We have demonstrated that several human states are characterized by hyperactivity or hypoactivity of the central stress system, which explains not only mood changes but also the propensity of patients with such disorders to develop developmental, metabolic, cardiovascular or autoimmune complications. These changes in the central nervous system also influences fertility by affecting various components of the reproductive system. We have previously reported that CLOCK/BMAL1, the self-oscillating transcription factors that generate circadian rhythms both in the central nervous system and periphery, rhythmically repressed GR-induced transcriptional activity, indicating that CLOCK/BMAL1 functions as a reverse phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing the biologic actions of diurnally fluctuating circulating glucocorticoids. We performed one human study and revealed that this negative regulation on GR transcriptional activity by CLOCK was also functional in humans. As an extension of this circadian rhythm project, we screened microRNAs regulated in a circadian fashion in granulosa cells of mouse ovaries. MicroRNAs are short hairpin-like RNAs that demonstrate strong biological actions on reproduction, and specifically, granulosa cells by influencing proliferation and apoptosis, as well as steroidogenesis of these cells. Granulosa cells, on the other hand, are components of ovarian follicles required for their proper development and steroid hormone production. We have found that primary granulosa cells obtained from mouse ovaries showed circadian oscillation of several CLOCK-related genes, such as Per1/2 and Cry1/2. We found that expression of 10% of over 600 known microRNA we screened were in circadian rhythms. We further focused on miR-196a, as it showed the most significant circadian oscillation. We found that its target gene HOX8B, a transcriptional repressor important for the regulation of cell growth and apoptosis, demonstrated diurnal expression through miR-196a. We are now examining importance of our finding in the biologic action of granulosa cells. Aging is an important factor for reducing the chance of successful pregnancy, eventually developing ovarian failure and menopause, but the biological mechanisms underlying this physiologic process have not completely been elucidated as yet. It is also possible that pathologic infertility, such as by malnutrition, stress and exercise, might share part of the mechanisms responsible for aging-dependent ovarian failure. To examine impact of aging on ovarian functions, we again examined microRNA expression in primary granulosa cells obtained from mouse ovaries. We obtained granulosa cells from 2-year old mice and tested expression of 600 microRNAs by employing the cells of young mice (6-9 week old) as controls. In this screening, we found that 37 microRNAs were significantly regulated in aged cells. We focused on miR-503 and -322, as these microRNAs showed the most significant reduction (over 20-fold) in aged granulosa cells and form a gene complex on chromosome X regulated by the shared promoter. We found in the subsequent protein expression profile analysis that several subunits of the ATP synthase, a component of the mitochondrial respiratory chain, were down-regulated by inhibition of these microRNAs, suggesting that miR-503 and -322 are required for mitochondrial activity. Recent publication indicated that these miRNAs down-regulate expression of mitophagy transcripts, such as Nix/Bnip3L, Ulk1, Gabarapl2, Sh3glb1, Atg12, Becn1, and Bcl2l1, thus these microRNAs maintain mitochondrial biogenesis and energy production by suppressing expression of mitophagy-inducing genes. Since reduction of mitochondria is a key feature of aged ovaries with reduced reproductive activity, it is likely that miR-503 and -322 are mediators of mitochondrial dysfunction observed in aged ovaries, and further, underlying factors for increased incidence of infertility in older subjects through modulation of mitochondrial biogenesis/activity. Crosstalk between the gonadotropin-regulating cAMP signaling system and that of the steroid hormone receptors, such as progesterone (PR), estrogen (ER) and glucocorticoid receptor (GR), are important for the regulation of ovarian functions. To elucidate their crosstalk at the transcriptional regulation, we focused on the CREB-regulated transcriptional coactivator 2 (CRTC2), a cofactor known to be specific for CREB (thus cAMP signaling)-mediated transcriptional regulation, and tested its effect on PR- or GR-induced transcriptional activity. We found that CRTC2 functions as a coregulator of GR and PR, stimulating former transcriptional activity, while repressing latter activity. We further found that CRTC2 physically interacts with the ligand-binding domain of PR and GR through its C-terminal portion harboring the transactivation domain in vitro and in vivo. We are now examining biologic importance of this crosstalk in granulosa cells.

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Nicolaides, Nicolas C; Charmandari, Evangelia; Chrousos, George P et al. (2014) Circadian endocrine rhythms: the hypothalamic-pituitary-adrenal axis and its actions. Ann N Y Acad Sci 1318:71-80
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