Estradiol and the feeding state modulate the interaction between leptin and the nitrergic system in female rats.

Leptin mediates the interaction between reproductive function and energy balance. However, leptin receptors are not expressed in neurons that produce gonadotropin-releasing hormone (GnRH), likely indicating an indirect action through interneurons. Among likely neurons that modulate the secretion of GnRH are NO (nitric oxide) neurons. We assessed whether estradiol and feeding conditions modulate a possible interaction between leptin and NO in brain areas related to the control of reproductive function. Estradiol-treated and untreated ovariectomized rats were normally fed or fasted for 48 h. Then, saline (control) or leptin (3 µg/1 µl) intracerebroventricular microinjections were administered, and after thirty minutes, the brains collected subsequent to the decapitation or transcardially perfusion. Leptin and estradiol increased NO synthase (nNOS) gene expression (RT-PCR) and content (Western blotting) in the medial preoptic area (MPOA) and medial basal hypothalamus (MBH) only in fasted rats. Leptin increased: 1-phosphorylated-signal transducer and activator of transcription-3(pSTAT3) (immunohistochemistry) in the MPOA and various hypothalamic nuclei [arcuate (ARC); ventromedial (VMH); dorsal/ventral dorsomedial (dDMH/vDMH); premammilar ventral (PMV)], effects potentiated by estradiol/fasting interaction; 2- nNOS/pSTAT3 coexpression in the MPOA only in estradiol-treated, fasted rats; 3- nNOS-immunoreactive cell expression in the VMH, DMH and PMV (areas related to reproductive function control) of estradiol -treated rats. Thus, when leptin is reduced during fasting, leptin replacement effectively increased the expression of nitric oxide, which activated the HPG axis only in the presence of estradiol. Estradiol modulates the nitrergic system, leptin sensitivity and consequently leptin's effects on the nitrergic system in hypothalamus and in particular vDMH and PMV.

Leptin actions through the nitrergic system to modulate the hypothalamic expression of the kiss1 mRNA in the female rat.

Gonadotrophin-releasing hormone (GnRH) is the main controller of the reproductive axis and stimulates the synthesis and secretion of gonadotrophins. Estrogen is the main peripheral factor controlling GnRH secretion, and this action is mainly mediated by the transsynaptic pathway through nitric oxide, kisspeptin, leptin, among other factors. Kisspeptin is the most potent factor known to induce GnRH release. Nitric oxide and leptin also promote GnRH release; however, neurons expressing GnRH do not express the leptin receptor (OB-R). Leptin seems to modulate the expression of genes and proteins involved in the kisspeptin system. However, few kisspeptin-synthesizing cells in the arcuate nucleus (ARC) and few cells, if any, in the preoptic area (POA) express OB-R; this indicates an indirect mechanism of leptin action on kisspeptin. Nitric oxide is an important intermediate in the actions of leptin in the central nervous system. Thus, this work aimed to verify the numbers of nNOS cells were activated by leptin in different hypothalamic areas; the modulatory effects of the nitrergic system on the kisspeptin system; and the indirect regulatory effect of leptin on the kisspeptin system via nitric oxide. Ovariectomized rats were treated with estrogen or a vehicle and received an intracerebroventricular (i.c.v.) injection of a nitric oxide donor, leptin or neuronal nitric oxide synthase (nNOS) enzyme inhibitor. Thirty minutes after the injection, the animals were decapitated. Leptin acts directly on nitrergic neurons in different hypothalamic regions, and the effects on the ventral premammillary nucleus (PMV) and ventral dorsomedial hypothalamus (vDMH) are enhanced. The use of a nitric oxide donor or the administration of leptin stimulates the expression of the kisspeptin mRNA in the ARC of animals with or without estrogenic action; however, these changes are not observed in the POA. In addition, the action of leptin on the expression of the kisspeptin mRNA in the ARC is blocked by a nitric oxide synthesis inhibitor. We concluded that the effects of leptin on the central nervous system are at least partially mediated by the nitrergic system. Also, nitric oxide acts on the kisspeptin system by modulating the expression of the kisspeptin mRNA, and leptin at least partially modulates the kisspeptin system through the nitrergic system, particularly in the ARC.

Gonadotrophic, prolactin, corticosterone, and gonadal hormones levels over 15 months in Giant Amazon River Turtles - Podocnemis expansa (Schweigger, 1812) (Testudines: Podocnemididae), in captive conditions / Gonadotrofinas, prolactina, corticosterona, e as hormonas sexuais durante 15 meses de Tartarugas da Amazônia - Podocnemis expansa (Schweigger, 1812) (Testudines: Podocnemididae), em condições de cativeiro

Abstract In order to achieve successful captive breeding the Podocnemis expansa, it is necessary to study their reproductive endocrinology. The purpose of this research was to evaluate and characterize plasma concentrations in gonadotrophic, gonadic, corticosterone and prolactin hormones from Giant Amazon Turtles under captive conditions. Blood samples were collected over a 15 month period. The samples were assayed by the use of radioimmunoassay, prolactin, corticosterone, LH, FSH, testosterone, 17β-estradiol and progesterone. We verified significant seasonal pattern increase in 17β-estradiol levels and decrease in progesterone levels in the course of a year, which indicates vitellogenesis. This is related to normal ovarian cycles and possibly to the functional integrity of the hypothalamus-pituitary-gonad axis of captive females. There were negative correlations between testosterone and corticosterone in the male samples, suggestive of stress (management stress) on the reproductive system. The plasma concentrations of gonadotrophic, gonadic, prolactin and corticosterone hormones may be used as a reference for further research and possible therapeutic approaches. The data collected during this research are unprecedented for this species and may serve as a reference for future research regarding the reproductive cycle of this turtle, also allowing reproductive management while in captivity. Information about these hormones must be gathered from wild populations during different periods of the year for better clarification of the reproductive physiology of this species.

Resumo Com o objetivo de obter reprodução em cativeiro de Podocnemis expansa, é necessário reunir o conhecimento a respeito de sua endocrinologia reprodutiva. O objetivo deste trabalho foi avaliar e caracterizar as concentrações plasmáticas de hormônios gonadotróficos, gonadais, corticosterona e prolactina em Tartarugas da Amazônia em condições de cativeiro. Amostras de sangue foram coletadas durante 15 meses. As amostras foram ensaiadas pelo uso de um radioimunoensáio, prolactina, corticosterona, LH, FSH, testosterona, 17β-estradiol e progesterona. Verificou-se aumento de padrão sazonal significativo nos níveis de 17β-estradiol e diminuição dos níveis de progesterona ao longo do ano, o que indica o recrutamento folicular. Isto está relacionado com ciclos ovarianos normais e possivelmente para a integridade funcional do eixo hipotálamo-hipófise-gônadas de fêmeas em cativeiro. Houve correlação negativa entre testosterona e corticosterona nas amostras do sexo masculino, sugestivos de efeito do estresse de manejo sobre o sistema reprodutivo. As concentrações plasmáticas de hormônios gonadotrofinas, gonadais, prolactina e hormônios corticosterona pode ser usado como referência para futuras pesquisas e possíveis abordagens terapêuticas. Os dados médios coletados durante a pesquisa são inéditos para a espécie e pode servir como referência para futuras pesquisas sobre o sistema reprodutivo da tartaruga, também permitindo manejo reprodutivo em cativeiro. Informações sobre esses hormônios devem ser recolhidas a partir de natureza selvagem em diferentes períodos do ano para melhor esclarecimento da fisiologia da reprodução desta espécie.

Gonadotrophic, prolactin, corticosterone, and gonadal hormones levels over 15 months in Giant Amazon River Turtles - Podocnemis expansa (Schweigger, 1812) (Testudines: Podocnemididae), in captive conditions.

In order to achieve successful captive breeding the Podocnemis expansa, it is necessary to study their reproductive endocrinology. The purpose of this research was to evaluate and characterize plasma concentrations in gonadotrophic, gonadic, corticosterone and prolactin hormones from Giant Amazon Turtles under captive conditions. Blood samples were collected over a 15 month period. The samples were assayed by the use of radioimmunoassay, prolactin, corticosterone, LH, FSH, testosterone, 17ß-estradiol and progesterone. We verified significant seasonal pattern increase in 17ß-estradiol levels and decrease in progesterone levels in the course of a year, which indicates vitellogenesis. This is related to normal ovarian cycles and possibly to the functional integrity of the hypothalamus-pituitary-gonad axis of captive females. There were negative correlations between testosterone and corticosterone in the male samples, suggestive of stress (management stress) on the reproductive system. The plasma concentrations of gonadotrophic, gonadic, prolactin and corticosterone hormones may be used as a reference for further research and possible therapeutic approaches. The data collected during this research are unprecedented for this species and may serve as a reference for future research regarding the reproductive cycle of this turtle, also allowing reproductive management while in captivity. Information about these hormones must be gathered from wild populations during different periods of the year for better clarification of the reproductive physiology of this species.

Pro-inflammatory cytokines, IL-1ß and TNF-α, produce persistent compromise in tonic immobility defensive behaviour in endotoxemia guinea-pigs.

AIM: Sepsis has been associated with acute behavioural changes in humans and rodents, which consists of a motivational state and an adaptive response that improve survival. However, the involvement of peripheral cytokines synthesized during systemic inflammation as modulators of the tonic immobility (TI) defensive behaviour remains a literature gap. Our purposes were to characterize the TI defensive behaviour in endotoxemia guinea-pigs at acute phase and after recovery from the initial inflammatory challenge. Furthermore, we investigated whether peri-aqueductal grey matter (PAG) exists as a brain structure related to this behaviour and also pro-inflammatory cytokines, tumour necrosis factor (TNF)-α and interleukin (IL)-1ß, act at this mesencephalic nucleus. METHODS: Endotoxemia was induced by lipopolysaccharide (LPS) administration in guinea-pigs. The parameters evaluated included TI defensive behaviour, survival, cytokines production, as well as neuronal activation and apoptosis in the PAG. RESULTS: Endotoxemia guinea-pigs exhibited a reduction in the duration of TI episodes, starting at 2 h after LPS administration and persisting throughout the experimental period evaluated over 7 days. Moreover, endotoxemia increased the c-FOS immunoreactivity of neurones in the ventrolateral PAG (vlPAG), as well as the caspase-3 expression. The LPS microinjection into vlPAG reproduces the same compromise, that is a decrease in the duration of TI defensive behaviour, observed after the peripheral administration. The immunoneutralization against IL-1ß and TNF-α into vlPAG reverts all the effects produced by peripheral LPS administration. CONCLUSION: Our findings confirm that vlPAG is an important brain structure involved in the behavioural alterations induced by endotoxemia, possibly changing the neuronal activity caused by pro-inflammatory cytokines produced peripherally.

Hypothalamic Effects of Tamoxifen on Oestrogen Regulation of Luteinising Hormone and Prolactin Secretion in Female Rats.

Oestradiol (E2) acts in the hypothalamus to regulate luteinising hormone (LH) and prolactin (PRL) secretion. Tamoxifen (TX) has been extensively used as a selective oestrogen receptor modulator, although its neuroendocrine effects remain poorly understood. In the present study, we investigated the hypothalamic effects of TX in rats under low or high circulating E2 levels. Ovariectomised (OVX) rats treated with oil, E2 or TX, or E2 plus TX, were evaluated for hormonal secretion and immunohistochemical analyses in hypothalamic areas. Both E2 and TX reduced LH levels, whereas TX blocked the E2 -induced surges of LH and PRL. TX prevented the E2 -induced expression of progesterone receptor (PR) in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), although it did not alter PR expression in OVX rats. TX blocked the E2 induction of c-Fos in AVPV neurones, consistent with the suppression of LH surge. However, TX failed to prevent E2 inhibition of kisspeptin expression in the ARC. In association with the blockade of PRL surge, TX increased the phosphorylation of tyrosine hydroxylase (TH) in the median eminence of OVX, E2 -treated rats. TX also precluded the E2 -induced increase in TH expression in the ARC. In all immunohistochemical analyses, TX treatment in OVX rats caused no measurable effect on the hypothalamus. Thus, TX is able to prevent the positive- but not negative-feedback effect of E2 on the hypothalamus. TX also blocks the effects of E2 on tuberoinfundibular dopaminergic neurones and PRL secretion. These findings further characterise the anti-oestrogenic actions of TX in the hypothalamus and provide new information on the oestrogenic regulation of LH and PRL.

Estrogen-dependent post-translational change in the nitric oxide system may mediate the leptin action on LH and prolactin secretion.

Gonadotrophin-releasing hormone (GnRH) neurons do not express the leptin receptor (OB-R) in the medial preoptic area (MPOA) and the medial basal hypothalamus (MBH). We assessed whether the effect of leptin on the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in proestrus could be mediated by nitric oxide (NO) under estrogen modulation. Female rats were treated with an estrogen antagonist (tamoxifen s.c. 3mg/rat) or vehicle during metestrus and diestrus. At proestrus, they received leptin (3 or 10 µg/µl) or intracerebroventricular saline at 11:00 am and were decapitated at 5:00 pm. The following were analyzed in this work: plasma LH, FSH and PRL levels (radioimmunoassay); neuronal NO-synthase (nNOS) and OB-R transcription (RT-PCR); nNOS and phosphorylated nNOS (pnNOS) translation levels (western blotting); and pSTAT3 immunoreactivity. Tamoxifen reduced the plasma LH and PRL levels and decreased the nNOS mRNA and pnNOS expression in the MPOA. Three micrograms of leptin increased the LH secretion and pnNOS protein levels in the MPOA and MBH. Ten micrograms of leptin decreased the transcription, translation and phosphorylation of nNOS in the MPOA. In the MBH, 10 µg of leptin increased the protein expression of nNOS but not the mRNA expression neither pnNOS protein. Tamoxifen did not change either the mRNA or protein expression of nNOS or the phosphorylation of nNOS but decreased the number of cells that contained pSTAT3 immunoreactivity in both areas. In conclusion, the stimulatory effect of leptin on the secretion of LH and PRL on the afternoon of proestrus may be mediated by estrogen-dependent post-translational changes in the nNOS in the MPOA and MBH.

Modulation of the activity of vasopressinergic neurons by estrogen in rats refed with normal or sodium-free food after fasting.

Feeding increases plasma osmolality and ovarian steroids may influence the balance of fluids. Vasopressin (AVP) neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) express estrogen receptor type ß (ERß), but not estrogen receptor type α (ERα). The circumventricular organs express ERα and project efferent fibers to the PVN and SON. Our aim was to assess whether interactions exist between food state-related osmolality changes and the action of estrogen on AVP neuron activity and estrogen receptor expression. We assessed plasma osmolality and AVP levels; fos-coded protein (FOS)- and AVP-immunoreactivity (-IR) and FOS-IR and ERα-IR in the median preoptic nucleus (MnPO) and organ vasculosum lamina terminalis (OVLT) in estrogen-primed and unprimed ovariectomized rats under the provision of ad libitum food, 48h of fasting, and subsequent refeeding with standard chow or sodium-free food. Refeeding with standard chow increased plasma osmolality and AVP as well as the co-expression of FOS-IR/AVP-IR in the PVN and SON. These responses were not altered by estrogen, with the exception of the decreases in FOS-IR/AVP-IR in the lateral PVN. During refeeding, estrogen modulates only a subpopulation of AVP neurons in the lateral PVN. FOS-ERα co-expression in the ventral median preoptic nucleus (vMnPO) was reduced by estrogen and increased after refeeding with standard chow following fasting. It appears that estrogen may indirectly modulate the activity of AVP neurons, which are involved in the mechanism affected by hyperosmolality-induced refeeding after fasting. This indirect action of estrogen can be at least in part via ERα in the vMnPO.

Kisspeptin regulates tuberoinfundibular dopaminergic neurones and prolactin secretion in an oestradiol-dependent manner in male and female rats.

Prolactin (PRL) secretion is inhibited by hypothalamic dopamine. Kisspeptin controls luteinising hormone (LH) secretion and is also involved in PRL regulation. We further investigated the effect of kisspeptin-10 (Kp-10) on the activity of tuberoinfundibular dopaminergic (TIDA) neurones and the role of oestradiol (E2 ) in this mechanism. Female and male rats were injected with i.c.v. Kp-10 and evaluated for PRL release and the activity of dopamine terminals in the median eminence (ME) and neurointermediate lobe of the pituitary (NIL). Kp-10 at the doses of 0.6 and 3 nmol increased plasma PRL and decreased 4-dihydroxyphenylacetic acid (DOPAC) levels in the ME and NIL of ovariectomised (OVX), E2 -treated rats but had no effect in OVX. In gonad-intact males, 3 nmol Kp-10 increased PRL secretion and decreased DOPAC levels in the ME but not in the NIL. Castrated males treated with either testosterone or E2 also displayed increased PRL secretion and reduced ME DOPAC in response to Kp-10, whereas castrated rats receiving oil or dihydrotestosterone were unresponsive. By contrast, the LH response to Kp-10 was not E2 -dependent in either females or males. Additionally, immunohistochemical double-labelling demonstrated that TIDA neurones of male rats contain oestrogen receptor (ER)-α, with a higher proportion of neurones expressing ERα than in dioestrous females. The dopaminergic neurones of periventricular hypothalamic nucleus displayed much lower ERα expression. Thus, TIDA neurones express ERα in male and female rats, and kisspeptin increases PRL secretion through inhibition of TIDA neurones in an E2 -dependent manner in both sexes. These findings provide new evidence about the role of kisspeptin in the regulation of dopamine and PRL.

Stress in neonatal rats with different maternal care backgrounds: monoaminergic and hormonal responses.

The first 2 weeks of life in rats are known as the stress hyporesponsive period because stress responses in pups are diminished as compared to adult animals. However, it is considered a critical period in development in which infant rats are susceptible to environmental events, such as stressful stimuli and quality of maternal care received. These early life events have long-lasting effects, shaping a variety of outcomes, such as stress responsivity. This study investigated the effects of maternal care and sex differences on the response to an aversive stimulus in rat pups from high (HL) and low licking (LL) mothers. Plasma corticosterone, oxytocin (OT), and central monoaminergic activity in 13-day-old rats submitted to cold stress were analyzed. Stress increased plasma corticosterone and marginally decreased hypothalamic dihydroxyphenylacetic acid/dopamine ratio. HL pups showed higher levels of plasma OT than LL pups. The maternal effect was also detected in the hippocampus, in which 5-hydroxyindole-3-acetic acid/serotonin ratio was increased in HL pups, independently of the sex and stress. Investigating the early life events is useful not only into understand the neurobiological and hormonal mechanisms underlying maternal and stressful influences on infant development into a healthy or psychopathological adult phenotype, but also to unveil the immediate outcomes on infancy.

Stress in Neonatal Rats with Different Maternal Care Backgrounds: Monoaminergic and Hormonal Responses.

The first 2 weeks of life in rats are known as the stress hyporesponsive period because stress responses in pups are diminished as compared to adult animals. However, it is considered a critical period in development in which infant rats are susceptible to environmental events, such as stressful stimuli and quality of maternal care received. These early life events have long-lasting effects, shaping a variety of outcomes, such as stress responsivity. This study investigated the effects of maternal care and sex differences on the response to an aversive stimulus in rat pups from high (HL) and low licking (LL) mothers. Plasma corticosterone, oxytocin, and central monoaminergic activity in 13-day-old rats submitted to cold stress were analyzed. Stress increased plasma corticosterone and marginally decreased hypothalamic dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratio. HL pups showed higher levels of plasma oxytocin than LL pups. The maternal effect was also detected in the hippocampus, in which 5-hydroxyindole-3-acetic acid/serotonin (5-HIAA/5-HT) ratio was increased in HL pups, independently of the sex and stress. Investigating the early life events is useful not only into understand the neurobiological and hormonal mechanisms underlying maternal and stressful influences on infant development into a healthy or psychopathological adult phenotype, but also to unveil the immediate outcomes on infancy.

Prevention of metabolic disorders and reproductive performance deficits by the blockade of Angiotensin II AT1 receptor in female rats fed with cafeteria diet.

Besides the well-known detrimental effects of obesity on cardiovascular and metabolic function, studies have shown that obesity is also associated with impaired reproductive function in women. Alterations in Angiotensin II (Ang II) have been associated with obesity and with female reproduction. The aim of the present study was to evaluate the reproductive and metabolic effects of Ang II AT1 receptor blockade with losartan in an animal model of obesity, in which female rats were offered a palatable, high calorie diet from weaning to adulthood. Sexual behavior, ovulation rates and preovulatory levels of the hormones estradiol, progesterone, LH and prolactin were analyzed. Retroperitoneal and perigonadal fat pads, triglycerides and cholesterol (total, HDL and LDL), and insulin resistance were analyzed. Losartan prevented increases in fat pad storage, insulin resistance, as well as triglycerides and LDL levels induced by cafeteria diet intake. Losartan also prevented ovulatory deficits and loss of preovulatory surges of progesterone and LH in cafeteria-fed female rats probably through the prevention of the increase in body weight and body fat. No alterations in sexual behavior were observed. These results suggest, for the first time, that Ang II contributes to the development of the deleterious effects of obesity on preovulatory surges of LH and progesterone and on the reduction of ovulation in obese female rats.

Lesions of the ventral premammillary nucleus disrupt the dynamic changes in Kiss1 and GnRH expression characteristic of the proestrus-estrus transition.

We have recently demonstrated that the ventral premammillary nucleus (PMV) plays a key role in the metabolic control of the female reproductive axis. However, whether PMV neurons modulate the reproductive neural circuitry and/or the expression of sexual behaviors has not been determined. Here, we showed that the expression of estrogen and progesterone receptors in the PMV is modulated by changing levels of sex steroids across the estrous cycle. We also showed that sexual behavior, not the high physiologic levels of sex steroids, induces Fos in PMV neurons. Bilateral lesions of the PMV caused no significant changes in proceptive behavior but a high percentage of PMV-lesioned rats failed to exhibit lordosis behavior when exposed to a sexually experienced male rat (50% vs. 18% in the control group). Notably, lesions of the PMV disrupted the physiologic fluctuations of Kiss1 and GnRH mRNA expression characteristic of the proestrus-to-estrus transition. This neurochemical imbalance may ultimately alter female reproductive behavior. Our findings suggest that the PMV is a component of the neural circuitry that modulates the physiologic fluctuations of key neuroendocrine players (i.e., Kiss1 and GnRH) in the control of the female reproductive physiology.

Snail1 controls TGF-ß responsiveness and differentiation of mesenchymal stem cells.

The Snail1 transcriptional repressor plays a key role in triggering epithelial-to-mesenchymal transition. Although Snail1 is widely expressed in early development, in adult animals it is limited to a subset of mesenchymal cells where it has a largely unknown function. Using a mouse model with inducible depletion of Snail1, here we demonstrate that Snail1 is required to maintain mesenchymal stem cells (MSCs). This effect is associated to the responsiveness to transforming growth factor (TGF)-ß1 that shows a strong Snail1 dependence. Snail1 depletion in conditional knockout adult animals causes a significant decrease in the number of bone marrow-derived MSCs. In culture, Snail1-deficient MSCs prematurely differentiate to osteoblasts or adipocytes and, in contrast to controls, are resistant to the TGF-ß1-induced differentiation block. These results demonstrate a new role for Snail1 in TGF-ß response and MSC maintenance.

Transitory activation of the central and ovarian norepinephrine systems during cold stress-induced polycystic ovary in rats.

Cold stress-induced ovarian sympathetic activation is associated with the development of ovarian cysts in rats. Although we have hypothesised that polycystic ovary (PCO) features induced by cold stress, as prevented by lesion of the noradrenergic nucleus locus coeruleus (LC), were a result of the increased activity of the ovarian norepinephrine (NE) system, this was not evident after 8 weeks of stress. In the present study, we investigated the temporal changes in LC and ovarian NE activities and steroid secretion in rats exposed to single (SS) or repeated (RS) cold stress. SS and 4 week (4W)-RS but not 8 week (8W)-RS increased c-Fos expression in the LC and ovarian NE release. Plasma oestradiol, testosterone and progesterone levels tended to increase in 4W-RS and were elevated in 8W-RS rats, which displayed PCO morphology. ß-adrenergic receptor agonist increased steroid hormone release from the ovary of unstressed (US) but not from 8W-RS rats. To determine whether increased activity of noradrenergic system during the initial 4 weeks of RS would be sufficient to promote PCO, rats were exposed to 4 weeks of cold stress and kept in ambient temperature for the next 4 weeks (4W-RS/4W-US). Accordingly, PCO morphology, increased steroid secretion and decreased ovulation rate were found in 4W-RS/4W-US rats, strengthening the hypothesis that the initial increase in NE release triggers the development of PCO. The correlated activity of LC neurones and ovarian noradrenergic terminals and the induction of PCO in 4W-RS/4W-US rats provide functional evidence for a major role of NE in disrupting follicular development and causing the long-lasting endocrine abnormalities found in stress-induced PCO.

Effect of the interaction between food state and the action of estrogen on oxytocinergic system activity.

Increased plasma osmolality by food intake evokes augmentation of plasma oxytocin (OT). Ovarian steroids may also influence the balance of body fluids by acting on OT neurones. Our aim was to determine if estrogen influences the activity of OT neurones in paraventricular nucleus (PVN) and supraoptic nucleus (SON) under different osmotic situations. Ovariectomized rats (OVX) were treated with either estradiol (E(2)) or vehicle and were divided into three groups: group I was fed ad libitum, group II underwent 48 h of fasting, and group III was refed after 48 h of fasting. On the day of the experiment, blood samples were collected to determine the plasma osmolality and OT. The animals were subsequently perfused, and OT/FOS immunofluorescence analysis was conducted on neurones in the PVN and the SON. When compared to animals which were fasted or fed ad libitum, the plasma osmolality of refed animals was higher, regardless of whether they were treated with vehicle or E(2). We observed neural activation of OT cells in vehicle- or E(2)-treated OVX rats refed after 48 h of fasting, but not in animals fed ad libitum or in animals that only underwent 48 h of fasting. Finally, the percentage of neurones that co-expressed OT and FOS was lower in both the PVN and the SON of animals treated with E(2) and refed, when compared to vehicle-treated animals. These results suggest that E(2) may have an inhibitory effect on OT neurones and may modulate the secretion of OT in response to the increase of osmolality induced by refeeding.

Co-expression of leptin and oestrogen receptors in the preoptic-hypothalamic area.

The interaction between the reproductive axis and energy balance suggests that leptin acts as a possible mediator. This hormone acts in the regulation of metabolism, feeding behaviour and reproduction. Animals homozygous for the gene 'ob' (ob/ob) are obese and infertile, and these effects are reversed after systemic administration of leptin. Thus, the present study aimed to determine: (i) whether cells that express leptin also express oestrogen receptors of type-alpha (ER-alpha) or -beta (ER-beta) in the medial preoptic area (MPOA) and in the arcuate (ARC), dorsomedial (DMH) and ventromedial hypothalamic nucleus and (ii) whether there is change in the gene and protein expression of leptin in these brain areas in ovariectomised (OVX) animals when oestrogen-primed. Wistar female rats with normal oestrous cycles or ovariectomised oestrogen-primed or vehicle (oil)-primed were utilised. To determine whether there was a co-expression, immunofluorescence was utilised for double staining. Confocal microscopy was used to confirm the co-expression. The technique of real-time polymerase chain reaction and western blotting were employed to analyse gene and protein expression, respectively. The results obtained showed co-expression of leptin and ER-alpha in the MPOA and in the DMH, as well as leptin and ER-beta in the MPOA, DMH and ARC. However, we did not detect leptin in the MPOA, ARC and DMH using western blotting and there was no statistical difference in leptin gene expression in the MPOA, DMH, ARC, pituitary or adipose tissue between OVX rats treated with oestrogen or vehicle. In conclusion, the results obtained in the present study confirm that the brain is also a source of leptin and reveal co-expression of oestrogen receptors and leptin in the same cells from areas related to reproductive function and feeding behaviour. Although these data corroborate the previous evidence obtained concerning the interaction between the action of brain leptin and reproductive function, the physiological relevance of this interaction remains uncertain and additional studies are necessary to elucidate the exact role of central leptin.

Actions of angiotensin II and dopamine in the medial preoptic area on prolactin secretion.

Dopamine (DA) is known as a primary regulator of prolactin secretion (PRL) and angiotensin II (Ang II) has been recognized as one brain inhibitory factor of this secretion. In this work, estrogen-primed or unprimed ovariectomized rats were submitted to the microinjection of saline or Ang II after previous microinjection of saline or of DA antagonist (haloperidol, sulpiride or SCH) both in the medial preoptic area (MPOA). Our study of these interactions has shown that 1) estrogen-induced PRL secretion is mediated by Ang II and DA actions in the MPOA, i.e. very high plasma PRL would be prevented by inhibitory action of Ang II, while very low levels would be prevented in part by stimulatory action of DA through D(2) receptors, 2) the inhibitory action of Ang II depends on estrogen and is mediated in part by inhibitory action of DA through D(1) receptors and in other part by inhibition of stimulatory action of DA through D(2) receptors.

Presence of oxytocin receptors in the gonadotrophin-releasing hormone (GnRH) neurones in female rats: a possible direct action of oxytocin on GnRH neurones.

Gonadotrophin-releasing hormone (GnRH) neurones constitute the final output pathway of a neuronal network that controls the preovulatory luteinising hormone (LH) surge and ovulation. Throughout the reproductive cycle, several neurotransmitters stimulate and inhibit the activity of GnRH neurones, including oxytocin. The central administration of oxytocin antiserum abolishes the pro-oestrous LH surge whereas oxytocin stimulates GnRH secretion from hypothalamic explants suggesting an oxytocin central action. Within the GnRH neuronal population in the rat, GnRH cells in the medial preoptic area (MPOA) are activated at the time of the LH surge. Thus, we hypothesised that GnRH neurones in the MPOA may express oxytocin receptors, and that oxytocin neurones in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) may be differentially activated during the oestrous cycle. Oxytocin receptors mRNA was detected in the MPOA using reverse transcription-polymerase chain reaction. In animals in either metoestrus or pro-oestrus, double-label immunofluorescence indicated that approximately 10% of GnRH neurones in the MPOA coexpressed oxytocin receptors and that a few oxytocin fibres are located in the vicinity of these GnRH neurones. However, other neurones positive for the oxytocin receptors were found near GnRH neurones. At both oestrous stages, double-label immunofluorescence revealed that approximately 30% of oxytocin neurones in the SON were Fos-positive whereas oxytocin neurones in the PVN were consistently Fos-negative. Together, these data suggest that oxytocin may directly control neuronal activity in a subpopulation of GnRH neurones. Moreover, both oxytocin neuronal activity and the oxytocin receptor expression on GnRH cells are not influenced by oestrogen.