesenchyme during Wolffian duct differentiation due to lowered testosterone production.

Molecular basis of androgen insensitivity

Androgens are important steroid hormones for expression of the male phenotype. They have characteristic roles during male sexual differentiation, during development and maintenance of secondary male characteristics, and during the initiation and maintenance of spermatogenesis. The two most important androgens in this respect are testosterone and 5α-dihydrotestosterone. Each androgen has its own specific role during male sexual differentiation, testosterone is involved in the development and differentiation of Wolffian duct derived structures, whereas 5α-dihydrotestosterone, a metabolite of testosterone, is the active ligand in the urogenital sinus and tubercle and their derived structures. The actions of androgens are mediated by the androgen receptor. This ligand dependent transcription factor belongs to the superfamily of nuclear receptors, including those for the other steroid hormones. The androgen receptor gene is located on the X-chromosome at Xq11–12 and codes for a protein with a molecular mass of approximately 110 kDa. Only one androgen receptor cDNA has been identified sofar, despite two different ligands. It is generally accepted that defects in the androgen receptor gene prevent the normal development of both internal and external male structures in 46, XY individuals. The end-organ resistance to androgens has been designated as androgen insensitivity syndrome (AIS) and is distinct from other forms of male pseudohermaphroditism like 17β-hydroxy-steroid dehydrogenase type 3 deficiency, leydig cell hypoplasia due to inactivating LH receptor mutations or 5α-reductase type 2 deficiency. Furthermore, two additional pathological situations are associated with abnormal androgen receptor structure and function — spinal and bulbar muscular atrophy (SBMA, or Kennedy's disease) and prostate cancer. In the AR gene, four different types of mutations have been detected in DNA from individuals with AIS — (i) single point mutations resulting in amino acid substitutions or premature stopcodons; (ii) nucleotide insertions or deletions most often leading to a frame shift and premature termination; (iii) complete or partial gene deletions; and (iv) intronic mutations in either splice donor or acceptor sites, which affect the splicing of AR RNA. The main phenotypic characteristics of individuals with the complete androgen insensitivity syndrome (CAIS) are, female external genitalia, a short, blind ending vagina, the absence of Wolffian duct derived structures, the absence of a prostate, development of gynecomastia and the absence of pubic and axillary hair. Usuallly testosterone levels are elevated at the time of puberty, while also elevated LH levels are found. In the partial androgen insensitivity syndrome (PAIS) several different phenotypes are evident, ranging from individuals with predominantly a female appearance to persons with ambiguous genitalia, or individuals with a predominantly male phenotype. At puberty, elevated LH, testosterone and estradiol levels are observed. Individuals with mild symptoms of undervirilization (mild androgen insensitivity syndrome (MAIS)) and infertility have been described as well. Phenotypic variation between individuals in different families has been described for several mutations. However, in cases of CAIS no phenotypic variation has been described within one single family, in contrast to families with individuals with PAIS. In general AIS, can be routinely analyzed and more than 150 different mutations have been reported now. Differential diagnosis of AIS is possible with syndromes presenting with almost similar phenotypes but with a completely different molecular cause.

Sexually dimorphic development of the mammalian reproductive tract requires Wnt-7a.

An important feature of mammalian development is the generation of sexually dimorphic reproductive tracts from the Müllerian and Wolffian ducts. In females, Müllerian ducts develop into the oviduct, uterus, cervix and upper vagina, whereas Wolffian ducts regress. In males, testosterone promotes differentiation of Wolffian ducts into the epididymis, vas deferens and seminal vesicle. The Sertoli cells of the testes produce Müllerian-inhibiting substance, which stimulates Müllerian duct regression in males. The receptor for Müllerian-inhibiting substance is expressed by mesenchymal cells underlying the Müllerian duct that are thought to mediate regression of the duct. Mutations that inactivate either Müllerian-inhibiting substance or its receptor allow development of the female reproductive tract in males. These pseudohermaphrodites are frequently infertile because sperm passage is blocked by the presence of the female reproductive system. Here we show that male mice lacking the signalling molecule Wnt-7a fail to undergo regression of the Müllerian duct as a result of the absence of the receptor for Müllerian-inhibiting substance. Wnt7a-deficient females are infertile because of abnormal development of the oviduct and uterus, both of which are Müllerian duct derivatives. Therefore, we propose that signalling by Wnt-7a allows sexually dimorphic development of the Müllerian ducts.

Cellular localization of immunoreactive epidermal growth factor during Wolffian duct differentiation of the fetal mouse.

Previous studies from this laboratory indicated a role for epidermal growth factor (EGF) in androgen-dependent male reproductive tract differentiation of the fetal mouse. Expression of an EGF-like protein during Wolffian duct differentiation was indicated from the determinations by radioimmunoassay (RIA) and radioreceptor assay. To further characterize the protein and to assess its role in male sexual differentiation, expression of the protein has been analyzed by Western blot assay and its tissue-specific cellular expression has been determined by immunocytochemical assay in the present study. Western blot analysis of the 18-day fetal male reproductive tract detected an immunoreactive band of the predicted 6-kDa size. Immunocytochemical analysis also detected EGF-specific immunostaining in the Wolffian duct derivatives. At day 18 of gestation, the staining was localized predominantly in the epithelial nuclei of the Wolffian duct derivatives whereas at days 14 and 16 of gestation, the staining was equally distributed in the mesenchymal and epithelial sites of the Wolffian duct derivative. The intensity of the staining increased with progression of differentiation during the 14th-18th days of gestation. Prenatal exposure to the antiandrogen flutamide significantly reduced the immunostaining of the duct. Thus, a role for EGF in Wolffian duct differentiation is indicated.

The role of growth hormone in fetal mouse reproductive tract differentiation.

Although GH plays a key role in postnatal growth and differentiation, its role in fetal differentiation is not clear at the present. The aim of the present study was to investigate whether GH plays a role in fetal sexual differentiation, and we used in vitro organ culture assay of sexual differentiation to determine this. The results showed that anti-rGH antibody blocked Wolffian duct differentiation specifically in the presence of fetal testes. Exogenous GH supplemented in the above experiment reversed the blocking effect of anti-GH. Among the other related products, insulin-like growth factor I was highly effective in reversing the anti-GH effect, insulin-like growth factor II was partially effective, but PRL was unable to reverse the anti-GH effect. GH itself was found to produce some masculinizing effect, as demonstrated by its ability to stabilize the Wolffian duct in female fetuses. The role of GH was further demonstrated by the observation that GH-immunoreactive material of the size of authentic GH was detected in the 18-day fetal reproductive tract, and the concentration of this material increased in response to progression of sexual differentiation. Determination of androgen-binding activity using Scatchard analysis on the cells isolated from the 18-day fetal reproductive tract indicated that androgen-binding activity increased after GH treatment of the cells. Thus, it may be concluded that GH influences male sexual differentiation and alters the androgen-binding activity of the fetal reproductive tract.

The role of epidermal growth factor receptor (EGFR) in male reproductive tract differentiation: stimulation of EGFR expression and inhibition of Wolffian duct differentiation with anti-EGFR antibody

We have shown previously that epidermal growth factor (EGF) plays a role in testosterone-dependent fetal Wolffian duct differentiation. To assess the role of EGF further, we examined EGF receptor (EGFR) expression during Wolffian duct differentiation and determined whether inhibition of EGFR results in inhibition of Wolffian duct differentiation. Using a polyclonal anti-EGFR antibody in the Western blot analysis, we have detected an immunoreactive band at the expected 180-kDa size, the molecular size for the EGFR protein, in the 18-day fetal male reproductive tract. The mouse 3T3 cell line, used as a positive control, also produced an immunospecific band at the same region. The intensity of the immunoreactive 180-kDa band in the 18-day male reproductive tract significantly decreased after pretreatment of the antibody with the 3T3 cell extract. Nonimmune serum produced no band at the 180- kDa region, suggesting EGFR specificity of the 180-kDa band. The level of the 180-kDa EGFR protein was found to be higher in the 18-day male than in the female reproductive tract. Testosterone increased expression of the EGFR level in the 18-day masculinized female reproductive tract, and it induced Wolffian duct morphogenesis. Under in vitro culture conditions, the anti-EGFR antibody prevented Wolffian duct differentiation, induced by the fetal testis, suggesting a direct role of EGFR in male reproductive tract differentiation. No such effect was found when tested with nonimmune serum. Anti-EGFR also blocked the development of the Wolffian duct in the female explant, induced by exogenous EGF. Thus, a role of EGFR mediating the effect of EGF during male reproductive tract differentiation is demonstrated.

The role of epidermal growth factor receptor (EGFR) in male reproductive tract differentiation: stimulation of EGFR expression and inhibition of Wolffian duct differentiation with anti-EGFR antibody.

We have shown previously that epidermal growth factor (EGF) plays a role in testosterone-dependent fetal Wolffian duct differentiation. To assess the role of EGF further, we examined EGF receptor (EGFR) expression during Wolffian duct differentiation and determined whether inhibition of EGFR results in inhibition of Wolffian duct differentiation. Using a polyclonal anti-EGFR antibody in the Western blot analysis, we have detected an immunoreactive band at the expected 180-kDa size, the molecular size for the EGFR protein, in the 18-day fetal male reproductive tract. The mouse 3T3 cell line, used as a positive control, also produced an immunospecific band at the same region. The intensity of the immunoreactive 180-kDa band in the 18-day male reproductive tract significantly decreased after pretreatment of the antibody with the 3T3 cell extract. Nonimmune serum produced no band at the 180- kDa region, suggesting EGFR specificity of the 180-kDa band. The level of the 180-kDa EGFR protein was found to be higher in the 18-day male than in the female reproductive tract. Testosterone increased expression of the EGFR level in the 18-day masculinized female reproductive tract, and it induced Wolffian duct morphogenesis. Under in vitro culture conditions, the anti-EGFR antibody prevented Wolffian duct differentiation, induced by the fetal testis, suggesting a direct role of EGFR in male reproductive tract differentiation. No such effect was found when tested with nonimmune serum. Anti-EGFR also blocked the development of the Wolffian duct in the female explant, induced by exogenous EGF. Thus, a role of EGFR mediating the effect of EGF during male reproductive tract differentiation is demonstrated.

Stimulation of epidermal growth factor gene expression during the fetal mouse reproductive tract differentiation: role of androgen and its receptor.

We have shown previously that epidermal growth factor (EGF) plays a role in testosterone-dependent fetal Wolffian duct differentiation. To further assess the role for EGF, we determined whether EGF gene expression was modulated in response to male reproductive tract differentiation. The expression of EGF messenger RNA (mRNA) was measured by an RT-PCR assay using primer pairs spanning the coding sequence of 3228 nucleotide (nt) to 3401 nt. Using the RT-PCR reaction, an amplimer of the expected size, 173 bp, was detected in the fetal male reproductive tract. The amplimer hybridized with a radioactive probe representing an internal sequence of the amplified product and was digested by the restriction enzyme HaeIII, which has an unique cleavage site at 3365 nt. The level of EGF mRNA at different stages of sexual differentiation was measured by a newly developed quantitative competitive RNA PCR (QCPCR) assay for EGF mRNA. The assay was sensitive and reproducible within a linear range of amplification with 5 x 10(4) to 140 x 10(4) copies of mRNA. Using the quantitative competitive RNA PCR we found that the level of EGF mRNA was higher in the male reproductive tract than that in the female reproductive tract. Exposure to testosterone (40 mg/kg.day) during days 13-17 of gestation induced the Wolffian duct in the female fetuses and resulted in stimulation of EGF-mRNA expression. Similarly, an antiandrogen receptor, flutamide (100 mg/kg.day) exposure during days 13-17 of gestation inhibited male reproductive tract differentiation and resulted in inhibition of EGF-mRNA expression. Moreover, during the differentiation of the male reproductive tract there was a biphasic increase in the level of EGF-mRNA, first at day 14 of gestation, the period of onset of testicular activity and Wolffian duct morphogenesis, and second at day 18 of gestation, corresponding to onset of differentiation of the urogenital sinus, epididymal duct, and seminal vesicle. Thus, it appears that testosterone-induced male sexual differentiation is accompanied by an increase in EGF gene expression.

Expression and regulation of steroid 5 alpha-reductase in the urogenital tract of the fetal rat.

Two androgens, testosterone and dihydrotestosterone, are required for the development of the male urogenital tract in the rat. Testosterone is secreted by the fetal testes and is thought to elicit differentiation of the Wolffian ducts into seminal vesicles, vas deferens, and epididymides. Testosterone is converted into dihydrotestosterone by steroid 5 alpha-reductase in the urogenital tract, and this conversion is necessary for the differentiation of the prostate and external genitalia. Genes encoding two 5 alpha-reductase isozymes, designated type 1 and type 2, have been identified. We examined the expression and regulation of these genes on days 17-21 in the urogenital tracts of male and female fetuses. Expression of the type 1 gene predominated in epithelial cells, whereas type 2 gene expression was limited to mesenchymal cells. Surprisingly, this expression pattern was detected in both testosterone-dependent and dihydrotestosterone-dependent anlagen of the urogenital tract and was the same in both male and female fetuses. Furthermore, transcripts encoding the two isozymes were present in their respective cell types before the overt differentiation of internal genitalia. Androgens stimulated expression of the type 2 gene in the urogenital tracts of both sexes, but did not effect expression of the type 1 gene or the cell type-specific expression patterns of the 5 alpha-reductase genes. In the adult prostate, 5 alpha-reductase gene expression is under feedforward control, in which the product of the enzyme, dihydrotestosterone, stimulates the expression of the gene. However, no evidence for feedforward regulation of either 5 alpha-reductase gene was detected in the fetus.

The 72-Kilodalton Protein of the Male Reproductive Tract is Differentially Expressed and Developmentally Regulated during Wolffian Duct Differentiation of the Fetal Mouse1

Differentiation of the mammalian reproductive tract into the male phenotype is dependent on the presence of functional testes secreting testosterone during in utero development. Recently, we identified a protein fraction (72 kDa) from the developing reproductive tract of the fetal male mouse that is able to mimic the effect of testosterone in stabilizing the Wolffian duct in vitro, suggesting a role played by this protein in mediating the effect of testosterone during Wolffian duct differentiation. In the present study, we further evaluated the masculinizing role of this protein by determining the specific appearance of the 72-kDa protein in relation to reproductive tract differentiation. Using Western blot and immunohistochemical analysis, we demonstrate that the protein was expressed specifically in the male reproductive tract and not in male non-reproductive organs, namely, lung, liver, or kidney. Within the male reproductive tract, the Wolffian duct derivatives demonstrated a high level of expression of the protein, whereas the urogenital sinus produced only a low level of reaction to this antibody in Western analysis and no reaction in the histochemical analysis. There was a gradual rise in expression of the protein in the male reproductive tract during the critical period of Wolffian duct differentiation. The female reproductive tract produced only a trace immune reaction in Western analysis and no reaction in immunohistochemical analysis. Thus, the results support the hypothesis that the 72-kDa protein plays a role in Wolffian duct differentiation.

Epidermal growth factor binding in the developing male reproductive duct and its regulation by testosterone.

It was shown previously that epidermal growth factor (EGF) plays a role in the testosterone-dependent fetal Wolffian duct differentiation. In this communication, the role of EGF was further investigated by determining whether EGF receptor plays a role in mediating the effect of EGF during Wolffian duct (male duct) differentiation, and experiments were designed to test this hypothesis. EGF-binding activity in cultured cells isolated from the reproductive ducts of 14- to 18-day-old fetal mouse was determined, and the results were evaluated by saturation/Scatchard analysis. Using this analysis, we demonstrate that the fetal mouse Wolffian duct contains EGF-saturable EGF-binding proteins with a binding affinity of 10(-10) M. The binding was temperature, time, and cell concentration dependent. Affinity cross-linking analysis of EGF binding of this tissue indicated the presence of an EGF receptor protein of 150 kilodaltons mol wt. The binding activity increased in parallel with the progression of Wolffian duct differentiation during the 14th to 18th day of gestation, which is the critical period of Wolffian duct differentiation. The binding activity was barely detectable at the onset of differentiation, i.e. on the 14th day of gestation. Binding activity in the male duct was higher than that in the female duct. Prenatal administration of testosterone during days 13-17 of gestation increased the EGF receptor concentration in the masculinized female fetuses, suggesting a role of fetal testicular testosterone in determining EGF-binding activity. Prenatal treatment of cyproterone acetate (an antiandrogen at the level of androgen receptor binding to androgen), however, produced no effect on EGF-binding activity in male fetuses. Thus, a role for the EGF receptor is indicated during Wolffian duct differentiation. Testosterone appears to play a role in modulating EGF-binding activity. Further work is necessary to determine the mechanism by which testosterone modulates EGF-binding activity in this tissue.

Epidermal growth factor binding in the developing male reproductive duct and its regulation by testosterone

It was shown previously that epidermal growth factor (EGF) plays a role in the testosterone-dependent fetal Wolffian duct differentiation. In this communication, the role of EGF was further investigated by determining whether EGF receptor plays a role in mediating the effect of EGF during Wolffian duct (male duct) differentiation, and experiments were designed to test this hypothesis. EGF-binding activity in cultured cells isolated from the reproductive ducts of 14- to 18-day-old fetal mouse was determined, and the results were evaluated by saturation/Scatchard analysis. Using this analysis, we demonstrate that the fetal mouse Wolffian duct contains EGF-saturable EGF-binding proteins with a binding affinity of 10(-10) M. The binding was temperature, time, and cell concentration dependent. Affinity cross-linking analysis of EGF binding of this tissue indicated the presence of an EGF receptor protein of 150 kilodaltons mol wt. The binding activity increased in parallel with the progression of Wolffian duct differentiation during the 14th to 18th day of gestation, which is the critical period of Wolffian duct differentiation. The binding activity was barely detectable at the onset of differentiation, i.e. on the 14th day of gestation. Binding activity in the male duct was higher than that in the female duct. Prenatal administration of testosterone during days 13-17 of gestation increased the EGF receptor concentration in the masculinized female fetuses, suggesting a role of fetal testicular testosterone in determining EGF-binding activity. Prenatal treatment of cyproterone acetate (an antiandrogen at the level of androgen receptor binding to androgen), however, produced no effect on EGF-binding activity in male fetuses. Thus, a role for the EGF receptor is indicated during Wolffian duct differentiation. Testosterone appears to play a role in modulating EGF-binding activity. Further work is necessary to determine the mechanism by which testosterone modulates EGF-binding activity in this tissue.

Immunological identification of the protein-producing masculine differentiation of the Wolffian duct in the fetal mouse: western blot analysis and determination of the biological activity.

Recently, we identified a protein in the developing male reproductive tract of the fetal mouse which induced Wolffian duct differentiation in vitro in the absence of testis or testosterone. In this paper, we further evaluated the masculinizing role of this protein using an immunological approach. Thus, we purified a 72K protein from the 18-day-old male fetal reproductive tracts, prepared an antibody against the protein, and determined the role of this protein in producing masculine differentiation of the Wolffian duct using the polyclonal antibody against the protein. We demonstrate that the antibody reacted with the purified 72K protein, producing only one immunoreactive band around the 72K region in Western blot analysis. However, it reacted with 72K and 63K proteins present in the 18-day-old male reproductive tract. Both of these immunoreactive bands disappeared when the antibody was pretreated with the purified antigen. Nonimmune immunoglobulin G (IgG) produced no reactive bands with the extract of the male reproductive tracts. The IgG preparation of the immune serum specifically prevented Wolffian duct differentiation when tested in organ culture containing 13-day-old fetal male or female reproductive tracts in the presence of testis or testosterone (1 micrograms/ml) in a dose-dependent manner. No effect was found on the development of other organs, namely testis, Mullerian duct, and urogenital sinus, that were included in the organ culture. Nonimmune IgG, as expected, produced no effect on Wolffian duct differentiation. Western blot analysis of male and female reproductive tracts indicated a difference in their reactivities with this antibody. In females, the reactivity to the proteins around the 72K and 63K regions was very weak compared to that found with male proteins in those regions. Both of the above bands of the female reproductive tract disappeared when the antibody was pretreated with an excess of female reproductive tract extract, but in males, only the 63K band disappeared, whereas its 72K band remained. In conclusion, it appears that a 72K protein of the male reproductive tract plays a role in the Wolffian duct differentiation of the fetal mouse.