Differential Expression Of MiR-34b/c And Predicetd Target Genes Of Testicular Tissue Among Different Development Stages Of Swine

MicroRNAs (miRNAs) are short (～22nucleotides) non-coding RNA moleculesthat can play important gene regulatory roles by cleaving RNA transcripts orrepressing translation. In recent years, there are reports that the expression of miRNAsin germ cells has obvious tissue specificity and timing. Furthermore, miRNAs alsoplays an important role in regulating the primitive reproductive development.MiR-34b and miR-34c are members of miR-34family. Studies have shown that theexpression of miR-34b/c has extremely significant differences between mature andimmature mice. Therefore, miR-34b/c plays an important role in the regulation ofmale germ cell development process. The purpose of the study is to investigate thedifferential expression of miR-34b/c and predicted target genes of swine testiculartissue among different development stages.The morphology of testicular tissue in Junmu NO.1White Pig among differentdevelopment stages were analyzed by HE staining of paraffin sections in theexperiment. The result showed that the interstitial connective tissue gradually reducedand the spermatic cord of the testes became larger as increasing months in visual fieldof100times. We also can see the maximum lumen in9-month-old. In400magnifications, no mature lumen was found, but sertoli cells and spermatogonia orsermatogonial stem cells were gradually moving to the basement membrane in2-day-old~3-month-old period. In the field of4-month-old, spermatogonia graduallydeveloped to mature sperm, mass of immature round spermatids and a spot of maturesperms were found. In5-month-old, more mature sperms appeared.According to theses morphology results of swine testicular tissue amongdifferent development stages, we collected some testicular tissue of2-day-old,3,4,5,6,7,9and12-month-old in the second experiment. Two microRNAs(miR-34c andmiR-34b) that keeping the great difference expression quantities between mature andimmature stages of testis were selected basing on the results of the previous Solexa sequencing technology in our research group. Four predicted target genes ZNF148,KLF4, PDGFRA and UBP1, which maybe related to the spermatogenesis and malereproductive development were selected by miRanda, PicTar and TargetscanS onlinesoftware. The results of the fluorescence quantitative showed that only PDGFRA keptthe highest correlation with miR-34c, so we inferred PDGFRA may be target gene ofmiR-34c. In order to verify the above results, the expression of PDGFRA in testiculartissues of2-day-old,3,4and5-month-old were tested by immunohistochemicalmethods in the third experiment. More factual evidences showed PDGFRA with highexpression in sertoli cells、spermatogonia and sermatogonial stem cells during theearly development, and decreased in4and5-month-old. We inferred somerelationship exist in PDGFRA and sertoli cells and spermatogonia or sermatogonialstem cells. It was possible that PDGFRA had the function to promote the growth ofconvoluted seminiferous tubule, but the function was weaking duringspermatogenesis.