Identification of Putative Biomarkers for the Early Stage of Porcine Spermatogonial Stem Cells Using Next-Generation Sequencing

• Published in: PloS one Vol. 11; no. 1; p. e0147298
• Main Authors: Lee, Won-Young, Do, Jeong Tae, Park, Chankyu, Kim, Jin Hoi, Chung, Hak-Jae, Kim, Kyung-Woon, Gil, Chang-Hyun, Kim, Nam-Hyung, Song, Hyuk
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 22.01.2016; Public Library of Science (PLoS)
• Subjects: Adult Stem Cells - cytology; Animal sciences; Animals; Base Sequence; Biological markers; Biology and life sciences; Biomarkers; Biotechnology; CC chemokine receptors; CCR1 protein; CD14 antigen; Cell culture; Cell Differentiation - physiology; Clustering; Colonies; Dendritic cells; Deoxyribonucleic acid; DNA; Endocrinology; Gelatinase B; Gene expression; Gene Expression Regulation - genetics; Gene sequencing; Genes; Genetic aspects; Genetic Markers - genetics; Genomics; Glutathione; Glutathione peroxidase; High-Throughput Nucleotide Sequencing; Identification and classification; Immunohistochemistry; Insulin; Insulin-like growth factor-binding protein 3; Insulin-like growth factors; Krueppel-like factor; Low temperature; Male; Medicine and Health Sciences; Meiosis; Meiosis - genetics; Metalloproteinase; Methods; Peroxidase; Proteins; Research and Analysis Methods; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA sequencing; Sequence Analysis, RNA; Spermatogenesis; Spermatogenesis - physiology; Spermatogonia; Spermatogonia - cytology; Stem cell transplantation; Stem cells; Swine; Testes; Testis - cytology; Zoology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0147298

To identify putative biomarkers of porcine spermatogonial stem cells (pSSCs), total RNA sequencing (RNA-seq) analysis was performed on 5- and 180-day-old porcine testes and on pSSC colonies that were established under low temperature culture conditions as reported previously. In total, 10,184 genes were selected using Cufflink software, followed by a logarithm and quantile normalization of the pairwise scatter plot. The correlation rates of pSSCs compared to 5- and 180-day-old testes were 0.869 and 0.529, respectively and that between 5- and 180-day-old testes was 0.580. Hierarchical clustering data revealed that gene expression patterns of pSSCs were similar to 5-day-old testis. By applying a differential expression filter of four fold or greater, 607 genes were identified between pSSCs and 5-day-old testis, and 2118 genes were identified between the 5- and 180-day-old testes. Among these differentially expressed genes, 293 genes were upregulated and 314 genes were downregulated in the 5-day-old testis compared to pSSCs, and 1106 genes were upregulated and 1012 genes were downregulated in the 180-day-old testis compared to the 5-day-old testis. The following genes upregulated in pSSCs compared to 5-day-old testes were selected for additional analysis: matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 1 (MMP1), glutathione peroxidase 1 (GPX1), chemokine receptor 1 (CCR1), insulin-like growth factor binding protein 3 (IGFBP3), CD14, CD209, and Kruppel-like factor 9 (KLF9). Expression levels of these genes were evaluated in pSSCs and in 5- and 180-day-old porcine testes. In addition, immunohistochemistry analysis confirmed their germ cell-specific expression in 5- and 180-day-old testes. These finding may not only be useful in facilitating the enrichment and sorting of porcine spermatogonia, but may also be useful in the study of the early stages of spermatogenic meiosis.