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Spermatogonial stem cell (SSC) technologies that are currently under clinical development to reverse human infertility hold the potential to be adapted and applied for the conservation of endangered and vulnerable wildlife species. The biobanking of testis tissue containing SSCs from wildlife species, aligned with that occurring in pediatric human patients, could facilitate strategies to improve the genetic diversity and fitness of endangered populations. Approaches to utilize these SSCs could include spermatogonial transplantation or testis tissue grafting into a donor animal of the same or a closely related species, or in vitro spermatogenesis paired with assisted reproduction approaches. The primary roadblock to progress in this field is a lack of fundamental knowledge of SSC biology in non-model species. Herein, we review the current understanding of molecular mechanisms controlling SSC function in laboratory rodents and humans, and given our particular interest in the conservation of Australian marsupials, use a subset of these species as a case-study to demonstrate gaps-in-knowledge that are common to wildlife. Additionally, we review progress in the development and application of SSC technologies in fertility clinics and consider the translation potential of these techniques for species conservation pipelines., (© The Author(s) 2024. Published by Oxford University Press behalf of Society for the Study of Reproduction.)

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.)

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).)

Platelet-Rich Plasma (PRP) is a regenerative therapy that has gained interest in recent years, being the subject of various studies and applications. In the field of human reproduction specifically, there are growth factors (GFs) present in PRP that have shown an impact on sperm quality and function. The main objective of this article is to conduct a literature review on the use of PRP for the treatment of male infertility. To achieve this, a bibliographic search of articles and reviews in scientific journals about the possible use of PRP for the treatment of male factor published between 2018 and 2023 has been carried out. Nine publications were identified, proposing the application of PRP in the following areas: sperm cryopreservation, oxidative stress, culture of spermatogonial stem cells, and secretory azoospermia. According to the literature, supplementing the cryopreservation medium with PRP improves sperm parameters, and samples incubated with PRP show an increase in their antioxidant capacity. Finally, PRP can increase the proliferation and vitality of spermatogonial stem cells and sperm recovery in cases of non-obstructive azoospermia. PRP shows therapeutic potential in various aspects of the male factor. Further research with a larger sample size is needed to define both its protocols and its clinical efficacy., Competing Interests: Los autores no declaran conflictos de intereses., (©2024 The Author(s). Published by MRE Press.)

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.)

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.)

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.

Spermatogonial stem cell (SSC) self-renewal is regulated by reciprocal interactions between Sertoli cells and SSCs in the testis. In a previous study, microtubule-associated serine/threonine kinase 4 (MAST4) has been studied in Sertoli cells as a regulator of SSC self-renewal. The present study focused on the mechanism by which MAST4 in Sertoli cells transmits the signal and regulates SSCs, especially cell cycle regulation. The expression of PLZF, CDK2 and PLZF target genes was examined in WT and Mast4 KO testes by Immunohistochemistry, RT-qPCR and western blot. In addition, IdU and BrdU were injected into WT and Mast4 KO mice and cell cycle of SSCs was analysed. Finally, the testis tissues were cultured in vitro to examine the regulation of cell cycle by MAST4 pathway. Mast4 KO mice showed infertility with Sertoli cell-only syndrome and reduced sperm count. Furthermore, Mast4 deletion led to decreased PLZF expression and cell cycle progression in the testes. MAST4 also induced cyclin-dependent kinase 2 (CDK2) to phosphorylate PLZF and activated PLZF suppressed the transcriptional levels of genes related to cell cycle arrest, leading SSCs to remain stem cell state. MAST4 is essential for maintaining cell cycle in SSCs via the CDK2-PLZF interaction. These results demonstrate the pivotal role of MAST4 regulating cell cycle of SSCs and the significance of spermatogenesis., (© 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Continuity of spermatogenesis in mammals is underpinned by spermatogenic (also called spermatogonial) stem cells (SSCs) that self-renew and differentiate into sperm that pass on genetic information to the next generation. Despite the fundamental role of SSCs, the mechanisms underlying SSC homeostasis are only partly understood. During homeostasis, the stem cell pool remains constant while differentiating cells are continually produced to replenish the lost differentiated cells. One of the outstanding questions here is how self-renewal and differentiation of SSCs are balanced to achieve a constant self-renewing pool. In this review, we shed light on the regulatory mechanism of SSC homeostasis, with focus on the recently proposed mitogen competition model in a facultative (or open) niche microenvironment.

Cytochrome P45011A1, encoded by Cyp11a1, converts cholesterol to pregnenolone (P5), the first and rate-limiting step in steroidogenesis. In zebrafish, cyp11a1 is maternally expressed and cyp11a2 is considered the ortholog of Cyp11a1 in mammals. A recent study has shown that depletion of cyp11a2 resulted in steroidogenic deficiencies and the mutants developed into males with feminized secondary sexual characteristics. Here, we independently generated cyp11a2 mutants in zebrafish and showed that the mutants can develop into males and females in the juvenile stage, but finally into infertile males with defective mating behavior in the adult stage. In the developing ovaries, the cyp11a2 mutation led to stage I oocyte apoptosis and final sex reversal, which could be partially rescued by treatment with P5 but not estradiol. In the developing testes, depletion of cyp11a2 resulted in dysfunction of Sertoli cells and lack of functional Leydig cells. Spermatogonial stem cells (SSCs) in the mutant testes underwent active self-renewal but no differentiation, resulting in a high abundance of SSCs in the testis, as revealed by immunofluorescence staining with Nanos2 antibody. The high abundance and differentiation competence of SSCs in the mutant testes were verified by a novel testicular cell transplantation method developed in this study, by transplanting mutant testicular cells into germline-depleted wild-type (WT) fish. The transplanted mutant SSCs efficiently differentiated into functional spermatids in WT hosts. Overall, our study demonstrates the functional importance of cyp11a2 in early oogenesis and differentiation of SSCs., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.)

The process of spermatogenesis is complex and systemic, requiring the cooperation of many regulators. However, little is known about how micro RNAs (miRNAs) regulate spermatogenesis in poultry. In this study, we investigated key miRNAs and their target genes that are involved in spermatogenesis in chickens. Next-generation sequencing was conducted to determine miRNA expression profiles in five cell types: primordial germ cells (PGCs), spermatogonial stem cells (SSCs), spermatogonia (Spa), and chicken sperm. Next, we analyzed and identified several key miRNAs that regulate spermatogenesis in the four germline cell miRNA profiles. Among the enriched miRNAs, miRNA-301a-5p was the key miRNA in PGCs, SSCs, and Spa. Through reverse transcription quantitative PCR (RT-qPCR), dual-luciferase, and miRNA salience, we confirmed that miR-301a-5p binds to transforming growth factor-beta 2 ( TGFβ2 ) and is involved in the transforming growth factor-beta (TGF-β) signaling pathway and germ cell development. To the best of our knowledge, this is the first demonstration of miR-301a-5p involvement in spermatogenesis by direct binding to TGFβ2 , a key gene in the TGF-β signaling pathway. This finding contributes to the insights into the molecular mechanism through which miRNAs regulate germline cell differentiation and spermatogenesis in chickens.

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.)

Spermatogonial stem cells (SSC), the foundation of spermatogenesis and male fertility, possess lifelong self-renewal activity. Aging leads to the decline in stem cell function and increased risk of paternal age-related genetic diseases. In the present study, we performed a comparative genomic analysis of mouse SSC-enriched undifferentiated spermatogonia (Oct4-GFP+/KIT-) and differentiating progenitors (Oct4-GFP+/KIT+) isolated from young and aged testes. Our transcriptome data revealed enormous complexity of expressed coding and non-coding RNAs and alternative splicing regulation during SSC differentiation. Further comparison between young and aged undifferentiated spermatogonia suggested these differentiation programs were affected by aging. We identified aberrant expression of genes associated with meiosis and TGF-β signaling, alteration in alternative splicing regulation and differential expression of specific lncRNAs such as Fendrr. Epigenetic profiling revealed reduced H3K27me3 deposition at numerous pro-differentiation genes during SSC differentiation as well as aberrant H3K27me3 distribution at genes in Wnt and TGF-β signaling upon aging. Finally, aged undifferentiated spermatogonia exhibited gene body hypomethylation, which is accompanied by an elevated 5hmC level. We believe this in-depth molecular analysis will serve as a reference for future analysis of SSC aging., Competing Interests: The authors have declared that no competing interests exist.

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.

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O 2 ) environment, whereas, their in-vitro expansion is typically performed under normoxia (20-21% O 2 ). The comparative information about the effects of low and normal O 2 levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O 2 ) and normoxia (21% O 2 ). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p < 0.05) improved, thus, yielding a significantly (p < 0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O 2 conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Gametogenesis is one of the most extreme cellular differentiation processes that takes place in Drosophila male and female germlines. This process begins at the germline stem cell, which undergoes asymmetric cell division (ACD) to produce a self-renewed daughter that preserves its stemness and a differentiating daughter cell that undergoes epigenetic and genomic changes to eventually produce haploid gametes. Research in molecular genetics and cellular biology are beginning to take advantage of the continually advancing genomic tools to understand: (1) how germ cells are able to maintain their identity throughout the adult reproductive lifetime, and (2) undergo differentiation in a balanced manner. In this review, we focus on the epigenetic mechanisms that address these two questions through their regulation of germline-soma communication to ensure germline stem cell identity and activity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) are essential to the initiation of spermatogenesis. Cryopreservation, long-term maintenance, and auto-transplantation of SSCs could be a new treatment for infertility. The aim of this study was to add melatonin to the basic freezing medium and to evaluate its effect on the efficiency of the thawed SSCs after transplantation into the testicles of azoospermic mice. SSCs were isolated from newborn NMRI mice, and the cells were enriched to assess morphological features. The thawed SSCs were evaluated for survival, apoptosis, and ROS level before transplantation, and the proliferation (MVH and ID4) and differentiation (c-Kit, SCP3, TP1, TP2, and Prm1) markers of SSCs were examined using immunofluorescence, western blot, and quantitative real-time polymerase chain reaction (PCR) after transplantation. It was found that the survival rate of SSCs after thawing was significantly higher in the melatonin group compared with the cryopreservation group containing basic freezing medium, and the rate of apoptosis and level of ROS production also decreased significantly in the cryopreservation group with melatonin (p < 0.05). The expression of proliferation and differentiation markers after transplantation was significantly higher in the cryopreservation group with melatonin compared to the cryopreservation group (p < 0.05). The results suggest that adding melatonin to the basic freezing medium can effectively protect the SSCs by increasing the viability and reducing the ROS production and apoptosis and improve the transplantation efficiency of SSCs after cryopreservation, which will provide a significant suggestion for fertility protection in the clinic.

Decellularized scaffolds have been found to be excellent platforms for tissue engineering applications. The attempts are still being made to optimize a decellularization protocol with successful removal of the cells with minimal damages to extracellular matrix components. We examined twelve decellularization procedures using different concentrations of Sodium dodecyl sulfate and Triton X-100 (alone or in combination), and incubation time points of 15 or 30 min. Then, the potential of the decellularized scaffold as a three-dimensional substrate for colony formation capacity of mouse spermatogonial stem cells was determined. The morphological, degradation, biocompatibility, and swelling properties of the samples were fully characterized. The 0.5%/30 SDS/Triton showed optimal decellularization with minimal negative effects on ECM (P ≤ 0.05). The swelling ratios increased with the increase of SDS and Triton concentration and incubation time. Only 0.5%/15 and 30 SDS showed a significant decrease in the SSCs viability compared with other groups (P < 0.05). The SSCs colony formation was clearly observed under SEM and H&E stained slides. The cells infiltrated into the subcutaneously implanted scaffold at days 7 and 30 post-implantation with no sign of graft rejection. Our data suggest the %0.5/30 SDS/Triton as an excellent platform for tissue engineering and reproductive biology applications.

Investigating the complex interactions between stem cells and their native environment requires an efficient means to image them in situ. Caenorhabditis elegans germline stem cells (GSCs) are distinctly accessible for intravital imaging; however, long-term image acquisition and analysis of dividing GSCs can be technically challenging. Here we present a systematic investigation into the technical factors impacting GSC physiology during live imaging and provide an optimized method for monitoring GSC mitosis under minimally disruptive conditions. We describe CentTracker, an automated and generalizable image analysis tool that uses machine learning to pair mitotic centrosomes and that can extract a variety of mitotic parameters rapidly from large-scale data sets. We employ CentTracker to assess a range of mitotic features in a large GSC data set. We observe spatial clustering of mitoses within the germline tissue but no evidence that subpopulations with distinct mitotic profiles exist within the stem cell pool. We further find biases in GSC spindle orientation relative to the germline's distal-proximal axis and thus the niche. The technical and analytical tools provided herein pave the way for large-scale screening studies of multiple mitotic processes in GSCs dividing in situ, in an intact tissue, in a living animal, under seemingly physiological conditions.

The microenvironment in the seminiferous tubules of buffalo changes with age, which affects the self-renewal and growth of spermatogonial stem cells (SSCs) and the process of spermatogenesis, but the mechanism remains to be elucidated. RNA-seq was performed to compare the transcript profiles of pre-pubertal buffalo (PUB) and adult buffalo (ADU) seminiferous tubules. In total, 17,299 genes from PUB and ADU seminiferous tubules identified through RNA-seq, among which 12,271 were expressed in PUB and ADU seminiferous tubules, 4,027 were expressed in only ADU seminiferous tubules, and 956 were expressed in only PUB seminiferous tubules. Of the 17,299 genes, we identified 13,714 genes that had significant differences in expression levels between PUB and ADU through GO enrichment analysis. Among these genes, 5,342 were significantly upregulated and possibly related to the formation or identity of the surface antigen on SSCs during self-renewal; 7,832 genes were significantly downregulated, indicating that genes in PUB seminiferous tubules do not participate in the biological processes of sperm differentiation or formation in this phase compared with those in ADU seminiferous tubules. Subsequently, through the combination with KEGG analysis, we detected enrichment in a number of genes related to the development of spermatogonial stem cells, providing a reference for study of the development mechanism of buffalo spermatogonial stem cells in the future. In conclusion, our data provide detailed information on the mRNA transcriptomes in PUB and ADU seminiferous tubules, revealing the crucial factors involved in maintaining the microenvironment and providing a reference for further in vitro cultivation of SSCs., (© 2021 Wiley-VCH GmbH.)

The spermatogonial stem cell (SSC) is a unique adult stem cell that requires tight physiological regulation during development and adulthood. As the foundation of spermatogenesis, SSCs are a potential tool for the treatment of infertility. Understanding the factors that are necessary for lifelong maintenance of a SSC pool in vivo is essential for successful in vitro expansion and safe downstream clinical usage. This review focused on the current knowledge of prepubertal testicular development and germ cell metabolism in different species, and implications for translational medicine. The significance of metabolism for cell biology, stem cell integrity, and fate decisions is discussed in general and in the context of SSC in vivo maintenance, differentiation, and in vitro expansion.

Spermatogonial stem cells (SSCs) sustain spermatogenesis by balancing self-renewal and initiation of differentiation to produce progenitor spermatogonia committed to forming sperm. To define the regulatory logic among SSCs and progenitors, we performed single-cell RNA velocity analyses and validated results in vivo. A predominant quiescent SSC population spawns a small subset of cell-cycle-activated SSCs via mitogen-activated protein kinase (MAPK)/AKT signaling. Activated SSCs form early progenitors and mTORC1 inhibition drives activated SSC accumulation consistent with blockade to progenitor formation. Mechanistically, mTORC1 inhibition suppresses transcription among spermatogonia and specifically alters expression of insulin growth factor (IGF) signaling in early progenitors. Tex14 -/- testes lacking intercellular bridges do not accumulate activated SSCs following mTORC1 inhibition, indicating that steady-state mTORC1 signaling drives activated SSCs to produce progenitor clones. These results are consistent with a model of SSC self-renewal dependent on interconversion between activated and quiescent SSCs, and mTORC1-dependent initiation of differentiation from SSCs to progenitor clones., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Most sturgeon and paddlefish are critically endangered; therefore, effective measures to conserve these genetic resources are required. Cryopreservation of gonad tissues containing germline stem cells could be an effective strategy for long term preservation and restoration of fish species using germ cell transplantation procedure. The aim of this study was to develop an optimal procedure for long-term cryopreservation of American paddlefish gonads using a slow-freezing method. Through optimization of permeating cryoprotectants, nonpermeating cryoprotectants, and supplementation of proteins, gonad tissues were frozen with a cryomedium containing 1.3 M dimethyl sulfoxide, 0.1 M trehalose, and 10 % fetal bovine serum at a cooling rate of -1 °C/min. This method was also successfully utilized for the cryopreservation of Yangtze sturgeon testes. Viability of gonadal cells isolated from frozen gonads was not different from cells isolated from fresh gonadal tissues, while the number of gonadal cells dissociated from frozen gonads was less. Germline stem cells dissociated from long-term (1 year) cryopreserved gonads were labeled with PKH26 fluorescent dye and intraperitoneally transplanted into larvae of Yangtze sturgeon. The colonization of transplanted germline stem cells was confirmed by the presence of PKH26-labeled donor germline stem cells and donor-derived mtDNA sequence in the recipient gonads, providing evidence that germline stem cells from sturgeon and paddlefish gonads that had been preserved for a long period maintained their functions. The results of present study indicate the procedures used are effective for long-term preservation of critically endangered species within the Acipenseriformes order which can later be regenerated using surrogate broodstock technology., (Copyright © 2020. Published by Elsevier B.V.)

Stem-cell niche signaling is short-range in nature, such that only stem cells but not their differentiating progeny receive self-renewing signals. At the apical tip of the Drosophila testis, 8 to 10 germline stem cells (GSCs) surround the hub, a cluster of somatic cells that organize the stem-cell niche. We have previously shown that GSCs form microtubule-based nanotubes (MT-nanotubes) that project into the hub cells, serving as the platform for niche signal reception; this spatial arrangement ensures the reception of the niche signal specifically by stem cells but not by differentiating cells. The receptor Thickveins (Tkv) is expressed by GSCs and localizes to the surface of MT-nanotubes, where it receives the hub-derived ligand Decapentaplegic (Dpp). The fate of Tkv receptor after engaging in signaling on the MT-nanotubes has been unclear. Here we demonstrate that the Tkv receptor is internalized into hub cells from the MT-nanotube surface and subsequently degraded in the hub cell lysosomes. Perturbation of MT-nanotube formation and Tkv internalization from MT-nanotubes into hub cells both resulted in an overabundance of Tkv protein in GSCs and hyperactivation of a downstream signal, suggesting that the MT-nanotubes also serve a second purpose to dampen the niche signaling. Together, our results demonstrate that MT-nanotubes play dual roles to ensure the short-range nature of niche signaling by (1) providing an exclusive interface for the niche ligand-receptor interaction; and (2) limiting the amount of stem cell receptors available for niche signal reception., Competing Interests: The authors declare no competing interests.

Mouse spermatogenesis is supported by spermatogenic stem cells (SSCs). SSCs maintain their pool while migrating over an open (or facultative) niche microenvironment of testicular seminiferous tubules, where ligands that support self-renewal are likely distributed widely. This contrasts with the classic picture of closed (or definitive) niches in which stem cells are gathered and the ligands are highly localized. Some of the key properties observed in the dynamics of SSCs in the testicular niche in vivo, which show the flexible and stochastic (probabilistic) fate behaviors, are found to be generic for a wide range of, if not all, tissue stem cells. SSCs also show properties characteristic of an open niche-supported system, such as high motility. Motivated by the properties of SSCs, in this review, I will reconsider the potential unity and diversity of tissue stem cell systems, with an emphasis on the varying degrees of ligand distribution and stem cell motility., (Copyright © 2020 Cold Spring Harbor Laboratory Press; all rights reserved.)

NOD-like receptors (NLRs) are traditionally recognized as major inflammasome components. The role of NLRs in germ cell differentiation and reproduction is not known. Here, we identified the gonad-specific Nlrp14 as a pivotal regulator in primordial germ cell-like cell (PGCLC) differentiation in vitro. Physiologically, knock out of Nlrp14 resulted in reproductive failure in both female and male mice. In adult male mice, Nlrp14 knockout (KO) inhibited differentiation of spermatogonial stem cells (SSCs) and meiosis, resulting in trapped SSCs in early stages, severe oligozoospermia, and sperm abnormality. Mechanistically, NLRP14 promoted spermatogenesis by recruiting a chaperone cofactor, BAG2, to bind with HSPA2 and form the NLRP14-HSPA2-BAG2 complex, which strongly inhibited ChIP-mediated HSPA2 polyubiquitination and promoted its nuclear translocation. Finally, loss of HSPA2 protection and BAG2 recruitment by NLRP14 was confirmed in a human nonsense germline variant associated with male sterility. Together, our data highlight a unique proteasome-mediated, noncanonical function of NLRP14 in PGCLC differentiation and spermatogenesis, providing mechanistic insights of gonad-specific NLRs in mammalian germline development., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Temperature affects animal physiology, including aging and lifespan. How temperature and biological systems interact to influence aging and lifespan has been investigated using model organisms, including the nematode Caenorhabditis elegans . In this review, we discuss mechanisms by which diverse cellular factors modulate the effects of ambient temperatures on aging and lifespan in C. elegans. C. elegans thermosensory neurons alleviate lifespan-shortening effects of high temperatures via sterol endocrine signaling and probably through systemic regulation of cytosolic proteostasis. At low temperatures, C. elegans displays a long lifespan by upregulating the cold-sensing TRPA channel, lipid homeostasis, germline-mediated prostaglandin signaling, and autophagy. In addition, co-chaperone p23 amplifies lifespan changes affected by high and low temperatures. Our review summarizes how external temperatures modulate C. elegans lifespan and provides information regarding responses of biological processes to temperature changes, which may affect health and aging at an organism level.

Juvenile hormone (JH) signaling plays crucial roles in insect metamorphosis and reproduction. Function of JH signaling in germline stem cells (GSCs) remains largely unknown. Here, we found that the number of GSCs significantly declined in the ovaries of Met, Gce and JHAMT mutants. Then we inhibited JH signaling in selected cell types of ovaries by expressing Met and Gce or Kr-h1 double-stranded RNAs (dsRNAs) using different Gal4 drivers. Blocking of JH signaling in muscle cells has no effect on GSC numbers. Blocking of JH signaling in cap cells reduced GSCs cells. Inductive expression of Met and Gce dsRNA but not Kr-h1 by Nos-Gal4 increased GSC cells. These results indicate that JH signaling plays an important role in GSC maintenance., (© 2019 Institute of Zoology, Chinese Academy of Sciences.)

Sox2 is an embryonal stem cell Ag essential for early embryonic development, tissue homeostasis and immune regulation. In the current study, one complete Sox2 cDNA sequence was cloned from freshwater bivalve Anodonta woodiana and named AwSox2. Histological changes of testis derived from Bisphenol A (BPA) treatment were analyzed by hematoxylin and eosin staining. Expressions of AwSox2 derived from BPA, LPS and polyinosinic:polycytidylic (Poly I:C) challenge were measured by quantitative real-time PCR. The full-length cDNA of AwSox2 contained an open reading frame of 927 nucleotides bearing the typical structural features of Sox2 family. Obvious degeneration, irregular arrangement of spermatids, and clotted dead and intertwined spermatids were observed in BPA-treated groups. Administration of BPA could result in a dose-dependent up-regulation of AwSox2 expression in the male gonadal tissue of A. woodiana . In addition, expression of AwSox2 was significantly induced by LPS and Poly I:C treatment in the hepatopancreas, gill and hemocytes, compared with that of control group. These results indicated that up-regulations of AwSOx2 are closely related to apoptosis of spermatogonial stem cells derived from BPA treatment as well as enhancement of immune defense against LPS and Poly I:C challenge in A. woodiana .

Infertility caused by chemotherapy or radiation treatments negatively impacts patient-survivor quality of life. The only fertility preservation option available to prepubertal boys who are not making sperm is cryopreservation of testicular tissues that contain spermatogonial stem cells (SSCs) with potential to produce sperm and/or restore fertility. SSC transplantation to regenerate spermatogenesis in infertile adult survivors of childhood cancers is a mature technology. However, the number of SSCs obtained in a biopsy of a prepubertal testis may be small. Therefore, methods to expand SSC numbers in culture before transplantation are needed. Here we review progress with human SSC culture., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) are a rare group of cells in the testis that undergo self-renewal and complex sequences of differentiation to initiate and sustain spermatogenesis, to ensure the continuity of sperm production throughout adulthood. The difficulty of unequivocal identification of SSCs and complexity of replicating their differentiation properties in vitro have prompted the introduction of novel in vivo models such as germ cell transplantation (GCT), testis tissue xenografting (TTX), and testis cell aggregate implantation (TCAI). Owing to these unique animal models, our ability to study and manipulate SSCs has dramatically increased, which complements the availability of other advanced assisted reproductive technologies and various genome editing tools. These animal models can advance our knowledge of SSCs, testis tissue morphogenesis and development, germ-somatic cell interactions, and mechanisms that control spermatogenesis. Equally important, these animal models can have a wide range of experimental and potential clinical applications in fertility preservation of prepubertal cancer patients, and genetic conservation of endangered species. Moreover, these models allow experimentations that are otherwise difficult or impossible to be performed directly in the target species. Examples include proof-of-principle manipulation of germ cells for correction of genetic disorders or investigation of potential toxicants or new drugs on human testis formation or function. The primary focus of this review is to highlight the importance, methodology, current and potential future applications, as well as limitations of using these novel animal models in the study and manipulation of male germline stem cells.

The objective of this study was to investigate effects of selenium (Se) on proliferation and apoptosis of sheep spermatogonial stem cells (SSC) in vitro. The SSC were assigned to five treatment groups (0, 2.0, 4.0, 8.0 and 16.0 μmol/L Se). After treatment with Se for 96 h, cell proliferation and apoptosis were evaluated. The relative abundance of P53 mRNA transcript and protein, cell cycle and apoptosis-related genes were detected using real-time PCR and Western blot quantifications, respectively. The results indicate there were the least cell proliferation rates in the Se 16.0 group. Treatments with relatively greater Se concentrations (8.0 and 16.0 μmol/L) resulted in a greater percentage of apoptotic cells, which was consistent with the relative abundances of P53, P21, P27 and pro-apoptosis mRNA transcripts. There were relatively greater ROS concentrations in the control, Se 8.0 and Se 16.0 groups. Compared with the control group, treatment with the Se concentration of 16.0 μmol/L resulted in an increased abundance of P53, P21, P27 and BAX proteins. Treatment with Pifithrin-α suppressed the increase in abundance of P53 and P21 proteins induced by the relatively greater concentration of Se (16.0 μmol/L), however, did not result in a change in abundances of P27 and BAX proteins. These results indicate the regulatory functions of Se on proliferation and apoptosis of sheep SSC is associated with the P21-mediated P53 signalling pathway. The P27 and BAX proteins have limited functions during the apoptotic process of SSC induced by the relatively greater concentrations of Se., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the effects of different oxygen (O 2 ) concentrations on the growth of mouse spermatogonial stem cells (SSCs) and the possible mechanisms of cell proliferation in vitro. The SSCs from testicular cells were cultured in various O 2 concentrations (1%, 2.5%, 5%, and 20% O 2 ) for 7 days. Colonies of SSCs were identified morphologically and by immunofluorescence. The number of mouse SSC colonies and the area covered by them were measured. Cell cycle progression of the SSCs was analyzed to identify the state of cell proliferation. The effects of O 2 concentrations on the levels of intracellular reactive oxygen species (ROS) and expression of ATP binding cassette subfamily G member 2 (ABCG2) were also analyzed in the SSCs. Following culturing for 7 days, the SSCs were treated with Ko143 (a specific inhibitor of ABCG2) for 1 h, and the ROS level and expression of bcl-2, bax, and p53 were analyzed. The results showed that mouse SSCs formed compact colonies and had unclear borders in different O 2 concentrations for 7 days, and there were no major morphologic differences between the O 2 treatment groups. The expression of the SSC marker, GFR α1 was studied in each O 2 treatment group. The number and area of SSC colonies, and the number of GFR α1 positive cells were the highest in the 2.5% O 2 treatment group. Compared with other O 2 concentrations, the number of cells in G 0 cycle was significantly higher, while the level of intracellular ROS was lower at 1% O 2 . Moreover, the intracellular ROS levels gradually increased with increasing O 2 concentration from 1% to 20%. The expression of ABCG2 in the SSCs cultured at 2.5% O 2 was higher than in the other O 2 groups. Inhibition of ABCG2 increased intracellular ROS generation, and the expression of the pro-apoptotic genes bax and p53, and decreased the expression of the anti-apoptotic gene bcl-2. In conclusion, moderate to low O 2 tension increases ABCG2 expression to maintain mild ROS levels, triggers the expression of the anti-apoptotic genes, suppresses the proapoptotic gene pathway, and further promotes the proliferation of mouse SSCs in vitro., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Currently, the systems for culturing buffalo spermatogonial stem cells (SSCs) in vitro are varied, and their effects are still inconclusive. In this study, we compared the effects of culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system ( p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 ( p < 0.05), DDX4 ( p < 0.01) and UCHL1 ( p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3⁺ subset in the cells cultured in the defined materials system. The expression levels of Stra8 ( p < 0.05) and Rec8 ( p < 0.01) were significantly increased, and the expression levels of ZBTB16 ( p < 0.01) and DDX4 ( p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro ., Competing Interests: The authors have no conflicts of interest to declare., (© 2020 The Korean Society of Veterinary Science.)

Genetic causes of male infertility that is associated with aging are largely unknown. This study was designed to identify novel pathogenic variants of FOXP3 gene causing azoospermia. One homozygous (c.155 G > T) pathogenic variant of FOXP3 was identified in nine non-obstructive azoospermia patients, and one heterozygous (c.691 C > A) of FOXP3 was found in one non-obstructive azoospermia patient. Pedigrees studies indicated that the homozygous (c.155 G > T) FOXP3 pathogenic variant was inherited, while heterozygous (c.691 C > A) FOXP3 pathogenic variant was acquired. Human testis carrying pathogenic variant exhibited abnormal spermatogenesis. FOXP3 protein was expressed at a lower level or undetected in spermatocytes of mutant testis of non-obstructive azoospermia patients compared to obstructive azoospermia patients. FOXP3 stimulated the proliferation and inhibited the apoptosis of human spermatogonial stem cells, and we further analyzed the targets of FOXP3. We have identified two new pathogenic variants of FOXP3 in non-obstructive azoospermia patients with high incidence, and FOXP3 silencing inhibits the proliferation and enhances the apoptosis of human spermatogonial stem cells. This study provides new insights into the etiology of azoospermia and offers novel pathogenic variants for gene targeting of male infertility.

The current strategy to preserve fertility of male prepubertal cancer patients consists of cryopreservation of a testicular tissue biopsy containing spermatogonial stem cells (SSCs). While in humans, fertility restoration strategies from prepubertal testicular tissues are still under investigation and have not yet resulted in complete germ cell differentiation, in mice various studies have described production of sperm and offspring through testicular organ culture and transplantation of in vitro propagated SSCs. Organ culture has shown to be successful in generating mature spermatozoa when using testicular fragments from various mouse strains, including CD1 and C57BL/6 J. Conversely, in vitro proliferation of SSCs from C57BL/6 J mice is highly inefficient when compared to other strains such as DBA2 or hybrid mice of C57BL/6 J and DBA2 with 75% C57BL/6 J background (B6D2F2). In this study, we investigated in vitro spermatogenesis by organ culture using testicular tissue from C57BL/6 J and B6D2F2 mice. Whereas spermatogenesis was initiated and completed in C57BL/6 J fragments, it could not be effectively supported in B6D2F2 testicular tissue. While maturation of Sertoli cells and Leydig cells functionality appeared to be identical between the two strains, in B6D2F2 tissue spermatogenesis did not proceed past the spermatocyte step, followed by a rapid decline of the number of all germ cells in the fragments. This suggests that the spermatogenic potential in vitro is dependent on specialized sites in the genome and therefore the organ culture conditions suboptimal for some strains of mice., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The intricate molecular and cellular interactions between spermatogonial stem cells (SSCs) and their cognate niche form the basis for life-long sperm production. To maintain long-term fertility and sustain sufficiently high levels of spermatogenesis, a delicate balance needs to prevail between the different niche factors that control cell fate decisions of SSCs by promoting self-renewal, differentiation priming or spermatogenic commitment of undifferentiated spermatogonia (Aundiff). Previously the SSC niche was thought to be formed primarily by Sertoli cells. However, recent research has indicated that many distinct cell types within the testis contribute to the SSC niche including most somatic cell populations and differentiating germ cells. Moreover, postnatal testis development involves maturation of somatic supporting cell populations and onset of cyclic function of the seminiferous epithelium. The stochastic and flexible behavior of Aundiff further complicates the definition of the SSC niche. Unlike in invertebrate species, providing a simple anatomical description of the SSC niche in the mouse is therefore challenging. Rather, the niche needs to be understood as a dynamic system that is able to serve the long-term reproductive function and maintenance of fertility both under steady-state and during development plus regeneration. Recent data from us and others have also shown that Aundiff reversibly transition between differentiation-primed and self-renewing states based on availability of niche-derived cues. This review focuses on defining the current understanding of the SSC niche and the elements involved in its regulation.

In vitro gamete differentiation could revolutionize animal production by decreasing generation intervals, increasing the number of gametes per animal and facilitating the dissemination of elite genetics. In addition, it could help to develop new strategies for the conservation of endangered species. The recent in vitro reconstitution of germ cell development in mice has inspired researchers to invest their best efforts into reproducing this achievement in livestock species. With this goal in mind, multiple differentiation approaches and cell sources have been evaluated. The degree of success in these evaluations varies according to the species and the stage of development studied, but, in general, partially positive results have been obtained. Evidence suggests that although functional gametes with true reproductive potential are still to be obtained, it is a matter of time before this goal is achieved., (© 2019 Blackwell Verlag GmbH.)

Background: Male infertility is a serious social problem in modern society. Nonobstructive azoospermia (NOA) caused by germ cell gene defects is an important reason for male infertility, but effective clinical treatment for this disease has not been established., Methods: We choose Kit w /Kit wv mouse as a research model and try to develop a new treatment strategy and "cure" its infertility. Mutant spermatogonial stem cells (SSCs) were isolated from one single unilateral testis of a 14-day-old Kit w /Kit wv mouse and propagated in vitro. The C to T point mutation on Kit wv site of these SSCs was corrected through CRISPR-Cas9-mediated homology-directed repair (HDR) in vitro. Then, the repaired SSCs were screened out, proliferated, and transplanted into the remaining testis, and complete spermatogenesis was established in the recipient testis., Results: Healthy offsprings with wild type Kit gene or Kit w mutation were obtained through natural mating 4 months after SSC transplantation., Conclusion: In this study, we established an effective new treatment strategy for NOA caused by germ cell gene defects through a combination of SSC isolation, CRISPR-Cas9-mediated gene editing, and SSC transplantation, which brought hope for these NOA patients to restore their natural fertility.

Keeping the self-renewal and differentiation of spermatogonial stem cell (SSC) in balance is essential for maintaining spermatogenesis. However, whether the cell death of SSC also plays a vital role in the human being remains unknown. To explore the necroptosis of SSC, the activation marker of necroptosis, phosphorylated mixed lineage kinase domain-like protein (pMLKL) in testes was evaluated by immunofluorescence staining and Western blot. Meanwhile, a total of 81 semen samples were divided based on the sperm concentration (>15, 10-15, 5-10 and 0-5 million/ml) to study the relationships between pMLKL levels and sperm counts. We found that the pMLKL was increased in the ageing human testes (p < 0.05). Moreover, the seminal pMLKL expression was decreased in groups with sperm concentration 0-5 and 5-10 million/ml when compared with normal sperm concentration in young men (p < 0.05). Further analysis revealed that pMLKL showed an age-related increased expression in men aged 22-60 years with normal sperm concentration. These data demonstrated that the necroptosis of SSC was important for the spermatogenic function and would raise in advance on the point of testicular hypofunction. In conclusion, the pMLKL may serve as a potential biologically seminal indicator for the spermatogenic function in men., (© 2019 Blackwell Verlag GmbH.)

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in male due to their capability to multiply in numbers by self-renewal and subsequent meiotic processes. However, as SSCs are present in a very small proportion in the testis, in vitro proliferation of undifferentiated SSCs will facilitate the study of germ cell biology. In this study, we investigated the effectiveness of various cell lines as a feeder layer for rat SSCs. Germ cells enriched for SSCs were cultured on feeder layers including SIM mouse embryo-derived thioguanine and ouabain-resistant cells, C166 cells, and mouse and rat testicular endothelial cells (TECs) and their stem cell potential for generating donor-derived colonies and offspring was assessed by transplantation into recipient testes. Rat germ cells cultured on TECs showed increased mRNA and protein levels of undifferentiated spermatogonial markers. Rat SSCs derived from these germ cells underwent spermatogenesis and generated offspring when transplanted into recipients. Collectively, TECs can serve as an effective feeder layer that enhances the proliferative and self-renewal capacity of cultured rat SSCs while preserving their stemness properties., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

The identification system of spermatogonial stem cell (SSC) was established in alpaca using the molecular expression as well as the reactivity pattern to Dolichos biflorus agglutinin (DBA) by flow cytometry. Twenty-four testicles with their epididymis were recovered from adult alpacas at the slaughterhouse of Huancavelica-Perú. Samples were transported to the Laboratory of Reproductive Physiology at Universidad Nacional Mayor de San Marcos. Testes were selected for our study when the progressive motility of epididymal spermatozoa (ESPM) was above 30%. Isolation of SSC was performed with two enzymatic digestions. Finally, sperm viability was evaluated by means of the trypan blue vital stain in spermatogonial round cells. Samples with more than 80% viability were selected. Isolated cells cultured for 2 days were used for identifying the presence of SSCs by the expression of integrin β1 (116 bp) and PLZF (206 bp) genes. Spermatogonia were classified according to the DBA reactivity. Spermatogonia with a strong positive to DBA (sDBA + ) were classified as SSC (Mean ± SEM=4.44 ± 0.68%). Spermatogonia in early differentiation stages stained weakly positive with DBA (wDBA + ) (Mean ± SEM=37.44 ± 3.07%) and differentiated round cells as DBA negative (Mean ± SEM=54.12 ± 3.18%). With the use of molecular and DBA markers, it is possible to identify easily the spermatogonial stem cells in alpaca., (© 2019 Blackwell Verlag GmbH.)

Spermatogonial stem cell (SSC) is known for its self-renewal capacity. We have studied the in vitro proliferation of isolated SSC from adult alpaca (Vicugna pacos) testes. A total of 107 samples were evaluated of which 31 were evaluated at baseline, 36 were cultivated in DMEM and 40 in STEMPRO media. Half of the cultivated samples was analysed after 14 days, and the rest after 21 days. Round cell subpopulations were identified with FITC-DBA by flow cytometry: strongly positive DBA (sDBA+) as SSC, weakly positive DBA (wDBA+) as in early differentiation and negative DBA as differentiated. At the beginning, 4.16% of the cells were SSC, 37.61% wDBA+ while 54.12% were DBA-. After 14 days, 42.28% of SSC, 44.68% wDBA+ and 11.07% DBA- were found in DMEM while 47.09% of SSC, 32.57% wDBA+ and 18.48% DBA- in STEMPRO. After 21 days 38.66% were SSC, 52.78% wDBA and 7.65% DBA- in DMEM and on STEMPRO media 47.92% SSC, 44.20% wDBA+, 4.93% DBA-. There is a significant difference between the number of initial and SSC cultivated, as well as between DBA- (p < 0.05), while there is no significant difference between the wDBA+ (p > 0.05). Our results suggest that both culture media are appropriate for the in vitro proliferation of alpacas SSC., (© 2019 Blackwell Verlag GmbH.)

Continuity, robustness, and regeneration of cell lineages relies on stem cell pools that are established during development. For the mammalian spermatogenic lineage, a foundational spermatogonial stem cell (SSC) pool arises from prospermatogonial precursors during neonatal life via mechanisms that remain undefined. Here, we mapped the kinetics of this process in vivo using a multi-transgenic reporter mouse model, in silico with single-cell RNA sequencing, and functionally with transplantation analyses to define the SSC trajectory from prospermatogonia. Outcomes revealed that a heterogeneous prospermatogonial population undergoes dynamic changes during late fetal and neonatal development. Differential transcriptome profiles predicted divergent developmental trajectories from fetal prospermatogonia to descendant postnatal spermatogonia. Furthermore, transplantation analyses demonstrated that a defined subset of fetal prospermatogonia is fated to function as SSCs. Collectively, these findings suggest that SSC fate is preprogrammed within a subset of fetal prospermatogonia prior to building of the foundational pool during early neonatal development.

During suboptimal growth conditions, Caenorhabditis elegans larvae undergo a global developmental arrest called "dauer." During this stage, the germline stem cells (GSCs) become quiescent in an AMP-activated Protein Kinase (AMPK)-dependent manner, and in the absence of AMPK, the GSCs overproliferate and lose their reproductive capacity, leading to sterility when mutant animals resume normal growth. These defects correlate with the altered abundance and distribution of a number of chromatin modifications, all of which can be corrected by disabling components of the endogenous small RNA pathway, suggesting that AMPK regulates germ cell integrity by targeting an RNA interference (RNAi)-like pathway during dauer. The expression of AMPK in somatic cells restores all the germline defects, potentially through the transmission of small RNAs. Our findings place AMPK at a pivotal position linking energy stress detected in the soma to a consequent endogenous small RNA-mediated adaptation in germline gene expression, thereby challenging the "permeability" of the Weismann barrier., Competing Interests: The authors have declared that no competing interests exist.

Spermatogonial stem cells (SSCs) are adult stem cells that are slowly cycling and self-renewing. The pool of SSCs generates very large numbers of male gametes throughout the life of the individual. SSCs can be cultured in vitro for long periods of time, and established SSC lines can be manipulated genetically. Upon transplantation into the testes of infertile mice, long-term cultured mouse SSCs can differentiate into fertile spermatozoa, which can give rise to live offspring. Here, we show that the testicular soma of mice with a conditional knockout (conKO) in the X-linked gene Tsc22d3 supports spermatogenesis and germline transmission from cultured mouse SSCs upon transplantation. Infertile males were produced by crossing homozygous Tsc22d3 floxed females with homozygous ROSA26-Cre males. We obtained 96 live offspring from six long-term cultured SSC lines with the aid of intracytoplasmic sperm injection. We advocate the further optimization of Tsc22d3-conKO males as recipients for testis transplantation of SSC lines., (© 2019 The Authors. Genesis published by Wiley Periodicals, Inc.)

Accumulating evidence indicates that paternal age correlates with disease risk in children. De novo gain-of-function mutations in the FGF-RAS-MAPK signaling pathway are known to cause a subset of genetic diseases associated with advanced paternal age, such as Apert syndrome, achondroplasia, Noonan syndrome, and Costello syndrome. It has been hypothesized that adult spermatogonial stem cells with pathogenic mutations are clonally expanded over time and propagate the mutations to offspring. However, no model system exists to interrogate mammalian germline stem cell competition in vivo. In this study, we created a lineage tracing system, which enabled undifferentiated spermatogonia with endogenous expression of HrasG12V, a known pathogenic gain-of-function mutation in RAS-MAPK signaling, to compete with their wild-type counterparts in the mouse testis. Over a year of fate analysis, neither HrasG12V-positive germ cells nor sperm exhibited a significant expansion compared to wild-type neighbors. Short-term stem cell capacity as measured by transplantation analysis was also comparable between wild-type and mutant groups. Furthermore, although constitutively active HRAS was detectable in the mutant cell lines, they did not exhibit a proliferative advantage or an enhanced response to agonist-evoked pERK signaling. These in vivo and in vitro results suggest that mouse spermatogonial stem cells are functionally resistant to a heterozygous HrasG12V mutation in the endogenous locus and that mechanisms could exist to prevent such harmful mutations from being expanded and transmitted to the next generation., Competing Interests: The authors have declared that no competing interests exist.

The properties and behaviour of stem cells rely heavily on signaling from the local microenvironment. At the apical end of Drosophila testis, self-renewal and differentiation of germline stem cells (GSCs) are tightly controlled by distinct somatic cells that comprise a specialised stem cell niche known as the hub. The hub maintains GSC homeostasis through adhesion and cell signaling. The Salvador/Warts/Hippo (SWH) pathway, which suppresses the transcriptional co-activator YAP/Yki via a kinase cascade, is a known regulator of stem cell proliferation and differentiation. Here, we show that increasing YAP/Yki expression in the germline, as well as reducing Warts levels, blocks the decrease of GSC numbers observed in aging flies, with only a small increase on their proliferation. An increased expression of YAP/Yki in the germline or a reduction in Warts levels also stymies an age-related reduction in hub cell number, suggesting a bilateral relationship between GSCs and the hub. Conversely, RNAi-based knockdown of YAP/Yki in the germline leads to a significant drop in hub cell number, further suggesting the existence of such a SC-to-niche relationship. All together, our data implicate the SWH pathway in Drosophila GSC maintenance and raise questions about its role in stem cell homeostasis in aging organisms., Competing Interests: The authors have declared that no competing interests exist.