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

Abstract

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 μm) or a soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ); 100 or 250 μm) for another 30 min. Finally, the incubation continued for a further 30 min, but in the presence of MnCl₂ (50 or 250 μm) to assess the effect of the blockers on stimulated LHRH secretion. Both 50 and 250 μm MnCl₂ stimulated LHRH release (P < 0.05 and P < 0.01, respectively). The addition of 300–500 μm 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 μm 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 μm) 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.

Mn²⁺ is an abundant, naturally occurring essential element that is required for normal mammalian physiological events, including those related to normal growth and development of bone and cartilage (Hurley, 1981), connective tissue and the reproductive system (Greger, 1999; Keen et al. 1999). It has been known for many years that Mn²⁺ deficiencies in laboratory animals are associated with impaired growth and reproduction in both sexes (Boyer et al. 1942; Smith et al. 1944), thus suggesting a role for Mn²⁺ in the reproductive process. Because Mn²⁺ crosses the blood–brain barrier over four times more efficiently in young versus adult animals (Deskin et al. 1980), and because it can accumulate in the hypothalamus (Deskin et al. 1980; Pine et al. 2005), we hypothesized that it may influence the neuroendocrine system just prior to puberty. Therefore, we have recently conducted studies to assess specifically whether Mn²⁺ contributes to the secretion of puberty-related hormones and the timing of puberty. In this regard, we used both in vitro and in vivo studies to demonstrate that Mn²⁺ can stimulate the secretion of luteinizing hormone-releasing hormone (LHRH) and luteinizing hormone (LH), respectively, in immature female rats (Pine et al. 2005). In that same study, we also assessed the effects of supplementing the diet of immature female rats with a low dose of MnCl₂ from day 12 until day 29, or, in some animals until vaginal opening (VO). This diet regimen resulted in a significant elevation in the serum levels of LH, follicle-stimulating hormone (FSH) and oestradiol (E₂), and a moderate but significant advancement in the age at VO. More recently, we have observed similar elevations in puberty-related hormones in immature males, as well as increases in both daily sperm production and efficiency of spermatogenesis in age-matched male controls. While this indicates that Mn²⁺ can accelerate maturation in males (Lee et al. 2006), it is important to note that the MnCl₂ dose required for this was 2.5-fold greater than that which we showed to accelerate female puberty (Pine et al. 2005), hence suggesting that the female is more sensitive in this regard. Taken together, the above studies indicate clearly that Mn²⁺ is capable of acting within the hypothalamus to influence LHRH release during prepubertal development.

At the present time, the hypothalamic mechanism by which Mn²⁺ acts to facilitate prepubertal LHRH secretion is not known. Because NO stimulates LHRH release (Rettori et al. 1993), and because of the ability of NO to activate soluble guanylyl cyclase (sGC) and the fact that the Mn²⁺ is the preferred cofactor for activation of this enzyme (Murad, 1994), we questioned whether this pathway was the site of action for Mn²⁺-induced LHRH secretion. As it is not known whether Mn²⁺ can effectively stimulate hypothalamic NO directly, or whether the principal action may reside in regulating activation of sGC, we investigated whether Mn²⁺-induced stimulation of LHRH secretion involves an action within the NO–GC–cGMP–protein kinase G (PKG) signalling pathway.

Methods

Animals

Immature female Sprague-Dawley rats raised in our colony at the Texas A & M University Department of Comparative Medicine were used for these experiments. The animals were housed under controlled conditions of photoperiod (lights on, 06.00 h; lights off, 18.00 h) and temperature (23°C), with ad libitum access to food and water. The diet (Harlan Teklad 2016) contained 94.7 mg kg⁻¹ manganese and 149.8 mg kg⁻¹ iron as analysed by the Heavy Metal Analysis Laboratory, Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A & M University. On the day of experiment, animals were killed using decapitation without anaesthesia. All procedures used were approved by the University Animal Care and Use Committee at Texas A & M University and in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.

Chemicals

N-monomethyl-l-arginine (NMMA), a nitric oxide synthase (NOS) inhibitor, KT5823, a PKG inhibitor, and MnCl₂ were purchased from Sigma (St Louis, MO, USA). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific sGC inhibitor, was purchase from EMD Biosciences Inc. (San Diego, CA, USA). Both KT5823 and ODQ were dissolved in dimethyl sulphoxide and subsequent dilutions were made using Locke solution. NMMA and MnCl₂ were diluted in Locke solution.

Experimental protocol

Female rats (30 days old) were decapitated, and the brain removed. The medial basal hypothalamus (MBH) was dissected by vertical cuts along the posterior side of the optic chiasm and the anterior edge of the mammillary bodies and laterally at the tuberoinfundibular sulci of the hypothalamus. The tissue was incubated as previously described (Nyberg et al. 1993) with minor modifications. Briefly, each MBH was incubated in a vial containing 350 μl Locke solution containing (mm): Hepes 2, NaCl 154, KCl 5.6, MgCl₂ 1, NaHCO₃ 6, glucose 10 and CaCl₂ 1.25, and 1 mg ml⁻¹ bovine serum albumin, pH 7.4; inside a Dubnoff shaker (80 cycles min⁻¹) at 37°C in an atmosphere of 95% O₂–% CO₂. After a 30 min equilibration period, the initial incubation medium was discarded, and all MBHs were incubated in fresh medium for 30 min to establish basal LHRH release. The medium was replaced by medium alone or medium plus NMMA (300 or 500 μm; Karanth et al. 2004), ODQ (100 or 250 μm; Karanth et al. 2004) or KT5823 (10 μm) for 30 min, then removed and stored in microcentrifuge tubes. The medium was then replaced by medium containing 50 or 250 μm MnCl₂ (Pine et al. 2005) or medium with MnCl₂ plus NMMA, ODQ or KT5823. The MBHs were incubated for an additional 30 min before these samples of medium were collected. All of the samples were boiled for 10 min, and then stored at −80°C until assayed for LHRH. MBHs were weighed to the nearest 0.1 mg.

In two other experiments, the medium was removed after equilibration and replaced by fresh medium for a 30 min incubation in order to establish basal secretion of total nitrite (NO₂⁻) and LHRH, or cGMP and LHRH. In each case, the medium was saved, then replaced with medium containing 50 or 250 μm MnCl₂ and the tissues incubated for a final 30 min. The samples of medium were collected, divided into aliquots, boiled for 10 min and then stored frozen until assayed for NO₂⁻ and LHRH or cGMP and LHRH, respectively. As above, the tissues were weighed to the nearest 0.1 mg.

Hormone analysis

LHRH was measured as previously described (Nyberg et al. 1993) using Antiserum R11B73 kindly provided by Dr V. D. Ramirez. Synthetic LHRH used for the standards and iodinations was purchased from Sigma. The sensitivity of the assay was 0.2 pg tube⁻¹, and the intra-assay and inter-assay coefficients of variation were < 10%. Total NO₂⁻ and cGMP were measured by kits purchased from Caymen Chemical (Ann Arbor, MI, USA). The sensitivity of the NO₂⁻ and non-acetylated cGMP assays were 1 μm and 1 pmol ml⁻¹, respectively.

Statistical analysis

All values are expressed as the means (± s.e.m.). Differences between treatment groups were analysed by Kruskal–Wallis non-parametric analysis of variance (ANOVA) followed by post hoc testing using Student–Newman–Keuls multiple range test and, where appropriate, the Student t test. P values less than 0.05 were considered significant. The IBM PC programs INSTAT and PRISM 3.0 (GraphPad, San Diego, CA, USA) were used to calculate and plot the results.

Results

Effect of NOS inhibition on Mn²⁺-induced LHRH release

Figure 1 demonstrates that the basal release of LHRH from the MBH was not altered by medium alone (Fig. 1A and C) or the medium containing 300 μm NMMA (Fig. 1B and D). The addition of 50 μm MnCl₂ to the medium caused an increase in the amount of LHRH released from both control tissues (Fig. 1A, P < 0.05) and tissues exposed to 300 μm NMMA (Fig. 1B, P < 0.05). Furthermore, the addition of 250 μm MnCl₂ likewise caused increased LHRH release from control tissues (Fig. 1C, P < 0.01), as well as from tissues exposed to both 300 μm NMMA (Fig. 1D, P < 0.05) and 500 μm NMMA (P < 0.05, not shown).

Figure 1.

Effect of nitric oxide synthase (NOS) inhibition using N-monomethyL-L-arginine (NMMA) on MnCl₂-induced luteinizing hormone-releasing hormone (LHRH) release Open bars represent basal LHRH release. Grey bar represents LHRH release in the presence of NMMA alone. Filled bars represent LHRH released following stimulation with 50 μm MnCl₂ in the absence (A) or presence (B) of NMMA. Hatched bars represent LHRH released following stimulation with 250 μm MnCl₂ in the absence (C) or presence (D) of the inhibitor. Note that Mn²⁺ stimulated LHRH release in control tissues after both doses (A and C), and that this action was not altered by the presence of the NOS blocker (B and D). *P < 0.05 versus medium alone or medium plus NMMA; **P < 0.01 versus medium alone. Each bar represents the mean (± s.e.m.). n = 8, 9, 11 and 10 for A, B, C and D, respectively.

Effect of Mn²⁺ on NO²⁺ production

In order to assess more directly a potential effect of Mn²⁺ on tissue NO formation, we measured the amount of total NO₂⁻, an indicator of NO generation, and LHRH release from the same tissue incubates. The 50 μm MnCl₂ dose failed to increase NO₂⁻ levels into the medium (Fig. 2A), but did stimulate a marked increase in LHRH released (Fig. 2B, P < 0.001). Identical results were noted following a 100-μm dose of MnCl₂ (not shown). The 250 μm dose of MnCl₂ did, however, increase the production of both NO₂⁻ (Fig. 2C, P < 0.001) and LHRH (Fig. 2D, P < 0.001).

Figure 2.

Effect of MnCl₂ on NO_2– production and luteinizing hormone-releasing hormone (LHRH) release from the same tissues Open bars represent basal NO₂⁻ and LHRH released into the medium. Filled or hatched bars represent NO₂⁻ and LHRH released following stimulation by MnCl₂ at 50 and 250 μm, respectively. Note that 50 μm MnCl₂ did not increase NO₂⁻ (A), but markedly induced LHRH release (B). Similar effects were observed with 100 μm MnCl₂ (not shown). The 250 μm MnCl₂ dose caused increases in both NO₂⁻ (C) and LHRH (D). Together, these data suggest that MnCl₂ can only stimulate NO production at higher concentrations. Each bar represents the mean (± s.e.m.). The number of tissues per group is depicted within each panel. ***P < 0.001 versus respective medium. n = 9 for A and B, and n = 14 for C and D.

Effect of GC inhibition on Mn²⁺-induced LHRH release

Figure 3 shows that the basal secretion of LHRH was not affected by medium alone or medium containing doses of 100 and 250 μm ODQ. The subsequent addition of MnCl₂ to the medium elicited a marked increase (P < 0.001) in LHRH released from control tissues (Fig. 3A), but this action was blunted markedly by 100 μm ODQ and blocked by 250 μm dose of the inhibitor (Fig. 3B and C, respectively).

Figure 3.

Effect of guanylyl cyclase (GC) inhibition using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on MnCl₂-induced luteinizing hormone-releasing hormone (LHRH) release Open bars represent basal LHRH release. Grey bars represent LHRH released in the presence of ODQ. Hatched bars represent LHRH released following stimulation with 250 μm MnCl₂ in the absence (A) or presence (B and C) of the inhibitor. Note that the MnCl₂ stimulated LHRH release from control tissues (A), but this stimulatory effect was dose-dependently blocked (B and C) by the presence of the soluble GC inhibitor in the medium. ***P < 0.01 versus medium alone. *P < 0.05 versus medium alone and medium plus ODQ. Each bar represents the mean (± s.e.m.). n = 20, 11 and 15 for A, B and C, respectively.

Effect of Mn²⁺ on cGMP release

Basal and Mn²⁺-stimulated release of both cGMP and LHRH were assessed from the same MBH tissue incubates. The 50 μm dose of MnCl₂ induced an increase (P < 0.05) in the amount cGMP released above basal levels (Fig. 4A), an effect that was accompanied by an increase (P < 0.01) in the release of LHRH (Fig. 4B). Similar stimulations were observed for both cGMP and LHRH following addition of the 250 μm dose of MnCl₂ (not shown).

Figure 4.

MnCl₂ stimulates cGMP and luteinizing hormone-releasing hormone (LHRH) release Open bar represents basal cGMP and LHRH release. Filled bars represent cGMP and LHRH released following addition of 50 μm MnCl₂ into the medium. Note that MnCl₂ stimulated both cGMP (A) and LHRH (B) secreted from the same tissue incubates. *P < 0.05 and **P < 0.01 versus basal levels. Each bar represents the mean (± s.e.m.). n = 8 for both A and B.

Effect of PKG inhibition on Mn²⁺-induced LHRH release

Figure 5A and B shows that basal secretion of LHRH was not affected by medium alone or medium containing KT5823. The addition of 250 μm MnCl₂ to the medium elicited an increase (P < 0.05) in LHRH released from control tissues, but failed to induce stimulation of the peptide in the tissues exposed to the inhibitor.

Figure 5.

Effect of protein kinase G (PKG) inhibition using KT5823 on MnCl₂-induced luteinizing hormone-releasing hormone (LHRH) release Open bars represent basal LHRH release. Grey bar represents LHRH released in the presence of 10 μm KT5823. Hatched bars represent LHRH released following stimulation with 250 μm MnCl₂ in the absence (A) or presence (B) of the inhibitor. Note that MnCl₂ stimulated LHRH release in control tissues (A), but was unable to stimulate LHRH release when the PKG blocker was present in the medium. *P < 0.05 versus medium alone. Each bar represents the mean (± s.e.m.). n = 8 for both A and B.

Discussion

We showed previously that Mn²⁺ is capable of acting at the hypothalamic level to stimulate LHRH release in prepubertal animals. Specifically, in our initial study, we showed in prepubertal female rats that Mn²⁺, when administered into the third ventricle, caused a dose-dependent release in LH. Furthermore, prior exposure to acyline, an LHRH receptor antagonist, blocked the central action of Mn²⁺ to stimulate LH, and Mn²⁺ dose-dependently stimulated LHRH release directly from MBHs incubated in vitro (Pine et al. 2005). In that study, we also demonstrated that the serum levels of LH, FSH and E₂ were elevated significantly, and that the day of VO was advanced when the diet of developing female rats was supplemented with a low dose of MnCl₂ beginning when the rats were 12 days old.

The present results demonstrate the site and mechanism by which Mn²⁺ induces prepubertal LHRH secretion in female rats. In this regard, we showed that blocking NOS with NMMA was ineffective in blocking LHRH release following doses of 50 and 250 μm MnCl₂. Furthermore, these doses of 50 and 100 μm MnCl₂ did not increase total NO₂⁻, a marker of NO production, yet were capable of stimulating LHRH release. Only 250 μm caused increased NO₂⁻ accumulation. Thus, taken together these results suggest that low doses of MnCl₂ do not induce LHRH release by acting first to stimulate NOS/NO. Conversely, ODQ, a specific blocker of sGC (Zhao et al. 2000), inhibited the release of LHRH induced by Mn²⁺ in a dose-dependent manner, demonstrating that sGC is the site of action of Mn²⁺ to facilitate LHRH secretion. Furthermore, Mn²⁺ stimulates directly the release of cGMP and LHRH from the same MBH tissue, and finally, a downstream PKG inhibitor, KT5823, blocked Mn²⁺-induced LHRH release. Overall, these results demonstrate that the principal action of Mn²⁺ is to facilitate activation of sGC, subsequently stimulating the cGMP–PKG pathway controlling LHRH secretion in prepubertal female rats. The fact that Mn²⁺ can stimulate sGC in prepubertal animals is supportive of an earlier report indicating that Mn²⁺ is a natural element that is particularly important during development and capable of activating more than 50 enzyme systems, including GC and protein kinases (Wedler, 1993). Furthermore, even though it has been known since the 1970s that Mn²⁺ is the preferred cofactor for GC and that it can increase GC activity either directly or as a cofactor with NO (Garbers, 1979; Murad, 1994), to our knowledge, the present report is the first to demonstrate stimulation of sGC by MnCl₂ in a physiological setting relevant to neuropeptide hormone secretion.

The results generated by this study further indicate that Mn²⁺, through its ability to stimulate prepubertal LHRH secretion, may contribute to events leading to puberty. The age at which normal puberty occurs depends on a complex series of events within the hypothalamus that culminate in the increased release of LHRH. This timely increase appears to require the interactive participation of neuronal circuitries and glial cells within the hypothalamus (Ojeda & Urbanski, 1994) which are likely to be influenced by specific metabolic signals, as well as genetic and environmental influences. Signals that can activate excitatory amino acid receptors (Gay & Plant, 1987; Urbanski & Ojeda, 1990; Nyberg et al. 1993), and the peptides insulin-like growth factor-1 (Hiney et al. 1996; Wilson, 1998; Danilovich et al. 1999) and kisspeptin (Navarro et al. 2004a; Shahab et al. 2005), are all examples of influences capable of stimulating LHRH release and advancing signs of sexual maturity. We have indicated here, and in previous work (Pine et al. 2005), that Mn²⁺ is an essential nutrient that can stimulate LHRH release and advance puberty, suggesting that it may represent an important environmental component of the pubertal process.

In addition to the above puberty-related substances, GPR54, a novel G-protein-coupled receptor (Lee et al. 1999), has received increasing attention during the last few years for its role in the onset of puberty. In this regard, a mutation of GPR54 in humans, and a deletion of the GPR54 locus in mice, caused hypogonadotrophic hypogonadism and delayed puberty (de Roux et al. 2003; Seminera et al. 2003; Funes et al. 2003). Kisspeptins, which are products of the metastasis suppressor gene, KiSS-1, have been shown to be ligands to this receptor (Navarro et al. 2004a). Increases in KiSS-1 and GPR54 gene expressions occur during pubertal development (Navarro et al. 2004b). Furthermore, kisspeptins can act at the hypothalamic level to stimulate LHRH/LH release in immature rats and rhesus monkeys (Navarro et al. 2004a; Thompson et al. 2004; Shahab et al. 2005) and advance VO in rats (Navarro et al. 2004b). Taken together, these data demonstrate that GPR54 activation by its ligands contributes to LHRH release at the time of puberty. Even though there has been no evidence presented to date demonstrating that Mn²⁺ can bind to or activate this protein receptor, and while this was not part of the present study, this potential action cannot be ruled out and warrants further discussion. GPR54 is localized in the MBH (Gottsch et al. 2004; Shahab et al. 2005), as well as on the LHRH neuronal soma in the preoptic area (POA) (Parhar et al. 2004; Irwig et al. 2005). Although this is speculative, should Mn²⁺ be shown to have the capability to activate this protein receptor by some mechanism, then it is possible that the metal would be able to act at both the terminals in the MBH and the neuronal soma in the POA. The present study was designed to assess only the effect of Mn²⁺ on LHRH secretory mechanisms within the MBH, and we showed conclusively that the metal stimulates LHRH release via a NO-independent, activation of cGMP within the LHRH nerve terminals. Further work will be needed to assess whether Mn²⁺ is capable of activating GPR54 and, if so, whether it will also produce similar effects on the LHRH soma in the POA.

Mn²⁺ is able to enter the brain either through the cerebral vasculature or via the cerebral spinal fluid (CSF). The mechanism by which Mn²⁺ crosses the blood–brain barrier is not well understood, but is thought to involve binding of the metal to transport systems such as transferrin (Aschner & Aschner, 1990; Aschner & Aschner, 2000). As the blood levels of Mn²⁺ rise, its influx into the CSF rises and entry across the choroid plexus becomes more important (Murphy et al. 1991). The POA and hypothalamus, which are adjacent to the third ventricle, receive Mn²⁺ from both capillaries and the CSF. Mn²⁺ crosses the blood–brain barrier over four times more efficiently in the young than old animals (Mena, 1974), and younger animals do not have the full capacity to eliminate Mn²⁺ (Fechter, 1999). Furthermore, the element accumulates in the hypothalamus (Deskin et al. 1980; Pine et al. 2005) and is known to be taken up by both neurons and glial cells (Tholey et al. 1990), thus suggesting a possible role in neuronal-glial communications within the developing hypothalamus.

Evidence has been presented in recent years suggesting that puberty may be occurring at an earlier age, especially in females (Herman-Giddings et al. 1997; Parent et al. 2003). The cause of this apparent trend is not known, but pubertal onset before 8 years of age in girls and 9.5 years in boys is considered precocious (Lee, 1996). The central form of precocious puberty is LHRH-dependent, and is characterized by hormonal changes similar to those occurring at the normal time of puberty. Of importance, in central precocious puberty there is a premature activation of the LHRH secretory system. In boys, this is usually accounted for by hypothalamic harmatomas, other CNS lesions or familial disease, with less than 10% of cases considered idiopathic. Conversely, in girls, over 65% of the cases of precocious puberty are considered idiopathic. Thus far, research suggests that in addition to a possible action of Mn²⁺ during normal pubertal events, a risk to certain individuals for precocious puberty could develop should they be exposed to elevated levels of the element during the juvenile or early adolescent years. This possibility is supported by several lines of evidence. As mentioned above, we have shown that Mn²⁺ acts centrally to stimulate LHRH secretion and it causes elevated gonadotropin and gonadal steroid levels, and can advance puberty in both sexes, although females appear to be more sensitive to low but elevated levels (Pine et al. 2005; Lee et al. 2006). Environmental sources of Mn²⁺ are abundant, with it found in water, food, soil and air, and some regions having higher levels than others, and some cultures consuming greater amounts in their diets than others. Infants and children have been classified as being more sensitive to Mn²⁺ exposure (Environmental Protection Agency, 2002), mainly because the minimum level of exposure is not well defined (Greger, 1999). We suggest that should moderately elevated levels of Mn²⁺ accumulate in the hypothalamus too early in life and reach levels not normally attained until later, then there is the potential for precocious development to occur. Epidemiological research in children and experimental studies in primates may be able to further address this issue.

In summary, our results clearly show that Mn²⁺ can induce prepubertal LHRH secretion via a hypothalamic action to activate sGC, causing stimulation of the cGMP–PKG pathway. Because of this hypothalamic action, and the fact that Mn²⁺ administration can advance puberty, we suggest that this essential nutrient is a potential environmental factor involved in the pubertal process.