186 RELIABILITY OF HOECHST 33342 STAINING UNDER STANDARD EPIFLUORESCENCE MICROSCOPY FOR EVALUATION OF THE NUCLEAR STATUS OF LIVING DOG OOCYTES

Abstract

Hoechst 33342 staining detected by standard epifluorescence microscopy (Epi) is widely used for nuclear stage determination of oocytes in numerous species. Because it maintains cell viability, observed oocytes can be further processed for assisted reproduction. This sorting would be of great interest in the canine, where oocytes resuming meiosis in vitro remain scarce. But because of the cytoplasmic opacity of the canine oocyte, the accuracy of this technique is questionable. The objective of this study was to evaluate the accuracy of Epi by comparison to a reference technique (confocal microscopy; Conf). Oocytes were obtained from anestrus ovaries of 46 pubertal bitches. In vitro maturation was conducted with 20 oocytes/well in 500 μL M199 with 20% fetal calf serum in a humidified atmosphere of 5% CO₂ at 39°C for 72 h. After Hoechst 33342 staining (20 min at RT), oocytes were observed without fixation under Epi (IX70, Olympus, France) with the excitation beam attenuated by 150-fold and emission detected by a high sensitivity camera (CCD, Olympus). The mean time for capture was 300 ms. For Conf, oocytes were fixed, stained with Ethidium Homodimer-2 (2 μM for 20 min at RT) and examined with an Ion-Argon laser (LSM 310, Carl Zeiss, Germany). For each oocyte, the nuclear stage was successively determined by Epi and by Conf. The results of both observations were compared by chi-square analysis. The optimal concentration of Hoechst dye was first determined on 401 oocytes (200 ng, 500 ng, 1 μg, or 2 μg mL^–1). The 1 μg mL^–1 was the lowest concentration allowing the identification of the nuclear stage by Epi in the largest proportion of living oocytes seen with DNA by Conf. At this dye concentration, Epi did not significantly impact degeneration or meiosis resumption rates: 33.3 and 22.4%, respectively for 183 oocytes submitted to Epi before Conf, compared to 37.4 and 13.6% for 147 oocytes observed only under Conf. Finally, the concordance of the nuclear stage diagnosis was tested on 379 oocytes observed individually with Epi and Conf successively. On Epi, 149 oocytes were seen without DNA. Observed on Conf, 37.5% of these were also seen without DNA, 59.7% were in the germinal vesicle stage and 2% were in metaphase. On Epi, 89 oocytes were considered as having resumed meiosis. On Conf, meiosis resumption was confirmed for 88.7% of them, but Epi failed to detect 31.4% of the oocytes having resumed meiosis among the 379 cultured oocytes. Although 80% of the metaphase II oocytes identified by Epi were confirmed to have reached this stage, 23% of metaphase II oocytes present in the culture dishes were missed. In conclusion, probably because of the high density in lipids of the cytoplasm, Hoechst staining detected by Epi for canine oocytes sorting after in vitro maturation leads to erroneously discarding a high proportion of oocytes.