Only COCs with homogenous cytoplasm and at least three layers of cumulus cells were used in the experiments. In a glass tube, a stock solution (SS) with 1 g of methyl-β-cyclodextrin was dissolved in 2 mL of methanol and stored at −20 °C [10]. To load cholesterol

from FCS, the SS was diluted with different concentrations (1, 2 or 3 mg) of MβCD in 1 mL of HEPES-buffered TCM-199 (GIBCO® BRL) supplemented with 20% FCS. The solution was incubated overnight at 38.5 °C. Oocyte vitrification was performed as previously described [12] PLX-4720 with slight modifications. The holding medium (HM), which was used to handle oocytes during vitrification and warming, was composed of HEPES-buffered TCM-199 (GIBCO® BRL) supplemented

with 20% FCS. For vitrification, groups were first washed three times in an equilibrium solution composed of 7.5% ethylene glycol and 7.5% dimethylsulfoxide (Me2SO) dissolved in HM for a total of 9 min. Oocytes were transferred BIBF1120 to a vitrification solution of 15% ethylene glycol, 15% Me2SO and 0.5 M of sucrose in HM where they were incubated for 45–60 s. Next, the oocytes were placed into the cryotop device in sets of 3–5 under a stereomicroscope. Before vitrification, most of the solution that was transferred with the oocytes was removed from the device, and only a thin layer (<0.1 μl) remained to cover the oocytes. Subsequently, the cryotop device was immediately submerged into liquid nitrogen. Warming was performed immediately after vitrification by immersing the cryotop end into a drop of HM supplemented with 1 M of sucrose for 1 min pre-warmed at 37 °C. The oocytes were transferred to HM medium supplemented with 0.5 M of sucrose for 3 min, respectively, and finally to the original holding medium.

Afterwards, the oocytes were placed in the culture dishes to mature or were fixed for maturational stage evaluation. After selleck compound warming, COCs were washed and transferred (groups of 25–30) to a 200 μL drop of maturation medium under silicone oil and incubated for 22 h at 39 °C in 5% CO2 in air. The maturation medium was TCM-199 supplemented with 10% FCS (v/v), 10 mg/mL of FSH and antibiotics (100 IU/mL of penicillin and 50 mg/mL of streptomycin). CCOs were distributed into 4 groups, each group represented one maturation period. The first one was fixed immediately after selection, before IVM; the second group was fixed with 8 h of IVM; the third was fixed 22 h of IVM and the fourth group completed IVM period and was fixed with 24 h of IVM. For meiotic progression evaluation, oocytes were denuded and fixed for at least 48 h with acetic alcohol (1:3). On the day of the evaluation, these oocytes were placed on a slide, covered with a coverslip and were stained with 1% lacmoid in 45% glacial acetic acid. The maturational stage of each oocyte was determined using phase contrast microscopy.