APPS November 2002 Meeting Abstract 2443

Alice A. Lim ¹, Martin H. Johnson², Margot L. Day¹, 1 Department of Physiology, University of Sydney, NSW, 2006, Australia, 2 Department of Anatomy, University of Cambridge, CB2 3DY, UK.

The circadian rhythm of the suprachiasmatic nucleus (SCN) of the brain is entrained by light. Upon input of light, the circadian rhythm is generated as follows: the transcription factors CLOCK and BMAL1 heterodimerise to upregulate transcription of the genes Per1-3 and Cry1-2. PER and CRY proteins heterodimerise, then translocate to the nucleus where they down-regulate CLOCK/BMAL1 activity. This reduces Per and Cry transcription and PER and CRY decay, releasing the inhibition on BMAL1. Eventually BMAL1 levels increase, initiating the next 24 h cycle of transcription. The circadian clock in the SCN coordinates circadian clocks in peripheral tissues¹. In the female reproductive tract and embryo, timers control when developmental events occur². It is possible that the circadian clock acts as one such timer as we have shown that the circadian genes, Per1-3, Cry1-2, Bmal1 and Clock, are expressed in the female reproductive tract (uteri and oviducts) and in preimplantation embryos of the mouse³. To determine whether a circadian clock exists in these tissues, we have quantified mRNA using real-time PCR.

Female Quakenbush Swiss (QS) mice, housed on a 12/12 h light/dark cycle, were superovulated and mated with mature male QS mice. Tissues were obtained from mice euthanised by cervical dislocation at circadian times 5, 11, 17, 23 h, on each day of the first four preimplantation days of pregnancy. Whole tissues were snap frozen in liquid nitrogen and total RNA was purified. The RNA was reverse transcribed and quantified via real-time PCR using Sybr Green I.

In preliminary experiments using RNA from liver, we have verified that real-time PCR is a valid method for quantification of circadian gene expression. Quantification of Per2, Cry1 and Bmal1 mRNA in the uterus via real-time PCR is in progress and will elucidate whether a circadian clock exists in this reproductive tissue.