冷冻保存对精子质膜蛋白PLCζ的损伤作用

The detrimental effect of cryopreservation on human sperm surface protein⁃PLCζ

目的 比较精液冷冻前、复苏后精子参数以及精子质膜蛋白磷脂酶C-zeta(Phospholipase C-zeta, PLCζ)表达水平的变化,探究冷冻保存对人类精子PLCζ的损伤作用。方法 选取2022年2月—2023年7月在本中心进行辅助生殖技术(Assisted reproductive technology, ART)治疗的不育男性患者30份正常精液标本。将每份精液均分为冻前新鲜组和复苏后冷冻组。比较分析冷冻前、复苏后精子的活力(PR级),存活率和形态变化;密度梯度离心处理两组精液标本,通过免疫荧光和Western blot技术检测精子PLCζ表达水平。结果 与新鲜精子相比,冷冻保存明显降低精子的前向运动精子百分率(31.8%±5.5%)vs(59.3%±7.4%)(P<0.01),精子活动率(38.7%±4.6%)vs(69.07%±6.7%)(P<0.01)和精子存活率(48.2%±6.1%)vs(83.4%±7.2%)(P<0.01),精子正常形态率也发生改变(3.4%±0.7%)vs(12.4%±3.3%)(P<0.01),尾部卷曲的异常形态比例增多;冷冻复苏精子PLCζ荧光免疫反应减弱(33.08±9.74)vs(114.1±12.92)(P<0.01),染色为散在红色点状荧光,且特征性的染色定位缺失;Western blot实验中,精子PLCζ蛋白表达明显减弱。结论 冷冻保存不仅对精子参数有负面影响,同时对精子质膜蛋白PLCζ有损伤作用。

Objective To comparatively analyze the changes in sperm parameters and sperm surface protein⁃phospholipase C⁃zeta(PLCζ)expression between fresh and frozen⁃thawed semen samples,and explore the detrimental effect of cryopreservation on human sperm PLCζprotein.Methods Thirty normal semen samples from infertile male patients who received assisted reproductive technology(ART)in our reproductive center from February 2022 to July 2023 were collected in this study.Each semen sample was divided into a fresh group before freezing and a frozen group after resuscitation.Sperm parameters of fresh and frozen⁃thawed semen samples including progressive and total motility,viability and morphology were examined.After semen samples of two groups were treated using density⁃gradient centrifuging,their PLCζexpressions were detected by immunofluorescence and Western blot.Results Compared with those of fresh semen samples,progressive motility(31.8%±5.5%)vs(59.3%±7.4%)(P<0.01)and total motility(38.7%±4.6%)vs(69.07%±6.7%)(P<0.01),viability(48.2%±6.1%)vs(83.4%±7.2%)(P<0.01),and normal morphology(3.4%±0.7%)vs(12.4%±3.3%)(P<0.01)of frozen⁃thawed semen samples were all significantly decreased,whereas the number of tail defects,such as coiled and looped tails was obviously increased.A significant decrease or absence in PLCζexpression and distribution was found in frozen⁃thawed sperms(33.08±9.74)vs(114.1±12.92)(P<0.01)and the protein expression of PLCζwas also reduced.Conclusion Our results suggest the cryopreservation has not only has detrimental effects on sperm parameters but also downregulates protein expression of PLCζ.