Progesterone blockade of estrogen activation of mu-opioid receptors regulates reproductive behavior.

The mu-opioid receptor (MOR), a G-protein-coupled receptor, is internalized after endogenous agonist binding. Although receptor activation and internalization are separate events, internalization is a good assay for activation because endogenous opioid peptides all induce internalization. Estrogen treatment of ovariectomized rats induces MOR internalization, providing a neurochemical signature of estrogen activation of the medial preoptic nucleus. MOR activation appears to be the mechanism via which estrogen acts in the medial preoptic area to prevent the display of female reproductive behavior during the first 20-24 hr after estrogen treatment. Naltrexone, an alkaloid universal opioid receptor antagonist, prevented MOR internalization, suggesting that estrogen induces the release of endogenous opioid peptides that in turn activate the MOR. Enkephalins and beta-endorphin are nonselective endogenous MOR ligands. The most selective endogenous MOR ligands are the endomorphins. Infusions of selective MOR agonists, H-Tyr-d-Ala-Gly-N-Met-Phe-glycinol-enkephalin (DAMGO) or endomorphin-1, into the medial preoptic nucleus attenuated lordosis, and their effects were blocked with the MOR antagonist H-d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP). Infusion of endomorphin-1 internalized MOR. To determine whether progestin also acts via the MOR system to facilitate reproductive behavior, ovariectomized rats were primed with 17beta-estradiol and progesterone. Progestin facilitation of lordosis was correlated with a reduction of estrogen-induced MOR internalization. Progestin reversed estrogen-induced MOR internalization, suggesting that progesterone blocked estrogen-induced endogenous opioid release, relieving estrogen inhibition and facilitating lordosis. These results indicate a central role of MOR in the mediation of sex steroid activation of the CNS to regulate female reproductive behavior.

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.

Levels of serum steroids, aromatase activity, and estrogen receptors in preoptic area, hypothalamus, and amygdala of B6D2F1 male house mice that differ in the display of copulatory behavior after castration.

Most male B6D2F1 hybrid house mice continue to copulate after castration (continuers), whereas others do not (noncontinuers). Copulation in continuers appears estrogen dependent. Serum testosterone (T), 17 beta-estradiol (E2), and dihydrotestosterone (DHT), as well as aromatase activity (AA) and estrogen receptor (ER) levels in preoptic area (POA), hypothalamus (HYP), and amygdala (AM) were measured to determine if continuers and noncontinuers differ in estrogen physiology. In general, continuers and noncontinuers did not differ in serum steroid levels, AA, or ER levels. Castration reduced AA in the POA, HYP, and AM. Castration did not affect nuclear ER levels in the POA and HYP but reduced nuclear ER in AM. The data demonstrate that castrated B6D2F1 male mice continue to be under the influence of circulating nongonadal E2 that is important for copulation.

Orphanin FQ/nociceptin in the ventromedial nucleus facilitates lordosis in female rats.

The localization of opioid receptor-like (ORL-1) orphan receptor in the ventromedial nucleus of the hypothalamus (VMH) suggested a role for this opioid system in the regulation of lordosis behavior. Recently, the ligand for ORL-1, orphanin FQ/nociceptin (OFQ/N), has been characterized and also demonstrated to be present in the VMH. The present experiments examined whether OFQ/N in the VMH facilitates lordosis behavior in estrogen-primed, sexually unreceptive female rats, and whether estrogen regulates ORL-1 levels in the VMH. Estrogen was shown to increase ORL-1 immunoreactivity in the VMH, and microinfusions of OFQ/N into the VMH facilitated lordosis behavior in a dose-dependent manner.

Estrogen and endogenous opioids regulate CCK in reproductive circuits.

This review focuses on the interaction of estrogen with the cholecystokinin (CCK) and endogenous opioid peptide systems in the medial preoptic nucleus, and how these interactions result in alterations of a stereotypic female reproductive behavior--lordosis. The medial preoptic nucleus is an integral part of a circuit controlling lordosis that extends from the limbic system through the hypothalamus. Estrogen alters the integration of sensory information in the circuit that results in the display of sexually receptive behavior. Estrogen determines the activity of CCK and endogenous opioid peptide systems through regulation of expression, release and interaction with specific receptors. Studies of each system individually have indicated that they are pivotal to the expression of lordosis. Recent studies demonstrate an estrogen-dependent interaction between endogenous opioid and CCK systems that control reproductive behavior.

Preproenkephalin mRNA levels are regulated by acute stress and estrogen stimulation.

Enkephalins facilitate female reproductive behavior. Within the limbic system and hypothalamus, estrogen induced the expression of preproenkephalin (PPE) mRNA. Estrogen injection caused a biphasic increase in the PPE mRNA levels within the ventromedial hypothalamic nucleus and posterodorsal medial amygdala. The first peak of PPE mRNA levels occurred within an hour, and the second 24 to 48 h after subcutaneous injection of estrogen. The present studies indicated that the rapid first peak of PPE mRNA expression was stress induced, whereas the second peak was estrogen induced. In the posterodorsal medial amygdala but not in the ventromedial hypothalamic nucleus, the antiestrogen, tamoxifen, did not inhibit the first peak, but blocked the second peak of PPE mRNA expression. Subcutaneous oil injection induced a 1-h peak of PPE mRNA levels but not a 24-h peak. Peak levels of plasma corticosterone were coincident with peak PPE mRNA levels. Adrenalectomy plus a constant, low level of corticosterone eliminated the injection-induced increase of corticosterone levels and the subsequent increase in PPE mRNA expression in the ventromedial hypothalamic nucleus and posterodorsal medial amygdala. The present results indicate that both stress steroids and estrogen positively regulate PPE mRNA levels in the ventromedial hypothalamic nucleus and posterodorsal medial amygdala. These results are consistent with the hypothesis that acute, mild stress may contribute to the activation of circuits that facilitate reproductive behavior in the female.

Gonadal steroids differentially modulate the actions of orphanin FQ/nociceptin at a physiologically relevant circuit controlling female sexual receptivity.

Orphanin FQ/nociceptin (OFQ/N) inhibits the activity of pro-opiomelanocortin (POMC) neurones located in the hypothalamic arcuate nucleus (ARH) that regulate female sexual behaviour and energy balance. We tested the hypothesis that gonadal steroids differentially modulate the ability of OFQ/N to inhibit these cells via presynaptic inhibition of transmitter release and postsynaptic activation of G protein-gated, inwardly-rectifying K(+) (GIRK)-1 channels. Whole-cell patch clamp recordings were performed in hypothalamic slices prepared from ovariectomised rats. OFQ/N (1 µm) decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs), and also caused a robust outward current in the presence of tetrodotoxin, in ARH neurones from vehicle-treated animals. A priming dose of oestradiol benzoate (EB; 2 µg) increased basal mEPSC frequency, markedly diminished both the OFQ/N-induced decrease in mEPSC frequency and the activation of GIRK-1 currents, and potentiated the OFQ/N-induced decrease in mIPSC frequency. Steroid treatment regimens that facilitate sexual receptivity reinstate the basal mEPSC frequency, the OFQ/N-induced decrease in mEPSC frequency and the activation of GIRK-1 currents to levels observed in vehicle-treated controls, and largely abolish the ability of OFQ/N to decrease mIPSC frequency. These effects were observed in an appreciable population of identified POMC neurones, almost one-half of which projected to the medial preoptic nucleus. Taken together, these data reveal that gonadal steroids influence the pleiotropic actions of OFQ/N on ARH neurones, including POMC neurones, in a disparate manner. These temporal changes in OFQ/N responsiveness further implicate this neuropeptide system as a critical mediator of the gonadal steroid regulation of reproductive behaviour.

Temporal and concentration-dependent effects of oestradiol on neural pathways mediating sexual receptivity.

The acceptance of oestradiol signalling through receptors found in the cell membrane, as well as, the nucleus, has provided for a re-examination of the timing and location of the actions of oestradiol on neural circuits mediating sexual receptivity (lordosis). Oestradiol membrane signalling involves the transactivation of metabotrophic glutamate receptors (mGluRs) that transduce steroid information through protein kinase C signalling cascades producing rapid activation of lordosis-regulating circuits. It has been known for some time that oestradiol initially produces an inhibition of the medial preoptic nucleus. We have demonstrated that underlying this inhibition is oestradiol acting in the arcuate nucleus to induce ß-endorphin release, which inhibits the medial preoptic nucleus through a µ-opioid receptor mechanism. This transient inhibition is relieved by either subsequent progesterone treatment or longer exposure to higher doses of oestradiol to facilitate lordosis behaviour. We review recent findings about oestradiol membrane signalling inducing dendritic spine formation in the arcuate nucleus that is critical for oestradiol induction of sexual receptivity. Moreover, we discuss the evidence that, in addition to oestrogen receptor α, several other putative membrane oestrogen receptors facilitate lordosis behaviour through regulation of the arcuate nucleus. These include the GRP30 and the STX activated Gq-mER. Finally, we report on the importance of GABA acting at GABAB receptors for oestradiol membrane signalling that regulates lordosis circuit activation and sexual receptivity.