Can in vitro culture systems and media modulate apoptosis?

Abstract

Objective: Apoptosis or programmed cell death has been observed in preimplantation embryos. Apoptosis may play a major role in regulating ICM and TE lineage during preimplantation development. Growth factors have been shown to act as “survival” factors in vitro and prevent apoptosis. Understanding apoptosis and its modulation by culture environment may aid in designing optimal culture media for embryonic growth. In this study we characterize apoptosis in preimplantation mouse embryos grown in a variety of culture systems. Design: The TUNEL assay was used to assess apoptosis in mouse embryos. Materials and Methods: Frozen one-cell mouse embryos were purchased from Conception Technologies. Thawed embryos were pooled and cultured overnight. Embryos cleaving to the two-cell stage were randomly allocated to different treatment regimens and cultured for an additional 72 hours. The experiments were divided into three parts to facilitate handling of embryos. The following treatment regimens were tested: A) MEM alpha/10% SSS vs. coculture on Vero cells, B) Sequential media (G2.1/G2.3) vs. coculture, C) Growth factors GM-CSF, IGF I and II, insulin, LIF, FGF- 4 and TGF alpha. Embryo morphology and blastocyst development were assessed. At termination of the experiment, embryos were assessed for apoptosis by the TdT-mediated dUTP nick end labeling (TUNEL) assay using In-Situ Cell Death Detection kit – TMR-Red (Boehringer Mannheim) followed by counterstaining with DAPI. Statistical analysis was performed using the Mann-Whitney paired T test. Embryos not reaching at least the morula stage (> 32 cells) were excluded when calculating average cell number and apoptosis. Apoptotic indices were calculated for all treatments. Results were expressed as mean ± SD and p values less than 0.05 were considered significant. Results: During the first phase of experiments, we compared embryonic growth and apoptosis in control MEM alpha medium to coculture on Vero cells. This coculture system consistently yields good quality blastocysts with high cell number. The average cell number was significantly higher in the coculture versus the medium alone control (87.5 ± 21.4 and 68.5 ± 22.2; p<0.001). No difference was however seen in overall rate of blastulation. A greater percentage of embryos exhibited at least one apoptotic cell in the control vs. the coculture group (63 % Vs 52 %) but the overall apoptotic index was not different between groups. Very few of the control embryos (8%) stained for more than two apoptotic cells. This was quite different from the coculture group where 19% of embryos had 2–6 apoptotic cells. The average cell number in this subset of embryos was 98.7 ± 16.8 and number of apoptotic cells was 4.2 ± 1.1. A similar characterization is underway for embryos grown in sequential media or augmented with growth factor additives. Conclusion: Understanding the role of apoptosis during the course of embryo development requires characterization and comparison of embryos cultivated under different culture conditions. To date apoptosis in cocultured embryos has not been studied. The increased presence of apoptosis in blastocysts of high cell number may reflect the regulatory role of apoptosis in balancing ICM: TE ratios. Apoptosis in blastocysts of low cell number may have a totally different implication.