Martin Guillaume, Sabido Odile, Durand Philippe, Levy Rachel
Laboratoire de Biologie de la Reproduction-GIMAP, Hopital Nord, 42055 Saint-Etienne, France.
Biol Reprod. 2004 Jul;71(1):28-37. doi: 10.1095/biolreprod.103.024281. Epub 2004 Feb 18.
Cryopreservation induces many changes in sperm cells, including membrane disorders and cell death. We tested the hypothesis that apoptosis, a form of programmed cell death, can contribute to the fatal effect of cryopreservation on sperm cells. A multiparametric study of apoptosis on bovine sperm is proposed, using flow cytometry, including mitochondrial membrane potential (DeltaPsi(m)), caspase activation, membrane permeability, nucleus condensation, DNA fragmentation, and phosphatidylserine (PS) externalization. The relevance of each test was first validated on a human somatic cell line, U937. Cryopreservation and/or thawing induced significant changes in all apoptotic markers in living bull sperm cells except those concerning the nucleus. After cryopreservation, 44.9% +/- 17% (vs. 11.3% +/- 10.6% before cryopreservation) of sperm cells showed low DeltaPsi(m), 12% +/- 6.3% (vs. 2.2% +/- 1.0% before) contained active caspases, and 10.8% +/- 5.8% (vs. 1.4% +/- 1.1% before) exhibited high membrane permeability. However, cryopreservation had no effect on DNA fragmentation (9.1% +/- 7.7% before vs. 11.1% +/- 5.7% after cryopreservation) or on nucleus condensation (46% +/- 12.7% before vs. 43.8% +/- 13.1% after). Cryopreservation acts as an apoptotic mechanism inducer in bovine sperm cells, where the earliest but not the latest features of cells undergoing apoptosis occur. We have named this abortive process an apoptosis-like phenomenon.
冷冻保存会在精子细胞中引发许多变化，包括膜紊乱和细胞死亡。我们检验了这样一个假设，即凋亡（一种程序性细胞死亡形式）可能导致冷冻保存对精子细胞产生致命影响。本研究提出使用流式细胞术对牛精子凋亡进行多参数分析，包括线粒体膜电位（ΔΨm）、半胱天冬酶激活、膜通透性、细胞核凝聚、DNA片段化以及磷脂酰丝氨酸（PS）外化。每个检测方法的相关性首先在人U937体细胞系上得到验证。冷冻保存和/或解冻在活的公牛精子细胞中引起了所有凋亡标志物的显著变化，但与细胞核相关的标志物除外。冷冻保存后，44.9%±17%（相对于冷冻保存前的11.3%±10.6%）的精子细胞显示出线粒体膜电位降低，12%±6.3%（相对于之前的2.2%±1.0%）含有活性半胱天冬酶，10.8%±5.8%（相对于之前的1.4%±1.1%）表现出高膜通透性。然而，冷冻保存对DNA片段化（冷冻保存前为9.1%±7.7%，冷冻保存后为11.1%±5.7%）或细胞核凝聚（冷冻保存前为46%±12.7%，冷冻保存后为43.8%±13.1%）没有影响。冷冻保存可作为牛精子细胞凋亡机制的诱导剂，在此过程中，细胞凋亡的早期而非晚期特征会出现。我们将这个流产的过程命名为凋亡样现象。
