Identification of the ZPC Oligosaccharide Ligand Involved in Sperm Binding and the Glycan Structures of Xenopus laevis Vitelline Envelope Glycoproteins1

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Abstract
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in the female reproductive tract during early pregnancy and can promote the growth and development of preimplantation embryos in several species. We have demonstrated with in vitro experiments that the incidence of blastulation in human embryos is increased approximately twofold when GM-CSF is present in the culture medium. In the present study, we investigated the mechanisms underlying the embryotrophic actions of GM-CSF. Using reverse transcription-polymerase chain reaction and immunocytochemistry, expression of mRNA and protein of the GM-CSF-receptor alpha subunit (GM-Rα) was detected in embryos from the first-cleavage through blastocyst stages of development, but the GM-CSF-receptor beta common subunit (βc) could not be detected at any stage. When neutralizing antibodies reactive with GM-Rα were added to embryo culture experiments, the development-promoting effect of GM-CSF was ablated. In contrast, GM-CSF activity in embryos was not inhibited either by antibodies to βc or by E21R, a synthetic GM-CSF analogue that acts to antagonize βc-mediated GM-CSF signaling. Unexpectedly, E21R was found to mimic native GM-CSF in promoting blastulation. When embryos were assessed for apoptosis and cell number by confocal microscopy after TUNEL and propidium iodine staining, it was found that blastocysts cultured in GM-CSF contained 50% fewer apoptotic nuclei and 30% more viable inner cell mass cells. Together, these data indicate that GM-CSF regulates cell viability in human embryos and that this potentially occurs through a novel receptor mechanism that is independent of βc.

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