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With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 ( BLOC1S1 ) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes ( PCNA , CDK2 , CCND1 ), and EIF2S3Y , a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1 . These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.

Double sex and mab-3 related transcription factor 1 (DMRT1) encodes a double sex/mab-3 (DM) domain, which is the most conserved structure that involved in sex determination both in vertebrates and invertebrates. This study revealed important roles of DMRT1 in maintaining self-renewal of male germline stem cells (mGSCs). Our results showed that insufficient expression of DMRT1 in mice testes resulted in decreased number of spermatogonial cells and collapse of testicular niche in vivo. Self-renewal and proliferation of mGSCs were inhibited. Based on the bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) assay, it was finally revealed that the interaction between DMRT1 and promyelocytic leukemia zinc finger (PLZF) protein was essential for maintaining self-renewal of mGSCs. Moreover, BTB domain of PLZF, DM and DMRT1 domain of DMRT1 were indispensable in mGSC, which were responsible for preserving the quantity of germ cells. Our research provided a new scientific basis for studying the mechanism of self-renewal and spermatogenesis in goat mGSCs.

Eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) gene, the gene encoding eIF2γ protein, is globally expressed in all tissues and plays important roles in regulating global and gene-specific mRNA translation initiation. It has been noticed that Eif2s3y plays crucial roles in spermatogenesis, however, the mechanism remains unclear. In the current study, transgenic Eif2s3y mice were generated to test our hypothesis that the Eif2s3y promotes the proliferation of spermatogonial stem cells (SSCs). Transgenic Eif2s3y mouse had enhanced SSCs proliferation rate when compared to WT mouse. Interesting, the testes from transgenic Eif2s3y mouse had increased Active-β-catenin protein expression and higher expression pattern of Wnt ligand Wnt6 when compared to testes from WT mouse. This study revealed novel roles of Eif2s3y in the activation Wnt6/β-catenin signal pathway in SSCs. Taken together, we identified Eif2s3y-Wnt6-β-catenin as a critical pathway in the regulation of spermatogenesis, which provides a platform for investigating the molecular mechanisms of male reproduction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

MicroRNAs were reported to be able to regulate mGSCs' self-renewal through post-transcriptional inhibition of gene expression. miR-302 worked as one important microRNA family existed mainly in human ESCs, and its role in mGSCs has not been reported yet. To elucidate the role of miR-302 in dairy goat mGSCs, the expression profile of miR-302 was explored through qPCR and FISH. Furthermore, to detect the function of miR-302, the expression vector containing miR-302 was transfected into mGSCs, and then, the cell cycle, the cell apoptosis and the genes associated with mGSCs' self-renewal and differentiation were examined. The results showed that miR-302 expressed in testis moderately and located on the basement of seminiferous tubes which shared the same location as mGSCs. Transfection of the vector containing miR-302 fragment into the immortalized mGSCs obviously enhanced the cell proliferation ability and the attachment ability, also, promoted the expression level of CD49f and OCT4. Also, miR-302 reduced the cell apoptosis and downregulated the expression of P21. miR-302 sustained mGSCs' proliferation in vitro., (© 2018 Blackwell Verlag GmbH.)

Self-renewal and differentiation of male germline stem cells (mGSCs) provide the basic function for continual spermatogenesis. Studies of in vitro culture of germline stem cells are important and meaningful for basic biological research and practical application. Growth factors, such as GDNF, bFGF, CSF1, and EGF, could maintain the self-renewal of mGSCs. Insulin-like growth factor 1 (IGF-1), an important growth factor, and its pathway have been reported to maintain the survival of several types of stem cells and play important roles in male reproduction. However, the mechanism through which the IGF-1 pathway acts to regulate the self-renewal of mGSCs remains unclear. We analyzed the effect of IGF-1 on the proliferation and apoptosis of bovine mGSCs. We evaluated the expression profile of long noncoding RNA (LncRNA) H19 in bovine and mouse tissues. Moreover, we investigated whether LncRNA H19 could regulate the IGF-1 pathway. Results showed that IGF-1 could activate the phosphorylation of AKT and ERK signaling pathways, and the IGF-1 pathway played an important role in regulating the proliferation and apoptosis of bovine mGSCs. The proliferation rate of mGSCs decreased, whereas the apoptosis rate of mGSCs increased when the IGF-1 receptor (IGF-1R) was blocked using the IGF-1R-specific inhibitor (picropodophyllin). LncRNA H19 could regulate the IGF-1 signaling pathway and, consequently, the proliferation and apoptosis of mGSCs. The number of cells in the seminiferous tubule decreased when H19 was interfered by injecting a virus-containing supernatant. Hence, LncRNA H19 participated in the regulation of the proliferation and apoptosis of mGSCs via the IGF-1 signaling pathway., (© 2018 Wiley Periodicals, Inc.)

Porcine pancreatic stem cells (PSCs) are seed cells with potential use for diabetes treatment. Stem cell differentiation requires strict control of protein turnover and lysosomal digestion of organelles. Autophagy is a highly conserved process that controls the turnover of organelles and proteins within cells and contributes to the balance of cellular components. However, whether autophagy plays roles in PSC differentiation remains unknown. In this study, we successfully induced porcine PSCs into insulin-producing cells and found that autophagy was activated during the second induction stage. Inhibition of autophagy in the second stage resulted in reduced differentiational efficiency and impaired glucose-stimulated insulin secretion. Moreover, the expression of active β-catenin increased while autophagy was activated but was suppressed when autophagy was inhibited. Therefore, autophagy is essential to the formation of insulin-producing cells, and the effects of autophagy on differentiation may be regulated by canonical Wnt signalling pathway., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: 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., Materials and Methods: Here, the mouse mGSCs were cultured and their identity was verified by semi-RT-PCR, alkaline phosphatase (AP) staining and immunofluorescence staining. Then, the p38 MAPK pathway was blocked by p38 MAPK-specific inhibitor SB202190. mGSC self-renewal ability was then analysed by observation of morphology, cell number, cell growth analysis, TUNEL incorporation assay and cell cycle analysis., Results: Results showed that mouse mGSC self-renewal ability was significantly inhibited by SB202190., Conclusions: This study showed for the first time that the p38 MAPK pathway plays a key role in maintaining self-renewal capacity of mouse mGSCs, which offers a new self-renewal pathway for these cells and contributes to overall knowledge of the mechanisms of mGSC self-renewal., (© 2016 John Wiley & Sons Ltd.)

Lin28a is a conserved RNA-binding protein that plays an important role in development, pluripotency, stemness maintenance, proliferation and self-renewal. Early studies showed that Lin28a serves as a marker of spermatogonial stem cells (SSCs) and promotes the proliferation capacity of mouse SSCs. However, there is little information about Lin28a in livestock SSCs. In this study, we cloned Capra hircus Lin28a CDS and found that it is evolutionarily conserved. Lin28a is widely expressed in different tissues of Capra hircus, but is expressed at a high level in the testis. Lin28a is specifically located in the cytoplasm of Capra hircus spermatogonial stem cells and may also be a marker of dairy goat spermatogonial stem cells. Lin28a promoted proliferation and maintained the self-renewal of GmGSCs-I-SB in vivo and in vitro. Lin28a-overexpressing GmGSCs-I-SB showed an enhanced proliferation rate, which might be due to increased PCNA expression. Moreover, Lin28a maintained the self-renewal of GmGSCs-I-SB by up-regulating the expression of OCT4, SOX2, GFRA1, PLZF and ETV5. Furthermore, we found that Lin28a may activate the AKT, ERK, and mTOR signaling pathways to promote the proliferation and maintain the self-renewal of GmGSCs-I-SB.

Porcine pancreatic stem cells (PSCs) are one kind of the potential cells for treatment of human diabetes. Autophagy is a highly conserved cellular degradation process in which it helps to maintain the balance between the synthesis, degradation and subsequent recycling of cellular components. However, how autophagy contributes to PSCs has not yet been investigated. Here, we established GFP-LC3 transfected porcine PSC lines in which the accumulation of autophagosomes can be efficiently visualized to evaluate the autophagic activity. Moreover, we observed that starved PSCs which showed increased autophagic activity exhibited an increased tendency to proliferate through the results of BrdU, flow cytometry and western blotting. Furthermore, increased expression of active β-catenin after inducing autophagy indicated that it might be the canonical Wnt signaling that autophagy activated to exert the function on the stimulation of PSCs proliferation. Collectively, these results demonstrated that autophagy stimulated proliferation of PSCs might be regulated by the canonical Wnt signaling pathway. Our results for the first time shed light on a role of autophagy for stimulating the proliferation of porcine PSCs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives: miR-375 is one of the highly expressed microRNAs (miRNAs) found in pancreatic islets of both humans and mice. In this study, we investigated functions of miRNA miR-375 in porcine pancreatic stem cells (PSC)., Materials and Methods: We transfected mimic and inhibitor of miR-375 in PSCs to measure functional roles of the microRNA and its effects on cell cycle proliferation and cell differentiation were determined. Luciferase assays were also performed to reveal the target gene of miR-375., Results: Overexpression of miR-375 suppressed proliferation, promoted apoptosis and inhibited differentiation into islet-like cells. PDK1 was identified as being a target of miR-375. Furthermore, we found that overexpression of miR-375 inhibited activation of the PDK1-AKT signalling pathway., Conclusion: miR-375 directly targeted PDK1 in porcine PSCs, suppressing cell proliferation and differentiation into islet-like cells., (© 2016 John Wiley & Sons Ltd.)

The regulation of spermatogonial stem cell (SSC) proliferation and self-renewal is a complex process. Several studies on the microRNA regulation of mammalian spermatogenesis have been reported. Here, we predicted miRNA targeting of Sirt1, and a dual luciferase experiment confirmed that miR-204 interacted with the Sirt1 3'-untranslated region (3'-UTR). The expression of miR-204 and Sirt1 in dairy goat testicles was investigated, and the results showed that the expression pattern of Sirt1 was similar to that of miR-204 in the temporal-spatial distribution. The over-expression of Sirt1 in goat SSCs can promote SSCs' self-renewal gene expression and cell proliferation. Furthermore, miRNA sequencing results showed that Sirt1 had a higher expression level in dairy goat CD49f(+) and CD90(+) SSCs, but the expression level of miR-204 was lower. In an in vitro assay, Sirt1 was significantly down-regulated in dairy goat SSCs when transfected with miR-204 mimics, indicating that Sirt1 was a target of miR-204 in the dairy goat. On the basis of the results of RT-qPCR, fluorescence-activated cell sorting (FACS), and western blotting, we found that the over-expression of Sirt1 in goat SSCs can promote cellular proliferation and change self-renewal and pluripotent gene expression. Thus, miR-204 was involved in the regulation of dairy goat SSCs proliferation via Sirt1.

Objectives: 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., Materials and Methods: CD49f expression patterns were investigated in dairy goats. CD49f positive cells were purified and enriched using magnetic-activated cell sorting (MACS), and characteristics of the cultured cells were assayed using alkaline phosphatase (AP) analysis, quantitative real-time PCR (QRT-PCR) and immunofluorescence analysis. Furthermore, the exogenous CD49f gene was transfected into mGSCs and its effects were analysed., Results: CD49f was found to be conserved in both mRNA and amino acid sequences and that it was an efficient marker for dairy goat mGSC identification, enrichment and purification. CD49f positive cells expressed higher levels of mGSC-specific markers, and proliferated faster than CD49f negative cells. Overexpression CD49f promoted proliferation of dairy goat mGSCs, and Oct4 expression was upregulated; histone H3-lysine 9 dimethylation (H3K9me2) was reduced., Conclusions: Taken together, our data suggest that CD49f plays novel and dynamic roles in regulating maintenance of pluripotency in mGSCs via Oct4 crosstalk and histone methylation dynamics,which may provide new solutions for mGSCs stability in vitro., (© 2016 John Wiley & Sons Ltd.)

Objectives: Porcine pancreatic stem cells (PSCs) are highly valuable in transplantation applications for type II diabetes. However, there are still many problems to be solved before they can be used in the clinic, such as insufficient cell number availability and low secretion level of insulin. It has been reported that Wnt3a plays pivotal roles during cell proliferation and differentiation. Here, we have aimed to establish an ideal research platform using the T-REx(™) system, to study mechanisms of Wnt3a during PSC proliferation and differentiation., Materials and Methods: Construction of the recombinant plasmid and cell transfection were used for establishment of a porcine PSC line. Related gene expressions were examined using quantitative real-time PCR (QRT-PCR), western blotting, immunostaining and flow cytometry. BrdU incorporation assay and cell cycle analysis were used to investigate Wnt3a roles in PSCs., Results: Wnt3a-expressing clones regulated by T-REx(™) were successfully obtained. Wnt3a and GFP expression were strictly regulated by Dox in a time- and dose-dependent manner. Furthermore, we found that Wnt3a-expressing porcine PSCs induced by Dox exhibited raised proliferative potential. After Dox stimulation, expression of PCNA, C-MYC and active β-catenin were higher, but were down-regulated after Dkk1 addition., Conclusion: We established a porcine PSC line that dynamically expressed Wnt3a, and we found that Wnt3a promoted PSC proliferative potential. This inducible expression system thus provides an important tool for further study on porcine PSC development and differentiation., (© 2015 John Wiley & Sons Ltd.)

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., (© 2015 Society for Reproduction and Fertility.)

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.

The mitosis-meiosis switch is a key event in the differentiation of germ cells. Meiosis is important in development biology, however, it has not been clear what is the regulation mechanism in mammals. Our previous study showed that Boule could activate Stra8 directly and result in the meiosis initiation of dairy goat male germline stem cells (mGSCs). Nanos2, a RNA-binding protein, plays critical roles in the suppression of meiosis by preventing Stra8 expression and maintain the male germ cell development. The main purpose of this study was to explore whether Nanos2 represses Stra8 transcription through Boule or not. We found ectopic over-expression of Nanos2 in GC-1 and mGSCs down-regulated Stra8 transcription and translation, and Boule expression was not affected. It was in consistent with our expectation that RA could up-regulate Boule and Stra8 expression, but down-regulate Nanos2 expression in mGSCs. In dairy goat, the expression levels of Boule and Stra8 would rise with the increase of age, but the expression level of Nanos2 in 90 dpp and adult testis had not shown a clear change. In conclusion, Nanos2 represses Stra8 expression but not through Boule in dairy goat mGSCs.

Reproduction is required for the survival of all mammalian animals. Spermatogenesis is an essential and complex developmental process that ultimately results in production of haploid spermatozoa. Recent studies demonstrated that Boule and stimulated by retinoic acid 8 (Stra8) played important roles in initiation meiosis in male germ cells. miR-34c is indispensable in the late steps of spermatogenesis; remarkably, the main function of miR-34c is to reduce cell proliferation potentiality and promote cellular apoptosis. The objectives of this study were to investigate the expression patterns of Boule, Stra8, P53 and miR-34c in dairy goat testis and their relationship in male germ line stem cells (mGSCs). The results first revealed the expression patterns of Boule, Stra8, P53 and miR-34c in 30 dpp, 90 dpp and adult testes of dairy goats. The expression levels of Boule, Stra8, P53 and miR-34c in adult dairy goat testes were significantly higher than that of 30 dpp. Overexpression of Boule and Stra8 promoted the expression of miR-34c in dairy goat mGSCs. In our previous study, we showed that miR-34c was P53 dependent in mGSCs. These results have shown that the up-regulation of miR-34c was not due to P53 protein activation but which might be caused by the up-regulation of Boule and Stra8 promoting the advance of meiosis. In addition, we found retinoic acid would decrease the expression of P53 and miR-34c, however, did not change the expression of c-Myc greatly. It suggested that the function of driving differentiation of dairy goat mGSCs by retinoic acid might not be caused by P53., (Copyright © 2013 John Wiley & Sons, Ltd.)

A small molecular chemical-Reversine has been shown to promote cell reprogramming and induce dedifferentiation of multiple terminally differentiated mesodermal origin cells, and then differentiate into other cell types within mesodermal lineages as well as neuroectodermal. However, the possibilities of these cells to give rise to germ cell lineages have not been examined. The objective of the current study was to detect the effect of Reversine on PMDSCs differentiation into germ cells. PMDSCs from fetal porcine skeletal muscle and their potential of differentiation into germ cells in vitro were investigated. The phenotype, proliferation potential, characteristic markers of the first adhesion cells (pp1), and the purified 2 times cells (pp3) were analyzed by growth curve, FACS, and RT-PCR, respectively. Then, the purified cells were induced with 10% or 20% bovine follicular fluid (FF), the results showed that some of the induced pp3 cells were similar as porcine oocyte, and expressed germ cell and oocyte markers analyzed by semi-quantitative RT-PCR and immunofluorescent staining. Reversine clearly increased the potentiality of PMDSCs differentiation into large round germ-like cells in FF induction medium analyzed by morpholgogy, QRT-PCR and immunofluoresce. The BrdU labeled PMDSCs might differentiate into female germ-like cells in recipient's kidney capsule, which were positive for germ cell and meiotic markers (Dazl, Vasa, Figla, Stra8, Scp3) and oocyte markers (Zp2, Zp3). These findings provided an efficient model to study the mechanism of cell proliferation and germ cell differentiation in livestock promoted by Reversine., (Copyright © 2012 Wiley Periodicals, Inc.)

The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precursors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement therapy for impairing ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells after transplanting, we transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. GLSC-V also were positive for anti-P63 and anti-Integrinbeta1 antibody by immunofluorescent staining. We founded neither GLSC-V nor GLSCs expressed keratin3 (k3) and keratinl2 (k12). However, GLSC-V had higher levels in expression of p63, pcna and venus compared with GLSCs. Further, we cultivated the cells on denude amniotic membrane to construct tissue engineered fluorescent corneal epithelial sheets. Histology and HE staining showed that the constructed fluorescent corneal epithelial sheets consisted of 5-6 layers of epithelium. Only the lowest basal cells of fluorescent corneal epithelial sheets expressed P63 analyzed by immunofluorescence, but not superficial epithelial cells. These results showed that our constructed fluorescent corneal epithelial sheets were similar to the normal corneal epithelium in structure and morphology. This demonstrated that they could be transplanted for patents with corneal impair, also may provide a foundation for the study on the mechanisms of corneal epithelial cell regeneration after LSCT.

Studies on ESRRB-regulating porcine-induced pluripotent stem cells (piPSCs) converted to trophoblast-like stem cells (TLSCs) contribute to the understanding of early embryo development. However, the epigenetic modification regulation network during the conversion is poorly understood. Here, the global change in histone H3 Lysine 4, 9, 27, 36 methylation and Lysine 27 acetylation was investigated in piPSCs and TLSCs. We found a high modification profile of H3K36me2 in TLSCs compared to that of piPSCs, whereas the profiles of other modifications remained constant. KDM4C, a H3K36me3/2 demethylase, whose gene body region was combined with ESRRB, was upregulated in TLSCs. Moreover, KDM4 inhibitor supplementation rescued the AP-negative phenotype observed in TLSCs, confirming that KDM4C could regulate the pluripotency of TLSCs. Subsequently, KDM4C replenishment results show the significantly repressed proliferation and AP-positive staining of TLSCs. The expressions of CDX2 and KRT8 were also upregulated after KDM4C overexpression. In summary, these results show that KDM4C replaced the function of ESRRB. These findings reveal the unique and crucial role of KDM4C-mediated epigenetic chromatin modifications in determination of piPSCs' fate and expand the understanding of the connection between piPSCs and TSCs. [ABSTRACT FROM AUTHOR]

Porcine spermatogonia stem cells (pSSCs) are the only type of somatic stem cell that can pass genetic information to the successive generations. Little is known about pSSCs vitality in vitro, and due to their increasing importance in stem cell research, here, we optimized a protocol to culture pSSCs and explored their potential fate in vitro. Utilizing a feeder‐free culture system with a 2D peptide‐coating and small chemical molecules (including CHIR99021, Repsox, vitamin C, folic acid, and CD lipid concentrate), we were able to prolong the culture time of pSSCs by at least three months compared with previous methods. Moreover, we found that pSSCs could proliferate and self‐renew in the seminiferous tubules of infertile mice. However, they could not perform meiosis. Our study shows that this feeder‐free culture system optimizes cell culture and may facilitate advanced research on SSC biology and genetic manipulation of germ cells. [ABSTRACT FROM AUTHOR]

Orchitis is one of the leading causes of male animal infertility and is associated with inflammatory reactions caused by the bacterium. It has been reported that there is a mutual coupling effect between endoplasmic reticulum stress (ERS) and inflammatory response. Our studies showed that lipopolysaccharide (LPS) could cause testicular damages, apoptosis, ERS, and inflammatory responses in spermatogonial stem cells (SSCs); ERS‐related apoptosis proteins were activated and the expression of ERS genes was significantly upregulated; meanwhile, the expression of Toll‐like receptor 4 and inflammation factors was apparently increased with LPS treatment. Moreover, melatonin (MEL) could rescue testicular damage, and significantly inhibited the expression of ERS‐related apoptosis genes, ERS markers, and inflammatory factors in SSCs and MEL played repairing and anti‐infection roles in LPS‐induced testicular damage. Therefore, MEL may be used as a drug to prevent and control bacterial infections in male reproductive systems. However, the specific molecular mechanism of MEL to resist ERS and inflammatory response remains to be further studied. [ABSTRACT FROM AUTHOR]

Spermatogenesis is a complex process that originates from and depends on the spermatogonial stem cells (SSCs). The number of SSCs is rare, which makes the separation and enrichment of SSCs difficult and inefficient. The transcription factor PAX7 maintains fertility in normal spermatogenesis in mice. However, for large animals, much less is known about the SSCs' self‐renewal regulation, especially in dairy goats. We isolated and enriched the CD49f‐positive and negative dairy goat testicular cells by magnetic‐activated cell sorting strategies. The RNA‐ sequencing and experimental data revealed that cells with a high CD49f and PAX7 expression are undifferentiated spermatogonia in goat testis. Our findings indicated that ZBTB16 (PLZF), PAX7, LIN28A, BMPR1B, FGFR1, and FOXO1 were expressed higher in CD49f‐positive cells as compared to negative cells and goat fibroblasts cells. The expression and distribution of PAX7 in dairy goat also have been detected, which gradually decreased in testis tissue along with the increasing age. When the PAX7 gene was overexpressed in dairy goat immortal mGSCs‐I‐SB germ cell lines, the expression of PLZF, GFRα1, ID4, and OCT4 was upregulated. Together, our data demonstrated that there is a subset of spermatogonial stem cells with a high expression of PAX7 among the CD49f+ spermatogonia, and PAX7 can maintain the self‐renewal of CD49f‐positive SSCs. [ABSTRACT FROM AUTHOR]

The future fertility of males with cancer may be irreversibly compromised by chemotherapy and/or radiotherapy. Spermatogonial stem cell transplantation is believed to be a way to restore fertility in men. However, the survival efficiency of transplanted cells is still low. Eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) located on the Y chromosome of male animals is a coding gene of eIF2γ which mainly functions in translation initiation. Recently, the emerging role of Eif2s3y in spermatogenesis has been emphasized in several studies. However, the underlying mechanism is still unclear. In addition, how Eif2s3y functions in large animals remains largely unknown. In this study, we obtained the CDS sequence of the Eif2s3y gene from the testis of dairy goats and found that this gene was highly expressed in the testis and was evolutionarily conserved among different species. Interestingly, overexpression of Eif2s3y promoted the proliferation of spermatogonial stem cells of dairy goats by activating the ERK signaling pathway. In animal experiments, overexpressing Eif2s3y promoted transplanted goat spermatogonial stem cells and produced more colonies after microinjection into the seminiferous tubules of infertile mice. In conclusion, our study highlights an undiscovered role of Eif2s3y in dairy goat reproduction. This finding may provide an important basis for future works regarding male spermatogenic cell restoration and represent a major advance toward surrogate sires becoming a tool for disseminating and regenerating germplasm in all mammals. [ABSTRACT FROM AUTHOR]

Male germline stem cells (mGSCs) can transmit genetic materials to the next generation and dedifferentiate into pluripotent stem cells. However, in livestock, mGSC lines are difficult to establish, because of the factors that affect their isolation and culture. The extracellular matrix serves as a substrate for attachment and affects the fate of these stem cells. Poly‐L‐lysine (PL), an extracellular matrix of choice, inhibits and/or kills cancer cells, and promotes the attachment of stem cells in culture. However, how it affects the characteristics and potentials of these stem cells in culture needs to be elucidated. Here, we isolated, enriched and cultured dairy goat mGSCs on five types of extracellular matrices. To explore the best extracellular matrix to use for culturing them, the characteristics and proliferation ability of the cells were determined. Results showed that the cells shared several characteristics with previously reported mGSCs, including the poor effect of PL on their proliferative and colony‐forming abilities. Further examination showed upregulation of p53 expression in these cells, which could be inhibiting their proliferation. When a p53 inhibitor was included in the culture medium, it was confirmed to be responsible for the inhibition of proliferation in mGSCs. Optimal concentration of the inhibitor in the culture of these cells was 5 µM. Furthermore, addition of the p53 inhibitor increased the expression of the markers of self‐renewal and cell cycle in goat mGSCs. In summary, suppressing p53 is beneficial for the proliferation of dairy goat mGSCs, cultured on PL. [ABSTRACT FROM AUTHOR]

Promyelocytic leukaemia zinc finger (Plzf), also known as zinc finger and BTB domain containing 16 (ZBTB16) or zinc-finger protein 145 (ZFP145), is a critical zinc finger protein of male germline stem cells (mGSCs). Multiple lines of evidence indicate that Plzf has a central role in the development, differentiation and maintenance of many stem cells, including mGSCs, and Plzf has been validated as an essential transcription factor for mammalian testis development and spermatogenesis. This review summarises current literature focusing on the significance of Plzf in maintaining and regulating self-renewal and differentiation of mGSCs, especially goat mGSCs. The review summarises evidence of the specificity of Plzf expression in germ cell development stage, the known functions of Plzf and the microRNA-mediated mechanisms that control Plzf expression in mGSCs. Multiple lines of evidence indicate that Plzf has a central node in the development, differentiation and maintenance of many stem cells, including male germline stem cells (mGSCs). This review summarises evidence showing tight regulation of cell type- and stage-specific expression of Plzf, that Plzf is activated with heterochromatin formation and that it may be controlled by specific microRNAs during germ cell development. [ABSTRACT FROM AUTHOR]

LIN28A serves as a crucial marker of dairy goat male germline stem cells (GmGSCs). In our previous study, we demonstrated that LIN28A promotes proliferation, self‐renewal, and maintains the stemness of GmGSCs. Here, we found that LIN28A could activate the transcription of NANOG in a let‐7g independent manner. We cloned the 5′ upstream of two NANOG genes which were located on chromosome 15 (NANOG‐ch15) and chromosome 5 (NANOG‐ch5), respectively, and then examined their promoter activities and promoter methylation levels. Results showed that NANOG‐ch15 is a pseudogene whereas NANOG‐ch5 is active in Capra hircus. Bioinformatics analysis indicated that the 5′ upstream region of NANOG‐ch5 does not have typical CpG islands but contains several CG enrichment regions and several LIN28A binding sites. Deletion analysis suggested that NANOG‐ch5 promoter can be activated by LIN28A directly binding to the site ‐210 but not by the indirect effect from the inhibition of let‐7g, which is known to be downregulated by LIN28A. Mechanistically, LIN28A recruits and interacts with 5‐methylcytosine‐dioxygenase Ten‐Eleven translocation 1 (TET1) to NANOG‐ch5 gene promoter binding sites to orchestrate 5‐methylcytosine and 5‐hydroxymethylcytosine dynamics. These results revealed the role of LIN28A in NANOG transcriptional regulation via epigenetic DNA modifications to maintain the stemness of GmGSC. In this study, we found that LIN28A in a let‐7g independent manner activating the transcription of NANOG that located on chromosome 5 via binding the −573/+210 5′ upstream region. Mechanistically, LIN28A recruits and interacts with 5‐methylcytosine‐dioxygenase Ten‐Eleven translocation 1 (TET1) to NANOG gene promoter binding sites to orchestrate 5‐methylcytosine and 5‐hydroxymethylcytosine dynamics. These results reveal the role of LIN28A in NANOG transcriptional regulation via epigenetic DNA modifications to maintain stemness of goat male germline stem cells (GmGSCs). [ABSTRACT FROM AUTHOR]

Abstract: Pluripotent mesenchymal stem-like cell lines were established from lungs of 3–4 months old aborted fetus. The cells present the high ex vivo expansion potential of MSC, a typical fibroblast-like morphology and proliferate up to 15 passages without displaying clear changes in morphology. Immunological localization and flow cytometry analyses showed that these cells are positive for OCT4, c-Kit, CD11, CD29, CD44, telomerase, CD106, CD105, CD166, and SSEA1, weakly expression or negative for SSEA1, SSEA3, SSEA4, CD34, CD105 and CD106. These cells can give rise to the adipogenic as evidenced by accumulation of lipid-rich vacuoles within cells identified by Oil-red O when they were induced with 0.5mM isobutylmethylxanthine, 200μM indomethacin, 10−6 M dexamethasone, and 10μg/ml of insulin in high-glucose DMEM. Osteogenic lineage cells were generated in 0.1μM dexamethasone, 50μg/ml ascorbic acid, 10mM β-glycerophosphate, which are shaped as the osteoblastic morphology, expression of alkaline phosphatase (AP), and the formation of a mineralized extracellular matrix identified by Alizarin Red staining. Neural cells are observed when the cultures were induced with 2-mercapometal, which are positive for nestin, NF-100, MBP and GFAP. Additionally, embryoid bodies (EBs) and sperm like cells are obtained in vitro differentiation of these lung MSCs induced with 10−5 M retinoic acid (RA). These results demonstrated that these MSCs are pluripotent and may provide an in vitro model to study germ-cell formation and also as a potential source of sperms for male infertility. [Copyright &y& Elsevier]

XBP1 is a transcription factor that plays a central role in controlling cellular responses to endoplasmic reticulum stress (ERS). Under stress conditions, the transcriptionally active form of XBP1 is generated by splicing of XBP1 mRNA by the ER-resident protein inositol-requiring enzyme-1α (IRE1α). This study aimed to investigate the role of XBP1 in male reproductive disorders. XBP1s-overexpressing goat spermatogonial stem cells (gSSCs) showed higher proliferative ability in vitro and in vivo. These cells also showed higher antioxidant capacity. In comparison, XBP1 knockdown significantly suppressed proliferation. Further analysis showed that XBP1 could stimulate the secretion of IL-6 from macrophages. Overall, the results indicate that XBP1s functions to enhance the proliferation ability and antioxidant capacity of gSSCs, potentially through a mechanism involving the regulation of gSSCs by macrophages. • The male reproductive dysfunction caused by LPS mainly damages germ cells except for spermatogonial stem cells (SSCs). • XBP1 was highly expressed in SSCs in LPS model. • The XBP1s-overexpressing gSSC (goat spermatogonial stem cell) presents the higher proliferative ability in vitro and in vivo. • XBP1 also affected the SSC by regulating the secretion of macrophages IL-6. [ABSTRACT FROM AUTHOR]

Abstract Jak-Stat pathway is the first pathway identified to stimulate spermatogonial stem cells (SSCs) self-renewal and maintenance activity. Recent studies have showed that stat3 a crucial gene implicated in this pathway can regulate self-renewal in male germline stem cell. In our previous study, we demonstrated that miR-19b-3p induces cell proliferation and reduces heterochromatin through Plzf which also regulates the balance between cell self-renewal and differentiation. Because miRNA can target several genes and to understand more about Plzf, a crucial transcription factor of SSCs, we performed microarray and found that miR-19b-3p integrate Jak-Stat through Plzf to regulate cell self-renewal. Our results demonstrated that miR-19b-3p induces Jak-Stat when Plzf is downregulated; overexpression of Plzf reversed the trend and shown an existence of feedback (-/+) between Plzf and GHR. The cell self-renewal markers CD49f, GFRα1, Oct4 and cKIT analyzed in the both groups miR-19b-3p and Plzf-overexpressing compared to their respective control confirm miR-19b-3p regulates cell pluripotency and self-renewal in goat male germline stem cells through Plzf. Together our finding revels that miR-19b-3p control Jak-Stat signaling through Plzf. [ABSTRACT FROM AUTHOR]

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 goat's 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. [ABSTRACT FROM AUTHOR]

Abstract: In mammalian testis, male germline stem cells (mGSCs) are originated from primordial germ cells and developed into spermatocyte and spermatid. In our previous studies, we had isolated a pluripotent mGSCs from goat testes and tested their pluripotency and differentiation potential in vitro and in vivo, which revealed that the isolated and cultured dairy goat mGSCs maintained the characteristics of mGSCs. However, Thy1, a marker of mGSCs, was not examined in detail. In this study, the dairy goat mGSCs were purified by differential plating followed by magnetic-activated cell sorting (MACS) using Thy1 antibody. The quantitative reverse transcription polymerase chain reaction and immunofluorescence analyses revealed that the transcription and expression of Thy1, CD49f, Plzf, Oct4, Gfra1, and Vasa were higher in Thy1-positive cells when compared with Thy1-negative cells. The detection results of culturing dairy goat mGSCs indicated that the Thy1-positive cells maintained the characteristics of mGSCs, grew relatively faster than Thy1-negative cells, and the percentage of alkaline phosphatase-positive cells and colonies were significantly higher in Thy1-positive mGSCs than Thy1-negative cells. Collectively, these results indicate that THY1 is a marker of undifferentiated spermatogonia in goat testes, the technique of magnetic-activated cell sorting using Thy1 antibody could be an efficient method to enrich mGSCs in goat. [Copyright &y& Elsevier]

• Folic acid promotes the proliferation of pPSCs by binding to FOLRα. • Folic acid increases the efficiency of directed differentiation of pPSCs into insulin-producing cells in vitro. • Canonical Wnt and ERK signaling pathway participate in the regulation of folic acid on proliferation and differentiation of pPSCs. Porcine pancreatic stem cells (pPSCs) can be induced to differentiate into insulin-producing cells in vitro and thus serve as a major cells source for β-cell regeneration. However, this application is limited by the weak cell proliferation ability and low insulin induction efficiency. In this study, we explored the role of folic acid in the proliferation of pPSCs and the formation of insulin-secreting cells. We found that FA-treated pPSCs cells had a high EDU positive rate, and the proliferation marker molecules PCNA, CyclinD1 and c-Myc were up-regulated, while the expression of folate receptor α (FOLRα) was up-regulated. In further research, interference FOLRα or adding canonical Wnt signaling pathway or ERK signaling pathway inhibitors could significantly inhibit the effect of FA on pPSCs proliferation. Meanwhile, during the differentiation of pPSCs into insulin-secreting cells, we found that the maturation marker genes Insulin, NKX6.1, MafA, and NeuroD1 was upregulated in insulin-secreting cell masses differentiationed from pPSCs after FA treatment, and the functional molecules Insulin and C-peptide were increased, the ability to secrete insulin in response to high glucose was also increased. With the addition of Wnt and ERK signaling pathway inhibitors, the pro-differentiation effect of FA was weakened. In conclusion, FA promotes the proliferation of pPSCs by binding to folate receptor α (FOLRα) and increase the efficiency of directed differentiation of pPSCs into insulin-producing cells by regulating canonical Wnt and ERK signaling pathway. This study lays theoretical foundation for solving the bottleneck in the treatment of diabetes with stem cell transplantation in future. [ABSTRACT FROM AUTHOR]

Porcine pancreatic stem cells (pPSCs) can be induced to insulin-secreting cells and therefore considered the most promising seeding cells for curing human diabetes in future. However, insufficient pPSCs number is one of the bottleneck problems before its clinical application. SerpinB1 is a serine protease inhibitor in neutrophils and can directly promote the proliferation of β cells. Whether SerpinB1 is involved in pPSC proliferation and differentiation remains unknown. The effects of SerpinB1 on pPSCs proliferation were measured by Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine, qRT-PCR, western blot, and flow cytometry assays. We found that pPSCs did not efficiently reach the S phase when SerpinB1 expression was knocked down with short hairpin RNA (sh-SerpinB1), the expression of Cyclin D1 , CDK-2 , and PCNA also decreased. Meanwhile, cell viability and proliferation ability were both declined. Further analyses showed that the expression level of phosphorylated STAT3/STAT3was downregulated, along with an upregulation of p53 and p21. We used a two-step induction method to induce pPSCs to insulin-secreting cells and found that SerpinB1 expression in insulin-secreting cells was higher than in pPSCs. Meanwhile, the protein expression level of phosphorylated STAT3/STAT3 was increased while p53 and p21 was decreased in induced insulin-secreting cells in comparison with control cells. The insulin-secreting cells derived from the sh-SerpinB1 cells secreted less insulin and showed poor sensitivity to high glucose than control group. However, the insulin-secreting cells derived from the ov-SerpinB1 cells has a quite contrary tendency. In conclusion, this study demonstrates that SerpinB1 promotes the proliferation of pPSCs through the STAT3 signaling pathway, and SerpinB1 is a key factor for maintaining the viability of pPSCs during the transition to insulin-secreting cells. [ABSTRACT FROM AUTHOR]