Matsuo et al. reported that circadian clock genes regulate the timing of cell division in mouse regenerating liver cells (2003). Their results suggested the importance of circadian clock genes for organs or tissues for which functions are characterized by cell division, such as pre-implantation embryos. To obtain basic information on the molecular functions of circadian clock genes in pre-implantation embryos, we investigated the expression profiles of transcripts and proteins of some circadian clock genes, clock, bmal1, cry1, and per2, in mouse germinal vesicle oocytes (GV), MII oocytes (MII), and pre-implantation embryos using real-time PCR and immunocytochemistry (ICC). Germinal vesicle oocytes were collected from ICR females at 48 h after PMSG priming. The mouse at 48 h after PMSG priming was primed with hCG, and MII were collected at 15 h after hCG priming. The pre-implantation embryos were collected at 6, 12, 24, 36, 48, 60, 72, 84, and 96 h after insemination, and they corresponded to early 1-cell, late 1-cell, early 2-cell, late 2-cell, 4-cell, 8-cell, early morula, late morula, and blastocyst stages, respectively. cDNA was produced by mRNA isolated from 20 oocytes or embryos using oligo dT and was subjected to real-time PCR using a TaqMan Probe system (ABI). Three sets of 20 oocytes or embryos at each developmental stage were applied to mRNA extraction and real-time PCR analysis to ensure equal mRNA extraction efficiency between samples. The level of mRNA of each clock gene contained in 3 samples from each developmental stage was almost the same. Statistical analysis of the transcripts of each gene were done by ANOVA. Germinal vesicles, MII and embryos collected at each time point were subjected to ICC using antibodies of CLOCK, BMAL1, CRY1, and PER2. The oocytes or embryos treated with only secondary antibody did not produce any signal. All of the examined genes except per2 were expressed in oocytes and pre-implantation embryos. The transcript level of clock, bmal1, and cry1 in MII were significantly lower than those in GV (P < 0.05). After fertilization, transcript levels of clock, bmal1, and cry1 significantly decreased from early 1-cell stage to late 2-cell stage (P < 0.05). These decreased transcript levels were maintained until the blastocyst stage after the late 2-cell stage. Immunocytochemistry analysis showed the nuclear localization of CLOCK and BMAL1 in early and late 2-cell embryos and of CRY1 in early 2-cell embryos but no signals of PER2 in oocytes or pre-implantation embryos. Because mouse oocytes and 1- to 2-cell embryos are transcriptionally inert, the abundant transcripts of clock, bmal1, and cry1 in these stages seemed to indicate that they were synthesized and stored during the oocyte growth phase. Moreover, the nuclear localization of CLOCK, BMAL1, and CRY1 in the oocytes and 1- to 2-cell stage embryos suggested that some clock genes were translated and worked for oocyte maturation and early embryogenesis.
This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.
