Peter B. Connolly

Progestins affect reproductive behavior and androgen receptor dynamics in male guinea pig brain.

The present study extends previous studies of ours by comparing the anti-androgenic effect of a progestin agonist (R5020) with progesterone (P). Intact male guinea pigs treated with P (1 and 10 mg/day) and R5020 (100 micrograms/day) had greater latency to mount and lower numbers of mounts and intromissions compared to controls. Ejaculation and plasma testosterone concentration were not affected. Specific brain regions were analyzed for androgen receptor (AR) content. Progestins produced fewer (P less than 0.01) nuclear AR in hypothalamus-preoptic area and pituitary without associated changes in cytosolic AR. These data are best interpreted by postulating an effect of P on AR dynamics mediated through the P receptor and not by competition for androgen binding to its receptor.

Testosterone 5α-reductase activity in neural tissue of fetal rhesus macaques

Abstract After development of a 5α-reductase activity (5α-RA) assay based on the capacity of microsomes to convert [3H]testosterone (T) to [3H]dihydrotestosterone (DHT), we analyzed 5α-RA in neural tissues of fetal rhesus macaques at 50, 80 and 150 days of gestation. This method allowed us to collect kinetic data on the properties of the 5α-reductase resident in fetal brain at 150 days of gestation. The Km and Vmax calculated from these data were 4.32μM and 22.6nmol·mg protein−1· h−1, respectively. Analyses of 5α-RA in microsomes from the hypothalamic-preoptic area-amygdala (HPA) at dilutions of 1 25 and 1 50 indicated higher enzyme activity with increasing dilution of the microsomes. Measurement of 5α-RA using concentrations of [3H]T which saturated the enzyme in diencephalon (DIEN), brain stem (B.STEM), temporal (TCTX) and frontal cortex (FCTX) of six 50-day old fetuses (3 males and 3 females) revealed no obvious sex differences in 5α-RA, however, a significant difference (P Significant increases in 5α-RA were observed in FCTX and TCTX with time of gestation (50, 80 and 150 days). Other tisues, including amygdala, hippocampus, cerebellum, tegmentum and septum also change with fetal age. These data demonstrate the existence of 5α-reductase in the fetal monkey brain. Significant changes in cortical 5α-RA suggest some role for 5α-reductase in development.

Androgen-Dependent and -Independent Aromatase Activity Coexists with Androgen Receptors in Male Guinea-pig Brain

Using a microdissection technique we localized androgen receptors and aromatase activity (AA) in the brain of male guinea‐pigs. In addition, we evaluated the effects of castration and androgen replacement on androgen receptor dynamics and induction of AA. In the castrate animal, cytosolic androgen receptor content was highest in the basal hypothalamus, specifically in the median eminence‐arcuate nucleus (> 15 fmol mg protein 1), while lesser levels were found in the preoptic regions and amygdala. Nuclear receptor content was highest (> 150 fmol mg DNA −1) in the median eminence‐arcuate nucleus, periventricular region of the preoptic area and cortical amygdala. All regions investigated showed a significant decrease in nuclear receptors following castration and an increase with androgen replacement. However, reciprocal changes in cytosolic androgen receptors were not always evident. Aromatase activity was high in the cortical amygdala, medial amygdala, periventricular region of the preoptic area and bed nucleus of the stria terminalis. Castration and androgen replacement had significant stimulatory effects on AA in the ventral medial hypothalamus, median eminence‐arcuate nucleus, cortical amygdala and periventricular regions of the preoptic area and anterior hypothalamus. Thus, androgen receptors and AA are unevenly distributed throughout the subcortical regions of the male guinea‐pig brain and respond differently to endocrine stimuli. Our data demonstrate that AA is androgen‐dependent in some subcortical regions which contain androgen receptors. Even though nuclear receptors in all brain regions were affected by castration and dihydrotestosterone treatment, the events were not always linked to AA regulation. Due to this difference in regulation, AA may serve divergent functions in guinea‐pig brain.

Role of Steroid 5α-Reductase Activity in Sexual Differentiation of the Guinea Pig

The possible role of 5α-reduction of steroids in the sexual differentiation of guinea pigs was determined by treating pregnant guinea pigs with a 5α-reductase activity (5αRA) inhibitor (17β-N, N-dieth

Effects of Exogenous Androgen on Brain Androgen Receptors of the Fetal Rhesus Monkey

Testosterone secreted by the fetal testes masculinizes and defeminizes the nonhuman primate brain during a defined prenatal critical period. We previously demonstrated the presence of high-affinity, specific androgen receptors (AR) in the developing rhesus monkey brain, but did not present data concerning their capacity for activation. To achieve this end, we analyzed the AR content in brains from intact and gonadectomized rhesus monkey fetuses at approximately 125 days of gestation, 2 h after injection of either 500 micrograms dihydrotestosterone (DHT) or vehicle directly into the fetus. After treatment, plasma DHT concentrations increased five-fold in the fetal circulation. In gonad-intact fetuses, cytosolic AR decreased in preoptic area, medial basal hypothalamus, and septum following DHT treatment. No significant effect of DHT treatment on nuclear AR was seen. In contrast, the increased level of DHT in the maternal circulation decreased cytosolic AR and increased nuclear AR of the maternal myometrium. In gonadectomized fetuses, DHT treatment decreased cytosolic AR as it did in the intact group. In contrast, a significant increase in nuclear AR was seen in preoptic area, medial basal hypothalamus, and tegmentum of these fetuses. Thus AR in fetal rhesus brain can be activated by DHT when the gonads are removed, but not in the intact fetuses. These data suggest that AR in the developing nervous system of rhesus macaques can be activated by exogenous androgen and hence are probably functional.

Androgen Regulation of Androgen Receptor Messenger Ribonucleic Acid Differs in Rat Prostate and Selected Brain Areas

Androgens bind to specific high-affinity receptors (AR), thereby initiating gene transcription. We investigated the effects of testosterone (T), dihydrotestosterone (DHT), and 17beta-estradiol (E(2)) on AR transcription and binding in prostate, medial basal hypothalamus (MBH), preoptic area (POA), amygdala, hippocampus, and cortex in the rat. Androgen receptor mRNA was measured by a ribonuclease protection assay. Cytosolic and nuclear AR binding (ARc and ARn, respectively) were measured by in vitro binding assays. In the prostate AR mRNA levels were low in intact animals. Castration produced a fourfold elevation of AR mRNA which was reduced to intact values by treatment with T or DHT (P < 0.05; n = 4). E(2) had no effect compared to castrate levels. In contrast to the prostate, no treatment effect was observed on the expression of AR gene in the MBH, POA, amygdala, hippocampus, or cortex. On the premise that treatment effects on AR mRNA in the brain may require longer than 48 h, we treated rats for 4 and 7 days and found no treatment effect on the expression of AR mRNA in MBH, POA, or amygdala. Next, we compared AR binding with its mRNA between prostate and various brain areas. Castration significantly increased ARc and reduced ARn compared to intact levels, and androgen treatments restored both ARc and ARn to intact values in prostate and brain areas (P < 0.05; n = 5). Changes in AR mRNA levels in prostate corresponded to changes in ARc but not ARn in castrated and androgen-treated males, which suggests that ARc is newly synthesized receptor. In contrast, ARc differed quantitatively between prostate and neural tissues. These results show that DHT regulates AR transcription in rat prostate as effectively as T. Our data also suggest that AR gene transcriptional activity in prostate and selected brain areas may be subjected to differential regulatory mechanisms. This may be due to the presence of tissue-specific regulatory proteins.

Androgen binding in peripheral tissues of fetal rhesus macaques: Effects of androgen metabolism in liver ☆

In rhesus monkeys sexual differentiation of the brain and reproductive tract (RT) is androgen-dependent. Presumably these effects are mediated through the androgen receptor (AR). The AR has not been characterized in fetal tissues such as liver, kidney, heart, spinal cord and RT in this species. We characterized AR binding using [3H]R1881 as the ligand in cytosols from tissues obtained on days 100-138 of gestation. Scatchard analyses revealed a single, saturable, high affinity AR in liver, kidney, heart, spinal cord and RT. The apparent dissociation constant (Kd) ranged from 0.52 to 0.85 nM with no significant tissue differences. The number of AR (Bmax; fmol/mg protein) differed significantly (P less than 0.01) between tissues (liver greater than RT much greater than kidney greater than or equal to heart greater than or equal to spinal cord). Radioinert testosterone (T) and 5 alpha-dihydrotestosterone (DHT) but not androstenedione, progesterone, estradiol-17 beta, estrone or cortisol in a 50-fold molar excess inhibited [3H]R1881 binding to the AR in spinal cord, heart, kidney and RT. However, in liver only DHT competed significantly (P less than 0.01) for binding. This difference in binding of DHT vs T in the liver was further investigated by incubating liver and kidney cytosols with [3H]DHT and [3H]T at 4 degrees C. We identified the metabolic products by mobility on Sephadex LH-20 columns and reverse isotope dilution. Liver cytosols metabolized [3H]DHT to 5 alpha-androstane- 3 alpha,17 beta-diol (5 alpha-diol) and [3H]T to 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-diol) at 4 degrees C. In contrast, kidney cytosols metabolized [3H]DHT while [3H]T remained unchanged. Further studies indicated that a 50-fold molar excess of 5 alpha-diol inhibited the binding of [3H]R1881 in liver cytosols by about 50% whereas the same molar concentration of 5 beta-diol had no effect. These data demonstrate the presence of AR in peripheral tissues of fetal rhesus monkeys and suggest that androgens through their receptors may affect development of these tissues. Liver cytosols are capable of metabolizing T and DHT at 4 degrees C at conditions similar to those used for measuring cytosolic AR. However, T and DHT are metabolized differently, generating different isomers which have different affinities for hepatic AR.

Estrone sulfatase activity in rat brain and pituitary: effects of gonadectomy and the estrous cycle

Estrone sulfatase activity was characterized in microsomal preparations from rat brain and anterior pituitary. No differences in apparent Km were found in hypothalamic-preoptic area between male (7.5 microM) and female (7.4 microM) rats. Apparent Kms of anterior pituitaries from males (14.5 microM) and females (22.5 microM) were higher than those found in brain. Estrone sulfatase activity was equally inhibited by estradiol-17 beta-3-sulfate, dehydroepiandrosterone-3-sulfate and estrone-3-sulfate indicating a broad range of substrate specificity for this enzyme. Sulfatase activity in female anterior pituitary was found to be twice that of male. Sulfatase activity was distributed similarly in brain tissues between sexes with cerebellum greater than or equal to medial basal hypothalamus greater than preoptic area = cortex. Following gonadectomy, sulfatase activity in anterior pituitary of males was significantly greater than activity found in intact animals (P less than 0.05). This increase in activity, however, was unaffected by treatment with testosterone, dihydrotestosterone or estradiol-17 beta. Gonadectomy did not change sulfatase activity in brains of males or females or in pituitaries of females. However, sulfatase activity in pituitary glands of females changed significantly (P less than 0.05) with stages of the estrous cycle (metestrus less than diestrus less than proestrus less than estrus). These data indicate sulfatase activity in rat anterior pituitary gland may be controlled by gonadal factors while sulfatase activity in brain is regulated differently.

Androgen metabolism by hepatic and renal tissues of the fetal rhesus monkey

Liver and kidney from fetal monkeys (day 125 of gestation) were fractionated into low speed pellets, microsomal and cytosolic fractions. Liver cytosols converted as much testosterone (T) to 5 beta-androstane-3 alpha,17 beta-diol (5 beta-diol) at 0 degrees C as at 4 degrees-45 degrees C without exogenous cofactors. The principal product formed from 5 alpha-dihydrotestosterone (5 alpha-DHT) was 5 alpha-diol. A 1000-fold molar excess of radioinert 5 beta- or 5 alpha-DHT inhibited 5 beta-diol formation from [3H]T by cytosols and increased 5 beta-DHT formation. Similarly, using 5 alpha-DHT as substrate, 5 alpha-diol formation was inhibited. Microsomal and low speed pellets with added cofactors formed products which recrystallized with either etiocholanolone or androsterone from [3H]T or [3H]DHT, respectively. Little product was formed without cofactor. Whole liver homogenates produced 5 beta-reduced products from [3H]T in the presence of an NADPH generating system whereas kidney homogenates produced 5 alpha-reduced products. These data provide new information on the capacity of fetal monkey liver and kidney to metabolize androgens. The 3 alpha-reductases are cytosolic. The 5 alpha- and 5 beta-reductases are mostly in the low speed pellet but are sufficiently represented in cytosols to mediate diol formation. The 17-hydroxysteroid dehydrogenases are in the microsomal fraction. Our results suggest that 5 alpha-DHT is the active androgen in fetal liver since testosterone is metabolized to 5 beta-DHT and 5 beta-diol which are inactive androgens.

Wood duck hatch date: relationship to pairing chronology, plasma luteinizing hormone, and steroid hormones during autumn and winter.

We examined variation in courtship activity and hormone levels of male and female wood ducks (Aix sponsa) in relation to hatch date. Young wood ducks were assigned in groups of 8 (4 males and 4 females) to 4 experimental pens; 2 pens contained early-hatched ducks (3-12 April) and 2 pens contained late-hatched ducks (7-16 June). Courtship behaviors occurred less frequently in October and November than in December and January-February for both early- and late-hatched groups. Early-hatched wood ducks participated in courtship more frequently and formed pair bonds sooner than late-hatched individuals. Testosterone (T) and androstenedione (AD) levels of males did not differ between treatment groups; however, average levels of luteinizing hormone (LH) were greater for early-hatched males. Differences in LH occurred in 2 of 12 samples (6 October and 3 November); otherwise, LH did not vary by treatment. Levels of T and LH in males varied temporally, but there was no significant temporal variation in AE levels. Temporal variation in hormone levels was similar for early- and late-hatched males. Estradiol (E), progesterone (P), and LH levels of females did not differ between treatment groups. There was temporal variation in levels of E and LH, but not of P; this variation was similar for early- and late-hatched females. These data indicate that behavioral differences occurring temporally and between early- and late-hatched wood ducks were not related to corresponding differences in hormone levels.