210 Post-Translational Modification of Histone H4 Acetylation During in Vitro Maturation of Canine Oocytes

Abstract

Proper in vitro oocyte maturation, crucial for in vitro fertilisation, is poorly understood and inefficient in dogs. Post-translational modifications of histones are involved in the meiotic process and preparation of oocytes for fertilisation. The present study aimed to evaluate post-translational histone H4 acetylation at lysine 12 (H4K12ac) and 16 (H4K16ac) in canine oocytes during in vitro maturation (IVM). Ovaries were retrieved from 1–7 year-old bitches undergoing routine ovariohysterectomy (OH). Grade I cumulus-oocyte complexes (COC) were recovered and evaluated before and after IVM in TCM 199 + 0.025 mM sodium pyruvate + 100 000 IU mL–1 penicillin G sodium + 100 mg mL–1 streptomycin sulfate, 0.003 g mL–1 BSA + 50 μL mL–1 Pluset® (500 UI FSH + 500 UI LH) + 1 μL mL–1 insulin-like growth factor (IGF) at 38.5°C in 5% CO2 for 72 h. After IVM, the nuclear stage of oocytes was established using Hoechst 33342 staining, and the acetylation patterns were investigated by indirect immunofluorescence staining of fixed immature and post-IVM oocytes (4% paraformaldehyde) with specific antibodies against the acetylated lysine residues, H4K12ac and H4K16ac. All stages were collected and evaluated simultaneously, and the experiment was repeated 4 times, with a total of 5–14 of oocytes evaluated per stage. Immunofluorescence signal was quantified using the NIH ImageJ software 1.4, and data are presented as a percentage of the average fluorescence intensity of each specific antibody over the intensity of DNA, as determined by Hoescht staining. Immunofluorescence values were analysed using the least square ANOVA by the general linear model procedures of SAS (SAS Institute, Cary, NC, USA), and comparisons of means were further performed by Fisher’s Exact test. A probability level of P < 0.05 was considered significant. Our results indicate a variation in acetylation levels of H4K12 (P < 0.02) and H4K16 (P < 0.04) during meiosis. Fluorescence levels of H4K12ac decreased significantly when comparing immature (2.19 ± 0.62) with oocytes in metaphase I/II (MI/MII) (0.52 ± 0.14). Similarly, H4K16ac displayed a greater acetylation pattern in immature oocytes (1.20 ± 0.27) when compared to oocytes in germinal vesicle breakdown (GVBD; 0.58 ± 0.16) and MI/MII (0.62 ± 0.07) stages. These results support the hypothesis that, similar to other domestic species, post-translational modification of histone acetylation takes place during the meiotic process of oocyte IVM. Whether these histone modifications take place in a similar fashion during IVM remains to be investigated.

Changes in acetylation of lysine 5 on histone H4 in canine oocytes following in vitro maturation

“…Global deacetylation has been described during the progression of meiosis in mice (Akiyama et al., , ; De La Fuente et al., ; Kim et al., ; Manosalva & Gonzalez, ; Spinaci et al., ), pigs (Endo et al., ; Wang, ; Wang et al., ), cows (Maalouf et al., ; Racedo et al., ), horses (Franciosi et al., ) and humans (van den Berg et al., ). As for canine oocytes, we have demonstrated in a previous study that other lysine residues, namely H4K12 and H4K16, were also deacetylated after the resumption of meiosis (Motheo et al., ). Interestingly, similar findings were described in the present study, where we observed a significant decrease in fluorescence intensity of H4K5 between immature and post‐IVM canine oocytes.…”

“…On average, only 15–20% of the canine oocytes are capable of resuming meiosis and achieving metaphase II (MII) after 48–72 hr of in vitro culture (Songsasen & Wildt, ). Many factors are attributed to such low efficiency, such as the uniqueness of the canine reproductive physiology, hormonal environment, role of the oviduct on oocyte maturation, oocyte morphology and metabolism, and incorrect gene activity and epigenetic marks during the IVM process (Apparício et al., ; Luvoni, Chigioni, Allievi, & Macis, ; Motheo et al., ; Songsasen & Wildt, ).…”