Centrifugation stress reduces the responsiveness of spermatozoa to a capacitation stimulus in in vitro-aged semen
Centrifugation stress reduces the responsiveness of spermatozoa to a capacitation stimulus in in vitro-aged semen
复制标题
DOI：
10.1111/andr.12064
复制
发表时间：
2015-09-01
期刊：
影响因子：
4.5
通讯作者：
Waberski, D.
中科院分区：
文献类型：
--
作者：
Henning, H.;Ngo, T. T.;Waberski, D.
精液密度梯度离心通常用于许多辅助生殖技术。尽管梯度分离具有分离和富集活动精子和活精子的潜力，但离心力对细胞器和细胞膜产生了应力因素。本研究的目的是评价密度梯度离心应激对精子获能动力学、细胞稳定性和精子对体外老化延长精液中碳酸氢盐特异性反应的能力的影响。将延长的公猪精液（n = 7）分别在17 ℃下储存12、24、72和120 h，然后离心并在体外获能培养基的变体中孵育。通过计算机辅助精液分析评估的活精子、顶体完整精子的数量和活力参数在储存期间没有变化。活力（质膜完整性）和细胞内钙离子水平（钙离子流入）在体外获能过程中的动力学变化进行了评估后，精液样品的制备与Percoll和蔗糖梯度离心，无论是只有Percoll，只有蔗糖离心或没有离心。测定了可明确归因于对碳酸氢盐或钙的反应的活精子群的变化。在体外老化（> 12 h储存）的精子中，离心降低了对获能刺激碳酸氢盐特异性反应的精子比例。同时，离心增加了精子的比例响应钙在不存在碳酸氢盐，从而表明增加了敏感性孵育本身。在精液制备过程中没有离心步骤，揭示了体外老化精子对碳酸氢盐特异性反应的高度保守能力。总之，密度梯度离心改变了精子的生理特性，以进行受控获能，这可能会影响辅助生殖技术中离心精液的成功率，并混淆获能试验的解释。
Density gradient centrifugation of semen is commonly used in many assisted reproduction techniques. Although gradients have the potential to isolate and enrich motile and viable spermatozoa, the centrifugation force presents a stress factor to cell organelles and membranes. The objective of the study was to evaluate the impact of density gradient centrifugation stress on sperm capacitation dynamics, cell stability and the ability of spermatozoa to specifically respond to bicarbonate in extended semen undergoing in vitro ageing. Extended boar semen (n = 7) was stored for 12, 24, 72 and 120 h respectively at 17 degrees C before centrifugation and incubation in variations of an in vitro capacitation medium. The number of viable, acrosome intact sperm and motility parameters as assessed by computer-assisted semen analysis did not change during storage. Kinetic changes in viability (plasma membrane integrity) and intracellular calcium levels (calcium influx) during in vitro capacitation were assessed after preparation of semen samples with both, a Percoll and a sucrose gradient centrifugation, either only Percoll, only sucrose centrifugation or no centrifugation. Changes in the viable sperm population that could be specifically attributed as a response to either bicarbonate or calcium were determined. In in vitro-aged (> 12 h stored) spermatozoa, centrifugation reduced the proportion of spermatozoa which specifically responded to the capacitating stimulus bicarbonate. Concomitantly, centrifugation increased the proportion of spermatozoa responding to calcium in absence of bicarbonate, thus indicating an increased sensitivity to incubation per se. Absence of centrifugation steps during semen preparation, revealed a highly conserved ability of in vitro-aged spermatozoa to specifically respond to bicarbonate. In conclusion, density gradient centrifugation alters the physiological property of spermatozoa for controlled capacitation, which may influence the success rates of centrifuged semen in assisted reproductive technologies and confound interpretation of capacitation assays.