Host neuro- immuno-endocrine responses in periodontal disease.

Periodontitis is a chronic inflammatory complex disease caused by microorganisms. It may be influenced by diverse systemic disorders, environmental, genetic and socio-psychological factors with the ability to alter the balance of the host neuro-immunoendocrine responses. It is characterized by the progressive destruction of the tooth supporting apparatus leading to tooth loss, with possible impact on general health. Starting with a brief description of the periodontium, etiopathogenesis, repair processes and several physiological mechanisms and their disarray on periodontium response to bacterial challenge. Following, the negative effects of stress on the disease and some remarks on the recently discovered effects of oxytocin that modulate stress response and its role in individual coping mechanisms to stress. We also focus on the participation of components and functions of endocannabinoid system with anti-inflammatory actions on gingiva. Finally, a discussion that may link between diabetes, cardiovascular diseases, stroke and metabolic syndrome associated with periodontal disease; all of them sharing a common denominator that is inflammation and oxidative stress.

Cilostazol blocks pregnancy in naturally cycling mice.

BACKGROUND: Administration of a phosphodiesterase three enzyme inhibitor (PDE3-I) in rodents and primates results in ovulation of immature oocytes. Concerns regarding inhibition of PDE3 enzymes that are expressed in heart and blood vessels discouraged further development of PDE3-Is as nonsteroidal contraceptives. Cilostazol (CLZ) is a PDE3A-I that is approved for medical indications in humans and has an additional effect of adenosine uptake inhibition that is believed to counterbalance the undesirable outcomes resulting from PDE inhibition. STUDY DESIGN: Cycling mature female mice were treated with 7.5 or 15 mg CLZ, dimethyl sulfoxide or water beginning on the day of proestrus. Animals were placed with fertility-proven males after 3 days of treatment. Treatments were continued until 1 day after detection of a vaginal plug, and then females were monitored up to 30 days postbreeding to assess the effects of the compounds on pregnancy. Each of the treated female with CLZ was then remated with the same male and again monitored up to 30 days. RESULTS: None of the CLZ-treated mice produced offspring, whereas all of the control animals maintained pregnancy and delivered normal pups (p<.0001). Remating of the previously CLZ-treated females exhibited normal pregnancies and gave birth to live offspring that were not different from the controls. CONCLUSION: CLZ is a potential nonsteroidal contraceptive agent that merits further evaluation in other mammals.

Hypothalamic glial-to-neuronal signaling during puberty: influence of alcohol.

Mammalian puberty requires complex interactions between glial and neuronal regulatory systems within the hypothalamus that results in the timely increase in the secretion of luteinizing hormone releasing hormone (LHRH). Assessing the molecules required for the development of coordinated communication networks between glia and LHRH neuron terminals in the basal hypothalamus, as well as identifying substances capable of affecting cell-cell communication are important. One such pathway involves growth factors of the epidermal growth factor (EGF) family that bind to specific erbB receptors. Activation of this receptor results in the release of prostaglandin-E(2) (PGE(2)) from adjacent glial cells, which then acts on the nearby LHRH nerve terminals to elicit release of the peptide. Another pathway involves novel genes which synthesize adhesion/signaling proteins responsible for the structural integrity of bi-directional glial-neuronal communication. In this review, we will discuss the influence of these glial-neuronal communication pathways on the prepubertal LHRH secretory system, and furthermore, discuss the actions and interactions of alcohol on these two signaling processes.

Prepubertal ethanol exposure alters hypothalamic transforming growth factor-α and erbB1 receptor signaling in the female rat.

Glial-derived transforming growth factor alpha (TGFα) activates the erbB1/erbB2 receptor complex on adjacent glial cells in the medial basal hypothalamus (MBH). This receptor activation stimulates the synthesis and release of prostaglandin-E(2) (PGE(2)) from the glial cells, which then induces the release of prepubertal luteinizing hormone-releasing hormone (LHRH) secretion from nearby nerve terminals; thus, showing the importance of glial-neuronal communications at the time of puberty. Ethanol (EtOH) is known to cause depressed prepubertal LHRH secretion and delayed pubertal development. In this study, we assessed whether short-term EtOH exposure could alter the hypothalamic glial to glial signaling components involved in prepubertal PGE(2) secretion. Immature female rats began receiving control or EtOH diets beginning when 27 days old. The animals were killed by decapitation after 4 and 6 days of treatment and confirmed to be in the late juvenile stage of development. Blood and brain tissues were collected for gene, protein, and hormonal assessments. Real-time polymerase chain reaction (PCR) analysis demonstrated that EtOH did not affect basal levels of erbB1 gene expression in the MBH. Expression of total erbB1 protein was also unaffected; however, the EtOH caused suppressed phosphorylation of erbB1 protein in the MBH at both 4 and 6 days (P<.01) as revealed by Western blotting. Phosphorylation and total protein levels of erbB2 receptor were not affected by EtOH exposure. Because this receptor is critical for PGE(2) synthesis/release, which mediates the secretion of LHRH, we assessed whether in vivo EtOH exposure could affect the release of PGE(2). EtOH exposure for 6 days suppressed (P<.01) basal levels of PGE(2) released into the medium. The effects of 4- and 6-day EtOH exposure on gene and protein expressions of TGFα, an upstream component in the activation of erbB1/erbB2, were also studied. The levels of TGFα mRNA were increased markedly at 4 days (P<.001), but declined to near basal levels by 6 days in the EtOH-treated animals. The EtOH caused increases in TGFα protein expression at both 4 (P<.001) and 6 (P<.01) days; hence, suggesting that the EtOH inhibited release of the peptide. We confirmed this inhibition by showing decreased (P<.01) TGFα released from MBHs incubated in vitro following 6 days of EtOH exposure in vivo. Thus, these results demonstrate that EtOH is capable of interfering with hypothalamic glial to glial signaling processes involved in prepubertal PGE(2) secretion.

Parity-induced decrease in systemic growth hormone alters mammary gland signaling: a potential role in pregnancy protection from breast cancer.

Early full-term pregnancy is an effective natural protection against breast cancer in both humans and experimental rodents. The protective effect of an early pregnancy is, in part, linked to changes in circulating hormones that are involved in both normal breast development and breast cancer. For example, a reduction in circulating growth hormone (GH) has been shown to protect rats from carcinogen-induced mammary tumors. We examined the ability of a full-term pregnancy to alter the endocrine GH/insulin-like growth factor-I (IGF-I) axis and how this change affected normal mammary gland function in two commonly used rat models (Sprague-Dawley and Wistar Furth). Circulating GH and IGF-I were measured in blood drawn every 30 minutes from parous and age-matched virgin female rats. Mean serum GH levels were significantly decreased (P < 0.01) in parous compared with age-matched virgin rats for both strains. Changes in GH levels were independent of estrous cycle, indicated by a significant (P < 0.05) reduction in circulating levels of GH during estrus and diestrus in both parous strains. Despite the decrease in circulating GH, pituitary GH mRNA levels were unaltered in parous rats. Circulating IGF-I and hepatic IGF-I mRNA were also unaltered by parity in either rat strain. Immunoblot analysis of mammary glands showed decreases in phosphorylation of signal transducer and activator of transcription 5A and Janus-activated kinase 2, suggesting reduced action of GH in the mammary gland. Therefore, although the parity reduction in circulating GH does not affect circulating IGF-I levels, it is possible that reduced GH acts directly at the mammary gland and may play a role in pregnancy protection from breast cancer.

Actions and interactions of alcohol and insulin-like growth factor-1 on female pubertal development.

Alcohol (ALC) is a drug that is capable of disrupting reproductive function in adolescent humans, as well as immature rhesus monkeys and rats. Critical to determining the mechanism(s) of the effects of ALC on the pubertal process is to have a better understanding of the important events involved in the initiation of puberty. For years it has been hypothesized that there may be metabolic signals capable of linking somatic growth to the activation of the reproductive system at the time of puberty. In recent years it has been shown that insulin-like growth factor-1 (IGF-1) is one such signal that plays an early role in the pubertal process. In this review, we will describe the actions and interactions of ALC and IGF-1 on molecular and physiological processes associated with pubertal development.

Short-term alcohol administration alters KiSS-1 gene expression in the reproductive hypothalamus of prepubertal female rats.

BACKGROUND: Kisspeptins bind to the G-protein-coupled receptor (GPR54) to activate hypothalamic luteinizing hormone releasing hormone (LHRH) secretion at the time of puberty. Alcohol (ALC) causes depressed prepubertal LHRH release, resulting in depressed luteinizing hormone (LH) secretion and delayed puberty. Because KiSS-1 and GPR54 are important to the onset of puberty, we assessed the effects of chronic ALC administration on basal expression of these puberty-related genes within the reproductive hypothalamus, as well as hormones and transduction signaling pathways contributing to their activity. METHODS: Immature female rats were fed a liquid diet containing ALC for 6 days beginning when 27 days old. Controls received either companion isocaloric liquid diet or rat chow and water. Animals were decapitated on day 33, in the late juvenile stage of development. Blood was collected for the assessment of serum hormone levels. Brain tissues containing the anteroventral periventricular (AVPV) and arcuate (ARC) nuclei were obtained for assessing expression of specific puberty-related genes and proteins. RESULTS: KiSS-1 mRNA levels in the AVPV and ARC nuclei were suppressed (p < 0.001) in the ALC-treated rats. GPR54 gene and protein expressions were both modestly increased (p < 0.05) in AVPV nucleus, but not in ARC nucleus. Alcohol exposure also resulted in suppressed serum levels of insulin-like growth factor-1 (IGF-1), LH, and estradiol (E(2)). As IGF-1, in the presence of E(2), can induce expression of the KiSS-1 gene, we assessed the potential for ALC to alter IGF-1 signaling in the reproductive hypothalamus. IGF-1 receptor gene and protein expressions were not altered. However, protein expression of phosphorylated Akt, a transduction signal used by IGF-1, was suppressed in the AVPV (p < 0.05) and ARC (p < 0.01) nuclei. CONCLUSIONS: Alcohol causes suppressed KiSS-1 gene expression in the reproductive hypothalamus; hence, contributing to this drug's ability to cause suppressed LHRH secretion and disruption of the pubertal process. We suggest that this action, at least in part, is through altered IGF-1 signaling.

The pyrethroid pesticide esfenvalerate suppresses the afternoon rise of luteinizing hormone and delays puberty in female rats.

BACKGROUND: One of the most widely used classes of insecticides is the synthetic pyrethroids. Although pyrethroids are less acutely toxic to humans than to insects, in vitro studies have suggested that pyrethroids may be estrogenic. OBJECTIVES: We assessed pubertal effects by orally administering 0.5, 1.0, and 5.0 mg/kg/day of the type II pyrethroid esfenvalerate (ESF) to female rats beginning on postnatal day (PND) 22 until vaginal opening. ESF administration suppresses serum estradiol and delays pubertal onset. MATERIALS AND METHODS: To assess possible hypothalamic and/or pituitary effects, animals received 0.5 or 1.0 mg/kg ESF or corn oil on PNDs 22-29. On PND30, we drew three blood samples (200 microL) from each rat at 15-min intervals beginning at 1000 hours, and again at 1500 hours. To test hypothalamic responsiveness, after the third afternoon sample, all animals received an intravenous injection of N-methyl-d,l-aspartic acid (NMA; 40 mg/kg), and then we drew two more samples. We performed a second experiment as above except that animals received luteinizing hormone-releasing hormone (LHRH; 25 ng/rat) to test pituitary responsiveness. RESULTS: Basal levels of luteinizing hormone (LH) in the afternoon hours were higher in control animals than in animals treated with 1.0 mg/kg ESF (p < 0.05). Furthermore, NMA- and LHRH-stimulated LH release was similar in control and ESF-treated animals, indicating that both hypothalamic and pituitary responsiveness, respectively, were unaffected. CONCLUSIONS: Although the hypothalamus is able to respond to exogenous stimuli, absence of a normal afternoon rise in LH would indicate a hypothalamic deficit in ESF-treated animals.

Acute effect of manganese on hypothalamic luteinizing hormone releasing hormone secretion in adult male rats: involvement of specific neurotransmitter systems.

Manganese chloride (MnCl2) is capable of stimulating luteinizing hormone releasing hormone (LHRH) secretion in adult male Sprague-Dawley rats through the activation of the hypothalamic nitric oxide/cyclic guanosine monophosphate (cGMP)/protein kinase G pathway. The present study aimed to determine the involvement of specific neurotransmitters involved in this action. Our results indicate that dopamine, but not glutamic acid and prostaglandins, mediates the MnCl2 stimulated secretion of LHRH from medial basal hypothalami in vitro, as well as increases the activity of nitric oxide synthase. Furthermore, a biphasic response was observed in that gamma aminobutyric acid (GABA) release was also increased, which acts to attenuate the MnCl2 action to stimulate LHRH secretion. Although it is clear that manganese (Mn+2) can acutely induce LHRH secretion in adult males, we suggest that the additional action of MnCl2 to release GABA, a LHRH inhibitor, may ultimately contribute to suppressed reproductive function observed in adult animals following exposure to high chromic levels of Mn+2.

The alcohol-induced suppression of ovarian insulin-like growth factor-1 gene transcription is independent of growth hormone and its receptor.

BACKGROUND: Insulin-like growth factor-1 (IGF-1) plays an important role in ovarian development and function. Alcohol (ALC) is a gonadal toxin and capable of causing depressed ovarian IGF-1 and suppressed estradiol. The mechanism by which ALC affects IGF-1 transcription is not well understood, and more information is needed to better understand the interrelationships between ALC, growth hormone (GH) and its ovarian receptor, and the gene expression of ovarian IGF-1. METHODS: Prepubertal transgenic mice carrying the bovine GH (bGH) gene were fed either a liquid diet containing ALC, pair-fed the companion isocaloric control liquid diet, or fed chow and water. A fourth group consisted of normal (nontransgenic) littermates fed chow and water. Mice received their diets for 5 days, were then killed and tissues collected and frozen. RESULTS: Alcohol did not alter circulating levels of bGH held constant by the promoter. Real-time polymerase chain reaction (PCR) showed elevated (p<0.05) ovarian IGF-1 mRNA levels in both groups of transgenic control mice, compared with normal mice. Insulin-like growth factor-1 expression in the ALC-treated transgenic mice was suppressed (p<0.01) compared with both transgenic controls. Insulin-like growth factor-1 receptor (IGF-1R) gene expression was also decreased (p<0.01) in ALC-treated transgenic mice compared with transgenic controls. Growth hormone-receptor (GH-R) synthesis revealed that all transgenic mice, including those exposed to ALC, showed increased (p<0.05) GH-R mRNA compared with normal controls, and ALC did not alter protein levels of the GH-R. CONCLUSIONS: These results suggest that the ALC-induced suppression of ovarian IGF-1 gene transcription is independent of alterations in serum GH.

Effects of alcohol on intraovarian nitric oxide synthase and steroidogenic acute regulatory protein in the prepubertal female rhesus monkey.

OBJECTIVE: In addition to affecting hypothalamic-pituitary function, alcohol is a gonadal toxin capable of inhibiting ovarian function and suppressing circulating levels of estradiol (E2) in female rats, rhesus monkeys, and adolescent girls. Both nitric oxide (NO) and steroidogenic acute regulatory protein (StAR) are intraovarian substances that influence steroidogenesis in opposite directions. This study was undertaken to determine whether alcohol exposure affects prepubertal ovarian steroidogenesis in female rhesus monkeys by altering nitric oxide synthase (NOS), StAR, or both. METHOD: At 20 months of age, monkeys received a single intragastric dose of alcohol (2.4 g/kg) or an equal volume of a saline/sucrose solution daily until they were 36 months old. Blood and ovaries were then collected for assessment of serum hormone levels and tissue gene and protein expression. RESULTS: Alcohol caused depressed levels of serum E2 (p < .05) and luteinizing hormone (p < .05) but not follicle-stimulating hormone. Real-time polymerase chain reaction (RT-PCR) assessment of ovarian mRNA encoding the three isoforms (i.e., neuronal [n] NOS, endothelial [e] NOS, and inducible [i] NOS) of NOS revealed that alcohol exposure did not alter gene expression of nNOS but caused increased basal levels of eNOS (p < .05) and iNOS (p < .01) mRNA expression compared with control ovaries. Alcohol also increased expression of eNOS (p < .01) and iNOS (p < .05) proteins. In contrast, ovaries from monkeys exposed to alcohol showed decreased (p < .05) StAR gene expression compared with controls. CONCLUSIONS: We showed previously that alcohol exposure during adolescence suppressed E2 and delayed development of regular monthly menstruation patterns in rhesus monkeys. The present results suggest that the combined action of alcohol to elevate ovarian NOS and suppress StAR synthesis contributes to these abnormalities.

Manganese stimulates luteinizing hormone releasing hormone secretion in prepubertal female rats: hypothalamic site and mechanism of action.

We have shown recently that Mn2+ stimulates gonadotropin secretion via an action at the hypothalamic level, and a diet supplemented with a low dose of the element is capable of advancing the time of female puberty. In this study, we used an in vitro approach to investigate the mechanism by which Mn2+ induces luteinizing hormone-releasing hormone (LHRH) secretion from prepubertal female rats. The medial basal hypothalamus from 30-day-old rats was incubated in Locke solution for 30 min to assess basal LHRH secretion, then incubated with buffer alone or buffer plus either a nitric oxide synthase (NOS) inhibitor (N-monomethyl-L-arginine (NMMA); 300 or 500 microM) or a soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ); 100 or 250 microM) for another 30 min. Finally, the incubation continued for a further 30 min, but in the presence of MnCl2 (50 or 250 microM) to assess the effect of the blockers on stimulated LHRH secretion. Both 50 and 250 microM MnCl2 stimulated LHRH release (P < 0.05 and P < 0.01, respectively). The addition of 300-500 microM NMMA to the medium did not block Mn2+-stimulated release of LHRH, even with the higher dose of MnCl2. Furthermore, while 50, 100 and 250 microM MnCl2 all significantly induced LHRH release, the two lowest doses did not stimulate total nitrite released from the same tissue, an effect only observed with the highest dose. Taken together, these data suggest that Mn2+ is not an effective stimulator of NO. Conversely, inhibiting sGC with ODQ blocked the Mn2+-stimulated secretion of LHRH in a dose-dependent manner, indicating that GC is the site of action of Mn2+. Additionally, we showed that Mn2+ stimulated cGMP and LHRH from the same tissues, and that downstream blocking of protein kinase G formation with KT5823 (10 microM) inhibited Mn2+-induced LHRH release. These data demonstrate that the principal action of Mn2+ within the hypothalamus is to activate sGC directly and/or as a cofactor with available NO, hence generating cGMP and resulting in prepubertal LHRH release.

Origin and ontogeny of mammalian ovarian neurons.

Mammalian ovaries contain sympathetic neurons expressing the low affinity neurotropin receptor (p75NTR). To date neither the role these neurons might play in ovarian physiology nor their embryological origin is known. Immunohistochemistry was used to detect postnatal changes in distribution and number of both p75NTR-positive and tyrosine hydroxylase-positive neurons in rhesus monkey ovaries. Pig fetuses were used to map the pathway of ovarian neuronal migration during embryonic development. Antiserum to p75NTR revealed the presence of isolated neurons and neurons clustered into ganglia in 2-month-old monkey ovaries. After 8 months, the neurons exhibited well-developed processes, and other than being more extensively interlaced, the localization and morphology did not change after 2 yr of age. Total number of p75NTR-positive neurons present decreased gradually between 2 months and 12 yr of age and declined markedly with reproductive aging. Conversely, the subpopulation of neurons immunoreactive to anti-tyrosine hydroxylase increased significantly at puberty and then declined with the loss of reproductive capacity. By d 21 of fetal life in the pig, p75NTR neurons had migrated medially from the neural crest to form the paraaortic autonomic ganglia. Some neurons migrated ventrally from the ganglia and then continued ventrolaterally to enter the genital ridge. By d 27, neurons had entered the developing ovary, and by d 35, the migration was complete with neurons demonstrating immunoreactivity to NeuN, a neuron-specific marker. Results demonstrate that p75NTR-expressing ovarian neurons originate from the neural crest and that a catecholaminergic subset is associated with pubertal maturation of the ovary and subsequent reproductive function.

Manganese acts centrally to activate reproductive hormone secretion and pubertal development in male rats.

Manganese (Mn) is an important element for normal growth and reproduction. Because Mn accumulates in the hypothalamus and is capable of stimulating puberty-related hormones in female rats, we assessed whether this metal could cause similar effects in male rats. We have demonstrated that MnCl2, when administered acutely into the third ventricle of the brain, acts dose dependently to stimulate luteinizing hormone (LH) release. Furthermore, there was a dose dependent stimulation in the secretion of LH-releasing hormone (LHRH) from the medial basal hypothalamus in vitro, and administration of an LHRH receptor antagonist in vivo blocks Mn-induced LH release. To assess potential chronic effects of the metal, male pups were supplemented with 10 or 25 mg MnCl2 per kg by gastric gavage from day 15 until days 48 or 55, at which times developmental signs of spermatogenesis were assessed. Results demonstrate that while significant effects were not observed with the 10 mg/kg dose, the animals receiving the 25 mg/kg dose showed increased LH (p<0.05), FSH (p<0.01) and testosterone (p<0.01) levels at 55 days of age. Furthermore, there was a concomitant increase in both daily sperm production (p<0.05) and efficiency of spermatogenesis (p<0.05), demonstrating a Mn-induced acceleration in spermatogenesis. Our results suggest Mn is a stimulator of prepubertal LHRH/LH secretion and may facilitate the normal onset of male puberty. These data also suggest that the metal may contribute to male precocious pubertal development should an individual be exposed to low but elevated levels of Mn too early in life.

IGF-1 administration to prepubertal female rats can overcome delayed puberty caused by maternal Pb exposure.

Because prepubertal female rats maternally exposed to lead (Pb) exhibit suppressed serum levels of insulin-like growth factor-1 (IGF-1) and delayed puberty, we investigated the ability of centrally administered IGF-1 to stimulate luteinizing hormone (LH) release in vivo and LH-releasing hormone (LHRH) release in vitro from maternally Pb-exposed prepubertal female rats. Additionally, we assessed whether IGF-1 replacement could affect the timing of female puberty. Results demonstrated that IGF-1 stimulated significantly LH release in both control and Pb-exposed animals. When median eminences from control and Pb-exposed females were incubated with rat IGF-1 in vitro, they responded similarly with significant peptide-induced LHRH release. Lastly, we showed IGF-1 replacement reversed the delay in puberty caused by Pb. These results indicate the central LHRH response to IGF-1 is intact and that Pb-induced delayed puberty is due, at least in part, to suppressed circulating IGF-1 available to the hypothalamus.

Lipopolysaccharide-induced leptin synthesis and release are differentially controlled by alpha-melanocyte-stimulating hormone.

OBJECTIVE: Since alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits the synthesis and release of proinflammatory cytokines and stimulates the synthesis and release of anti-inflammatory cytokines, and leptin is a cytokine that has anti-inflammatory actions in the presence of lipopolysaccharide (LPS), we hypothesized that alpha-MSH increases leptin synthesis and release. METHODS: alpha-MSH or 0.9% NaCl (saline) were injected intraperitoneally 15 min prior to intravenous injection of 0.5 ml of saline or LPS (0.15 mg/kg). Thereafter, repeated blood samples were withdrawn over a period of 6 h and plasma leptin concentrations determined. The rats were sacrificed at 6 h and leptin mRNA was measured in epididymal fat pads. RESULTS: Plasma leptin concentrations of the saline-injected control group were unaltered during the 6 h, whereas in the LPS group, leptin was unaltered between 0 and 30 min and thereafter progressively increased between 30 and 360 min by 2.5-fold. alpha-MSH slightly increased plasma leptin concentrations by 15 min and then increased them further by 120 min, after which they declined towards baseline. The pattern of plasma leptin concentrations in the alpha-MSH + LPS group was similar to that of the LPS group, except that higher concentrations were observed at 120 min in the rats injected with alpha-MSH + LPS. LPS increased leptin mRNA by 3-fold at 6 h, whereas it was unaffected in the MSH-treated animals. On the contrary, alpha-MSH completely blocked the LPS-induced leptin mRNA. CONCLUSIONS: Our results suggest that alpha-MSH increased leptin release without altering its synthesis, but when LPS increased release and synthesis of leptin, alpha-MSH, although further increasing release, blocked the enhanced synthesis of leptin elicited by LPS.

Alcohol alters insulin-like growth factor-1 activated oct 2 POU domain gene expression in the immature female hypothalamus.

OBJECTIVE: The Oct 2 POU homeodomain gene has been shown to increase during late juvenile development; the upstream control of Oct 2 is not known, however. The insulin-like growth factor-1 (IGF-1) is known to act centrally to stimulate luteinizing hormone (LH)-releasing hormone (LHRH) release and advance female puberty. We therefore sought to determine if this peptide induces transcription of Oct 2 genes as an early pubertal event. Furthermore, as alcohol (ALC) blocks IGF-1-induced LHRH and LH release acutely, we aimed to determine if it could affect the ability of IGF-1 to stimulate Oct 2 gene expression. METHOD: Female rats, 25 days old, were administered saline or IGF-1 (rat IGF-1 20 ng/3 microI) in the third ventricle at 0900 hours and killed 2, 4 and 6 hours later for assessment of Oct 2 gene expression in the preoptic area (POA) and the medial basal hypothalamus (MBH). In another experiment, we determined whether ALC (3 g/kg) could block IGF-1-induced Oct 2 gene expression. RESULTS: In the POA, IGF-1 did not affect the expression of Oct 2a, but it increased the Oct 2c mRNA levels at 2 hours. In the MBH, both transcripts were elevated 4 hours after IGF-1 stimulation. ALC did not alter basal expression of either of the Oct 2 gene isoforms. In both regions, however, ALC blocked IGF-1-induced gene expression. CONCLUSIONS: IGF-1 induced Oct 2 genes prior to the normal increase during the late juvenile period, indicating this IGF- 1 induction may be an early event in the activation of the LHRH secretory pathway. ALC blocks this action, suggesting the Oct 2 POU gene is a likely target by which ALC can interfere with glial-neuronal interactions and interrupt LHRH secretion during prepubertal development.

Manganese acts centrally to stimulate luteinizing hormone secretion: a potential influence on female pubertal development.

Manganese (Mn), an essential element considered important for normal growth and reproduction, has been shown in adults to be detrimental to reproductive function when elevated. Because Mn can cross the blood-brain barrier and accumulate in the hypothalamus, and because it has been suggested that infants and children are potentially more sensitive to Mn than adults, we wanted to determine the effects of Mn exposure on puberty-related hormones and the onset of female puberty. We demonstrated that MnCl(2) when administered acutely into the third ventricle of the brain acts dose-dependently to stimulate luteinizing hormone (LH) release in prepubertal female rats. Incubation of hypothalami in vitro showed that this effect was due to a Mn-induced stimulation of luteinizing hormone releasing hormone (LHRH). Further demonstration that this is a hypothalamic site of action was shown by in vivo blockade of LHRH receptors and lack of a direct pituitary action of Mn to stimulate LH in vitro. To assess potential short-term effects, animals were supplemented with MnCl(2) (10 mg/kg) by gastric gavage from day 12 until day 29, or, in other animals, until vaginal opening (VO). Mn caused elevated serum levels of LH, follicle stimulating hormone, and estradiol, and it initiated a moderate but significant advancement in age at VO. Our results are the first to show that Mn can stimulate specific puberty-related hormones and suggest that it may facilitate the normal onset of puberty. They also suggest that Mn may contribute to precocious puberty if an individual is exposed to elevated levels of Mn too early in development.

Effect of ethanol on follicle stimulating hormone-induced steroidogenic acute regulatory protein (StAR) in cultured rat granulosa cells.

Steroidogenic acute regulatory protein (StAR) plays a critical role in trophic hormone-stimulated steroid biosynthesis by facilitating the transfer of cholesterol across the mitochondrial membrane, where the cytochrome P450scc enzyme resides to initiate steroid hormone biosynthesis. Because follicle stimulating hormone (FSH) is a critically important regulator of estradiol (E2) synthesis in granulosa cells and because ethanol is known to suppress gonadotropin-stimulated ovarian steroidogenesis, we evaluated the effects of ethanol on FSH-stimulated StAR in ovarian granulosa cells. Granulosa cells from immature rats pretreated with pregnant mare serum gonadotropin were cultured for 24 h in serum-free medium, either alone (medium only) or with FSH (25 ng/ml) in the presence or absence of ethanol (50 mM). Real-time polymerase chain reaction (PCR) analysis showed increased (p < 0.01) expression of the StAR transcript in FSH-treated cells, when compared with cells that received medium only. The FSH stimulation of StAR transcript was blocked (p < 0.01) by the presence of ethanol. This effect coincided with a decrease in E2 secretion into the culture medium. We also examined whether ethanol could affect the production of cyclic AMP (cAMP), the main second messenger that mediates gonadotropin action within the ovary. FSH treatment of granulosa cells markedly increased (p < 0.001) cAMP levels, an effect that was not altered by ethanol. Importantly, FSH induced an increase (p < 0.01) in the release of prostaglandin E2 (PGE2), an effect that was blocked by ethanol. Real-time PCR analysis showed that ethanol had no effect on the expression of cyclooxygenase-1 (COX-1), but blocked (p < 0.01) FSH-stimulated expression of COX-2. These results demonstrate that ethanol is capable of inhibiting FSH-induced ovarian StAR and thus, contributing to suppressed E2 secretion, at least in part, through an inhibitory action on the COX-2-PGE2 pathway.

Alcohol alters luteinizing hormone secretion in immature female rhesus monkeys by a hypothalamic action.

We determined whether the effect of alcohol (ALC) to suppress LH secretion in immature female monkeys is due to a hypothalamic or pituitary site of action. Beginning at 20 months of age, four monkeys received a single intragastric dose of ALC (2.4 g/kg), and four monkeys received an equal volume of a saline/sucrose solution daily until they were 36 months old. For the hypothalamic response test, two basal samples (3.5 ml) were collected at 15-min intervals via the saphenous vein, and then N-methyl-D-L-aspartic acid (NMA; 20 mg/kg) was given iv and four more blood samples collected. Three weeks later, this protocol was repeated except LH-releasing hormone (LHRH) (5 microg/kg) was used to test pituitary responsiveness. NMA or LHRH was administered 3 h after the ALC. After the pituitary challenge, each monkey was ovariectomized and 6 wk later, implanted with an indwelling subclavian vein catheter. Blood samples were drawn every 10 min for 8 h to assess effects of ALC on post-ovariectomy LH levels and the profile of LH pulsatile secretion. The hypothalamic challenge showed NMA stimulated LH release in control monkeys, an action that was blocked by ALC. The pituitary challenge revealed that LHRH stimulated LH release equally well in control and ALC-treated monkeys. A post-ovariectomy rise in LH was observed in both groups, but levels were 45% lower in ALC-treated monkeys. This reduction was attributed to an ALC-induced suppression of both baseline and amplitude of pulses. Results demonstrate that the ALC-induced suppression of LH in immature female rhesus monkeys is due to an inhibitory action of the drug at the hypothalamic level.