Cleavage kinetics is a better indicator of embryonic developmental competency than brilliant cresyl blue staining of oocytes

Nix, Jada; Marrella, Mackenzie A.; Oliver, Mary Ali; Ealy, Alan D.; Biase, Fernando H.

doi:10.1016/j.anireprosci.2022.107174

Cited by 6 publications

(2 citation statements)

References 39 publications

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“…All procedures for oocyte maturation, embryo culture and media composition were described in detail elsewhere (Ortega et al 2017; Nix et al 2023a; Tríbulo et al 2019). Bos taurus ovaries were collected from a commercial slaughterhouse and transported to the laboratory in a 0.9% saline solution and antibiotic and antimycotic solution (1x, ThermoFisher Scientific, Waltham, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Identification of novel genes in cattle (Bos taurus) and biological insights into their function in embryo development

Schettini,

Morozyuk,

Biase

2024

Preprint

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Appropriate regulation of genes expressed in oocytes and embryos is essential for acquisition of developmental competence in mammals. Here, we hypothesized that several genes expressed in oocytes and pre-implantation embryos remain unknown. Our goal was to reconstruct the transcriptome of oocytes (germinal vesicle and metaphase II) and pre-implantation cattle embryos (blastocysts) using short-read and long-read sequences to identify putative new genes. We identified 274,342 transcript sequences, and 3,033 of those transcripts do not match a gene present in an annotation, thus are potential new genes. Notably, 63.67% (1,931/3,033) of potential novel genes exhibited coding potential. Also noteworthy, 97.92% of the putative novel genes overlapped annotation with transposable elements. Comparative analysis of transcript abundance identified that 1,840 novel genes (recently added to the annotation) or potential new genes were differentially expressed between developmental stages (FDR<0.01). We also determined that 522 novel or potential new genes (448 and 34 respectively) were upregulated at eight-cell embryos compared to oocytes (FDR<0.01). In eight-cell embryos, 102 novel or putative new genes were co-expressed (|r|>0.85, P<1x10-8) with several genes annotated with gene ontology processes related to pluripotency maintenance and embryo development. CRISPR-Cas9 genome editing confirmed that the disruption of one of the novel genes highly expressed in eight-cell embryos reduced blastocyst development (ENSBTAG00000068261, P=1.55x10-7). In conclusion, our results revealed several putative new genes that need careful annotation. Many of the putative new genes have dynamic regulation during pre-implantation development and are important components of gene regulatory networks involved in pluripotency and blastocyst formation.

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Section: Methodsmentioning

confidence: 99%

Identification of novel genes in cattle (Bos taurus) and biological insights into their function in embryo development

Schettini,

Morozyuk,

Biase

2024

Preprint

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“…All procedures and culture media composition for in vitro production of embryos are described in detail elsewhere (94,95). Briefly, we obtained cattle ovaries from an abattoir (Brown Packing, Gaffney, SC) and washed them with anti-biotic anti-mycotic (Antibiotic-Antimycotic 100X, Thermofisher Scientific, Waltham, MA) and 0.9% saline solution.…”

Section: In Vitro Production Of Embryosmentioning

confidence: 99%

Ablation ofOCT4function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART)

Nix

Schettini

Speckhart

et al. 2023

Preprint

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CRISPR-Cas ribonucleoproteins are important tools for gene editing in pre-implantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes. Here, we aimed to improve the efficiency of biallelic deletions and deplete specific maternal RNAs in cattle zygotes using CRISPR-Cas editing technology. Two electroporation sessions with Cas9D10A ribonucleoproteins targeting exon 1 and the promoter of OCT4 produced biallelic deletions in 91% of the embryos tested. In most cases, the deletions were longer than 1000 nucleotides long. Electroporation of Cas13a ribonucleoproteins prevents the production of the corresponding proteins. We electroporated Cas9D10A ribonucleoproteins targeting exon 1, including the promoter region, of OCT4 in two sessions with inclusion of Cas13a ribonucleoproteins targeting OCT4 mRNAs in the second session to ablate OCT4 function in cattle embryos. A lack of OCT4 resulted in embryos arresting development prior to blastocyst formation at a greater proportion (13%) than controls (31.6%, P<0.001). The few embryos that developed past the morula stage did not form a normal inner cell mass. Transcriptome analysis of single blastocysts, confirmed to lack exon 1 and promoter region of OCT4, revealed a significant (FDR<0.1) reduction in transcript abundance of many genes functionally connected to stemness, including markers of pluripotency (CADHD1, DPPA4, GNL3, RRM2). The results confirm that OCT4 is key regulator of genes that modulate pluripotency and is required to form a functional blastocyst in cattle.

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The impact of cumulus cell viability and pre-culture with the healthy cell mass on brilliant cresyl blue (BCB) staining assessment and meiotic competence of suboptimal porcine oocytes

Fonseka,

Do,

Van

et al. 2024

Theriogenology

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Cleavage kinetics is a better indicator of embryonic developmental competency than brilliant cresyl blue staining of oocytes

Cited by 6 publications

References 39 publications

Identification of novel genes in cattle (Bos taurus) and biological insights into their function in embryo development

Identification of novel genes in cattle (Bos taurus) and biological insights into their function in embryo development

Ablation ofOCT4function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART)

The impact of cumulus cell viability and pre-culture with the healthy cell mass on brilliant cresyl blue (BCB) staining assessment and meiotic competence of suboptimal porcine oocytes

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