Biomedical subjects

R M Bigsby

Publications and source records attributed to R M Bigsby.

At least 37 records · Page 2Linked to original sources

PubMed1998

The xenoestrogen bisphenol A induces growth, differentiation, and c-fos gene expression in the female reproductive tract.

The xenoestrogen bisphenol A (BPA) has been shown to mimic estrogen both in vivo and in vitro. BPA stimulates PRL secretion and the expression of a PRL regulating factor from the posterior pituitary in the estrogen-sensitive Fischer 344 rat (F344), but not in Sprague-Dawley (SD) rats. The goal of the present studies was to examine the in vivo actions of BPA on the reproductive tract. The specific objectives were 1) to characterize the short term effects of BPA on cell proliferation and c-fos expression in the uterus and vagina, and 2) to compare the effects of prolonged exposure to low doses of BPA on the reproductive tract of F344 and SD rats. Treatment with single high doses of BPA induced cell proliferation in the uterus and vagina of ovariectomized F344 rats, as determined by bromodeoxyuridine immunostaining. This proliferation was dose dependent (from 37.5-150 mg/kg) and followed a time course similar to that of estradiol (E2). Quantitative RT-PCR revealed that both BPA and E2 increased c-fos messenger RNA levels in the uterus 14- to 16-fold within 2 h, which returned to basal levels after 6 h. In the vagina, BPA-induced c-fos expression remained elevated for up to 6 h, compared with the transient increase caused by E2. Treatment of F344 rats for 3 days with continuous release capsules that supplied a much lower dose of BPA (approximately 0.3 mg/kg x day) resulted in hypertrophy, hyperplasia, and mucus secretion in the uterus and hyperplasia and cornification of the vaginal epithelium. The reproductive tract of SD rats did not respond to this treatment paradigm with BPA. These studies demonstrate that 1) the molecular and morphological alterations induced by BPA in the uterus and vagina are nearly identical to those induced by estradiol; 2) the vagina appears to be especially sensitive to the estrogenic actions of BPA; 3) the reproductive tract of the inbred F344 rat appears more sensitive to BPA than that of the outbred SD rat; and 4) continuous exposure to microgram levels of BPA is sufficient for exerting estrogenic actions.

Animals↗

PubMed1997

Altered hematopoiesis, behavior, and sexual function in mu opioid receptor-deficient mice.

The mu opioid receptor is thought to be the cellular target of opioid narcotics such as morphine and heroin, mediating their effects in both pain relief and euphoria. Its involvement is also implicated in a range of diverse biological processes. Using a mouse model in which the receptor gene was disrupted by targeted homologous recombination, we explored the involvement of this receptor in a number of physiological functions. Mice homozygous for the disrupted gene developed normally, but their motor function was altered. Drug-naive homozygotes displayed reduced locomotor activity, and morphine did not induce changes in locomotor activity observed in wild-type mice. Unexpectedly, lack of a functional receptor resulted in changes in both the host defense system and the reproductive system. We observed increased proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in both bone marrow and spleen, indicating a link between hematopoiesis and the opioid system, both of which are stress-responsive systems. Unexpected changes in sexual function in male homozygotes were also observed, as shown by reduced mating activity, a decrease in sperm count and motility, and smaller litter size. Taken together, these results suggest a novel role of the mu opioid receptor in hematopoiesis and reproductive physiology, in addition to its known involvement in pain relief.

Animals↗

PubMed1997

Xenobiotics released from fat during fasting produce estrogenic effects in ovariectomized mice.

The pesticide residues 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p'-DDT) and beta-hexachlorocyclohexane (beta-HCH) act as weak estrogens, producing uterotrophic responses in ovariectomized rodents and stimulating human breast cancer cells in culture. Such activity suggests that these compounds may act as tumor promoters in estrogen-responsive tissues. Organochlorine compounds such as o,p'-DDT and beta-HCH are concentrated in body fat. The present report tests whether sufficient compound can be released from fat depots to produce estrogenic effects in uteri of ovariectomized mice. Adult animals were "loaded" with test compound by three daily injections of vehicle (DMSO), 17beta-estradiol (E2), beta-HCH, or o,p'-DDT. Uterotrophic effects were assessed at 24 h after the last loading dose of test compound and at 2 weeks after the loading regimen, with or without a prior 2-day period of fasting. The initial 3-day treatment with either beta-HCH or o,p'-DDT doubled the relative dry weight of the uterus: 102 +/- 8.6 mg/kg body weight (BW) and 104 +/- 4.4 mg/kg BW for beta-HCH and o,p'-DDT, respectively, compared to 49 +/- 1.9 mg/kg BW for vehicle-treated animals. E2-treated animals had uterine dry weights of 228 +/- 11 mg/kg BW. After 2 weeks without further treatment, a 2-day fast produced a decrease in body mass of 4.1 g/animal (fasted, 25.9 +/- 1.89 g versus fed, 30.0 +/- 2.82 g). Animals that had been loaded with beta-HCH and fasted had uterine weights (88 +/- 12 mg/kg BW) significantly greater (P < 0.05) than those of vehicle-loaded, fasted animals (51 +/- 2.9 mg/kg BW) or of beta-HCH-loaded, fed animals (59 +/- 4.6 mg/kg BW). The uterine weights of the fasted and fed o,p'-DDT-loaded or E2-loaded animals were not different from those of control weights. The difference between wet and dry weights showed that fasting of beta-HCH-loaded animals also increased water imbibition in the uterus; there was no effect from fasting in the other groups. Generally, epithelial cell height reflected the same responses as uterine weight with the exception that cell heights of beta-HCH-loaded, fed animals were slightly higher (P < 0.05) than corresponding controls, indicating that there may have been some active compound available to the tissues even without fasting. The effects of fasting show that during periods of lipolysis beta-HCH can be released in quantities sufficient to stimulate estrogen target tissues, suggesting a novel mechanism linking obesity and the progression of estrogen-responsive tumors. The lack of effect from fasting in o,p'-DDT-loaded animals indicates that these compounds are differentially mobilized from fat depots.

Adipose Tissue↗

PubMed1997

Collagenase expression in ovarian cancer cell lines.

BACKGROUND: There are several distinct forms of the matrix metalloproteinases (MMP) that degrade collagen in the extracellular matrix. Most involved in the metastatic process are collagenases exhibiting specificity for type IV collagen, the collagen that makes up the backbone of the basement membrane. We chose to examine the expression of one type IV collagenase, MMP-9, in several ovarian cancer cell lines. METHODS: Under the influence of phorbol esters such as TPA, the fibrosarcoma cell line HT1080 is known to express MMP-9. We examined the effect of TPA on MMP-9 expression with Northern analysis, and on MMP-9 collagenase activity with polyacrylamide gel electrophoresis-zymography, in HT1080 cells (positive control) and in six ovarian cancer cell lines: Hey, HeyA8, CAOV3, PA-1, SKOV3, and OVCAR3. Cells were grown to 70% confluence and the growth medium was changed to a serum-free medium for 24 hr. Cells were then treated with 12 ng/mL TPA or its vehicle for an additional 24 hr. Medium was collected for PAGE-zymographic analysis. The cells were lysed and their RNA collected for Northern analysis. RESULTS: PA-1, a teratocarcinoma, expressed more than 10-fold the amount of RNA for MMP-9 than the other lines following TPA treatment. Both Northern analysis and zymography showed that four of the six ovarian cell lines responded to TPA with increased collagenase expression and activity. The other two cell lines showed no MMP-9 activity either before or after TPA treatment. CONCLUSIONS: These results suggest that ovarian cancer cell lines express collagenase and that this expression may be regulated by phorbol esters which activate the protein kinase C pathway.

Base Sequence↗

PubMed1997

The environmental estrogen bisphenol A stimulates prolactin release in vitro and in vivo.

Environmental estrogens (xenoestrogens) are a diverse group of chemicals that mimic estrogenic actions. Bisphenol A (BPA), a monomer of plastics used in many consumer products, has estrogenic activity in vitro. The pituitary lactotroph is a well established estrogen-responsive cell. The overall objective was to examine the effects of BPA on PRL release and explore its mechanism of action. The specific aims were to: 1) compare the potency of estradiol and BPA in stimulating PRL gene expression and release in vitro; 2) determine whether BPA increases PRL release in vivo; 3) examine if the in vivo estrogenic effects are mediated by PRL regulating factor from the posterior pituitary; and 4) examine if BPA regulates transcription through the estrogen response element (ERE). BPA increased PRL gene expression, release, and cell proliferation in anterior pituitary cells albeit at a 1000- to 5000-fold lower potency than estradiol. On the other hand, BPA had similar efficacy to estradiol in inducing hyperprolactinemia in estrogen-sensitive Fischer 344 (F344) rats; Sprague Dawley (SD) rats did not respond to BPA. Posterior pituitary cells from estradiol- or BPA-treated F344 rats strongly increased PRL gene expression upon coculture with GH3 cells stably transfected with a reporter gene. Similar to estradiol, BPA induced ERE activation in transiently transfected anterior and posterior pituitary cells. We conclude that: a) BPA mimics estradiol in inducing hyperprolactinemia in genetically predisposed rats; b) the in vivo action of estradiol and BPA in F344 rats is mediated, at least in part, by increasing PRL regulating factor activity in the posterior pituitary; c) BPA appears to regulate transcription through an ERE, suggesting that it binds to estrogen receptors in both the anterior and posterior pituitaries. The possibility that BPA and other xenoestrogens have adverse effects on the neuroendocrine axis in susceptible human subpopulations is discussed.

Animals↗

PubMed1997

A novel estrogen-enhanced transcript identified in the rat uterus by differential display.

Estrogen exerts its physiological effects in the uterus by inducing a cascade of transcriptional events; however, the number of genes known to be directly activated by estrogen in the uterus is small. In this study, immature ovariectomized rats were treated with estrogen or vehicle, and 3 h later the uterine horns were flushed to extract epithelial RNA. This RNA was used in the differential display technique to search for estrogen-responsive genes. Products of reverse transcriptase-PCR, made with pairs of arbitrary and oligo-deoxythymidine primers, were separated on denaturing polyacrylamide gels; candidate bands were excised and reamplified to produce probes for use in Northern blot analysis and screening of a lambda gt10 complementary DNA library made from rat uterus. A novel estrogen-enhanced transcript, designated EET-1, was identified from a differential display band, and the estrogen sensitivity of its expression was verified in Northern analysis. Characterization of EET-1 expression in the uterus showed that estrogen treatment resulted in a rapid and transient increase in EET-1 messenger RNA; steady state levels peaked between 2-3 h, returning to basal levels by 6 h. This increase was not abolished by pretreatment with cycloheximide, indicating that induction of EET-1 is a primary response to estrogen. Induction was specific to estrogen when extracts of whole uterus were examined; in the epithelium, there was also a slight response to progesterone. Expression of the gene was found in all organs surveyed; however, hormonal regulation was observed only in tissues of the reproductive tract and in the kidney. Analysis of cloned EET-1 complementary DNA revealed a 2008-base sequence that showed 61% identity with a reported transcript that encodes a protein that plays a role in phorbol ester-induced regulation of the tumor necrosis factor-alpha gene. Potential casein kinase-2 and protein kinase C phosphorylation sites and a cysteine-rich region were identified in the amino acid sequence deduced from EET-1. Thus, it appears that EET-1 represents a primary estrogen response gene that may code for a phosphorylated protein involved in gene regulation through a protein kinase C-activated pathway.

Amino Acid Sequence↗

PubMed1997

Mesenchymal-epithelial interactions in an in vitro model of neonatal mouse uterus.

Stromal factors have been implicated in epithelial growth of the fetal and neonatal mouse uterus, as well as in uterine epithelial proliferation in the adult. In the neonate, uterine growth is independent but responsive to estrogen, while epithelial proliferation in the adult uterus is hormonally regulated. A co-culture model was developed to study interactions between uterine epithelium and stroma. Uteri of neonatal mouse were enzymatically separated into epithelial and mesenchymal cell fractions, and these were cultured for 5 days on Millipore well inserts, either separately or in co-culture on opposite sides of the insert membrane. When epithelial cells were grown alone, inclusion of serum in the culture medium tripled the number of cells present after 3-5 days compared with serum-free medium. Co-culture in the presence or absence of serum resulted in a 5-fold increase in epithelial cell number after 5 days. Addition of estrogen had no significant effect on epithelial cell number regardless of the presence of mesenchyme. Epithelial cultures grown in medium conditioned by mesenchymal cells exhibited an intermediate increase in cell number. We therefore conclude that uterine mesenchyme from neonatal mouse produces a diffusible factor that enhances the growth of the overlying epithelium; however, whether the mesenchyme has a role in estrogen-stimulated epithelial proliferation has not been definitively ascertained.

Animals↗

PubMed1996

Novel estrogenic action of the pesticide residue beta-hexachlorocyclohexane in human breast cancer cells.

The estrogenic action of some persistent organochlorine pesticide residues may play a role in the progression of hormonally responsive tumors of the breast and uterus. The prototypical xenoestrogen o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT) acts by binding and activating the estrogen receptor (ER). The present study focuses attention on the mechanisms through which another organochlorine compound, beta-hexachlorocyclohexane (beta-HCH), exerts estrogen-like effects in human breast cancer cells. Both o,p'DDT and beta-HCH stimulated proliferation in a dose-dependent manner in the ER-positive cell lines MCF-7 and T47D but not in the ER-negative lines MDA-MB231, MDA-MB468, and HS578T. Both compounds produced an increase in the steady state level of pS2 mRNA in MCF-7 cells. These responses were equal in magnitude to the maximal effect of estradiol, and they were inhibited by inclusion of the antiestrogen ICI164384. On the other hand, when tested in a competitive binding assay, beta-HCH did not displace 17beta-[3H]estradiol from the ER even at a concentration that was 40,000-fold higher than the tracer steroid. Furthermore, nuclear retention of the ER during homogenization procedures was induced by a 2- or 24-h treatment of MCF-7 cells with o,p'-DDT and 17beta-estradiol but not by treatment with beta-HCH; this indicates that beta-HCH nether activates the ER, nor is it converted intracellularly to an ER ligand. Transcriptional activation by beta-HCH occurs in estrogen-responsive GH3 rat pituitary tumor cells transfected with a luciferase reporter construct driven by a complex 2500-bp portion of the PRL gene promoter; this trans-activation response is inhibited by inclusion of ICI164384. However, beta-HCH is ineffective in stimulating a reporter construct driven only by a consensus estrogen response element and a minimal promoter derived from the herpes simplex virus thymidine kinase gene. Thus, beta-HCH cannot act on a simple, single estrogen response element; rather, it requires the combinatorial regulation found in a complex promoter. These data are consistent with the notion that beta-HCH stimulation of cell proliferation and gene expression is ER dependent, but its action is not through the classic pathway of binding and activating the ER. beta-HCH may represent a new class of xenobiotic that produces estrogen-like effects through nonclassic mechanisms and, therefore, may be of concern with regard to breast and uterine cancer risk.

Adenocarcinoma↗

PubMed1994

Estrogenic effects of the antiprogestin onapristone (ZK98.299) in the rodent uterus.

OBJECTIVE: Our purpose was to assess the estrogenic action of onapristone (ZK299). STUDY DESIGN: Three rodent models of estrogen action in the uterus were used. Deoxyribonucleic acid synthesis in the uterine epithelium of neonatal mice was determined by thymidine autoradiography. In adult ovariectomized mice uterine wet weight and progesterone receptor and estrogen receptor concentrations were determined. In immature rats uterine deoxyribonucleic acid synthetic activity was determined by thymidine autoradiography, epithelial hypertrophy and stromal edema were assessed histomorphometrically, and complement C3 protein synthesis was assessed by metabolic labeling in vitro. The effects of ZK299 were challenged with the antiestrogens ICI164,384 and tamoxifen. The ability of ZK299 to displace tritiated estradiol from the estrogen receptor was assessed in cytosolic preparations from mouse uterus. RESULTS: In the neonatal mouse ZK299 stimulated epithelial deoxyribonucleic acid synthesis; two other antiprogestins, RU486 and ZK98.734, had no effect. Three daily injections of ZK299 at 10 micrograms/gm body weight to 6-week-old ovariectomized mice increased uterine progesterone receptor 42%; this effect was blocked by ICI164,384. In another experiment three daily doses of ZK299 (20 micrograms/gm) to 10-week-old ovariectomized mice increased progesterone receptor concentration by 63% and uterine wet weight by 42%. In 21-day-old rats a single injection of ZK299 increased uterine epithelial deoxyribonucleic acid synthesis; this effect was blocked by tamoxifen. Both ZK299 and tamoxifen increased epithelial cell height and thymidine labeling in the stroma. ZK98.734 had no effect on epithelium or stroma. ZK299 also stimulated synthesis of complement C3 by uteri of immature rats. In competitive binding assays ZK299 exhibited weak relative binding affinity (0.05%) for mouse uterine estrogen receptor. CONCLUSIONS: ZK299 can act as a weak estrogen in the rodent uterus, most likely through a direct, low-affinity interaction with the estrogen receptor. Because estrogens may increase the risk for endometrial, breast, and liver cancer, caution is warranted in long-term administration of this drug to women.

Animals↗

PubMed1994

Differentially regulated immediate early genes in the rat uterus.

Estrogen stimulates cellular proliferation in the luminal epithelium, stroma, and smooth muscle of immature rat uterus. Progesterone administered concurrently with estrogen blocks the stimulatory effect of estrogen specifically in the epithelium, whereas progesterone administered alone stimulates proliferation in the endometrial stroma and myometrium. The present studies determined the effects of estrogen and progesterone on expression of the growth-associated, immediate early genes c-fos, c-jun, and jun-B in the luminal epithelium of the uterus. Hormonal effects were quantitated by Northern analysis of RNA extracted directly from the uterine luminal epithelium. Estrogen stimulated c-fos and jun-B expression, but repressed c-jun mRNA levels in the epithelium. In contrast, when whole organ RNA extracts were analyzed, estrogen increased mRNA levels for all three genes. Although progesterone administered alone showed no effect on mRNA levels in either epithelial or whole uterus extracts, it did attenuate the estrogen-induced increase in c-fos mRNA by 50% in whole uterus extracts and by 23% in epithelial extracts. The estrogen-induced increase in epithelial jun-B mRNA was not affected by progesterone pretreatment. Thus, in the immature rat uterus, no simple correlation exists between cellular proliferation and increased expression of the genes studied. However, progesterone completely blocked the repressive effect of estrogen on epithelial c-jun, suggesting a link between decreased c-jun expression and induction of cell proliferation in the uterine luminal epithelium. Estrogen repression of epithelial c-jun expression was hormone specific and sensitive to antiestrogen blockade. After estrogen treatment, epithelial c-jun mRNA decreased with a rate similar to its half-life, as determined in primary cultures of rat uterine cells. These results suggest that estrogen, acting through its receptor, directly represses transcription of c-jun in the uterine epithelium. Differences in hormonal regulation of immediate early genes between epithelial and nonepithelial uterine tissues probably results from tissue-specific transactivating factors that control the expression of these genes.

Animals↗

PubMed1994

Activation of the prolactin promoter in transfected GH3 cells by posterior pituitary cells.

UNLABELLED: We previously reported that PRL production is significantly enhanced by a PRL-releasing/regulating factor derived from the posterior pituitary (PP). Specifically, the levels of PRL messenger RNA synthesis and release were dramatically increased in cocultures of GH3 and PP cells. The present objectives were to: 1) determine whether PP cells activate PRL gene transcription in a promoter- and cell-specific manner; 2) compare promoter activation by PP cells with that caused by selected substances that regulate the PRL gene; and 3) examine which region of the promoter (proximal and/or distal) mediates the action of the PP. In Exp 1, GH3 cells, transfected either with luciferase reporter plasmids containing a wild type PRL promoter, a GH promoter, or a glycoprotein alpha-subunit promoter, were cocultured with PP cells. Luciferase activity was used as an index for promoter activation. PP cells induced an 18-fold stimulation of the PRL promoter, as compared with a 2-fold stimulation of the GH promoter and no effect on the glycoprotein alpha-subunit promoter. In Exp 2, GH3 cells transfected with the wild type PRL promoter were cocultured with PP, anterior pituitary, uterine, or PC12 cells for 24 h. PP cells caused a 20-fold stimulation of the PRL promoter, whereas anterior pituitary cells showed a moderate 5-fold stimulation; uterine and PC12 cells caused minimal (< 2-fold) increases in luciferase activity. In Exp 3, GH3 cells were transfected with either the wild type PRL promoter (-2500 PRLluc) or with a truncated promoter (-425PRLluc) containing the proximal region only and were incubated with PP cells, TRH, vasoactive intestinal peptide (VIP), epidermal growth factor (EGF), or estradiol (E2) for 24 h. Compared with the induction of the wild type promoter, PP cells activated the truncated promoter by 30% only. Stimulation of the promoter by relatively high concentrations of TRH, VIP, EGF, or E2, either alone or in combination, was significantly less effective than that caused by PP cells. CONCLUSIONS: 1) PP cells stimulate PRL gene transcription in a tissue- and promoter-specific manner; 2) the magnitude of induction caused by PP cells far exceeds that caused by high concentrations of TRH, VIP, EGF, or E2; and 3) the distal enhancer region of the rat PRL gene is necessary for maximal responsiveness of the PRL promoter to PP cells.

Animals↗

PubMed1993

Progesterone and dexamethasone inhibition of uterine epithelial cell proliferation: studies with antiprogesterone compounds in the neonatal mouse.

Progestins and glucocorticoids inhibit uterine epithelial cell proliferation. Earlier studies showed that in the neonatal mouse, dexamethasone (Dex) was at least 100-fold more potent than progesterone (P) as an inhibitor of epithelial DNA synthesis. Using steroid autoradiography, we now show that the uterine cells of the neonatal mouse have receptors for both progestins (PR) and glucocorticoids (GR). Since it is known that P can interact with the GR it is possible that even a weak interaction might mediate the inhibitory effects of large doses of P. Therefore, we examined the receptor pathway specificity of the P response in neonatal mice using antiP steroids with reportedly strong antiglucocorticoid activity, RU486, or weak antiglucocorticoid activity, ZK98.734 (ZK734). Both RU486 and ZK734 exhibited strong binding activity in a PR assay performed on cytosolic preparations from adult mouse uteri. For murine thymic GR, the relative binding activity of RU486 was 12-fold that of ZK734. The high PR binding activity was reflected by the in vivo dose-response of the antagonists administered in conjunction with P; either antagonist completely blocked the inhibitory effect of P on uterine epithelial DNA synthesis when it was administered at one-tenth the dose of P. On the other hand, blockade of the inhibitory effect of Dex only occurred when the dose of antagonist was 10-fold the dosage of Dex. There were no significant differences between the two antagonists in blocking the effects of either P or Dex. These results indicate that progestins and glucocorticoids act through their own receptor systems to inhibit DNA synthesis in the uterine epithelium of the neonatal mouse. However, because of the considerable antiglucocorticoid activity of ZK734, a potential role for the interaction between P and the GR cannot be ruled out by these experiments.

Animals↗

PubMed1993

Progesterone and dexamethasone inhibition of estrogen-induced synthesis of DNA and complement in rat uterine epithelium: effects of antiprogesterone compounds.

Progesterone (P) blocks estrogen induction of cell proliferation and synthesis of complement C3 in the epithelium of the immature rat uterus. Here it is shown that dexamethasone (Dex) exerts a similar inhibitory effect on these two parameters. Furthermore, analysis of the newly synthesized, secreted proteins produced during a 20 h explant culture period showed that not only does the uterus synthesize complement C3 but it is capable of proteolytically cleaving complement into its biologically active peptides. Since large doses of P are required for its inhibitory effects and since P can interact with the glucocorticoid receptor (GR), the role of the GR in mediating the P effect was questioned. Two antiprogesterone compounds with reportedly differing antiglucocorticoid activity, ZK98.734 and RU486, were tested for their ability to antagonize the inhibitory actions of P and Dex. Attenuation of estrogen-induced epithelial DNA synthesis by either P or Dex was fully overcome by concurrent administration of either ZK98.734 or RU486. On the other hand, while either antagonist was effective against the inhibitory action of P on estrogen-induced complement C3 synthesis, only ZK98.734 was fully effective in blocking inhibition by Dex. Thus, unlike its activity in rat hepatic cells, ZK98.734 is a potent antiglucocorticoid in the immature rat uterus. Because of this antiglucocorticoid activity, differential antagonism of the P response was not possible using these two steroid analogs. Although these observations support the notion of GR mediated inhibition of estrogen action in the uterus, they do not answer the question of whether P might act through a GR mediated pathway.

Animals↗

PubMed1992

Production of mouse placental lactogen-I and placental lactogen-II by the same giant cell.

In previous studies, mouse placental lactogen I (mPL-I) and mPL-II were localized to trophoblast giant cells in the placenta at midpregnancy. The present study was undertaken to determine whether mPL-I and mPL-II are produced by two distinct populations of giant cells or by the same cells. A heterogeneous population of cells that included trophoblast giant cells was obtained by enzymatic dispersion and Percoll gradient centrifugation of placentas from days 7 and 9 of pregnancy. Cells from day 7 of pregnancy were cultured in serum-free medium for 5 days, and cells that contained mPL-I, mPL-II, or both mPL-I and mPL-II were identified by double-staining immunocytochemistry. The percentage of PL cells that contained both mPL-I and mPL-II increased from about 30% on the first day of culture to about 90% on the third, and then declined to zero by day 5. Between 50% and 60% of the PL cells contained only mPL-I on the first 2 days of culture, and then the percentage of PL cells containing only mPL-I declined. The percentage of cells that contained only mPL-II was low for 3 days (<10%) and then increased to about 80% of the PL-containing cells by day 5. Cells from day 9 of pregnancy were analyzed for the release of mPL-I and/or mPL-II by sequential reverse hemolytic plaque assay. Cells that released only one of the PLs, as well as those that released both PLs, were identified. A shift was present in the type of PL released by the cells when they were followed for two consecutive days of culture. On day 1, most of the plaque-forming cells released only mPL-I, but by day 2, the fraction of plaque-forming cells that released only mPL-I declined whereas the fraction that released only mPL-II increased. Cells that released only mPL-I on the first day of culture and both mPL-I and mPL-II or only mPL-II on the second day of culture were observed. These data suggest that under these culture conditions, PL cells follow a pathway in which they initially produce only mPL-I, then both mPL-I and mPL-II, and finally only mPL-II. In vivo, there is a shift at midpregnancy in the type of PL that is produced by the mouse placenta, and these data suggest that this shift results, at least partly, from a change in gene expression in one population of giant cells.

Animals↗

PubMed1992

Immunohistochemical study of HER-2/neu, epidermal growth factor receptor, and steroid receptor expression in normal and malignant endometrium.

HER-2/neu oncogene protein, epidermal growth factor receptor, progesterone receptor, and estrogen receptor were examined immunohistochemically in specimens of normal and neoplastic endometrium. Tissues obtained at the time of hysterectomy were snap-frozen at liquid nitrogen temperature and serially sectioned at 4 microns. Normal endometrial epithelial cells stained with anti-epidermal growth factor receptor and anti-HER-2/neu with intensities graded from 0 to 3+. Of the 49 endometrial malignancies studied, seven (14%) contained tissue exhibiting HER-2/neu staining in excess (4+) of any of the normal tissues or the other 42 cancer specimens. Expression of both HER-2/neu and steroid receptors was heterogeneous within these seven tumors. To examine this heterogeneity more closely, sections of these and other tumors were double-stained for HER-2/neu and progesterone receptor. It was found that the cells exhibiting 4+ HER-2/neu staining were progesterone receptor-negative. Conversely, cells that were progesterone receptor-positive within the same specimen exhibited HER-2/neu immunostaining equal to or less than 3+. All specimens containing 4+ HER-2/neu tissue were graded 1 or 2 adenocarcinomas, stage I. Thus, there is an inverse relationship between overexpression of HER-2/neu and progesterone receptor in endometrial cancer. On the other hand, overexpression of HER-2/neu in endometrial cancer does not seem to be related to loss of other differentiated characteristics. The prognostic value of these observations awaits continued study.

Biomarkers, Tumor↗

PubMed1991

Effects of progestin antagonists, glucocorticoids and estrogen on progesterone-induced protein secreted by rabbit endometrial stromal cells in culture.

Progesterone enhances the synthesis of a 42 kDa protein secreted by rabbit endometrial stromal cells in primary culture. The duration of that response, the effects of estrogen and the inhibitory ability of antiprogestin steroid analogs, RU486, ZK98.299 and ZK98.734, were tested. Although there was a progressive decrease in the amount of the 42 kDa protein synthesized during a 6-day culture period, progesterone stimulated its rate of synthesis greater than 2-fold throughout that period. The addition of estrogen did not prevent the progressive decrease in the amount of the protein synthesized, nor did it enhance the progesterone effect when the culture medium contained phenol red. Estrogen alone did slightly induce 42 kDa protein synthesis by cells grown in phenol red-free medium, and the progesterone response was accentuated to the same degree. When present in a concentration that was 100-fold that of the progesterone, RU486, ZK98.299 and ZK98.734 each abolished stimulation. This antagonistic effect was overcome by addition of an equimolar concentration of progesterone. Deoxycorticosterone (DOC) also stimulated 42 kDa protein synthesis. The antiprogestins blocked this stimulatory effect, even when both steroids were in equimolar concentrations. There was no difference in the ability of ZK98.299 or ZK98.734 to block DOC stimulation, even though ZK98.734 exhibits no antiglucocorticoid activity [J. Steroid Biochem. 25 (1986) 835]. Therefore, it is likely that the DOC effect is mediated by the progesterone receptor system. These studies indicate that enhanced synthesis of the 42 kDa protein represents a progesterone receptor mediated event and that the cell culture system described can be used as a bioassay for determination of antiprogestin activity.

Animals↗

PubMed1990

Strain differences in the ontogeny of estrogen receptors in murine uterine epithelium.

The expression of estrogen receptor (ER) in the reproductive tracts of neonatal mice was examined using immunocytochemical and autoradiographic methods. Two strains of mice used in previous studies that reported contradictory results showed different rates of uterine epithelial development. In the inbred strain, BALB/c, the epithelium was devoid of receptor from birth through 5 days of age, while uterine epithelial cells of the outbred strain, CD-1, expressed ER as early as 3 days of age. Oviductal epithelium and cervical epithelium expressed ER on the day of birth in CD-1 mice. Glandular ontogeny in the uteri of CD-1 animals was also advanced by 3 days compared to that of BALB/c mice. These observations reconcile the conflicting reports of ER ontogeny in the neonatal mouse. More importantly, these results confirm our earlier observations, indicating that the cells lining uteri of 2- and 4-day-old BALB/c mice lack ER at a time when estrogen induces their proliferation.

Aging↗

PubMed1988

Progesterone and dexamethasone inhibition of uterine epithelial proliferation in two models of estrogen-independent growth.

It is believed that progestins and glucocorticoids block estrogen-induced growth of the uterus by perturbing the mechanism of estrogen stimulation. We have examined the effect of progesterone or dexamethasone on two rodent models of estrogen-independent proliferation of uterine epithelium. Tissues of the neonatal mouse uterus exhibit high rates of deoxyribonucleic acid synthesis in the absence of endogenous steroids. Progesterone and dexamethasone inhibit deoxyribonucleic acid synthesis in the uterine epithelium of 4-day-old mice. When grafted beneath the kidney capsule of an ovariectomized adult, the tissues of the neonatal uterus continue to grow without the addition of estrogen. Both progesterone and dexamethasone blocked uterine epithelial mitotic activity in these allografts. A true growth response can be stimulated in the uterus of an ovariectomized rat by intraluminal instillation of cholera toxin. In the present study, autoradiographic analysis showed that cholera toxin stimulation of deoxyribonucleic acid synthesis was confined to the epithelial compartment in ovariectomized, vehicle-treated rats. When ovariectomized rats were pretreated with progesterone, cholera toxin induced deoxyribonucleic acid synthesis in the stroma but not in the epithelium. Thus progesterone and dexamethasone inhibit uterine epithelial proliferation that is not driven by estrogen stimulation. Therefore in these estrogen-independent model systems, the inhibitory effects of these steroids cannot be due to antagonism of estrogen receptor--mediated events. These observations suggest that inhibition in estrogen-induced uterine epithelial proliferation may also work through a mechanism unrelated to antagonism of estrogen receptor action.

Animals↗