Guangpeng Li

CD49f-Positive Testicular Cells in Saanen Dairy Goat Were Identified as Spermatogonia-Like Cells by miRNA Profiling Analysis

miRNAs, a type of small RNA, play critical roles in mammalian spermatogenesis. Spermatogonia are the foundation of spermatogenesis and are valuable for the study of spermatogenesis. However, the expression profiling of the miRNAs in spermatogonia of dairy goats remains unclear. CD49f has been one of the surface markers used for spermatogonia enrichment by magnetic activated cell sorting (MACS). Therefore, we used a CD49f microbead antibody to purify CD49f‐positive and ‐negative cells of dairy goat testicular cells by MACS and then analysed the miRNA expression in these cells in depth using Illumina sequencing technology. The results of miRNA expression profiling in purified CD49f‐positive and ‐negative testicular cells showed that 933 miRNAs were upregulated in CD49f‐positive cells and 916 miRNAs were upregulated in CD49f‐negative cells with a twofold increase, respectively; several miRNAs and marker genes specific for spermatogonial stem cells (SSCs) in testis had a higher expression level in CD49f‐positive testicular cells, including miR‐221, miR‐23a, miR‐29b, miR‐24, miR‐29a, miR‐199b, miR‐199a, miR‐27a, and miR‐21 and CD90, Gfra1, and Plzf. The bioinformatics analysis of differently expressed miRNAs indicated that the target genes of these miRNAs in CD49f‐positive cells were involved in cell‐cycle biological processes and the cell‐cycle KEGG pathway. In conclusion, our comparative miRNAome data provide useful miRNA profiling data of dairy goat spermatogonia cells and suggest that CD49f could be used to enrich dairy goat spermatogonia‐like cells, including SSCs. J. Cell. Biochem. 115: 1712–1723, 2014.

Silencing of fat-1 transgene expression in sheep may result from hypermethylation of its driven cytomegalovirus (CMV) promoter.

The fat-1 gene was isolated from roundworm Caenorhabditis elegans, and built into pIRES2-EGFP expression vectors driven by cytomegalovirus (CMV) promoter or cytomegalovirus enhancer and chickenβ-actin (CAG) promoter. Both CMV- and CAG-driven expression vectors were transfected to sheep fetal fibroblast cells. Positive transfected cells were used as donors for somatic cell nuclear transfer (SCNT) and the cloned embryos were transferred into the oviducts of synchronized recipient sheep. Two lambs derived from CMV vector and three lambs derived from CAG vector developed to term. Although Southern analyses using tissues from the two lambs derived from CMV vectors indicated integration of fat-1 gene into the genome, fat-1 mRNAs were not detected by RT-PCR. However, there was fat-1 expression (detected by RT-PCR) in tissues from transgenic lambs driven by CAG vectors. To investigate potential mechanisms involved in the two transgene models, methylation state of the vector promoters were examined. In CMV-driven transgenics, CMV promoters had almost no methylation in transfected cells and the resultant cloned embryos, whereas high methylations were detected in tissues and organs in transgenic lambs. In the CAG-driven transgenics, there were almost no methylations in transgenic cells and transgenic cloned embryos, and cloned lambs expressed fat-1 mRNA (detected by RT-PCR). Moreover, although SV40 promoters which drove neo/kan marker gene in CMV vectors were highly methylated in tissues from transgenic lambs, they were without methylation in cells and embryos. Therefore, we concluded that highly methylated CMV promoters induced the silence of fat-1 transgene expression in sheep. Furthermore, CAG promoter, but not CMV promoter was suitable for generation of fat-1 transgenic sheep.

PRMT5 enhances generation of induced pluripotent stem cells from dairy goat embryonic fibroblasts via down‐regulation of p53

Protein arginine methyltransferase 5 (PRMT5), is thought to play a role in epigenetic reprogramming of mouse germ cells. However, up to now there has been little information concerning its expression profile and effects on generation of induced pluripotent stem cells (iPSCs) from somatic cells, in livestock. Here, we have explored PRMT5 expression profiles in dairy goats and its consequences to derivation of iPSCs from dairy goat embryonic fibroblasts (GEFs).

PLZF‐Induced Upregulation of CXCR4 Promotes Dairy Goat Male Germline Stem Cell Proliferation by Targeting Mir146a

Previous studies have shown that promyelocytic leukemia zinc finger (PLZF), chemokine (C‐X‐C motif) receptor 4 (CXCR4) and mir146a were associated with the self‐renewal of mouse spermatogonial stem cells (SSCs); however, there is little information on their effects on the fate of livestock SSCs. Here, we have identified a regulatory pathway in dairy goat mGSCs, involving PLZF, mir146a and the SDF‐1 receptor CXCR4. PLZF overexpression downregulated mir146a and simultaneously upregulated the expression of CXCR4 protein, whereas PLZF knockdown (siPLZF) induced the specifically opposite effects. The in vitro assays demonstrated that PLZF specifically interacts with and suppresses the mir146a promoter, and mir146a targets CXCR4 to impede its translation. The levels of ERK1/2 phosphorylation in the mGSCs overexpressed CXCR4 and PLZF were upregulated, respectively, whereas mir146a expression was decreased and CXCR4 protein was increased. Mir146a overexpression and siPLZF impaired mGSC proliferation and differentiation, however, Mir146a knockdown induced the opposite effects. The effects of PLZF and mir146a were mediated regulation by mir146a and CXCR4, respectively. Overexpression of CXCR4 or addition of CXCL12 in cultures of dairy goat mGSCs resulted in the upregulation of their signaling, and the phosphorylation of ERK1/2 was increased. Collectively, these findings indicate that PLZF is an important transcription factor in the regulation of the expression of CXCR4 to promote dairy goat mGSC proliferation by targeting mir146a. J. Cell. Biochem. 117: 844–852, 2016.

miR‐544 Regulates Dairy Goat Male Germline Stem Cell Self‐Renewal via Targeting PLZF

The balance between the self‐renewal and differentiation of male germline stem cells (mGSCs) is critical for the initiation and maintenance of mammalian spermatogenesis. The promyelocytic leukemia zinc finger (PLZF), a zinc finger protein, is a critical factor for maintaining the self‐renewal of mGSCs, so, evaluation of the PLZF pathway in mGSCs may provide a deeper insight into mammalian spermatogenesis. miRNA was also an important regulating factor for the self‐renewal and differentiation of mGSCs; however, there is currently no data indicating that which miRNA regulate the self‐renewal and differentiation of mGSCs via PLZF. Here, we predicted the prospective miRNA targeting to PLZF using the online Bioinformatics database‐Targetscan, and performed an analysis of the dual‐luciferase recombinant vector, psiCHCEKTM‐2‐PLZF‐3′UTR. miR‐544 mimics (miR‐544m), miR‐544 inhibitors (miR‐544i), Control (NC, scrambled oligonucleotides transfection), pPLZF‐IRES2‐EGFP or PLZF siRNA were transfected into mGSCs; the cells proliferation was evaluated by BRDU incorporation assay and flow cytometry, and the mGSC marker, GFRa1, PLZF, KIT, DAZL, and VASA expression were analyzed by RT‐qPCR, immunofluorescence and Western blot. The results showed that miR‐544 regulates dairy goat male germline stem cell self‐renewal via targeting PLZF. Our study identifies a new regulatory pathway for PLZF and expands upon the PLZF regulatory network in mGSCs. J. Cell. Biochem. 116: 2155–2165, 2015.

Conservation and function of Dazl in promoting the meiosis of goat male germline stem cells

Dazl (deleted in azoospermia-like) is a conserved gene in mammalian meiosis, which encodes RNA binding protein required for spermatocyte meiosis. Up to date, the expression and function of Dazl in the goat testis are unknown. The objectives of this study were to investigate the expression pattern of Dazl in dairy goat testis and their function in male germline stem cells (mGSCs). The results first revealed that the expression level of Dazl in adult testes was significantly higher than younger and immature goats, and azoospermia and male intersex testis. The dairy goat Dazl is highly conserved analysed by several online and bioinformatics software, respectively. Over-expression of Dazl promoted the expression of meiosis-related genes in dairy goat mGSCs. The expression of Stra8 was up-regulated by over-expression of Dazl analysed by Luciferase reporter assay. Taken together, results suggest the Dazl plays an important role in dairy goat spermatogenesis and that over-expression of Dazl may promote Stra8 expression in dairy goat mGSCs.

Expression pattern of Ngn3 in dairy goat testis and its function in promoting meiosis by upregulating Stra8

Ngn3 is a typical transcription factor and marker of differentiating spermatogonial stem cells (SSCs) in mouse, belonging to the basic helix‐loop‐helix (bHLH) family. Its gene is specifically expressed in A type spermatogonia in mouse testis, thus plays a critical role in controlling differentiation of SSCs. However, roles of Ngn3 and its protein in dairy goat testis remain unknown.

The function of Msx1 gene in promoting meiosis of dairy goat male germline stem cells (mGSCs).

During sequential stages of meiosis, numerous cytoplasmic and nuclear events take place in which many germline and non‐germline genes involved. It is demonstrated that the germline gene Stra8 and synaptonemal complex protein 3 (Scp3) play an important role in the meiosis. Recently, studies showed Msx1, a DNA‐binding protein taking part in the skeletal development, also having a functional attractive factor to Stra8 and Scp3 in the meiosis. In this study, we cloned the gene Msx1 then transfected the Msx1 constructed recombination plasmid, pMsx1‐Ires2‐AcGFP, into the dairy goat germline stem cells (male germline stem cells) and analysed the effects of Msx1 on the expression of Stra8 and Scp3. The results showed that Msx1 could enhance the expression of Stra8 and Scp3 and promote the meiosis in goat testicular cells. Bmp4 activated the expression of Msx1 and Stra8. This study suggests that Msx1 plays an important role in spermatogenesis and meiosis. Copyright