Abstract
BackgroundPrevious studies have shown that circadian genes might be involved in the development of polycystic ovarian syndrome (PCOS). Hyperandrogenism is a hallmark feature of PCOS. However, the effect of hyperandrogenism on circadian gene expression in human granulosa cells is unknown, and the general expression pattern of circadian genes in the human ovary is unclear.MethodsExpression of the circadian proteins CLOCK and PER2 in human ovaries was observed by immunohistochemistry. The mRNA expression patterns of the circadian genes CLOCK, PER2, and BMAL1, and the steroidogenesis-related genes STAR, CYP11A1, HSD3B2, and CYP19A1 in cultured human luteinized granulosa cells were analyzed over the course of 48 h after testosterone treatment by quantitative polymerase chain reaction.ResultsImmunostaining of CLOCK and PER2 protein was detected in the granulosa cells of dominant antral follicles but was absent in the primordial, primary, or preantral follicles of human ovaries. After testosterone stimulation, expression of PER2 showed an oscillating pattern, with two peaks occurring at the 24th and 44th hours; expression of CLOCK increased significantly to the peak at the 24th hour, whereas expression of BMAL1 did not change significantly over time in human luteinized granulosa cells. Among the four steroidogenesis-related genes evaluated, only STAR displayed an oscillating expression pattern with two peaks occurring at the 24th and 40th hours after testosterone stimulation.ConclusionsCircadian genes are expressed in the dominant antral follicles of the human ovary. Oscillating expression of the circadian gene PER2 can be induced by testosterone in human granulosa cells in vitro. Expression of STAR also displayed an oscillating pattern after testosterone stimulation. Our results indicate a potential relationship between the circadian clock and steroidogenesis in the human ovary, and demonstrate the effect of testosterone on circadian gene expression in granulosa cells.
Highlights
Previous studies have shown that circadian genes might be involved in the development of polycystic ovarian syndrome (PCOS)
Expression of circadian proteins in the human ovary Immunohistochemistry results showed positive expression of Circadian locomotor output cycles kaput (CLOCK) in the cumulus and mural granulosa cells in dominant antral follicles and in interstitial cells, but no CLOCK expression was observed in the primordial follicles, primary follicles, and preantral follicles, and in the theca cells of antral follicles
Expression patterns of circadian genes in human granulosa cells stimulated by testosterone Treatment of testosterone induced oscillations in PER2 expression, with the first peak and bottom occurring at the 24th and 32nd hours, respectively, and the second peak occurring at the 44th hour (P4 vs. 24 = 0.028, P24 vs. 32 = 0.041, P24 vs. 48 = 0.039, P 4 vs. 44 = 0.024)
Summary
Previous studies have shown that circadian genes might be involved in the development of polycystic ovarian syndrome (PCOS). A circadian clock is a biochemical mechanism that oscillates with a period of 24 h and is coordinated with the day–night cycle. Light signals perceived by the retina are transmitted via the retino-hypothalamic tract to the suprachiasmatic nuclei (SCN), which contain the master pacemaker for the generation of circadian rhythms [1]. The basis for maintaining the circadian rhythm is a molecular clock consisting of interlocked transcriptional/translational feedback loops. The proteins encoded by the genes circadian locomotor output cycles kaput (Clock) and brain and muscle arnt-like protein 1 (Bmal1) heterodimerize and promote the rhythmic transcription of the period (Per, Per2) and cryptochrome (Cry, Cry2) gene families, whereas modified PER–CRY complexes repress the activity of the CLOCK–BMAL1 complex. PER–CRY complexes are degraded, and the CLOCK–BMAL1 complex is eventually released from feedback inhibition [3,4,5,6]
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