Demonstrating the effect of circadian RHYTHM on the uterus of the mouse via the hippo signaling pathway

The circadian rhythm coordinates many physiological functions in all mammals, including humans. The female reproductive system is regulated by the central circadian system. Studies have shown that the synchronization of circadian rhythm genes is critical for reproductive functions, and that any disruption in the light and dark cycle may adversely affect the mother's sleep pattern, early embryonic development, and the development of the fetus. In our thesis, we hypothesized that the relationship between the circadian rhythm and the uterus may be related to the Hippo signaling pathway and telomerase activity. Therefore, we aimed to show the Hippo signaling pathway components and mTERT expression in the uterus of animals with impaired circadian rhythm. In our study, we created two groups (n=10) using a total of 20 BalbC 6-8 weeks old female mice. In the control group, mouse caged on a 12:12 hour light/dark cycle for one week. In the experimental group mice, the circadian rhythm was disrupted by keeping the mice on a 12:12 hour light/light cycle for one week. At the end of the one-week experimental period, mice were dissected and uterine tissues were isolated. While the right uterine horn of the uterus was placed in fixative for tissue processing for morphological analysis and immunofluorescence staining, the left uterus tissue was isolated for use in quantitative real-time polymerase chain reaction (qRT-PCR). While PER2, YAP1, TEAD4 and mTERT localization were determined by immunofluorescence method, YAP1, TEAD4 and mTERT expressions and circadian rhythm genes BMAL1, CLOCK, PER2 and CRY were detected at mRNA level by qRT-PCR. All the comparisions between groups were performed using ImageJ and GraphPad programs. The obtained data were statistically evaluated with Student's t test (p<0.05). We determined that the thickness of the lumen epithelium of the endometrium decreased in the experimental group compared to the control group, the number of glands and lumen diameter increased, the thickness of the myometrium decreased, and there was no significant difference in endometrial thickness and diameter of vessel lumen. We showed that the expression of the Hippo signaling pathway proteins YAP1 and TEAD4 were increased in the experimental group and the expression of mTERT was decreased. When the expressions of YAP1, TEAD4, mTERT, BMAL1, CLOCK, PER2 and CRY were evaluated at the mRNA level, it was determined that YAP1, TEAD4, BMAL1, CLOCK and PER2 expressions did not change between groups, CRY expression increased and mTERT expression decreased in experimental group. In conclusion, we showed that the effect of circadian rhythm disruption on the mouse uterus may be mediated through the Hippo signaling pathway. We though that the Hippo signaling pathway may be important in regulating the circadian rhythm of the uterus in cases such as night shifts workers, repetitive light/dark changes or jetlag-like conditions in women. We suggested that Hippo signaling pathway components may be considered as therapeutic targets in unexplained infertility as a result of circadian rhythm disruption. Keywords: Circadian rhythm, Hippo signaling pathway, mTERT, Uterus.