钙离子介导的卵母细胞冻融性损伤及拮抗剂治疗研究

Zona pellucida hardening, DNA injury and spindle abnormality induced by oocyte cryopreservation are closely related with low fertilization of cryopreserved oocytes through in vitro fertilization (IVF). The recent studies showed these injuries were mainly contributed by the increase of Ca2+ content of oocytes. In this study, in order to understand the exact contribution mechanism and further inhibit the increase of Ca2+ content and improve these injuries, the exact roles of the release of endogenic calcium and influx of exogenous Ca2+ for zona pellucida hardening, DNA injury and spindle abnormality are firstly investigated. Then, the specific calcium ion antagonist would be selected and the corresponding inhibition role for the increase of Ca2+ content, alleviation of those injuries and improvement of fertilization are studied. Meanwhile, the study also includes the toxicity of calcium ion antagonist on oocyte developmental potential. This study would provide theoretical basis and technique support for the improvement of the result of oocyte freeze-thaw protocol and fertilization of cryopreserved oocytes. Meanwhile, it is beneficial to reduce the application and its corresponding risk of intracytoplasmic sperm injection (ICSI) technology in cryopreserved oocyte and the medical fee of patients. Furthermore, it would lay a foundation for the clinical application of oocyte cryopreservation.

卵母细胞冷冻保存引起的透明带硬化、DNA损伤以及纺锤体异常等损伤与冻融卵母细胞体外受精（IVF）能力下降密切相关，而冷冻过程中细胞内钙离子浓度异常是诱发这些损伤的重要原因。本项目拟通过研究冷冻中卵母细胞内钙池钙离子释放和细胞外钙离子内流变化及其与透明带硬化、DNA损伤以及纺锤体异常的相关性，揭示钙离子浓度升高导致卵母细胞冻融性损伤的机制；进而选用特异性钙离子拮抗剂，研究其对卵母细胞内钙离子浓度升高的抑制作用以及对卵母细胞冷冻损伤和冷冻复苏后细胞质量的改善作用；同时进行钙离子拮抗剂对卵母细胞发育潜力毒性的研究。本项目的实施可为改善冷冻复苏方案、提高冻融卵母细胞IVF受精能力提供理论依据和技术支持，减少卵胞浆内单精子注射（ICSI）技术在冻融卵母细胞受精中的应用，降低因ICSI技术带来的各种风险，同时也可减少病人治疗费用，进一步为卵母细胞冷冻保存的临床应用奠定基础。

本研究主要针对卵母细胞冷冻保存后冷冻损伤造成的透明带硬化和受精能降低等问题进行研究玻璃化冷冻能有效实现卵母细胞冷冻保存，但在冷冻中仍存在卵母细胞发育潜力和超微结构损伤等问题，损伤机制尚不清楚，尤其对细胞内钙离子变化与损伤之间的关系研究较少。本研究主要以小鼠卵母细胞作为材料，对玻璃化冷冻导致卵母细胞受精损伤及钙离子抑制剂2-APB和钙离子耗竭剂TG诱导的细胞内钙离子变化对卵母细胞超微结构、发育能力影响进行了研究，结果显示玻璃化冷冻可使卵母细胞通过IVF受精的正常受精率降低（23.53% vs. 70.00%）（P＜0.001)，降低穿透透明带精子数(2.27±0.95 vs. 1.53±0.88)( P＜0.05)；冷冻前后卵母细胞膜CD9蛋白I、II型在冷冻复苏卵母细胞显著低于新鲜组和冷冻保护剂处理组（1.1% vs.100%；68.4% vs. 22.9%）；扫描电镜对卵母细胞细胞透明带超微结构进行检测，玻璃化冷冻可显著改变透明带超微结构，使粗面透明带在玻璃化冷冻组显著低于新鲜和冷冻保护剂处理组 (44.8% vs. 92.9% and 84.8%)（P＜0.001) ；通过透射电镜观察卵母细胞冷冻前后的超微结构变化，结果发现冻后MII期卵母细胞透明带、微绒毛、细胞膜损伤甚至消失、皮质区皮质颗粒分布减少，脂滴部分溶解，脂滴周围伴随大量肿胀呈圆形的线粒体；而单独TG处理不经过冷冻的卵母细胞即可导致大部分微绒毛消失，皮质颗粒减少，线粒体聚集和卵周隙变窄；TG处理并冷冻卵母细胞进一步卵母细胞超微结构损伤；2-APB处理缓解冷冻造成的卵母细胞超微结构损伤。TG处理冷冻组精子穿透数低于常规冷冻组（1.17±1.03 vs. 2.75±1.29）（P＜0.05),而2-APB处理则可改善冷冻后精子穿透（4.17±1.11 vs. 2.75±1.29）（P＜0.05)；卵母细胞内钙瞬变研究显示冷冻保护剂可诱发卵母细胞钙离子瞬时升高，其稳态水平亦高于对照组，TG使瞬时钙离子浓度升高峰值和稳态水平高于冷冻保护剂组，而2-APB可有效稳定卵母细胞内钙离子水平，使钙瞬变峰值和稳态水平值低于冷冻保护剂所诱发钙离子变化，且在稳态水平，其钙离子浓度更接近对照组钙离子水平。研究提示玻璃化冷冻造成的卵母细胞损伤与细胞内钙离子浓度升高有关，采用内质网钙离子抑制剂可稳定细胞内钙离子变化并缓解冷冻损。