Study on the role of JAK/STAT signaling pathway during chicken spermatogonial stem cells generation based on RNA-Seq

Spermatogonia! stem cells（SSCs） form the foundation for spermatogenesis and sustain male fertility.To explore the regulatory mechanisms of chicken SSCs generation,we obtained highly purified chicken embryonic stem cells（ESCs）,primordial germ cells（PGCs） and SSCs by fluorescence-activated cell sorting（FACS）.High-throughput analysis methods（RNA-Seq） were used to sequence the transcriptome level of these cells.Gene ontology and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment were used to analyze RNA-Seq results.BMP4 was used to induce chicken ESCs differentiation to SSCs-like cells in vitro.The quantitative real-time（qRT）-PCR was used to detect the expression changes of the key genes.The results showed that 22 relevant critical pathways were found by RNA-Seq,one of them was the Janus kinase/signal transducer and activator of transcription（JAK/STAT） signaling pathway.Total of 103 related genes were detected in this pathway.Protein-protein interactions analysis found that 87 proteins were significantly related to 19 key proteins in this pathway.These 87 proteins were enriched in 21 biological processes and 18 signaling pathways.Moreover,during the differentiation of chicken ESCs to SSCs-like cells induced by BMP4 in vitro,JAK2 and STAT3 were activated.The qRT-PCR results showed that the expression trends of JAK2 and STAT3 were basically the same as in vivo.We concluded that JAK/STAT signaling pathway plays an important role in the process of chicken SSCs generation both in vivo and in vitro;it may achieve its function through multiple biological processes and other related pathways.

Genetic assessment of inbred chicken lines indicates genomic signatures of resistance to Marek's disease

Background: Marek’s disease(MD) is a highly contagious pathogenic and oncogenic disease primarily affecting chickens. However, the mechanisms of genetic resistance for MD are complex and not fully understood. MD-resistant line 63and MD-susceptible line 72are two highly inbred progenitor lines of White Leghorn. Recombinant Congenic Strains(RCS) were developed from these two lines, which show varied susceptibility to MD.Results: We investigated genetic structure and genomic signatures across the genome, including the line 63and line72, six RCSs, and two reciprocally crossed flocks between the lines 63and 72(F1 63× 72and F1 72× 63) using Affymetrix~? Axiom~? HD 600 K genotyping array. We observed 18 chickens from RCS lines were specifically clustered into resistance sub-groups distributed around line 63. Additionally, homozygosity analysis was employed to explore potential genetic components related to MD resistance, while runs of homozygosity(ROH) are regions of the genome where the identical haplotypes are inherited from each parent. We found several genes including SIK, SOX1, LIG4, SIK1 and TNFSF13B were contained in ROH region identified in resistant group(line 63and RCS), and these genes have been reported that are contribute to immunology and survival. Based on FSTbased population differential analysis, we also identified important genes related to cell death and anti-apoptosis, including AKT1, API5, CDH13, CFDP and USP15,which could be involved in divergent selection during inbreeding process.Conclusions: Our findings offer valuable insights for understanding the genetic mechanism of resistance to MD and the identified genes could be considered as candidate biomarkers in further evaluation.

Marek’s disease virus challenge induced immune-related gene expression and chicken repeat 1 (CR1) methylation alterations in chickens

Marek’s disease virus (MDV) challenge induces lymphoma in susceptible chickens. Host genes, especially immune related genes, are activated by the virus. DNA methylation is an epigenetic mechanism that governs gene transcription. In the present study, we found that expression of signal transducer and activator of transcription 1 (STAT1) was upregulated at 10 days post infection (dpi) in MD susceptible chickens, whereas interleukin 12A (IL12A) was elevated in both resistant and susceptible chickens. However, we did not observe MDV-induced DNA methylation variations at the promoter CpG islands (CGIs) in STAT1 and IL12A. Interestingly, the methylation levels at Chicken Repeat 1 (CR1), the transposable elements (TEs) located upstream of two genes, were different between resistant and susceptible chickens. Furthermore, a mutation was identified in the CR1 element near IL12A. The impact of the point mutation in transcriptional factor binding is to be examined in the near future.