Reduced estradiol production by a substituted triazole results in delayed ovulation in rats

Ovulation in the rat is delayed by a single administration of the substituted triazole R151885 (1,1-di(4-fluorophenyl)-2-(1,2,4-triazol-1-yl)-ethanol). This delay results from a 24-hr shift in the preovulatory luteinizing hormone (LH) surge since administration of chorionic gonadotrophin on proestrus restores ovulation. Plasma levels of estradiol are markedly reduced (42-45%) 6-12 hr after administration of R151885. The restoration of ovulation in R151885-pretreated rats, by administration of exogenous estradiol benzoate, indicates that the reduced estradiol levels play a pivotal role in the delay of ovulation. Granulosa cells isolated from rat ovaries produce estradiol and progesterone in vitro in the presence of both follicle-stimulating hormone and testosterone. The addition of R151885 to such cultures results in a dose-dependent inhibition of estradiol production (69% by 1 microM) without a significant effect on progesterone production. This inhibition occurs at concentrations of R151885 similar to those measured in vivo. R151885 is a competitive inhibitor of human placental aromatase (apparent Ki with androstenedione substrate of 410 nM) and produces a type II spectral perturbation of cytochrome P-450 from placental microsomes. Pituitaries isolated from R151885-treated rats have reduced LH output in response to gonadotrophin-releasing hormone stimulation compared with those of controls. It is proposed that R151885 competitively inhibits aromatase activity in developing ovarian follicles. The resultant temporary reduction of plasma estradiol levels at a critical time in the estrous cycle, and consequent inadequate pituitary sensitization, produces a 24-hr delay in the preovulatory LH surge and hence ovulation is delayed by 24 hr.     

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.     

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.     

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.     

Suppression of the luteinizing hormone surge by chlordimeform in ovariectomized, steroid-primed female rats.

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.     

Effects of octamethylcyclotetrasiloxane (D4) on the luteinizing hormone (LH) surge and levels of various reproductive hormones in female Sprague-Dawley rats

The objectives of this study were to assess the potential for D₄ to suppress the pre-ovulatory lutenizing hormone (LH) surge, to block or delay ovulation, and to evaluate potential effects on reproductive hormones in rats. Female Sprague–Dawley Crl:CD^® (SD) IGS BR rats received whole-body vapor inhalation exposure to D₄ (0, 700, or 900 ppm) 6 h per day for 3 days. Trunk blood obtained on proestrus at 10 a.m. was evaluated for levels of follicle stimulating hormone (FSH), estradiol (E2), estrone (E1), and progesterone (P4). Other rats had serial blood samples collected via cannula at 2, 4, 6, 8, and 10 p.m. on the day of proestrus and plasma evaluated for LH and prolactin (PRL). Trunk blood was collected at 8 a.m. of estrus and plasma evaluated for FSH, E2, E1, and P4. At 10 a.m. on proestrus, significant increases in E1 levels in the 700 and 900 ppm groups and significant increases in P4 levels in the 900 ppm group were noted. At 8 a.m. on estrus, significant increases in E1, E2, in the E1/E2 ratio and decreases in FSH were noted in the 700 and 900 ppm groups. The major effect on the LH profile was observed most clearly when the rats were grouped by ovulatory status, animals that did or did not ovulate. Regardless of treatment, suppression of the LH surge correlated with blocked ovulation. The percentage of rats that ovulated was (700 ppm, 42%; 900 ppm, 31%) compared to controls (79%). Overall, the data indicate that high exposures to D₄ attenuated the pre-ovulatory LH surge and significantly decreased the portion of female rats that ovulated.     

Endocrine disruption by indole-3-carbinol and tamoxifen: blockage of ovulation

Immature Sprague-Dawley rats received daily doses of indole-3-carbinol (I3C, 0-1.5 g/kg/day), 3,3'-diindolymethane (DIM, 0-400 mg/kg/day), tamoxifen (TAM, 0-0.5 mg/kg/day), or vehicle to determine if their antiestrogenic effects occur by the same mechanism and whether I3C's action is mediated by DIM. Follicular development was induced on day 24 of age by equine chorionic gonadotropin (eCG, 5 IU) 1 day after the initial dose. In a hormone replacement study, human chorionic gonadotropin (hCG, 10 IU sc, 48 h post-eCG) was used to mimic a normal preovulatoy luteinizing hormone (LH) surge following treatment with either I3C or TAM. Blood and ovaries were collected throughout follicular development and the number of ova shed was measured on the morning following expected ovulation (72 h post-eCG). I3C but not TAM reduced body weight gain at higher doses after 4 days of dosing. Ovarian weight gain and ovulation were inhibited by both I3C and TAM in a dose-dependent fashion. During the preovulatory period, both I3C and TAM blocked normal LH and follicle-stimulating hormone (FSH) surges and suppressed serum progesterone (P(4)) profoundly without changing circulating levels of estrogen (E(2)). At the time of expected ovulation, serum E(2) was increased in rats receiving I3C or tamoxifen, whereas serum P(4) was dose-dependently decreased. DIM exerted no significant effects on any of the endpoints studied, even at the highest dose, indicating that the antiestrogenic effects of I3C are not mediated by this metabolite of I3C. hCG successfully restored ovarian weight gain and ovulation in TAM-treated rats. However, hCG only partially reversed the blockage of ovulation by I3C, although ovarian weight gain was restored to normal. In summary, both I3C and TAM block ovulation by altering preovulatory concentrations of LH and FSH, but I3C appears to exert its effect(s) by (a) different mechanism(s) of action. I3C seems to act at both the ovarian and hypothalamic levels by mechanisms similar to those seen in TCDD-treated rats, whereas TAM appears to act only on the hypothalamic-pituitary axis as an anti-estrogen.     

Ovarian effects of an anti-inflammatory-immunomodulatory drug in the rat

The purpose of this study was to determine whether a 30-day administration of SK&F 86002-A2, an inhibitor of cyclooxygenase and 5-lipoxygenase pathways of arachidonate metabolism, adversely affected reproductive cycles, ovarian structure, and/or pituitary/ovarian hormone secretion. Cyclooxygenase and 5-lipoxygenase enzymes catalyze the reactions leading to the synthesis of prostaglandins and leukotrienes, respectively, which are physiological regulators of ovarian function. Female rats were dosed once daily by gavage with 0, 1, 5, 10, 30, or 60 mg (base)/kg/day of SK&F 86002-A2 for 30 consecutive doses beginning on the day of vaginal proestrus. Vaginal smears were then examined daily until necropsy, when ovaries and uteri were collected for macroscopic and histological examination. In addition, serum concentrations of estradiol, progesterone, luteinizing hormone, follicle stimulating hormone, and prolactin were estimated by radioimmunoassay. Estrous cycle irregularity, resulting from a dose-related lengthening of the interestrous interval, significantly (p less than 0.05) reduced the number of cycles in rats receiving 60 mg/kg/day of SK&F 86002-A2 compared to controls. Furthermore, the ovaries from this group of rats weighed significantly more (p less than 0.05) than controls, apparently due to an increased occurrence of enlarged, cystic follicles that occasionally contained blood. Luteinized follicles with entrapped ova were also detected during histological examination. Dilatation of the uterine lumen was observed in some rats receiving doses of SK&F 86002-A2 greater than 1 mg/kg/day. Serum progesterone in rats receiving 60 mg/kg/day of SK&F 86002-A2 was significantly (p less than 0.05) lower than controls. In contrast, mean levels of serum estradiol were elevated in rats receiving 30 mg/kg/day of SK&F 86002-A2. Serum concentrations of FSH, LH, and prolactin were not significantly different in any group. The results of this study suggest that SK&F 86002-A2 disrupts cyclic ovarian function by a local, cumulative action that inhibits ovulation and alters steroid secretion.     

利洛司酮对子宫内膜异位症大鼠血清黄体生成素和雌二醇水平的影响

目的 :观察利洛司酮对子宫内膜异位症大鼠血清黄体生成素 (LH)和雌二醇 (E2 )水平的影响。方法 :2 0只子宫内膜异位症大鼠 ,随机分成 4组 ,灌胃给予利洛司酮 2 0 ,80mg·kg- 1,米非司酮 5 0mg·kg- 1和精制花生油 4mL·kg- 1,每日 1次 ,共 2 1d。采集用药d 19,2 0 ,2 1及停药 2 4h的血样本 ,用大鼠睾丸间质细胞睾酮法 (RICT法 )及放射免疫分析(RIA)法分别测定血清LH和E2 的水平。结果 :对照组大鼠给药d 19,2 0 ,2 1及停药 2 4h ,这 4d的血清LH和E2 水平均有周期性变化 ,出现一个动情前期的LH峰和E2 峰 ;利洛司酮 2 0 ,80mg·kg- 1及米非司酮 5 0mg·kg- 1组大鼠血清LH和E2 水平未见周期性变化 ,也未见动情前期的LH峰和E2 峰。结论 :和米非司酮一样 ,利洛司酮可抑制LH和E2 的分泌 ,取消动情前期的LH峰和E2 峰     

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.     

Phenytoin inhibits both the first ovulation and uterine development in gonadotropin-primed immature rats

This study was planned to determine the effects and possible mechanism of action of phenytoin on development of the reproductive tract and first ovulation in immature rats. Rats were injected s.c. with 5 IU of equine chorionic gonadotrophin (equine CG) on day 26 to induce ovarian and uterine development. Treatment with phenytoin (140 mg/kg) at 1200 h on day 28, which induces serum levels approximately twice those reached with the clinical dose as anticorvulsant drug for humans, was effective for inhibiting the first ovulation and normal secretion of serum follicle - stimulating hormone and luteinizing hormone (LH) on day 29 as well as the preovulatory gonadotrophin surge on day 28. The block of ovulation was overcome by administration of human chorionic gonadotrophin or LH-releasing hormone on day 28. Simultaneous treatment with equine CG and phenytoin at 0800 h on day 26 did not affect either ovarian weight or ovarian hormones secretion, whereas phenytoin clearly inhibited the normal increase in uterine weight on day 27. Furthermore, phenytoin suppressed uterine growth after 17beta-oestradiol injection. These results indicate that phenytoin inhibits the first ovulation by inhibiting the gonadotrophin surge and further, that the drug impairs the stimulatory effects of oestrogen on uterine proliferation in the gonadotropin-induced ovulation model.     

2,3,7,8-tetrachlorodibenzo-p-dioxin decreases responsiveness of the hypothalamus to estradiol as a feedback inducer of preovulatory gonadotropin secretion in the immature gonadotropin-primed rat

Sprague-Dawley rats (23-day-old) were dosed with TCDD (32 microg/kg) in corn oil or vehicle alone. Equine chorionic gonadotropin (eCG) was injected (5 IU, sc) 24 h later to induce follicular development. Another 24 h later, half of TCDD- or corn oil-treated rats were injected (sc) with 17 beta-estradiol-cypionate (ECP, at 0.004 to 0.5 mg/kg). Blood and ovaries were collected on expected proestrous (preovulatory period) at 51, 54, and 58 h after eCG injection as well as in the morning after ovulation (72 h after eCG). Serum concentrations of 17 beta-estradiol (E), progesterone (P), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were determined by radioimmunoassay. The number of ova shed was measured at 72 h after injection of eCG by irrigating ova from oviducts. During the preovulatory period (approximately 58 h after eCG injection), a circulating level of 70-100 pg E/ml coincided with LH and FSH surges and later normal ovulation of 10 to 12 ova/rat was observed in controls. However, the same concentration of E was not associated with LH and FSH surges in rats treated with TCDD (32 microg/kg), resulting in reduced ovarian weight gain and reduction of ovulation by 70 to 80% (2-3 ova/rat). Blockage of the gonadotropin surge, reduced ovarian weight gain, and ovulation were all reversed completely by the lowest effective dose of ECP (0.1 mg/kg). At 72 h after eCG, serum P secretion was reduced and serum E levels were significantly increased compared to those of corn oil-treated controls. ECP alone had no effect on serum P levels at any time point, but in rats treated with TCDD and ECP, both the reduction of P (at 58 and 72 h) and the increase in E secretion (72 h) were completely reversed. Further studies confirmed that restoration by ECP of gonadotropin surges and associated ovulation could not be attained until circulating levels of E rose sufficiently high to trigger the LH and FSH surges. The new action threshold of E for inducing gonadotropin surges in rats treated with TCDD (32 microg/kg) was determined to be eight- to 10-fold higher than that in controls. Thus, it is apparent that TCDD decreased the responsiveness of the hypothalamus to E as a feedback inducer of preovulatory gonadotropin secretion.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin Decreases Responsiveness of the Hypothalamus to Estradiol as a Feedback Inducer of Preovulatory Gonadotropin Secretion in the Immature Gonadotropin-Primed Rat

Sprague–Dawley rats (23-day-old) were dosed with TCDD (32 μg/kg) in corn oil or vehicle alone. Equine chorionic gonadotropin (eCG) was injected (5 IU, sc) 24 h later to induce follicular development. Another 24 h later, half of TCDD- or corn oil-treated rats were injected (sc) with 17β-estradiol-cypionate (ECP, at 0.004 to 0.5 mg/kg). Blood and ovaries were collected on expected proestrous (preovulatory period) at 51, 54, and 58 h after eCG injection as well as in the morning after ovulation (72 h after eCG). Serum concentrations of 17β-estradiol (E), progesterone (P), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were determined by radioimmunoassay. The number of ova shed was measured at 72 h after injection of eCG by irrigating ova from oviducts. During the preovulatory period (58 h after eCG injection), a circulating level of 70–100 pg E/ml coincided with LH and FSH surges and later normal ovulation of 10 to 12 ova/rat was observed in controls. However, the same concentration of E was not associated with LH and FSH surges in rats treated with TCDD (32 μg/kg), resulting in reduced ovarian weight gain and reduction of ovulation by 70 to 80% (2–3 ova/rat). Blockage of the gonadotropin surge, reduced ovarian weight gain, and ovulation were all reversed completely by the lowest effective dose of ECP (0.1 mg/kg). At 72 h after eCG, serum P secretion was reduced and serum E levels were significantly increased compared to those of corn oil-treated controls. ECP alone had no effect on serum P levels at any time point, but in rats treated with TCDD and ECP, both the reduction of P (at 58 and 72 h) and the increase in E secretion (72 h) were completely reversed. Further studies confirmed that restoration by ECP of gonadotropin surges and associated ovulation could not be attained until circulating levels of E rose sufficiently high to trigger the LH and FSH surges. The new action threshold of E for inducing gonadotropin surges in rats treated with TCDD (32 μg/kg) was determined to be eight- to 10-fold higher than that in controls. Thus, it is apparent that TCDD decreased the responsiveness of the hypothalamus to E as a feedback inducer of preovulatory gonadotropin secretion.     

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.     

The reversible effects of raloxifene on luteinizing hormone levels and ovarian morphology in mice

Raloxifene is a selective estrogen receptor modulator that has estrogen agonist effects on bone and serum lipids and estrogen antagonist effects on breast and uterine tissues. This study assessed the effects of raloxifene hydrochloride (HCl) treatment on circulating luteinizing hormone (LH) levels and ovarian morphology in sexually mature, 15-week-old, female CD-1 mice. Mice were maintained on diets providing average daily doses of 0 or 233 mg/kg raloxifene for 2 weeks (Study 1) or 0, 7.9, or 236 mg/kg raloxifene for 4 weeks (Study 2). At the end of the treatment period, blood samples were collected every 2 hours for 24 h in Study 1 (5 mice per group) and at 10:00 a.m. and 10:00 p.m. in Study 2 (8 mice per group). Serum LH levels were measured by radioimmunoassay. Ovarian histomorphology was evaluated in the 10 mice per group (Study 1) and the 8 mice per group (Study 2). For the reversibility phase (Study 2), mice were fed untreated diets for 3 weeks; serum LH levels and ovarian histomorphology were then assessed. Raloxifene treatment at 233 mg/kg/day for 2 weeks (Study 1) significantly elevated circulating LH levels by 4- to 7-fold compared with control. Raloxifene-treated mice had elevated LH levels sustained over the 24-h sampling period and did not exhibit the preovulatory LH surge evident in some control mice at the 4:00 p.m., 6:00 p.m., and 8:00 p. m. time points. Mice treated with 236 mg/day raloxifene for 4 weeks (Study 2) had elevated LH levels (4.4-fold compared to control), whereas mice exposed to 7.9 mg/kg/day raloxifene had a slight, nonsignificant increase in LH (2-fold compared to control). In both dose groups, LH levels were indistinguishable from controls 3 weeks after raloxifene treatment was discontinued. The ovaries in six of the eight mice treated with 7.9 mg/kg/day raloxifene had dilated and/or anovulatory follicles. One mouse in this group had a single hemorrhagic follicle; however, corpora lutea distribution was normal, indicating that ovulation was occurring. Raloxifene-treated mice in Study 1 and mice treated with a comparable raloxifene dose (236 mg/day) in Study 2 had histomorphological changes in the ovary indicative of arrested follicular maturation, including anovulatory hemorrhagic follicles, some developing follicles, and very few corpora lutea. At the end of the reversibility phase, hemorrhagic follicles were no longer evident and follicular maturation and corpora lutea distribution were normal. Raloxifene treatment in mice produces a dose-dependent, sustained elevation in serum LH levels and is associated with changes in ovarian follicular morphology. These changes are reversible upon discontinuation of raloxifene treatment.     

Studies on metabolism and effects of estrogen on pituitary prolactin and LH secretion.

The effect of a subcutaneous injection of estradiol on the secretion of pituitary prolactin in the rat and the relationship between serum estradiol level and luteinizing hormone (LH) secretion in mare were reviewed. In addition, the effect of estradiol injection on LH secretion and the metabolism of [14C] estradiol in intact and bile duct fistulated pony mares were studied. Low (0.1 mug/day/rat) to moderate dose (5 mug/day/rat) of estradiol benzoate injected subcutaneously to mature or immature rats significantly increased pituitary content of prolactin and serum prolactin level five- to tenfold. On the other hand, high dose of estradiol (10 mug/day/rat or more) was less effective in stimulating prolactin secretion, and it appeared that progesterone injected concurrently with estradiol had some inhibitory action on the stimulatory effect of estradiol. Studies in pony mares showed that the physiologic level of serum estradiol during proestrus was important for the induction of the ovulatory surge of LH. Intramuscular injection of a low dose (2 or 4 mg/mare) of estradiol was stimulatory, whereas a high dose (8 mg/mare) was inhibitory for LH secretion in pony mares. Results of the estradiol metabolism studies indicated a relatively long half-life for estradiol in the mare. The majority of the [14C] estradiol metabolites appeared in the urine within 24 hr following intravenous injection. Enterohepatic circulation appeared to be important for estradiol metabolism in mare.     

Is inhibition by diazepam and beta-carbolines of estrogen-induced luteinizing hormone secretion related to sedative effects?

The effects of diazepam (DZ) and the beta-carbolines ZK-91296 and ZK-93423 on luteinizing hormone (LH) release evoked by beta-estradiol were studied in estradiol-primed ovariectomized rats. Acute treatment with 2.5, 5 and 10 mg/kg DZ and ZK-91296 significantly blunted the LH response. ZK-93423 (1, 5 and 10 mg/kg) produced a similar effect. The inverse agonist DMCM (2 mg/kg) was unable to modify plasma LH levels in estrogen-primed, ovariectomized rats. Sedative effects were observed with doses of DZ (1 mg/kg) ineffective to blunt the LH response. In contrast, ZK-91296 (5 mg/kg) produced inhibition of LH surges in nonsedative doses. These results indicate that diazepam and beta-carboline agonists prevent the LH surge evoked by estrogen. They suggest, in addition, that the actions on LH release and their sedative effects are not directly related.     

Effects of alcohol on E2 beta-stimulated luteinizing hormone in ovariectomized rhesus monkeys.

Anovulation is a frequent concomitant of alcohol abuse, but it has been difficult to assess the acute effects of alcohol on ovulation. Estradiol benzoate (E2 beta) can stimulate a luteinizing hormone (LH) surge in ovariectomized monkeys that appears to be associated with increased luteinizing hormone-releasing hormone (LHRH) pulse frequency and amplitude. The acute effects of alcohol (2.5 and 3.5 g/kg) and an isocaloric sucrose control solution on LH and follicle-stimulating hormone (FSH) secretory activity were studied in five ovariectomized monkeys 41 to 51 hours after administration of E2 beta (42 micrograms/kg, IM). Integrated plasma samples were collected at 20-minute intervals over 10 hours. Under sucrose control conditions, LH increased to 445 and 584 ng/ml within 46 to 49.3 hours after E2 beta administration in two monkeys and high-amplitude LH pulses were evident in three monkeys. Alcohol (2.5 and 3.5 g/kg) significantly decreased the number of LH peaks and valleys (p < 0.01). Peak blood alcohol levels averaged 195 and 291 mg/dl. After 2.5 g/kg alcohol, there was no LH surge or LH pulses in four of five monkeys. A delayed LH surge occurred in one monkey 48 to 50.6 hours after E2 beta when blood alcohol levels decreased to 62 mg/dl. After 3.5 g/kg alcohol, no monkey had an LH surge and pulsatile LH release was significantly reduced in comparison to control conditions (p < 0.01). FSH levels remained stable across alcohol and control conditions. These data suggest that alcohol attenuates pituitary release of LH in response to E2 beta stimulation. These findings are consistent with menstrual cycle disruptions observed in alcohol-dependent women, social drinkers, and in a primate model of alcoholism.     

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.     

Effect of lindane on hormonal control of reproductive function in the female rat

The effect of the gamma isomer of 1,2,3,4,5,6-hexachlorocyclohexane, lindane, on reproductive function in the female rat was examined in two experiments. In the first experiment, chronic treatment with 0, 5, 10, 20, and 40 mg/kg lindane delayed vaginal opening and disrupted ovarian cyclicity until approximately 110 days of age. Thereafter, regular ovarian cycles were present in the majority of females in all dose groups. When killed on the day of vaginal proestrus, the females receiving the two higher doses of lindane had smaller pituitary and uterine weights, lower serum and pituitary luteinizing hormone (LH) and prolactin, and higher pituitary follicle stimulating hormone (FSH) concentrations than the oil-treated control females. Serum estrogen concentrations were not different from controls in the 5 and 20 mg/kg groups, significantly greater than the controls in the 10 mg/kg group, and significantly less than the controls in the group receiving 40 mg/kg. In a second experiment, the uterine weight and pituitary hormone response of 28-day-old, lindane-treated females to a 10-micrograms injection of estradiol benzoate (EB) were investigated. The uteri of the lindane-treated prepubertal females were smaller than controls at 30 hr after EB injection. Furthermore, the EB-induced increase in serum luteinizing hormone, observed at 30 hr after EB injection, was lower in the lindane-treated animals. Similarly, the reduction in pituitary LH, FSH, and prolactin induced by EB was not as great in the lindane-treated animals as in the controls. Serum estrogen concentrations in the lindane-treated animals were not different from controls. These data indicate that lindane may effectively block the response of estrogen-dependent tissues to this ovarian steroid hormone and that this apparent antiestrogenic effect of lindane is responsible for the disturbances observed in the neuroendocrine control of ovarian function in the rat.