163 SEARCH FOR GENES OF WHICH THE AMOUNTS OF TRANSCRIPTS OSCILLATE EVERY 24 h IN THE MOUSE OVARY

Perturbation of circadian rhythm is believed to be detrimental to the physiology of organs, including the mammalian ovary. However, the molecular mechanisms that are regulated by circadian rhythm in the ovary have not been identified. To identify the molecular mechanisms that are regulated by circadian rhythm and to speculate on the physiologies that are likely to be damaged by perturbation of circadian rhythm in the ovary, we searched for genes in which the amount of transcripts oscillates every 24 h in the mouse ovary. To achieve this, expression profiles of circadian genes (per1, per2, and bmal1) that code transcription-regulation factors for which transcription activities are known to oscillate every 24 h in almost all organs, and wee1, the transcription activity of which circadian genes regulate and which is known to elongate the G2 phase in the cell cycle, were analyzed in this study. Six-week-old female ICR mice were kept individually under a lighting schedule with lights on for 14 h followed by lights off for 10 h. A vaginal smear of each mouse was collected every day to determine its estrous cycle. Ovaries of 3 mice were collected continuously every 4 h over a 4-day period from the start of the light period on the day of proestrus. Total RNA was extracted from each ovary, and 500 ng each was used for cDNA synthesis. Transcripts of each gene and of tbp were quantified by real-time PCR, and the amount of the transcripts of each gene in each sample was divided by the amount of tbp transcripts. The obtained relative values in each sample were used as the representative data of the amount of transcripts of each gene. The amounts of per1, per2, and bmal1 clearly oscillated every 24 h. The maximum and minimum values of per1 and per2 were observed at 16 and 4 h, respectively, after onset of the light period each day. The maximum and minimum values of bmal1 were observed at the time of onset of the light period and at 12 h after onset of the light period each day. Averages of the maximum values of per1, per2, and bmal1 each day were significantly greater than averages of the minimum values (per1, 3.60 � 0.10 and 1.38 � 0.09; per2, 0.82 � 0.08 and 0.27 � 0.06; bmal1, 0.61 � 0.05 and 0.17 � 0.01; P < 0.05). The cyclicity in the oscillation of the amount of wee1 transcripts was weaker than that observed in circadian genes, but the average of values that were obtained from 12 to 20 h after onset of the light period each day was significantly greater than that obtained from 0 to 8 h (0.29 � 0.02 and 0.22 � 0.01; P < 0.05). Our results suggested that the cell cycle of ovarian cells is regulated in a circadian manner through wee1 transcription, which is regulated by circadian genes of which the amounts of transcripts oscillate every 24 h. Because an abnormal cell cycle seems to trigger the development of tumors or follicular cysts, perturbation of circadian rhythm may cause those ovarian diseases.