Mitogenic and anti-apoptotic activity of insulin on bovine embryos produced in vitro

Insulin improves development of mammalian preimplantation embryos and, in addition to the regulation of glucose transport, it exerts mitogenic and anti-apoptotic activities. The expression of glucose transporters (Glut) mediating the uptake of this essential energy substrate is critical for embryo survival. An impaired expression of Glut leads to an increase in apoptosis at the blastocyst stage and involves Bax. The various effects of insulin were unravelled by supplementing the in vitro culture medium with insulin (1.7 micromol l(-1)) and (i) the rates of cleavage and blastocyst development were recorded; (ii) mitogenic activity was studied by determining the total number of blastocyst cells and the ratio between trophectoderm and inner cell mass (ICM) cells; (iii) the frequency of apoptosis in blastocysts was determined by the TdT-mediated duTP nick-end labelling (TUNEL) assay and by quantification of the relative amounts of mRNA for Bax and Bcl-XL; and (iv) expression for Glut1, Glut3 and Glut8 transcripts was compared between embryos cultured in the presence or absence of insulin. Insulin increased rates of cleavage (81.2+/-2.2 (control) to 86.0+/-2.5) and blastocyst development (24.7+/-1.9 to 31.3+/-1.2), and number of blastocyst cells (123.7+/-6.0 to 146.3+/-6.6); the increase in the number of blastocyst cells was due to a significantly higher number of trophectoderm cells (82.3+/-5.0 versus 100.3+/-5.5). Blastocysts derived from cultures supplemented with insulin showed a significant decrease in apoptosis as determined by the TUNEL assay (14.8+/-0.9 to 12.2+/-0.7). No effects of insulin on the mRNA expression of Glut isoforms and Bax and Bcl-XL were found. These results demonstrate that the mitogenic and anti-apoptotic effects of insulin on bovine preimplantation embryos did not correlate with changes in the amounts of mRNA for the glucose transporter isoforms Glut1, -3 and -8, or transcripts for Bax and Bcl-XL.