Department of Biotechnology, BIT-Campus, Anna University, Tiruchirappalli, Tamil Nadu, India.
Department of Bioinformatics, Alagappa University, Karaikudi, Tamil Nadu, India.
Mol Hum Reprod. 2022 Aug 29;29(9). doi: 10.1093/molehr/gaac025.
During ejaculation, cauda epididymal spermatozoa are suspended in a protein-rich solution of seminal plasma, which is composed of proteins mostly secreted from the seminal vesicle. These seminal proteins interact with the sperm cells and bring about changes in their physiology, so that they can become capacitated in order for the fertilization to take place. Sulfhydryl oxidase (SOX) is a member of the QSOX family and its expression is found to be high in the seminal vesicle secretion (SVS) of mouse. Previously, it has been reported to cross-link thiol-containing amino acids among major SVS proteins. However, its role in male reproduction is unclear. In this study, we determined the role of SOX on epididymal sperm maturation and also disclosed the binding effect of SOX on the sperm fertilizing ability in vitro. In order to achieve the above two objectives, we constructed a Sox clone (1.7 kb) using a pET-30a vector. His-tagged recombinant Sox was overexpressed in Shuffle Escherichia coli cells and purified using His-Trap column affinity chromatography along with hydrophobic interaction chromatography. The purified SOX was confirmed by western blot analysis and by its activity with DTT as a substrate. Results obtained from immunocytochemical staining clearly indicated that SOX possesses a binding site on the sperm acrosome. The influence of SOX on oxidation of sperm sulfhydryl to disulfides during epididymal sperm maturation was evaluated by a thiol-labeling agent, mBBr. The SOX protein binds onto the sperm cells and increases their progressive motility. The effect of SOX binding on reducing the [Ca2+]i concentration in the sperm head was determined using a calcium probe, Fluo-3 AM. The inhibitory influence of SOX on the sperm acrosome reaction was shown by using calcium ionophore A32187 to induce the acrosome reaction. The acrosome-reacted sperm were examined by staining with FITC-conjugated Arachis hypogaea (peanut) lectin. Furthermore, immunocytochemical analysis revealed that SOX remains bound to the sperm cells in the uterus but disappears in the oviduct during their transit in the female reproductive tract. The results from the above experiment revealed that SOX binding onto the sperm acrosome prevents sperm capacitation by affecting the [Ca2+]i concentration in the sperm head and the ionophore-induced acrosome reaction. Thus, the binding of SOX onto the sperm acrosome may possibly serve as a decapacitation factor in the uterus to prevent premature capacitation and acrosome reaction, thus preserving their fertilizing ability.
在射精过程中，附睾尾部的精子悬浮在富含蛋白质的精液中，这些蛋白质主要来自精囊的分泌。这些精液蛋白与精子相互作用，改变精子的生理状态，使精子获能，从而发生受精。硫氧还蛋白氧化酶（SOX）是 QSOX 家族的一员，其在小鼠精囊分泌物（SVS）中的表达水平较高。先前的研究表明，它可以使 SVS 中的含硫氨基酸之间发生交联。然而，其在男性生殖中的作用尚不清楚。在这项研究中，我们确定了 SOX 在附睾精子成熟过程中的作用，并揭示了 SOX 在体外对精子受精能力的结合效应。为了实现上述两个目标，我们使用 pET-30a 载体构建了 Sox 克隆（1.7kb）。His 标记的重组 Sox 在 Shuffle 大肠杆菌细胞中过表达，并通过 His-Trap 柱亲和层析和疏水相互作用层析进行纯化。通过 Western blot 分析和 DTT 作为底物的活性对纯化的 SOX 进行了确认。免疫细胞化学染色的结果清楚地表明，SOX 在精子顶体上具有结合位点。通过巯基标记剂 mBBr 评估 SOX 对附睾精子成熟过程中精子巯基氧化为二硫键的影响。SOX 蛋白与精子结合并增加其运动能力。通过钙探针 Fluo-3 AM 测定 SOX 结合对精子头部[Ca2+]i 浓度的影响。通过使用钙离子载体 A32187 诱导顶体反应来显示 SOX 对顶体反应的抑制作用。用 FITC 标记的花生凝集素（花生）染色检查顶体反应的精子。此外，免疫细胞化学分析显示，SOX 在雌性生殖道转运过程中，在子宫中与精子结合，但在输卵管中消失。上述实验结果表明，SOX 与精子顶体结合通过影响精子头部的[Ca2+]i 浓度和离子载体诱导的顶体反应，阻止精子获能。因此，SOX 与精子顶体的结合可能作为子宫中的脱能因子，防止精子过早获能和顶体反应，从而保持其受精能力。
