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

• Published in: Human reproduction (Oxford) 2019-02, Vol.34 (2), p.297-307
• Main Authors: Maidarti, Mila, Clarkson, Yvonne L, McLaughlin, Marie, Anderson, Richard A, Telfer, Evelyn E
• Format: Article
• Language: English
• Subjects: Animals; Cattle; DNA Damage - drug effects; DNA Repair - drug effects; Female; Granulosa Cells - drug effects; Granulosa Cells - metabolism; Oocytes - drug effects; Oocytes - metabolism; Original; Ovarian Follicle - cytology; Ovarian Follicle - drug effects; Ovarian Follicle - growth & development; Ovarian Follicle - metabolism; PTEN Phosphohydrolase - antagonists & inhibitors; PTEN Phosphohydrolase - metabolism; Tissue Culture Techniques; Vanadium Compounds - pharmacology
• ISSN: 0268-1161; 1460-2350
• DOI: 10.1093/humrep/dey354

Abstract STUDY QUESTION Does 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, METHODS Bovine 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 fo