Cristiane M. Leite

Kisspeptin Regulates Prolactin Release through Hypothalamic Dopaminergic Neurons

Prolactin (PRL) is tonically inhibited by dopamine (DA) released from neurons in the arcuate and periventricular nuclei. Kisspeptin plays a pivotal role in LH regulation. In rodents, kisspeptin neurons are found mostly in the anteroventral periventricular and arcuate nuclei, but the physiology of arcuate kisspeptin neurons is not completely understood. We investigated the role of kisspeptin in the control of hypothalamic DA and pituitary PRL secretion in adult rats. Intracerebroventricular kisspeptin-10 (Kp-10) elicited PRL release in a dose-dependent manner in estradiol (E2)-treated ovariectomized rats (OVX+E2), whereas no effect was found in oil-treated ovariectomized rats (OVX). Kp-10 increased PRL release in males and proestrous but not diestrous females. Associated with the increase in PRL release, intracerebroventricular Kp-10 reduced Fos-related antigen expression in tyrosine hydroxylase-immunoreactive (ir) neurons of arcuate and periventricular nuclei in OVX+E2 rats, with no effect in OVX rats. Kp-10 also decreased 3,4-dihydroxyphenylacetic acid concentration and 3,4-dihydroxyphenylacetic acid-DA ratio in the median eminence but not striatum in OVX+E2 rats. Double-label immunofluorescence combined with confocal microscopy revealed kisspeptin-ir fibers in close apposition to and in contact with tyrosine hydroxylase-ir perikarya in the arcuate. In addition, Kp-10 was not found to alter PRL release from anterior pituitary cell cultures regardless of E2 treatment. We provide herein evidence that kisspeptin regulates PRL release through inhibition of hypothalamic dopaminergic neurons, and that this mechanism is E2 dependent in females. These findings suggest a new role for central kisspeptin with possible implications for reproductive physiology.

Locus Coeruleus Mediates Cold Stress-Induced Polycystic Ovary in Rats

Previous reports about the rat ovary have shown that cold stress promotes ovarian morphological alterations related to a polycystic ovary (PCO) condition through activation of the ovarian sympathetic nerves. Because the noradrenergic nucleus locus coeruleus (LC) is activated by cold stress and synaptically connected to the preganglionic cell bodies of the ovarian sympathetic pathway, this study aimed to evaluate the LCs role in cold stress-induced PCO in rats. Ovarian morphology and endocrine and sympathetic functions were evaluated after 8 wk of chronic intermittent cold stress (4 C, 3 h/d) in rats with or without LC lesion. The effect of acute and chronic cold stress upon the LC neuron activity was confirmed by Fos protein expression in tyrosine hydroxylase-immunoreactive neurons. Cold stress induced the formation of follicular cysts, type III follicles, and follicles with hyperthecosis alongside increased plasma estradiol and testosterone levels, irregular estrous cyclicity, and reduced ovulation. Considering estradiol release in vitro, cold stress potentiated the ovarian response to human chorionic gonadotropin. Ovarian norepinephrine (NE) was not altered after 8 wk of stress. However, LC lesion reduced NE activity in the ovary of cold-stressed rats, but not in controls, and prevented all the cold stress effects evaluated. Cold stress increased the number of Fos/tyrosine hydroxylase-immunoreactive neurons in the LC, but this effect was more pronounced for acute stress as compared with chronic stress. These results show that cold stress promotes PCO in rats, which apparently depends on ovarian NE activity that, under this condition, is regulated by the noradrenergic nucleus LC.

Role of sex steroids in progesterone and corticosterone response to acute restraint stress in rats: sex differences

Abstract Adrenal progesterone secretion increases along with corticosterone in response to stress in male and female rats to modulate some stress responses. Here we investigated the role of sex steroids in sex differences in the progesterone response to 60 min of restraint stress in adult male and female rats. Comparisons between males and females in the progesterone response were evaluated in parallel with corticosterone responses. From day 5 to 7 after gonadectomy, female and male rats were treated with estradiol or testosterone, respectively (OVX-E and ORCH-T groups), or oil (OVX and ORCH groups). Female rats in proestrus, intact and 7 d adrenalectomized (ADX) male rats were also studied. At 10:00 h, blood samples were withdrawn via an implanted jugular cannula before (−5 min), during (15, 30, 45, 60 min) and after (90 and 120 min) restraint stress to measure plasma progesterone and corticosterone concentrations by radioimmunoassay. Intact male and proestrus female rats exhibited similar progesterone responses to stress. Gonadectomy did not alter the amount of progesterone secreted during stress in female rats but decreased secretion in male rats. Unlike corticosterone, the progesterone response to stress in females was not influenced by estradiol. In males, testosterone replacement attenuated the progesterone and corticosterone responses to stress. Basal secretion of progesterone among intact, ORCH and ADX males was similar, but ADX-stressed rats secreted little progesterone. Hence, the gonads differently modulate adrenal progesterone and corticosterone responses to stress in female and male rats. The ovaries enhance corticosterone but not progesterone secretion, while the testes stimulate progesterone but not corticosterone secretion.

Release of norepinephrine in the preoptic area activates anteroventral periventricular nucleus neurons and stimulates the surge of luteinizing hormone.

The role of norepinephrine (NE) in regulation of LH is still controversial. We investigated the role played by NE in the positive feedback of estradiol and progesterone. Ovarian-steroid control over NE release in the preoptic area (POA) was determined using microdialysis. Compared with ovariectomized (OVX) rats, estradiol-treated OVX (OVX+E) rats displayed lower release of NE in the morning but increased release coincident with the afternoon surge of LH. OVX rats treated with estradiol and progesterone (OVX+EP) exhibited markedly greater NE release than OVX+E rats, and amplification of the LH surge. The effect of NE on LH secretion was confirmed using reverse microdialysis. The LH surge and c-Fos expression in anteroventral periventricular nucleus neurons were significantly increased in OVX+E rats dialyzed with 100 nm NE in the POA. After Fluoro-Gold injection in the POA, c-Fos expression in Fluoro-Gold/tyrosine hydroxylase-immunoreactive neurons increased during the afternoon in the A2 of both OVX+E and OVX+EP rats, in the locus coeruleus (LC) of OVX+EP rats, but was unchanged in the A1. The selective lesion of LC terminals, by intracerebroventricular N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, reduced the surge of LH in OVX+EP but not in OVX+E rats. Thus, estradiol and progesterone activate A2 and LC neurons, respectively, and this is associated with the increased release of NE in the POA and the magnitude of the LH surge. NE stimulates LH secretion, at least in part, through activation of anteroventral periventricular neurons. These findings contribute to elucidation of the role played by NE during the positive feedback of ovarian steroids.

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.

Alpha-oestrogen and progestin receptor expression in the hypothalamus and preoptic area dopaminergic neurones during oestrous in cycling rats.

A secretory surge of prolactin occurs on the afternoon of oestrous in cycling rats. Although prolactin is regulated by ovarian steroids, plasma oestradiol and progesterone levels do not vary during oestrous. Because prolactin release is tonically inhibited by hypothalamic dopamine and modulated by dopamine transmission in the preoptic area (POA), the present study aimed to evaluate whether oestrogen receptor (ER)‐α and progestin receptor (PR) expression in the dopaminergic neurones of arcuate (ARC), periventricular, anteroventral periventricular (AVPe) and ventromedial preoptic (VMPO) nuclei changes during the day of oestrous. Cycling rats were perfused every 2 h from 10–20 h on oestrous. Brain sections were double‐labelled to ERα or PR and tyrosine hydroxylase (TH). The number of TH‐immunoreactive (ir) neurones did not vary significantly in any area evaluated. ERα expression in TH‐ir neurones increased at 14 and 16 h in the rostral‐ARC and dorsomedial‐ARC, 14 h in the caudal‐ARC and 16 h in the VMPO, whereas it was unaltered in the ventrolateral‐ARC, periventricular and AVPe. PR expression in TH‐ir neurones of the periventricular and rostral, dorsomedial, ventrolateral and caudal‐ARC decreased transitorily during the afternoon, showing the lowest levels between 14 and 16 h; but it did not vary in the AVPe and VMPO. Plasma oestradiol and progesterone concentrations were low and unaltered during oestrous, indicating that the changes in receptors expression were probably not due to variation in ligand levels. Thus, our data suggest that variations in ERα and PR expression may promote changes in the activity of medial basal hypothalamus and POA dopaminergic neurones, even under unaltered secretion of ovarian steroids, which could facilitate the occurrence and modulate the magnitude of the prolactin surge on oestrous.

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. β‐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.

The Increase in Signaling by Kisspeptin Neurons in the Preoptic Area and Associated Changes in Clock Gene Expression That Trigger the LH Surge in Female Rats Are Dependent on the Facilitatory Action of a Noradrenaline Input

In rodents, kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) of the preoptic area are considered to provide a major stimulatory input to the GnRH neuronal network that is responsible for triggering the preovulatory LH surge. Noradrenaline (NA) is one of the main modulators of GnRH release, and NA fibers are found in close apposition to kisspeptin neurons in the RP3V. Our objective was to interrogate the role of NA signaling in the kisspeptin control of GnRH secretion during the estradiol induced LH surge in ovariectomized rats, using prazosin, an α1-adrenergic receptor antagonist. In control rats, the estradiol-induced LH surge at 17 hours was associated with a significant increase in GnRH and kisspeptin content in the median eminence with the increase in kisspeptin preceding that of GnRH and LH. Prazosin, administered 5 and 3 hours prior to the predicted time of the LH surge truncated the LH surge and abolished the rise in GnRH and kisspeptin in the median eminence. In the preoptic area, prazosin blocked the increases in Kiss1 gene expression and kisspeptin content in association with a disruption in the expression of the clock genes, Per1 and Bmal1. Together these findings demonstrate for the first time that NA modulates kisspeptin synthesis in the RP3V through the activation of α1-adrenergic receptors prior to the initiation of the LH surge and indicate a potential role of α1-adrenergic signaling in the circadian-controlled pathway timing of the preovulatory LH surge.

Activity of Hypothalamic Dopaminergic Neurones During the Day of Oestrus: Involvement in Prolactin Secretion

A secretory surge of prolactin occurs on the afternoon of oestrus in cycling rats. Pituitary prolactin is inhibited by dopamine. We evaluated the activity of the neuroendocrine dopaminergic neurones during oestrus and dioestrus, as determined by dopaminergic activity in the median eminence and neurointermediate lobe of the pituitary, as well as Fos‐related antigen expression in tyrosine hydroxylase (TH)‐immunoreactive (ir) neurones of the arcuate nucleus (ARC) and periventricular nucleus (Pe). During oestrus, the 4‐dihydroxyphenylacetic acid/dopamine ratio in the median eminence decreased at 16.00 h, coinciding with the increase in plasma prolactin levels. Similarly, the expression of Fos‐related antigen in TH‐ir neurones of Pe and rostral‐, dorsomedial‐ and caudal‐ARC also decreased at 16.00 h. On dioestrus, 4‐dihydroxyphenylacetic acid/dopamine ratio in the median eminence and Fos‐related antigen expression in TH‐ir neurones of Pe and rostral‐ARC decreased at 18.00 h, whereas prolactin levels were unaltered. No variation in dopaminergic activity was found in the neurointermediate lobe of the pituitary on either oestrus or dioestrus. The number of TH‐ir neurones in the ARC and parameters of dopaminergic activity were found to be generally lower on oestrus compared to dioestrus. The transitory decrease in the activity of neuroendocrine dopaminergic neurones temporally associated with the prolactin surge on the afternoon of oestrus suggests a role for dopamine in the generation of the oestrous prolactin surge.

Progesterone increased β-endorphin innervation of the locus coeruleus, but ovarian steroids had no effect on noradrenergic neurodegeneration

With the decline of ovarian steroids levels at menopause, many women experience an increase in anxiety and stress sensitivity. The locus coeruleus (LC), a central source of noradrenaline (NE), is activated by stress and is inhibited by β-endorphin. Moreover, increased NE has been implicated in pathological anxiety syndromes. Hormone replacement therapy (HRT) in menopause appears to decrease anxiety and vulnerability to stress. Therefore, we questioned the effect of HRT on the inhibitory β-endorphin innervation of the LC. In addition, we found that progesterone protects serotoninergic neurons in monkeys, leading us to question whether ovarian steroids are also neuroprotective in LC neurons in monkeys. Adult Rhesus monkeys (Macaca mulatta) were ovariectomized, and either treated with Silastic capsules that contained estradiol, estradiol+progesterone, progesterone alone or that were empty (ovariectomized; control). After 1month, the LC was obtained and processed for immunohistochemistry for β-endorphin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL). The density of β-endorphin axons was determined with image analysis using ImageJ. The TUNEL-positive neurons were counted in the entire LC. Progesterone-alone significantly increased the density of the β-endorphin axons in the LC (p<0.01). No significant differences between groups in the number of TUNEL-positive cells in the LC were found. In conclusion, we found that HRT increases the inhibitory influence of β-endorphin in the LC, which could, in turn, contribute to reduce anxiety and increase stress resilience. In addition, we did not find compelling evidence of neurodegeneration or neuroprotection by HRT in the LC of Rhesus monkeys.