CircKDM5B sponges miR-128 to regulate porcine blastocyst development by modulating trophectoderm barrier function.

Circular RNAs (circRNAs), which exert critical functions in the regulation of transcriptional and post-transcriptional gene expression, are found in mammalian cells but their functions in mammalian preimplantation embryo development remain poorly understood. Here, we showed that circKDM5B mediated miRNA-128 (miR-128) to regulate porcine early embryo development. We screened circRNAs potentially expressed in porcine embryos through an integrated analysis of sequencing data from mouse and human embryos, as well as porcine oocytes. An authentic circRNA originating from histone demethylase KDM5B (referred to as circKDM5B) was abundantly expressed in porcine embryos. Functional studies revealed that circKDM5B knockdown not only significantly reduced blastocyst formation but also decreased the number of total cells and trophectoderm (TE) cells. Moreover, the knockdown of circKDM5B resulted in the disturbance of tight junction assembly and impaired paracellular sealing within the TE epithelium. Mechanistically, miR-128 inhibitor injection could rescue the early development of circKDM5B knockdown embryos. Taken together, the findings revealed that circKDM5B functions as a miR-128 sponge, thereby facilitating early embryonic development in pigs through the modulation of gene expression linked to tight junction assembly.

Emamectin benzoate exposure impaired porcine oocyte maturation.

Emamectin benzoate (EB) is a widely used insecticide that can damage the central nervous and immune systems. EB exposure significantly reduced the number of eggs laid, hatching rate, and developmental rate of lower organisms such as nematodes. However, effects of EB exposure on the maturation of higher animals such as porcine oocytes remains unknown. Here we reported that EB exposure severely impaired porcine oocyte maturation. EB exposure with 200 µM prevented cumulus expansion and reduced the rates of first polar body (pb1) extrusion, cleavage and blastocyst after parthenogenetic activation. Moreover, EB exposure disrupted spindle organization, chromosome alignment, and polymerization of microfilaments, but also apparently decreased the levels of acetylated α-tubulin (Ac-Tub) in oocytes. In addition, EB exposure perturbed mitochondria distribution and increased levels of reactive oxygen species (ROS), but did not affect the distribution of cortical granules (CGs) in oocytes. Excessive ROS caused DNA damage accumulation and induced early apoptosis of oocytes. EB exposure led to the abnormal expression of cumulus expansion and apoptosis-associated genes. Altogether, these results demonstrate that EB exposure impaired nuclear and cytoplasmic maturation of porcine oocytes probably through oxidative stress and early apoptosis.

Inhibitory Effects of 3-Methylcholanthrene Exposure on Porcine Oocyte Maturation.

3-methylcholanthrene (3-MC) is a highly toxic environmental pollutant that impairs animal health. 3-MC exposure can cause abnormal spermatogenesis and ovarian dysfunction. However, the effects of 3-MC exposure on oocyte maturation and embryo development remain unclear. This study revealed the toxic effects of 3-MC exposure on oocyte maturation and embryo development. 3-MC with different concentrations of 0, 25, 50, and 100 µM was applied for in vitro maturation of porcine oocytes. The results showed that 100 µM 3-MC significantly inhibited cumulus expansion and the first polar body extrusion. The rates of cleavage and blastocyst of embryos derived from 3-MC-exposed oocytes were significantly lower than those in the control group. Additionally, the rates of spindle abnormalities and chromosomal misalignments were higher than those in the control group. Furthermore, 3-MC exposure not only decreased the levels of mitochondria, cortical granules (CGs), and acetylated α-Tubulin, but also increased the levels of reactive oxygen species (ROS), DNA damage, and apoptosis. The expression of cumulus expansion and apoptosis-related genes was abnormal in 3-MC-exposed oocytes. In conclusion, 3-MC exposure disrupted the nuclear and cytoplasmic maturation of porcine oocytes through oxidative stress.

Vitrification of Pronuclear Zygotes Perturbs Porcine Zygotic Genome Activation.

Zygotic genome activation (ZGA) plays an essential role in early embryonic development. Vitrification is a common assisted reproductive technology that frequently reduces the developmental competence of embryos. However, the effect of vitrification on porcine ZGA and gene expression during ZGA remains largely unclear. Here, we found that vitrification of pronuclear zygotes derived from parthenogenetic activation (PA) and in vitro fertilization (IVF) resulted in a significant reduction in the rates of 2-cell, 4-cell, and blastocysts, but did not affect the quality of blastocysts. Functional research revealed that RNA polymerase II Inhibitor (α-amanitin) treatment significantly reduced global transcriptional activity and developmental efficiency of both 4-cell and 8-cell embryos, implying an essential role of ZGA in porcine early embryonic development. Furthermore, vitrification did not affect the synthesis of nascent mRNA of 2-cell embryos, but significantly inhibited global transcriptional activity of both 4-cell and 8-cell embryos, suggesting an impaired effect of vitrification on porcine ZGA. Correspondingly, the single-cell analysis showed that vitrification caused the downregulation or upregulation expression of maternal genes in 4-cell embryos, also significantly decreased the expression of zygotic genes. Taken together, these results indicated that vitrification of pronuclear zygotes impairs porcine zygotic genome activation.

Chromatin remodeler INO80 mediates trophectoderm permeability barrier to modulate morula-to-blastocyst transition.

Inositol requiring mutant 80 (INO80) is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells. However, the roles and mechanisms of INO80 in porcine pre-implantation embryonic development remain largely unknown. Here, we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development. The INO80 protein is highly expressed in the nuclei during morula-to-blastocyst transition. Functional studies revealed that RNA interference (RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm. Mechanistically, single-embryo RNA sequencing revealed that INO80 regulates multiple genes, which are important for lineage specification, tight junction assembly, and fluid accumulation. Consistent with the altered expression of key genes required for tight junction assembly, a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts. Importantly, aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium. Taken together, these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification, tight junction assembly, and fluid accumulation.

Histone Arginine Methyltransferase CARM1-Mediated H3R26me2 Is Essential for Morula-to-Blastocyst Transition in Pigs.

Coactivator-associated arginine methyltransferase 1 (CARM1) is involved in both establishment of first pluripotent lineage and pluripotency maintenance of embryonic stem cells (ESCs) in mice. However, the histone substrates and role of CARM1 in early embryonic development remain largely unknown. Here, we show that CARM1 specifically catalyzes H3R26me2 to promote porcine blastocyst formation. The putative histone substrates of CARM1, including H3R2me2, H3R17me2, and H3R26me2, are present in pig early embryos. The changes of CARM1 mRNA during early embryogenesis parallel that of H3R26me2. Functional studies using a combinational approach of chemical inhibition and RNA interference (RNAi) showed that catalytic activity inhibition of CARM1 protein or knockdown (KD) of CARM1 mRNA did not alter the levels of both H3R2me2 and H3R17me2, but significantly reduced H3R26me2 levels in porcine embryos. Furthermore, CARM1 inhibition or KD did not affect embryo development to the 2-cell, 4-cell, 8-cell, and morula stages, but severely compromised blastocyst development. CARM1 knocked down embryos that developed to the blastocyst stage had fewer total cells, inner cell mass (ICM), and trophectoderm (TE) cells. Mechanistically, single embryo RNA-sequencing analysis revealed that CARM1 KD altered the transcriptome characterized by downregulation of key genes associated with Hippo and PI3K-AKT signaling pathways. Taken together, these results demonstrate that CARM1 specifically catalyzes H3R26me2 in porcine embryos and participates in blastocyst development.