Gualtieri Roberto, Iaccarino Mirella, Mollo Valentina, Prisco Marina, Iaccarino Stefania, Talevi Riccardo
Department of Structural and Functional Biology, University of Naples Federico II, Monte S Angelo, Naples, Italy.
Fertil Steril. 2009 Apr;91(4):1023-34. doi: 10.1016/j.fertnstert.2008.01.076. Epub 2008 Mar 25.
To identify the damages caused by slow cooling human metaphase II (MII) oocytes comparing the ultrastructure, inner mitochondrial membrane potential (DeltaPsim), and apoptotic status of fresh and cryopreserved oocytes.
Experimental study.
University biology research unit and private IVF unit.
PATIENT(S): Fresh and cryopreserved supernumerary MII oocytes donated from women undergoing IVF cycles.
MAIN OUTCOME MEASURE(S): Ultrastructure was assessed by transmission electron microscopy (TEM), mitochondrial function by means of the fluorescent DeltaPsim reporter JC-1, and apoptotic status through fluorescent labeling with the pan-caspase inhibitor fluorescein isothiocyanate conjugate (FITC)-VAD FMK, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
RESULT(S): Compared to fresh oocytes, frozen/thawed (F/T) oocytes showed reduced cortical granule densities (F/T 3.35 +/- 1.94/10 microm vs. fresh 10.30 +/- 3.9/10 microm), swelling of smooth endoplasmic reticulum (F/T 0.084 +/- 0.03 microm(2) vs. fresh 0.040 +/- 0.02 microm(2)), decreased electron density of the mitochondrial matrix and damage to the mitochondrial membranes, low DeltaPsim of pericortical mitochondria, but no signs of apoptosis.
CONCLUSION(S): Slow cooling is associated with cortical granule exocytosis, swelling of smooth endoplasmic reticulum vesicles, and mitochondrial damage, but does not induce early or late apoptotic events. The observed injuries might be responsible for the reduced developmental competence of cryopreserved oocytes.
通过比较新鲜和冷冻保存的人中期 II（MII）卵母细胞的超微结构、线粒体内膜电位（ΔΨm）和凋亡状态，确定缓慢冷却对其造成的损害。
实验研究。
大学生物学研究单位和私立体外受精单位。
接受体外受精周期的女性捐赠的新鲜和冷冻保存的多余 MII 卵母细胞。
通过透射电子显微镜（TEM）评估超微结构，利用荧光 ΔΨm 报告染料 JC-1 评估线粒体功能，通过用泛半胱天冬酶抑制剂异硫氰酸荧光素缀合物（FITC）-VAD FMK 进行荧光标记以及末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记评估凋亡状态。
与新鲜卵母细胞相比，冻融（F/T）卵母细胞显示皮质颗粒密度降低（F/T 为 3.35±1.94/10μm vs.新鲜为 10.30±3.9/10μm），滑面内质网肿胀（F/T 为 0.084±0.03μm² vs.新鲜为 0.040±0.02μm²），线粒体基质电子密度降低以及线粒体膜受损，皮质周线粒体的 ΔΨm 较低，但无凋亡迹象。
缓慢冷却与皮质颗粒胞吐、滑面内质网囊泡肿胀和线粒体损伤有关，但不诱导早期或晚期凋亡事件。观察到的损伤可能是冷冻保存的卵母细胞发育能力降低的原因。
