Molecular Cloning, Characterization And Expression Analyses Of POU5F1, SOX2, KLF4 And MYC Genes In The Ovine Testis

【Objective】This study was to analyze the expressions of POU domain, class 5, transcription factor 1(POU5F1), SRY(sex determining region Y)-box 2 [SOX2], myelocytomatosis oncogene(MYC) and Kruppel-like factor 4(gut) [KLF4] genes in ovine testis tissues, identify these genes’ molecular sequences and construct their retroviral gene transfer expression vectors, aiming to provide the material basis for future source of sheep Leydig cells-derived induced pluripotent stem cell(iPSC) lines. Furthermore, this study was to determine the morphological characteristics of adult mouse Leydig cells in culture by using HSD3 B staining method in order to provide the technical basis for a better method to identify ovine Leydig cells in culture.【Methods】 ⑴ Complementary DNA templates were derived from one-week-old and six-month-old testis tissues of the Chinese Hu sheep. The expression patterns in Hu testis tissues and the mRNA sequences of sheep POU5F1, SOX2 and MYC genes were identified by RT-PCR and sequencing. ⑵ Sheep genomic templates were derived from the cultured ovine fetal fibroblast cells from the Mongolian sheep fetus at day 30-40 of gestation. Ovine KLF4 genomic sequence was identified by PCR and sequencing. ⑶ The coding sequences, amino acid sequences and protein structures were predicted by using bioinformatics analysis tools. ⑷ Construct four retroviral gene transfer expression vectors that all carry ovine reprogramming factor genes. They are pMXs-oPOU5F1, pMXs-oSOX2, pMXs-oKLF4 and pMXs-oMYC vectors. The sequences of plasmids were vertified by restriction enzyme digestion and sequencing. ⑸ The control-group pMXs-GFP plasmid and the four constructed plasmids were separately transfected into the Phoenix Amphotropic cells with calcium phosphate transfection. Forty-eight hours and 72 hours after transfection, the transfection efficiency of pMXs-GFP plasmid was observed under a fluorescence microscope, and the viral supernatants were harvested and used to infect the newborn ovine Leydig cells. Four days after infection, the infection efficiency of pMXs-GFP plasmid was observed under the fluorescence condition, and exogenous-gene expressions carried by the four constructed vectors were dectected by RT-PCR. ⑹ The testes of two-month-old mice were decapsulated and then the Leydig cells were isolated by collagenase digestion.【Results】 ⑴ POU5F1 transcripts were detected in both neonatal and viripotent sheep testes. Ovine POU5F1 mRNA contains a 1080 bp open reading frame(ORF) that encodes 359 amino acids. Ovine POU5F1 protein contains a POU domain and a homeobox domain. Ovine POU5F1 lacks a tryptophan between lysine 206 and valine 207, which is unique among mammals. ⑵ SOX2 transcripts were detected in both neonatal and viripotent sheep testes. Ovine SOX2 mRNA contains a 963 bp ORF that encodes 320 amino acids. Ovine SOX2 protein contains a high mobility group(HMG) domain. ⑶ MYC transcripts were detected in neonatal sheep testes. Ovine MYC mRNA contains a 1320 bp ORF that encodes 439 amino acids. Ovine MYC protein contains a helix-loop-helix(HLH) domain. ⑷ There are 3159 bp of nucleotides from the initiation codon to the termination codon of ovine KLF4 genomic sequence. Sheep KLF4 gene is comprised of four exons and three introns, which the sheep KLF4 gene conatins a 1434-bp coding sequence that encodes 477 amino acids. Ovine KLF4 protein contains three cysteine2/histidine2-type zinc fingers at its carboxyl terminus. ⑸ By using pMXs-GFP plasmid as the control, it was predicted that the four constructed plasmids could be effectively transfected into the AMPHO cell lines. RT-PCR results showed that the exogenous genes carryied by the four retroviral plasmids were able to integrate into the genome of ovine Leydig cells and can be expressed. ⑹ After 48 hours of Leydig cell culture, both the nucleus and the cytoplasm were very clear under the optical microscope. The nucleus was big and round and the cytoplasm was filled with abundant lipid drops with a strong refractivity. After 5 days of culture, Leydig cells were fully elongated with multiple shapes. Some cells grew in aggregation, and some cells grew independently. Leydig cells in aggregation elongated many cellular tentacles for intercellular connections, which formed an epithelium-like appearance. RT-PCR results showed that Leydig cells in culture after 5 days could express Leydig cell-specific transcriptions, HSD3B6, CYP17A1 and StAR. 【Conclusions】POU5F1, SOX2 and MYC transcripts were detected in neonatal ovine testes, but KLF4 transcription was not detected. The coding mRNA sequences of sheep POU5F1, SOX2 and MYC genes and sheep KLF4 genomic sequence were identified. We constructed four retroviral gene-transfer expression vectors that all carry ovine reprogramming factor genes, which are pMXs-oPOU5F1, pMXs-oSOX2, pMXs-oKLF4 and pMXs-oMYC vectors. We also determined the morphological characteristics of adult mouse Leydig cells in culture by using HSD3 B staining method, which will help up explore a better method to identify the Leydig cells in culture. In conclusion, these results will lay a foundation for future source of ovine Leydig cells-derived iPSC lines.