Transforming growth factors beta 1, beta 2, and beta 3 messenger RNA and protein expression in mouse uterus and vagina during estrogen-induced growth: a comparison to other estrogen-regulated genes.

Increasing evidence suggests that the differential regulation of multiple peptide growth factors by steroid hormones contributes significantly to the pleiotropic effects elicited in target tissues. We report here an evaluation of the effects of the potent estrogen, diethylstilbestrol, on the expression of the three mammalian transforming growth factor beta (TGF beta) isoforms, TGF beta 1, TGF beta 2, and TGF beta 3, in both the uterus and the vagina of the prepubescent mouse. Immunohistochemical protein detection, in situ hybridization, and Northern RNA analyses demonstrate overlapping but distinct time-dependent and site-specific induction of all three TGF beta genes in the reproductive tract in response to estrogen. Temporal analysis of steady-state levels of the TGF beta mRNAs in the uterus by Northern blotting clearly demonstrates that diethylstilbestrol significantly but transiently up-regulates TGF beta 3 mRNA within 30 min and TGF beta 1 and TGF beta 2 mRNAs by 3 h with decreases to/or below control levels by 6 h. The vagina also responds to diethylstilbestrol with similar kinetics of induction for TGF beta 2 and TGF beta 3 mRNAs as that observed in the uterus; however, TGF beta 1 mRNA levels increase gradually and peak around 16 h after treatment. Investigation of the steroid specificity demonstrates predominant estrogen specificity in the control of TGF beta expression in the immature mouse reproductive tract. In situ hybridization localizes the mRNAs for all three TGF beta isoforms, primarily to the uterine and vaginal epithelium. Unlike the transient nature of TGF beta mRNA induction elicited by estrogen, immunohistochemistry demonstrates that estrogen treatment results in a more prolonged elevation of the proteins for TGF beta 1, TGF beta 2, and TGF beta 3 in the epithelium of both tissues. Investigation of specific binding of 125I-TGF beta 1 by affinity labeling reveals the existence of the receptor/binding proteins (types I, II, and III) in the uterus. Estrogen treatment significantly reduces binding to each of these components in the uterus, which suggests that estrogen may modulate TGF beta responsiveness at the receptor level. A comparison of TGF beta mRNA expression to the induction of other estrogen-regulated genes, TGF alpha, insulin-like growth factor-1, c-myc, progesterone receptor and lactotransferrin reveals that, in general, the TGF beta transcript levels are regulated in a more transient manner by estrogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of epidermal growth factor in suprabasal cells of stratified squamous epithelia: implications for a role in differentiation.

There is a disparity in the fact that epidermal growth factor (EGF) can stimulate proliferation of a wide variety of cells in vitro, yet sites of synthesis for this polypeptide in vivo are generally associated with nonproliferative or differentiated cell populations. Unlike other known mitogenic members of the EGF family of ligands, EGF derives from a large (M(r) approximately 130,000) protein that contains multiple EGF-like domains; some of these domains contain putative Ca(2+)-binding sites. In the present study, the mouse vaginal epithelium was used as a prototype tissue to investigate expression of the EGF precursor by stratified squamous epithelia. Stratification and cornification in this tissue are estrogen dependent. In the fully differentiated epithelium, EGF precursor transcripts were detected by in situ hybridization in suprabasal cells; labeling was not observed in the proliferative basal cell compartment. Amplified transcripts of predicted size were detected in the vaginal organ by the reverse transcription-polymerase chain reaction and Northern hybridization of polyadenylated RNA. Antisera to mature EGF or to the COOH-terminal (cytoplasmic) domain of the precursor localized reactivity exclusively to cells of the granular layer. The staining with the precursor antiserum was localized along cell borders, which supports that this protein is translocated to the cell membrane following synthesis, a known property for precursors of EGF-like ligands. EGF expression was not apparent in the atrophic vaginal epithelium of castrates, which did not present a granular layer. Other stratified squamous epithelia in organs such as skin, esophagus, and tongue also revealed EGF/EGF precursor immunoreactivity in the granular layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-alpha is a potential mediator of estrogen action in the mouse uterus.

To better understand the role of peptide growth factors in sex steroid hormone-mediated growth of the female reproductive tract, the effect of estrogen on the expression of transforming growth factor-alpha (TGF alpha) in mouse uterus was investigated. Our results show that estrogen induces the expression of TGF alpha mRNA in the mouse uterus in a dose- and time-dependent manner. The up-regulation of TGF alpha transcripts occurs predominantly in uterine epithelial cells. RIA and Western blot analysis demonstrate that immunoreactive TGF alpha protein is secreted at high levels into mouse uterine luminal fluid after estrogen treatment. The induction of uterine TGF alpha mRNA is specific to estrogen; nonestrogenic steroids did not induce expression. Antibody specific to TGF alpha significantly reduces estrogen-mediated uterine growth, which supports the concept that TGF alpha is a mitogen for the reproductive tract. Analysis of TGF alpha/EGF receptors by binding, affinity labeling, and phosphorylation studies indicates that functional receptors are present in the mouse uterus after estrogen exposure. Thus, our data support a physiological role for TGF alpha and its receptor pathway in the female mouse reproductive tract.

Reproductive toxicity of triethylene glycol and its diacetate and dimethyl ether derivatives in a continuous breeding protocol in Swiss CD-1 mice.

Triethylene glycol and two of its derivatives were evaluated for reproductive toxicity in a continuous breeding protocol with Swiss CD-1 mice. Triethylene glycol (TEG: 0, 0.3, 1.5, and 3%), triethylene glycol diacetate (TGD: 0, 0.75, 1.5, and 3%), and triethylene glycol dimethyl ether (TGDME: 0, 0.25, 0.5, and 1%) were administered in drinking water to breeding pairs (20 pairs per treatment group, 40 control pairs) during a 98-day cohabitation period. Reproductive function was assessed by the number of litters per pair, live pups per litter, proportion of pups born alive, and pup weight. There were no apparent effects on reproductive function in the animals receiving TEG or TGD at doses up to 3% in the drinking water (representing 6.78 or 5.45 g/kg, respectively). However, some developmental toxicity was demonstrated for both TEG and TGD. Continuous exposure of dams to 1.5 or 3% TEG significantly reduced live pup weight at birth compared to control and 0.3% TEG, while exposure to 3% TGD during lactation significantly (but reversibly) reduced pup body weights on Postnatal Days 14 and 21. In contrast, TGDME was toxic to the reproductive system as evidenced by decreases at the highest dose (1% TGDME; 1.47 g/kg) in the proportion of pairs that produced at least one litter, live pups per litter, and proportion of pups born alive, with dose-related trends seen in the latter two parameters. A crossover mating trial showed that TGDME was more toxic to the female than the male reproductive system. These data indicate that TGDME (1.47 g/kg) is a reproductive toxicant in Swiss mice while reproductive toxicity was not demonstrated in mice receiving TEG or TGD (at doses up to 6.78 or 5.45 g/kg, respectively).

Human granulosa-luteal and cumulus cells express transforming growth factors-beta type 1 and type 2 mRNA.

Human granulosa-luteal cells and cumulus cells obtained from women undergoing in vitro fertilization and embryo transfer (IVF-ET) were examined for the presence of TGF-beta 1 and TGF-beta 2 mRNA by reverse transcription-polymerase chain reaction (RT-PCR) analysis. RT-PCR analysis revealed that both follicle cell types express mRNA for both TGF-beta subtypes. Verification of RT-PCR products was done by restriction enzyme digestion analysis. These results suggest a role(s) for TGF-beta 1 and TGF-beta 2 in the development of human granulosa-luteal cells and the oocyte-cumulus cell complex.

Epidermal growth factor replaces estrogen in the stimulation of female genital-tract growth and differentiation.

The in vivo studies presented here demonstrate that epidermal growth factor (EGF) is an important autocrine and/or paracrine mediator of estrogen-induced growth and differentiation in mouse uterus and vagina. An antibody specific for EGF significantly inhibited estrogen-induced uterine and vaginal growth, thereby implicating EGF involvement in estrogen action. Furthermore, EGF administered via slow-release pellets in ovariectomized mice acted as a potent uterine and vaginal mitogen as well as an inducer of vaginal keratinization. Experiments with ovariectomized, adrenalectomized, hypophysectomized mice indicated that EGF mitogenesis does not require pituitary or adrenal hormones. Treatment with EGF also mimicked estrogen in the induction of uterine lactoferrin (a major estrogen-inducible secretory protein) mRNA and protein. These data suggest that EGF has estrogen-like effects in the promotion of cell growth in the reproductive tract and that EGF may serve as an important mediator of estrogen action in vivo.

Epidermal growth factor binding and receptor distribution in the mouse reproductive tract during development.

The ontogeny of the epidermal growth factor (EGF) receptor in the different cell types in the neonatal and immature mouse uterus and vagina was examined. Immunohistochemical examination of prenatal and neonatal reproductive tracts with a polyclonal antibody to the EGF receptor shows immunoreactive EGF receptors as early as Day 13 of gestation. Autoradiographic analysis of tissue sections at 3 to 17 days of age (the day of birth is Day 1) demonstrates that both uterine and vaginal epithelial and stromal cells are capable of binding 125I-labeled EGF. Both the 125I-labeled EGF autoradiography and immunohistochemistry in whole tissue show higher EGF receptor levels in the uterine epithelium than the uterine stroma. The presence of EGF receptors was also confirmed by affinity labeling and Scatchard analysis of isolated uterine cell types at 7 and/or 17 days of age. However, in contrast to the autoradiography and immunohistochemistry data of intact tissue, the affinity labeling and Scatchard data of isolated cells indicate that the uterine stroma contains higher levels of EGF receptor than that of the uterine epithelium. The reason for this discrepancy between the different techniques is, as yet, unknown. Regardless of the differences in the actual numbers of EGF receptors obtained, our data demonstrate that the developing mouse reproductive tract contains immunoreactive EGF receptors that are capable of binding 125I-labeled EGF.

Protein alterations associated with N-methyl-N-nitrosourea exposure of preimplantation mouse embryos transferred to surrogate mothers.

Mouse preimplantation embryo functions have been shown to be disrupted by in vitro exposure to N-methyl-N-nitrosourea (MNU) with subsequent transfer to the uteri of pseudopregnant surrogate mothers. Increased gross malformations and decreased fetal body lengths in the midgestational period have been previously documented. Protein extracts were isolated from day 12 mouse fetuses developed from MNU- or solvent-exposed blastocysts and analyzed by two-dimensional electrophoresis. The electrophoretic patterns reveal six protein alterations in day 12 fetal tissue induced by MNU treatment at the blastocyst stage. Five of these alterations involve shifts in isoelectric point (pI) and the other alteration involves a quantitative increase in a protein. The possibility that two of the proteins which exhibit a shift in pI following MNU exposure represent the cell adhesion molecules, N-CAM and L-CAM (based on similar Mr values), was investigated by Western blot analysis. No pI alteration in L-CAM or N-CAM expression is seen after MNU exposure. These results demonstrate that in vitro MNU exposure of preimplantation embryos results in protein alterations in midgestational fetuses. Thus, the effects of MNU exposure on preimplantation embryos may be manifest long after exposure, and subtle, non-lethal mutations may play a role in poor fetal outcome after early chemical exposure.

Disruption of embryonic and fetal development due to preimplantation chemical insults: a critical review.

Descriptive teratology has developed several fundamental precepts, two of which can now be challenged on the basis of experimental evidence. The first is that prior to implantation the developing embryo is not susceptible to survivable defects from chemical injury. The second is that developmental defects cannot be due to mutational events since rare events seem unlikely to explain alterations in large populations of cells. This review presents current experimental evidence demonstrating that the effects of chemical exposure on blastocyst stage embryos may be manifest long after the time of insult and that subtle nonlethal mutations may have a role in poor fetal performance after early chemical exposures.

Midgestational abnormalities associated with in vitro preimplantation N-methyl-N-nitrosourea exposure with subsequent transfer to surrogate mothers.

Mouse blastocyst functions have been shown to be disrupted by in vitro exposure to N-methyl-N-nitrosourea (MeNU). After exposure, the chemically treated blastocysts were transferred to the uteri of pseudopregnant surrogate mothers. Implantation rate and birth rate have been shown previously to decrease in a concentration-dependent manner. Because of the large progressive decrease in the 50% effective concentration (EC50) for cytotoxicity, implantation rate, and live birth rate, we have investigated the midgestational effects of preimplantation exposure to MeNU after the transfer of treated embryos to surrogate mothers. A concentration-dependent decrease in normal implantation and a concurrent concentration-dependent increase in resorption number was observed in surrogates sacrificed at gestational age day 12 or day 15. Gross malformations were significantly increased by preimplantation exposure, in vitro, to MeNU. Fetal body length did not differ between fetuses developed from solvent-treated blastocysts and those that developed from natural pregnancies (nontransferred control) at either gestational age examined. Fetal body length was significantly shorter in fetuses developed from MeNU-treated blastocysts.