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

Abstract

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.

CD14 is a unique membrane marker of porcine spermatogonial stem cells, regulating their differentiation

“…Previously, we have identified putative cell surface biomarkers of undifferentiated porcine spermatogonia, including chemokine receptor 1 (CCR1), CD14, and CD209, using next-generation sequencing technologies 15 . In mice, flow cytometric analysis confirmed that CCR2 and CD14 were expressed in a subpopulation of cryptorchid testicular cells enriched for SSCs 12 .…”

“…CD14 (1:50 dilution; AbFrontier, Seoul, Korea) 15 and PGP9.5 (1:500 dilution, 7863-0504; Serotec, Oxford, UK), followed by incubation with Alexa Fluor® 568-conjugated donkey anti-rabbit IgG and Alexa Fluor® 488-conjugated goat anti-mouse IgG (both Life Technologies, Carlsbad, CA, USA). To identify nuclei, samples were incubated with 1 µg/mL of 4′,6-diamidino-2-phenylindol (DAPI; Sigma–Aldrich) for 10 min and washed with PBS twice.…”

“…The pSSC isolation and in vitro culture were performed as described previously 15 . Briefly, testis samples were collected from 5-day-old crossbred piglets (Sam Woo breeding farm), the tunica albuginea were removed, digested with an enzyme mixture solution (DNase I and collagenase IV in PBS) at 37 °C for 15 min, and filtration through a 40-µm nylon mesh.…”

“…Porcine testicular cells were isolated using our routine cell culture techniques as previously described 15 . Briefly, testes from 5-day-old crossbred piglets were detunicated and digested with an enzyme mixture solution (collagenase IV and DNase I in PBS) at for 15 min at 37 °C and strained through a 40-µm nylon mesh to obtain single testicular cells for FACS.…”

“…The expression of CD14 mRNA were also observed in the human and rat testes expressing Toll-like receptors (TLRs) 13,14 , although the role of CD14 in the testes is unclear. We have previously found that CD14 is expressed in porcine SSCs (pSSCs) using a next-generation sequencing approach; however, the role of CD14 in the testis have not been established 15 .…”