Inhibition of PTEN activates bovine non-growing follicles in vitro but increases DNA damage and reduces DNA repair response.

study questionDoes ovarian follicle activation by phosphatase homologue of chromosome-10 (PTEN) inhibition affect DNA damage and repair in bovine oocytes and granulosa cells? SUMMARY ANSWER: PTEN inhibition promotes bovine non-growing follicle activation but results in increased DNA damage and impaired DNA repair capacity in ovarian follicles in vitro. WHAT IS KNOWN ALREADY: Inhibition of PTEN is known to activate primordial follicles but may compromise further developmental potential. In breast cancer cells, PTEN inhibition represses nuclear translocation of breast cancer susceptibility 1 (BRCA1) and Rad51; this impairs DNA repair resulting in an accumulation of damaged DNA, which contributes to cell senescence. STUDY DESIGN, SIZE, DURATION: Bovine ovarian tissue fragments were exposed to control medium alone or containing either 1 or 10 μM bpv(HOpic), a pharmacological inhibitor of PTEN, in vitro for 24 h. A sub-group of tissue fragments were collected for Western blot analysis after bpv(HOpic) exposure. The remainder were incubated in control medium for a further 5 days and then analysed histologically and by immunohistochemistry to detect DNA damage and repair pathways. PARTICIPANTS/MATERIALS, SETTING,

methodsBovine ovaries were obtained from abattoir-slaughtered heifers. Tissue fragments were exposed to either control medium alone or medium containing either 1 μM or 10 μM bpv(HOpic) for 24 h. Tissue fragments collected after 24 h were subjected to Akt quantification by Western blotting (six to nine fragments per group per experiment). Follicle stage and morphology were classified in remaining fragments. Immunohistochemical analysis included nuclear exclusion of FOXO3 as a marker of follicle activation, γH2AX as a marker of DNA damage, meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM), Rad51, breast cancer susceptibility 1 (BRCA1) and breast cancer susceptibility 2 (BRCA2) as DNA repair factors. A total of 29 550 follicles from three independent experiments were analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Tissue fragments exposed to bpv(HOpic) had increased Akt phosphorylation at serine 473 (pAkt/Akt ratio, 2.25- and 6.23-fold higher in 1 and 10 μM bpv(HOpic) respectively compared to control, P < 0.05). These tissue fragments contained a significantly higher proportion of growing follicles compared to control (78.6% in 1 μM and 88.7% in 10 μM versus 70.5% in control; P < 0.001). The proportion of morphologically healthy follicles did not differ significantly between 1 μM bpv(HOpic) and control (P < 0.001) but follicle health was lower in 10 μM compared to 1 μM and control in all follicle types (P < 0.05). DNA damage in oocytes, indicated by expression of γH2AX, increased following exposure to 1 μM bpv(HOpic) (non-growing, 83%; primary follicles, 76%) and 10 μM (non-growing, 77%; primary, 84%) compared to control (non-growing, 30% and primary, 59%) (P < 0.05 for all groups). A significant reduction in expression of DNA repair proteins MRE11, ATM and Rad51 was observed in oocytes of non-growing and primary follicles of treatment groups (primary follicles in controls versus 10 μM bpv(HOpic): MRE, 68% versus 47%; ATM, 47% versus 18%; Rad51, 48% versus 24%), P < 0.05 for all groups. Higher dose bpv(HOpic) also resulted in lower expression of BRCA1 compared to control and 1 μM bpv(HOpic) (P < 0.001) in non-growing and primary follicles. BRCA2 expression was increased in oocytes of primary follicles in 1 μM bpv(HOpic) (36%) compared to control (20%, P = 0.010) with a marked decrease in 10 μM (1%, P ≤ 0.001). Granulosa cells of primary and secondary follicles in bpv(HOpic) groups showed more DNA damage compared to control (P < 0.05). However, bpv(HOpic) did not impact granulosa cell DNA repair capacity in secondary follicles, but BRCA1 declined significantly in higher dose bpv(HOpic). LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study focuses on non-growing follicle activation after 6 days culture and may not reflect DNA damage and repair capacity in later stages of oocyte and follicle growth. WIDER IMPLICATIONS OF THE

findingsIn vitro activation of follicle growth may compromise the bidirectional signalling between oocyte and granulosa cells necessary for optimal oocyte and follicle health. This large animal model may be useful in optimising follicle activation protocols with a view to transfer for clinical application. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Indonesia endowment fund for education. No competing interest. TRIAL REGISTRATION NUMBER: Not applicable.