The role of gonadal steroid receptor activation in the restoration of sociosexual behavior in adult male rats

This work tested the hypothesis that gonadal steroid receptor activation was necessary for the restoration of several sociosexual behaviors (such as copulatory behavior, partner preference, 50-kHz vocalizations, and scent marking) in testosterone-treated gonadectomized male rats. Gonadal steroid receptors were blocked by systemic administration of the antiandrogen hydroxyflutamide, the antiestrogen RU 58668, or both antagonists simultaneously in a restoration paradigm. Inhibiting androgen receptors with hydroxyflutamide blocked the restoration of male copulatory behavior, partner preference (time spent with a sexually receptive female over a nonreceptive female), 50-kHz ultrasonic vocalizations, and scent marking. On the other hand, we did not find that blocking estrogen receptors with RU 58668 inhibited the restoration of copulatory behavior or partner preference in testosterone-treated gonadectomized male rats, even though the level of brain nuclear estrogen receptor occupation was significantly reduced to the level found in gonadectomized males. However, the restoration of scent marking and 50-kHz vocalizations were impaired by RU 58668. Blocking both nuclear androgen and estrogen receptors with the two antagonists simultaneously did not have a greater inhibitory effect than treatment with each antagonist alone. Therefore, the activation of nuclear estrogen receptors is necessary for the restoration of some, but not all, sociosexual behaviors, which are also androgen receptor-dependent. Besides nuclear estrogen receptors, there are additional, but unknown, targets of estradiol that play a role in mediating copulatory behavior in adult male rats. Moreover, the signals from multiple gonadal steroid signaling pathways converge in the regulation of some sociosexual behaviors in adult male rats.     

Reentrainment of motor activity and spontaneous neuronal activity in the suprachiasmatic nucleus of Djungarian hamsters

Neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus exhibit a daily rhythm in spontaneous electrical activity. Essentially two methods have been employed to record this circadian rhythm: (1) an in vitro brain slice technique and (2) in vivo multiunit recordings. Reentrainment of a circadian output to a shifted light:dark cycle commonly takes several cycles (depending on the amount of shift) until completed. Such a resetting kinetic has also been shown to be valid for SCN electrical activity if recorded in vivo. In an in vitro slice preparation, however, pharmacologically induced resetting is much faster and lacks transients; that is, a shift is completed within one cycle. This study was designed to probe for the presence of transients in the neuronal activity of the SCN in a brain slice preparation. The authors exposed Djungarian hamsters to an 8-h advanced or delayed light:dark cycle and monitored wheel-running activity during reentrainment. Additional groups of identically treated hamsters were used to record the pattern of spontaneous neuronal activity within the SCN using the brain slice preparation. Neuronal activity exhibited the usual rhythm with high firing rates during the projected day and low firing rates during the projected night. However, following 1 day of exposure to the 8-h advanced light:dark cycle, this rhythm disappeared in 6 of 7 slices. Rhythmicity was still absent following 3 days of exposure to the advanced light:dark cycle (n = 4). By contrast, 3 of 7 slices prepared from hamsters exposed to a delayed light:dark cycle for 3 days exhibited a daily rhythm in electrical activity. Although pharmacological agents reset the in vitro SCN neuronal activity almost instantaneously and in in vivo studies a stable phase relationship to a shifted light:dark cycle occurs gradually over several cycles, the authors did not detect either of these patterns. Such differences in resetting kinetics (e.g., rapid resetting, gradual reentrainment, temporary lack of measurable rhythmicity) may be due to (a) application of a resetting stimulus in vivo versus in vitro, (b) duration of the resetting stimulus, (c) the nature of the resetting stimulus, or (d) the recording technique employed.     

Changes in the content of progesterone receptor isoforms and estrogen receptor alpha in the chick brain during embryonic development

Progesterone and estradiol participate in the regulation of several reproductive functions through interaction with intracellular progesterone receptors (PR) and estrogen receptors (ER), respectively. In this work, we determined PR and ER-alpha isoforms content in the brain of chicks of both sexes on days 8 and 13 of embryonic development as well as on the day of hatching by Western blot analysis. PR isoforms protein content increased during embryonic development in both female and male chick brain. The highest PR isoforms content was observed on the day of hatching in both sexes. Interestingly, PR-A content was higher in the brain of chick males than in that of females on day 8 of embryonic development. PR-A/PR-B ratio was higher in the brain of males than in that of females at all ages. We found two ER-alpha isoforms of 66 and 52 kDa; the content of both isoforms was higher in the brain of females than in that of males on days 8 and 13 of embryonic development. An opposite pattern of ER-alpha isoforms content was observed. In males, ER-alpha content increased during embryonic development whereas in the females it decreased during this process. These results indicate that the content of PR and ER-alpha isoforms is related to the degree of brain development in chicks, and suggest that PR and ER-alpha isoforms should exhibit sexual dimorphism in the brain of chicks during embryonic development.     

Effect of fasting and immobilization stress on estrogen receptor immunoreactivity in the brain in ovariectomized female rats

The present study examined the effect of 48-h fasting and 1-h immobilization on estrogen receptor immunoreactivity in selected hypothalamic areas and the nucleus of the solitary tract (NTS) in ovariectomized rats. Fasting induced an increase in ER-immunoreactive cells in the paraventricular nucleus (PVN), periventricular nucleus (PeVN) and NTS compared with the unfasted control group. Similarly, immobilization caused an increase in ER-positive cells in the same areas, PVN, PeVN and NTS, versus the non-immobilized group. There was no significant increase in the number of ER-immunoreactive cells in the preoptic area (POA), arcuate nucleus (ARC) or ventromedial hypothalamic nucleus (VMH) following fasting and immobilization. Our previous work in ovariectomized rats with estrogen microimplants in the brain revealed that the PVN and A2 region of the NTS are the feedback sites of estrogen in activating the neural pathway to suppress pulsatile LH secretion during 48-h fasting. The result in the food-deprived rats suggests that estrogen modulation of the suppression of LH secretion during fasting is partly due to the increase in estrogen receptors in the PVN and A2 region. The physiological significance of the increase in neural ER following immobilization remains to be elucidated.     

Steroid hormones and their receptors in the brain

Steroid hormones regulate several important functions of the brain by altering the expression of particular genes through their receptors. First in this paper the localization of glucocorticoid receptor immunoreactivity and mRNA in the brain was examined. Second biphasic effects of glucocorticoid on the hippocampus was described and particular emphasis was given on the apoptosis. Third the significance of estrogen receptor in the sexually dimorphic areas was discussed. These results suggest that steroids modulate the gene expression along with the alteration of cell structures in a different manner in a tissue-specific pattern.     

Creatine kinase-B mRNA levels in brain regions from male and female rats

The creatine kinase-B (CKB) enzyme is proposed to have a pivotal role in the regeneration of ATP in the nervous system. In the present study, the steady-state levels of CKB mRNA were determined by RNase protection assay in seventeen separate brain regions obtained from rats during the initial interval of the light period or period of inactivity in rats. The antisense probe used specifically hybridizes to CKB mRNA and discriminates CKB from CKM mRNA. The results show that brain regions from Wistar rats differ in CKB mRNA content. Highest levels of CKB mRNA were detected in the male and female cerebellum. High levels of CKB mRNA were observed in the spinal cord, brain stem and its structures (medulla, pons and midbrain) and olfactory bulb of the male rats. Female rats also contained high levels of CKB mRNA in the brain stem. In both male and female rats, the frontal cortex, occipital cortex, hippocampus and striatum exhibited lower levels of CKB mRNA relative to the complete brain. Statistical analyses demonstrated a significant difference between the male and female CKB mRNA profiles. However, CKB mRNA levels in brain regions with estrogen receptors (hypothalamus, hippocampus) were similar in male and female rats. Differential CKB mRNA levels in various brain regions may suggest diverse physiological significance of the CKB system in the regulation of brain energy metabolism.     

Corneal asphericity in eye bank eyes implanted with the intrastromal corneal ring

The endogenous circadian rhythm of melatonin in humans provides information regarding the resetting response of the human circadian timing system to changes in the light-dark (LD) cycle. Alterations in the LD cycle have both acute and chronic effects on the observed melatonin rhythm. Investigations to date have firmly established that the melatonin rhythm can be reentrained following an inversion of the LD cycle. Exposure to bright light and darkness given over a series of days can rapidly induce large-magnitude phase shifts of the melatonin rhythm. Even single pulses of bright light can shift the timing of the melatonin rhythm. Recent data have demonstrated that lower light intensities than originally believed are capable of resetting the melatonin rhythm and that stimulation of photopically sensitive photoreceptors (i.e., cones) is sufficient to reset the endogenous circadian melatonin rhythm. In addition to phase resetting, exposure to light of critical timing, strength, and duration can attenuate the amplitude of the endogenous circadian rhythm of melatonin. Measurement of melatonin throughout resetting trials provides a dynamic view of the resetting response of the human circadian pacemaker to light. Future studies of the melatonin rhythm in humans may further characterize the resetting response of the human circadian timing system to light.     

Genetic resistance to parasitic infection

Intraventricular administration of carbachol can induce phase shifts in wheel-running activity in rodents, which depend on circadian phase and are mediated via muscarinic cholinergic receptors in Syrian hamsters. We studied the circadian variation in binding of [3H]-N-methylscopolamine ([3H]NMS), a hydrophilic muscarinic receptor antagonist, in micropunches obtained from the anterior hypothalamus and occipital cortex of Syrian hamsters housed in a 14:10 light:dark cycle. Binding sites were characterized on cells contained within 1 mm punches (obtained from slices 300 microm thick), using a method to selectively detect cell surface (functional) receptors. Atropine sulphate was used to determine nonspecific binding. Cortex showed a significant daily rhythm in [3H]NMS binding with a peak occurring late in the light phase and a trough at lights on, while the hypothalamus showed no detectable rhythm. Following suprachiasmatic nucleus (SCN) ablation or maintenance in constant darkness, the rhythm in the cortex was abolished. These findings suggest that photic information conveyed via the SCN is responsible for the receptor binding rhythm in the cortex. Autoradiographic studies ([3H]NMS; 2 nM, 3 weeks exposure) clearly revealed both M1 and M2 subtypes of muscarinic receptors in the region of the SCN and the visual cortex.     

Regulation mechanism of melatonin rhythm in the pineal gland by light: experimental studies by in vivo microdialysis

Light has dual effects on the pineal melatonin; one is the entrainment of the circadian rhythm and the other is suppression of the melatonin synthesis. It is not known whether the entraining and suppressing effects of light are mediated by the same pathway or not. To elucidate the mechanism of the dual effects of light, (1) the sensitivity of the retina, (2) effects of acetylcholine agonist and, (3) the arrhythmicity induced by longterm continuous light, were studied by measuring melatonin continuously from a single rat by means of in vivo microdialysis. Pineal melatonin was suppressed by light more strongly at the late dark phase than at midnight, and by green light (520nm) than by red light (660nm). Pineal melatonin measured by microdialysis was decreased rapidly by a short light exposure and the melatonin rhythm was shifted on the following days. Microinjection of cholinergic agonist, carbachol, into the suprachiasmatic nucleus neither suppressed nor entrained the pineal melatonin rhythm. Immediately after the blinding, rats showed the circadian rhythm in pineal melatonin which had been abolished under long-term continuous light. While, it took several days for the locomotor rhythm to reappear. It is concluded that, (1) suppression of the pineal melatonin by light depends on the circadian phase and on the wavelength of light, (2) the threshold for light suppression is lower than that for phase-shift, (3) the melatonin rhythm starts to phase-shift on the following day of light pulse. (4) Acetylcholine is unlikely to be involved in the photic transmission both to the circadian clock and to the pineal, (5) arrhythmicity induced by long-term continuous light seems to be due to masking for the melatonin rhythm, and to uncoupling from the clock for the locomotor rhythm.     

Photic entrainment of the mammalian rhythm in melatonin production

This review summarizes studies on the photic entrainment of the circadian rhythm in the rat pineal melatonin production, namely of the rhythm in N-acetyltransferase (NAT) activity, and compares the NAT rhythm resetting with preliminary results on the resetting of an intrinsic rhythmicity in the suprachiasmatic nucleus (SCN) of the hypothalamus, namely with the entrainment of the rhythm in the light-induced c-fos gene expression. Phase delaying of the NAT rhythm after various light stimuli proceeds within 1 day with almost no transients, whereas during phase advancing of the rhythm only the morning NAT decline is phase advanced within 1 day and the evening rise phase shifts through transients. A light stimulus encompassing the middle of the night may phase delay the evening NAT rise, phase advance the morning decline, compress the rhythm waveform, and eventually lower its amplitude. Similarly, a long photoperiod compresses the NAT rhythm waveform. The magnitude of phase shifts of the NAT rhythm, as well as their direction, depends on a previous photoperiod. Phase shifts of the evening rise in c-fos gene photoinduction in the SCN and of the morning decline are similar to those of the pineal NAT rhythm after all light stimuli studied so far. The data indicate that the resetting of the rhythm in melatonin production in the rat pineal gland reflects changes in the SCN functional state and suggest that the underlying SCN pacemaking system is complex.     

NADPH diaphorase activity and nitric oxide synthase immunoreactivity in lordosis-relevant neurons of the ventromedial hypothalamus

The distribution of the enzymes NADPH diaphorase and nitric oxide synthase in the ventromedial nucleus of the hypothalamus of cycling and ovariectomized/estrogen-treated and control female rats was demonstrated using histochemical and immunocytochemical methods. Serial section analysis of vibratome sections through the entire ventromedial nucleus showed that NADPH diaphorase cellular staining was localized primarily in the ventrolateral subdivision. NADPH diaphorase staining was visible in both neuronal perikarya and processes. Light microscopic immunocytochemistry using affinity-purified polyclonal antibodies to brain nitric oxide synthase revealed a similar pattern of labelling within the ventromedial nucleus and within neurons of the ventrolateral subdivision of the ventromedial nucleus. Control experiments involved omitting the primary antibodies; no labelling was visible under these conditions. Some, but not all, neurons in the ventrolateral subdivision of the ventromedial nucleus contained both NADPH diaphorase and brain nitric oxide synthase as demonstrated by co-localization of these two enzymes in individual cells of this area. That NADPH diaphorase and brain nitric oxide synthase were found in estrogen-binding cells was shown by co-localization of NADPH diaphorase and estrogen receptor and brain nitric oxide synthase and estrogen receptor at the light and ultrastructural levels, respectively. Our studies suggest that brain nitric oxide synthase is present and may be subject to estrogenic influences in lordosis-relevant neurons in the ventrolateral subdivision of the ventromedial nucleus. The hypothalamus is a primary subcortical regulatory center controlling sympathetic function. Therefore, not only is nitric oxide likely to be important for reproductive behavior, but also for the regulation of responses to emotional stress and other autonomic functions.     

Deficits in conditioned avoidance responding following adrenalectomy and central norepinephrine depletion are dependent on postsurgical recovery period and phase of the diurnal cycle.

Four experiments with 284 Wistar rats showed that Ss who had undergone combined dorsal noradrenergic bundle lesion (DNBL) and bilateral adrenalectomy were impaired in acquiring a conditioned avoidance response when tested 1 wk following surgery. Normal acquisition was observed, however, when testing occurred 3 wks or more after surgery, despite low levels of both plasma corticosterone and brain norepinephrine. Neither neonatal systemic administration of 6-hydroxy-dopamine to deplete forebrain norepinephrine, combined with the corticosterone inhibitor metyrapone, nor the pharmacological blockade of noradrenergic receptors, combined with adrenalectomy, disrupted acquisition of the avoidance response. Thus, the combination of forebrain norepinephrine loss and low plasma corticosterone did not inevitably impair avoidance acquisition; rather, the determining factor for such impairment was the interval between surgery and testing. The impairment at 1 wk following DNBL and adrenalectomy occurred only for Ss tested during the dark phase of their light cycle. The DNBL abolished the effect of the light–dark cycle on posttraining plasma corticosterone. Findings demonstrate the importance of the phase of diurnal rhythm on both the hormonal and the behavioral effects of altering the pituitary-adrenal axis and/or forebrain norepinephrine. (45 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Localization of estrogen-receptor-related antigen in human odontoblasts

Estrogen receptors have been demonstrated in many osteogenic cell lines. Recently, we showed that estrogen deficiency induced by ovariectomy caused enhanced dentin formation in adult rats, suggesting that estrogen receptors may be present in dental tissues. Nothing is known about estrogen receptors in human teeth. We used immunohistochemical staining and immuno-blotting to demonstrate the presence of estrogen receptors in human pulp and/or the pulpo-dentinal border. Unerupted human wisdom teeth were surgically removed, frozen in liquid nitrogen, and prepared for immunological studies. Western blot analysis with monoclonal antibodies specific for human estrogen-receptor-related antigens demonstrated an approximately 29-kDa clear double band in the material scraped from the predentin-odontoblast border and in the fluid that emerged into the pulpal chamber, evidently from the odontoblasts. A weaker double band was also present in pulpal tissue samples. By immunohistochemical staining, estrogen-receptor-related antigens were visualized in the predentinal-odontoblast region and in the pulpal blood vessels. Our results suggest the presence of estrogen receptors in human teeth, and thus the previously reported enhancement of the dentin formation in rats after ovariectomy may be mediated via these receptors.     

Paraneoplastic demyelinating disorder in the brain of a patient with seminoma

OBJECTIVE: To evaluate the estrogen and progesterone receptor status of glassy cell carcinoma of the cervix to assess the possible implications of hormone replacement therapy in these patients. METHODS: The estrogen and progesterone receptor status of 13 glassy cell carcinomas was evaluated by immunohistochemistry using commercial monoclonal antibodies and a streptavidin-biotin detection system. RESULTS: No immunohistochemically detectable estrogen or progesterone receptor protein was present in tumor cells, although both receptors were identified in the adjacent normal cervical tissue. CONCLUSION: The absence of estrogen and progesterone receptors in glassy cell carcinoma suggests that this tumor would not be hormonally responsive and that these patients can be safely treated with hormone replacement therapy. Further studies are indicated to confirm this observation.     

The GnRH system of seasonal breeders: anatomy and plasticity

Seasonal breeders, such as sheep and hamsters, by virtue of their annual cycles of reproduction, represent valuable models for the study of plasticity in the adult mammalian neuroendocrine brain. A major factor responsible for the occurrence of seasonal reproductive transitions is a striking change in the responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the inhibitory effects of gonadal steroids. However, the neural circuitry mediating these seasonal changes is still relatively unexplored. In this article, we review recent findings that have begun to define that circuitry and its plasticity in a well-studied seasonal breeder, the ewe. Tract tracing studies and immunocytochemical analyses using Fos and FRAs as markers of activation point to a subset of neuroendocrine GnRH neurons in the MBH as potential mediators of pulsatile GnRH secretion. Because the vast majority of GnRH neurons lack estrogen receptors, seasonal changes in responsiveness to estradiol are most probably conveyed by afferents. Two possible mediators of this influence are dopaminergic cells in the A14/A15 cell groups of the hypothalamus, and estrogen receptor-containing cells in the arcuate nucleus that project to the median eminence. The importance of GnRH afferents in the regulation of season breeding is underscored by observations of seasonal changes in the density of synaptic inputs onto GnRH neurons. Thyroid hormones may participate in this remodeling, because they are important in seasonal reproduction, influence the morphology of other brain systems, and thyroid hormone receptors are expressed within GnRH neurons. Finally, in the hamster, neonatal hypothyroidism affects the number of caudally placed GnRH neurons in the adult brain, suggesting that thyroid hormones may influence development of the GnRH system as well as its reproductive functions in the adult brain.     

Use of computer interview data to test associations between risk factors and pregnancy outcomes

Estrogens are an important class of steroid hormones, involved in the development of brain, skeletal, and soft tissues. These hormones influence adult behaviors, endocrine state, and a host of other physiological functions. Given the recent cloning of a second estrogen receptor (ER) cDNA (the ER beta), work on alternate spliced forms of ER alpha, and the potential for membrane estrogen receptors, an animal with a null background for ER alpha function is invaluable for distinguishing biological responses of estrogens working via the ER alpha protein and those working via another ER protein. Data generated to date, and reviewed here, indicate that there are profound ramifications of the ER alpha disruption on behavior and neuroendocrine function. First, data on plasma levels of estradiol (E2), testosterone (T), and luteinizing hormone (LH) in wild-type (WT) versus ER alpha- mice confirm that ER alpha is essential in females for normal regulation of the hypothalamic-pituitary gonadal axis. Second, ovariectomized female ER alpha- mice do not display sexual receptivity when treated with a hormonal regime of estrogen and progesterone that induces receptivity in WT littermates. Finally, male sexual behaviors are disrupted in ER alpha- animals. Given decades of data on these topics our findings may seem self-evident. However, these data represent the most direct test currently possible of the specific role of the ER alpha protein on behavior and neuroendocrinology. The ER alpha- mouse can be used to ascertain the specific functions of ER alpha, to suggest functions for the other estrogen receptors, and to study indirect effects of ER alpha on behavior via actions on other receptors, neurotransmitters, and neuropeptides.     

Behavioral effects of estrogen receptor gene disruption in male mice

Gonadal steroid hormones regulate sexually dimorphic development of brain functions and behaviors. Their nuclear receptors offer the opportunity to relate molecular events in neurons to simple instinctive mammalian behaviors. We have determined the role of estrogen receptor (ER) activation by endogenous estrogen in the development of male-typical behaviors by the use of transgenic estrogen-receptor-deficient (ERKO) mice. Surprisingly, in spite of the fact that they are infertile, ERKO mice showed normal motivation to mount females but they achieved less intromissions and virtually no ejaculations. Aggressive behaviors were dramatically reduced and male-typical offensive attacks were rarely displayed by ERKO males. Moreover, ER gene disruption demasculinized open-field behaviors. In the brain, despite the evident loss of functional ER protein, the androgen-dependent system appears to be normally present in ERKO mice. Together, these findings indicate that ER gene expression during development plays a major role in the organization of male-typical aggressive and emotional behaviors in addition to simple sexual behaviors.     

Androgen and estrogen receptors in the developing mouse brain

Specific binding of the androgens, 5alpha-dihydrotestosterone (DHT) and testosterone, and of 17beta-estradiol by brain cytosol from mice at 3-5,9-11, and 18-23 days of age was measured by charcoal assay and glycerol gradient centrifugation and analyzed by Scatchard plots. The immature mouse brain contains putative receptors for these steroids which migrate at 8 S in gradients at low ionic strength and at 5 S in 0.5 M KCl. Investigation of estradiol binding was complicated by the presence in cytosol from 3-5 day-old mice, and to a lesser extent from 9-11 day-old mice, of the high capacity, fetoneonatal estradiol binding protein (FEBP) which is no longer detectable at 3 weeks. The rapid dissociation of the FEBP-estradiol complex under non-equilibrium conditions probably led to over-estimation of free steroid concentration and thus to an apparent increase in the affinity of 8 S receptor for estradiol with age (for female brain cytosol KD=9.5 X 10(-10)M at 3-5 days and 2.7 X 10(-10)M at 18-23 days). The number of estradiol binding sites remains relatively constant during the first 3 weeks at 7-9 fmol/mg protein, while the number of DHT binding sites in female brain increases from 3.2+/-0.3 to 6.6+/-0.9 to 9.6+/-0.3 (mean+/-SE) fmol/mg protein in the 3 age groups. Dissociation constants and numbers of sites for both DHT and estradiol binding are similar in brain cytosol from male and female mice. Testosterone and DHT compete for the same binding site, but its affinity for DHT is about twice that for testosterone. The high affinity of the brain receptor for DHT (KD=4-5 X 10(-10)M) may reflect the slow metabolism of DHT to 5alpha-androstanediols, amounting to less than 10% after 2 h at 0 C. Binding of DHT and estradiol to cytosol from brain regions was also investigated. DHT receptors increase in parallel in various regions with age; the concentration of sites in the hypothalamus-preoptic area (HPOA) is 1.2-3.4 times that in the cerebral cortex (C). The concentration of estradiol binding sites in HPOA to that in C increases about 12-fold from neonatal to adult stages, reflecting both an increase in HPOA sites and a decrease in C sites, while the concentration in the remainder of the brain shows little change. Androgen and estrogen receptors in brain cytosol from immature mice can be distinguished by their different specificities and developmental patterns in whole brain and brain regions. The presence and properties of these receptors in the brain of neonatal mice are discussed with respect to their possible role in sexual differentiation of the brain.