Hailong Mu

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.

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.

BMP4 promotes SSEA-1(+) hUC-MSC differentiation into male germ-like cells in vitro.

Recent studies have demonstrated that primordial germ cells (PGC) can be differentiated from human umbilical cord mesenchymal stem cells (hUC‐MSCs), and embryonic stem cells (ESCs) in vitro. Nevertheless, efficiencies were low and unstable. Here, whether hUC‐MSCs can be induced to differentiate into germ‐like cells with the aid of bone morphogenetic protein (BMP4) was investigated.

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.

The Tet1 and histone methylation expression pattern in dairy goat testis

DNA methylation and histone methylation are critical for mammalian development. Ten-eleven translocation (Tet1), a key regulator of DNA methylation, has been identified as a key enzyme for the activation of DNA demethylation; histone H3 lysine 9 (H3K9) and 27 (H3K27) methylation repress gene expression. Significant progress on the biological functions of Tet proteins has been made in mice and humans. However, their expression pattern and function in the male germ cells in the dairy goat testis are still unclear. The present study described the expression pattern of Tet1, H3K9, and H3K27 in the dairy goat testis and cultured goat spermatogonia stem cells (gSSCs). The results showed that Tet1 was weakly expressed in the dairy goats testis compared to other organ tissues. Tet1, 5-hydroxymethylcytosine, H3K9, and H3K27 expressions were positive and dynamically changing during spermatogenesis; however, they showed weak expression in neonate stage in vivo. Tet1 and 5-hydroxymethylcytosine showed low expression in gSSCs in vitro in differentiated cultures. These will provide new perspectives for DNA methylation/demethylation and better regulation of epigenetic modifications in gSSCs.

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.

EIF2S3Y suppresses the pluripotency state and promotes the proliferation of mouse embryonic stem cells

Eukaryotic translation initiation factor 2, subunit 3, and structural gene Y-linked (EIF2S3Y) is essential for spermatogenesis in mouse models. However, its effect on embryonic stem (ES) cells remains unknown. In our observation, differentiated ES cells showed higher levels of EIF2S3Y. To further elucidate its role in ES cells, we utilized ES-derived EIF2S3Y-overexpressing cells and found that EIF2S3Y down-regulated the pluripotency state of ES cells, which might be explained by decreased histone methylation levels because of reduced levels of ten-eleven translocation 1 (TET1). Moreover, EIF2S3Y-overexpressing cells showed an enhanced proliferation rate, which might be due to increased Cyclin A and Cyclin E levels. This study highlighted novel roles of EIF2S3Y in the pluripotency maintenance and proliferation control of ES cells, which would provide an efficient model to study germ cell generation as well as cancer development using ES cells, thus providing valuable target for clinical applications of ES cells.

p38 MAPK pathway is essential for self-renewal of mouse male germline stem cells (mGSCs)

Male germline stem cells (mGSCs), also called spermatogonial stem cells (SSCs), constantly generate spermatozoa in male animals. A number of preliminary studies on mechanisms of mGSC self‐renewal have previously been conducted, revealing that several factors are involved in this regulated process. The p38 MAPK pathway is widely conserved in multiple cell types in vivo, and plays an important role in cell proliferation, differentiation, inflammation and apoptosis. However, its role in self‐renewal of mGSCs has not hitherto been determined.