Patrick J. Thomas

An androgen receptor in rat brain and pituitary.

Dihydrotestosterone (DHT) binding was measured in cytosols from brain regions and pituitary of adult female rats and, with the addition of ventral prostate, in adult male rats. Two types of binding were distinguished: one, saturable at concentration of DHT greater than or equal to 5 X 10(-9) M and an unsaturable component. In intact males saturable (limited capacity) binding was detected only in ventral prostate cytosol; 3 days after orchidectomy the saturable binding sites increase 3-fold in prostate and in pituitary, hypothalamus, amygdala and cortex to detectable levels in approximately the same abundance as in females. There were significant differences in the affinities of the limited capacity binding reactions in cytosols of different tissues though all were in the order of magnitude, 10(-9) M DHT. The affinity in pituitary cytosol was lower than in brain regions with the single exception of female amygdala in which the affinity was significantly lower than in cytosol of the same region from 3-day castrate males. The specificity of the limited capacity binding was investigated by competition between [3h]DHT and unlabelled steroids; the most effective competitors were potent androgen agonists and antagonists.

The specificity of oestrogen receptor in brain, pituitary and uterus.

1 The specificity of oestrogen receptor in rat brain regions (hypothalamus, amygdala and cortex), pituitary and uterus was studied by measurement of the inhibition of 17β‐oestradiol high affinity binding in cytosol, in the presence of unlabelled putative inhibitors of binding. 2 Binding was inhibited only by those of the compounds tested that possessed oestrogen agonist or antagonist activity. The affinities were estimated and the ranking order of the compounds was the same in all tissue sources of cytosol and corresponded to the ranking order of agonist or antagonist potency. 3 There were some significant differences between some of the estimated affinities in different tissues, these being seen most commonly between pituitary and hypothalamus on one hand and uterus on the other. 4 The possibility of heterogeneity of oestrogen receptor is discussed.

Effect of estradiol on neurotransmitter sensitive adenylate cyclase. Its possible role in ‘sexual differentiation’

Adenylate cyclase activity is lower in the hypothalamus of 5-day-old male rats than in females. This may be due to the presence of estradiol (E2) in this area of the male but not in the female since castration of the newborn male leads to an enzyme activity in the hypothalamus that is indistinguishable from that of the female and androgenization of the newborn female causes an enzyme activity level comparable to that of the male. In the adult, enzyme activity is highest when the concentration of E2 is at its lowest level; adenylate cyclase activity in the hypothalamus-preoptic area was found to be higher in the metestrus female than in proestrus or in the adult male. In vitro E2 was shown to reduce enzyme activity and this reduction was found to be dependent on induction of protein biosynthesis. In addition, there appears to be a requirement for Ca2+ in the E2-induced reduction of cyclase activity. Although F- activates the enzyme in all of the cases studied, the reduction in enzyme activity brought about by E2 is not reversed by F-, suggesting that the effect of E2 is not on the receptor. As sexual differentiation is brought about by the action of E2 during the first week after birth, it seems plausible to suggest that this interaction between hormone and enzyme is an early step in the sexual differentiation of the brain.

Sex differences in the distribution of cytoplasmic oestrogen receptors in rat brain and pituitary: Effects of gonadectomy and neonatal androgen treatment

High affinity binding of 17 beta-oestradiol was measured in cytosols of hypothalamus, amygdaloid region, pituitary and (in females) uterus of adult male and female rats. There were no differences between intact or gonadectomised male and female animals in any of the tissues in the equilibrium dissociation constants (Kd). The number of available binding sites (n) in brain and pituitary in intact females at metoestrus is higher than at proestrus but only in hypothalamus is n greater than in ovariectomised animals. Binding sites in male hypothalamus, amygdala and pituitary are significantly less than in metoestrous females; the sex difference is seen also in gonadectomised rats but is significant only in pituitary. In all female animals the highest concentration of binding sites in the hypothalamus is in the anterior part and the lowest in the posterior part. The distribution of binding sites and Kd values in adult females treated neonatally with testosterone propionate were not different from those of intact proestrous rats. In intact males the highest level of n was in mid-hypothalamus; after gonadectomy the pattern reverted to that in females. It is suggest that these results support the concept that testicular androgen is conversted in brain to a substance with affinity for cytosol oestrogen receptor.

ANDROGEN‐INDUCED SEXUAL DIMORPHISM IN HIGH AFFINITY DOPAMINE BINDING IN THE BRAIN TRANSCENDS THE HYPOTHALAMIC‐LIMBIC REGION

1 High affinity binding of [3H]‐dopamine and [3H]‐5‐hydroxytryptamine ([3H]‐5‐HT) was measured in membrane fractions prepared from cerebral cortex, amygdala, hypothalamus, thalamus and brain stem of rats of either sex and of rats which had been either neonatally castrated or androgenized. 2 Binding was measured in rats of 8, 20 and 30 days old as well as in adults. 3 [3H]‐dopamine bound with approximately 30 nm affinity and [3H]‐5‐HT with approximately 10 nm affinity to all areas of the brain tested. The relative inhibitory effects of haloperidol, apomorphine, cis‐flupenthixol, unlabelled dopamine, noradrenaline, spiroperone, (+)‐butaclamol, fluphenazine, pimozide and 5‐HT on [3H]‐dopamine binding in the cerebral cortex was consistent with receptor status for the binding components there as were the relative inhibitory effects of methysergide, dopamine, fluoxetine and ouabain on [3H]‐5‐HT binding in the fore brain. 4 Neither [3H]‐dopamine nor [3H]‐5‐HT binding varied with the state of the sexual cycle in females. 5 There were no sexual differences in [3H]‐5‐HT binding in any of the brain areas tested nor was it affected by neonatal androgenization or neonatal castration. 6 [3H]‐dopamine binding was greater in the cerebral cortex and amygdala of male than of female rats. These differences could be mimicked artificially by neonatal castration of males (female type development) or neonatal androgenization of females (male type development). Sexual dimorphism did not become overt until 20 days of age and did not extend to hypothalamus, thalamus or brain stem. 7 It is concluded that neonatal sex differences in exposure to steroid hormones has permanent effects on the number of dopamine binding sites in the cerebral cortex and is suggested that this sexual dimorphism extends to the amygdala.

OCCURRENCE AND PROPERTIES OF 17β-OESTRADIOL RECEPTORS IN RAT BRAIN

Publisher Summary This chapter describes the occurrence and properties of 17β-oestradiol receptors in rat brain. The ratio of estrogen specific high affinity binding site concentration in adult cortex-amygdalahypothalamus is in the order 1:5:11 in mixed cycling females and 1:3:6 in intact males. In 5-day-old rats, the diethyl stilboestrol (DES) suppressible binding sites are distributed in roughly equal concentration in both brain areas and at levels similar to those found in adult hypothalamus or amygdala. Values for the reaction affinities for E2 obtained in the experiments with cytosol from brain of 5-day-old rats invariably were 3–4 times less than in adult preparations; however, for reasons to be given later, one believe that the difference is artifact. The neonatal brain E2 binding activity is, in most other ways, indistinguishable from the binding moiety from adult tissues and they are broadly estrogen specific, readily saturable with DES. There is a consistent difference in affinity for E2 between pituitary cytosol and cytosol from brain region or uterus.