臺灣博碩士論文加值系統

下視丘中的視前區（preoptic area）在調節生殖內分泌之功能中扮演著十分重要的角色，如促性腺釋放激素（gonadotropin-releasing hormone, GnRH）分泌的調節及交配行為的調控。中樞神經系統富含的麩胺酸鹽(glutamate)，透過活化N-甲基-D-天門冬胺酸鹽受器（N-methyl-D-aspartate receptor, NMDAR）的作用可促進視前區所調節生殖內分泌功能。然而大量的麩胺酸鹽反而會造成下視丘的神經損傷，繼而導致生殖內分泌功能的受損，且雄鼠生殖行為損傷較雌鼠嚴重。故本論文將於第一部份探討NMDA受器在視前區性別分化過程中所扮演的藥理性角色。首先，利用影像分析儀探討麩胺酸鹽的神經毒性對視前區造成的形態學上的變化。並以西方墨點法分析出生第一天及第三天視前區NMDA受器的含量，以瞭解NMDA受器與麩胺酸鹽神經毒性的關聯。並分別偵測細胞鈣離子的動力變化及乳酸去氫酉每（lactate dehydrogenase, LDH）的釋出量以探究麩胺酸鹽所產生的即時性及延遲性毒性訊息。實驗的結果顯示（1）雄鼠視前區中的性別差異性神經核(sexually dimorphic nucleus of preoptic area, SDN-POA)在麩胺酸鹽處理後神經細胞皺縮數目增加及神經細胞密度下降，而雌鼠則否，（2）出生第一天及第三天雄鼠視前區中NMDA受器表現皆較雌鼠多，（3）麩胺酸鹽處理後會引發雄鼠視前區神經細胞內鈣離子驟增並釋出大量LDH。總括以上的結果證實雄鼠視前區NMDA受器的表現較多、經由過度引發細胞內鈣離子上升的現象，可能是雄鼠對麩胺酸鹽引發的神經毒性敏感度較雌鼠高的主因。此外，我們利用胸腺口票口令結合量測定（thymidine incorporation）、細胞計數及細胞週期分析（cell cycle analysis）探討麩胺酸鹽抑制視前區星狀細胞增殖之影響，並測定calcium/calmodulin-dependent kinase II (CaM kinase II)及蛋白激酉每C (protein kinase C, PKC)的活性，以瞭解麩胺酸鹽影響星狀細胞增殖的可能機制。並測定麩胺酸鹽刺激星狀細胞NO的釋出量及NO合成酉每（NO synthase, NOS）的表現。藉以瞭解星狀細胞在麩胺酸鹽造成視前區性別差異性神經損傷的角色。實驗結果顯示：（i）麩胺酸鹽顯著抑制雄鼠星狀細胞的之增殖並使細胞停滯於細胞週期之S期，（ii）麩胺酸鹽抑制雄鼠星狀細胞之CaM kinase II及PKC的活性較雌鼠顯著，（iii）麩胺酸鹽促使雄鼠星狀細胞產生的一氧化氮及NOS的表現皆較雌鼠多。因此星狀細胞之數量多寡亦可能修飾麩胺酸鹽造成視前區的性別差異性神經毒性。
雄鼠視前區中的SDN-POA約為雌鼠的三至七倍。一般認為雄鼠視前區中SDN-POA體積大於雌鼠乃因出生前後的分化關鍵期中，僅雄鼠暴露於睪固酮（testosterone）高峰的環境，而睪固酮可保護神經細胞免於出生第七至十天自然發生的細胞凋亡（natural occurring neuronal apoptosis），故雄鼠可維持其核區中的細胞數，而雌鼠的神經細胞數則明顯減少。在出生前後雄鼠的視前區中所含的NMDA 受器較雌鼠多，且NMDA受器的活化為神經細胞存活所必須，因此雄鼠視前區中含量較高的NMDA受器可能於SDN-POA的性別分化中扮演重要角色。故本論文在第二部份探討NMDA受器在視前區性別分化的過程中所扮演的生理性角色。實驗分為兩單元，第一單元取雄性素雄性化（androgen sterilized, ASR）之雌胎鼠模擬內源性睪固酮高峰或出生前承受壓力（prenatal stress, PNS）之雄胎鼠為消除內源性睪固酮高峰，取其視前區定量NMDA受器之mRNA及蛋白含量，以探討關鍵期睪固酮高峰與雄鼠高NMDA受器表現之關聯。進一步利用含有NMDA受器及雌二醇受器之GT1-7細胞（immortalized GnRH-secreting neurons）分別培養於睪固酮（可環化）、二氫睪固酮（不可環化）及雌二醇的情況下，定量NMDA受器之mRNA及蛋白含量及計數細胞存活率，以探討睪固酮是否需環化成雌二醇方可調節NMDA受器之表現，及細胞存活。並經MK-801（NMDA受器之非競爭拮抗劑）處理後計數細胞存活率，以瞭解性類固醇促進細胞的存活是否經由NMDA受器的活化。實驗結果證實（a）ASR雌鼠NMDA受器的蛋白含量接近控制雄鼠組，而PNS雄鼠NMDA受器的蛋白含量接近控制雌鼠組，（b）雄鼠、ASR雌鼠、PNS雄鼠及雌鼠之mRNA表現並無顯著差異(c) 僅有雌二醇可調節NMDA受器的表現及促進細胞存活，（d）雌二醇促進細胞存活的作用則受到MK-801所阻斷。以上結果顯示大鼠胚胎晚期的睪固酮環化成雌二醇後確實可經由調節NMDA受器於轉錄後的表現(post-transcriptional modification)而促進細胞存活。在第二單元的實驗中，於胚胎第十七天經母鼠投與MK-801，於胚胎第十八天取胎鼠血清，偵測血清中睪固酮的濃度，以觀察NMDA受器受阻斷後對血清中睪固酮的濃度的影響。此外，並測定神經細胞凋亡及Bcl-2家族蛋白的含量以探究NMDA受器與視前區性別分化時之細胞凋亡的關係。實驗結果顯示，於胚胎第十七天投與MK-801（NMDA受器之拮抗劑）可阻斷睪固酮高峰的產生。此外MK-801亦可減少雄鼠視前區Bcl-2的表現及增加細胞凋亡的現象。因此NMDA受器表現較多可能經由調控睪固酮高峰或Bcl-2家族蛋白的表現來保護視前區的神經免於凋亡。
整體而言，雄鼠視前區NMDA受器的表現較多、經由過度引發細胞內鈣離子上升的現象，可能是雄鼠對麩胺酸鹽神經毒性的敏感度較雌鼠為高的主因。更重要的在生理功能上NMDA受器可能與出生前睪固酮高峰之引發密切相關，且對於保護視前區神經細胞免於自然發生的神經凋亡扮演重要角色。

The preoptic area (POA) is involved in regulation of reproductive function, such as GnRH release and copulatory behavior. Glutamate plays an important role in GnRH secretion, through acting on N-methyl-D-aspartate (NMDA) receptor in the POA. However, a large dose of glutamate is neurotoxic. Previous result indicated that neonatal male rats show a higher susceptibility to glutamate-induced reproductive dysfunction than that of females. Thus, the pharmacological role of NMDA receptor in the POA was studied during sexual development. First, glutamate-induced morphological changes of POA were estimated using the image analytic system. Then, the relationships between the NMDA receptor and sex-specific neurotoxicity of glutamate on the POA of neonatal rats were investigated. The NMDA receptor was semiquantified by western blot analysis. The kinetic changes of intracellular Ca2+ and LDH efflux were monitored as rapid and delayed toxic signals, respectively. The results showed that (1) Only the SDN-POA of male rats showed significant increase of pyknosis and decrease of neuronal density after neonatal monosodium glutamate treatment. (2) The protein expression of NMDA receptor in POA of male rat is higher than that of female at both postnatal day 1 and 3. (3) After glutamate-treatment, the elevations of intracellular Ca2+ and LDH efflux in neurons derived form male rat were more significant than those of females, respectively. These results suggested that the quantitative difference of NMDA receptor between male and female rats might contribute to the glutamate-induced sex-specific reproductive dysfunction.
Additionally, the antiproliferative effect of glutamate on astrocytes was measured by thymidine incorporation, cell count and cell cycle analysis. The activities of CaM kinase II and PKC were assayed and the NO release and expression of NO synthase (NOS) after glutamate treatment were also estimated. The results showed that (i) Glutamate inhibited the proliferation of astrocytes derived from male rats and (ii) The cell cycle progression of male astrocyte was accumulated in S phase. (iii) The activities of CaM kinase II and PKC were markedly declined more, while the release of NO and expression of NO synthase (NOS) were more significantly increased in astrocytes derived from male rats than those from females after glutamate treatment. It seemed that the amount of astrocytes after glutamate-treatment might modulate the sex-specific neurotoxicity of glutamate on the POA.
The sexual dimorphic nucleus of preoptic area (SDN-POA) of male rats exhibits seven-fold greater nucleus volume than that of females. During sexual development, only male rats showed prenatal testosterone peak. The natural occurring neuronal apoptosis, which can be prevented by testosterone, in SDN-POA of female rats during postnatal day 7-10 may contribute to the sexual difference of SDN-POA. Since the expression of NMDA receptor in POA of male rat was higher than that in females and activation of NMDA receptor was essential for the neuronal survival, the high expression of NMDA receptor in POA of male rat might play an important role in the sexual development of SDN-POA. Thus, two parts of experiments were designed to study the physiological role of NMDA receptor in POA during development.
In the first part of the experiment, the testosterone milieu was simulated in androgen sterilized (ASR) female fetus or diminished by prenatal stressed (PNS) in male fetus. The expressions of NMDAR mRNA and protein in POA were quantified to study the relationship between prenatal testosterone peak and expression of NMDAR. The immortalized GnRH-secreting neurons (GT1-7 cell) which express steroid and NMDA receptors were cultured with testosterone (aromatizable), DHT (unaromatizable), or estradiol to understand whether the testosterone or estradiol modulates the expression of NMDA receptor. Furthermore, the cell viability was also counted by cytometry when GT1-7 cells were co-cultured with or without MK-801 (non-competitive NMDA receptor antagonist) and sex-steroid. The results showed that (a) The protein expression of NMDA receptor in POA of ASR female rats was similar to that of intact male rats, whereas the expression of NMDA receptor in POA of PNS male rats was closed to that of intact females. (b) There was no significant difference of mRNA expression among intact males, ASR female, PNS male, and intact female. (c) Only estradiol increased the NMDA receptor expression and increased the cell viability in GT1-7 cells. (d) The enhance effect of estradiol on cell viability was blocked by MK-801 co-cultured. These results suggested that prenatal testosterone, after being converted into estradiol, increased the cell viability through modulating NMDA receptor expression at the post-transcriptional level.
In the second part of the experiment, the male fetuses were pretreated with MK-801 to block the NMDA receptor at embryonic day (ED) 17. Serum level of testosterone was estimated at ED18 to observe the effect of NMDA receptor blockage on the serum testosterone level. Moreover, the neuronal apoptosis and expression of Bcl-2 family proteins in POA were estimated to find out the relationship between the NMDA receptor activation and the natural occurring neuronal death. The results showed that the level of testosterone was lower in MK-801 pretreated male rats than untreated males. The expression of Bcl-2 in POA of male rats was significantly decreased, while apoptotic bodies were increased after MK-801 pretreated. It suggested that the NMDA receptor highly expressed in male rats might protect POA neurons from apoptosis by modulating prenatal testosterone level and/or Bcl-2 protein expression.
In conclusion, the sex-specific expression of NMDA receptor in POA between male and female rats might contribute to the glutamate-induced sex-specific neurotoxicity. Importantly, the NMDA receptor highly expressed in the POA of male rat might be necessary for the elevation of prenatal testosterone level and play an important role in the protection of neurons from the natural occurring cell death during sexual development.

封面
目錄
第一章、總緒論
第二章、研究動機及研究設計
第一部分、NMDA受器的藥理性角色探討
第三章、初生大花鼠經過麩胺酸鹽處理後對其成熟後之下視丘內側視前區神經受損之探討
一、摘要
二、緒論
三、材料與方法
四、結果
五、討論
第四章、麩胺酸鹽造成視前區性別差異性神經毒性機制之探討
一、摘要
二、緒論
三、材料與方法
四、結果
五、討論
第五章、星狀細胞在麩胺酸鹽造成大花鼠視前區性別差異性神經毒性中之角色探討
第一節、麩胺酸鹽抑制視前區星狀細胞增殖之性別差異
第二節、麩胺酸鹽引發視前區星狀細胞釋出一氧化氮之性別差異
第二部分、NMDA受器的生理性角色探討 : 雄鼠視前區高 N-甲基-D-天門冬胺酸鹽受器與性別分化關鍵期睪固酮高峰之關係
第六章、雄鼠視前區高 N-甲基-D-天門冬胺酸鹽受器與性別分化關鍵期睪固酮高峰之關係 : 關鍵期睪固酮高峰改變之動物
一、摘要
二、緒論
三、材料與方法
四、結果
五、討論
第七章、雄鼠視前區高 N-甲基-D-天門冬胺酸鹽受器與性別分化關鍵期睪固酮高峰之關係 : N-甲基-D-天門冬胺酸鹽受器拮抗劑處理之動物
一、摘要
二、緒論
三、材料與方法
四、結果
五、討論
第八章、總結論
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