180 DETECTION OF CASPASE-3 AND -7 IN NORMAL AND SLOW DEVELOPING BOVINE IN VITRO EMBRYOS

Abstract

The incidence of apoptosis is an important indicator of embryo quality that can be measured by detection of active caspase-3 and -7 at various stages of in vitro development. The aim of the present study was to investigate the expression of these apoptotic markers in bovine in vitro embryos at different developmental stages using a fluorescent staining method. Additionally, expression of both caspases was compared between normal and slow-developing embryos. A total of 2640 immature bovine oocytes (four replicates) were matured and fertilized in vitro. Presumed zygotes (n = 2400) were denuded 24 h post-insemination (hpi) after fertilization and cultured in 50-μL droplets of modified SOF medium with 10% fetal calf serum at 39.0°C in 5% CO₂, 5% O₂ and 90% N₂. Cleavage rate was determined at 45 hpi for all embryos, except 400 that were used as positive controls for the caspase staining. At different time points, embryos were allocated to the normal developing group if they reached the 5–8-cell stage at 45 hpi; the 9–16-cell stage at 80 hpi, the morula stage at 117 hpi and the expanded blastocyst stage or beyond at 168 hpi. Embryos that lagged one cell cycle behind at each time point were designated as slow-developing embryos. All selected embryos were stained using CaspaTag Pan-Caspase In Situ Assay kit, Fluorescein^® (Chemicon International, Temecula, CA, USA). Positive controls were incubated in 0.5 μM staurosporine (Sigma, Belgium) for 24 h. The apoptotic cell ratio (ACR) was determined for each embryo by means of confocal laser scanning microscopy (ACR = number caspase-positive cells/total cell number × 100). Control embryos (n = 400) were cultured undisturbed until 168 hpi and evaluated for blastocyst yield. Mixed model analyses of variance with group as fixed factor and replicate as random factor were used to evaluate the ACR at 45, 80, 117, and 168 hpi, respectively. The mean cleavage rates at 45 hpi and the blastocyst yield at 168 hpi were 60.5% and 26.9%, respectively. The ACR over all time points and at each individual time point was significantly lower in normal-developing embryos compared with slow-developing embryos, as determined by caspase staining (P < 0.01) (Table 1). In conclusion, this study demonstrated that slow-developing embryos express a higher level of apoptosis. Further research is necessary to investigate the cause of the higher ACR in slow developing embryos, which may be the result of altered gene expression or environmental influences.

Table 1. Apoptotic cell ratio (ACR; mean % ± SD) at four time points in normal and slow-developing embryos

This study was supported by a grant of Research Foundation – Flanders (aspirant 1.1.084.04N00).