Effect of manipulating PI3K pathway components on primordial follicle activation and DNA damage response in bovine ovarian follicles in vitro

Abstract

Developing immature follicles either from fresh or cryopreserved ovarian tissue to attain competent and fertilisable oocytes could provide an option for some patients undergoing fertility preservation to avoid the risk of ovarian micro-metastases following ovarian tissue transplantation. The first stage of an in vitro growth system (IVG) is activation of primordial follicles. Regulation of this process is critical as uncontrolled and precocious growth initiation of primordial follicles during in vitro activation (IVA) has been a major concern. A delicate balance between inhibitory and stimulatory signals is required to achieve activation but this can also be regulated by manipulating key signalling pathways associated with follicle activation. Phosphatase and tensin homolog of chromosome 10 (PTEN), expressed by the oocyte, is a negative regulator of the Phosphoinositide 3-kinase (PI3K) pathway and has been utilised to initiate primordial follicle growth both in vivo and in vitro in a range of species. Pregnancies have been achieved after grafting small ovarian cortical fragments exposed to PI3K/protein kinase B (Akt) activators to reinitiate the growth of residual follicles in the ovarian tissue of premature ovarian insufficiency (POI) patients. However, activating growth in this way may be damaging to the ovarian follicles. PTEN also has a role in maintaining genomic integrity. Its effects on DNA double strand breaks (DSBs) repair capacity has been debatable. Notably, unrepaired DNA damage is related to ovarian ageing. Meiotic errors are also more likely, leading to chromosomal abnormalities and impacting on oocyte quality. Therefore, we hypothesised that inhibiting PTEN to increase the activation of primordial follicles could result in increased DNA damage and compromised DNA repair capacity in oocytes and granulosa cells. This technique may also affect further growth of isolated preantral follicles selected for culture. The overall aim of this thesis was to determine the collective effects of PI3K/PTEN/Akt/mammalian target of rapamycin (mTOR) modulation pathway, either by inhibiting or activating the signals, on primordial follicle activation and DNA damage response (DDR) of bovine ovarian follicles in vitro. These experiments demonstrated that short-term incubation of ovarian cortex with low (1PM) and high dose (10PM) dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate (V) (bpv(HOpic)), a PTEN inhibitor, increased primordial follicle activation but resulted in a reduction in the proportion of morphologically healthy follicles in the high dose group. In parallel, DNA damage increased with limited DNA repair function. This was observed both in low and high dose. The mTOR signalling pathway is a master regulator of cell growth and metabolism and its inhibition attenuates follicle growth activation. In the second part, the potential benefit of inhibiting PI3K/Akt/mTOR signalling on the regulation of in vitro follicular activation was investigated. The addition of a low dose rapamycin to bpv(HOpic) or rapamycin on its own reduced DNA damage and improved DNA repair capacity of the oocytes. In the last part, experiments were extended to isolated preantral follicles. None of the treatments had an effect on promoting isolated follicle growth and survival. Although DNA repair protein ataxia telangiectasia mutated (ATM) was significantly upregulated in the presence of rapamycin, it appeared that cumulative effects of increased gamma H2A histone family member X (JH2AX) and upregulation of ATM and Rad51 were not sufficient to support follicle growth. Altogether, these data provide unimproved understanding into the regulation of the follicular activation and its relation with DDR, highlighting the significance of getting closer to physiological conditions to maintain follicle integrity. This may be a promising strategy for the derivation of mature oocytes in vitro. However, further investigations at the stage of isolated preantral follicle culture onwards are essential.