Gallardo Bolaños J M, Balao da Silva C, Martín Muñoz P, Plaza Dávila M, Ezquerra J, Aparicio I M, Tapia J A, Ortega Ferrusola C, Peña F J
Laboratory of Equine Reproduction and Equine Spermatology, Faculty of Veterinary Medicine, University of Extremadura, Cáceres, Spain.
Faculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
Reprod Domest Anim. 2014 Aug;49(4):657-664. doi: 10.1111/rda.12343. Epub 2014 Jun 13.
To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h decrease in the percentages of motile and progressive motile sperm occurred. The decrease observed in sperm functionality after 6 h of incubation was accompanied by a significant increase in the production of hydrogen peroxide and the greatest increase in caspase 3 activity. Additionally, the percentage of phosphorylated Akt reached a minimum after 6 h of incubation. These results provide evidences that sperm death during in vitro incubation is largely an apoptotic phenomena, probably stimulated by endogenous production of hydrogen peroxide and the lack of prosurvival factors maintaining Akt in a phosphorylated status. Disclosing molecular mechanisms leading to sperm death may help to develop new strategies for stallion sperm conservation.
为了研究射精后诱导精子死亡的机制，将种马精液在37°C的BWW培养基中孵育6小时。在孵育期开始时以及1、2、4和6小时后，评估精子活力和运动学参数（计算机辅助精子分析）、线粒体膜电位以及膜通透性和完整性（流式细胞术）。此外，在相同的时间间隔，评估活化的半胱天冬酶3、过氧化氢、超氧阴离子（流式细胞术）和Akt磷酸化（流式细胞术）。孵育6小时后精子功能出现显著下降，不过在孵育1小时后，活动精子和进行性活动精子的百分比就出现了下降。孵育6小时后观察到的精子功能下降伴随着过氧化氢产生的显著增加以及半胱天冬酶3活性的最大增加。此外，孵育6小时后磷酸化Akt的百分比达到最低。这些结果表明，体外孵育期间精子死亡在很大程度上是一种凋亡现象，可能是由过氧化氢的内源性产生以及缺乏将Akt维持在磷酸化状态的生存因子所刺激。揭示导致精子死亡的分子机制可能有助于开发种马精子保存的新策略。
