The present study assessed changes in epigenetic markers and pre- and postimplantation development in somatic cell nuclear transfer (SCNT) porcine embryos after treatment with the inhibitor of histone deacetylases (HDACi), Trichostatin A (TSA). Embryos were generated using in vitro matured oocytes and nuclei from either a male fetal fibroblast (FF) cell line or bone marrow cells (BMC) from three adult sows. After nuclear transfer, oocytes were either exposed or not to 10 ng/mL TSA for 10 h starting 1 h after cell fusion. Samples of one-cell stage and cleaved (two- to four-cell stage) embryos were fixed at 15 to 18 h or 46 to 48 h after cell fusion and immunocytochemically processed to detect histone H3 acetylation at lysine 14 (H3K14ac) or histone H3 dimethylation at lysine 9 (H3K9m2) using specific primary antibodies. TSA treatment increased the immunofluorescent signal for H3K14ac in cleaved embryos derived from both FF and BMC but did not affect H3K9m2. Development to the blastocyst stage was increased by TSA treatment (45.2 vs. 23.9%) in embryos produced from FF cells but not in those produced from BMC (30.6 vs. 27.4%). Cloned piglets were produced from both treatments when day 5 to 6 blastocyst-stage embryos derived from FF cells were transferred into the uterus of recipient females. Cloned piglets were also produced after the transfer of TSA-treated blastocysts derived from BMC of adult sows but not from control embryos. These findings suggest that the inhibition of histone deacetylases have similar effects on enhancing H3K14ac in SCNT embryos reconstructed from different cell types but the effect on in vitro and in vivo development seems to differ according to the nuclear donor cell type.

Studies in different species, including human, mice, bovine, and swine, demonstrated that early-cleaving embryos have higher capacity to develop to the blastocyst stage and produce better quality embryos with superior capacity to establish pregnancy than late-cleaving embryos. It has also been shown that experimentally induced DNA damage delays embryo cleavage kinetics and reduces blastocyst formation. To gain additional insights into the effects of genome damage on embryo cleavage kinetics and development, the present study compared the occurrence of DNA double-strand breaks (DSBs) with the expression profile of genes involved in DNA repair and cell cycle control between early- and late-cleaving embryos. Porcine oocytes matured in vitro were activated, and then early-cleaving (before 24 h) and late-cleaving (between 24 and 48 h) embryos were identified and cultured separately. Developing embryos, on Days 3, 5, and 7, were used to evaluate the total cell number and presence of DSBs (by counting the number of immunofluorescent foci for phosphorylated histone H2A.x [H2AX139ph] and RAD51 proteins) and to quantify transcripts of genes involved in DNA repair and cell cycle control by quantitative RT-PCR. Early-cleaving embryos had fewer DSBs, lower transcript levels for genes encoding DNA repair and cell cycle checkpoint proteins, and more cells than late-cleaving embryos. Interestingly, at the blastocyst stage, embryos that developed from early- and late-cleaving groups had similar number of DSBs as well as transcript levels of genes induced by DNA damage. This indicates that only embryos with less DNA damage and/or superior capacity for DNA repair are able to progress to the blastocyst stage. Collectively, findings in this study revealed a negative correlation between the occurrence of DSBs and embryo cleavage kinetics and embryo developmental capacity to the blastocyst stage.

Epigenetics is a fundamental regulator underlying many biological functions, such as development and cell differentiation. Epigenetic modifications affect key chromatin regulation, including transcription and DNA repair, which are critical for normal embryo development. In this study, we profiled the expression of epigenetic modifiers and patterns of epigenetic changes in porcine embryos around the period of embryonic genome activation (EGA). We observed that Brahma-related gene 1 (BRG1) and Lysine demethylase 1A (KDM1A), which can alter the methylation status of lysine 4 in histone 3 (H3K4), localize to the nucleus at Day 3-4 of development. We then compared the abundance of epigenetic modifiers between early- and late-cleaving embryos, which were classified based on the time to the first cell cleavage, to investigate if their nuclear localization contributes to developmental competence. The mRNA abundance of BRG1, KDM1A, as well as other lysine demethylases (KDM1B, KDM5A, KDM5B, and KDM5C), were significantly higher in late- compared to early-cleaving embryos near the EGA period, although these difference disappeared at the blastocyst stage. The abundance of H3K4 mono- (H3K4me) and di-methylation (H3K4me2) during the EGA period was reduced in late-cleaving and less developmentally competent embryos. By contrast, BRG1, KDM1A, and H3K4me2 abundance was greater in embryos with more than eight cells at Day 3-4 of development compared to those with fewer than four cells. These findings suggest that altered epigenetic modifications of H3K4 around the EGA period may affect the developmental capacity of porcine embryos to reach the blastocyst stage. Mol. Reprod. Dev. 84: 19-29, 2017. © 2016 Wiley Periodicals, Inc.

This study assessed the presence of cleaved caspase 3 (CC3) during the in vitro development of swine embryos produced by parthenogenetic activation (PA). Embryos with high and low capacity to develop into blastocysts and the exposure to a caspase inhibitor (z-DEVD-fmk) were used to investigate the effect of CC3 on embryo development. The blastocyst rate (64.3% vs. 16.4%) and the average number of nuclei per blastocyst (39.7 vs. 19.8) were significantly higher (P < 0.05) in early- (before 24 hr) compared to late- (between 24 and 48 hr) cleaving embryos after PA. CC3 was mainly detected in the cytoplasm of Day-2 and -4 embryos, but was primarily localized in the nucleus of Day-5 and -6 embryos. The fluorescence signal for CC3 relative to negative controls was significantly higher (P < 0.05) in early- (2.42-fold) compared to late-cleaving (1.39-fold) embryos at Day 2 of culture. Treatment with z-DEVD-fmk during the first 24 or 48 hr of the culture period resulted in more embryos developing into blastocysts compared to the control group (55.8% and 55.1% vs. 37%, respectively; P < 0.05). This study confirmed the presence of CC3 in PA embryos from the two-cell to the blastocyst stage, and revealed that CC3 cellular-localization changed during embryo development. CC3 was shown to be more abundant in early-cleaving and more developmentally competent embryos compared to late-cleaving and less developmentally competent embryos. The inhibition of caspase activity at the beginning, but not at the end, of the culture period affected development of PA embryos.