JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):109
Poster Presentation
29^th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263725

P-97. Female aging causes DNA hypomethylation in mouse oocytes

Caroline Alencar Imaeda-Carvalho¹, Fabíola Freitas Paula-Lopes¹

¹ Universidade Federal de São Paulo - Diadema - SP - Brasil

Objective: The ovarian reserve consists of a non-renewable pool of quiescent primordial follicles, each containing an oocyte, formed before birth in humans and shortly after birth in mice. With advancing age, the ovarian reserve undergoes a progressive decline. Due to the prolonged storage period, oocytes become particularly susceptible to damage, leading to cellular, molecular, and functional alterations. As a result, aging is characterized by a reduction in both oocyte quantity and quality, thereby decreasing female fertility. Among the molecular mechanisms, epigenetic alterations have been extensively studied, as they regulate the expression of genes essential for oocyte survival, growth, and maturation, as well as subsequent early embryonic development. Therefore, the aim of this study was to investigate the effect of aging on global DNA methylation in murine oocytes.
Methods: For this purpose, young (6–10 weeks) and aged (49–54 weeks) C57BL/6J female mice were intraperitoneally injected with 5 IU of eCG and euthanized 46 h later for oocyte collection, followed by immunofluorescence assay. Oocytes were fixed, permeabilized, subjected to DNA denaturation in HCl, neutralization in Tris-HCl, blocking with goat serum, incubation with a rabbit anti-mouse monoclonal 5-methylcytosine primary antibody (1:1000) and a goat anti-rabbit Alexa Fluor 488 polyclonal secondary antibody (1:500). Oocytes were examined under a fluorescence microscope (Zeiss), and fluorescence intensity was quantified using ImageJ software. Data were analyzed using normality (Shapiro–Wilk) and homogeneity of variance (Levene) tests, followed by Student's t-test or Wilcoxon test.
Results: As expected, the mean number of oocytes per female was higher in young mice (35.08±2.84; N=12 females) compared to aged mice (13.57±2.15; N=36 females; p=0.0003). In addition, DNA methylation was higher in young oocytes (74.36±4.07; N=20 oocytes) than in aged oocytes (64.71±4.78; N=20 oocytes; p=0.0285).
Conclusion: Therefore, aging is associated with global DNA hypomethylation and a reduction in oocyte quantity in mice.