GM-CSF. A regulator of pre-implantation embryo development

Abstract

GM-CSF is a multifunctional cytokine expressed in abundance in the female reproductive tract during early pregnancy. We have investigated the regulatory influence of GM-CSF in development of pre-implantation mouse and human embryos. RT-PCR and radio-labeled ligand binding studies show that mouse embryos express the low affinity alpha chain of the GM-CSF receptor (GM-Ra), but not the affinity-converting beta chain (bc). Expression was evident from the 1-cell stage of development and in blastocysts GM-Ra was present in both inner cell mass and trophectoderm cells. In vitro culture of embryos in recombinant GM-CSF accelerated development of blastocysts to hatching and implantation stages. Blastocysts from GM-CSF deficient mice showed retarded development, with diminished inner cell mass size. Incubation of blastocysts with recombinant GM-CSF stimulated glucose uptake, suggesting a role in regulation of metabolism.In human embryos, addition of GM-CSF to in vitro culture increased the proportion that developed to the blastocyst stage from 30% to 76%. Blastocysts formed more rapidly, and their developmental competence as assessed by hatching and attachment to culture dishes was improved. Blastocysts grown in GM-CSF were found to contain approximately 35% more cells, due primarily to an increase in the size of the inner cell mass. Using RT-PCR and immunocytochemistry, expression of mRNA and protein of GM-Ra but not bc was detected in human embryos from first cleavage through to blastocyst stage. Neutralising antibodies reactive with GM-Ra, but not antibodies to bc nor the bc-dependent antagonist E21R, ablated the development-promoting effect of GM-CSF in culture. Using TUNEL labeling, it was found that blastocysts cultured in GM-CSF contained 50% fewer apoptotic nuclei and 30% more viable inner cell mass cells.In embryo transfer experiments, exposure to GM-CSF during pre-implantation development was found to increase the proportion of embryos yielding viable implantation sites, and enhance subsequent fetal survival and development. While in vitro culture was associated with a significant reduction in fetal weight, this was alleviated by addition of GM-CSF to culture media, which gave rise to fetuses that were comparable in weight to fetuses from in vivo formed embryos. Furthermore, progeny from cultured embryos had an altered growth trajectory with increased total body mass and relative fat mass in adulthood, and exposure to GM-CSF normalised these parameters.These experiments implicate a physiological role for GM-CSF in regulating pre-implantation embryo development in mice and humans through a novel receptor mechanism independent of the bc subunit of the GM-CSF receptor. The data demonstrate that GM-CSF can alleviate the detrimental influence of ex vivo culture on fetal growth and developmental programming in mice. Together, our findings support the postulate that the cytokine environment experienced by an embryo during the pre-implantation period is a key determinant of blastomere proliferation, viability and metabolic activity, and can influence post-implantation growth and development, and long-term outcomes in adult progeny. The discovery that GM-CSF can improve cell number and inhibit apoptosis in human embryos may have clinical significance, but its use in reproductive medicine needs to be undertaken cautiously with regard for the consequences for fetal viability and health.