Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency.

The emergence of the first three lineages during development is orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system with a robust efficiency by introducing premature codon with cytosine base editors in bovine early embryos. By using this approach, we have determined the functional consequences of three critical lineage-specific genes (SOX2, OCT4 and CDX2) in bovine embryos. In particular, SOX2 knockout results in a failure of the establishment of pluripotency in blastocysts. Indeed, OCT4 level is significantly reduced and NANOG barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. RNA-seq analysis of single blastocysts (day 7.5) reveals dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 level is sustained in the trophectoderm in absence of CDX2. However, OCT4 knockout does not affect the expression of SOX2. Overall, we propose that SOX2 is indispensable for OCT4 and NANOG expression and CDX2 represses the expression of SOX2 in the trophectoderm in cattle, which are all in sharp contrast with results in mice.

Functional role of GATA3 and CDX2 in lineage specification during bovine early embryonic development.

In brief: The lineage specification during early embryonic development in cattle remains largely elusive. The present study determines the effects of trophectoderm-associated factors GATA3 and CDX2 on lineage specification during bovine early embryonic development. Abstract: Current understandings of the initiation of the trophectoderm (TE) program during mammalian embryonic development lack evidence of how TE-associated factors such as GATA3 and CDX2 participate in bovine lineage specification. In this study, we describe the effects of TE-associated factors on the expression of lineage specification marker genes such as SOX2, OCT4, NANOG, GATA6, and SOX17, by using cytosine base editor system. We successfully knockout GATA3 or CDX2 in bovine embryos with a robust efficiency. However, GATA3 or CDX2 deletion does not affect the developmental potential of embryos to reach the blastocyst stage. Interestingly, GATA3 deletion downregulates the NANOG expression in bovine blastocysts. Further analysis of the mosaic embryos shows that GATA3 is required for NANOG in the TE of bovine blastocysts. Single blastocyst RNA-seq analysis reveals that GATA3 deletion disrupts the transcriptome in bovine blastocysts. Altogether, we propose that GATA3 plays an important role in maintaining TE lineage program in bovine embryos and the functional role of GATA3 is species-specific.

The nucleolar protein NOP2 is required for nucleolar maturation and ribosome biogenesis during preimplantation development in mammals.

The maternal nucleolus plays an indispensable role in zygotic genome activation (ZGA) and early embryonic development in mice. During oocyte-to-embryo transition, the nucleolus is subject to substantial transformation. Despite the primary role of the nucleolus is ribosome biogenesis, accumulating evidence has uncovered its functions in various other cell processes. However, the regulation of nucleolar maturation and ribosome biogenesis and the molecules involved remain unclear during early embryonic development. In this study, we observed that nucleolar protein 2 (NOP2) is restrictedly localized within the nucleolus, first detected in the late two-cell embryos, and increases to a peak level at the eight-cell stage in mice. RNAi-mediated NOP2 depletion leads to a developmental arrest during the morula-to-blastocyst transition. RNA-seq analyses reveal that 208 genes are differentially expressed, including multiple lineage-specific genes and several genes encoding ribosome proteins. Indeed, we observe a failure of the first lineage specification with reduced TEA domain transcription factor 4(TEAD4) (trophectoderm-specific), tir na nog (NANOG), and kruppel-like factor 4 (KLF4) (inner cell mass-specific). Importantly, by Transmission Electron Microscopy (TEM), we noted a decrease in the ratio of the nucleolus size and an increase in the ratio of the size of the nucleolus precursor body, suggesting the nucleolar maturation is disrupted. Moreover, both qPCR and Fluorescence In Situ Hybridization (FISH) data showcase a significant decrease in the abundance of ribosome RNAs. Similarly, NOP2 depletion causes reduced developmental potential and decreased rRNA level in bovine early embryos, suggesting a functional conservation of NOP2 in mammals. Taken together, these results suggest that NOP2 is required for mammalian preimplantation development, presumably by regulating nucleolar maturation and ribosome biogenesis.

Transcriptomic Prediction of Pig Liver-Enriched Gene 1 Functions in a Liver Cell Line.

The newly identified liver-enriched gene 1 (LEG1) encodes a protein with a characteristic domain of unknown function 781 (DUF781/LEG1), constituting a protein family with only one member in mammals. A functional study in zebrafish suggested that LEG1 genes are involved in liver development, while the platypus LEG1 homolog, Monotreme Lactation Protein (MLP), which is enriched in the mammary gland and milk, acts as an antibacterial substance. However, no functional studies on eutherian LEG1s have been published to date. Thus, we here report the first functional prediction study at the cellular level. As previously reported, eutherian LEG1s can be classified into three paralogous groups. Pigs have all three LEG1 genes (pLEG1s), while humans and mice have retained only LEG1a. Hence, pLEG1s might represent an ideal model for studying LEG1 gene functions. RNA-seq was performed by the overexpression of pLEG1s and platypus MLP in HepG2 cells. Enrichment analysis showed that pLEG1a and pLEG1b might exhibit little function in liver cells; however, pLEG1c is probably involved in the endoplasmic reticulum (ER) stress response and protein folding. Additionally, gene set enrichment analysis revealed that platypus MLP shows antibacterial activity, confirming the functional study in platypus. Therefore, our study showed from the transcriptomic perspective that mammalian LEG1s have different functions in liver cells due to the subfunctionalization of paralogous genes.

Rapid detection of avian leukosis virus using a fluorescent microsphere immunochromatographic test strip assay.

We developed a rapid fluorescent microsphere immunochromatographic test strip (FM-ICTS) assay for the quantitative detection of avian leukosis virus (ALV). A monoclonal antibody specific for the ALV major capsid protein encoded by the gag gene was coupled to label fluorescent microspheres. ALV antibodies were coated on a nitrocellulose membrane to prepare a test line for sample detection. The fluorescence signals of the test and control lines can be read either visually by exposure to UV light or using a fluorescence analyzer. ALV could be detected quantitatively using the ratio of fluorescence signals of the test and control lines (T/C). The assay threshold was determined as a T/C value of 0.0606. The fitting curve equation was established between 1 and 2,048 ng/mL P27 protein with an r2 value of 0.9998. The assay showed no cross reactivity with Newcastle disease virus, infectious laryngotracheitis virus, infectious bronchitis virus, Marek's disease virus, infectious bursal disease, Reoviridae virus, or avian influenza virus. The repeatability was satisfactory with an overall average CV of 8.65%. The Kappa coefficient between a commercial ELISA kit was 0.7031 using clinical chicken meconium samples. Thus, a simple, rapid, sensitive, and specific fluorescent microsphere immunochromatographic test strip was developed based on specific anti-capsid protein p27 monoclonal antibodies.

Protective effects of Panax notoginseng saponins on PME-Induced nephrotoxicity in mice.

Polymyxin E (PME) plays an important role in fighting against Gram-negative bacterial infections; however, it causes nephrotoxicity, which limits its clinical use. The aim of this study was to investigate the protective effects of a plant extract Panax notoginseng saponins (PNS) on PME-induced nephrotoxicity in mice. In vivo studies showed that PNS significantly reduced blood urea nitrogen (BUN), serum creatinine (CRE) and number of apoptotic cells in kidney, as well as renal histopathological damage which increased in the presence of PME, and suppressed PME-induced oxidative stress in kidney, as shown by the up-regulation of superoxide dismutase (SOD) and the down-regulation of malondialdehyde (MDA) levels. Furthermore, PNS inhibited the expression of Bax, while increased the expression of Bcl-2 compared to the PME-treated group. In vitro studies showed that PNS decreased intracellular reactive oxygen species (ROS) and MDA levels, increased glutathione (GSH) levels, and enhanced the activity of SOD and glutathione peroxidase (GSH-Px) in murine renal tubular epithelial cells (TCMK-1 cells). In addition, PNS enhanced cell viability and the expression of Bcl-2, restored the mitochondrial membrane potential, inhibited the expression of Bax, inhibited the activity of caspase-3 and caspase-9, and reduce apoptotic rate in PME-treated TCMK-1 cells. PNS could reduce PME-induced nephrotoxicity. The protective effects could result from inhibition of oxidative stress, and prevention of cell apoptosis via the mitochondrial pathway. These findings highlight the potential of PNS as a safe adjunct for ameliorating the nephrotoxicity.