Characterization of a hepatic protein in nonhuman primates that binds mibolerone but not dihydrotestosterone or methyltrienolone.

During our studies of the hepatic androgen receptor in cynomolgus monkeys, tritiated mibolerone +/- a 200-fold excess of unlabeled mibolerone has been used to determine specific binding in cytosol. During time-course studies, high-capacity, unsaturable binding of [3H]mibolerone was noted after short-term incubations (4 h, 4 degrees C). When hepatic cytosol from male monkeys was incubated for 18 h at 4 degrees C, the high-capacity binding disappeared; saturable, high-affinity binding with characteristics consistent with the androgen receptor then could be identified. The characterization of [3H]mibolerone binding in molybdate-stabilized hepatic cytosol using sucrose density gradients and gel filtration yielded an unstable binding peak in addition to that of the androgen receptor. This lower molecular weight protein identified by gel filtration did not bind other androgens, including methyltrienolone, and did not have characteristics of other binding proteins that have been identified previously. This protein was not precipitated from 30% ammonium sulfate, which allowed it to be separated from the androgen receptor. Binding to this protein in ovariectomized female monkeys did not disappear with extended incubation at 4 degrees C, suggesting greater stability or a higher capacity. The function of this protein is not known, but both triamcinolone acetonide and contraceptive progestins appeared to displace tritiated mibolerone that was bound to it. This high-capacity binding of mibolerone interferes in the assessment of androgen receptor levels in these females unless it is eliminated. The synthetic androgen methyltrienolone does not bind to this protein and is a better choice for defining binding to the androgen receptor in these tissues.

Noradrenergic vasorelaxation of porcine coronary arteries is enhanced by direct, acute exposure to 17 beta-estradiol.

1. We tested whether responses of isolated coronary arteries to adrenergic agents are altered by overnight (18-22 hr) or acute exposure to physiological levels of 17 beta-estradiol. 2. Sensitivity to relaxation by norepinephrine (-log M ED50) was enhanced significantly in isolated arteries after acute exposure to 10(-9) M 17 beta-estradiol. Neither alpha-adrenergic contraction to norepinephrine, nor beta-adrenergic relaxation to isoproterenol was affected by acute exposure to 17 beta-estradiol. 3. Responses of coronary arteries to relaxation or contraction by norepinephrine, as well as relaxation by isoproterenol, were not altered by overnight incubation with 17 beta-estradiol or by removal of the endothelium. 4. We conclude that relaxation of coronary arteries by norepinephrine is enhanced by acute, direct exposure to physiological levels of 17 beta-estradiol. This enhancement does not result from effects of estradiol to suppress alpha-adrenergic contraction to norepinephrine or increase coronary beta-adrenergic receptor sensitivity.

17 beta-estradiol treatment increases the levels of estrogen receptor and its mRNA in male rat liver.

This study examined estrogen receptor dynamics in the livers of male obese rats (SHHF/Mcc-cp) treated for two weeks with a continuous, low dose of 17 beta-estradiol compared with untreated controls. An increased binding capacity for tritiated 17 beta-estradiol in the cytosol, consistent with binding to the estrogen receptor, was demonstrated in treated males relative to control males (P < 0.01). These observations were confirmed using curve-peeling techniques with saturation analysis, ammonium sulfate precipitation/fractionation of cytosol protein, and chromatographic techniques to isolate the high-affinity binding from other interfering factors. Increased hepatic nuclear estrogen receptor levels in treated males (112.3 +/- 8.3 fmol/g liver) compared with controls (64.1 +/- 6.8 fmol/g liver) suggested that the liver was under estrogenic influences. This interpretation was supported by an increase in serum triglyceride levels, reflecting increased very low density lipoprotein secretion by the liver. Reductions in testosterone levels and in the weights of seminal vesicles and the testes in treated males indicated detrimental effects on reproduction. An interpretation of increased synthesis of estrogen receptor with 17 beta-estradiol treatment was supported by the observation of an increase in the mRNA for estrogen receptor. Taken together, these observations indicate that continuous, low-dose 17 beta-estradiol treatment induces estrogenic action in the livers of male rats and also increases hepatic estrogen receptor, probably indirectly, via an increase in its mRNA.

Estrogen pretreatment directly potentiates endothelium-dependent vasorelaxation of porcine coronary arteries.

We tested whether vasorelaxation of coronary arteries is altered after overnight (18-22 h) exposure to physiological levels of 17 beta-estradiol. Ring segments of left circumflex coronary artery from six female and six castrated male pigs were incubated in vials of sterile Dulbecco's modified Eagle's medium with 1 nM 17 beta-estradiol, 1 nM 17 beta-estradiol + 10 nM tamoxifen, 1 nM 17 alpha-estradiol, or estrogen vehicle (ethanol) under normoxic conditions in an O2-CO2 incubator at 37 degrees C for 18-22 h. Coronary rings, with and without endothelium, were then suspended in vessel baths for measurement of isometric force. Vasorelaxation responses to the calcium ionophore A-23187, ADP, and nitroglycerin were examined in the rings after prostaglandin synthesis blockade and precontraction with U-46619. Sensitivity to A-23187 (-log M concentration required for 50% of maximal relaxation) was significantly enhanced in coronary rings with endothelium from females and castrated males when rings were incubated with 17 beta-estradiol but not when they were incubated with 17 alpha-estradiol or 17 beta-estradiol+tamoxifen. Acute (2h) exposure of coronary arteries to 1 nM 17 beta-estradiol did not alter responses to A-23187. 17 beta-Estradiol (1 nM) was not itself directly vasoactive in coronary arteries with or without prior incubation with the steroid. Vasorelaxation of rings with and without endothelium to ADP and nitroglycerin was not significantly different among the treatment groups. Relaxation to A-23187, but not ADP, was abolished by removal of the endothelium or exposure to 100 microM NO2-L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of obesity and fertility status on sex steroid levels in men.

Endocrine studies were performed on fertile and infertile obese men and compared with fertile and infertile nonobese men in order to determine the independent and codependent effects of obesity and fertility status on the male hypothalamic-pituitary gonadal axis. The obese infertile group exhibited significant endocrinologic changes as compared with fertile nonobese control group which was not observed in any of the other three groups. Serum testosterone was significantly lower. The testosterone/estradiol ratio was significantly lower despite a lack of significant change in serum estradiol levels. Serum steroid hormone binding globulin (SHBG) was significantly lower which correlated with elevated bioavailability of both testosterone and estradiol in the obese infertile group. Serum luteinizing hormone levels were no different, suggesting that free testosterone levels were unchanged. Obese infertile men exhibit endocrinologic changes that are not observed in men with either obesity or infertility alone. Reduction of serum SHBG, total testosterone, and testosterone/estradiol ratio appear to be a marker of infertility among obese men.

Inhibition of coronary artery atherosclerosis by 17-beta estradiol in ovariectomized monkeys. Lack of an effect of added progesterone.

Although controversy continues, the preponderance of evidence indicates that estrogen replacement therapy favorably influences the risk of coronary heart disease in postmenopausal women. It remains uncertain how this effect is mediated and whether the cyclic addition of a progestin may influence adversely an estrogen-related cardioprotective effect. We investigated the influence of sex hormone replacement therapy on diet-induced coronary artery atherosclerosis in estrogen-deficient (ovariectomized) adult female cynomolgus monkeys. Monkeys were assigned randomly to one of three treatment groups: 1) no hormone replacement (n = 17), 2) continuously administered 17-beta estradiol plus cyclically administered progesterone (n = 20), and 3) continuously administered 17-beta estradiol (n = 18). The physiologic patterns of plasma estradiol and progesterone concentrations were maintained by administering the hormones in sustained-release subcutaneous Silastic implants. The experiment lasted 30 months. At necropsy, coronary artery atherosclerosis was inhibited similarly (reduced by approximately one-half) in animals in both hormone replacement groups (p less than or equal to 0.05). Antiatherogenic effects of hormone replacement were independent of variation in total plasma cholesterol, lipoprotein cholesterol, apoprotein A-1 and B concentrations, high density lipoprotein subfraction heterogeneity, and low density lipoprotein molecular weight. We conclude that physiologic estrogen replacement therapy with or without added progesterone inhibits atherosclerosis progression in ovariectomized monkeys. This may explain why estrogen replacement therapy results in reduced risk of coronary heart disease in postmenopausal women.

Progesterone and estradiol concentrations in nonpregnant and pregnant human myometrium. Effect of progesterone and estradiol on cyclic adenosine monophosphate-phosphodiesterase activity.

We measured the concentration of progesterone and estradiol and calculated the progesterone:estradiol ratio in nonpregnant and pregnant human myometrium. Progesterone, estradiol and the progesterone:estradiol ratio were higher in pregnant than in nonpregnant myometrium. There was no difference in the concentration in the presence of labor. The progesterone:estradiol ratio showed a similar pattern. We also investigated the effect of the ovarian steroids on the activity of cyclic adenosine monophosphate-phosphodiesterase (cAMP-PDE). Progesterone in pharmacologic doses inhibited the activity of the high-affinity enzyme as much as 72% and the low-affinity form as much as 34%. High-affinity phosphodiesterase from nonpregnant myometrium was the least sensitive to inhibition, and the enzyme from pregnant myometrium obtained from laboring women was the most sensitive. Low-affinity phosphodiesterase from nonpregnant myometrium was less sensitive to inhibition than enzyme from pregnant women with or without labor. The degree of inhibition of the low-affinity enzyme in the two pregnant groups was not different. The type of inhibition was competitive in both the high- and low-affinity forms. Estradiol at similar concentrations did not have any effect on the activity of the enzyme. Progesterone in part may exert its effect on the human myometrium by its effect on cyclic adenosine monophosphate-PDE activity and the metabolism of cAMP.

Hormonal determinants of apolipoprotein B,E receptor expression in human liver. Positive association of receptor expression with plasma estrone concentration in middle-aged/elderly women.

The relationships of the expression of hepatic low-density lipoprotein (LDL) receptors (apo B,E receptors) to several plasma hormone concentrations were examined in 15 fasted women aged 37-75 years (mean, 57 years), who were undergoing laparotomy for non-neoplastic disease. No subject had clinical or biochemical evidence of familial hypercholesterolemia, renal disease, hepatic disease, or endocrine disease. Hepatic apo B,E receptor expression was quantified in vitro as the EDTA-suppressible binding of 125I-labeled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C; values were 23-75 ng LDL protein/mg cell protein (mean, 47 ng/mg). Receptor expression was strongly correlated with plasma estrone concentration (rs = +0.70, P = 0.035), but was unrelated to the concentrations of testosterone, thyroxine, free triiodothyronine, cortisol, sex hormone-binding globulin (SHBG) or cortisol-binding globulin. Insulin and estradiol concentrations were mostly very low. The correlation of receptor expression with plasma total estrone concentration reflected associations with both the albumin-bound (rs = +0.78, P = 0.014) and unbound (rs = +0.80, P = 0.009) fractions, but not with the SHBG-bound fraction (rs = -0.22, P = 0.574), of this hormone. As the non-SHBG-bound fractions of gonadal steroids are considered to be the biologically active components, these results are consistent with experimental evidence that the synthesis of apo B,E receptors in hepatocytes is stimulated by estrogens, and suggest that circulating estrone may be the major hormonal determinant of receptor expression in fasted middle-aged/elderly women.

Differential effects of oral estrone versus 17 beta-estradiol on lipoproteins in postmenopausal women.

Toward the definition of optimal postmenopausal estrogen replacement we compared the effects of three graduated doses of two oral estrogens, estrone sulfate and 17 beta-estradiol, on the lipid profiles of two groups of six postmenopausal women. Because of metabolic interconversions equivalent serum concentrations of estrone and estradiol were produced with these regimens. However, differential effects were noted in lipoproteins. 17 beta-Estradiol caused an increase in total plasma cholesterol (from 5.71 +/- 0.36 to 5.99 +/- 0.57 mmol/L, baseline to high dose; P less than 0.02), high density lipoprotein (HDL) cholesterol (from 1.45 +/- 0.15 to 1.78 +/- 0.36 mmol/L; P less than 0.02), HDL2 cholesterol concentration (from 0.41 +/- 0.08 to 0.62 +/- 0.26 mmol/L; P less than 0.01), and triglyceride concentration (from 1.09 +/- 0.29 to 1.24 +/- 0.30 mmol/L; P less than 0.01) without affecting low density lipoprotein (LDL) cholesterol concentration. By contrast, estrone sulfate caused a decrease in total plasma cholesterol (from 6.51 +/- 0.85 to 5.87 +/- 0.41 mmol/L; P less than 0.05) and LDL cholesterol concentration (from 4.34 +/- 0.57 to 3.67 +/- 0.44 mmol/L; P less than 0.01) and an increase in HDL cholesterol (from 1.37 +/- 0.20 to 1.50 +/- 0.26 mmol/L; P less than 0.05) and HDL2 cholesterol concentration (from 0.34 +/- 0.18 to 0.49 +/- 0.18 mmol/L; P less than 0.01), but no change in total triglyceride concentration. We deduce that the differential effect of orally administered estrogens on lipoprotein metabolism in postmenopausal women may be attributed to a first pass effect on hepatic metabolism.

Oral contraceptive administration, interfemale relationships, and sexual behavior in Macaca fascicularis.

The effects of oral contraceptive administration on the social relationships of adult female cynomolgus monkeys (Macaca fascicularis) were examined. Ten females were administered ethinyl estradiol/ethynodiol diacetate (Demulen), 10 were administered ethinyl estradiol/norgestrel (Ovral), and 10 served as a control group. The monkeys lived in social groups of 5 females each, and patterns of social interaction and social status were recorded. Interfemale relationships were also observed when a vasectomized male was placed in each social group for 50 min, once/week. During the latter observations, preliminary data on the effects of oral contraceptive treatment on sexual interaction were also collected. In the absence of the male, interfemale agonistic interactions and time spent alone were influenced by social status but not by oral contraceptive treatment. Time spent in passive body contact, an affiliative state, was reduced by Ovral treatment. In the presence of the male, dominant females aggressively interfered with the sexual interactions of subordinates. This aggression resulted in the termination of a greater proportion of the sexual interactions of subordinates than dominants in the control group only, indicating suppression of this type of interaction by oral contraceptive treatment. Other effects included a decreased frequency of ejaculation with Ovral-treated females. These results suggest that oral contraceptives may suppress certain types of female agonistic behavior (e.g., in the context of mate competition) and some oral contraceptives may interfere with sexual activity. More broadly, these findings indicate that intrasexual competition for access to mates may occur in females as well as males.

Oral contraceptives and coronary artery atherosclerosis of cynomolgus monkeys.

Studies of both human and nonhuman primates show an inverse relationship between high-density lipoprotein (HDL) cholesterol concentrations and coronary artery atherosclerosis. For this reason, there has been concern that the HDL cholesterol-lowering effect of oral contraceptives might exacerbate coronary artery atherosclerosis. We studied three groups of adult female cynomolgus macaques fed a moderately atherogenic diet: a control group, a group given ethinyl estradiol and norgestrel, and another group given ethinyl estradiol and ethynodiol diacetate. Norgestrel and ethynodiol diacetate, co-administered with ethinyl estradiol, lowered the plasma concentrations of HDL cholesterol. However, the extent of coronary artery atherosclerosis was lessened by both contraceptives, especially among females at high risk based on their plasma lipid profiles.

Cardiovascular and testosterone responses to contested dominance in women.

Examined cardiovascular and testosterone responses of dominant and submissive women to an acute stressor involving contested dominance. Twenty (10 dominant, 10 submissive) university students rated their dominance to a predefined reference group, participated in a task that involved debating with a trained technician, and were continuously provided with bogus feedback throughout the task concerning biochemical substances in their blood that purportedly covaried with dominance. Covariance analyses revealed that dominant women were much more reactive to the stressor in several cardiovascular parameters than those classified as submissive. Serum testosterone did not vary as a function of the experimental manipulation, and at each measurement point, dominant and submissive subjects had essentially identical serum levels of this hormone. Interestingly, however, baseline testosterone for the dominant group was related to changes in systolic blood pressure (i.e., gain scores computed as task response minus baseline values).

Treatment of oophorectomized guinea pigs with intrauterine 17 beta-estradiol pellets may modulate myometrial beta-adrenergic receptor binding properties.

Intrauterine 17 beta-estradiol pellets can induce an up-regulation of guinea pig myometrial beta-adrenergic receptor density and l-isoproterenol-dependent adenylate cyclase activity. Does 17 beta-estradiol influence the ability of beta-adrenergic receptors to form a "high affinity" state with l-isoproterenol, which is a necessary step for adenylate cyclase activation? Nonpregnant, oophorectomized guinea pigs received intrauterine pellets of either placebo, 17 beta-estradiol, progesterone, or 17 beta-estradiol plus progesterone for 1 week. 17 beta-Estradiol resulted in pharmacologic, whereas progesterone resulted in physiologic plasma 17 beta-estradiol and progesterone concentrations, respectively. The affinity of myometrial beta-adrenergic receptors for l-isoproterenol was measured by percentage of inhibition of -[125I]cyanopindolol binding. In all groups, the competition curves in the presence of magnesium chloride could be resolved into two affinity states of the beta-adrenergic receptor, "high" and "low," respectively. The ratio of their dissociation constants was not influenced by hormonal treatment. However, the relative concentration of beta-adrenergic receptors in the high affinity state was significantly higher in the 17 beta-estradiol-treated group than that in the control group. This correlates with the up-regulation in myometrial adenylate cyclase activity and suggests that myometrial beta-adrenergic receptor-adenylate cyclase function may be modulated by 17 beta-estradiol.

Up-regulation of guinea pig myometrial adenylate cyclase activity by intrauterine estradiol and progesterone pellets.

The effects of intrauterine implantation of 17 beta-estradiol and progesterone on basal and stimulated adenylate cyclase activity in guinea pig myometria were studied in nonpregnant, previously oophorectomized guinea pigs receiving intrauterine implants of either estradiol, progesterone, a combination of the two hormones, or placebo for 7 days. Guanine nucleotides resulted in a significant increase in basal enzymatic activity. The extent of enzymatic stimulation in estradiol-treated animals was significantly higher than that observed in either controls, animals receiving progesterone, or a combination of estradiol and progesterone. Sodium fluoride stimulation occurred in all treatment groups to a similar degree. However, guanine nucleotides resulted in a significant decrease in percent stimulation of maximal sodium fluoride-stimulated enzymatic activity. Finally, beta-adrenergic receptor-mediated enzymatic activity, as assayed by isoproterenol stimulation, was higher in those animals that received estradiol implants than in controls or the other two hormonally treated groups. Intrauterine administration of these sex steroid hormones, directly or indirectly, modulates myometrial adenylate cyclase activity.

From menarche to menopause: coronary artery atherosclerosis and protection in cynomolgus monkeys.

The effects on atherogenesis of stress, pregnancy, and oral contraceptive therapy were studied in a nonhuman primate model. The stress of social subordination was associated with ovarian dysfunction, unfavorable lipoprotein changes, and increased coronary artery atherosclerosis compared with nonstressed (socially dominant) or normal monkeys. Although pregnant animals exhibited lower high-density lipoprotein cholesterol concentrations, they had only one half as much diet-induced coronary artery atherosclerosis as their nonpregnant counterparts. Monkeys treated with an Ovral-like regimen also exhibited adverse lipoprotein changes. Nevertheless, prevalence and extent of coronary artery plaques decreased. We conclude that estrogen is an important factor in the animals' "female protection" against diet-induced atherosclerosis. We also suggest that the lowering of high-density lipoproteins by the progestin component of higher-dose contraceptives is not necessarily atherogenic if a sufficiently potent exogenous estrogen is administered concomitantly.

Up-regulation of guinea pig myometrial beta-adrenergic receptors by intrauterine estradiol and progesterone pellets.

The effect of intrauterine implantation of 17 beta-estradiol and progesterone on the concentration and affinity of myometrial beta-adrenergic receptor were studied in nonpregnant, previously oophorectomized guinea pigs receiving intrauterine implants of either 17 beta-estradiol, progesterone, a combination of the two hormones, or placebo for 7 days. Myometrial beta-adrenergic receptors were characterized by use of (-)-iodine 125-cyanopindolol as the specific beta-adrenergic receptor ligand. On comparison with the control group, administration of 17 beta-estradiol or progesterone resulted in a severalfold increase in the concentration (Bmax) of myometrial beta-adrenergic receptor and a lesser but significant increase in the dissociation constant, KD. Although a combination of 17 beta-estradiol and progesterone treatment increased the concentration and the dissociation constant of beta-adrenergic receptors, it did not result in any synergistic or additive effect. We conclude that intrauterine administration of these sex steroid hormones, directly or indirectly, modulates myometrial beta-adrenergic receptor concentrations and affinity.

Acute stress reactivity from contested dominance in dominant and submissive males.

We examined the cardiovascular and testosterone responses of dominant and submissive males to an interpersonal challenge of dominance status. Twenty college-aged students rated their dominance to predefined reference groups and engaged in a debate against a trained technician. Throughout the task, they were continuously given false feedback regarding biochemical substances in the plasma that purportedly covaried with dominance. Covariance analyses revealed that the task created greater heart rate reactivity and lower testosterone levels on the part of submissive subjects when contrasted with those scoring high in self-reported dominance. Interpretation of these data emphasizes the importance of an interactionist perspective in research dealing with acute stress reactivity.