Abstract Study question Does verteporfin, an inhibitor of YAP/TAZ activity, prevent from the impacts of chemotherapy exposure on signaling pathways governing follicle activation and survival? Summary answer Verteporfin treatment prevents from the chemotherapy-induced impairments of the Hippo pathway but also of PI3K and survival pathways. What is known already The Hippo pathway is a crucial regulator of the ovarian reserve. Disruption of this pathway occurring in non-physiological contexts, such as ovarian processing during ovarian tissue preservation or after chemotherapy exposure, leads to massive uncoordinated follicular growth and depletion of the ovarian stockpile. To prevent from the harmful impact of chemotherapy exposure, several studies assessed the potential of inhibition of the PI3K pathway, the major signaling pathway involved in physiological follicle activation. However, the protective effect of this inhibition appeared to be moderate in vitro, suggesting the involvement of other disrupted mechanisms, as Hippo pathway. Study design, size, duration This study was performed on post-natal day 3 mouse ovaries. Half-cut ovaries were first treated with 0, 0.2, 1.5 and 3 µM Verteporfin (VERT) during 3 hours in vitro culture to establish the efficient dose of VERT. Whole ovaries were then cultured for 24 and 48 hours and exposed to 10 µM 4-hydroperoxycyclophosphamide (4HC) and/or 3 µM VERT to assess the effect of this inhibitor on follicle activation and survival pathways. (N = 3-5) Participants/materials, setting, methods To assess the impacts of sectioning and VERT treatment on follicle activation, gene expression analyses (RT-qPCR) were used to assess Hippo, PI3K/AKT/mTOR and apoptosis signaling pathways. The potential preventive effect of VERT co-treatment with 4HC exposure was evaluated by gene (RT-qPCR) and protein expression (western blot) analyses of these three pathways, and completed with histological staining for DNA damages (TUNEL). Main results and the role of chance Following exposure to increased concentration of VERT for 3 hours, a significant impact of the inhibitor was observed at 3µM on Ccn2 (p = 0.003) and Cmyc (p = 0.045) expression levels compared to control, without impacting on apoptosis genes expression. Exposure to 10 µM 4HC induced a higher YAP/pYAP protein ratio and Ccn2 expression level compared to control after 24 and 48 hours of culture, confirming the Hippo pathway disruption. VERT co-treatment was able to prevent from this increase of protein levels and gene expression, although the impact on Ccn2 expression was moderated after 48 hours of culture. Assessment of PI3K signaling revealed an induction of mTOR signaling following 4HC exposure at both time of culture as shown by the higher pRPS6/RPS6 ratio compared to control. Surprisingly, VERT co-treatment significantly decreased this level compared to 4HC alone at 24 and 48 hours of culture. This result suggests an indirect impact of this inhibitor on the PI3K pathway. Despite Bax and Bcl2 gene levels remained stable among conditions, ovaries exposed to 4HC had a significant higher level of DNA damages at both culture timepoints compared to control. Notably, VERT co-treatment was able to decrease the 4HC-induced gonadotoxicity at 48 hours of culture. Limitations, reasons for caution This study evaluated the impact of an inhibitor to control follicle activation in mouse model, and was limited to the assessment of two main signaling. Although we completed our study with apoptosis analyses, the results should be interpreted with caution as other pathways may be involved into the activation process. Wider implications of the findings Our results suggest that verteporfin is a promising inhibitor to control acute signaling pathway impairments under unphysiological conditions. Moreover, this study sustains the presence of a potential interaction between the two main signaling pathways regulating follicle activation, PI3K and Hippo pathways. Trial registration number Not applicable
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Sep 10, 2024
Ziwen Ding + 2
Open Access