探討副睪中第一型硫氫氧化酶的分泌調控機制

Abstract

哺乳類動物的精子經由睪丸製造釋出後尚未完全成熟，精子要在副睪中進行不同形式與程度的蛋白質轉譯後修飾才會獲得完整的受孕能力與活動力，眾多修飾過程中，雙硫鍵催化過程對於精子的蛋白質結構穩定和頭部染色體濃縮扮演著很重要的角色。過去我們實驗室之前透過蛋白質鑑定，找到了一個參與雙硫鍵催化過程的關鍵蛋白質: 第一型精漿蛋⽩硫氫氧化（quiescin sulfhydryl oxidase 1, QSOX1），並且發現此蛋白質在副睪的不同區段 (前、中、後) 有不同的含量，此外，也發現此蛋白質可附著於副睪前端精子的頂體區域，然而此蛋白質在精子成熟過程中扮演的角色尚未被完全瞭解。 本論文希望藉由探討QSOX1的分泌調控機制，讓我們更加了解此蛋白在副睪中的特性，進而在未來能進一步探討QSOX1在精子成熟過程中的所扮演的角色。透過西方墨點法及免疫螢光染⾊法，我們發現QSOX1的分泌在30天大的小鼠副睪中有顯著的上升， 此結果說明調控此蛋白質分泌的機制可能是透過體內睪固酮的增加或是透過副睪管腔中精子的刺激而導致，藉由二維共培養系統的建立，我們進一步測試上述兩個者對QSOX1的分泌是否具有影響，結果顯示，加入睪固酮對於QSOX1的分泌並沒有顯著的影響，而在精子共培養的組別中，培養液中的QSOX1有顯著性的增加。此外，相較於副睪後段較成熟的精子，副睪前端的精子能更有效的刺激QSOX1的分泌。透過高效液相層析串聯質譜儀分析和比較蛋白質體分析，我們進一步從培養液中鑑定出額外的582個分泌型蛋白質，推測其中的一些 （258個）蛋白質可能由精子釋放並刺激QSOX1的分泌。 綜合以上結果，QSOX1的分泌主要是經由精子的刺激而不是由睪固酮，但其作用分子與機制與切確的刺激因子為何有待進一步的研究。

Spermatozoa are not fully mature after being released from the testis. They gradually become mature and acquire the capacity for progressive motility and fertilization ability through post-translational modifications that occur in different segments of the epididymis. Among these modifications, disulfide bond formation is essential for sperm protein and structure stability, including chromatin condensation, sperm midpiece, and tail stabilization. The quiescin sulfhydryl oxidase 1 (QSOX1), which catalyzes the thiol-oxidation reaction has been identified in the epididymis. We have demonstrated that QSOX1 exhibits a region-specific distribution in the epididymis, and appears at the acrosome region of the caput sperm. However, the functional role and the regulation of QSOX1 secretion in the epididymis are still unknown. This thesis focuses on the understanding of secretory regulation of QSOX1 in the epididymis. Through Western blotting and indirect immunofluorescent studies, we showed that mouse secretory QSOX1 was upregulated at postnatal day 30, suggesting the potential involvement of testosterone and/or sperm cells on the regulation and stimulation of QSOX1 secretion. To closely mimic in vivo situation, we set up a 2D polarized co-culture system to investigate the effects of these stimuli and to measure the level of QSOX1 secretion in vitro. Our data showed that QSOX1 secretion was increased after co-incubating with sperm cells (especially in caput sperm cells) but not when co-incubating with testosterone or its metabolites, DHT. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and comparing proteome analysis were used to identify and characterize the possible regulatory molecules that were being released from sperm. We identified 582 secretory proteins that were exclusively present in the sperm-enriched group and further categorized 258 caput sperm proteins that might be responsible for stimulating QSOX1 secretion in the epididymis. In conclusion, our study provides solid evidence that QSOX1 secretion is likely regulated by sperm-epididymis epithelium interaction rather than by testosterone stimulation. This result may further explain the role of QSOX1 upon sperm maturation.