This study was conducted to investigate mitochondrial, nuclear chromatin and cytoskeletal organisation of vitrified embryos based on timing of the first zygotic cleavage. Embryos were retrieved from superovulated ICR mice, 28 hours after hCG injection. Two-cell stage embryos were categorised as earlycleaving (EC), while zygotes with 2-pronuclei as late-cleaving (LC) embryos. Embryos were cultured overnight in M16 medium supplemented with 3% bovine serum albumin (BSA) in carbon dioxide incubator. After 20 hours, the embryos were vitrified for one hour and warmed to room temperature. They were then fixed and immunostained to visualise distribution and intensity of mitochondria, nuclear chromatin and cytoskeleton. Finally, the embryos were mounted on glass slides and examined under a Confocal Laser Scanning Microscope (CLSM). Fluorescence intensities were analysed using LAS-AF-Lite Software. Results showed that EC embryos had significantly higher mitochondria (39.22 ± 12.50 versus 35.42 ± 14.61 pixel, p
The quality of RNA is crucial when performing microarray experiments. This is particularly important when dealing with preimplantation embryos, from which a minimum yield of RNA of good quality can be produced. We report the optimization of several RNA extraction methods applied to preimplantation embryos at different stages of development. The quality of the samples was confirmed using a microarray and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. A total of 30 cultured two-cell stage embryos of ICR mice were pooled at the 8-cell, morula, and blastocyst stages. The embryos were divided into two groups comprising DNase-treated and non-DNase-treated RNA samples. Total RNA was extracted using a Pico Pure RNA Isolation Kit following the manufacturer protocol, with some modifications. Lysed samples were bound to a silica-based filter, treated with deoxyribonuclease I (DNase I), and washed several times before elution. RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer and an RNA 6000 Pico Assay kit. Although concentrations of non-DNase-treated RNAs were higher than DNase-treated RNA, DNase-treated RNA gave a higher RNA integrity number compared with non-DNase-treated RNA. Inclusion of DNase treatment in the RNA extraction procedure gave the best quality RNA samples from preimplantation embryos, as validated by microarray and RT-qPCR quality control.