95 OBSERVATION OF CHROMOSOME DECONDENSATION, HISTONE H3 MODIFICATION, AND HP1 PROTEIN IN MOUSE CLONED EMBRYOS FOLLOWING INHIBITION OF HISTONE DEACETYLATIONS AND CYCLIN-DEPENDENT KINASE

Abstract

Low chromosome decondensation and hypoacetylation and hypermethylation of histones characterize the incomplete reprogramming of cloned embryos during the first mitotic prophase. Our study looks at the effect of histone deacetylase inhibitors (HDACi), Trichostatin A (TSA), APHA Compound 8 (APHA), and cyclin-dependent kinase inhibitor (CDKi) roscovitin (ROS), on nuclear reprogramming in cloned mouse embryos during the first mitotic prophase. Oocytes were collected from female B6D2F1 mouse cumulus cells collected from female B6D2F1 and ICR, and fibroblasts from male GFP-ICR. We performed somatic cell nuclear transfer (SCNT) using a piezo-actuated micromanipulator system; the SCNT oocytes were subsequently activated for 6 h with 5 mM SrCl₂ in Ca²⁺-free CZB medium (CZB) supplemented individually with 100 nM TSA, 250 nM APHA, or 100 nM ROS or in combination with TSA+ROS or APHA+ROS. Activated SCNT embryos were then cultured in KSOM medium with the same concentrations of TSA, APHA, ROS, TSA+ROS, or APHA+ROS for 2 h. Following treatment, we cultured the cloned embryos in KSOM for in vitro development. In the first experiment, the levels of chromosome decondensation during the first mitotic prophase in these cloned embryos were performed, based on the ratio of nuclear volume, at 6 h and 10 h after activation. We calculated the average nuclear volume ± SD for each treatment, and these were subsequently compared to control values (100%, without treatment). We next turned to examining the intensity of methyl H3-K9, acetyl H3-K9, and HP1β of cloned embryos at 6 h and 10 h after activation by immunostaining. Images of immunostained embryo nuclei were acquired using an OLYMPUS Fluoview FV 1000 confocal system. The distribution of fluorescence intensities from 5 different regions of the nucleus were determined using Fluoview FV 1.4a software. Student's t-test was used to calculate the significance of differences between groups in the experiment. We repeated each experiment 4 times to obtain 40 nuclear transfer embryos per treatment. The present results show that global histone hyperacetylation and hypomethylation can be characterized by an increase in H3-K9 acetylation and a decrease in H3-K9 methylation in the presence of HDAC inhibitors. The levels of all isoforms of HP1β, however, were not reduced following inhibition of HDAC. This was in contrast to the high decondensation observed in interphase chromatin, leading to a significant increase of pronuclear volumes and a decrease in H3-K9 methylation in cloned embryos in the presence of ROS, although we were unable to obtain hyperacetylation and HP1β did not change. From these results, we conclude that the combined inhibition of histone deacetylases and cyclin-dependent kinase for 8 h following NT caused an increase in somatic chromosome decondensation, hyperacetylation, and demethylation of histone H3-K9 during the first mitotic cell cycle in cloned mouse embryos. Our findings strongly suggest that there is no correlation of HP1β involved in hyperacetylation and demethylation of H3-K9.