Atrazine suppression of the LH surge slowly develops over time and peaks after 4 days; sensitivity to atrazine decreases after 8 or 14 days of dosing. Adaptation of the LH response was correlated with increased phase I and phase II liver enzyme activity/expression.

The paraventricular nucleus of the hypothalamus (PVH) plays a central role in regulating the hypothalamic-pituitary-adrenal (HPA) axis. Medial parvocellular neurons of the PVH (mpPVH) integrate sensory and humoral inputs to maintain homeostasis. Humoral inputs include glucocorticoids secreted by the adrenals, which down-regulate HPA activation. A primary glucocorticoid target is the population of mpPVH neurons that synthesize and secrete corticotropin-releasing factors, the most potent of which is corticotropin-releasing hormone (CRH). Although CRH gene (crh) expression is known to be down-regulated by glucocorticoids, the mechanisms by which this process occurs are still poorly understood. To begin this study we postulated that glucocorticoid repression of crh involves HDAC recruitment to the region of the crh proximal promoter. To evaluate this hypothesis, we treated hypothalamic cells that express CRH with the HDAC inhibitor trichostatin A (TSA). As predicted, treatment with TSA led to increased CRH mRNA levels and crh promoter activity. Although co-treatment with Dex (10(-7)M) reduced the TSA effect on mRNA levels, it failed to reduce promoter activity; however co-transfection of HDAC1 but not 3 restored Dex inhibition. A distinction between HDAC1 and 3 was also apparent with respect to crh promoter occupancy. Dex led to increased HDAC1 but not HDAC3 occupancy. In vivo studies revealed that CRH-immunoreactive (-ir) neurons contained HDAC1- and HDAC3-ir. Collectively, these data point to a role for HDAC1 in the physiologic regulation of crh.

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.

Atrazine (ATR) blunts the hormone-induced luteinizing hormone (LH) surge, when administered by gavage (50-100 mg/kg/day for 4 days), in ovariectomized rats. In this study, we determined if comparable doses delivered either by gavage (bolus dose) or distributed in diet would reduce the LH surge and subsequently affect fertility in the intact female rat. ATR was administered daily to intact female Sprague-Dawley (SD) or Long Evans (LE) rats by gavage (0, 0.75 1.5, 3, 6, 10, 12, 50, or 100 mg/kg/day) or diet (0, 30, 100, 160, 500, 660, or 1460 ppm) during one complete 4-day estrous cycle, starting on day of estrus. Estrous status, corpora lutea, ova, and LH plasma concentrations were evaluated. A second cohort of animals was mated on the fourth treatment day. Fertility metrics were assessed on gestational day 20. A higher portion of LE rats had asynchronous estrous cycles when compared to SD rats both during pretreatment and in response to ATR (≥50 mg/kg). In contrast, bolus doses of ATR (≥50 mg/kg) inhibited the peak and area under the curve for the preovulatory LH surge in SD but not LE animals. Likewise, only bolus-treated SD, not LE, rats displayed reduced mean number of corpora lutea and ova. There were no effects of ATR administered by gavage on mating, gravid number, or fetus number. Dietary administration had no effect on any reproductive parameter measured. These findings indicate that short duration, high-bolus doses of ATR can inhibit the LH surge and reduce the number of follicles ovulated; however, dietary administration has no effect on any endocrine or reproductive outcomes.

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 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.

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.

Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus (ARC) play a key role in gonadotropin-releasing hormone (GnRH) pulse generation and gonadal steroid feedback, with kisspeptin driving GnRH release and neurokinin B and dynorphin acting as pulse start and stop signals, respectively. A separate cell group, expressing RFamide-related peptide-3 (RFRP-3) has been shown to be a primary inhibitor of GnRH release. Very little is known regarding these cell groups in the bovine. In this study, we examined the relative immunoreactivity of kisspeptin, dynorphin, and RFRP-3 and their possible connectivity to GnRH neurons in the hypothalami of periestrus and diestrus bovine. While GnRH and RFRP-3 immunoreactivity were unchanged, kisspeptin and dynorphin immunoreactivity levels varied in relation to plasma progesterone concentrations and estrous status. Animals with higher plasma progesterone concentrations in diestrus had lower kisspeptin and increased dynorphin immunoreactivity in the ARC. The percentage of GnRH cells with kisspeptin or RFRP-3 fibers in close apposition did not differ between estrous stages. However, the proportions of GnRH cells with kisspeptin or RFRP-3 contacts (∼49.8% and ∼31.3%, respectively) suggest direct communication between kisspeptin and RFRP-3 cells to GnRH cells in the bovine. The data produced in this work support roles for kisspeptin and dynorphin, within the KNDy neural network, in controlling GnRH release over the ovarian cycle and conveying progesterone-negative feedback onto GnRH neurons in the bovine.

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.

Glucocorticoids have short- and long-term effects on adrenal gland function and development. RNA sequencing (RNA-seq) was performed to identify early transcriptomic responses to the synthetic glucocorticoid, dexamethasone (Dex), in vitro and in vivo. In total, 1711 genes were differentially expressed in the adrenal glands of the 1-h Dex-treated mice. Among them, only 113 were also considered differentially expressed genes (DEGs) in murine adrenocortical Y-1 cells treated with Dex for 1 h. Gene ontology analysis showed that the upregulated DEGs in the adrenal gland of the 1-h Dex-treated mice were highly associated with the development of neuronal cells, suggesting the adrenal medulla had a rapid response to Dex. Interestingly, only 4.3% of Dex-responsive genes in the Y-1 cell line under Dex treatment for 1 h were differentially expressed under Dex treatment for 24 h. The heatmaps revealed that most early responsive DEGs in Y-1 cells during 1 h of treatment exhibited a transient response. The expression of these genes under treatment for 24 h returned to basal levels similar to that during control treatment. In summary, this research compared the rapid transcriptomic effects of Dex stimulation in vivo and in vitro. Notably, adrenocortical Y-1 cells had a transient early response to Dex treatment. Furthermore, the DEGs had a minimal overlap in the 1-h Dex-treated group in vivo and in vitro.