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(Ⅰ)過氧化氫的暴露影響大鼠精子染色質DNA完整性之研究
摘要
反應性氧化物質被認為是損害精子的重要物質。體內過多反應性氧化物質的產生與人類精子的異常有關。DNA的破碎與脂質過氧化已知與精子暴露於反應性氧化物質有關。本研究的研究目的是在評估過氧化氫的暴露對大鼠精子染色質DNA完整性之影響。
研究方法，是取雄性SD鼠的精子並暴露在不同濃度的過氧化氫與溫度中，其暴露的最終濃度分別為0, 20uM, 1mM, 50mM, 500mM和1000mM，暴露溫度分別為370C與500C。精子染色質DNA的分析均是以AO染劑進行螢光染色，AO為一具有核酸結合專一特性之螢光染劑，當精子染色質DNA是正常的雙股螺旋，AO染劑與雙股螺旋結合呈現綠色螢光；若染色質DNA損壞裸露出單股DNA，AO染劑與單股螺旋結合呈現紅色螢光，再利用流式細胞儀做精子染色質結構分析以瞭解樣本間的變化。
研究結果發現，隨著過氧化氫暴露濃度的增加，大鼠精子染色質DNA異常的百分比呈現增加趨勢，且在溫度較高的情況下會特別顯著。本研究所得到的結論為，在體外試驗研究中我們證實過氧化氫的暴露會對大鼠精子染色質DNA造成損害，此損害呈現劑量關係，在溫度較高的情況下會特別顯著。

(Ⅱ)維生素C保護過氧化氫引起人類精子染色質DNA損害之研究
摘要
反應性氧化物質被認為是損害精子的重要物質。體內過多反應性氧化物質的產生與人類精子的異常有關。DNA的破碎與脂質過氧化已知與精子暴露於反應性氧化物質有關，飲食中維生素C的補充已被發現能減少人類精子染色質DNA的氧化傷害。本研究的研究目的是在評估人類精子與過氧化氫暴露前先以抗氧化劑維生素C處理是否能保護過氧化氫引起的人類精子染色質DNA之損害。
研究方法，是取8位年齡20-30歲的健康男性之精子，實驗分成兩大組，一組精子樣本不事先處理，另一組精子樣本先以26 mM的維生素C培養30分鐘後，之後各大組中的精子分別暴露在不同濃度的過氧化氫，使最終濃度分別為0mM, 20mM, 2mM, 和200mM。精子染色質DNA的分析均是以AO染劑進行螢光染色，AO為一具有核酸結合專一特性之螢光染劑，當精子染色質DNA是正常的雙股螺旋，AO染劑與雙股螺旋結合呈現綠色螢光；若染色質DNA損壞裸露出單股DNA，AO染劑與單股螺旋結合呈現紅色螢光，再利用流式細胞儀做精子染色質結構分析以瞭解樣本間的變化。
研究結果發現，先以26mM的維生素C處理後的人類精子，在過氧化氫暴露濃度為200 mM時，對精子染色質DNA有顯著的保護作用。
本研究所得到的結論為，在體外試驗研究中我們證實預先外加抗氧化劑維生素C能減少因過氧化氫暴露所誘導的精子DNA損害。

(Ⅰ)Effects of Hydrogen Peroxide Exposure on Chromatin DNA Integrity In Rats Spermatozoa
ABSTRACT
Reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2) have been found to have advert effects on human and mammalian spermatozoa. ROS may affect DNA integrity of human spermatozoa. The aim of this study was to evaluate the effect of hydrogen peroxide (H2O2) exposure on rats sperm chromatin DNA integrity.
Rat spermatozoa were incubated with H2O2 at the final concentration of 0, 20mM, 1, 50, 500, and 1000mM. Sperm were incubation at 370C and 500C. Sperm DNA integrity was assessed by the sperm chromatin structure assay using flow cytometry with acridine orange (AO) staining to follow changes in DNA packaging.
Generation of highly reactive oxygen species (ROS) such as superoxide radicals (O2.-), hydrogen peroxide (H2O2), and hydroxyl radicals (.OH) is known to damage various cellular components including proteins, membrane lipids and nucleic acids. In rets study, the percentage of chromatin DNA abnormality was following the increase of H2O2 concentrations, respectively, especially in high temperature.
In conclusion, in vitro rats sperm incubation with H2O2 induced the damage of DNA integrity in a dose-response fashion, especially in high temperature.

(Ⅱ)Protection of Ascorbic Acid on Hydrogen Peroxide-induced Human Sperm DNA Damage
ABSTRACT
The aim of this study was to evaluate the effect of hydrogen peroxide (H2O2) exposure on human sperm chromatin DNA integrity and the beneficial role of antioxidant nutrients, ascorbic acid in these adverse effects.
Spermatozoa samples were pretreated with ascorbic acid at the final concentration of 0 mM and 26 mM. Then spermatozoa sample were separated and incubated with H2O2 at the final concentration of 0, 20 mM, 2 mM, and 200 mM. Sperm DNA integrity was assessed by flow cytometry with acridine orange (AO) staining to follow changes in DNA packaging. The extent of DNA denaturation per cell is quantified as alpha-t (aT), which is express as a ratio or percent of the quantify of [red/(green +red)] fluorescence and the percentage of sperm falling outside of the main population (COMP) representing the percentage of cells with increased sensitivity to denaturation compared with the normal cells in a sample.
Generation of highly reactive oxygen species (ROS) such as superoxide radicals (O2.-), hydrogen peroxide (H2O2), and hydroxyl radicals (.OH) is known to damage various cellular components including proteins, membrane lipids and nucleic acids. Dietary ascorbic acid supplementation might produce a decrease in human sperm oxidative DNA damage. In non-ascorbic acid treated groups, the fractions of spermatozoa with abnormal chromatin (COMP at) were 7.24%, 7.29%, 23.01%, and 26.78% (P<0.001) following the increase of H2O2 concentrations, respectively. Pretreatment of 26mM ascorbic acid before exposure of H2O2, the percentage of DNA damage were 6.72%, 6.2%, 11.6%, and 12.75% (P=0.0844) following the increase of H2O2 concentrations, respectively.
We demonstrated that pretreatment of ascorbic acid significantly decrease the hydrogen peroxide-induced sperm DNA damage in vitro.

中文摘要Ⅰ---------------------------------------------------------------------------i
中文摘要Ⅱ--------------------------------------------------------------------------ii
英文摘要Ⅰ-------------------------------------------------------------------------iv
英文摘要Ⅱ-------------------------------------------------------------------------vi
誌謝---------------------------------------------------------------------------------viii
目錄----------------------------------------------------------------------------------ix
表目錄------------------------------------------------------------------------------xiii圖目錄-----------------------------------------------------------------------------xiv
壹、緒論----------------------------------------------------------------------------1
一、 前 言------------------------------------------------------------------------1
二、 研究方向 ------------------------------------------------------------------2
貳、文獻探討------------------------------------------------------------------------4
一、 雄性生殖系統簡介-----------------------------------------------------4
(一) 雄性生殖機能 -------------------------------------------------------4
(二) 精子生成之步驟------------------------------------------------------4
(三) 精液---------------------------------------------------------------------6
二、DNA ------------------------------------------------------------------------7
(一)DNA在生物學上的重要性----------------------------------------7
(二)DNA的基本結構----------------------------------------------------8
(三) DNA的物理及化學構造-------------------------------------------9
三、反應性氧化物質----------------------------------------------------------9
(一) 反應性氧化物質之簡介-------------------------------------------9
(二) 生物體內反應性氧化物質的生成與生理作用----------------10
(三) 反應性氧化物質對精子的影響----------------------------------11
四、 維生素C------------------------------------------------------------------13
(一) 維生素C在生理上的功能-----------------------------------------13
(二) 維生素C的抗氧化特性--------------------------------------------13
五、流式細胞儀 -------------------------------------------------------------14
(一) 流式細胞儀的工作原理-------------------------------------------15
(二) 流式細胞儀的分析原理-------------------------------------------16
(三)流式細胞儀各系統功能簡介------------------------------------18
六、流式細胞儀精子染色質結構分析-----------------------------------23

參、材料與方法--------------------------------------------------------------------25
一、過氧化氫的暴露影響大鼠精子染色質DNA完整性之研究------------------------------------------------------------------------25
(一) 研究設計---------------------------------------------------------------25
(二) 動物來源---------------------------------------------------------------26
(三) 大鼠精子的前處理---------------------------------------------------26
(四) 反應性氧化物質( ROS)的偵測-------------------------------------28
(五) 流式細胞儀精子染色質結構分析---------------------------------32
二、維生素C保護過氧化氫引起人類精子染色質DNA損害之研究------------------------------------------------------------------------42
(一) 研究設計---------------------------------------------------------------42
(二) 人類精子樣本的來源------------------------------------------------42
(三) 人類精子樣本的前處理---------------------------------------------43
(四) 流式細胞儀精子染色質結構分析---------------------------------44
肆、 結果與討論------------------------------------------------------------------54
一 、 過氧化氫的暴露影響大鼠精子染色質DNA完整性之研究------------------------------------------------------------------------54
(一) 流式細胞儀精子染色質結構分析所得的典型圖---------------54
(二) JMP統計分析所得的結果-------------------------------------------58
(三) COMP aT函數與aT值之相關性-----------------------------------69
(四) 過氧化氫的暴露影響大鼠精子染色質DNA完整性之討論------------------------------------------------------------------------69
二、 維生素C保護過氧化氫引起人類精子染色質DNA
損害之研究----------------------------------------------------------71
(一) 精子濃度與活動力的觀察------------------------------------------71
(二) 流式細胞儀精子染色質結構分析所得的典型圖形------------ 71
(三) JMP統計分析所得的結果-------------------------------------------76
(四) COMP aT函數與aT值之相關性-----------------------------------84
(五) 暴露過氧化氫引起人類精子染色質DNA損害之討論---------84
(六) 維生素C保護暴露過氧化氫引起人類精子染色質DNA損
害之討論----------------------------------------------------------------85
伍、 結論---------------------------------------------------------------------------86
參考文獻----------------------------------------------------------------------------87




表目錄
表2-1 反應性氧化物質對精液參數和男性不孕之影響-------------------12
表3-1 以Evenson et al. 在1995年的研究為例，來比較由List-View軟體所分析的aT值與人工計算所得的aT值之間的關係------------40
表4-1 反應性氧化物質(ROS)的含量-----------------------------------------59
表4-2 370C條件下過氧化氫的暴露對大鼠精子染色質影響之偵測，流式細胞儀精子染色質結構分析-------------------------------------62
表4-3 500C條件下過氧化氫的暴露對大鼠精子染色質影響之偵測，流式細胞儀精子染色質結構分析------------------------------------63
表4-4 8位自願捐贈者的新鮮精子樣品以顯微鏡觀察精子的濃度與活動力，按編號如所示------------------------------------------------72
表4-5 A組不加入維生素C保護，並以不同濃度的過氧化氫暴露所得的結果---------------------------------------------------------------78
表4-6 B組以最終暴露濃度26mM的維生素C暴露30分鐘，並以不同濃度的過氧化氫暴露所得的結果------------------------------78




圖目錄
圖2-1 大鼠精子的構造圖--------------------------------------------------------5
圖2-2 人類精子的構造圖--------------------------------------------------------6
圖2-3 核苷酸的概要結構--------------------------------------------------------8
圖2-4 流式細胞儀的工作原理-------------------------------------------------15
圖2-5 樣品的進流與偵測-------------------------------------------------------16
圖2-6 信號的收集與濾片的配置----------------------------------------------17
圖2-7 液流系統-------------------------------------------------------------------19
圖2-8 分選系統-------------------------------------------------------------------22
圖3-1 大鼠精子前處理流程圖-------------------------------------------------29
圖3-2 反應性氧化物質( ROS)的偵測----------------------------------------31
圖3-3 流式細胞儀精子染色質結構分析-------------------------------------35
圖3-4 綠色螢光與紅色螢光強度關係圖-------------------------------------37
圖3-5 細胞顆粒大小與細胞複雜度的關係圖-------------------------------37
圖3-6 COMP aT函數的定義---------------------------------------------------38
圖3-7 紅色螢光強度圖----------------------------------------------------------39
圖3-8 綠色螢光強度圖----------------------------------------------------------39
圖3-9 流式細胞儀精子染色質結構分析-------------------------------------48
圖3-10 綠色螢光與紅色螢光強度關係圖-------------- ---------------------49
圖3-11 細胞顆粒大小與細胞複雜度的關係圖-----------------------------49
圖3-12 COMP aT函數的定義-----------------------------------------------51
圖3-13 紅色螢光強度圖--------------------------------------------------------51
圖3-14 綠色螢光強度圖--------------------------------------------------------52
圖4-1 流式細胞儀精子染色質結構分析所得紅色螢光強度與綠色螢光強度關係的典型圖形---------------------------------------------------55
圖4-2 流式細胞儀精子染色質結構分析所得的典型綠色螢光強度圖形------------------------------------------------------------------------------56
圖4-3 流式細胞儀精子染色質結構分析所得的典型紅色螢光強圖形------------------------------------------------------------------------------57
圖4-4 過氧化氫暴露濃度與ROS關係圖------------------------------------60
圖4-5 過氧化氫暴露濃度與COMPaT函數關係圖------------------------64
圖4-6 過氧化氫暴露濃度與綠色螢光強度值關係圖----------------------64
圖4-7 過氧化氫暴露濃度與紅色螢光強度值關係圖 --------------------65
圖4-8 過氧化氫暴露濃度與aT值關係圖------------------------------------65
圖4-9 COMP aT函數與aT值之相關性------------------------------------69
圖4-10 在不同條件下流式細胞儀精子染色質結構分析所得紅色螢光強度與綠色螢光強度關係的典型圖形------------------------------73
圖4-11 不同條件下流式細胞儀精子染色質結構分析所得紅色螢光強度的典型圖-------------------------------------------------------------74
圖4-12 不同條件下流式細胞儀精子染色質結構分析所得綠色螢光強度的典型圖形----------------------------------------------------------75
圖4-13 過氧化氫暴露濃度與COMPaT函數關係圖---------------------79
圖4-14 過氧化氫暴露濃度與紅色螢光強度值關係圖-------------------79
圖4-15 過氧化氫暴露濃度與綠色螢光強度值關係圖-------------------80
圖4-16 過氧化氫暴露濃度與aT值關係圖----------------------------------80
圖4-17過氧化氫暴露濃度與精子活動力關係圖--------------------------81
圖4-18 COMP aT函數與aT值之相關性----------------------------------83

1.李權益，1998，分子生物學，合記圖書出版。2.林建城，2001，氧化壓力分析培訓講義，美商必帝股份有限公司台灣分公司。3.許昺奇，1998，鉛暴露引起大鼠精子產生反應性氧化物質並影響精子功能，國立成功大學，博士論文。4.Agarwal, A., Ikemoto, I., Loughlin, K. R., 1994. Relationship of sperm parameters with levels of reactive oxygen species in semen specimens. J. Urol. 152, 107-110. 5.Aitken, R.J., 1995. Free radicals, Lipid peroxidation and sperm function. Reprod. Fertil. 7, 657-668.6.Aitken, R.J., Fisher, H., 1994. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioassays. 16, 259-267.7.Aitken, R.J., Clarkson, J.S., 1988. Significance of reactive oxygen species and antioxidants in defining the dfficiency of sperm preparation techniques. J. Androl. 9, 367-3768.Alvarez, J.G., Storey, B.T., 1989. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res. 23, 77-90.9.Armand Zini, M.D., Ryszard Bielecki, D. V. M., Donna Phang, M. Sc., Maria Teresa Zenzes, 2001. Correlation between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertility and Sterility. 75, 674-677.10.Auger, J., Kunstmann, J.M., Czyblik, F., Jouannet, P., 1995. Decline in semen quality among fertile men in Paris during the past 20 years. N. Engl. J. Med. 332, 281-285.11.Aurich, J.E., Schonherr, U., Hoppe, H., Aurich, C., 1997. Effect of antioxidants on motility and membrane integrity of chilled-stored stallion semen. Theriogenology. 48, 185-192.12.Becton Dickinson, 1996. FACSCalibur system users guide. pp. 1-271.13.Carten, W.O., Robinson, J.P, 1994. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J. Leukocyte Biol. 55, 253-258.14.Chen, J., He, J., Ogden, LG., Batuman, V., Wheltin, PK., 2002. Relationship of serum antioxidant vitamins to serum creatinine in the US population. American Journal of Kidney Diseases. 39, 460-468.15.Chandley, A.C., Kofman-Alfaro, S., 1971. Unscheduled DNA synthesis in human germ cells following UV irradiation. Exp. Cell Res. 69, 45-48.16.Chen, C.S., Chao, H. T., Pan, R. L., Wei, Y. H., 1997. Hydroxyl radical-induced decline in motility and increase in lipid peroxidation and DNA modification in human sperm. Biochem. Mol. Biol. Int. 43, 291-303.17.Donnelly, E.T., Mcclure, N., Lewis, S.E.M., 1999. The effect of ascorbate and a-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa. Mutagenesis.14, 505-511.18.Esterbauer, H., Striegl, G., Puhl, H., Rotheneder, M., 1989.Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radical Res. Com. 6, 67-75.19.Evenson, D. P., Darzynkiewicz, Z., Melamed, M. R., 1980. Relation of Mammalian Sperm Chromatin Heterogeneity to Fertility. Science. Vol. 210, 1131~1133.20.Evenson, D.P., Higgins, P.J, Grueneberg, D., Ballachey, B.E., 1985. Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea. Cytometry. 238-253.21.Evenson, D.P., Jost, L., Gandour, D., Rhodes, L., Stanton, B., Clausen, O.P., Angelis, P.D., Coico, R., Daley, A., Becker, K., Yopp, T., 1995. Comparative sperm chromatin structure assay measurements on epiillumination and orthogonal axes flow cytometers. Cytometry. 295-303. 22.Fraga, C.G., Motchnik, P.A., Shigenaga, H.J., Helbock, R.A., Jacob, R.A., Ames, B.N., 1991. Ascorbic acid protecs against endogenous oxidative DNA damage in human sperm. Proc. Natl. Acad. Sci. 88, 11003-11006.23.Hoshi, K., Katayose, H., Yanagida, K., 1996. The relationship between acridine orange fluorescence of sperm nuclei and the fertilizing ability of human sperm. Fertility and Sterility. 66, 634-639.24.Hughes, C.M., Lewis, S.E.M., Mckelvey-Martin, V.J., Thompson, W., 1997. Reproducibility of human sperm DNA measurement using the alkaline single cell gel electrophoresis assay. Mutat. Res. 374, 261~268.25.Langlois, M., Duprez, D., Delanghe, J., De Buyzere, M., Clement, DL., 2001. Serum vitamin C concentration is low in peripheral arterial disease and is associated with inflammation and severity of atherosclerosis. Circulation. 103, 1863-1868.26.Lopes, S., Jurisicova, A., Sun, J.G. , Casper, R.F., 1998. Reactive oxygen species potential cause for DNA fragmentation in human spermatozoa. Hum. Reprod.13, 896-900.27.Mann, T., Jones, R., Sherins, R., 1980. Oxygen damage, Lipid peroxidation, and motility of spermatozoa, in: A. Steinberger, E. Steinberger, Testicular Development, Structure and Function, Raven Press, New York, USA, pp.497-501.28.Potts, R.J., Notarianni, L.M., Jefferies, T.M., 1999. Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation. Mutation Research. 447, 249-256.29.Sharma, R.K., Agarwal, A., 1996.Role of reactive oxygen species in male infertility. Copyright. Urology. 48, 835-850.30.Spano, Marcello, Jens, P. B., Henrik, I. H., Kolstad, A., 2000. Sperm chromatin damage impairs human fertility. Fertility and Sterility. vol. 73, 43-49.31.Spano, M., Cordelli, E., Lombardo, F., Lenzi, A., Gandini, G., 1998. Nuclear Chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay. European Society of human reproduction and embryology. 29-37.32.Twigg, J., Lrvine, D.S., Houston, P., Fulton, N., Michael, L., Aitken, R.J., 1998. Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol. Hum. Reprod. 4, 439-444.33.Vcrma, A., Kanwar, K.G., 1998. Human sperm motility and lipid peroxidation in different ascorbic acid concentration: an in vitro analysis. Andrologia. 30, 325~329.34.Weissenberg, R. et al, 1995. Changes during puberty in chromatin condensation morphology and fertilizing ability of epididymal spermatozoa of the golden hamster. Andrologia.27, 341~344.35.World Health Organization, 1992. in: WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, Cambridge University Press, Cambridge, UK, cus Interaction, Cambridge University Press, Cambridge,UK.36.Yossefi, S., Oschry, Y., Lewin, L.M., 1994.Chromation Condenastion in Hamster Sperm: A Flow Cytometric Investigation. Molecular reproduction and development.37, 93-98.