Pharmacological inhibition of the PI3K/PTEN/Akt and mTOR signalling pathways limits follicle activation induced by ovarian cryopreservation and in vitro culture

Abstract

Background Cryopreservation and transplantation of ovarian tissue (OTCTP) represent a promising fertility preservation technique for prepubertal patients or for patients requiring urgent oncological management. However, a major obstacle of this technique is follicle loss due to, among others, accelerated recruitment of primordial follicles during the transplantation process, leading to follicular reserve loss in the graft and thereby potentially reducing its lifespan. This study aimed to assess how cryopreservation itself impacts follicle activation. Results Western blot analysis of the PI3K/PTEN/Akt and mTOR signalling pathways showed that they were activated in mature or juvenile slow-frozen murine ovaries compared to control fresh ovaries. The use of pharmacological inhibitors of follicle signalling pathways during the cryopreservation process decreased cryopreservation-induced follicle recruitment. The second aim of this study was to use in vitro organotypic culture of cryopreserved ovaries and to test pharmacological inhibitors of the PI3K/PTEN/Akt and mTOR pathways. In vitro organotypic culture-induced activation of the PI3K/PTEN/Akt pathway is counteracted by cryopreservation with rapamycin and in vitro culture in the presence of LY294002. These results were confirmed by follicle density quantifications. Indeed, follicle development is affected by in vitro organotypic culture, and PI3K/PTEN/Akt and mTOR pharmacological inhibitors preserve primordial follicle reserve. Conclusions Our findings support the hypothesis that inhibitors of mTOR and PI3K might be an attractive tool to delay primordial follicle activation induced by cryopreservation and culture, thus preserving the ovarian reserve while retaining follicles in a functionally integrated state.

Comprehensive Review of In Vitro Human Follicle Development for Fertility Restoration: Recent Achievements, Current Challenges, and Future Optimization Strategies

“…It is essential to replace culture medium after a fixed amount of time in order to prevent nutrient depletion and ensure elimination of toxic waste products, like ammonia and lactic acid. In studies involving IVC for more than four days, standard practice is to replace half of the culture medium every other day [ 10 , 21 , 38 , 50 , 51 , 52 , 53 ].…”

Circulating exosomal miR-21 mediates HUVEC proliferation and migration through PTEN/PI3K/AKT in Crohn’s disease

“…Therefore, the ability of miRNA to target PTEN has attracted the attention of many researchers. Multiple studies have shown that exosomal miR-21 can regulate the proliferation of multiple cells (33,37,38). In our study, we simulated the process of miRNA delivery through the lipid shell of exosomes and transferred miR-21 mimics and inhibitors into HUVECs for observation.…”

Mechanical stress-induced Hippo signaling in respect to primordial follicle development and polycystic ovary syndrome pathogenesis

“…The development of primordial follicles requires two-way signals to communicate with surrounding somatic cells (i.e., granulosa cells), where particular cytokines and growth factors are involved [8] . PI3K/PTEN/serine/threonine kinase (AKT) signaling, for instance, is related to the fate of primordial follicles [9,10] . Moreover, rapamycin, an inhibitor of mTOR, is known to suppress excessive activation of primordial follicles and protect the ovarian reserve by receiving regulatory signals from upstream AKT [11,12] .…”