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Seasonal spermatogenesis in fish is driven by spermatogonial stem cells (SSCs), which undergo a complex cellular process to differentiate into mature sperm. In this study, we characterized spermatogenesis in the large yellow croaker (Larimichthys crocea), a marine fish of significant commercial value, based on a high-resolution single-cell RNA-sequencing atlas of testicular cells from three distinct developmental stages: juvenile, adult differentiating and regressed testes. We detailed a continuous developmental trajectory of spermatogenic cells, from spermatogonia to spermatids, elucidating the molecular events involved in spermatogenesis. We uncovered dynamic heterogeneity in cellular compositions throughout the annual reproductive cycle, accompanied by strong molecular signatures within specific testicular cells. Notably, we identified a distinct population of SSCs and observed a critical metabolic transition from glycolysis to oxidative phosphorylation, enhancing our understanding of the biochemical and molecular characteristics of SSCs. Additionally, we elucidated the interactions between somatic cells and spermatogonia, illuminating the mechanisms that regulate SSC development. Overall, this work enhances our understanding of spermatogenesis in seasonal breeding teleosts and provides essential insights for the further conservation and culture of SSCs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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.

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.

Spermatogonial stem cells (SSCs) are essential for sustained sperm production, but SSC regulatory mechanisms and markers remain poorly defined. Studies have suggested that the Id family transcriptional regulator Id4 is expressed in SSCs and involved in SSC maintenance. Here, we used reporter and knockout models to define the expression and function of Id4 in the adult male germline. Within the spermatogonial pool, Id4 reporter expression and inhibitor of DNA-binding 4 (ID4) protein are found throughout the GFRα1+ fraction, comprising the self-renewing population. However, Id4 deletion is tolerated by adult SSCs while revealing roles in meiotic spermatocytes. Cultures of undifferentiated spermatogonia could be established following Id4 deletion. Importantly, ID4 loss in undifferentiated spermatogonia triggers ID3 upregulation, and both ID proteins associate with transcription factor partner TCF3 in wild-type cells. Combined inhibition of IDs in cultured spermatogonia disrupts the stem cell state and blocks proliferation. Our data therefore demonstrate critical but functionally redundant roles of IDs in SSC function., Competing Interests: Declaration of interests F.J.R. receives institutional support as a co-investigator and is subcontracted by Peter MacCallum Cancer Centre for an investigator-initiated trial, which receives funding from Sanofi/Regeneron Pharmaceuticals., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Organoids play pivotal roles in uncovering the molecular mechanisms underlying organogenesis, intercellular communication, and high-throughput drug screening. Testicular organoids are essential for exploring the genetic and epigenetic regulation of spermatogenesis in vivo and the treatment of male infertility. However, the formation of testicular organoids with full spermatogenesis has not yet been achieved. In this study, neonatal mouse testicular cells were isolated by two-step enzymatic digestion, and they were combined with Matrigel and transplanted subcutaneously into nude mice. Histological examination (H&E) staining and immunohistochemistry revealed that cell grafts assembled to form seminiferous tubules that contained spermatogonial stem cells (SSCs) and Sertoli cells, as illustrated by the co-expression of PLZF (a hallmark for SSCs) and SOX9 (a marker for Sertoli cells) as well as the co-expression of UCHL1 (a hallmark for SSCs) and SOX9, after 8 weeks of transplantation. At 10 weeks of transplantation, SSCs could proliferate and differentiate into spermatocytes as evidenced by the expression of PCNA, Ki67, c-Kit, SYCP3, γ-HA2X, and MLH1. Notably, testicular organoids were seen, and spermatids were observed within the lumen of testicular organoids after 16 weeks of transplantation, as shown by the presence of TNP1 and ACROSIN (hallmarks for spermatids). Collectively, these results implicate that we successfully established testicular organoids with spermatogenesis in vivo. This study thus provides an excellent platform for unveiling the mechanisms underlying mammalian spermatogenesis, and it might offer valuable male gametes for treating male infertility.

The actions of spermatogenic stem cells (SSCs) provide the foundation for continual spermatogenesis and regeneration of the cognate lineage following cytotoxic insult or transplantation. Several decades of research with rodent models have yielded knowledge about the core biology, morphological features, and molecular profiles of mammalian SSCs. Translation of these discoveries to utilities for human fertility preservation, improving animal agriculture, and wildlife conservation are actively being pursued. Here, we provide overviews of these aspects covering both historical and current states of understanding., (© 2024 The Author(s). Molecular Reproduction and Development published by Wiley Periodicals LLC.)

Study Question: What is the molecular landscape underlying the functional decline of human testicular ageing?, Summary Answer: The present study provides a comprehensive single-cell transcriptomic atlas of testes from young and old humans and offers insights into the molecular mechanisms and potential targets for human testicular ageing., What Is Known Already: Testicular ageing is known to cause male age-related fertility decline and hypogonadism. Dysfunction of testicular cells has been considered as a key factor for testicular ageing., Study Design, Size, Duration: Human testicular biopsies were collected from three young individuals and three old individuals to perform single-cell RNA sequencing (scRNA-seq). The key results were validated in a larger cohort containing human testicular samples from 10 young donors and 10 old donors., Participants/materials, Setting, Methods: scRNA-seq was used to identify gene expression signatures for human testicular cells during ageing. Ageing-associated changes of gene expression in spermatogonial stem cells (SSCs) and Leydig cells (LCs) were analysed by gene set enrichment analysis and validated by immunofluorescent and functional assays. Cell-cell communication analysis was performed using CellChat., Main Results and the Role of Chance: The single-cell transcriptomic landscape of testes from young and old men was surveyed, revealing age-related changes in germline and somatic niche cells. In-depth evaluation of the gene expression dynamics in germ cells revealed that the disruption of the base-excision repair pathway is a prominent characteristic of old SSCs, suggesting that defective DNA repair in SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of ageing-associated transcriptional changes demonstrated that stress-related changes and cytokine pathways accumulate in old somatic cells. Age-related impairment of redox homeostasis in old LCs was identified and pharmacological treatment with antioxidants alleviated this cellular dysfunction of LCs and promoted testosterone production. Lastly, our results revealed that decreased pleiotrophin signalling was a contributing factor for impaired spermatogenesis in testicular ageing., Large Scale Data: The scRNA-seq sequencing and processed data reported in this paper were deposited at the Genome Sequence Archive (https://ngdc.cncb.ac.cn/), under the accession number HRA002349., Limitations, Reasons for Caution: Owing to the difficulty in collecting human testis tissue, the sample size was limited. Further in-depth functional and mechanistic studies are warranted in future., Wider Implications of the Findings: These findings provide a comprehensive understanding of the cell type-specific mechanisms underlying human testicular ageing at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism., Study Funding/competing Interest(s): This work was supported by the National Key Research and Development Program of China (2022YFA1104100), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 82371611, 82371609, 82301796), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921, 2022A1515111201), and the China Postdoctoral Science Foundation (2021M703736). The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Extracellular membrane proteins are crucial for mediating cell attachment, recognition, and signal transduction in the testicular microenvironment, particularly germline stem cells. Cadherin 18 (CDH18), a type II classical cadherin, is primarily expressed in the nervous and reproductive systems. Here, we investigated the expression of CDH18 in neonatal porcine prospermatogonia (ProSGs) and murine spermatogonial stem cells (SSCs). Disruption of CDH18 expression did not adversely affect cell morphology, proliferation, self-renewal, or differentiation in cultured porcine ProSGs, but enhanced cell adhesion and prolonged cell maintenance. Transcriptomic analysis indicated that the down-regulation of CDH18 in ProSGs significantly up-regulated genes and signaling pathways associated with cell adhesion. To further elucidate the function of CDH18 in germ cells, Cdh18 knockout mice were generated, which exhibited normal testicular morphology, histology, and spermatogenesis. Transcriptomic analysis showed increased expression of genes associated with adhesion, consistent with the observations in porcine ProSGs. The interaction of CDH18 with β-catenin and JAK2 in both porcine ProSGs and murine SSCs suggested an inhibitory effect on the canonical Wnt and JAK-STAT signaling pathways during CDH18 deficiency. Collectively, these findings highlight the crucial role of CDH18 in regulating cell adhesion in porcine ProSGs and mouse SSCs. Understanding this regulatory mechanism provides significant insights into the testicular niche.

Background: Spermatogonial stem cells (SSCs) are essential for the maintenance and initiation of male spermatogenesis. Despite the advances in understanding SSC biology in mouse models, the mechanisms underlying human SSC development remain elusive., Results: Here, we analyzed the signaling pathways involved in SSC regulation by testicular somatic cells using single-cell sequencing data (GEO datasets: GSE149512 and GSE112013) and identified that Leydig cells communicate with SSCs through pleiotrophin (PTN) and its receptor syndecan-2 (SDC2). Immunofluorescence, STRING prediction, and protein immunoprecipitation assays confirmed the interaction between PTN and SDC2 in spermatogonia, but their co-localization was observed only in approximately 50% of the cells. The knockdown of SDC2 in human SSC lines impaired cell proliferation, DNA synthesis, and the expression of PLZF, a key marker for SSC self-renewal. Transcriptome analysis revealed that SDC2 knockdown downregulated the expression of GFRA1, a crucial factor for SSC proliferation and self-renewal, and inhibited the HIF-1 signaling pathway. Exogenous PTN rescued the proliferation and GFRA1 expression in SDC2 knockdown SSC lines. In addition, we found downregulation of PTN and SDC2 as well as altered localization in non-obstructive azoospermia (NOA) patients, suggesting that downregulation of PTN and SDC2 may be associated with impaired spermatogenesis., Conclusions: Our results uncover a novel mechanism of human SSC regulation by the testicular microenvironment and suggest a potential therapeutic target for male infertility., (© 2024. The Author(s).)

Male infertility can be caused by chromosomal abnormalities, mutations and epigenetic defects. Epigenetic modifiers pre-program hundreds of spermatogenic genes in spermatogonial stem cells (SSCs) for expression later in spermatids, but it remains mostly unclear whether and how those genes are involved in fertility. Here, we report that Wfdc15a, a WFDC family protease inhibitor pre-programmed by KMT2B, is essential for spermatogenesis. We found that Wfdc15a is a non-canonical bivalent gene carrying both H3K4me3 and facultative H3K9me3 in SSCs, but is later activated along with the loss of H3K9me3 and acquisition of H3K27ac during meiosis. We show that WFDC15A deficiency causes defective spermiogenesis at the beginning of spermatid elongation. Notably, depletion of WFDC15A causes substantial disturbance of the testicular protease-antiprotease network and leads to an orchitis-like inflammatory response associated with TNFα expression in round spermatids. Together, our results reveal a unique epigenetic program regulating innate immunity crucial for fertility., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Spermatogonial stem cells (SSCs) play a crucial role in the male reproductive system, responsible for maintaining continuous spermatogenesis. The microenvironment or niche of SSCs is a key factor in regulating their self-renewal, differentiation and spermatogenesis. This microenvironment consists of multiple cell types, extracellular matrix, growth factors, hormones and other molecular signals that interact to form a complex regulatory network. This review aims to provide an overview of the main components of the SSCs microenvironment, explore how they regulate the fate decisions of SSCs, and discuss the potential impact of microenvironmental abnormalities on male reproductive health., (© 2024. The Author(s).)

Human spermatogonial stem cells (SSCs) have significant applications in reproductive medicine and regenerative medicine because of their great plasticity. Nevertheless, it remains unknown about the functions and mechanisms of long non-coding RNA (LncRNA) in regulating the fate determinations of human SSCs. Here we have demonstrated that LncRNA ACVR2B-as1 (activin A receptor type 2B antisense RNA 1) controls the self-renewal and apoptosis of human SSCs by interaction with ALDOA via glycolysis activity. LncRNA ACVR2B-as1 is highly expressed in human SSCs. LncRNA ACVR2B-as1 silencing suppresses the proliferation and DNA synthesis and enhances the apoptosis of human SSCs. Mechanistically, our ChIRP-MS and RIP assays revealed that ACVR2B-as1 interacted with ALDOA in human SSCs. High expression of ACVR2B-as1 enhanced the proliferation, DNA synthesis, and glycolysis of human SSCs but inhibited their apoptosis through up-regulation of ALDOA. Importantly, overexpression of ALDOA counteracted the effect of ACVR2B-as1 knockdown on the aforementioned biological processes. Collectively, these results indicate that ACVR2B-as1 interacts with ALDOA to control the self-renewal and apoptosis of human SSCs by enhancing glycolysis activity. This study is of great significance because it sheds a novel insight into molecular mechanisms underlying the fate decisions of human SSCs and it may offer innovative approaches to address the etiology of male infertility., (© 2024. The Author(s).)

Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene-targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid-mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock-in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site-specific knock-in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samples Basic Protocol 2: Preparation of plasmids for gene knock-in using the CRISPR/Cas9 system Basic Protocol 3: Establishment of gene knock-in mGSC lines by electroporation gene delivery., (© 2024 Wiley Periodicals LLC.)

This study explores the effects of Trib3 gene knockout on adult male rat spermatogenesis. Using CRISPR/Cas9, we knocked out the Trib3 gene in Wistar rats. Results indicate altered expression of PLZF, ID4, and c-KIT in knockout rats, suggesting impaired spermatogonial stem cell proliferation and differentiation. Histological analysis reveals reduced seminiferous tubule area and decreased spermatocyte numbers. Mating experiments demonstrate reduced offspring rates after the second self-mating in knockout rats. SYCP3, a meiosis marker, shows elevated expression in knockout rat testes at 14 days postpartum, suggesting an impact on reproductive processes. ELISA results indicate decreased testosterone, FSH, and FGF9 levels in knockout rat testicular tissues. In conclusion, Trib3 gene deletion may impede spermatogonial self-renewal and promote differentiation through the FSH-FGF9- c-KIT interaction and p38MAPK pathway, affecting reproductive capacity. These findings contribute to understanding the molecular mechanisms regulating spermatogenesis., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Infertility is intricately linked with alterations in circadian rhythms along with physiological decline and stem cell senescence. Yet, the direct involvement of circadian mechanisms in nicotine-induced injury to the testes, especially the senescence of spermatogonia stem cells (SSCs), is not well comprehended. This study revealed that nicotine exposure induced testis injury by triggering SSCs senescence along with the upregulation of senescence marker genes and senescence-associated secretory phenotype components. Moreover, nicotine treatment caused mitochondrial hyper-fusion, increased oxidative stress, and DNA damage. Exposure to nicotine was found to suppress the expression of sirtuin 6 (SIRT6), which accelerated the senescence of spermatogonia stem cells (SSCs). This acceleration led to increased acetylation of brain and muscle ARNT-like protein (Bmal1), consequently reducing the expression of Bmal1 protein. Conversely, the overexpression of Bmal1 alleviated mitochondrial hyper-fusion and senescence phenotypes induced by nicotine. Overall, this study unveiled a novel molecular mechanism behind nicotine-induced disorders in spermatogenesis and highlighted the SIRT6/Bmal1 regulatory pathway as a potential therapeutic target for combating nicotine-associated infertility., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organization that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitle., (Copyright © 2024. Published by Elsevier Inc.)

Spermatogonial stem cells (SSCs) possess the characteristics of self-renewal and differentiation, as well as the ability to generate functional sperm. Their unique stemness has broad applications in male infertility treatment and species preservation. In rodents, research on SSCs has been widely reported, but progress is slow in large livestock such as cattle and pigs due to long growth cycles, difficult proliferation in vitro, and significant species differences. Previously, we showed that histone 3 (H3) lysine 9 (K9) trimethylation (H3K9me3) is associated with the proliferation of bovine SSCs. Here, we isolated and purified SSCs from calf testicular tissues and investigated the impact of different H3K9me3 levels on the in vitro proliferation of bovine SSCs. The enriched SSCs eventually formed classical stem cell clones in vitro in our feeder-free culture system. These clones expressed glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα1, specific marker for SSCs), NANOG (pluripotency protein), C-KIT (germ cell marker), and strong alkaline phosphatase (AKP) positivity. qRT-PCR analysis further showed that these clones expressed the pluripotency genes NANOG and SOX2, and the SSC-specific marker gene GFRα1 . To investigate the dynamic relationship between H3K9me3 levels and SSC proliferation, H3K9me3 levels in bovine SSCs were first downregulated using the methyltransferase inhibitor, chaetocin, or transfection with the siRNA of H3K9 methyltransferase suppressor of variegation 3-9 homologue 1 (SUV39H1). The EDU (5-Ethynyl-2'-deoxyuridine) assay revealed that SSC proliferation was inhibited. Conversely, when H3K9me3 levels in bovine SSCs were upregulated by transfecting lysine demethylase 4D (KDM4D) siRNA, the EDU assay showed a promotion of cell proliferation. In summary, this study established a feeder-free culture system to obtain bovine SSCs and explored its effects on the proliferation of bovine SSCs by regulating H3K9me3 levels, laying the foundation for elucidating the regulatory mechanism underlying histone methylation modification in the proliferation of bovine SSCs.

Spermatogonial stem cells (SSCs) are essential for the maintenance of male fertility and survival of species. Environmental conditions, notably heat stress, have been identified as important causes of male infertility and have a negative impact on SSCs. Animals with cryptorchid testes (CT) are optimal models for the study of long-term heat stress-related changes in germ cells. The effect of heat stress on germ cells differs depending on the spermatogenesis stage. Thus, verifying whether the specific phase of spermatogenesis is dependent or independent of heat stress in stallions is important. We evaluated the heat stress-related response of SSCs by comparing the relative abundance of mRNA transcripts and expression patterns of the undifferentiated embryonic cell transcription factor 1 (UTF-1) and deleted in azoospermia-like (DAZL) in the seminiferous tubules of CT and normal testes (NT) of stallions using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blotting. We also analyzed the relative abundance of mRNA of different proliferative markers, including minichromosome maintenance 2 (MCM2), marker of proliferation Ki-67 (MKI-67), and proliferating cell nuclear antigen (PCNA). Testicular tissues from four Thoroughbred unilateral cryptorchid postpubertal stallions were used in this study during the breeding season. The relative abundance of the mRNA transcripts of UTF-1 and MCM2 was significantly upregulated in the CT group than that of those in the NT group. In contrast, the relative abundance of the mRNA transcripts of DAZL was significantly downregulated in the CT group than that of those in the NT group. Western blot quantification showed that the relative intensity of UTF-1 protein bands was significantly higher, while that of DAZL protein bands was significantly lower in the CT group than in the NT group. Immunofluorescence studies showed that the number of germ cells immunostained with UTF-1 was significantly higher while immunostained with DAZL was significantly lower in the CT group than that in the NT group. The higher expression level of UTF-1 in the CT group shows that undifferentiated SSCs are not affected by long-term exposure to heat stress. These results also indicate that germ cells after differentiation phase are directly affected by heat-stress conditions, such as cryptorchidism, in stallions., Competing Interests: Declaration of competing interest No conflicts of interest have been disclosed by the authors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: This study examined SSC proliferation on an epididymosome-enriched decellularized testicular matrix (DTM) hydrogel and spermatogenesis induction in azoospermic mice., Methods: Epididymosomes were extracted and characterized using SEM and western blotting. After cryopreservation, thawed SSCs were cultured in a hydrogel-based three-dimensional (3D) culture containing 10 ng/mL GDNF or 20 µg/mL epididymosomes. SSCs were assessed using the MTT assay, flow cytometry, and qRT-PCR after two weeks of culture. The isolated SSCs were microinjected into the efferent ducts of busulfan-treated mice. DiI-labeled SSCs were followed, and cell homing was assessed after two weeks. After 8 weeks, the testes were evaluated using morphometric studies and immunohistochemistry., Results: The expression of PLZF, TGF-β, and miR-10b did not increase statistically significantly in the 3D + GDNF and 3D + epididymosome groups compared to the 3D group. Among the groups, the GDNF-treated group exhibited the highest expression of miR-21 (*P < 0.05). Caspase-3 expression was lower in the epididymosome-treated group than in the other groups (***P < 0.001). Compared to the 3D and negative control groups, the 3D + epididymosomes and 3D + GDNF groups showed an increase in spermatogenic cells. Immunohistochemical results confirmed the growth and differentiation of spermatogonial cells into spermatids in the treatment groups., Conclusion: The DTM hydrogel containing 20 µg/mL epididymosomes or 10 ng/mL GDNF is a novel and safe culture system that can support SSC proliferation in vitro to obtain adequate SSCs for transplantation success. It could be a novel therapeutic agent that could recover deregulated SSCs in azoospermic patients., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

RNA-binding proteins (RBPs), as key regulators of mRNA fate, are abundantly expressed in the testis. However, RBPs associated with human male infertility remain largely unknown. Through bioinformatic analyses, we identified 62 such RBPs, including an evolutionarily conserved RBP, DEAD-box helicase 20 (DDX20). Male germ-cell-specific inactivation of Ddx20 at E15.5 caused T1-propsermatogonia (T1-ProSG) to fail to reenter cell cycle during the first week of testicular development in mice. Consequently, neither the foundational spermatogonial stem cell (SSC) pool nor progenitor spermatogonia were ever formed in the knockout testes. Mechanistically, DDX20 functions to control the translation of its target mRNAs, many of which encode cell-cycle-related regulators, by interacting with key components of the translational machinery in prospermatogonia. Our data demonstrate a previously unreported function of DDX20 as a translational regulator of critical cell-cycle-related genes, which is essential for cell-cycle reentry of T1-ProSG and formation of the SSC pool., Competing Interests: Declaration of interests A patent for a screening method for male infertility genes has been issued to W.S., D.Z., L.S., and K.L. (patent number: ZL 2023 1 1149986.4; issue number: CN 116884488 B)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Context A population of sperm progenitor cells, known as Asingle spermatogonia, has been described in mammalian testes. During division cycles in spermatogenesis, some cells will form part of the Asingle spermatogonia group, while others form primary spermatocytes. Thus, during spermatogenesis, spermatogonia are the progenitor cells of spermatozoa. Aims In this study, we characterise the spermatogonial stem cells (SSCs) in the testicles of Artibeus jamaicensis and Sturnira lilium bats. The knowledge generated from this will contribute to the understanding of the biology of germ cells and the mechanisms of spermatogenesis in mammals, generating information on wildlife species that are important for biodiversity. Methods Testes were analysed by light and electron microscopy. Likewise, the expression of specific factors of stem cells (Oct4 and C-kit), germ cells (Vasa), cell proliferation (pH3 and SCP1) and testicular somatic cells (MIS, 3βHSD and Sox9) was characterised by immunofluorescence and western blot. Key results The histological analysis enabled the location of type Asingle, Apaired and Aaligned spermatogonia in the periphery of the seminiferous tubules adjacent to Sertoli cells. The expression of genes of stem and germ cells made it possible to corroborate the distribution of the SSCs. Conclusions Results indicate that type Asingle spermatogonia were not randomly distributed, since proliferative activity was detected in groups of cells adjacent to the seminiferous tubules membrane, suggesting the localisation of spermatogonial niches in a specific region of testes. Implications This study provides evidence for the existence of SSCs in the testis of chiropterans that contribute to the renewal of germline progenitor cells to maintain the reproduction of the organisms.

Spermatogonial stem cells (SSCs) undergo self-renewal division to sustain spermatogenesis. Although it is possible to derive SSC cultures in most mouse strains, SSCs from a 129 background never proliferate under the same culture conditions, suggesting they have distinct self-renewal requirements. Here, we established long-term culture conditions for SSCs from mice of the 129 background (129 mice). An analysis of 129 testes showed significant reduction of GDNF and CXCL12, whereas FGF2, INHBA and INHBB were higher than in testes of C57BL/6 mice. An analysis of undifferentiated spermatogonia in 129 mice showed higher expression of Chrna4, which encodes an acetylcholine (Ach) receptor component. By supplementing medium with INHBA and Ach, SSC cultures were derived from 129 mice. Following lentivirus transduction for marking donor cells, transplanted cells re-initiated spermatogenesis in infertile mouse testes and produced transgenic offspring. These results suggest that the requirements of SSC self-renewal in mice are diverse, which has important implications for understanding self-renewal mechanisms in various animal species., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

In Brief: Oogonial stem cells in the adult ovary can generate oocytes, but they are usually quiescent. TGFB1 is key in stimulating the proliferation of OSC, thereby ensuring the sustained reproductive potential in poultry species., Abstract: Oogonial stem cells (OSCs) are a type of germ stem cell present in the adult ovary. They have the ability to self-renew through mitosis and differentiate into oocytes through meiosis. We have previously identified a population of OSCs in the chicken ovary, but the underlying mechanisms controlling their activation and proliferation were unclear. In this study, we observed that OSCs showed robust proliferation when cultured on a layer of chicken embryo fibroblasts (CEF), suggesting that CEF may secrete certain crucial factors that activate OSC proliferation. We further detected TGFB1 as a potent signaling molecule to promote OSC proliferation. Additionally, we revealed the signaling pathways that play important roles downstream of TGFB1-induced OSC proliferation. These findings provide insights into the mechanisms underlying OSC proliferation in chickens and offer a foundation for future research on in situ activation of OSC proliferation in ovary and improvement of egg-laying performance in chickens.

Purpose: The use of 3D (3-Dimensional) culture systems supported cell-to-cell and cell-to-extracellular matrix (ECM) interactions, proliferation, and differentiation of SSCs (Spermatogonial stem cells). The potential advantages of ECM-based scaffolds for in vitro spermatogenesis have been indicated in human and animal experiments. Furthermore, the strong antioxidant and anti-inflammatory activities of melatonin have improved in vitro manipulation of human SSCs in culture conditions., Materials and Methods: SSCs were isolated from the testis of three dead-brain patients and then propagated for four weeks. The characterization of SSC colonies was done using real-time PCR (Polymerase chain reaction), ICC (Immunocytochemistry), and xenotransplantation to mice model. Decellularization of the human testis was performed using 0.3% sodium dodecyl sulfate (SDS) solution and 1% Triton X-100. Also, various characterizations of DTM (Decellularized testicular matrix ) were carried out using histological staining and DNA content analysis. The optimum dose of melatonin was selected by MTT (Methyl thiazol tetrazolium). SSCs were cultured in 4 groups: control, melatonin, ECM, and ECM-melatonin in a differentiation medium for four weeks. The expression of differentiation genes was evaluated by real-time polymerase chain reaction. In addition, the viability of cultured cells was assessed by MTT assay., Results: The results of ICC and real-time PCR showed the expression of undifferentiated SSC markers (PLZF and GRFA1) in SSC colonies following the 2D culture of isolated SSCs. The presence of testicular ECM components after different staining methods; and the reduction of DNA content confirmed the proper decellularization process. Germ cell apoptosis significantly decreased in melatonin and ECM groups, and the higher viability of SSCs was seen in the ECM-melatonin group. The relative expression of GFRA1 and PRM2 decreased and increased in ECM and ECM-melatonin groups, respectively., Conclusion: Our study showed that the addition of melatonin to the human naturally-derived ECM scaffold could provide a suitable platform for inducing the differentiation and preserving the viability of SSCs.

Normal testicular development ensures the process of spermatogenesis, which is a complex biological process. The sustained high productivity of spermatogenesis throughout life is predominantly attributable to the constant proliferation and differentiation of spermatogonial stem cells (SSCs). The self-renewal and differentiation processes of SSCs are strictly regulated by the SSC niche. Therefore, understanding the developmental pattern of SSCs is crucial for spermatogenesis. The Shaziling pig is a medium-sized indigenous pig breed originating from central China. It is renowned for its superior meat quality and early male sexual maturity. The spermatogenic ability of the boars is of great economic importance to the pig industry. To investigate testicular development, particularly the pattern of SSC development in Shaziling pigs, we used single-cell transcriptomics to identify gene expression patterns in 82,027 individual cells from nine Shaziling pig testes at three key postnatal developmental stages. We generated an unbiased cell developmental atlas of Shaziling pig testicular tissues. We elucidated the complex processes involved in the development of SSCs within their niche in the Shaziling pig. Specifically, we identified potential marker genes and cellular signaling pathways that regulate SSC self-renewal and maintenance. Additionally, we proposed potential novel marker genes for SSCs that could be used for SSC isolation and sorting in Shaziling pigs. Furthermore, by immunofluorescence staining of testicular tissues of different developmental ages using marker proteins (UCHL1 and KIT), the developmental pattern of the spermatogonia of Shaziling pigs was intensively studied. Our research enhances the comprehension of the development of SSCs and provides a valuable reference for breeding Shaziling pigs.

Spermatogonial stem cells (SSCs) are capable of transmitting genetic information to the next generations and they are the initial cells for spermatogenesis. Nevertheless, it remains largely unknown about key genes and signaling pathways that regulate fate determinations of human SSCs and male infertility. In this study, we explored the expression, function, and mechanism of USP11 in controlling the proliferation and apoptosis of human SSCs as well as the association between its abnormality and azoospermia. We found that USP11 was predominantly expressed in human SSCs as shown by database analysis and immunohistochemistry. USP11 silencing led to decreases in proliferation and DNA synthesis and an enhancement in apoptosis of human SSCs. RNA-sequencing identified HOXC5 as a target of USP11 in human SSCs. Double immunofluorescence, Co-immunoprecipitation (Co-IP), and molecular docking demonstrated an interaction between USP11 and HOXC5 in human SSCs. HOXC5 knockdown suppressed the growth of human SSCs and increased apoptosis via the classical WNT/β-catenin pathway. In contrast, HOXC5 overexpression reversed the effect of proliferation and apoptosis induced by USP11 silencing. Significantly, lower levels of USP11 expression were observed in the testicular tissues of patients with spermatogenic disorders. Collectively, these results implicate that USP11 regulates the fate decisions of human SSCs through the HOXC5/WNT/β-catenin pathway. This study thus provides novel insights into understanding molecular mechanisms underlying human spermatogenesis and the etiology of azoospermia and it offers new targets for gene therapy of male infertility., (© 2024. The Author(s).)

Spermatogenesis involves a complex process of cellular differentiation maintained by spermatogonial stem cells (SSCs). Being critical to male reproduction, it is generally assumed that spermatogenesis starts and ends in equivalent transcriptional states in related species. Based on single-cell gene expression profiling, it has been proposed that undifferentiated human spermatogonia can be subclassified into four heterogenous subtypes, termed states 0, 0A, 0B, and 1. To increase the resolution of the undifferentiated compartment and trace the origin of the spermatogenic trajectory, we re-analysed the single-cell (sc) RNA-sequencing libraries of 34 post-pubescent human testes to generate an integrated atlas of germ cell differentiation. We then used this atlas to perform comparative analyses of the putative SSC transcriptome both across human development (using 28 foetal and pre-pubertal scRNA-seq libraries) and across species (including data from sheep, pig, buffalo, rhesus and cynomolgus macaque, rat, and mouse). Alongside its detailed characterisation, we show that the transcriptional heterogeneity of the undifferentiated spermatogonial cell compartment varies not only between species but across development. Our findings associate 'state 0B' with a suppressive transcriptomic programme that, in adult humans, acts to functionally oppose proliferation and maintain cells in a ready-to-react state. Consistent with this conclusion, we show that human foetal germ cells-which are mitotically arrested-can be characterised solely as state 0B. While germ cells with a state 0B signature are also present in foetal mice (and are likely conserved at this stage throughout mammals), they are not maintained into adulthood. We conjecture that in rodents, the foetal-like state 0B differentiates at birth into the renewing SSC population, whereas in humans it is maintained as a reserve population, supporting testicular homeostasis over a longer reproductive lifespan while reducing mutagenic load. Together, these results suggest that SSCs adopt differing evolutionary strategies across species to ensure fertility and genome integrity over vastly differing life histories and reproductive timeframes., Competing Interests: The authors declare that there are no conflicts of interest.

The continuous regeneration of spermatogonial stem cells (SSCs) underpins spermatogenesis and lifelong male fertility, but the developmental origins of the SSC pool remain unclear. Here, we document that hnRNPU is essential for establishing the SSC pool. In male mice, conditional loss of hnRNPU in prospermatogonia (ProSG) arrests spermatogenesis and results in sterility. hnRNPU-deficient ProSG fails to differentiate and migrate to the basement membrane to establish SSC pool in infancy. Moreover, hnRNPU deletion leads to the accumulation of ProSG and disrupts the process of T1-ProSG to T2-ProSG transition. Single-cell transcriptional analyses reveal that germ cells are in a mitotically quiescent state and lose their unique identity upon hnRNPU depletion. We further show that hnRNPU could bind to Vrk1, Slx4, and Dazl transcripts that have been identified to suffer aberrant alternative splicing in hnRNPU-deficient testes. These observations offer important insights into SSC pool establishment and may have translational implications for male fertility., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Spermatogenesis is a systematically organized process that ensures uninterrupted sperm production in which the spermatogonial stem cells (SSCs) play a crucial role. However, the existing absence of teleost-specific molecular markers for SSCs presents a notable challenge. Herein we characterized phenotypically the spermatogonial stem cells using specific molecular markers and transmission electron microscopy. Moreover, we also describe a simple method to suppress common carp spermatogenesis using the combination of Busulfan and thermo-chemical treatment, and finally, we isolate and enrich the undifferentiated spermatogonial fraction. Our results showed that C-kit, GFRα1, and POU2 proteins were expressed by germ cells, meanwhile, undifferentiated spermatogonial populations preferentially expressed GFRα1 and POU2. Moreover, the combination of high temperature (35 °C) and Busulfan (40 mg/kg/BW) effectively suppressed the spermatogenesis of common carp males. Additionally, the amh expression analysis showed differences between the control (26 °C) when compared to 35 °C with a single or two Busulfan doses, confirming that the testes were depleted by the association of Busulfan at high temperatures. In an attempt to isolate the undifferentiated spermatogonial fraction, we used the Percoll discontinuous density gradient. Thus, we successfully dissociated the carp whole testes in different cellular fractions; subsequently, we isolated and enriched the undifferentiated spermatogonial population. Therefore, our results suggest that probably both GFRα-1 and POU2 are highly conserved factors expressed in common carp germinative epithelium and that these molecules were well conserved along the evolutionary process. Furthermore, the enriched undifferentiated spermatogonial population developed here can be used in further germ cell transplantation experiments to preserve and propagate valued and endangered fish species., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Meiosis, a key step in spermatogenesis, is affected by many factors. Current studies have shown that long noncoding RNAs (lncRNAs) are potential factors regulating meiosis, and their regulatory mechanisms have received much attention. However, little research has been done on its regulatory mechanism in the spermatogenesis of roosters. Here, we found that lncRNA involved in meiosis and spermatogenesis (lncRNA-IMS) was involved in the regulation of Stra8 by gga-miR-31-5p and hindered the inhibition of Stra8 by gga-miR-31-5p. The acquisition and loss of function experiments demonstrated that lncRNA-IMS was involved in meiosis and spermatogenesis. In addition, we predicted and determined the core promoter region of lncRNA-IMS. Prediction of transcription factors, deletion/overexpression of binding sites, knockdown/overexpression of Jun, and dual-luciferase reporter analysis confirmed that Jun positively activated transcription of lncRNA-IMS. Our findings further enrich the TF-lncRNA-miRNA-mRNA regulatory network during male meiosis and provide new ideas for studying the molecular mechanism of meiosis and spermatogenesis in chicken spermatogonial stem cells., (© 2023 Wiley Periodicals LLC.)

Spermatogenesis is sustained by homeostatic balance between the self-renewal and differentiation of spermatogonial stem cells, which is dependent on the strict regulation of transcription factor and chromatin modulator gene expression. Chromodomain helicase DNA-binding protein 4 is highly expressed in spermatogonial stem cells but roles in mouse spermatogenesis are not fully understood. Here, we report that the germ-cell-specific deletion of chromodomain helicase DNA-binding protein 4 resulted in complete infertility in male mice, with rapid loss of spermatogonial stem cells and excessive germ cell apoptosis. Chromodomain helicase DNA-binding protein 4-knockdown in cultured spermatogonial stem cells also promoted the expression of apoptosis-related genes and thereby activated the tumor necrosis factor signaling pathway. Mechanistically, chromodomain helicase DNA-binding protein 4 occupies the genomic regulatory region of key apoptosis-related genes, including Jun and Nfkb1. Together, our findings reveal the determinant role of chromodomain helicase DNA-binding protein 4 in spermatogonial stem cells survival in vivo, which will offer insight into the pathogenesis of male sterility and potential novel therapeutic targets., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Spinal cord injury (SCI) induces neuronal death and disrupts the nerve fiber bundles, which leads to partial or complete sensorimotor function loss of the limbs. Transplantation of exogenous neurons derived from stem cells to the lesion site becomes a new neurorestorative strategy for SCI treatment. Spermatogonial stem cells (SSCs) can attain pluripotency features by converting to embryonic stem-like cells in vitro . However, differentiating SSCs into lineage-specific neurons is quite difficult and low efficiency. Methods: Immunofluorescence, immunohistochemistry, Western blotting, whole-cell patch clamp, and behavioral tests were performed to verify that self-assembled hydrogels could improve the directional differentiation efficiency of SSCs and the feasibility of SSC-derived neurons in the treatment of spinal cord injury. Results: We developed a novel self-assembled peptide Nap-FFGEPLQLKMCDPGYIGSR (Nap-E7-YIGSR) coated with aligned electrospun PCL fibers to enhance neuronal differentiation of SSCs. The Nap-E7-YIGSR peptide could evenly self-assemble on the surface of PCL fibers, enhanced the materials's hydrophilicity, and improved the SSC affinity of PCL fibers through the stem cell adhesion peptide sequence EPLQLKM domain. In addition, Nap-E7-YIGSR could effectively induce SSC neuron differentiation by activating the integrin β1/GSK3β/β-catenin signaling pathway. Moreover, implanting the induced neurons derived from SSCs into SCI lesion sites in rats resulted in the formation of new relay circuits, myelination, and synapse formation. Furthermore, SSC-derived neurons could survive and function in the spinal cord injury microenvironment, boosting the recovery of locomotion. Conclusion: The combination of the multifunctional peptide and aligned fibers can potentially trigger SSC differentiation to neurons, facilitating neuronal replacement therapy and promoting functional recovery after SCI., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The most serious condition of male infertility is complete Sertoli cell-only syndrome (SCOS), which refers to the lack of all spermatogenic cells in the testes. The genetic cause of SCOS remains to be explored. We aimed to investigate the genetic cause of SCOS and assess the effects of the identified causative variant on human male germ cells., Methods: Whole-exome sequencing was performed to identify potentially pathogenic variants in a man with complete SCOS, and Sanger sequencing was performed to verify the causative variant in this man and his father and brother. The pathogenic mechanisms of the causative variant were investigated by in vitro differentiation of human-induced pluripotent stem cells (hiPSCs) into germ cell-like cells., Results: The homozygous loss-of-function (LoF) variant p.His244ArgfsTer31 (c.731&#95;732delAT) in PIWIL2 was identified as the causative variant in the man with complete SCOS, and the same variant in heterozygosis was confirmed in his father and brother. This variant resulted in a truncated PIWIL2 protein lacking all functional domains, and no PIWIL2 expression was detected in the patient's testes. The patient and PIWIL2 -/- hiPSCs could be differentiated into primordial germ cell-like cells and spermatogonial stem cell-like cells (SSCLCs) in vitro, but the formation and maintenance of SSCLCs were severely impaired. RNA-seq analyses suggested the inactivation of the Wnt signaling pathway in the process of SSCLC induction in the PIWIL2 -/- group, which was validated in the patient group by RT-qPCR. The Wnt signaling pathway inhibitor hindered the formation and maintenance of SSCLCs during the differentiation of normal hiPSCs., Conclusions: Our study revealed the pivotal role of PIWIL2 in the formation and maintenance of human spermatogonial stem cells. We provided clinical and functional evidence that the LoF variant in PIWIL2 is a genetic cause of SCOS, which supported the potential role of PIWIL2 in genetic diagnosis. Furthermore, our results highlighted the applicability of in vitro differentiation models to function validation experiments., (© 2022. The Author(s).)

Studies on gene regulation and signaling transduction pathways of human spermatogonial stem cells (SSCs) are of the utmost significance for unveiling molecular mechanisms underlying human spermatogenesis and gene therapy of male infertility. We have demonstrated, for the first time, that RNF144B stimulated cell proliferation and inhibited the apoptosis of human SSCs. The target of RNF144B was identified as FCER2 by RNA sequencing. We revealed that RNF144B interacted with FCER2 by immunoprecipitation. Consistently, overexpression of FCER2 reversed the phenotype of proliferation and apoptosis of human SSCs caused by RNF144B knockdown. Interestingly, FCER2 pulled down N2ICD (NOTCH2 intracellular domain), while N2ICD could bind to FCER2 in human SSCs. The levels of NOTCH2, FCER2, HES1, and HEY1 were reduced by RNF144B siRNA in human SSCs. Significantly, RNF144B was expressed at a lower level in nonobstructive azoospermia (NOA) patients than in the obstructive azoospermia (OA) patients with normal spermatogenesis, and 52 patients with heterozygous mutations of RNF144B were detected in 1,000 NOA patients. These results implicate that RNF144B promotes the proliferation of human SSCs and suppresses their apoptosis via the FCER2/NOTCH2/HES1 pathway and that the abnormality of RNF144B is associated with spermatogenesis failure. This study thus provides novel molecular mechanisms regulating the fate determinations of human SSCs, and it offers new biomarkers for the diagnosis and treatment of male infertility., (© 2022 Wiley Periodicals LLC.)

Detailed analysis of the regulatory mechanism of spermatogonia stem cell (SSCs) genesis can provide a novel strategy for the application of SSCs in the fields of transgenic animal production and regenerative medicine. Previous studies in this study showed that WNT signaling can positively regulate the formation of SSCs, but the exact regulatory mechanism is not clear. Here, we predicted the target gene of the Wnt/TCF7L2 pathway, namely TDRD1, by bioinformatics analysis. Functional studies revealed that overexpression of TDRD1 during RA-induced SSCs formation in vitro significantly upregulated the expression of reproductive marker genes (Integrinβ1 and Integrinα6), and further flow cytometric analysis also confirmed that the formation efficiency of SSCs was significantly increased after overexpression of TDRD1; while interference with TDRD1 showed the exact opposite result. The in vivo experiments were consistent with the results of the in vitro experiments. Interestingly, although Wnt/TCF7L2 can promote the formation of SSCs, its function must be dependent on the expression of TDRD1, which was also repeatedly demonstrated as a target gene of the Wnt/TCF7L2 signaling pathway. Mechanistically, we found a large number of CpG sites in the TDRD1 promoter, and BSP analysis also confirmed that DNA methylation modifications in the TDRD1 promoter were significantly higher in embryonic stem cells than in SSCs, and further dual-luciferase reporter system assays revealed that low DNA methylation modification levels could enhance TDRD1 promoter activity; although previous studies demonstrated that TCF7L2 could enrich in the TDRD1 promoter region, the binding of the two was dependent on low DNA methylation modification. Taken together, we confirmed that low DNA methylation mediates Wnt/TCF7L2 regulation of TDRD1 to promote the formation of SSCs, providing a basis for SSCs in improving animal productivity., (© 2022 Wiley Periodicals LLC.)

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. Systematically exploring the critical factors associated with the formation of SSCs will provide new insight to improve the formation efficiency, and their practical application. Here we explore the regulatory mechanism of the ECM-receptor interaction signaling pathway and related genes during differentiation of SSCs in chicken. Firstly, the positive cell rate of SSCs protein marker was detected by immunofluorescence and flow cytometry and qRT-PCR was used to identify, the expression of related marker genes after 10 days of RA-induction. Secondly, the ESCs on 0d/ 4d /10d after RA- induction/self-differentiation were collected, and the total RNA was then extracted from cells. Finally, high-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. After PCA analysis of the RNA-seq data, Venny analysis, GO and KEGG enrichment were further used to find the key signaling pathways and genes in the RA-induction process. The results showed that on day 10 of RA-induction, grape cluster growth cells expressed integrinβ1, the specific marker protein of SSCs cells, and the integrinβ1 positive rate was 35.1%. Also, SSCs marker genes CVH, Integrinβ1, Integrinα6 were significantly up-regulated during RA-induction. Moreover, the significantly enriched pathway, ECM-receptor interaction signaling, in current study may play a crucial role in RA-induction. Then, JASPAR was used to predict the differential gene transcription factors in the signaling pathway, finding that RA receptor was a transcription factor of COL5A1, COL5A2 and COL3A1. The qRT-PCR results showed that the expression levels of RA receptors (RXRA, RARA and RXRG) and the predicted genes (COL5A1, COL5A2 and COL3A1) were both significantly increased during RA-induction. Also, dual-luciferase reporter assay showed that RA could affect the luciferin activities of COL5A1, COL5A2 and COL3A1. These results suggest that RA plays a crucial role in the formation of chicken spermatogonial stem cells via the transcription levels of COL5A1, COL5A2 and COL3A1 to regulate the ECM-receptor interaction signaling pathway. Additionally, knockdown of COL5A1/COL5A2/COL3A1 could effectively reduce the formation efficiency of SSCs. This indicated that the interference of RA receptor binding genes in the ECM-receptor interaction signaling pathway could decrease the efficiency of RA induced SSCs formation. Therefore, this study concludes that RA promotes formation of chicken spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In the mouse testis, sperm originate from spermatogonial stem cells (SSCs). SSCs give rise to spermatogonial progenitors, which expand their population until entering the differentiation process that is precisely regulated by a fixed time-scaled program called the seminiferous cycle. Although this expansion process of progenitors is highly important, its regulatory mechanisms remain unclear. NANOS3 is an RNA-binding protein expressed in the progenitor population. We demonstrated that the conditional deletion of Nanos3 at a later embryonic stage results in the reduction of spermatogonial progenitors in the postnatal testis. This reduction was associated with the premature differentiation of progenitors. Furthermore, this premature differentiation caused seminiferous stage disagreement between adjacent spermatogenic cells, which influenced spermatogenic epithelial cycles, leading to disruption of the later differentiation pathway. Our study suggests that NANOS3 plays an important role in timing progenitor expansion to adjust to the proper differentiation timing by blocking the retinoic acid (RA) signaling pathway., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Non-obstructive azoospermia is one of the most common causes of male infertility, but there is still no specific treatment drug. Given that the Oct4 (Octamer-binding transcription factor 4) has an important regulatory effect on spermatogenesis, activating it can effectively promote spermatogenesis, so it is of great value to develop Oct4-targeted drug design and elucidating its mechanism of action. Here, we screened out the Oct4-targeted drug molecule NBMA (N-benzyl-4-methoxy-2-(1-(4-(trifluoromethyl)phenyl)vinyl)aniline) by computer-assisted technology, and found that it has a significant promoting effect on spermatogenesis in the established mouse azoospermia model. Subsequently, through transcriptome sequencing and enrichment analysis, real-time fluorescent quantitative PCR (qPCR) and western blot experiments revealed that NBMA promotes the differentiation of spermatogonial stem cells by activating the Oct4 pathway, thereby promoting spermatogenesis. This study proves that NBMA is a molecule with great potential to be developed as a therapeutic drug for azoospermia. It also shows that computer-assisted, chemical and biological multidisciplinary methods play a very important role in innovative drug discovery., (© 2022 The Authors. Published by Wiley-VCH GmbH.)

Spermatogonial stem cells (SSCs) are able to undergo both self-renewal and differentiation. Unlike self-renewal, which replenishes the SSC and progenitor pool, differentiation is an irreversible process committing cells to meiosis. Although the preparations for meiotic events in differentiating spermatogonia (Di-SG) are likely to be accompanied by alterations in chromatin structure, the three-dimensional chromatin architectural differences between SSCs and Di-SG, and the higher-order chromatin dynamics during spermatogonial differentiation, have not been systematically investigated. Here, we performed in situ high-throughput chromosome conformation capture, RNA-seq, and chromatin immunoprecipitation-sequencing analyses on porcine undifferentiated spermatogonia (which consist of SSCs and progenitors) and Di-SG. We identified that Di-SG exhibited less compact chromatin structural organization, weakened compartmentalization, and diminished topologically associating domains in comparison with undifferentiated spermatogonia, suggesting that diminished higher-order chromatin architecture in meiotic cells, as shown by recent reports, might be preprogrammed in Di-SG. Our data also revealed that A/B compartments, representing open or closed chromatin regions respectively, and topologically associating domains were related to dynamic gene expression during spermatogonial differentiation. Furthermore, we unraveled the contribution of promoter-enhancer interactions to premeiotic transcriptional regulation, which has not been accomplished in previous studies due to limited cell input and resolution. Together, our study uncovered the three-dimensional chromatin structure of SSCs/progenitors and Di-SG, as well as the interplay between higher-order chromatin architecture and dynamic gene expression during spermatogonial differentiation. These findings provide novel insights into the mechanisms for SSC self-renewal and differentiation and have implications for diagnosis and treatment of male sub-/infertility., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Many child cancer patients endure anticancer therapy containing alkylating agents before sexual maturity. Busulfan (BU), as an alkylating agent, is a chemotherapy drug, causing DNA damage and cytotoxicity in germ cells. In the present study, we aimed to investigate the protective effect of astaxanthin (AST), as a potent antioxidant and powerful reactive oxygen species (ROS) scavenger, on BU-induced toxicity in human spermatogonial stem cells. For this purpose, testes were obtained from four brain-dead donors. After tissue enzymatic digestions, testicular cells were cultured for 3 weeks for spermatogonial stem cell (SSC) isolation and purification. K562 cell line was cultured to survey the effect of AST on cancer treatment. The cultured SSCs and K562 cell line were finally treated with AST (10μM), BU (0.1nM), and AST+BU. The expression of NRF-2, HO-1, SOD2, SOD3, TP53, and apoptotic genes, including CASP9, CASP3, BCL2, and BAX, were assayed using real-time PCR. Moreover, ROS level in different groups and malondialdehyde level and total antioxidant capacity in cell contraction of SSCs were measured using ELISA. Data showed that AST significantly upregulated the expression of NRF-2 gene (P<0.001) and protein (P<0.005) and also significantly decreased the production of BU-induced ROS (P<0.001). AST activated the NRF-2/HO-1 pathway that could remarkably restrain BU-induced apoptosis in SSCs. Interestingly, AST upregulated the expression level of apoptosis genes in the K562 cell line. The results of this study indicated that AST reduces the side effects of BU on SSCs without interference with its chemotherapy effect on cancerous cells through modulation of the NRF-2/HO-1 and mitochondria-mediated apoptosis pathways., (© 2021. Society for Reproductive Investigation.)

Niches maintain a finite pool of stem cells via restricted space and short-range signals. Stem cells compete for limited niche resources, but the mechanisms regulating competition are poorly understood. Using the Drosophila testis model, we show that germline stem cells (GSCs) lacking the transcription factor Chinmo gain a competitive advantage for niche access. Surprisingly, chinmo -/- GSCs rely on a new mechanism of competition in which they secrete the extracellular matrix protein Perlecan to selectively evict non-mutant GSCs and then upregulate Perlecan-binding proteins to remain in the altered niche. Over time, the GSC pool can be entirely replaced with chinmo -/- cells. As a consequence, the mutant chinmo allele acts as a gene drive element; the majority of offspring inherit the allele despite the heterozygous genotype of the parent. Our results suggest that the influence of GSC competition may extend beyond individual stem cell niche dynamics to population-level allelic drift and evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Perinatal exposures to endocrine disrupting chemicals (EDCs) alter the male reproductive system. Infants are exposed to genistein (GEN) through soy-based formula, and to Mono(2-ethylhexyl) Phthalate (MEHP), metabolite of the plasticizer DEHP. Spermatogonial stem cells (SSCs) are formed in infancy and their integrity is essential for spermatogenesis. Thus, understanding the impact of EDCs on SSCs is critical. Prostaglandins (PGs) are inflammatory mediators synthesized via the eicosanoid pathway starting with cyclooxygenases (Coxs), that regulate physiological and pathological processes. Our goal was to study the eicosanoid pathway in SSCs and examine whether it was disrupted by GEN and MEHP, potentially contributing to their adverse effects. The mouse C18-4 cell line used as SSC model expressed high levels of Cox1 and Cox2 genes and proteins, and eicosanoid pathway genes similarly to levels measured in primary rat spermatogonia. Treatments with GEN and MEHP at 10 and 100 μM decreased Cox1 gene and protein expression, whereas Cox2, phospholipase A2, prostaglandin synthases transcripts, PGE2, PGF2a and PGD2 were upregulated. Simultaneously, the transcript levels of spermatogonia progenitor markers Foxo1 and Mcam and differentiated spermatogonial markers cKit and Stra8 were increased. Foxo1 was also increased by EDCs in primary rat spermatogonia. This study shows that the eicosanoid pathway is altered during SSC differentiation and that exposure to GEN and MEHP disrupts this process, mainly driven by GEN effects on Cox2 pathway, while MEHP acts through an alternative mechanism. Thus, understanding the role of Cox enzymes in SSCs and how GEN and MEHP exposures alter their differentiation warrants further studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

RNA N6-methyladenosine (m6A) is essential for many bioprocesses in many species, but its role in goat testis development remains elusive, especially alkB homolog 5 (ALKBH5), one of the m6A demethylases. To this end, nine healthy Haimen goats of different ages were chosen randomly to provide testes. The results showed that the expression level of ALKBH5 was increased significantly (P < 0.05) in the 9-month group compared with the 0-day and 3-month groups, and ALKBH5 was located in goat spermatocytes with the highest expression level compared with Leydig cells and Sertoli cells. Thus, pcDNA3.1-ALKBH5 was constructed to explore the influences of the ALKBH5 increase in goat spermatogonial stem cells (SSC) in vitro. The results showed that the expression level of ALKBH5 in SSC transfected with pcDNA3.1-ALKBH5 (OE&#95;ALKBH5) was significantly increased (P < 0.001) compared with that in SSC transfected with pcDNA3.1-EGFP (EGFP). With ALKBH5 overexpression in SSC, flow cytometry analysis showed that cells at G1 phase were significantly reduced (P < 0.01), while cells at S phase significantly increased (P < 0.01), and cell apoptosis was inhibited. Accordingly, the mRNA degradation of CCND1, CCNE1, and BCL2 was suppressed with ALKBH5 overexpression in SSC after treatment with actinomycin D. Furthermore, the mRNA levels of pluripotency maintenance- and cell differentiation-associated genes were changed between the two groups. Overall, the results indicated the crucial role of ALKBH5 during Haimen goat testis development. The results of this study provide a theoretical basis and technical means for RNA methylation participating in goat testis development., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mouse CD90 + SSCs were enriched using the MACS technique and incubated with different doses of estradiol, ranging from 0.01 ng/mL to 500 μg/mL, for 7 days. The viability of SSCs was determined using an MTT assay. The combined effects of estradiol plus Sertoli cell differentiation medium on the orientation of SSCs toward Sertoli-like cells were also assessed. Using immunofluorescence imaging, we monitored protein levels of Oct3/4 after being exposed to estradiol. In addition, protein levels of testosterone, TF, and ABP were measured using ELISA. The expression of Sertoli cell-specific genes such as SOX9, GATA4, FSHR, TF, and ESR-1 and -2 was monitored using real-time PCR assay, and the effects of 14-day injection of estradiol on sperm parameters and Oct3/4 positive progenitor cells in a model of mouse were determined. Data showed that estradiol increased the viability of mouse SSCs in a dose-dependent manner compared to the control (p < 0.05). Along with these changes, cells displayed morphological changes and reduced Oct3/4 transcription factor levels compared to the control SSCs. 7-day incubation of SSCs with estradiol led to the up-regulation of SOX9, GATA4, FSHR, TF, and ESR-1 and -2, and levels of testosterone, TF, and ABP were increased compared to the control group (p < 0.05). The in-vivo examination noted that estradiol reduced sperm parameters coincided with morphological abnormalities (p < 0.05). Histological examination revealed pathological changes in seminiferous tubules and reduction of testicular Oct3/4 + progenitor cells. In conclusion, estradiol treatment probably can induce Sertoli cell differentiation of SSCs while exogenous administration leads to testicular progenitor cell depletion and infertility in long term., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells (SSCs). However, such therapy for infertility has not been experimentally investigated yet. In this study, a mouse model with an X-linked testis-expressed 11 (TEX11) mutation (Tex11 PM/Y ) identified in azoospermia patients exhibited meiotic arrest due to aberrant chromosome segregation. Tex11 PM/Y SSCs could be isolated and expanded in vitro normally, and the mutation was corrected by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated endonuclease 9 (Cas9), leading to the generation of repaired SSC lines. Whole-genome sequencing demonstrated that the mutation rate in repaired SSCs is comparable with that of autonomous mutation in untreated Tex11 PM/Y SSCs, and no predicted off-target sites are modified. Repaired SSCs could restore spermatogenesis in infertile males and give rise to fertile offspring at a high efficiency. In summary, our study establishes a paradigm for the treatment of male azoospermia by combining in vitro expansion of SSCs and gene therapy., Competing Interests: None

The centromere is the constricted chromosomal region required for the correct separation of the genetic material at cell division. The kinetochore protein complex assembles at the centromere and captures microtubules emanating from the centrosome to orchestrate chromosome segregation in mitosis and meiosis. Asymmetric cell division (ACD) is a special type of mitosis that generates two daughter cells with different fates. Epigenetic mechanisms operating at the centromere have been proposed to contribute to ACD. Recent studies have shown that an asymmetric distribution of CENP-A-the centromere-specific histone H3 variant-between sister chromatids can bias chromosome segregation in ACD. In stem cells, this leads to non-random sister chromatid segregation, which can affect cell fate. These findings support the 'silent sister' hypothesis, according to which the mechanisms of ACD are epigenetically regulated through centromeres. Here, we review the recent data implicating centromeres in ACDs and cell fate in Drosophila melanogaster female and male germline stem cells.

Background: Studying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on SSCLC induction., Methods: We examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. Haploid cells and cells expressed meiotic genes were also detected. RNA-seq analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols., Results: The combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. The high concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-seq analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and decreased expression of genes in Wnt signaling pathway., Conclusions: VPA robustly promoted the differentiation of human pluripotent stem cells into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that SSC depletion in azoospermia testes might be associated with inactivation of Wnt signaling pathway. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility., (© 2021. The Author(s).)

In mouse testis, a heterogeneous population of undifferentiated spermatogonia (A undiff ) harbors spermatogenic stem cell (SSC) potential. Although GFRα1 + A undiff maintains the self-renewing pool in homeostasis, the functional basis of heterogeneity and the implications for their dynamics remain unresolved. Here, through quantitative lineage tracing of SSC subpopulations, we show that an ensemble of heterogeneous states of SSCs supports homeostatic, persistent spermatogenesis. Such heterogeneity is maintained robustly through stochastic interconversion of SSCs between a renewal-biased Plvap + /GFRα1 + state and a differentiation-primed Sox3 + /GFRα1 + state. In this framework, stem cell commitment occurs not directly but gradually through entry into licensed but uncommitted states. Further, Plvap + /GFRα1 + cells divide slowly, in synchrony with the seminiferous epithelial cycle, while Sox3 + /GFRα1 + cells divide much faster. Such differential cell-cycle dynamics reduces mitotic load, and thereby the potential to acquire harmful de novo mutations of the self-renewing pool, while keeping the SSC density high over the testicular open niche., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

More than a decade ago, the ENCODE and NIH Epigenomics Roadmap consortia organized large multilaboratory efforts to profile the epigenomes of >110 different mammalian somatic cell types. This generated valuable publicly accessible datasets that are being mined to reveal genome-wide patterns of a variety of different epigenetic parameters. This consortia approach facilitated the powerful and comprehensive multiparametric integrative analysis of the epigenomes in each cell type. However, no germ cell types were included among the cell types characterized by either of these consortia. Thus, comprehensive epigenetic profiling data are not generally available for the most evolutionarily important cells, male and female germ cells. We discuss the need for reproductive biologists to generate similar multiparametric epigenomic profiling datasets for both male and female germ cells at different developmental stages and summarize our recent effort to derive such data for mammalian spermatogonial stem cells and progenitor spermatogonia., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The importance of spermatogonial stem cells (SSCs) in spermatogenesis is crucial and intrinsic factors and extrinsic signals mediate fate decisions of SSCs. Among endogenous regulators, microRNAs (miRNAs) play critical role in spermatogenesis. However, the mechanisms which individual miRNAs regulate self- renewal and differentiation of SSCs are unknown. The aim of this study was to investigate effects of miRNA-30a-5p inhibitor on fate determinations of SSCs., Methods: SSCs were isolated from testes of neonate mice (3-6 days old) and their purities were performed by flow cytometry with ID4 and Thy1 markers. Cultured cells were transfected with miRNA- 30a-5p inhibitor. Evaluation of the proliferation (GFRA1, PLZF and ID4) and differentiation (C-Kit & STRA8) markers of SSCs were accomplished by immunocytochemistry and western blot 48 h after transfection., Results: Based on the results of flow cytometry with ID4 and Thy1 markers, percentage of purity of SSCs was about 84.3 and 97.4 % respectively. It was found that expression of differentiation markers after transfection was significantly higher in miRNA-30a- 5p inhibitor group compared to other groups. The results of proliferation markers evaluation also showed decrease of GFRA1, PLZF and ID4 protein in SSCs transfected with miRNA-30a-5p inhibitor compared to the other groups., Conclusions: It can be concluded that inhibition of miRNA-30a-5p by overexpression of differentiation markers promotes differentiation of Spermatogonial Stem Cells.