跳到主要內容

臺灣博碩士論文加值系統

(3.236.225.157) 您好!臺灣時間:2022/08/15 23:45
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:侯媄菱
研究生(外文):Mei-Ling Hou
論文名稱:膠達納黴素增加公豬精子產生一氧化氮和促進獲能作用
論文名稱(外文):Geldanamycin Augments Nitric Oxide Production and Promotes Capacitation in Boar Spermatozoa
指導教授:黎耀基黎耀基引用關係
指導教授(外文):Yiu-Kay Lai
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
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:英文
論文頁數:44
中文關鍵詞:公豬精子熱休克蛋白質90一氧化氮一氧化氮合成酵素膠達納黴素
外文關鍵詞:boarspermatozoaheat shock protein 90nitric oxidenitric oxide synthasegeldanamycin
相關次數:
  • 被引用被引用:0
  • 點閱點閱:135
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
獲能作用(capacitation)和頂體作用(acrosome reaction)是精子能使卵子受精的兩個重要過程。獲能作用是由一系列功能性和生物物理性修飾所組成的過程,此過程使射出的精子具有與卵子受精的能力,一氧化氮(nitric oxide, NO)已被證實在雄性繁殖系統具多重功能。本研究之目的在於探討公豬精子是否能產生NO,以及探討NO的產生與熱休克蛋白質90 (heat-shock protein 90, HSP90)對公豬精子獲能作用之效應。主要研究結果如下:利用西方墨點分析法(Western blot analysis)證實公豬精子具有內皮細胞型的一氧化氮合成酵素(endothelial nitric oxide synthase, eNOS);利用免疫化學(immunocytochemistry)染色法可證實此酵素位於公豬精子的頂體和中片部;進一步利用NO檢驗試劑得知精子本身能產生NO,並對NOS有關的調節物質有反應。在非獲能條件下,精子產生少量的NO,而在獲能條件下,精子能產生相對於基本量兩倍量的NO (P<0.001)。當精子與NOS抑制劑(L-NAME)或膠達納黴素(geldanamycin,GA)共同培養在獲能培養基中4小時後,L-NAME處理可使獲能作用中精子NO的產生降低30%至40% (P<0.05),反之,GA處理卻能增加精子獲能作用中NO的產生23%至75% (P<0.05),而且NO產生的量與GA濃度有劑量相關關係。若精子與一氧化氮釋放化合物SNP (sodium nitroprusside)、L-NAME或GA共同培養於獲能培養基中4小時,再以A23187處理30分鐘,可以誘使公豬精子頂體作用的發生,藉螢光顯微鏡觀察可測定精子進行頂體反應的百分比;結果顯示,L-NAME降低了精子頂體反應的百分比,而SNP和GA皆可增加頂體反應(P<0.05)。由於GA處理並不是如預期的透過抑制HSP90而降低NO的產生,反而增加NO的量,因此,HSP90非常有可能在公豬體外獲能過程中,不直接參與調節NOS的活性。另外,GA誘使NO產生增加的現象,類似A23187的作用,由此我們推論在獲能條件下,GA增加精子頂體作用,可能是經由公豬精子細胞內鈣離子的提昇和增加NO的量所達成的。

Capacitation and acrosome reaction are of fundamental importance in the fertilization of oocyte by spermatozoon. The process of capacitation consists of a series of functional and biophysical modifications that render the ejaculated spermatozoa competent for fertilization of the oocyte. Nitric oxide (NO) has been shown to have multifunction in male reproduction system. The objectives of the present study were to investigate whether boar spermatozoa can generate NO and the effects of NO and heat-shock protein 90 (HSP90) on the capacitation of boar spermatozoa. The major results were as follows: Boar spermatozoa possess endothelial nitric oxide synthase (eNOS) in the acrosome and middle piece, and spermatozoa can generate NO. Under noncapacitating conditions, spermatozoa produced low levels of NO. However, there was an up to 2-fold increase of NO generation under capacitating conditions (P<0.001). When boar spermatozoa were incubated with capacitation medium in the presence of NOS inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) or geldanamycin (GA) for 4 h, L-NAME treatment reduced 30% to 40% of NO production in capacitating spermatozoa (P<0.05). However, GA promoted 23% to 75% of NO production in a dose-dependent manner (P<0.05). When spermatozoa were capacitated in the presence or absence of NO-releasing compound (SNP), L-NAME or GA, and then the percentage of acrosome reaction induced by calcium ionophore (A23187) was determined. The results indicated that L-NAME decreased the percentage of acrosome reaction, whereas SNP and GA increased the percentage of acrosome reaction (P<0.05). Since GA treatment did not decrease but increase NO production as expected, HSP90 may not directly involve in regulating NOS activity during boar spermatozoa capacitation in vitro. On the other hand, the phenomenon of increasing NO production is similar to the effect of A23187, we suggest that acrosome reaction induced by GA may be triggered the increase of intracellular calcium concentration and NO production.

一、 中文摘要(Abstract in Chinese)……………………1
二、 英文摘要(Abstract in English)……………………2
三、 縮寫(Abbreviations)………………………………. 4
四、 前言(Introduction)………………………………….5
五、 材料與方法(Materials and Methods)……………..9
六、 結果(Results)…………………………………………15
七、 討論(Discussion)…………………………………..18
八、 參考文獻(References)……………………………..23
九、 圖例說明(Figure Legends)………………………..30
十、 圖(Figures)………………………………………...34

Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 1987; 81:459-469.
Austin CR. Observation on the penetration of the sperm into the mammalian egg. Aust J Biol Sci 1951; 4:581-596.
Bender AT, Silverstein AM, Demady DR, Kanelakis KC, Noguchi S, Pratt WB, Osawa Y. Neuronal nitric-oxide synthase is regulated by the Hsp90-based chaperone system in vivo. J Biol Chem 1999; 274:1472-1478.
Bucci M, Roviezzo F, Cicala C, Pinto A, Cirino G. 17-beta-oestradiol-induced vasorelaxation in vitro is mediated by eNOS through hsp90 and akt/pkb dependent mechanism. Br J Pharmacol 2002; 135:1695-1700.
Bucci M, Roviezzo F, Cicala C, Sessa WC, Cirino G. Geldanamycin , an inhibitor of heat shock protein 90 (HSP90) mediated signal transduction has anti-inflammatory effects and interacts with glucocorticoid receptor in vivo. Br J Pharmacol 2000; 131:13-16.
Chang MC. Fertilizing capacity of spermatozoa deposited into fallopian tubes. Nature 1951; 168:697-698.
Creech MM, Arnold EV, Boyle B, Muzinich MC, Montville C, Bohle DS, Atherton RW. Sperm motility enhancement by nitric oxide produced by the oocytes of fathead minnows, Pimephelas promelas. J Androl 1998; 19:667-674.
Cross NL, Morales P, Overstreet JW, Hanson FW. Two simple methods for detecting acrosome reacted human sperm. Gamete Res 1986; 15:235-244.
de Lamirande E, Gagnon C. Capacitation-associated production of superoxide anion by human spermatozoa. Free Radic Biol Med 1995a; 18:487-495.
de Lamirande E, Gagnon C. Human sperm hyperactivation and capacitation as parts of an oxidative process. Free Radic Biol Med 1993; 14:157-166.
de Lamirande E, Gagnon C. Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum Reprod 1995b; Suppl 1:15-21.
de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate plays an important role in the inhibition of sperm motility. Reactive oxygen species and human spermatozoa. I. Effects on the motility of intact spermatozoa and on sperm axonemes. J Androl 1992; 13: 368-386.
de Lamirande E, Jiang H, Zini A, Kodama H, Gagnon C. Reactive oxygen species and sperm physiology. Rev Reprod 1997; 2:48-54.
Francavilla F, Santucci R, Macerola G, Ruvolo G, Romano R. Nitric oxide synthase inhibition in human sperm affects sperm-oocyte fusion but not zona pellucida binding. Biol Reprod 2000; 63:425-429.
Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetropoulos A, Sessa WC. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature 1998; 392:821-824.
Gratton JP, Fontana J, O’Connor DS, Garcia-Cardena G, McCabe TJ, Sessa WC. Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro. Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1. J Biol Chem 2000; 275:22268-22272.
Griveau JF, Renard P, Le Lannou D. An in vitro promoting role for hydrogen peroxide in human sperm capacitation. Int J Androl 1994; 17:300-307.
Gross SS, Wolin MS. Nitric oxide: pathophysiological mechanisms. Annu Rev Physiol 1995; 57:737-769.
Gusev NB. Some properties of caldesmon and calponin and the participation of these proteins in regulation of smooth muscle contraction and cytoskeleton formation. Biochemistry (Mosc) 2001; 66:1112-1121.
Hancock JK, Hovell GLR. The collection of boar semen. Vet Res 1959; 71:664-669.
Harrison RA. Capacitation mechanisms, and the role of capacitation as seen in eutherian mammals. Reprod Fertil Dev 1996; 8:581-94.
Hellström WJ, Bell M, Wang R, Sikka SC. Effect of sodium nitroprusside on sperm motility, viability and lipid peroxidation. Fertil Steril 1994; 61:1117-1122.
Herrero MB, Cebral E, Boquet M, Viggiano JM, Vitullo A, Gimeno MA. Effect of nitric oxide on mouse sperm hyperactivation. Acta Physiol Pharmacol Ther Latinoam 1994; 44:65-69.
Herrero MB, Chatterjee S, Lefievre L, de Lamirande E, Gagnon C. Nitric oxide interacts with the cAMP pathway to modulate capacitation of human spermatozoa. Free Radic Biol Med 2000; 29:522-536.
Herrero MB, de Lamirande E, Gagnon C. Nitric oxide regulates human sperm capacitation and protein-tyrosine phosphorylation in vitro. Biol Reprod 1999; 61:575-581.
Herrero MB, Gagnon C. Nitric oxide: a novel mediator of sperm function. J Androl 2001; 22:349-356.
Herrero MB, Goin JC, Boquet M, Canteros MG, Franchi, MF, Perez Martinez S, Polak JM, Viggiano JM, Gimeno MF. The nitric oxide synthase of mouse spermatozoa. FEBS Lett 1997; 411:39-42.
Herrero MB, Perez Martinez S, Viggiano JM, Polak JM, Gimeno MF. Localization by indirect immunofluorescence of nitric oxide synthase in mouse and human spermatozoa. Reprod Fertil Dev 1996; 8:931-934.
Huang SY, Kuo YH, Lee WC, Tsou HL, Lee YP, Chang HL, Wu JJ, Yang PC. Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa. Theriogenology 1999; 51:1007-1016.
Huang SY, Kuo YH, Tsou HL, Lee YP, King YT, Huang HC, Yang PC, Lee WC. The decline of porcine sperm motility by geldanamycin, a specific inhibitor of heat-shock protein 90 (HSP90). Theriogenology 2000; 53:1177-1184.
Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J 1994; 298:249-258.
Köhn FM, Mack SR, Schill WB, Zaneveld LJ. Detection of human sperm acrosome reaction: comparison between methods using double staining, Pisum sativum agglutinin, concanavalin A and transmission electron microscopy. Hum Reprod 1997; 12:714-721.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-685.
Lewis SE, Donnelly ET, Sterling ES, Kennedy MS, Thompson W, Chakravarthy U. Nitric oxide synthase and nitrite production in human spermatozoa: evidence that endogenous nitric oxide is beneficial to sperm motility. Mol Hum Reprod 1996; 2:873-878.
Moncada S, Higgs EA. Molecular mechanisms and therapeutic strategies related to nitric oxide. FASEB J 1995; 9:1319-1330.
Nims RW, Cook JC, Krishna MC, Christodoulou D, Poore CM, Miles AM, Grisham MB, Wink DA. Colorimetric assays for nitric oxide and nitrogen oxide species formed from nitric oxide stock solutions and donor compounds. Methods Enzymol 1996; 268:93-105.
O’Bryan MK, Zini A, Cheng CY, Schlegel PN. Human sperm endothelial nitric oxide synthase expression: correlation with sperm motility. Fertil Steril 1998; 70:1143-1147.
Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 1988; 333:664-666.
Pritchard KA Jr, Ackerman AW, Gross ER, Stepp DW, Shi Y, Fontana JT, Baker JE, Sessa WC. Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase. J Biol Chem 2001; 276; 21:17621-17624.
Revelli A, Soldati G, Costamagna C, Pellery O, Aldieri E, Massobrio M, Bosia A, Ghigo D. Follicular fluid proteins stimulate nitric oxide (NO) synthesis in human sperm: a possible role for NO in acrosomal reaction. J Cell Physiol 1999; 178:85-92.
Richter K, Buchner J. Hsp90: chaperoning signal transduction. J Cell Physiol 2001; 188:281-290.
Russell KS, Haynes MP, Caulin-Glaser T, Rosneck J, Sessa WC, Bender JR. Estrogen stimulates heat shock protein 90 binding to endothelial nitric oxide synthase in human vascular endothelial cells. Effects on calcium sensitivity and NO release. J Biol Chem 2000; 275:5026-5030.
SAS Institute. SAS User’s Guide: Statistics, release 6.03. Gary NC: SAS Institute Inc, 1989.
Sessa WC. The nitric oxide synthase family of proteins. J Vasc Res 1994; 31:131-143.
Sikka SC. Relative impact of oxidative stress on male reproductive function. Curr Med Chem 2001; 8:851-862.
Storey BT. Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa. Mol Hum Reprod 1997; 3:203-213.
Tardif S, Dube C, Chevalier S, Bailey JL. Capacitation is associated with tyrosine phosphorylation and tyrosine kinase-like activity of pig sperm proteins. Biol Reprod 2001; 65; 3:784-792.
Tardif S, Sirard MA, Sullivan R, Bailey JL. Identification of capacitation-associated phosphoproteins in porcine sperm electroporated with ATP-γ-32P. Mol Reprod Dev 1999; 54:292-302.
Tomlinson MJ, East SJ, Barratt CL, Bolton AE, Cooke ID. Preliminary communication: possible role of reactive nitrogen intermediates in leucocyte-mediated sperm dysfunction. Am J Reprod Immunol 1992; 27:89-92.
Uma Devi K, Jha K, Patil SB, Padma P, Shivaji S. Inhibition of motility of hamster spermatozoa by protein tyrosine kinase inhibitors. Andrologia 2000; 32:95-106.
Visconti PE, Galantino-Homer H, Ning X, Moore GD, Valenzuela JP, Jorgez CJ, Alvarez JG, Kopf GS. Cholesterol efflux-mediated signal transduction in mammalian sperm. Beta-cyclodextrins initiate transmembrane signaling leading to an increase in porcine tyrosine phosphorylation and capacitation. J Biol Chem 1999; 274:3235-3242.
Weinberg JB, Doty E, Bonaventura J, Haney AF. Nitric oxide inhibition of human sperm motility. Fertil Steril 1995; 64:408-413.
Whitesell L, Mamnaugh EG, De Costa B, Myers CE, Neckers LM. Inhibition of heat-shock protein 90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: Essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci USA 1994; 91:8324-8328.
Wink DA, Mitchell JB. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 1998; 25: 434-456.
Wolfe CA, James PS, Mackie AR, Ladha S, Jones R. Regionalized lipid diffusion in the plasma membrane of mammalian spermatozoa. 1998; 59:1506-1514.
Yanagimachi R. Mammalian fertilzation. In: Knobil E.; Neill JD, eds. The physiology of reproduction. New York: Raven Press. 1994:189-317.
Yeoman RR, Jones WD, Rizk BM. Evidence for nitric oxide regulation of hamster sperm hyperactivation. J Androl 1998; 19:58-64.
Zini A, de Lamirande E, Gagonon C. Low levels of nitric oxide promote human sperm capacitation in vitro. J Androl 1995; 16:424-431.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top