不孕症 (infertility) 影響全球近八千萬的夫妻已成為全球關注的議題，其中男性因素 (male factor) 占不孕症發生的原因近50％，而其中又有55％是與精子無力症有關。已知葡萄糖進入精子細胞內會經由糖解作用產生的腺苷三磷酸 (ATP) 作為精子活動能量的最主要來源，而 ATP 經酵素轉化產生的環狀腺核苷單磷酸 (cAMP)，則將經由 protein kinase activator (PKA) 訊息傳遞進而促進精子的活動。然而，葡萄糖濃度過高時是否會影響精子活動力仍待釐清。因此，本研究利用電腦輔助精子分析儀 (computer assisted sperm analysis, CASA) 分析高濃度葡萄糖對精子活動力的影響並探討其可能的生化路徑。方法：將小鼠精子分別在含有正常葡萄糖濃度 (5.5 mM)、高葡萄糖濃度 (25 mM) 的緩衝液培養，分別在第 0、30、60、90、120 分鐘利用精子輔助分析儀分析精子八項運動參數包括精子的活動力 (Motility; M)、精子活動路徑平均速度 (VAP)、精子直線速度 (VSL)、精子曲線速度 (VCL)、精子側頭位移之振幅 (ALH)、精子搏動頻率 (BCF)、精子向前性 (STR)、精子直線性 (LIN)，進而探討各時間點間各項精子運動參數的差異再以 one-way ANOVA 檢測兩組間的統計差異；高葡萄糖所造成的改變之生化路徑則利用已知具調控糖平衡的水飛薊 (silymarin；5、10、20 ug/ml) 進一步分析。結果：在高濃度葡萄糖緩衝液中，參數 M 值在第 90 分鐘較培養於正常葡萄糖的精子高 (52.0 ± 10.4% vs. 28.7 ± 10.1%；p < 0.05)；參數 VAP 值、參數 VSL 值、參數 VCL 值在 60、90 分鐘時間點亦較正常葡萄糖的精子高，且具統計上顯著差異 (p < 0.05)；而高葡萄緩衝液中添加或未添加 PKA 抑制劑 (H-89) 在第 30 分鐘參數 M 值分別為 (42.0 ± 7.8% vs. 55.7 ± 19.9%)。前述結果顯示：在高葡萄糖濃度下精子有較高的活動力，推論可能與過量葡萄糖能提供額外糖解訊息傳遞經 PKA 路徑有關。生化路徑的探討部分：在正常葡萄糖緩衝液下，參數 M 值在 20 ug/ml silymarin 培養 90 分鐘時間點較未含有silymarin 培養組別高 (52.3 ± 10.8% vs. 28.7 ± 10.1%, p < 0.05)；而參數 VAP值、參數VSL值、參數 VCL 值也有相同趨勢，但無統計上顯著差異。但在高濃度葡萄糖下，參數 M 值、參數 VAP 值、參數 VSL 值、參數 VCL 值隨著 silymarin 濃度 (5、10、20 ug/mL) 的上升而下降；其第 60 分鐘時活動力分別為 (40.7 ± 6.5% vs. 33.7 ± 5.5% vs. 29.3 ± 9.1%)，相較於高葡萄糖組具顯著差異 (60.7 ± 10.4%； p < 0.05 )；然而在參數 ALH 值、參數 BCF 值、參數 STR 值、參數 LIN 值，經藥物處理過後均無明顯變化；而比較含 silymarin 之高葡萄緩衝液測試組中添加或未添加 PKA 抑制劑在第 30 分鐘參數 M 值分別為 (36.5 ± 4.9% vs. 59.3 ± 16.3%)。結果顯示：在正常濃度緩衝液下20 ug/ml silymarin 可維持較長時間的精子活動力，而在高葡萄糖緩衝液下且含有silymarin時，精子活動力會有下降的情形。而 PKA 抑制劑在高葡萄糖環境下具抑制精子活動力作用但在含 20 ug/ml silymarin 的高葡萄糖環境參數 M 值則低於無silymarin組別 (36.5 ± 4.9% vs. 42 ± 7.8%)。結論：高濃度葡萄糖 (25 mM) 可以增加精子活動力，且可能與活化 PKA 路徑作用有關；20 ug/ml silymarin 可以延長精子活動力情形，但 silymarin 減緩高葡萄糖所促進的精子活動力結果。Silymarin 對 PKA 以外之其他促進精子活動力訊息傳遞，以及將葡萄糖補充或 silymarin 應用在人工受孕中以增進精子活動力的可行性，則需要進一步的研究。

Infertility affecting nearly 80 million couples has become a global concern. Among those, male factors accounts for 50 percent cause of the infertility, while 55% male infertility is related to the asthenozoospermia. Adenosine triphosphate (ATP) is known to be produced by glycolysis while glucose enters into sperm cell, which is mainly dedicated to maintain sperm motility. Cyclic adenosine monophosphate (cAMP) derived from ATP further stimulate protein kinase activator (PKA) pathway to boost motility. However, the effect of high concentration glucose on mouse spermatozoa is still unclear. Here, we aimed to determine the effect of high concentration glucose on mouse spermatozoa motility parameters and its possible biochemical mechanisms. Methods: Prepared sperm samples were incubated in HM buffer containing normal glucose (NG; 5.5 mM) or high concentration glucose (HG; 25 mM), then analysize at 0, 30, 60, 90, 120 mins by using computer assisted semen analysis (CASA) to assess sperm motility parameters. In this study, the primary CASA measurements assessed were percent motility, average pathway velocity (VAP), straightline velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), straightness (STR) and linearity (LIN). The one-way ANOVA was used to test variances between groups at different time periods. Furthermore, the influence of silymarin (5, 10, 20 ug/mL) and its possible biochemical pathway involved in the effects of high concentration of glucose on sperm motility parameters was then investigated. Results: Sperms incubated in HG had higher motility compared to NG at 90 mins (52.0 ± 10.4% vs. 28.7 ± 10.1%, p < 0.05). VAP, VSL, VCL had similar pattern at 60, 90 mins (p < 0.05). Incubated in HG condition with or without PKA inhibitor (H-89), sperm motility at 30 mins was 42.0 ± 7.8%, 55.7 ± 19.9%, respectively. This result indicates that higher percent of sperms had higher motility rate in HG which may due to extra energy provided by additional glucose source for spermatozoa via PKA pathway. Presence of 20 ug/ml silymarin resulted in higher motility compared to control group at 90 mins (52.3 ± 10.8% vs. 28.7 ± 10.1%, p < 0.05). VAP, VSL, VCL of sperm treated with 20 ug/ml silymarin also showed the similar trend but did not reach statistical differences. However, presence of silymarin resulted in decreased motility parameters (motility, VAP, VSL, VCL) in HG condition while the concentration of silymarin (5, 10, 20 ug/ml) increased. At 60 mins while compared the control (60.7 ± 10.4%), the motility of sperms treated with various concentrations of silymarin (5, 10, 20 ug/ml) was 40.7 ± 6.5%, 33.7 ± 5.5%, 29.3 ± 9.1%, respectively (p < 0.05). On the other hand, ALH, BCF, STR, LIN was not affected by glucose and silymarin. Incubated in HG condition with or without H-89, sperm motility treated with 20 ug/ml silymarin at 30 mins was 36.5 ± 4.9% vs. 59.3 ± 16.3%, respectively. Those results indicate that 20 ug/ml silymarin prolonged sperm motility. In contrasts, silymarin decreased sperm motility induced by HG. Those observations suggest that silymarin increased sperm motility via PKA activation. Conclusion: High concentration of glucose (25 mM) increased sperm motility and such effects may be related to PKA pathway activation. Silymarin at 20 ug/ml prolongs sperm motility, but depress sperm motility induced by HG. The mechanisms of how silymairn affects cell signal pathway other than PKA pathway and whether supplementation of glucose or silymarin can be applied to in vitro fertilization need further study.

目錄 I
縮寫表 II
摘要.............................................................................................................................. III
Abstract IV
表目錄 V
圖目錄 VI
第一章、緒論 1
一、精子的發生與成熟 2
二、精子構造 3
三、精子游動力參數介紹 5
四、精子活動的訊息傳遞機制 6
五、醣類對精子之影響 8
六、研究動機與目的 12
第二章、研究設計 13
一、藥品、試劑、分析材料與儀器設備 14
二、精子的製備 17
三、控制組與實驗組的製備 18
四、精子活動速度評估 19
五 、資料分析與統計 21
第三章、研究結果 22
一、精子活動力 23
二、前進游動精子百分率 24
三、精子活動路徑平均速度 25
四、精子曲線速度 26
六、精子直線速度 27
七、其他運動參數 28
第四章、討論 29
第五章、結論與展望 41
第六章、圖表 45
參考文獻 113

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