Paraquat exposure impairs porcine oocyte meiotic maturation.

Paraquat (PQ) is a heterocyclic pesticide that not only damages the testicular development and reduces the quality of semen, but also disturbs the secretion of hormones in the reproductive system. However, the effects of PQ on oocyte maturation and its toxic mechanism have not been yet fully clarified. Here we showed that PQ exposure could have toxic effects on porcine oocyte maturation. PQ exposure with 100 µM inhibited cumulus cell expansion and significantly reduced the rate of first polar body extrusion during oocyte maturation. PQ-exposed oocytes could not develop to the 2-cell and blastocyst stage. PQ exposure with 100 µM significantly increased abnormal spindle rate (65.2% ± 1.0%) and misaligned chromosome rate (63.2% ± 3.4%) compared to the control group (38.3% ± 1.0% and 38.4% ± 1.0%, respectively; P < 0.05). F-actin also exhibited reduced distribution in PQ-exposed oocytes (10.3% ± 1.0%) compared to the control group (14.4% ± 1.0%, P < 0.05). In addition, PQ exposure reduced the active mitochondria levels, but apparently increased the reactive oxygen species (ROS), rH2AX, and LC3 (autophagy marker) levels. qPCR analyses showed that PQ exposure caused the aberrant expression of genes associated with cumulus cell expansion, but did not affect the expression of apoptosis-related genes. Taken together, these results indicate that PQ exposure impaired oocyte nuclear and cytoplasmic maturation probably through oxidative stress.

Expression Analysis of Circular RNAs in Young and Sexually Mature Boar Testes.

Testicular development is critical for male animals' reproduction and is tightly regulated by epigenetic factors. Circular RNAs (circRNAs) were recently identified in the testes of humans and bulls. However, the expression profile of circRNAs and their potential biological functions in boar testicular development remain unclear. We identified 34,521 and 31,803 circRNAs in piglet (30 d) and adult (210 d) boar testes by high-throughput sequencing, respectively. Bioinformatics analysis revealed that these circRNAs are widely distributed on autosomes and sex chromosomes. Some of the host genes can generate multiple circRNAs. A total of 2326 differentially expressed circRNAs (DECs) derived from 1526 host genes was found in testicular development, of which 1003 circRNAs were up-regulated in adult boar testes and 1323 circRNAs were down-regulated. Furthermore, gene ontology analysis of host genes of DECs revealed that these circRNAs are mainly involved in regulating spermatogenesis, cilia motility, and hormone biosynthesis. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that the DECs are markedly enriched to stem cell pluripotency regulation, tight junctions, adhesion junctions, and cAMP signaling pathway. These results indicate that circRNAs are abundantly expressed in boar testes and exhibit dynamic changes during testicular development.

CLAUDIN7 modulates trophectoderm barrier function to maintain blastocyst development in pigs.

Trophectoderm (TE) barrier function is an essential prerequisite for blastocyst development. CLAUDIN7 (CLDN7), a member of CLAUDINS family, is involved in regulating intercellular exchange and cell polarity in epithelium cells. However, the role of CLDN7 in porcine early embryo development is yet to be explored. Here, we found that CLDN7 was highly conserved in different species and was widely expressed in different tissues. Remarkably, CLDN7 expression maintained a low level from GV oocyte to 4-cell stage whereas its expression exhibited a higher level from 8-cell stage onwards. Microinjection of siRNA into cytoplasm effectively knocked down expression of CLDN7 mRNA and protein in porcine embryos. CLDN7 knockdown not only significantly reduced blastocyst rates of embryos derived from parthenogenetic activation and in vitro fertilization, but also reduced number of total cells and TE cells in the resulting blastocysts. Furthermore, CLDN7 knockdown led to a significant reduction in expression of multiple genes associated with tight junction assembly and fluid accumulation. A permeability assay revealed that CLDN7 knockdown disrupted tight junction assembly and paracellular sealing in the TE epithelium. Taken together, these results demonstrate that CLDN7 regulates porcine blastocyst development via modulating trophectoderm barrier function.

NHC-Catalyzed Asymmetric Formal [4 + 2] Annulation To Construct Spirocyclohexane Pyrazolone Skeletons.

A chiral N-heterocyclic carbene (NHC)-catalyzed [4 + 2] annulation of γ-chloroenals and α-arylidene pyrazolinones was developed in the absence of expensive oxidants. The reaction proceeds smoothly via a vinyl enolate intermediate to afford spirocyclohexane pyrazolones in moderate to good yield (up to 86%) with high diastereoselectivities (up to 15:1 dr) and excellent enantioselectivities (up to >99% ee).