W. Les Dees

Effects of lead (Pb) exposure during gestation and lactation on female pubertal development in the rat.

Lead (Pb) can delay sexual maturation; however, the mechanism and critical time of insult are not clearly defined. Therefore, we assessed maternal Pb levels during low-level gestational and/or lactational exposure, as well as blood and tissue Pb in developing fetuses in relation to the subsequent detrimental effects of Pb on puberty-related hormones and the onset of female puberty. Adult Fisher 344 female rats were gavaged daily with either a 1-ml solution of PbAc containing 12 mg/ml Pb or an equal volume of sodium acetate (NaCl), for the controls, from 30 days prior to breeding until their pups were weaned at 21 days. By cross-fostering at the time of birth, the pups were either exposed to PbAc or NaAc during gestation only, lactation only, or during both gestation and lactation. Pb delayed the timing of puberty and this delay was associated with suppressed serum levels of insulin-like growth factor-1 (IGF-1), luteinizing hormone (LH), and estradiol (E(2)). Liver IGF-1 mRNA was not affected, suggesting that Pb altered translation and/or secretion of IGF-1. We reported previously that peripherally derived IGF-1 acts at the hypothalamic level to facilitate LH release at puberty; hence, we suggest that the action of Pb in decreasing circulating IGF-1 contributes to the delayed puberty. The detrimental effects occurred regardless of the developmental time of exposure, although gestational exposure appeared more sensitive to the effects of Pb. Also, the effects noted were with blood Pb levels less than previously reported and these levels are relevant to human health concerns.

Control of Gonadotropin Secretion by Follicle-Stimulating Hormone-Releasing Factor, Luteinizing Hormone-Releasing Hormone, and Leptin

Fractionation of hypothalamic extracts on a Sephadex G-25 column separates follicle-stimulating hormone-releasing factor (FSHRF) from luteinizing hormone-releasing hormone (LHRH). The FSH-releasing peak contained immunoreactive lamprey gonadotropin-releasing hormone (lGnRH) by radioimmunoassay, and its activity was inactivated by an antiserum specific to lGnRH. The identity of lGnRH-III with FSHRF is supported by studies with over 40 GnRH analogs that revealed that this is the sole analog with preferential FSH-releasing activity. Selective activity appears to require amino acids 5-8 of lGnRH-III. Chicken GnRH-II has slight selective FSH-releasing activity. Using a specific lGnRH-III antiserum, a population of lGnRH-III neurons was visualized in the dorsal and ventral preoptic area with axons projecting to the median eminence in areas shown previously to control FSH secretion based on lesion and stimulation studies. Some lGnRH-III neurons contained only this peptide, others also contained LHRH, and still others contained only LHRH. The differential pulsatile release of FSH and LH and their differential secretion at different times of the estrous cycle may be caused by differential secretion of FSHRF and LHRH. Both FSH and LHRH act by nitric oxide (NO) that generates cyclic guanosine monophosphate. lGnRH-III has very low affinity to the LHRH receptor. Biotinylated lGnRH-III (10(-9) M) labels 80% of FSH gonadotropes and is not displaced by LHRH, providing evidence for the existence of an FSHRF receptor. Leptin has equal potency as LHRH to release gonadotropins by NO. lGnRH-III specifically releases FSH, not only in rats but also in cows.

An Interleukin-1-Alpha-Like Neuronal System in the Preoptic-Hypothalamic Region and Its Induction by Bacterial Lipopolysaccharide in Concentrations Which Alter Pituitary Hormone Release

We studied the effect of intravenous injection of lipopolysaccharide (LPS) (30-250 micrograms) on the release of several anterior pituitary hormones as indicated by changes in their concentrations in plasma. Within 30 min after intravenous injection of LPS there was a dose-related stimulation of ACTH release; prolactin (PRL) release was induced only by the highest LPS dose injected (250 micrograms). Even the lowest dose of LPS (30 micrograms) decreased plasma growth hormone (GH) by 60 min. Higher doses lowered plasma GH by 30 min, but thyroid-stimulating hormone release was only significantly inhibited by the highest dose of LPS. The action of LPS seems to be primarily exerted on the central nervous system, since incubation of hemipituitaries with LPS for 3 h in doses ranging from 0.001 to 10 micrograms/ml had no effect on ACTH release. LPS is thought to induce its effects on hormones either by release of cytokines from immune cells which subsequently induce the hormonal changes or possibly by direct action within the hypothalamus. In this report we demonstrate the immunocytochemical localization of a population of interleukin-1 alpha (IL-1 alpha)-like cells in a region extending from the basal forebrain at the level of the diagonal band of Broca, caudally and dorsally to the dorsolateral preoptic region and the hypothalamus at the level of the paraventricular nucleus. Further caudally, IL-1 alpha-like immunoreactive cells were located in the midportion of the amygdala. Two hours after injection of the 125-micrograms dose of LPS, the number of these immunoreactive cells was dramatically increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-Like Growth Factor-I Activates KiSS-1 Gene Expression in the Brain of the Prepubertal Female Rat

KiSS-1 gene expression has been shown to increase as puberty approaches, and its peptide products, kisspeptins, are involved in LHRH secretion at puberty. Factors contributing to increased KiSS-1 expression, however, have not been identified; thus, the purpose of this study was to assess whether IGF-I could induce transcription of this gene in prepubertal female rats. IGF-I or saline was centrally administered to immature rats that were killed 2, 4, and 6 h later. Real-time PCR revealed that IGF-I induced (P < 0.01) KiSS-1 gene expression at 6 h in a tissue fragment that contained both the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei. Subsequently, the AVPV and ARC nuclei were separated to assess whether region-specific effects could be identified. IGF-I stimulated (P < 0.01) KiSS-1 gene expression in the AVPV nucleus at 6 h after injection, with no change observed in the ARC nucleus. Serum estradiol (E2) levels were not altered at any time point after IGF-I, demonstrating that the increased KiSS-1 expression observed was not caused by an elevation in E2. Additionally, the IGF-I action to induce KiSS-1 gene expression in the AVPV nucleus was further demonstrated when the IGF-I was administered systemically. E2 appears to play an important permissive role because 1-d ovariectomized rats responded to IGF-I with increased (P < 0.01) KiSS-1 expression, whereas, 20 d after ovariectomy, when the E2 levels had fallen below assay sensitivity, the IGF-I was unable to induce KiSS-1 expression. The IGF-I effect was further demonstrated by showing that the IGF-I receptor antagonist, JB-1, blocked the IGF-I-induced increase in KiSS-1 expression. Collectively, these data indicate that IGF-I is an activator of the KiSS-1 gene in the prepubertal female rat.

Actions of ethanol on hypothalamic and pituitary hormones in prepubertal female rats.

To determine the effects of prepubertal ethanol (ETOH) exposure on hypothalamic and pituitary hormones known to be involved in the onset of female puberty, we have chronically exposed female rats to either a liquid-diet containing ETOH or an isocaloric control liquid-diet. An additional set of controls consisted of animals maintained on Lab Chow, and water provided ad lib. Our results indicate that the feeding regimen employed produced no differences with regard to body and reproductive organ weights, as well as any of the hormones measured between the two control groups. Conversely, ETOH-treated animals showed significantly lower body and reproductive organ weights than the control animals and although no differences were detected between ETOH-treated and control animals with regard to the hypothalamic content of somatostatin (SRIF), there was a significant increase in the hypothalamic content of growth hormone releasing hormone (GHRH), with a concomitant and significant decrease in the serum concentration of growth hormone (GH). Furthermore, the ETOH-treated animals showed a significant increase in the hypothalamic content of luteinizing hormone releasing hormone (LHRH) with a significant decrease in the serum concentration of luteinizing hormone (LH), but not follicle stimulating hormone (FSH). These results demonstrate for the first time that chronic, prepubertal ETOH administration alters the concentrations of specific hypothalamic and pituitary hormones which are known to be involved in the female pubertal process.

Expression of β-nerve growth factor in cultured cells derived from the hypothalamus and cerebral cortex

Although the synthesis of nerve growth factor (NGF) in brain regions innervated by magnocellular cholinergic neurons of the basal forebrain is well documented, the cell type(s) able to produce NGF in the central nervous system (CNS) remain only partially characterized. Moreover, little is known regarding the ability of brain areas not innervated by magnocellular cholinergic neurons to express NGF protein. The hypothalamus, which controls the endocrine system, is one of such regions. Primary culture of mixed populations of cells from the fetal hypothalamus were used to identify the presence of NGF in this brain area. Immunocytochemistry revealed that hypothalamic oligodendrocytes and a subpopulation of neurons expressed the NGF protein. In contrast, astrocytes were either immunonegative or equivocally stained. To define whether synthesis of NGF is restricted to a particular cell type, cultures of purified astrocytes, oligodendrocyte progenitor (oligoP) cells and neurons were utilized. They were obtained from the neonatal cerebral cortex to ensure an adequate yield of glial cells. Virtually the entire population of cerebral oligoP cells were found to express NGF protein. In contrast, and similar to hypothalamic astrocytes, cerebral type I astrocytes isolated at the same time as oligoP cells exhibited little or no NGF staining. When type I astrocytes were induced to differentiate in the presence of a serumless, chemically defined medium, a subpopulation of the culture became more robustly positive for the NGF protein. Contrasting with these differences in NGF immunoreactivity, Northern analysis of RNA isolated from purified cerebral type I astrocytes, oligoP cells and neurons demonstrated that NGF mRNA was expressed in each of these cell types at approximately the same levels. The results indicate that: (a) when placed in culture, each of the major cell types within the CNS has the capability of transcribing the NGF gene, and (b) despite similar NGF mRNA levels the cellular content of NGF protein is greater in a subpopulation of neurons and in oligodendrocytes than in astrocytes, suggesting differences in NGF post-transcriptional regulation between these cell types. In addition, the presence of NGF in hypothalamic cells suggests that NGF may be involved in the regulation of specific hypothalamic neuronal systems.

Neural and Neurotrophic Control of Ovarian Development

This chapter discusses the involvement of the ovarian innervation and neurotrophic factors in the control of follicular development and ovulation, and provides a succinct account of relevant aspects of early follicular development, the distribution of ovarian nerves, and some of the important features that define the neurotrophins (NTs) family and its receptors. The mammalian ovary is a prominent terminal field for sympathetic and sensory neurons. The neurons projecting to the ovary innervate the different structural components of the gland, including the vasculature, interstitial tissue, and developing follicles, with various degrees of complexity. Before the 1990s, several reports appeared documenting the distribution of intraovarian nerve fibers, their presence in different species, and their neurotransmitter identity. During the early 1990s, the availability of simplified culture procedures and new biochemical and molecular techniques provided insights into the specific contribution that ovarian nerves may have to the regulation of ovarian function. Thus, a number of reports appeared indicating an involvement of neurotransmitters contained in ovarian nerves, such as norepinephrine (NE) and vasoactive intestinal peptide (VIP), in the control of follicular steroidogenesis and suggesting the facilitatory participation of the sympathetic innervation in follicular development.

INSULIN-LIKE GROWTH FACTOR-1 STIMULATION OF HYPOTHALAMIC KiSS-1 GENE EXPRESSION IS MEDIATED BY Akt: EFFECT OF ALCOHOL

Kisspeptin, as well as insulin-like growth factor-1 (IGF-1), act centrally to stimulate luteinizing hormone-releasing hormone (LHRH) secretion at puberty. IGF-1 can induce KiSS-1 gene expression as an early pubertal event; however, the signaling pathway mediating this effect is not known. Since alcohol (ALC) blocks IGF-1 induced LHRH release acutely, we assessed whether this drug could affect IGF-1 stimulated prepubertal KiSS-1 gene expression following a binge type of exposure. Immature female rats were administered either ALC (3 g/kg) or water via gastric gavage at 07.30 h. At 09.00 h the ALC and control groups were subdivided where half received either saline or IGF-1 (200 ng) into the third ventricle. A second dose of ALC (1.5, 2 and 3 g/kg) or water was administered at 11.30 h. These regimens produced moderate blood alcohol concentrations of 77, 89 and 117 mg/dl, respectively, over the time course of the experiment. Rats were sacrificed 6 h after the IGF-1 injection and tissues containing the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei were collected. IGF-1 stimulated (P<0.01) KiSS-1 gene expression in the AVPV nucleus at 6 h, but did not affect expression of the kisspeptin receptor, GPR54. While ALC did not alter basal expression of either gene, its dose dependently blocked IGF-1-induced KiSS-1 gene expression in the AVPV nucleus. No changes were observed in the ARC nucleus. Assessment of IGF-1 signaling indicated that the acute administration of IGF-1, ALC, or both did not alter the basal expression of IGF-1 receptor protein. However, IGF-1 stimulated (P<0.05) phosphorylated Akt protein over basal levels, an action blocked by ALC. Our results indicate that the IGF-1 induction of KiSS-1 gene expression is mediated by Akt activation, and that ALC alters this important prepubertal action of IGF-1.

Ethanol blocks the central action of IGF-1 to induce luteinizing hormone secretion in the prepubertal female rat.

Insulin-like growth factor-1 (IGF-1) is emerging as a signal of peripheral origin capable of acting centrally to induce luteinizing hormone (LH) secretion and accelerate the initiation of female puberty. Since we have shown previously that ethanol (ETOH) can suppress prepubertal LH release and delay female puberty, we hypothesized that these detrimental effects might be due, at least in part, to the drugs ability to alter the central actions of IGF-1. Thus, we have investigated the effects of ETOH on IGF-1 induced LH release in vivo, and on IGF-1 induced prostaglandin-E2 (PGE2) and LH-releasing hormone (LHRH) release in vitro from rats during the juvenile phase of development as well as from rats during the early stage of first proestrus. For the in vivo experiment three initial jugular blood samples were taken at 10-min. intervals from all rats, then the animals received either a 3g/Kg dose of ETOH or an equal volume of saline by gastric gavage. The rats were subsequently left undisturbed for 90 min. to allow time for ETOH absorption, then a single blood sample was drawn from each rat. Finally, IGF-1 (200 ng/3 microl) was microinjected into the third ventricle of all animals and five more blood samples were withdrawn at 10 min. intervals. We demonstrated that IGF-1 induced the release of LH (p<0.01) in the saline controls from rats in both phases of pubertal development. Conversely, this effect of IGF-1 was blocked by ETOH in both developmental groups. For the in vitro experiment, median eminences (MEs) were dissected, then incubated in the presence or absence of ETOH (50 mM). The amount of PGE2 and LHRH released was measured from the same samples following the addition of IGF-1 (100 ng/ml). As above, similar responses were observed from rats in both developmental phases. IGF-1 stimulated the release of PGE2 (p<0.001) and LHRH (p<0.01) from the incubated nerve terminals in the absence of ETOH; however, these effects were blocked by the presence of ETOH. Thus, these combined in vivo and in vitro results suggest that ETOH can acutely block IGF-1 induced LH release during the juvenile-peripubertal transition period, and that this is a centrally mediated action which is due to the diminished formation of PGE2 resulting in decreased LHRH release.

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.