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.

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.

Changes in ontogenetic expression of estrogen receptor alpha and not of estrogen receptor beta in the female rat reproductive tract.

To evaluate ontogenetic expression and localization of estrogen receptor (ER) alpha and beta in fetal female rat reproductive tract, competitive RT-PCR and immunohistochemistry were performed. Expression levels for Müllerian ERalpha, ERbeta1 and ERbeta2 mRNAs were determined by competitive RT-PCR. ERalpha expression on gestational day (GD) 15 x 5 increased 4 x 4-fold by GD 21 x 5, whereas both ERbeta1 and ERbeta2 gene expression were maintained at lower constant levels compared with ERalpha during development. ER immunolocalization was evaluated within three regions along the Müllerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. Nuclear ERalpha was localized predominantly in proximal Müllerian epithelium, and middle and caudal Müllerian mesenchyme on GDs 15 x 5-21 x 5. Staining intensity for ERalpha increased with development in all regions. However, ERbeta immunoreactivity was not detected in any region during prenatal life after separate staining with three different polyclonal anti-rat ERbeta antibodies. These findings provide fundamental information critical for clarifying the species-specific physiological roles of ER subtypes during fetal development and for investigating the tissue-specific mechanisms underlying the prenatal response to estrogen and estrogen receptor agonists.

Effect of diethylstilbestrol on cell proliferation and expression of epidermal growth factor in the developing female rat reproductive tract.

To evaluate mechanisms of cell proliferation in the fetal female rat reproductive tract, diethylstilbestrol (DES) effects on cell division and estrogen receptor (ER), epidermal growth factor (EGF) and EGF receptor (EGF-R) expressions were determined from gestational day (GD) 15.5 to 21.5. Reproductive tracts were evaluated within three regions along the Müllerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. In fetuses from non-treated dams, epithelial and mesenchymal proliferation, as evaluated by 5-bromo-2'-deoxyuridine incorporation, was decreased with development in all regions of the Müllerian duct. EGF levels were determined by immunohistochemistry. Müllerian epithelial EGF immunoreactivity was intense in the proximal and middle regions on GDs 15.5 and 17.5. EGF staining remained intense only in the proximal epithelia by GD 19.5 and was weak in the caudal epithelium, but substantially reduced throughout epithelia in all regions by GD 21.5. Thus, decreased cell proliferation correlated with decreased EGF expression in the developing Müllerian duct. DES (100 microg/kg body weight) was injected from GD 15 to 19 and female fetuses were collected on GD 19.5. DES increased Müllerian duct cell proliferation in the proximal epithelium and mesenchyme but decreased it in the caudal epithelium compared with oil-treated controls. No proliferative DES effect was observed in any cell type in the middle region. Müllerian duct EGF immunoreactivity was suppressed by DES compared with oil. Competitive RT-PCR indicated DES also decreased mRNAs for EGF, ERbeta1 and ERbeta2, but not ERalpha and EGF-R. These results indicate EGF may be an important regulatory factor of Müllerian duct cell proliferation, and that DES may alter cell proliferation by disrupting normal EGF, ERbeta1 and ERbeta2 expression in the developing female rat reproductive tract.

Antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse uterus: critical role of the aryl hydrocarbon receptor in stromal tissue.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the role of aryl hydrocarbon receptor (AhR) in estradiol (E(2))-induced uterine epithelial mitogenic activity and secretory protein mRNA expression were determined. Ovariectomized wild-type (wt) and AhR-knockout (AhRKO) mice received oil, E(2), or 5 microg/kg TCDD+E(2). E(2) stimulated similar large increases in the uterine epithelial labeling index (LI) and mRNA abundance for the E(2)-dependent epithelial secretory protein, lactoferrin (LF), in both wt and AhRKO mice. However, uterine epithelial LI and LF mRNA were significantly reduced by TCDD+E(2) in wt but not AhRKO mice. To determine the roles of stromal and epithelial AhR in the TCDD effect, uterine stroma and epithelium from AhRKO and wt mice were enzymatically separated and recombined into four types of tissue recombinants that either contained or lacked AhR in one or more tissue compartments. Tissue recombinants were grafted into nude mice, which were later ovariectomized and given oil, E(2), or TCDD+E(2). Epithelial LI was significantly reduced by TCDD in grafts containing stromal AhR, regardless of epithelial AhR status. However, LI was unaffected by TCDD in grafts lacking stromal AhR, even when epithelial AhR was present. Thus, TCDD inhibits E(2)-induced uterine epithelial mitogenic and secretory activity, and this requires AhR. Anti-proliferative effects of TCDD on uterine epithelia appear to be mediated indirectly through stromal AhR, suggesting that liganded AhR alters epithelial function by disrupting normal E(2)-induced stromal activity. This is the first demonstration that TCDD impairs uterine epithelial function by altering normal stromal-epithelial interactions in vivo.

Inhibin B levels in plasma of the male rat from birth to adulthood: effect of experimental manipulation of Sertoli cell number.

Sertoli cells undergo important changes in their number and function at different ages in the rat and may be the primary source of circulating inhibin B. The aims of this study were 1) to establish the profile of inhibin B levels from birth to adulthood in normal rats and 2) to identify whether experimental manipulation of Sertoli cell numbers was able to alter this profile. Levels of inhibin B, measured by a specific two-site assay, increased fivefold in normal Wistar rats between day 3 and days 10-15, plateaued, and then declined in late puberty to reach adult levels which were approximately 60% of those observed on days 10-15. The increase in inhibin B levels in the neonatal period coincided with the period of Sertoli cell multiplication as indicated by incorporation of bromodeoxyuridine. Neonatal treatment of rats with a GnRH antagonist (GnRHa) reduced Sertoli cell number and adult testis weight by 48% and significantly reduced plasma levels of inhibin B at all ages through to adulthood. Induction of neonatal hypothyroidism in Sprague-Dawley rats by administration of propylthiouracil (PTU) up to day 25 of age increased final testis weight by 41% (indicative of increased Sertoli cell numbers) and resulted in elevation of plasma levels of inhibin B at all ages beyond 7 days of age. The degree of change in inhibin B levels in adult rats in the two experimental treatment groups was approximately proportional to the change in final testis weight. Plasma follicle-stimulating hormone (FSH) showed changes opposite to inhibin B, with levels being lowered in PTU-treated rats and elevated (beyond day 25) in GnRHa-treated animals. The present results suggest that final Sertoli cell number per testis exerts an important effect on the circulating level of inhibin B (and FSH) in the rat. These findings are compared to the emerging data for the human male.

Role of the aryl hydrocarbon receptor in the development of control and 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed male mice.

Experiments were conducted to determine the role of the aryl hydrocarbon receptor (AhR) in the development of control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed C57Bl/6 male mice. Male and female mice heterozygous for the AhR (Ahr+/-) were mated, and pregnant females were dosed orally on gestation day 13 with corn oil vehicle or TCDD (5 microg/kg). Pups were necropsied on postnatal day (PND) 21, 35, and 90. Comparison of vehicle-exposed wild-type (Ahr+/+) pups with vehicle-exposed AhR knockout (AhRKO; Ahr-/-) pups confirmed and extended previous reports that development of the liver, heart, spleen, thymus, and kidney is affected by absence of the AhR. Lung, submandibular gland, testis, and epididymis weights were also affected, indicating that the AhR plays a role in normal development of these organs as well. The presence or absence of the AhR had no effect on the incidence of hydronephrosis, daily sperm production, or cauda epididymal sperm numbers in vehicle-exposed mice. TCDD caused numerous effects in wild-type mice that were absent in AhRKO mice; specifically, hydronephrosis, increases in relative liver and heart weight, decreases in absolute heart and lung weight, and decreases in absolute and relative thymus, submandibular gland, epididymis, and testis weight. In several cases, TCDD produced one effect in wild-type mice (reductions in body weight and absolute thymus, submandibular gland, and epididymis weight on PND 21; and reductions in absolute and relative submandibular gland and absolute testis weight on PND 35) but caused the opposite effect in AhRKO mice. In yet other cases (reduced relative spleen weight on PND 21 and reductions in absolute and relative thymus weight on PND 35), TCDD produced similar effects in wildtype and AhRKO mice. There were also cases in which TCDD significantly affected AhRKO mice without significantly altering the same endpoint in wild-type mice; absolute liver, lung, and kidney weight were increased and relative submandibular gland weight was decreased on PND 21; relative heart weight was reduced and absolute lung weight increased on PND 35; and relative liver weight was decreased on PND 90. Although many effects of TCDD required the presence of the AhR, these results provide evidence either for multiple forms of the AhR in mice (one or more of which are still present in AhRKO mice), or for AhR-independent effects of low-level TCDD exposure.

Gluteal plication closure of sacral pressure ulcers.

A technique for closure of gluteal pressure sores is described. It has been successfully used on 19 of 21 patients during the last 58 months. Median follow-up time is 23 months. Muscular and vascular integrity are preserved for future myocutaneous flap coverage if required. It is recommended as a relatively conservative method of early repair of sacral pressure defects not in excess of 8 cm in diameter.

Electric current arterial injury: a laboratory model.

A laboratory model capable of creating reproducible, nonlethal, fourth-degree type electrical extremity burns in rats was developed. Fifty-seven rats were studied immediately after burn to 90 days after burn with microdissections, light microscopy, microangiography, and flow studies. Progressive necrosis was not observed. Minimal major vessel arterial injury was demonstrated. This significant finding supports the concept of early debridement and reconstruction to include possible free tissue transfer.

Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium.

Estradiol-17beta (E2) acts through the estrogen receptor (ER) to regulate uterine growth and functional differentiation. To determine whether E2 elicits epithelial mitogenesis through epithelial ER versus indirectly via ER-positive stromal cells, uteri from adult ER-deficient ER knockout (ko) mice and neonatal ER-positive wild-type (wt) BALB/c mice were used to produce the following tissue recombinants containing ER in epithelium (E) and/or stroma (S), or lacking ER altogether: wt-S + wt-E, wt-S + ko-E, ko-S + ko-E, and ko-S + wt-E. Tissue recombinants were grown for 4 weeks as subrenal capsule grafts in intact female nude mice, then the hosts were treated with either E2 or oil a week after ovariectomy. Epithelial labeling index and ER expression were determined by [3H]thymidine autoradiography and immunohistochemistry, respectively. In tissue recombinants containing wt-S (wt-S + wt-E, wt-S + ko-E), E2 induced a similar large increase in epithelial labeling index compared with oil-treated controls in both types of tissue recombinants despite the absence of epithelial ER in wt-S + ko-E tissue recombinants. This proliferative effect was blocked by an ER antagonist, indicating it was mediated through ER. In contrast, in tissue recombinants prepared with ko-S (ko-S + ko-E and ko-S + wt-E), epithelial labeling index was low and not stimulated by E2 despite epithelial ER expression in ko-S + wt-E grafts. In conclusion, these data demonstrate that epithelial ER is neither necessary nor sufficient for E2-induced uterine epithelial proliferation. Instead, E2 induction of epithelial proliferation appears to be a paracrine event mediated by ER-positive stroma. These data in the uterus and similar studies in the prostate suggest that epithelial mitogenesis in both estrogen and androgen target organs are stromally mediated events.

A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior.

Two chemicals previously shown to have estrogenic activity, bisphenol A and octylphenol, were examined for their effects on accessory reproductive organs and daily sperm production in male offspring of mice fed these chemicals during pregnancy. These chemicals are used in the manufacture of plastics and other products, and have been detected in food and water consumed by animals and people. From gestation day 11-17 female mice were fed an average concentration (dissolved in oil) of bisphenol A or octylphenol of 2 ng/g body weight (2 ppb) and 20 ng/g (20 ppb). The 2 ppb dose of bisphenol A is lower than the amount reported to be swallowed during the first hour after application of a plastic dental sealant (up to 931 micrograms; 13.3 ppb in a 70 kg adult). We found that the 2 ng/g dose of bisphenol A permanently increased the size of the preputial glands, but reduced the size of the epididymides; these organs develop from different embryonic tissues. At 20 ng/g, bisphenol A significantly decreased efficiency of sperm production (daily sperm production per g testis) by 20% relative to control males. The only significant effect of octylphenol was a reduction in daily sperm production and efficiency of sperm production at the 2 ng/g dose. A new approach to studying physiologically relevant doses of environmental endocrine disruptors is discussed, particularly with regard to the development of the reproductive organs, the brain, and behavior.

Altered prostate growth and daily sperm production in male mice exposed prenatally to subclinical doses of 17alpha-ethinyl oestradiol.

Approximately 2 million women in the USA and Europe continue taking oral contraceptives each year during undetected pregnancy due primarily to non-compliance and also to individual variation in sensitivity to hormones in the contraceptives. Prenatal exposure to oral contraceptives containing 17alpha-ethinyl oestradiol (EE) has generally not been associated with an increased incidence of externally observable malformations at birth. The purpose of this study was to assess effects on reproductive organs in adult male mice that had been exposed during gestation day 0 through 17 (equivalent to gestation week 16 in humans) to clinically relevant (approximately 0.5 microg/kg/day) and lower doses of EE. Doses used in this study ranged from 0.002 to 2 microg/kg/day. By 5 months of age, prostate weight was significantly (P < 0.05) higher than controls in most treatment groups of EE (0.02-2 microg/kg). Prostatic androgen receptor populations were significantly elevated only in the 0.02 microg/kg group, suggesting different mechanisms for the increase in prostate weight at different doses. Daily sperm production (DSP) and DSP per gramme of testis were reduced in all treatment groups during adolescence, but not later in adulthood. These findings are consistent with prior studies showing that prenatal exposure of mice to very low doses of a number of oestrogenic chemicals can alter the adult male reproductive system without causing gross external malformations.