Direct Effects Of Estrogen Research Articles

Estrogen receptor-α signaling in osteoblast progenitors stimulates cortical bone accrual

The detection of estrogen receptor-α (ERα) in osteoblasts and osteoclasts over 20 years ago suggested that direct effects of estrogens on both of these cell types are responsible for their beneficial effects on the skeleton, but the role of ERα in osteoblast lineage cells has remained elusive. In addition, estrogen activation of ERα in osteoclasts can only account for the protective effect of estrogens on the cancellous, but not the cortical, bone compartment that represents 80% of the entire skeleton. Here, we deleted ERα at different stages of differentiation in murine osteoblast lineage cells. We found that ERα in osteoblast progenitors expressing Osterix1 (Osx1) potentiates Wnt/β-catenin signaling, thereby increasing proliferation and differentiation of periosteal cells. Further, this signaling pathway was required for optimal cortical bone accrual at the periosteum in mice. Notably, this function did not require estrogens. The osteoblast progenitor ERα mediated a protective effect of estrogens against endocortical, but not cancellous, bone resorption. ERα in mature osteoblasts or osteocytes did not influence cancellous or cortical bone mass. Hence, the ERα in both osteoblast progenitors and osteoclasts functions to optimize bone mass but at distinct bone compartments and in response to different cues.

Estradiol Treatment and Hormonal Fluctuations During the Estrous Cycle Modulate the Expression of Estrogen Receptors in the Auditory System and the Prepulse Inhibition of Acoustic Startle Response

Estrogens' effects on hearing are documented across species, but the responsible molecular mechanisms remain unknown. The presence of estrogen receptors (ER) throughout the auditory system offers a potential pathway of direct estrogenic effects on auditory function, but little is known about how each ER's expression is regulated by the overall hormonal status of the body. In the present study, we determined the effects of ovariectomy and chronic 17β-estradiol treatment on mRNA and protein expression of ERα and ERβ in peripheral (cochlea) and central (inferior colliculus) auditory structures of mice, as well as on auditory-related behavior using the acoustic startle response (ASR), prepulse inhibition (PPI), and habituation of the startle response. 17β-Estradiol treatment down-regulated ERα but not ERβ and increased PPI and latency of the ASR. Neither the magnitude nor the habituation of ASR was affected. Furthermore, ER's mRNA and protein expression in the inner ear were analyzed throughout the estrous cycle (proestrus, estrus, metestrus, and diestrus), revealing a negative correlation of circulating estrogens with ERα expression, whereas ERβ was stable. Our findings show that ER not only are present in both the peripheral and central auditory system but also that circulating estrogen levels down-regulate ERα expression in the auditory system and affect PPI and the latency of ASR, suggesting a key role of ERα as a hormone-induced modulator of the auditory system and behavior.

Estrogen-dependent activation of neutral cholesterol ester hydrolase underlying gender difference of atherogenesis in apoE−/− mice

ObjectiveMechanisms underlying gender difference of atherogenesis were investigated focusing on direct effects of estrogen on the artery. MethodsFirst, male and female apoE−/− mice were fed an atherogenic diet for 16 weeks from 10 weeks of age. Second, female apoE−/− mice were ovariectomized (ovx) or sham operated at 8 weeks of age, and 2-weeks afterwards, one-third of each ovx-group received conjugated equine estrogens (CEE) (0, 2.5 or 5.0μg/day) for 16 weeks. Atherosclerotic lesions were examined after experimental periods. To clarify anti-atherogenic effect of 17β-estradiol (E2) on artery, neutral cholesteryl ester hydrolase (N-CEase) activity in aorta and peritoneal macrophages, and E2-treated J774A.1 cells were measured. ResultsFirst, atherosclerotic lesion in female mice was significantly less than male mice without any changes in serum lipids and lipoprotein profile. N-CEase activity in aorta and peritoneal macrophages in female mice was significantly higher than male mice. Second, atherosclerotic lesion in ovx-group was significantly greater than sham-group. CEE-replacement to ovx-group decreased atherosclerotic lesion in a dose-dependent manner. N-CEase activity in aorta and peritoneal macrophages was decreased in ovx-group compared to sham-group, and restored by CEE-replacement in macrophages. To study detailed mechanisms, J774A.1 cells were treated with E2. E2 significantly increased N-CEase activity, and cAMP-dependent protein kinase (A-kinase) type II activity and the protein in cytosol fraction without any changes of total protein of A-kinase type II. ConclusionThese results suggest that gender difference of atherogenesis is partly accounted for activation of N-CEase through estrogen-dependent translocation of A-kinase type II in macrophages.

Estrogenic effects of dissolved organic matter and its impact on the activity of 17β-estradiol

The ubiquitous dissolved organic matter (DOM) is actually not inert as we always think, and the hormone-like effects of DOM have been reported. The objective of this study was to investigate the estrogenic effects of DOM and its impact on the activity of the natural estrogen 17β-estradiol (E2). DOM of three different sources, HA sodium salt, Suwannee River natural organic matter (NOM), and Nordic Reservoir NOM, were used. The estrogenic activity was detected by using the yeast estrogen screen (YES) assay. Estrogenic effects of DOM without and after solar irradiation were tested. Influences on the action of E2 by DOM were also investigated. No direct estrogenic effects of the DOM used were observed in the YES assay. However, the estrogenic activities after 24 h of irradiation increased to 0.0288, 0.0178, and 0.0195 μM of E2 equivalents for HA sodium salt, Suwannee River NOM, and Nordic Reservoir NOM, respectively. After incubation of DOM, the estrogenic activity of E2 was increased by low concentrations (8.33 and 83.3 μM) of DOM while decreased by higher concentrations (8.33 × 10(2) and 8.33 × 10(3) μM) of DOM. Though direct estrogenic effects of DOM were not observed, increase in the estrogenic activity of DOM after irradiation was significant. DOM shows amphoteric influence on the natural estrogen E2, which depends on the concentration of DOM used. Because of its ubiquity, DOM may be of great ecological significance, playing an important role in regulating the reproduction of aquatic organisms.

Anterior prostate epithelial AR inactivation modifies estrogen receptor expression and increases estrogen sensitivity

Androgens influence prostate growth and development, so androgen withdrawal can control progression of prostate diseases. Although estrogen treatment was originally used to induce androgen withdrawal, more recently direct estrogen effects on the prostate have been recognized, but the nature of androgen-estrogen interactions within the prostate remain poorly understood. To characterize androgen effects on estrogen sensitivity in the mouse prostate, we contrasted models of castration-induced androgen withdrawal in the prostate stromal and epithelial compartments with a prostate epithelial androgen receptor (AR) knockout (PEARKO) mouse model of selective epithelial AR inactivation. Castration markedly increased prostate epithelial estrogen receptor (ER)α immunoreactivity compared with very low ERα expression in intact males. Similarly, strong basal and luminal ERα expression was detected in PEARKO prostate of intact males, suggesting that epithelial AR activity regulated epithelial ERα expression. ERβ was strongly expressed in intact, castrated, and PEARKO prostate. However, strong clusters of epithelial ERβ positivity coincided with epithelial stratification in PEARKO prostate. In vivo estrogen sensitivity was increased in PEARKO males, with greater estradiol-induced prostate growth and epithelial proliferation leading to squamous metaplasia, featuring markedly increased epithelial proliferation, thickening, and keratinization compared with littermate controls. Our results suggest that ERα expression in the prostate epithelial cells is regulated by local, epithelia-specific, androgen-dependent mechanisms, and this imbalance in the AR- and ER-mediated signaling sensitizes the mature prostate to exogenous estrogens.

Thyroid Function and Growth Regulation under Normal and Abnormal Conditions

The thyroid gland exerts a major control on the physiology of the whole body, and it raises fundamental questions about molecular physiological processes. Moreover, an increased proliferation of thyroid cells is associated with several pathologies, and several mechanisms may be involved. Thyroid disorders are very common, affecting millions of people. These include hypothyroidism, hyperthyroidism, thyroid nodules, thyroid cancer, and so forth, but they are also associated with other nonthyroid disorders. This special issue is devoted to illustrate the particular richness of current investigations in the field of thyroid function and growth regulation under normal and abnormal conditions. Although thyroid gland function is mainly under the control of pituitary TSH in normal conditions, other factors may also play a role in this process. Thyroid disease is more common in women than in men. Tania Weber Furlanetto and Ana Paula Santin reviewed the direct effects of estrogens on thyroid function and growth regulation in the paper titled “Role of estrogen in thyroid function and growth regulation.” Thyroid cancer is the most common endocrine malignancy, and its incidence has significantly risen in the last decades in the world. The knowledge how thyroid cancer develops is expanding rapidly. The sequential acquisition of mutations which arise as a consequence of damage to the genome is required in order to transform a normal cell into a malignant one. The understanding of the process of thyroid carcinogenesis at the molecular level will improve not only the diagnostic but also the treatment of this pathology. Ioannis Legakis and Konstantinos Syrigos describe the molecular events associated with the progression and dedifferentiation of thyroid carcinoma in the paper titled “Recent advances in molecular diagnosis of thyroid cancer.” Thyroid-specific transcription factors regulate thyroid-specific gene expression and organogenesis. Their possible role in thyroid cancer as well as in the maintenance and/or activity of stem cells is discussed by Shioko Kimura in the paper titled “Thyroid-specific transcription factors and their roles in thyroid cancer.” MicroRNAs (miRNAs) are short ribonucleic acid molecules (around 22 nucleotides) found in eukaryotic cells. miRNAs are posttranscriptional regulators that bind to complementary sequences on messenger RNA transcripts inducing the translational repression or messenger RNA degradation. Several miRNAs have been found to have links with some types of cancer. Francesca Marini, Ettore Luzi, and Maria Luisa Brandi reviewed the role of miRNAs in thyroid cancer development in the paper titled “MicroRNA role in thyroid cancer development.” Growth factors play a role in thyroid proliferation and function, while EGF acts as a mitogen for thyroid cells inhibiting also thyroid differentiation. TGF-β is a potent inhibitor of thyroid cell growth. However, in some transformed thyroid cells this inhibition is lost. The role of TGF-β and EGF on thyroid carcinogenesis and the crosstalk between these growth factors are discussed by Gabriella Mincione Maria Carmela Di Marcantonio, Chiara Tarantelli, Sonia D'Inzeo, Arianna Nicolussi, Francesco Nardi, Caterina Francesca Donini, and Anna Coppa in the paper titled “EGF and TGF-β1 effects on thyroid function.” Grave's disease and Hashimoto's thyroiditis are the two main types of autoimmune thyroid disease. Occasionally they are also associated with other autoimmune diseases. Emina Kasumagic-Halilovic, Asja Prohic, Begler Begovic, and Nermina Ovcina-Kurtovic bring additional support to the existence of a significant association between vitiligo and thyroid autoimmunity in the paper titled “Association between vitiligo and thyroid autoimmunity.” Although some authors have found an association between an abnormal thyroid condition and bipolar disorder, little is known about the implication of the hypothalamo-pituitary-thyroid in neuropsychological deficits. Subho Chakrabarti review the last findings on this topic including genetic and neuroimaging investigations (Thyroid Functions and Bipolar Affective Disorder). Guillermo Juvenal Daniel Christophe Pierre Roger Mario Pisarev

Direct effects of estrogen on differentiation and apoptosis of osteoclasts

Osteoclasts play a crucial role in bone resorption. Since osteoclast differentiation/activation is involved in orthodontic tooth movement at the compression sites, the investigation on osteoclasts is very important to the field of orthodontics. It is well known that estrogen has the protective effect on bone. However, the mechanisms by which estrogen prevents bone loss remain to be elucidated. Although estrogen was recently reported to induce apoptosis of osteoclasts, the precise mechanisms of estrogen-induced osteoclast apoptosis remained controversial with regard to whether estrogen affects osteoclasts directly or not. Here we investigated whether estrogen directly induces differentiation and apoptosis of osteoclasts in vitro using mouse monocytic RAW264 cells differentiated into osteoclasts by RANKL. It was observed that estrogen inhibited RANKL-induced osteoclast differentiation of RAW264 cells in a dose-dependent manner. Estrogen suppressed p38 phosphorylation while it enhanced ERK phosphorylation induced by RANKL, suggesting that modulation of MAPK signaling may be involved in inhibition of osteoclast differentiation by estrogen. Next, it was shown that estrogen dose-dependently augmented caspase-3 activation in osteoclasts differentiated from RAW264 cells by RANKL, demonstrating that estrogen directly enhanced apoptosis of osteoclasts. Estrogen-induced caspase-3 activation was attenuated by ICI 182,780, suggesting that the effects of estrogen on osteoclast apoptosis is mediated through estrogen receptors. Thus, these results suggest that estrogen may directly inhibit differentiation and induce apoptosis of osteoclasts.

Estrogens and bone disease in chronic kidney disease: role of FGF23

To describe the direct and indirect effects of estrogen on bone with special emphasis on the analysis of the recent findings related to the putative role of FGF23 in the estrogen-dependent parathyroid hormone (PTH) suppression. Estrogens act directly on bone cells, downregulating osteoclast precursors and differentiation, increasing osteoclast apoptosis and stimulating osteoblast proliferation and differentiation. However, estrogens can also act indirectly on bone, modulating the calcium-phosphorus-vitamin D-PTH axis. It has been recently demonstrated that estrogens suppress PTH synthesis and secretion in a dose-dependent manner and reduce serum calcitriol and phosphorus levels by an indirect mechanism. In-vivo and in-vitro experiments demonstrated that FGF23 positively correlated, also in a dose-dependent manner, with the dose of estrogens and with the observed changes in calcitriol and phosphorus. These new findings support the importance of the indirect effects of estrogens on bone, suggesting a role for FGF23 in the regulation of PTH by estrogens.

Effect of oestrogen on T cell apoptosis in patients with systemic lupus erythematosus

Defective control of T cell apoptosis is considered to be one of the pathogenetic mechanisms in systemic lupus erythematosus (SLE). Oestrogen has been known to predispose women to SLE and also to exacerbate activity of SLE; however, the role of oestrogen in the apoptosis of SLE T cells has not yet been documented. In this study, we investigated the direct effect of oestrogen on the activation-induced cell death of T cells in SLE patients. The results demonstrated that oestradiol decreased the apoptosis of SLE T cells stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in a dose-dependent manner. In addition, oestradiol down-regulated the expression of Fas ligand (FasL) in activated SLE T cells at the both protein and mRNA levels. In contrast, testosterone increased FasL expression dose-dependently in SLE T cells stimulated with PMA plus ionomycin. The inhibitory effect of oestradiol on FasL expression was mediated through binding to its receptor, as co-treatment of tamoxifen, an oestrogen receptor inhibitor, completely nullified the oestradiol-induced decrease in FasL mRNA expression. Moreover, pre-treatment of FasL-transfected L5178Y cells with either oestradiol or anti-FasL antibody inhibited significantly the apoptosis of Fas-sensitive Hela cells when two types of cells were co-cultured. These data suggest that oestrogen inhibits activation-induced apoptosis of SLE T cells by down-regulating the expression of FasL. Oestrogen inhibition of T cell apoptosis may allow for the persistence of autoreactive T cells, thereby exhibiting the detrimental action of oestrogen on SLE activity.

Estrogen receptor-mediated enhancement of venous relaxation in female rat: Implications in sex-related differences in varicose veins

A greater incidence of varicose veins has been reported in premenopausal women than in men. We hypothesized that the sex differences in venous function reflect reduced constriction and enhanced venous dilation in women due to direct venous relaxation effects of estrogen on specific estrogen receptors (ER). Circular segments of inferior vena cava (IVC) from male and female Sprague-Dawley rats were suspended between two wires, and isometric contraction (in mg/mg tissue) to phenylephrine, angiotensin II (AngII), and 96 mM KCl was measured. To investigate sex differences in venous smooth muscle, Ca(2+) release from the intracellular stores, and Ca(2+) entry from the extracellular space, the transient phenylephrine contraction in 0 Ca(2+) Krebs was measured. Extracellular CaCl(2) (0.1, 0.3, 0.6, 1, 2.5 mM) was added, and the [Ca(2+)](e)-dependent contraction was measured. To investigate sex differences in venous endothelial function, acetylcholine-induced relaxation was measured. To test the role of specific ERs, the amount of venous tissue ERs was measured using Western blots, and the venous relaxation in response to 17beta-estradiol (E2, activator of most ERs), 4,4,'4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT; ERalpha agonist), 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ERbeta agonist), and ICI 182,780 (ERalpha/ERbeta antagonist, and G protein-coupled receptor 30 [GPR30] agonist) was measured in IVC segments nontreated or treated with the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). Phenylephrine caused concentration-dependent contraction that was less in female (max 104.2 +/- 16.2) than male IVC (172.4 +/- 20.4). AngII (10(-6))-induced contraction was also less in female (81.0 +/- 11.1) than male IVC (122.5 +/- 15.0). Phenylephrine contraction in 0 Ca(2+) Krebs was insignificantly less in female (4.8 +/- 1.8) than male IVC (7.2 +/- 1.7), suggesting little difference in the intracellular Ca(2+) release mechanism. In contrast, the [Ca(2+)](e)-dependent contraction was significantly reduced in female than male IVC. Also, contraction to membrane depolarization by 96 mM KCl, which stimulates Ca(2+) influx, was less in female (129.7 +/- 16.7) than male IVC (319.7 +/- 30.4), supporting sex differences in Ca(2+) entry. Acetylcholine relaxation was greater in female (max 80.6% +/- 4.1%) than male IVC (max 48.0% +/- 6.1%), suggesting sex differences in the endothelium-dependent relaxation pathway. Western blots revealed greater amounts of ERalpha, ERbeta, and GPR30 in female than male IVC. ER agonists caused concentration-dependent relaxation of phenylephrine contraction in female IVC. E2-induced relaxation (max 76.5% +/- 3.4%) was more than DPN (74.8% +/- 9.1%), PPT (71.4% +/- 12.5%), and ICI 182,780 (67.4% +/- 7.8%), and was similar in L-NAME-treated and nontreated IVC. The reduced alpha-adrenergic, AngII, depolarization-induced, and [Ca(2+)](e)-dependent venous contraction in female rats is consistent with sex differences in the Ca(2+) entry mechanisms, possibly due to enhanced endothelium-dependent vasodilation and increased ER expression/activity in female rats. E2/ER-mediated venous relaxation in female rats is not prevented by NOS blockade, suggesting activation of an NO-independent relaxation pathway. The decreased venous contraction and enhanced E2/ER-mediated venous relaxation would lead to more distensible veins in female rats.

Estrogenic in vitro assay on mouse embryonic Leydig cells

We and others have reported that mouse embryonic testes contain a subpopulation of somatic cells expressing estrogen receptor alpha (ERalpha). In order to provide evidence for a possible direct estrogen effect on mammalian testes from the early stage of their differentiation, here we devised a method for the in vitro culture of the ERalpha-expressing cells from 12.5 days post coitum mouse testes and their transfection with plasmids containing the classical estrogen responsive element (ERE) or the alternative estrogen AP-1 responsive element upstream of the luciferase reporter gene (ERE-Luc and AP-1-Luc). StAR immunopositivity of the most part of the ERalpha+ cells grown in culture and subjected to the estrogenic assay, allowed their identification as embryonic Leydig cells. Maximum induction of the ERE-Luc activity was achieved with 10 nM 17-beta estradiol (E2), from 1.7 to 3-fold in such cells and from 2.3 to 5.7-fold in MCF-7 cells used for comparison; the anti-estrogen ICI 182.780 abolished such effects. AP-1-Luc was less sensitive to E2 in both cell types (10 nM E2, 1.2 to 2.7-fold increase in embryonic Leydig cells; about 3-fold in MCF-7 cells) and the effect was not ICI-dependent. Eventually, we stimulated the transfected cells with various xenoestrogens such as lindane, bisphenol A or mono-(2-ethylhexyl) pthalate and with the phytoestrogen zeralenone obtaining evidence for ERE-Luc, but not AP-1-Luc stimulation in embryonic Leydig cells. These results represent evidence of functional ERalpha-dependent genomic pathways in embryonic Leydig cells and describe an in vitro assay suitable for evaluating the activity of putative estrogenic compounds on such cells.

Estrogens and progestins: molecular effects on brain cells.

Sex steroids are known to regulate brain function and their role is so important that several diseases are strictly correlated with the onset of menopause when estrogen-progesterone deficiency makes neural cells much more vulnerable to toxic stimuli. Although in the past years several scientists have focused their studies on in vitro and in vivo effects of sex steroids on the brain, we are still far from complete knowledge. Indeed, contrasting results from large clinical trials have made the entire issue much more complicated. Currently we know that protective effects exerted by sex steroids depend on several factors among which the dose, the health of the cells and the type of molecule being used. In this review, we present an overview of the direct and indirect effects of estrogen and progesterone on the brain with specific focus on the molecular mechanisms by which these molecules act on neural cells.

Estrogen modulates iodoacetate‐induced gene expression in bovine cartilage explants

Estrogen loss may be involved in onset or progression of osteoarthritis. Estrogen receptors are present in chondrocytes, thus estrogen may exert effects directly on cartilage. However, studies on direct estrogen effects on cartilage are limited. We investigated, in an in vitro cartilage explant model, whether estrogen prevents damage or stimulates repair after damage induced by addition of iodoacetate (IA), as an experimental model for osteoarthritis. We used healthy bovine cartilage explants. Prevention experiment: Explants precultured with/without estradiol (E) for 3 days were cultured with IA for 4 h on day 0, and subsequently cultured as in preculture: with/without E. Explants were harvested at day 2 for gene expression analysis. Repair experiment: At day 0, explants were cultured with IA for 4 h on day 0, and subsequently cultured without E or with E. Explants were harvested at days 2, 10, and 14 for gene expression analysis. IA transiently downregulated most genes tested, whereas vascular endothelial growth factor (VEGF) was upregulated on day 2. On day 14, transforming growth factor beta (TGFB)1 and TGFB3 were upregulated, and matrix metalloproteinase (MMP)13 and VEGF downregulated. Estradiol affected gene expression of aggrecan (AGC)1, MMP2, MMP14, tissue inhibitor of metalloproteinase (TIMP)2, TGFB2, and TGFB3. Prevention experiment: Estradiol did not significantly affect IA-induced changes in gene expression (no significant interaction). Repair experiment: Estradiol affected IA-induced changes in expression of collagen (COL)2, MMP2, MMP3, MMP13, MMP14, TIMP2, TGFB2, TGFB3, and VEGF. Estradiol affects expression of anabolic and catabolic genes in bovine cartilage explants and modulates the effects of IA. These effects of estradiol may be beneficial for cartilage maintenance and repair.

Effect of estradiol benzoate on rat testis and adrenal.

The effects of in vivo administration of estradiol benzoate at a dose of 50 micrograms/rat/day for 15 days on testosterone production of testis and adrenal in vitro were investigated in rats. The data revealed that the androgen synthesis of both the steroidogenic organs were reduced by the treatment. The decrease in androgen production by testis was related to its reduced sperm count and weight. On the contrary, accumulation of cholesterol occurred in treated rat testis indicating inhibition of its steroidogenesis. This data confirm our earlier reports on the direct effect of estrogen on testis.

The Dexamethasone Suppression Test and Long-Term Contraceptive Treatment: Measurement of ACTH or Salivary Cortisol Does Not Improve the Reliability of the Test

Under the influence of high estrogen levels, the suppression of total serum cortisol in the dexamethasone test has often been found to be incomplete. Its measurement for the purpose of excluding Cushing's disease or adrenal tumors in women taking oral contraceptives is, therefore, considered unreliable. This study was designed to compare the reliability of measurements of total cortisol, unbound cortisol and ACTH suppression during chronic hyperestrogenaemia. An overnight suppression test with 2 mg of dexamethasone was performed in 19 women receiving long-term contraceptive treatment (group A) and in 12 controls (group C). Baseline and post-dexamethasone morning levels of ACTH, total serum cortisol and unbound salivary cortisol were determined by RIA. In addition, unbound serum cortisol was measured by equilibrium dialysis. Mean baseline levels of all four parameters were significantly higher in group A. This result points towards the possibility of a direct stimulatory effect of estrogens upon the corticotroph axis which is independent from CBG-mediated increase of total cortisol. Post-dexamethasone values of total and unbound cortisol showed no statistically significant differences, while ACTH suppression in group A was even slightly better than in group C. From these data it is concluded that there is no need for post-dexa routine measurement of ACTH or unbound cortisol under contraceptive treatment since neither one of these parameters provides any additional information in comparison to total cortisol.

Changes in Content of Prolactin mRNA during the Rat Estrous Cycle

An attempt was made to clarify the relationship between the synthesis of prolactin (PRL) and its secretion during the rat estrous cycle by examining the changes in PRL messenger RNA (mRNA); PRL secretion and anterior pituitary PRL content. At various stages of the estrous cycle, pituitary and serum levels of PRL and growth hormone (GH) were measured by radioimmunoassay, while their respective mRNA contents were determined by the hybridization method. The content of pituitary PRL mRNA did not increase in parallel with the increment of the serum level of the hormone. Significant increase in PRL mRNA were observed as three peaks; 1st peak at 14:00 on the proestrus (PE) day, 2nd peak at 4:00 and 3rd peak at 22:00 on the estrus (E) day. The GH levels in the pituitary and serum, or the change in PRL mRNA appeared to be unrelated to the rat estrous cycle. From the present results, we speculate that the three peaks of PRL mRNA content might be ascribable to the direct effect of estrogen on transcription, indirect activation of transcription followed by rapid and massive secretion of PRL, and mammotrope proliferation, respectively.

Effect of Cytokines and Steroid Hormones on Nitric Oxide Generating System in Bovine Luteal Endothelial Cells.

Injection of a luteolytic dose of prostaglandin (PG) F2α induces an acute increase in plasma concentration of nitric oxide (NO) metabolites (nitrite/nitrate). Furthermore, NO suppresses progesterone (P4) production and induces apoptosis in bovine luteal cells. These findings suggest that NO plays important roles in luteolysis. However, the regulation of NO generating system in bovine CL is not fully understood. Although tumor necrosis factor α (TNF) and interferon γ (IFNG) produced by immune cells have been demonstrated stimulate NO production in extragonadal cells, the roles of TNF and IFNG in the regulation of NO production, NO synthase (NOS) expression and NOS activity in the bovine corpus luteum (CL) remain unclear. To elucidate whether NO generating system in luteal endothelial cells (LECs) is regulated by cytokines, the effects of TNF or IFNG on the expression of NOS (inducible NO synthase; iNOS and endothelial NO synthase; eNOS) mRNA, protein and NO production were examined. In addition, the direct effects of estrogen (E2) and P4 on NOS proteins and NOS activity were examined. LECs were obtained from mid CL (Days 8-12 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. Cultured bovine LECs were treated for 24 h with TNF (2.9 nM), IFNG (2.5 nM), E2 (10 nM) or P4 (1 μM). Both TNF and IFNG stimulated iNOS mRNA, protein expression and NO production. Furthermore, E2 stimulatediNOS protein expression, whereas P4 inhibited iNOS protein expression. In contrast to iNOS, eNOS protein expression was not affected by treatments with TNF, IFNG, E2 or P4. By observing the conversion of [3C]L-arginine to [3C]L-citrulline, we found that TNF, IFNG and E2 stimulated the NOS activity in cultured LECs (P<0.05). The overall results demonstrate that TNF, IFNG and E2 accelerate NO production by stimulating iNOS protein expression as well as NOS activity. On the other hand, P4 was able to suppress iNOS expression in LECs. This research was supported by JSPS Grants (No. 19580326 and No. 18380166), and by a research fund from the Livestock Technology Association (LTA), Japan. (poster)

Hormones and Hormonal Target Cells in the Testis*

Studies over the last few years have greatly increased our knowledge about target cells for sex hormones and gonadotropins in the testis. A diagram illustrating our present state of knowledge is given in Fig. 1. LH is the principle stimulus of testosterone secretion by the Leydig cells. Direct effects of estrogens and androgens on these cells might modulate the response to LH. Androgens are apparently influencing the differentiation and the contractility of peritubular cells. FSH is the principle stimulus for Sertoli cell secretory function before puberty, although androgens are acting synergistically with FSH. After puberty, androgens alone are capable of maintaining on optimal secretory function of the Sertoli cells. In addition to normal secretory activity of the Sertoli cells, direct stimulation of the germ cells by androgens is needed in order maintain spermatogenesis.

The ultrastructural response of human Leydig cells to exogenous estrogens.

The effect of long term treatment with estrogens alone or along with medroxyprogesterone acetate on the Leydig cell ultrastructure was studied in testes from males undergoing surgery for sexual reassignment. The testes were fixed for electron microscopy by a perfusion method to insure uniform preservation. The morphological features were not the same in all the treated testes. Therefore, the cells found in the intertubular region were classified into three groups: (A) Leydig cells very similar to controls; (B) Absence of typical Leydig cells, but with cells having increased microfilaments, abundant smooth endoplasmic reticulum and some lipid droplets; (C) Absence of any cell type possessing abundant smooth endoplasmic reticulum, but having varying amounts of microfilaments and pigmentation. It is suggested that some of the cell types found in the intertubular region are dedifferentiated Leydig cells. This study indicates that the human testis from transsexuals of reproductive age is an appropriate model to study the indirect and direct effects of estrogens on the ultrastructure of cell types found in the human testes.

The inhibitory effect of diarylpropionitrile, a selective agonist of estrogen receptor beta, on the growth of MC38 colon cancer line

The protective role of estrogens in the colon carcinogenesis has been suggested for many years and attributed mainly to estrogen receptor beta (ERβ). However, the direct effect of estrogens and their action through ERβ on the growth of colon cancer have been rarely studied. The aim of this study was to examine the effect of various concentrations (10 −4–10 −12 M) of diarylpropionitrile (DPN) – a selective agonist of ERβ – on the growth of murine MC38 colon cancer line. Moreover, the aim of this paper was the immunohistochemical assessment of estrogen and progesterone receptor expression in human colon tissues and in MC38 cells (only ERβ). We found that DPN induced a growth inhibition of MC38 cancer (50–94% of control group) at the highest (10 −4 M) and two lowest concentrations (10 −11 and 10 −12 M). Furthermore, we detected a nuclear-cytoplasmic expression of ERβ in human normal and neoplastic colon tissues and in the studied MC38 cancer cells. The inhibitory effect of DPN on the growth of MC38 colon cancer line suggests a possibility of using a selective estrogen receptor agonist in the treatment of colon cancer.