Transdifferentiation of adult rat stem Leydig cells into prostatic and uterine epithelium, but not epidermis.

Stem Leydig cells (SLCs), precursors of testicular Leydig cells that secrete testosterone required for male sexual differentiation, spermatogenesis, and fertility, were recently identified in rat testes. Various types of stem cells have shown the ability to differentiate into other tissues, but there is no information on the plasticity of adult rat SLCs (rSLCs). This study investigated the ability of rSLCs to transdifferentiate into cell types from all three germ layers-prostatic epithelium (endoderm), uterine epithelium (mesoderm), and epidermis (ectoderm)-under the influence of inductive mesenchyme from fetal and neonatal tissues. To differentiate rSLCs into cells of other lineages, mesenchyme from green fluorescent protein (GFP)-expressing mice was used. Tissue recombinants of urogenital sinus mesenchyme (a potent prostate inducer) and rSLCs grafted into adult male hosts formed ductal structures resembling prostate after 5 weeks. Prostate epithelium was of rSLC origin as determined by absence of GFP expression, and expressed characteristic markers of prostatic epithelium. Similarly, uterine mesenchyme + rSLCs tissue recombinants contained a simple columnar epithelium that was histologically similar to normal uterine epithelium and expressed typical uterine epithelial markers, but was of rSLC origin. In contrast, epidermal tissue was absent in fetal dermis + rSLCs recombinants, suggesting rSLCs did not form skin epithelium. Thus, rSLCs can transdifferentiate into uterine and prostatic epithelium, mesodermal, and endodermal derivatives, respectively, but they may have a limited transdifferentiation potential, as shown by their inability to form epidermis, an ectodermal derivative.

Role of insulin in the growth of fetal rat tissues.

The effect of insulin on the growth of fetal rat tissues was investigated using a transplant system. Paws from 15-day-old fetal rats were transplanted under the kidney capsule of 1-month-old syngeneic hosts, where they grew and differentiated normally. After 11 days of incubation, growth of transplants in hosts made diabetic by streptozotocin injection was reduced by 37% compared to growth in nondiabetic controls, but tissue differentiation and bone formation were normal in the absence of insulin. Injections of insulin (2 U, twice daily) into diabetic hosts restored paw growth to normal. Growth of transplants in hypophysectomized (HX) and in HX-diabetic hosts was reduced to the same degree (i.e. by 65%). Thus, the growth decrements produced by host hypophysectomy and diabetes are not additive. In contrast to the results with insulin-deficient hosts, the transplants failed to differentiate normally in the HX hosts. Injections of exogenous insulin (3 U, twice daily) to produce transient hyperinsulinemia failed to increase transplant growth in intact hosts over 11 days of incubation. The transplants were exposed to frequent periods of hyperglycemia and hyperinsulinemia by injecting 0.66 g glucose/100 g BW four times per day into intact hosts during 6 days of incubation. This treatment also failed to stimulate transplant growth. These results indicate that normal growth of transplanted fetal paw tissue is partially dependent on insulin, but whether the insulin acts directly or indirectly to support growth is not known. Supranormal insulin levels or frequent periods of hyperglycemia with hyperinsulinemia are not capable of producing overgrowth of the fetal paws. The HX, diabetic, and HX-diabetic host rats did not grow, as judged by tail length increase, and they lost weight. Accordingly, the juvenile host tissues have an obligatory dependence on insulin and GH for normal growth, but the fetal tissue is only partially dependent, because the paw transplants continued to grow in an internal milieu that was severely deficient in insulin and/or pituitary hormones. Overall, the results indicate that insulin is necessary for normal growth of the skeletal and connective tissues of the transplanted paw, but its effects are more growth supporting than growth promoting in these tissues.

Early hypothyroidism in rats causes increased adult testis and reproductive organ size but does not change testosterone levels.

The role of thyroid hormones in the testis is unclear, although recent evidence indicates they may be important for testicular development. Here we describe a novel method for increasing adult testicular size in the rat by induction of transient hypothyroidism during neonatal life. Rats were treated with a reversible goitrogen, 6-propyl-2-thiouracil from birth to day 25 when treatment was stopped, allowing return to a euthyroid state. At days 90, 135, 160, and 180, wt and DNA content of the testis, epididymis, ventral prostate, seminal vesicle, and those of some nonreproductive organs were determined, as well as serum levels of testosterone (T) and thyroid hormones. Despite decreased body wts in 90-day and older 6-propyl-2-thiouracil-treated rats, testis wt was increased by 40% and 60% at 90 and 135 days, respectively; maximal increase (80%) occurred at 160 days. These wt increases were accompanied by proportional changes in DNA content. Significant enlargements were also seen in other reproductive organs, but they occurred after a time lag and were smaller in magnitude. Interestingly, serum T levels showed no increase at any age. Weight and DNA content of nonreproductive organs, like body wts, were less than controls at all ages but thyroid hormone levels were normal. Thus, transient hypothyroidism in neonatal rats is associated with lasting enlargements in the ultimate size of testis and other reproductive organs in the adult. These changes are not related to excess T levels. The results indicate early critical influences of thyroid hormones on growth and development of the reproductive system and suggest an experimental model for inducing lasting enlargements in testis and reproductive organs. The model may also be useful for studying regulation of reproductive growth and final size.

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.

Thyroid hormone and follicle-stimulating hormone regulate Müllerian-inhibiting substance messenger ribonucleic acid expression in cultured neonatal rat Sertoli cells.

Thyroid hormone is a major regulator of Sertoli cell development, and the present study sought to determine the role of T3 in Müllerian-inhibiting substance (MIS) messenger RNA (mRNA) expression. MIS, a Sertoli cell secretory protein that induces Müllerian duct regression and also may be critical for germ and Leydig cell development, is maximal perinatally, then decreases as Sertoli cells mature. The fall in MIS mRNA expression is delayed by hypothyroidism in vivo, indicating that T3 could regulate MIS mRNA. However, understanding of the hormonal regulation of MIS has been limited due partly to the lack of a primary Sertoli cell culture system in which sustained expression of MIS or its mRNA can be obtained. We have developed a Sertoli cell culture system for examining hormonal regulation of MIS mRNA. We then tested the effects of T3 and/or FSH treatment on MIS mRNA levels in this new system. Initial studies indicated that MIS mRNA production by 5-day-old rat Sertoli cells was minimal in vitro. Therefore, Sertoli cells from 2-day-old rats were cultured for 2 or 4 days. After 2 days in vitro, steady state MIS mRNA levels were decreased to 36% of the levels seen in freshly isolated Sertoli cells from 2-day-old rats. However, by day 4 of culture, steady state MIS mRNA production had recovered to 67% of that seen in freshly isolated 2-day-old Sertoli cells, which closely paralleled the decrease seen in MIS production in vivo from days 2-6. MIS mRNA levels were decreased 53%, 64%, and 86% in cultures treated with 0.01, 0.1, and 1.0 nM T3 (P < 0.05), respectively. This decrease in Sertoli cell MIS mRNA did not reflect a nonspecific effect on cell viability and/or activity, as shown by a dose-responsive increase in inhibin-alpha mRNA in these same cultures. FSH (2.5-100 ng/ml) also produced a dose-responsive decrease in MIS mRNA levels, and FSH and T3 together had an additive inhibitory effect on MIS mRNA levels, indicating that these hormones may act through distinct mechanisms. In summary, this is the first primary culture system in which sustained MIS mRNA production can be demonstrated, and it should prove useful for understanding the regulation of MIS in developing Sertoli cells. In addition, T3 and FSH are major regulators of the postnatal decrease in MIS production by the rat Sertoli cell, and these hormones may act through separate pathways.

Adult testicular enlargement induced by neonatal hypothyroidism is accompanied by increased Sertoli and germ cell numbers.

Our previous studies have shown that transient neonatal hypothyroidism, induced by treatment with the reversible goitrogen 6-propyl-2-thiouracil (PTU), increases testicular size and daily sperm production in the adult rat by up to 82% and 136%, respectively. The objective of the present study was to examine morphological and functional changes in adult seminiferous tubules associated with PTU-induced increases in testicular size and sperm production. Sprague-Dawley rats were treated with PTU from birth to day 25 or left untreated; for morphometry, all testes were fixed by vascular perfusion at 90 days of age. Although testicular weight was increased 62% in treated rats, gross pathological changes were not evident in these organs, and spermatogenesis appeared morphologically normal. The percent area of testis occupied by seminiferous tubules was equal in control and treated testes, but mean seminiferous tubule diameter and length were increased in the PTU-treated testis. The adult number of Sertoli cells in treated testes was increased by 157%, and the numbers of leptotene spermatocytes and round spermatids were increased 84% and 93%, respectively. These results demonstrate that increases in Sertoli cell numbers result in increased sperm production and support the idea that Sertoli cells are the major regulators of the magnitude of sperm production. Although the round spermatid to Sertoli cell ratio was reduced by nearly 30%, the number of round spermatids per g testis was increased by 14%. This increased efficiency of sperm production was accomplished by an increased density of Sertoli cells along the basement membrane and an increased height of the seminiferous epithelium. Despite the large increase in Sertoli cell numbers in treated rats, Northern blot analysis using Sertoli cell-specific cDNA probes for transferrin and androgen-binding protein indicated that relative steady state levels of mRNAs per Sertoli cell for these two secretory proteins were similar in control and treated rats at 90 days of age.

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.

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.

Development of thyroid hormone dependence for growth in the rat: a study involving transplanted fetal, neonatal, and juvenile tissues.

The role of thyroid hormones (TH) in the growth of rat tissue of different ages was investigated in a transplant system. Paws from 14- to 15-day-old fetal rats or pieces of xiphoid cartilage from 18- to 20-day fetuses and 2-, 9-, or 30-day-old rats were transplanted under the kidney capsule of control or thyroidectomized (TX) syngeneic adult female hosts and grown there for 11 days. Paw transplants grew equally well, and tissue differentiation was similar in control and TX hosts. The degree of ossification was measured by a histometric procedure and was found to be equivalent in paws that developed in situ until 4 days postpartum and in paw transplants of equivalent age grown for 11 days on the kidneys of euthyroid or TX hosts. These results show that growth and differentiation of the skeletal and other tissues of the paw are not dependent on TH during the transplant period, which encompassed the equivalent of the late fetal and first few days of neonatal life. Transplanted xiphoid cartilage was used to analyze growth and the development of TH dependence of a differentiated tissue which is relatively uniform in animals of different ages. In control and TX hosts, the growth capacity of transplanted xiphoids declined progressively with increasing donor age. The tissue also showed an age-related increase in dependence on TH for growth. The xiphoid transplants from the fetal rats grew equally well in the euthyroid and hypothyroid hosts, but those from 30-day-old rats showed a near-total dependence on TH for growth. Overall, the results indicate that rat supportive tissues begin to develop a dependence on TH for growth during the second week postpartum.

The internal milieu of the pregnant rat is not growth promoting for maternal tissues and it reduces growth of fetal paw transplants.

We have investigated whether the internal milieu of the pregnant female rat is growth promoting to fetal or adult tissues. Adult female littermates were divided into two groups, one of which remained virginal while the other was bred repeatedly. Four successive pregnancies did not affect skeletal growth (i.e. tail length increase) or BW gain. Three-month-old females were hypophysectomized (HX) or sham HX on day 13 of pregnancy. Age-matched virgins were treated similarly. Eight days after surgery, the width of the tibial epiphysial plate of the HX virgins had decreased from 146.2 +/- 5.6 mu to 98.4 +/- 2.5 mu. In the pregnant rats, the width of the plate in the shams was reduced to 120.8 +/- 7.6 mu, and hypophysectomy tended to further reduce its width to 103.2 +/- 8.4 mu. Paws from 14- to 15-day fetal rats were transplanted under the kidney capsule of adult nonpregnant or pregnant hosts and grown there for 6 or 11 days. In the nonpregnant hosts the paws grew substantially during both periods, but in gravid females their growth was inhibited by about 35%. Paws were also transplanted to the kidney of HX pregnant and HX nonpregnant females to determine whether the internal milieu of the gravid female retains more growth-promoting/growth-supporting activity after pituitary removal than does that of HX virgin hosts. In both types of HX hosts, transplant growth was inhibited to the same degree (65-70%) relative to growth in intact hosts. The growth-inhibiting properties of the internal environment of the gravid hosts were not due to ovarian steroids because injections of estradiol plus progesterone had no effect on transplant growth. We also tested the effects of placental transplants and placental extracts on the growth of transplanted paws in juvenile female hosts. Both placental treatments caused inhibition of transplant growth by 25-30%. Overall, our results indicate that the internal milieu of the pregnant rat is not somatotrophic for her tissues, and it actually suppresses growth of fetal tissue transplants. The placenta may be responsible for this growth-inhibitory effect.

A transplant system for studying hormonal control of growth of fetal rat tissues: effects of hypophysectomy growth hormone, prolactin, and thyroxine.

Much uncertainty still exists about whether mammalian fetuses are dependent on and/or responsive to hormones that are growth promoting postnatally. To allow this problem to be investigated more meaningfully, a transplant system was developed. Paws from 14- to 15-day-old fetal rats were incubated under the kidney capsules of 28- to 30-day-old syngeneic female hosts for up to 11 days. After an initial lag period, the growth rate of the transplants was similar to that of in situ paws. The implants differentiated normally, and their histological structure as well as various biochemical indices were similar to those of in situ paws. Growth of paws in hypophysectomized (HX) hosts was reduced to 35% of that of transplants in intact hosts, but the HX hosts themselves showed total cessation of growth. Injections of T4 or PRL into HX hosts did not improve transplant growth, but GH restored paw growth almost completely. The combination of GH plus T4 was no more effective than GH alone. Doses of GH (in HX hosts) that restored growth of the fetal tissue to 80-95% of that of paws in intact hosts caused only a 20-60% restoration of growth of the HX hosts themselves. Our results indicate that the internal milieu of juvenile female rats is as suitable for the growth of fetal paws as the normal intrauterine environment. Furthermore, GH is needed to maintain that internal milieu for optimal growth of fetal tissue. Although the tissues of the fetal paw are less dependent on GH than are those of the juvenile hosts, they appear to be more responsive to the hormone.

Increased sperm production in adult rats after transient neonatal hypothyroidism.

In the preceding paper it was shown that transient neonatal hypothyroidism induced by treatment of rats from birth to day 25 with the goitrogen 6-propyl-2-thiouracil (PTU) is associated with increases in testis wt and DNA content of up to 80% during adulthood. The testis changes were accompanied by similar, though less marked, increases in the wt and DNA content of epididymis and accessory organs. The purpose of this study was to assess sperm production in these enlarged testes and measure changes in sperm reserves in the epididymis. Testes and epididymides were obtained from control rats or rats given PTU from birth to day 25 (designated "treated") at 90, 135, 160, and 180 days of age. Daily sperm production (DSP), efficiency of sperm production (DSP/g testis), and epididymal sperm reserves were measured in all animals. Compared to controls, DSP of the treated rats was increased by 83%, 86%, 136%, and 132% at 90, 135, 160, and 180 days, respectively. Thus, in the treated rats, DSP, like testis wt, plateaued at day 160. In addition, efficiency of sperm production was increased by 15%-30% at all ages in treated animals. Epididymal sperm reserves were also increased in treated rats at all ages, but the correlation between DSP and epididymal sperm reserves was weak. Sperm motility and concentration in caudal epididymal fluid of adult males treated from birth to day 25 with PTU were normal. These males were fertile and sired litters in which pup wt and pup number were normal. These results indicate that neonatal hypothyroidism in rats is associated not only with increased testis size but also with increased efficiency of sperm production, resulting in increases in DSP of up to 140% in these animals during adulthood. Maximal sperm production is reached at 160 days of age in treated rats (compared to 100 days in controls), coinciding with the attainment of final testicular size. This system represents the first experimental model in which such large increases in sperm production can be produced. The neonatal PTU treatment does not appear to impair fertility or alter sperm characteristics when these animals become adults and may be a useful system with which to study factors which normally regulate sperm production.

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).

Leydig cells increase their numbers but decline in steroidogenic function in the adult rat after neonatal hypothyroidism.

Administration of the goitrogen, 6-propyl-2-thiouracil (PTU), to suckling rat pups from birth through day 24 postpartum as a 0.1% solution in the mother's drinking water increases adult testis size and sperm production by about 80% and 140%, respectively, without affecting peripheral testosterone levels. The objectives of this study were to determine whether adult Leydig cell numbers were altered in PTU-treated rats and whether the steroidogenic function of these cells was normal. The number of Leydig cells per testis at 180 days increased by 69% in PTU-treated compared to control rats, whereas the average Leydig cell volume declined by about 20%. Steroidogenic function assessed in isolated adult Leydig cells decreased after neonatal PTU treatment. LH-stimulated testosterone production was reduced by 55% in Leydig cells from treated rats, commensurate with a 50% decline in the number of hCG-binding sites in these cells. The difference in steroidogenic potential was even more striking after incubations with saturating concentrations of steroid substrate, 22(R)-hydroxycholesterol; Leydig cells from treated males produced 73% less testosterone than controls. Therefore, this decrease in testosterone production may be partially due to a reduction in the numbers of LH receptors, but also reflects the impaired steroidogenic potential of these cells. These results clearly show that the dramatic increase in adult Leydig cell number after neonatal PTU treatment is counterbalanced by a permanent decline in Leydig cell steroidogenic function, producing no net change in peripheral testosterone levels.

Androgen receptor expression and 5 alpha-reductase activity along the proximal-distal axis of the rat prostatic duct.

The rat prostate consists of a series of branched ducts that eminate from the urethra. Heterogeneity of rat prostatic growth, secretory activity, and cell turnover has been observed along the proximal-distal axis of the branched ductal network. In addition, there are regional differences in androgen sensitivity along the ducts, with the distal ductal tips being highly androgen dependent and the proximal regions being relatively androgen independent. To determine the underlying mechanisms that may regulate these regional differences in androgen responsiveness, androgen receptor (AR) levels and 5 alpha-reductase activity were examined along the proximal-distal axis of microdissected ventral prostatic ducts from 15-, 30-, and 100-day-old rats. As in the murine prostate, DNA synthetic activity was concentrated in the distal tip region of the 15- and 30-day ducts. Immunocytochemistry and autoradiography with [3H] dihydrotestosterone were used to examine AR expression and functional ability to bind ligand, respectively. The results revealed no discernable differences in AR levels or binding activity in any cell type along the ductal length in prepubertal, pubertal, or adult rats. In addition, 5 alpha-reductase activity was the same in the distal and proximal ductal regions. We conclude that regional heterogeneity in prostatic growth and function is not a result of differences in levels of AR and 5 alpha-reductase. Rather, other region-specific structural, intracellular, or paracrine factors may be responsible for the differences in androgen responsiveness along the prostatic duct.

Developmental hormonal profiles accompanying the neonatal hypothyroidism-induced increase in adult testicular size and sperm production in the rat.

Neonatal treatment with the reversible goitrogen 6-N-propyl-2-thiouracil (PTU) results in a near doubling of testicular size and a 25% increase in the efficiency of spermatogenesis, without affecting circulating testosterone (T) levels in adult rats. The objectives of the present study were to examine the effects of neonatal PTU treatment on the pattern of testicular growth and circulating levels of anterior pituitary (FSH, LH, PRL, GH, and TSH), gonadal [immunoreactive inhibin-alpha (irI alpha) and T], and thyroid (T3 and T4) hormones over the first 100 days of life. Treatment of rats with PTU from birth to 24 days of age significantly reduced testicular weights between 10 and 60 days of age. However, the duration of testicular growth was extended in treated males, resulting in a 68% increase at 100 days of age. Serum gonadotropin levels in treated males were reduced throughout the experimental period, typically remaining between 50-70% of control levels. The characteristic robust prepubertal FSH peak was absent in PTU-treated males. Initially high until 20 days of age, irI alpha levels characteristically declined to adult levels (200-300 pg/ml) in control males. In treated males, irI alpha levels were reduced during the period of hypothyroidism, increased between 30 and 60 days, and then declined, but remained significantly higher (1.7- to 2-fold greater) than those observed in control males. Serum T levels were similar in treated and control males. Control males demonstrated increased T levels beginning at 45 days of age, earlier than observed in treated males; however, similar peak T levels were observed in all males. PTU treatment significantly suppressed serum GH and PRL and led to a 14-fold increase in circulating TSH during the period of treatment. However, unlike the gonadotropins, these hormones returned to control levels after PTU treatment, suggesting that the reduced levels of FSH and LH observed are not due to a generalized reduction in pituitary function. Serum T4 and T3 levels returned to control levels within 15 days after the removal of PTU. These results demonstrate that the neonatal PTU treatment-induced increases in adult testicular size and sperm production were not due to increased levels of FSH at any point in development. On the contrary, the observed increases occur in spite of chronically reduced FSH levels.

The differential fate of mesonephric tubular-derived efferent ductules in estrogen receptor-alpha knockout versus wild-type female mice.

We investigated mesonephric tubular-derived efferent ductules in female wild-type (WT) and estrogen receptor-alpha knockout (ERalphaKO) mice from late fetal to adult life. On gestational day 17, efferent ductules in both fetal WT and ERalphaKO females were well developed and morphologically similar, although one third the size of the male counterpart. Unexpectedly, efferent ductules with a ciliated epithelium were still present on postnatal day 10 in WT and ERalphaKO females. By day 23, however, marked phenotypic differences occurred in efferent ductules of WT and ERbetaKO vs. ERalphaKO female mice. In the latter, efferent ductules became hypertrophied and dilated, whereas only small tubules remained in WT and ERbetaKO adult mice. The serum testosterone concentrations were similar in 21- to 25-day-old ERalphaKO, heterozygous, and WT female mice, suggesting that increased testosterone was not inducing enlargement of efferent ductules in ERalphaKO females. In conclusion, remnants of efferent ductules persisted in normal adult female mice, although these structures were greatly reduced in size compared with efferent ductules in ERalphaKO female mice. The underlying mechanism inducing hypertrophy and dilation of efferent ductules in ERalphaKO females is not clear, but secretory and/or reabsorptive function of female efferent ductules may involve ERalpha.

Role of stromal and epithelial estrogen receptors in vaginal epithelial proliferation, stratification, and cornification.

Estradiol 17-beta (E2) induces epithelial proliferation, stratification, and cornification in vaginal epithelium. Our aim was to determine the respective roles of epithelial and stromal estrogen receptor-alpha (ER alpha) in these E2-induced events. Vaginal epithelium (E) and stroma (S) from adult ER alpha knockout (ko) and wild-type (wt) neonatal Balb/c mice were enzymatically separated and used to produce four types of tissue recombinants in which epithelium, stroma, or both lack functional ER alpha. Tissue recombinants were grafted into female nude mice, which were subsequently ovariectomized and treated with oil or E2. In response to E2 treatment, grafts prepared with wt-S (wt-S + wt-E and wt-S + ko-E) showed similar large increases in epithelial labeling index, indicating that E2 stimulated epithelial proliferation despite a lack of epithelial ER alpha in wt-S + ko-E tissue recombinants. Conversely, in tissue recombinants prepared with ko-S (ko-S + wt-E and ko-S + ko-E), epithelial labeling index remained at baseline levels after E2 or oil treatment, even though epithelial ER alpha were detected in ko-S + wt-E grafts. Epithelial cornification was present in wt-S + wt-E grafts from E2-treated hosts, whereas epithelium in all other tissue recombinants failed to cornify. Grafts composed of wt-S + wt-E from E2-treated hosts had highly stratified epithelium, whereas epithelial thickness was reduced almost 60% in wt-S + ko-E tissue recombinants grown in E2-treated hosts and was atrophic in all other tissue recombinants. In addition, cytokeratin 10, a marker of epithelial differentiation, was strongly expressed in wt-S + wt-E tissue recombinants grown in E2-treated hosts but was markedly reduced or absent in all other tissue recombinants. These results indicate that E2-induced vaginal epithelial proliferation is mediated indirectly through stromal ER alpha, consistent with our recent findings in uterus. Conversely, both epithelial and stromal ER alpha are required for E2-induced cornification and normal epithelial stratification. These are the first known functions attributed to epithelial ER alpha in vivo and the first time any epithelial response to E2 has been shown to involve both stromal and epithelial ER alpha.

Tissue compartment-specific estrogen receptor-alpha participation in the mouse uterine epithelial secretory response.

17Beta-estradiol (E2) acts through the estrogen receptor (ER) to regulate uterine epithelial cell growth, proliferation, differentiation, and secretory protein production. We have previously shown that E2-induced uterine epithelial proliferation is mediated indirectly by ER alpha-positive stroma; epithelial ER alpha is neither necessary nor sufficient for E2-induced uterine epithelial mitogenesis. In the present study, we addressed the question of whether production of uterine epithelial secretory proteins and their messenger RNAs (mRNAs) requires ER alpha in stroma, epithelium, or both by analyzing tissue recombinations composed of uterine tissue from adult ER alpha knockout (ko) and neonatal BALB/c (wt) mice. Stroma (S) and epithelium (E) were separated by trypsinization, and four types of uterine tissue recombinants were prepared: wt-S + wt-E, wt-S + ko-E, ko-S + wt-E, and ko-S + ko-E. These tissue recombinants were grown as subrenal capsule grafts in intact female nude mice for 4 weeks, at which time the hosts were ovariectomized. To assess the production of secretory proteins and their mRNAs, 1 week after ovariectomy the hosts were given three daily injections of oil or E2 (100 ng), and then 24 h later the grafts were recovered and used for either ER or lactoferrin (LF) immunohistochemistry. To assess steady state mRNA levels by Northern blotting, hosts received one injection of oil or E2 24 h before harvest. ER immunohistochemistry was used to monitor the completeness of tissue separation. In wt-S + wt-E tissue recombinants from E2-treated hosts, the epithelium stained intensely for LF (an abundant E2-dependent uterine secretory protein), whereas similar tissue recombinants from oil-treated hosts showed minimal immunostaining. Conversely, LF immunostaining was minimal in wt-S + ko-E grafts from both oil- and E2-treated hosts. LF staining was also minimal in ko-S + ko-E and ko-S + wt-E tissue recombinants regardless of hormone treatment. For Northern analyses, the epithelial content of the tissue recombinants was monitored using the reference epithelial transcript, E-cadherin. While all tissue recombinant groups expressed E-cadherin mRNA, wt-S + wt-E tissue recombinants from E2-treated hosts produced a strong, single 2.6-kb band of LF mRNA. LF transcripts were minimal or absent in all other tissue recombinant types. Northern blotting results identical to those seen for LF were also observed for the uterine secretory protein complement component C3. Our data demonstrate that both stromal and epithelial ER alpha are required for the production of LF protein and of LF or C3 mRNAs in response to E2. Thus, in contrast to E2-induced epithelial mitogenesis, which requires only stromal ER alpha, both epithelial and stromal ER alpha are necessary for the production of E2-dependent epithelial secretory proteins.

Neonatal hypothyroidism permanently alters follicle-stimulating hormone and luteinizing hormone production in the male rat.

Transient neonatal hypothyroidism, induced with the goitrogen 6-n-propyl-2-thiouracil (PTU), results in dramatic increases in both testis size and sperm production in the adult rat. The observed increases in testis size and function occur in the presence of normal circulating testosterone levels. However, circulating gonadotropin levels are chronically reduced by 30-50% at all times in treated males. To better understand the permanent reduction in serum gonadotropin levels following transient neonatal hypothyroidism, we conducted a series of experiments to evaluate pituitary and hypothalamic function in the adult male PTU-treated rat. PTU treatment led to a significant reduction in GnRH-stimulated LH production. Castration resulted in 3.9- to 8.5-fold increases in circulating gonadotropin levels in both treated and control males; however, the absolute increases were significantly reduced in treated males. In contrast to circulating levels, pituitary gonadotropin contents did not increase in treated males after castration. PTU treatment did not lead to a reduction in the density of either luteotropes or folliculotropes, and both cell types increased in size and density after castration. The relative concentrations of both gonadotropin beta-subunit messenger RNAs increased more slowly in treated males than in controls after castration. Thus, although treated rats have the intrinsic ability to produce normal circulating levels of LH and FSH, gonadal feedback and an overall reduction in gonadotrope synthetic ability combine to produce the chronically reduced circulating levels of these hormones.