| Spermatogonial stem cells(SSCs), locatd the base membrane of seminiferous tubule intestis, are a member of adult stem cells which could maintain their own population stability byself-renewal and differentiate into mature spermatids. SSCs, retaining the characteristics ofstem cells and male germline cells, are an ideal cellular model for the study ofspermatogenesis, meiosis regulation and cellular reprogramming in vitro.microRNAs(miRNAs) are a kind of small noncoding RNA molecules coding the length of18-24nt in eukaryotic genomes, which play a critical role in cell proliferation, differentiation,apoptosis, germ cells formation and sex determination. Increasing evidences showed thatseveral classes of miRNAs were expressed in male germ cells and played essential roles insmall RNA-mediated regulation of spermatogenesis. With the improvement of the secondgeneration sequencing technology, the testicular tissue microRNA expression profiles ofhuman, rat, mice, cows, and pig had been fully analyzed, respectively. However, the miRNAsprofiling in dairy goat testicles had not been investigated.miR-204specifically expressed in the reproductive organs with high levels in human andmouse testicles, and involved in the regulation of cell proliferation and differentiation. Sirt1was a member of the sirtuin family with a high level in mouse SSCs, functions as anicotinamide adenine dinucleotide-dependent protein deactylase. The initial investigations ofSirt1deficient mice have revealed a phenotype that significantly reduced Oct4-positive SSCsnumber, attenuated spermatogenesis and an increased frequency of abnormal sperm. It hadreported that miR-204regulated cell migration, apoptosis and reprogrammed in mice gastriccancer cells and fibroblasts by targeted Sirt1. So, it was necessary to validate the function ofmiR-204-Sirt1axis in the self-renewal and proliferation in dairy goat SSCs.In the study, we analyzed the specifically expressed miRNAs profiling in dairy goatCD49f-positive and negative testicular cells, and ensured that the CD49f-positive testicularcells were SSC-like cells based on the expression characteristics of spermatogonial stem cellsspecific miRNAs and genes in the two kinds of testicular cells. Furthermore, it was found thatmiR-204regulated the expression of SSCs self-renewal genes Oct4and Plzf, inhibited the self-renewal and proliferation of dairy goat SSCs by investigating the function ofmiR-204-Sirt1axis in the regulation of dairy goat SSCs in vitro.1. The miRNA expression profiling of dairy goat spermatogoniaThe differentially expressed miRNAs profiling of CD49f-positive and negative cellspurified by MACS (Magnetic Activated Cell Sorting) were in-depth analyzed by Illuminahigh-throughput sequencing technology. The results showed that, among the identified noveland known miRNAs,933and916miRNAs were upregulated with a2-fold increase inCD49f-positive and negative spermatogonia, respectively. Some spermatogonial stemcells(SSCs) specific miRNAs and marker genes in testis had a higher level expression inCD49f-positive spermatogonia, such as miR-221, miR-21, miR-23, miR-29a, miR-29b,miR-24, miR-199a, miR-199b, miR-27a and Gfra1, Plzf. Immunofluorescence assay on adultgoat testes revealed that CD49f was primarily localized in the plasma membrane of goatspermatogonia-like cells lying adjacent to the basement membrane of the seminiferoustubules. These results suggested that the isolated CD49f-positive spermatogonia might be apopulation of dairy goat spermatogonia stem cells. The bioinformatics analysis indicated that40target genes of differentially expressed miRNAs in CD49f-positive cells were involved inthree GO Biological Process term: transcription, cell cycle and DNA damage processes, and10genes in KEGG cell cycle pathway. It was shown that there were near80%cells ofCD49f-positive spermatogonia in G0/G1phase by FACS-mediated cell-cycle analysis, thedata of FACS and bioinformatics analysis of target genes of differently expressed miRNAssuggested that the differently expressed miRNAs played a role in regulating the cell-cycle ofdairy goat CD49f-positive SSCs.2. The expression of miR-204and Sirt1in dairy goat testiclesmiR-204was mainly expressed in dairy goat reproductive organ, and had a peakexpression level in6-month old dairy goat testicles. The fluorescence immunostain intesticular sections of dairy goat indicated that the expression pattern of Sirt1was similar tomiR-204in the temporal-spatial distribution. Sirt1was expressed with a peak level in6-month old dairy goat testicles and then decreased in the next months, the expression of theSSCs self-renewal genes Oct4and Plzf were expressed followed Sirt1. Dual luciferaseexperiment confirmed that miR-204were interacted with Sirt1-3’UTR. Furthermore, Sirt1waswith a higher expression level in dairy goat CD49f+and CD90+SSCs, miR-204was opposite.These results suggested that miR-204involved in regulation of dairy goat SSCs via Sirt1.3. Sirt1promoted SSCs’ self-renewal genes expression and cell proliferationSirt1could regulate self-renewal and pluripotency of some stem cells.WhenmGSCs-I-SB cells were overexpressed the Sirt1gene, it were found that the expression of the SSCs’ self-renewal genes Oct4and Plzf were increased and the cell proliferation-associatedgenes Myc, PCNA and CCNA1were also increased. The FACS analysis of mGSCs-I-SBcells showed that the cells were increased in S phase and decreased in G1/G0phase whenSirt1overexpressed. So, these data suggested that the expression of the SSCs’ self-renewalgenes and cell proliferation-associated genes could increasely expressed and the cellproliferation was improved when Sirt1overexpressed.4. miR-204regulated the self-renewal and proliferation of dairy goat SSCs via Sirt1The mRNA and protein levels of Sirt1were significantly down regulated in dairy goatimmortalized spermatogonial stem cell lines(mGSCs-I-SB) when transfected with miR-204mimics, this indicated that Sirt1was a target of miR-204in dairy goat. The SSCs’ self-renewalgenes Oct4and Plzf were also significantly decreased when miR-204mimics transfected. TheFACS and immunofluorescent staining analysis of mGSCs-I-SB cells showed that the cellswere increased in G1/G0phase and decreased in S phase with less BrdU+cells, whenmiR-204mimics transfected. All the results indicated that miR-204inhibited the expressionof SSCs’ self-renewal genes Oct4and Plzf, regulated the self-renewal and proliferation ofdairy goat SSCs. |
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