Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, Spain.
Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
Biol Reprod. 2019 Apr 1;100(4):1090-1107. doi: 10.1093/biolre/ioy241.
We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 μM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.
我们假设巯基，特别是谷胱甘肽（GSH）对于调节种马精子功能至关重要。为了验证这一假设，我们最初研究了精子功能与 GSH 含量之间的关系，发现 GSH 与精子活力、运动性和速度参数之间存在高度显著的相关性（P<0.001）。此外，使用 menadione 和 1,3 二甲氧基 1,4,萘醌（DMNQ）耗尽 GSH 会导致精子膜受到损害，但添加半胱氨酸（但不是其他抗氧化剂）可以防止这种情况。半胱氨酸孵育可防止 menadione 和 DMNQ 在孵育 1 小时后（P<0.001；P<0.05）和 3 小时后（P<0.001，P<0.05）诱导的精子膜损伤。半胱氨酸孵育可减轻 menadione 和 DMNQ 诱导的 4-羟基壬烯醛（P<0.001）的增加。由于半胱氨酸是 GSH 的前体，我们假设种马精子能够使用外源性半胱氨酸合成这种三肽。为了验证这一假设，我们研究了合成 GSH（GSH 和 GCLC）所需的两种酶的存在，并通过 Western 印迹和免疫细胞化学检测到这两种酶都存在于种马精子中。GCLC 的抑制作用降低了耗尽后添加半胱氨酸时 GSH 的恢复，表明种马精子可能使用外源性半胱氨酸来调节 GSH。其他支持这一假设的发现是 BSO 处理后精子功能的变化以及使用 HPLC-MS 验证的 GSH 和 GSSG 的变化，表明 BSO 阻止了在添加半胱氨酸时 GSH 的增加，尽管发生了重要的种马间变异性，并且表明谷氨酸半胱氨酸连接酶的表达存在差异。种马精子中 GSH 的平均浓度为 8.2±2.1 μM/109 个精子，远高于其他哺乳动物报道的每十亿个精子纳摩尔范围内的浓度。
