Analysis of prostatic bud induction by brief androgen treatment in the fetal rat urogenital sinus

The androgen dependency of prostatic bud formation in fetal rat urogenital sinuses was studied using brief treatments with androgen, and the incorporation of androgens by the sinus mesenchyme was followed by steroid autoradiography. Urogenital sinuses from 16.5-day fetuses of both sexes were grown in organ culture and treated with androgens for periods ranging from 4 to 72 h and then transferred to control medium. A minimum treatment of 24 h was required to induce prostatic buds in male sinuses and of 36 h in all female sinuses. This difference in response disappeared after more prolonged treatment. In both sexes the number of prostatic buds increased with the time of exposure to androgens. Prostatic bud formation continued for 24-36 h after transfer to control medium. Steroid autoradiographic analysis showed that the labelled androgen was concentrated in the mesenchymal nuclei. The rate of incorporation rose steeply during the first 12 h and then more slowly. After transfer to control medium the amount of labelled androgen decreased rapidly to half within 12 h and then decreased more slowly. In the competition experiments a 200-fold excess of unlabelled testosterone or dihydrotestosterone in the labelling medium greatly reduced the nuclear labelling with [3H]testosterone.     

Age-dependent loss of sensitivity of female urogenital sinus to androgenic conditions as a function of the epithelia-stromal interaction in mice.

The ability of the female urogenital sinus to respond to androgens in forming prostate was determined by growing 13- to 18-day old embryonic female urogenital sinuses and vaginas from 1- to 30-day old mice as grafts to male hosts. All embryonic unrogenital sinuses as well as vaginas from 1-day old mice were responsive to androgens and formed prostate, whereas vaginas from mice 5- or more days old never formed prostate. To determine which tissue, the epithelium or stroma, accounts for the age-dependent loss in responsiveness of the vagina to androgens in forming prostate, recombinations composed of epithelium and stroma from 16-day old embryonic urogenital sinuses and vaginas from 1- to 20-day old mice were grown as grafts to male hosts. The developmental response of these recombinants demonstrated that the age-dependent loss in responsiveness of the intact vagina to androgens results from an age-dependent loss in the ability of vaginal stroma to participate in prostatic morphogenesis. These data emphasize the importance of stromal factors during prostatic morphogenesis and the relationship of temporal factors to developmental properties of urogenital stroma.     

Estrogen receptor expression in developing epididymis, efferent ductules, and other male reproductive organs

The distribution of estrogen receptors (ER) in developing reproductive organs of male BALB/c mice was determined by 3H-estradiol steroid autoradiography. Efferent ductules, urogenital sinus and Wolffian ducts, and their derivatives, the epididymis, ductus deferens, seminal vesicles, coagulating glands, prostate, and bulbouretheral glands (BUGs), were examined from 16 days fetal (gestation = 19-20 days) to 10 days postnatal. All fetal reproductive organs strongly expressed mesenchymal ER. Stromal cells of these organs remained ER+ at later times. However, smooth muscle cells in organs such as the ductus deferens, BUG, prostate, and caudal epididymis were only weakly ER+ or ER- after their differentiation from mesenchyme, although fibroblasts interspersed within the smooth muscle remained strongly ER+. Efferent ductules were the first site of epithelial ER expression in the developing male tract; this organ expressed epithelial ER on day 16 of gestation and subsequently. Wolffian ducts and urogenital sinus did not contain epithelial ER on day 16 of gestation. Epididymis began expressing epithelial ER soon after its differentiation, on day 19 of gestation. A clear gradient of ER expression was noted in the regions of the developing epididymis, with the efferent ducts and the initial segment of the epididymis containing 3-fold more silver grains per epithelial cell than more distal regions of the epididymis. Epithelium of the seminal vesicle and coagulating gland was initially ER-, but became weakly ER+ at day 6 postnatal and later. The epithelium of all other organs (ductus deferens, prostate, and BUGs) never expressed ER at any time.     

Androgen receptors are similar in fetal and adult rabbits

In an effort to explain the separate roles of testosterone and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) in virilizing the male fetus, we compared the binding of these androgens to cytosolic receptors from urogenital tract tissues of fetal and adult male rabbits. As measured by a direct binding assay, fetal and adult androgen receptors are similar in respect to specificity, affinity, and amount of binding. Apparent dissociation constants for dihydrotestosterone binding averaged 1.1 nM for fetal receptor and 0.8 nM for adult androgen receptors. Average apparent dissociation constants for testosterone binding were 4- to 24-fold higher than those for dihydrotestosterone in fetal and adult tissues. Nonradioactive dihydrotestosterone and testosterone competed for [3H]dihydrotestosterone binding to the androgen receptor in both adult prostate and fetal urogenital sinus in a manner consistent with their affinity for binding, whereas estradiol, progesterone, and cortisol were weak competitors for [3H]dihydrotestosterone. On sucrose density gradients, both testosterone and dihydrotestosterone were bound to a protein with a sedimentation coefficient of approximately 8S. Although androgen receptors were detectable in urogenital tubercle and urogenital sinus of both male and female fetuses on days 18 and 29 of gestation, we were unable to characterize androgen binding in fetal Wolffian ducts. The nature of the androgen receptor in this tissue remains unresolved. These findings are consistent with the hypothesis that dihydrotestosterone formation acts to amplify the androgenic signal in both the fetus and adult, but is not absolutely required for virilization.     

Metabolism of testosterone by the epithelium and mesenchyme of the rat urogenital sinus

Testosterone metabolism was measured in separated epithelium and mesenchyme from the urogenital sinuses of 17- and 19-day-old male and female rat embryos and compared with testosterone metabolism in the intact sinus. Both the epithelium and the mesenchyme converted testosterone to 5 alpha-dihydrotestosterone. The epithelium produced much more androstanedione and androsterone but less 3 alpha, 17 beta-androstanediol than did the mesenchyme. The whole sinus synthesized all four metabolites, but in different proportions, producing relatively more androsterone than either of its two component tissues. These data suggest that androsterone is formed by the joint action of epithelium and mesenchyme. Metabolism of testosterone did not differ with sex or foetal age in either of the separated tissues or in the intact sinus, implying that the failure of urogenital mesenchyme from 19-day-old female foetuses to induce prostatic morphogenesis is not due to the loss of 5 alpha-reductase. It is suggested that this lack of inductive capacity may be attributable to a decline in androgen levels with age in female mesenchyme.     

Change of mosaic pattern by androgens during prostatic bud formation in XTfm/X+ heterozygous female mice

The testicular feminization (Tfm) gene, which is characterized by a deficiency in androgen receptors, is located on the X-chromosome. Using steroid autoradiography, the mosaicism of the Tfm gene has been demonstrated in the androgen target tissues of XTfm/X+ heterozygous female mouse fetuses and the effects of androgens on the mosaic pattern analysed. In the mesenchyme of urogenital sinuses of wild-type female fetuses (X+/X+), more than 95% of the cells were androgen-receptor positive (labelled with [3H]testosterone) while in that of heterozygous fetuses (XTfm/X+), only half of the cells were receptor positive (Tfm gene inactive), and receptor-positive cells and -negative cells formed small irregular patches. When the heterozygous sinuses were cultured in vitro in the presence of androgens, the sinuses underwent male sexual development and formed epithelial buds (prostate gland rudiments) projecting into the surrounding mesenchyme. Autoradiographic analysis revealed that the mosaicism of the mesenchyme disappeared around the developing epithelial buds: almost all the mesenchymal cells in close vicinity to the buds were receptor positive while in the outer layers receptor-positive and -negative cells coexisted. The proportion of receptor-positive cells was greatly increased in the mesenchyme beneath the non-budding area of the sinus epithelium. This androgen-induced increase was observed before the onset of bud formation. The results obtained in the thymidine incorporation experiments suggest that the increase of receptor-positive cells beneath the sinus epithelium might be explained by the migratory behaviour of the androgen-incorporating cells rather than by their selective proliferation.     

Androgen dependence of growth and epithelial morphogenesis in neonatal mouse bulbourethral glands

The early development of the mouse bulbourethral gland (BUG) and the role of testosterone (T) in the normal growth and epithelial morphogenesis of this male accessory sex gland were examined. The mouse BUG differentiates from the urogenital sinus on day 17 of gestation (vaginal plug = day 0; birth = day 19), and initially consists of a solid epithelial rudiment encased in a large condensed capsular mesenchyme. The epithelium begins to branch and canalize on day 1 postnatally, and the branches enlarge and become more numerous on days 2 and 3. On day 4, secondary branches appear, and by day 6, the epithelium has become extensively arborized and almost fills the mesenchymal capsule. The BUG increases 3.9-fold in DNA content from day 0 (day of birth) to day 6 postnatally; the epithelium grows proportionately more than the mesenchyme during this period (12-fold vs. 2.3-fold). Growth of BUGs in mice castrated at birth or castrated and then treated with cyproterone acetate, an antiandrogen, over the first 6 days of life was reduced by 80%, but not abolished. Thus, the growth of the BUG is partially independent of androgens during early neonatal life. However, morphogenesis of the BUG epithelium is totally abolished in neonatally castrated mice. T replacement given to neonatally castrated mice during days 0-6 restored development to normal. T injections also reinitiated growth and morphogenesis in developmentally retarded BUGs from 6-day-old neonatally castrated mice. The partial dependence of the neonatal BUG on androgens for growth is similar to that seen in the prostate, which is also derived from the urogenital sinus. In contrast to the prostate, where neonatal castration reduces but does not abolish epithelial morphogenesis, androgen deprivation completely abolished epithelial morphogenesis in the neonatal BUG. (Endocrinology 121: 2153-2160, 1987).     

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 vivo regeneration of murine prostate from dissociated cell populations of postnatal epithelia and urogenital sinus mesenchyme

The existence of a postnatal prostate stem cell is supported by several types of evidence. Withdrawal of androgen leads to involution of the gland, but readdition can rapidly stimulate regeneration. Tissue fragments derived from mouse or rat prostatic epithelia from midgestation embryos or adult mice, when combined with tissue fragments from urogenital sinus mesenchyme and grafted under the kidney capsule, can regenerate prostatic structures. Indirect evidence supports that the stem cell population is contained within the basal layer. Purified prostatic stem cell preparations would be useful to define the physical and functional properties required for regeneration and to compare with cells that accumulate during abnormal growth states, like prostate cancer. We have developed a regeneration system using dissociated cell populations of postnatal prostate epithelia and embryonic urogenital sinus mesenchyme. Efficient in vivo regeneration of prostatic structures in the subcapsular space of the kidney was observed within 4-8 wk with as few as 103 epithelial cells from prostates derived from donors 10 d to 6 wk of age. The regenerated structures show a branching tubular epithelial morphology, with expression of a panel of markers consistent with prostate development. Donor epithelial populations can be readily infected with GFP expressing lentiviral vectors to provide integration markers and easy visualization. The cell preparations of urogenital sinus mesenchyme can be expanded in short-term in vitro culture while their inductive capabilities are retained. Further definition of the subpopulation of prostate epithelial cells containing the regeneration activity should be possible with such technologies.     

Estrogen-initiated transformation of prostate epithelium derived from normal human prostate stem-progenitor cells

The present study sought to determine whether estrogens with testosterone support are sufficient to transform the normal human prostate epithelium and promote progression to invasive adenocarcinoma using a novel chimeric prostate model. Adult prostate stem/early progenitor cells were isolated from normal human prostates through prostasphere formation in three-dimensional culture. The stem/early progenitor cell status and clonality of prostasphere cells was confirmed by immunocytochemistry and Hoechst staining. Normal prostate progenitor cells were found to express estrogen receptor α, estrogen receptor β, and G protein-coupled receptor 30 mRNA and protein and were responsive to 1 nm estradiol-17β with increased numbers and prostasphere size, implicating them as direct estrogen targets. Recombinants of human prostate progenitor cells with rat urogenital sinus mesenchyme formed chimeric prostate tissue in vivo under the renal capsule of nude mice. Cytodifferentiation of human prostate progenitor cells in chimeric tissues was confirmed by immunohistochemistry using epithelial cell markers (p63, cytokeratin 8/18, and androgen receptor), whereas human origin and functional differentiation were confirmed by expression of human nuclear antigen and prostate-specific antigen, respectively. Once mature tissues formed, the hosts were exposed to elevated testosterone and estradiol-17β for 1-4 months, and prostate pathology was longitudinally monitored. Induction of prostate cancer in the human stem/progenitor cell-generated prostatic tissue was observed over time, progressing from normal histology to epithelial hyperplasia, prostate intraepithelial neoplasia, and prostate cancer with local renal invasion. These findings provide the first direct evidence that human prostate progenitor cells are estrogen targets and that estradiol in an androgen-supported milieu is a carcinogen for human prostate epithelium.     

Androgen receptor expression in the developing rat prostate is not altered by castration, flutamide, or suppression of the adrenal axis

Mesenchymal epithelial interactions are believed to be important to the growth and development of the neonatal prostate. Prior studies in the rat ventral prostate, using autoradiography and tritiated dihydrotestosterone, indicate that androgen receptors are present in the prostatic stroma on day 3 and are detected in the epithelium by the tenth postnatal day. These findings suggested that androgen stimulation of the prostatic mesenchyme is a crucial step in the growth and development of the prostate. We have examined this developmental program directly using polyclonal antibodies that recognize specific epitopes of the androgen receptor to examine the pattern of androgen receptor expression in intact and neonatally castrate animals. In keeping with previous studies, androgen receptors are present in the prostate stroma at birth and subsequently appear in the prostatic epithelium by the 10th postnatal day. Development of androgen receptor expression in the epithelium was not changed when the animals were castrated at birth, castrated and blocked by flutamide, or castrated and given hydrocortisone to suppress the production of adrenal androgens. These findings suggest that the appearance of androgen receptors in the prostatic epithelium is programmed by androgens before birth or that factors other than testicular or adrenal androgens control the development of epithelial androgen receptors.     

Prostate formation in a marsupial is mediated by the testicular androgen 5 alpha-androstane-3 alpha,17 beta-diol

Development of the male urogenital tract in mammals is mediated by testicular androgens. It has been tacitly assumed that testosterone acts through its intracellular metabolite dihydrotestosterone (DHT) to mediate this process, but levels of these androgens are not sexually dimorphic in plasma at the time of prostate development. Here we show that the 3 alpha-reduced derivative of DHT, 5 alpha-androstane-3 alpha,17 beta-diol (5 alpha-adiol), is formed in testes of tammar wallaby pouch young and is higher in male than in female plasma in this species during early sexual differentiation. Administration of 5 alpha-adiol caused formation of prostatic buds in female wallaby pouch young, and in tissue minces of urogenital sinus and urogenital tubercle radioactive 5 alpha-adiol was converted to DHT, suggesting that circulating 5 alpha-adiol acts through DHT in target tissues. We conclude that circulating 5 alpha-adiol is a key hormone in male development.     

Induction of androgen receptor formation by epithelium-mesenchyme interaction in embryonic mouse mammary gland.

The role of tissue interaction in the development of hormone responsiveness was studied in the embryonic mammary gland of the mouse, which becomes sensitive to testosterone on day 14. Previously, the mesenchyme had been identified as the sole target tissue for the hormone, although it was also demonstrated that its response to testosterone required the presence of mammary epithelium. Using autoradiography, we now show that [3H]testosterone or [3H]5 alpha-dihydrotestosterone is bound only by those mesenchymal cells closest to the epithelial mammary bud. When mammary epithelia were experimentally associated with mesenchyme of the mammary region and cultured together for 3 days in vitro, they also became surrounded by several layers of [3H]testosterone-binding mesenchymal cells. Correspondingly, this tissue association was accompanied by a substantial increase of androgen-binding sites in the explants. No hormone-building mesenchymal cells were seen in combinations with epidermis or pancreas epithelium; only salivary epithelium showed a weak positive effect. From these results we conclude that mammary epithelium induces the formation of androgen receptors in adjacent mesenchyme and thereby controls the development of androgen responsiveness in this tissue.     

Visualization of X-chromosome inactivation mosaicism of Tfm gene in XTfm/X+ heterozygous female mice

The testicular feminization (Tfm) locus, which produces a deficiency in androgen receptors, is located on the X-chromosome. Steroid autoradiographic techniques were used to demonstrate the mosaicism of the X-chromosome inactivation in two androgen target tissues of XTfm/X+ heterozygous female mice. In the mesenchyme of urogenital sinuses of wild-type female fetuses (X+/X+), more than 95% of the cells were androgen-receptor positive (labelled with [3H]testosterone) while in that of heterozygous fetuses (XTfm/X+), about half of the cells were receptor positive (Tfm gene inactive). Statistical analysis of coherent clone size was applied to the heterozygous mesenchyme of the urogenital sinus and the coherent clone size of receptor-positive cells was estimated to be two or three cells per clone. This small clone size suggests that considerable cell mixing occurred in the tissue during embryonic development. Androgen binding in the mammary gland rudiments was restricted to the mesenchymal cells only in close vicinity to the epithelial mammary bud. In the wild-type rudiments most of the mesenchymal cells beneath the epithelium were receptor positive, while in heterozygous rudiments, receptor-positive and -negative cells intermingled. This observation suggests that in the wild-type mammary gland rudiments the epithelial bud may induce the formation of androgen receptors in adjacent mesenchymal cells rather than attract pre-existing receptor-rich mesenchymal cells.     

Regulation of overgrowth and expression of prostatic binding protein in rat chimeric prostate gland

Enlargement of the chimeric ventral prostate gland (VP) was induced by directly implanting either fetal urogenital sinus mesenchyme (UGM) or intact fetal urogenital sinus (UGS) into the VP of intact adult rats. The macromolecular content in the chimeric prostate increased from 40-100% (UGM implants) to 200-300% (UGS implants) above control levels. The enlargement of the prostate gland was the result of growth from both the donor tissue and the host gland. Growth of the donor fetal UGS within the host prostate gland may account for the difference observed between the growth induced by fetal UGS and fetal UGM implants. Because fetal UGM regressed when implanted and grown under the renal capsules, the observation of growth in the adult rat VP induced by fetal UGM, either by implanting UGM in situ or forming tissue recombinants of UGM and the adult VP, suggests that fetal UGM requires close association with the VP for the induction of growth to occur. The concentration of an epithelial androgen-dependent protein, the prostatic binding protein (PBP), expressed by the enlarged lobe of the rat VP was similar to that of the control lobe of rat VP. The adult host gland, rather than donor implants, appeared to determine the levels of expression of PBP within the chimeric prostate gland. Immunofluorescence data indicated that PBP was distributed evenly throughout most of the prostatic acini. PBP also accumulated in the lumen of the prostatic acini. Positive immunofluorescence, although less intense, was detected in the UGS remnant, suggesting that fetal UGS was induced by the intact adult VP environment to express PBP. We observed a developmental restriction in the ability of donor prostatic tissues to induce enlargement of the host prostate gland. Fetal UGS was the most effective inducer, whereas neonatal prostatic tissue was marginally effective, and adult prostatic tissue or stromal cells derived from adult VP were completely ineffective.     

Androgen aromatization and 5 alpha-reduction in ferret brain during perinatal development: effects of sex and testosterone manipulation

Ferrets of both sexes were killed 8 or 5 days before expected parturition as well as 7, 15, 30, or 51 days after birth, and the activities of aromatase (using 19-[3H]hydroxyandrostenedione as substrate) and of 5 alpha-reductase (using [3H] testosterone as substrate) were assayed in whole homogenates of preoptic area plus anterior hypothalamus (POA), mediobasal hypothalamus (MBH), temporal lobe (TL), and cerebral cortex. Aromatase and testosterone 5 alpha-reductase activities were also measured in these regions in adult gonadectomized male and female ferrets. Compared with adults of both sexes in which aromatase activity was low in all brain regions studied, fetal ferrets had high levels of aromatase activity in POA plus MBH and in TL. At these prenatal ages, aromatase activity in POA plus MBH was significantly higher in males than in females. Aromatase activity in POA, MBH, and TL remained high in both sexes on postnatal days 7, 15, and 30, before declining by postnatal day 51. Cortical aromatase activity was uniformly low across all perinatal ages. The existence of a sex difference in aromatase activity in fetal POA plus MBH cannot be explained by a concurrent sex difference in circulating testosterone. Administration of testosterone to pregnant female ferrets over days 30-41 of gestation caused 150- to 350-fold increases in maternal plasma concentrations of testosterone and 2- to 5-fold increases in fetal plasma testosterone. However, aromatase activity was not affected in the POA and MBH of fetuses or mothers, although activity was significantly increased in the TL of mothers given testosterone. Furthermore, castration of neonatal or adult breeding males decreased plasma androgen levels by factors of 8 and 480, respectively, but resulted in only modest reductions in POA, MBH, and TL aromatase activity (a significant reduction occurred only in the adult male TL). Relatively high levels of testosterone 5 alpha-reductase activity were found in all brain regions across all perinatal ages, as well as in gonadectomized adult ferrets; there was no sex differences at any postnatal age studied. Prenatally, males had higher levels of 5 alpha-reductase activity than females only on day -8 in the POA plus MBH. The results show that estrogen and 5 alpha-reduced androgens can be synthesized in the brains of ferrets of both sexes during the perinatal period of sexual differentiation. A functional role for this neural metabolism of androgen remains to be demonstrated in this carnivorous species.     

Testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone

Testosterone and dihydrotestosterone are believed to exert their androgenic effects by interacting with a single intracellular receptor protein in androgen target tissues. During fetal life, however, testosterone mediates the virilization of the Wolffian ducts into the epididymis, vas deferens, and seminal vesicles, whereas the urogenital sinus and external genitalia require the in situ conversion of testosterone to dihydrotestosterone to undergo male development. The reason why the signal provided by testosterone needs to be amplified in some androgen target tissues but not in others remains an enigma. To provide insight into the different actions of these androgens we studied their interaction with the human androgen receptor in fibroblasts cultured from the genital skin of a patient with 5 alpha-reductase deficiency. Dihydrotestosterone was formed in negligible amounts in these cells, and in some experiments the residual 5 alpha-reductase activity was further blocked with the 5 alpha-reductase inhibitor finasteride. Saturation analysis in fibroblast monolayers disclosed similar amounts of binding with testosterone and dihydrotestosterone, and the affinity of binding of dihydrotestosterone was, on the average, about 2-fold greater than that of testosterone. [3H]Testosterone also exhibited a 5-fold faster dissociation rate from the receptor than [3H]dihydrotestosterone. In thermolability experiments the [3H]testosterone-receptor complex displayed marked instability at 42 C with 2 nM [3H] testosterone, whereas with 20 nM [3H]testosterone, receptor stability was similar to that seen with [3H]dihydrotestosterone. In up-regulation experiments, 2 nM [3H]testosterone produced a 34% increase in specific androgen receptor binding after 24 h, whereas 20 nM [3H]testosterone produced an average increase of 64%. Our results suggest that the weaker androgenic potency of testosterone compared to that of dihydrotestosterone resides in its weaker interaction with the androgen receptor, most clearly demonstrable as an increase in the dissociation rate of testosterone from the receptor. When present in relatively high concentrations, however, testosterone overcomes this defect by mass action.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of human recombinant mullerian inhibiting substance on isolated epithelial and mesenchymal cells during mullerian duct regression in the rat.

The effect of human recombinant Mullerian Inhibiting Substance (MIS) on the regression of the Mullerian duct (MD) of female rat fetuses was examined in vitro to determine whether MIS acts on MD epithelium and/or mesenchyme at the critical periods of sexual differentiation. Urogenital ridges (URs) of female rat fetuses at 14.5- to 18.5-days of gestation (plug day = 0) were cultured for 3 days with or without recombinant human MIS in CMRL 1066 medium with 10% female fetal calf serum. In URs from 14.5- and 15.5-day-old fetuses, the cranial portion of the MD regressed almost completely during the 3-day culture period in the presence of MIS, whereas the caudal half to third of the MD remained intact but tapered to a fine point cranially. MDs survived in URs from 16.5-day-old fetuses cultured in the presence of MIS except that the cranial portion of the MDs was deformed. MIS did not elicit regression of MDs in URs obtained from 17.5- and 18.5-day-old fetuses, but instead caused the MD epithelium to form bulges projecting into the mesenchyme. MD epithelium at 15.5-days of gestation was separated from the surrounding UR mesenchyme, and both components (MD epithelium and mesenchyme) were cultured separately for 3 days in the presence or absence of MIS. Both epithelial and mesenchymal cells survived in the presence or absence of MIS. MD epithelium formed typical epithelial colonies, whereas UR mesenchyme spread as fibroblastic cells. Analysis of labeling index after incorporation of [3H] thymidine demonstrated that MD epithelial DNA synthesis was not influenced by MIS. In contrast, mesenchymal labeling index was reduced significantly by MIS. This effect of MIS on UR mesenchyme in conjunction with earlier histological observations of mesenchymal condensation during MD regression and an absence of direct effects of MIS on the epithelium suggests that MIS elicits its effect on the MD epithelium via the surrounding mesenchyme.     

Effects of steroid androgens on the uptake and distribution of zinc in slices from human prostates

Slices of human hyperplastic prostates were superfused with magnitude of [6 5Zn] Cl2 in the absence or in the presence of testosterone or 5 alpha - dihydrotestosterone or 5 alpha-androstane-3 alpha, 17 beta-diol. Uptake of magnitude of [6 5Zn] Cl2 was not significantly changed by steroid androgens but the binding of magnitude of [6 5Zn] to the citrate of prostatic cytosol was generally significantly decreased by the tested steroids. The 5 alpha-reduced androgens were more efficient that testosterone in decreasing bound zinc levels and increasing free zinc concentrations. Implications of these findings on the metabolism and differentiation of prostatic cells are discussed.