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研究生:翁筱晶
研究生(外文):Hsiao-Ching Weng
論文名稱:磷酸比哆醛對過氧化氫誘發人類精子損傷之研究
論文名稱(外文):Study on the Pyridoxal-5-phosphate in Human Spermatozoa Damage Induced by Hydrogen Peroxide
指導教授:張素瓊張素瓊引用關係
指導教授(外文):Sue-Joan Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:153
中文關鍵詞:磷酸比哆醛反應性氧化物質精子功能
外文關鍵詞:spermatozoa functionreactive oxygen speciespyridoxal-5-phosphate
相關次數:
  • 被引用被引用:2
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  • 收藏至我的研究室書目清單書目收藏:0
摘 要
調查顯示,不孕症患者中約有40~50 %為男性因素所造成,而男性精子功能損傷是造成男性不孕症的主要原因之一。近年研究指出,反應性氧化物質(reactive oxygen species, ROS)會誘導精子功能損傷。ROS所造成的精子氧化傷害包括精子頂體膜脂質過氧化而失去膜完整性,粒線體基質與膜氧化而使ATP生成量降低,染色質氧化而使DNA斷裂或去濃縮,因而導致精子活動力及對卵穿透能力下降。因此,降低精子ROS可改善精子功能受氧化壓力損害所導致的男性不孕症。
本研究室近年對細胞訊息的研究顯示,磷酸口比哆醛(pyridoxal-5-phosphate; PLP)可抑制胞內鈣離子濃度增加,維持細胞完整性。此外,PLP是合成cysteine(參與抗氧化系統-glutathione合成)之重要的輔因子。因此,本研究針對PLP的抗氧化性與細胞訊息傳導的角色,對H2O2誘發ROS的生成,及其對精子的功能,探討:(一)精子活動力及其影響因子:利用冷光儀偵測精子ATP濃度、以流式細胞儀分析精子胞內ROS濃度與粒腺體膜電位(mitochondrial membrane potential; MMP)、電腦輔助精子分析系統偵測精子活動速度。(二)精子對卵穿透能力的影響因子:利用流式細胞儀分析精子胞內Ca2+濃度、精子染色質結構與頂體反應。
PLP對H2O2誘發下精子活動能力的結果顯示,隨H2O2處理濃度與時間增加會導致精子粒腺體膜電位與活動速度下降。經PLP再以H2O2處理75及105分鐘之精子粒線體膜電位比不以PLP只以2與20 mM H2O2處理者高。PLP可抑制不同濃度H2O2所導致精子活動速度因子的下降。500 nM PLP可顯著緩和20 mM H2O2處理105分鐘時所導致的精子ATP生成量下降。
PLP對H2O2誘發下精子對卵穿透能力的結果顯示,隨H2O2處理濃度與時間增加會導致精子胞內鈣離子濃度提升,且會顯著使精子頂體反應與染色質變性增加。經PLP處理之精子胞內鈣離子濃度、DNA變性細胞比例(COMPαT)與DNA變性值(αT value)皆比以0.2 mM H2O2處理105分鐘者低。然而,PLP對不同濃度與不同時間處理之H2O2所導致的精子頂體反應無顯著影響。
PLP對H2O2誘發精子胞內ROS之結果顯示,PLP可抑制不同濃度H2O2在不同處理時間所誘發的精子胞內ROS濃度。
本研究結果亦發現,500 nM PLP對H2O2導致精子功能損傷之抗氧化效果與生理濃度維生素C(1.5 mg/dl)類似。高粒腺體膜電位精子百分比與胞內ROS濃度呈顯著負相關(r = -0.726 , P<0.01),而與精子活動力(r = 0.613, P<0.01)和前進速度佳之精子百分比呈顯著正相關(r = 0.617, P<0.01)。顯示精子胞內ROS過高會誘發精子MMP下降,導致精子活動能力降低。
精子頂體反應細胞比例與受精率呈顯著負相關(r = - 0.5, P<0.05),精子頂體反應細胞比例與其胞內鈣離子濃度呈正相關(r = 0.47, P=0.076),但未有統計顯著。精子胞內鈣離子濃度(r = - 0.64, P<0.01),DNA變性值(αT value)(r = - 0.545, P<0.01)及DNA變性細胞比例(COMPαT)(r = - 0.649, P<0.01)皆與受精率呈顯著負相關。顯示精子本身胞內鈣離子濃度過高、染色質結構不正常與早發性頂體反應皆會導致精子對卵穿透能力下降。
本研究發現,不同濃度與不同處理時間的H2O2會導致不同程度的精子功能損害。0.2、2與20 mM H2O2處理5分鐘後即可使精子胞內鈣離子濃度增加;30分鐘後可誘發精子頂體反應;105分鐘可使精子染色質結構變性。0.2 mM H2O2處理105分鐘對精子粒腺體膜電位無顯著影響,2與20 mM H2O2分別在75及30分鐘時可顯著使精子MMP下降。因此,低濃度H2O2需長時間處理(105分鐘以上)與高濃度H2O2需短時間(30分鐘),可使精子粒腺體膜電位下降。
本研究結論,生理濃度之PLP可保護不同劑量與不同處理時間之H2O2誘發ROS所導致的精子功能損傷。本研究建議PLP可能可用於臨床上改善氧化壓力所導致的男性不孕症。
關鍵字:精子功能、反應性氧化物質、磷酸口比 哆醛
Abstract
It has been estimated that up to 40~50 % of infertility cases are caused exclusively by a male factor. Spermatozoa damage is thought to be the major cause of male infertility. Recent studies have indicated that damage to spermatozoa functions are induced by reactive oxygen species (ROS). The ROS-induced spermatozoa damage, via the lipid peroxidation of acrosome membrane leading to the loss of membrane integrity, oxidation of both mitochondria matrix and membrane leading to a decreased ATP production and oxidation of chromatin leading to DNA strand breaks or decondensation, results in a loss of spermatozoa motility and a decreased capacity of sperm-oocyte penetration. Therefore, it appears that male infertility can be improved by decreasing spermatozoa ROS to reduce spermatozoa damage.
Previous studies in our laboratory have shown that pyridoxal- 5-phosphate (PLP) decreases the intracellular calcium concentration and maintains the cellular integrity. In addition, PLP acts as cofactor in the synthesis of cysteine which participates in the glutathione status, an endogenous antioxidant. Therefore, the objective of this study was to investigate the anti-oxidative and signaling roles of PLP on the human spermatozoa function under excess ROS induced by varying concentrations of H2O2. The spermatozoa functions studied included spermatozoa motility and sperm-oocyte penetration capacity. Motility related factors including ATP production measured by luminometer, mitochondrial membrane potential (MMP) measured by flow cytometry and spermatozoa velocity determined by computer-assisted spermatozoa analysis (CASA) were also investigated. Sperm-oocyte related factors including spermatozoa 【Ca2+】i, spermatozoa chromatin structure assay (SCSA) and acrosome reaction (AR), all determined by flow cytometry were also studied.
The results of PLP treatments on spermatozoa motility induced by H2O2 showed that MMP and velocity of spermatozoa were decreased with increasing incubation time and concentrations of H2O2. Spermatozoa pretreated with PLP and H2O2 had higher MMP than spermatozoa treated with H2O2 alone (at concentrations of 2 and 20 mM of H2O2) incubated for 75 and 105 min. PLP inhibited the reduction of spermatozoa velocity induced by different levels of H2O2. 500 nM of PLP significantly alleviated the decreased production of ATP in spermatozoa induced by 20 mM H2O2 incubated for 105 min.
The results of PLP on H2O2-induced sperm-oocyte penetration showed that 【Ca2+】i in spermatozoa was increased with increasing incubation time and concentrations of H2O2. Additions of H2O2 significantly increased the AR and denatured chromatin in spermatozoa. Spermatozoa pretreated with PLP and H2O2 had lower 【Ca2+】i, COMPαT (denatured DNA cells) andαT values (DNA denaturation) than that pretreated with only 0.2 mM H2O2 incubated for 105 min. However, PLP did not significantly affect the AR of spermatozoa treated with different levels of H2O2 for different durations.
The results of PLP additions on H2O2-induced spermatozoa ROS status showed that PLP significantly inhibited the intracellular ROS concentrations of spermatozoa treated with different levels of H2O2 for different incubation durations.
In addition, the anti-oxidative effect of 500 nM PLP on spermatozoa functions damaged by H2O2 additions was found to be similar to that of vitamin C at a concentration of 1.5 mg/dl. The percentage of high MMP spermatozoa was negatively correlated to the intracellular ROS levels (r = - 0.726, P<0.01) and positively correlated to spermatozoa motility (r = 0.613, P<0.01) and the percentage of good progressive motile spermatozoa (r = 0.617, P<0.01). The results indicated that high intracellular ROS led to a decrease of spermatozoa MMP resulting in a decreased spermatozoa motility.
The percentage of AR spermatozoa was negatively correlated to the fertilization rate (r = - 0.5, P<0.05). No significant relationship between the percentage of AR spermatozoa and basal 【Ca2+】i (r = 0.47, P=0.076) was found. The fertilization rate was negatively correlated to basal【Ca2+】i (r = - 0.64, P<0.01), the αT values (r = - 0.545, P<0.01) and COMPαT (r = - 0.649, P<0.01). These results indicated that a decreased capacity in the sperm-oocyte penetration of spermatozoa was resulted from high basal 【Ca2+】I levels, abnormal chromatin structure and premature AR.
Our study indicated that different concentrations and incubation time of H2O2 caused different degrees of spermatozoa damage. Treatment using 0.2, 2 and 20 mM H2O2 for 5 min led to an increase of 【Ca2+】i. At 30 min, AR was induced, and at 105 min, denaturation of the spermatozoa chromatin structure resulted. MMP of spermatozoa was not significantly affected by 0.2 mM H2O2 addition incubated up to 105 min. MMP of spermatozoa treated with 2 and 20 mM H2O2 for 75 and 30 min, respectively, were significantly decreased. Therefore, low concentrations of H2O2 needed long-term incubation (over 105 min) and high concentrations of H2O2 needed short-term incubation (30 min) to decrease the MMP of spermatozoa.
In conclusion, the physiological concentrations of PLP prevented spermatozoa damage against ROS induced by various concentrations of H2O2 and incubated for different durations. We suggested that PLP could be use clinically for male infertility resulting from oxidative stress.

Key words: spermatozoa function, reactive oxygen species, pyridoxal-5-phosphate
目 錄
中文摘要 Ⅰ
英文摘要 Ⅳ
誌謝 Ⅷ
目錄 Ⅸ
表目錄 Ⅹ
圖目錄 ⅩⅠ
第一章:前言 1
第二章:文獻探討 4
第三章:研究材料及方法 18
第四章:結果 40
第五章:討論 56
第六章:參考文獻 69
表 83
圖 89
自述 153
參考文獻
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