雄性生殖道中硫氫氧化酶的特性化與純化

Abstract

背景:在台灣,因不孕症尋求繁殖醫學門診,求助人工受孕技術(Assisted Reproductive Technology, ART)治療的比例逐年提高。然而,在人工受孕技術大量 介入的同時,成功受孕及生育率仍舊無法顯著提高,代表篩選精卵品質的標準有待 改善。現今,精液品質分析多以精子活動力、形態、數量作為主要判定標準,然而 內部因子如:配子的去氧核醣核酸(Deoxyribonucleic Acid, DNA)和染色體 (chromosome)、精子表面膜蛋白組成是否符合受孕標準,無法在執行人工受孕技 術前得知。 目的:精漿成份以及精子表面蛋白質組成和不孕症息息相關,本論文希望藉由探討 精漿蛋白硫氫氧化酶(quiescin sulfhydryl oxidase, QSOXs)和精子在附睪成熟過程、 生殖道中移動到與卵子受精前的交互作用,進一步了解硫氫氧化酶修飾精子表面的 機制,增加配子篩選的參考標準。 方法:(1)以免疫組織染色法以及免疫螢光染色法,,探究小鼠以及豬在雄性生殖 道中兩種硫氫氧化酶(QSOX1C 與 QSOX2)的表現和分佈,(2)從小鼠及豬的儲 精囊分泌液,以離子交換法結合高效液相色譜(High Performance Liquid Chromatography, HPLC)及親和層析純化硫氫氧化酶,(3)藉由人類胚胎腎臟細胞 株(HEK 293 cell line) 建立表現人類硫氫氧化酶-2 (QSOX2)重組蛋白的穩定 細胞株,並以硫酸銨沈澱執行鹽溶鹽析、免疫沈澱法以及親和層析純化硫氫氧化酶, (4)將純化完成的硫氫氧化酶和精子共同培養觀察硫氫氧化酶對精子造成的影響。 結果與結論:(1)在小鼠和豬的雄性生殖道,硫氫氧化酶-1C(QSOX1C)主要分佈 表現在管腔內的分泌液,而硫氫氧化酶-2(QSOX2)以囊狀小泡分佈在上皮細胞前端,(2)透過離子交換法成功純化小鼠儲精囊分泌液中的硫氫氧化酶-1C 可使小鼠 精子產生顯著凝集現象,(3)兩型硫氫氧化酶在雄性生殖道不同區域的表現,可能 代表其在精子成熟過程以及受精前參與的角色不同。透過優化兩型硫氫氧化酶的純 化步驟,未來取得大量純化蛋白後,能夠更深入探討硫氫氧化酶和精子的交互作用, 以了解硫氫氧化酶於精子繁殖生理之影響。

In Taiwan, the number of infertile patients seeking for medical assistance via assisted reproductive technology (ART) increases exponentially each year. Despite ART has been used to achieve pregnancy, discrepancies between up-going demands on ART and down- going fertility rate might be explained partly by the lack of thorough evaluations on gamete quality. The commonly used sperm quality assessment systems measure sperm concentration and sperm motility-based parameters without monitoring the non-motility related factors (e.g. sperm DNA or chromosomal abnormality, sperm membrane integrity...etc.). Male infertility is the major cause of conception failure in about 25% of all infertile couples. Many proteins have been proven to be the sperm motility (e.g. spermatic protein DDX4/VASA) or quality marker within the seminal fluid or on the sperm membrane. This research focus on one of the seminal plasma enriched and epididymal segment-specific proteins namely Quiescin Q6 Sulfhydryl Oxidase (QSOX). Sulfhydryl oxidase belongs to the family of oxidoreductase, containing the sulfhydryl (- SH) functional group and can catalyze oxidation of –SH group into stable disulfides (S-S) with the reduction of hydrogen and hydrogen peroxidase (thio-disulfide exchange). There are two isoforms of QSOX express in mammalian cells: QSOX1 and QSOX2. Both QSOX1 and QSOX2 express in most tissues, but QSOX2 is much less abundant than QSOX1. QSOX2 shares 40% identity with QSOX1 having the same overall domain structure and containing a transmembrane domain that confers membrane localization. To investigate the functions of QSOXs on sperm membrane surface modifications, we first purified endogenous QSOX1C proteins from both mouse and porcine. Mouse QSOX1C was purified from seminal vesicle secretions by a series of purification steps including ion exchange chromatography on a DEAE-Sephacel column and ion exchange high- performance liquid chromatography on a sulfopropyl column. Porcine QSOX1C was purified from seminal vesicle and epididymis tissue homogenates as well as from the seminal fluids by ammonium sulfate protein precipitation in combination with antibody- affinity column. To minimize the use of animals, we also established stable cell line overexpressed polyhistidine- and V5-tagged recombinant human QSOX2 using HEK293. A 65 kDa QSOX1C was detected in mouse, while two distinct bands at 50 and 75 kDa were observed in porcine. QSOX2 presents in a vacuole-like structure at the apical region of the epithelium in the reproductive tracts and the amount varies from caput to cauda of mouse and porcine epididymis. Moreover, mouse QSOX1C increases the formation of sperm aggregates by constant recruiting surrounding free-swimming sperm. By successful establishment of stable cell line expresses recombinant human QSOX2 and the natural QSOX1C, we could have the ability to understand the interactions of QSOXs and spermatozoa in the reproductive tract.