Actions of ethanol on epidermal growth factor receptor activated luteinizing hormone secretion.

OBJECTIVE: Activation of the epidermal growth factor receptor (EGF-R) stimulates prepubertal luteinizing hormone-releasing hormone (LHRH) release, which in turn induces luteinizing hormone (LH) secretion. Although ethanol (ETOH) diminishes LHRH secretion and delays the onset of female puberty, its actions following EGF-R activation are unknown. We therefore investigated the effects of ETOH on EGF-induced LHRH and LH release, both in vivo and in vitro. METHOD: Basal blood samples were taken every 15 minutes from immature female rats, which then received ETOH (3 g/kg) or saline by gastric gavage. After a 90-minute ETOH absorption period, a single blood sample was drawn from each rat. Finally, EGF (200 ng/3 microl) was injected into the third ventricle of all animals and postinjection samples were drawn every 15 minutes. A similar experimental design was performed except no blood samples were taken. After ETOH exposure, EGF was administered, and 45 minutes later the animals were killed and their medial basal hypothalamus and pituitary were removed and analyzed for EGF-R and cyclooxygenase (COX) 1 and 2 by western blot analysis. For in vitro experiment, median eminence fragments from immature female rats were exposed to ETOH in a static incubation system, and prostaglandin-E, (PGE2) and LHRH were measured. RESULTS: EGF stimulated LH release, and this release was blunted (p < .05) by ETOH. Western blot analysis revealed that ETOH did not alter the EGF-R protein levels in the hypothalamus. ETOH blocked EGF-induced PGE2 and LHRH released from isolated median eminences. EGF administration increased both COX-1 (p < .001) and COX-2 (p < .01), but both enzymes were blocked by ETOH. CONCLUSIONS: The ETOH-induced decrease in EGF-stimulated LH release is due to a reduction in the formation of hypothalamic PGE2 and, subsequently, suppressed LHRH release.

Acute effects of ethanol on steroidogenic acute regulatory protein (StAR) in the prepubertal rat ovary.

BACKGROUND: Steroidogenic acute regulatory protein (StAR) is a 30 kDa mitochondrial protein that plays an essential role in steroid hormone biosynthesis by facilitating delivery of cholesterol across the mitochondrial membrane, where side chain cleavage occurs to initiate ovarian steroidogenesis. Because ethanol (EtOH) suppresses estradiol secretion in prepubertal female rats, we evaluated the effects of EtOH on prepubertal ovarian StAR. METHODS: At 0700 hr, 28-day-old female rats were gavaged with saline or a 3 g/kg dose of EtOH. At 0800 hr, half of each of these two groups was treated with 15 IU of pregnant mare serum gonadotropin (PMSG). At 1000 hr, a 2 g/kg dose was administered to maintain moderately elevated blood alcohol levels. At 1600 hr, all of the animals were killed by decapitation, and blood and ovaries were collected for measurement of serum pregnenolone and estradiol and for ovarian StAR gene and protein expression. RESULTS: Northern blot analysis showed two major transcripts of 3.8 and 1.7 kb of ovarian StAR mRNA. The ovaries from EtOH-treated rats showed decreased (p < 0.01) basal expression of both 3.8 and 1.7 kb StAR transcripts. PMSG-stimulated animals showed a more than 4-fold increase (p < 0.001) in the levels of both transcripts, when compared with ovaries from animals that received saline or EtOH only. Conversely, in EtOH-treated animals, the PMSG-stimulated expression of the 1.7 kb transcript was blocked, and the increase in the 3.8 kb StAR transcript was blunted (p < 0.05 vs. PMSG). Western blot analysis revealed that EtOH exposure also depressed (p < 0.01) the basal expression of StAR protein. PMSG-stimulated animals showed an increase (p < 0.001) in levels of StAR protein, and this was blocked (p < 0.01) by EtOH. These changes observed in ovarian StAR mRNA and protein were paralleled by changes in serum pregnenolone and estradiol. Specifically, acute EtOH exposure suppressed (p < 0.05) the basal levels of both steroids. Furthermore, PMSG-stimulated animals showed an increase in the production of pregnenolone (p < 0.05) as well as estradiol (p < 0.01), and EtOH blocked this stimulatory action of PMSG on both steroids. CONCLUSION: These results demonstrate for the first time that EtOH is capable of altering ovarian StAR expression, which contributes to the detrimental effect this drug has on ovarian steroidogenesis during prepubertal development.

Lamprey gonadotropin-releasing hormone-III selectively releases follicle stimulating hormone in the bovine.

Recent studies have shown that lamprey gonadotropin-releasing hormone (l-GnRH) is localized in the mammalian brain, and that l-GnRH-III, can selectively induce FSH secretion in the rat both in vivo and in vitro. Consequently, the purpose of this study was to determine if l-GnRH-III could elicit selective FSH release in cattle and compare this response with that to mammalian luteinizing hormone releasing hormone (m-LHRH). Cattle were chosen as the animal model because previous studies have demonstrated that FSH and LH are secreted by separate gonadotropes in that species. For these studies, crossbred cycling heifers were implanted with jugular cannulae and l-GnRH-III was infused either between Days 9-14 or on Day 20 of the estrous cycle. Blood samples were collected both before and following peptide infusion. Our results demonstrate that during Days 9-14 of the estrous cycle (luteal phase), when progesterone levels averaged between 4 and 5 ng/ml, a dose of 0.25 mg of l-GnRH-III induced the release of FSH (P < 0.05), but not LH. A 0.5 mg dose of l-GnRH-III caused a greater release of FSH (P < 0.01), but still did not induce LH release. Higher doses of the peptide were capable of significantly releasing both gonadotropins. Importantly, during the luteal phase, doses of 0.5 and 2 mg of m-LHRH were ineffective in stimulating FSH, but did elicit marked increases (P < 0.001) in LH. Again, progesterone levels averaged 4-5 pg/ml. In order to assess gonadotropin releasing ability of l-GnRH-III at a different phase of the estrous cycle, some animals were administered the peptide on Day 20, when progesterone levels were below 1.0 pg/ml. At this time, the l-GnRH-III induced the release of LH (P < 0.01), but not FSH. Overall, our results demonstrate that l-GnRH-III can selectively induce FSH in cattle during the luteal phase, whereas m-LHRH was ineffective in that regard. Furthermore, the fact that l-GnRH-III can selectively stimulate FSH when serum progesterone is high, and LH when serum progesterone is low, suggests its actions are under strong control of this steroid. We suggest the FSH releasing capacity of l-GnRH-III in cattle could render this peptide useful for enhancement of reproductive efficiency in this species.

Leptin acts centrally to induce the prepubertal secretion of luteinizing hormone in the female rat.

Recent data generated from adult male and female rats indicates that leptin is capable of stimulating luteinizing hormone (LH) secretion via a hypothalamic action. Consequently, we hypothesized that this peptide may similarly play a role in controlling LH secretion during late juvenile and peripubertal development; hence, contributing to hypothalamic-pituitary function during sexual maturation. Therefore, this study was conducted to determine if leptin is capable of stimulating LH release during this critical time of development and, if so, to determine whether this action is due to an effect at the hypothalamic level. Results showed that leptin, when administered directly into the brain third ventricle (3V), can stimulate (P < 0. 01) LH release in late juvenile animals at doses of 0.01-1.0 microg. A higher dose of 10 microg was ineffective in stimulating LH release. Immunoneutralization of luteinizing hormone-releasing hormone (LHRH) via 3V administration of LHRH antiserum to late juvenile animals indicated a hypothalamic site of action, since the leptin-induced LH release was blocked in the animals that received anti-LHRH, but not in the control animals that received normal rabbit serum. Leptin did not significantly stimulate LH release from animals in first proestrus, estrus, or diestrus. We also report that the serum levels of leptin increase (P < 0.05) during the late juvenile period of development, then decrease (P < 0.05) once the animal enters the peripubertal period. Collectively, our results show that leptin is capable of acting centrally to stimulate LH release, but only during late juvenile development; thus, we suggest the peptide likely plays a facilitatory role on late juvenile LH secretion, but does not drive the LHRH/LH releasing system to first ovulation and hence, sexual maturity.

Effects of ethanol on leptin secretion and the leptin-induced luteinizing hormone (LH) release from late juvenile female rats.

BACKGROUND: Chronic ethanol (EtOH) exposure lowers serum insulin-like growth factor-1 (IGF-1) and luteinizing hormone (LH) levels and also delays female puberty, similar to the deficits in the reproductive system that occur during leptin deficiency. Leptin administration restores fertility and gonadotropin secretion in the ob/ob mouse and can induce recovery of reproductive function in food-restricted animals. This study assessed the effects of EtOH on serum leptin levels, and whether exogenous leptin administration could restore IGF-1 and LH levels in the EtOH-treated animals. METHODS: In the first study, 29-day-old female rats were divided into control and EtOH-treated groups, each of which received their respective diet regimen for 5 consecutive days. The EtOH-treated animals were subdivided and received an intraperitoneal injection of either leptin (100 microg/0.1 ml) or saline twice daily. Control animals also received intraperitoneal saline injections twice daily. On day 34, animals were killed, and serum leptin, LH, and IGF-1 were measured by RIA. In a second study we assessed the acute effects of a single 3 g/kg dose of EtOH on the ability of leptin to act centrally to induce LH release. For this, leptin (1 microg) was administered via a third ventricular (3V) cannula and blood sampling via jugular cannula. In a third experiment, animals were again subjected to a chronic feeding regimen. When 34 days old, they were killed and the anterior pituitaries removed and incubated in a static incubation system for 60 min to establish basal LH release, then for an additional 60 min in medium containing leptin (10(-7) M). RESULTS: Chronic EtOH exposure lowered serum leptin (p < 0.01), IGF-1 (p < 0.01), and LH (p < 0.05) levels. Leptin administration to EtOH-treated animals did not restore serum IGF-1 levels. This peptide did, however, effectively restore LH levels to normal, but did not advance the timing of puberty. Acute EtOH administration was found to block leptin-induced LH release following central administration of the peptide. Conversely, anterior pituitaries from control and 5-day EtOH-treated animals that were incubated in vitro released (p < 0.01) equal amounts of LH in response to leptin (10(-7) M). CONCLUSIONS: These data demonstrate that EtOH administration not only can suppress peripheral levels of leptin, but also blocks its central action to facilitate LH secretion. Although replacement of leptin can reverse the EtOH-induced suppression of LH by a direct action at the level of the pituitary, it cannot elevate serum IGF-1; a peripheral signal that acts centrally to stimulate LH releasing-hormone (LHRH)/LH release during the juvenile-peripubertal transition period, and thus accelerates the initiation of female puberty. These results demonstrate further the complex actions and interactions of multiple hormones involved in the pubertal process and the vulnerability of their actions to the toxic effects of EtOH.