92 DOES THE SEX OF THE EMBRYO AFFECT DEVELOPMENTAL AND APOPTOTIC RATES AT THE BLASTOCYST STAGE?

Abstract

Several studies have demonstrated differences in developmental rates and metabolism between bovine female and male embryos after IVF. Such differences seem related to the activity of both X chromosomes in female embryos up to the blastocyst stage and can lead to a shift in sex ratio. Developmental differences between male and female embryos are influenced by culture conditions. The objective of this study was to evaluate developmental and apoptotic rates of male and female bovine embryos in 2 SOF-based culture media, one with 5% FCS and the other with 4 mg mL^–1 of BSA. Sex-sorted semen of one bull was used to produce cohorts of embryos of the desired sex. In preliminary experiments, IVF procedures were adapted to the use of sexed semen, and the purity of the sexed semen was verified through embryo sexing. The levels of apoptosis were assessed in Day-7 blastocysts using 2 techniques on the same embryos: TUNEL and detection of cleaved caspase-3 by immunostaining (caspase staining). Analysis by confocal microscopy and subsequent 3-D reconstruction allowed a precise cell count. A higher blastocyst rate on cleaved embryos was observed at Day 8 post-insemination for male than for female embryos in both media (BSA: male: 36.7 ± 4.0%, female: 28.6 ± 3.7%; FCS: male: 41.7 ± 2.9%, female: 31.7 ± 4.7%; ANOVA 3, P = 0.01). No significant difference in cell number was observed between male and female blastocysts (BSA: male: 188 ± 9, female: 170 ± 9; FCS: male: 186 ± 6, female: 177 ± 7; ANOVA 3, P = 0.14). In both media a higher proportion of cells showing caspase staining was observed in female than in male embryos (BSA: male: 7.3 ± 1.3%, female: 9.4 ± 2.1%; FCS: male: 9.2 ± 0.6%, female: 14.2 ± 1%; ANOVA 3, P = 0.01), whereas the proportion of stained cells was higher in FCS than in BSA medium whatever the sex (ANOVA 3, P = 0.02). The same tendency, although not significant, was obtained for the proportion of cells showing TUNEL staining with higher values in female than in male embryos (BSA: male: 9.3±2.1%, female: 10.5 ± 2.6%; FCS: male: 13.1 ± 0.9%, female: 16.5 ± 1.1%; ANOVA 3, P = 0.07) and higher values in FCS than in BSA medium whatever the sex (ANOVA 3, P = 0.05). A tendency for a higher proportion of double-stained cells (TUNEL and caspase-positive) was also observed in female embryos whatever the medium (BSA: male: 3.8 ± 1.1%, female: 5 ± 1.6%; SOF: male: 4.8 ± 0.5%, female: 7.0 ± 0.7%; ANOVA 3, P = 0.08). Intriguingly, only about half of the stained cells showed the double staining (TUNEL and caspase). This could be explained by the fact that caspase-3 activation can appear before DNA fragmentation during the apoptosis process and caspase staining disappear when TUNEL staining is still visible. But both caspase-3 activation and DNA fragmentation can also occur independently of apoptosis. In conclusion, male embryos seem to show a higher developmental rate in both media and could be less affected by apoptosis than female ones, particularly when cultured with FCS. Those experiments have to be repeated with the sexed sperm of another bull to draw final conclusions.