PAC1 receptors mediate pituitary adenylate cyclase-activating polypeptide- and progesterone-facilitated receptivity in female rats

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as a feed-forward, paracrine/autocrine factor in the hypothalamic ventromedial nucleus (VMN) for receptivity and sensitizes pituitary hormone release for ovulation. The present study examined receptor(s) and signaling pathway by which PACAP enhances rodent lordosis. PACAP binds to PACAP (PAC1)- and vasoactive intestinal peptide-preferring receptors (VPAC1, VPAC2). Ovariectomized rodents primed with estradiol (EB) were given PACAP or vasoactive intestinal peptide directly onto VMN cells. Only PACAP facilitated receptivity. Pretreatment with VPAC1 and VPAC2 inhibitors blocked both PACAP- and progesterone (P)-induced receptivity. Antisense (AS) oligonucleotides to PAC1 (not VPAC1 or VPAC2) inhibited the behavioral effect of PACAP and P. By real-time RT-PCR, EB, P and EB+P enhanced VMN mRNA expression of PAC1. Within the total PAC1 population, EB and EB+P induced expression of short form PAC1 and PAC1hop2 splice variants. Finally, blocking cAMP/protein kinase A signaling cascade by antagonists to cAMP activity and protein kinase A or by antisense to dopamine- and cAMP-regulated phosphoprotein of 32 kDa blocked the PACAP effect on behavior. Collectively, these findings provide evidence that progesterone receptor-dependent receptivity is, in part, dependent on PAC1 receptors for intracellular VMN signaling and delineate a novel, steroid-dependent mechanism for a feed-forward reinforcement of steroid receptor-dependent reproductive receptivity.     

Mechanisms of ovarian steroid regulation of norepinephrine receptor-mediated signal transduction in the hypothalamus: implications for female reproductive physiology

In many mammalian species, the ovarian steroid hormones estradiol (E(2)) and progesterone (P) act in the hypothalamus and preoptic area to coordinate the timing of female sexual receptivity with ovulation. We study lordosis behavior, an important component of sexual receptivity in rats, and its regulation by E(2) and P as a model system for understanding how hormonal modulation of synaptic neurotransmission influences reproductive physiology and behavior. Our findings suggest that E(2) and P extensively regulate synaptic communication involving the catecholamine norepinephrine (NE) in the hypothalamus. Estrogen priming shifts the balance of postsynaptic NE receptor signaling in the hypothalamus and preoptic area away from beta-adrenergic activation of cAMP synthesis toward alpha(1)-adrenergic signaling pathways. Attenuation of beta-adrenergic signal transduction is achieved by receptor-G-protein uncoupling, apparently due to stable receptor phosphorylation. E(2) modification of alpha(1)-adrenergic signaling includes both increased expression of the alpha(1B)-adrenoceptor subtype and a dramatic, P-induced reconfiguration of the biochemical responses initiated by agonist activation of alpha(1)-adrenoceptors. Among these is the emergence of alpha(1)-adrenergic receptor coupling to cGMP synthesis. We also present evidence that estrogen promotes novel, functional interactions between insulin-like growth factor-1 (IGF-1) and alpha(1)-adrenergic receptor signaling in the hypothalamus and preoptic area. Thus, estrogen amplification of signaling mediated by alpha(1)-adrenoceptors is multifaceted, involving changes in gene expression (of the alpha(1B)-adrenoceptor), switching of receptor linkage to previously inactive intracellular pathways, and the promotion of cross talk between IGF-1 and NE receptors. We propose that this hormone-dependent remodeling of hypothalamic responses to NE maximizes reproductive success by coordinating the timing of the preovulatory release of gonadotropins with the period of behavioral receptivity in female rodents.     

Stimulation of the hypothalamic ventromedial nuclei by pituitary adenylate cyclase-activating polypeptide induces hypophagia and thermogenesis

Numerous studies have demonstrated that the hypothalamic ventromedial nuclei (VMN) regulate energy homeostasis by integrating and utilizing behavioral and metabolic mechanisms. The VMN heavily express pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptors (PAC1R). Despite the receptor distribution, most PACAP experiments investigating affects on feeding have focused on intracerebroventricular administration or global knockout mice. To identify the specific contribution of PACAP signaling in the VMN, we injected PACAP directly into the VMN and measured feeding behavior and indices of energy expenditure. Following an acute injection of PACAP, nocturnal food intake was significantly reduced for 6 h after injections without evidence of malaise. In addition, PACAP-induced suppression of feeding also occurred following an overnight fast and could be blocked by a specific PAC1R antagonist. Metabolically, VMN-specific injections of PACAP significantly increased both core body temperature and spontaneous locomotor activity with a concurrent increase in brown adipose uncoupling protein 1 mRNA expression. To determine which signaling pathways were responsive to PACAP administration into the VMN, we measured mRNA expression of well-characterized hypothalamic neuropeptide regulators of feeding. One hour after PACAP administration, expression of pro-opiomelanocortin mRNA was significantly increased in the arcuate nuclei (ARC), with no changes in neuropeptide Y and agouti-related polypeptide mRNA levels. This suggests that PAC1R expressing VMN neurons projecting to pro-opiomelanocortin neurons contribute to hypophagia by involving melanocortin signaling. While the VMN also abundantly express PACAP protein, the present study demonstrates that PACAP input to the VMN can influence the control of energy homeostasis.     

Effects of ventromedial nucleus lesions on estradiol induced ovulation in the rat

Small bilateral stereotaxic lesions were made in the hypothalamic ventromedial nucleus (SVMN) to determine: (i) whether estrogen would restore early receptivity in unreceptive SVMN lesioned female rats and (ii) whether SVMN lesions would suppress estrogen induced ovulation in the rat. SVMN lesions were shown to completely suppress spontaneous early receptivity and seriously impair estrous receptivity in 5-day cyclic female Wistar rats. A loss of early receptivity in response to 10 μg estradiol benzoate (EB) was also observed in SVMN lesioned females, in comparison to unoperated, sham VMN and dorsomedial nucleus (DMN) lesioned animals. Isolated SVMN lesioned females exhibited a weak ovulatory response to 10 μg EB, but, where shown to be unreceptive prior to estrogen injection, they never ovulated. On the contrary, ovulation occured in about 50% of cases in isolated unoperated and in sham VMN and DMN lesioned females following estrogen administration. The mechanisms whereby EB brought about precocious ovulation in 5-day cyclic female rats were therefore concluded to be dependent on VMN functional integrity and thereby on the degree of early sexual receptivity in the rat.     

A contributory role for midbrain progesterone in the facilitation of female sexual behavior in rats

Progesterone (P) facilitation of sexual receptivity in rodents has been achieved by intracranial administration to the ventral hypothalamus; the preoptic area; and midbrain areas such as central gray, mesencephalic reticular formation, and ventral tegmental nucleus. In our laboratory, by far the most effective site in rats has been the ventromedial nucleus of the hypothalamus (VMN). However, several reports of sensitivity to P in the midbrain of rats and other rodent species led us to investigate whether stimulation of the ventral midbrain of female rats might contribute to facilitation of sexual receptivity. Ovariectomized Long-Evans rats received one cannula aimed at the VMN, and another aimed at the contralateral ventral mesencephalon. P in both cannulae, following a priming dose of estradiol, caused significantly higher lordosis quotients (LQ) than blank tubes. Controls with bilateral cannulae in the VMN responded when both tubes were filled with P, but did not respond to unilateral VMN P stimulation. P in the VMN and contralateral anterior preoptic area did not result in a greater degree of receptivity than did the empty tubes. These studies indicate that although progesterone stimulation in the midbrain alone is not sufficient to facilitate receptivity in female rats with our methods, the midbrain may play an auxiliary role. P implants in the midbrain appear to facilitate receptivity in the case of VMN implant treatments that are subthreshold for stimulating lordosis. The results are discussed in light of similar studies in other rodent species, and in the context that more than one brain site may be important in the natural stimulation of sexual receptivity by gonadal hormones.     

Long-term ovariectomy and hormone-induced sexual behavior, progestin receptors, and hypothalamic morphology in female rats

Long-term ovariectomy reduces the ability of estradiol and progesterone treatment to induce sexual receptivity in female rats. Previous researchers suggested that this effect may be due to a decreased induction of neural progestin receptors by estradiol in the long-term ovariectomized rats. The present study was designed to replicate and extend this finding, and to search for neuroanatomical correlates by measuring the volume of the ventromedial nucleus (VMN) of the hypothalamus, a putative site of action of estradiol and progesterone for the induction of female sexual behavior. Long-term ovariectomy (5 to 6 weeks) as compared to short-term ovariectomy (1 week) reduced the ability of estradiol-17 beta and progesterone treatment to induce sexually receptive and proceptive behaviors. Consistent with previous reports, our data show that the reduced levels of cytosol progestin receptors after long-term ovariectomy and estradiol treatment are related to a reduced ability of estradiol to induce the receptors. Long-term ovariectomy did not affect the concentration of cytosol progestin receptors in the preoptic area, suggesting a neuroanatomical specificity to this effect. Contrary to our predictions, long-term ovariectomy did not affect the volume of the VMN. In fact, estradiol treatment, while blocking the effect of long-term ovariectomy on sexual behavior, decreased the volume of the VMN. Therefore, the measurement of the volume of the VMN is not a good predictor of the responsiveness to steroid hormone induction of sexual behavior.     

Visualizing activation of opioid circuits by internalization of G protein-coupled receptors

Mu-opioid receptor (MOR) and opioid receptor-like receptor (ORL-1) circuits in the limbic hypothalamic system are important for the regulation of sexual receptivity in the female rat. Sexual receptivity is tightly regulated by the sequential release of estrogen and progesterone from the ovary suggesting ovarian steroids regulate the activity of these neuropeptide systems. Both MOR and ORL-1 distributions overlap with the distribution of estrogen and progesterone receptors in the hypothalamus and limbic system providing a morphological substrate for interaction between steroids and the opioid circuits in the brain. Both MOR and ORL-1 are receptors that respond to activation by endogenous ligands with internalization into early endosomes. This internalization is part of the mechanism of receptor desensitization or down regulation. Although receptor activation and internalization are separate events, internalization can be used as a temporal measure of circuit activation by endogenous ligands. This review focuses on the estrogen and progesterone regulation of MOR and ORL-1 circuits in the medial preoptic nucleus and ventromedial nucleus of the hypothalamus that are central to modulating sexual receptivity.     

Estrogen receptor and progesterone receptor-immunoreactive cells are not co-localized with gonadotropin-releasing hormone in the brain of the female mink (Mustela vison)

The distribution of gonadal steroid (estrogen, progesterone) receptors in the brain of the adult female mink was mapped by immunocytochemistry. Using a monoclonal rat antibody raised against human estrogen receptor (ER), the most dense collections of ER-immunoreactive (IR) cells were found in the preoptic/anterior hypothalamic area, the mediobasal hypothalamus (arcuate and ventromedial nuclei), and the limbic nuclei (amygdala, bed nucleus of the stria terminalis, lateral septum). Immunoreactivity was mainly observed in the cell nucleus and a marked heterogeneity of staining appeared from one region to another. A monoclonal mouse antibody raised against rabbit uterine progesterone receptor (PR) was used to identify the PR-IR cells in the preoptic/anterior hypothalamic area and the mediobasal hypothalamus (arcuate and ventromedial nuclei). This study also focused on the relationship between cells containing sex-steroid receptors and gonadotropin-releasing hormone (GnRH) neurons on the same sections of the mink brain using a sequential double-staining immunocytochemistry procedure. Although preoptic and hypothalamic GnRH neurons were frequently in close proximity to perikarya containing ER or PR, they did not themselves possess receptor immunoreactivity. The present study provides neuroanatomical evidence that GnRH cells are not the major direct targets for gonadal steroids and confirms for the first time in mustelids the results previously obtained in other mammalian species.     

Relevance of mating-associated stimuli, ovulation, and the progesterone receptor for the post-coital inhibition of estrous behavior in the female rabbit

Estrous female domestic rabbits (Oryctolagus cuniculus) display scent marking (“chinning”) and sexual receptivity. Mating induces ovulation, which occurs approximately 12 h later, and also decreases chinning and receptivity. In the present study, we explored the participation of mating-associated stimuli, ovulation, and the progesterone receptor (PR) in mediating such behavioral effects. We found that copulatory stimuli were not necessary, and that ovulation alone was sufficient, as these behavioral changes were replicated in unmated females by intravenous administration of human chorionic gonadotropin (hCG). The post-mating administration (s.c.) of 5 μg/day estradiol benzoate (EB), prevented the decline in chinning and receptivity. A lower dose of EB (1 μg/day) had no effect, nor did the antiprogestin RU486 (20 mg, s.c., administered 3 h before mating). However, the combination of a single pre-mating administration of RU486 plus the post-mating administration of 1 μg/day EB completely blocked the decline in estrous behavior. We propose that PR activation around the time of mating and a post-mating decline in ovarian estradiol secretion and/or estradiol responsiveness act in parallel to terminate estrus in this species.     

Hormonal Regulation of Female Reproductive Behavior in Fish

SYNOPSIS. Fundamentally different mechanisms regulate femalesexual behavior in the ovoviviparous guppy and the oviparousgoldfish. In the female guppy, ovarian estrogen evidently synchronizescycles of sexual receptivity with endogenous cycles of ovarianmaturation and also increases female attractivity at the timeof maximum receptivity by stimulating the release of a sexualpheromone. In the goldfish, it appears that prostaglandin, releasedfrom the ovary or oviduct in conjunction with ovulation andthe presence of ovulated eggs, acts on the brain to stimulatespawning behavior. In contrast to the situation in the guppy,steroid treatments alone (in the absence of ovulated eggs) failto stimulate spawning behavior in the goldfish. It isproposedthat endocrine mechanisms regulating female sexual behaviorin the teleosts and in other vertebrates are less related tophylogeny than to the mode of reproduction employed. In thegoldfish and several other externally fertilizing teleosts,where sexual behavior involves oviposition, female sexual behaviorapparently is synchronized with ovulation by mechanisms whichrespond to elevated plasma prostaglandin as an indicator ofthe presence of ovulated eggs. In internally fertilizing species(guppy, reptiles, birds, mammals), where sexual behavior andfertilizationare temporally dissociated, female sexual behavior is synchronizedwith ovulation by mechanisms which anticipate either an imminentspontaneous ovulation, or the potential for reflex ovulation,by responding to increases in plasma estrogen associated withfolliculardevelopment.     

Changes in the levels of protein and steroid hormones in the plasma and steroid hormone receptors in the uterus of normal cycling guinea pigs

This study deals with the estrous cycle of guinea pigs in relation to sexual behavior, uterine weight, levels of gonadotropins, steroid hormones, and steroid hormone receptors in the uterus. The guinea pigs in this study showed cyclic changes in various reproductive functions broadly similar to other laboratory species studied. The increase in the uterine weight coincided with high concentration of steroid hormones (estradiol and progesterone) secreted during proestrus and estrus. The elevated levels of steroid hormone receptor concentrations in the uterus during these periods also confirm the role of these hormones. The rise in progesterone level from day 14 of the cycle was associated with lordosis and its related behavior. It was noted that the "estrus behavior" is the most accurate external marker for ovulation and sexual receptivity to males. It was also observed that there is an association between follicle-stimulating hormone and luteinizing hormone during the preovulatory period that was not demonstrated in previous studies.     

Progesterone versus estrogen facilitation of female sexual behavior by intracranial administration to female rats

The CNS sites of action for progesterone facilitation of female sexual behavior are disputed. Among the areas most often cited are the ventromedial hypothalamus and the ventral midbrain. There is also a controversy about whether estradiol may substitute for progesterone for the facilitation of receptive behavior when given systemically or intracranially. We tested VMH and ventral midbrain applications of estradiol versus progesterone for the facilitation of female sexual behavior in estrogen-primed, ovariectomized female rats. Subjects were implanted with bilateral guide tubes aimed at ventral hypothalamic or midbrain sites. Estrogen-primed rats received either 28-gauge insert cannulae filled at the lumen with pure progesterone, estradiol, or cholesterol, or empty tubes, and were tested for receptivity with intact, experienced stud males just before, and 1 and 4 hr after, intracranial hormone administration. Significant estrous responsiveness was seen only in the 4-hr test after progesterone was implanted in the VMN in the first intracranial cannula test. We conclude, in contrast to some previous reports, that administration of progesterone to the VMN is more effective in the facilitation of female sexual behavior than when it is implanted in the ventral midbrain, and that administration of estradiol to either site is ineffective.     

Genomic regulation of sexual behavior

Estrogen receptors are distributed in discrete areas of the hypothalamus, preoptic area and amygdala of the rat brain, and in some of these areas estrogens induce progestin receptor sites. Estradiol (E), followed by progesterone (P), induce feminine sexual behavior in female, but not in male, rats. This induction takes time (on the order of hours, not minutes, so that the hormone may be cleared from the body) and is dependent on RNA and protein synthesis. Within the hypothalamic ventromedial nuclei (VMN), E and P induce changes in RNA and protein synthesis and also induce morphological changes indicative of cellular growth, genomic activation, and either new synapse formation or morphological rearrangement of existing synapses. Neurochemically, a number of neurotransmitter systems are implicated in the control of feminine sexual behavior, including acetylcholine, serotonin, GABA, and the neuropeptides, oxytocin and CCK. One of the means by which E and P may exert their influence on sexual behavior, aside from the morphological alterations, is by regulating levels of receptors for certain of these neurotransmitters. The critical differences which underlie the inability of male rats to display high levels of feminine sexual behavior after E plus P priming may depend on sex differences in the ability of E to induce particular neurochemical products as well as P receptors and upon differences in neural circuitry in the VMN.     

Rapid inhibition of female sexual behavior by gonadotropin-inhibitory hormone (GnIH)

Gonadotropin-releasing hormone (GnRH) is largely responsible for the initiation of sexual behaviors; one form of GnRH activates a physiological cascade causing gonadal growth and gonadal steroid feedback to the brain, and another form is thought to act as a neurotransmitter to enhance sexual receptivity. In contrast to GnRH, gonadotropin-inhibitory hormone (GnIH) inhibits gonadotropin release. The distribution of GnIH in the avian brain suggests that it has not only hypophysiotropic actions but also unknown behavioral actions. GnIH fibers are present in the median eminence (ME) and are in apparent contact with chicken GnRH (cGnRH)-I and -II neurons and fibers. In birds, cGnRH-I regulates pituitary gonadotropin release, whereas cGnRH-II enhances copulation solicitation in estradiol-primed females exposed to male song. In the present study, we determined the effects of GnIH administered centrally to female white-crowned sparrows. A physiological dose of GnIH reduced circulating LH and inhibited copulation solicitation, without affecting locomotor activity. Using rhodaminated GnIH, putative GnIH binding sites were seen in the ME close to GnRH-I fiber terminals and in the midbrain on or close to GnRH-II neurons. These data demonstrate direct effects of GnIH upon reproductive physiology and behavior, possibly via separate actions on two forms of GnRH.     

Release of orphanin FQ/nociceptin in the medial preoptic nucleus and ventromedial nucleus of the hypothalamus facilitates lordosis

Opioid regulation of reproduction has been widely studied. However, the role of opioid receptor-like 1 receptor (NOP; also referred to as ORL-1 and OP4) and its endogenous ligand orphanin FQ/nociceptin (OFQ/N) have received less attention despite their extensive distribution throughout nuclei of the limbic-hypothalamic system, a circuit that regulates reproductive behavior in the female rat. Significantly, the expression of both receptor and ligand is regulated in a number of these nuclei by estradiol and progesterone. Activation of NOP in the ventromedial nucleus of the hypothalamus (VMH) of estradiol-primed nonreceptive female rats facilitates lordosis. NOPs are also expressed in the medial preoptic nucleus (MPN), however, their roles in reproductive behavior have not been studied. The present experiments examined the role of NOP in the regulation of lordosis in the MPN and tested whether endogenous OFQ/N in the MPN and VMH mediates reproductive behavior. Activation of NOP by microinfusion of OFQ/N in the MPN facilitated lordosis in estradiol-primed sexually nonreceptive female rats. Passive immunoneutralization of OFQ/N in either the MPN or the VMH reduced lordosis in estradiol-primed females, but had no effect on lordosis in estradiol+progesterone-primed sexually receptive rats. These studies suggest that OFQ/N has a central role in estradiol-only induced sexual receptivity, and that progesterone appears to involve additional circuits that mediate estradiol+progesterone sexual receptivity.