探討於雄性生殖系統中 QSOX2 對於氧化壓力之角色

Abstract

氧化壓力 (oxidative stress) 源自於抗氧化能力與活性氧物質 (ROS) 之間量與活性的失衡。雖然適量的 ROS 能維持許多正常的生理功能，然而過多的 ROS會導致脂質、蛋白質及 DNA 之氧化傷害。精子帶有來自父系的遺傳訊息，但是精細胞膜富含多元不飽和脂肪酸以及極少量的細胞質，造成內生性抗氧化能力與相關修復蛋白不足，導致精細胞非常容易受到氧化壓力傷害。因此雄性生殖器官-睪丸與附睪之抗氧化與修復氧化傷害的能力至關重要。Quiescin-sulfhydryl oxidase 2 (QSOX2) 能夠利用硫醇 (thiol) 形成雙硫鍵，並將氧氣代謝為過氧化氫(hydrogen peroxide)，過氧化氫作為內生性抗氧化酵素的受質。然而，QSOX2在雄性生殖系統中是否扮演調控氧化能力的角色尚未明確，因此本研究欲使用 Qsox2 基因剔除的小鼠 (QSOX2-/-)，透過 cisplatin 誘導產生系統性氧化壓力之動物模式以探討 QSOX2 於 ROS 產生與雄性生殖系統抗氧化能力的作用。本研究結果發現 Qsox2 基因剔除小鼠與野生型小鼠相比，具有相似的睪丸重量與組織型態，但於附睪中觀察到上皮細胞有型態上（高度）的改變。在 cisplatin 誘導氧化壓力後，Qsox2 基因剔除小鼠相較野生型小鼠出現較嚴重的睪丸損傷，並伴隨著附睪管腔縮小。此外，我們也觀察到Qsox2 基因剔除小鼠其DNA氧化傷害的感受性提升以及 γ-H2AX 的趨化作用受損。雖然本研究目前因樣本數目不足，於統計學上不具有顯著差異，但上述結果皆顯示 Qsox2 基因剔除對於小鼠生殖系統調控氧化壓力具有負面的影響。另外，我們在 cisplatin 誘導氧化壓力後也發現野生型與 Qsox2 基因剔除小鼠在負責調控homologous recombination (HR) 與 non-homologous end joining (NHEJ) 之DNA 損傷調節蛋白（mediator of DNA damage checkpoint 1, MDC1) 及Ki-67 之表現皆出現受損情形，顯示 cisplatin導致生精作用下降，且在Qsox2 基因剔除小鼠身上更為嚴重。綜上所述我們證實 cisplatin 對於生殖系統之傷害，同時也發現Qsox2 基因剔除可能會增加 cisplatin 對於睪丸的傷害、改變附睪型態以及增加 DNA 氧化傷害易感性。

Oxidative stress (OS) originates from the imbalance between the capacity of antioxidant enzymes and the production of reactive oxygen species (ROS). The proper level of ROS is essential for biological reactions; however, the overproduced ROS would cause oxidative damage in lipids, proteins, and DNA. Spermatozoa keep genetic material from the male and are susceptible to oxidative stress due to the high proportion of polyunsaturated fatty acid content in the plasma membrane and restricted repair proteins and antioxidant enzymes owing to the minimal cytoplasm. Testis and epididymis as the reproductive organ responsible for producing functional fertile sperm, the repair and antioxidant capacity in these organs are crucial to defense against oxidative stress-induced damage. Quiescin-sulfhydryl oxidase 2 (QSOX2) catalyzes thiol groups into disulfide bonds and transforms the oxygen molecule into hydrogen peroxide, which as a substrate for the endogenous antioxidant enzyme system; however, the role of QSOX2 in the regulation of oxidative stress in the male reproductive system is unclear. Therefore, we used Qsox2-/- mice and cisplatin administration to create systemic oxidative stress and explore the functional involvement of QSOX2 in regulating the homeostasis between ROS production and antioxidant capacity. In this study, the Qsox2 knockout mice exhibit a similar testis weight and histology but altered epididymal epithelium height. With cisplatin-induced oxidative damage, we observed severe testicular lesions and shrinkage of epididymal tubule in the Qsox2 knockout mice. Furthermore, we observed that Qsox2-/- mice are more sensitive to oxidative stress-induced DNA damage and showed impaired recruitment of γ-H2AX. These results showed a clear trend of negative outcomes in Qsox2 knockout in mice upon ROS attacks, despite the fact that we could not confirm with the statistical differences due to a low sample number. Under the cisplatin administration, we detected severe damage in DNA damage response in both wild-type and Qsox2-/- mice. The mediator of DNA damage checkpoint 1 (MDC1), which regulates the repair mechanism homologous recombination (HR) and non-homologous end joining (NHEJ), showed an intensive decrease in protein expression under cisplatin administration. In line with our finding of the proliferation ability in spermatogonia, Ki-67 decreased excessively in the same trend as MDC1 expression, which indicates the decline of spermatogenesis. In this study, using Qsox2-/- mice, we showed the role of QSOX2 under oxidative stress conditions and also confirm the cisplatin-induced damage in the male reproductive system. We demonstrated that Qsox2 knockout exacerbates cisplatin-induced damage in the testis, alters the epididymis morphology, and increases the sensitivity of oxidative DNA damage in the testis. Nevertheless, the limited sample number in the present study led to no solid conclusions at this moment, yet, we still detected the potential critical effect of QSOX2 in the regulation of oxidation balance in the male reproductive system.