Subclones of normal clonal prolactin cells of the rat

Summary We recently established a clone (2B8) of normal rat prolactin cells that secretes only prolactin into the medium. When grown in the presence of thyrotrophin-releasing hormone (TRH), estradiol (E₂) or arginine vasotocin (AVT), the cells show increased production of prolactin. Subclones of single cell origin were developed from 2B8 cells exposed for 1 week to TRH, E₂ or TRH plus E₂. These subclones differ in their response to TRH, E₂ or AVT and therefore may possess different receptors for these hormones. Presented in preliminary from at the meeting of the Endocrine Society in Chicago, June 9, 1977. This work was supported by USPHS Grant AM12583.     

Inhibitory effect of obesity on gonadotropin, estradiol, and inhibin B levels in fertile women

Objective: To examine whether obesity and insulin resistance have an independent effect on the gonadotropin, estradiol, and inhibin B serum levels and follicle count in the early follicular phase of fertile women with a wide range of BMI and without signs of hyperandrogenism. Research Methods and Procedures: Twenty‐two overweight and obese (BMI ≥25.0 kg/m²) women and 10 normal‐weight (BMI <25.0 kg/m²) women, all having apparently normal fertility, were studied. Serum concentrations of follicle‐stimulating hormone (FSH), luteinizing hormone (LH), estradiol, inhibin B, and insulin, level of insulin resistance (estimated by homeostasis model assessment for insulin resistance), and follicle count were measured during the early follicular phase (Days 2 to 5 of the menstrual cycle). Results: Overweight women showed lower FSH (p < 0.001), LH (p < 0.001), and inhibin B (p < 0.05) levels compared with normal‐weight women, whereas estradiol concentrations and follicle count were not significantly different between the two groups. When normal‐weight and overweight women were examined as a group and multiple regression analyses were performed, estradiol showed a negative association with BMI (or waist circumference) (p < 0.05) and a positive correlation with LH (p < 0.05) and FSH (p < 0.05); inhibin B maintained a positive association only with estradiol (p < 0.05); and FSH and LH showed a negative correlation with BMI (or waist circumference) (p < 0.001 and p < 0.01, respectively). Discussion: Overweight and obese fertile women have lower FSH, LH, inhibin B, and estradiol levels in the early follicular phase, with a possible direct inhibitory effect of body mass on gonadotropin and estradiol production, independently of age, insulin (concentrations and sensitivity), and other hormones. By contrast, the number of ovary follicles does not seem to be influenced by insulin and body mass in these patients.     

Tissue selectivity and potential clinical applications of trenbolone (17β-hydroxyestra-4,9,11-trien-3-one): A potent anabolic steroid with reduced androgenic and estrogenic activity

Recently, the development of selective androgen receptor modulators (SARMs) has been suggested as a means of combating the deleterious catabolic effects of hypogonadism, especially in skeletal muscle and bone, without inducing the undesirable androgenic effects (e.g., prostate enlargement and polycythemia) associated with testosterone administration. 17β-Hydroxyestra-4,9,11-trien-3-one (trenbolone; 17β-TBOH), a synthetic analog of testosterone, may be capable of inducing SARM-like effects as it binds to androgen receptors (ARs) with approximately three times the affinity of testosterone and has been shown to augment skeletal muscle mass and bone growth and reduce adiposity in a variety of mammalian species. In addition to its direct actions through ARs, 17β-TBOH may also exert anabolic effects by altering the action of endogenous growth factors or inhibiting the action of glucocorticoids. Compared to testosterone, 17β-TBOH appears to induce less growth in androgen-sensitive organs which highly express the 5α reductase enzyme (e.g., prostate tissue and accessory sex organs). The reduced androgenic effects result from the fact that 17β-TBOH is metabolized to less potent androgens in vivo; while testosterone undergoes tissue-specific biotransformation to more potent steroids, dihydrotestosterone and 17β-estradiol, via the 5α-reductase and aromatase enzymes, respectively. Thus the metabolism of 17β-TBOH provides a basis for future research evaluating its safety and efficacy as a means of combating muscle and bone wasting conditions, obesity, and/or androgen insensitivity syndromes in humans, similar to that of other SARMs which are currently in development.     

Evidence on the operation of ATP-induced capacitative calcium entry in breast cancer cells and its blockade by 17beta-estradiol

Little is known about the regulation of cytosolic calcium Ca(2+) levels ([Ca(2+)](i)) in breast cancer cells. We investigated the existence of capacitative calcium entry (CCE) in the tumorigenic cell line MCF-7 and its responsiveness to ATP. MCF-7 cells express purinergic receptors as well as estrogen receptors (ER). Depletion of calcium stores with thapsigargin (TG, 500 nM) or ATP (10 microM) in the absence of extracellular Ca(2+), resulted in a rapid and transient elevation in [Ca(2+)](i). After recovery of basal levels, Ca(2+) readmission (1.5 mM) to the medium increased Ca(2+) influx (twofold over basal), reflecting pre-activation of a CCE pathway. Cells pretreated with TG were unable to respond to ATP, thus indicating that the same Ca(2+) store is involved in their response. Moreover, IP(3)-dependent ATP-induced calcium mobilization and CCE were completely blocked using compound U-73122, an inhibitor of phospholipase C. Compound 2-APB (75 microM) and Gd(3+) (10 microM), antagonists of the CCE pathway, completely prevented ATP-stimulated capacitative Ca(2+) entry. CCE in MCF-7 cells was highly permeable to Mn(2+) and to the Ca(2+) surrogate Sr(2+). Mn(2+) entry sensitivity to Gd(3+) matched that of the Ca(2+) entry pathway. 17Beta-estradiol blocked ATP-induced CCE, but was without effect on TG-induced CCE. Besides, the estrogen blockade of the ATP-induced CCE was completely abolished by preincubation of the cells with an ER monoclonal antibody. ER alpha immunoreactivity could also be detected in a purified plasma membrane fraction of MCF-7 cells. These results represent the first evidence on the operation of a ATP-responsive CCE pathway in MCF-7 cells and also indicate that 17beta-estradiol interferes with this mechanism by acting at the cell surface level.     

Supraphysiological estradiol promotes human T follicular helper cell differentiation and favours humoural immunity during in vitro fertilization

During pregnancy, humoural immunity is essential for protection against many extracellular pathogens; however, autoimmune diseases may be induced or aggravated. T follicular helper (Tfh) cells contribute to humoural immunity. The aim of this study was to test whether Tfh cell function can be manipulated via hormones. Seventy-four women who underwent in vitro fertilization were recruited and divided into four groups: menstrual period (MP), controlled ovarian hyperstimulation (COH), embryo transfer (ET) and pregnant after embryo transfer (P). A flow cytometry analysis was performed to identify Tfh cells in peripheral blood mononuclear cells (PBMCs). Bioinformatics analysis revealed a possible pathway between Tfh and B cells. Enzyme-linked immunosorbent assays were used to detect interleukin (IL)-21 and IL-6. The quantitative polymerase chain reaction was performed to quantify BCL-6, BACH2, XBP-1, IRF-4 and G protein-coupled (GP)ER-1 mRNA expression. Compared with the MP group, the COH, ET and P groups showed more Tfh and B cells, as well as higher IL-21, IL-6, BCL-6 and BACH2 expression. Furthermore, Tfh cell frequency in PBMCs, as well as serum IL-21 and IL-6 levels, were all positively correlated with serum estradiol (E₂) levels; the B cell percentage also correlated positively with Tfh cells in PBMCs. Combined with the bioinformatics analysis, XBP-1, IRF-4 and GPER-1 expression was related to E₂ levels, both in vivo and in vitro. We speculate that E₂ augments Tfh cells and favours humoural immunity. This study indicates that Tfh cell regulation may be a novel target in maintaining the maternal-foetal immune balance.     

Modulating the binding properties of an anti-17beta-estradiol antibody by systematic mutation combinations

The anti-17beta-estradiol antibody 57-2 has been a subject for several protein engineering studies that have produced a number of mutants with improved binding properties. Here, we generated a set of 16 antibody 57-2 variants by systematically combining mutations previously identified from phage display-derived improved antibody mutants. These mutations included three point mutations in the variable domain of the light-chain and a heavy-chain variant containing a four-residue random insertion in complementarity determining region CDR-H2. The antibody variants were expressed as Fab fragments, and they were characterized for affinity toward estradiol, for cross-reactivity toward three related steroids, and for dissociation rate of the Fab/estradiol complex by using time-resolved fluorescence based immunoassays. The double-mutant cycle method was used to address the cooperativity effects between the mutations. The experimental data were correlated with structural information by using molecular modeling and visual analysis of the previously solved antibody 57-2 crystal structures. These analyses provided information about the steroid-binding mode of the antibody, the potential mechanisms of individual mutations, and their mutual interactions. Furthermore, several combinatorial mutants with improved affinity and specificity were obtained. The capacity of one of these mutants to detect estradiol concentrations at a clinically relevant range was proved by establishing a time-resolved fluorescence based immunoassay.     

Cellular localization of the receptor-dependent and receptor-independent uptake of human LDL in the liver of normal and 17 alpha-ethinyl estradiol-treated rats

The cellular localization in the liver of the receptor-dependent and -independent uptake of human low density lipoprotein (LDL) in normal and 17 alpha-ethinyl estradiol-treated rats was investigated by the simultaneous in vivo injection of human 131I-LDL and human reductive methylated 125I-LDL. The cells were subsequently isolated by a low temperature method. In untreated rats, after 30 min of in vivo circulation of human LDL, 57% of the receptor-dependent liver-association of human LDL occurs in non-parenchymal cells and 43% in parenchymal cells. Estradiol treatment of rats for 3 days selectively increases the receptor-dependent cell-association of human LDL with hepatocytes (17-fold), while the receptor-dependent cell-association with non-parenchymal cells is not affected.     

Rapid estrogen regulation of DHEA metabolism in the male and female songbird brain

In the songbird brain, dehydroepiandrosterone (DHEA) is metabolized to the active and aromatizable androgen androstenedione (AE) by 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD). Thus, brain 3β-HSD plays a key role in regulating the steroidal milieu of the nervous system. Previous studies have shown that stress rapidly regulates brain 3β-HSD activity in a sex-specific manner. To elucidate endocrine regulation of brain 3β-HSD, we asked whether 17β-estradiol (E₂) regulates DHEA metabolism in adult zebra finch ( Taeniopygia guttata ) and whether there are sex-specific effects. Brain tissue was homogenized and centrifuged to obtain supernatant lacking whole cells and cell nuclei. Supernatant was incubated with [³H]DHEA and radioinert E₂ in vitro . Within only 10 min, E₂ significantly reduced 3β-HSD activity in both male and female brain. Interestingly, the rapid effects of E₂ were more pronounced in females than males. These are the first data to show a rapid effect of estrogens on the songbird brain and suggest that rapid estrogen effects differ between male and female brains.     

LC3 as a potential therapeutic target in hypoxia-induced pulmonary hypertension

Recent research suggests that microtubule-associated protein 1 light chain 3B (LC3B) confers protection against hypoxia-induced pulmonary hypertension (HPH) by inhibiting proliferation of pulmonary artery (PA) wall cells. We recently demonstrated that 17β-estradiol (E2), a sex hormone with known protective properties in HPH, increases lung LC3-II expression in chronically hypoxic male Sprague-Dawley rats. Stimulatory E2 effects on LC3-II were recapitulated in isolated hypoxic (1% O₂ for 48 h), but not room air-exposed primary rat PA endothelial cells (PAECs), and were accompanied by hypoxia-specific inhibitory effects on other parameters involved in proproliferative signaling (MAPK3/ERK1-MAPK1/ERK2 activation, VEGF secretion), as well as inhibitory effects on PAEC proliferation. Taken together, these results suggest that E2 mediates hypoxia-specific antiproliferative effects in PAECs, and that stimulation of autophagy may be one of the underlying mechanisms of E2-mediated protection in HPH. Viewed in the context of previously published data, these results indicate that LC3 1) exerts protective effects in the pathogenesis of HPH, and 2) may represent a potential target for future therapeutic interventions in HPH.     

Relationship between gonadal steroid hormones and vulvar bleeding in southern tamandua, Tamandua tetradactyla

This study aimed at demonstrating the profiles of circulating gonadal steroid hormones during the estrous cycle and pregnancy in a southern tamandua (Tamandua tetradactyla). Additionally, this study clarified the relationship between vulvar bleeding and hormonal changes. The concentrations of serum progesterone (P(4)) and estradiol-17β (E(2)) were determined by enzyme immunoassays. Serum P(4) and E(2) concentrations changed cyclically and the estrous cycle length (± SD) based on the E(2) cycles was 44.3 ± 4.5 days. Vulvar bleeding started to be seen at the decreasing of P(4). The cycle length for vulvar bleeding was 43.3 ± 4.2 days. Interval from the first day of bleeding to the peak of E(2) concentration was 23.1 ± 3.1 days. Serum P(4) during pregnancy remained high and E(2) increased 8 weeks after conception and remained high until parturition. The female delivered normally after a 165 day-pregnancy period and reared the offspring well. Approximately 3 weeks after parturition, serum E(2) and P(4) cycles resumed. Visual bleeding may be useful as a real-time indicator for understanding the ovarian cycle of southern tamanduas, and estrus could be expected approximately 3 weeks after the first bleeding.