Pubertal development of estradiol-induced hypothalamic progesterone synthesis.

In females, a hallmark of puberty is the luteinizing hormone (LH) surge that triggers ovulation. Puberty initiates estrogen positive feedback onto hypothalamic circuits, which underlie the stimulation of gonadotropin releasing hormone (GnRH) neurons. In reproductively mature female rodents, both estradiol (E2) and progesterone (P4) signaling are necessary to stimulate the surge release of GnRH and LH. Estradiol membrane-initiated signaling facilitates progesterone (neuroP) synthesis in hypothalamic astrocytes, which act on E2-induced progesterone receptors (PGR) to stimulate kisspeptin release, thereby activating GnRH release. How the brain changes during puberty to allow estrogen positive feedback remains unknown. In the current study, we hypothesized that a critical step in estrogen positive feedback was the ability for estradiol-induced neuroP synthesis. To test this idea, hypothalamic neuroP levels were measured in groups of prepubertal, pubertal and young adult female Long Evans rats. Steroids were measured with liquid chromatography tandem mass spectrometry (LC-MS/MS). Hypothalamic neuroP increases from pre-puberty to young adulthood in both gonad-intact females and ovariectomized rats treated with E2. The pubertal development of hypothalamic E2-facilitated progesterone synthesis appears to be one of the neural switches facilitating reproductive maturation.

Neurosteroids and female reproduction: estrogen increases 3beta-HSD mRNA and activity in rat hypothalamus.

A central event in mammalian reproduction is the LH surge that induces ovulation and corpus luteum formation. Typically, the LH surge is initiated in ovariectomized rats by sequential treatment with estrogen and progesterone (PROG). The traditional explanation for this paradigm is that estrogen induces PROG receptors (PR) that are activated by exogenous PROG. Recent evidence suggests that whereas exogenous estrogen is necessary, exogenous PROG is not. In ovariectomized-adrenalectomized rats, estrogen treatment increases hypothalamic PROG levels before an LH surge. This estrogen-induced LH surge was blocked by an inhibitor of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD), the proximal enzyme for PROG synthesis. These data indicate that estrogen induces de novo synthesis of PROG from cholesterol in the hypothalamus, which initiates the LH surge. The mechanism(s) by which estrogen up-regulates neuro-PROG is unknown. We investigated whether estrogen increases 1) mRNA levels for several proteins involved in PROG synthesis and/or 2) activity of 3beta-HSD in the hypothalamus. In ovariectomized-adrenalectomized rats, estrogen treatment increased 3beta-HSD mRNA in the hypothalamus, as measured by relative quantitative RT-PCR. The mRNAs for other proteins involved in steroid synthesis (sterol carrier protein 2, steroidogenic acute regulatory protein, and P450 side chain cleavage) were detectable in hypothalamus but not affected by estrogen. In a biochemical assay, estrogen treatment also increased 3beta-HSD activity. These data support the hypothesis that PROG is a neurosteroid, produced locally in the hypothalamus from cholesterol, which functions in the estrogen positive-feedback mechanism driving the LH surge.

Peripubertal ontogeny and estrogen stimulation of cholecystokinin and preproenkephalin mRNA in the rat hypothalamus and limbic system.

The neuropeptide cholecystokinin (CCK) is expressed in limbic system and hypothalamic nuclei that form a circuit that regulates the display of the female rodent reproductive behavior, lordosis. CCK mRNA and peptide levels fluctuate across the estrous cycle and have been shown to be modulated by estrogen exposure. The objective of these experiments was to examine the expression of CCK mRNA during postnatal development of this limbic-hypothalamic, lordosis regulating circuit, and to determine the age at which CCK mRNA expression becomes responsive to estrogen stimulation, by using quantitative in situ hybridization histochemistry. CCK mRNA levels were below the level of detectability within the circuit during the postnatal period, but increased during the peripubertal period. Rats were injected with either estradiol benzoate (EB), EB and progesterone, progesterone, or oil, and were killed 48 hours later on postnatal day (PND) 15, 20, and 25. Alternate brain sections were processed for CCK and preproenkephalin (PPE) mRNA in situ hybridization histochemistry. EB treatment induced CCK mRNA expression in the central portion of the medial preoptic nucleus and posterodorsal medial amygdala at PND 20 and 25, respectively. However, EB treatment increased PPE mRNA levels within the nuclei of the circuit at all ages examined. Progesterone had neither an independent nor additive effect on the EB induction of these neuropeptide messages. The estrogenic induction of CCK mRNA appears to be dependent on estrogen sensitive pathways of neurotransmission, or components of second messenger pathways which regulate CCK mRNA expression in the adult limbic-hypothalamic lordosis regulating circuit, which are not functional before PND 18-25.

Opiate receptors modulate estrogen-induced cholecystokinin and tachykinin but not enkephalin messenger RNA levels in the limbic system and hypothalamus.

Cholecystokinin, substance P and methionine enkephalin all regulate the display of reproductive behaviour. Their expression is exquisitely regulated by estrogen in the limbic-hypothalamic circuit, a circuit that regulates the display of estrogen-sensitive female reproductive behavior. Relatively little is known, however, about the interaction of endogenous opioid peptides with cholecystokinin and substance P in the limbic-hypothalamic circuit. Opiates antagonize the release of cholecystokinin and substance P in the hypothalamus and periaqueductal gray and stimulate cholecystokinin messenger RNA levels in the amygdala. To determine the effect of endogenous opioid input on estrogen-induced cholecystokinin, enkephalin and substance P expression, in situ hybridization histochemistry was used to examine estrogen-induced messenger RNA levels of these neuropeptides in specific nuclei of the limbic system and hypothalamus in the presence of opiate receptor antagonists. Estrogen treatment of ovariectomized rats significantly elevated cholecystokinin messenger RNA levels in the central portion of the medial preoptic nucleus, the encapsulated portion of the bed nucleus of the stria terminalis and the posterodorsal medial amygdala, as well as increased preproenkephalin and preprotachykinin messenger RNA levels in the ventromedial hypothalamic nucleus and the posterodorsal medial amygdala. The universal opiate receptor antagonist naltrexone and the delta-opiate receptor antagonist naltrindole each potentiated the estrogen-induced increase and elevated cholecystokinin messenger RNA levels an additional 1.9- to 2.8-fold depending on the nucleus examined, but had no effect on the estrogen-induced expression of either preproenkephalin or preprotachykinin messenger RNA. beta-Funaltrexamine, a mu-opiate receptor antagonist, had no effect on the medial preoptic or medial amygdaloid cholecystokinin messenger RNA levels or on the estrogen-induced expression of preproenkephalin messenger RNA but did cause a decrease in estrogen-induced cholecystokinin messenger RNA levels in the bed nucleus of the stria terminalis and a decrease in the preprotachykinin messenger RNA levels in the ventromedial hypothalamic nucleus. These results indicate that endogenous opioids, acting on the delta-opiate receptor within nuclei of the limbic-hypothalamic circuit, restrain the estrogen-induced increase of cholecystokinin messenger RNA expression. Activation of the mu-opiate receptor, however, may facilitate cholecystokinin messenger RNA expression in the bed nucleus of the stria terminalis and preprotachykinin messenger RNA expression in the ventromedial hypothalamic nucleus. Thus, endogenous opioid peptides may act in a site- and receptor-specific manner to modulate estrogen-induced neuropeptide levels in the limbic system and hypothalamus.

Estrogen modulation of opioid and cholecystokinin systems in the limbic-hypothalamic circuit.

The display of lordosis behavior has been correlated with the estrogen-induced expression of cholecystokinin (CCK) and enkephalin within the limbic-hypothalamic circuit. These neuropeptides have opposing effects on lordosis; for example, in the medial preoptic nucleus, CCK facilitates and opiates inhibit lordosis. Antisense oligodeoxynucleotide blockade of receptor expression indicated that CCK modulates lordosis in the medial preoptic nucleus through the CCK(A)-receptor. Sequence-specific antibodies directed against delta- and mu-opiate receptor proteins labeled fibers in the medial preoptic nucleus. Estrogen treatment of ovariectomized rats or etorphine (a nonselective opiate agonist) treatment altered the appearance of the immunoreactivity from a diffuse pattern to one of distinctly stained mu-opiate receptor immunoreactive cells and varicose fibers in the medial preoptic nucleus. Such a pattern of staining reflects an internalization of mu-opiate receptors following agonist stimulation. This type of internalization has been used as an indication of synaptic activity. The distribution of receptor internalization surrounds the distribution of CCK cells in the medial preoptic nucleus, suggesting that endogenous opioid peptides may modulate estrogen-induced CCK mRNA expression. Interestingly, nonselective and delta-opiate receptor selective antagonists potentiated the estrogen-induced CCK mRNA expression in the medial preoptic nucleus. Together, these results suggest that endogenous opioid peptides may modulate the estrogenic upregulation of CCK mRNA expression and demonstrate an important level of regulation of gene expression in which synaptic activity modifies hormonal input.

Distribution of choline acetyltransferase mRNA in the efferent vestibular neurons of the chinchilla.

The distribution of choline acetyltransferase messenger RNA (mRNA) among efferent vestibular neurons in the chinchilla was investigated. mRNA coding for choline acetyltransferase, the enzyme that synthesizes acetylcholine, was used as a marker for the cholinergic system. In order to retrogradely label the efferent vestibular neurons, Fluoro-gold was injected through the oval window into the inner ear of anesthetized young male chinchillas (6 to 12 months old). The animals were anesthetized and perfused through the heart 2 days post injection with 4% paraformaldehyde in phosphate buffer. Retrogradely labeled efferent vestibular neurons were mapped in brainstem sections prior to processing for in situ hybridization histochemistry using radiolabeled ribonucleic acid probes complementary to the 3' end of the choline acetyltransferase mRNA. At the levels of the ascending facial nerve and the genu of the facial nerve, we found that approximately 90% of the Fluoro-gold labeled cells in group E1 contained choline acetyltransferase mRNA. All of the group E2 cells that were labeled with Fluoro-gold were found to be cholinergic (contain choline acetyltransferase mRNA). Finally, 60% of the Fluoro-gold-labeled cells in the caudal pontine reticular nucleus contained choline acetyltransferase mRNA.

Estrogen regulates preproenkephalin-A mRNA levels in the rat ventromedial nucleus: temporal and cellular aspects.

Numerous studies suggest that the ventromedial nucleus of the hypothalamus is a primary locus of control for reproductive behavior in the female rat. The display of lordosis behavior is estrogen dependent and its regulation appears to involve the activity of endogenous opioid circuits in the mediobasal hypothalamus and brainstem. Hypothalamic levels of preproenkephalin-A mRNA and the neuropeptide which it encodes, methionine-enkephalin, are dramatically up-regulated by estrogen. To characterize the temporal and cellular aspects of the effect of an acute exposure to estrogen on preproenkephalin-A mRNA levels in the ventromedial nucleus and arcuate nucleus of the hypothalamus, ovariectomized female rats were injected with 50 micrograms estradiol benzoate and used for quantitative in situ hybridization histochemistry of preproenkephalin-A mRNA. In the ventromedial nucleus, estrogen treatment caused a biphasic increase in the number of preproenkephalin-A mRNA cells. Maximal numbers of cells expressing preproenkephalin-A mRNA were observed at 1 and 48 h after estrogen injection. At 4 and 96 h after estrogen injection, the numbers of preproenkephalin-A mRNA cells were similar to that which was seen in the ovariectomized female. The induction of preproenkephalin-A mRNA expression in cells of the arcuate nucleus followed a similar pattern that peaked 48 h after exposure to estrogen. Levels of preproenkephalin-A mRNA per cell did not change in either nucleus as a function of estrogen treatment. Thus, acute estrogen treatment induces expression of preproenkephalin-A mRNA in populations of cells in the ventromedial hypothalamus and arcuate nucleus in which preproenkephalin-A mRNA expression is undetectable by in situ hybridization in the absence of circulating estrogen.

Temporal regulation by estrogen of beta-preprotachykinin mRNA expression in the rat ventromedial nucleus of the hypothalamus.

In the rat, reproduction and sexual behavior are controlled by the gonadal steroid regulation of synaptic interactions within the sexually dimorphic limbic-hypothalamic system. The effects of estrogen on the ventromedial nucleus of the hypothalamus, one nucleus within the circuit, are central to the modulation of this behavior. Involvement of the neuropeptide substance P, a member of the tachykinin family of neuropeptides, has been implicated in the regulation of both lordosis behavior and gonadotropin release. However, previous studies have provided conflicting evidence as to whether levels of substance P in the ventromedial nucleus of the hypothalamus are modulated by circulating estrogens. To study this question further, in situ hybridization histochemistry was used to examine levels of beta-preprotachykinin mRNA, which encodes substance P and other tachykinins, in the ventrolateral subdivision of the ventromedial hypothalamus at 10 consecutive timepoints over a 4 day period subsequent to an acute administration of estrogen. Following estrogen treatment, beta-preprotachykinin mRNA expression was increased in cells of the ventrolateral portion of the ventromedial nucleus of the hypothalamus which constitutively express beta-preprotachykinin mRNA; however, there were no statistically significant changes in the number of cells that express detectable levels of beta-preprotachykinin mRNA in the ventrolateral portion of the ventromedial nucleus. Estrogen treatment produced two peaks of beta-preprotachykinin mRNA expression, the first at 2 h and the second at 48 h after the injection of estrogen. These data indicate that estrogen has both rapid and prolonged effects on beta-preprotachykinin mRNA levels, suggesting that estrogen may affect different cellular mechanisms relevant to the induction of beta-preprotachykinin mRNA expression.

Gonadal steroid control of preprocholecystokinin mRNA expression in the limbic-hypothalamic circuit: comparison of adult with neonatal steroid treatments.

The neuropeptide cholecystokinin (CCK) is involved in the regulation of female, but not male, reproductive behavior. In both sexes, estrogen regulates the expression of CCK in adulthood within the bed nucleus of the stria terminalis and medial amygdaloid nucleus. These areas are parts of an interconnected limbic system-hypothalamic circuit, the development of which is influenced by estrogen during the early postnatal period. This is the same period during which central nervous system (CNS) expression of CCK is dramatically increased, suggesting that the male and female patterns of CCK expression may be the result of early postnatal exposure to estrogen. In the present experiment, the expression of preprocholecystokinin (pCCK) mRNA was determined by in situ hybridization with an isotopically labeled pCCK complementary RNA and emulsion autoradiography in animals whose neonatal and adult gonadal steroid levels had been manipulated. The number of pCCK-expressing cells in animals that were gonadectomized as adults was determined by neonatal estrogen, but stimulation with steroids in adulthood induced a similar number of pCCK-expressing cells in both sexes in the medial amygdala and bed nucleus of the stria terminalis. Neonatal treatment of females with estrogen or testosterone, followed by ovariectomy in adulthood, eliminated the sex difference in pCCK mRNA expression. Males treated neonatally with the aromatase inhibitor androstenedione (to block metabolism of testosterone to estrogen) and orchidectomized in adulthood had a level of pCCK mRNA expression that was similar to that of ovariectomized females. These data suggest that, during neonatal development, estrogen determines the constitutive expression of pCCK mRNA in the medial amygdala and bed nucleus of the stria terminalis, resulting in higher levels of pCCK mRNA expression in males than in females. However, exogenous gonadal steroids induce the same levels of pCCK mRNA expression in adult females, indicating that the levels of gonadal steroids and the patterns of their secretion are the predominant influences on the sexually dimorphic adult levels of pCCK mRNA expression.

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.

Distribution of cholecystokinin-immunoreactive cell bodies in the male and female rat: I. Hypothalamus.

The hypothalamic distribution of cholecystokinin-immunoreactive (CCKI) cell bodies in colchicine-treated male and female rats was studied. Immunoreactive neurons were visualized along the anterior two-thirds of the third ventricle but were especially numerous in the preoptic periventricular nucleus. Dense aggregations of CCKI cells were found in the anterior magnocellular, posterior magnocellular, medial parvicellular, and posterior parvicellular divisions of the paraventricular nucleus. Both the supraoptic nucleus and the central, cell-dense part of the dorsomedial nucleus contained large numbers of CCKI cells. CCKI cells in the preoptic periventricular nucleus were more numerous in the female, as was a population of labeled cells in the dorsal medial preoptic area. However, CCKI cell bodies in this part of the medial preoptic area were larger in males than in females. Males had more CCKI cells in the central part of the medial preoptic nucleus and in the posterior magnocellular subdivision of the paraventricular nucleus. Both males and females had similar numbers of immunoreactive cells in the anterior magnocellular and the parvicellular divisions of the paraventricular nucleus as well as in the anterior hypothalamus, dorsal areas, dorsomedial nucleus, and supramammillary region. These data provide morphological evidence for a sexually differentiated hypothalamic CCKI system.

Effect of opiates on the release of cholecystokinin from in vitro hypothalamus and frontal cortex of Zucker lean (Fa/-) and obese (fa/fa) rats.

Opiates, morphine and [D-Ala2-D-Leu5]-enkephalin (DADLE), inhibited the K+-stimulated release of cholecystokinin (CCK) from the hypothalamus of both Zucker obese (fa/fa) and lean (Fa/-) rats, in vitro. Morphine and DADLE did not inhibit the K+-stimulated release of CCK from frontal cortex from either strain. The opiates did not affect basal efflux of CCK and their effects were all blocked by equimolar concentrations of naloxone. These studies indicate a regional specificity for the effect of opiates on CCK release, and may provide evidence for a cellular mechanism by which endogenous opiates modulate feeding behavior.

Studies on the opiate receptor-mediated inhibition of K+-stimulated cholecystokinin and substance P release from cat hypothalamus in vitro.

The addition of 50 mM K+ to the perfusate of cat hypothalamic slices results in a 3.4- and 5.5-fold increase in the levels of cholecystokinin (CCK) and substance P (sP) like immunoreactivity, respectively. The addition of morphine (10(-11)-10(-8) M; a mu receptor agonist) and D-Ala2-D-Leu5-enkephalin (DADL: 10(-12)-10(-10) M; a delta receptor agonist) resulted in a dose-dependent suppression of the K+-evoked release. SKF10047 (a sigma receptor ligand) and U50488H (a kappa receptor ligand) had no effect in doses up to 10(-6) M. Naloxone added with the lowest dose of agonist producing a maximal inhibition produced a dose-dependent reversal of the anti-release effects of morphine and DADL. The IC50 of naloxone for the antagonism by DADL and morphine of the release of CCK were similar, whereas the naloxone IC50 was lower for morphine than DADL in the reversal of the effects of the agonist in sP release. Within the constraints of receptor selectivity of the several ligands, these data suggest that at least two populations of opioid receptors (mu and delta) may be discriminated which govern the release of hypothalamic sP.

In vitro, release of cholecystokinin from hypothalamus and frontal cortex of Sprague-Dawley, Zucker lean (Fa/-) and obese (fa/fa) rats.

Cholecystokinin (CCK) has been suggested as a putative satiety factor, whose site of action is in the hypothalamus. The genetically obese (fa/fa) Zucker rat has been proposed as a model of human obesity. Though hypothalamic tissue levels of CCK did not vary between the fa/fa rat and age-matched lean littermates (25.5 +/- 5.7 vs. 27.6 +/- 5.2 pmoles/g tissue) we sought to determine if the releasability of hypothalamic and cortical CCK was the same in lean and obese rats. The in vitro superfusion paradigm was used to study the release of CCK and substance P (sP) from hypothalamus, and CCK and vasoactive intestinal polypeptide (VIP) from frontal cortex. The potassium stimulated release of CCK from obese rat hypothalamic tissue was significantly higher than from lean rat hypothalamus (3.62 +/- 0.3 vs. 1.91 +/- 0.3 fmole equivalents CCK-8/mg tissue/10 min). Similarly, sP release was exaggerated in obese rats in a parallel fashion (5.56 +/- 0.44 vs. 2.761 +/- 0.46 fmoles/mg tissue/10 min). However, the potassium stimulated release of CCK and VIP from cortical tissue was the same in all three groups of rats. The obese Zucker rat thus, may have an anomalous release of CCK and sP from the hypothalamus, but not from the frontal cortex, an area not presumably associated with satiety.

Opiate-mediated inhibition of the release of cholecystokinin and substance P, but not neurotensin from cat hypothalamic slices.

The neuroactive peptides neurotensin (NT), substance P (SP) and cholecystokinin (CCK) have been shown to be distributed in the hypothalamus. These peptides may be part of hypothalamic mechanisms which regulate the release of pituitary hormones and feeding behavior. Numerous experiments have demonstrated opiate modulation of anterior pituitary hormone release. These effects have been reported to be mediated via a hypothalamic mechanism, which modulates the secretion of releasing, release inhibiting factors or other neuroactive peptides such as SP, CCK and NT. We have examined the effects of morphine on the potassium-stimulated, calcium-dependent release of SP, CCK and NT from cat hypothalamic slices. The potassium-stimulated release of SP and CCK was profoundly depressed by the addition of morphine (10(-5) M) in a naloxone-reversible manner. This morphine inhibition was shown to be stereospecific, levorphanol (10(-7) M) depressed the release, while dextrophan (10(-7) M) was inactive. Gel filtration chromatography of the potassium-stimulated release was determined to be isographic with authentic NT, SP and CCK-8, respectively. There was no indication of any gastrin-like activity. These data may suggest a regulatory mechanism through which opiates exert some of their neuroendocrine or feeding regulatory effects.

Neurons containing alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity in the cat hypothalamus.

Immunohistochemical studies were conducted on sections of cat hypothalamus in order to determine the distribution of neurons containing alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity. A large number of neurons in the arcuate nucleus were stained after incubation of sections with antisera to either substance. Analysis of serial sections suggested that each neuron revealed with one antiserum was also revealed with the other antiserum, indicating the co-existence of alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity within these arcuate neurons. In contrast, a more diffuse group of lateral hypothalamic neurons which extended from the retrochiasmatic level to the posterior hypothalamus were stained only with the antiserum directed against alpha-melanocyte stimulating hormone. The present results largely confirm findings in the rat hypothalamus, although the lateral hypothalamic group of alpha-melanocyte stimulating hormone immunoreactive neurons appears to be more extensive in the cat.