The volume of the ovine sexually dimorphic nucleus of the preoptic area is independent of adult testosterone concentrations.

The ovine sexually dimorphic nucleus (oSDN) is characterized by high levels of aromatase mRNA expression which can be used to delineate its boundaries. The volume of the oSDN is approximately 2 to 3-fold larger in rams that mate with ewes (female-oriented rams) than in rams that mate with other rams (male-oriented rams) and ewes. The sex difference in oSDN volume is present in late gestation fetuses and can be eliminated before birth by exposing genetic females to exogenous testosterone during midgestation, suggesting that early exposure to androgen masculinizes volume of the oSDN. The present study was performed to determine whether differences in oSDN volume are influenced by the adult hormonal environment. Adult rams, behaviorally characterized as female-oriented or male-oriented, and ewes were gonadectomized and treated with subcutaneous implants of testosterone to achieve physiologic concentrations of serum testosterone. Three weeks after implant placement brain tissue was prepared for histological assessment of oSDN volume using in situ hybridization for detection of aromatase mRNA expression. Quantitative analysis revealed that despite similar serum testosterone levels among the groups, the volume of the oSDN was greater in female-oriented rams than in male-oriented rams and ewes (P<0.05). Differences in oSDN volume were specific and not reflective of differences in preoptic area height or brain size. These results suggest that differences in the size of the oSDN in adult sheep were not influenced by adult exposure to testosterone.

The effect of aromatase inhibition on the sexual differentiation of the sheep brain.

This study tested the hypothesis that aromatization of testosterone to estradiol is necessary for sexual differentiation of the sheep brain. Pregnant ewes (n = 10) were treated with the aromatase inhibitor 1,4,6- androstatriene-3,17-dione (ATD) during the period of gestation when the sheep brain is maximally sensitive to the behavior-modifying effects of exogenous testosterone (embryonic d 50-80; 147 d is term). Control (n = 10) ewes received vehicle injections. Fifteen control lambs (7 males and 8 females) and 17 ATD-exposed lambs (7 males and 10 females) were evaluated for sexually dimorphic behavioral and neuroendocrine traits as adults. Prenatal ATD exposure had no significant effect on serum concentrations of androgen at birth, growth rates, expression of juvenile play behaviors, or the onset of puberty in male and female lambs. Rams exposed to ATD prenatally exhibited a modest, but significant, decrease in mounting behavior at 18 mo of age. However, prenatal ATD exposure did not interfere with defeminization of adult sexual partner preferences, receptive behavior, or the LH surge mechanism. In summary, our results indicate that aromatization is necessary for complete behavioral masculinization in sheep. However, before we can conclude that aromatization does not play a role in defeminization of the sheep brain, it will be necessary to evaluate whether intrauterine exposure of male fetuses to higher doses of ATD for a more extended period of time can disrupt normal neuroendocrine and behavioral development.

Role of aromatization in anticipatory and consummatory aspects of sexual behavior in male rats.

The present study was designed to test the hypothesis that aromatization is involved in the maintenance by testosterone of the appetitive component of male sexual behavior. We measured appetitive sexual behavior by administering behavioral tests in bilevel chambers and quantifying anticipatory level changes during a 5-min period prior to introduction of a stimulus female. In addition, we recorded standard measures of consummatory male sexual behavior after the female was introduced. Following 3 weekly tests, level-changing behavior reached a plateau and remained stable for up to 10 weeks. After 10 bilevel tests, rats were given subcutaneous testosterone capsules to clamp circulating androgen at physiological levels. Rats were tested and divided into two groups that were matched for measures of sexual behavior. One group was then treated with the nonsteroidal aromatase inhibitor, Fadrozole (2.5 mg/kg/day), given subcutaneously in beta-cyclodextrin and the other group was treated with vehicle. Within 1 week of Fadrozole treatment, the number of anticipatory levels changes was significantly reduced, but not the latency to begin searching. Fadrozole treatment also significantly reduced all measures of copulatory behavior over the period of treatment and increased latencies to first mount, intromission, and ejaculation. After 8 weeks, both treatment groups were given an additional Silastic capsule filled with estradiol and tested for 4 additional weeks. Estrogen treatment partially restored level-changing behavior, mounts, and intromissions but had little effect on ejaculations. These results support the view that aromatization is important for maintaining both the appetitive and the consummatory aspects of sexual behavior in male rats.

Cytochrome P450 aromatase in testis and epididymis of male rhesus monkeys.

To understand the role of estrogen in testicular and epididymal function of rhesus monkeys, we measured steroids in the spermatic and peripheral venus circulation and aromatase activity and its mRNA in testis and epididymis. Testosterone, estradiol-17beta, and estrone, but not androstenedione, were elevated in the spermatic vein serum compared to the peripheral circulation. Aromatase activity in testis and in caput epididymis (259+/-16 [SEM] vs 274+/-47 fmol of 3H2O/mg of protein/h [n = 10], respectively) was significantly higher (p < 0.01) than in corpus and cauda (124+/-28 and 113+/-33 fmol of 3H2O/mg of protein/h [n = 10], respectively). In the ribonuclease protection assay, two P450arom mRNA transcripts were identified in testis and epididymis. One corresponded with the aromatase full-length transcript and the other was a truncated isoform. The latter was significantly more abundant than the former (p < 0.01). Our results demonstrate that the monkey testis and, to a lesser extent, the epididymis can aromatize androgens. However, in the epididymis, like in some areas of the brain, there was a discrepancy between the aromatase activity and the mRNA. The fact that P450arom mRNA and aromatase activity do not correlate in the epididymis may indicate that aromatase activity is not strictly regulated at the level of RNA expression and that other mechanisms for this regulation should be considered.

Cellular observations and hormonal correlates of feedback control of luteinizing hormone secretion by testosterone in long-term castrated male rhesus monkeys.

Testosterone at physiological levels cannot exert negative feedback action on LH secretion in long-term castrated male monkeys. The cellular basis of this refractoriness is unknown. To study it, we compared two groups of male rhesus macaques: one group (group 1, n = 4) was castrated and immediately treated with testosterone for 30 days; the second group (group 2, n = 4) was castrated and treated with testosterone for 9 days beginning 21 days after castration. Feedback control of LH by testosterone in group 1 was normal, whereas insensitivity to its action was found in group 2. Using the endpoints of concentrations of aromatase activity (P450(AROM) messenger RNA [mRNA]) and androgen receptor mRNA in the medial preoptic anterior hypothalamus and in the medial basal hypothalamus, we found that aromatase activity in both of these tissues was significantly lower, P: < 0.01, in group 2 compared with group 1 males. P450(AROM) mRNA and androgen receptor mRNA did not differ, however. Our data suggest that the cellular basis of testosterone insensitivity after long-term castration may reside in the reduced capacity of specific brain areas to aromatize testosterone. Because P450(AROM) mRNA did not change in group 2 males, we hypothesize that an estrogen-dependent neural deficit, not involving the regulation of the P450(AROM) mRNA, occurs in long-term castrated monkeys.

Region-specific regulation of cytochrome P450 aromatase messenger ribonucleic acid by androgen in brains of male rhesus monkeys.

We demonstrated previously that testosterone regulates aromatase activity in the anterior/dorsolateral hypothalamus of male rhesus macaques. To determine the level of the androgen effect, we developed a ribonuclease protection assay to study the effects of testosterone or dihydrotestosterone (DHT) on aromatase (P450(AROM)) mRNA in selected brain areas. Adult male rhesus monkeys were treated with testosterone or DHT. Steroids in serum were quantified by RIA. Fourteen brain regions were analyzed for P450(AROM) mRNA. Significant elevations of its message over controls (P<0.05) were found in the medial preoptic area/anterior hypothalamus of both androgen treatment groups and the medial basal hypothalamus of the testosterone-treated males. Other brain areas were not affected by androgen treatment. We conclude that testosterone and DHT regulate P450(AROM) mRNA in brain regions that mediate reproductive behaviors and gonadotropin release. The P450(AROM) mRNA of other brain areas is not androgen dependent. Brain-derived estrogens may also be important for maintaining neural circuitry in brain areas not related to reproduction. The control of P450(AROM) mRNA in these areas may differ from what we report here, but it is equally important to understand the function of in situ estrogen formation in these areas.

Effect of estrogen on androgen receptor dynamics in female rat pituitary.

We examined the role of estrogen in regulating the number of androgen receptors (AR) in the pituitary gland of the female rat. Female Sprague-Dawley rats (240-280 g) that were ovariectomized for 3 days were used in this study. AR numbers were determined in pituitary cytosol and nuclear fractions by binding and exchange assays using the synthetic ligand 17 alpha-methyl-3H-trienolone. The administration of a single dose of estradiol benzoate [(EB) 10 micrograms/100 g BW] to ovariectomized female rats resulted in a 60% increase in cytosolic AR which was significantly (P less than 0.01) elevated above that of oil-treated controls by 12 h post EB. Cytosolic AR levels remained elevated for as long as 48 h post EB (213% of controls). Saturation analysis of pituitary cytosolic AR revealed a single, high affinity binding site for 17 alpha-methyl-3H-trienolone exhibiting an apparent dissociation constant (Kd) of 0.5 X 10(-10) M in both EB- and oil-treated animals. The administration of cycloheximide (1 mg/kg BW) before EB administration prevented the EB-induced rise in AR when measured 8 h post EB. When dihydrotestosterone (1.5 mg) was injected 24 h after EB or oil, there was a rapid increase in nuclear AR accompanied by a rapid decrease in cytosolic AR. The increase in nuclear AR was significantly greater (P less than 0.05) in EB-pretreated animals vs. oil-treated controls. These observations show that a potential synergism exists between androgen and estrogen in the female rat pituitary and suggest that androgens may play an important role in regulating cyclic pituitary hormone release.

Regulation of androgen metabolism and luteinizing hormone-releasing hormone content in discrete hypothalamic and limbic areas of male rhesus macaques.

The conversion of androgens to active metabolites by neural tissue is believed to be an essential component in the cellular mechanism of androgen-induced neuroendocrine responses. In this study, we measured the in vitro aromatization and 5 alpha-reduction of androgens in incubations of microdissected brain regions from four intact and five castrated (6 weeks) adult male rhesus monkeys. Individual nuclei were microdissected from 600-microns frozen brain sections and homogenized in a potassium phosphate buffer. Aromatase activity was measured by a radiometric assay that uses the incorporation of tritium from [1 beta-3H]androstenedione into 3H2O as an index of estrogen formation. We estimated 5 alpha-reductase activity by isolating 5 alpha-dihydrotestosterone on two different chromatography systems and measuring the amount of this product formed from [1 alpha,2 alpha-3H]testosterone. We acidified a portion of each homogenate and determined LHRH content by RIA. Between brain nuclei, aromatase activity varied 1500-fold, whereas 5 alpha-reductase activity varied only 3-fold. Both enzyme activities were highest in amygdaloid, medial preoptic, and medial diencephalic nuclei and lowest in the caudate nucleus. Aromatase activities in the supraoptic nucleus, periventricular area, medial preoptic area-anterior hypothalamus, and lateral hypothalamus were significantly (P less than 0.05) lower in castrated males. Castration did not significantly affect 5 alpha-reductase activity, except for an increase in the basolateral amygdala. The highest concentrations of LHRH were in the infundibular nucleus-median eminence and were 30 times greater than amounts measured in preoptic and medial hypothalamic nuclei. The LHRH contents of the infundibular nucleus-median eminence, ventral medial nucleus, and lateral hypothalamus were significantly lower in castrated males (P less than 0.05). In addition, we observed a significant correlation between aromatase activity and LHRH content in the basal hypothalamus of intact males (r = 0.947; P less than 0.05; n = 8), but not in the preoptic-anterior hypothalamus (r = 0.068; P greater than 0.05; n = 10). No correlation was observed between 5 alpha-reductase activity and LHRH content in either area. These data indicate that castration selectively affects androgen metabolism and LHRH content in discrete regions of the brain of male monkeys and suggest that aromatase and 5 alpha-reductase are regulated differentially in the primate brain.

Control of 5 alpha-reduction of testosterone in neuroendocrine tissues of female rats.

An assay that involved generating [3H] dihydrotestosterone from [1 alpha,2 alpha-3H] testosterone by a microsomal preparation was developed to measure 5 alpha-reductase (5 alpha R) activity in brain and pituitary tissues of female rats. A major part of the activity was located within the microsomes and was linear, with protein concentrations ranging from 0.01 to 0.23 mg. The apparent Michaelis-Menten constants for pituitary and hypothalamic-preoptic areas were 2.37 and 2.69 microM respectively. Using this assay, we studied changes in 5 alpha R activity in brains and pituitaries of female rats ovariectomized 3 days prior to treatment and treated with either vehicle (oil) or estradiol benzoate (E2B, 10 micrograms/100 g of body weight). Groups of 5-17 animals were killed at 0, 12, 24, 48 and 72 h after treatment. In the pituitary gland, 5 alpha R activity 48 and 72 h after treatment was twice the value obtained at time 0 (p less than 0.05). A single injection of E2B maintained the 5 alpha R at pretreatment levels (p less than 0.05). The 5 alpha R values for intact females were significantly less than the values obtained from pituitaries of animals treated with estrogen (p less than 0.05). This probably indicates that the ovaries control 5 alpha R through mechanisms other than E2 secretion. In the preoptic area and the hypothalamus, ovariectomy did not produce marked elevations in 5 alpha R activity (p greater than 0.05). Thus, the responsiveness of the brain to estrogen treatment differed from the responsiveness of the pituitary. These results confirm the work of others on the effects of ovariectomy and estrogen treatment on 5 alpha R activity in the brain and pituitary. In addition, the data establish a time course for estrogen action that can be correlated with data on estrogen in the circulation. New data are also provided for understanding short-term effects of estrogen on the brain, effects that may be applicable to the control of gonadotropin secretion in rats.

Experimental induction of estradiol positive feedback in intact male monkeys: absence of inhibition by physiologic concentrations of testosterone.

Estradiol benzoate (E2B) induces an increase in serum luteinizing hormone (LH) in gonadectomized macaques of both sexes and in intact females but not males. To determine the interaction of testosterone (T) with E2B, we treated 6 intact male cynomolgus macaques as follows: 1) with E2B alone; 2) E2B after pretreatment with estradiol-17 beta (E2) for 2 wk; and 3) Treatment 2 plus exogenous T along with the E2. In Experiment 4, 6 female cynomolgus macaques were treated with large doses of T for 10 days prior to E2B. Serum levels of T and E2 were quantified by radioimmunoassay; LH by bioassay. In Experiment 1, LH decreased from 5.20 +/- 0.95 micrograms/ml (mean +/- SEM) to 0.47 +/- 0.04 micrograms/ml. In Experiment 2, E2 pretreatment depressed LH and T, and these animals responded to E2B with an increase in LH. Replacement of T (Experiment 3) did not inhibit positive feedback. Intact females treated with T responded to E2B with an increase in LH. These results suggest that a testicular product other than T is involved in the inhibition of the positive feedback response to E2B in intact male macaques, and that E2 pretreatment overcomes this effect.

Small doses of human chorionic gonadotropin prevent estradiol-induced luteolysis.

The manner in which exogenous 17 beta-estradiol (E2) induces premature luteolysis in primates is unclear. In an effort to determine whether exogenous luteotropic hormone inhibits E2-induced luteolysis, E2 capsules were implanted subcutaneously in 11 cynomolgus macaques during the early luteal phase; six animals received injections of human chorionic gonadotropin (hCG; 7.5, 10, or 15 IU/day) for 10 days, and the remaining monkeys received saline. Blood was collected once daily for measurement of E2, progesterone, and bioactive luteinizing hormone (LH). Peak progesterone concentrations were between 0.7 and 5.0 ng/ml and declined prematurely in monkeys given E2 plus saline; the luteal phase was 11.5 +/- 0.6 days (mean +/- SE). With E2 plus hCG treatment, serum progesterone continued to increase after E2 capsule placement and reached peak levels of 4.0-13.0 ng/ml; the luteal phase was 15.3 +/- 0.5 days. Therefore, E2-induced luteolysis was overcome by concurrent administration of hCG. These results suggest that exogenous tropic hormone circumvents the inhibitory influence of E2 on luteal function, but the details of the interaction remain unknown.

Cryptorchid rhesus macaques: long term studies on changes in gonadotropins and gonadal steroids.

We studied the relationship of testosterone (T), dihydrotestosterone (DHT), and 27 beta-estradiol (E2) to FSH and LH in the systemic circulation of six rhesus macaques with surgically induced cryptorchidism at selected times over 420 days. We measured these hormones by RIA and compared their concentrations with those of four sham-operated controls. Midway through the experimental procedure the animals were electroejaculated, and on day 425 the testes were removed and analyzed histologically. The cryptorchid monkeys did not have sperm in their ejaculates, and treatment adversely affected spermatogenesis. For the first 30 days after treatment none of the hormones differed significantly between cryptorchid monkeys and controls. Gonadotropins in three castrated males, however, gradually rose to postcastration levels within approximately 2 weeks post operation. After castration, all three steroids declined significantly within 24 h. Beginning on days 60, 150, 215, 320, and 420 post operation the animals were bled for 5 days on a diurnal regimen. The steroids as well as LH concentrations varied diurnally. The concentration of FSH never showed diurnal variation in any of the groups at any of the periods studied. Concentrations of T and LH did not differ significantly between control and cryptorchid groups at any time. With longer time periods (beginning 215 days after cryptorchidism had been induced), DHT in cryptorchid monkeys was significantly higher than in controls (P < 0.01). Although E2 was significantly lower in cryptorchid monkeys on days 60 and 150 post operation, this difference disappeared with time. Changes in steroid concentrations were not associated with the elevations in FSH that occurred in cryptorchid monkeys by days 60 and 420. Therefore, we assumed that they were independent phenomena. Significant FSH elevations in cryptorchid monkeys occurred only in the fall of the year. Testicular homogenates from cryptorchid and control monkeys produced similar quantities of T, DHT, and androstenedione in vitro. Very little E2 or estrone was found. Although significant amounts of progesterone were quantified in the incubation media of control testes, little or no progesterone was found in media from cryptorchid testes. Similar results were obtained when these steroids were measured in plasma collected from the testicular veins. In testicular venous plasma, 20 alpha-dihydroprogesterone concentrations were not elevated after cryptorchidism. These data suggest that there is similar negative feedback control of gonadotropins in crytorchid and control rhesus monkeys. The absence of dramatic differences in systemic concentrations of FSH between the two groups suggests that the seminiferous tubule does not play a major role in the negative feedback control of gonadotropins in this species or that the tubular component of this control system is not perturbed by the cryptorchid condition...

Estrogens in fetal and maternal plasma of the rhesus monkey,.

The quantities of estrone and estradiol were determined by radioimmunoassay in maternal and fetal plasma of the rhesus monkey from day 59 to 163 of gestation. A two way analysis of variance of data classified according to fetal sex and 3 pooled gestational ages for each hormone and for mother or fetus (4 analyses) revealed significant elevations in fetal estradiol and maternal estrone concentrations with age. All other comparisons were not significant by these analyses. The concentrations of estradiol were greater in maternal than in fetal plasma [769 +/- 64 (SE) pg/ml, N = 63 VS 57 +/- 6 (SE) pg/ml, N = 77, P less than 0.01] by a t test. Estrone, on the other hand, was similar in mother and fetus [265 +/- 30 (SE) pg/ml, N = 60 vs 318 +/- 37 (SE) pg/ml, N = 73, P greater than 0.05]. No sex differences in the concentrations of these hormones were observed except in the fetus after 150 days of gestation. At this time plasma from female fetuses contained significantly more estradiol than plasma from male fetuses [118 +/- 20 (SE) pg/ml, N = 7 vs 61 +/- 10 (SE) pg/ml, N = 19, P less than 0.01]. Except for estradiol in female fetuses, the concentrations of estrogen were significantly higher in the umbilical vein than in the umbilical artery, an indication that the placenta is a major source of fetal estrogen in this species. Estrone and estradiol were significantly correlated in both the fetal and maternal circulation, r = 0.58, P less than 0.001 and r = 0.39, P less than 0.01 respectively. The results provide quantitative data about the estrogen miliue in which the monkey fetus develops and suggest mechanisms for controlling fetal estrogen in this species.