Suppression of the Luteinizing Hormone Surge by Chlordimeform in Ovariectomized,Steroid-Primed Female Rats

Abstract: The midcycle surge of luteinizing hormone (LH) from the pituitary provides the physiological trigger in the mammalian female for the process of ovulation. Accordingly, any agent that compromises the LH surge could function as a reproductive toxicant. Since ovariectomized (OVX) rats implanted with oestradiol capsules will exhibit daily afternoon surges, such animals can serve as a useful model for the investigation of toxicant-induced alterations in this functional hormonal event. The acaricide chlordimeform (CDF) has previously been found to decrease serum LH, probably by altering the hypothalamic noradrenergic transmitter control of LH secretion. Consequently, the present study focused on the effect of acute CDF administration on the appearance of the induced LH surge. Single intraperitoneal injections of CDF (0, 10, 25, 50 mg/kg) in OVX, oestradiol-implanted female Long-Evans rats approximately 5 hr prior to the expected surge caused a complete suppression at 25 and 50 mg/kg. Ten mg/kg had no effect on surge amplitude, but advanced the LH peak by 2 hr. The observed suppression did not persist beyond the day of CDF administration. Earlier dosing at 11 or 18 hr prior to the surge was without effect. Since CDF has been found to elevate serum corticosterone (CORT), 10 mg CORT/rat were given at different times prior to the surge. Twenty hr after administration only a partial lowering was seen; 5 hr exposure were ineffective. This indicates that an indirect adrenal effect was not the principal route, but may accompany an action of CDF on the hypothalamic mechanisms regulating the surge and becomes evident after more prolonged exposure.     

Evidence that atrazine and diaminochlorotriazine inhibit the estrogen/progesterone induced surge of luteinizing hormone in female Sprague-Dawley rats without changing estrogen receptor action.

High oral doses of atrazine (ATRA) disrupt normal neuroendocrine function, resulting in suppression of the luteinizing hormone (LH) surge in adult, ovariectomized (OVX) estrogen-primed female rats. While the mechanism by which ATRA inhibits LH secretion is not known, current data indicate that ATRA does have anti-estrogenic properties in vitro and in vivo. In the body, ATRA is rapidly converted to diaminochlorotriazine (DACT). The present study was conducted to investigate the effects of ATRA and DACT on the estradiol benzoate (EB)/progesterone (P) induced LH surge and to determine if such changes correlate with impaired estrogen receptor (ER) function. ATRA, administered by gavage for five consecutive days to adult OVX, female Sprague-Dawley rats, caused a dose-dependent suppression of the EB/P induced LH surge. Although to a lesser degree than ATRA, DACT significantly suppressed total plasma LH and peak LH surge levels in EB/P primed animals by 60 and 58%, respectively. DACT treatment also decreased release of LH from the pituitary in response to exogenous gonadotropin releasing hormone (GnRH) by 47% compared to control. Total plasma LH secretion was reduced by 37% compared to control, suggesting that in addition to potential hypothalamic dysfunction, pituitary function is altered. To further investigate the mechanism by which hypothalamic function might be altered, potential anti-estrogenicity of ATRA and DACT were assessed by evaluating ER function treated rats. Using an in vitro receptor binding assay, ATRA, but not DACT, inhibited binding of [(3)H]-estradiol to ER. In contrast, ATRA, administered to female rats under dosing conditions which suppressed the LH surge, neither changed the levels of unoccupied ER nor altered the estrogen induced up-regulation of progesterone receptor mRNA. Collectively, these results indicate that although ATRA is capable of binding ER in vitro, the suppression of LH after treatment with high doses of ATRA is not due to alterations of hypothalamic ER function.     

Persistent vaginal estrus and serum hormones after chlordecone (Kepone) treatment of adult female rats

The effects of chlordecone on vaginal estrus and neuroendocrine responses were examined in adult ovariectomized and intact females. Persistent vaginal estrus was seen in females given 50 mg/kg or more of chlordecone. The development of vaginal estrus was similar to that seen in ovariectomized females after treatment with estrogen. Since chlordecone is known to have estrogen-like effects on the reproductive system, the persistent vaginal estrus probably results from this estrogen-like action and chlordecone's long half-life in the organism. Neuroendocrine effects of chlordecone also resembled, to some degree, estrogen's effects on pituitary secretions. Chlordecone increased serum prolactin and decreased serum luteinizing hormone (LH) in ovariectomized females 30 to 36 hr after a single exposure to 50 mg/kg of the pesticide. Basal LH levels were not altered in intact females, but the proestrous LH surge was suppressed by 36 hr after treatment. Both basal and proestrous levels of prolactin were suppressed. Unlike the effects of estrogen, serum follicle stimulating hormone was not altered by chlordecone. These results indicate an effect on the hypothalamic pituitary axis by chlordecone treatment and offer a possible explanation for the reduced fertility seen in adult females after chlordecone exposure. However, blockage of the proestrous LH surge was not obligatory for the appearance of vaginal estrus. Although chlordecone produced peripheral changes in the vaginal smear pattern as well as neuroendocrine alterations, the peripheral changes were not always indicative of the neuroendocrine events.     

Acute exposure to molinate alters neuroendocrine control of ovulation in the rat.

Molinate, a thiocarbamate herbicide, has been reported to impair reproductive capability in the male rat and alter pregnancy outcome in a two-generation study. Published data are lacking on the effects of acute exposure to molinate in the female. Based on this work and our previous observations with related dithiocarbamate compounds, we hypothesized that a single exposure to molinate during the critical window for the neural trigger of ovulation on the day of proestrus (PRO) would block the luteinizing hormone (LH) surge and delay ovulation. To examine the effect of molinate on the LH surge, ovariectomized (OVX) rats were implanted with Silastic capsules containing estradiol benzoate to mimic physiological levels on proestrus. Doses of 25 and 50 mg/kg molinate significantly suppressed LH and prolactin secretion. Intact regularly cycling females gavaged with 0, 25, or 50 mg/kg molinate at 1300 h on PRO were examined on estrus or estrus +1 day for the presence of oocytes in the oviduct. All control females had oocytes in the oviduct on estrus. Molinate doses of 6.25 to 50 mg/kg delayed ovulation for 24 h. Estrous cyclicity was examined after daily exposure to 50 mg/kg (21 days). Estrous cyclicity was irregular in the molinate group, showing extended days in estrus. Two experiments were conducted to determine whether molinate blocked the LH surge via a central nervous system (CNS) mode of action or via an alteration in pituitary response. In the first experiment, we evaluated the release of LH in control and molinate-treated rats after a bolus dose of exogenous GnRH. Luteinizing hormone release was comparable in the two groups, suggesting that the effect of molinate is centrally mediated. To further examine the potential role of the CNS, we examined the pulsatile release of LH present in the long-term OVX females. In this model, the pulsatile pattern of LH secretion is directly correlated with GnRH release from the hypothalamus. A significant decrease in the LH pulse frequency was observed in molinate-treated females. These results indicate that molinate is able to delay ovulation by suppressing the LH surge on the day of proestrus and that the brain is the primary target site for the effects on pituitary hormone secretion.     

Dibromoacetic acid-induced elevations in circulating estradiol: effects in both cycling and ovariectomized/steroid-primed female rats

Oral exposures to high concentrations of the drinking water disinfection by-product dibromoacetic acid (DBA) over the course of 14 days have been found to disrupt estrous cyclicity in the female rat. In order to investigate possible alterations in the relevant hormonal regulatory mechanisms, female Sprague-Dawley rats were gavaged for 2 weeks with 270 mg/kg DBA, ovariectomized (OVX) and implanted with estradiol capsules. For these females, the induced luteinizing hormone (LH) surge in these animals showed a borderline suppression in peak LH concentrations that was accompanied by a marked increase in circulating estradiol. This elevation in estradiol was DBA dose-related and, for intact, normally cycling females receiving lower doses of DBA (60 and 120 mg/kg, 14 days), was present on the day of estrus, at a time when a dramatic fall from proestrous concentrations is normally evident. Evaluations of liver microsomal cytochrome p450 activity in OVX/estradiol-implanted rats showed a suppression in ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-deethylase (PROD) activity (indications of the activity of CYP1A and 2B, respectively-two key enzymes in estradiol oxidative metabolism). Phenobarbital (PhB) exposure in these animals did show induction of this activity, but was unable to lower E2 concentrations. This suggests that a DBA-induced suppression in estradiol catabolism is present and may either involve a targeted effect on the estrogen binding site on the CYP2B1/2 and CYP1A genes apart from the PhB-responsive unit, or a second pathway (possibly sulfation) that is not PhB-inducible.     

Moderating influence of the drinking water disinfection by-product dibromoacetic acid on a dithiocarbamate-induced suppression of the luteinizing hormone surge in female rats

The disinfection by-product dibromoacetic acid (DBA) has been found in female rats to increase circulating concentrations of both estradiol (E2) and estrone (E1). This effect is apparently due, at least in part, to a suppression in hepatic catabolism. The present study investigated whether DBA, by increasing sex steroid levels, is able either to augment the hypothalamic up-regulation involved in triggering a luteinizing hormone (LH) surge, or to affect the ability of the neurotoxicant sodium dimethyldithiocarbamate (DMDC) to block the surge. Sprague–Dawley rats were gavaged for 14 days with DBA (0–150 mg/kg) and ovariectomized on dosing day 11, and at the same time implanted with an estradiol capsule to generate daily LH surges. An injection of 0.1 mM/kg DMDC was administered at 13:00 h on day 14 and blood was sampled over the afternoon. DBA induced a dose-related increase in total estrogens. For identified surges, areas under the LH curve partitioned into two groups, comprising the two lower (0 and 37.5 mg/kg DBA) and the two higher (75 and 150 mg/kg) treatment groups. Consequently, low and high DBA groups were compared and found to be significantly different. At 150 mg DBA/0.1 mM DMDC, the timing of an identifiable LH peak was comparable to non-DMDC females, unlike the 37.5 mg DBA/0.1 mM DMDC group in which the appearance of peak concentrations was delayed. A significant effect with DBA treatment alone was not present. Results indicated that this exposure to DBA induced a dose-related increase in total estrogen concentrations that paralleled a diminished DMDC blockade of the LH surge. The effect appeared to be attributable to an augmentation in the estrogen-associated up-regulation in brain mechanisms stimulating the surge.     

Further evidence for effects of ethanol on gonadotrophins and prolactin secretion in female rats

The effect of different doses of ethanol (0.5, 1.0, 2.0 and 4.0 g/kg) on LH, FSH and prolactin levels has been studied in female rats. Ethanol was administered in preovulatory periods (18 hr of diestrous or 9 hr of proestrous) and hormonal levels were measured at the 18 hr of proestrous. Ethanol administered at the 18 hr of diestrous produces a biphasic effect on serum LH levels. High doses of alcohol significantly decreased LH levels, whereas low doses (0.5 g/kg) increased the hormonal levels. When ethanol-treatment was at the 9 hr of proestrous, it only decreased LH levels with the dose of 4.0 g/kg. Serum FSH levels were unaffected by the preovulatory administration of ethanol. Serum prolactin concentrations were significantly elevated after i.p. administration of ethanol at the 18 hr of diestrous and the 9 hr of proestrous. The hyperprolactinemia is more pronounced in the rats treated at the 9 hr of proestrous. The results of these studies suggest that the ability of ethanol to modify LH and prolactin levels is due to a central depression caused for alcohol. These effects of ethanol could be mediated by the hypothalamic releasing factors and/or could be due to a direct action on the pituitary function. The sum of these effects produces important failures of the reproductive function in the female rat.     

Antagonism of ethanol-induced decrease in LH by para-chlorophenylalanine: Lack of correlation with altered serotonergic mechanisms

Acute administration of ethanol lowers plasma levels of luteinizing hormone (LH) in several species. Since ethanol may interact with central serotonergic (5HT) neurons, and since 5HT systems have been found to play a role in modulating LH release, we examined the possible role of central serotonergic neurons in the ethanol-induced depression of LH. Acute PCPA (400 mg/kg, 20 hr before 2.0 g/kg ethanol) was effective in preventing the ethanol-induced depression of LH, suggesting that ethanol activates 5HT systems to lower LH. In support of this, the central 5HT agonist 5-methoxy-N, N-dimethyltryptamine (5MDMT) depressed LH in a dose-dependent manner. However, while the effects of a sub-maximal dose of 5MDMT were blocked by prior administration of methysergide, this 5HT receptor antagonist was unable to prevent the post-ethanol fall in LH. Additionally, because other doses of PCPA (250 mg/kg 20 hr prior to ethanol, and 100 mg/kg P.O. × 3 days before ethanol) produced similar reductions in hypothalamic 5HT but did not block the ethanol effect, and because electrolytic lesions of the median raphe nucleus were also ineffective in preventing the post-ethanol depression of LH, we conclude that activation of serotonergic systems does not play a major role in the ethanol induced depression of LH.     

Changes in serum LH and FSH following preovulatory administration of ethanol in rats

The effect of different single doses of ethanol (1.0, 2.0 and 4.0 g/kg) on serum LH and FSH has been studied in rats treated during preovulatory periods (18th h of diestrous). High doses of ethanol (2.0 and 4.0 g/kg) decreased serum LH levels at the 18th h of proestrous, 24 h after ethanol administration, inhibiting the preovulatory LH surge. No changes were observed in FSH levels. These effects could be mediated through the inhibition of the hypothalamic releasing factors.     

The effects of atrazine on the sexual maturation of female rats

The mammalian hazard assessment of the herbicide atrazine (ATR) has focused on the induction of mammary tumors and accelerated reproductive aging of adult rats, and the relationship of these effects to the inhibition of leutinizing hormone (LH) release from the pituitary, an effect itself caused by inhibition of GnRH signaling by the adult rat hypothalamus. In earlier studies, Laws et al. (Toxicol. Sci., 58, 366-376, 2000) demonstrated a delay in female rat sexual maturation induced by ATR, effects that could equally have been caused by inhibition of hypothalamic GnRH release. The present studies were designed to compare the doses that interfere with GnRH signaling seen in previous studies in adult Sprague-Dawley (SD) rats (LH surge suppression) with doses that impair GnRH signaling in peripubertal rats, as indicated by delayed sexual maturation. The studies evaluated the effects of ATR treatment on the timing of uterine growth and vaginal opening (VO) in peripubertal female Wistar (Alderley Park, AP) and SD rats. Doses of 10, 30, and 100 mg/kg ATR were administered daily from postnatal day (pnd) 21 to up to pnd 46. Determinations of uterine weight were made at pnd 30, 33, 43 (AP), and 46 (SD) and the timing of VO was also assessed in the last two of these experiments. The centrally acting GnRH antagonist Antarelix (ANT) was used as a positive control agent as it has previously been shown to prevent uterine growth and to delay VO in peripubertal AP rats. Uterine growth and VO were completely prevented in AP rats exposed to ANT. Uterine growth was delayed at pnd 30 and 33 in AP rats exposed to 100 mg/kg ATR, but this growth inhibition had been overcome by pnd 43. VO was significantly delayed in AP rats for the 100 mg/kg ATR dose. By pnd 46, VO was significantly delayed in SD rats exposed to both 30 and 100 mg/kg ATR, but uterine weights were unaffected by that time (as for AP rats). It is concluded that the no-effect level for the effects of ATR on sexually immature rats (10 mg/kg in SD; 30 mg/kg AP) is approximately the same as reported previously by Laws et al. in peripubertal Wistar rats (25 mg/kg). However, the no-effect level in peripubertal female SD rats is nearly an order of magnitude greater than the no-observed effect level observed in female SD rats fed ATR for 6 months (1.8 mg/kg) where LH suppression was used as an indicator of effect on the pituitary/hypothalamic axis (USEPA, Atrazine-DACT Fourth Report of the Hazard Identification and Review Committee, April 5, 2002). These results support the conclusion that the pituitary/hypothalamic axis in peripubertal female SD rats is less sensitive than that in adult female SD rats.     

Influence of chlordimeform on alpha-adrenergic receptor-associated mechanisms of hormonal regulation in the rat: pituitary and adrenocortical secretion.

The acaricide chlordimeform (CDF) has been reported to have effects on the central nervous system that appear to involve an interaction with alpha-adrenergic receptor-mediated mechanisms of neurotransmission. The present study examined the effects of CDF on adrenocortical and pituitary prolactin secretion, which are known to involve central adrenergic receptors. Male Long-Evans rats were injected i.p. with 20 or 50 mg/kg CDF and killed after 1, 4, 8 or 24 h. Both noninjected and saline-injected controls were included. Dosing was structured so that trunk blood could be collected during the morning nadir of circulating corticosterone (CORT). Assays for plasma adrenocorticotropic hormone (ACTH), CORT and prolactin (PRL) showed that with 50 mg/kg, all three hormones rose sharply by 1 h. CORT increased in a dose-dependent fashion and declined over the ensuing 8 h. Other rats were treated with the alpha-adrenergic antagonist phenoxybenzamine (PBZ, 20 mg/kg) or the alpha-agonist clonidine (CLON, 0.6 mg/kg) 40 min before and killed 1 h after CDF (25 mg/kg) injection. CLON was found to completely suppress the CDF-induced rise in CORT, while PBZ enhanced the CORT/ACTH response to CDF. CLON also significantly elevated PRL, an alteration not seen in the CLON-pretreated CDF rats. Dexamethasone was able to block the CDF-induced rise in CORT and significantly suppressed PRL levels in both saline- and CDF-treated groups. These effects indicate that CDF is interfering with a regulatory signal mediated by alpha-adrenergic receptor-associated activity.     

The effects of morphine sulphate on ovulation in the immature rat treated with PMSG

Two experiments were carried out on the effects on ovulation of morphine sulfate administered prior to the preovulatory LH surge in the immature rat treated with PMSG. At the commencement of the experiments, rats were 30 days old. In Experiment 1 all rats were injected subcutaneously with 12 IU of PMSG at 1200 hr on day 30. Doses of 6, 12, 24 and 36 mg/kg of morphine were given IP at 1555 hr on day 32. Examination of oviducts on the morning of day 33 enabled the verification of ovulation as well as oocyte counts. Results suggest that the effect of morphine on ovulation is biphasic resulting in the stimulation of ovulation at low doses (6 mg/kg) and inhibition of ovulation at high doses (24 and 36 mg/kg). In Experiment 2, rats injected with a low dose of PMSG sufficient to result in ovarian maturation but not in a preovulatory LH surge, were injected on the eve of day 32 with either saline, 6 or 24 mg/kg morphine. The treatment of rats with 6 mg/kg morphine significantly increased mean ovulatory values compared with control and 24 mg/kg morphine conditions. Further, the percentage of 6 mg/kg treated rats ovulating was more than that of both control and 24 mg/kg morphine conditions. The failure of rats treated with 24 mg/kg morphine to display increments in ovulatory response similar to 6 mg/kg morphine injected rats suggests that increased ovulation is not due to the ability of morphine to cause adrenal progesterone release but is more probably the result of LH release at low doses of morphine.     

Chlordimeform-induced alterations in endocrine regulation within the male rat reproductive system

The acaricide chlordimeform has been reported to have adverse effects in mammals that may be mediated by an interaction with alpha-adrenergic receptors. Since the hormonal signals involved in the regulation of reproductive function are themselves under hypothalamic adrenergic control, the present study was designed to investigate the effects of acute exposure to this compound on the hypothalamic-pituitary-testicular axis. Male rats given two intraperitoneal injections of chlordimeform-HCl (20 or 50 mg/kg) spaced 12 hr apart showed 24-hr declines in serum gonadotropins at 50 mg/kg that were paralleled by a drop in testosterone. These changes returned to control levels by 96 hr. Thyroid-stimulating hormone exhibited a dose-response decline that was accompanied by a similar decrease in serum thyroid hormone levels. The norepinephrine-stimulated secretion in vitro of gonadotropin-releasing hormone from hypothalamic explants was suppressed at the higher dose, while LH release from pituitary fragments in culture was unaffected. Although measurements of the in vitro release of other pituitary hormones suggest that there could be some direct pituitary effects of the compound, it appears likely that chlordimeform is able to influence endocrine regulation adversely within the reproductive system by interfering with hypothalamic alpha-adrenergic activity.     

Ovulation in rats is delayed by a substituted triazole

When a single oral dose of 5 or 25 mg/kg of the substituted triazole R151885 [1,1-di(4-fluorophenyl)-2-(1,2,4-triazol-1-yl)-ethanol] was administered at midday on diestrus-2 to rats with regular 4-day estrous cycles, the subsequent ovulation was delayed by 24 or 48 hr, respectively. There was no evidence of toxicity at the doses used. The only morphological changes detected in the reproductive tract were a delay in accumulation of uterine fluid and prolonged but normal follicular maturation prior to the delayed ovulation. The delayed follicles were slightly larger than normal follicles at the time of ovulation. The preovulatory peak plasma concentrations of progesterone, follicle stimulating hormone (FSH), and luteinizing hormone (LH) were delayed by 24 hr in rats treated with 5 mg/kg of R151885 on diestrus-2. Although there was a normal preovulatory peak plasma concentration of estradiol, values were reduced by between 30 to 50% late on diestrus-2 and early on proestrus. Additionally, in ovariectomized rats, 3 daily doses of 25 mg/kg of R151885 antagonized the action of estradiol on the uterus by 45%. We suggest that the reductions in plasma estradiol concentrations during diestrus-2 and proestrus, combined with some antagonism of estradiol's action, may prevent adequate priming of the pituitary thereby suppressing the preovulatory LH surge required for ovulation. The suppression of this LH surge is of a temporary nature indicating a reversible effect of R151885 on the hormonal control system.     

Low dose 4-MBC effect on neuroendocrine regulation of reproductive axis in adult male rats

4-Methylbenzylidene camphor (4-MBC) is an ultraviolet absorbent. The objective of this paper was to evaluate the effect of 4-MBC low-dose exposure on the neuroendocrine reproductive regulation in male rats. Wistar male adult rats were injected sc. with 4-MBC during 5 days with a dose of 2 and 10mg/kg or during 2 days with a dose of 2 and 20mg/kg. In all rats serum prolactin, LH and FSH concentration were assayed. The hypothalamus of rats injected during 2 days were also dissected to study GnRH release. Rats that received 2 and 10mg/kg of 4-MBC during 5 days showed a decrease in the LH and FSH serum concentration. In rats injected during 2 days, serum LH decreased with 2 and 20mg/kg and FSH decreased with 2mg/kg of 4-MBC. In vitro hypothalamic GnRH release also decreased in these animals. These results show that low doses of 4-MBC inhibit the reproductive axis in adult male rats.     

Reproductive functions and hypothalamic catecholamines in response to the soil fumigant metam sodium: adaptations to extended exposures

Metam sodium (MS) is a soil fumigant and Category II pesticide with a relatively low toxicity in mammals. Previous data have shown an ability to impair reproductive mechanisms in ovariectomized, estradiol-primed rats. A single i.p. injection blocked the luteinizing hormone (LH) surge that in gonadal-intact females initiates the final stages of follicular and oocytic maturation and serves as the trigger for ovulation. The effect paralleled a fall in hypothalamic norepinephrine (NE) and rise in hypothalamic dopamine (DA) that was likely due to a suppression in dopamine-beta-hydroxylase activity. In addition to determining the influence on catecholamine (CA) concentrations from a single oral exposure to MS, the present study explored effects of longer, 3-week treatments on estrous cyclicity, the LH surge, ovulation and hypothalamic CAs. Normally cycling 90 d S-D rats were administered MS (0-200 mg/kg/d, oral) and cyclicity was monitored daily. At the end of the 3rd week, proestrous blood was sampled over the afternoon from regular 4-day cyclers for a determination of LH. These animals were then killed on the following day of estrus (treatment days 21-26) for oocyte retrieval and assessment of hypothalamic CAs. Results showed that shortly after treatment began there occurred a dose-related period of persistent diestrus that typically lasted 8-16 d before regular cycles were reinstated. After 3 weeks, no effects were seen on the magnitude/timing of the LH surge or ovulated oocyte numbers. Anterior and posterior hypothalamic NE and DA were not significantly different from controls, although DA turnover (reflected by the ratio of DOPAC {3,4-dihydroxy-phenylacetic acid} to DA) in both anterior hypothalamic and caudate regions was decreased at all dosages. The data indicate that a 3 week oral exposure to MS induced an initial period of extended diestrus before the resumption of apparently normal reproductive activity, with previously reported CA alterations (apart from a persistent alteration in the DOPAC/DA ratio) being normalized by the end of dosing.     

Atrazine disrupts the hypothalamic control of pituitary-ovarian function.

The chloro-S-triazine herbicides (i.e., atrazine, simazine, cyanazine) constitute the largest group of herbicides sold in the United States. Despite their extensive usage, relatively little is known about the possible human-health effects and mechanism(s) of action of these compounds. Previous studies in our laboratory have shown that the chlorotriazines disrupt the hormonal control of ovarian cycles. Results from these studies led us to hypothesize that these herbicides disrupt endocrine function primarily through their action on the central nervous system. To evaluate this hypothesis, we examined the estrogen-induced surges of luteinizing hormone (LH) and prolactin in ovariectomized Sprague-Dawley (SD) and Long-Evans hooded (LE) rats treated with atrazine (50-300 mg/kg/day, by gavage) for 1, 3, or 21 days. One dose of atrazine (300 mg/kg) suppressed the LH and prolactin surge in ovariectomized LE, but not SD female rats. Atrazine (300 mg/kg) administered to intact LE females on the day of vaginal proestrus was without effect on ovulation but did induce a pseudopregnancy in 7 of 9 females. Three daily doses of atrazine suppressed the estrogen-induced LH and prolactin surges in ovariectomized LE females in a dose-dependent manner, but this same treatment was without effect on serum LH and prolactin in SD females. The estrogen-induced surges of both pituitary hormones were suppressed by atrazine (75-300 mg/kg/day) in a dose-dependent manner in females of both strains evaluated after 21 days of treatment. Three experiments were then performed to determine whether the brain, pituitary, or both organs were the target sites for the chlorotriazines. These included examination of the ability of (1) the pituitary lactotrophs to secrete prolactin, using hypophyosectomized females bearing pituitary autotransplants (ectopic pituitaries); (2) the synthetic gonadotropin-releasing hormone (GnRH) to induce LH secretion in females treated with high concentrations of atrazine for 3 days; and (3) atrazine (administered in vivo or in vitro) to suppress LH and prolactin secretion from pituitaries, using a flow-through perifusion procedure. In conclusion, the results of these studies demonstrate that atrazine alters LH and prolactin serum levels in the LE and SD female rats by altering the hypothalamic control of these hormones. In this regard, the LE female appeared to be more sensitive to the hormone suppressive effects of atrazine, as indicated by the decreases observed on treatment-day 3. These experiments support the hypothesis that the effect of atrazine on LH and prolactin secretion is mediated via a hypothalamic site of action.     

Potentiative action of alpha- and beta-adrenergic receptor stimulation in inducing lordosis behavior

Ovariectomized (OVX) and ovariectomized-adrenalectomized (OVX-ADX) rats were injected with estradiol benzoate (EB, 4.0 micrograms/rat) and received 44 hr by infusion into the ventromedial hypothalamic area one of the following treatments: norepinephrine (NE), clonidine (an alpha-agonist), isoproterenol (a beta-agonist) or a combination of clonidine and isoproterenol. Infusion of NE (200 ng/rat) induced lordosis in both OVX and OVX-ADX rats 15 minutes after its administration. NE-induced lordosis was blocked by systemic treatment with either the alpha-antagonist, prazosin (1.0 mg/kg), or the beta-antagonist, propranolol (4.0 mg/kg). Intrahypothalamic infusion of clonidine (200 ng/rat) or isoproterenol (200 ng/rat) induced lordosis behavior in OVX, but not in OVX-ADX rats, suggesting the involvement of adrenal secretions in this response. Combined administration of clonidine (100 ng/rat) and isoproterenol (100 ng/rat) induced lordosis behavior 15 minutes after its intrahypothalamic infusion in OVX-ADX animals. Results are discussed in relation to a model proposed for the induction of lordosis behavior involving steroid-NE interactions.     

Impact of the UV-B filter 4-(Methylbenzylidene)-camphor (4-MBC) during prenatal development in the neuroendocrine regulation of gonadal axis in male and female adult rats

4-(Methylbenzylidene)-camphor (4-MBC), a UV-B ray filter, is an endocrine disruptors (ED). Our goal was to study the effect of 4-MBC on the neuroendocrine parameters that regulate reproduction in adult female and male rats that received this disrupter during prenatal development. The 4-MBC was administered (sc) to female rats (FO) since pregnancy onset, in doses of 100 mg/kg every other day. The litters (F1) were sacrificed at 70 days to determine gonadotrophin serum levels and also GnRH and the amino acids glutamate, aspartate and GABA release from the hypothalamus. The male litter rats (F1) present at adult age a decrease in serum LH and FSH concentration and so also GnRH, excitatory amino acids and GABA hypothalamic secretion. The female litters (F1) rats present at adult age an increase in serum LH and FSH concentration, whereas hypothalamic GnRH release was not modified. In these animals a significant increase of hypothalamic aspartate release as well as GABA secretion decrease were observed. Glutamate secretion was not modified. All these changes were accompanied by an advance (3 days) on the vaginal opening in 4-MBC rats group. In conclusion, prenatal administration of 4-MBC disrupts the gonadal axis in a sexual dimorphic mode that could be connected with the physiological sexual differences in the development of gonadotrophin secretion hypothalamic control mechanisms.     

Reduction of the Amplitude of Preovulatory LH and FSH Surges and of the Amplitude of the In Vitro GnRH-induced LH Release by Substance P. Reversal of the Effect by RP 67580

The effects of Substance P (SP) and of a specific nonpeptide antagonist of the NK1 receptor (RP 67580) on preovulatory gonadotropin surges and on the in vitro GnRH induced LH surge were investigated in cycling female rats. A subcutaneous injection of SP (0.5 mg/kg body weight) at 12.00h on the proestrous day significantly decreased the LH preovulatory surge. RP 67580 (1.5 mg/kg) significantly increased this LH surge. However, when SP and its antagonist were administered together, LH preovulatory surge was normal. The FSH preovulatory surge at 18.00h and also at 19.00h was significantly inhibited by SP administration. RP 67580 alone had no effect on the FSH preovulatory surge. When SP and RP 67580 were both administered, there was no diminution of FSH plasma levels at 18.00h and 19.00h. In vitro perifusions of anterior pituitaries showed that SP inhibits GnRH-induced LH release via a NK1 receptor. Thus, SP inhibits the LH preovulatory surge via NK1 receptors and SP modulation of gonadotropin surges is at least partly exerted at the pituitary. © 1997 Elsevier Science Ltd. All rights reserved.