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. 2019 Nov;14(11):1088-1101.
doi: 10.1080/15592294.2019.1633864. Epub 2019 Jun 24.

The histone lysine demethylase KDM7A is required for normal development and first cell lineage specification in porcine embryos

Vitor Braga Rissi  1 , Werner Giehl Glanzner  2 , Mariana Priotto De Macedo  2 , Karina Gutierrez  2 , Hernan Baldassarre  2 , Paulo Bayard Dias Gonçalves  1 , Vilceu Bordignon  2
Affiliations

The histone lysine demethylase KDM7A is required for normal development and first cell lineage specification in porcine embryos

Vitor Braga Rissi et al. Epigenetics. 2019 Nov.

Abstract

There is growing evidence that histone lysine demethylases (KDMs) play critical roles in the regulation of embryo development. This study investigated if KDM7A, a lysine demethylase known to act on mono-(me1) and di-(me2) methylation of H3K9 and H3K27, participates in the regulation of early embryo development. Knockdown of KDM7A mRNA reduced blastocyst formation by 69.2% in in vitro fertilized (IVF), 48.4% in parthenogenetically activated (PA), and 48.1% in somatic cell nuclear transfer (SCNT) embryos compared to controls. Global immunofluorescence (IF) signal in KDM7A knockdown compared to control embryos was increased for H3K27me1 on D7, for H3K27me2 on D3 and D5, for H3K9me1 on D5 and D7, and for H3K9me2 on D5 embryos, but decreased for H3K9me1, me2 and me3 on D3. Moreover, KDM7A knockdown altered mRNA expression, including the downregulation of KDM3C on D3, NANOG on D5 and D7, and OCT4 on D7 embryos, and the upregulation of CDX2, KDM4B and KDM6B on D5 embryos. On D3 and D5 embryos, total cell number and mRNA expression of embryo genome activation (EGA) markers (EIF1AX and PPP1R15B) were not affected by KDM7A knockdown. However, the ratio of inner cell mass (ICM)/total number of cells in D7 blastocysts was reduced by 45.5% in KDM7A knockdown compared to control embryos. These findings support a critical role for KDM7A in the regulation of early development and cell lineage specification in porcine embryos, which is likely mediated through the modulation of H3K9me1/me2 and H3K27me1/me2 levels, and changes in the expression of other KDMs and pluripotency genes.

Keywords: Lysine demethylases; cell differentiation; embryo development; histone methylation; porcine.

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Figures

Figure 1.
Figure 1.
Developmental rates and total number of cells in parthenogenetic (PA), in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos injected with si-CT (black bars) and si-KDM7A (white bars). Results are presented as means ± SEM, and P < 0.05 was considered statistically significant. Different letters indicate statistical significance between groups on the same day. Three independent replicates were performed using 30–40 embryos per group in each replicate.
Figure 2.
Figure 2.
Immunofluorescence staining for H3K27me1, me2 and me3 on day 3, 5 and 7 PA-derived embryos injected with si-CT or si-KDM7A. (a) Representative pictures of H3K27me1, me2 and me3 staining in si-CT and si-KDM7A embryos. (b) Quantification of the pixel intensity for H3K27me1 and H3K27me2 in si-CT (black bars) and si-KDM7A (white bars). Results are presented as means ± SEM, and P < 0.05 was considered statistically significant. Different letters indicate statistical significance between groups on the same day. Values were corrected to 1 (dashed line) in si-CT groups and results are shown as relative differences between groups. Three independent replicates were performed and 10–15 embryos per group were used for quantification of pixel intensities.
Figure 3.
Figure 3.
mmunofluorescence staining for H3K9me1, me2 and me3 on day 3, 5 and 7 PA-derived embryos injected with si-CT or si-KDM7A. (a) Representative pictures of H3K9me1, me2 and me3 staining in si-CT and si-KDM7A embryos. (b) Quantification of the pixel intensity for H3K9me1, me2 and me3 in si-CT (black bars) and si-KDM7A (white bars). Results are presented as means ± SEM, and P < 0.05 was considered statistically significant. Different letters indicate statistical significance between groups on the same day. Values were corrected to 1 (dashed line) for si-CT groups and results are shown as relative differences between groups. Three independent replicates were performed and 10–15 embryos per group were used for quantification of pixel intensities.
Figure 4.
Figure 4.
Relative mRNA expression of H3K9 (a), H3K27 (b) and H3K4 (c) demethylases on day 3 and day 5 PA embryos injected with si-CT (black bars) or si-KDM7A (white bars). Results are presented as means ± SEM, and P < 0.05 was considered statistically significant. Different letters indicate statistical significance between groups on the same day. Three independent replicates were performed and pools of 10–15 embryos per group from each replicate were used for mRNA extraction.
Figure 5.
Figure 5.
(a) Total number of cells on day 3, 5 and 7 PA embryos from si-CT (black bars) and si-KDM7A (white bars) groups. (b) Representative pictures of Sox2 immunofluorescence staining for differential cell count in blastocysts. (c) Ratio of ICM/total number of cells for si-CT (black bars) and si-KDM7A (white bars) blastocysts. Results are presented as means ± standard error of the mean (S.E.M), and P < 0.05 was considered statistically significant. Three independent replicates were performed and 10–15 embryos per group were used to evaluate total cell number and ratio of ICM/total number of cells.
Figure 6.
Figure 6.
Relative mRNA expression of genes involved on the regulation of cell pluripotency on day 5 and 7 PA embryos injected with si-CT (black bars) or si-KDM7A (white bars). Results are presented as means ± SEM, and P < 0.05 was considered statistically significant. Different letters indicate statistical significance between groups on the same day. * indicates p = 0.0592 value. Three independent replicates were performed and pools of 10–15 embryos per group from each replicate were used for mRNA extraction.
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This study was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada [RGPIN/04910-2016], the Brazilian National Council for Scientific and Technological Development (CNPq), and the Research Support Foundation of the State of Rio Grande do Sul (FAPERGS). V.B.R, W.G.G, and K.G. were supported by a scholarship from the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES).

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