The experimental protocol was approved by the Animal Care and Usage Committee of Ankara University (protocol no: 2024—16—138). The female Balb/C mice (n = 75) at 4–5 weeks of age were purchased from the Research Animal Laboratory Unit of Ankara University. All mice were hosted with free access to food and water and kept in a 12 h light/dark cycle.

Cumulus-enclosed germinal vesicle (GV)-stage oocytes were collected from the ovaries of 4-week-old Balb/C female micethat were superovulated with 5 IU pregnant mare’s serum gonadotropin (PMSG, Intervet, Milton Keynes, UK) and sacrificed through cervical dislocation. For this purpose, after removing adipose tissue surrounding the ovaries, cumulus-enclosed oocytes complexes (COC) containing fully grown GV-stage oocytes encircled by cumulus cells were released by puncturing the ovaries with a 23-gauge needle in morpholinepropanesulfonic acid (MOPS)-buffered medium (G-MOPS™) (Vitrolife, Göteborg, Sweden) (Fig. 1). The attached cumulus cells were removed by repetitive pipetting of COCs. Denuded GV oocytes were transferred to the culture medium (G-TL™; Vitrolife, Göteborg, Sweden) as 50 μL volumes of culture drops in 35 mm culture dishes (Corning, Corning, NY, USA) that were overlaid by approximately 3 mL of Ovoil (10,029, Göteborg,Vitrolife). GV oocytes (0 h) were cultured up to metaphase II (MII) oocytes (14–16 h) at 37 °C in 5% CO2. The criteria used to identify MII oocytes was the presence of polar body between the zona pellucida and oocyte.

Experimental design. Following PMSG injection, GV-stage oocytes were collected from the ovaries and divided into control and experimental groups. Subsequently, MII-stage oocytes were retrieved, and oocyte maturation rates were evaluated. Immunofluorescence staining was performed, and global DNA methylation levels as well as protein expression levels were analyzed

Lipid-based transfection method was used for siRNA delivery. Oocytes were transferred into 50 μL-drops of culture medium (G-TL™; Vitrolife, Göteborg, Sweden) containing 50 nM small interfering RNA (siRNA) oligonucleotides against DNMT3B (ON-TARGET plus SMART pool siRNAs L-044164—00—0005, Dharmacon, Lafayette, CO, USA) and against DNMT3L (ON-TARGET plus SMART pool siRNAs L-063056—01—0005, Dharmacon, Lafayette, CO, USA) versus nontargeting control siRNA duplexes for 14–16 h using DharmaFECT (Dharmacon, Lafayette, CO, USA) as a transfection reagent. Nontargeting siRNA duplex served as the control for siRNA. Transfection medium preparations were performed according to DharmaFECT™ Transfection Reagents—siRNA transfection protocol (Dharmacon, Lafayette, CO, USA).

Following fixation of MII oocytes for 20 min in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), a permeabilization step was performed using 1% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature (RT). Subsequently, blocking was carried out for 1 h using a blocking solution containing 20% normal goat serum (Vector Laboratories, Newark, CA, USA). Immunofluorescence (IF) was applied to detect the relative quantity and cellular distribution profiles of the DNMT1, DNMT3A, DNMT3B, and DNMT3L proteins, and the relative levels of 5-methylcytosine (5mC) for global DNA methylation in the MII stage oocytes. Briefly, oocytes were incubated overnight at +4 °C with rabbit polyclonal antibody against DNMT1 (Abcam, ab87654, Cambridge, UK; reactive with either oocyte-specific (DNMT1o) or somatic (DNMT1s) isoforms), rabbit monoclonal antibodies against DNMT3A (Abcam, ab188470, Cambridge, UK), DNMT3B (Cell Signaling, 48,488, Danvers, MA, USA), DNMT3L (Abcam, ab3493, Cambridge, UK), or 5mC (Cell Signaling, 28692S, Danvers, MA, USA). Antibody specificity was validated by the manufacturers, and validation data are provided in the technical datasheets supplied by Abcam and Cell Signaling. For 5mC staining, oocytes were incubated in 2 M HCl for 30 min. Subsequent to an initial triple wash using 1 × PBS containing 2% bovine serum albumin (BSA) for a duration of 10 min each (PBS-BSA; Sigma-Aldrich, St. Louis, MO, USA), oocytes were subjected to an incubation with anti-rabbit IgG Alexa 488 secondary antibody (Invitrogen, California, USA) for a span of 1 h at RT. This was succeeded by another round of triple washes, utilizing the 1 × PBS-BSA solution, for 10 min each. For the negative control group, the primary antibodies were omitted to assess nonspecific binding of secondary antibody. The staining procedures were executed within miniwell trays (Thermo Fisher Scientific, Waltham, MA, USA) placed in a humidified chamber. Multiple 4 μL droplet of a PBS-based mounting medium, each containing 1 μg/mL of Hoechst dye (Thermo Fisher Scientific, Waltham, MA, USA) for DNA labeling, were prepared onto glass-bottomed 35 mm petri dishes. These droplets were then covered with paraffin oil. The stained oocytes were carefully placed within these droplets. All fluorescently tagged oocytes were kept intact in terms of their three-dimensional (3D) spherical shape and then were examined and imaged using a Zeiss LSM-880 Airyscan® system (Zeiss, Oberkochen, Germany). Oocytes were classified as MII-stage based on standard morphological criteria and maturation timing under in vitro culture conditions. For signal intensity measurement, fluorescence channels were separated as green for DNMTs and 5mC and blue for Hoechst, and images were converted into 32-bit format. Total signal intensities were measured through pixel-based method by Image J software (National Institutes of Health, Bethesda, MD, USA). Fluorescence intensity measurements were performed within the oocyte cytoplasm while excluding the zona pellucida to avoid potential background signals. All images were acquired using identical microscope settings, and negative controls without primary antibody were included to verify staining specificity. Negative control signal intensities were subtracted, then signal intensity values normalized to the control group, and the relative staining intensity levels of the DNMT proteins and global DNA methylation have been quantified. For each experimental group, at least three independent replicates were performed, and representative images were selected from multiple fields. Quantitative analyses were conducted on a minimum of 150 oocytes per group.

Images obtained from the immunofluorescence analyses were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Statistical analyses were performed using a computer-based program (IBM SPSS) employing one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test to identify differences among groups. A p-value of < 0.05 was considered statistically significant.