The endocannabinoid system expression in the female reproductive tract is modulated by estrogen.

The endocannabinoid system (ECS) is involved in several physiological events that resulted in a growing interest in its modulation. Moreover, the uterine levels of anandamide (AEA), the major endocannabinoid, must be tightly regulated to create proper embryo implantation conditions. However, there are no evidences about the regulation of AEA in uterus by estrogen. Thus, the aim of this study is to elucidate whether estradiol benzoate (EB) and tamoxifen (TAM) administration to ovariectomized (OVX) rats can induce changes in the expression of cannabinoid receptors and AEA-metabolic enzymes in uterus by evaluating gene transcription and protein levels by qPCR, Western blot and immunohistochemistry. Moreover, the plasmatic and uterine levels of AEA and of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), the major cyclooxygenase-2 (COX-2) products, were determined by UPLC-MS/MS. The immunohistochemistry showed that cannabinoid receptors, as well as AEA-metabolic enzymes are mainly located in the epithelial cells of both lumen and glands and, to a lesser extent, in the muscle cells. Moreover, EB administration to OVX rats significantly increased CB1, CB2, NAPE-PLD, FAAH and COX-2 expression and transcription. These effects were absent in TAM and TAM+EB treatments showing that this response is estrogen receptor dependent. Additionally, although uterine levels of AEA remained unchanged in EB or TAM treated animals, they showed a rise with EB treatment in plasma. The latter also produced a decrease in uterine PGE2 levels. In summary, these data collectively indicate that the expression of ECS components, as well as, the AEA and PGE2 levels in rat uterus is modulated by EB. Thus, estradiol may have a direct regulatory role in the modulation of ECS in female reproductive tissues.

Effects of gonadal steroids and of estrogen receptor agonists on the expression of estrogen receptor alpha in the medial preoptic nucleus of female rats.

The medial preoptic nucleus (MPN) is a sexually dimorphic cell group of the medial preoptic area that plays a central role in the integration of olfactory and hormonal stimuli that modulate sexually differentiated behaviors. The influence of sex steroids in these behaviors is mediated through activation of estrogen receptors (ERs), which are highly expressed in this nucleus. Little is known about the effects of progesterone (P) or the selective activation of each ER subtype on the expression of estrogen receptor alpha (ERα) in the MPN of female rats. We have addressed this subject in the current investigation by estimating, using stereological tools, the total number of MPN neurons that express ERα in rats at each phase of the estrous cycle and in ovariectomized rats treated with estradiol benzoate (EB), P or the ERα- and estrogen receptor beta (ERß)-specific agonists. Results show that the total number of ERα-immunoreactive neurons does not change over the estrous cycle, except at proestrus when the number is reduced. A similar effect was observed after the administration of EB, but not of P. Results also show that the estradiol-induced down-regulation of the ERα is mediated by activation of both ER subtypes, and that ERß activation leads to a reduction in the total number of ERα-immunoreactive neurons that is twice that resulting from ERα activation. Present data suggest that ERα activation triggers a sort of negative feedback mechanism in MPN neurons that reduces its own expression, which might be of importance for the regulation of estradiol-dependent physiological and behavioral responses.

Role of estrogen receptor α and ß in the induction of progesterone receptors in hypothalamic ventromedial neurons.

The estrogen induction of progesterone receptors (PRs) in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) is critical for the regulation of female sexual behavior. VMNvl neurons express PRs and both types of estrogen receptors (ERα and ERß), and their sequential activation initiates the molecular mechanisms underlying sexual behavior. To assess the relative importance of each ER subtype in the induction of PRs, we have estimated the total number of PR-immunoreactive neurons and quantified the total amount of PR protein in the VMNvl of adult ovariectomized rats that were injected with either estradiol benzoate (EB) or the specific agonists of the ERα, propyl-pyrazole triol (PPT), and of the ERß, diaryl-propionitrile (DPN), in different doses and schedules. The administration of EB and of PPT alone, but not of DPN alone, increased the total number of PR-immunoreactive neurons and PR protein levels. When the specific agonists were administered sequentially, the total number of PR-immunoreactive neurons also increased, particularly when PPT was administered before DPN. Conversely, the concomitant administration of PPT and DPN did not increase the number of PR-immunoreactive neurons. The observation that PPT increases the number of PR-immunoreactive neurons and the levels of PR protein far less than EB shows that the estradiol induction of PRs in the VMNvl does not involve solely the activation of the ERα and suggests that it might also implicate the activation of membrane receptors. The present results also show that ERß activation averts the action of ERα in the induction of PRs.

Effects of estrogens and progesterone on the synaptic organization of the hypothalamic ventromedial nucleus.

The majority of the studies on the actions of estrogens in the ventrolateral part of the hypothalamic ventromedial nucleus (VMNvl) concern the factors that modulate the receptive component of the feminine sexual behavior and the expression of molecular markers of neuronal activation. To further our understanding of the factors that regulate synaptic plasticity in the female VMNvl, we have examined the effects of estradiol and progesterone, and of estrogen receptor (ER) subtype selective ligands on the number of dendritic and spine synapses established by individual VMNvl neurons and on sexual behavior. In contrast to earlier studies that analyzed synapse densities, our results show that exogenous estradiol increases the number of spine as well as of dendritic synapses, irrespective of the dose and regimen of administration. They also reveal that an effective dose of estradiol administered as one single pulse induces the formation of more synapses than the same dose administered as two pulses on consecutive days. Our results further show that both ER subtypes are involved in the mediation of the synaptogenic effects of estrogens on VMNvl neurons since the administration of the selective ERalpha, propyl-pyrazole-triol (PPT), and ERbeta, diarylpropionitrile (DPN), agonists induced a significant increase in the number of synapses that, however, was more exuberant for PPT. Despite its relevant role in feminine sexual behavior, progesterone had no synaptogenic effect in the VMNvl as no changes in synapse numbers were noticed in rats treated with progesterone alone, with estradiol followed by progesterone or with the antiprogestin mifepristone (RU486). Except for the sequential administration of estradiol and progesterone, none of the regimens was associated with lordosis response to vaginocervical stimulation. Therefore, from the sex steroids that undergo cyclic variations over the estrous cycle, only estrogens, acting through both ERalpha and ERbeta, play a key role in the activation of the neural circuits involving the ventromedial nucleus of the hypothalamus.

Neuronal organelles and nuclear pores of hypothalamic ventromedial neurons are sexually dimorphic and change during the estrus cycle in the rat.

Neurons in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) become hypertrophied when exposed to high estrogen levels, an effect that has been observed after estrogen treatment of ovariectomized rats as well as during the proestrus stage of the ovarian cycle. In an attempt to examine whether the neuronal hypertrophy noticed in these conditions reflects metabolic activation of the neurons we have examined, using quantitative methods, the cytoplasmic organelles involved in protein synthesis and the nuclear pores of VMNvl neurons from females on proestrus, when estrogen levels are high, and on diestrus, when estrogen levels are low. Because VMNvl neurons are sexually dimorphic with respect to their size we have performed, in parallel, similar analyses in neurons from age-matched male rats. Our results show that the volume and the surface area of the rough endoplasmic reticulum (RER) and Golgi apparatus are increased at proestrus. They also show that the density of nuclear pores is greater in males than in females whereas the volume and the surface area of the RER and Golgi apparatus are sexually dimorphic only at specific phases of the ovarian cycle: the male-female differences are notorious in the RER when females are on diestrus and in the Golgi apparatus when they are on proestrus. Given that the size of the RER and of the Golgi apparatus correlates with the level of neuronal protein synthesis, data obtained in this study suggest that the sex-related differences and the estrus cycle variations in neuronal size reflect corresponding differences and fluctuations in the metabolic activity of VMNvl neurons.

Simultaneous determination of dextropropoxyphene napsylate, caffeine, aspirin and salicylic acid in pharmaceutical preparations by reversed-phase HPLC.

The official compendial method for the determination of dextropropoxyphene napsylate, caffeine, aspirin and salicylic acid involves a lengthy extraction by gas chromatography and spectrophotometry. The analytical scheme reported here provides a fast, sensitive, and stability-indicating reversed-phase HPLC assay for all these components concurrently. The total elution time is 10 min. The accuracy of the method has been tested on commercial products. The method can easily detect low levels of salicylic acid.