Determination of the effect of circadian rhythm disruption on follicular development in the ovary in mice via the hippo signaling pathway

The circadian rhythm serves to control many physiological and biological cycles in humans and mammals. The circadian rhythm system consists of two parts: the central clock (SCN) and the peripheral clock. The rhythmic functioning of the central circadian clock is important for the development of the female reproductive system and the regulation of events that occur there. In the female reproductive system this rhythmicity is very tightly linked to the light-dark cycle. In the ovary, circadian clock function is often associated with steroidogenesis, folliculogenesis, cellular differentiation, response to gonadotropins and ovulation. In our study, we hypothesized that the effect of circadian rhythm disruption on ovarian and follicle development may be related to the hippo signaling pathway and mTERT. We aimed to show the effect of circadian rhythm disruption on follicular development in the ovary through the hippo signaling pathway and to show mTERT expression in mice. In our thesis study, 20 female Balb/c mice of 6-8 weeks of age entering the estrous cycle were used. We designed two expremental groups: The control group (n=10) with 12:12 h light/dark cycle and in the experimental group (n=10) with 12:12 h light/dark cycle. At the end of the experimental period, the mice were dissected and ovarian tissues were isolated. The left ovaries of the subjects were placed in fixative for morphological analysis and immunofluorescence staining, while the left ovarian tissue was isolated for quantitative real-time polymerase chain reaction (qRT-PCR). The localization of PER2, YAP1, TEAD4 and mTERT was determined by immunofluorescence staining, while the expression of YAP1, TEAD4 and mTERT and the expression of circadian rhythm genes BMAL1, CLOCK, PER2 and CRY were determined at mRNA level by qRT-PCR. All evaluations between 12:12 h light/dark cycle control and 12:12 h light/dark cycle experimental groups were performed using ImageJ and GraphPad programs. The data obtained were statistically evaluated by Student's t test (p<0.05). Immunofluorescence results showed that PER2 expression was decreased, indicating that the circadian rhythm was disrupted. Hippo signaling pathway proteins YAP1 expression was decreased and TEAD4 expression was increased in the experimental group compared to the control group. We also showed that mTERT expression was increased. When the expressions of YAP1, TEAD4, mTERT, BMAL1, CLOCK, PER2 and CRY1 were evaluated at the mRNA level, no significant change was found. In conclusion, we demonstrated that the effect of circadian rhythm disruption on ovarian and follicle development in mice may be mediated through the Hippo signaling pathway at the translational level, suggesting that there may be an inverse relationship between circadian rhythm disruption and mTERT. In conclusion, we suggested that the Hippo signaling pathway and its relationship with mTERT may have important role to understanding ovarian patologies depend on the circadien clock changes. Key words: Circadian Rhythm, Ovary, Hippo Signaling Pathway, mTERT,