Detecção de proteínas envolvidas no remodelamento da cromatina de embriões bovinos produzidos in vitro a partir de oócitos oriundos de folículos antrais pequenos e grandes

Abstract

It has been demonstrated that oocytes of several species acquire the capacity to complete meiotic maturation and support early embryonic development during the final stages of follicular growth. Aiming to investigate molecular differences between bovine embryos produced from oocytes derived from small (1 to 2-mm) and large (4 to 8-mm) follicles, two experiments were designed to evaluate by immunocytochemistry the presence on chromatin of three regulatory factors involved mainly in DNA transcription and repair. In the first experiment, we evaluated whether the expression pattern of the High- Mobility Group N2 (HMGN2) and acetylated histone H3 Lysine 14 (Ac.H3K14) were affected by the origin (from small vs. large follicles) and time of first cleavage (< 24 h vs. > 24 h) of parthenogenetically activated (PA) oocytes. Early (until 24 hr) and late (after 24 hr) cleaved embryos were fixed at 36, 50, 60, 70 and 80 h after PA and processed to detect HMGN2 or Ac.H3K14. The rate of nuclear maturation (81% vs. 59%), early cleavage (47% vs. 39%), and blastocyst (34% vs. 19%) were significantly higher (P<0.05) in oocytes from large compared to small follicles. The rate of Ac.H3K14 (61% vs. 38%) and HMGN2 (74% vs. 56%) positively stained nuclei at 60 h post PA was higher (P<0.05) in embryos derived from small compared to large follicles. However, more HMGN2 positively stained nuclei (94% vs. 75%; P<0.05) were detected in embryos from large follicles at 80 h post PA. We concluded that the temporal proportion of embryonic nuclei with positive signal to HMGN2 and Ac.H3K14 is affected by both follicle size and time to complete first cleavage of oocytes. In the second experiment, we investigated whether the expression pattern of the phosphorylated histone H2A.X (γH2A.X) protein, which is an indicator of DNA double-strand breaks, is different in embryos produced from oocytes derived from small or large follicles. Oocytes were PA or in vitro fertilized (IVF) for 18 h and then cultured. Cleavage was assessed at 24 and 36 h after PA and at 32 and 42 h after IVF. Cleaved embryos were fixed at 36 h after PA and 42 h after IVF, and then processed to detect the γH2A.X. Most of the cleaved embryos produced from PA and IVF oocytes had detectable amounts of γH2A.X, ranging from few foci to a complete diffuse staining of the nuclei. γH2A.X was detected in 64% vs. 76% (P<0.05) of nuclei in PA embryos and in 76% vs. 80% (P>0.05) of nuclei in IVF embryos produced from oocytes derived from small and large follicles, respectively. IVF embryos presenting less than 4 nuclei at 42 h showed higher rates (P<0.05) of γH2A.X positive nuclei (89% and 85%) than those with ≥4 nuclei (72% and 62%, for large and small follicles, respectively). We found that γH2A.X is highly detected but not differently expressed in early bovine embryos produced from PA and IVF oocytes derived from small and large follicles. In general, the present experiments demonstrate that HMGN2, Ac.H3K14 and γH2A.X proteins are expressed during early bovine embryogenesis.