SIRT1-dependent modulation of methylation and acetylation of histone H3 on lysine 9 (H3K9) in the zygotic pronuclei improves porcine embryo development

Abstract

Background: The histone code is an established epigenetic regulator of early embryonic development in mammals. The lysine residue K9 of histone H3 (H3K9) is a prime target of SIRT1, a member of NAD⁺-dependent histone deacetylase family of enzymes targeting both histone and non-histone substrates. At present, little is known about SIRT1-modulation of H3K9 in zygotic pronuclei and its association with the success of preimplantation embryo development. Therefore, we evaluated the effect of SIRT1 activity on H3K9 methylation and acetylation in porcine zygotes and the significance of H3K9 modifications for early embryonic development.

Results: Our results show that SIRT1 activators resveratrol and BML-278 increased H3K9 methylation and suppressed H3K9 acetylation in both the paternal and maternal pronucleus. Inversely, SIRT1 inhibitors nicotinamide and sirtinol suppressed methylation and increased acetylation of pronuclear H3K9. Evaluation of early embryonic development confirmed positive effect of selective SIRT1 activation on blastocyst formation rate (5.2 ± 2.9% versus 32.9 ± 8.1% in vehicle control and BML-278 group, respectively; P ≤ 0.05). Stimulation of SIRT1 activity coincided with fluorometric signal intensity of ooplasmic ubiquitin ligase MDM2, a known substrate of SIRT1 and known limiting factor of epigenome remodeling.

Conclusions: We conclude that SIRT1 modulates zygotic histone code, obviously through direct deacetylation and via non-histone targets resulting in increased H3K9me3. These changes in zygotes lead to more successful pre-implantation embryonic development and, indeed, the specific SIRT1 activation due to BML-278 is beneficial for in vitro embryo production and blastocyst achievement.

Keywords: Embryonic development; Epigenetics; H3K9 methylation; SIRT1; Sirtuin.

Fig. 1

Representative image of SIRT1 immunofluorescence in a 22-h zygote. a SIRT1 is exclusively localized in zygote pronuclei. b Yellow arrows indicate the level of signal intensity profiles in the respective maternal and paternal pronuclei. NC: negative control where the primary antibody was omitted. Scale bar represents 50 μm

Fig. 2

The effect of resveratrol on H3K9 methylation and acetylation in zygotic pronuclei. a Fluorescent signal intensities relative to signal intensity of vehicle control (= 1) and b representative images of H3K9me3 and H3K9ac in both pronuclei. ^a,b,c;1,2,3Significant differences in H3K9me3 and H3K9ac, respectively, among experimental groups (P ≤ 0.05). Asterisks indicate paternal pronucleus. Scalebar represents 25 μm

Fig. 3

The effect of nicotinamide on H3K9 methylation and acetylation in zygotic pronuclei. a Fluorescent signal intensities relative to vehicle control (= 1) and b representative images of H3K9me3 and H3K9ac in both pronuclei. ^a,b,c;1,2,3Significant differences in H3K9me3 and H3K9ac, respectively, among experimental groups (P ≤ 0.05). Asterisk indicates paternal pronucleus. Scalebar represents 25 μm

Fig. 4

The effect of BML-278 on H3K9 methylation and acetylation in the zygotic pronuclei. a Fluorescent signal intensities relative to vehicle control (= 1) and b representative images of H3K9me3 and H3K9ac in both pronuclei. *^#Significant difference between control and treated group (P ≤ 0.05). Asterisk indicates paternal pronucleus. Scalebar represents 25 μm

Fig. 5

The effect of sirtinol on H3K9 methylation and acetylation in zygotic pronuclei. a Fluorescent signal intensities relative to vehicle control (= 1) and b representative images of H3K9me3 and H3K9ac in both pronuclei. *^#Significant differences between control and treated group (P ≤ 0.05). Asterisk indicates paternal pronucleus. Scalebar represents 25 μm

Fig. 6

Quantification of MDM2 signal intensity in 22 h zygotes after treatment with BML-278. a Fluorescent signal intensity of MDM2 was normalized against housekeeping protein GAPDH. b Representative images show MDM2 and GADPH in control and treated zygotes. *Significant difference between control and treated group (P ≤ 0.05). Scalebar represents 50 μm