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Mammalian spermatogenesis is a complex, highly productive process generating millions of sperm per day. Spermatogonial stem cells (SSCs) are at the foundation of spermatogenesis and can either self-renew, producing more SSCs, or differentiate to initiate spermatogenesis and produce sperm. The biological potential of SSCs to produce and maintain spermatogenesis makes them a promising tool for the treatment of male infertility. However, translating knowledge from rodents to higher primates (monkeys and humans) is challenged by different vocabularies that are used to describe stem cells and spermatogenic lineage development in those species. Furthermore, while rodent SSCs are defined by their biological potential to produce and maintain spermatogenesis in a transplant assay, there is no equivalent routine and accessible bioassay to test monkey and human SSCs or replicate their functions in vitro. This chapter describes progress characterizing, isolating, culturing, and transplanting SSCs in higher primates., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: In male pre-pubertal cancer patients, radiation and chemotherapy impact future fertility by eradication of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but only a small percentage of spermatozoa produced were of donor origin. Transient hormone suppression with a GnRH antagonist (GnRH-ant) enhanced spermatogenic recovery from transplanted SSCs., Objectives: To evaluate donor-derived and endogenous spermatogenic recovery after SSC transplantation into irradiated monkeys and to test whether hormone suppression around the time of transplantation facilitates spermatogenic recovery., Materials and Methods: Testes of 15 adult rhesus monkeys were irradiated with 7 Gy and 4 months later transplanted, to one of the testes, with cryopreserved testicular cells containing SSCs from unrelated monkeys. Monkeys were either treated with GnRH-ant for 8 weeks before transplantation, GnRH-ant from 4 weeks before to 4 weeks after transplantation, or with no GnRH-ant. Tissues were harvested 10 months after transplantation., Results: Two of the 15 monkeys, a control and a pre-transplantation GnRH-ant-treated, showed substantially higher levels of testicular spermatogenesis and epididymal sperm output in the transplanted side as compared to the untransplanted. Over 84% of epididymal spermatozoa on the transplanted side had the donor genotype and were capable of fertilizing eggs after intracytoplasmic sperm injection forming morulae of the donor paternal origin. Low levels of donor spermatozoa (~1%) were also identified in the epididymis of three additional monkeys. Transplantation also appeared to enhance endogenous spermatogenesis., Discussion and Conclusion: We confirmed that SSC transplantation can be used for restoration of fertility in male cancer survivors exposed to irradiation as a therapeutic agent. The success rate of this procedure, however, is low. The success of filling the tubules with the cell suspension, but not the GnRH-ant treatment, was related to the level of colonization by transplanted cells., (© 2020 American Society of Andrology and European Academy of Andrology.)

Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for treating male infertility, which afflicts >100 million men world-wide. While much has been learned about rodent SSCs, human SSCs remain poorly understood. Here, we molecularly characterize human SSCs and define conditions favoring their culture. To achieve this, we first identified a cell-surface protein, PLPPR3, that allowed purification of human primitive undifferentiated spermatogonia (uSPG) highly enriched for SSCs. Comparative RNA-sequencing analysis of these enriched SSCs with differentiating SPG (KIT + cells) revealed the full complement of genes that shift expression during this developmental transition, including genes encoding key components in the TGF-β, GDNF, AKT, and JAK-STAT signaling pathways. We examined the effect of manipulating these signaling pathways on cultured human SPG using both conventional approaches and single-cell RNA-sequencing analysis. This revealed that GDNF and BMP8B broadly support human SPG culture, while activin A selectively supports more advanced human SPG. One condition-AKT pathway inhibition-had the unique ability to selectively support the culture of primitive human uSPG. This raises the possibility that supplementation with an AKT inhibitor could be used to culture human SSCs in vitro for therapeutic applications., Competing Interests: The authors declare no competing interest.

Spermatogonial stem cells (SSCs) undergo continuous self-renewal division in response to self-renewal factors. The present study identified ephrin type-A receptor 2 (EPHA2) on mouse SSCs and showed that supplementation of glial cell-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), which are both SSC self-renewal factors, induced EPHA2 expression in cultured SSCs. Spermatogonial transplantation combined with magnetic-activated cell sorting or fluorescence-activated cell sorting also revealed that EPHA2 was expressed in SSCs. Additionally, ret proto-oncogene (RET) phosphorylation levels decreased following the knockdown (KD) of Epha2 expression via short hairpin ribonucleic acid (RNA). Although the present immunoprecipitation experiments did not reveal an association between RET with EPHA2, RET interacted with FGFR2. The Epha2 KD decreased the proliferation of cultured SSCs and inhibited the binding of cultured SSCs to laminin-coated plates. The Epha2 KD also significantly reduced the colonization of testis cells by spermatogonial transplantation. EPHA2 was also expressed in human GDNF family receptor alpha 1-positive spermatogonia. The present results indicate that SSCs express EPHA2 and suggest that it is a critical modifier of self-renewal signals in SSCs., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Impact Statement: This study developed and characterized human testis extracellular matrix (htECM) and porcine testis ECM (ptECM) for testing in human spermatogonial stem cell (hSSC) culture. Results confirmed the hypothesis that ECM from the homologous species (human) and homologous tissue (testis) is optimal for maintaining hSSCs. We describe a simplified feeder-free, serum-free condition for future iterative testing to achieve the long-term goal of stable hSSC cultures. To facilitate analysis and understand the fate of hSSCs in culture, we describe a multiparameter, high-throughput, quantitative flow cytometry approach to rapidly count undifferentiated spermatogonia, differentiated spermatogonia, apoptotic spermatogonia, and proliferative spermatogonia in hSSC cultures.

A major challenge in stem cell differentiation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus (r) macaque PGCLCs (rPGCLCs) from induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late rPGCs that initiate epigenetic reprogramming but do not complete the conversion into ENO2-positive spermatogonia.

Undifferentiated spermatogonia comprise a pool of stem cells and progenitor cells that show heterogeneous expression of markers, including the cell surface receptor GFRα1. Technical challenges in isolation of GFRα1+ versus GFRα1- undifferentiated spermatogonia have precluded the comparative molecular characterization of these subpopulations and their functional evaluation as stem cells. Here, we develop a method to purify these subpopulations by fluorescence-activated cell sorting and show that GFRα1+ and GFRα1- undifferentiated spermatogonia both demonstrate elevated transplantation activity, while differing principally in receptor tyrosine kinase signaling and cell cycle. We identify the cell surface molecule melanocyte cell adhesion molecule (MCAM) as differentially expressed in these populations and show that antibodies to MCAM allow isolation of highly enriched populations of GFRα1+ and GFRα1- spermatogonia from adult, wild-type mice. In germ cell culture, GFRα1- cells upregulate MCAM expression in response to glial cell line-derived neurotrophic factor (GDNF)/fibroblast growth factor (FGF) stimulation. In transplanted hosts, GFRα1- spermatogonia yield GFRα1+ spermatogonia and restore spermatogenesis, albeit at lower rates than their GFRα1+ counterparts. Together, these data provide support for a model of a stem cell pool in which the GFRα1+ and GFRα1- cells are closely related but show key cell-intrinsic differences and can interconvert between the two states based, in part, on access to niche factors., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The developmental origins of most adult stem cells are poorly understood. Here, we report the identification of a transcription factor-RHOX10-critical for the initial establishment of spermatogonial stem cells (SSCs). Conditional loss of the entire 33-gene X-linked homeobox gene cluster that includes Rhox10 causes progressive spermatogenic decline, a phenotype indistinguishable from that caused by loss of only Rhox10. We demonstrate that this phenotype results from dramatically reduced SSC generation. By using a battery of approaches, including single-cell-RNA sequencing (scRNA-seq) analysis, we show that Rhox10 drives SSC generation by promoting pro-spermatogonia differentiation. Rhox10 also regulates batteries of migration genes and promotes the migration of pro-spermatogonia into the SSC niche. The identification of an X-linked homeobox gene that drives the initial generation of SSCs has implications for the evolution of X-linked gene clusters and sheds light on regulatory mechanisms influencing adult stem cell generation in general., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans., (© 2015 Pech et al.; Published by Cold Spring Harbor Laboratory Press.)

Objective: To illustrate the step-by-step protocol followed to assay germ cell transplantation into the seminiferous epithelium of mouse testes., Design: Video presentation of an animal model for research in reproductive and regenerative medicine., Setting: Research laboratory., Animal(s): Male nude mice (NU-Foxn1(nu))., Intervention(s): Mice were chemically sterilized with alkylant compounds (busulfan) followed by gonadal microsurgery to inject donor germ cells., Main Outcome Measure(s): Donor cells should be labeled with reporter genes, such as green fluorescent protein (GFP), lactose operon (LacZ), or alternatively design an effective strategy with specific antibodies to track them within the recipient testes. Sperm detection in the ejaculate can also be used as a read out. However, in this case detection of the donor genotype in the sperm is mandatory to elucidate their origin., Result(s): In the present study we describe the complete protocol for germ cell transplant by efferent duct injection, including the preparation of recipient mice, surgery for the germ cell transplant, and analysis of recipient testes. The main strength of this technique is that it constitutes the gold standard for a functional test of the germ cell potential as only spermatogonial stem cells are able to properly colonize the seminal lumen. Both fresh and frozen/thawed testicular cells are suitable for this technique as donor germ cells. Also, enrichment of living spermatogonial stem cells, previous to the transplant, seems to improve the efficiency of colonization. For proper colonization of germ cells, the niche should be available and thus mouse strains that lack endogenous spermatogenesis such as W/W(v) mutant mice are usually used. In the case of nonmatched donor cells, seminiferous epithelium of immune-suppressed recipient mice should be germ cell depleted before the transplant. One limitation of this technique is that the procedure can take up to 3 months. Also, in contrast to the full recovery of spermatogenesis in mouse-to-mouse transplants, xenotransplantation of germ cells from phylogenetically distant species, such as humans into mouse recipients, results in colonization of donor cells and spermatogonial expansion, but fail in their spermatogenic progression due to evolutive incompatibilities with the recipient niche. Xenografting of pieces of donor testis tissue under the skin of mouse hosts is an alternative approach that is currently being investigated to try to solve this limitation., Conclusion(s): Transplantation of spermatogonial stem cells into the seminal lumen of mouse testes is a functional assay that defines this cellular subpopulation by its ability to colonize it. This technique can be used as a model to elucidate the insights of spermatogonial stem cells, to produce transgenic animals by genetically manipulating donor cells before transplantation, but also it has potential applications in fertility preservation in cattle and humans as it is feasible in large animals, as recent reports have demonstrated with rhesus monkeys, that recovered spermatogenesis after allogenic transplantation, and even from human cadaver testes. Therefore spermatogonial stem cells isolated from prepuberal boys, who are treated with alkylant chemotherapy, could be returned to their testis to regenerate spermatogenesis in the future., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the molecular characteristics of human spermatogonia and optimize methods to enrich spermatogonial stem cells (SSCs)., Design: Laboratory study using human tissues., Setting: Research institute., Patient(s): Healthy adult human testicular tissue., Intervention(s): Human testicular tissue was fixed or digested with enzymes to produce a cell suspension. Human testis cells were fractionated by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS)., Main Outcome Measure(s): Immunostaining for selected markers, human-to-nude mouse xenotransplantation assay., Result(s): Immunohistochemistry costaining revealed the relative expression patterns of SALL4, UTF1, ZBTB16, UCHL1, and ENO2 in human undifferentiated spermatogonia as well as the extent of overlap with the differentiation marker KIT. Whole mount analyses revealed that human undifferentiated spermatogonia (UCHL1+) were typically arranged in clones of one to four cells whereas differentiated spermatogonia (KIT+) were typically arranged in clones of eight or more cells. The ratio of undifferentiated-to-differentiated spermatogonia is greater in humans than in rodents. The SSC colonizing activity was enriched in the THY1dim and ITGA6+ fractions of human testes sorted by FACS. ITGA6 was effective for sorting human SSCs by MACS; THY1 and EPCAM were not., Conclusion(s): Human spermatogonial differentiation correlates with increased clone size and onset of KIT expression, similar to rodents. The undifferentiated-to-differentiated developmental dynamics in human spermatogonia is different than rodents. THY1, ITGA6, and EPCAM can be used to enrich human SSC colonizing activity by FACS, but only ITGA6 is amenable to high throughput sorting by MACS., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

SHP2 is a widely expressed protein tyrosine phosphatase required for signal transduction from multiple cell surface receptors. Gain and loss of function SHP2 mutations in humans are known to cause Noonan and LEOPARD syndromes, respectively, that are characterized by numerous pathological conditions including male infertility. Using conditional gene targeting in the mouse, we found that SHP2 is required for maintaining spermatogonial stem cells (SSCs) and the production of germ cells required for male fertility. After deleting SHP2, spermatogenesis was halted at the initial step during which transit-amplifying undifferentiated spermatogonia are produced from SSCs. In the absence of SHP2, proliferation of SSCs and undifferentiated spermatogonia was inhibited, thus germ cells cannot be replenished and SSCs cannot undergo renewal. However, germ cells beyond the undifferentiated spermatogonia stage of development at the time of SHP2 knockout were able to complete their maturation to become sperm. In cultures of SSCs and their progeny, inhibition of SHP2 activity reduced growth factor-mediated intracellular signaling that regulates SSC proliferation and cell fate. Inhibition of SHP2 also decreased the number of SSCs present in culture and caused SSCs to detach from supporting cells. Injection of mice with an SHP2 inhibitor blocked the production of germ cells from SSCs. Together, our studies show that SHP2 is essential for SSCs to maintain fertility and indicates that the pathogenesis of infertility in humans with SHP2 mutations is due to compromised SSC functions that block spermatogenesis., (© AlphaMed Press.)

Spermatogonial stem cells (SSCs) and undifferentiated progenitor spermatogonia in mammalian seminiferous tubules are organized in chains, connected by intracellular bridges. Clone size is generally related to stem cell potential, with shorter chains containing the majority of the stem cell population. Immunofluorescence detection of spermatogonia-specific proteins in whole-mount seminiferous tubule preparations is the only method that allows researchers to relate clone size with the molecular phenotype in spermatogenic lineage development. Here we describe in detail the method used to detect nuclear, cytoplasmic, and cell surface molecules in seminiferous tubules isolated from mouse, monkey, and human testes.

Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4-contaminated human testicular cell suspensions was performed to isolate EpCAM+/HLA-ABC-/CD49e- (putative spermatogonia) and EpCAM-/HLA-ABC+/CD49e+ (putative MOLT-4) cell fractions. The EpCAM+/HLA-ABC-/CD49e- fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to-nude mouse xenotransplantation. The EpCAM-/HLA-ABC+/CD49e+ fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.

The spermatogenic lineage is established after birth when gonocytes migrate to the basement membrane of seminiferous tubules and give rise to spermatogonial stem cells (SSC). In adults, SSCs reside within the population of undifferentiated spermatogonia (A(undiff)) that expands clonally from single cells (A(single)) to form pairs (A(paired)) and chains of 4, 8 and 16 A(aligned) spermatogonia. Although stem cell activity is thought to reside in the population of A(single) spermatogonia, new research suggests that clone size alone does not define the stem cell pool. The mechanisms that regulate self-renewal and differentiation fate decisions are poorly understood due to limited availability of experimental tools that distinguish the products of those fate decisions. The pluripotency factor SALL4 (sal-like protein 4) is implicated in stem cell maintenance and patterning in many organs during embryonic development, but expression becomes restricted to the gonads after birth. We analyzed the expression of SALL4 in the mouse testis during the first weeks after birth and in adult seminiferous tubules. In newborn mice, the isoform SALL4B is expressed in quiescent gonocytes at postnatal day 0 (PND0) and SALL4A is upregulated at PND7 when gonocytes have colonized the basement membrane and given rise to spermatogonia. During steady-state spermatogenesis in adult testes, SALL4 expression overlapped substantially with PLZF and LIN28 in A(single), A(paired) and A(aligned) spermatogonia and therefore appears to be a marker of undifferentiated spermatogonia in mice. In contrast, co-expression of SALL4 with GFRα1 and cKIT identified distinct subpopulations of A(undiff) in all clone sizes that might provide clues about SSC regulation. Collectively, these results indicate that 1) SALL4 isoforms are differentially expressed at the initiation of spermatogenesis, 2) SALL4 is expressed in undifferentiated spermatogonia in adult testes and 3) SALL4 co-staining with GFRα1 and cKIT reveals distinct subpopulations of A(undiff) spermatogonia that merit further investigation.

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a man's life and may have application for treating some cases of male infertility, including those caused by chemotherapy before puberty. We performed autologous and allogeneic SSC transplantations into the testes of 18 adult and 5 prepubertal recipient macaques that were rendered infertile with alkylating chemotherapy. After autologous transplant, the donor genotype from lentivirus-marked SSCs was evident in the ejaculated sperm of 9/12 adult and 3/5 prepubertal recipients after they reached maturity. Allogeneic transplant led to donor-recipient chimerism in sperm from 2/6 adult recipients. Ejaculated sperm from one recipient transplanted with allogeneic donor SSCs were injected into 85 rhesus oocytes via intracytoplasmic sperm injection. Eighty-one oocytes were fertilized, producing embryos ranging from four-cell to blastocyst with donor paternal origin confirmed in 7/81 embryos. This demonstration of functional donor spermatogenesis following SSC transplantation in primates is an important milestone for informed clinical translation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Chemotherapy and radiation treatments for cancer and other diseases can cause male infertility. There are currently no options to preserve the fertility of prepubertal boys who are not yet making sperm. Cryopreservation of spermatogonial stem cells (SSCs, obtained via testicular biopsy) followed by autologous transplantation back into the testes at a later date may restore fertility in these patients. However, this approach carries an inherent risk of reintroducing cancer., Methods: To address this aspect of SSC transplantation safety, prepubertal non-human primate testis cell suspensions were inoculated with MOLT4 T-lymphoblastic leukemia cells and subsequently sorted for cell surface markers CD90 (THY-1) and CD45., Results: Cancer cells segregated to the CD90-/CD45+ fraction and produced tumors in nude mice. Nearly all sorted DEAD box polypeptide 4-positive (VASA+) spermatogonia segregated to the CD90+/CD45- fraction. In a preliminary experiment, a purity check of the sorted putative stem cell fraction (CD90+/CD45-) revealed 0.1% contamination with cancer cells, which was sufficient to produce tumors in nude mice. We hypothesized that the contamination resulted from mis-sorting due to cell clumping and employed singlet discrimination (SD) in four subsequent experiments. Purity checks revealed no cancer cell contamination in the CD90+/CD45- fraction from three of the four SD replicates and these fractions produced no tumors when transplanted into nude mouse testes., Conclusions: We conclude that spermatogonia can be separated from contaminating malignant cells by fluorescence-activated cell sorting prior to SSC transplantation and that post-sorting purity checks are required to confirm elimination of malignant cells.

This article will provide an updated review of spermatogonial stem cells and their role in maintaining the spermatogenic lineage. Experimental tools used to study spermatogonial stem cells (SSCs) will be described, along with research using these tools to enhance our understanding of stem cell biology and spermatogenesis. Increased knowledge about the biology of SSCs improves our capacity to manipulate these cells for practical application. The chapter concludes with a discussion of future directions for fundamental investigation and practical applications of SSCs.

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout the reproductive life of mammals. While A(single) spermatogonia comprise the rodent SSC pool, the identity of the stem cell pool in the primate spermatogenic lineage is not well established. The prevailing model is that primate spermatogenesis arises from A(dark) and A(pale) spermatogonia, which are considered to represent reserve and active stem cells respectively. However, there is limited information about how the A(dark) and A(pale) descriptions of nuclear morphology correlate with the clonal (A(single), A(paired), and A(aligned)), molecular (e.g. GFRalpha1 (GFRA1) and PLZF), and functional (SSC transplantation) descriptions of rodent SSCs. Thus, there is a need to investigate primate SSCs using criteria, tools, and approaches that have been used to investigate rodent SSCs over the past two decades. SSCs have potential clinical application for treating some cases of male infertility, providing impetus for characterizing and learning to manipulate these adult tissue stem cells in primates (nonhuman and human). This review recounts the development of a xenotransplant assay for functional identification of primate SSCs and progress dissecting the molecular and clonal characteristics of the primate spermatogenic lineage. These observations highlight the similarities and potential differences between rodents and primates regarding the SSC pool and the kinetics of spermatogonial self-renewal and clonal expansion. With new tools and reagents for studying primate spermatogonia, the field is poised to develop and test new hypotheses about the biology and regenerative capacity of primate SSCs.

Background: The spermatogonial stem cell (SSC) pool in the testes of non-human primates is poorly defined., Methods: To begin characterizing SSCs in rhesus macaque testes, we employed fluorescence-activated cell sorting (FACS), a xenotransplant bioassay and immunohistochemical methods and correlated our findings with classical descriptions of germ cell nuclear morphology (i.e. A(dark) and A(pale) spermatogonia)., Results: FACS analysis identified a THY-1+ fraction of rhesus testis cells that was enriched for consensus SSC markers (i.e. PLZF, GFRalpha1) and exhibited enhanced colonizing activity upon transplantation to nude mouse testes. We observed a substantial conservation of spermatogonial markers from mice to monkeys [PLZF, GFRalpha1, Neurogenin 3 (NGN3), cKIT]. Assuming that molecular characteristics correlate with function, the pool of putative SSCs (THY-1+, PLZF+, GFRalpha1+, NGN3+/-, cKIT-) comprises most A(dark) and A(pale) and is considerably larger in primates than in rodents. It is noteworthy that the majority of A(dark) and A(pale) share a common molecular phenotype, considering their distinct functional classifications as reserve and renewing stem cells, respectively. NGN3 is absent from A(dark), but is expressed by some A(pale) and may mark the transition from undifferentiated (cKIT-) to differentiating (cKIT+) spermatogonia. Finally, the pool of transit-amplifying progenitor spermatogonia (PLZF+, GFRalpha1+, NGN3+, cKIT+/-) is smaller in primates than in rodents. CONCLUSIONS These results provide an in-depth analysis of molecular characteristics of primate spermatogonia, including SSCs, and lay a foundation for future studies investigating the kinetics of spermatogonial renewal, clonal expansion and differentiation during primate spermatogenesis.

Gene expression and consequent biological activity of adult tissue stem cells are regulated by signals emanating from the local microenvironment (niche). To gain insights into the molecular regulation of spermatogonial stem cells (SSCs), gene expression was characterized from SSCs isolated from their cognate niches of cryptorchid (stem cell-enriched), wild-type, and busulfan-treated (stem cell-depleted) mouse testes. Quantitative assessment of stem cell activity in each testis model was determined using an in vivo functional assay and correlated with gene expression using Affymetrix MGU74Av2 microarrays and the ChipStat algorithm optimized to detect gene expression from rare cells in complex tissues. We identified 389 stem/progenitor spermatogonia candidate genes, which exhibited significant overlap with genes expressed by embryonic, hematopoietic, and neural stem cells; enriched spermatogonia; and cultured SSCs identified in previous studies. Candidate cell surface markers identified by the microarray may facilitate the isolation and enrichment of stem and/or progenitor spermatogonia. Flow cytometric analyses confirmed the expression of chemokine receptor 2 (Ccr2) and Cd14 on a subpopulation cryptorchid testis cells (alpha6-integrin+, side scatter(lo)) enriched for SSCs. These cell surface molecules may mark progenitor spermatogonia but not SSCs because Ccr2+ and Cd14+ fractions failed to produce spermatogenesis upon transplantation to recipient testes. Functional annotation of candidate genes and subsequent immunohistochemistry revealed that proteins involved in post-transcriptional regulation are overrepresented in cryptorchid testes that are enriched for SSCs. Comparative analyses indicated that this is a recurrent biological theme among stem cells.

Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. Whereas rare A(single) spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant A(dark) and A(pale) spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year following high-dose busulfan treatment, sperm counts remained undetectable, and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFRalpha1), and cells expressing these markers were depleted following high-dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high-dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell-depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes.

Spermatozoa produced from spermatogonial stem cells (SSCs) are the vehicle by which genes of a male are passed to the next generation. A single SSC has the ability to self-renew and produce thousands of spermatozoa; therefore, it is an ideal target for genetic modification to efficiently generate transgenic animals in mammalian species. Rats are an important model organism for biological research; however, gene function studies have been difficult because of a limited ability to generate transgenic animals. Transgenic rat production through SSCs offers a means to overcome this obstacle. Because SSCs divide slowly both in vivo and in vitro, lentiviral vectors may be an ideal method for introducing stable genetic modification. Using a lentiviral vector, an enhanced green fluorescent protein (eGFP) transgene was introduced into the genome of cultured rat SSCs, which were microinjected into testes of immunodeficient mice to assess transduction efficiency. Approximately 40% of rat SSCs exposed to the lentiviral vector overnight carried the eGFP transgene and generated colonies of spermatogenesis. When transduced SSCs were transplanted into recipient rat testes, in which endogenous germ cells had been decreased but not eliminated by busulfan treatment, approximately 6% of offspring were transgenic. The transgene was stably integrated into the donor SSC genome and transmitted to and expressed by progeny in subsequent generations. Thus, lentiviral transduction of SSCs followed by transplantation is an effective means for generating transgenic rats through the male germline, and this approach may be applicable to other species in which existing methods are inadequate or not applicable.

Aging is evident in most tissues and organ systems, but the mechanisms of aging are difficult to identify and poorly understood. Here, we test the hypothesis that aging results in uncorrected defects in stem cell and/or niche function, which lead to system failure. We used the spermatogonial stem cell (SSC) transplantation assay to determine the effect of aging on testis stem cell/niche function in mice. Between 12 and 24 months of age, male mice experienced a declining level of fertility associated with decreased testis weight, level of spermatogenesis, and total stem cell content. However, when stem cells were consecutively passaged at 3-month intervals to testes of young males, these stem cells continued to produce spermatogenesis for more than 3 years. Thus, SSC self-renewal continues long past the normal life span of the animal when the stem cell is continually maintained in a young niche/microenvironment. Moreover, these data suggest that infertility in old males results from deterioration of the SSC niche and failure to support an appropriate balance between stem cell self-renewal and differentiation.

Spermatogonial stem cells (SSCs) are essential for spermatogenesis, and these adult tissue stem cells balance self-renewal and differentiation to meet the biological demand of the testis. The developmental dynamics of SSCs are controlled, in part, by factors in the stem cell niche, which is located on the basement membrane of seminiferous tubules situated among Sertoli cells. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), and disruption of GDNF expression results in spermatogenic defects and infertility. The GDNF signals through a receptor complex that includes GDNF family receptor alpha1 (GFRA1), which is thought to be expressed by SSCs. However, expression of GFRA1 on SSCs has not been confirmed by in vivo functional assay, which is the only method that allows definitive identification of SSCs. Therefore, we fractionated mouse pup testis cells based on GFRA1 expression using magnetic activated cell sorting. The sorted and depleted fractions of GFRA1 were characterized for germ cell markers by immunocytochemistry and for stem cell activity by germ cell transplantation. The GFRA1-positive cell fraction coeluted with other markers of SSCs, including ITGA6 and CD9, and was significantly depleted of KIT-positive cells. The transplantation results confirmed that a subpopulation of SSCs expresses GFRA1, but also that the stem cell pool is heterogeneous with respect to the level of GFRA1 expression. Interestingly, POU5F1-positive cells were enriched nearly 15-fold in the GFRA1-selected fraction, possibly suggesting heterogeneity of developmental potential within the stem cell pool.

The existence of spermatogonial stem cells in the testis offers clinically relevant options for preservation and restoration of male fertility. New approaches based on male germ cell transplantation and testicular tissue grafting can be applied to generate a limited number of sperm cells and could therefore be considered important new avenues for restoration of fertility in oncological patients. We have developed approaches to infuse germ cells into rodent and primate testes and shown that germ cell transplantation is a procedure for restoration of spermatogenesis in the testis that might be adaptable to primates. As a promising alternative, grafting of testicular tissue has been used to produce fertile sperm. The rapid progress in the development of novel experimental strategies to generate sperm by transplantation of spermatogonial stem cells or by grafting of testicular tissue should stimulate oncologists to consider the cryopreservation of testicular tissue. This review introduces the reader to the physiology of spermatogonial stem cells and summarizes the current and potential future options for fertility preservation in male oncological patients.

Spermatogonial stem cells (SSCs) are at the foundation of the highly productive spermatogenic process that continuously produces male gametes throughout postnatal life. However, experimental evaluation of SSCs in postnatal testes is complicated because these cells are extremely rare and few defining morphology or biochemical characteristics are known. In this study, we used the spermatogonial transplantation functional assay, combined with fluorescence-activated cell sorting (FACS) analysis to identify cellular, biochemical and surface antigenic characteristics of SSCs in rat testes during development. Our results demonstrated that forward scatter (FSc)(hi), side scatter (SSc)(hi), mitochondria membrane potential (DeltaPsim)(lo), Ep-CAM(+), Thy-1(+), beta3-integrin(+) stem cells in neonate rat testes become SSc(lo), DeltaPsim(hi), Ep-CAM(+), Thy-1(lo), beta3-integrin(-) stem cells in pup rat testes. Furthermore, prospective identification of rat testis cell populations (Ep-CAM(+)), highly enriched for SSCs (1 in 13 for neonate; 1 in 8.5 for pup) enabled us to predict the Thy-1 and beta3-integrin status of stem cells in neonate and pup testes, which was subsequently confirmed by transplantation analyses. Systematic characterization of SSCs enabled the production of testis cell populations highly enriched (up to 120-fold) for SSCs and will facilitate future investigations of functional and genomic characteristics.

Little is known of the molecular mechanisms whereby spermatogonia, mitotic germ cells of the testis, self-renew and differentiate into sperm. Here we show that Zfp145, encoding the transcriptional repressor Plzf, has a crucial role in spermatogenesis. Zfp145 expression was restricted to gonocytes and undifferentiated spermatogonia and was absent in tubules of W/W(v) mutants that lack these cells. Mice lacking Zfp145 underwent a progressive loss of spermatogonia with age, associated with increases in apoptosis and subsequent loss of tubule structure but without overt differentiation defects or loss of the supporting Sertoli cells. Spermatogonial transplantation experiments revealed a depletion of spermatogonial stem cells in the adult. Microarray analysis of isolated spermatogonia from Zfp145-null mice before testis degeneration showed alterations in the expression profile of genes associated with spermatogenesis. These results identify Plzf as a spermatogonia-specific transcription factor in the testis that is required to regulate self-renewal and maintenance of the stem cell pool.

The ability to isolate, manipulate, and transplant spermatogonial stem cells provides a unique opportunity to modify the germline. We used the rat-to-nude mouse transplantation assay to characterize spermatogonial stem cell activity in rat testes and in culture. Our results indicate that rat spermatogonial stem cells can survive and proliferate in short-term culture, although a net loss of stem cells was observed. Rat spermatogonial stem cells also were susceptible to transduction with a retroviral vector carrying a lacZ reporter transgene. Using a 3-day periodic infection protocol, 0.5% of stem cells originally cultured were transduced and produced transgenic colonies of spermatogenesis in recipient mouse testes. The level of transgenic donor-derived spermatogenesis observed in the rat-to-mouse transplantation was similar to levels that produced transgenic progeny in the mouse-to-mouse transplantation. This work provides a basis for understanding the biology of rat spermatogonial stem cells. Development of an optimal rat recipient testis model and application of these methods for germline modification will enable the production of transgenic rats, potentially valuable tools for evaluating genes and their functions. In addition, these methods may be applicable in other species where existing transgenic methods are inefficient or not available.

STUDY QUESTION Twenty years after the inception of the first fertility preservation programme for pre-pubertal boys, what are the current international practices with regard to cryopreservation of immature testicular tissue? SUMMARY ANSWER Worldwide, testicular tissue has been cryopreserved from over 3000 boys under the age of 18 years for a variety of malignant and non-malignant indications; there is variability in practices related to eligibility, clinical assessment, storage, and funding. WHAT IS KNOWN ALREADY For male patients receiving gonadotoxic treatment prior to puberty, testicular tissue cryopreservation may provide a method of fertility preservation. While this technique remains experimental, an increasing number of centres worldwide are cryopreserving immature testicular tissue and are approaching clinical application of methods to use this stored tissue to restore fertility. As such, standards for quality assurance and clinical care in preserving immature testicular tissue should be established. STUDY DESIGN, SIZE, DURATION A detailed survey was sent to 17 centres within the recently established ORCHID-NET consortium, which offer testicular tissue cryopreservation to patients under the age of 18 years. The study encompassed 60 questions and remained open from 1 July to 1 November 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS Of the 17 invited centres, 16 completed the survey, with representation from Europe, Australia, and the USA. Collectively, these centres have cryopreserved testicular tissue from patients under the age of 18 years. Data are presented using descriptive analysis. MAIN RESULTS AND THE ROLE OF CHANCE Since the establishment of the first formal fertility preservation programme for pre-pubertal males in 2002, these 16 centres have cryopreserved tissue from 3118 patients under the age of 18 years, with both malignant (60.4%) and non-malignant (39.6%) diagnoses. All centres perform unilateral biopsies, while 6/16 sometimes perform bilateral biopsies. When cryopreserving tissue, 9/16 centres preserve fragments sized ≤5 mm3 with the remainder preserving fragments sized 6–20 mm3. Dimethylsulphoxide is commonly used as a cryoprotectant, with medium supplements varying across centres. There are variations in funding source, storage duration, and follow-up practice. Research, with consent, is conducted on stored tissue in 13/16 centres. LIMITATIONS, REASONS FOR CAUTION While this is a multi-national study, it will not encompass every centre worldwide that is cryopreserving testicular tissue from males under 18 years of age. As such, it is likely that the actual number of patients is even higher than we report. Whilst the study is likely to reflect global practice overall, it will not provide a complete picture of practices in every centre. WIDER IMPLICATIONS OF THE FINDINGS Given the research advances, it is reasonable to suggest that cryopreserved immature testicular tissue will in the future be used clinically to restore fertility. The growing number of patients undergoing this procedure necessitates collaboration between centres to better harmonize clinical and research protocols evaluating tissue function and clinical outcomes in these patients. STUDY FUNDING/COMPETING INTEREST(S) K.D. is supported by a CRUK grant (C157/A25193). R.T.M. is supported by an UK Research and Innovation (UKRI) Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health at the University of Edinburgh is supported by MRC (MR/N022556/1). C.L.M. is funded by Kika86 and ZonMW TAS 116003002. A.M.M.v.P. is supported by ZonMW TAS 116003002. E.G. was supported by the Research Program of the Research Foundation—Flanders (G.0109.18N), Kom op tegen Kanker, the Strategic Research Program (VUB_SRP89), and the Scientific Fund Willy Gepts. J.-B.S. is supported by the Swedish Childhood Cancer Foundation (TJ2020-0026). The work of NORDFERTIL is supported by the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115), the Swedish Research Council (2018-03094; 2021-02107), and the Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2021-00073; 2022-00317; 2023-00353). C.E is supported by the Health Department of the Basque Government (Grants 2019111068 and 2022111067) and Inocente Inocente Foundation (FII22/001). M.P.R. is funded by a Medical Research Council Centre for Reproductive Health Grant No: MR/N022556/1. A.F. and N.R. received support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. K.E.O. is funded by the University of Pittsburgh Medical Center and the US National Institutes of Health HD100197. V.B-L is supported by the French National Institute of Cancer (Grant Seq21-026). Y.J. is supported by the Royal Children's Hospital Foundation and a Medical Research Future Fund MRFAR000308. E.G. N.N. S.S. C.L.M. A.M.M.v.P. C.E. R.T.M. K.D. M.P.R. are members of COST Action CA20119 (ANDRONET) supported by COST (European Cooperation in Science and Technology). The Danish Child Cancer Foundation is also thanked for financial support (C.Y.A.). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]