The dichotomous anxiogenic and anxiolytic properties of estrogens have been reported to be mediated by two distinct neural estrogen receptors (ER), ERα and ERβ, respectively. Using a combination of pharmacological and genetic approaches, we confirmed that the anxiolytic actions of estradiol are mediated by ERβ and extended and these observations to demonstrate the neuroanatomical targets involved in ERβ activation in these behavioral responses. We examined the effects of the biologically active S-enantiomer of diarylpropionitrile (S-DPN) on anxiety-related behavioral measures, the corresponding stress hormonal response to hypothalamo-pituitary-adrenal axis reactivity, and potential sites of neuronal activation in mutant female mice carrying a null mutation for ERβ gene (βERKO). S-DPN administration significantly reduced anxiety-like behaviors in the open field, light-dark exploration, and the elevated plus maze (EPM) in ovariectomized wild-type (WT) mice, but not in their βERKO littermates. Stress-induced corticosterone (CORT) and ACTH were also attenuated by S-DPN in the WT mice but not in the βERKO mice. Using c-fos induction after elevated plus maze, as a marker of stress-induced neuronal activation, we identified the anterodorsal medial amygdala and bed nucleus of the stria terminalis as the neuronal targets of S-DPN action. Both areas showed elevated c-fos mRNA expression with S-DPN treatment in the WT but not βERKO females. These studies provide compelling evidence for anxiolytic effects mediated by ERβ, and its neuroanatomical targets, that send or receive projections to/from the paraventricular nucleus, providing potential indirect mode of action for the control of hypothalamo-pituitary-adrenal axis function and behaviors.

Neurobehavioral effects of progesterone are mediated primarily by its interaction with neural progesterone receptors (PRs), expressed as PR-A and PR-B protein isoforms. Whereas the expression of two isoforms in the neural tissues is suggestive of their selective cellular responses and modulation of distinct subsets of PR-induced target genes, the role of individual isoforms in brain and behavior is unknown. We have previously demonstrated a critical role for PRs as transcriptional mediators of progesterone (ligand-dependent), and dopamine (ligand-independent)-facilitated female reproductive behavior in female mice lacking both the isoforms of PR. To further elucidate the selective contribution of the individual PR isoforms in female sexual receptive behavior, we used the recently generated PR-A and PR-B isoform-specific null mutant mice. We present evidence for differential responses of each isoform to progesterone and dopamine agonist, SKF 81297 (SKF), and demonstrate a key role for PR-A isoform in both hormone-dependent and -independent facilitation of sexual receptive behavior. Interestingly, whereas both the isoforms were essential for SKF-facilitated sexual behavior, PR-A appeared to play a more important role in the 8-bromo-cAMP-facilitated lordosis response, raising the possibility of distinct intracellular signaling pathways mediating the responses. Finally, we also demonstrate that antiprogestin, RU38486, was an effective inhibitor of PR-A-mediated, progesterone-dependent, but not SKF or 8-bromo-cAMP-dependent sexual receptivity. The data reveal the selective contributions of individual isoforms to the signaling pathways mediating female reproductive behavior.

Although several studies have reported the localization of membrane progesterone (P(4)) receptors (mPR) in various tissues, few have attempted to describe the distribution and regulation of these receptors in the brain. In the present study, we investigated expression of two mPR subtypes, mPRα and mPRβ, within regions of the brain, known to express estradiol (E(2))-dependent [preoptic area (POA) and hypothalamus] and independent (cortex) classical progestin receptors. Saturation binding and Scatchard analyses on plasma membranes prepared from rat cortex, hypothalamus, and POA demonstrated high-affinity, specific P(4)-binding sites characteristic of mPR. Using quantitative RT-PCR, we found that mPRβ mRNA was expressed at higher levels than mPRα, indicating that mPRβ may be the primary mPR subtype in the rat brain. We also mapped the distribution of mPRβ protein using immunohistochemistry. The mPRβ-immunoreactive neurons were highly expressed in select nuclei of the hypothalamus (paraventricular nucleus, ventromedial hypothalamus, and arcuate nucleus), forebrain (medial septum and horizontal diagonal band), and midbrain (oculomotor and red nuclei) and throughout many areas of the cortex and thalamus. Treatment of ovariectomized female rats with E(2) benzoate increased mPRβ immunoreactivity within the medial septum but not the medial POA, horizontal diagonal band, or oculomotor nucleus. Together, these findings demonstrate a wide distribution of mPRβ in the rodent brain that may contribute to functions affecting behavioral, endocrine, motor, and sensory systems. Furthermore, E(2) regulation of mPRβ indicates a mechanism through which estrogens can regulate P(4) function within discrete brain regions to potentially impact behavior.

Previous studies in our laboratory demonstrated that high-performance liquid chromatography (HPLC) analysis of ground corncob bedding extracts characterized two components (peak I and peak II) that disrupted endocrine function in male and female rats and stimulated breast and prostate cancer cell proliferation in vitro and in vivo. The active substances in peak I were identified as an isomeric mixture of 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid, collectively designated tetrahydrofurandiols (THF-diols). Studies presented here describe the purification and identification of the HPLC peak II component as 9,10-dihydroxy-12-octadecenoic acid (leukotoxin diol; LTX-diol), a well-known leukotoxin. A synthetic mixture of LTX-diol and 12,13-dihydroxy-9-octadecenoic acid (isoleukotoxin diol; i-LTX-diol) isomers was separated by HPLC, and each isomer stimulated (p < 0.001) MCF-7 cell proliferation in an equivalent fashion. The LTX-diol isomers failed to compete for [3H]estradiol binding to the estrogen receptor or nuclear type II sites, even though oral administration of very low doses of these compounds (>> 0.8 mg/kg body weight/day) disrupted estrous cyclicity in female rats. The LTX-diols did not disrupt male sexual behavior, suggesting that sex differences exist in response to these endocrine-disruptive agents.

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