Henry L. Stadelman

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-17β, 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.

Region-Specific Regulation of Cytochrome P450 Aromatase Messenger Ribonucleic Acid by Androgen in Brains of Male Rhesus Monkeys

Abstract 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 (P450AROM) 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 P450AROM 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 P450AROM mRNA in brain regions that mediate reproductive behaviors and gonadotropin release. The P450AROM 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 P450AROM 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.

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.

Separate critical periods exist for testosterone-induced differentiation of the brain and genitals in sheep.

Sheep exposed to testosterone during a critical period from gestational day (GD) 30 to GD 90 develop masculine genitals and an enlarged male-typical ovine sexually dimorphic nucleus of the preoptic area (oSDN). The present study tested the hypothesis that separate critical periods exist for masculinization of these two anatomical end points. Pregnant ewes were treated with testosterone propionate (TP) either from GD 30 to GD 60 (early TP) or GD 60 to GD 90 (late TP). Control (C) pregnant ewes were treated with corn oil. Fetuses were delivered at GD 135 and the volume of the oSDN was measured. Early TP females possessed a penis and a scrotum devoid of testes, whereas late TP and C females had normal female genitals. Neither period of TP exposure grossly affected the genitals of male fetuses. Despite masculinized genitals, the mean volume of the oSDN in early TP females (0.32 ± 0.06 mm³) was not different from C females (0.24 ± 0.02 mm³) but was significantly enlarged in late TP females (0.49 ± 0.04 mm³; P < 0.05 vs. C) when the genitals appeared normal. In contrast, the volume of the oSDN in late TP males (0.51 ± 0.02 mm³) was not different from C males (0.51 ± 0.04 mm³) but was significantly smaller in the early TP males (0.35 ± 0.04 mm³; P < 0.05 vs. C). These results demonstrate that the prenatal critical period for androgen-dependent differentiation of the oSDN occurs later than, and can be separated temporally from, the period for development of masculine genitals.

17β-Hydroxysteroid dehydrogenase activity in the pituitary gland and neural tissue of rhesus monkeys

Ovariectomized rhesus monkeys were treated with estradiol-17β (E2) for 24 days; E2 for 24 days and progesterone (P) for the last 12 days; or P alone for 12 days. Parts of the brain (hypothalamus, preoptic area, amygdala, frontal cortex, and cerebellum) and anterior pituitary gland were analyzed for activity of the 17β-hydroxy steroid dehydrogenase (17β-HSDH) by quantification of the amount of [14C]-estrone (14C-E1) or [3H]-androstenedione ([3h]-a) formed from [14C]-E2 or [3H]-testosterone ([3H]-T) by minced tissue in a 2-h incubation period at pH 7.4. No significant effect of treatment was observed. Therefore, the between treatment data were pooled and analyzed together. The anterior pituitaries, both in the amount of [14C]-E1 formed from [14C]-E2 and in the amount of [3H]-A formed from [3H]-t, had a significantly higher level of enzyme activity (P < 0.001) than any of the neural tissue analyzed. The difference between the activity of the 17β-HSDHs in anterior pituitary and brain tissue was confirmed after we incubated the 800 g supernatant of these tissues and used radioimmunoassayable E1 as an end point of 17β-HSDH activity. With this technique, the activity was lost after freezing, was greater at pH 8.0 than at 7.0, and was linear at protein concentrations from 0.3 to 1.0 mg/ml of incubation medium. The fact that this enzyme is localized with such intensity within the anterior pituitary may indicate that it participates in E2 action at the pituitary level in this species.

Prolactin expression in the sheep brain.

Accumulating evidence in rodents suggests that a prolactin locally synthesized and released within the brain can act together with that taken up from the circulation to modulate neuroendocrine responses. The present study was designed to identify the regional patterns of prolactin expression in the adult and developing sheep brain. Specifically, we tested the hypothesis that prolactin is expressed in regions of the adult and fetal sheep brain that are critical in the development of neuroendocrine homeostatic and behavioral functions. The expression of prolactin protein in sheep brain was demonstrated by Western blot analysis and brain prolactin mRNA was detected and sequenced using RT-PCR. In situ hybridization histochemistry revealed that prolactin mRNA was expressed in the medial preoptic area, periventricular preoptic nucleus, bed nucleus of the stria terminalis, and in the paraventricular nucleus of the hypothalamus, particularly the ventral region. The neuroanatomical distribution of prolactin mRNA was best visualized in the fetus and prolactin-immunoreactive neurons could also be identified in late gestation fetuses. Brain prolactin mRNA was expressed as early as day 60 of gestation and increased as the fetus aged and peaked at day 135 (term = 147 days). Prolactin mRNA expression did not exhibit a sex difference in the preoptic area, but in the amygdala prolactin mRNA was significantly higher in females than in males at day 100 of gestation. We conclude that prolactin expressed in adult and fetal sheep brain could be involved in neurodevelopment and/or modulation of the neuroendocrine stress axis, although it is too early to rule out other possibilities given the diverse actions that have been attributed to prolactin.

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.

Cellular Observations and Hormonal Correlates of Feedback Control of Luteinizing Hormone Secretion by Testosterone in Long-Term Castrated Male Rhesus Monkeys

Abstract 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 (P450AROM 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. P450AROM 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 P450AROM mRNA did not change in group 2 males, we hypothesize that an estrogen-dependent neural deficit, not involving the regulation of the P450AROM mRNA, occurs in long-term castrated monkeys.

17β-Hydroxysteroid Dehydrogenase Activity in Tissues of Fetal Rhesus Macaques

Abstract 17β-Hydroxysteroid dehydrogenase (17β-HSDH) activity was measured in tissues (anterior pituitary, diencephalon, frontal cortex, liver, whole blood, and uterus) of fetal rhesus macaques on Days 80, 120, and 150 of gestation. The production of estrone (E1 (quantified by radioimmunoassay) by an 800g supernatant incubated with excess substrate (estradiol-17β [E2]) was used as an index of 17β-HSDH activity. Over a 30-min incubation period E1 was produced in a linear fashion by all the tissues that we studied. The pH optimum for this activity in the pituitary gland was ~9. Boiling of the tissue for 1 min reduced its activity significantly. Significant changes in 17β-HSDH activity during development were observed for two of the six tissues studied. Activity in fetal anterior pituitary glands was greatest on Day 80 of gestation but changed to significantly lower levels by Day 120 (P < 0.01). This low level continued on Day 150 of gestation. The pattern of activity in the liver was different from that in the pituitary gland. High activity was found on Day 80, and this increased significantly by Day 120 (P < 0.05). The levels of activity observed on Day 150 were similar to those found on Day 120. Low levels of 17β-HSDH activity were found in the central nervous system, whole blood, and uterus. In rhesus adults as in fetuses we have found relatively high levels of 17β-HSDH activity in the anterior pituitary gland. The biological significance of changing levels of this activity during gestation is not known.