38. Impact of microwave processing on dehydration and structural integrity of germinal vesicles from domestic cat oocytes

Abstract

The ability to compact and inject the cat germinal vesicle (GV) into a recipient cytoplast allows exploration of a new fertility preservation strategy that avoids whole oocyte freezing and could enable drying and storage of GV at supra-zero temperatures. Previous studies demonstrated that (1) 60% of GVs can survive simple air-drying in the presence of trehalose followed by 4 weeks of storage at 4 °C and (2) 10% of reconstructed oocytes with rehydrated GV can achieve nuclear maturation (Graves-Herring et al., 2011). However, air-drying is an uncontrolled process that often results in non-uniform distributions of water within and between samples. Compared to existing techniques, microwave assisted dehydration appears to be a simple and reliable approach to dry biological samples at ambient temperatures (Chakraborty et al., 2008). The objective of the study therefore was to (1) explore the feasibility of drying GV exposed to trehalose using microwave processing and (2) assess the impact on GV structural integrity (chromatin configuration and incidence of DNA damage). Immature cat oocytes from adult ovaries were denuded from the cumulus cells and permeabilized with hemolysin. Groups of five denuded oocytes were exposed to Tris–HCL EDTA (pH 8.0) buffer containing 200 mM trehalose for 10 min and then transferred into 10-μl droplets placed on small pieces of Millipore Isopore Membrane filters (n = 182 total oocytes in 5 replicates). A maximum of eight droplets were arranged on the turntable of a CEM SAM 255 microwave system. Drying was conducted at 20% power for 5, 8, 20, 25 or 30 min. After each time point, samples were weighed immediately to establish the extent of water loss and then were rehydrated before fixation in 4% paraformaldehyde followed by GV chromatin configuration assessment (Hoechst staining) and DNA fragmentation assay (TUNEL). Fresh denuded oocytes (n = 60 total oocytes) were fixed as controls. The average ambient humidity during all measurements was 43.2 ± 1.1%. From time 0 through 30 min of microwave treatment, moisture contents (g water/g dry weight) progressively decreased (0 min: 12.3 ± 0.5; 5 min: 8.3 ± 1.2; 8 min: 8.9 ± 1.2; 20 min: 5.0 ± 0.5; 25 min: 1.9 ± 0.3; 30 min: 1.3 ± 0.4). GV chromatin configuration was not affected by the microwave treatment. The proportion of degenerated oocytes (absence of GV) ranged from 10% to 15% after the different time points and was not different (P > 0.05; Chi-square test) from the fresh controls. Interestingly, pooled proportions of GV with DNA damage following 5–8 min of microwave dehydration were not different (P > 0.05) from fresh controls (14.3% and 10.9%, respectively). However, the incidence of DNA fragmentation was higher (43.5%; P