Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development.  In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment.  At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated.  From 0.1 to 10 μmol L^–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal.  When the LPA concentration was 15 μmol L^–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst.  Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2

and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol

L^–1.  The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L^–1 to LPA-10 μmol L^–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L^–1 LPA-treated embryos compared with LPA-10 μmol L^–1 group (P<0.05).  The inner cell mass in 15 μmol L^–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal.  Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells.  The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells.  They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L^–1, while 15 μmol L^–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells.  The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L^–1 group and LPA-10 μmol L^–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L^–1 group compared with LPA-10 μmol L^–1 group (P<0.05).  Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L^–1 concentration.  This study references the IVF embryo production and embryonic stem cell research of domestic animals. 

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene knock-ins with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) in bovine and dairy goat fetal fibroblasts.  To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin (MSTN) gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2.  Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in the GFP-PGK-NeoR plasmid background, including a 5´ and 3´ homologous arm flanking the genes humanized Fat-1 (hFat-1) or enhanced green fluorescent protein (eGFP).  Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and the hFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts.  After G418 (Geneticin) selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove.  The gene knock-in events were screened by PCR across the homologous arms.  The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP and hFat-1 gene knock-ins were 13.68 and 0%, respectively.  The efficiencies of CRISPR/Cas9-mediated eGFP and hFat-1 gene knock-ins were 77.02 and 79.01%, respectively.  The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system.  Additionally, the hFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system.  The difference of knock-in efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant (P<0.01).  In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP and hFat-1 gene knock-ins were 32.35 and 26.47%, respectively.  The efficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively.  The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant (P<0.01).  This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs.  The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines. 

Spermatogonial stem cells (SSCs) are the key to maintaining production of the sperms and healthy offsprings, and also treating breeding livestock’s reproductive damage and infertility. MicroRNAs act a decisive role in regulating gene expression in many cells and tissues, including in processes such as proliferation, self-renewal, differentiation, and apoptosis of stem cells. However, the miRNA mechanism in regulation of SSCs is still unclear. Here, high-throughput sequencing was used to identify specific miRNAs. We confirmed that miR-21-5p was concentrated in both goat and mouse SSCs, and enhanced the proliferation and antiapoptotic ability of SSCs. In vivo experiments have shown that miR-21-5p resisted the damage of the chemotherapy drug Busulfan to germ cells, ameliorated Busulfan-induced testicular dysfunction, and maintained spermatogenesis. Further RNA-seq and target gene prediction revealed that SPRY1 and FASLG are targets of miR-21-5p, thereby activating downstream signaling pathways such as MAPK/ERK, PI3K-AKT, and apoptosis. In summary, miR-21-5p is crucial for the self-renewal and maintenance of SSCs. This study provides new avenues for treating breeding livestock’s reproductive damages, infertility, oligospermia, and other conditions.