Low-Intensity pulsed ultrasound reduces ovarian cryopreservation injury through the Notch signaling pathway

The cryopreservation of ovarian tissue can induce oxidative damage. In this study, we investigated whether low-intensity pulsed ultrasound (LIPUS) can mitigate this oxidative damage and examined the underlying molecular mechanisms. Ovarian tissues were randomly divided into control, cryopreservation, and cryopreservation + LIPUS group. The cryopreserved tissues were stored in liquid nitrogen. The cryopreservation + LIPUS group received LIPUS stimulation immediately after thawing, followed by 24 h of in vitro culture before sample collection. We utilized WB, immunohistochemistry, and TUNEL staining to assess tissue damage. Proteomic and phosphoproteomic analyses were performed to identify differentially expressed proteins and phosphorylation sites. The mechanisms were further validated using pathway inhibitors, point mutation, and coimmunoprecipitation. Immunohistochemical staining for 3-NT, 4-HNE, and 8-OHdG; WB analysis of Nrf2 and HO-1; and TUNEL staining confirmed that ovarian tissue cryopreservation induces oxidative damage, whereas LIPUS treatment alleviates such damage. The differentially expressed proteins were enriched mainly in the Notch pathway, and there were differences in the phosphorylation levels of the TLEs. We also confirmed that the effects of LIPUS on ovarian tissues were consistent with those achieved by Notch pathway inhibitors. Coimmunoprecipitation demonstrated that LIPUS reduced the phosphorylation of TLE1. This modification weakens the formation of the HES1-TLE1 transcriptional repressor complex, leading to increased HO-1 expression and decreased oxidative damage. In conclusion, LIPUS can ameliorate oxidative damage during the cryopreservation of ovarian tissues associated with the Notch pathway and may be a promising adjuvant treatment.