152 DIELECTROPHORETIC BEHAVIOR OF BOVINE OOCYTES AND ZYGOTES IN RELATION TO DEVELOPMENT AND TRANSCRIPTIONAL ABUNDANCE

Abstract

Selection of developmentally competent oocytes and zygotes based on external morphology has been employed to increase in vitro embryo production. However, this method is more often influenced by personal judgments and lacks universal standards. Hence, this study was aimed at investigating the dielectrophoretic behavior of oocytes and zygotes in relation to their development and mRNA abundance. To achieve the objective, 2 experiments were conducted. In the first experiment, matured bovine oocytes (PB⁺) and zygotes were subjected to a dielectrophoresis (DEP) procedure designed as follows: 4 MHz AC, 450-µm electrode distance, 5 V, and 80-µs cm^-1 medium conductivity. The time elapsed for each of the oocytes and zygotes to reach one of the electrodes from the center was recorded. To accomplish this, 457 PB⁺ oocytes were subjected to the DEP procedure and 152, 121, 90, and 94 were classified as very fast, fast, slow, and very slow, respectively. Moreover, 152 oocytes were used as controls. Similarly, a total of 940 zygotes were subjected to the DEP procedure and classified as very fast (n = 329), fast (n = 329), slow (n = 97), and very slow (n = 245). In addition, 323 zygotes were used as the control group. The oocyte DEP groups were parthenogenetically activated, and the zygote DEP groups were further in vitro-cultured in a CR1 culture medium supplemented with 10% estrous cow serum, 20 mL mL^-1 ²-mercaptoethanol, and 10 mL mL^-1 minimal essential medium. In PB⁺ DEP groups, the results show that the blastocyst rate (mean ± SEM) at 7 days post-parthenogenetic activation was significantly (P < 0.05) lower in the very slow moving group (7.2 ± 4.9) compared to the very fast (21.8 ± 3.2), fast (21.5 ± 4.6), slow (23.3 ± 4.5), and the control (19.7 ± 3.7) groups. In zygotes, the blastocyst rate at 7 days post-insemination was significantly (P < 0.05) higher in the very fast (16.1 ± 2.7) compared to the slow (9.1 ± 2.7) and very slow (10.6 ± 2.7) groups, but it was not significantly higher than the fast (12.2 ± 2.7) and control (12.3 ± 2.7) groups. To investigate whether the transcriptional level of DEP separated very fast and very slow oocytes and zygotes, mRNA expression level was analyzed using a bovine cDNA microarray from a pool of 30 zygotes and oocytes in 6 replications including the dyeswap. The data analyzed using statistical analysis for microarray revealed that 31 and 5 genes were up- and down-regulated, respectively, in very fast compared to very slow PB⁺ DEP groups. The up-regulated genes are known to be involved in RNA binding and protein biosynthesis (RPL2, RPL8, and RPLPO), ion binding (PTGS2 and ANXA2), and cell cycle regulation (CDC91L1, NUSAP1, and CKS1B). Similarly, 25 and 17 genes were up- and down-regulated, respectively, in the very fast compared to the very slow zygote DEP groups. Some of these genes enriching the very fast zygotes are involved in ion binding (ZNF85, ZNF519, and NANOS1), regulation of cell cycle (NASP, SMARCA5, and AURKA), and signal transduction (RALA). Therefore, from this experiment we can conclude that dielectrophoretically separated oocytes and zygotes show differences in the rate of blastocyst development accompanied by differences in transcriptional abundances.