Inhibition of Apoptotic Pathways Improves DNA Integrity but Not Developmental Competence of Domestic Cat Immature Vitrified Oocytes.
Inhibition of Apoptotic Pathways Improves DNA Integrity but Not Developmental Competence of Domestic Cat Immature Vitrified Oocytes.
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DOI：
10.3389/fvets.2020.588334
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发表时间：
2020
影响因子：
3.2
通讯作者：
Luvoni GC
中科院分区：
文献类型：
--
作者：
Colombo M;Zahmel J;Jänsch S;Jewgenow K;Luvoni GC
家猫卵母细胞作为濒危野生猫科动物的模型，其冷冻保存技术正在不断发展。未成熟的玻璃化冷冻卵母细胞（VO）是一种宝贵的资源，生育力保存计划，但他们往往在升温后退化，其体外发育差。由于确切的机制尚不清楚，本研究评估了玻璃化是否可能触发两种凋亡标志物（DNA片段化和半胱天冬酶活性，实验I）和化学抑制剂（即，泛半胱天冬酶抑制剂Z-VAD-FMK）对相同标记物（实验II）和对体外发育的VO（实验III）的作用。主要目的是检查是否凋亡抑制可能是一种策略，以提高猫卵母细胞的低温耐受性。在实验I中，与新鲜对照组（9.68%; 199.6 ± 178.3; p = 0.02）相比，玻璃化冷冻诱导了加热后孵育24 h的VO中的DNA片段化并增加了caspase活性（DNA片段化：59.38%; caspase活性：414.6 ± 326.8）。在实验II中，与对照（未处理的）VO（69.44%; 434.5 ± 248.3; p < 0.001）相比，向玻璃化-加温和孵育培养基中添加Z-VAD-FMK降低了DNA片段化和半胱天冬酶活性（8.82%; 243.7 ± 106.9）。在实验III中，Z-VAD-FMK使处理的VO的成熟率接近新鲜卵母细胞的成熟率（分别为53.13和65.38%，p = 0.057），但VO胚胎发育没有差异（卵裂率; Z-VAD-FMK处理的VO：34.38%;对照VO：31.78%; p = 0.69）。总之，玻璃化冷冻增加了猫VO中的凋亡标志物，虽然Z-VAD-FMK能够阻碍DNA损伤和半胱天冬酶活性，但它的加入并不是胚胎发育的决定性因素。为了充分利用VO，应研究其他卵母细胞体外成熟和胚胎培养策略，如添加其他抑制剂或延长其使用时间。
Being a model for endangered wild felids, cryopreservation protocols for domestic cat oocytes are under continuous development. Immature vitrified oocytes (VOs) are a valuable resource for fertility preservation programs, but they often degenerate after warming and their in vitro development is poor. Since the exact mechanisms are not clear, this study assessed whether vitrification might trigger two apoptotic markers (DNA fragmentation and caspase activity, Experiment I) and the effects of a chemical inhibitor (i.e., the pan-caspase inhibitor Z-VAD-FMK) on the same markers (Experiment II) and on VOs in vitro development (Experiment III). The overarching aim was to check whether apoptosis inhibition might be a strategy to improve cat oocytes cryotolerance. In Experiment I, vitrification induced DNA fragmentation and increased caspase activity in VOs incubated for 24 h after warming (DNA fragmentation: 59.38%; caspase activity: 414.6 ± 326.8) compared to a fresh control (9.68%; 199.6 ± 178.3; p = 0.02). In Experiment II, the addition of Z-VAD-FMK to vitrification-warming and incubation media decreased DNA fragmentation and caspase activity (8.82%; 243.7 ± 106.9) compared to control (untreated) VOs (69.44%; 434.5 ± 248.3; p < 0.001). In Experiment III, Z-VAD-FMK brought maturation rates of treated VOs close to those of fresh oocytes (53.13 and 65.38%, respectively, p = 0.057), but there were no differences in VOs embryo development (cleavage rates; Z-VAD-FMK-treated VOs: 34.38%; control VOs: 31.78%; p = 0.69). In summary, vitrification increased apoptotic markers in cat VOs, and while Z-VAD-FMK was able to hinder DNA damage and caspase activity, its addition was not determinant for embryo development. To make the best use of VOs, other oocyte in vitro maturation and embryo culture strategies, such as the addition of other inhibitors or their prolonged use, should be investigated.
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