Interaction between PI3K/AKT and Hippo pathways during in vitro follicular activation and response to fragmentation and chemotherapy exposure using a mouse immature ovary model

Abstract

Understanding and control of the massive and accelerated follicular growth that occurs during in vitro culture of ovarian tissue is a crucial step toward the development of efficient culture systems that offer an attractive alternative to ovarian tissue transplantation for fertility restoration in cancer survivors. One outstanding question focuses on processes that occur prior to cryopreservation, such as tissue sectioning or chemotherapeutic treatment, might exacerbate this follicular activation. Although the PI3K/AKT/mTOR pathway is well known as a major trigger of physiological and chemotherapy-induced follicular activation, studies have shown that disruption of Hippo pathway due to ovarian fragmentation acts as an additional stimulator. This study aimed to characterize the possible interactions between these pathways using post-natal day 3 mouse ovaries cultured for 4 or 48 h. Morphology, gene transcription, and protein levels were assessed to investigate the impact of sectioning or chemotherapy exposure (4-hydroperoxycyclophosphamide [4HC], 3 and 20 μM). The effect of an mTORC1 inhibitor, Everolimus, alone or as a 4HC co-treatment to prevent follicle activation was evaluated. The results showed that organ removal from its physiological environment was as effective as sectioning for disruption of Hippo pathway and induction of follicle activation. Both PI3K/AKT/mTOR and Hippo pathways were involved in chemotherapy-induced follicular activation and responded to fragmentation. Surprisingly, Everolimus was able to prevent the activation of both pathways during chemotherapy exposure, suggesting cross-talk between them. This study underscores the major involvement of PI3K/AKT/mTOR and Hippo pathways in in vitro follicle activation and provides evidence that both can be regulated using mTORC1 inhibitor.

In vitro activation of ovarian cortex and autologous transplantation: A novel approach to primary ovarian insufficiency and diminished ovarian reserve

“…In recent years, certain novel technologies have been introduced to promote the growth and maturation of primordial follicles in vitro , becoming a promising alternative approach to fertility restoration ( Devos et al , 2020 ). In order to achieve a complete development of oocytes, a dynamic culture system that adapts to each stage of transition is required.…”

“…mTORC1 promotes protein translation and ribosomal biogenesis in oocytes, by phosphorylating S6K1-rpS6 (P70 S6 kinase 1-ribosomal protein S6) and 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) ( Adhikari and Liu, 2010 ). Studies performed in mice and human models suggest that transient culture of ovarian tissue with mTORC1 inhibitors, such as everolimus, partially prevents spontaneous activation by limiting growing follicle counts, without impairing the ability of growing follicles to develop and synthesize hormones ( Grosbois and Demeestere, 2018 ; Grosbois et al , 2019 ; Devos et al , 2020 ).…”

Nitric Oxide Synthase Is Involved in Follicular Development via the PI3K/AKT/FoxO3a Pathway in Neonatal and Immature Rats

“…As reported, NOS/cGMP activated the PI3K/AKT pathway [55], which is involved in chemotherapy-induced follicular activation and responds to fragmentation [56]. In addition, the NOS/cGMP pathway is related to the angiotensin (HIF-1) in endothelial cells [57].…”

Ovarian Fragmentation and AKT Stimulation for Expansion of Fertile Lifespan

“…Consequently, increases in YAP nuclear translocation stimulated the expression of CCN growth factors and BIRC apoptosis inhibitors, resulting eventually in follicle growth (5, 7, 44) ( Figure 2). Furthermore, other studies confirmed that the Hippo signaling pathway works in concert with PI3K/Akt activators to accelerate primordial follicle recruitment (6,45,46). Using the animal model, several studies demonstrated thoroughly the mechanism of this intracellular signaling pathway.…”

“…Among the pathways modulating early folliculogenesis, the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays a crucial role in the activation of primordial follicles (4)(5)(6). Subsequent experiments suggested that the Hippo signaling pathway is also important in the development of follicles (7)(8)(9).…”