IMPACT OF RECONSTRUCTION BY GERMINAL VESICLE TRANSFER ON NUCLEAR MATURATION OF DOMESTIC CAT OOCYTES

Abstract

Cytoplasmic defects as a result of aging or cryopreservation can be circumvented in immature oocytes by transferring the germinal vesicle (GV) into a previously enucleated good quality oocyte. We are interested in oocyte reconstruction by GV transfer in felids to provide options for propagating rare domestic cat biomedical models as well as endangered species. Objectives of this study were to assess the impact of (1) cumulus cell removal, (2) treatments that allow viewing the GV, and (3) enucleation and fusion on subsequent oocyte meiotic resumption. In Experiment 1 (3 replicates), grade I immature cumulus-oocytes complexes (COC) collected from adult cat ovaries were denuded in 0.2% hyaluronidase and matured in vitro (28 h) alone (n = 56 total denuded oocytes; DO) or in co-culture (n = 52 DO with equal number of COC). Nuclear status of DO and intact COC was assessed by fixation in ethanol and Hoechst 33341 staining. The proportions of metaphase II stage (MII) oocytes was less (P < 0.05; ANOVA) when oocytes were denuded and matured alone (23.8 ± 2.7%; Mean ± SD) compared to the co-cultured DO (76.7 ± 4.3%) or intact COC (82.3 ± 3.1%). In Experiment 2 (3 replicates), oocytes were denuded and either treated to visualize the GV (n = 74; incubation in 7.5 μg/ml Cytochalasin B for 15 min at 38.5°C, centrifugation at 15,000 × g for 10 min, and exposure to 0.1 M sucrose for 10 min at 38.5°C) or not treated (n = 75; control). Nuclear status of treated and control DO was assessed after in vitro maturation (IVM) with intact COC (co-culture; ratio 1:1). Percentages of MII were similar (P > 0.05) between DO treated to visualize the GV (79.7 ± 3.5%) and controls (82.7 ± 4.6%). In Experiment 3 (4 replicates), oocytes were denuded and treated as described above. After partial zona dissection of DO and enucleation, GV surrounded by a small amount of cytoplasm (karyoplasts) were fused by two electrical pulses (1.8 kV/cm DC for 30 μs in 0.15 M sorbitol) with the same or different cytoplasts (n = 93 homoplasmic, 91 heteroplasmic reconstructions, respectively). Control oocytes (n = 85) were denuded, treated to visualize the GV and subjected to electric pulses alone. Nuclear status of reconstructed and control oocytes was assessed after IVM with intact COC (1:1). Percentages of fused oocytes after homo- (71.7 ± 6.3%) and heteroplasmic reconstruction (69.0 ± 4.2%) were similar (P > 0.05). The proportion of fused oocytes reaching MII after homo- (46.5 ± 2.2%) versus heteroplasmic (47.7 ± 1.4%) reconstruction also did not differ (P > 0.05), but was less (P < 0.05) than controls subjected to electric pulses alone (80.0 ± 3.5%). This is the first report in carnivores of successful oocyte reconstruction by GV transfer. Results clearly demonstrate that removing cumulus cells is detrimental to nuclear maturation, but can be circumvented by co-culturing DO with intact COC. Furthermore, treatments that allow viewing the GV have no impact on oocyte nuclear maturation. Although enucleation reduces the overall proportion of oocytes able to reach MII, neither electric pulses nor heteroplasmic reconstruction appear to have any influence on GV-cytoplasm interactions during meiotic resumption. (Supported by the National Institutes of Health K01 RR020045). (platform)