Search

Summary Enrichment and propagation of gonocytes or spermatogonial stem cells (SSCs) from cryopreserved testicular tissue is essential to apply SSCs-related techniques in large domestic animals. We previously reported the cryopreservation of adult bovine testicular tissue. Here, we conducted the enrichment and culture of putative gonocytes from cryopreserved testicular tissues of post-natal 1-day-old bulls. The testicular structure was well maintained after freezing and thawing. Higher mRNA levels of gonocyte/SSCs markers (PLZF, GFRα1, and UCHL-1) than those of pluripotency genes (Oct4, Sox2, and Nanog) were detected in the frozen–thawed sex cords. GFRα1 was specifically detected in the membrane and cytoplasm of gonocytes by immunostaining. Differential plating provided 40–50% enrichment of putative gonocytes. They were single, paired-, aligned-cells, or grape cluster-like colonies in minimum essential medium (MEM) containing 2.5% FBS + 2 mM glutamine + 100 IU/mL penicillin-streptomycin + 40 μg/mL gentamycin + 15 mM HEPES + 10 mM β-mercaptoethanol + 0.1 mM non-essential amino acids + 1 mM sodium pyruvate. On day 3, gonocyte progeny increased and the contaminated somatic cells spread and concurrently divided slowly. On day 5, gonocyte progeny proliferated continuously and typical intercellular bridges formed by incomplete cytokinesis in paired-cells or aligned-cysts were observed. Immunochemically, they were still GFRα1 and PLZF positive. These cells expressed significantly higher gonocyte/SSCs marker mRNAs than pluripotency gene mRNAs, concomitant with a higher level of differentiated spermatogonia marker c-kit. With time, gonocyte progeny colonies appeared in varied sizes and expanded dramatically on day 7. After cultured for 9–10 days, however, large colonies collapsed and dispersed as some single cells and small syncytial cysts. Together, MEM containing 10% dimethyl sulfoxide + 2.5% newborn calf serum provides efficient cryoprotection for the testicular tissue from 1-day-old neonatal bulls. Putative gonocytes enriched from these nascent tissues present robust proliferation capacity, conserved gonocyte/SSCs markers, and SSCs-like in vitro features.

DNA methylation and histone modification play important roles in the development of mammalian embryos. Cytochalasin B (CB) is an actin polymerization inhibitor that can significantly affect cell activity and is often used in studies concerning cytology. In recent years, CB is also commonly being used inin vitroexperiments on mammalian embryos, but few studies have addressed the effect of CB on the epigenetic modification of embryonic development, and the mechanism underlying this process is also unknown. This study was conducted to investigate the effects of CB on DNA methylation and histone modification in the development of parthenogenetically activated porcine embryos. Treatment with 5 μg/mL CB for 4 h significantly increased the cleavage rate, blastocyst rate and total cell number of blastocysts. However, the percentage of apoptotic cells and the expression levels of the apoptosis-related genesBCL-XL,BAXandCASP3were significantly decreased. Treatment with CB significantly decreased the expression levels ofDNMT1,DNMT3a,DNMT3b,HAT1andHDAC1at the pronuclear stage and promoted the conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). After CB treatment, the level of AcH3K9 was upregulated and the level of H3K9me3 was downregulated. When combined with Scriptaid and 5-Aza-Cdr, CB further improved the embryonic development competence and decreased the expression ofBCL-XL,BAXandCASP3. In conclusion, these results suggest that CB could improve embryonic development and the quality of the blastocyst by improving the epigenetic modification during the development of parthenogenetically activated embryos.