Estrogens modulate the circadian rhythm of hypothalamic beta-endorphin contents in female rats

The aim of the present study was to evaluate the changes in the diurnal rhythm of the hypothalamic beta-endorphin (beta-EP) contents in female rats as a function of circulating estrogens. With this purpose we evaluated the diurnal hypothalamic beta-EP changes (1) during the estrous cycle, and (2) in ovariectomized rats with and without acute and chronic estrogen replacement. Ovariectomized rats were treated either acutely with 10 micrograms of estradiol benzoate (EB) or chronically with 2 micrograms/day of EB for 15 days. beta-EP concentrations were measured in acid extracts of medial basal hypothalamus by a specific radioimmunoassay. During the estrous cycle, hypothalamic beta-EP concentrations showed a significant nocturnal increase, with no difference between the 4 days of the cycle. On the day of estrus, beta-EP concentrations between 12.00 and 18.00 h resulted significantly lower than in the other days of the cycle. After ovariectomy, the night-related changes in hypothalamic beta-EP disappeared. The acute administration of EB induced a significant increase in hypothalamic beta-EP after 21 h (18.00 h). On the other hand, the chronic replacement restored the nocturnal peak of hypothalamic beta-EP (18.00, 21.00, 24.00 h). The present data emphasize the role of central beta-EP in regulating the reproductive functions. Moreover, the effect of estrogen in modulating the circadian changes in hypothalamic beta-EP supports the important role of estrogens in brain function.     

Effect of androgens on hypothalamic pro-opiomelanocortin

Testosterone and estradiol have been shown to affect the hypothalamic content of several pro-opiomelanocortin (POMC)-derived peptides in castrated male and female rats, respectively. It was unclear, however, whether the effects of testosterone on hypothalamic POMC were due to conversion by aromatization to estradiol or whether there were independent androgen actions on hypothalamic POMC. In order to answer this question, the effect of treatment with the nonaromitizable androgen 5-alpha-dihydrotestosterone (DHT) on the concentration of beta-endorphin (beta-EP) in the medial basal hypothalamus (MBH) was studied in castrated male rats and compared to the effect of treatment with testosterone or estradiol. The concentrations of two other POMC-derived peptides, corticotropin-like intermediate lobe peptide (CLIP) and alpha-MSH were measured as well. Adult male rats were castrated and received either no treatment or treatment with subcutaneously implanted silastic capsules, containing either DHT, testosterone or estradiol, designed to produce steroid levels in a physiological range. After 4 weeks the mean concentration of beta-EP in the MBH of the untreated castrated rats was 1,640 +/- 56 fmol/mg protein. This was reduced significantly to 1,184 +/- 74 fmol/mg protein after DHT treatment (p less than 0.001). Similar reductions to 1,340 +/- 95 and 1,130 +/- 85 fmol/mg protein were noted after testosterone and estradiol treatment, respectively. The mean CLIP concentration of 1,870 +/- 73 fmol/mg protein in the untreated animals fell to 1,390 +/- 95 after DHT (p less than 0.001) compared to 1,520 +/- 105 and 1,260 +/- 101 after testosterone and estradiol treatment, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The regulation of oxytocin receptor binding in the ventromedial hypothalamic nucleus by testosterone and its metabolites.

Oxytocin (OT) receptor binding in the ventromedial hypothalamic nucleus is regulated by testosterone (T) in male rats. However, T is metabolized in the brain, and many of the central effects of T are mediated by its metabolites. The experiments reported here were designed to determine whether T affects OT receptor binding directly or through the action of its metabolites 17 beta-estradiol and 5 alpha-dihydrotestosterone. Adult male rats were either sham operated or castrated and treated 1 week later with T propionate (TP), 17 beta-estradiol benzoate (EB), dihydrotestosterone benzoate (DHTB), DHTB plus EB, or oil. OT receptor binding was assessed autoradiographically using [125I]d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT. In addition, seminal vesicle weights were measured as an index of androgenic activity. These experiments showed that TP and DHTB plus EB increased OT receptor binding in the ventromedial hypothalamic nucleus to the levels in intact males. Treatment with EB alone partially reinstated binding to the levels in intact males, while DHTB treatment was without effect. Castrated males treated with either TP or DHTB had seminal vesicle weights comparable to those of gonadally intact males and greater than those of animals in all other steroid conditions, indicating that sufficient levels of circulating steroids were attained in these groups. These data suggest that the induction of hypothalamic OT receptor binding by T is the result of the combined actions of estradiol and dihydrotestosterone. However, the mechanism underlying this interaction is unknown.     

The in-vitro transformation of [3H]dehydroepiandrosterone into its principal metabolites in the adrenal cortex of adult castrated male rats and following steroid treatment

The adrenal gland of castrated adult male rats metabolized [3H]dehydroepiandrosterone in vitro to delta 4-androsten-3,17-dione (4AD), testosterone, dihydrotestosterone (DHT) and 5 alpha-androstane-3,17-dione (5 alpha AD). Despite the low testosterone values, DHT and 5 alpha AD were higher 30 and especially 60 days after castration, with raised 4AD:testosterone and decreased testosterone:DHT ratios. The 5 alpha-reductase activity thus appears to increase with time after castration. Fourteen days after castration, 4AD was the only metabolite that was raised compared with intact animals, and testosterone was comparable in sham-operated and castrated rats. The administration of testosterone propionate to castrated rats restored testosterone values to those of intact rat adrenals, whereas 4AD values were greater. The administration of dihydrotestosterone propionate also yielded higher levels of 4AD, in the presence of a lower testosterone value. After administration of oestradiol benzoate, 4AD values were lower especially compared with the other hormone-treated groups, and there was an unexpectedly high testosterone value. These data indicate that the adrenal gland contributes to the production of androgens, as previously noted by Andò, Canonaco, Beraldi et al. (1988) who showed increased plasma 4AD and testosterone levels in adult male rats 30 days after castration. Furthermore, adrenal androgen production in castrated animals is differentially regulated by sex steroids.     

Multihormonal control of pre-pro-somatostatin mRNA levels in the periventricular nucleus of the male and female rat hypothalamus

The influence of sex steroids as well as the possible involvement of dopaminergic pathways in the modulation of pre-pro-somatostatin (SS) mRNA levels was investigated by quantitative in situ hybridization in the hypothalamic periventricular nucleus (PeN) in adult male and female rats. In situ hybridization was performed using a [35S]-labeled cDNA probe encoding pre-proSS mRNA. Gonadectomy performed 14 days earlier decreased the mean number of silver grains/neuron corresponding to the relative pre-proSS mRNA levels by 22% in male and by 18-28% in female rats. A 14-day treatment with the nonaromatizable androgen dihydrotestosterone (DHT) increased the mean number of silver grains/neuron by 34-40% in gonadectomized animals of both sexes. Moreover, administration of 17 beta-estradiol (E2, 0.25 microgram twice daily) increased pre-proSS mRNA levels by 40% in ovariectomized (OVX) animals. Such treatment with E2 or DHT changed the frequency distribution profile of the hybridization signal intensity, thus increasing the percentage of highly labeled neurons (greater than or equal to 61 grains/neuron) by 10 to 12-fold. A 14-day treatment with the D2 dopamine receptor agonist bromocriptine (BRO) increased pre-proSS mRNA levels by 15 and 28% in intact female and OVX animals, respectively, while the dopaminergic antagonist haloperidol (HAL) decreased the value of this parameter by 20 and 30%. Furthermore, BRO increased pre-proSS mRNA levels by 10 and 20% in intact and castrated male rats, respectively, whereas HAL decreased pre-proSS mRNA levels by 25 and 14% in the same groups of animals. Administration of E2 in combination with HAL in OVX animals increased pre-proSS mRNA levels by 70% compared to those measured in OVX animals treated with HAL alone. In HAL-treated castrated male rats, administration of DHT increased the relative pre-proSS mRNA levels by 35% compared to those measured in castrated animals treated with HAL alone. The present data clearly demonstrate that androgens and estrogens as well as dopamine-mediated mechanisms could play a regulatory role in pre-proSS mRNA levels in somatostatinergic neurons in the hypothalamic PeN in both male and female rats.     

Estradiol regulation of proopiomelanocortin gene expression and peptide content in the hypothalamus.

Previous studies have shown that the hypothalamic concentrations of beta-endorphin (beta-EP) and other proopiomelanocortin (POMC)-derived peptides change in the female rat following castration and gonadal steroid replacement. In this study we have measured POMC mRNA by solution hybridization assay in the medial basal hypothalamus (MBH) of ovariectomized rats treated with a regimen of estradiol (E2) that we have previously shown alters brain beta-EP peptide content. In addition the effect of progesterone (P) was also studied. In the first experiment the concentration of beta-EP and alpha-melanocyte-stimulating hormone (alpha-MSH) in the MBH of castrated rats decreased significantly after 3 weeks of E2 treatment compared to castrated unreplaced rats: beta-EP decreased from 6.00 +/- 0.46 to 4.32 +/- 0.38 ng/mg protein and alpha-MSH decreased from 3.00 +/- 0.23 to 2.35 +/- 0.15 ng/mg protein (p less than 0.05). A similar decrease in peptide content was noted in the anterior hypothalamus/preoptic area. A parallel reduction in the concentration of POMC mRNA was measured in the MBH of the E2-replaced animals: 1.17 +/- 0.14 vs. 0.72 +/- 0.08 pg/microgram RNA (p less than 0.02). In a second study castrated rats were studied after 2 weeks of E2 or E2 plus P treatment. After 2 weeks, POMC peptide levels did not change significantly in the MBH of either the E2- or E2 plus P-treated rats. POMC mRNA, however, was significantly reduced from 1.10 +/- 0.10 pg/micrograms RNA in the unreplaced rats to 0.58 +/- 0.05 and 0.61 +/- 0.06 pg/microgram RNA after E2 or E2 plus P, respectively (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sexual differentiation of positive feedback: effect of hour of castration at birth on estradiol-induced luteinizing hormone secretion in immature male rats

In the male rat, a dramatic increase in hypothalamic testosterone and estradiol concentrations occurs during the first few hours of postnatal life. These experiments sought to determine whether such increases participate in the defeminization of positive estrogen feedback effects on LH secretion. Newborn male rats were castrated either in utero (0 h males), or 10 or 24 h after birth. Some males were castrated at 0 h in utero and injected at the time of surgery with 1,2.5, or 5 micrograms testosterone propionate. A group of females was ovariectomized at 0 h in utero (0 h females). The control group consisted of male and female rats sham gonadectomized at 0 h in utero which were either gonadectomized at 21 days of age or left intact. The experimental groups were challenged before puberty to determine if estrogen induced a release of LH using two different types of estrogen treatment. The first treatment consisted of an injection of 0.2 microgram estradiol benzoate (EB) on day 28 followed by a second 10 micrograms injection of EB on day 29. This treatment resulted on the afternoon of day 30 in a surge of LH in intact females. Normal males, 0 h males, or females castrated at 21 days did not have a significant LH surge. The second test consisted of the daily injection of 0.05 microgram EB on days 23-27; on day 28 the rats were injected with 2.5 micrograms EB. Zero hour male and female rats showed a large LH surge on the afternoon of day 29; sham castrated males never responded to this treatment. No sex difference was observed in the mean size of the LH surge providing the males were castrated at 0 h in utero. The effect of the hour of castration on the day of birth also was studied. Males castrated at 10 or 24 h after birth showed either no LH surge or the magnitude of the surge was greatly reduced compared to that obtained in the 0 h males (P less than 0.001). The fact that 0 h males injected with 1 microgram testosterone propionate never showed an LH surge after prepuberal treatment with estrogen suggests that 0 h is a time during which the newborn is sensitive to the defeminizing effect of androgens. These results are consistent with the idea that the testicular hyperactivity which occurs at the time of birth could influence the defeminization of the LH surge mechanisms.     

Steroid induction of gonadotropin surges in the immature rat. I. Priming effects of androgens.

Sexually immature female rats were either primed with estradiol benzoate on day 23 or given daily injections of various androgens on days 23--25. Plasma for LH and FSH determinations was collected on day 26, 5 h after an injection of progesterone. Massive gonadotropin surges were found after priming with estradiol benzoate or treatment with dehydroepiandrosterone (DHEA), delta 4-androstenedione, and testosterone, but not with ring A-reduced androgens (5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol, its 3 beta-epimer, and androsterone) or the nonaromatizable 11 beta-hydroxy- and 11-ketoderivatives of delta 4-androstenedione. Rats bearing DHEA-containing Silastic implants also produced LH surges in response to progesterone. A single injection of an antiestrogen antiserum abolished gonadotropin surges in rats primed with estradiol benzoate or DHEA and greatly reduced the accompanying uterine hypertrophy. DHEA and delta 4-androstenedione were barely uterotrophic in ovariectomized rats but sustained progesterone-induced gonadotropin surges. The results indicate that certain (adrenal?) androgens are able to induce maturation of the steroid-sensitive surge system via extragonadal aromatization, whereas their uterotrophic effect is largely mediated by the ovaries. Coordinated increased conversion of androgens at central and peripheral sites may be of physiological importance for the triggering of puberty.     

Cyclophenil, a non-steroidal compound with a higher central than peripheral oestrogenic activity: study of its effects on uterine growth and on some central parameters in castrated female rats

The oestrogenic activity of cyclophenil, a non-steroidal compound which has structural analogies with both stilbene and triphenylethylene, has been reevaluated utilizing both central and peripheral parameters. The central parameters considered were LH, FSH, prolactin secretion and two enzymatic systems known to be oestrogen-sensitive: hypophyseal 5alpha-reductase and hypothalamic aromatase. The uterine growth test was used to determine oestrogenic peripheral activity. The compound was administered at various doses in comparison with oestradiol benzoate (EB) to long-term castrated female rats. Cyclophenil has an activity 1/8110 times that of EB on uterine growth, and 1/1660 and 1/550 times that of EB in inhibiting LH and FSH, respectively. The hypophyseal 5 alpha-reductase (expressed as DHT formation) was inhibited 1710 times less by cyclophenil than by EB. The other parameters considered were unsuitable to provide a statistically reliable estimate of the potency ratios between the two compounds. The data show that cyclophenil is an oestrogenic compound with peculiar characteristics. This substance is more effective in expressing its oestrogenic activity in central structures than in the peripheral ones.     

Nuclear accumulation of androgens in perfused rat accessory sex organs and testes.

The uptake of androgens into the nuclei of caput epididymis, ventral prostate, seminal vesicle and testis was studied by recirculating physiological and pharmacological concentrations of [3H]testosterone in an artificial medium through the lower half (hemicorpus) of castrated or hypophysectomized rats. The accumulation of dihydrotestosterone in accessory sex organ nuclei was saturable, inhibited by perfusion of excess testosterone or cyproterone acetate, and associated with binding to 3S salt-extractable molecules. In castrated preparations the mean saturation levels (pmol/mg DNA) were different in the three organs: seminal vesicle, 2.8; ventral prostate, 1.8; caput epididymis, 0.9. The saturation level was significantly lower in ventral prostate of hypophysectomized rats (1.2) treated with testosterone to regenerate the accessory sex organs. Testosterone was the major nuclear androgen in the testes of mature hypophysectomized preparations perfused with testosterone. Although there was a large amount of nonspecific accumulation, testosterone binding to 3S molecules was shown by sucrose gradient centrifugation. Binding of dihydrotestosterone to 3S molecules in testicular nuclei was also demonstrated. The ratio of dihydrotestosterone to testosterone was different in immature and mature testicular nuclei and was altered by treatments known to affect testicular 5 alpha-reductase activity. The results suggest that in rat accessory sex organs and immature testis the major active androgen is dihydrotestosterone, whereas in mature testis it is testosterone. The shift in the predominant nuclear androgen in the testis from dihydrotestosterone to testosterone is most simply explained by the maturational change in 5 alpha-reductase activity.     

Plasma lipoprotein profile in the male cynomolgus monkey under normal, hypogonadal, and combined androgen blockade conditions

In men, orchiectomy (GDX) produces an atherogenic lipid profile, whereas combined androgen blockade (CAB) induces a favorable lipid pattern. To better understand the opposite effects of GDX and CAB on lipid metabolism, we have compared the changes in plasma lipoproteins, mesenteric fat metabolism, as well as serum and intratissular sex steroid concentrations in intact, GDX, and GDX+FLU [GDX male cynomolgus monkeys treated for 3 months with flutamide (FLU)]. Serum concentrations of dehydroepiandrosterone, dehydroepiandrosterone sulfate (DHEA-S), and androstenediol remained stable after GDX. Serum androstenedione (-40%), testo (-97%), dihydrotestosterone (-89%), androsterone-glucuronide (-75%), and androstane-3alpha,17beta-diol-glucuronide (-80%) levels decreased similarly in both GDX and GDX+FLU animals. Intratissular dihydrotestosterone (-59 to -99%), estradiol (-31 to -53%), and androsterone-glucuronide (-28 to -85%) concentrations also decreased after GDX. GDX induced significant increases in plasma low-density lipoprotein (LDL) (+78%) and high-density lipoprotein (+34%) cholesterol as well as in LDL-apoB (+58%) and high-density lipoprotein-apoAI (+32%). In the GDX+FLU group, except for the LDL-apoB that showed a tendency to decrease, lipid and apoprotein parameters remained unchanged compared with baseline values measured in intact animals. It is worth noting that these differences in the lipid profile could not be explained by changes in the metabolism of mesenteric adipose tissue. In summary, in the cynomolgus monkey, GDX and CAB induced opposite effects on the plasma lipoprotein profile. These differences possibly result from differences in the specific activity of the androgens and estrogens derived from adrenal precursors. Such data support the suggestion that androgens and estrogens produced from adrenal precursors in peripheral intracrine tissues could have important, but so-far unsuspected, effects on the homeostasis of lipid and lipoprotein metabolism.     

Estrogen regulation of the nuclear 1,25-dihydroxyvitamin D3 receptor in rat liver and kidney.

We previously identified a receptor protein for 1,25-dihydroxyvitamin D3 in rat liver nuclei. The present studies were undertaken to investigate the ontogenesis of the hepatic nuclear vitamin D receptor (nVDR) and the estrogen regulation of this receptor in the liver, small intestine, and kidneys. The hepatic nVDR was significantly elevated in adult female rats compared to prepubertal female rats, while in male rats, this increase was not observed. Oophorectomized rats contained significantly less hepatic nVDR than did intact female rats. Administration of estradiol to castrated male or oophorectomized rats increased the hepatic nVDR. Further studies demonstrated that the increase in the hepatic nVDR was observed only after 2 weeks of estradiol treatment and was positively correlated with circulating estradiol concentrations. Castration of male rats did not alter the hepatic nVDR compared to intact male rats nor did testosterone administration to castrated male rats for 4 weeks change the hepatic nVDR concentration. Unlike the liver, intact female rats contained significantly less renal nVDR than did kidneys from intact male or castrated male rats. Estradiol administration to oophorectomized rats significantly decreased the renal nVDR. Renal nVDR concentrations correlated inversely with the serum concentration of estradiol. Castration of male rats had no effect on the renal nVDR. Intestinal nVDR concentrations were unaffected by castration of male rats or by treatment of castrated male rats with estrogen for up to 4 weeks. These results indicate that estradiol increases the nVDR in liver, decreases the nVDR in kidney and does not change the nVDR in the intestine. Physiological concentrations of testosterone do not regulate the nVDR in these tissues. Estradiol regulation of this receptor is organ specific and, therefore, conclusions about the regulation of the nVDR in one tissue cannot be extrapolated to other tissues.     

Role of the hypophysis in estrogen-mediated induction of enzymes in the regenerating liver from castrated male rats.

Perfusions with corticosterone, of isolated regenerated livers from adult male rats, subjected to castration, partial hepatectomy and hypophysectomy with or without estradiol treatment during parenchymal regeneration, yielded very similar patterns of biliary steroid metabolites. The degree of steroid conjugation was lower than that seen in livers from normal, untreated, adult male rats. In operated animals, with or without estradiol benzoate treatment, ring A-reduced 20-keto metabolites constituted about 20%, whereas metabolites with a 20-hydroxy group made up approximately 80% of the corticosterone metabolites formed. Furthermore, no 15-hydroxylated metabolites derived from corticosterone, quantitatively the most important compounds in bile from female rats, could be detected in bile from these treated male animals. However, livers from male rats which had been castrated, hepatectomized and treated with estradiol benzoate during liver regeneration, produced 15-hydroxy-tetrahydrocorticosterone to the same extent as female rat livers, when perfused with corticosterone. The results obtained indicate that the effects of estradiol on the induction and differentiation of steroid metabolizing enzymes in the regenerating liver are pronounced and manifested only in the presence of an intact hypophysis.     

Gene expression changes in rat prostate after activation or blocking of the androgen and estrogen receptor

Several endpoints of different molecular complexity were studied in the Hershberger assay in order to evaluate the specificity and suitability of this test as a broad screening model. Androgen and estrogen receptors were activated or blocked, and expression of typical estrogen- or androgen responsive genes (complement C3, ERalpha, ERbeta, AR, TRPM-2, PBP C3, ODC, and IGF-1 mRNA) was analyzed in rat ventral prostate by real time RT-PCR. Administration of estradiol benzoate (EB) to castrated testosterone-treated rats had no effect on reproductive organ weights or gene expression levels and the anti-estrogen, ICI 182780, only affected ODC expression. Therefore, estrogenic or anti-estrogenic compounds would not be expected to seriously affect the outcome of a Hershberger test. However, EB given alone to castrated rats resulted in various effects. EB increased seminal vesicle weight, an effect reversed by ICI 182780, and affected TRPM-2, PBP C3, ODC, IGF-1, AR, and ERalpha mRNA levels. AR expression in the prostate seemed to be under regulation of both estrogens and androgens, as ICI 182780 inhibited the testosterone-induced AR expression, and flutamide inhibited the EB-induced AR expression. These data indicate that estrogens have various effects in castrated male rats and that expression of several genes is under multi-hormonal control in the ventral prostate. However, interactions between estrogens and androgens do not play a major role in the Hershberger assay, as simultaneous TP administration abolished the effects of EB. First choice of gene expression profiles in the Hershberger assay to study androgenic or anti-androgenic effects would be the traditional, TRPM-2 and PBP C3, supplemented with the new complement C3.     

Steroid-sensitive gene-1 is an androgen-regulated gene expressed in prostatic smooth muscle cells in vivo

Steroid-sensitive gene-1 (SSG1) is a novel gene we cloned, found regulated by 17beta-estradiol in the rat uterus and mammary gland, and over-expressed in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. We show here that SSG1 mRNA and protein expression are regulated by androgens in the rat ventral prostate. Increases in SSG1 mRNA levels were detected by Northern blotting after 24 h and reached a 27-fold peak 96 h following castration, relative to SSG1 mRNA expression in sham-operated rats. Dihydrotestosterone or testosterone supplementation of castrated rats prevented this rise in SSG1 mRNA. In contrast with SSG1 mRNA expression, SSG1 protein was decreased 16-fold 2 weeks following castration but was at control levels in the prostates of castrated rats receiving dihydrotestosterone or testosterone. Although SSG1 is regulated by androgens in vivo, treatment of LnCap cells with dihydrotestosterone, cyproterone acetate or flutamide did not result in the regulation of SSG1 protein levels in vitro. Immunofluorescence studies show that SSG1 is mainly expressed in prostatic smooth muscle cells. These results indicate that SSG1 is an androgen-regulated gene that is expressed in the smooth muscle component of the rat ventral prostate in vivo.     

Regulation of beta-endorphin, corticotropin-like intermediate lobe peptide, and alpha-melanotropin-stimulating hormone in the hypothalamus by testosterone

Ovarian steroids have previously been shown to regulate the hypothalamic content of beta-endorphin (beta EP) and its release into hypophyseal portal blood. Although the hypothalamic content of beta EP in cycling female rats was unchanged by ovariectomy, chronic treatment of ovariectomized rats with estradiol lowered hypothalamic beta EP levels. In this study, the hypothalamic content of beta EP was compared in male and cycling female rats, and the effects of orchiectomy and testosterone replacement on hypothalamic beta EP were examined. The beta EP content of the medial basal hypothalamus (MBH) was significantly higher in female rats compared to that in males of either the same weight (175-200 g) or the same age (65 days; P less than 0.025). When male rats were studied 4 weeks after castration, the beta-EP content of the MBH increased from a value of 2100 +/- 103 fmol in the controls to 2680 +/- 126 fmol (P less than 0.005). The hypothalamic beta EP content in the castrated males was similar to that in the intact females (2700 +/- 158 fmol). The increase in hypothalamic beta EP induced by castration was blocked by testosterone replacement. When orchiectomized animals were treated for 4 weeks with Silastic capsules filled with testosterone, there was a significant fall in the hypothalamic content of beta EP compared to that in the unreplaced animals. beta EP fell from 3180 +/- 115 to 2033 +/- 53 fmol in the MBH (P less than 0.001), from 1693 +/- 122 to 934 +/- 80 fmol in the anterior hypothalamus (P less than 0.001), and from 148 +/- 26 to 90.3 +/- 11 fmol in the median eminence (P less than 0.05). Testosterone replacement was also associated with a significant decline in the hypothalamic content of corticotropin-like intermediate lobe peptide and alpha MSH. Corticotropin-like intermediate lobe peptide fell from 2400 +/- 53 to 1560 +/- 84 fmol in the MBH (P less than 0.001) and from 1200 +/- 74 to 805 +/- 94 fmol in the anterior hypothalamus (P less than 0.01). alpha MSH fell from 1660 +/- 162 to 884 +/- 75 fmol in the MBH (P less than 0.001) and from 823 +/- 106 to 544 +/- 92 fmol in the anterior hypothalamus (P less than 0.05). Thus, testosterone, as well as estradiol, affects the hypothalamic content of several proopiomelanocortin-derived peptides. The effect on brain peptide content, however, depends on whether the steroids are secreted relatively constantly, as in the male, or fluctuate, as in the cycling female.     

Relationship between the pituitary gland and gonadal steroids: involvement of a hypophysial factor in reduced alpha 2u-globulin and increased transcortin concentrations in rat serum.

Evidence is presented that the level of alpha 2u-globulin in the serum of male rats depends, at least in part, on neonatal androgens. After castration of adult animals the concentration of this protein falls but remains measurable, whereas in intact or ovariectomized female rats alpha 2u-globulin cannot be detected. Moreover, alpha 2u-globulin is found in adult male and female rats gonadectomized at birth and treated with a single injection of testosterone propionate immediately thereafter. The mechanism by which neonatal androgens increase the concentration of alpha 2u-globulin has been investigated. Transplantation of a supplementary pituitary gland under the renal capsule of male rats resulted in reduced levels of alpha 2u-globulin and increased levels of transcortin. The changes discussed here were observed only in those animals in which the transplant was functional and they were amplified or reversed by modulators of prolactin secretion such as oestrogens or bromocriptine respectively. The hypothesis is advanced that neonatal androgens stimulate the production of a hypothalamic inhibitory factor that controls the secretion of prolactin, or another hypophysial hormone subjected to similar neuroendocrine control. Measurements in gonadectomized animals and in rats receiving both oestradiol benzoate and bromocriptine indicate that, besides these pituitary-mediated effects, both oestrogens and androgens exert direct effects on the level of alpha 2u-globulin.     

Immunoreactive dynorphin is regulated by estrogen in the rat anterior pituitary

The pituitary and hypothalamic content of dynorphin was determined by radioimmunoassay and characterized by high-performance liquid chromatography (HPLC) in adult female Sprague-Dawley rats, intact and ovariectomized with and without estrogen treatment. Animals were given estradiol benzoate, or vehicle (oil) by six daily intramuscular injections. Anterior pituitary content of immunoreactive (ir)-dynorphin in ovariectomized rats was approximately twice that of intact animals, and consisted of a single HPLC peak co-eluting with dynorphin 32. Administration of estradiol benzoate (0.06-6 micrograms/day) caused a marked decrease of ir-dynorphin in the anterior lobe of castrate female rats, with a half-maximal effect at 0.2 microgram/day; levels were restored to those seen in intact animals with 6 micrograms estradiol benzoate per day, an effect which was not influenced by concomitant administration of progesterone (1 mg/day), or bromocriptine (100 micrograms/day). In the hypothalamus and neuro-intermediate lobe multiple peaks of immunoreactive dynorphin were seen, coeluting with dynorphin A 1-8, dynorphin A 1-17 and dynorphin 32. Neither castration nor estrogen treatment altered ir-dynorphin content in these tissues. These findings suggest that the ovary exerts a specific modulating influence on AP ir-dynorphin in the rat, and that in addition this inhibition appears to be mediated by ovarian estrogen.     

Sexual differentiation of the external genitalia and the timing of puberty in the presence of an antiandrogen in sheep

Testicular steroids during midgestation sexually differentiate the steroid feedback mechanisms controlling GnRH secretion in sheep. To date, the actions of the estrogenic metabolites in programming neuroendocrine function have been difficult to study because exogenous estrogens disrupt maternal uterine function. We developed an approach to study the prenatal actions of estrogens by coadministering testosterone (T) and the androgen receptor antagonist flutamide, and tested the hypothesis that prenatal androgens program estradiol inhibitory feedback control of GnRH secretion to defeminize (advance) the timing of the pubertal increase in LH. Pregnant sheep were either untreated or treated with T, dihydrotestosterone (DHT) (a nonaromatizable androgen), or T plus flutamide from d 30-90 of gestation. To study the postnatal response to steroid negative feedback, lambs were gonadectomized and estradiol-replaced, and concentrations of LH were monitored in twice-weekly blood samples. Although T and DHT produced penile and scrotal development in females, the external genitalia of T plus flutamide offspring remained phenotypically female, regardless of genetic sex. Untreated females and females and males treated with T plus flutamide exhibited a pubertal increase in circulating LH at 26.4+/-0.5, 26.0+/-0.7, and 22.4+/-1.6 wk of age, respectively. In females exposed to prenatal androgens, the LH increase was advanced (T: 12.0+/-2.6 wk; DHT: 15.0+/-2.6 wk). These results demonstrate the usefulness of combining T and antiandrogen treatments as an approach to increasing prenatal exposure to estradiol. Importantly, the findings support our hypothesis that prenatal androgens program sensitivity to the negative feedback actions of estradiol and the timing of neuroendocrine puberty.