In vitro Compaction of Germinal Vesicle Chromatin is Beneficial to Survival of Vitrified Cat Oocytes

Introduction - Using the domestic cat as an experimental model for rare and endangered felids, we have previously demonstrated that immature oocytes denuded and then pre- incubated in cytochalasin B survive the vitrification process better than intact cumulus-oocyte complexes [1]. However, the immature cat oocyte contains a large-sized germinal vesicle (GV) with decondensed chromatin that is prone to freeze damage. It is known that enhancement of histone deacetylations results in GV chromatin compaction without microtubule polymerization [2]. The aim of the present study therefore was to examine the influence of in vitro compaction of cat GV chromatin using resveratrol (Res), a histone deacetylase enhancer [3], on its survival during vitrification.

In Experiment 1, grade I oocytes were denuded in 0.2 % hyaluronidase and then exposed to 0, 0.5, 1.0 or 1.5 mM Res in the presence of 7.5 μM cytochalasin B for 1.5 h at 38.5°C. After each treatment, oocytes were extensively rinsed and: 1) fixed and stained with anti-acetylated histone antibodies (FITC-labeled) and Hoechst 33341 to evaluate acetylation status and GV diameter; 2) matured in vitro for 30 h before fixation and Hoechst-staining to determine incidence of nuclear maturation; or 3) matured, inseminated with frozen-thawed sperm and then cultured in vitro for 7 days before fixation and Hoechst-staining to assess embryo status.
In Experiment 2, denuded oocytes were exposed to identical conditions as in Experiment 1. After each treatment, half of the oocytes were directly cultured in vitro whereas the other half was vitrified and stored 1 day in liquid nitrogen [1]. Fresh or vitrified oocytes were maturedin vitro and then: 1) fixed to evaluate nuclear maturation status; or 2) inseminated in vitro for 18 h before fixation and Hoechst-staining to assess pronuclear formation and apposition in fertilized oocytes. In Experiments 1 and 2, an oocyte subset (12 to 15 oocytes/treatment) was matured and cultured 18 h without sperm before fixation and Hoechst-staining to assess level of spontaneous activation (parthenogenesis).

Results - In Experiment 1, GV chromatin was entirely deacetylated after exposures to 1.0 or 1.5 M Res. GV diameter was reduced (P<0.01) after exposure to Res concentrations of 0.5- 1.5 mM (Table 1). The 1.0 or 1.5 mM Res treatments induced the most significant GV compaction (P<0.01), but with no difference (P>0.05) between these two concentrations (Table 1). However, exposure to 1.5 mM Res compromised oocyte ability to reach the metaphase II (MII) or to form cleaved embryos or blastocysts. In contrast, these same measures were not adversely affected by 0.5 and 1.0 mM Res treatments. [...]