Granulocyte-macrophage colony stimulating factor (GM-CSF) and co-culture can affect post-thaw development and apoptosis in cryopreserved embryos

Abstract

The objective of this study was to evaluate the effects of growth factor supplementation and Vero cell co-culture on apoptosis and development of frozen thawed one-cell mouse embryos. The following treatment regimens were assessed: (a) control medium (b) Vero cell co-culture and (c) growth factor supplemented medium. The individual growth factors tested were: GM-CSF, IGF-I, IGF-II, TNF-alpha, FGF-4, LIF, TGF-alpha, TGF-beta, IL-6, PDGF and EGF. Blastocyst development and differentiation were monitored. At termination of the experiments, overall blastomere number and apoptosis were assessed using the TUNEL assay. No differences were observed in blastulation and hatching rates. ICM differentiation in thawed embryos was notably improved with either co-culture or growth factor supplementation. The only growth factor significantly modulating apoptosis in thawed embryos was granulocyte-macrophage colony stimulating factor (GM-CSF). GM-CSF enhanced continued cell survival and prevented apoptosis but did not influence overall cell number in developing blastocysts. Vero cell co-culture significantly increased cell number in blastocysts (124+/-42 vs 100+/-44 in control; P<0.05). Embryonic apoptosis was higher in the co-cultured embryos. The increased presence of apoptotic cells in blastocysts of high cell number may reflect the regulatory role of apoptosis in balancing ICM: TE ratios. These data indicate that culture conditions can modulate post-thaw embryonic development and apoptosis.