The effect of the presence of cumulus cells during IVM on lipid droplets and mitochondria destribution, the number of transzonal projection and IVM rate of bovine and murine oocytes

Immature COCs (immCOCs) obtained from bovine or murine ovaries were randomly allocated into three experimental groups: 1‒ intact COCs, 2 ‒ denuded oocytes (DOs), and 3 ‒ DOs supplemented with a suspension of CCs (DOs+CCs). All groups were subjected to a 24 h IVM. Bovine ovaries were transported within two hours after collection in a 35 °C sodium chloride solution supplemented with penicillin/streptomycin. COCs were aspirated from ovarian follicles measuring 2-8 mm in diameter using an 18-gauge needle attached to a 20 mL syringe. After aspiration, bovine COCs were collected, washed, and maintained before IVM or fixation in wash medium based on Medium 199 with L-glutamine and HEPES (Gibco, Thermo Fisher Scientific, USA), supplemented with 5% FBS, sodium pyruvate, and penicillin/streptomycin. Only COCs surrounded by at least three layers of CCs were selected for further procedures. After collection, the COCs were either fixed in paraformaldehyde (PFA) or randomly assigned to one of the three experimental groups and directed to the IVM process. Murine immCOCs were collected from ovaries of hormonally unstimulated mice (BALB/C strain) aged 6-10 weeks using a scraping technique with a 30-gauge needle. Murine immature COCs were collected from ovaries of hormonally unstimulated BALB/C mice aged 6–10 weeks using a scraping technique with a 30-gauge needle. During collection and sorting, murine COCs were maintained in M2 medium (Sigma-Aldrich, USA). After retrieval, the COCs were either fixed in 4% paraformaldehyde (PFA) or randomly distributed into the experimental groups and subjected to IVM. Species-adapted IVM media were used for bovine and murine oocytes. For bovine oocytes, COCs, DOs, and DOs+CCs were matured in 100 μL drops of commercial bovine IVM medium (Stroebech, Denmark) under mineral oil for 22–24 h at 38.5°C in a humidified atmosphere containing 5% CO₂. For murine oocytes, COCs, DOs, and DOs+CCs were matured in M16 medium (Sigma-Aldrich, USA) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, USA), 10 μg/mL follicle-stimulating hormone (FSH), and 10 μg/mL luteinizing hormone (LH) for 18 h at 37°C in a humidified atmosphere containing 5% CO₂. The murine M16-based IVM condition was selected based on previously described mouse oocyte culture protocols using M16 medium (Sigma-Aldrich, USA) supplemented with serum and gonadotropins . Transzonal projections (TZPs) were visualized by fluorescent F-actin labeling using rhodamine–phalloidin (cat. #ab235138, Abcam, Cambridge, UK) diluted 1:1000 in 10× Blocking Buffer. Mitochondrial staining of murine and bovine oocytes was performed using MitoTracker™ Red CMXRos (M7512, Invitrogen, USA), applied directly to the culture medium for 30 minutes at 37 °C (mouse) or 38.5 °C (bovine) in 5% CO₂. Following staining, cells were briefly washed, fixed in 4% PFA for 60 minutes, washed again in PBST, and counterstained with DAPI (ThermoFisher Scientific, USA) for 15 minutes before mounting on µ-Slide 18 Well chambers. Mitochondrial distribution patterns were evaluated using the same ImageJ Fiji software. Quantitative parameters included the central-to-peripheral mitochondrial ratio, central, peripheral, and total heterogeneity indices, and integrated mitochondrial signal. Lipid droplets were visualized using Nile Red (N1142, Invitrogen, USA). Mouse and bovine oocytes were fixed in 4% PFA for 60 minutes, washed in PBST, and stained with Nile Red for 30 minutes. After a final wash, samples were mounted on µ-Slide 18 well chambers for imaging. Lipid parameters, including total lipid content (intensity, a.u.), number of lipid droplets, droplet size (area, µm2), and the oocyte area occupied by lipid droplets (µm2), were quantified using ImageJ Fiji version 1.53c (NIH, Bethesda, MD, USA).