Clock genes and female reproduction
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The involvement of clock genes in the temporal regulation of the function and lifespan of the corpus luteum (CL) has not been investigated in detail. Immunohistochemistry and real-time quantitative PCR techniques were used to examine the expression of the canonical clock genes: period1, period2, period3, cryptochrome1, cryptochrome2, clock and bmal1, at protein and mRNA levels respectively. The expression of the clock genes was examined in the human CL, cultured luteinised granulosa cells, cultured luteal fibroblast-like cells and the ovine CL. The main findings were that clock genes are expressed in the human and ovine CL; that this expression is manifest at mRNA and protein level in all discernible cell types within the human and ovine CL, and that the pattern of mRNA expression differs between the early luteal phase compared to the late luteal phase. The circadian expression of the clock genes was established in the ovine CL during the late luteal phase and could not be determined in the human CL, although indications from cultured luteinised granulosa cells and luteal fibroblast-like cells suggest that this may also be the case in humans. With the exception of per2, the circadian pattern of clock gene expression emerged in the late luteal phase CL when the early luteal phase CL did not demonstrate circadian clock gene expression. This emergence later in the lifespan of the CL was akin to that observed in embryonic development, where the clock genes are initially non-rhythmic but then acquire circadian rhythmicity with age. In this case, the clock genes have been proposed to perform a non-classical circadian timing role in the timing of embryonic development. The per2 gene was also found to be special, in its loss rather than gain of rhythmic gene expression across the luteal lifespan and in its protein localisation in the cytoplasm of some granulosa-lutein cells. The exceptional behaviour of per2 is consistent with a growing body of evidence supporting its role as a unique clock gene in many respects, able to maintain circadian protein levels in the absence of circadian gene expression, integrating peripheral clock inputs and outputs and acting as a tumour suppressor gene. The CL was also found to be a potential target of melatonin regulation, based on its possession of melatonin MT1 receptors and the timing of circadian cry1 gene expression in the late luteal phase. The expression of cry1 is known to be directly melatonin-induced in the PT and appeared to be similarly activated, downstream of a melatonin signal, in the CL. This supports the evolving view of a hierarchical organisation of the central and peripheral clocks, which are integrated in order to establish information feedback loops that maintain circadian homeostasis, and which can regulate seasonal physiology.