Manganese induces IGF-1 and cyclooxygenase-2 gene expressions in the basal hypothalamus during prepubertal female development

Abstract

Precocious puberty is a significant child health problem, especially in girls, because 95% of cases are idiopathic. Our earlier studies demonstrated that low-dose levels of manganese (Mn) caused precocious puberty via stimulating the secretion of luteinizing hormone-releasing hormone (LHRH). Because glial-neuronal communications are important for the activation of LHRH secretion at puberty, we investigated the effects of prepubertal Mn exposure on specific glial-derived puberty-related genes known to affect neuronal LHRH release. Animals were supplemented with MnCl(2) (10 mg/kg) or saline by gastric gavage from day 12 until day 22 or day 29, then decapitated, and brains removed. The site of LHRH release is the medial basal hypothalamus (MBH), and tissues from this area were analyzed by real-time PCR for transforming growth factor α (TGFα), insulin-like growth factor-1 (IGF-1), and cyclooxygenase-2 (COX-2) messenger RNA levels. Protein levels for IGF-1 receptor (IGF-1R) were measured by Western blot analysis. LHRH gene expression was measured in the preoptic area/anteroventral periventricular (POA/AVPV) region. In the MBH, at 22 days, IGF-1 gene expression was increased (p < 0.05) with a concomitant increase (p < 0.05) in IGF-1R protein expression. Mn also increased (p < 0.01) COX-2 gene expression. At 29 days, the upregulation of IGF-1 (p < 0.05) and COX-2 (p < 0.05) continued in the MBH. At this time, we observed increased (p < 0.05) LHRH gene expression in the POA/AVPV. Additionally, Mn stimulated prostaglandin E(2) and LHRH release from 29-day-old median eminences incubated in vitro. These results demonstrate that Mn, through the upregulation of IGF-1 and COX-2, may promote maturational events and glial-neuronal communications facilitating the increased neurosecretory activity, including that of LHRH, resulting in precocious pubertal development.

FIG. 1.

Effect of low-dose Mn exposure on TGFα (A) and IGF-1 (B) gene expression in the MBH of 22-day-old female rats as determined by real-time PCR. Panel (A) shows the gene expression of TGFα in 22-day-old rats was not affected by Mn exposure (solid bar) versus those that received saline (open bar). Panel (B) shows that in these same animals, Mn exposure markedly increased expression of the IGF-1 gene (solid bar) compared with the controls (open bar). The respective bars illustrate the mean (± SE) of an N of 5 per group. *p < 0.05 versus control.

FIG. 2.

Effect of low-dose Mn exposure on IGF-1R protein expression in the MBH of 22-day-old female rats. (A) Representative Western immunoblot of IGF-1R and β-actin proteins in the MBH isolated from control (lanes 1–3) and Mn-treated (lanes 4–6) animals. (B) Densitometric quantitation of all the bands from two blots assessing IGF-1R protein expression in the MBH. These data were normalized to the internal control β-actin protein, and the densitometric units represent the IGF-1R/β-actin ratio. Note that IGF-1R protein expression was increased following Mn exposure (solid bar) of these 22-day-old animals when compared with the control animals treated with saline (open bar). The respective bars illustrate the mean (± SE) of an N of 8 per group. *p < 0.05 versus control.

FIG. 3.

Effect of low-dose Mn exposure on COX-2 gene expression in the MBH of 22-day-old female rats as determined by real-time PCR. Note that Mn exposure significantly induced COX-2 gene expression (solid bar) in the MBH of 22-day-old animals when compared with the control animals that received saline (open bar). The respective bars illustrate the mean (± SE) of an N of 5 per group. **p < 0.01 versus control.

FIG. 4.

Effect of low-dose Mn on IGF-1 (A) and COX-2 (B) gene expression in the MBH of 29-day-old animals as determined by real-time PCR. Panel (A) shows that IGF-1 gene expression was increased following Mn exposure (solid bar) when compared with control animals exposed to saline (open bar). Panel (B) shows that in these same animals, Mn exposure also increased COX-2 gene expression (solid bar) compared with the controls (open bar). The respective bars illustrate the mean (± SE) of an N of 9 per group. *p < 0.05 versus control.

FIG. 5.

Effect of Mn on the in vitro release of PGE₂ from the MEs obtained from 29-day-old female rats. MEs were incubated in medium only or medium containing 50μM MnCl_2. Note that the presence of Mn in the medium caused a marked increase in the release of PGE₂ (hatched bar) compared with the basal release of PGE₂ from the medium-only controls (open bar). The respective bars illustrate the mean (± SE) of an N of 8 per group. **p < 0.01 versus control.

FIG. 6.

Effect of low-dose Mn exposure on LHRH gene expression in the POA/AVPV region of 29-day-old female rats as determined by real-time PCR. Note that at 29 days of age, the LHRH gene expression is increased in those animals exposed to Mn (solid bar) compared with control animals that received saline (open bar). The respective bars illustrate the mean (± SE) of an N of 5 per group. *p < 0.05 versus control.