IGF-1 Influences Gonadotropin-Releasing Hormone Regulation of Puberty.

The pubertal process is initiated as a result of complex neuroendocrine interactions within the preoptic and hypothalamic regions of the brain. These interactions ultimately result in a timely increase in the secretion of gonadotropin-releasing hormone (GnRH). Researchers for years have believed that this increase is due to a diminished inhibitory tone which has applied a prepubertal brake on GnRH secretion, as well as to the gradual development of excitatory inputs driving the increased release of the peptide. Over the years, insulin-like growth factor-1 (IGF-1) has emerged as a prime candidate for playing an important role in the onset of puberty. This review will first present initial research demonstrating that IGF-1 increases in circulation as puberty approaches, is able to induce the release of prepubertal GnRH, and can advance the timing of puberty. More recent findings depict an early action of IGF-1 to activate a pathway that releases the inhibitory brake on prepubertal GnRH secretion provided by dynorphin, as well as demonstrating that IGF-1 can also act later in the process to regulate the synthesis and release of kisspeptin, a potent stimulator of GnRH at puberty.

How alcohol affects insulin-like growth factor-1's influences on the onset of puberty: A critical review.

Alcohol (ALC) is capable of delaying signs associated with pubertal development in laboratory animals, as well as in humans. The normal onset of puberty results from a timely increase in gonadotropin-releasing hormone (GnRH) secretion, which is associated with a gradual decline in prepubertal inhibitory influences, and the establishment of excitatory inputs that increase GnRH release, which together drive pubertal development. In recent years, insulin-like growth factor-1 (IGF-1) has emerged as a pivotal contributor to prepubertal GnRH secretion and pubertal development, whose critical actions are interfered with by ALC abuse. Here we review the neuroendocrine research demonstrating the important role that IGF-1 plays in pubertal development, and describe the detrimental effects and mechanisms of action of ALC on the onset and progression of pubertal maturation.

Alcohol Delays the Onset of Puberty in the Female Rat by Altering Key Hypothalamic Events.

BACKGROUND: Because alcohol (ALC) delays signs of pubertal development, we assessed the time course of events associated with the synthesis of critical hypothalamic peptides that regulate secretion of luteinizing hormone-releasing hormone (LHRH), the peptide that drives the pubertal process. METHODS: Immature female rats were administered either laboratory chow or BioServe isocaloric control or ALC-liquid diets from 27 through 33 days of age. On days 28, 29, 31, and 33, animals were killed by decapitation and tissue blocks containing the medial basal hypothalamus (MBH) and the rostral hypothalamic area (RHA) were isolated and stored frozen until assessed by Western blot analysis. RESULTS: Synthesis of dynorphin (DYN), a prepubertal inhibitor of LHRH secretion, was increased (p < 0.05) in the MBH of ALC-treated animals by day 29. DYN was further elevated (p < 0.01) on day 33 and was associated with an increase (p < 0.01) in DYN receptor expression. ALC did not affect synthesis of neurokinin B (NKB), a prepubertal stimulator of LHRH; however, it did suppress (p < 0.05) NKB receptor expression in the MBH by day 31. The most potent stimulator of prepubertal LHRH secretion, kisspeptin (Kp), was also decreased (p < 0.05) in the MBH as early as day 29, with continued suppression (p < 0.01) through day 33. Similar timely suppressions of mammalian target of rapamycin (mTOR), an immediate upstream regulator of Kp, were also noted. These decreases in mTOR and Kp were consistent with ALC stimulating (p < 0.05) the p-AMP-activated protein kinase/Raptor inhibitory pathway to mTOR on day 29, then later suppressing (p < 0.001) an Akt-mediated induction pathway to mTOR by day 31. In the RHA, ALC affected the pathways regulating Kp in a manner similar to that described in the MBH; however, these effects were not noted until day 33. CONCLUSIONS: ALC acts within the MBH as early as 29 days to induce inhibitor and repressor inputs to LHRH, while depressing stimulatory inputs to the peptide. Collectively, these events lead to delayed signs of pubertal development.

Regulation of Kisspeptin Synthesis and Release in the Preoptic/Anterior Hypothalamic Region of Prepubertal Female Rats: Actions of IGF-1 and Alcohol.

BACKGROUND: Alcohol (ALC) causes suppressed secretion of prepubertal luteinizing hormone-releasing hormone (LHRH). Insulin-like growth factor-1 (IGF-1) and kisspeptin (Kp) are major regulators of LHRH and are critical for puberty. IGF-1 may be an upstream mediator of Kp in the preoptic area and rostral hypothalamic area (POA/RHA) of the rat brain, a region containing both Kp and LHRH neurons. We investigated the ability of IGF-1 to stimulate prepubertal Kp synthesis and release in POA/RHA, and the potential inhibitory effects of ALC. METHODS: Immature female rats were administered either ALC (3 g/kg) or water via gastric gavage at 0730 hours. At 0900 hours, both groups were subdivided where half received either saline or IGF-1 into the brain third ventricle. A second dose of ALC (2 g/kg) or water was administered at 1130 hours. Rats were killed 6 hours after injection and POA/RHA region collected. RESULTS: IGF-1 stimulated Kp, an action blocked by ALC. Upstream to Kp, IGF-1 receptor (IGF-1R) activation, as demonstrated by the increase in insulin receptor substrate 1, resulted in activation of Akt, tuberous sclerosis 2, ras homologue enriched in brain, and mammalian target of rapamycin (mTOR). ALC blocked the central action of IGF-1 to induce their respective phosphorylation. IGF-1 specificity and ALC specificity for the Akt-activated mTOR pathway were demonstrated by the absence of effects on PRAS40. Furthermore, IGF-1 stimulated Kp release from POA/RHA incubated in vitro. CONCLUSIONS: IGF-1 stimulates prepubertal Kp synthesis and release following activation of a mTOR signaling pathway, and ALC blocks this pathway at the level of IGF-1R.

Differential Effects of Alcohol on Excitatory and Inhibitory Puberty-Related Peptides in the Basal Hypothalamus of the Female Rat.

BACKGROUND: An increase in development of excitatory inputs along with a decline in inhibitory inputs ultimately govern the timely increased secretion of hypothalamic luteinizing hormone-releasing hormone (LHRH) at the time of puberty. As chronic alcohol (ALC) exposure acts at the hypothalamic level to suppress LHRH secretion and delay puberty, we assessed its ability to differentially affect the expression of key puberty-related proteins. METHODS: ALC was administered to female rats from days 27 to 33, at which time animals were killed and tissues collected for protein expression. In the medial basal hypothalamus (MBH), we assessed kisspeptin (Kp) 10, an excitatory peptide critical for prepubertal LHRH secretion, and Lin28b, a peptide with an inhibitory influence on puberty. As a direct mechanism of action of Lin28b was not known, we determined whether its central administration could induce dynorphin (DYN), a peptide that is inhibitory on LHRH secretion. Also, ALC's effect on DYN protein expression was assessed, as well as its effect on DYN release in vitro. RESULTS: ALC markedly suppressed (p < 0.01) the expression of the excitatory Kp protein, while at the same time increased (p < 0.001) the expression of inhibitory Lin28b protein. Subsequently, we showed for the first time that the central administration of Lin28b stimulated (p < 0.01) the synthesis of DYN. Finally, ALC also induced (p < 0.01) the protein expression and stimulated (p < 0.01) the in vitro release of DYN from the MBH. CONCLUSIONS: These results indicate that ALC can simultaneously and differentially alter both excitatory and inhibitory influences governing pubertal development, show for the first time a mechanism of action by which Lin28b exerts its prepubertal inhibitory tone, and further demonstrate the negative influences of ALC on the pubertal process.

Actions and interactions of alcohol and transforming growth factor ß1 on prepubertal hypothalamic gonadotropin-releasing hormone.

BACKGROUND: Alcohol (ALC) diminishes gonadotropin-releasing hormone (GnRH) secretion and delays puberty. Glial transforming growth factor ß1 (TGFß1) plays a role in glial-neuronal communications facilitating prepubertal GnRH secretion. We assessed the effects of acute ALC administration on TGFß1-induced GnRH gene expression in the brain preoptic area (POA) and release of the peptide from the medial basal hypothalamus (MBH). Furthermore, we assessed actions and interactions of TGFß1 and ALC on an adhesion/signaling gene family involved in glial-neuronal communications. METHODS: Prepubertal female rats were administered ALC or water via gastric gavage at 7:30 am. At 9:00 am, saline or TGFß1 (100 ng/3 µl) was administered into the third ventricle. At 3:00 pm, the POA was removed and frozen for gene expression analysis and repeated for protein assessments. In another experiment, the MBH was removed from ALC-free rats. After equilibration, tissues were incubated in Locke's medium only or medium containing TGFß1 with or without 50 mM ALC for measurement of GnRH peptide released in vitro. RESULTS: TGFß1 induced GnRH gene expression in the POA, and this effect was blocked by ALC. We also described the presence and responsiveness of the TGFß1 receptor in the POA and showed that acute ALC exposure not only altered the TGFß1-induced increase in TGFß-R1 protein expression but also the activation of receptor-associated proteins, Smad2 and Smad3, key downstream components of the TGFß1 signaling pathway. Assessment of an adhesion/signaling family consisting of glial receptor protein tyrosine phosphatase beta and neuronal contactin-associated protein-1 (Caspr1) and contactin showed that the neuronal components were induced by TGFß1 and that ALC blocked these effects. Finally, TGFß1 was shown to induce release of the GnRH peptide in vitro, an action that was blocked by ALC. CONCLUSIONS: We have demonstrated glial-derived TGFß1 induces GnRH gene expression in the POA and stimulates release of the peptide from the MBH, actions necessary for driving the pubertal process. Importantly, ALC acted at both brain regions to block stimulatory effects of TGFß1. Furthermore, ALC altered neuronal components of an adhesion/signaling family previously shown to be expressed on GnRH neurons and implicated in glial-GnRH neuronal communications. These results further demonstrate detrimental effects of ALC at puberty.

Alcohol alters insulin-like growth factor-1-induced transforming growth factor ß1 synthesis in the medial basal hypothalamus of the prepubertal female rat.

BACKGROUND: Insulin-like growth factor-1 (IGF-1) and transforming growth factor ß1 (TGFß1) are produced in hypothalamic astrocytes and facilitate luteinizing hormone-releasing hormone (LHRH) secretion. IGF-1 stimulates release by acting directly on the LHRH nerve terminals and both peptides act indirectly through specific plastic changes on glial/tanycyte processes that further support LHRH secretion. Because the relationship between these growth factors in the hypothalamus is not known, we assessed the ability of IGF-1 to induce TGFß1 synthesis and release and the actions of alcohol (ALC) on this mechanism prior to the onset of puberty. METHODS: Hypothalamic astrocytes were exposed to medium only, medium plus IGF-1 (200 ng/ml), or medium plus IGF-1 with 50 mM ALC. After 18 hours, media were collected and assayed for TGFß1. For the in vivo experiment, prepubertal female rats were administered either ALC (3 g/kg) or water via gastric gavage at 07:30 hours and at 11:30 hours. At 09:00 hours, saline or IGF-1 was administered into the third ventricle. Rats were killed at 15:00 hours and the medial basal hypothalamus (MBH) was collected for assessment of TGFß1, IGF-1 receptor (IGF-1R), and Akt. RESULTS: IGF-1 induced TGFß1 release (p < 0.01) from hypothalamic astrocytes in culture, an action blocked by ALC. In vivo, IGF-1 administration caused an increase in TGFß1 protein compared with controls (p < 0.05), an action blocked by ALC as well as a phosphatidylinositol 3 kinase/Akt inhibitor. IGF-1 stimulation also increased both total (p< 0.01) and phosphorylated (p)-IGF-1R (p < 0.05) protein levels, and phosphorylated (p)-Akt levels (p < 0.01), which were also blocked by ALC. CONCLUSIONS: This study shows that ALC blocks IGF-1 actions to stimulate synthesis and release of hypothalamic TGFß1, total and p-IGF-1R, and p-Akt levels further demonstrating the inhibitory actions of ALC on puberty-related events associated with hypothalamic LHRH release.

In vitro effects of cilostazol, a phosphodiesterase 3A inhibitor, on mouse oocyte maturation and morphology.

Inhibition of phosphodiesterase 3A (PDE3A) in oocytes has been reported to arrest oocyte maturation and to increase intra-oocyte cyclic adenosine monophosphate levels. Although many PDE3A inhibitors have been found to arrest oocyte maturation in different species, including humans, the most commonly prescribed PDE3A inhibitor named cilostazol (CLZ) has not yet been fully evaluated in reproduction. The present study was designed to investigate the potential inhibitory effects of CLZ on oocyte maturation and morphology in vitro. Antral oocytes were recovered from hyperstimulated mice and allocated to 10 different CLZ concentrations (0.00-67.66 µmol/L). Oocytes were then assessed after 24 and 48 h of incubation for maturation and morphology. Some of the evaluated CLZ concentrations (1.06-4.23 µmol/L) were made similar to those observed in human clinical trials. CLZ arrested oocyte maturation at the germinal vesicle (GV) stage at concentrations as low as 1.06 µmol/L (P < 0.0001). A selective degenerative impact of CLZ targeting arrested oocytes at the GV stage was observed during 24 h of incubation (r = -0.781, P < 0.0001). This was not the case with non-arrested oocytes (r = -0.082, P = 0.64). Such degenerative impact was dose-dependent (P < 0.0001), suggesting a role for cyclic adenosine monophosphate in this degenerative process. The degenerated oocytes were of distorted oolema or fragmented cytoplasm. Based on the experiments, it is concluded that CLZ can inhibit oocyte maturation in vitro, at concentrations similar to those observed in humans taking CLZ, and under such conditions the prolonged maintenance of oocytes at the GV stage is harmful.

Hypothalamic actions and interactions of alcohol and IGF-1 on the expression of glial receptor protein tyrosine phosphatase-ß during female pubertal development.

BACKGROUND: Hypothalamic glial-neuronal communications are important for the activation of luteinizing hormone releasing hormone (LHRH) secretion at the time of puberty. As we have shown that alcohol (ALC) diminishes prepubertal LHRH secretion and delays puberty, we first assessed the effects of short-term ALC administration on the basal expression of a specific gene family involved in glial-neuronal communications. Second, as insulin-like growth factor-1 (IGF-1) is a critical regulator of LHRH secretion and the pubertal process, we then assessed whether IGF-1 could induce the expression of these signaling genes and determine whether ALC can block this affect. METHODS: Immature female rats were fed a liquid diet containing ALC for 6 days beginning when 27 days old. Control animals received either the companion isocaloric liquid diet or rat chow and water. Animals were decapitated on day 33, in the late juvenile stage of development. Medial basal hypothalamic (MBH) tissues were obtained for gene and protein analyses of glial receptor protein tyrosine phosphatase-ß (RPTPß) and the 2 neuronal components, contactin and contactin-associated protein 1 (Caspr1). In the second experiment, IGF-1 was administered into the third ventricle (3V) and the MBH removed 6 hours after peptide delivery, and the above-mentioned 3 genes were analyzed by real-time PCR. To determine whether this action was affected by ALC, immature female rats were administered either ALC (3 g/kg) or water via gastric gavage at 0900 hours. At 1030 hours, the ALC and control groups were subdivided such that half of the animals were injected into the 3V with IGF-1 and the other half with an equal volume of saline. Rats were killed 6 hours after the IGF-1 injection and MBHs collected. RESULTS: Real-time PCR showed that when compared with control animals, ALC caused a marked decrease (p < 0.001) in the basal expression of the RPTPß gene, but did not affect the expression of either contactin or Caspr1. Likewise, analysis by Western blotting demonstrated that ALC caused suppressed (p < 0.001) levels of the RPTPß protein, with the expressions of both contactin and Caspr1 proteins being unaltered. In the second experiment, results showed that only the RPTPß gene was stimulated (p < 0.05) by IGF-1 in the MBH 6 hours after peptide delivery. Assessments revealed that the IGF-1 induced increase (p < 0.01) in the expression of the RPTPß gene was blocked by the presence of ALC. CONCLUSIONS: Prepubertal ALC exposure is capable of interfering with hypothalamic glial-neuronal communications by suppressing the synthesis of the glial product, RPTPß, which is required for binding to the contactin-Caspr1 complex on LHRH neuronal terminals, thus suggesting that this action of ALC contributes to its detrimental effects on the pubertal process.

Regulation of prepubertal dynorphin secretion in the medial basal hypothalamus of the female rat.

The onset of puberty is the result of an increase in secretion of hypothalamic gonadotrophin-releasing hormone (GnRH). This action is a result of not only the development of stimulatory inputs to its release, but also the gradual decrease in inhibitory inputs that restrain release of the peptide prior to pubertal onset. Dynorphin (DYN) is one of the inhibitory inputs produced in the medial basal hypothalamus (MBH); however, little is known about what substance(s) control its prepubertal synthesis and release. Because neurokinin B (NKB) increases in the hypothalamus as puberty approaches, we considered it a candidate for such a role. An initial study investigated the acute effects of an NKB agonist, senktide, on the secretion of DYN from MBH tissues incubated in vitro. In other experiments, central injections of senktide were administered to animals for 4 days then MBHs were collected for assessment of DYN synthesis or for the in vitro secretion of both DYN and GnRH. Because insulin-like growth factor (IGF)-1 has been shown to play an important role at puberty, additional animals received central injections of this peptide for 4 days to assess NKB and DYN synthesis or the in vitro secretion of NKB. The results obtained show that senktide administration up-regulates the NKB receptor protein, at the same time as suppressing the DYN and its receptor. Senktide consistently suppressed DYN and elevated GnRH secretion in the same tissue incubates from both the acute and chronic studies. IGF-1 administration caused an increase in NKB protein, at the same time as decreasing DYN protein. Furthermore, the central administration of IGF-1 caused an increase in NKB release, an action blocked by the IGF-1 receptor blocker, JB-1. These results indicate that the IGF-1/NKB pathway contributes to suppressing the DYN inhibitory tone on prepubertal GnRH secretion and thus facilitates the puberty-related increase in the release of GnRH to accelerate the onset of puberty.

Influences of manganese on pubertal development.

The onset of puberty is the result of complex neuroendocrine interactions within hypothalamic region of the brain, as well as from genetic and environmental influences. These interactions ultimately result in the increased synthesis and release of luteinizing hormone-releasing hormone (LHRH). Manganese (Mn) is an essential environmental element known for years to be involved in numerous mammalian physiological processes, including growth and reproductive function. Studies in recent years have shown the ability of Mn to cross the blood-brain barrier and act within the hypothalamus to influence the timing of puberty. This review will depict research showing the molecular and physiological actions of Mn in the control of prepubertal LHRH and discuss the potential for the element to cause either helpful or harmful outcomes on the developmental process depending upon the age and accumulation of Mn within the hypothalamus.

Alcohol and Puberty: Mechanisms of Delayed Development

Adolescence represents a vulnerable period for developing youth. Alcohol use and misuse are especially problematic behaviors during this time. Adolescents are more sensitive to alcohol and less tolerant of its detrimental effects than are adults. Research in humans and animals has revealed that early alcohol consumption can result in delayed pubertal development. Animal studies have shown that alcohol detrimentally affects neuroendocrine systems within the hypothalamic region of the brain that are associated with the normal, timely onset of the pubertal process. To effectively restore development and shorten recovery time associated with the adverse effects of alcohol on puberty, researchers must first understand the molecular and physiological mechanisms by which alcohol interferes with critical hypothalamic functions.

Improvements in oocyte competence in superovulated mice following treatment with cilostazol: Ovulation of immature oocytes with high developmental rates.

Exogenous administration of superovulatory hormones negatively affects oocyte competence in mammals. Phosphodiesterase 3A inhibitors were found to improve competence of oocytes matured in vitro in several species, including humans. This study was therefore designed to define oocyte maturation synchronization and competence, in vivo, using superovulated mice treated with cilostazol, a selective phosphodiesterase 3A inhibitor. Swiss Webster mice were superovulated and treated orally with 7.5mg cilostazol once or twice to result in ovulation of immature oocytes at the metaphase I (MI) or germinal vesicle (GV) stage, respectively. Control immature oocytes were recovered from preovulatory follicles of superovulated mice not treated with cilostazol. Treated GV oocytes had significantly higher rates of synchronized and advanced chromatin configuration and cortical granule distribution than did control GV oocytes. Treated GV oocytes had a moderate increase in cAMP levels and consequently higher rates of meiotic maturation, IVF, and blastocyst formation than did control GV oocytes (P<0.0001). Treated MI oocytes had higher rates of normal spindles and chromosomes aligned at the metaphase plate than did control MI oocytes (P<0.003). Treated mice ovulating MI oocytes produced litter sizes larger than those observed in control mice ovulating mature oocytes (P<0.002). This study reveals that synchronization of oocyte maturation in superovulated mice improves oocyte development and competence. The capability of cilostazol, a clinically approved medication, to improve mouse oocyte competence suggests the potential benefit of including this compound in ovarian hyperstimulation programs to improve in vitro fertilization outcomes in infertile women.

Manganese protects against the effects of alcohol on hypothalamic puberty-related hormones.

AIMS: Since manganese (Mn) is capable of stimulating the hypothalamic-pituitary unit and advancing female puberty, we assessed the possibility that this element might overcome some of the detrimental effects of prepubertal alcohol (ALC) exposure on the hypothalamic control of pituitary function. MAIN METHODS: Rats received either saline or Mn (10mg/kg) daily by gastric gavage from day 12 to day 31. After weaning, all rats were provided Lab Chow diet ad libitum until day 27 when they began receiving either the Bio Serv control or ALC diet regime. On day 31, the medial basal hypothalamus (MBH) was collected to assess luteinizing hormone-releasing hormone (LHRH) and cyclooxygenase 2 (COX2) protein levels. Release of prostaglandin-E2 (PGE2), LHRH and serum luteinizing hormone (LH) were also assessed. Other animals were not terminated on day 31, but remained in study to assess timing of puberty. KEY FINDINGS: Short-term ALC exposure caused elevated hypothalamic LHRH content, suggesting an inhibition in peptide release, resulting in a decrease in LH. Both actions of ALC were reversed by Mn supplementation. COX2 synthesis, as well as PGE2 and LHRH release were suppressed by ALC exposure, but Mn supplementation caused an increase in COX2 synthesis and subsequent PGE2 and LHRH release in the presence of ALC. Mn supplementation also ameliorated the action of ALC to delay puberty. SIGNIFICANCE: These results suggest that low level Mn supplementation acts to protect the hypothalamus from some of the detrimental effects of ALC on puberty-related hormones.

Manganese-Stimulated Kisspeptin Is Mediated by the IGF-1/Akt/Mammalian Target of Rapamycin Pathway in the Prepubertal Female Rat.

Low-dose administration of manganese chloride (MnCl2) causes release of hypothalamic LH-releasing hormone (LHRH) and advances puberty in rat. Recently, this element was shown to up-regulate mammalian target of rapamycin (mTOR), kisspeptin gene (KiSS-1), and LHRH gene expressions in the brain preoptic area (POA)/anteroventral periventricular (AVPV) nucleus. Because these genes are critical for puberty, this study was conducted to identify the upstream mechanism by which Mn activates the mTOR/KiSS-1 pathway. On day 12, immature female rats began receiving a daily supplemental dose of 10 mg/kg of MnCl2 or saline by gavage, and POA/AVPV tissues were collected on day 29 for specific protein assessments. Another experiment assessed in vitro IGF-1 release in response to Mn and assessed signal transduction pathways in the POA/AVPV region after Mn delivery into the third ventricle. Chronic Mn exposure increased (P < .05) basal expressions of mTOR and kisspeptin proteins. Mn increased protein kinase B (Akt) and Ras homolog enriched in brain, both capable of activating mTOR. Central Mn delivery increased expressions of phosphorylated IGF-1 receptor (IGF-1R) (P < .05) and Akt (P < .01) in the POA/AVPV region. The previous central delivery of JB1, an IGF-1R antagonist, blocked Mn-induced expressions of both phosphorylated IGF-1R and Akt. Downstream to Akt, centrally administered Mn increased tuberous sclerosis complex 2 (P < .05), Ras homolog enriched in brain (P < .01), mTOR (P < .05), and kisspeptin (P < .05). Finally, we observed that the early puberty induced by Mn was blocked by the administration of an mTOR inhibitor. These results suggest that Mn acts, at least in part, through the IGF-1/Akt/mTOR pathway to influence prepubertal kisspeptin and LHRH.

Prepubertal exposure to elevated manganese results in estradiol regulated mammary gland ductal differentiation and hyperplasia in female rats.

Evidence suggests that environmental substances regulating estrogenic pathways during puberty may be detrimental to the developing mammary gland (MG). Manganese (Mn) is a trace mineral required for normal physiological processes. Prepubertal exposure to Mn induces precocious puberty in rats, an event associated with early elevations in puberty-related hormones, including estradiol (E2). However, until now the effect of Mn-induced precocious MG development has not been determined. Therefore, we assessed the ability of prepubertal Mn exposure to advance normal MG development and alter E2 driven pathways involved in tumorigenesis. Sprague Dawley female rats were gavaged daily with either 10 mg/kg manganese chloride (MnCl2) or saline (control) from postnatal day (PND) 12 through PND 30. Blood and MGs were collected on PNDs 30 and 120. Compared to controls, serum E2 levels on PND 30 were elevated (p < 0.05) in the Mn-treated group. Mn exposure significantly increased differentiated MG terminal ductal structures and the percentage of MG epithelial cells that stained positive for the proliferative marker, Ki67, at PND 30 (p < 0.001) and PND 120 (p < 0.001). Levels of Mn (ppm) were not elevated in these MGs. Mn-treated animals (40%) exhibited reactive stroma and intra-luminal focal hyperplasia in hemotoxylin and eosin stained MGs at PND 120. Furthermore, Mn exposure resulted in elevated protein expression levels of estrogen receptor α, activator protein 2α, phosphorylated (p)-Akt, and p53 in MGs on PND 120, but not on PND 30. Collectively, these data show that exposure to a supplemental dose of Mn causes accelerated pubertal MG growth which can progress to adult hyperplasia; thus, providing evidence that early life Mn exposure may increase susceptibility to breast cancer.

Cilostazol administered to female mice induces ovulation of immature oocytes: a contraceptive animal model.

AIMS: Both Cilostamide and Org 9935 are phosphodiesterase 3A (PDE3A) inhibitors that were evaluated in rodents and monkeys for their non-steroidal contraceptive properties. Although both compounds inhibited oocyte maturation, an adverse effect on heart rate was observed. Cilostazol (CLZ, Pletal) is a safe PDE3A inhibitor that was recently reported to block pregnancy in naturally cycling mice. In this study, the dose, frequency, time of administration, and reversibility effects of CLZ on oocyte maturation were defined using superovulated mice. MAIN METHODS: Superovulated mice were gavaged once or twice with 0, 7.5, or 15 mg CLZ at various times around the ovulatory stimulus. Ovulated oocytes were then evaluated for maturational stages. KEY FINDINGS: CLZ resulted in mice ovulating significant numbers of immature oocytes when administered anytime between 9h before the ovulatory stimulus and 2 h after the stimulus. This inhibitory effect was greater when CLZ dose was increased, administered twice or closer to the time of the ovulatory stimulus. Conversely, ovulated immature oocytes resumed maturation in oviducts when CLZ dose was reduced, administered once and away from the time of the stimulus. SIGNIFICANCE: Controlling CLZ dose, frequency, and time of administration produced mice ovulating immature oocytes at different stages, which may be an advantage over the conventional collection of immature oocytes from ovaries. More importantly, the capability of a clinically approved PDE3A inhibitor, with a reasonable margin of safety, to arrest oocyte maturation over a wide range of administration times and at doses extrapolated from human therapeutic doses demonstrates the contraceptive capacity of CLZ.

Early life manganese exposure upregulates tumor-associated genes in the hypothalamus of female rats: relationship to manganese-induced precocious puberty.

Prepubertal exposure to low, but elevated levels of manganese (Mn) can induce increased secretions of puberty-related hormones resulting in precocious pubertal development in female rats. These events are due to an action of the element within the hypothalamus to induce the secretion of gonadotropin-releasing hormone (GnRH). Because of these prepubertal effects of Mn and because precocious puberty is a serious neuroendocrine disorder, we have assessed whether early life exposure to this environmental element is capable of precociously upregulating the expression of a select group of genes previously associated with tumor growth or suppression, and that have more recently been shown to increase at the normal time of puberty. Female rat pups received a daily dose of either 10mg/kg manganese(II) chloride or an equal volume of saline by gastric gavage from postnatal day 12 through day 22 or 29. At this time, blood was collected for estradiol analysis and hypothalamic brain tissue frozen on dry ice until assessed for gene expressions. Rats exposed to the elevated levels of Mn showed a precocious increase in GnRH gene expression in the preoptic area and rostral hypothalamus on day 29, an action associated with precociously increased expressions of specific tumor-associated, puberty-related genes. These results demonstrate for the first time that prepubertal Mn exposure is capable of activating specific upstream genes regulating hypothalamic GnRH and suggest that these actions are involved in the mechanism by which this element can induce precocious puberty.