Identification of radioactivity in cell nuclei from brain, pituitary gland and genital tract of male rhesus monkeys after the administration of [3H]testosterone

Enzymes are present in the primate brain that convert testosterone into 17 beta-hydroxy-5 alpha-androstan-3-one (dihydrotestosterone), estradiol-17 beta and 4-androstene-3,17-dione. To identify the metabolites of testosterone that accumulate in cell nuclei obtained from different regions of the brain, 9 adult castrated male rhesus monkeys were injected with 5 mCi [3H]testosterone as an intravenous bolus. After 1 h, brains were rapidly removed and the left halves were used for autoradiography while the right halves were dissected to provide 14 samples. Radioactive metabolites in cell nuclei were identified by high-performance liquid chromatography (HPLC) and by repeated recrystallization. In autoradiograms of brain, most of the labeled neurons were in the hypothalamus, preoptic area and amygdala. These three regions also had the highest levels of radioactivity. The major form of this radioactivity was [3H]estradiol-17 beta (Type I tissues) and the major radioactive androgen present was [3H]testosterone. In all other brain regions and pituitary gland, the major form of radioactivity was unchanged [3H]testosterone (Type II tissues). In genital tract structures, [3H]dihydrotestosterone predominated (Type III tissues). These results suggested that, in contrast to its actions on genital tract structures, testosterone acts on neuronal nuclei mainly in unmetabolized form or after conversion to estradiol-17 beta.     

Autoradiographic localization of 3Hdihyrotestosterone in the preoptic area,hypothalamus, and amygdala of a male rhesus monkey

In a preliminary study, autoradiography was used to localize target cells for ³Hdihydrotestosterone (DHT), a non-aromatizable androgen, in the brain of the rhesus monkey. One castrated male was injected intravenously with 2 mCi of ³HDHT (0.42 μg/kg), and was killed one hour later. Neurons that concentrated radioactivity in their nuclei were located in widespread areas of the brain, which included the medial and suprachiasmatic preoptic nuclei, bed nucleus of the stria terminalis, lateral septal nucleus, anterior hypothalamic area, ventromedial, arcuate, or dorsemedial, and paraventricular hypothalamic nuclei, ventral premammillary nucleus, and medial, cortical, basal accessory, and lateral amygdaloid nuclei. These results indicate that the topographic distribution of androgen target neurons is considerably wider than that observed in a study using ³Htestosterone (T) in the male rhesus monkey (1). However, further work is needed to elucidate these differences before attempting correlations between behavioral activity and androgen receptors in the brain.     

Pre-optic and hypothalamic neurons accumulate [3H]medroxyprogesterone acetate in male cynomolgus monkeys

Medroxyprogesterone acetate (MPA) is a synthetic progestin that is reported to be effective in the treatment of paraphilic behavior, including paraphilic aggression, in men. The mechanisms and sites of action for its behavioral effects are not known. Thaw-mount autoradiography was used to help identify sites in the brain at which MPA may act in a male primate. Two adult, castrated male cynomolgus monkeys were administered [3H]MPA and killed one hour later. Radioactivity was concentrated in the nuclei of many neurons in the medial preoptic nucleus (n.), anterior hypothalamic area, ventromedial hypothalamic n., and arcuate n. Virtually no labeled cells were observed in the bed n. of the stria terminalis, lateral septal n., or amygdala. Analysis by high performance liquid chromatography of brain samples from the same animals demonstrated that 84% of the extractable radioactivity in cell nuclei from the hypothalamus and preoptic area was in the form of unmetabolized [3H]MPA. The localization of MPA-concentrating neurons in regions of the brain known to be implicated in regulating both sexual behavior and pituitary function suggests that, among other sites of action, MPA may act directly upon the brain.     

Medroxyprogesterone acetate and the nuclear uptake of testosterone and its metabolites by brain, pituitary gland and genital tract in male cynomolgus monkeys.

The synthetic progestin, medroxyprogesterone acetate (MPA), is used to treat male sex offenders, and it is also suppresses sexual activity in male monkeys. To examine the possibility that MPA may act as an anti-androgen in the primate brain, 4 intact male cynomolgus monkeys were given MPA (40 mg i.m.) once a week for 16 weeks, while 4 control males received i.m. injections of vehicle. All males were then castrated and 3 days later were given 3 mCi [3H]testosterone ([3H]T) i.v.; 1 h after injection males were killed, and radioactivity in nuclear pellets obtained from the hypothalamus (HYP), preoptic area (POA), amygdala (AMG), septum, pituitary gland and genital tract was analyzed by HPLC. Concentrations of [3H]T and [3H]dihydrotestosterone in nuclear pellets were 65-96% lower in MPA-treated males than in controls (P less than 0.001), but the aromatized metabolite, [3H]estradiol, which was the major form of radioactivity present in nuclear pellets from HYP, POA and AMG, was unchanged. There were no differences in concentrations of [3H]T in supernatants from the tissues of MPA-treated and control males. Because the reduced nuclear uptake of androgen in brain occurred in males whose androgen-dependent behavior had been suppressed by MPA treatments, it is proposed that MPA may have anti-androgenic effects at the level of the cell nucleus in brain regions that control behavior.     

News from the molecular frontier. Review of Paternity in Primates: Genetic Tests and Theories R. D. Martin,A. F. Dixson,E. J. Wickings eds. Karger, 1992, xi + 287 pp, $198.50

We have previously demonstrated that the exogenous administration of estradiol-17β (E₂) to rhesus monkeys induces atresia of the dominant preovulatory follicle (DF); and that this effect is mediated centrally, via the inhibition of follicle-stimulating hormone, and is also exerted directly at the level of the ovarian granulosa cell. We wished to investigate whether the local effect of E₂ is transduced through interaction with the nuclear receptor for estrogen, particularly in light of certain evidence that suggests a general lack of estrogen receptor (E-R) in the rhesus monkey ovary, except in the germinal epithelium. In the present study, we evaluated the presence of E-R by both autoradiographic and immunocytochemical techniques. Frozen sections of ovaries from rhesus females were incubated in experiment 1 with either ³H-E₂ or ¹²⁵I-E₂, in the presence or absence of excess, non-radioactive ligand or analogues diethylstilbestrol (DES) or the receptor antagonist 4-OH-tamoxifen (TAM). ³H-E₂ binding was most intense over functional corpora lutea, and was reduced to background with excess DES; label was also evident over antral follicles, Image analysis showed specific binding of ¹²⁵I-E₂ by ovaries. In experiment 2, cryostat sections were processed for immunocytochemical staining using the per-oxidase-anti-peroxidase (PAP) method and the H222 monoclonal antibody to the E-R. Intense, specific label was observed over nuclei of germinal epithelium, but, additionally, for the first time, to granulosa cells of antral follicles and other compartments of the ovary. In this paper, we report the first evidence for estrogen binding to rhesus monkey ovary; tins binding is specific, apparently receptor mediated, and corroborated independently by autoradiographic and immunocytochemical means. We herein provide substantial support for estrogen's dramatic effects being exerted directly at the level of the monkey ovary. © 1993 Wiley-Liss, Inc.     

Cellular observations and hormonal correlates of feedback control of luteinizing hormone secretion by testosterone in long-term castrated male rhesus monkeys

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 (P450(AROM) 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. P450(AROM) 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 P450(AROM) mRNA did not change in group 2 males, we hypothesize that an estrogen-dependent neural deficit, not involving the regulation of the P450(AROM) mRNA, occurs in long-term castrated monkeys.     

Neuroanatomical distribution of aromatase mRNA in the rat brain: indications of regional regulation

Aromatase, the enzyme responsible for the conversion of testosterone to estradiol, is found in the rat brain and is present in regions of the preoptic area, hypothalamus, and limbic system. Gonadal steroid hormones regulate aromatase activity levels in many brain regions, but not all. Using in situ hybridization, we examined the distribution of aromatase mRNA in the adult male forebrain, as well as the levels of aromatase mRNA in the brains of males and females, and the regulation by gonadal steroid hormones. In the adult male, many heavily labelled cells were found in the encapsulated bed nucleus of the stria terminalis (BNST), the medial preoptic nucleus (MPN), the ventro-medial nucleus (VMN), the medial amygdala (mAMY) and the cortical amygdala (CoAMY). The regional distribution of aromatase mRNA was similar in males and females, but males tended to have a greater number of aromatase mRNA-expressing cells in each region compared to females. Aromatase mRNA levels in the BNST, MPN, VMN and mAMY tended to be lower in castrated males than in intact males, whereas aromatase mRNA levels were unaltered by castration in the CoAMY. Further analysis of individual cells expressing aromatase mRNA suggests that aromatase mRNA may be regulated by steroid hormones differentially in specific populations of cells in regions where enzyme activity levels are steroid-hormone-dependent.     

Localization and mechanism of stimulatory feedback action of estrogen: effect of limbic forebrain implantation of estradiol benzoate on advancement of ovulation.

The sites and mechanism of the ovulation-inducing action of estradiol benzoate (EB) were studied by brain implantation of the crystalline steroid through chronically implanted outer cannula at 12:00 on diestrus day 2 in the 5-day cyclic rat. EB implantation in the medial amygdala or the bed nucleus of stria terminalis advanced cyclic changes in vaginal smears, timing of ovulatory LH release, and ovulation by 1 day, resulting in 4-day cycle. When implants in the bed nucleus of stria terminalis were placed for a shorter period of time on diestrus day 2, from 12:00 to 20:00, advancement of these parameters were similarly observed. Serum concentration of FSH and that of prolactin were significantly elevated at 20:00 on the same day in the rats implanted with EB in the medial amygdala or the bed nucleus of stria terminalis, compared with those in the non-treated controls. LH was not affected. The implantation in the arcuate nucleus was also effective to advance ovulation, but the anterior deafferentation prevented the effect. In contrast, EB implantation in the medial septal nucleus, the medial preoptic area, or the medial basal prechiasmatic area was consistently ineffective to advance vaginal cycle and ovulation. Multiunit activity in the arcuate nucleus showed an afternoon elevation on the day of implantation in these areas and as well on the day following, while it did not show such elevation on the day of implantation in the medial preoptic area. It is concluded that EB acts on the medial amygdala and the bed nucleus of stria terminalis in the mid-diestrus in 5-day cycle to stimulate FSH and prolactin release without affecting LH, which changes trigger a chain of reproductive events inducing early release of ovarian steroid responsible for early ovulatory gonadotropin release. The arcuate nucleus in one of the sites of stimulatory action of estrogen, but it requires the neural influence presumably from the medial amygdala and the bed nucleus of stria terminalis via the preoptic area for stimulating the ovulatory hormone release. EB exposure is considered to be endowed with the increase of its responsibility to this neural influence.     

Cellular and subcellular localization of 3H-diethylstilboestrol in the central nervous system

Summary The cellular and subcellular localization of radioactivity in the brain of immature female rats was determined by dry-mount autoradiography 2 h after iv injection of 1.0 g of (monethyl-³H) diethylstilboestrol per 100 g body weight. A specific topographic pattern of nuclear concentration of the synthetic oestrogen was obtained similar to that for ³H-oestradiol-17 in specific neurons of the basal hypothalamus, preoptic region and amygdala. In competition experiments, the nuclear concentration of radioactivity in all areas studied was inhibited by unlabeled oestradiol, while unlabeled testosterone had no effect. These data suggest that although oestradiol can bind to androgen receptors, the oestrogen receptor itself can account for the localization seen after the injection of ³H-oestradiol.This research was supported in part by US PHS Grant No. NS12933NIH Career Development Awardee No. NS00164The expert technical assistance of Ms. Riki Ison and Ms. Linda Furr is gratefully acknowledged.     

Effect of testosterone and season on proenkephalin messenger RNA expression in the preoptic area of the hypothalamus in the ram

Enkephalin appears to exert an inhibitory action on LH secretion, but whether testosterone regulates enkephalin gene expression is unknown. This study tested the hypothesis that testosterone and/or season modulate preproenkephalin mRNA expression in specific areas of the hypothalamus. Romney Marsh rams were castrated (wethers) either during the breeding season or nonbreeding season and received intramuscular injections of either oil or testosterone propionate (five/group). Blood samples were taken for the assay of plasma LH and testosterone. Preproenkephalin mRNA expression was quantified in hypothalamic sections by in situ hybridization. Mean plasma LH concentrations were reduced and the interpulse interval for LH pulses was greater in testosterone propionate-treated wethers compared with oil-treated wethers, with no change in LH pulse amplitude. Testosterone propionate treatment reduced proenkephalin expression in the diagonal band of Broca, the caudal preoptic area, and the bed nucleus of the stria terminalis. Seasonal differences in proenkephalin expression were observed in the bed nucleus of the stria terminalis, lateral septum, periventricular nucleus, and paraventricular nucleus. No differences were observed between treatments in seven other regions examined. We conclude that testosterone and season regulate proenkephalin mRNA levels in the preoptic area/hypothalamus in the ram in a region-specific manner.     

Neurons in the brain of fetal rhesus monkeys accumulate 3H-testosterone or its metabolites

Autoradiography was used to map sites in the primate brain at which testosterone may have sexual differentiating actions on brain function and behavior during fetal development. Two female rhesus monkey fetuses were injected in utero on days 112 and 114 of gestation respectively with 3H-testosterone, and were killed 30 and 60 minutes later. Thaw-mount autoradiography of the brains revealed the accumulation of radioactivity, representing 3H-testosterone or its metabolites, in neurons of the medial preoptic-anterior hypothalamic area, bed nucleus (n.) of the stria terminalis, ventromedial hypothalamic n., and corticomedial amygdala. Thus, it appears that steroid receptors are present in a circumscribed system in the brain of the primate fetus at this stage of development.     

Oxytocin immunoreactivity in the hypothalamus of female hamsters

In Syrian hamsters (Mesocricetus auratus), oxytocin (OXT) activity within the medial preoptic-anterior hypothalamus (MPOA-AH) and the ventromedial hypothalamus (VMH) plays an important role in the expression of sexual receptivity. Immunocytochemical analysis with OXT-specific antibodies was used to identify the distribution of OXT-containing cell bodies and fibers in female hamster brain and to determine the possible sources of OXT important for sexual receptivity. Oxytocin-immunoreactive cell bodies and fibers were found in several regions of the preoptic area, including the medial preoptic area, the medial preoptic nucleus, and the bed nucleus of the stria terminalis. Large numbers of cell bodies and fibers were localized within the paraventricular and supraoptic nuclei, and in anterior hypothalamus. OXT-immunoreactive fibers were observed in the VMH and the ventral tegmental area. The anatomical data from the present study support the hypothesis that OXT activity in the MPOA-AH and the VMH plays an important role in the regulation of sexual receptivity in hamsters.     

LHRH-systems in the brain of the golden hamster

Summary Vibra tome sections of male hamster brains were treated immunohistochemically with LHRH antiserum, and the anatomical distribution of LHRH immunoreactive cells and nerve fibers was assessed. LHRH-cell bodies are found in the ventral hypothalamus that includes its preoptic, anterior and central parts, in the septum, the olfactory tubercle, the main and accessory olfactory bulb, and the prepiriform cortex. In addition, extracerebral LHRH-neurons and ganglia exist in LHRH-positive nerves at the ventromedial surface of the olfactory tubercle and bulb as well as in olfactory nerves. Dense networks of LHRH-immunoreactive fibers are found in all regions where LHRH-cell bodies exist. Intraseptal connections reach the organum vasculosum of the lamina terminalis, the subfornical organ, and the lateral ventricle. Dorsolateral projections from the septum can be traced via the fimbria hippocampi and alveus to the ventral hippocampus, via the stria terminalis to the amygdala and piriform cortex. Ventrolateral projections extend from the level of the olfactory tubercle and preoptic-anterior hypothalamic area via the ventral amygdalofugal pathway to the prepiriform and piriform cortex as well as the amygdala. Dorsal supracallosal projections via the stria longitudinalis are seen in the induseum griseum and the cingulate cortex. Caudal efferents reach the habenula, interpeduncular nucleus, midbrain raphe, and central gray of the rostral fourth ventricle via the stria medullaris and fasciculus retroflexus and by a ventral projection via the periventricular and subventricular hypothalamus. A major portion of this ventrocaudal projection gives rise to a dense network in the median eminence. Anatomical relationships of LHRH-fibers to certain regions of the inner ventricular and outer brain surface are noted.Postdoctoral fellow of the Deutsche ForschungsgemeinschaftSupported by US PHS grant NS09914 and NRCHD grant HD03110     

1,25(OH)2 vitamin D3 sites of action in the brain

Summary After injection of ³H 1,25(OH)₂ vitamin D₃ to adult rats and mice, under normal or vitamin D deficient diet, the hormone was found to be accumulated in nuclei of neurons in certain brain regions. Nuclear concentration was prevented or diminished, when excess unlabeled 1,25 (OH)₂ vitamin D₃ was injected before ³H 1,25(OH)₂ vitamin D₃, while excess 25 (OH) vitamin D₃ did not prevent nuclear labeling.Highest nuclear concentration of ³H 1,25 (OH)₂ vitamin D₃ is observed in certain neurons in the nucleus interstitialis striae terminalis, involving its septo-preoptic pars dorsolateralis and its anterior hypothalamic-thalamic portion, and in the nucleus centralis of the amygdala, all constituting a system of target neurons linked by a component of the stria terminalis. Nuclear concentration of ³H 1,25 (OH)₂ vitamin D₃ is also found in neurons in the periventricular nucleus of the preoptic-hypothalamic region, including its extensions, the parvocellular paraventricular and arcuate nucleus, in the ventromedial nucleus, supramammillary nucleus, reticular nucleus of the thalamus, ventral hippocampus, caudate nucleus, pallium, in the midbrain-pontine central gray, dorsal raphe nucleus, parabrachial nuclei, cranial motor nuclei, substantia gelatinosa of the sensory nucleus of the trigeminus, Golgi type II cells of the cerebellum, and others.The extensive distribution of target neurons suggests that 1,25(OH)₂ vitamin D₃ regulates the production of several aminergic and peptidergic messengers, and influences the activity of certain endocrine-autonomic, sensory and motor systems.     

Evidence for an absence of estrogen-concentration by CCK-immunoreactive neurons in the hypothalamus of the female rat

It is becoming increasingly clear that the neuropeptide cholecystokinin (CCK), widely distributed in the rat hypothalamus and limbic system, is subject to both organizational and activational influences of steroid hormones. Sex differences in numbers of CCK-immunoreactive elements have been demonstrated in sexually dimorphic structures such as the bed nucleus of the stria terminalis, medial preoptic nucleus, and ventromedial nucleus of the hypothalamus. Steroid activation of CCK has been indicated by findings that hypothalamic CCK levels and binding capacity vary over the estrous cycle. These studies, in combination with evidence of CCK mediation of sexually differentiated functions, prompted us to test for estrogen concentration among CCK-containing cells of the female rat hypothalamus by combining the techniques of immunohistochemistry and autoradiography. A method employing 2-week ovariectomies and perfusion fixation with 4% paraformaldehyde was compatible with the localization of both estrogen-accumulating and CCK-immunoreactive cell bodies. The maintenance of numbers of CCK-positive cells after gonadectomy suggested that expression of this peptide may not be directly regulated by ovarian steroids in female rats. This suggestion was substantiated by the finding that, with rare exceptions, CCK-immunoreactive cells did not concentrate estrogen in tissues collected from the anterior-posterior extent of the bed nucleus of the stria terminalis, medial preoptic nucleus, anterior hypothalamic area, and paraventricular nucleus.     

Peptide immunoreactive neurons in the amygdala and the bed nucleus of the stria terminalis project to the midbrain central gray in the rat.

The central nucleus of the amygdala, bed nucleus of the stria terminalis, and central gray are important components of the neural circuitry responsible for autonomic and behavioral responses to threatening or stressful stimuli. Neurons of the amygdala and bed nucleus of the stria terminalis that project to the midbrain central gray were tested for the presence of peptide immunoreactivity. To accomplish this aim, a combined immunohistochemical and retrograde tracing technique was used. Maximal retrograde labeling was observed in the amygdala and bed nucleus of the stria terminalis after injections of retrograde tracer into the caudal ventrolateral midbrain central gray. The majority of the retrogradely labeled neurons in the amygdala were located in the medial central nucleus, although many neurons were also observed in the lateral subdivision of the central nucleus. Most of the retrogradely labeled neurons in the BST were located in the ventral and posterior lateral subdivisions, although cells were also observed in most other subdivisions. Retrogradely labeled neurotensin, corticotropin releasing factor (CRF), and somatostatin neurons were mainly observed in the lateral central nucleus and the dorsal lateral BST. Retrogradely labeled substance P-immunoreactive cells were found in the medial central nucleus and the posterior and ventral lateral BST. Enkephalin-immunoreactive retrogradely labeled cells were not observed in the amygdala or bed nucleus of the stria terminalis. A few cells in the hypothalamus (paraventricular and lateral hypothalamic nuclei) that project to the central gray also contained CRF and neurotensin immunoreactivity. The results suggest the amygdala and the bed nucleus of the stria terminalis are a major forebrain source of CRF, neurotensin, somatostatin, and substance P terminals in the midbrain central gray.     

Agonistic encounters and brain activation in dominant and subordinate male greater long-tailed hamsters

During an agonistic encounter test, dominant male greater long-tailed hamsters (Tscheskia triton) initiated attacks sooner and displayed higher levels of aggression and flank marking behavior than their subordinate counterparts. Accordingly, subordinate males exhibited more defensive behavior than dominant ones. Specific patterns of neuronal activation, measured by Fos-immunoreactive staining (Fos-ir), were found in the hamster brain following agonistic interactions. Increased Fos-ir was observed in the bed nucleus of the stria terminalis (BST), ventromedial hypothalamus (VMH), and medial (MeA) and anterior cortical (ACo) nuclei of the amygdala (AMYG) in both dominant and subordinate males. In contrast, dominant males had significantly higher Fos-ir densities in the medial preoptic area (MPOA) than subordinate males, whereas subordinate males expressed higher densities of Fos-ir in the anterior hypothalamus (AH) and central nucleus of the amygdala (CeA). Additionally, Fos-ir levels in the MPOA were significantly correlated with aggression and Fos-ir levels in the AH and CeA were correlated with defensive behavior. Together, our data indicate distinct patterns of neuronal activation associated with agonistic encounters in a behavior-specific manner in male greater long-tailed hamsters.     

Amygdaloid lesion-induced obesity: relation to sexual behavior, olfaction, and the ventromedial hypothalamus

Lesions of the amygdala have long been known to produce hyperphagia and obesity in cats, dogs, and monkeys, but only recently have studies with rats determined that the effective site is the posterodorsal amygdala (PDA)-the posterodorsal medial amygdaloid nucleus and the intra-amygdaloid bed nucleus of the stria terminalis. There is a sex difference; female rats with PDA lesions display greater weight gain than male rats. In the brains of female rats with obesity-inducing PDA lesions, there is a dense pattern of axonal degeneration in the capsule of the ventromedial hypothalamus (VMH) and other targets of the stria terminalis. Transections of the dorsal component of the stria terminalis also result in hyperphagia and obesity in female rats. Similar to rats with VMH lesions, rats with PDA lesions are hyperinsulinemic during food restriction and greatly prefer high-carbohydrate diets. The PDA is also a critical site for some aspects of rodent sexual behavior, particularly those that depend on olfaction, and the pattern of degeneration observed after obesity-inducing PDA lesions is remarkably parallel to the circuit that has been proposed to mediate sexual behavior. Medial amygdaloid lesions disrupt the normal feeding pattern and result in impaired responses to caloric challenges, and there is evidence that these behavioral changes are also due to a disruption of olfactory input. With its input from the olfactory bulbs and connections to the VMH, the PDA may be a nodal point at which olfactory and neuroendocrine stimuli are integrated to affect feeding behavior.