Long non-coding RNAs (lncRNA) play a key role in the orchestration of transcriptional regulation during development and many other cellular processes. The importance of the regulatory co-expression network was highlighted in the identification of the mechanism of these processes in humans and mice. However, elucidation of the properties of porcine lncRNAs involved in the regulatory network during pre-implantation embryonic development and fibroblast reprogramming to induced pluripotent stem cell (iPSC) has been limited to date. Using a weighted gene co-expression network analysis, we constructed the regulatory network and determined that the novel lncRNAs were functionally involved in key events of embryonic development during the pre-implantation period; moreover, reprogramming could be delineated by a small number of potentially functional modules of co-expressed genes. These findings indicate that lncRNAs may be involved in the transcriptional regulation of zygotic genome activation, first lineage segregation and somatic reprogramming to pluripotency. Furthermore, we performed a conservation and synteny analysis with the significant lncRNAs involved in these vital events and validated the results via experimental assays. In summary, the current findings provide a valuable resource to dissect the protein coding gene and lncRNA regulatory networks that underlie the progressive development of embryos and somatic reprogramming.

Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs), are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs) under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX). These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM) of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal–epithelial transition (MET) through the cAMP/PKA/CREB signal pathway and upregulates the E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation.

Cell-cultivated meat has emerged as an alternative approach for sustainable protein production, but replicating the texture and complex composition of conventional meat remains a critical challenge. Here, we developed an efficient approach to produce multitissue engineered meat by leveraging a serum- and animal-component-free differentiation system to direct porcine pregastrulation epiblast stem cells toward muscle, adipose, and endothelium with synergistic functions. These three types of cell progenitors demonstrated autonomous intercellular recognition and coculture compatibility in a scaffold-free 3D suspension system, enabling the spontaneous formation of tissue-mimetic spheroids with enhanced cellular multiplication efficiency. The products of multitissue cultivated meat recapitulated the textural properties of conventional pork and allowed for nutritional modulation. This platform overcomes key limitations in currently employed cultured meat technologies by integrating scalable 3D suspension culture with serum-free, species-specific stem cell multidirectional differentiation, providing an avenue for the development of multitissue cell-cultivated meat.

Capturing different pluripotent state stem cells from epiblast in vitro helps understand embryonic development and provides invaluable cell sources for basic research and regenerative medicine. Sheep are not only one of the most important livestock species in agriculture but also serve as an ideal preclinical model for studying human disease. Single‐cell transcriptome analysis of sheep preimplantation embryos from embryonic day (E) 1 to E14 is performed to investigate the pluripotency changes of epiblast and elucidate the pluripotent regulation signaling. By combination of growth factors or inhibitors of JAK/STAT3, FGF, WNT, and TGF‐β pathways in the culture medium, sheep formative and primed pluripotent stem cells (sfPSCs and spPSCs) are established respectively. The newly derived PSCs could maintain over 100 passages and differentiate into three germ layers. In addition, sfPSCs and spPSCs exhibit different molecular features, and sfPSCs have the ability of contribution to ICM and can be used as donor cells for producing cloned embryos efficiently. A cross‐species comparison of early embryo development in mouse, pig and sheep illustrates the conservation of the epiblast naïve to primed state transition process and the divergence of the developmental events time points, the specific gene expression patterns and pluripotent regulation signaling. These studies are expected to improve our understanding of mammal early embryo development and present a reference for defining pluripotency.

Long noncoding RNAs (lncRNAs) are crucial in porcine preimplantation embryonic development, yet their regulatory role during zygote genome activation (ZGA) is poorly understood. We analyzed transcriptome data from porcine fetal fibroblasts (PEF), induced pluripotent stem cells (iPS), and preimplantation embryos, identifying ZGA-specific lncRNAs like XLOC-040580 , and further predicted its potentially interacting genes TPRA1 and BCL2L1 via co-expression network. XLOC-040580 was knocked down by siRNA microinjection and the expression of ZGA-related genes was detected by qRT-PCR. After microinjecting siRNA targeting TPRA1 and BCL2L1 at the one-cell stage, we counted the blastocyst development rate. The blastocyst development rate was consistent with the results from si-XLOC-040580 after si- TPRA1 . Through dual-luciferase reporter assays, we found that XLOC-040580 was a downstream target of TPRA1 . To further elucidate the mechanism of XLOC-040580 , Single-cell mRNA sequencing after XLOC-040580 knockdown revealed its regulatory network involved in embryonic developmental defects. Transcriptome analysis revealed that XLOC-040580 was specifically expressed during zygote activation. Knockdown of XLOC-040580 decreased the blastocyst development rate and reduced both the total blastocyst cell number and TE cell number. TPRA1 and BCL2L1 were specifically co-expressed with XLOC-040580 during ZGA stage, and TPRA1 could interact with the promoter region of XLOC-040580 and regulate its expression. Knockdown of TPRA1 or XLOC-040580 blocked porcine embryonic development by affecting the expression of ZGA-related genes. We found and validated that lncRNA XLOC-040580 played a key role in the ZGA process, which was regulated by TPRA1 . These results implied that the functional axis of TPRA1 - XLOC-040580 -downstream genes involved in ZGA-related functions also coordinated early embryonic development in porcine.

Negative energy balance (NEB) in postpartum dairy cows elevates systemic non-esterified fatty acid (NEFA) levels, including palmitic acid (PA), which impair oocyte quality and contribute to reduced reproductive performance. Salidroside (SAL) has attracted attention for its prominent role in maintaining mitochondrial homeostasis. However, whether SAL can alleviate PA-induced oocyte damage remains unclear. Therefore, the present study was designed to investigate the protective effects of SAL against PA-induced oocyte damage and to explore the underlying mechanisms. We found that 0.2 mM PA triggered oxidative stress in oocytes, as evidenced by fluorescent staining results showing elevated ROS levels, reduced GSH content, and single-cell transcriptomic data revealing transcriptional suppression of HSPE1, ROMO1, and GSTM3. Meanwhile, PA treatment impaired mitochondrial function, reflected by decreased mitochondrial membrane potential and ATP levels, along with significant downregulation of mitochondrial function related genes, including NDUFA4, NDUFA6, ATP5PO, ATP5ME, COX5A, and UQCRH. These changes help explain why PA exposure reduces oocyte maturation rate and embryonic developmental potential. Notably, supplementation with 5 μM SAL effectively reversed the PA induced damage and restored oocyte developmental competence to control level to levels comparable to untreated controls. These findings suggest that SAL can restore oocyte developmental potential by mitigating the lipotoxic microenvironment induced by PA administration.