3 HISTONE H4 ACETYLATION AT LYSINE 12 AND Cdc2a EXPRESSION ARE DECREASED IN AGED MOUSE GERMINAL VESICLE-STAGE OOCYTES

Abstract

Ageing is a phenomenon related to mammalian female infertility. One cause of ageing-induced infertility is the abnormal meiotic maturation from germinal vesicle stage oocytes (GVs). GVs are immature oocytes, which stay arrested in the ovaries during the life span. The abnormal oocyte maturation in aged animals is partially originated from molecular changes, not well defined, such as chromatin modifications and differential gene expression. Here, we analyze chromatin modifications such as histone acetylation and the corresponding gene expression changes induced by ageing in mouse GVs. We measured by immunofluorescence histone H4 acetylation at lysine residues 5 (H4-K5), 8 (H4-K8), and 12 (H4-K12) in GVs collected from young (1 month old) and aged (12–18 months old) CDC1 female mice. Immunofluorescence was analyzed with a microscope (Leica TCS SP2 AOBS) and its image analysis software. Whereas H4-K5 and H4-K8 show similar acetylation levels in both young and old mice, significant lower acetylation of H4-K12 is detected in GVs from old mice. Since H4-K12 acetylation has been related to Cdc2a expression during oocyte maturation (Akiyama et al. 2004 Mol. Reprod. Dev. 69, 222–227; Minuzzo et al. 2005 Mol. Pharmacol. 68, 1496–1503), we investigated whether Cdc2a mRNA levels change in aged mice. Cdc2a expression was measured by RT-PCR and quantified with a densitometer (BioRad GS800). We observed a decrease of Cdc2a expression in GVs of old mice. This result is further confirmed by an immunofluorescence analysis where lower levels of Cdc2a protein in old mouse GVs was observed. In conclusion, we find that the levels of H4-K12 acetylation and Cdc2a mRNA are lower in old compared to young mouse GVs. Our observations suggest that ageing affects histone modifications such as H4-K12, which might induce chromatin remodelling and gene expression changes like that of Cdc2a.