The Effect of Sex Steroid on the Glutamate-induced Neurotocixity in the Preoptic Area of the Postnatal Rats

• Main Authors: Shih, Huei-Chuan, 施慧娟
• Other Authors: Hsu Chin
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/07514512701686710303

碩士 === 高雄醫學大學 === 醫學研究所 === 88 === Glutamate plays an important role in neuroendocrine regulation of reproduction through acting on the N-methyl D-aspartate receptor (NMDAR) in the preoptic area (POA). However, a larger dose of glutamate is neurotoxic. The susceptibility to glutamate-induced neuron loss in medial preoptic area of neonatal male rats was higher than that of females. Our previous finding suggested that the higher expression of NMDAR in the POA of neonatal male rats may contribute to the more sever neurotoxicity of glutamate on the POA of male rats than that of females. In the prenatal period, only male rats exhibited testosterone peaks on embryonic day 17-19. In order to know whether prenatal hormone milieu affects the glutamate-induced neurotoxicity through modulating NMDAR expression. Four groups of Long-Evan rats: (1) normal male (2) androgen sterilized (ASR) female (3) prenatal stressed (PNS) male and (4) normal female were designed. The testosterone peak was simulated or diminished by testosterone injection or prenatal stress, respectively. Serum level of testosterone was measured by RIA on embryonic day 18. Protein and mRNA of the NMDAR were quantified by western blotting analysis and RT-PCR, respectively. Glutamate was administered to the pups on postnatal day 1 (PND 1) and PND 3. The glutamate-induced neuronal apoptosis was assessed on PND 4 by in situ TUNEL staining and gel electrophoresis. The results showed that (1) The testosterone level of the normal male fetus was significantly higher than that of the normal female, and the testosterone level of the ASR female or PNS male was similar to that of the normal male or normal female, respectively. (2) After glutamate treatment, the incidence of apoptosis in the POA of the normal male was significantly higher than that of the normal female, whereas no significant difference of apoptotic incidence was observed between the ASR female and the normal male or between the PNS male and the normal female. (3) Severe DNA fragmentation was observed in the glutamate-treated normal male but not in the female. A lesser degree of DNA laddering was observed in the PNS male than in that of normal male. (4) Protein expression of NMDAR in the normal male was significantly higher than that in the normal female on PND 1, while no significant difference was noted between the ASR female and the normal male or between the PNS male and the normal female. (5) No significant difference of RT-PCR product of NMDAR mRNA was found among the normal male, the ASR female, the PNS male and the normal female. These results suggested that prenatal testosterone exposure might enhance the glutamate-induced neurotoxicity through increasing the expression of the NMDAR at the post-transcriptional level. Previous report indicated that testosterone influenced POA sexual differentiation after being converted into estradiol which can increase the number of NMDAR binding sites and enhance the expression of NMDAR1 subunit protein. It is possible that testosterone affects the NMDAR mediated neurotoxicity after being converted into estradiol. Three groups of rats (1) normal male (2) ATD (aromatase inhibitor)-treated male (3) normal female were designed to test this hypothesis. ATD (1,4,6 androstatriene-3,17 dione) (5 mg / 0.1 ml / day), an aromatase inhibitor, was administered from ED 10 to ED 22 to inhibite the conversion of testosterone to estradiol. The results showed that (1) The NMDAR protein expression of the ATD-treated male was similar to that of the normal female. (2) There was no significant difference of the RT-PCR product of NMDAR mRNA among the normal male, the ATD-treated male, and the normal female. (3) After glutamate treatment, no significant difference of apoptotic incidence was observed between the ATD-treated male and the normal female. (4) Sever DNA fragmentation was observed in the glutamate-treated normal male, while only mild DNA fragmentation was observed in the ATD-treated male. These results suggested that estradiol converted from prenatal testosterone enhances the glutamate-induced neurotoxicity through increasing the protein expression of the NMDAR. Estradiol could produce long term effects through classical genomic action on intracellular receptor or produce rapid membrane effects through non-classical mechanism. In order to investigate whether estradiol modulates the neurotoxic effect of glutamate on the neurons through estrogen receptor. The immortalized GT1-7 cells which express estrogen receptor and NMDA receptor were used. The in vitro treatment of GT 1-7 cells were divided into 6 groups (1) control groups: no treatment (2) 10 nM E2 treatment for 3 days (3) 10 mM tamoxifen (estrogen receptor antagonist) treatment for 3 days (4) co-treatment with 10 nM E2 and 10 mM tamoxifen for 3 days (5) 1.0 mM MK-801 (non-competitive NMDAR antagonist) treatment for 3 days (6) co-treatment with 10 nM E2 and 1.0 mM MK-801 for 3 days. NMDAR expression were first semiquantitated by Western blot analysis in groups (1)(2)(3)(4). Afterward, cell viability and LDH efflux were analysed in all groups with or without 1.0 nM glutamate treatment for 16 hours. Also cell necrosis and apoptosis were rated by phosphatidylserine translocation and DNA laddering. The results showed that (1) The expression of NMDAR of the E2-pretreated (10 nM, for 3 day) GT1-7 cells was higher than that of non-E2-pretreated cells. (2) The percentages of apoptosis after glutamate treatment in the E2-pretreated group were higher than those in non-E2-treated group. (3) The decrease of viability and increase of LDH efflux after glutamate treatment were more significant in E2-pretreated cells than in the non-E2-treated cells. (4) Glutamate-induced DNA laddering was enhanced by E2-pretreatment. (5) Higher dose of MK-801 (2 mM) was needed in E2-pretreated cells than in non-E2-pretreated group to block the glutamate-induced neurotoxicity. (6) Tamoxifen abolished the estradiol-enhanced NMDAR expression and neurotoxicity of glutamate. These results suggested that pretreatment of estradiol might enhance the expression of NMDAR and glutamate-induced neurotoxicity on the GT1-7 cells through acting on estrogen receptor. In conclusion, prenatal testosterone, after being converted into estradiol and acting on estrogen receptor, enhances the glutamate-induced neurotoxicity through modulating NMDAR expression at the post-transcription level.