In vitro development of bovine embryos in conditioned media from bovine granulosa cells and vero cells cultured in exogenous protein- and amino acid-free chemically defined human tubal fluid medium.

Abstract

We have investigated the effect of protein- and amino acid-free simple human tubal fluid (HTF) medium conditioned with bovine granulosa cells (BGC) or Vero cells on the development of early bovine embryos to the blastocyst stage. Serum-containing medium (SCM) and serum-free medium (CM) conditioned by BGC (BGC-SCM, BGC-CM) and by Vero cells (VC-SCM, VC-CM) were prepared. Early embryos (1-cell stage and 5- to 8-cell stage) were obtained by in vitro maturation and fertilization of oocytes from slaughtered cows. Embryos were randomly divided into 7 culture groups as follows: culture with BGC-SCM, BGC-CM, VC-SCM, or VC-CM; coculture with BGC or Vero cells; or culture with fresh HTF medium without serum. The proportion of 5- to 8-cell embryos developing to the blastocyst stage in BGC-CM (16%) and VC-CM (12%) was significantly lower (p < 0.05) than in BGC-SCM (41%) and in VC-SCM (29%) and after coculture with BGC (48%) and Vero cells (30%). Similarly, the percentages of 1-cell embryos developing to blastocyst in BGC-CM and VC-CM were significantly lower than in BGC-SCM and VC-SCM and after coculture. Cell numbers per blastocyst developed from 5- to 8-cell embryos in BGC-CM (96.8 cells) and in VC-CM (94.0 cells) were somewhat lower than those in BGC-SCM (128.5 cells) and VC-SCM (117.1 cells) and after coculture with BGC (124.2 cells) and Vero cells (115.3 cells). These results suggest that BGC and Vero cells cultured in a protein- and amino acid-free simple HTF medium synthesize and secrete factor(s) promoting blastocyst formation in vitro. Physiochemical analysis indicated that the embryotrophic substances in BGC-CM were distributed in two molecular size ranges, one between 10 kDa and 30 kDa and another greater than 30 kDa.