A new model system for studying androgen-induced growth and morphogenesis in vitro: the bulbourethral gland

An organ culture system was devised for neonatal mouse bulbourethral glands (BUGs) in which androgen-dependent development parallels that in vivo. BUGs from 0-day-old (day of birth) mice were grown on Millipore filters placed on metal grids in petri dishes for 3 or 6 days in Dulbecco's Modified Eagle's Medium-Ham's F-12 medium (1:1) containing 10% fetal bovine serum. In medium supplemented with testosterone (T; 10(-7) or 10(-8) M), growth and epithelial morphogenesis of the cultured BUGs were comparable to those of the gland in situ. At lower doses of T (10(-9) -10(-12) M), BUGs showed dose-dependent decreases in the rate of growth and degree of epithelial morphogenesis. BUGs cultured without T contained only 38% as much DNA as those in T-supplemented medium, and epithelial morphogenesis did not occur. Thus, BUG development in vitro was dependent on androgens. The continued, albeit reduced, growth in cultures without T indicates that growth is also partially independent of androgens, but epithelial branching morphogenesis is totally dependent on this hormone. Growth and epithelial morphogenesis were reinitiated in glands that had developed in the absence of T, either in vivo or in vitro, by culturing the BUGs for 3 days in T-containing medium. The growth of BUGs in a serum-free medium with or without T paralleled that in comparable serum-containing cultures in vitro and in normal and castrated animals in situ. Coincubation of BUGs with T and 390 MSD (17 beta-N,N-diisopropylcarbamoyl-4-aza-5 alpha-androstan-3-one), an inhibitor of the enzyme 5 alpha-reductase, resulted in retarded development, indicating that T must be converted to 5 alpha-dihydrotestosterone (DHT) to promote normal BUG growth. Additionally, DHT (10(-8) M) alone could substitute for T in promoting BUG development. Thus, DHT must be the proximal androgen for BUG growth. The BUG is an excellent model system for examining androgen-dependent development and should be useful for studying epithelial morphogenesis, growth, and hormonal effects in vitro.     

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.     

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.     

Androgens lower prostaglandin E2 levels in neonatal mouse bulbourethral gland in in vitro cultures.

The neonatal mouse bulbourethral gland (BUG) in vitro culture model is useful to study hormone-induced genitourinary (GU) tract growth and differentiation. Like the prostate, the BUG is a derivative of the urogenital sinus and may have relevance to understanding growth processes involved in normal and pathological GU tract development. Previous studies have reported androgen-induced elevation of prostaglandin E2 (PgE2) levels in mouse GU tract in vivo. PgE2 has been proposed to mediate neonatal GU tract masculinization. In our studies, tissues were obtained from neonatal male mice and cultured in serum-free Dulbecco's Modified Eagle's Medium-Ham's F-12 Medium (1:1) supplemented with varying concentrations of androgen. PgE2 levels were measured by RIA in the medium, and tissue specimens were cultured for 7 days or less. During this period, androgens induced proliferation and glandular morphogenesis in the BUGs. In the absence of androgen, tissue and medium PgE2 levels increased over 7 days. Significant (P < 0.05) PgE2 increases over day 1 control values were observed from days 5-7 in tissues and on day 7 in media. During this same time period, androgen supplementation decreased PgE2 levels. Significant (P < 0.05) PgE2 decreases from day 1 cultures were observed from days 3-7 in tissues and on day 7 in media. PgE2 was decreased significantly (P < 0.05) by androgen compared to control values from days 3-7 in tissues and from days 5-7 in media. On day 7 of culture, PgE2 levels were significantly (P < 0.05) inhibited by androgen in a concentration-dependent fashion in tissues and media. Maximal androgen-induced inhibition of PgE2 levels was 96% and 99% in tissues and media, respectively. Although the addition of indomethacin to control cultures markedly inhibited PgE2 production, BUG morphology was unaffected. In addition, the morphology of androgen-stimulated BUGs does not appear to be affected by the addition of exogenous PgE2. We conclude that although androgens induce development and decrease PgE2 levels, PgE2 does not appear to play a major role in in vitro BUG postnatal growth and morphogenesis. The BUG in vitro culture model may mimic growth and morphogenetic processes occurring in the human GU tract. Further understanding of the role of steroid hormones and PG metabolism may yield additional insight into developmental and proliferative GU tract disorders.     

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.     

Proliferative pattern of uterine cells from birth to adulthood in intact, neonatally castrated, and/or adrenalectomized mice, assayed by incorporation of [125I]iododeoxyuridine

The proliferative pattern of uterine cells from birth to adulthood was investigated in mice. The uptake of 5-[125I] iodo-2'-deoxyuridine [( 125I]dUrd) by the whole uterus was used as an index of cell proliferation. Nearly parallel changes in uterine growth were found from days 0-25 after birth in both intact and neonatally castrated mice. In both groups of mice, the weight of the uterus increased (0.7-6 mg) and high [125I] IdUrd uptake values were found from days 0-15, while the weight remained nearly constant, and very low uptake values were found in the next 10 days. Additionally, we could demonstrate that adrenalectomy plus ovariectomy caused no significant effect on neonatal growth of the uterus. After day 25, the weight of uterus (6-50 mg) and [125I]IdUrd uptake increased again in the intact mice, but remained low in the neonatally castrated mice. The proliferative response of the uterine cells to exogenous 17 beta-estradiol was then examined. The injection of 17 beta-estradiol on days 0 (20 micrograms/mouse) and 10 (5 micrograms/mouse) induced significant increases in [125I]IdUrd uptake the next day. Neonatal castration had no significant effect on the responsiveness of the uterus to estrogen-induced growth in adult mice. These findings suggest that sex steroids secreted from both the ovaries and adrenals of neonatal and prepubertal mice play no significant role in the proliferation of uterine cells, and that a quiescent interval of cell proliferation occurs around day 20 after birth between the autonomous (days 0-15) and the ovary-dependent (after day 25) proliferation of mouse uterine cells.     

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.     

Morphogenetic and proliferative effects of testosterone and insulin on the neonatal mouse seminal vesicle in vitro

Effects of testosterone (T) and insulin on epithelial branching morphogenesis were investigated in cultured seminal vesicles (SVs) of neonatal mice. SVs from 0-day-old male mice were cultured for 0.5-6 days in serum-free chemically defined medium in the presence of T (10(-7) M), insulin (10 micrograms/ml), or T plus insulin or in medium lacking both hormones. Without the addition of both hormones, SVs failed to grow, based on DNA and protein contents, and did not show any epithelial branching morphogenesis. T induced a 2.5-fold increase in protein content in SVs cultured for 6 days and elicited modest epithelial branching morphogenesis. Insulin increased the protein content of cultured SV rudiments as much as T, but failed to elicit epithelial branching morphogenesis. The combination of both hormones induced a 4.3-fold increase in protein content in cultured SVs and elicited more extensive epithelial branching morphogenesis than T alone. Epithelial and mesenchymal DNA contents in SVs declined slightly during the first 12 h of culture in all treatment groups. The epithelial DNA content in SVs grown with insulin alone remained constant thereafter, while that of SVs grown with T alone or in combination with insulin increased 1.2- and 2.6-fold, respectively. In the absence of both hormones, the mesenchymal DNA content remained constant in SVs grown for 6 days after the initial decline in DNA content. In contrast, mesenchymal DNA content was increased to the same degree (1.4-fold) by either T or insulin alone or in combination. The labeling index with [3H]thymidine of SV epithelium and mesenchyme grown under the hormonal conditions described above corroborated the results of epithelial and mesenchymal DNA contents. These data indicate that insulin by itself has no effect on epithelial proliferation and branching morphogenesis in the neonatal mouse SV, but, instead, amplifies the morphogenetic and proliferative effects of androgen on the developing mouse SV, thus eliciting extensive branching morphogenesis. Both insulin and T have a slight (nonsynergistic) effect on proliferation of SV mesenchyme. Analysis of androgen metabolism in developing mouse SVs indicated that dihydrotestosterone was the major product when T was used as substrate in SVs grown under all hormonal conditions. The rate of DHT production per 100 mg protein was not significantly different among the different treatment groups. Higher levels of 5 alpha-androstane-3 alpha,17 beta-diol were detected in SVs grown in the absence vs. the presence of T regardless of the presence or absence of insulin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inability of Tfm (testicular feminization) epithelial cells to express androgen-dependent seminal vesicle secretory proteins in chimeric tissue recombinants

To assess the role of androgen receptors (ARs) in the expression of androgen-dependent seminal vesicle (SV) secretory proteins, tissue recombinants were prepared with rat seminal vesicle mesenchyme plus ureter epithelium of wild-type or Tfm mice (rat SVM plus wild-type mouse URE and rat SVM plus Tfm mouse URE, respectively). After growth in male hosts, both the wild-type and Tfm ureter epithelia were induced by SVM to differentiate into a simple columnar epithelium exhibiting the complex folded morphology characteristic of the SV. In SVM plus wild-type mouse URE recombinants, epithelial ARs were induced, and the epithelium expressed the full spectrum of SV secretory proteins. By contrast, in SVM plus Tfm mouse URE recombinants, the Tfm epithelium was genetically incapable of producing functional ARs and failed to produce SV secretory proteins. These data demonstrate in vivo that the induction of SV secretory proteins by androgens is an event requiring intraepithelial ARs. In contrast, androgen-dependent epithelial morphogenesis, columnar cytodifferentiation, and probably also proliferation can be expressed in Tfm epithelium grown in association with wild-type mesenchyme, strongly suggesting that these events are indirect effects on the epithelium mediated by mesenchymal ARs.     

The in vitro effect of triamcinolone acetonide on branching morphogenesis in the fetal rat lung.

We have studied the effect of triamcinolone acetonide (TAC) on airway morphogenesis of the Sprague-Dawley fetal rat in whole organ lung cultures from day 15 to day 21 of equivalent gestational age (6 days in culture). TAC produced an increased number of peripheral buds from day 18 onward and the airway and airspaces had larger lumens. Airway branching was increased compared to controls, and there was a higher proportion of airway epithelium and a lower proportion of mesenchyme. Cell height was significantly lower in TAC treated lungs except on day 17. This was due to accumulation of glycogen prior to the increased branching activity. In both controls and TAC-treated lungs, peripheral bud number and volume proportion of epithelium increased with time in culture, whereas volume proportion of mesenchyme, mean chord length of airways and airspaces, and epithelial cell height decreased. These changes were more pronounced in the TAC-treated group and were significant. However, TAC-treated lungs were morphologically irregular. We conclude that TAC has a direct effect on airway morphogenesis and it promotes growth of morphologically abnormal lungs. TAC also appears to enhance airway branching and morphologic changes interpreted as increased epithelial maturation.     

Autoradiographic studies of androgen-binding sites in the rat urogenital sinus and postnatal prostate

Binding sites of [3H]testosterone and [3H]dihydrotestosterone in the rat fetal urogenital sinus and postnatal prostate and vagina grown in vitro were examined by steroid autoradiography. Distinct nuclear incorporation of both androgens appeared between 14.5 and 16.5 days of gestation in rat fetuses. Nuclear labelling in the sinus was restricted to the mesenchyme surrounding the epithelium which showed no nuclear labelling. A similar distribution of labelled cells was observed in male and female sinuses up to 18.5 days of gestation. By 20.5 days of gestation, the labelling in the ventral mesenchyme of female urogenital sinuses became less intense but persisted in the mesenchyme of the dorsal sinus wall from which the vagina is formed. In the postnatal prostate, the epithelium showed nuclear [3H]testosterone labelling at 10 days coinciding with the onset of its functional differentiation. Epithelial labelling became more intensive at 4 weeks post partum while that of the mesenchyme declined. The results suggest two phases of androgen action: formation of the prostatic buds mediated by the androgen-activated mesenchyme of the fetal urogenital sinus and the differentiation of the postnatal prostatic epithelium directly stimulated by androgens.     

The content of a specific cell product in the vaginal epithelium of normal and neonatally estrogenized mice: its dependence on an estradiol-prolactin interaction.

The amount of an immunological marker (CVA) in mucified cells of the mouse vaginal epithelium was quantified by a mixed hemagglutination technique for tissue sections. Immature mice, adult mice which had been estrogenized neonatally (5 mug diethylstilbestrol daily for the first five days after birth), and adult non-estrogenized mice were studied. All adult animals were castrated 7-10 days before starting the experiments. Injections of 5 mug estradiol-17beta (48 and 24 h before killing the animals) increased the amount of CVA in all three groups of animals, but most markedly in the neonatally estrogenized mice. The amount of CVA found following estradiol treatment was decreased in adult animals injected with the ergot alkaloid CB154 (0.5 mg twice daily for 6 days) in addition to the hormone. This partial block of the estradiol-induced CVA response by CB154 was relieved by exogenous rat prolactin. The CVA content in immature animals was not influenced by CB154, given alone or together with estradiol. Combined treatment with estradiol and rat prolactin (3 mug every 8 h for 6 days) increased more efficiently than estradiol alone the amount of CVA in immature and adult nonestrogenized animals. Prolactin injected alone had no effect on the CVA content. These data strongly suggest a synergistic action of estradiol and prolactin in augmenting the epithelial CVA content. Explants of the vaginal wall from normal and neonatally estrogenized mice were grafted into the thigh muscles of newborn mice, every host carrying one graft from both types of animals. The CVA content in the epithelium of the two grafts increased to the same level in response to estradiol. When the hosts were injected with estradiol and prolactin, the CVA content was higher in grafts from estrogenized donors than in those from nonestrogenized animals. Our results demonstrate that the mucified vaginal cells in adult, neonatally estrogenized mice have a content of CVA which is higher than in nonestrogenized animals. This difference may be ascribed to hormonal factors (estradiol-prolactin) as well as to persistent effects in the vaginal cells as a result of the neonatal estrogen treatment.     

Quantitative analysis of the development of genital organs from the urogenital sinus of the fetal male mouse treated prenatally with a 5 alpha-reductase inhibitor

The role of 5 alpha-dihydrotestosterone (DHT) in the development of the genital organs and in the differentiation of the genital tract into prostate, coagulating gland (CG), bulbo-urethral gland (BUG) and seminal vesicle (SV) in male mice exposed prenatally to the 5 alpha-reductase inhibitor 6-methylene-4-pregnene-3,20-dione (6-MP) has been examined quantitatively. Female ICR mice were given 7 daily s.c. injections of the inhibitor (400 mg/day) starting on day 12 of gestation and the experiment was terminated on day 19 when the fetuses were removed by Caesarean section. In the prenatally 6-MP-exposed male mice the anogenital distance was significantly shorter than in the controls. Feminization of the nipples and hypospadias of the phallic urethra were noted. Development of prostate, CG and BUG was significantly suppressed. SV and testis development were not affected. These results lend further support to the conclusion that DHT is necessary for the development of the urogenital sinus (prostate, CG and BUG) and penis, and for the regression of the nipples in male mice. Reproductive abnormalities were not found in 90-day-old mice of both sexes exposed to 6-MP in utero. The 6-MP-exposed male and female mice had a normal reproductive capacity when mated with normal mice. These results show that 6-MP-induced growth retardation of reproductive organs is evident on day 19 of gestation, but that such retardation is no longer apparent in the adult.     

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.     

Suppression of the development of female hamster behaviour by implants of testosterone and non-aromatizable androgens administered neonatally.

Testosterone in its free form, and dihydrotestosterone (DHT) and androsterone, both androgens which are not aromatizable to oestrogen, injected in oil during the neonatal period have been reported not to modify the development of female sexual behaviour. This failure might be due to the short period of activity of these substances when injected in liquid vehicles. In the current study, a Silastic pellet containing 9% of its weight of testosterone, androsterone, or DHT was implanted subcutaneously in 42 female and 38 neonatally castrated male hamsters on day 2 of life and removed on day 10. Pellets of pure Silastic were implanted in 36 control animals. Males were gonadectomized on day 5 and females on day 45. Female sexual behaviour induced by oestradiol benzoate and progesterone was measured in a series of 10-min mating tests with vigorous males, starting at 55 days of age. The duration of lordosis was consistently reduced below control levels in females implanted with testosterone, DHT, and androsterone, and in males, with testosterone and DHT. Thus the free form of testosterone, and some non-aromatizable androgens, when present for a sufficiently long period after birth, can permanently suppress development of female reproductive behaviour.     

Androgen dependence during development of the mouse vas deferens protein mRNA.

The mRNA encoding a major protein of the mouse vas deferens (MVDP) was first detected in 10-day-old males and its concentration increased sharply between 10 and 20 days, reaching adult levels at 40 days. This increase was not associated with an increase in tissular androgen concentrations. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP mRNA levels were not abolished and were similar to those measured in 10- and 20-day-old controls. These results suggest that the neonatal expression of MVDP gene is independent of androgens. In addition, precocious accumulation of MVDP mRNA could be induced by injection of excess amounts of androgens in 20- but not in 10-day-old animals. The prepubertal increase in MVDP mRNA levels is androgen-dependent but other factors may be necessary for MVDP expression.     

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.     

Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development.     

Comparison of the effect of oestradiol,tamoxifen and raloxifene on nerve growth factor-alpha expression in specific neonatal mouse uterine cell types using laser capture microdissection

Oral dosing of CD-1 mice on days 2-5 after birth with tamoxifen but not raloxifene disrupts the development of the myometrium, resulting in adult uterine adenomyosis. Using laser capture microdissection and RT-PCR we have investigated nerve growth factor (NGF) and cognate receptor expression in uterine cells of 6-day-old pups that may be important in early developmental changes that give rise to adenomyosis. NGF down-regulation is known to occur during terminal myogenic differentiation. NGF was found exclusively in endometrial luminal epithelium of controls. It was up-regulated 18-fold in the luminal epithelium following dosing with tamoxifen but not raloxifene. Western blotting for NGF protein in the whole uterus showed a 25-fold increase after tamoxifen treatment. Expression of the low affinity p75 neutrophin receptor (p75(NTR)) was twofold higher in the myometrium compared with luminal epithelium or stroma. This was not altered following tamoxifen treatment. There was no detectable expression of high affinity tyrosine kinase receptor (trkA(NGFR)). This study shows luminal epithelial cells of the endometrium primarily form NGF. This suggests that NGF normally regulates the differentiation of the mesenchyme into uterine myocytes through paracrine mechanisms and that an early disturbance of this process plays a key role in the subsequent development of adenomyosis.