Wencong Song

Promyelocytic leukaemia zinc finger maintains self-renewal of male germline stem cells (mGSCs) and its expression pattern in dairy goat testis.

Previous studies have shown that promyelocytic leukaemia zinc finger (PLZF) is a spermatogonia‐specific transcription factor in the testis, required to regulate self‐renewal and maintenance of the spermatogonia stem cell. Up to now, expression and function of PLZF in the goat testis has not been known. The objectives of this study were to investigate PLZF expression pattern in the dairy goat and its effect on male goat germline stem cell (mGSC) self‐renewal and differentiation.

Characterization of Immortalized Dairy Goat Male Germline Stem Cells (mGSCs)

Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals. Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs self‐renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the results showed that the proliferation of these cells that were named mGSCs‐I‐SB was improved significantly. They maintained the typical characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm‐like cells in vitro. Additionally, mGSCs‐I‐SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos. Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine mGSCs. Conclusively, our results demonstrate that mGSCs‐I‐SB present the characteristics of mGSCs and may promote the future study on goat mGSCs. J. Cell. Biochem. 115: 1549–1560, 2014.

Rapamycin inhibits acrolein-induced apoptosis by alleviating ROS-driven mitochondrial dysfunction in male germ cells.

Acrolein (Acr) is a highly reactive α, β‐unsaturated aldehyde, which can induce reactive oxygen species (ROS) generation. Several factors, including lipid peroxidation, clinical use of cyclophosphamide, fried foods, automobile exhausts, smoking and aging can increase its concentration in blood serum. Mounting evidence has suggested that Acr‐induced ROS might reduce quality of sperm. Thus, the aim of this study was to examine reproductive toxicity of Acr‐caused ROS in vitro and find a means to alleviate it.

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.

Platelet-derived growth factor promotes the proliferation of human umbilical cord-derived mesenchymal stem cells

This study was designed to investigate the effect of platelet‐derived growth factor (PDGF) on the proliferation of human umbilical cord mesenchymal stem cells (UC‐MSCs) and further explore the mechanism of PDGF in promoting the proliferation of UC‐MSCs. The human UC‐MSCs were treated with different concentrations of PDGF, and the effects were evaluated by counting the cell number, the cell viability, the expression of PDGF receptors analyzed by RT‐PCR, and the detection of the gene expression of cell proliferation, cell cycle and pluripotency, and Brdu assay by immunofluorescent staining and Quantitative real‐time (QRT‐PCR). The results showed that PDGF could promote the proliferation of UC‐MSCs in vitro in a dose‐dependent way, and 10 to 50 ng/ml PDGF had a significant proliferation effect on UC‐MSCs; the most obvious concentration was 50 ng/ml. Significant inhibition on the proliferation of UC‐MSCs was observed when the concentration of PDGF was higher than 100 ng/ml, and all cells died when the concentration reached 200 ng/ml PDGF. The PDGF‐treated cells had stronger proliferation and antiapoptotic capacity than the control group by Brdu staining. The expression of the proliferation‐related genes C‐MYC, PCNA and TERT and cell cycle–related genes cyclin A, cyclin 1 and CDK2 were up‐regulated in PDGF medium compared with control. However, pluripotent gene OCT4 was not significantly different between cells cultured in PDGF and cells analyzed by immunofluorescence and QRT‐PCR. The PDGF could promote the proliferation of human UC‐MSCs in vitro. Copyright

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.

Ras/ERK1/2 pathway regulates the self-renewal of dairy goat spermatogonia stem cells

Spermatogonia stem cells (SSCs), also named the male germline stem cells (mGSCs), which is located at the base of the seminiferous tubules of testis, is the basis for generating sperm steadily in male animals. Currently, there are some preliminary study on the self-renewal and differentiation of SSCs, but further mechanism, especially in large animals, has not been clearly understood. Ras/ERK1/2 pathway is widely distributed in multiple cells in vivo. It plays an important role in cell proliferation, differentiation and so on. However, the study on the function for the self-renewal of dairy goats SSCs has not been investigated. In this study, the dairy goat SSCs characterization were evaluated by semi-RT-PCR, alkaline phosphatase (AP) staining, and immunofluorescence staining. Then, Ras/ERK1/2 pathway was blocked by specific MEK1/2 inhibitor PD0325901. We analyzed the proliferation by cell number, cell growth curve, Brdu incorporation assay, and cell cycle analysis. The results showed that the proliferation was significantly inhibited by PD0325901. Cell apoptosis induced by blocking the Ras/ERK1/2 pathway was analyzed by TUNEL. The expression of ETV5 and BCL6B, the downstream gene of Ras/ERK1/2 pathway, was downregulated. This study suggest that the Ras/ERK1/2 pathway plays a critical role in maintaining the self-renewal of dairy goat SSCs via regulation of ETV5 and BCL6B. This study laid a foundation for insights into the mechanism of SSCs self-renewal comprehensively.

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.

CD49f promotes proliferation of male dairy goat germline stem cells.

CD49f enhances multipotency and maintains stemness in embryonic stem cells (ESCs), however, whether it would be effective in mGSCs has remained unclear. Moreover, better standards for mGSC enrichment and purification are necessary. The present study was conducted to determine roles of CD49f in mGSC enrichment and regulation.