Spermatogonial Stem Cells Function Research Articles

Functionally redundant roles of ID family proteins in spermatogonial stem cells

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.

Spermatogonial stem cell technologies: applications from human medicine to wildlife conservation†.

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.

Retraction: Cellular functions of spermatogonial stem cells in relation to JAK/STAT signaling pathway

Retraction: Cellular functions of spermatogonial stem cells in relation to JAK/STAT signaling pathway

RETRACTED: Cellular functions of spermatogonial stem cells in relation to JAK/STAT signaling pathway

This manuscript comprehensively reviews the interrelationship between spermatogonial stem cells (SSCs) and the JAK/STAT signaling pathway. Spermatogonial stem cells in the testes of male mammals, characterized by their self-renewal and pluripotential differentiation capabilities, are essential for tissue regeneration, immunomodulation, and advancements in regenerative medicine. This review delves into the historical background and biological characteristics of SSCs, with a particular emphasis on the pivotal role of the JAK/STAT signaling pathway in their proliferation, maturation, and differentiation processes. Research indicates that the JAK/STAT pathway extensively influences various functionalities of spermatogonial stem cells, encompassing immunomodulation, tissue differentiation, homing, and adaptation to the microenvironment. Herein, we collate and dissect related studies, shedding light on the intricate dynamics between SSCs and the JAK/STAT signaling pathway, and examine the implications of these interactions on the biological attributes and functionalities of SSCs. Furthermore, the review discusses the profound implications of these findings for preclinical research and the domain of cellular engineering. It is acknowledged that, despite advancements in the research of SSCs and the JAK/STAT signaling pathway, investigations in humans and larger mammals remain inadequate, necessitating more in-depth exploration to establish a comprehensive theoretical framework. Overall, this review offers an invaluable reference for deciphering the mechanisms of the spermatogonial stem cell signaling pathways and establishes a theoretical groundwork for related preclinical research.

The hypoxia-inducible factor EPAS1 is required for spermatogonial stem cell function in regenerative conditions

The hypoxia-inducible factor EPAS1 is required for spermatogonial stem cell function in regenerative conditions

Spermatogonial fate in mice with increased activin A bioactivity and testicular somatic cell tumours.

Adult male fertility depends on spermatogonial stem cells (SSCs) which undergo either self-renewal or differentiation in response to microenvironmental signals. Activin A acts on Sertoli and Leydig cells to regulate key aspects of testis development and function throughout life, including steroid production. Recognising that activin A levels are elevated in many pathophysiological conditions, this study investigates effects of this growth factor on the niche that determines spermatogonial fate. Although activin A can promote differentiation of isolated spermatogonia in vitro, its impacts on SSC and spermatogonial function in vivo are unknown. To assess this, we examined testes of Inha KO mice, which feature elevated activin A levels and bioactivity, and develop gonadal stromal cell tumours as adults. The GFRA1+ SSC-enriched population was more abundant and proliferative in Inha KO compared to wildtype controls, suggesting that chronic elevation of activin A promotes a niche which supports SSC self-renewal. Intriguingly, clusters of GFRA1+/EOMES+/LIN28A- cells, resembling a primitive SSC subset, were frequently observed in tubules adjacent to tumour regions. Transcriptional analyses of Inha KO tumours, tubules adjacent to tumours, and tubules distant from tumour regions revealed disrupted gene expression in each KO group increased in parallel with tumour proximity. Modest transcriptional changes were documented in Inha KO tubules with complete spermatogenesis. Importantly, tumours displaying upregulation of activin responsive genes were also enriched for factors that promote SSC self-renewal, including Gdnf, Igf1, and Fgf2, indicating the tumours generate a supportive microenvironment for SSCs. Tumour cells featured some characteristics of adult Sertoli cells but lacked consistent SOX9 expression and exhibited an enhanced steroidogenic phenotype, which could arise from maintenance or acquisition of a fetal cell identity or acquisition of another somatic phenotype. Tumour regions were also heavily infiltrated with endothelial, peritubular myoid and immune cells, which may contribute to adjacent SSC support. Our data show for the first time that chronically elevated activin A affects SSC fate in vivo. The discovery that testis stromal tumours in the Inha KO mouse create a microenvironment that supports SSC self-renewal but not differentiation offers a strategy for identifying pathways that improve spermatogonial propagation in vitro.

Lineage Tracing of Spermatogonial Stem Cells Within the Male Germline.

Spermatogonial stem cells (SSCs) are the fundamental units from which continuous spermatogenesis arises. Although our knowledge regarding the basic properties of SSCs has grown, driven primarily through the advancement of techniques and technologies to study SSCs, the mechanisms controlling their fate remain largely unknown. Among the modern strategies to evaluate SSCs, lineage tracing is among the few established approaches that allow for functional assessment of stem cell capacity. As a result, lineage tracing continues to forge new discoveries underlying the basic attributes of SSCs as well as the molecular factors that govern SSC function. Traditional approaches to lineage tracing with dyes or radioactive labels suffer from progressive loss after successive cell divisions or unintentional label transfer to neighboring cells. To address these limitations, genetic approaches primarily leveraging transgenic technologies have prevailed as the preferred avenue for modern lineage tracing. This chapter will discuss current protocols for effective genetic lineage tracing and address applications of this technology, considerations when designing lineage tracing experiments, and the methods involved in utilizing lineage tracing to study SSCs and other cell populations.

Screening of Potential Plasticizer Alternatives for Their Toxic Effects on Male Germline Stem Cells.

Plasticizers give flexibility to a wide range of consumer and medical plastic products. Among them, phthalate esters are recognized as endocrine disruptors that target male reproductive functions. With this notion, past studies designed and produced alternative plasticizers that could replace phthalates with limited toxicity to the environment and to male reproductive functions. Here, we focused on one reproductive cell type that was not investigated in past studies-spermatogonial stem cells (SSCs)-and examined in vitro the effects on 22 compounds (seven plasticizers currently in use and 15 newly synthesized potential alternative plasticizers) for their effects on SSCs. Our in vitro compound screening analyses showed that a majority of the compounds examined had a limited level of toxicity to SSCs. Yet, some commercial plasticizers and their derivatives, such as DEHP (di-(2-ethylhexyl) phthalate) and MEHP (mono-(2-ethylhexyl) phthalate), were detrimental at 10-5 to 10-4 M. Among new compounds, some of maleate- and fumarate-derivatives showed toxic effects. In contrast, no detrimental effects were detected with two new compounds, BDDB (1,4 butanediol dibenzoate) and DOS (dioctyl succinate). Furthermore, SSCs that were exposed to BDDB and DOS in vitro successfully established spermatogenic colonies in testes of recipient mice after transplantation. These results demonstrate that SSC culture acts as an effective platform for toxicological tests on SSC function and provide novel information that two new compounds, BDDB and DOS, are alternative plasticizers that do not have significant negative impacts on SSC integrity.

Metabolic transitions define spermatogonial stem cell maturation.

Do spermatogonia, including spermatogonial stem cells (SSCs), undergo metabolic changes during prepubertal development? Here, we show that the metabolic phenotype of prepubertal human spermatogonia is distinct from that of adult spermatogonia and that SSC development is characterized by distinct metabolic transitions from oxidative phosphorylation (OXPHOS) to anaerobic metabolism. Maintenance of both mouse and human adult SSCs relies on glycolysis, while embryonic SSC precursors, primordial germ cells (PGCs), exhibit an elevated dependence on OXPHOS. Neonatal porcine SSC precursors reportedly initiate a transition to an adult SSC metabolic phenotype at 2 months of development. However, when and if such a metabolic transition occurs in humans is ambiguous. To address our research questions: (i) we performed a meta-analysis of publicly available and newly generated (current study) single-cell RNA sequencing (scRNA-Seq) datasets in order to establish a roadmap of SSC metabolic development from embryonic stages (embryonic week 6) to adulthood in humans (25 years of age) with a total of ten groups; (ii) in parallel, we analyzed single-cell RNA sequencing datasets of isolated pup (n = 3) and adult (n = 2) murine spermatogonia to determine whether a similar metabolic switch occurs; and (iii) we characterized the mechanisms that regulate these metabolic transitions during SSC maturation by conducting quantitative proteomic analysis using two different ages of prepubertal pig spermatogonia as a model, each with four independently collected cell populations. Single testicular cells collected from 1-year, 2-year and 7-year-old human males and sorted spermatogonia isolated from 6- to 8-day (n = 3) and 4-month (n = 2) old mice were subjected to scRNA-Seq. The human sequences were individually processed and then merged with the publicly available datasets for a meta-analysis using Seurat V4 package. We then performed a pairwise differential gene expression analysis between groups of age, followed by pathways enrichment analysis using gene set enrichment analysis (cutoff of false discovery rate < 0.05). The sequences from mice were subjected to a similar workflow as described for humans. Early (1-week-old) and late (8-week-old) prepubertal pig spermatogonia were analyzed to reveal underlying cellular mechanisms of the metabolic shift using immunohistochemistry, western blot, qRT-PCR, quantitative proteomics, and culture experiments. Human PGCs and prepubertal human spermatogonia show an enrichment of OXPHOS-associated genes, which is downregulated at the onset of puberty (P < 0.0001). Furthermore, we demonstrate that similar metabolic changes between pup and adult spermatogonia are detectable in the mouse (P < 0.0001). In humans, the metabolic transition at puberty is also preceded by a drastic change in SSC shape at 11 years of age (P < 0.0001). Using a pig model, we reveal that this metabolic shift could be regulated by an insulin growth factor-1 dependent signaling pathway via mammalian target of rapamycin and proteasome inhibition. New single-cell RNA sequencing datasets obtained from this study are freely available through NCBI GEO with accession number GSE196819. Human prepubertal tissue samples are scarce, which led to the investigation of a low number of samples per age. Gene enrichment analysis gives only an indication about the functional state of the cells. Due to limited numbers of prepubertal human spermatogonia, porcine spermatogonia were used for further proteomic and in vitro analyses. We show that prepubertal human spermatogonia exhibit high OXHPOS and switch to an adult-like metabolism only after 11 years of age. Prepubescent cancer survivors often suffer from infertility in adulthood. SSC transplantation could provide a powerful tool for the treatment of infertility; however, it requires high cell numbers. This work provides key insight into the dynamic metabolic requirements of human SSCs across development that would be critical in establishing ex vivo systems to support expansion and sustained function of SSCs toward clinical use. This work was funded by the NIH/NICHD R01 HD091068 and NIH/ORIP R01 OD016575 to I.D. K.E.O. was supported by R01 HD100197. S.K.M. was supported by T32 HD087194 and F31 HD101323. The authors declare no conflict of interest.

Roles of Spermatogonial Stem Cells in Spermatogenesis and Fertility Restoration.

Spermatogonial stem cells (SSCs) are a group of adult stem cells in the testis that serve as the foundation of continuous spermatogenesis and male fertility. SSCs are capable of self-renewal to maintain the stability of the stem cell pool and differentiation to produce mature spermatozoa. Dysfunction of SSCs leads to male infertility. Therefore, dissection of the regulatory network of SSCs is of great significance in understanding the fundamental molecular mechanisms of spermatogonial stem cell function in spermatogenesis and the pathogenesis of male infertility. Furthermore, a better understanding of SSC biology will allow us to culture and differentiate SSCs in vitro, which may provide novel stem cell-based therapy for assisted reproduction. This review summarizes the latest research progress on the regulation of SSCs, and the potential application of SSCs for fertility restoration through in vivo and in vitro spermatogenesis. We anticipate that the knowledge gained will advance the application of SSCs to improve male fertility. Furthermore, in vitro spermatogenesis from SSCs sets the stage for the production of SSCs from induced pluripotent stem cells (iPSCs) and subsequent spermatogenesis.

Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells.

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1−/− mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1−/− mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1−/− compared to wild-type mice. Furthermore, cultured Uch-l1−/− SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

A regulatory role for CHD4 in maintenance of the spermatogonial stem cell pool.

SummaryMaintenance and self-renewal of the spermatogonial stem cell (SSC) population is the cornerstone of male fertility. Here, we have identified a key role for the nucleosome remodeling protein CHD4 in regulating SSC function. Gene expression analyses revealed that CHD4 expression is highly enriched in the SSC population in the mouse testis. Using spermatogonial transplantation techniques it was established that loss of Chd4 expression significantly impairs SSC regenerative capacity, causing a ∼50% reduction in colonization of recipient testes. An scRNA-seq comparison revealed reduced expression of “self-renewal” genes following Chd4 knockdown, along with increased expression of signature progenitor genes. Co-immunoprecipitation analyses demonstrated that CHD4 regulates gene expression in spermatogonia not only through its traditional association with the remodeling complex NuRD, but also via interaction with the GDNF-responsive transcription factor SALL4. Cumulatively, the results of this study depict a previously unappreciated role for CHD4 in controlling fate decisions in the spermatogonial pool.

Gestational Exposure to Bisphenol A Affects Testicular Morphology, Germ Cell Associations, and Functions of Spermatogonial Stem Cells in Male Offspring.

Exposure to bisphenol A (BPA) in the gestational period damages the reproductive health of offspring; detailed evidence regarding BPA-induced damage in testicular germ cells of offspring is still limited. In this study, pregnant mice (F0) were gavaged with three BPA doses (50 μg, 5 mg, and 50 mg/kg body weight (bw)/day; tolerable daily intake (TDI), no-observed-adverse-effect-level (NOAEL), and lowest-observed-adverse-effect level (LOAEL), respectively) on embryonic days 7 to 14, followed by investigation of the transgenerational effects of such exposure in male offspring. We observed that the NOAEL- and LOAEL-exposed F1 offspring had abnormalities in anogenital distance, nipple retention, and pubertal onset (days), together with differences in seminiferous epithelial stages and testis morphology. These effects were eradicated in the next F2 and F3 generations. Moreover, there was an alteration in the ratio of germ cell population and the apoptosis rate in germ cells increased in F1 offspring at the LOAEL dose. However, the total number of spermatogonia remained unchanged. Finally, a reduction in the stemness properties of spermatogonial stem cells in F1 offspring was observed upon LOAEL exposure. Therefore, we provide evidence of BPA-induced disruption of physiology and functions in male germ cells during the gestational period. This may lead to several reproductive health issues and infertility in offspring.

Gene expression dynamics during the gonocyte to spermatogonia transition and spermatogenesis in the domestic yak

BackgroundSpermatogenesis is a cellular differentiation process that includes three major events: mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis. Steady-state spermatogenesis relies on functions of spermatogonial stem cells (SSCs). Establishing and maintaining a foundational SSC pool is essential for continued spermatogenesis in mammals. Currently, our knowledge about SSC and spermatogenesis is severely limited in domestic animals.ResultsIn the present study, we examined transcriptomes of testes from domestic yaks at four different stages (3, 5, 8 and 24 months of age) and attempted to identify genes that are associated with key developmental events of spermatogenesis. Histological analyses showed that the most advanced germ cells within seminiferous tubules of testes from 3, 5, 8 and 24 months old yaks were gonocytes, spermatogonia, spermatocytes and elongated spermatids, respectively. RNA-sequencing (RNA-seq) analyses revealed that 11904, 4381 and 2459 genes were differentially expressed during the gonocyte to spermatogonia transition, the mitosis to meiosis transition and the meiosis to post-meiosis transition. Further analyses identified a list of candidate genes than may regulate these important cellular processes. CXCR4, a previously identified SSC niche factor in mouse, was one of the up-regulated genes in the 5 months old yak testis. Results of immunohistochemical staining confirmed that CXCR4 was exclusively expressed in gonocytes and a subpopulation of spermatogonia in the yak testis.ConclusionsTogether, these findings demonstrated histological changes of postnatal testis development in the domestic yak. During development of spermatogonial lineage, meiotic and haploid germ cells are supported by dynamic transcriptional regulation of gene expression. Our transcriptomic analyses provided a list of candidate genes that potentially play crucial roles in directing the establishment of SSC and spermatogenesis in yak.

Regulation of long non-coding RNAs and circular RNAs in spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are one of the most significant stem cells with the potentials of self-renewal, differentiation, transdifferentiation and dedifferentiation, and thus, they have important applications in reproductive and regenerative medicine. They can transmit the genetic and epigenetic information across generations, which highlights the importance of the correct establishment and maintenance of epigenetic marks. Accurate transcriptional and post-transcriptional regulation is required to support the highly coordinated expression of specific genes for each step of spermatogenesis. Increasing evidence indicates that non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), play essential roles in controlling gene expression and fate determination of male germ cells. These ncRNA molecules have distinct characteristics and biological functions, and they independently or cooperatively modulate the proliferation, apoptosis and differentiation of SSCs. In this review, we summarized the features, biological function and fate of mouse and human SSCs, and we compared the characteristics of lncRNAs and circRNAs. We also addressed the roles and mechanisms of lncRNAs and circRNAs in regulating mouse and human SSCs, which would add novel insights into the epigenetic mechanisms underlying mammalian spermatogenesis and provide new approaches to treat male infertility.

Mechanisms regulating mammalian spermatogenesis and fertility recovery following germcell depletion.

Mammalian spermatogenesis is a highly complex multi-step process sustained by a population of mitotic germ cells with self-renewal potential known as spermatogonial stem cells (SSCs). The maintenance and regulation of SSC function are strictly dependent on a supportive niche that is composed of multiple cell types. A detailed appreciation of the molecular mechanisms underpinning SSC activity and fate is of fundamental importance for spermatogenesis and male fertility. However, different models of SSC identity and spermatogonial hierarchy have been proposed and recent studies indicate that cell populations supporting steady-state germline maintenance and regeneration following damage are distinct. Importantly, dynamic changes in niche properties may underlie the fate plasticity of spermatogonia evident during testis regeneration. While formation of spermatogenic colonies in germ-cell-depleted testis upon transplantation is a standard assay for SSCs, differentiation-primed spermatogonial fractions have transplantation potential and this assay provides readout of regenerative rather than steady-state stemcell capacity. The characterisation of spermatogonial populations with regenerative capacity is essential for the development of clinical applications aimed at restoring fertility in individuals following germline depletion by genotoxic treatments. This review will discuss regulatory mechanisms of SSCs in homeostatic and regenerative testis and the conservation of these mechanisms between rodent models and man.

The DNA methylation profile of human spermatogonia at single-cell- and single-allele-resolution refutes its role in spermatogonial stem cell function and germ cell differentiation.

Human spermatogonial stem cells (hSSCs) have potential in fertility preservation of prepubertal boys or in treatment of male adults suffering from meiotic arrest. Prior to therapeutic application, in vitro propagation of rare hSSCs is mandatory. As the published data points to epigenetic alterations in long-term cell culture of spermatogonia (SPG), an initial characterisation of their DNA methylation state is important. Testicular biopsies from five adult normogonadotropic patients were converted into aggregate-free cell suspensions. FGFR3-positive (FGFR3+) SPG, resembling a very early stem cell state, were labelled with magnetic beads and isolated in addition to unlabelled SPG (FGFR3-). DNA methylation was assessed by limiting dilution bisulfite pyrosequencing for paternally imprinted (H19 and MEG3), maternally imprinted (KCNQ1OT1, PEG3, and SNRPN), pluripotency (POU5F1/OCT4 and NANOG), and spermatogonial/hSSC marker (FGFR3, GFRA1, PLZF, and L1TD1) genes on either single cells or pools of 10 cells. Both spermatogonial subpopulations exhibited a methylation pattern largely equivalent to sperm, with hypomethylation of hSSC marker and maternally imprinted genes and hypermethylation of pluripotency and paternally imprinted genes. Interestingly, we detected fine differences between the two spermatogonial subpopulations, which were reflected by an inverse methylation pattern of imprinted genes, i.e. decreasing methylation in hypomethylated genes and increasing methylation in hypermethylated genes, from FGFR3+ through FGFR3- SPG to sperm. Limitations of this study are due to it not being performed on a genome-wide level and being based on previously published regulatory gene regions. However, the concordance of DNA methylation between SPG and sperm implies that hSSC regulation and germ cell differentiation do not occur at the DNA methylation level.

Influence of testicular tissue and testicular cell suspension cryopreservation on spermatogonial stem cell function

Influence of testicular tissue and testicular cell suspension cryopreservation on spermatogonial stem cell function

In silico analysis of single-cell RNA sequencing data from 3 and 7 days old mouse spermatogonial stem cells to identify their differentially expressed genes and transcriptional regulators.

Spermatogonial stem cells (SSCs), which are at the basis of spermatogenesis process, are valuable cells with different applications in biotechnology and regenerative medicine. Understanding the molecular basis of SSC self-renewal and differentiation at various developmental stages of the male organism is crucial to find key factors in the SSCs fate and function. Therefore, this study was aimed to use single-cell RNA-sequencing dataset analysis for identification of differentially expressed genes (DEGs) and their regulators in 3 and 7 days old mouse-derived single SSCs (mSSCs). Results showed 68 upregulated and 203 downregulated genes in 7 days old mouse-derived SSCs compared to 3 days old mSSCs, which were associated with 1493 and 3077 biological processes, respectively. It also found that DAZL, FKBP6, PAIP2, DDX4, H3F3B, TEX15, XRN2, MAEL, and SOD1 are important factors with the higher gene expression pattern, which may be pivotal for mSSCs fate and function during development of germ cells. Moreover, NR3C1, RXRA, NCOA, ESR1, PML, ATF2, BMI1, POU5F1, and CHD1 were the main central regulators for the upregulated DEGs, while HNF1A, C/EBPα, and NFATC1 were the master regulators for the downregulated DEGs. In this regard, two significant protein complexes were found in the protein-protein interactions network for the upregulated DEGs regulators. Furthermore, 24 protein kinases detected upstream of the main central regulators of DEGs. In conclusion, this study presents DEGs and their transcriptional regulators that are crucial for inducing and regulating SSCs commitment during development, and for developing efficient protocols to identify and isolate SSCs for different applications.

In Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated Viruses

SummaryAdeno-associated virus (AAV) penetrates the blood-brain barrier, but it is unknown whether AAV penetrates other tight junctions. Genetic manipulation of testis has been hampered by the basement membrane of seminiferous tubules and the blood-testis barrier (BTB), which forms between Sertoli cells and divides the tubules into basal and adluminal compartments. Here, we demonstrate in vivo genetic manipulation of spermatogonial stem cells (SSCs) and their microenvironment via AAV1/9. AAV1/9 microinjected into the seminiferous tubules penetrated both the basement membrane and BTB, thereby transducing not only Sertoli cells and SSCs but also peritubular cells and Leydig cells. Moreover, when congenitally infertile KitlSl/KitlSl-d mouse testes with defective Sertoli cells received Kitl-expressing AAVs, spermatogenesis regenerated and offspring were produced. None of the offspring contained the AAV genome. Thus, AAV1/9 allows efficient germline and niche manipulation by penetrating the BTB and basement membrane, providing a promising strategy for the development of gene therapies for reproductive defects.