An estrogen receptor genetic polymorphism and the risk of primary and secondary recurrent spontaneous abortion.

OBJECTIVE: A case-control study was undertaken to assess the association between an estrogen receptor gene variant and the risk of recurrent spontaneous abortions. STUDY DESIGN: The frequency of the estrogen receptor gene variant in blood lymphocyte deoxyribonucleic acid and other selected maternal characteristics was compared among 60 primary recurrent aborters, 61 secondary recurrent aborters, and 43 women who had had at least two live births but no spontaneous abortions. RESULTS: No association was evident between the estrogen receptor gene variant and the risk of either primary or secondary recurrent abortion. There were data suggesting that primary recurrent aborters in particular were more likely to report a family history of recurrent abortion and a family history of breast cancer. CONCLUSIONS: These findings indicate that the estrogen receptor polymorphism is not a genetic marker for recurrent spontaneous abortions. Therefore, as suggested by previous investigations, this polymorphism appears to be a marker for breast cancer risk only among the subgroups who have had a history of repeated abortions.

Temperature effect on the action of corticotropin-releasing hormone (CRH) on secretion of immunoreactive beta-endorphin (IR beta EP) in TM3 and AtT-20 cell lines.

Testicular function is sensitive to chemical and thermal stresses. To investigate the effects of small temperature changes on CRH-stimulated beta EP release, we employed TM3 cells, a mouse prepubertal Leydig cell line that secretes ir beta EP. To monitor beta EP secretion from these cells we used the reverse hemolytic plaque assay. After 3.5 hr incubation of cells with hormone, the EC50 of the CRH dose-response curve at 34 degrees C and 37 degrees C were 0.1 nM and 1 nM, respectively. For comparison, we also investigated the effect of temperature on CRH-stimulated beta EP release from a non-testicular cell line, AtT-20, a mouse anterior pituitary cell line. Using radioimmunoassay to measure ir beta EP levels in the media of AtT-20 cells, the EC50s for the CRH dose-response curve at 34 degrees C and 37 degrees C were 0.2 nM and 2 nM, respectively, at 1 h. After 3.5 h this temperature dependent difference in EC50 was still observed. These results suggest that CRH receptors or post-receptor actions in Leydig cells and anterior pituitary corticotropes are sensitive to small temperature changes.

Association between pregnancy-induced hypertension and asthma during pregnancy.

OBJECTIVE: Pregnancy-induced hypertension is an important cause of maternal mortality, intrauterine growth retardation, and perinatal mortality. We examined the relationship between pregnancy-induced hypertension and asthma. STUDY DESIGN: The study population consisted of 24,115 women without a history of chronic systemic hypertension who were delivered of live born and stillborn infants at Mount Sinai Medical Center between January 1987 and December 1991. Pregnancy-induced hypertension was defined as blood pressure of at least 140/90 mm Hg or an increase of > or = 30 mm Hg in systolic pressure or > or = 15 mm Hg in diastolic pressure. RESULTS: There was a significant association between pregnancy-induced hypertension and asthma during pregnancy (chi 2 = 17.86, p < 0.001). In addition, there was a significant upward trend in the incidence of asthma during pregnancy in women without, with moderate, and with severe pregnancy-induced hypertension (Mantel-Haenszel chi 2 = 11.8, p = 0.001). Logistic regression analysis demonstrated that the association between pregnancy-induced hypertension and asthma during pregnancy persisted after adjustment for the confounding factors of race or ethnicity, maternal age, parity, and prepregnancy weight (adjusted odds ratio 2.52, 95% confidence interval 1.47 to 4.35, p = 0.0008). An association between pregnancy-induced hypertension and a history of asthma was also found (chi 2 = 11.2, p = 0.001). However, after adjustment for potential confounders, this association failed to achieve statistical significance (adjusted odds ratio 1.2, 95% confidence interval 0.97 to 1.53, p = 0.083). CONCLUSION: Both pregnancy-induced hypertension and asthma might be caused by a third factor affecting smooth muscle reactivity.

Estrogen receptor variant and hypertension in women.

Eighty-eight women visiting a gynecologist were tested for an estrogen receptor B-variant allele. The women were ethnically and racially homogeneous to a large degree. They were from a suburb of Long Island, and most were white. The 12% incidence of hypertension in women with the estrogen receptor wild-type allele is comparable to the 13-32% incidence in the general population of women aged 55-64 years. However, the 48% incidence of hypertension in women with the estrogen receptor B-variant allele is considerably higher than in the general population of women in this age group. We conclude that the presence of the estrogen receptor B-variant allele might have increased the prevalence of hypertension in the women in this study.

An estrogen receptor genetic polymorphism and a history of spontaneous abortion--correlation in women with estrogen receptor positive breast cancer but not in women with estrogen receptor negative breast cancer or in women without cancer.

We previously identified a polymorphism in the human estrogen receptor gene. In a preliminary study on women with estrogen receptor positive (ER+) breast tumors, we found that the presence of the rarer of the two alleles, the B' allele, is correlated with a history of spontaneous abortion. Because that study evaluated only women with estrogen receptor positive (ER+) breast cancer, it was unknown whether the observed correlation was restricted to the cancer group or was independent of breast cancer. We have now extended our analysis to include not only additional women with ER+ breast cancer, but also those with estrogen receptor negative (ER-) breast cancer and women without cancer. Results of the current study continue to show an association between the B' allele and a history of spontaneous abortion in the ER+ breast cancer group. There was no such correlation either in the ER- breast cancer group or in the group without cancer. Also, we continue to observe, in the ER+ breast cancer group, a significantly higher concentration of ER protein in tumors from homozygous wild type women (genotype BB), than in the tumors from women who are heterozygous for the rarer allele (genotype BB'). We conclude that the combination of spontaneous abortion and the BB' ER genotype may be a marker for breast cancer susceptibility.

Stimulation of angiotensinogen mRNA levels in rat pituitary by estradiol.

Angiotensin II (ANG II) is a putative paracrine hormone in the anterior pituitary. Angiotensinogen mRNA, however, is not detectable by Northern blot hybridization, suggesting that ANG II may not be synthesized within the pituitary. An alternative explanation may be that angiotensinogen gene activity is low under normal conditions, with angiotensinogen mRNA being below the level of detection. Utilizing a sensitive solution hybridization method, we sought to determine whether angiotensinogen mRNA could be detected in pituitaries from normal male rats or ovariectomized (OVX) rats treated with estradiol (E2) for 4 days. Very low levels of angiotensinogen mRNA were detected from male or OVX rat pituitaries, but E2 treatment resulted in a marked dose-dependent increase in pituitary angiotensinogen mRNA levels. Levels of angiotensinogen within the pituitary were not significantly different after the E2 treatment. Angiotensinogen mRNA levels in liver and brain were much higher than in the pituitary but were not altered significantly by the chronic E2 treatment. These results are consistent with the local synthesis of angiotensinogen in the pituitary and further suggest that pituitary angiotensinogen gene transcription is regulated by estrogen.

Up-regulation of the uterine estrogen receptor and its messenger ribonucleic acid during the mouse estrous cycle: the role of estradiol.

Uterine estrogen receptor (ER) and ER mRNA were measured in cycling and ovariectomized (OVX) estrogen-treated mice to probe the physiological regulation of the intracellular distribution and biosynthesis of ER. On proestrus, when plasma estradiol (E2) levels are highest, the cell nuclear ER concentration was 2.4-fold greater than on metestrus. This increase was primarily attributable to an increase in total cellular ER (cytosolic plus nuclear ER) and only secondarily to an activation of ER, as measured by its redistribution from the cytosolic (i.e. nuclear-extractable) to the nuclear (nonextractable) fraction. Total cellular ER concentration was 1.8-fold higher on proestrus than on metestrus, whereas the fraction of total ER in the nuclear compartment (i.e. the percentage activated) was only 1.3-fold higher. The concentration of cellular ER mRNA was 3-fold greater on proestrus than on the other days of the estrous cycle, suggesting that the increased concentration of ER on proestrus was a consequence of increased ER gene expression. In OVX mice, physiological and, to a lesser extent, supraphysiological levels of E2 increased cell nuclear ER. As in proestrous mice, the increased ER content contributed more than ER activation to the increased cell nuclear ER concentration. Physiological, but not supraphysiological, concentrations of E2 increased ER mRNA in OVX mice. Together, these results suggest that up-regulation by E2 of ER mRNA and ER accounts for most of the increased nuclear binding of ER on proestrus. E2-dependent activation and consequent DNA binding of ER presumably initiate this process, but quantitatively account for only a small fraction of the increased nuclear binding of ER.

Regulation of beta-endorphin secretion by corticotropin-releasing factor in the intact rat testis.

CRF stimulates beta-endorphin (beta EP) secretion from adult rat Leydig cells in vitro. To evaluate the relevance of this action of CRF in a physiological context, we studied the effect of CRF on beta EP secretion in the testis in whole animals. In the pubertal rat, intratesticular infusion of CRF resulted in a 3-fold stimulation of immunoreactive beta EP (ir beta EP) levels in testicular interstitial fluid (TIF) compared with levels in contralateral saline-infused testes. Coadministration of the antagonist CRF-(12-41) resulted in TIF ir beta EP concentrations that were reduced compared to levels in paired saline controls. Infusion of the competitive antagonist CRF-(9-41) alone slightly stimulated ir beta EP concentrations in TIF. In adult animals, all of the peptides tested were without effect. These results suggest a developmental regulation of the action of CRF on beta EP levels in the pubertal rat testis. Our studies in conjunction with documented results demonstrate that in the testis, beta EP is actively secreted. These results imply that testicular ir beta EP is derived from a POMC mRNA that encodes a signal peptide containing preprohormone similar or identical to pituitary POMC. Previous studies of rodent testicular mRNA have found only 5'-truncated forms of POMC message. The current study provides direct evidence for a low abundance POMC transcript that could encode the preprohormone. Therefore, the major components of a pituitary-like CRF/POMC stimulus/secretion system appear to be present in the rat testis, including the full-length POMC mRNA and release of POMC-derived beta EP that is stimulated by CRF.

Stimulation of beta-endorphin secretion by corticotropin-releasing factor in primary rat Leydig cell cultures.

CRF, a hypothalamic peptide, is a potent stimulator of POMC synthesis and secretion in the pituitary. POMC biosynthesis has been documented in the testis, specifically in Leydig cells, and recent studies suggest that CRF is synthesized locally in the testis. A reverse hemolytic plaque assay and immunocytochemistry with Leydig cell-specific antibodies were used to study the effect of CRF on secretion of the POMC peptide beta-endorphin (beta EP) from normal rat primary Leydig cell cultures. In enriched Leydig cell preparations incubated with beta EP antiserum (diluted 1:50) then with complement (diluted 1:25), approximately 15% of immunocytochemically identified Leydig cells formed plaques. Preabsorption of the antiserum with beta EP (2 micrograms/microliters antiserum) overnight at 4 C abolished the formation of plaques. Increasing concentrations of CRF (from 10(-1) to 10(-7) M) resulted in an approximately 80% increase in both the percentage of plaque-forming cells and the mean plaque size. When the CRF antagonist CRF-(9-41) (10(-6) M) was added in the presence of CRF, the increases in plaque number and average size did not occur. These results demonstrate that Leydig cells have functional CRF receptors and that beta EP secretion from these cells is stimulated by CRF.

Distribution and partial characterization of immunoreactive prolactin in the rat brain.

Immunoreactive (IR) prolactin was localized immunocytochemically in cell bodies in the mediobasal hypothalamus and in fibers in many regions of the rat brain. The cell bodies were found in the arcuate nuclei and the adjacent areas ventral to the ventromedial nuclei. Fiber projections extended rostrally to and/or through the anterior hypothalamus, preoptic area, nucleus accumbens, septum, diagonal bands of Broca, caudate-putamen, frontal cortex and accessory olfactory bulb; laterally to the amygdala, especially the central nucleus and some parts of the medial nucleus; caudally to and/or through the midbrain central gray, reticular formation, parabrachial region, and several portions of the lower brain stem and spinal cord extending to sacral levels. The system appears to be essentially identical to that containing proopiomelanocortin (POMC) and its processed peptides, as shown by double immunocytochemistry. Preabsorption of the antiprolactin antiserum with either prolactin or the 16,000-dalton N-terminus of POMC eliminated immunoreactivity in the brain. Preabsorption with other POMC-derived peptides, including beta-lipotropic hormone, beta-endorphin, met-enkephalin, adrenocorticotrophic hormone (1-24), corticotropin-like intermediate lobe peptide, alpha- and gamma-melanocyte-stimulating hormones and an octapeptide region of the N-terminus of POMC bearing some homology with prolactin, did not eliminate immunoreactivity in the brain. Similarly, preabsorption with growth hormone, luteinizing hormone, follicle-stimulating hormone, motilin or fetuin did not eliminate immunoreactivity in the brain. The antiprolactin antiserum also recognized all cells in the intermediate lobe and a subset of cells in the anterior lobe of the Snell dwarf mouse pituitary. This immunoreactivity was eliminated by preabsorption of the antiserum with prolactin or with the 16,000-dalton N-terminus of POMC. These results suggest that IR prolactin in the brain may be related to the N-terminus of POMC. Additional results based on one- and two-dimensional gel electrophoresis and immunoblotting indicate that the antiprolactin antiserum used in the majority of the immunocytochemical studies recognized a number of proteins.

Age-related changes in proopiomelanocortin messenger ribonucleic acid levels in hypothalamus and pituitary of female C57BL/6J mice.

Concentrations of POMC-derived neuropeptides are reduced in hypothalami of aged rodents, whereas levels of POMC products in the pituitary are usually either unchanged or increased. Whether these changes reflect altered synthesis or processing of POMC or altered expression of the POMC gene has not been established. We, therefore, measured POMC mRNA in hypothalami and pituitaries of young (7-month-old), middle-aged (15-month-old), and old (31-month-old) female mice, using slot blot hybridization of total RNA to riboprobes synthesized from cloned POMC DNA fragments. Concentrations of poly(A) and ribosomal RNA were also determined, using probes synthesized from poly(T) and a cloned ribosomal DNA fragment, respectively. Hypothalamic POMC mRNA content and concentration were 30% lower in old than in young and middle-aged mice (P less than 0.01). This change was not due to a general decline in hypothalamic mRNA or rRNA levels, neither of which changed with age. In contrast to the hypothalamus, the relative concentration of POMC mRNA in the pituitary nearly doubled in old mice (P less than 0.01). This increase was secondary to a decrease in other RNA species, however, since the total pituitary content of POMC mRNA did not differ between young and old mice. These results indicate that the regulation of POMC gene expression during aging differs in hypothalamus and pituitary, and that reduced levels of hypothalamic POMC mRNA may account for the previously reported reductions in concentrations of hypothalamic POMC peptides during aging.

Localization of HLA-DR alpha-chain messenger ribonucleic acid in normal and autoimmune human thyroid using in situ hybridization.

We studied the cellular location of HLA-DR alpha-chain synthesis within the human thyroid gland using the technique of in situ hybridization. Tritium-labeled cRNA probes for the HLA-DR alpha-chain DNA sequence revealed significant HLA-DR alpha-chain gene expression in thyroid glands from patients with Graves' hyperthyroidism and Hashimoto's thyroiditis. Hybridization signals, which were RNA strand specific, were widely distributed over thyroid follicular epithelial cells as well as over areas of lymphoid infiltration, with the highest concentration of HLA-DR alpha-chain mRNA within epithelial cells in Graves' disease follicles adjacent to areas of lymphoid infiltration. Qualitatively similar results were obtained with immunoperoxidase staining of thyroid sections for DR antigen. Thyroid tissue obtained from patients who did not have autoimmune thyroid disease contained no detectable HLA-DR antigen, but in situ hybridization revealed variable levels of HLA-DR alpha-chain mRNA in thyroid epithelial cells in such glands. Some glands had no detectable HLA-DR alpha-chain mRNA levels above background, whereas other adult glands had significant DR-specific mRNA levels. Fetal thyroid tissue, however, was negative for strand-specific HLA-DR alpha-chain transcripts as well as for HLA-DR antigen. These results indicate that thyroid epithelial cells are capable of synthesizing HLA class II antigens. The DR genes were expressed to the highest degree within the thyroid glands of patients with autoimmune thyroid disease, where expression was strongly associated with lymphoid infiltration.

HLA-DR alpha chain expression in human thyroid cells.

Using a cDNA probe encoding the human major histocompatibility Class II antigen HLA-DR alpha chain, we have detected a single DR alpha chain-specific transcript in total cellular RNA prepared from human thyroid tissue. The hybridizable RNA in thyroid samples was indistinguishable in size from the DR mRNA in the Raji human B lymphoblastoid cell line. Of the thyroid glands examined, samples from patients with autoimmune thyroid disease consistently demonstrated the highest DR alpha chain transcript levels, with a mean +/- SEM OF 62 +/- 13% of levels found in DR-positive Raji cells. Cytoplasmic dot blot analyses of 5-day thyroid cell cultures depleted of lymphocytes and monocytes indicated that normal thyrocytes contain readily-detectable levels of DR alpha chain-mRNA. These transcript levels varied from individual to individual, with a mean of 16 +/- 9% relative to Raji cell control values and were shown to correlate after lectin or gamma interferon stimulation with enhanced numbers of immunoreactive DR antigen-positive cells. Such findings demonstrate expression of HLA Class II antigen genes in normal unstimulated human thyroid cells and suggest that quantitative variation in thyroid Class II antigen (DR) gene expression may be a major factor in thyroid immunoregulation.

Combination of immunocytochemistry and in situ hybridization in the same tissue section of rat pituitary.

A procedure is described for combining avidin-biotinylated horseradish peroxidase immunocytochemistry, for localizing peptides or proteins, with in situ hybridization for localizing mRNA autoradiographically in the same cryostat section of paraformaldehyde-fixed rat pituitary. Protection against enzymatic degradation of target mRNAs during the immunocytochemical step was necessary and was accomplished by including an RNase inhibitor, 0.04% diethylpyrocarbonate, in primary and secondary antisera. This combination of methods may be useful in other tissues, as well, for (a) determining the relation of protein content to the concentration of its encoding mRNA, (b) proving the synthetic capacity of a cell in which a protein has been localized, (c) determining immunological or nucleic acid probe specificity, or (d) as an alternative to double-labeling immunocytochemical methods.

Characterization and localization of proopiomelanocortin messenger RNA in the adult rat testis.

Northern blot analysis of total RNA and polyadenylated RNA isolated from adult rat testes showed that a proopiomelanocortin (POMC)-like messenger RNA molecule is present in these extracts. The testicular POMC messenger RNA is comparable in length to amygdala and midbrain POMC messenger RNA and appears to be at least 200 nucleotides shorter than POMC messenger RNA found in the hypothalamus and anterior and intermediate lobes of the pituitary gland. Hybridization in situ showed that POMC messenger RNA is located in Leydig cells, which are the only testicular cells that contain immunostainable POMC-derived peptides. These results suggest that local synthesis of POMC occurs in the testis.

Prolactin mRNA exists in rat hypothalamus.

Using radiolabeled DNA complementary to rat pituitary prolactin mRNA, we probed RNA gel blots from three rat tissues: pituitary, hypothalamus, and liver. Poly (A)-enriched RNA from male and female hypothalami contained a hybridizable RNA which was the same size as, though less abundant than, mature pituitary PRL mRNA. These results support the proposal that the rat brain synthesizes PRL.

Intraneuronal alpha-fetoprotein and albumin are not synthesized locally in developing brain.

Alpha-Fetoprotein and albumin are found intraneuronally in developing mammalian brains. To investigate whether this results from local biosynthesis, the existence of the corresponding brain messenger RNAs and the ability of brain cultures to synthesize these proteins were studied in the mouse. The findings show that neither the proteins nor the mRNAs coding for them are made in sufficient quantities to account for the material detected immunologically, supporting an extracellular origin.

Differential regulation by glucocorticoids of proopiomelanocortin mRNA levels in the anterior and intermediate lobes of the rat pituitary.

Glucocorticoids negatively regulate de novo synthesis of proopiomelanocortin (POMC) and the steady state POMC mRNA levels in the rat pituitary anterior lobe. The POMC system of the intermediate lobe is far less sensitive to alterations in circulating glucocorticoid levels. Possible mechanisms for this differential regulation of POMC gene expression in the two tissues are discussed.