Vitrification of immature oocytes is crucial for wild animals’ conservation and is applicable in field conditions. In the domestic cat, model for endangered felids, the viability of vitrified oocytes (VOs) is usually high after warming, but many VOs degenerate during in vitro maturation and struggle to develop into embryos. We aimed to assess whether vitrification induces apoptosis and whether an inhibitor might block it (Exp.1) supporting VOs development (Exp.2). In Exp.1, DNA fragmentation (TUNEL assay) and caspase (i.e. apoptosis effectors) activity were assessed with commercial kits as apoptosis markers in the following immature oocytes: fresh (FOs, negative control, n=31), 100µM H2O2-treated (HOs, positive control, n=33), conventionally vitrified (CVOs, n=36, Cryotop® method) and vitrified with the addition of pan-caspase inhibitor ZVAD(OMe)-FMK 20µM (ZVOs, n=34). In Exp.2, FOs (n=59, control), CVOs (n=67) and ZVOs (n=66) were in vitro matured (24h), fertilized (18h) and cultured (7 days) to assess their embryonic development (daily observations). Data were analyzed by non-parametric one-way-ANOVA (caspase) or Fisher’s exact test (TUNEL, embryonic development); significance: p<0.05. In Exp.1, CVOs had higher caspase activity (fluorescence intensity mean±SD, 434.5±248.3) and DNA fragmentation (TUNEL positive oocytes, 69.4%) than FOs (199.6±178.3; 9.7%; p<0.001), whereas caspase activity was similar in CVOs and HOs (420.1±346.1; p=0.9), suggesting apoptotic activation in CVOs. The inhibitor lowered apoptosis markers in ZVOs (243.7±106.9; 8.8%; p=0.001 vs CVOs), but its presence in vitrificationwarming and maturation media did not influence (Exp.2) maturation (ZVOs vs CVOs; 51.5% vs 47.8%; p=0.7), cleavage on day 2 of culture (31.8% vs 37.3%; p=0.6) or cumulative degeneration (34.9% vs 35.8%; p=0.9). As expected, FOs had higher rates (74.6% maturation; 64.4% cleavage; p=0.004). The addition of Z-VAD(OMe)-FMK did not influence VOs development, but vitrification remains fundamental for germplasm preservation. The use of other apoptosis inhibitors, after the identification of specific apoptotic pathways involved, might improve VOs outcomes.
Vitrification-induced apoptosis and pan-caspase inhibition in domestic cat immature oocytes / M. Colombo, J. Zahmel, S. Jänsch, K. Jewgenow, G.C. Luvoni. - In: CRYOBIOLOGY. - ISSN 0011-2240. - 97(2020 Jul), pp. 538.263-538.263. ((Intervento presentato al 57. convegno Cryo2020: Annual Meeting of the society for Cryobiology tenutosi a Online nel 2020.
|Titolo:||Vitrification-induced apoptosis and pan-caspase inhibition in domestic cat immature oocytes|
COLOMBO, MARTINA (Primo) (Corresponding)
LUVONI, GAIA CECILIA (Ultimo)
|Parole Chiave:||feline; cryopreservation; gametes|
|Settore Scientifico Disciplinare:||Settore VET/10 - Clinica Ostetrica e Ginecologia Veterinaria|
|Data di pubblicazione:||lug-2020|
|Appare nelle tipologie:||01 - Articolo su periodico|