A Single-Cell Atlas of an Early Mongolian Sheep Embryo.

Cell types have been established during organogenesis based on early mouse embryos. However, our understanding of cell types and molecular mechanisms in the early embryo development of Mongolian sheep has been hampered. This study presents the first comprehensive single-cell transcriptomic characterization at E16 in Ujumqin sheep and Hulunbuir short-tailed sheep. Thirteen major cell types were identified at E16 in Ujumqin sheep, and eight major cell types were identified at E16 in Hulunbuir short-tailed sheep. Function enrichment analysis showed that several pathways were significantly enriched in the TGF-beta signaling pathway, the Hippo signaling pathway, the platelet activation pathway, the riboflavin metabolism pathway, the Wnt signaling pathway, regulation of the actin cytoskeleton, and the insulin signaling pathway in the notochord cluster. Glutathione metabolism, glyoxylate, and dicarboxylate metabolism, the citrate cycle, thyroid hormone synthesis, pyruvate metabolism, cysteine and methionine metabolism, thermogenesis, and the VEGF signaling pathway were significantly enriched in the spinal cord cluster. Steroid biosynthesis, riboflavin metabolism, the cell cycle, the Hippo signaling pathway, the Hedgehog signaling pathway, the FoxO signaling pathway, the JAK-STAT signaling pathway, and the Wnt signaling pathway were significantly enriched in the paraxial mesoderm cluster. The notochord cluster, spinal cord cluster, and paraxial mesoderm cluster were found to be highly associated with tail development. Pseudo-time analysis demonstrated that the mesenchyme can translate to the notochord in Ujumqin sheep. Molecular assays revealed that the Hippo signaling pathway was enriched in Ujumqin sheep. This comprehensive single-cell map revealed previously unrecognized signaling pathways that will further our understanding of the mechanism of short-tailed sheep formation.

Reprogramming efficiency and pluripotency of mule iPSCs over its parents†.

The mule is the interspecific hybrid of horse and donkey and has hybrid vigor in muscular endurance, disease resistance, and longevity over its parents. Here, we examined adult fibroblasts of mule (MAFs) compared with the cells from their parents (donkey adult fibroblasts and horse adult fibroblasts) (each species has repeated three independent individuals) in proliferation, apoptosis, and glycolysis and found significant differences. We subsequently derived mule, donkey, and horse doxycycline (Dox)-independent induced pluripotent stem cells (miPSCs, diPSCs, and hiPSCs) from three independent individuals of each species and found that the reprogramming efficiency of MAFs was significantly higher than that of cells of donkey and horse. miPSCs, diPSCs, and hiPSCs all expressed the high levels of crucial endogenous pluripotency genes such as POU class 5 homeobox 1 (POU5F1, OCT4), SRY-box 2 (SOX2), and Nanog homeobox (NANOG) and propagated robustly in single-cell passaging. miPSCs exhibited faster proliferation and higher pluripotency and differentiation than diPSCs and hiPSCs, which were reflected in co-cultures and separate-cultures, teratoma formation, and chimera contribution. The establishment of miPSCs provides a unique research material for the investigation of "heterosis" and perhaps is more significant to study hybrid gamete formation.

Reprogramming barriers in bovine cells nuclear transfer revealed by single-cell RNA-seq analysis.

Many progresses have recently been achieved in animal somatic cell nuclear transfer (SCNT). However, embryos derived from SCNT rarely result in live births. Single-cell RNA sequencing (scRNA-seq) can be used to investigate the development details of SCNT embryos. Here, bovine fibroblasts and three factors bovine iPSCs (3F biPSCs) were used as donors for bovine nuclear transfer, and the single blastomere transcriptome was analysed by scRNA-seq. Compared to in vitro fertilization (IVF) embryos, SCNT embryos exhibited many defects. Abnormally expressed genes were found at each stage of embryos, which enriched in metabolism, and epigenetic modification. The DEGs of the adjacent stage in SCNT embryos did not follow the temporal expression pattern similar to that of IVF embryos. Particularly, SCNT 8-cell stage embryos showed failures in some gene activation, including ZSCAN4, and defects in protein association networks which cored as POLR2K, GRO1, and ANKRD1. Some important signalling pathways also showed incomplete activation at SCNT zygote to morula stage. Interestingly, 3F biPSCNT embryos exhibited more dysregulated genes than SCNT embryos at zygote and 2-cell stage, including genes in KDM family. Pseudotime analysis of 3F biPSCNT embryos showed the different developmental fate from SCNT and IVF embryos. These findings suggested partial reprogrammed 3F biPS cells as donors for bovine nuclear transfer hindered the reprogramming of nuclear transfer embryos. Our studies revealed the abnormal gene expression and pathway activation of SCNT embryos, which could increase our understanding of the development of SCNT embryos and give hints to improve the efficiency of nuclear transfer.

Development and Application of a High-Resolution Melting Analysis with Unlabeled Probes for the Screening of Short-Tailed Sheep TBXT Heterozygotes.

The short-tailed phenotype has long been considered one of the best traits for population genetic improvement in sheep breeding. In short-tailed sheep, not only is tail fat eliminated but also the pubic area is exposed due to the lack of a tail covering, giving them an advantage in reproduction. Recent studies have shown that two linked mutations in sheep TBXT at nucleotides 333 and 334 are associated with the short-tailed phenotype. In the population of short-tailed sheep, several heterozygous mutants of this gene are found. In our research, we used high-resolution melting (HRM) to identify homozygous and heterozygous genotypes in a flock of short-tailed sheep and compared the results with those of Sanger sequencing, which were identical. This demonstrates that our established HRM method, a rapid and inexpensive genotyping method, can be used to identify homozygous and heterozygous individuals in short-tailed sheep flocks.

S100A8 expression in oviduct mucosal epithelial cells is regulated by estrogen and affects mucosal immune homeostasis.

Chronic inflammation can cause oviduct mucosal damage and immune dysfunction, leading to infertility, early pregnancy loss, ectopic pregnancy, tumors, and a decrease in reproductive capacities in female animals. Estrogen can suppress immune responses in different tissues and oviducts, and regulate the oviduct immune balance; however, the underlying mechanisms remain unclear. The objective of this study was to explore the mechanism of estrogen-regulated oviduct mucosal immunity and discover new estrogen targets for regulating oviduct mucosal immune homeostasis. Sheep oviduct epithelial cells (SOECs) were treated with 17-ß estradiol (E2). Transcriptome sequencing and analysis showed differentially expressed S100 calcium-binding protein A (S100A) genes that may participate in the oviduct mucosa immunoregulation of estrogen. Quantitative polymerase chain reaction and immunocytochemistry analysis showed that S100A8 expression changed dynamically in E2-treated SOECs and peaked after 7 h of treatment. Estrogen nuclear receptors and G protein-coupled membrane receptors promoted E2-dependent S100A8 upregulation. The S100A8 gene was disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 method. Levels of inflammatory factors interleukin (IL)-1ß and IL-4 were significantly upregulated in S100A8-knockdown SOECs, whereas those of the anti-inflammatory factor IL-10 was downregulated. Following S100A8 knockdown in SOECs treated with E2 for 7 h, IL-10 levels increased significantly. Estrogen affected oviduct mucosa immune function and dynamically regulated S100A8 in SOECs. S100A8 knockdown caused an excessive immune response, indicating that S100A8 is beneficial for maintaining immune homeostasis in the oviduct mucosa. Moreover, estrogen can compensate for the effect of S100A8 knockdown by upregulating IL-10.

Establishment of bovine expanded potential stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.

Generation of Sheep Induced Pluripotent Stem Cells With Defined DOX-Inducible Transcription Factors via piggyBac Transposition.

Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development. Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro. In this study, sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology, pluripotent marker expression, and differentiation ability, delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4, SOX2, KLF4, cMYC, porcine NANOG, human LIN28, SV40 large T antigen, and human TERT. Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro. A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs. This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep.

Whole Genome Sequencing of Hulunbuir Short-Tailed Sheep for Identifying Candidate Genes Related to the Short-Tail Phenotype.

The Hulunbuir short-tailed sheep (Ovis aries) is a breed native to China, in which the short-tail phenotype is the result of artificial and natural selection favoring a specific set of genetic mutations. Here, we analyzed the genetic differences between short-tail and normal-tail phenotypes at the genomic level. Selection signals were identified in genome-wide sequences. From 16 sheep, we identified 72,101,346 single nucleotide polymorphisms. Selection signals were detected based on the fixation index and heterozygosity. Seven genomic regions under putative selection were identified, and these regions contained nine genes. Among these genes, T was the strongest candidate as T is related to vertebral development. In T, a nonsynonymous mutation at c.G334T resulted in p.G112W substitution. We inferred that the c.G334T mutation in T leads to functional changes in Brachyury-encoded by this gene-resulting in the short-tail phenotype. Our findings provide a valuable insight into the development of the short-tail phenotype in sheep and other short-tailed animals.

Lipopolysaccharide induces SBD-1 expression via the P38 MAPK signaling pathway in ovine oviduct epithelial cells.

BACKGROUND: Beta defensins are secreted from ovine oviduct epithelial cells (OOECs) in response to microbial infection, and are potential alternatives to antibiotic agents in the treatment of microorganism infection, particularly given the abuse of antibiotic agents and the increasing number of drug-resistant bacteria. The aberrant expression of defensins may result in disorders involving organ and oviduct inflammation, such as salpingitis. METHODS: In the present study, we investigated the effects of LPS on the mRNA expression levels of sheep ß-defensin-1 (SBD-1) in ovine oviduct epithelial cells. The OOECs in vitro culturing system were established and treated with different concentrations of LPS for indicated time. In addition, MAPK inhibitors and TLR4 antibodies were pretreated to investigate the potential mechanism which involves in LPS regulating SBD-1 expression. RESULTS: LPS markedly upregulated SBD-1 expression in a concentration- and time-dependent manner. Treatment with 100 ng/mL LPS resulted in the phosphorylation of JNK, ERK and P38 MAPK. Interestingly, the LPS stimulated SBD-1 expression was attenuated by pretreatment with the P38 MAPK inhibitors SB203580 and SB202190 but not the JNK inhibitor SP600125, while the ERK inhibitor PD98059 had a minor effect. Furthermore, treatment with a Toll-like receptor 4 (TLR4) neutralizing antibody significantly decreased P38 MAPK phosphorylation and LPS induced SBD-1 expression. CONCLUSIONS: Together, these findings suggest that SBD-1 is upregulated by LPS via the TLR4 receptor, mainly through the P38 MAPK signaling pathway in ovine oviduct epithelial cells to protect the ovine oviduct epithelium from pathogen invasion.