OBJECTIVES: To use the protein-free medium blastocyst antihatching model to characterize physiological events that mediate hatching. DESIGN: In a series of four prospective experiments, our aims were to [1] test the efficacy of the antihatching model and assisted hatching; [2] exam the influence of initial in vivo developmental stage and late serum supplementation on hatching inhibition; [3] discount the role of zona hardness and physical expansion directly affecting hatching; and [4] provide evidence that the trophectoderm is directly responsible for secreting a zona lysin. SETTING: University-based research laboratory. RESULTS: Culturing two- to eight-cell mouse embryos in serum-free human tubal fluid (HTF) medium significantly reduced hatching levels to < or = 2%, however, hatching increased to 10.7% when initially culturing morula-stage embryos. Hatching was effectively rescued to control levels when embryos were placed in HTF with serum at the early blastocyst stage. There was no difference in blastocyst total cell numbers or zona pellucida digestion intervals between culture treatments. Finally, we showed that trophectodermal vesicles, devoid of inner cell mass, are capable of hatching under control conditions. CONCLUSIONS: The primary mechanism of blastocyst hatching is not physical expansion and abnormal zona hardness is not responsible for hatching inhibition. Certain extracellular precursors found in serum (e.g., amino acids) are required in culture medium upon cellular determination of trophectoderm (i.e., morula to blastocyst stage) to facilitate the intrinsic secretion of an undefined hatching factor.
OBJECTIVES: To use the protein-free medium blastocyst antihatching model to characterize physiological events that mediate hatching. DESIGN: In a series of four prospective experiments, our aims were to [1] test the efficacy of the antihatching model and assisted hatching; [2] exam the influence of initial in vivo developmental stage and late serum supplementation on hatching inhibition; [3] discount the role of zona hardness and physical expansion directly affecting hatching; and [4] provide evidence that the trophectoderm is directly responsible for secreting a zona lysin. SETTING: University-based research laboratory. RESULTS: Culturing two- to eight-cell mouse embryos in serum-free human tubal fluid (HTF) medium significantly reduced hatching levels to < or = 2%, however, hatching increased to 10.7% when initially culturing morula-stage embryos. Hatching was effectively rescued to control levels when embryos were placed in HTF with serum at the early blastocyst stage. There was no difference in blastocyst total cell numbers or zona pellucida digestion intervals between culture treatments. Finally, we showed that trophectodermal vesicles, devoid of inner cell mass, are capable of hatching under control conditions. CONCLUSIONS: The primary mechanism of blastocyst hatching is not physical expansion and abnormal zona hardness is not responsible for hatching inhibition. Certain extracellular precursors found in serum (e.g., amino acids) are required in culture medium upon cellular determination of trophectoderm (i.e., morula to blastocyst stage) to facilitate the intrinsic secretion of an undefined hatching factor.