The Differential Effect of Atrazine on Luteinizing Hormone Release in Adrenalectomized Adult Female Wistar Rats 1

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1 BIOLOGY OF REPRODUCTION 85, (2011) Published online before print 15 June DOI /biolreprod The Differential Effect of Atrazine on Luteinizing Hormone Release in Adrenalectomized Adult Female Wistar Rats 1 Chad D. Foradori, 2,3 Laura R. Hinds, 3 Alicia M. Quihuis, 3 Anthony F. Lacagnina, 3 Charles B. Breckenridge, 4 and Robert J. Handa 3 Department of Basic Medical Sciences, 3 University of Arizona, College of Medicine, Phoenix, Arizona Syngenta Crop Protection, LLC, 4 Greensboro, North Carolina ABSTRACT 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-pituitarygonadal 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 shamoperated 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. corticosterone, luteinizing hormone (LH/LH receptor), stress, toxicology INTRODUCTION Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine; ATR) is a commonly used herbicide that inhibits the Hill reaction in the photosynthesis of broadleaf grasses [1]. ATR is used widely throughout the world, where it has been applied to control weeds in a wide range of crops, including corn and sorghum fields, both prior to and following emergence [2]. 1 Supported by Syngenta Crop Protection, Inc. 2 Correspondence: Chad D. Foradori, Department of Basic Medical Sciences, University of Arizona, College of Medicine, 425 North Fifth St., ABC 1, Phoenix, AZ FAX: ; chad.foradori@auburn.edu Received: 23 March First decision: 22 April Accepted: 2 June Ó 2011 by the Society for the Study of Reproduction, Inc. eissn: ISSN: In rodents, ATR has been shown to inhibit both the luteinizing hormone (LH) surge and pulsatile LH release [3 5]. ATR can also prolong the estrous cycle [6, 7], delay the onset of puberty in both sexes [8, 9], and reduce basal testosterone levels [10, 11]. We have shown that ATR treatment decreases LH pulse frequency and increases pulse amplitude in ovariectomized rats without changing pituitary sensitivity to a gonadotropin-releasing hormone (GnRH) receptor agonist [5]. In addition, using FOS (c-fos) as a measure of neuronal activity, we have shown that ATR inhibits the hormoneinduced activation of GnRH neurons in concert with its effect on the LH surge [4]. However, the mechanisms by which ATR exposure elicits changes in hypothalamo-pituitary-gonadal (HPG) output remain unclear. Recent studies have shown that ATR treatment causes an increased release of adrenal hormones in rodents [12 14]. Given that corticosterone can act to inhibit LH secretion, increases in CORT could contribute to the well-characterized attenuation of LH release following ATR exposure [15]. In the studies described in this report, we tested the hypothesis that ATR treatment causes a rise in plasma CORT and that adrenal activation mediates the ATR-induced alteration of both the phasic and pulsatile patterns of LH release. MATERIALS AND METHODS Animals All animal surgeries and experimental protocols were approved by the Animal Care and Use Committees at Arizona State University under subcontract with the University of Arizona College of Medicine, Phoenix, and carried out in accordance with the guidelines of the National Institutes of Health and the Association for Assessment and Accreditation of Laboratory Animal Care International. Young adult female Wistar rats (60 90 days old) were obtained from Charles River Laboratories (Wilmington, MA). Animals were double housed at the Arizona State University vivarium and maintained on a 12L:12D light schedule (lights on at 0700 h) with ad libitum access to food (Harlan Teklad Global Rodent Diet 2018) and water. Experiment 1: Effects of a Single ATR Gavage on Corticosterone To determine the effects of ATR treatment on plasma corticosterone (CORT) levels, 75 adult female Wistar rats were ovariectomized. One week after ovariectomy, animals were given a single dose of ATR by gavage (50 or 200 mg/kg body weight [BW] in 1% carboxymethlcellulose sodium salt [CBC]; n ¼ 3 5 per group/time point). These doses of ATR have previously been shown to inhibit the LH surge and pulsatile LH secretion [4, 5]. Therefore, they were used to determine the effects of ATR on CORT levels. Control animals received CBC only. Animals were sacrificed at 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, or 12 h postgavage by rapid anesthesia with isoflurane followed by decapitation. Trunk blood was collected, and plasma was removed and stored ( 808C). Radioimmunoassay (RIA) was performed to measure CORT levels. Care was taken to reduce exposure to any additional stressors during the interval between gavage and blood sample. All samples were taken between 0800 and 1200 h to avoid the diurnal rise in CORT. 684

2 ATRAZINE S EFFECTS ON LH SECRETION IN ADX RATS 685 Experiment 2: Effects of ATR on the Hormone-Induced LH Surge in Adrenalectomized Animals To determine if the adrenal glands are necessary for ATR to inhibit the estradiol plus progesterone (P)-induced LH surge, 70 adult Wistar rats were ovariectomized. Seven to 10 days after surgery, half the animals were bilaterally adrenalectomized (ADX). Sham surgery was performed on the remaining animals. To help maintain sodium balance and plasma volume, ADX animals were given 0.9% NaCl in their drinking water and injected twice daily with sterile saline (1 ml, 0.9%, s.c.). The day after adrenalectomy, animals were administered ATR (once daily for 4 consecutive days between 1000 and 1100 h; doses of 0, 10, 100, or 200 mg/kg BW; n ¼ 7 11/group) via gavage in 1% CBC. ATR doses were chosen on the basis of previous studies showing ATR inhibition of the hormone-induced LH surge [4]. On the second day of ATR treatment, intra-atrial cannulae were implanted. To induce an LH surge, animals were injected with 17b-estradiol-3-benzoate (EB; s.c.; 40 lg/kg BW in safflower oil between 1000 and 1100 h) for 3 consecutive days beginning on the second day of ATR treatment. On the third day of EB treatment and the final day of ATR treatment, animals were also injected with P (s.c.; 2 mg/animal in safflower oil) at 1100 h. This hormone treatment paradigm has been utilized previously and has been shown to effectively induce a preovulatory-like surge of LH [4, 16]. On the afternoon following P administration (the fourth day of ATR treatment), blood samples (200 ll) were taken at 1-h intervals in conscious freely moving rats using the intra-atrial cannulae. Rats were kept in their home cages during the sampling session, which lasted for 9 h, commencing at 1300 h (lights out at 1900 h). Blood samples were centrifuged (2000 rpm for 15 min at 48C), and plasma was removed. Following the third sample, red blood cells were suspended in sterile saline and were infused into the same animal from which it came to maintain blood volume and hematocrit level. Plasma was frozen for later analysis of LH using RIA. Plasma from the initial blood sample was assayed for CORT to validate the completeness of bilateral adrenalectomy. Experiment 3: Effects of ATR on the Pulsatile LH in Adrenalectomized Animals To determine if the presence of the adrenal glands are necessary for ATR s ability to alter the pulsatile secretory release pattern of LH in freely moving rats, 75 adult Wistar rats were ovariectomized. Seven to 10 days postsurgery, half the animals were bilaterally adrenalectomized. Sham surgeries were performed on the remaining animals. To maintain sodium balance, adrenalectomized animals were given 0.9% NaCl in their drinking bottles and injected twice daily with sterile saline (1 ml, 0.9%, s.c.). The day after adrenalectomy, animals were administered ATR (once daily for 4 consecutive days between 1000 and 1100 h; doses of 0, 50, or 200 mg/kg BW; n ¼ 9 13/group) via gavage in 1% CBC. ATR doses were chosen to mirror previous studies showing ATR inhibition of basal pulsatile LH release [5]. Approximately 3 h after the final dose of ATR, blood (200 ll) was sampled via an intra-atrial cannula at 5-min intervals for 3 h. To maintain hematocrit and electrolyte balance following repeated blood sampling, a rat red blood cell preparation was infused into animals following each sample [17]. In brief, citrate-phosphate-dextrose (CPD) was made fresh and filter sterilized (89.4 mm trisodium citrate, 15.5 mm citric acid, 142 mm dextrose, 16 mm monosodium phosphate). CPD (2 ml) was added to a sterile 10-ml syringe with an 18-gauge needle that was then used to perform a heart puncture of an adult isoflurane anesthetized rat to harvest donor blood. The blood/cpd mix was transferred into sterile 50 ml conical tubes through two layers of sterile gauze to filter out any clots and kept on ice. Blood was centrifuged for 10 min at g, then plasma was removed. An equal volume of sterile saline (0.9% NaCl) was added, and blood was gently resuspended. Blood was washed twice more with saline, hematocrit was measured, and lactated Ringer was added to adjust to a final hematocrit of 45%. Heparin was added to 20 U/ml, gentamycin was added to 0.03 mg/ml, and glucose was added to 0.5 mg/ml of blood substitute. Blood substitute was made the day of the procedure and kept on ice until shortly before use when it was warmed to an approximate rodent body temperature (388C). Equal volume of replacement blood was administered to each animal as was removed from the animal in the previous sample. Plasma was removed from each blood sample and frozen for later analysis of LH using RIA. Plasma from the initial blood sample was also assayed for CORT to validate the completeness of bilateral adrenalectomy. Radioimmunoassay Plasma levels for CORT and LH were determined by RIA. For the CORT assay, plasma samples were diluted in 0.01 M PBS (1:25), and corticosteroid binding globulin (CBG) was inactivated by incubation at 658C for 1 h. Samples and standards (5 700 ng/ml) were incubated overnight at 48C with antiserum (rabbit anti-cort; MP Biomedicals, Solon, OH) and [ 3 H] CORT (Perkin-Elmer, Boston, MA) in 0.1% gelatin 0.01 M PBS. Free CORT was separated from antibody-bound CORT with 1.0 ml dextran-coated charcoal. After centrifugation, the supernatant containing antibody-bound CORT was mixed with scintillation fluid and counted with a Packard 2900 TR liquid scintillation counter (Packard, Meriden, CT). The intra-assay variance as measured by internal quality controls was 4.9%. LH was measured using reagents provided by the National Hormone and Peptide Program. Rat LH-RP3 was used for constructing the respective standard curves. Ovine LH (olh) was iodinated using the chloramine T method by the Colorado State University peptide assay core. Plasma samples (50 ll) were incubated overnight at RT with antiserum (NIDDK-Anti-rLH-SII, diluted 1: ). Following incubation, iodinated olh was added to each tube (;10,000 cpm/tube) and incubated overnight at RT. Bound LH was separated from free LH by incubation with goat anti-rabbit c globin (Calbiochem, catalog no ; 1:1000) in a 5% polyethylene glycol solution. Bound 125 I-LH was counted with a Packard Cobra II gamma counter. The intra- and interassay coefficients of variation for LH assays were 4.32% and 6.2%, respectively. Intra-Atrial Cannulation To determine the dynamic changes in plasma LH, we used a chronic indwelling cannulation procedure to discretely remove blood samples from freely moving rats while they were residing in their own home cage. As described previously [5], this procedure is a modification of that by Harms and Ojeda [18]. Briefly, a cannula of Silastic tubing (0.037-inch outer diameter and inch inner diameter; Technical Products of Georgia, Decatur, GA) was inserted into the right atrium via the right jugular vein. The cannula was fixed to the pectoralis major muscle and externalized at the back by tunneling under the skin and allowing the end to hang free. The cannula was sealed with a piece of 23-gauge copper wire. For blood sampling, polyethylene 50 tubing was attached to the free end of the cannula, and the animal was infused with sodium heparin (100 U; Baxter Healthcare Corp., Deerfield, IL). The animal was left undisturbed for 1 h before sampling commenced. Samples of 200 ll of blood were taken at discrete intervals via the cannula during the sampling session. We have previously used this paradigm to sample blood from freely moving animals under nonstressful conditions. There was no evidence of dramatic increases in stress hormones, such as adrenocorticotropic hormone, during sampling [19]. Data Analysis CORT data were analyzed by analysis of variance (ANOVA) using Prism 5 software (GraphPad Software, San Diego, CA) for treatment, time, and treatment 3 time interactions. In cases where a significant treatment effect (P, 0.05) was observed, the dose-response data within each time point were further evaluated using the Bonferroni multiple-comparison test. For LH data from experiment 2, a two-way ANOVA (treatment 3 time) was performed on all data with repeated measures across the time factor. The peak LH surge amplitude was calculated by determining the peak concentration during the sampling period regardless of timing. The LH levels were plotted using all time points, and the area under the curve (AUC) for each hormone was determined using Prism 5 software. For AUC determinations, the mean of the first and last samples were used as baseline values. If all samples were not present, the animal was removed from AUC analysis. For peak and AUC, a two-way ANOVA was performed to examine ATR dose and adrenalectomy interaction. When significant differences were observed, Bonferroni post hoc test was used to determine which experimental groups differed from the controls. The level of statistical significance was set at P For LH data from experiment 3, concentrations were analyzed for the presence of pulses using a method similar to that described by Gallo [20]. A pulse was defined as a group of at least three data points whose values were two deviations from mean basal level. The pulse amplitude was calculated by subtracting the preceding nadir value from the greatest pulse value. The pulse period was determined by subtracting the time of consecutive pulses. Two-way ANOVA was performed (treatment 3 adrenalectomy factors), and when significant differences were observed, Bonferroni post hoc test was used. The level of statistical significance was set at P All values are reported as the mean 6 SEM. RESULTS Experiment 1: Effects of a Single Gavage Dose of ATR on Plasma Corticosterone A single dose of ATR delivered via gavage resulted in a significant dose-dependent increase in circulating CORT (F 2,54

3 686 FORADORI ET AL. FIG. 1. Histogram depicting the mean (6SEM) corticosterone levels in female Wistar rats after a single gavage of vehicle (white bar; n ¼ 3 5) or one of two doses of ATR: 50 mg/kg (gray bar; n ¼ 4 5) or 200 mg/kg (black bar; n ¼ 3 4). *Groups that are significantly different from vehicle-treated control at the same time point, P, ¼ 30.21; P, ; Fig. 1). At the 20-min postgavage time point, both doses of ATR (50 and 200 mg/kg) caused significant increases compared to vehicle-treated animals. CORT levels in the 200-mg/kg-ATR-treated group were elevated above vehicle-treated animals for all the remaining time points measured (1, 3, 6, 12 h). Moreover, at the 3- and 6- h postgavage times, CORT levels in the 200-mg/kg group were also significantly (P, 0.05) different from the 50-mg/kgtreated group of animals. In contrast, the elevated levels of CORT seen in the 50-mg/kg-treated animals were not sustained as they were not different from control levels at any other time point examined. CORT levels in vehicle-treated animals were increased 20 min postgavage compared to control groups at 5 min and 12 h (F 5,23 ¼ 3.54; P, 0.05), presumably representing the stress of the gavage procedure. FIG. 2. LH levels of adrenalectomized or sham animals after 4 days of ATR treatment. A and B) Line graphs showing the mean 6 SEM LH levels of sham (A) or adrenalectomized (B) animals over time following vehicle treatment or ATR given at 10, 100, or 200 mg/kg BW. C and D) Histograms showing the mean 6 SEM peak (C) and AUC (D) parameters of the LH surge after ATR treatment in sham (black bars) and adrenalectomized (white bars) animals. n ¼ 8 11/group. *Significant difference (P, 0.05) vs. control group. Experiment 2: Effects of ATR on the Hormone-Induced LH Surge in Adrenalectomized Animals Given that the ATR-induced increase in plasma CORT could be mediating the effects of ATR on LH secretion in female rats, we next examined the effect of adrenalectomy on ATR s inhibition of the LH surge. ATR treatment at a dose of 100 or 200 mg/kg inhibited the EBþP-induced LH surge in both sham and ADX animals when compared to the respective control groups (Fig. 2, A and B). The ATR-treated animals at 100 and 200 mg/kg demonstrated lowered peak amplitude in the LH rise both in sham (P, 0.002; F 3,24 ¼ 7.11) and ADX (P, 0.003; F 3,31 ¼ 5.92) animals (Fig. 2C). Likewise, both sham (F 3,23 ¼ 7.25; P, 0.002) and ADX (F 3,27 ¼ 4.88; P, 0.008) ATR-treated animals demonstrated a reduced AUC for the LH surge compared with the controls (Fig. 2C). There was no interaction between adrenalectomy and ATR on peak LH levels (P ¼ 0.98; F 3,57 ¼ 0.06) or AUC (P ¼ 0.94; F 3,54 ¼ ). Using an unpaired t-test, there was no effect of adrenalectomy alone on vehicle-treated animals in peak LH (t ¼ 0.20; df ¼ 14; P ¼ 0.85) and AUC (t ¼ 0.36; df ¼ 14 P ¼ 0.725). Experiment 3: Effects of ATR on Pulsatile LH Secretion in Adrenalectomized Animals ATR treatment altered the pulsatile release of LH in sham but not in ADX animals (Fig. 3). Sham animals treated with ATR (200 mg/kg) demonstrated LH pulses with increased pulse amplitude (F 2,31 ¼ 11.65; P, ) and pulse period (F 2,30 ¼ 7.865; P, 0.002) compared to vehicle-treated sham controls. In contrast, ADX animals showed no measurable effect of ATR on LH pulse amplitude (F 2,34 ¼ 0.61; P ¼ 0.55) and period (F 2,30 ¼ 0.84; P ¼ 0.44) at any dose used. There was a significant interaction between ATR and adrenalectomy in pulse amplitude (P, ; F 2,61 ¼ 8.824). Using an unpaired t-test, there was no effect of adrenalectomy alone on vehicle-treated animals in LH pulse amplitude (t ¼ 0.77; df ¼ 19; P ¼ 0.45) or period (t ¼ 0.91; df ¼ 16 P ¼ 0.38).

4 ATRAZINE S EFFECTS ON LH SECRETION IN ADX RATS 687 FIG. 3. LH levels of adrenalectomized or sham animals after 4 days of ATR treatment. A and B) Line graphs showing representative patterns of LH secretion in sham (A) and adrenalectomized (B) animals treated with 200 mg/kg of ATR for 4 days. Samples were obtained every 5 min. Histograms depicting the mean (6SEM) LH pulse peak concentration (C) and the mean period (6SEM) of LH pulses (D) in sham (black bars) and adrenalectomized (white bars) animals treated with ATR (0, 50, or 200 mg/kg). n ¼ 9 13/group, *P, 0.05 for 200 mg/kg vs. all other groups; # P, 0.05 for 200 mg/kg vs. 0 mg/kg control group. DISCUSSION In these studies, we have characterized the temporal changes in circulating CORT in ovariectomized Wistar rats following a single dose of ATR (0, 50, and 200 mg/kg). Prior studies have reported that elevations in CORT can impair the HPG axis and result in alterations in LH secretory parameters that are similar to those reported in animals following ATR treatment [21, 22]. However, surgical removal of the adrenal glands to prevent CORT elevations by ATR administration failed to prevent the effect of ATR on the hormone-induced LH surge. In contrast, adrenalectomy was able to prevent the effects of ATR on pulsatile LH secretion. Our data showing elevated levels of CORT following a single gavage dose of ATR support previous findings in the mouse and rat of rapid elevations in CORT after both 50 and 200 mg/kg of ATR treatment [13, 23]. In our studies, CORT levels in animals treated with 50 mg/kg of ATR returned to control levels 1 h postgavage, while CORT levels in animals treated with 200 mg/kg of ATR remained elevated over a 12-h postgavage period. These findings are consistent with a previous study examining ATR effects on rat CORT levels; however, that study sampled CORT only for 3 h postgavage [13]. Taken together, these data indicate that a single high dose of ATR can cause an elevation in CORT above control levels for up to 12 h posttreatment. Studies in mice have shown a more rapid return to control levels (6 h), but this might be accounted for by species or delivery (gavage in the current study vs. i.p. injection) [23]. The primary mechanism of ATR-induced CORT secretion remains unclear. Adrenocorticotropin hormone (ACTH) is also elevated by ATR, but ACTH levels appear to wane after 4 days of ATR treatment, independent of the CORT response [14]. Therefore, ATR could also be acting directly at the level of the adrenal to directly increase CORT secretion. In support of this possibility, it has been reported that high doses of ATR increase adrenal gland weight [24], and in vitro studies suggest that ATR may directly affect adrenal steroidogenesis [25]. A related mechanism for ATR-induced elevations in CORT may be through its reported ability to induce cyclic adenosine monophosphate levels by the inhibition of phosphodiesterase activity in human adrenocortical carcinoma cells [26 29]. Since ACTH works to increase CORT secretion through a pathway involving camp, ATR treatment may circumvent ACTH stimulation by acting directly on pathways downstream of the ACTH receptor [30]. The possibility exists that activation of adrenal steroidogenesis could contribute to the observed alterations in LH release caused by ATR. The interaction between stress hormones and reproductive function has been known for many decades [31, 32]. High CORT levels cause an inhibition of the hormone-induced LH surge [22] and decrease LH pulse frequency with increased pulse amplitude [33]. Such changes in LH secretion are very similar to our findings reporting changes in LH secretion following ATR treatment [4, 5]. Although the mechanism by which the HPA axis modifies the function of the HPG axis is not completely understood, stress hormones have been reported to act at all levels of the HPG axis [21, 22, 34 36]. To determine if ATR-induced adrenal hormone secretion is responsible for its inhibition of LH release, we examined the effects of ATR on LH secretory patterns in the presence or absence of the adrenal gland. ADX had no discernible effect on alleviating ATR inhibition of the hormone-induced LH surge, whereas ADX prevented the effect of ATR on pulsatile LH secretion. A pulsatile pattern of GnRH and LH is necessary for reproductive function and is present in all vertebrate species examined to date [37, 38]. The present findings demonstrate that the presence of the adrenal gland is necessary for ATR to inhibit LH pulse secretion. The most likely factors conferring this inhibition are CORT and/or P. P, a precursor to CORT, has long been known to be secreted along with CORT during times

5 688 FORADORI ET AL. of adrenal activation and has recently been shown to be released after ATR treatment [13]. Given this possibility, an important next step is to identify which hormonal component of adrenal secretion enables ATR to reduce LH pulse frequency. However, both of these hormones could very well be acting in conjunction to inhibit GnRH/LH release. Since type II glucocorticoid receptor (GR, official symbol, NR3C1) and P receptor (PR, official symbol PGR) are not expressed in GnRH neurons [39, 40], it is likely that if CORT or P are the intermediaries of ATR action, they act indirectly via a common inhibitory interneuronal system to lower GnRH/ LH pulse frequency. Strong evidence indicates the primary inhibitory interneuronal system(s) involved in regulating GnRH pulse frequency includes endogenous opioid peptides [41 43]. Administration of opioid peptides have been shown to inhibit LH pulse frequency [44 47] similar to that which occurs following ATR treatment. In particular, dynorphin and b-endorphin neurons in the arcuate nucleus contain both GR and PR, and P has also been shown to increase the number of GR-immunoreactive cells in the arcuate [39, 48 50]. In addition, endogenous opioid peptides expressing neurons are activated during times of stress or accompanying elevations in glucocorticoids in a fashion that could suppress LH release [51 53]. Together, these finding suggest a scenario whereby high doses of ATR treatment might cause an increase in CORT and/or P levels leading to the activation of endogenous opioid neurons, most likely dynorphin, in the arcuate nucleus. The activation of the endogenous opioids system subsequently leads to the inhibition in GnRH pulsatility. Further work is needed to determine whether this population of opioidergic neurons provides a substrate for interaction between CORT and P and serves as a final common pathway by which ATR alters GnRH pulse frequency. The inability of ADX to prevent ATR inhibition of the hormone-induced LH surge suggests an alternate mechanism of action in the presence of steroid hormone priming beyond activation of adrenal steroid secretion. It remains unclear whether ATR acts directly on GnRH neurons or on one of many neurotransmitter-containing neurons in the brain that are known to be involved in the phasic patterns of GnRH release [54, 55]. The relevance to humans, however, is in doubt because of differences in the endocrine mechanisms connecting GnRH/LH and ovulation in rodents versus those found in nonhuman primate and women [56 58]. While atrazine and its metabolites are found in the soil, groundwater and drinking water, and foodstuffs [59], the concentrations of these compounds are in the range of parts per billion. This would imply that the levels of atrazine in humans and wildlife species are well below the concentrations used in the present study. It should be noted that while there are a few reports on the effects of ADX on the hormone-induced LH surge, this is only the second report on the effects of ADX on pulsatile LH release in ADX animals [19, 60 62]. The present findings suggest that the adrenal glands are not critical for the normal pattern of LH secretion. ADX animals treated with vehicle demonstrated no measurable difference in either LH pulse parameters or the hormone-induced LH surge when compared to vehicle-treated adrenal intact animals. However, it is unclear whether longterm ADX would result in alternate findings. Saline supplementation was given to alleviate water loss due to aldosterone deficiency in the adrenalectomized animals. However, glucocorticoids and mineralocorticoids were not replaced in ADX animals. Although unknown, it is predicted that the animals would be hypoglycemic. 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