Sex Steroid Regulation Research Articles

Cellular localization and sex steroid regulation of insulin-like growth factor binding protein messenger ribonucleic acids in the primate myometrium.

Insulin-like growth factors (IGF)-I and -II are abundant in the primate myometrium and are implicated in the sex steroid-induced growth of this tissue. To evaluate the potential for modulation of IGF action in the primate myometrium by locally produced IGF binding proteins (IGFBPs), we examined the cellular localization and sex steroid regulation of IGFBPs 1-5 in the nonhuman primate uterus. Ovariectomized rhesus monkeys were treated with placebo, estradiol (E2), or E2 and progesterone (P4) (E2 and P4) for 2 weeks, after which their uteri were removed and cut into thin serial sections for analysis by in situ hybridization. IGFBP-1 messenger RNA (mRNA) was not detected in control or E2-treated uteri but was found in a few unidentified cells in the E2 and P4-treated group. IGFBP-2, -3, -4, and -5 mRNAs were all expressed by myometrial smooth muscle cells but each displayed distinctive patterns of regulation by sex steroids. IGFBP-2 was barely detectable in control myometrium, was significantly increased by E2 and even more significantly by E2 and P4. IGFBP-4 and 5 mRNAs were readily detectable in control myometrium and significantly increased by E2 treatment. The addition of P4 to E2 treatment did not produce a significantly greater augmentation in IGFBP-4 or 5mRNA level compared with E2 alone. IGFBP-3 mRNA was abundant in the control myometrium, but in contrast to other IGFBPs was significantly reduced by approximately 75% in smooth muscle cells by E2 and by E2 and P4 treatment. Interestingly, however, IGFBP-3 mRNA was increased in the uterine vascular endothelium by E2 and by E2 and P4 treatment. In summary, this study has shown that four of the six known IGFBPs are highly expressed in the primate myometrium, and that their expression is differentially regulated by sex steroids. The cellular and sex steroid-regulated pattern of IGFBP-2 gene expression is very similar to that of IGF-I, as previously determined in these same myometrial samples. Both IGF-I and IGFBP-2 are dependent on E2 for significant myometrial expression, and both are further augmented by the addition of P4 to E2 treatment. Uterine smooth muscle IGFBP-3 mRNA levels are negatively regulated, whereas IGFBP-4 and -5 mRNA levels are positively regulated by E2: none of these myometrial IGFBPs appears sensitive to the effects of P4. The present observations, together with our previous data from the same animals, demonstrate that the primate myometrial smooth muscle cell expresses mRNAs for IGF-I and -II, IGF-I and -II receptors, as well as expresses mRNAs for IGFBP-2, -3, -4, and -5. These data provide evidence for complex local interactions between IGF system components regulated by estrogen and progesterone.

Cellular localization and sex steroid regulation of insulin-like growth factor binding protein messenger ribonucleic acids in the primate myometrium

Cellular localization and sex steroid regulation of insulin-like growth factor binding protein messenger ribonucleic acids in the primate myometrium

Sex steroid regulation of insulin-like growth factor system gene expression and proliferation in primate myometrium.

To investigate the role of locally produced insulin-like growth factors (IGFs) in sex steroid-induced growth in the primate uterus, ovariectomized rhesus monkeys were treated with placebo (control), estradiol (E2) alone, or E2 plus progesterone (P4). After 2 weeks, uteri were removed, and serial thin uterine sections were analyzed by in situ hybridization for IGF-I, IGF-II, and IGF-I and -II receptor messenger ribonucleic acids (mRNAs) and by immunocytochemistry for the cell proliferation-specific antigen Ki-67. IGF-I and IGF-II and both IGF receptor mRNAs are coexpressed by smooth muscle cells, supporting the possibility of autocrine/paracrine IGF action in stimulating myometrial growth. IGF-I mRNA is barely detected in control myometrium, is significantly increased by E2 treatment, and is augmented even more by the combination of E2 and P4 treatment, whereas little change is noted in myometrial IGF-II or IGF-I receptor mRNA levels. Ki-67-positive myometrial nuclei are also significantly increased by E2 and are augmented even more by E2 plus P4 treatment, with a correlation between local IGF-I mRNA concentration and local Ki-67-positive cell count of r = 0.891 (P = 0.003). These data provide direct experimental evidence for regulation of IGF-I gene expression by sex steroids in the primate uterus in vivo and implicate local IGF-I action in both estrogen- and P4-induced myometrial growth.

Sex steroid regulation of insulin-like growth factor system gene expression and proliferation in primate myometrium

Sex steroid regulation of insulin-like growth factor system gene expression and proliferation in primate myometrium

Sex steroid regulation of IGF gene expression and cell proliferation in primate myometrium

Sex steroid regulation of IGF gene expression and cell proliferation in primate myometrium

Sex steroid regulation of thromboxane A 2 receptors in cultured rat aortic smooth muscle cells

Thromboxane A 2 (TXA 2) has been implicated as an important mediator of cardiovascular diseases, and male rat aortas are reported to be more sensitive to it than female aortas. The effects of sex steroids to regulate the expression of TXA 2 receptors in cultured male rat aortic smooth muscle cells (RASMC) were determined. TXA 2 receptor density (B max) and affinity (K d) were determined via radioligand binding studies with [ 125I]BOP, a TXA 2 receptor agonist. Testosterone increased Bmax in a concentration-dependent manner without any significant change in K d. Cycloheximide, actinomycin D, and the 5α-reduc-tase inhibitor L645,390 significantly ( P < 0.01) blocked the effect of testosterone. Dihydrotestosterone, the active metabolite of testosterone, increased B max and was more potent than testosterone. To determine if there is a sex-related difference in response to testosterone, its effect in cultured female RASMC was assessed. Testosterone increased B max in female RASMC but the increase was significantly ( P < 0.001) less than that seen in male RASMC. These results indicate that androgenic steroids regulate the expression of vascular TXA 2 receptors.

Myometrial estrogen and progesterone receptor binding in pregnancy: Inhibition by the detergent action of phospholipids

We characterized the phospholipid inhibition of estradiol and progesterone binding to guinea-pig and human myometrial receptors. Of twelve compounds studied, phosphatidylinositol (PI), lysophosphatidic acid and lysophosphatidylcholine (lyso-PC) were the most active inhibitors (50% inhibition at 10(-5) M). Lyso-PC with fatty acid chain length C14:0 inhibited ligand binding both to estrogen receptor (ER) and progesterone receptor (PR), C16:0 only to PR and C18:0 neither to ER nor to PR. The lyso-derivates were more inhibitory than the parent compounds. The ionic detergent (sodium taurocholate) inhibited both ER and PR binding, but the non-ionic detergent (Triton X-100) only PR. Triton X-100 enhanced the PI-induced inhibition of ER binding by a factor of 10. PR was more sensitive to inhibition than ER in all cases. The type of inhibition was non-competitive. At term pregnancy, ligand binding to myometrial ER or PR was low or absent in humans, but moderate in the guinea-pig. Phospholipid extracts of human decidua and fetal membranes contained PI and phosphatidylserine rather than lyso-PC. The extract was a potent inhibitor of ligand binding to PR (50% inhibition at 10(-6) M phospholipid phosphorus), but not to ER. The physicochemical environment, modulated by phospholipids acting as detergents, may regulate sex steroid function also in vivo. This might have special significance for pregnancy maintenance.

Regulation of sex steroid receptor gene expression by progesterone and testosterone in cultured human endometrial stromal cells.

Progesterone (P) is known to regulate sex steroid receptors in uterine cells. However, its precise regulation at the messenger ribonucleic acid (mRNA) level is unclear. In this study we examined the effects of P and testosterone (T) on the regulation of sex steroid receptors in cultured human endometrial stromal cells (ESC), using the quantitative reverse transcriptase polymerase chain reaction method. We isolated ESC from human endometrial tissues and cultured them with or without P (10(-6) mol/L) or T (10(-8) mol/L) for 9 days. Incubation with P decreased progesterone receptor (PR), estrogen receptor, and androgen receptor mRNA levels in cultured human ESC to 0.56 +/- 0.04-, 0.53 +/- 0.08-, and 0.84 +/- 0.04-fold (mean +/- SE), respectively. T also decreased PR, estrogen receptor, and androgen receptor mRNA levels in cultured human ESC to 0.48 +/- 0.06-, 0.52 +/- 0.05-, and 0.82 +/- 0.04-fold (mean +/- SE), respectively. These decreases by P and T occurred in a dose-dependent manner. We also examined the sex steroid receptor levels in human ESC cultured for 0, 3, 6, and 9 days. The PR mRNA level in ESC without P was increased in a time-dependent manner. This increase was also inhibited by P, and the mRNA level in the presence of P was almost constant throughout the culture period. Our results demonstrated that P or T is a regulator of sex steroid receptors in ESC and that this regulation may influence the responsiveness to P of decidual change in ESC.

Sex steroid regulation of the hypothalamo-pituitary-adrenal axis activity in middle-aged mice during endotoxic shock.

Many studies have documented the presence of a sexually dimorphic response of neuroendocrine functions in response to immune signals. The aims of the present study were to evaluate the hypothalamopituitary-adrenal (HPA) axis response to inflammatory stress stimulus in 15-month-old mice, and to determine whether such a response depends on circulating sex steroids. Our results indicate that in the 15-month-old mice: (1) there is a sexual dimorphism in the HPA axis activity in basal condition and after endotoxin treatment with generally higher levels of several parameters of this axis in female than in male mice, (2) gonadectomy alone, followed by sex steroid therapy, modulates HPA axis function in both basal and stress conditions, and (3) whereas estradiol plays a stimulatory role on adrenal function, testosterone inhibits adrenal glucocorticoid production. This study further suggests a clear sexual dimorphism in middle-aged mice injected with endotoxin. These results may be relevant for the treatment of sepsis in aged patients.

A Phorbol Ester and Calcium Ionophore Regulate Sex Steroid and Prostaglandin Release by Follicles of the Anuran Rana esculenta and the Urodele Triturus carnifex

The aim of this work was to study the relationships among protein kinase C (PKC), calcium, prostaglandins (PGs), and sex steroids in follicles of Rana esculenta and Triturus carnifex. Follicles, oocytes, and wall cells of follicle (theca and granulosa cells) were incubated in vitro with an activator of PKC, phorbol-12-myristate-13-acetate (PMA), a calcium ionophore (A23187), an antagonist of calcium channel, verapamil, PMA + A23187, prostaglandin F 2α (PGF 2α), and prostaglandin E 2 (PGE 2). Progesterone, androgens, and 17βestradiol were assessed in incubation media of follicles and wall cells and PGs in incubation media of follicles, oocytes, and wall cells. In both species, PMA increased progesterone; A23187 increased progesterone, 17β-estradiol, and PGs; verapamil decreased progesterone and PGs; PMA + A23187 increased progesterone, 17β-estradiol, and PGs; PGF 2α increased 17β-estradiol: PGE 2 increased progesterone. These data suggest that PKC and calcium intervene in the regulation of steroidogenesis and PG synthesis by follicles of both R. esculenta and T. carnifex; in particular, calcium seems to regulate PGs synthesis, activating an enzymatic pathway which does not include PKC.

Sex steroid regulation of chin-marking behavior in male New Zealand rabbits

Chin-marking behavior (chinning) was evaluated daily in nine intact adult male rabbits. All subjects (Ss) displayed chinning (mean of means ± SE = 61 ± 7 marks/10 min) but the frequency of this behavior varied largely across them (range of mean chinning frequency = 19–84 marks/10 min). Chinning frequency showed abrupt variations at intervals of 2–3 days, but periodogram analysis did not reveal the existence of an endogenous rhythm in this behavior. Castration significantly decreased (mean of means ± SE = 29 ± 9 marks/10 min; p < 0.01) but did not suppress chinning. Testosterone propionate (TP; 1 mg/day for 16 days) restored chinning in castrated Ss to slightly below precastration levels (mean ± S.E. = 53 ± 13 marks/10 min). The daily administration of 1 μg estradiol benzoate (EB) plus 1 mg dihydrotestosterone propionate (DHTP) stimulated chinning within 2 days (mean increase = 147%; p < 0.005). DHTP (1 mg/day) given alone stimulated chinning only after 11 days of treatment (mean increase = 475%; p < 0.01). At higher doses, both DHTP (10 mg/day) and EB (10 or 50 μg/day) stimulated chinning by 450%, 80%, and 100%, respectively, over baseline values. Results indicate that chinning largely depends on testicular steroids. Androgen receptor occupation by T or DHT, which is enhanced by E, optimally activates chinning.

Dopamine and sex steroid regulation of POMC gene expression in the hypothalamus.

Previous studies have shown that POMC mRNA and peptide levels are increased in the medial basal hypothalamus (MBH) of the chronically castrated rat and are suppressed with sex steroid replacement. In a parallel time course, hypothalamic dopamine turnover similarly changes after chronic castration and sex steroid replacement. In this study we have examined the effects of dopamine on POMC in the MBH and questioned whether the increase in dopamine activity which occurs in the MBH of chronically castrated rats is responsible for the stimulation of POMC seen under these conditions. We have therefore measured POMC gene expression and peptide content in the MBH of chronically castrated male and female rats in response to the dopamine antagonist haloperidol, and in intact or sex steroid replaced animals in response to the dopamine agonist pergolide. Adult male and female rats were studied 3-4 weeks after castration with and without testosterone (T) or estradiol (E2) replacement. POMC mRNA was measured by a solution hybridization S1 nuclease protection assay; beta-endorphin (beta-EP) and alpha-MSH were measured by RIA. In the first study 4 groups of ovariectomized (OVX) rats were treated with saline, haloperidol, E2 or E2 + pergolide. The mean POMC mRNA concentration in the MBH was 0.85 +/- 0.04 pg/microgram RNA in the saline group and decreased to 0.62 +/- 0.06 pg/microgram with haloperidol (p < 0.01). A similar decrease to 0.53 +/- 0.03 pg/microgram was seen with E2 (p < 0.01); pergolide however prevented the E2 induced decrease in POMC mRNA. In the second study ORCX rats received saline or haloperidol and sham-ORCX rats received saline or pergolide.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of ovarian sex steroids in the regulation of thyrotropin (TSH) secretion of hypogonadal women.

The exact role of ovarian sex steroids in the neuroendocrine regulation of thyrotropin (TSH) release in women can only be accurately assessed in the absence of any considerable ovarian sex steroid feedback upon the hypothalamic-pituitary unit. Consequently, the unstimulated episodic and thyrotropin-releasing hormone (TRH) stimulated TSH secretion was evaluated in postmenopausal women before and during sequential ovarian sex steroid replacements. Seven euthyroid women (mean age: 59.4 years) were studied initially without any sex steroid replacement (control studies), then on the last day of a 21-day course of oral estradiol-valeriate (E2) administration (2 mg daily) and finally, on the last day of a 21-day course of oral estradiol-progesterone (E2/P4) replacement (2 mg E2 and 200 mg micronized P4 daily). During all study occasions, blood was sampled at 10 min intervals for 10 h, while TRH (200 micrograms iv) was administered 8 h after initiation of blood collections. Compared to the control conditions, serum E2 and P4 concentrations markedly increased (p less than 0.001) following oral E2 or E2/P4 treatments. Total triiodothyronine (T3) and thyroxine (T4) concentrations and free T3 and T4 equivalents remained unchanged during E2 and E2/P4 regimens. In the unstimulated secretory profiles, TSH was found to be episodically released, with little interindividual variability for each study condition. Since the TSH pulse attributes (pulse amplitudes, frequencies, interpulse intervals, mean TSH concentrations, by Cluster pulse algorithm) did not significantly change during E2 and E2/P4 replacements, the episodic character of TSH secretion virtually remained unchanged by sex steroid replacements.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex steroid regulation of autoimmunity

The immune response of males and females is not identical but instead has been shown to be dimorphic in its nature, with females generally demonstrating a greater overall response than males. This dimorphism extends to both the humoral and cell mediated systems and appears to be mechanistically based on the differences in type and concentration of sex steroids in males vs females. Furthermore, growth hormone and prolactin secretions which are different in males and females may also be partly responsible for the observed dimorphism. Because autoimmune disease results from a pathological perturbation of normal immune function, it follows that expression of these disease will also demonstrate a dimorphic pattern. Examples of this autoimmune dimorphism include (but are not limited to) lupus, rheumatoid arthritis and multiple sclerosis with the two former more prevalent in females than males and the latter more severe during pregnancy. To explain autoimmune dimorphism it therefore becomes necessary firstly to describe the cellular and hormonal interactions found in normal immune regulation and thereafter extrapolate these to autoimmune phenomena.

Maintenance of in Vivo-Like Keratin Expression, Sex Steroid Responsiveness, and Estrogen Receptor Expression in Cultured Human Ectocervical Epithelial Cells*

In the present manuscript we demonstrate that ectocervical epithelial cells (ECE cells) retain a high degree of differentiated function when cultured using feeder layers. We characterize the cultured cells with respect to morphology, expression of cytokeratins, responsiveness to sex steroids, and the presence of estrogen-binding sites. Like ectocervical cells in vivo, the cultured cells display a typical epithelial cell morphology and undergo extensive stratification and envelope (superficial cell) formation. Like the in vivo ectocervical epithelium, the cultured ECE cells express type I cytokeratins K13, K14, K16, K17, and K19 and type II cytokeratins K5 and K6. Under normal culture conditions, however, cytokeratins K1, K2, K4, K11, and K15, which are expressed in vivo, are not expressed. An interesting finding is that ECE cells, in contrast to endocervix and epidermis in vivo and cultured epidermal keratinocytes, express very abundant levels of K13. In fact, K13 appears to be a specific marker of ECE cells in the female reproductive tract. When incubated with 10 nM diethylstilbestrol, ECE cell envelope production increased 3-fold, while incubation with 100 nM progesterone decreased envelope formation 3.4-fold. Simultaneous incubation with progesterone antagonized the diethylstilbestrol stimulation. Thus, in vivo-like sex steroid regulation of ECE cell differentiation is maintained in culture. In addition, the cells possess a high affinity, limited capacity binding site for estradiol that has a Kd of 1.2 +/- 0.1 nM. This system is likely to provide a useful model for the study of sex steroid regulation of normal ectocervical epithelial cell function.

Sex steroid regulation of ß-adrenergic sensitive adenylate cyclase in rabbit myometrial cells in primary culture

Cyclic adenosine 3′–5′-monophosphate (cAMP) is the likely mediator of myometrial relaxation induced by agents like isoproterenol and relaxin. In vivo , the relaxation response to ß-adrenergic catecholamines is modulated by estradiol (E 2) and progesterone (P) during the estrous cycle and pregnancy. However, it is still debated whether that regulation occurs via a direct action on myometrial cells or indirectly through modulation of adrenergic neurotransmitters. We have studied the properties of adenylate cyclase in primary cultures of smooth muscle cells isolated from rabbit myometrium. Adenylate cyclase activity increased with time in culture reaching a maximum on day 6. The cultured cells had basal activity which could be stimulated by guanosine triphosphate (GTP, 2.3 fold), its non-hydrolysable analogue guanylyl-imido-diphosphate (GPP[NH]p, 8.3 fold) and sodium fluoride (NaF, 15.7 fold). Isoproterenol (Iso) and prostaglandin E 2 (PGE 2) stimulated adenylate cyclase activity to the same level (4 fold) and required the presence of exogenous GTP. Sex steroid treatment did not affect basal activity or its stimulation by guanyl nucleotides, PGE 2 or NaF. However, the adenylate cyclase response to Iso was significantly affected by E 2 and/or P treatment. E 2 treatment for 6 days diminihed the sensitivity 10 fold (from 10 nM to 100 nM) while E 2 for 3 days followed by P for 3 days increased it 10 fold (to 1 nM). Maximal response was not affected by treatment with E 2 for 6 days but was diminished by 43% (p < 0.001) when P was added alone and increased by 63% (p < 0.001) when P was added following E 2 pretreatment. Our results demonstrate that sex steroids can alter ß-adrenergic response in vitro by a direct action on myometrial cells.

Ultrastructural study of cultured smooth muscle cells from uterine leiomyoma and myometrium under the influence of sex steroids

Upon the hypothesis that the differentiation or development of smooth muscle cells in both myometrium and leiomyomas may be under the regulation of sex steroids, myometrial and myoma cells were cultured in the presence of sex steroids and their morphological changes were studied ultrastructurally. The cells from the explants of both leiomyoma and myometrium at the 14th day in either control or E (10 −8 M 17-β-estradiol)-added medium showed extremely well-developed cytoplasmic organelles with few filaments located at the marginal portion of the cytoplasm. These ultrastructural features resembled those of a dedifferentiated stage of smooth muscle cells in vitro. At the 21st day, these cells showed a moderate number of cytoplasmic filaments with dense bodies. These ultrastructural features resembled those of a redifferentiated stage of smooth muscle cells in vitro. In both E- and P (10 −7 M progesterone)-added medium, the cultured smooth muscle cells from both leiomyoma and myometrium showed an increased number of myofilaments with dense bodies even at the 14th day to the 21st day of culture in comparison to the cells either in control or estrogen-added medium. Therefore, the cultured smooth muscle cells in the medium with E and P lacked their dedifferentiated phase in vitro and/or the differentiation or maintenance of their filaments might be under the influence of both estrogen and progesterone. On the observation that the cultured smooth muscle cells from both leiomyoma and myometrium showed very similar ultrastructural changes under the influence of sex steroids, it was suggested that leiomyoma may have the same progenitor cells as myometrium.

Sex steroid regulation of urinary excretion of carnitine in rats

The concentration of acid soluble carnitine was determined in several body tissues and fluids in rats under various conditions of sex steroid regulation. Intact female rats had significantly greater liver carnitine concentrations and urinary excretion rates, and lower blood plasma and heart carnitine concentrations than intact male rats. Ovariectomy increased blood plasma carnitine concentrations ( P < 0.01) and the excretion of carnitine in the urine ( P < 0.05). The administration of either estradiol or testosterone to ovariectomized rats did not alter blood plasma concentrations or urinary excretion of carnitine. Orchidectomized rats had similar blood plasma carnitine concentrations when compared to intact males but excreted significantly ( P < 0.01) greater quantities of carnitine in their urine. Administration of testosterone to orchidectomized rats reduced ( P < 0.01), whereas estradiol stimulated ( P < 0.05) the excretion rate of carnitine in the urine; however, blood plasma carnitine concentrations were not affected by these hormones. These data suggest that a major site for modulation of body carnitine concentration in the male resides in the control of kidney excretion by androgens. Liver, heart and skeletal muscle carnitine concentrations were not altered by the administration of either estradiol or testosterone to orchidectomized or ovariectomized rats.

Demonstration of an androgen receptor in rat pancreas

[1,2,6,7-3H]Testosterone (250 muCi) was administered to castrated male rats; after 30 min a labelled testosterone-receptor protein complex with a pI of 5.1 was recovered from the pancreatic cytosol. A labelled testosterone-receptor complex with an identical pI was also extracted from the nuclear fraction of rat pancreas after incubation of minced pancreatic tissue with 0.1 muM-]1,2,6,7-3H]testosterone for 30 min at 37 degrees C. Studies in vitro showed that [1,2,6,7-3H]testosterone was bound to a receptor protein focusing at a pI of 5.1 and with a Kd of 2 nM and a number of binding sites of 4.7 fmol/mg of protein in castrated male rats. The testosterone-receptor complex sedimented at 3.5 S in high-salt sucrose-density gradients, was excluded from Sephadex G-200 and Ultragel ACA-34, was stable towards treatment with dextran-coated charcoal, was relatively sensitive to heat, and was stable to treatment with deoxyribonuclease and ribonuclease, but was sensitive to treatment which proteinase. It is suggested that the pancreatic androgen receptor, which was also present in castrated female rats, may play a role in sex-steroid regulation of pancreatic function.