Inhibition of Apoptotic Pathways Improves DNA Integrity but Not Developmental Competence of Domestic Cat Immature Vitrified Oocytes

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.

Comparison of Different Materials for Self-Pressurized Vitrification of Feline Oocytes—First Results

“…Here, a slightly higher result was found with titanium tubes obtaining 20% of cleavage with embryos developing until the 4-cell stage, but in the previous study we presented 28.6% of cleavage with the cryotop with 30% of embryos achieving the morula stage [10]. While IVM rates after warming are in accordance with previous publications in felids [7,11], embryo competence seems to be triggered as in other novel approaches [27].…”

Maturation and fertilization of African lion (Panthera leo) oocytes after vitrification

“…In this study ICSI has also been used, but mainly because of the lack of lion sperm in a suitable quality for IVF. We currently demonstrated again [12], as others before, that at least in domestic cat IVF of vitrified oocytes is possible and zona hardening is not an issue.…”

“…Among felids, cryopreservation of domestic cat oocytes has been tried several times but led to similar results as in bovine. With current protocols most of the oocytes survive vitrification, but maturation, fertilization and embryo developmental competences are severely affected [3,12,19,45].…”

Cryopreservation of Gametes and Embryos and Their Molecular Changes

“…Specifically, methylation levels of promoter, exon, and intron regions were reduced when compared with controls. These results suggest differential outputs of embryo development depending on whether they were cryopreserved or not [ 92 ]. Changes in histone modifications reveal further epigenetic modifications induced by cryopreservation methods.…”

“…This activation induces apoptotic pathways, which are related to poor oocyte development. Interestingly, the treatment with Z-VAD-FMK, an inhibitor of pan-caspase activity, ameliorates the DNA fragmentation in vitrified oocytes, but it does not restore the altered oocyte development [ 92 ]. In humans, vitrification of oocytes during the MII stage induces changes in chromosomal and mitotic spindle configuration.…”