Plasminogen activator inhibitor 1 and Antipain preserve acrosome integrity of bovine spermatozoa during cryopreservation

ABSTRACT Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.

摘要：精浆中含有丝氨酸蛋白酶（serine proteases）与丝氨酸蛋白酶抑制剂（serine protease inhibitor），二者均参与哺乳动物受精过程，其中该类抑制剂可用于预防冷诱导的精子获能。本研究以纤溶酶原激活物抑制剂1（Plasminogen activator inhibitor 1, PAI-1）（设置70ng、140ng及210ng三个浓度梯度）与抗痛素（Antipain）（设置10μg、50μg及100μg三个浓度梯度）这两种丝氨酸蛋白酶抑制剂作为牛精液冷冻保存稀释液的添加剂，分析了不同浓度抑制剂对牛精液冷冻保存效果的影响。随后，本研究在解冻后的精液样本中评估了上述抑制剂对各项精子参数的作用，包括精子运动学参数（计算机辅助精子分析，CASA）、顶体完整性、质膜完整性、线粒体膜电位、精子畸形率及顶体反应率。结果显示，采用添加抗痛素的方案进行精子冷冻保存，会降低解冻后精子的MP、VSL、LIN、SRT运动参数以及超激活精子占比；而210ng浓度的PAI-1仅会降低VSL与LIN两项参数。抗痛素与PAI-1对质膜完整性、线粒体膜电位及精子畸形率无显著影响。当添加70ng与140ng浓度的抗痛素及PAI-1时，冷冻保存的精子可维持顶体完整性，且能够完成体外顶体反应。综上，丝氨酸蛋白酶抑制剂抗痛素与70ng、140ng浓度的PAI-1可有效维持冷冻保存牛精子的顶体完整性。