Thymosin alpha-1 and FA-1 monoclonal antibody affect murine preimplantation embryo development by modulating protein phosphorylation

Abstract

The effects of thymosin α1 (Tα1) and FA-1 monoclonal antibody (anti-FA-1 mAb) on murine preimplantation embryonic development were investigated by performing 2-cell embryo bioassay and by studying ova/embryos protein phosphorylation pattern (by 32P metabolic labeling and by in vitro kinase assay) and protein synthesis (by in vitro [ 35S]methionine labeling). Tα1 treatment (0.1, 0.5 and 5 ng/100 μl) significantly increased blastulation rates ( P < 0.01), blastocyst hatching rate ( P < 0.0001), blastocyst diameter ( P < 0.001) and number of cells per blastocyst ( P < 0.0001) of the in vitro cultured 2-cell stage embryos. Anti-FA-1 mAb reduced blastulation rates ( P < 0.001) primarily due to an arrest of development at morula stage. In vitro metabolic labeling of murine ova/embryos showed 32P incorporation into 4 major protein bands of murine ova ( M r 125, 90, 68 and 31 kDa, respectively), 7 protein bands of 2-cell ( M r 90, 68 and 31; and 145, 52, 38 and 32 kDa, respectively), 10 protein bands of morula ( M r 150, 110, 92, 82, 70, 54, 39, 34, 30 and 29 kDa, respectively), and 15 protein bands of blastocyst (150, 110, 92, 70, 68, 54, 39, 34 and 30; and 131, 105, 52, 44, 43 and 33 kDa, respectively) stage embryos. Tα1 treatment (0.1–0.5 ng/100 μl) resulted in a general increase in 32P labeling in all proteins of 2-cell, morula and blastocyst stage embryos. Anti-FA-1 mAb completely blocked 32P labeling of various proteins of murine ova, 2-cell, morula and blastocyst stage embryos, whereas control mouse myeloma IgG did not affect phosphorylation of these proteins. In vitro kinase assay performed directly on various ova/embryos extracts revealed 6 phosphoproteins ( M r 105, 82, 55, 38, 34 and 33 kDa, respectively) that were common to ova and 2-cell embryos, besides a 43 kDa protein detected only in the ova extract. Of these phosphoproteins, Tα1 treatment specifically enhanced whereas anti-FA-1 mAb inhibited autophosphorylation of a 55 kDa protein of 2-cell embryos. Similarly, Tα1 treatment enhanced 6 phosphoproteins of morula ( M r 102, 60, 58, 49, 35 and 32 kDa, respectively) and 4 phosphoproteins of blastocyst ( M r 102, 60, 49 and 35 kDa, respectively) stage embryo extracts in the kinase assay. In [ 35S]methionine labeling experiments, Tα1 treatment did not affect labeling of any of the 5 proteins ( M r 170, 95, 86, 62 and 42 kDa, respectively) of 2-cell embryos, however, it enhanced labeling of 1 protein band of ova (46 kDa), 7 proteins of morula ( M r 95, 86, 70, 43, 42, 39 and 35 kDa, respectively) and 9 proteins of blastocyst ( M r 95, 86, 70, 43, 42, 39 and 35; and 51 and 33 kDa, respectively) stage embryos. Anti-FA-1 mAb did not affect 35S labeling of murine ova and 2-cell, morula or blastocyst proteins compared to control myeloma IgG. These results suggest that Tα1 enhance murine preimplantation embryonic development by modulating protein phosphorylation pattern as well as protein synthesis. On the other hand, anti-FA-1 mAb inhibit embryonic development by modulating protein phosphorylation pattern without affecting protein synthesis.