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The control of gonadotropin-releasing hormone (GnRH) secretion depends on the action of ovarian steroids and several substances, including nitric oxide (NO). NO in the medial preoptic area (MPOA) stimulates the proestrus surge of luteinizing hormone (LH). We studied the effect of estrogen (Tamoxifen-TMX) and progesterone (RU-486) antagonists on mRNA and protein expression of NO synthase (NOS), the enzyme that produces NO, as well as its activity within MPOA. Female rats received s.c. injections of TMX (3mg/animal) on first and second days of the estrous cycle (9 am), RU-486 (2mg/animal) on first, second, (8 am and 5 pm) and third days of the estrous cycle (8 am) or oil (controls) and were killed on the third day (5 pm). Real time-PCR and western blotting were performed to study NOS mRNA and protein expressions. The NOS activity was indirectly assessed by measuring the conversion from [(14)C]-L-arginine into [(14)C]-L-citrulline. TMX significantly decreased neuronal NOS (nNOS) mRNA expression (90%), and the activity of NOS, but did not alter nNOS protein expression. Also, TMX significantly decreased LH, FSH, estrogen and progesterone plasma levels. RU-486 nor affected NOS mRNA and protein expressions neither the NOS activity in the MPOA, but reduced FSH levels. The nitrergic system in the MPOA can be stimulated by estrogen whereas TMX decreased NOS activity and mRNA expression. In conclusion, the involvement of the nitrergic system in the MPOA to induce the surge of LH on proestrus depends on the estrogen action to stimulate the mRNA-nNOS expression and the activity of nNOS but it does not seem to depend on progesterone action., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Early-life environmental events that disrupt the mother-pup relationship may induce profound long-lasting changes on several behavioral and neuroendocrine systems. The neonatal handling procedure, which involves repeated brief maternal separations followed by experimental manipulations, reduces sexual behavior and induces anovulatory estrous cycles in female rats. On the afternoon of proestrus, neonatally handled females show a reduced surge of luteinizing hormone (LH) and an increased content of gonadotropin-releasing hormone in the medial preoptic area (MPOA). In order to detect the possible causes for the reduced ovulation and sexual behavior, the present study aimed to analyze the effects of neonatal handling on noradrenaline (NA) and nitric oxide (NO) levels in the MPOA on the afternoon of proestrus. Neonatal handling reduced MHPG (NA metabolite) levels and MHPG/NA ratio in the MPOA, indicating decreased NAergic activity. Additionally, neonatal handling decreased NO levels, as measured by the metabolites (NO(x)), nitrite and nitrate in the same period. We may conclude that the neonatal handling procedure decreased activity of the NAergic and NOergic systems in the MPOA during proestrus, which is involved in the control of LH and FSH secretion, and this may possibly explain the anovulatory estrous cycles and reduced sexual behavior of the neonatally handled female rats., ((c) 2007 S. Karger AG, Basel)

The neurons that produce gonadotrophin-releasing hormone (GnRH) are mainly found in the medial preoptic area (MPOA) and constitute a common final pathway to the control of luteinizing hormone (LH) surge on proestrus. The control of GnRH secretion depends on several neurotransmitters, such as serotonin (5-HT), noradrenaline (NA), dopamine (DA) and nitric oxide (NO). The aim of this work was to study the profile of 5-HT, catecholamines and their main metabolites in the MPOA throughout the estrous cycle and their interactions with NO system in this area to control LH surge. For this purpose, the following were evaluated: (I) the effect of pargyline (a monoamine oxidase inhibitor) acute treatment on plasma LH secretion throughout the estrous cycle, correlated with changes of 5-HT, DA and NA content as well as activity and expression of neuronal NO synthase (nNOS) within MPOA; (II) the effect of 5,7-dihydroxitriptamine (a drug that depletes 5-HT) microinjection into MPOA on plasma LH in ovariectomized rats treated with oil, estradiol (E(2)) or E(2) plus progesterone (P(4)). Pargyline prevented LH surge on proestrus without altering its basal secretion. Throughout the estrous cycle, pargyline augmented both 5-HT and DA contents in approximately 300% and NA content in 50% in the MPOA. During proestrus, pargyline stimulated nNOS activity at 9 h and inhibited it at 11 h. nNOS expression was inhibited by pargyline at 15 h. Depletion of 5-HT content in the MPOA increased LH secretion in ovariectomized rats treated with E(2) plus P(4), but it did not modify in rats treated with either oil or E(2). Therefore, the present data show that pargyline treatment can inhibit proestrus LH surge through a mechanism that may involve 5-HT and NO systems in the MPOA. Moreover, the effect of 5-HT in the MPOA for limiting LH surge seems to depend on plasma levels of E(2) and P(4).

We have previously shown that the locus coeruleus (LC) is essential for triggering surges of LH. Since LC neurons are responsive to estradiol, which induces progesterone receptor (PR) expression, this study aimed to investigate whether LC neurons express the alpha-estradiol receptor (alphaER) and PR as well as comparing such responses to that observed in the preoptic area (POA). Female rats were perfused at 10, 14 and 16 h on each day of the estrous cycle, and a blood sample was collected for estradiol, progesterone and LH measurements. alphaER- and PR immunoreactive (ir) neurons were detected in POA and LC by immunocytochemistry (ICC). Higher plasma estradiol levels were observed on the day of proestrus, when a smaller number of alphaER-ir POA neurons were detected. An increase in the number of alphaER-ir neurons were observed at 16 h of proestrus and estrus. The number of PR-ir neurons increased in POA only at 16 h of proestrus, and remained unchanged during all other days and times. The profile of alphaER-ir and PR-ir neurons in LC changed over the estrous cycle, with a lower expression on metestrus morning and reaching a peak on diestrus afternoon before declining on the day of proestrus. However, on estrus afternoon, alphaER-ir neurons increased, while PR-ir neurons decreased which may be related to the prolactin surge of estrus. These data show that LC neurons express alphaER and PR and seem to be more sensitive to variations in estradiol than POA. Also, the fluctuation in alphaER and PR observed for LC neurons seems to accompany the hormonal events that occur during the estrous cycle. This profile of alphaER and PR expression might be related to the ability of estradiol and progesterone in regulating the activity of LC neurons, which could be associated to the control mechanisms of LH and prolactin release.

Neonatal handling alters the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonads axis (HPG) in adult animals, and angiotensin II (Ang II) modulates the functions in these axes. We tested whether neonatal handling could change the density of Ang II receptors in some central areas in female rats. Results showed decreased density of the Ang II receptors in the medial preoptic area (MPOA) and hypothalamic paraventricular nucleus (PVN) of the neonatal handled group.

Angiotensin II (Ang II) receptors in specific brain areas and in the anterior pituitary are controlled by reproductive hormones. Since Ang II also plays a role in controlling reproductive functions, such as luteinizing hormone and prolactin secretion, the objective of the present study was to evaluate the regulation of Ang II receptors by estradiol (E(2)) and progesterone (P) in areas of the brain involved in homeostatic and reproductive functions, such as the locus coeruleus (LC), median preoptic nucleus (MnPO) and subfornical organ (SFO). Adult female rats were ovariectomized under anesthesia and divided into 2 groups after 2 weeks: OVX plus E(2)/P replacement (OVXE(2)P) and OVX plus oil vehicle (OVX). E(2) was injected for 3 consecutive days followed by an injection of P on the 4th day. Animals were killed by decapitation and the brains were removed and frozen. Consecutive coronal brain sections were cut in a cryostat and Ang II receptors were quantified by autoradiography in the MnPO, LC and SFO. Treatment of OVX rats with E(2) and P induced a significant increase in the Ang II receptor binding (fmol/mg protein) in the MnPO (OVX: 4.48 +/- 0.58 and OVXE(2)P: 9.89 +/- 1.65), LC (OVX: 2.72 +/- 0.37 and OVXE(2)P: 8.03 +/- 0.9) and SFO (OVX: 5.45 +/- 0.66 and OVXE(2)P: 10.73 +/- 1.79) compared to OVX animals treated with the vehicle, P < 0.05. In conclusion, these results show that Ang II receptors are upregulated by E(2) and P in the LC, MnPO and SFO of ovariectomized rats.

Our objective was to study in which situations the median preoptic nucleus (MnPO) interferes with the control of oxytocin secretion and salt intake and the possible mediation of angiotensin II (AII) through their AT1 receptors. Lesion of the MnPO by ibotenic acid in male rats did not change water and NaCl intake in conditions of ad libitum offer, water deprivation or salt load, but it did cause significant decrease of NaCl intake in sodium depleted animals. These animals presented higher water intake or lower NaCl intake after microinjection of AII or losartan into the MnPO, respectively. They decreased plasma oxytocin after microinjection of losartan into the MnPO, but not of AII or isotonic saline. Oxytocin secretion induced by hypertonic saline i.p. was reduced by microinjection of AII, but not losartan into the MnPO. On the other hand, microinjection of losartan in this area, but not AII, reduced plasma oxytocin in animals submitted to the isotonic saline i.p. Thus, it seems that the sodium intake control is performed by MnPO neurons through the stimulatory action of angiotensin II on AT1 receptors under sodium depletion, but not water deprivation or salt overload neither of ad libitum water and salt intake condition. On the other hand, in the high-sodium condition, endogenous angiotensin did not act on MnPO neurons to the control of oxytocin secretion while exogenous angiotensin inhibited oxytocin secretion. These results indicate two possible angiotensinergic neural circuits: one is stimulating and the other is inhibiting oxytocin secretion, depending on sodium balance.

Due to the stimulatory action of serotonin (5HT) and nitric oxide (NO) on the secretion of gonadotropins and PRL, this work aimed at investigating the participation of serotoninergic receptors 5HT(1) and 5HT(2) of the medial preoptic area (MPOA) in the control of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) secretion and the possible modulation by ovarian steroids as well as the possible participation of NO as a mediator of the stimulatory effects of serotonin in the MPOA on LH secretion. Microinjections of three different doses (0.02, 0.2, and 2 ug) of methiothepin, a serotoninergic 5HT(1) antagonist or ketanserin, a seretoninergic 5HT(2) antagonist, were carried out into the MPOA in ovariectomized rats treated or not with estrogen or estrogen plus progesterone. Other groups of ovariectomized rats treated with estrogen, estrogen plus progesterone or vehicle were prepared to evaluate NOS activity in the MPOA. Plasma LH, FSH, and PRL in ovariectomized rats were not altered by the microinjection of methiothepin or ketanserin in the MPOA. Methiothepin microinjection in the MPOA reduced LH but did not change plasma FSH and PRL in ovariectomized rats treated with estrogen or estrogen plus progesterone. On the other hand, ketanserin microinjection in the MPOA reduced plasma LH and FSH but did not change plasma PRL in the animals submitted to the same steroidal treatment. NOS activity in the MPOA was significantly reduced by methiothepin or ketanserin in ovariectomized rats treated with estrogen or estrogen plus progesterone. In conclusion, this work showed that in the studied conditions, serotonin in the MPOA: (1) does not work in the control of PRL secretion through 5HT(1) and 5HT(2) receptors; (2) integrates the control of FSH secretion by 5HT(2) receptors, but not 5HT(1); (3) in the presence of estrogen, stimulates LH secretion by 5HT(1) and 5HT(2) receptors, which can be differentially modulated by progesterone; (4) at least partly, stimulates LH secretion by nitric oxide activity.

We tested the hypothesis that Locus Coeruleus (LC) inputs to the medial preoptic area (MPOA) and median eminence (ME) are essential for gonadotropin release. Proestrus and ovariectomized (OVX) rats were decapitated at 16:00 h. LC electrolytic lesion was performed at 11:00 h during proestrus and 24h before decapitation in OVX rats. Plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured and MPOA and ME were microdissected for LHRH content measurement. In addition, FOS protein in LC and MPOA were studied in proestrus and OVX rats at 12:00, 15:00, and 17:00 h. On proestrus, LC lesion blocked the LH surge and only decreased plasma FSH; in OVX rats the lesion induced only a slight decrease on plasma LH without affecting FSH secretion. An increased content of LHRH in the MPOA and ME of both groups accompanied the decreases of plasma LH. In proestrus, the number of FOS-immunoreactive (FOS-ir) neurons increased from 12:00 to 17:00 h in the LC and MPOA. In OVX rats, there was an increase at 15:00 h in the LC and a decrease at 17:00 h in both areas. The number of FOS-ir neurons was lower in OVX than in proestrus animals. Thus, LC (1) is responsible, at least in part, for gonadotropin release through the activation of LHRH neurons, (2) is more closely related to the positive than the negative feedback, and (3) seems to show an intrinsic cyclic activity which is amplified by ovarian steroids.