Motheo T F, Arnold D R, Padilha-Nakaghi L C, Pires-Buttler E A, Alves A E, Apparicio M, Vicente Wrr, Lopes F L
Department of Animal Reproduction, Faculdade de Ciencias Agrarias e Veterinarias Campus de Jaboticabal, Universidade Estadual Paulista Julio de Mesquita Filho, Jaboticabal, Brazil.
In vitro Brasil S/A, Mogi Mirim, Brazil.
Reprod Domest Anim. 2017 Apr;52 Suppl 2:103-107. doi: 10.1111/rda.12897. Epub 2016 Dec 27.
Post-translational modifications of histones, such as acetylation, are involved in regulating chromatin remodelling and gene expression. Proper in vitro maturation (IVM) of canine oocytes, for many reasons, is up to now inefficient. This study aimed to evaluate the post-translational histone H4 acetylation at lysine 5 (H4K5) in immature and post-IVM canine oocytes. Oocyte nuclear stage was assessed using Hoechst 33342 staining. Acetylation patterns were determined by indirect immunofluorescence staining of immature and post-IVM oocytes, using an antibody against the acetylated lysine 5 residue on histone 4 (H4K5ac). The experiment was repeated four times, with a total of 7-17 oocytes evaluated per stage. Immunofluorescence signal was quantified using the NIHimagej software. Data were expressed as a percentage of the average fluorescence intensity of the specific antibody over the intensity of DNA, as determined by Hoescht staining. H4K5ac displayed a significantly higher acetylated pattern in immature oocytes (0.97 ± 0.08) when compared to post-IVM oocytes at different nuclear stages. There was a decrease in the fluorescence level of the matured oocytes with the progression of meiosis (GVBD: 0.47 ± 0.06 and MI/MII: 0.35 ± 0.04). Similarly to other domestic species, we hypothesized that post-translational modification of histone acetylation takes place during meiosis of in vitro matured canine oocytes. However, it remains to be investigated whether these changes occur during in vivo maturation.
组蛋白的翻译后修饰，如乙酰化，参与调节染色质重塑和基因表达。由于多种原因，犬类卵母细胞的体外成熟（IVM）至今效率仍不高。本研究旨在评估未成熟和IVM后犬类卵母细胞中赖氨酸5（H4K5）处的组蛋白H4乙酰化情况。使用Hoechst 33342染色评估卵母细胞核阶段。通过对未成熟和IVM后的卵母细胞进行间接免疫荧光染色，使用针对组蛋白4上乙酰化赖氨酸5残基（H4K5ac）的抗体来确定乙酰化模式。实验重复进行了4次，每个阶段共评估7 - 17个卵母细胞。使用NIHimagej软件对免疫荧光信号进行定量。数据表示为特定抗体的平均荧光强度与由Hoescht染色确定的DNA强度的百分比。与不同核阶段的IVM后卵母细胞相比，H4K5ac在未成熟卵母细胞中显示出显著更高的乙酰化模式（0.97±0.08）。随着减数分裂的进行，成熟卵母细胞的荧光水平降低（GVBD：0.47±0.06和MI/MII：0.35±0.04）。与其他家养物种类似，我们假设组蛋白乙酰化的翻译后修饰发生在体外成熟的犬类卵母细胞减数分裂过程中。然而，这些变化是否发生在体内成熟过程中仍有待研究。
