Abstract
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.
Highlights
IntroductionSince the domestic cat is used as a model for the development of cryopreservation strategies transferable to its wild relatives [1], survival and development of feline oocytes after vitrification and warming are critical steps to make gamete biobanks worth the effort
In Experiment I, to check whether Cryotop vitrification induces the activation of apoptotic pathways in cat vitrified oocytes (VOs), fresh (FOs, negative control), hydrogen peroxide-exposed (HPOs, positive control), and vitrified-warmed oocytes were analyzed by TUNEL assay and by a caspase activity assay
Caspase activity was higher in VOs and HPOs than in fresh oocytes (FOs) (p = 0.001), and there were no differences between VOs incubated for different time after warming (2hVOs vs. 24hVOs, p = 0.989) or between VOs and HPOs (p = 0.766)
Summary
Since the domestic cat is used as a model for the development of cryopreservation strategies transferable to its wild relatives [1], survival and development of feline oocytes after vitrification and warming are critical steps to make gamete biobanks worth the effort. Immature vitrified oocytes (VOs) experience several cryoinjuries, which affect their ability to resume meiosis and develop properly into embryos after warming. Both temperature decrease and high concentrations of cryoprotectants have a role in inducing damages. In domestic cat immature oocytes, vitrification affects mitochondria aggregation [15], and probably the permeability of gap junctions hemichannels, causing the loss of small essential metabolites, ionic imbalance, and the penetration of small, potentially toxic, molecules [16]. Information is still limited, and only one study tried to target the apoptosis pathways through an inhibitor of a signaling molecule known as ROCK (Rho-associated coiled-coil containing protein kinase), obtaining an improvement in the developmental competence of VOs in terms of normal fertilization and cleavage rates [4]
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