The Herbicide Atrazine Potentiates Angiotensin II-Induced Aldosterone Synthesis and Release From Adrenal Cells.

Atrazine is one of the most commonly used pre-emergence and early post-emergence herbicides in the world. We have shown previously that atrazine does not directly stimulate the pituitary or adrenal to trigger hormone release but acts centrally to activate a stress-like activation of the hypothalamic-pituitary-adrenal axis. In doing so, atrazine treatment has been shown to cause adrenal morphology changes characteristic of repeated stress. In this study, adrenals from atrazine treated and stressed animals were directly compared after 4 days of atrazine treatment or restraint stress. Both atrazine and stressed animals displayed reduced adrenocortical zona glomerulosa thickness and aldosterone synthase (CYP11B2) expression, indicative of repeated adrenal stimulation by adrenocorticotropic hormone. To determine if reduced CYP11B2 expression resulted in attenuated aldosterone synthesis, stressed and atrazine treated animals were challenged with angiotensin II (Ang II). As predicted, stressed animals produced less aldosterone compared to control animals when stimulated. However, atrazine treated animals had higher circulating aldosterone concentrations compared to both stressed and control groups. Ang II-induced aldosterone release was also potentiated in atrazine pretreated human adrenocortical carcinoma cells (H295R). Atrazine pretreated did not alter the expression of the rate limiting steroidogenic StAR protein or angiotensin II receptor 1. Atrazine treated animals also presented with higher basal blood pressure than vehicle treated control animals suggesting sustained elevations in circulating aldosterone levels. Our results demonstrate that treatment with the widely used herbicide, atrazine, directly increases stimulated production of aldosterone in adrenocortical cells independent of expression changes to rate limiting steroidogenic enzymes.

Expression of Rasd1 in mouse endocrine pituitary cells and its response to dexamethasone.

Dexamethasone-induced Ras-related protein 1 (Rasd1) is a member of the Ras superfamily of monomeric G proteins that have a regulatory function in signal transduction. Rasd1, also known as Dexras1 or AGS1, is rapidly induced by dexamethasone (Dex). While prior data indicates that Rasd1 is highly expressed in the pituitary and that the gene may function in regulation of corticotroph activity, its exact cellular localization in this tissue has not been delineated. Nor has it been determined which endocrine pituitary cell type(s) are responsive to Dex-induced expression of Rasd1. We hypothesized that Rasd1 is primarily localized in corticotrophs and furthermore, that its expression in these cells would be upregulated in response to exogenous Dex administration. Rasd1 expression in each pituitary cell type both under basal conditions and 1-hour post Dex treatment were examined in adult male mice. While a proportion of all endocrine pituitary cell types expressed Rasd1, a majority of corticotrophs and thyrotrophs expressed Rasd1 under basal condition. In vehicle treated animals, approximately 50-60% of corticotrophs and thyrotrophs cells expressed Rasd1 while the gene was detected in only 15-30% of lactotrophs, somatotrophs, and gonadotrophs. In Dex treated animals, Rasd1 expression was significantly increased in corticotrophs, somatotrophs, lactotrophs, and gonadotrophs but not thyrotrophs. In Dex treated animals, Rasd1 was detected in 80-95% of gonadotrophs and corticotrophs. In contrast, Dex treatment increased Rasd1 expression to a lesser extent (55-60%) in somatotrophs and lactotrophs. Corticotrophs of the pars intermedia, which lack glucocorticoid receptors, failed to display increased Rasd1 expression in Dex treated animals. Rasd1 is highly expressed in corticotrophs under basal conditions and is further increased after Dex treatment, further supporting its role in glucocorticoid negative feedback. In addition, the presence and Dex-induced expression of Rasd1 in endocrine pituitary cell types, other than corticotrophs, may implicate Rasd1 in novel pituitary functions.

Characterization of Activation of the Hypothalamic-Pituitary-Adrenal Axis by the Herbicide Atrazine in the Female Rat.

Atrazine (ATR) is a commonly used pre-emergence and early postemergence herbicide. Rats gavaged with ATR and its chlorometabolites desethylatrazine (DEA) and deisopropylatrazine (DIA) respond with a rapid and dose-dependent rise in plasma corticosterone, whereas the major chlorometabolite, diaminochlorotriazine (DACT), has little or no effect on corticosterone levels. In this study, we investigated the possible sites of ATR activation of the hypothalamic-pituitary-adrenal (HPA) axis. ATR treatment had no effect on adrenal weights but altered adrenal morphology. Hypophysectomized rats or rats under dexamethasone suppression did not respond to ATR treatment, suggesting that ATR does not directly stimulate the adrenal gland to induce corticosterone synthesis. Immortalized mouse corticotrophs (AtT-20) and primary rat pituitary cultures were treated with ATR, DEA, DIA, or DACT. None of the compounds induced an increase in ACTH secretion or potentiated ACTH release in conjunction with CRH on ACTH release. In female rats gavaged with ATR, pretreatment with the CRH receptor antagonist astressin completely blocked the ATR-induced rise in corticosterone concentrations, implicating CRH release in ATR-induced HPA activation. Intracerebroventricular infusion of ATR, DEA, and DIA but not DACT at concentrations equivalent to peak plasma concentrations after gavage dosing resulted in an elevation of plasma corticosterone concentrations. However, ATR did not induce c-Fos immunoreactivity in the paraventricular nucleus of the hypothalamus. These results indicate that ATR activates the HPA axis centrally and requires CRH receptor activation, but it does not stimulate cellular pathways associated with CRH neuronal excitation.

Changes in hepatic phase I and phase II biotransformation enzyme expression and glutathione levels following atrazine exposure in female rats.

1. To determine the effects of repeated atrazine (ATR) treatment on hepatic phase I and II enzymes, adult female rats were treated with vehicle or 100 mg/kg of ATR for 1, 2, 3 or 4 days. Glutathione-s-transferases (GST) mRNA expression, protein levels (mu, pi, alpha, omega), and activity (cytosolic and microsomal), along with bioavailable glutathione (GSH) were assayed. 2. GST expression, concentrations and activity were increased, along with GSH levels, in animals treated with ATR for 3 and 4 days. 3. A subsequent study was performed with animals treated with vehicle, 6.5, 50 or 100 mg/kg/day for 4, 8 or 14 days. Expression of hepatic phase I CYP 450 enzymes was evaluated in conjugation with GST expression, protein and activity. Nineteen of the 45 CYP enzymes assayed displayed increased mRNA levels after eight days of treatment in animals treated with 50 or 100 mg/kg/day. After 14 days of treatment, all CYP expression levels returned to control levels except for CYP2B2, CYP2B3, CYP2C7, CYP2C23, CYP2E1, CYP3A9, CYP4A3 and CYP27A1, which remained elevated. 4. Results indicate that there may be a habituation or adaptation of liver phase I and phase II expression following repeated ATR treatment.

Kisspeptin Stimulates Growth Hormone Release by Utilizing Neuropeptide Y Pathways and Is Dependent on the Presence of Ghrelin in the Ewe.

Although kisspeptin is the primary stimulator of gonadotropin-releasing hormone secretion and therefore the hypothalamic-pituitary-gonadal axis, recent findings suggest kisspeptin can also regulate additional neuroendocrine processes including release of growth hormone (GH). Here we show that central delivery of kisspeptin causes a robust rise in plasma GH in fasted but not fed sheep. Kisspeptin-induced GH secretion was similar in animals fasted for 24 hours and those fasted for 72 hours, suggesting that the factors involved in kisspeptin-induced GH secretion are responsive to loss of food availability and not the result of severe negative energy balance. Pretreatment with the neuropeptide Y (NPY) Y1 receptor antagonist, BIBO 3304, blocked the effects of kisspeptin-induced GH release, implicating NPY as an intermediary. Kisspeptin treatment induced c-Fos in NPY and GH-releasing hormone (GHRH) cells of the arcuate nucleus. The same kisspeptin treatment resulted in a reduction in c-Fos in somatostatin (SS) cells in the periventricular nucleus. Finally, blockade of systemic ghrelin release or antagonism of the ghrelin receptor eliminated or reduced the ability of kisspeptin to induce GH release, suggesting the presence of ghrelin is required for kisspeptin-induced GH release in fasted animals. Our findings support the hypothesis that during short-term fasting, systemic ghrelin concentrations and NPY expression in the arcuate nucleus rise. This permits kisspeptin activation of NPY cells. In turn, NPY stimulates GHRH cells and inhibits SS cells, resulting in GH release. We propose a mechanism by which kisspeptin conveys reproductive and hormone status onto the somatotropic axis, resulting in alterations in GH release.

Effect of Age, Duration of Exposure, and Dose of Atrazine on Sexual Maturation and the Luteinizing Hormone Surge in the Female Sprague-Dawley Rat.

Atrazine (ATZ) was administered daily by gavage to pregnant female Sprague Dawley rats at doses of 0, 6.25, 25 or 50 mg/kg/day, either during gestation, lactation and post-weaning (G/L/PW cohort) to F1 generation female offspring or only from postnatal day (PND 21) until five days after sexual maturation (vaginal opening) when the estrogen-primed, luteinizing hormone (LH) surge was evaluated (PW cohort). Additional subgroups of F1 females received the vehicle or ATZ from PND 21-133 or from PND 120-133. Slight reductions in fertility and the percentage of F1 generation pups surviving to PND 21 in the gestationally exposed 50 mg/kg dose group were accompanied by decreased food intake and body weight of dams and F1 generation offspring. The onset of puberty was delayed in of the F1 generation G/L/PW females at doses of 25 and 50 mg/kg/day. F1 generation females in the PW high-dose ATZ group also experienced a delay in the onset of puberty. ATZ had no effect on peak LH or LH AUC in ovariectomized rats 5 days after sexual maturation, irrespective of whether the F1 generation females were treated from gestation onward or only peripubertally. There was no effect of ATZ treatment on the estrous cycle, peak LH or LH AUC of F1 generation females exposed from gestation through to PND 133 or only for two weeks from PND 120-133. These results indicate that developing females exposed to ATZ are not more sensitive compared to animals exposed to ATZ as young adults.

Atrazine inhibits pulsatile gonadotropin-releasing hormone (GnRH) release without altering GnRH messenger RNA or protein levels in the female rat.

Atrazine (ATR) is a commonly used pre-emergence/early postemergence herbicide. Previous work has shown that exposure to high doses of ATR in rats results in blunting of the hormone-induced luteinizing hormone (LH) surge and inhibition of pulsatile LH release without significantly reducing pituitary sensitivity to a gonadotropin-releasing hormone (GnRH) agonist. Accompanying the reduction in the LH surge was an attenuation of GnRH neuronal activation. These findings suggest that ATR exposure may be acting to inhibit GnRH release. In this study, we examined GnRH directly to determine the effect of high doses of ATR on GnRH pulsatile release, gene expression, and peptide levels in the female rat. Ovariectomized adult female Wistar rats were treated with ATR (200 mg/kg) or vehicle for 4 days via gavage. Following the final treatment, GnRH release was measured from ex vivo hypothalamic explants for 3 h. In another experiment, animals were administered either vehicle or ATR (50, 100, or 200 mg/kg) daily for 4 days. Following treatment, in situ hybridization was performed to examine total GnRH mRNA and the primary GnRH heterogeneous nuclear RNA transcript. Finally, GnRH immunoreactivity and total peptide levels were measured in hypothalamic tissue of treated animals. ATR treatment resulted in no changes to GnRH gene expression, peptide levels, or immunoreactivity but a reduction in GnRH pulse frequency and an increased pulse amplitude. These findings suggest that ATR acts to inhibit the secretory dynamics of GnRH pulses without interfering with GnRH mRNA and protein synthesis.

Anxiolytic effects and neuroanatomical targets of estrogen receptor-ß (ERß) activation by a selective ERß agonist in female mice.

The dichotomous anxiogenic and anxiolytic properties of estrogens have been reported to be mediated by two distinct neural estrogen receptors (ER), ERα and ERß, respectively. Using a combination of pharmacological and genetic approaches, we confirmed that the anxiolytic actions of estradiol are mediated by ERß and extended and these observations to demonstrate the neuroanatomical targets involved in ERß 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 ERß gene (ßERKO). 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 ßERKO littermates. Stress-induced corticosterone (CORT) and ACTH were also attenuated by S-DPN in the WT mice but not in the ßERKO 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 ßERKO females. These studies provide compelling evidence for anxiolytic effects mediated by ERß, 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.

The differential effect of atrazine on luteinizing hormone release in adrenalectomized adult female Wistar rats.

High doses of atrazine (ATR), administered for 4 days, suppress luteinizing hormone (LH) release and increase adrenal hormones levels. Considering the known inhibitory effects of adrenal hormones on the hypothalamo-pituitary-gonadal axis, we investigated the possible role the adrenal gland has in mediating ATR inhibition of LH release. To determine the extant and duration of adrenal activation, ovariectomized Wistar rats were given a single dose of ATR (0, 50, or 200 mg/kg), and corticosterone (CORT) levels were assayed at multiple time points posttreatment. CORT levels were increased within 20 min and remained elevated over 12 h postgavage in 200-mg/kg animals. To determine the effects of adrenalectomy on ATR inhibition of the LH surge and pulsatile LH release, adrenalectomized (ADX) or sham-operated ovariectomized rats were treated for 4 days with ATR (0, 10, 100, or 200 mg/kg), and an LH surge was induced with hormone priming. In the afternoon following the last dose of ATR, blood was sampled hourly for 9 h. Another cohort of ovariectomized rats was examined for pulsatile patterns of LH secretion after ATR (0, 50, or 200 mg/kg) and sampled every 5 min for 3 h. ADX had no effect on ATR inhibition of the LH surge but prevented the ATR disruption of pulsatile LH release. These data indicate that ATR selectively affects the LH pulse generator through alterations in adrenal hormone secretion. Adrenal activation does not play a role in ATR's suppression of the LH surge, and therefore ATR may work centrally to alter the preovulatory LH surge in female rats.

Estrogen receptor beta activation prevents glucocorticoid receptor-dependent effects of the central nucleus of the amygdala on behavior and neuroendocrine function.

Neuropsychiatric disorders such as anxiety and depression have formidable economic and societal impacts. A dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis leading to elevated endogenous glucocorticoid levels is often associated with such disorders. Chronically high glucocorticoid levels may act upon the central nucleus of the amygdala (CeA) to alter normally adaptive responses into those that are maladaptive and detrimental. In addition to glucocorticoids, other steroid hormones such as estradiol and androgens can also modify hormonal and behavioral responses to threatening stimuli. In particular, estrogen receptor beta (ERbeta) agonists have been shown to be anxiolytic. Consequently, these experiments addressed the hypothesis that the selective stimulation of glucocorticoid receptor (GR) in the CeA would increase anxiety-like behaviors and HPA axis reactivity to stress, and further, that an ERbeta agonist could modulate these effects. Young adult female Sprague-Dawley rats were ovariectomized and bilaterally implanted via stereotaxic surgery with a wax pellet containing the selective GR agonist RU28362 or a blank pellet, to a region just dorsal to the CeA. Four days later, animals were administered the ERbeta agonist S-DPN or vehicle (with four daily sc injections). Anxiety-type behaviors were measured using the elevated plus maze (EPM). Central RU28362 implants caused significantly higher anxiety-type behaviors in the EPM and greater plasma CORT levels than controls given a blank central implant. Moreover, S-DPN treated animals, regardless of type of central implant, displayed significantly lower anxiety-type behaviors and post-EPM plasma CORT levels than vehicle treated controls or vehicle treated animals implanted with RU28362. These results indicate that selective activation of GR within the CeA is anxiogenic, and peripheral administration of an ERbeta agonist can overcome this effect. These data suggest that estradiol signaling via ERbeta prevents glucocorticoid-dependent effects of the CeA on behavior and neuroendocrine function.

Effects of atrazine and its withdrawal on gonadotropin-releasing hormone neuroendocrine function in the adult female Wistar rat.

High doses of the commonly used herbicide atrazine have been shown to suppress luteinizing hormone (LH) release. To determine whether atrazine alters the function of gonadotropin-releasing hormone (GnRH) neurons, we examined the effects of atrazine on GnRH neuronal activation and the subsequent release of LH normally associated with ovulation. Ovariectomized adult Wistar rats were administered atrazine (50, 100, or 200 mg/kg of body weight daily by gavage) or vehicle for 4 days. Animals were primed with estrogen and progesterone to induce an evening LH surge. Blood samples were obtained over the afternoon and evening using an indwelling right atrial cannula, and plasma was assayed for LH and FSH. Another cohort of animals was transcardially perfused in the afternoon to examine GnRH activation using FOS immunoreactivity. Results of these studies show that 4-day treatment with atrazine resulted in a significant reduction in the magnitude of the LH and FSH surges, and this corresponds to a decrease in GnRH neurons expressing FOS immunoreactivity. To determine if the effects of atrazine were long lasting, additional studies were performed examining LH levels and GnRH activation 2 days and 4 days after atrazine withdrawal. Within 4 days (but not 2 days) after cessation of atrazine treatment, measures of hypothalamic-pituitary-gonadal (HPG) activation returned to normal. These data indicate that atrazine affects neuroendocrine function in the female rat by actions at the level of the GnRH neuron and that the acute effects of high doses of atrazine can be reversed within 4 days after withdrawal of treatment.

Atrazine inhibits pulsatile luteinizing hormone release without altering pituitary sensitivity to a gonadotropin-releasing hormone receptor agonist in female Wistar rats.

Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-tri-azine] is one of the most commonly used herbicides in the United States. Atrazine has been shown to suppress luteinizing hormone (LH) release and can lead to a prolongation of the estrous cycle in the rat. The objectives of this study were to examine the effects of atrazine on normal tonic release of LH and to elucidate the site of action of atrazine in the hypothalamic-pituitary-gonadal axis. Episodic release of gonadotropin-releasing hormone (GnRH) and the corresponding release of LH from the anterior pituitary gland are required for normal reproductive function. To determine if atrazine affects pulsatile LH release, ovariectomized adult female Wistar rats were administered atrazine (50, 100, or 200 mg/kg of body weight daily by gavage) or vehicle control for 4 days. On the final day of atrazine treatment, blood samples were obtained using an indwelling right atrial cannula. In the group receiving 200 mg/kg, there was a significant reduction in LH pulse frequency and a concomitant increase in pulse amplitude. To determine if the effects of atrazine on LH release were due to changes at the level of the pituitary, animals were passively immunized against endogenous GnRH, treated with atrazine, and challenged with a GnRH receptor agonist. Atrazine failed to alter pituitary sensitivity to the GnRH receptor agonist at any dose used. Taken together, these findings demonstrate that high doses of atrazine affect the GnRH pulse generator in the brain and not at the level of gonadotrophs in the pituitary.

Biological and anatomical evidence for kisspeptin regulation of the hypothalamic-pituitary-gonadal axis of estrous horse mares.

The purpose of the present study was to evaluate the effects of kisspeptin (KiSS) on LH and FSH secretion in the seasonally estrous mare and to examine the distribution and connectivity of GnRH and KiSS neurons in the equine preoptic area (POA) and hypothalamus. The diestrous mare has a threshold serum gonadotropin response to iv rodent KiSS decapeptide (rKP-10) administration between 1.0 and 500 microg. Administration of 500 microg and 1.0 mg rKP-10 elicited peak, mean, and area under the curve LH and FSH responses indistinguishable to that of 25 microg GnRH iv, although a single iv injection of 1.0 mg rKP-10 was insufficient to induce ovulation in the estrous mare. GnRH and KiSS-immunoreactive (ir) cells were identified in the POA and hypothalamus of the diestrous mare. In addition, KiSS-ir fibers were identified in close association with 33.7% of GnRH-ir soma, suggesting a direct action of KiSS on GnRH neurons in the mare. In conclusion, we are the first to reveal a physiological role for KiSS in the diestrous mare with direct anatomic evidence by demonstrating a threshold-like gonadotropin response to KiSS administration and characterizing KiSS and GnRH-ir in the POA and hypothalamus of the diestrous horse mare.

Estrogen receptor beta in the brain: from form to function.

Estrogens have numerous effects on the brain, both in adulthood and during development. These actions of estrogen are mediated by two distinct estrogen receptor (ER) systems, ER alpha (ERalpha) and ER beta (ERbeta). In brain, ERalpha plays a critical role in regulating reproductive neuroendocrine function and behavior, however, a definitive role for ERbeta in any neurobiological function has been slow in forthcoming. Clues to the function of ERbeta in the central nervous system can be gleaned from the neuroanatomical distribution of ERbeta and the phenotypes of neurons that express ERbeta. ERbeta immunoreactivity has been found in populations of GnRH, CRH, vasopressin, oxytocin and prolactin containing neurons in the hypothalamus. Utilizing subtype-selective estrogen receptor agonists can help determine the roles for ERbeta in non-reproductive behaviors in rat models. ERbeta-selective agonists exert potent anxiolytic activity when animals were tested in a number of behavioral paradigms. Consistent with this, ERbeta-selective agonists also inhibited the ACTH and corticosterone response to stress. In contrast, ERalpha selective agonists were found to be anxiogenic and correspondingly increased the hormonal stress response. Taken together, our studies implicate ERbeta as an important modulator of some non-reproductive neurobiological systems. The molecular and neuroanatomical targets of estrogen that are mediated by ERbeta remain to be determined. A number of splice variants of ERbeta mRNA have been reported in brain tissue. Imaging of eGFP labeled chimeric receptor proteins transfected into cell lines shows that ERbeta splice variation can alter trafficking patterns and function. The originally described ERbeta (herein termed ERbeta1) is characterized by possessing a high affinity for estradiol. Similar to ERalpha, it is localized in the nucleus and is trafficked to nuclear sites termed "hyperspeckles" following ligand binding. In contrast, ERbeta2 contains an 18 amino acid insert within the ligand-binding domain and as a result can be best described as a low affinity form of ERbeta. A delta3 (delta3) variant of ERbeta has a deletion of the 3rd exon (coding for the second half of the DNA-binding domain) and as a result does not bind an estrogen response element in DNA. delta3 variants are trafficked to a unique low abundance and larger nuclear site following ligand binding. A delta4 (delta4) variant lacks exon 4 and as a result is localized to the cytoplasm. The amount of individual splice variant mRNAs varies depending upon brain region. Examination of neuropeptide promoter regulation by ERbeta splice variants demonstrates that ERbeta functions as a constitutively active transcription factor. Moreover, it appears that splice variation of ERbeta alters its ability to regulate transcription in a promoter-dependent and ligand-dependent fashion.

Corticotropin-releasing hormone heterogeneous nuclear RNA (hnRNA) and immunoreactivity are induced in extrahypothalamic brain sites by kainic-acid-induced seizures and are modulated by estrogen.

Corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are pivotal mediators of the hormonal response to stressors and are found within neurons of the paraventricular nucleus of the hypothalamus (PVN) and several extrahypothalamic sites where expression is activity-dependent. Previous work has shown increased CRH immunoreactivity in extrahypothalamic sites after kainic-acid (KA)-induced seizures in male rats. This study examined the induction of CRH heterogeneous nuclear RNA (hnRNA), AVP hnRNA and c-fos as a measure of gene transcription and cell activation following kainic-acid (KA)-induced seizures. KA or saline was administered to intact male rats, ovariectomized (OVX) females and OVX females treated with 17beta-estradiol (E2). Animals were sacrificed 0, 15, 60 or 120 min following KA treatment. In the PVN, CRH hnRNA levels were increased by KA treatment at 15, 60, and 120 min. AVP hnRNA and c-fos mRNA in the PVN were also significantly elevated above controls at all time points. Elevations in CRH hnRNA were also identified in hippocampus, the lateral bed nucleus of the stria terminalis (BNST) and globus pallidus at 60 and 120 min following KA and in the piriform cortex, and central nucleus of the amygdala at 120 min after KA. CRH hnRNA levels at 120 min in the PVN, amygdala, cingulate cortex, hippocampus (CA1), piriform cortex, and BNST were lower in OVX+E2 females compared to females without E2. To determine if the increases in CRH hnRNA translated to increased CRH peptide, immunocytochemistry was performed. CRH immunoreactivity was increased in the amygdala, BNST, cingulate cortex, PVN and globus pallidus within 3 h after KA treatment and in the piriform cortex and hippocampus by 6 h after KA. These results suggest a time-dependent activation of the CRH system following activation of kainate receptors, which may result in long-term changes in the expression of extrahypothalamic CRH.

Orphanin FQ: evidence for a role in the control of the reproductive neuroendocrine system.

Orphanin FQ (OFQ), also known as nociceptin, is a member of the endogenous opioid peptide family that has been functionally implicated in the control of pain, anxiety, circadian rhythms, and neuroendocrine function. In the reproductive system, endogenous opioid peptides are involved in the steroid feedback control of GnRH pulses and the induction of the GnRH surge. The distribution of OFQ in the preoptic area and hypothalamus overlaps with GnRH, and in vitro evidence suggests that OFQ can inhibit GnRH secretion from hypothalamic fragments. Using the sheep as a model, we examined the potential anatomical colocalization between OFQ and GnRH using dual-label immunocytochemistry. Confocal microscopy revealed that approximately 93% of GnRH neurons, evenly distributed across brain regions, were also immunoreactive for OFQ. In addition, almost all GnRH fibers and terminals in the external zone of the median eminence, the site of neurosecretory release of GnRH, also colocalized OFQ. This high degree of colocalization suggested that OFQ might be functionally important in controlling reproductive endocrine events. We tested this possibility by examining the effects of intracerebroventricular administration of [Arg(14), Lys(15)] OFQ, an agonist to the OFQ receptor, on pulsatile LH secretion. The agonist inhibited LH pulse frequency in both luteal phase and ovariectomized ewes and suppressed pulse amplitude in the latter. The results provide in vivo evidence supporting a role for OFQ in the control of GnRH secretion and raise the possibility that it acts as part of an ultrashort, autocrine feedback loop controlling GnRH pulses.

Progesterone increases dynorphin a concentrations in cerebrospinal fluid and preprodynorphin messenger ribonucleic Acid levels in a subset of dynorphin neurons in the sheep.

Recent studies suggest that the endogenous opioid peptide, dynorphin, is an important mediator of progesterone negative feedback on GnRH pulse frequency in the ewe. These experiments tested this hypothesis by examining the effects of progesterone on dynorphin A concentrations in cerebrospinal fluid (CSF) collected from the third ventricle and expression of preprodynorphin (PPD) mRNA in hypothalamic nuclei. CSF was collected every 10 min for 5 h in three groups of ewes: 1) ovary-intact ewes during the luteal phase (d 6-7 of estrous cycle); 2) ewes 6-7 d after ovariectomy (OVX); and 3) OVX ewes treated for 6-7 d with implants that produced luteal-phase progesterone levels. Diencephalic tissue from these ewes was then collected and processed for in situ hybridization using an ovine cDNA probe against PPD. Progesterone treatment increased dynorphin A concentrations in CSF over that observed in untreated OVX ewes; CSF dynorphin A concentrations in ovary-intact ewes were midway between the other groups. OVX significantly decreased the number of PPD mRNA-expressing cells in the preoptic area (POA), anterior hypothalamic area (AHA), and arcuate nucleus (ARC), with no change seen in any other PPD-expressing nuclei. Progesterone treatment of OVX ewes restored PPD expression in the POA and AHA to levels seen in luteal-phase animals but had no effect on PPD expression in the ARC. These results are consistent with the hypothesis that progesterone acts via dynorphin neurons to inhibit pulsatile GnRH secretion and point to dynorphin neurons in the POA, AHA, and ARC as potential mediators of this action during the luteal phase.

Colocalization of progesterone receptors in parvicellular dynorphin neurons of the ovine preoptic area and hypothalamus.

Recent evidence suggests that the dynorphin-kappa receptor opioid system acts to mediate the inhibitory effect of progesterone (P) on GnRH pulse frequency during the luteal phase of the ovine estrous cycle. It is known that progesterone receptors (PRs) are required for the actions of P on GnRH secretion. Therefore, if P acts directly on dynorphin (DYN) neurons, then these neurons should contain PRs. To test this hypothesis, we used a dual-label immunoperoxidase procedure to visualize PRs and DYN in the preoptic area (POA) and hypothalamus of ovary-intact ewes killed during the luteal phase of the estrous cycle. The PR was colocalized in more than 90% of parvicellular DYN neurons in the POA, anterior hypothalamus (AHA), and arcuate nucleus (ARC). By contrast, none of magnocellular DYN cells of the paraventricular and supraoptic nuclei coexpressed immunoreactive PRs. The high percentage of colocalization of PRs in parvicellular DYN cells of the POA, AHA, and ARC suggests that these cells are prime targets of P. In addition, DYN cells in the ARC, but not the POA or AHA, were found to receive synaptic inputs from DYN-positive axon terminals. This observation raises the possibility that an ultrashort feedback loop controls the release of DYN from ARC neurons.