Neurotransmitter modulation of steroid action in target cells that mediate reproduction and reproductive behavior.

Two major functional interactions between steroid hormones and neurotransmitters are generally recognized. First, steroids affect neurotransmission, and second, through effects on hypothalamic peptides that regulate anterior pituitary function neurotransmitters affect steroid secretion. In recent years, evidence has accumulated which indicates that neurotransmitters can also affect steroid action within postsynaptic steroid target cells. We review evidence for this relationship in pineal, uterus and hypothalamus and propose that the modulation of target cell responsiveness to steroids is an important mechanism by which neurotransmitters affect steroid-dependent processes. The operation of such a mechanism provides a means for environmental, behavioral and emotional events to rapidly and selectively alter steroid effects on behavior and physiology.

Relation between individual differences in sexual behavior and plasma testosterone levels in the guinea pig.

After 3 tests for male sex behavior, adult male guinea pigs were classified as either low-activity (LA, no ejaculations) or high-activity (HA, ejaculation during at least 2 behavior tests). In one experiment, resting levels of peripheral plasma testosterone (T), measured by radioimmunoassay, did not differ between the groups (LA=2.22 +/- 0.17 ng/ml, HA=2.09 +/- 0.11 ng/ml, X +/- SEM). However, plasma T levels were significantly higher in HA males 2 min after a sex test (2.27 +/- 0.24 ng/ml) than in LA males (1.40 +/- 0.20 ng/ml). A second experiment confirmed these results and also demonstrated that exposure of HA or LA males to an estrous female placed on the opposite side of a wire mesh barrier similarly led to higher T levels in HA than in LA males. These results indicate that a) behavioral differences between HA and LA guinea pigs are not attributable to differences in resting T levels, b) HA and LA males perceive the sex test situation differently, leading to slight increases in T in HA males and slight decreases in T in LA males, and c) these changes in T level are not dependent on copulation but can be induced by mere exposure to the sight smell and/or sound of an estrous female.

Hormones modulate the concentration of cytoplasmic progestin receptors in the brain of male ring doves (Streptopelia risoria).

A cytoplasmic progestin receptor has been characterized in the brain of castrated ring doves using an in-vitro assay that measures the binding of a synthetic progestin, [3H]17 alpha,21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione(promegestone; R5020). The affinity of the receptor was similar in both the hyperstriatum and the hypothalamus (Kd approximately equal to 4 X 10(-10) mol/l). Its concentration was higher in the anterior hypothalamus-preoptic area (63 +/- 4 fmol/mg (S.E.M.) protein) than in other brain regions (posterior hypothalamus, 33 +/- 5; hyperstriatum, 28 +/- 3; midbrain, 17 +/- 4 fmol/mg protein; n = 7). Progesterone and R5020 competed well for binding but oestradiol and 5 beta-dihydrotestosterone did not. Corticosterone and, to a lesser extent, testosterone and 5 alpha-dihydrotestosterone completed for binding but much higher concentrations were required than for progestins. Injections of testosterone (200 micrograms testosterone propionate daily for 7 days) significantly increased the concentration of progestin receptors in the anterior and posterior hypothalamus without having any significant effect on other brain areas. Shorter treatment, lasting for 2 days, with testosterone propionate (200 micrograms daily), 5 alpha-dihydrotestosterone (200 micrograms daily) or oestradiol benzoate (50 micrograms daily) did not always cause this increase but seven injections of oestradiol benzoate (50 micrograms daily for 7 days) were even more effective than seven injections of testosterone propionate (200 micrograms daily for 7 days). These data suggested that the sensitivity to progesterone of the brain of the bird changes as a consequence of increases in the level of testosterone in the circulation.

Idazoxan decreases estrogen-induced lordosis in female but not "hormone-independent" lordosis in male guinea pigs of an inbred strain.

This experiment examined whether the imidazoline idazoxan (which binds to alpha-noradrenergic receptors and to imidazoline-preferring sites) interferes with hormone-dependent or hormone-independent lordosis responses. Ovariectomized (ovx) Strain 2 female guinea pigs which were sexually receptive after receiving estradiol benzoate (EB; 3 micrograms/d for 3 days) were injected with either idazoxan (10 mg/kg) or with vehicle at 24 hr after the last EB injection. Idazoxan significantly decreased EB-facilitated lordosis responses in these females. Castrated Strain 2 males, which show lordosis behavior without gonadal hormone administration, were injected with the same dosage of idazoxan (10 mg/kg) or with vehicle. Idazoxan did not inhibit lordosis behavior in these males.

Estrogen receptor immunostaining in the preoptic area and medial basal hypothalamus of estradiol benzoate- and prazosin-treated female guinea-pigs.

Evidence has accumulated showing that the alpha 1-adrenergic antagonist prazosin decreases nuclear estrogen binding in the hypothalamus of the guinea-pig. In this study we asked if prazosin treatment alters estrogen receptor (ER) protein content as reflected by changes in ER-immunoreactivity. The monoclonal rat antibody H222 directed against ER was used to detect ER-immunoreactive (ER-ir) cells in eight specific preoptic and hypothalamic brain regions of ovariectomized Hartley strain guinea-pigs treated with estradiol benzoate and 1.0 mg/kg prazosin or vehicle. Immunocytochemical parameters which provided optimum conditions for detection of even modest changes in ER-immunoreactivity were first established. Then, using these optimum conditions, we compared 1) the mean number of ER-ir profiles, 2) the mean density of ER-ir staining, and 3) the distribution of ER-ir staining density readings, between conditions within each of the eight brain regions. No differences in any of these measures were found between prazosin- and vehicle-treated females. We also compared the percentage of ER-ir nuclear profiles across the eight cell groups investigated in estradiol benzoate- and vehicle-treated females. The medial preoptic area had by far the highest percentage (48%) of ER-ir profiles (P < 0.05) compared to all seven other brain regions (23% to 32% ER-ir cells). Our data, showing that ER-immunoreactivity is not reduced (6h) after prazosin treatment, suggests that mechanisms other than alterations in ER protein should be considered when interpreting the effects of prazosin on the retention of estradiol by nuclear or cytosolic extracts of hypothalamic lysates.

Sex differences in nuclear androgen receptors in guinea pig brain and the effects of an alpha 2 noradrenergic blocker on androgen receptors.

The binding of the synthetic androgen, [3H]methyltrienolone (R1881) to nuclear androgen receptors (NAR) was studied in various brain areas of gonadectomized male and female guinea pigs treated for 3 days with 2 mg testosterone propionate. The Scatchard analysis of salt-extracted NAR showed a single, high-affinity receptor with a Kd of 0.152 nM and maximum binding sites (Bmax) of 161.9 fmol/mg DNA. The concentration of NAR was highest in the hypothalamus (HYPO) and preoptic area (POA) in both males and females. Lower receptor levels were detected in the amygdala and cortex. NAR levels were significantly lower in the POA of females than in males. Systemic injection of prazosin, an alpha 1-adrenergic antagonist had no effect on NAR concentrations, but an alpha 2-antagonist, idazoxan, significantly reduced the binding of [3H]R1881 to NAR in both HYPO and POA. The reduction in binding of the ligand to receptor was not due to alterations in affinity of NAR, but rather to the decline in the number of receptors.

Progestin receptors in the ventromedial nucleus of the hypothalamus and arcuate nucleus-median eminence are decreased by idazoxan.

Steroid-dependent lordosis behavior in ovariectomized (OVX) guinea pigs is attenuated by alpha 1- and/or alpha 2-noradrenergic (NE) receptor antagonists. Correlated with the decrease in lordosis after alpha 1-NE receptor blockade by prazosin is a decrease in 'cytosol' progestin receptors in the ventromedial hypothalamic nucleus (VMN). We examined whether a presumed alpha 2-NE receptor blocker (idazoxan, IDA) also affects progestin receptors. A decrease in 'cytosol' progestin receptors was found after IDA treatment of OVX, estrogen-treated guinea pigs in the VMN and the arcuate nucleus-median eminence (ARC-ME). Apparently, either prazosin or IDA can inhibit lordosis behavior and decrease 'cytosol' progestin receptors in the VMN. In contrast, idazoxan but not prazosin, decrease 'cytosol' progestin receptors in the ARC-ME.

Alpha 1-noradrenergic regulation of hypothalamic progestin receptors and guinea pig lordosis behavior.

Experiments were conducted to determine whether alpha 1- or alpha 2-receptors mediate noradrenergic (NA) regulation of guinea pig lordosis behavior and hypothalamic progestin receptors. When infused into a lateral cerebroventricle at a dose that inhibits lordosis and that decreases the concentration of estradiol-inducible hypothalamic progestin receptors, phenoxybenzamine decreased binding of the alpha 1-ligand [3H]WB4101 but not the alpha 2-ligand [3H]clonidine to brain membranes. Thus, under the conditions used, phenoxybenzamine appears to block alpha 1-receptors with little or no effect on alpha 2-receptors. Experiments with the selective alpha 1-antagonist prazosin also indicated alpha 1-receptor regulation of lordosis and hypothalamic progestin receptors. Prazosin inhibited lordosis induced by estradiol benzoate (EB) plus progesterone and by EB + clonidine and decreased the concentration of cytoplasmic progestin receptors in hypothalamus (but not in preoptic area or frontal cortex) of EB-primed females. The inhibition of lordosis is apparently not due to some unknown side effect of prazosin because pretreatment with a high dose of clonidine attenuated the inhibition. The possibility that a causal relationship exists between effects of alpha 1-NA transmission on hypothalamic progestin receptors and lordosis was discussed. Also, because effects of NA transmission on hypothalamic progestin receptors are dependent on prior treatment with EB, it was suggested that NA transmission might influence estradiol action in addition to progestin action in hypothalamic cells.

Development of steroid-receptor systems in guinea pig brain. II. Cytoplasmic progestin receptors.

The development of the cytoplasmic progestin receptor (CPR) system in male and female guinea pigs was examined to determine if insensitivity to the lordosis promoting activity of estrogen-progestin treatment in adult males and neonatal guinea pigs is correlated with alterations in this receptor system. Gonadectomized neonatal (4-6 days old) and adult (50-65 days old) guinea pigs of both sexes were injected with estradiol benzoate (EB) and killed 40 h later. CPRs were measured in the hypothalamus (HYPO), preoptic area (POA) and cortex ( CORT ). Neonatal brain contained both high (Kd congruent to 0.1 nM) and low (Kd greater than 10 nM) affinity binders for [3H]R5020 similar to those found in adult brain. In the absence of EB-priming the concentration of CPR in HYPO, POA and CORT was lower in neonatal animals than adults. In both males and females, neither a low (1.6 micrograms) nor a high (10 micrograms) dose of EB resulted in a concentration of estrogen inducible CPR in neonatal HYPO greater than that found in adult HYPO after 1.6 micrograms EB. Even after treatment with 50 micrograms EB the concentration of CPR in neonatal female HYPO was not different from adult female HYPO after 1.6 micrograms EB. In neonatal POA, 10 micrograms EB resulted in a concentration of CPR greater than that found in adult POA after 1.6 micrograms EB. Finally, there was a sex difference in the concentration of estrogen inducible CPR in HYPO (female greater than male) but not POA of adult guinea pigs. No sex difference in CPR concentration was found in any neonatal brain area.

Development of steroid-receptor systems in guinea pig brain. III. Nuclear progestin receptors.

Nuclear progestin receptors (NPR) were measured, with an exchange assay, in neonatal (4-6 days old) and adult (50-60 days old) guinea pigs of both sexes in order to further examine deficits in the neonatal hypothalamic progestin receptor system. Both neonatal and adult hypothalamus (HYPO) and cortex ( CORT ) contained a KCl extractable high affinity (Kd congruent to 0.1 nM) binder for R5020. In neonatal guinea pigs, given 10 micrograms estradiol benzoate (EB), NPR accumulation in HYPO and CORT peaked 2-4 h after a 0.5 mg injection of progesterone (P). There was no dose response of NPR to EB in neonatal HYPO, CORT or adult CORT . In adult HYPO, 10 micrograms EB-priming resulted in a higher concentration of NPR than 1.6 micrograms EB-priming. Neonatal HYPO (1.6 or 10 micrograms EB) did not differ in concentration of NPR from adult HYPO (1.6 micrograms EB). Neonatal CORT (1.6 or 10 micrograms EB) showed a lower NPR concentration than adult CORT (1.6 micrograms EB). There was a sex difference (females greater than males) in NPR concentration in both neonatal and adult HYPO, but not CORT . These results are discussed with regard to deficits in cytoplasmic progestin receptors and behavioral insensitivity to estrogen-progestin treatment found in neonatal guinea pigs.

Development of steroid-receptor systems in guinea pig brain. I. Cytoplasmic estrogen receptors.

The development of the cytoplasmic estrogen receptor (CER) system in male and female guinea pigs was examined to determine whether attainment of sensitivity to estrogen action related to lordosis behavior is correlated with increases in brain CER concentrations. Ovariectomized neonatal (7 days old) guinea pigs failed to display lordosis after priming with 10 micrograms estradiol benzoate (EB) and 0.5 mg progesterone (P). The percentage of ovariectomized steroid-treated females displaying lordosis increased at 29-33 days of age. At 50-65 days of age this percentage increased even further. Plasma levels of estrogen in 4-6 day old animals at 4, 8 and 12 h after 10 micrograms EB were at least equal to those seen in 50-65 day old animals. Neither the concentration nor the affinity of CER in hypothalamus (HYPO), preoptic area (POA), amygdala (AMYG) and cortex ( CORT ) were different in 4-6 day olds vs 50-65 day olds. The rank order for concentrations of CER in brain regions was: POA greater than HYPO greater than AMYG greater than CORT . A sex difference in CER was found in HYPO (females greater than males), but not POA, AMYG or CORT of 4-6 day olds.

Noradrenergic transmission and female sexual behavior of guinea pigs.

Treatment with the dopamine beta-hydroxylase (DBH) inhibitor U-14,624 (50, 100, or 150 mg/kg) blocked the induction of lordosis behavior be estradiol benzoate (EB) and progesterone (P) in ovariectomized guinea pigs. After treatment with U-14,624 (100 mg/kg), norepinephrine (NE) content of medial basal hypothalamus, preoptic area and cortex was reduced (by 55%) and dopamine (DA) content of medial basal hypothalamus was increased (by 155%) during the period when females treated with EB and P normally display lordosis. Treatment with the NE receptor stimulator clonidine (1.0 mg/kg) restored lordosis behavior in females treated with EB, P, and U-14,624 (100 mg/kg), but the putative DA and serotonin (5-HT) receptor blockers pimozide (1.0 mg/kg) and methysergide (20.0 mg/kg) were ineffective in this respect. Thus, inhibition of lordosis after treatment with U-14,624 appeared to be attributable primarily to a reduction in NE neurotransmission, rather than to increase in DA or 5-HT activity. Because clonidine induced lordosis in females treated with EB, P, and U-14,624, it seemed unlikely that the facilitatory effects of clonidine on lordosis were mediated by activation of presynaptic alpha-adrenergic receptors (i.e. inhibitory NE autoreceptors) rather than by postsynaptic alpha-receptors. In addition, pretreatment with the postsynaptic alpha-adrenergic antagonist phenoxybenzamine (20.0 mg/kg) blocked the facilitation of lordosis by clonidine (1.0 mg/kg) in females primed with EB alone and with EB plus P. Thus, the facilitatory effects of clonidine on lordosis appear to be mediated by activation of postsynaptic alpha-adrenergic (i.e. NE) receptors. The results of this study provide further evidence that NE neurotransmission facilitates the expression of female sexual behavior in guinea pigs.

Cytoplasmic progestin-receptors in guinea pig brain: characteristics and relationship to the induction of sexual behavior.

The synthetic progestin, R 5020, was used to measure cytoplasmic progestin receptors in the brain and pituitary gland of ovariectomized guinea pigs. Progestin receptors with a dissociation constant of 0.1--0.3 nM were measured by gel filtration in all brain regions studied, pituitary gland and the uterus. The receptor is progestin-specific; biologically potent progestins compete well against [3H]R 5020 for binding, but androgens, glucocorticoids and estrogens do not. The concentration of the cytoplasmic progestin receptor in hypothalamus-preoptic area-septum and midbrain is decreased in vivo by behaviorally effective doses of progesterone. In the pituitary gland, hypothalamus, preoptic area-septum and midbrain, but not other brain regions, the concentration of progestin receptors increases after estradiol benzoate-priming. The increase in the concentration of cytoplasmic progestin receptors in hypothalamus-preoptic area-septum is dependent on the dose of estradiol benzoate injected. After a single injection of a dose of estradiol benzoate routinely used to facilitate the display of sexual receptivity (1.6 microgram estradiol benzoate/animal), the latency to an increase and subsequent decrease in cytoplasmic progestin receptors in the hypothalamus-preoptic area-septum correlates well with the previously reported time course for progesterone's facilitation of sexual receptivity after estradiol benzoate injection. The experiments are consistent with the notion that brain progestin receptors mediate at least some of the behavioral effects of progesterone.

Characteristics of [3H]prazosin binding to guinea pig brain alpha-adrenergic receptors: effects of estradiol and progesterone.

[3H]Prazosin was found to bind to sites on guinea pig brain membranes with alpha 1-adrenergic receptor characteristics. Treatment of ovariectomized guinea pigs with estradiol benzoate (EB) or EB followed by progesterone (P) did not affect [3H]prazosin binding to membranes from hypothalamus, preoptic area, amygdala or cerebral cortex. When added to the incubation mixture of the assay, estradiol, P, and other steroids decreased [3H]prazosin binding but only at high concentrations. These results do not support the idea that estrogen and progestin influence reproductive physiology through effects on brain alpha 1-receptors, although limitations of the methodology employed do not completely rule out this possibility.

Relation of uptake and metabolism of (1,2,6,7-3H)testosterone to individual differences in sexual behavior in male guinea pigs.

Male guinea pigs were given 3 tests for sexual behavior. Animals that never ejaculated were classified as low activity (LA), animals that ejaculated on one test were classified as medium activity (MA), and animals that ejaculated on two or more tests were classified as high activity (HA). Subsequently, animals from each group were castrated and given an s.c. injection of 43 muCi of [1,2,6,7-3H]testosterone and were killed 0.5, 1, or 4 h after injection. There were no significant differences in uptake or metabolism of radioactive testosterone among LA, MA, and HA males in homogenates of anterior and posterior hypothalamus, cerebral cortex, midbrain, or seminal vesicle. Thus, differences in sexual behavior could not be attributed to differences in testosterone uptake in tissue homogenates. At the 1 h time interval (time of peak plasma radioactivity), radioactivity in the seminal vesicles of all males was primarily in the form of steroids with the chromatographic mobility of dihydrotestosterone. In all males, anterior and posterior hypothalamus contained a higher proportion of steroids with the mobility of testosterone than did midbrain, and midbrain contained more testosterone zone radioactivity than cerebral cortex at 1 h. The highest proportion of dihydrotestosterone zone radioactivity in neural tissues was found in anterior hypothalamus. These results are discussed in terms of androgenic mediation of sex behavior by the anterior hypothalamus in guinea pigs.

Estradiol and progesterone influence L-5-hydroxytryptophan-induced myoclonus in male guinea pigs: sex differences in serotonin-steroid interactions.

The effects of castration in males and sex differences in the effects of estradiol and progesterone on L-5-hydroxytryptophan (L-5-HTP)-induced myoclonus in guinea pigs were examined. Castration had no effect on L-5-HTP-induced myoclonus in males. There were sex differences in sensitivity to L-5-HTP. In the absence of steroids, L-5-HTP-induced myoclonus was higher in gonadectomized males than females. A low dose of estradiol benzoate (EB; 3.5 micrograms) given 46 h before L-5-HTP (100 mg/kg) enhanced myoclonus in gonadectomized females but not males. However, at a higher dose of EB (10 micrograms) and a lower dose of L-5-HTP (80 mg/kg), myoclonic responding was enhanced in males. These findings indicate that estradiol has a similar effect on L-5-HTP-induced myoclonus in males and females, but do not rule out the possibility of sex differences in sensitivity to L-5-HTP when both sexes are given estradiol priming. When L-5-HTP was given 6 h after 0.5 mg progesterone in estradiol-primed males, myoclonus was enhanced. Progesterone treatment reverses the facilitative effect of EB on L-5-HTP-induced myoclonus in females. Therefore, progesterone has opposite effects on L-5-HTP-induced myoclonus in males and females. These findings were discussed with respect to the interaction of steroids and 5-HT transmission in the regulation of steroid-dependent reproductive behavior.

Estradiol and progesterone influence a serotonin mediated behavioral syndrome (myoclonus) in female guinea pigs: comparison with steroid effects on reproductive behavior.

L-5-hydroxytryptophan (L-5-HTP)-induced myoclonus was used as a behavioral index of central serotonergic activity. Estradiol benzoate (EB) and progesterone (P) influenced the induction of myoclonus by L-5-HTP. When L-5-HTP was injected 46 h after EB, myoclonus was enhanced. P blocked this effect on EB when 100 or 125 mg/kg L-5-HTP (but not 80 mg/kg) was given 6 h after P in EB-primed animals. When L-5-HTP was given 3 or 11-15 h after P in EB-primed animals, there was no inhibitory effect of P on myoclonus. In fact, at the lowest dose (80 mg/kg), L-5-HTP increased myoclonus when given 3 h after P in EB-primed animals. The inhibitory effects of P in EB-primed females on myoclonus were temporally correlated with the display of lordosis, suggesting that the neural progestin receptor mechanisms that have been proposed to mediate P effects on lordosis are also involved in the inhibitory effects of P on myoclonus.

Uptake of (6,7-3H)estradiol-17beta in ovariectomized rats, guinea pigs, and hamsters: correlation with species differences in behavioral responsiveness to estradiol.

PIP: The amount of estradiol benzoate with progesterone required to induce lordosis in ovariectomized hamsters was determined to compare the responsiveness of hamsters to estradiol benzoate with that of rats and guinea pigs. In addition, the uptake and metabolism of tritiated estradiol in ovariectomized rats, guinea pigs, and hamsters was examined in an attempt to correlate species differences in behavioral sensitivity to estradiol with possible differences in neural affinity for the steroid. A dose of nearly 90 mg/kg was required to induce lordosis in 100% of the hamsters compared with the 2-5 mcg/kg which is effective in rats and guinea pigs. In all 3 species, highest uptake of estradiol was in the uterus and anterior pituitary gland. In the rat and guinea pig brains, the hypothalamus took up more estradiol than either the cortex or midbrain. In the hamster, there were no consistent differences in brain uptake. The affinity of the uterus, anterior pituitary, and hypothalamus of rats and guinea pigs for estradiol was greater than that of hamsters. In all 3 species, estrone was the principal metabolite of estradiol found in the tissues. The authors suggest that the higher the endogenous levels of a steroid, the less sensitive the animal is to that steroid.^ieng

Cytosol androgen receptors in guinea pig brain and pituitary.

Cytosol androgen receptors were assayed in guinea pig brain and pituitary tissues, using [3H]R1881 as ligand. These receptors had an apparent Kd of 0.04 nM and were androgen-specific (R1881 greater than dihydrotestosterone greater than testosterone = estradiol greater than progesterone). Concentrations of cytosol androgen receptors in castrated adult male guinea pigs were 12.2, 11.6, 6.9, 2.6 and 1.3 fmol per mg protein in anterior pituitary, hypothalamus, medial preoptic area, amygdala and cortex, respectively. No significant differences in receptor levels were observed between castrated adult males and females. The concentration of androgen receptors was significantly lower in the hypothalamus, medial preoptic area and anterior pituitary of castrated neonatal males than in castrated adult male guinea pigs. The systemic injection of the alpha 1-adrenergic antagonist, prazosin, had no significant influence on androgen receptor levels in castrated males in any brain area.

Sex differences in cytosolic progestin receptors in microdissected regions of the hypothalamus/preoptic area of guinea pigs.

Cytosolic progestin receptors (CPRs) were measured in microdissected nuclei of the hypothalamus and preoptic area of male and female guinea pigs. Adult gonadectomized animals were given 3 daily injections of 20 micrograms/day estradiol benzoate (EB) or oil vehicle. 24 h later, animals were sacrificed and cytosolic progestin receptors were measured using the synthetic progestin 3H-R5020. CPR levels did not differ significantly between oil treated males and oil treated females in any brain areas examined. With EB treatment, males showed significant increases in CPRs in most of the brain areas in which females showed increases, i.e. in the medial preoptic area, the periventricular part of the preoptic area, the periventricular part of the anterior hypothalamus, the ventromedial nucleus of the hypothalamus, the periventricular part of the medial hypothalamus and the arcuate-median eminence. However, EB treated males showed significantly lower CPR levels than EB treated females in both the periventricular part of the preoptic area and the periventricular part of the medial hypothalamus.