250 DISTRIBUTION OF HISTONE macroH2A1 IN BOVINE PRE-IMPLANTATION EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

Abstract

It is known that heterochromatin is characterized by the presence of the histone variant of macroH2A1. MacroH2A1 is a core variant histone with a hybrid structure consisting of a domain that resembles a full-length histone H2A1 followed by a large nonhistone domain. We have previously studied the dynamic changes of macroH2A1 accumulation during the pre-implantation developmental period in the mouse. In the present study, we investigated the distribution of microH2A1 in bovine metaphase II oocytes and pre-implantation embryos at 2-, 4-, 8-, 16-cell, and morula stages as well as blastocysts harvested at Days 8, 9, 10, 11, 12, and 13 following activation and in vitro fertilization (IVF). To generate parthenotes, denuded and in vitro-matured oocytes were activated using a combined treatment of calcium ionophore A23187, cycloheximide (CHX), and 6-dimethylaminopurine (6-DMAP). Five oocytes and pre-implantation embryos at each stage of development were used to follow the development expression pattern of microH2A1 by immunocytochemistry. The cross-reactivity of the primary antibody against mouse microH2A1 was verified by Western blot analysis with bovine fibroblasts. Another staining control included immunostaining with antibody against histone molecules. The stained embryos were observed by laser-scanning confocal microscopy and epiflourescence microscopy. No microH2A1 stain was observed in bovine oocytes or pre-implantation embryos up to the expanded blastocyst stages. In the IVF group, the macroH2A1 was first found in elongated blastocysts (Day 11) after hatching. We observed different expression patterns of macroH2A1 in activated vs. IVF bovine embryos. In the parthenote group, we failed to find robust expression even when embryos were cultured for 13 days. Moreover, the pattern of macroH2A1 expression in bovine embryos was different fromn that in the mouse, in which the onset of macroH2A1 expression occurred by the 16-cell morula stage. These results suggest species differences in the establishment of epigenetic signals.

This work was supported by grants from USDA to X. Y. and X. C. T.