精子功能障礙為最常見之男性不孕症原因，而先前研究指出精子蛋白磷酸化的缺乏，可能為精子功能障礙的原因之一。過去曾發現鐵質過度負荷之病人的精子功能下降。具有正常活動力及型態的精子經獲能反應 (capacitation) 過程，進行蛋白磷酸化之轉譯後修飾 (post-translational modifications) 才具有完整之受孕能力，其中以蛋白酪胺酸磷酸化 (tyrosine phosphorylation) 對精子功能的調節最為重要。鐵質過度負荷病患其精子功能低下之病理機制被認為可能與鐵質引起之氧化壓力有關，但鐵質存在多寡對精子功能的影響尚未有定論。本研究目的主要在探討鐵質對小鼠精子蛋白質磷酸化情形之影響；並進一步探討鐵螯合劑deferasirox (DEFR) 的作用。研究方法：本研究所分析之精子由小鼠 (ICR品系) 副睪取出後經HEPES 培養液清洗而取得品質較好之精子。首先將小鼠精子分成預先暴露 (5分鐘) 與未預先暴露組，利用西方墨點法分析不同濃度之亞鐵溶液 (0 uM、20 uM、40 uM) 對小鼠精子獲能反應過程中酪胺酸磷酸化蛋白及AKAP4蛋白酪胺酸磷酸化比值 (酪胺酸磷酸化AKAP4表現量與AKAP4蛋白總表現量之比值) 的改變情形；研究結果：未預先暴露組小鼠精子在獲能反應進行30、60及90分鐘後，亞鐵離子組酪胺酸磷酸化蛋白表現 (分子量約100、82及70 kDa) 及AKAP4蛋白酪胺酸磷酸化比值皆低於無亞鐵離子組別，尤其在獲能反應60分鐘時間點隨亞鐵離子濃度上升而下降。而預先暴露組之精子細胞在亞鐵離子存在下其酪胺酸磷酸化蛋白表現 (分子量約100、82、70、60、50 kDa及35-40 kDa之間) 及AKAP4蛋白酪胺酸磷酸化比值皆低於無亞鐵離子組別，尤其在獲能反應60分鐘時間點隨亞鐵離子濃度上升而下降。分析精子細胞酪胺酸磷酸化蛋白結果顯示含90 uM DEFR及亞鐵之治療組於獲能反應90分鐘時酪胺酸磷酸化蛋白表現 (分子量約100、82、70、60、50 kDa及35-40 kDa之間) 及AKAP4蛋白酪胺酸磷酸化比值皆高於對照組 (40 uM亞鐵離子組) 但低於控制組。而DEFR組在分子量約70 kDa酪胺酸磷酸化蛋白表現皆高於控制組，且隨DEFR濃度上升而上升。結論：本研究首次證明亞鐵離子抑制小鼠精子酪胺酸磷酸化蛋白之表現，而鐵螯合劑DEFR具改善鐵質抑制小鼠精子獲能反應相關蛋白磷酸化的作用。未來需要更多研究來證實鐵質抑制精子磷酸化蛋白表現之詳細機轉以及進一步鑑定這些酪胺酸磷酸化蛋白的身份以期瞭解鐵質造成精子功能下降之可能機轉。

Sperm dysfunction is the most common cause of male infertility and sperm protein phosphorylation deficiency has been shown to be one of the possible causes of male infertility. Defective sperm function has been observed in patients with iron overload. Proteins of sperms with normal motility and morphology will need to be phosphorylated which is a post-translational modifications during capacitation. Among these, tyrosine phosphorylation acts as the most important mechanism for regulating sperm function to attain fertilizing capacity. The impaired sperm function in patients with iron overload may be associated with iron induced oxidative stress, the impact of various iron concentration on sperm function is still inconclusive. Current study aimed to investigate the effects of iron on mouse sperm protein phosphorylation and the protection of iron chelator, deferasirox (DEFR), will be further studied. Methods: Cauda epididymal sperm from male ICR mice were collected into HEPES medium and washed. Mouse sperms were divided into pre-incubation and co-incubation groups with various concentrations of ferrous ion (Fe2+; 0 uM, 20 uM, or 40 uM). Western blotting was used to determine protein tyrosine phosphorylation and phosphorylation ratio of AKAP4 protein (ratio of phosphorylated AKAP4 to total AKAP4) during calcium ion and bovine serum albumin induced capacitation. Results: In co-incubation groups, tyrosine phosphorylation proteins with molecular weights of ~100, 82 (A-kinase anchoring proteins 4, AKAP4) and 70 kDa proteins, and phosphorylation ratio of AKAP4 protein decreased while Fe2+ concentration increases, particularly at the 60-minute incubation time. In pre-incubation groups, tyrosine phosphorylation of proteins of ~100, 82, 70, 60, 50 and 35-40 kDa and phosphorylation ratio of AKAP4 protein decreased while Fe2+ concentration increases, particularly at the 60-minute incubation time. Compared to the group with 40 uM Fe2+, 90 uM DEFR ameliorated the decreased tyrosine phosphorylation in ~100, 82, 70, 60, 50 and 35-40 kDa proteins and decreased phosphorylation ratio of AKAP4 protein induced by 40 uM Fe2+ at the 90-minute incubation time under capacitation condition. Tyrosine phosphorylation protein of ~70 kDa increased with the increased concentration of DEFR. Conclusion: It is the first study demonstrates that Fe2+ inhibits protein phosphorylation of mouse sperm and this effect could be improved by DEFR. Further studies are required to elucidate the exact mechanism involved in the effect of iron on sperm protein phosphorylation. Identify these affected phosphorylated proteins will help understand the possible mechanism involved in iron caused sperm dysfunction.

目錄I
縮寫表III
摘要VI
AbstractVII
圖目錄VIII
表目錄X
第一章 緒論1
第一節 精子與其功能介紹2
第一項、精子生成與基本構造2
第二項、精子獲能反應 (capacitation)3
第三項、精子蛋白磷酸化 (protein phosphorylation) 與精子功能的關係6
第二節 精子功能障礙 (sperm dysfunction)12
第一項、氧化壓力 (oxidative stress) 與精子功能障礙12
第二項、鐵質 (iron) 與精子功能障礙14
第三項、蛋白質磷酸化與精子功能障礙16
第三節 研究目的與動機17
第二章 實驗材料與方法18
第一節 藥品試劑與材料19
第一項、實驗動物19
第二項、藥品試劑19
第三項、分析材料與實驗儀器22
第二節 實驗步驟與方法24
第一項、精子製備24
第二項、精子培養25
第三項、精子蛋白質分析28
第三章 研究結果32
第一節 精子獲能反應伴隨之酪胺酸磷酸化蛋白表現情形33
第二節 不同濃度亞鐵離子對精子蛋白質表現之影響34
第一項、未預先將小鼠精子暴露於不同濃度亞鐵離子溶液培養後對精子蛋白質表現之影響34
第二項、預先將小鼠精子暴露於不同濃度亞鐵離子溶液再與獲能反應之培養液培養後對精子蛋白質表現之影響41
第三節 鐵螯合劑對精子蛋白質表現之影響46
第一項、預先將小鼠精子暴露於亞鐵離子及鐵螯合劑之溶液再與獲能反應之培養液培養後對精子蛋白質表現之影響46
第二項、預先將小鼠精子暴露於不同濃度鐵螯合劑溶液再與獲能反應之培養液培養後對精子蛋白質表現之影響48
第四章 討論50
第一節 精子獲能反應伴隨之酪胺酸磷酸化蛋白表現情形51
第二節 不同濃度亞鐵離子對精子蛋白質表現之影響53
第三節 鐵螯合劑對精子蛋白質表現之影響56
第五章 結論與展望58
第六章 圖表62
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