Search Results (72)

Spermatogonial stem cells (SSCs) are pivotal in surrogate broodstock technology. However, species-specific protocols for the efficient enrichment and long-term preservation of SSCs in olive flounder (Paralichthys olivaceus) are not yet fully established. In this study, we evaluated and optimized methods for the isolation and cryopreservation of P. olivaceus SSCs. First, we compared two enrichment methods, including Percoll density gradient centrifugation (PDGC) and differential plating (DP). Although SSCs enriched by both methods showed increased expression of SSC-specific marker genes, PDGC resulted in significantly greater enrichment than DP. A combination of PDGC and DP did not further improve enrichment efficiency, suggesting that PDGC alone is sufficient in P. olivaceus. Second, we optimized cryopreservation conditions according to various cryoprotectants. Among the conditions, SSCs cryopreservation using 1.3 M propylene glycol (PG) as a permeating agent and 0.2 M raffinose (Raf) as a non-permeating cryoprotectant provided the highest cell viability (56.1%), demonstrating a synergistic protective effect. Finally, preliminary in vivo migration and localization ability of the cryopreserved SSCs was confirmed through xenotransplantation into zebrafish (Danio rerio) larvae. PKH26-labeled donor cells exhibited successful initial localization and short-term persistence within the presumptive gonadal ridge of the recipients at 5 days post-transplantation. These findings provide an optimized protocol for the handling and preservation of P. olivaceus germline resources, contributing to the technical advancement of surrogate reproduction strategies in this species. Full article

Spermatogonial stem cells (SSCs) are the only adult male germline stem cells capable of lifelong self-renewal and differentiation into spermatozoa. Scalable ex vivo survival is essential for endangered species germplasm banking, genetic resource conservation, and male infertility therapy. Here, chitosan (CO) or chitosan oligosaccharide (COS) was cross-linked into injectable, biodegradable 3D hydrogels loaded with the natural astaxanthin (AST). CCK-8 optimization identified 0.3% CO + 0.2% AST (CHAG) and 0.2% COS + 0.2% AST (COAG) as superior formulations. After 7 or 14 d of 3D culture, CHAG yielded significantly more colonies than controls (p < 0.01), with elevated EdU incorporation, alkaline phosphatase activity, and positive OCT4 and PLZF staining, confirming preserved stemness. Caspase-3 expression was markedly reduced, indicating the AST-mediated suppression of oxidative apoptosis. RNA-seq showed distinct transcriptome pathways (p < 0.01): CHAG up-regulated adhesion and ECM–receptor and cell cycle pathways, whereas COAG enriched immune-modulatory and signaling modules, enabling context-specific use. AST-loaded CO/COS hydrogels are inexpensive, cytocompatible, and scalable, doubling as a biomimetic niche that accelerates SSC proliferation while delaying senescence. The platform provides a robust, controllable 3D system for SSC expansion and establishes a pre-clinical basis for translating CO/COS/AST composites to reproductive stem cell biotechnology. Full article

The growing interest in improving the fertility-rate of livestock species, considering their high economic value, has prompted the development of new methodological approaches using male germline stem cells. Spermatogonial stem cells’ (SSCs) potential to self-renew and differentiate into mature spermatozoa holds promise for their transplantation into testicular tissue and use in new biotechnological methodologies. Moreover, SSCs’ ability to convey genetic information to the next generation is a property that could be exploited for gene targeting. The review provides an update on the main aspects of SSC biology, focusing on the genetic regulators of self-renewal and differentiation processes and different isolation methods. In addition, recent advancement in the cryopreservation of SSCs from domestic animals and their transplantation into recipients’ testes are also discussed. Finally, a section focused on canine SSCs (cSSCs), their biological aspects, and their potential clinical application in the field of reproduction is included. This represents an effective animal model for human reproduction, development, and disease, given that the reproductive anatomy and physiology of canine species and human are similar. We then report on the potential clinical transplantation of SSCs into recipient testicular tissue and suggest future topics to explore for significant advances in fertility preservation. Full article

Gametogenesis is a fundamental biological process that ensures both genetic recombination and the continuity of successive generations. Interspecific hybrids can reproduce through modified mechanisms, such as hybridogenesis, by transmitting clonal, unrecombined genomes of only one of the parental species via their gametes. Pelophylax grafi (RP) is a natural hybrid frog composed of mixed genomes (subgenomes) of two related species, Pelophylax perezi (P) and Pelophylax ridibundus (R), and coexists in populations with P. perezi. This study tested the involvement of programmed genome elimination in gamete production of P. grafi, providing new insight into reproductive mechanisms of hybrid vertebrates. Using comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH), we examined the genomic constitution of germline cells in tadpoles and adult male and female P. grafi. Controlled crosses between P. perezi and P. grafi produced F1 hybrid tadpoles, whose genotypes confirmed that P. grafi parents transmitted the R subgenome through their gametes. In the early germline cells (gonocytes) of these tadpoles, P chromosomes were selectively eliminated via micronuclei formation during interphase. The occasional presence of the R genome and mixed R/P genome micronuclei suggests variability and imperfect fidelity in the elimination process. In adult hybrids, the majority of diplotene oocytes, spermatogonial stem cells (SSC) and spermatocytes carried R subgenomes. We demonstrated that programmed genome rearrangement in Pelophylax hybrids is an evolutionarily conserved mechanism underlying this unique reproductive strategy. Full article

Spermatogonial stem cells (SSCs) are unipotent germline cells with emerging pluripotent potential under specific in vitro conditions. Understanding their capacity for reprogramming and the molecular mechanisms involved offers valuable insights into regenerative medicine and fertility preservation. SSCs were isolated from Oct4-GFP C57BL/6 transgenic mice using enzymatic digestion and cultured in defined media. Under these conditions, ES-like colonies emerged expressing pluripotency markers. These cells were characterized by immunocytochemistry, teratoma assays, and transcriptomic analyses using bulk and single-cell RNA sequencing datasets. Gene expression profiles were compared with ESCs and SSCs using datasets from GEO (GSE43850, GSE38776, GSE149512). Protein–protein interaction (PPI) networks and co-expression modules were explored through STRING, Cytoscape, and WGCNA. ES-like cells derived from SSCs exhibited strong expression of OCT4, DAZL, and VASA. Transcriptomic analysis revealed key differentially expressed genes and shared regulatory networks with ESCs. WGCNA identified key co-expression modules and hub regulatory RNA binding genes (Ctdsp1, Rest, and Stra8) potentially responsible for the reprogramming process. Teratoma assays confirmed pluripotency, and single-cell RNA-seq validated expression of critical markers in cultured SSCs. This study demonstrates that SSCs can acquire pluripotency features and be reprogrammed into ES-like cells. The integration of transcriptomic and network-based analyses reveals novel insights into the molecular drivers of SSC reprogramming, highlighting their potential utility in stem cell-based therapies and male fertility preservation. Full article

Fertility preservation and restoration using cryo-banked prepubertal testicular tissue is a pivotal part of the childhood hematological cancer care pathway. Estimations indicate that one in 900–1400 young adults is a childhood cancer survivor, underlying the urge to develop fertility restoration protocols as some of the patients have reached the age to father their own genetic child. While it has been reported that 39% of patients present cancer cells in their testes, no efficient decontamination technique has been identified to circumvent cancer reintroduction after autologous testicular cell transplantation. Obtaining single-cell suspensions and selecting only testicular cells might be an option. In this review, mechanical dissociation/enzymatic digestion protocols applied to human testicular tissue, as well as selection and enrichment strategies, and their outcome will be presented and discussed. While the literature revealed a plethora of mechanical dissociation/enzymatic digestion protocols, testicular tissue characteristics are often missing, precluding the comparison of protocols and their outcomes. Downstream selection and enrichment strategies showed promising results with flow cytometry reaching fractions with the highest purity. Future studies should focus on investigating digestion outcomes to elucidate potential influences on both the cell type-specific viability and the cell-to-cell interactions necessary for cell proliferation and differentiation of selected or enriched testicular cell types. Such research outputs will then also be crucial for further progress in in vitro spermatogenesis from testicular cell suspensions as another option for patients that banked testicular tissue at the time of a hematological cancer. Full article

Optimizing the cultivation system is crucial for tissue culture. The culture of cryopreserved testicular tissues is of great importance for the germplasm preservation of endangered animals and especially to ensure high-quality and high-output livestock. In this study, we compared two cultivation systems (Agarose-Supported system and Direct Adherent system) by evaluating their effects on tissue morphology, cell proliferation, apoptosis, gene expression, and endocrine function in cryopreserved testicular tissues from 30-day-old calves. The testicular tissues were cultured for 18 and 27 days with three biological replicates per group, aiming to identify which system better supports tissue preservation, cellular viability, and spermatogenic differentiation. This allowed us to clarify how different cultivation systems influence the structural maintenance and developmental potential of immature bovine testicular tissues. Histological and gene expression analyses revealed that the Agarose-Supported system better preserved the seminiferous cord architecture and supported the development of the seminiferous epithelium compared to the Direct Adherent system. The Agarose system significantly reduced the apoptosis and enhanced the expression of some key genes, including spermatogonial stem cell (SSC) markers (GFRα-1, UCHL1), meiotic marker (SYCP3), mature sperm marker (CRISP1), and testicular somatic cell markers (STAR, SOX9, ACTA2). The Agarose-Supported system also benefited spermatogenic differentiation and testosterone secretion. These findings demonstrate that the Agarose-Supported system facilitates the in vitro development of spermatogenic cells and Leydig cells in post-cryopreserved immature bovine testicular tissues. Full article

The extracellular matrix (ECM) supports spermatogonial stem cell (SSC) function by mimicking biochemical and structural features of the native niche. However, optimal feeder systems and ECM materials remain key limitations in porcine SSC (pSSC) cultures. We developed a porcine-derived ECM (pECM) from porcine feet and evaluated its effectiveness in supporting pSSC maintenance and proliferation under feeder-dependent conditions. We examined protein molecular weight distribution and pECM extract composition. Surface characterization was performed using scanning electron microscopy and atomic force microscopy. We compared pECM with conventional coatings, including gelatin and non-coated controls, using morphological analysis, WST-1 assay, cell cycle analysis, and gene/protein expression of SSC markers. pECM promoted larger, well-defined pSSC colonies and enhanced stemness-related marker expression, including PGP9.5, Thy-1, PLZF, GFRA1, NANOG, and VASA. Additionally, pECM facilitated active pSSC proliferation while suppressing feeder overgrowth, contributing to a stable and functional co-culture environment. Conversely, gelatin supported early feeder proliferation but led to growth saturation, whereas N/C showed delayed attachment and reduced viability. These findings suggest that pECM mimics the native SSC niche and improves pSSC culture. The dual function of pECM in regulating feeder behavior and enhancing pSSC maintenance highlights its potential as a biomaterial for species lacking established feeder-free protocols. Full article

Background: Oxidative stress is a critical factor contributing to male infertility, impairing spermatogonial stem cells (SSCs) and disrupting normal spermatogenesis. This study aimed to isolate and characterize human SSCs and to investigate oxidative stress-related gene expression, protein interaction networks, and developmental trajectories involved in SSC function. Methods: SSCs were enriched from human orchiectomy samples using CD49f-based magnetic-activated cell sorting (MACS) and laminin-binding matrix selection. Enriched cultures were assessed through morphological criteria and immunocytochemistry using VASA and SSEA4. Transcriptomic profiling was performed using microarray and single-cell RNA sequencing (scRNA-seq) to identify oxidative stress-related genes. Bioinformatic analyses included STRING-based protein–protein interaction (PPI) networks, FunRich enrichment, weighted gene co-expression network analysis (WGCNA), and predictive modeling using machine learning algorithms. Results: The enriched SSC populations displayed characteristic morphology, positive germline marker expression, and minimal fibroblast contamination. Microarray analysis revealed six significantly upregulated oxidative stress-related genes in SSCs—including CYB5R3 and NDUFA10—and three downregulated genes, such as TXN and SQLE, compared to fibroblasts. PPI and functional enrichment analyses highlighted tightly clustered gene networks involved in mitochondrial function, redox balance, and spermatogenesis. scRNA-seq data further confirmed stage-specific expression of antioxidant genes during spermatogenic differentiation, particularly in late germ cell stages. Among the machine learning models tested, logistic regression demonstrated the highest predictive accuracy for antioxidant gene expression, with an area under the curve (AUC) of 0.741. Protein oxidation was implicated as a major mechanism of oxidative damage, affecting sperm motility, metabolism, and acrosome integrity. Conclusion: This study identifies key oxidative stress-related genes and pathways in human SSCs that may regulate spermatogenesis and impact sperm function. These findings offer potential targets for future functional validation and therapeutic interventions, including antioxidant-based strategies to improve male fertility outcomes. Full article

Histone modifications play a critical role in regulating gene expression and maintaining the functionality of spermatogonial stem cells (SSCs), which are essential for male fertility and spermatogenesis. In this study, we integrated microarray and single-cell RNA-sequencing (scRNA-seq) data to identify key histone modification gene changes associated with SSC function and aging. Through differential expression analysis, we identified 2509 differentially expressed genes (DEGs) in SSCs compared to fibroblasts. Among these, genes involved in histone modification, such as KDM5B, SCML2, SIN3A, and ASXL3, were highlighted for their significant roles in chromatin remodeling and gene regulation. Protein–protein interaction (PPI) networks and gene ontology (GO) enrichment analysis revealed critical biological processes such as chromatin organization, histone demethylation, and chromosome structure maintenance. Weighted gene co-expression network analysis (WGCNA) further revealed three key modules of co-expressed genes related to spermatogonial aging. Additionally, ligand–receptor interaction scoring based on tumor microenvironment analysis suggested potential signaling pathways that could influence the stemness and differentiation of SSCs. Our findings provide new insights into the molecular mechanisms underlying SSC aging, highlighting histone modification genes as potential therapeutic targets for preserving male fertility and improving SSC-culturing techniques. This study advances our understanding of histone modification in SSC biology and will serve as a valuable resource for future investigations into male fertility preservation. Full article

The formation of mature spermatozoa originates from spermatogonial stem cells (SSCs) located near the basement membrane of the seminiferous tubules. This developmental process, known as spermatogenesis, is tightly regulated to ensure continuous sperm production. A critical aspect of this regulation is the balance between SSC differentiation and self-renewal, which is directed by various factors guiding SSCs in either of these two directions. The SSC niche, defined functionally rather than anatomically, includes all factors necessary for SSC maintenance. These factors are produced by cells surrounding the SSC niche, collectively creating the microenvironment of the seminiferous tubules. Coordination between the cells in this microenvironment is essential for the proper function of the SSC niche and successful spermatogenesis. Testicular mast cells (MCs) significantly influence the regulation of this niche, as they contain various biologically active substances that regulate a wide range of physiological processes and contribute to different pathological conditions affecting fertility. This review explores the effects of testicular MCs on SSCs, their role in regulating spermatogenesis under normal and pathological conditions, and their interactions with other components of the testicular microenvironment, with a focus on their potentially critical impact on spermatogenesis and male fertility. Full article

Background: Histone deacetylase 4 (HDAC4) is a member of the class II histone deacetylase family, whose members play a crucial role in various biological processes. An in-depth investigation of the transcriptional characteristics of chicken HDAC4 can provide fundamental insights into its function. Methods: We examined HDAC4 expression in chicken embryonic stem cells (ESC) and spermatogonial stem cells (SSC) and cloned a 444 bp fragment from upstream of the chicken HDAC4 transcription start site. Subsequently, we constructed pEGFP-HDAC4 and a series of 5′-deletion luciferase reporter constructs, which we transfected into DF-1 cells to measure their transcriptional activity. The regulatory mechanisms of chicken HDAC4 expression were investigated by performing trichostatin A (TSA) treatment, deleting putative transcription factor binding sites, and altering transcription factor expression levels. Results: HDAC4 exhibited higher expression in SSC than in ESC. We confirmed that the upstream region from −295 bp to 0 bp is the core transcriptional region of HDAC4. TSA effectively inhibited HDAC4 transcription, and bioinformatics analysis indicated that the chicken core HDAC4 promoter sequence exhibits high homology with those of other avian species. The myelocytomatosis viral oncogene homolog (MYC) and hypoxia-inducible factor 1 α (HIF1A) transcription factors were predicted to bind to this core region. Treatment with TSA for 24 h resulted in the upregulation of MYC and HIF1A, which repressed HDAC4 transcription. Conclusions: Our results provide a basis for subsequent investigations into the regulation of HDAC4 expression and biological function. Full article

Spermatogonial stem cells (SSCs) sustain and modulate spermatogenesis through intricate signaling pathways and transcription factors. Promyelocytic leukemia zinc-finger (Plzf, also known as Zbtb16) has been identified as a critical transcription factor influencing various signaling and differentiation pathways. Plzf plays a pivotal role in regulating the differentiation properties of SSCs and is essential for the proper maintenance of spermatogenesis. However, the transcription patterns of Plzf along the seminiferous tubules and its interaction network with adjacent partners still need to be fully elucidated. This study employed immunostaining techniques coupled with Fluidigm quantitative real-time polymerase chain reaction (Fluidigm qPCR) to quantify Plzf expression in undifferentiated and differentiated spermatogonia. Furthermore, we utilized bioinformatics analyses to identify Plzf partners and their associations with other regulatory factors. Immunohistostaining (IMH) revealed a high expression of Plzf in cells near the basal membrane of seminiferous tubules and a lower expression in the middle regions in vivo. Immunocytochemistry (ICC) demonstrated that undifferentiated spermatogonia exhibited significant Plzf positivity, whereas differentiated spermatogonia showed reduced Plzf expression in vitro. Fluidigm qPCR confirmed a significant differential expression of Plzf between undifferentiated and differentiated spermatogonia. In silico differential expression analysis between undifferentiated spermatogonia and spermatids indicated that Plzf is closely associated with Mycn, Lin28a, Kras, Ccnd1, and Jak1, highlighting the importance of these partnerships during spermatogenesis. Our findings suggest that the network of Plzf-related partners and their associated proteins involves differentiation, localization, apoptosis, and signal transduction. This comprehensive approach advances our understanding of Plzf transcription patterns and sheds light on its interactions with other cellular factors, revealing previously obscure pathways and interactions. These insights could lead to more effective diagnostic strategies for reproductive system-related diseases and inform the development of improved therapeutic and clinical applications. Full article

The in vitro generation of spermatogonial stem cells (SSCs) from embryonic stem cells (ESCs) offers a viable approach for addressing male infertility. A multitude of molecules participate in this intricate process, which requires additional elucidation. Despite the decline in SSCs in aged testes, SSCs are deemed immortal since they can multiply for three years with repeated transplantation. Nonetheless, the examination of aging is challenging due to the limited quantity and absence of precise indicators. Using a microarray, we assessed genome-wide transcripts (about 55,000 transcripts) of fibroblasts and SSCs. The WGCNA approach was then used to look for SSC-specific transcription factors (TFs) and hub SSC-specific genes based on ATAC-seq, DNase-seq, RNA-seq, and microarray data from the GEO databases as well as gene expression data (RNA-seq and microarray data). The microarray analysis of three human cases with different SSCs revealed that 6 genes were upregulated, and the expression of 23 genes was downregulated compared to the normal case in relation to aging genes. To reach these results, online assessments of Enrich Shiny GO, STRING, and Cytoscape were used to forecast the molecular and functional connections of proteins before identifying the master routes. The biological process and molecular function keywords of cell–matrix adhesion, telomerase activity, and telomere cap complex were shown to be significantly altered in upregulated differentially expressed genes (DEGs) by the functional enrichment analysis. According to our preliminary research, cell-specific TFs and TF-mediated GRNs are involved in the creation of SSCs. In order to maximize the induction efficiency of ESC differentiation into SSCs in vitro, hub SSC-specific genes and important SSC-specific TFs were identified, and sophisticated network regulation was proposed. According to our research, these genes and the hub proteins that they interact with may be able to shine a light on the pathophysiologies of infertility and aberrant germ cells. Full article

Background: The Jun proto-oncogene (JUN), also referred to as C-JUN, is an integral component of the JNK signaling pathway, which is crucial for the formation and differentiation of spermatogonial stem cells (SSCs). Investigations into the transcriptional regulation of chicken JUN can offer a molecular foundation for elucidating its mechanistic role in SSCs. Methods: In this study, we successfully cloned a 2000 bp upstream sequence of the JUN transcription start site and constructed a series of pGL3 recombinant vectors containing JUN promoters of varying lengths. Results: We verified the promoter activity of the 2000 bp upstream sequence by assessing the fluorescence intensity of DF-1 and identified the promoter activities of different regions via dual-luciferase assays. The transcription of JUN and its promoter region spanning −700 to 0 bp was modulated by an activator of the JNK signaling pathway. Bioinformatics analysis revealed that this −700 to 0 bp region was highly conserved among avian species and predicted the presence of binding sites for Wilms tumor 1 (WT1) and CCAAT/enhancer binding protein alpha (CEBPA). The JNK signaling pathway activator was found to upregulate the expression of these transcription factors in DF-1 cells. Through the deletion of binding sites and the overexpression of WT1 and CEBPA, we demonstrated that WT1 inhibited the transcription of JUN, while CEBPA promoted it. Conclusions: In conclusion, the −700 to 0 bp region is the key region of the JUN promoter, with WT1 inhibiting JUN transcription. The results of the study not only provide ideas for exploring the regulatory mechanism of JUN in chicken SSCs, but also lay an important foundation for the study of avian SSCs. Full article