Chad D. Foradori

Atrazine Inhibits Pulsatile Gonadotropin-Releasing Hormone (GnRH) Release Without Altering GnRH Messenger RNA or Protein Levels in the Female Rat

ABSTRACT 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.

The Effect of Atrazine Administered by Gavage or in Diet on the LH Surge and Reproductive Performance in Intact Female Sprague‐Dawley and Long Evans Rats

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

Hypothalamic Integration of Nutrient Status and Reproduction in the Sheep

Nutrient availability is a determinant of reproductive success. It is well known that inadequate nutrition results in reproductive failure due to a number of factors including delay of puberty or anoestrous in post-pubertal animals. The lack of nutrients is detected primarily by changes in circulating nutrient molecules and hormones and communicated directly or indirectly to the hypothalamus and brain stem for integration. The general effect is that low nutrition leads to increased appetite stimulation and reduced reproductive performance. When nutrition is adequate, the reverse is true. Both aspects will be the focus of this review. One result of the lack of nutrients is a reduction in luteinizing hormone (LH) concentrations and pulse frequency. Nutrient signals, such as glucose availability, hormonal signals, such as insulin and leptin, and neuroendocrine signals, such as neuropeptide Y and corticotropin-releasing hormone, have been clearly demonstrated to interact to produce changes in LH and reproductive success. Other signals, such as fatty acids, ghrelin, agouti-related peptide, melanin-concentrating hormone, orexin, melanocyte-stimulating hormone, kisspeptin, neurokinin, dynorphin and gonadotropin inhibitory hormone may also play a role in integrating nutrition and reproduction. This review will focus on the major features of the reciprocal control of appetite and reproduction in sheep.

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

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

Kisspeptin stimulates growth hormone release by utilizing neuropeptide y pathways and is dependent on the presence of ghrelin in the ewe

&NA; 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.

Distribution and regulation of gonadotropin-releasing hormone, kisspeptin, RF-amide related peptide-3, and dynorphin in the bovine hypothalamus

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.

Lack of immunotoxic effects of repeated exposure to atrazine associated with the adaptation of adrenal gland activation

&NA; T cell‐dependent IgM antibody production and natural killer cell (NKC) activity were assessed in SD rats orally administered atrazine for 28 days to males (0, 6.5, 25, or 100 mg/kg/day) or females (0, 3, 6, or 50 mg/kg/day), or 30 or 500 ppm in diet (3 or 51 mg/kg/day). Anti‐asialo GM1 antibodies (NKC) and cyclophosphamide (antibody‐forming cell assay [AFC]) served as positive controls. Pituitary (ACTH, prolactin), adrenal (corticosterone, progesterone, aldosterone), and gonadal (androgens, estrogens) hormones were assessed after 1, 7, and/or 28 days of treatment. Food intake and body weights were significantly reduced in the highest dosed males, and transiently affected in females. Urinary corticosterone levels were not increased in atrazine‐treated groups in either sex at any time point measured (10, 22, or 24 days). Corticosterone and progesterone were elevated in males after a single atrazine dose ≥6.5 mg/kg/day, but not after 7, 14, or 28 doses. There were no effects on adrenal, pituitary, or gonadal hormones in females. Atrazine did not suppress the AFC response or decrease NKC function after 28 days in males or females. Atrazine had no effect on spleen weights or spleen cell numbers in males or females, although thymus weights were elevated in males receiving the highest dose. The lack of immunotoxic effect of atrazine was associated with diminished adrenal activation over time in males, and no effects on adrenal hormones in females. HighlightsA single dose of atrazine (≥6.5 mg/kg) activated the HPA axis in male SD rats.HPA activation indicated by increased plasma corticosterone and progesterone.HPA activation habituated after 7 days and was also absent at 14 and 28 days.Cell‐mediated immunity (AFC) unaffected by atrazine at 28 days (males, females).Natural killer cell activity unaffected by atrazine at 28 days (males, females).

Reproduction and beyond, kisspeptin in ruminants

Kisspeptin (Kp) is synthesized in the arcuate nucleus and preoptic area of the hypothalamus and is a regulator of gonadotropin releasing hormone in the hypothalamus. In addition, Kp may regulate additional functions such as increased neuropeptide Y gene expression and reduced proopiomelanocortin (POMC) gene expression in sheep. Other studies have found a role for Kp to release growth hormone (GH), prolactin and luteinizing hormone (LH) from cattle, rat and monkey pituitary cells. Intravenous injection of Kp stimulated release LH, GH, prolactin and follicle stimulating hormone in some experiments in cattle and sheep, but other studies have failed to find an effect of peripheral injection of Kp on GH release. Recent studies indicate that Kp can stimulate GH release after intracerebroventricular injection in sheep at doses that do not release GH after intravenous injection. These studies suggest that Kp may have a role in regulation of both reproduction and metabolism in sheep. Since GH plays a role in luteal development, it is tempting to speculate that the ability of Kp to release GH and LH is related to normal control of reproduction.

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

Abstract 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.