Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication

Simmet, Kilian; Reichenbach, M.; Reichenbach, Horst-Dieter; Wolf, Eckhard

doi:10.1016/j.theriogenology.2015.08.012

Cited by 11 publications

(13 citation statements)

References 32 publications

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“…In contrast to CRISPR-Cas9-mediated editing of zygotes, this approach guarantees consistent modification of all cells of the embryo and allows efficient screening for off-target effects. We used one cell line with a PGK-EGFP reporter construct, to enable later chimeric complementation studies of OCT4-deficient blastomeres (34). To exclude possible effects of the reporter construct, we also used a wild-type fibroblast cell line.…”

Section: Discussionmentioning

confidence: 99%

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OCT4/POU5F1 is required for NANOG expression in bovine blastocysts

Simmet

Zakhartchenko

Philippou‐Massier

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Mammalian preimplantation development involves two lineage specifications: first, the CDX2-expressing trophectoderm (TE) and a pluripotent inner cell mass (ICM) are separated during blastocyst formation. Second, the pluripotent epiblast (EPI; expressing NANOG) and the differentiated primitive endoderm (PrE; expressing GATA6) diverge within the ICM. Studies in mice revealed that OCT4/POU5F1 is at the center of a pluripotency regulatory network. To study the role of OCT4 in bovine preimplantation development, we generated knockout (KO) fibroblasts by CRISPR-Cas9 and produced embryos by somatic cell nuclear transfer (SCNT). SCNT embryos from nontransfected fibroblasts and embryos produced by in vitro fertilization served as controls. In KO morulae (day 5), ∼70% of the nuclei were OCT4 positive, indicating that maternal mRNA partially maintains OCT4 protein expression during early development. In contrast, KO blastocysts (day 7) lacked OCT4 protein entirely. CDX2 was detected only in TE cells; OCT4 is thus not required to suppress CDX2 in the ICM. Control blastocysts showed a typical salt-and-pepper distribution of NANOG- and GATA6-positive cells in the ICM. In contrast, NANOG was absent or very faint in the ICM of KO blastocysts, and no cells expressing exclusively NANOG were observed. This mimics findings in OCT4-deficient human blastocysts but is in sharp contrast to-null mouse blastocysts, where NANOG persists and PrE development fails. Our study supports bovine embryogenesis as a model for early human development and exemplifies a general strategy for studying the roles of specific genes in embryos of domestic species.

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Section: Discussionmentioning

confidence: 99%

“…SCNT and IVP procedures were performed as described previously (44). At 5 or 7 d after activation of fused complexes or IVF, respectively, the zona pellucida was removed, and embryos were fixed in 2% paraformaldehyde (34) or stored at −80°C until RNA extraction.…”

Section: Methodsmentioning

confidence: 99%

OCT4/POU5F1 is required for NANOG expression in bovine blastocysts

Simmet

Zakhartchenko

Philippou‐Massier

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Embryo aggregation is widely used in the production of transgenic and chimeric animals in various mammals, including mice (Yamaguchi et al, 2017), bovine (Simmet et al, 2015) and monkeys (Tachibana et al, 2012), to improve the production efficiency by compensating for the developmental deficiency of IVP embryos. In particular, pigs have been reported to respond positively to embryo aggregation, with increases in blastocyst formation rate, total cell number, ICM/TE ratio, and cellular survival (Lee et al, 2007; Terashita et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Embryo aggregation regulates in vitro stress conditions to promote developmental competence in pigs

Jeong

Yoon

Lee

et al. 2019

PeerJ

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Embryo aggregation is a useful method to produce blastocysts with high developmental competence to generate more offspring in various mammals, but the underlying mechanism(s) regarding the beneficial effects are largely unknown. In this study, we investigated the effects of embryo aggregation using 4-cell stage embryos in in vitro developmental competence and the relationship of stress conditions in porcine early embryogenesis. We conducted aggregation using the well of the well system and confirmed that aggregation using two or three embryos was useful for obtaining blastocysts. Aggregated embryos significantly improved developmental competence, including blastocyst formation rate, blastomere number, ICM/TE ratio, and cellular survival rate, compared to non-aggregated embryos. Investigation into the relationship between embryo aggregation and stress conditions revealed that mitochondrial function increased, and oxidative and endoplasmic reticulum (ER)-stress decreased compared to 1X (non-aggregated embryos) blastocysts. In addition, 3X (three-embryo aggregated) blastocysts increased the expression of pluripotency, anti-apoptosis, and implantation related genes, and decreased expression of pro-apoptosis related genes. Therefore, these findings indicate that embryo aggregation regulates in vitro stress conditions to increase developmental competence and contributes to the in vitro production of high-quality embryos and the large-scale production of transgenic and chimeric pigs.

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“…Enzyme reaction was stopped by washing embryos in THP supplemented with 10% foetal calf serum (FCS) and the dissolved ZP was completely removed by gentle pipetting. Embryos were incubated in drops of PBS without Mg 2+ and Ca 2+ supplemented with 4 mg/ml polyvinylpyrrolidone under oil for 5–10 minutes and blastomeres were subsequently disaggregated by gentle pipetting 51 . Single cells were transferred individually to 0.5-µl drops of lysis buffer (Buffer A of Prelude Direct Lysis Module, NuGEN) under mineral oil, collected in a 384-well plate, and stored at −80 °C.…”

Section: Methodsmentioning

confidence: 99%

Single-cell RNA sequencing reveals developmental heterogeneity of blastomeres during major genome activation in bovine embryos

Lavagi¹,

Krebs²,

Simmet³

et al. 2018

Sci Rep

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Embryonic development is initially controlled by maternal RNAs and proteins stored in the oocyte, until gene products gradually generated by the embryo itself take over. Major embryonic genome activation (EGA) in bovine embryos occurs at the eight- to 16-cell stage. Morphological observations, such as size of blastomeres and distribution of microvilli, suggested heterogeneity among individual cells already at this developmental stage. To address cell heterogeneity on the transcriptome level, we performed single-cell RNA sequencing of 161 blastomeres from 14 in vitro produced bovine embryos at Day 2 (n = 6) and Day 3 (n = 8) post fertilization. Complementary DNA libraries were prepared using the Single-Cell RNA-Barcoding and Sequencing protocol and sequenced. Non-supervised clustering of single-cell transcriptome profiles identified six clusters with specific sets of genes. Most embryos were comprised of cells from at least two different clusters. Sorting cells according to their transcriptome profiles resulted in a non-branched pseudo-time line, arguing against major lineage inclination events at this developmental stage. In summary, our study revealed heterogeneity of transcriptome profiles among single cells in bovine Day 2 and Day 3 embryos, suggesting asynchronous blastomere development during the phase of major EGA.

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Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication

Cited by 11 publications

References 32 publications

OCT4/POU5F1 is required for NANOG expression in bovine blastocysts

OCT4/POU5F1 is required for NANOG expression in bovine blastocysts

Embryo aggregation regulates in vitro stress conditions to promote developmental competence in pigs

Single-cell RNA sequencing reveals developmental heterogeneity of blastomeres during major genome activation in bovine embryos

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