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Wilms' tumour 1 gene (WT1) is essential for the development of mammalian urogenital system. However, the expression pattern of WT1 in the development of porcine urogenital organs is still unclear. Here, we examined the expression of WT1 mRNA and protein in porcine kidneys, ovaries and testes from embryonic days 35 and 60 (E35d, E60d, n = 3) to the newborn (0d, n = 4) and adult (210d, n = 3) stages, using real-time PCR and immunofluorescent staining. Real-time PCR analysis showed that porcine kidneys, ovaries and testes all expressed high level of WT1 mRNAs, especially in adult testes (p < 0.05 or 0.01 vs. kidney and ovary, respectively). Morphologically, characteristic microstructures of the kidneys, ovaries and testes were observed and discerned at all four stages. Immunofluorescently, WT1 expression was detected in a dynamic and context-specific pattern during the development of these organs. Taken together, porcine urogenital organs express relatively high levels of WT1 mRNA. Dynamical and context-specific expression profile of WT1 in these organs occurs during their development, implying its close association with the development and function of porcine kidney, ovary and testis.

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.

Propagation of bovine spermatogonial stem cells (SSCs) from the cryopreserved testicular tissue is essential for the application of SSCs-related techniques. To explore the appropriate conditions for in vitro culture of bovine spermatogonia (containing putative SSCs), Sertoli cell monolayer and serum concentration were set as two main control factors. Morphological examination showed that the intactness and structure of adult bovine testicular tissue were well maintained after cryopreservation. The enriched bovine spermatogonia were large round CD9 and promyelocytic leukemia zinc finger protein (PLZF) positive cells, with high nucleocytoplasmic ratios and multiple types including single, paired-, aligned-cells or grape cluster-like colonies in vitro. In Sertoli cell co-culture system, bovine spermatogonia attached quickly and proliferated obviously faster than those in the system without Sertoli cells. Serum-free media was no good for the attachment and proliferation of bovine spermatogonia. When 2.5%, 5% and 10% fetal bovine serum (FBS) was employed in the media, spermatogonia attached easily and divided quickly to form paired-, chained-cells or grape cluster-like colonies with comparable percentages in all groups. However, the contaminated somatic cells proliferated robustly in groups containing 5% and 10% FBS. Together, bovine spermatognia isolated from cryopreserved adult testis tissue express CD9 and PLZF, can survive and proliferate conspicuously in Sertoli cell co-culture system, and low serum provides an optimal condition for the survival and proliferation of bovine spermatogonia because of avoiding the rapid growth of testis somatic cells.

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.