Fig. 3

Effect of the addition of pharmacological inhibitors of the PI3K/PTEN/Akt and mTOR signalling pathways during cryopreservation and/or in vitro organotypic culture on follicle activation. Ratio of phosphorylated to total Akt and rps6 for A ovaries cryopreserved without inhibitors and cultured for 24 h with LY or Ra; B ovaries cryopreserved with LY or Ra and cultured for 24 h without inhibitors; C ovaries cryopreserved and cultured for 24 h with inhibitors or D ovaries cryopreserved with Ra and cultured for 24 h with LY. E and F Computer-assisted quantification of the immunofluorescence staining density of phosphorylated forms of Akt and rps6 in the whole ovarian section. G and H Quantification of primordial follicles labelled or not by p-Akt or p-rps6. I Representative images of p-Akt (a-c) and p-rps6 (d-f) staining for the different groups. Red staining = DDX4; green staining = p-Akt or p-rps6. SF = slow-frozen/thawed ovaries with or without inhibitors followed by organotypic culture (cult) for 24 h with or without inhibitors. CT = control; LY = LY294002; Ra = rapamycin. a significant difference compared to the corresponding control; b significant difference compared to control not labelled primordial follicles. c significant difference compared to not labelled primordial follicles in ovaries cultured with LY294002; d significant difference compared to control labelled primordial follicles; e significant difference compared to labelled primordial follicles in ovaries cultured with LY294002. Ovaries from C57Bl/6, Nu/Nu and Rag mice, 4–8 weeks old. Numbers in columns represent the number of ovaries analysed per group. E-I n = 4–5 ovaries per group. 2–3 experimental replicates were performed. *p ≤ 0.05, **p ≤ 0.01. Scale bar represents 100 µm