A thorough characterization of cryoprotectant (CPA) sensitivity is required to formulate a successful cryopreservation protocol for any biomaterial. The aim of this study was to characterize the toxic impact of various CPA types, concentrations, and exposure temperatures on the immature domestic cat oocyte. In Experiment 1, grade I immature oocytes (n=561) were exposed (30min; 25°C or 0°C) to 0M, 0.75M, 1.5M, or 3M of propylene glycol (PrOH) or ethylene glycol (EG) in PBS+20% fetal calf serum (v/v). After exposure, CPA was removed step-wise by subjecting oocytes to decreased CPA concentrations. Oocytes were cultured (30h; 38.5°C, 5% CO2) in IVM medium as reported previously (Wolfe and Wildt 1996 J. Reprod. Fertil. 106, 135–141). Oocytes were then fixed and stained to examine nuclear status (Hoechst 33342) and spindle integrity (FITC-labeled anti-α-tubulin antibodies; Sigma Chemical Co., St. Louis, MO). Experiment 2 was designed on the basis of Experiment 1 results to assess the impact of the spindle abnormalities on subsequent embryo development. Oocytes (n=776) were exposed to CPA conditions yielding optimal nuclear maturation with either high (0.75M or 3.0M PrOH or 1.5M EG at 25°C) or low (1.5M PrOH at 25°C) proportions of abnormal spindle. After IVM, oocytes were inseminated with thawed semen (5×105 motile sperm mL−1 ) in Ham’s F-10 (Irvine Scientific, St-Anna, CA). At 16h post-insemination, oocytes were fixed and stained (Hoechst 33342) to assess IVF success (pronuclear formation) or cultured in vitro for 7 days to assess embryo development. Data were analyzed by ANOVA and Tukey’s multiple comparison test. In Experiment 1, CPA treatment had no effect (NS) on meiotic progression to metaphase I. However, percentage of oocytes reaching metaphase II (MII) was reduced (P<0.05) in 3.0M PrOH at 0°C (29.3±8.3%; mean±SD), 3.0M EG at 25°C (33.7±8.9%), and 0°C (29.4±11.0%) compared to all other conditions examined (range, 52.0% to 62.0%). All CPA treatments also increased (P<0.05) spindle abnormalities at MII (range, 40.3% to 75.9%) compared to control (13.8±8.6%), except 1.5M PrOH at 25°C (20.7±10.1%). None of the CPA treatments in Experiment 2 influenced IVF success (range, 55% to 63%; NS). However, percentage of cleaved embryos was reduced (P<0.05) in 0.75M PrOH (32.1±4.1%), 1.5M EG (33.4±4.0%), and 3.0M PrOH (29.3±3.8%) compared to control (50.1±4.0%) or 1.5M PrOH (50.6±4.9%). Developmental competence (number of blastocysts relative to number of cleaved embryos) also was impaired (P<0.05) in 1.5M EG (16.5±7.4%) and 3.0M PrOH (14.9±7.8%) compared to the other conditions (range, 32.5% to 38.5%), including 1.5 PrOH at 25°C (32.5±7.8%). In conclusion, exposure of immature oocytes to 1.5M PrOH at 25°C does not adversely impact oocyte maturation, MII spindle, fertilization, or embryo development in vitro in the domestic cat.
Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro
