Changes in gene expression associated with oocyte meiosis after Obox4 RNAi

Abstract

Objective: Previously, we found that oocyte specific homeobox (Obox) 4 plays significant role in completion of meiosis specifically at meiosis I-meiosis II (MI-MII) transition. The purpose of this study was to determine the mechanism of action of Obox4 in oocyte maturation by evaluating downstream signal networking.

Methods: The Obox4 dsRNA was prepared by in vitro transcription and microinjected into the cytoplasm of germinal vesicle oocytes followed by in vitro maturation in the presence or absence of 0.2 mM 3-isobutyl-1-metyl-xanthine. Total RNA was extracted from 200 oocytes of each group using a PicoPure RNA isolation kit then amplified two-rounds. The probe hybridization and data analysis were used by Affymetrix GeneChip® Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively.

Results: Total 424 genes were up (n=80) and down (n=344) regulated after Obox4 RNA interference (RNAi). Genes mainly related to metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathway was changed. Among the protein kinase C (PKC) isoforms, PKC-alpha, beta, gamma were down-regulated and especially the MAPK signaling pathway PKC-gamma was dramatically decreased by Obox4 RNAi. In the cell cycle pathway, we evaluated the expression of genes involved in regulation of chromosome separation, and found that these genes were down-regulated. It may cause the aberrant chromosome segregation during MI-MII transition.

Conclusion: From the results of this study, it is concluded that Obox4 is important upstream regulator of the PKC and anaphase-promoting complex action for maintaining intact germinal vesicle.

Keywords: Microarray Analysis; Obox4, Mouse; Oocyte Maturation; RNA Interference.

Figure 1

Venn diagram showing the number of transcripts up- and down-regulated more than 2-fold in comparisons between control and oocyte specific homeobox (Obox) 4 RNA interference, RNAi treated oocytes. Control group; germinal vesicles (GVs) cultured for 0 hour vs. GVs cultured for 4 hours in 3-isobutyl-1-metyl-xanthine (IBMX) supplemented medium (non-injected), Obox4 RNAi group; GVs cultured for 0 hour vs. GVs cultured for 4 hours in IBMX supplemented medium (Obox4 dsRNA-injected).

Figure 2

Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway displaying transcripts in mitogen-activated protein kinase (MAPK) signaling pathway. Selected genes are presented with bold characters in a small box and their expression was confirmed by real-time reverse transcription-polymerase chain reaction as summarized in Figure 3 [34]. PKC, protein kinase; Raf1, threonin-protein kinase; MEK, mitogen-activated protein kinase kinase; Erk, extracellular signal-regulated kinase.

Figure 3

Relative expression of genes related to mitogen-activated protein kinase pathway in germinal vesicle (GV) oocytes after oocyte specific homeobox 4 RNA interference. Expression of mRNA was confirmed by real-time reverse transcription-polymerase chain reaction analysis. The expression levels were calculated cycle threshold values, and then mRNA expression ratio was determined relative to that of control GV oocytes. Experiments were repeated least three times, and data was expressed as mean±SE. PKC, protein kinase; Raf1, threonin-protein kinase; MEK, mitogen-activated protein kinase kinase; Erk, extracellular signal-regulated kinase. ^aStatistical significance at p<0.05.

Figure 4

Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway display transcripts in cell cycle pathway. Selected genes are presented in bold characters in a small box and their expression was confirmed by real-time reverse transcription-polymerase chain reaction. Rad21 encodes cohesin, esp1 encodes separin, and PTTG encodes securin [35]. Cdc20, cell-division cycle 20; APC1, anaphase-promoting complex; PTTG, pituitary tumor-transforming gene 1; Espl, extra spindle poles-like; Rad21, cohesin.

Figure 5

Relative expression of genes related to cell cycle pathway in germinal vesicle (GV) oocytes after oocyte specific homeobox 4 RNAi. Expression of mRNA was confirmed by real-time reverse transcription-polymerase chain reaction analysis. The expression levels were calculated cycle threshold values, and then mRNA expression ratio was determined relative to that of control GV oocytes. Experiments were repeated least three times, and data was expressed as mean±SE. Cdc20, cell-division cycle 20; APC1, anaphase- promoting complex; PTTG, pituitary tumor-transforming gene 1; Espl, extra spindle poles-like; Rad21, cohesin. ^aStatistical significance at p<0.05.