臺灣博碩士論文加值系統

在工業化國家不孕率的持續攀升及男性精液品質的惡化之下，環境汙染物對生殖系統的負面影響日益受到重視，而空氣污染被認為極有可能是肇因之ㄧ。本研究目的在於探討機車廢氣是否為造成精子毒性的環境風險因子。
在模擬人類可能暴露環境的條件下， Wistar雄性大鼠在每天兩小時，每週五天持續四周呼吸暴露十倍稀釋的機車廢氣後，其睪丸重量下降30%，睪丸、副睪精子密度分別下降70%及90%。此外，副睪精子活動力則下降80%而輸精管精子的活動力則幾乎完全喪失。在相同實驗條件改以四行程機車引擎，結果也同樣發現精子毒性，但精子毒性強度較二行程機車為弱。四行程機車廢氣對睪丸重量沒有影響,對睪丸及副睪則造成精子密度下降20%及30%，並使得副睪、輸精管精子活動力分別下降40%及30%。在組織病理切片觀察下，二行程機車廢氣使睪丸細精管明顯萎縮、精細胞數目大幅下降，並使壞死精子形成的多核巨細胞數目大增，而副睪則呈現明顯的精子密度下降及壞死精子的增加。相較之下，四行程機車廢氣則無造成睪丸及副睪顯著的組織病理變化。
為釐清二行程機車廢氣造成精子毒性的可能機轉，我們探討呼吸暴露二行程機車廢氣大鼠其睪丸及副睪中（1）外生性物質代謝酵素及抗氧化酵素的活性變化；（2）參與類固醇生成相關基因、雄性素調控基因及發炎/細胞凋亡相關基因的表現變化，（3）脂質過氧化、雄性素受體表現及血中促濾泡成熟激素的濃度變化。實驗結果顯示，二行程機車廢氣能使睪丸中的一相酵素pentoxyresurufin O-dealkylase 和7-ethoxycoumarin O-deethylase 活性分別下降50%和60%，而aniline hydroxylase 及benzo(a)pyrene hydroxylase 的活性則無改變。就二相酵素而言，NAD(P)H:quninone oxidoreductase 在睪丸上升46%，在副睪下降20%，glutathione S-transferase 則不受影響。就抗氧化酵素而言，睪丸和副睪superoxide dismutase 活性分別下降30%和27%，而heme oxygenase活性則分別上升126%和650%。Glutathione peroxidase 活性在副睪上升28%，在

睪丸則無變化。以反轉錄聚合鏈反應探討基因mRNA的改變，發現二行程機車廢氣造成雄性素調控基因sulfated glycoprotein-1 和sulfated glycoprotein-2 在睪丸的表現增加，androgen receptor(AR)在副睪的表現減少，發炎/細胞凋亡相關基因Bax, COX-2在睪丸、副睪表現增加，睪丸中IL-1α、 IL-1β和副睪中IL-6也有表現增加的情況。而參與類固醇生成的基因包括steroid transport acute regulatory protein, CYP11A, CYP17, 3β-hydroxylsteroid dehydrogenase, CYP19, 5α-reductase等，及發炎/細胞凋亡相關基因TNF-α，其mRNA表現則沒有顯著改變。以測量thiobarbituric acid-reactive substances生成的方式探討二行程機車廢氣對脂質過氧化的影響，發現副睪的脂質過氧化程度上升20%，以AR binding assay, enzyme immunoassay方式分別探討二行程機車廢氣對雄性素受體表現及血中促濾泡成熟激素濃度的影響，結果顯示二行程機車廢氣並不會造成此二者的改變。
綜合以上實驗結果，我們的初步結論是二行程機車廢氣及四行程機車廢氣均會對雄性生殖系統造成不良影響。二行程機車廢氣造成精子毒性的可能機轉為增加氧化壓力而使抗氧化平衡失調，其內分泌干擾作用則需再更近一步的探討。

With the realization of increasing rate of infertility and deteriorating semen quality in many industrial countries, the ability of an environmental pollutant to affect reproductive system gains growing concern and air pollution has been indicated as a possible cause. The present study was conducted to test the hypothesis that motorcycle exhaust（ME）exposure is an environmental risk factor of spermatotoxicity.
Under environmentally realistic experimental conditions, adult male Wistar rats exposed to 10-fold diluted 2-stroke ME for one month showed a 30% decrease of testes weight, a 70% decrease of testicular sperm density, and a 90% decrease of epididymal sperm density. In addition, sperm motility was decreased by 80% in epididymis and there was an almost complete lost of motility in vas deferens. Under similar experimental conditions using a 4-stroke motorcycle engine, spermatotoxicity was also observed in male rats but the toxicity was less serious, compared to 2-stoke ME. 4-Stroke ME had no effects on testes weight, reduced testicular and epididymal sperm density by 20% and 30%, and decrease epididymal and vas deferens sperm motility by 40% and 30%, respectively. Histophathological study revealed that 2-stroke ME caused seminiferous tubule atrophy, decreased sperm cell number, increased multinucleated giant cell (necrotic sperm cells) number in testis, and decreased sperm densitiy and increased necrotic sperm in epididymis. On the contrary, 4-stoke ME did not cause marked histopathological changes in rat testis and epididymis. To elucidate the mechanism of ME-induced spermatotoxicity, we have investigated the effects of ME on（1）xenobiotic-metabolism and antioxidant enzyme activities;（2）steroidogenesis genes, androgen-responsive and apoptosis/inflammation -related genes mRNA levels; and（3）lipid peroxidation level, serum level of follicle stimulating hormone, and tissue androgen receptor concentration.
The results showed that 2-stroke ME decreased the activities of testicular xenobiotic-metabolizing enzymes, pentoxyresorufin O-dealkylase and 7-ethoxycoumarin O-deethylase by 50% and 60%, respectively, and aniline


hydroxylase and benzo(a)pyrene hydroxylase activities were not changed. Phase II enzyme glutathione S-transferase activity was not changed in testis and epididymis,
but NAD(P)H:quinone oxidoreductase activity showed a 46% increase in testis and a 20% decrease in epididymis. With respective to antioxidant enzymes activities, 2-stroke ME decreased superoxide dismutase by 30% and 27% and increased heme oxygenase by 126% and 650% in testis and epididymis, respectively. Glutathione peroxidase activity was increased by 28% in epididymis and not altered in testis. Gene expression changes studied by standard RT-PCR analysis showed that 2-stroke ME had no effects on steroidogenesis genes including steroid transport acute regulatory protein, CYP11A, CYP17, 3β-hydroxylsteroid dehydrogenase, CYP19, and 5α-reductase and an inflammation-related gene tumor necrosis factor-α.. ME had no effect on androgen-responsive genes androgen receptor interacting protein-3, complement 3, however, androgen-responsive sulfated glycoprotein-1 and sulfated glycoprotein-2 genes expression were up-regulated in testis. Androgen receptor (AR) was down-regulated in epididymis and not changed in testes. Apoptosis/ inflammation-related genes Bax, COX-2, IL-1α and IL-1β were upregulated in testis. IL-6 was up-regulated in epididymis but not changed in testes. Lipid peroxidation analysed by thiobarbituric acid-reactive substances formation showed that 2-stroke ME increased epididymal lipid peroxidation level by 20%. Testicular AR binding assay showed that the receptor concentration was not changed after 2-stroke ME exposure. Serum hormone studied by enzyme immunoassay showed no significant serum FSH alterations after 2-stroke ME exposure.
In conclusion, 2-strok and 4-stroke ME both have adverse effects on male reproductive system in rat. The 2-stroke ME induced-spermatotoxicity is probabaly due to antioxidant defense failure and the role of endocrine disrupting effect still needs further investigation.

中文摘要------------------------------------------------------------------------------------------1
英文摘要------------------------------------------------------------------------------------------3

第一章 前言
第一節、空氣污染－嚴肅的環境議題-------------------------------------------------------5
第二節、空氣污染所致的健康危害及其機制—氧化壓力所扮演的角色-------------6
第三節、機車廢氣的基礎性質及其研究背景----------------------------------------------7
第四節、空氣污染與精液品質惡化以及不孕之間的關連-------------------------------9
第五節、本篇研究目的------------------------------------------------------------------------10

第二章 研究方法與步驟
第一節、 呼吸暴露實驗
i. 實驗動物----------------------------------------------------------------------12
ii. 機車廢氣暴露裝置系統----------------------------------------------------12
iii. 大鼠呼吸暴露機車廢氣實驗----------------------------------------------13
第二節、 精子毒性評估
i. 精子活動力計算-------------------------------------------------------------14
ii. 精子密度計算----------------------------------------------------------------15
第三節、 酵素活性測試
i. 組織微粒體的製備----------------------------------------------------------15
ii. ㄧ相酵素活性測試
a. Benzo(a)pyrene Hydroxylase Activity Assay------------------------16
b. 7-Pentoxyresorufin O-dealkylase Activity Assay-------------------17
c. 7-Ethoxycoumarin O-dealkylase Activity Assay--------------------17
d. Aniline Hydroxylase Activity Assay----------------------------------19
iii. 二相酵素活性測試
Glutathione S-Transferase Acitivity Assay------------------------------20
iv. 抗氧化酵素活性測試
a. NAD(P)H:quinone Oxidoreductase Activity Assay-------------------20
b. Heme Oxygenase Activity Assay----------------------------------------21
c. Superoxide Dismutase Activity Assay----------------------------------22
d. Catalase Activity Assay---------------------------------------------------23
第四節、脂質過氧化分析---------------------------------------------------------------------23
第五節、組織中雄性素受體含量測定------------------------------------------------------24
第六節、血液中促濾泡成熟激素濃度測定------------------------------------------------25
第七節、睪丸及副睪基因mRNA表現分析
i. 組織RNA萃取--------------------------------------------------------------- 26
ii. 反轉錄-聚合酶連鎖反應--------------------------------------------------- 27
iii. 聚合酶連鎖反應之引子序列及反應條件------------------------------- 28

第三章 結果
第一節、二行程及四行程機車廢氣對雄性大鼠器官重量變化之影響---------------30
第二節、二行程及四行程機車廢氣對精子數目及活動力之影響---------------------31
第三節、二行程及四行程機車廢氣對睪丸、副睪組織病理變化之影響------------32
第四節、二行程機車廢氣對脂質過氧化程度、雄性素受體表現及血中
促濾泡成熟激素之影響---------------------------------------------------------- 33
第五節、二行程機車廢氣對睪丸一相代謝酵素活性之影響---------------------------34

第六節、二行程機車廢氣對睪丸、副睪二相代謝酵素及抗氧化酵素活性之影響-35
第七節、二行程機車廢氣對睪丸、副睪中參與類固醇生成相關基因
mRNA 表現之影響---------------------------------------------------------------36
第八節、二行程機車廢氣對睪丸、副睪中雄性素調控相關基因mRNA
表現之影響-------------------------------------------------------------------------37
第九節、二行程機車廢氣對睪丸、副睪中發炎/細胞凋亡相關基因mRNA
表現之影響-------------------------------------------------------------------------38
第四章 討論
第一節、二行程機車廢氣造成精子毒性的可能機制------------------------------------42
第二節、氧化壓力在二行程機車廢氣所導致的精子毒性所扮演的可能角色------43
第三節、血基質氧化酵素活性上升—細胞毒性與內分泌干擾的橫向關聯---------48
第四節、二行程機車廢氣的內分泌干擾作用---------------------------------------------50
第五節、二行程機車廢氣導致生殖細胞死亡的證據------------------------------------54
第六節、發炎反應及細胞凋亡在二行程機車廢氣所導致的精子毒性所扮演的可能角色------------------------------------------------------------------------------------55
第七節、結論------------------------------------------------------------------------------------58

參考文獻---------------------------------------------------------------------------------------- 60










22
Table 1 Oligonucleotide Primers for Standard RT-PCR Analysis of Androgen-Responsive Genes, Steroidogenesis Genes, Inflammation and Apoptosis Related Genes ------------------------------------------------------72
Table 2 Concentrations of Components of Exposure Atmosphere in 2-Stroke and 4-Stroke ME Inhalation Experiments-----------------------------------------74
Table 3 Effects of 2-Stroke ME Inhalation Exposure on Organ Weights in Adult Male Wistar Rats-----------------------------------------------------------------75
Table 4 Effects of 4-Stroke ME Inhalation Exposure on Organ Weights in Adult Male Wistar Rats-----------------------------------------------------------------76
Table 5 Effect of 2-Stroke ME Inhalation Exposure on Rat Sperm Density and Motility----------------------------------------------------------------------------77
Table 6 Effect of 4-Stroke ME Inhalation Exposure on Rat Sperm Density and Motility----------------------------------------------------------------------------78
Table 7 Effect of 2-Stroke ME on Androgen Receptor in Rat Testis -------------- 79
Table 8 Effects of 2-Stroke ME Inhalation Exposure on Phase I Enzyme Acitivities in Rat Testis and Epididymis--------------------------------------80
Table 9 Effects of 2-Stroke ME Inhalation Exposure on Phase II and Antioxidant Enzyme Acitivities in Rat Testis and Epididymis---------------------------81
Table 10 Effects of 2-Stroke ME Exposure on Androgen-Responsive Genes mRNA Expression in Rat Testis and Epididymis-------------------------------------82
Table 11 Effects of 2-Stroke ME Exposure on Steroidogenesis Genes mRNA Expression in Rat Testis and Epididymis-------------------------------------83
Table 12 Effects of 2-Stroke ME Exposure on Inflammation and Apoptosis Related Genes mRNA Expression in Rat Testis and Epididymis------------------- 84



圖目錄
Fig. 1 Effects of 2-Stroke ME Inhalation Exposure on Testicular Histopathological Changes in Rats-----------------------------------------------------------------------85
Fig. 2 Effects of 2-Stroke ME Inhalation Exposure on Epididymal Histopathological Changes in Rats ----------------------------------------------------------------------86
Fig. 3 Effects of 4-Stroke ME Inhalation Exposure on Testicular Histopathological Changes in Rats-----------------------------------------------------------------------87
Fig. 4 Effects of 4-Stroke ME Inhalation Exposure on Epididymal Histopathological Changes in Rats-----------------------------------------------------------------------88
Fig. 5 Effects of 2-Stroke ME Inhalation Exposure on Lipid Peroxidation in Rat Epididymis-----------------------------------------------------------------------------89
Fig. 6 Effect of 2-Stroke ME Inhaltion Exposure on Serum FSH Level of Male Rats------------------------------------------------------------------------------------- 90
圖解目錄
Scheme 1 Possible Mechanisms of 2-Stroke ME-Induced Spermatotoxicity---------91
附錄
附錄一、 利用空氣品質監測站資料估算通勤者的可能廢氣暴露量--------------92

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