Influence of protein supplements on the secretion of leukaemia inhibitory factor by mitomycin-pretreated Vero cells: possible application to the in vitro production of bovine blastocysts with high cryotolerance.

A co-culture system for bovine embryos using mitomycin-treated Vero cells and serum-supplemented modified synthetic oviduct fluid (mSOF) supports the development of in vitro maturation and fertilization-derived oocytes to hatched blastocysts. In this system, it has been suggested that one contribution made by the co-culture cells to embryo development is production of the cytokine leukaemia inhibitory factor (LIF). However, there are concerns about exposure of early embryos to serum due to its incompatibility with embryo cryosurvival. In this study, the influence of two protein supplements (synthetic serum substitute (SSS), a lipid-free human serum-derived product) and oestrous cow serum (ECS)) on Vero cell LIF secretion was compared, with the aim of designing a co-culture system that is supportive of bovine embryo cryopreservation. Vero cells cultured for 72 h in medium 199 + 5% fetal bovine serum (FBS) (recommended maintenance medium for this cell line) secreted detectable amounts of LIF (13.1 +/- 0.9 pg LIF per 10(5) cells). Culture in mSOF, the medium routinely used in this laboratory for embryo culture, also supported LIF secretion in Vero cells. However, the amount of LIF was tenfold higher (24.7 +/- 6.2 pg LIF per 10(5) cells; P < 0.05) when mSOF was supplemented with 10% (v/v) ECS compared with supplementation with 2% (v/v) SSS. Results of a second series of experiments in which supplementation with each protein was normalized to 10% revealed similar differences in LIF secretion, indicating that LIF secretion was affected by the type, not the amount, of protein. Time course analysis revealed stepwise increases (P < 0.05) in cumulative LIF secretion with every 24 h of culture in mSOF + either SSS or ECS. In terms of embryo development and post-cryopreservation viability, medium supplementation with 2% (v/v) SSS alone versus the two-step system of 2% (v/v) SSS (days 1-4) + 10% (v/v) ECS (days 4-10) had no influence (P > 0.05) on the ability of bovine blastocysts to hatch, with or without intervening cryostorage. However, the rate of blastocyst formation (expressed as the percentage of cleaved embryos) was only 27% in the presence of 2% (v/v) SSS, and increased almost twofold (P < 0.05) when ECS was added beginning on day 4 of co-culture. In summary, Vero cell LIF secretion was increased markedly by ECS. A two-step system of medium supplementation, in which embryos are exposed to ECS beginning on day 4 of in vitro development combined high rates of blastocyst formation with cryotolerance. This effect may be a result of limiting embryo exposure to serum-derived lipid until after the eight-cell stage and providing an increase in LIF during the critical developmental stages of compaction and cavitation.