As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus oocyte complexes (COCs) were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at Metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce Metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential spindle assembly checkpoint protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step towards improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.

中文翻译：

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溶酶体组织蛋白酶 B 的玻璃化诱导激活扰乱小鼠卵母细胞中的纺锤体组装检查点功能。

随着生育年龄的增加并与不孕症相关，女性配子的冷冻保存在 ART 中变得司空见惯。然而，玻璃化卵母细胞的发育能力仍然很低。导致玻璃化后卵母细胞质量降低的潜在机制在很大程度上是未知的。小鼠卵丘卵母细胞复合物 (COC) 使用冷冻环技术和二甲基亚砜、乙二醇和海藻糖的混合物作为冷冻保护剂进行玻璃化。比较新鲜和玻璃化/解冻卵母细胞的染色体排列、纺锤体形态、动粒-微管附着、纺锤体组装检查点 (SAC) 和非整倍体。虽然大多数玻璃化卵母细胞挤出第一极体（PB），但它们的染色体错位显着增加，中期 II 的异常纺锤体形成和非整倍体。与对照相比，玻璃化的卵母细胞在诺考达唑和依托泊苷存在的情况下挤出第一个 PB，这应该以 SAC 依赖性方式诱导中期 I 停滞。有丝分裂停滞缺陷 2 (MAD2)（一种必需的纺锤体组装检查点蛋白）在玻璃化卵母细胞中的动粒处的荧光强度降低，表明 SAC 在玻璃化/解冻后减弱。此外，我们发现玻璃化相关的压力会破坏溶酶体功能并刺激组织蛋白酶 B 活性，随后激活 caspase 3。用组织蛋白酶 B 或 caspase 3 抑制剂处理后，玻璃化卵母细胞中的 MAD2 定位和 SAC 功能得以恢复。这项研究是使用小鼠卵母细胞进行的，因此，在将这些发现应用于 IVF 诊所之前，在人类卵母细胞中确认这些结果是先决条件。在这里，我们发现了有助于理解玻璃化冷冻如何影响卵母细胞质量的潜在分子途径。调节这些途径将是提高玻璃化冷冻后卵母细胞质量的一步，并有可能提高玻璃化冷冻程序的效率。