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

In this study we explored the role of hypoxia and the hypoxia-inducible transcription factor EPAS1 in regulating spermatogonial stem cell (SSC) function in the mouse testis. We have demonstrated that SSCs reside in hypoxic microenvironments in the testis through utilization of the oxygen-sensing probe pimonidazole, and by confirming the stable presence of EPAS1, which is degraded at >5% O2. Through the generation of a germline-specific Epas1 knockout mouse line, and through modulation of EPAS1 levels in primary cultures of spermatogonia with the small drug molecule Daprodustat, we have demonstrated that EPAS1 is required for robust SSC function in regenerative conditions (post-transplantation and post-chemotherapy), via the regulation of key cellular processes such as metabolism. These findings shed light on the relationship between hypoxia and male fertility and will potentially facilitate optimization of in vitro culture conditions for infertility treatment pipelines using SSCs, such as those directed at pediatric cancer survivors.

Introduction: The Why's and How's for Studying Spermatogenesis and Spermatogonial Stem Cells.

Spermatogenesis is maintained throughout adulthood by a pool of adult stem cells termed spermatogonial stem cells (SSCs). Research investigations into spermatogenesis can provide insight into the etiology of certain types of male infertility (e.g., Sertoli cell only syndrome), elucidate means of improving food animal production, reveal new therapeutic avenues to address naturally occurring defects in sperm production, mitigate iatrogenic male infertility (e.g., arising from cancer therapy), and potentially intervene for male contraception. This chapter will serve as a commentary about why studying spermatogenesis is important, including a high-level overview of spermatogonia and SSCs, and make the case for a critical need for use of stringent definitions for SSCs and experimental platforms that allow for clear distinction of the multiple types of spermatogonia that exist in testes of mammals.

Perspectives: Approaches for Studying Livestock Spermatogonia.

At present, the knowledge base on characteristics and biology of spermatogonia in livestock is limited in comparison to rodents, yet the importance of studying these cells for comparative species analysis and enhancing reproductive capacity in food animals is high. Previous studies have established that although many core attributes of organ physiology and mechanisms governing essential cellular functions are conserved across eutherians, significant differences exist between mice and higher order mammals. In this chapter, we briefly discuss distinguishing aspects of testicular anatomy and the spermatogenic lineage in livestock and critical considerations for studying spermatogonial stem cell biology in these species.

ARRDC5 expression is conserved in mammalian testes and required for normal sperm morphogenesis.

In sexual reproduction, sperm contribute half the genomic material required for creation of offspring yet core molecular mechanisms essential for their formation are undefined. Here, the α-arrestin molecule arrestin-domain containing 5 (ARRDC5) is identified as an essential regulator of mammalian spermatogenesis. Multispecies testicular tissue transcriptome profiling indicates that expression of Arrdc5 is testis enriched, if not specific, in mice, pigs, cattle, and humans. Knockout of Arrdc5 in mice leads to male specific sterility due to production of low numbers of sperm that are immotile and malformed. Spermiogenesis, the final phase of spermatogenesis when round spermatids transform to spermatozoa, is defective in testes of Arrdc5 deficient mice. Also, epididymal sperm in Arrdc5 knockouts are unable to capacitate and fertilize oocytes. These findings establish ARRDC5 as an essential regulator of mammalian spermatogenesis. Considering the role of arrestin molecules as modulators of cellular signaling and ubiquitination, ARRDC5 is a potential male contraceptive target.

Culture of Kenyan Goat (Capra hircus) Undifferentiated Spermatogonia in Feeder-Free Conditions.

The undifferentiated spermatogonial population in mammalian testes contains a spermatogonial stem cell (SSC) population that can regenerate continual spermatogenesis following transplantation. This capacity has the potential to be exploited as a surrogate sires breeding tool to achieve widespread dissemination of desirable genetics in livestock production. Because SSCs are relatively rare in testicular tissue, the ability to expand a population in vitro would be advantageous to provide large numbers for transplantation into surrogate recipient males. Here, we evaluated conditions that would support long-term in-vitro maintenance of undifferentiated spermatogonia from a goat breed that is endemic to Kenyan livestock production. Single-cell suspensions enriched for undifferentiated spermatogonia from pre-pubertal bucks were seeded on laminin-coated tissue culture plates and maintained in a commercial media based on serum-free composition. The serum-free media was conditioned on goat fetal fibroblasts and supplemented with a growth factor cocktail that included glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), stromal cell-derived factor (SDF), and fibroblast growth factor (FGF) before use. Over 45 days, the primary cultures developed a cluster morphology indicative of in-vitro grown undifferentiated spermatogonia from other species and expressed the germ cell marker VASA, as well as the previously defined spermatogonial marker such as promyelocytic leukemia zinc finger (PLZF). Taken together, these findings provide a methodology for isolating the SSC containing undifferentiated spermatogonial population from goat testes and long-term maintenance in defined culture conditions.

A novel high throughput screen to identify candidate molecular networks that regulate spermatogenic stem cell functions†.

Spermatogenic regeneration is key for male fertility and relies on activities of an undifferentiated spermatogonial population. Here, a high-throughput approach with primary cultures of mouse spermatogonia was devised to rapidly predict alterations in functional capacity. Combining the platform with a large-scale RNAi screen of transcription factors, we generated a repository of new information from which pathway analysis was able to predict candidate molecular networks regulating regenerative functions. Extending from this database, the SRCAP-CREBBP/EP300 (Snf2-related CREBBP activator protein-CREB binding protein/E1A binding protein P300) complex was found to mediate differential levels of histone acetylation between stem cell and progenitor spermatogonia to influence expression of key self-renewal genes including the previously undescribed testis-specific transcription factor ZSCAN2 (zinc finger and SCAN domain containing 2). Single cell RNA sequencing analysis revealed that ZSCAN2 deficiency alters key cellular processes in undifferentiated spermatogonia such as translation, chromatin modification, and ubiquitination. In Zscan2 knockout mice, while spermatogenesis was moderately impacted during steady state, regeneration after cytotoxic insult was significantly impaired. Altogether, these findings have validated the utility of our high-throughput screening approach and have generated a transcription factor database that can be utilized for uncovering novel mechanisms governing spermatogonial functions.

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

Maintenance 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.

Proper timing of a quiescence period in precursor prospermatogonia is required for stem cell pool establishment in the male germline.

The stem cell-containing undifferentiated spermatogonial population in mammals, which ensures continual sperm production, arises during development from prospermatogonial precursors. Although a period of quiescence is known to occur in prospermatogonia prior to postnatal spermatogonial transition, the importance of this has not been defined. Here, using mouse models with conditional knockout of the master cell cycle regulator Rb1 to disrupt normal timing of the quiescence period, we found that failure to initiate mitotic arrest during fetal development leads to prospermatogonial apoptosis and germline ablation. Outcomes of single-cell RNA-sequencing analysis indicate that oxidative phosphorylation activity and inhibition of meiotic initiation are disrupted in prospermatogonia that fail to enter quiescence on a normal timeline. Taken together, these findings suggest that key layers of programming are laid down during the quiescent period in prospermatogonia to ensure proper fate specification and fitness in postnatal life.

Spermatogonial Stem Cell Numbers Are Reduced by Transient Inhibition of GDNF Signaling but Restored by Self-Renewing Replication when Signaling Resumes.

One cause of human male infertility is a scarcity of spermatogonial stem cells (SSCs) in testes with Sertoli cells that neither produce adequate amounts of GDNF nor form the Sertoli-Sertoli junctions that form the blood-testis barrier (BTB). These patients raise the issue of whether a pool of SSCs, depleted due to inadequate GDNF stimulation, will expand if normal signaling is restored. Here, we reduce adult mouse SSC numbers by 90% using a chemical-genetic approach that reversibly inhibits GDNF signaling. Signal resumption causes all remaining SSCs to replicate immediately, but they primarily form differentiating progenitor spermatogonia. Subsequently, self-renewing replication restores SSC numbers. Testicular GDNF levels are not increased during restoration. However, SSC replication decreases as numbers of SSCs and progenitors increase, suggesting important regulatory interactions among these cells. Finally, sequential loss of SSCs and then pachytene spermatocytes causes dissolution of the BTB, thereby recapitulating another important characteristic of some infertile men.

Unique Epigenetic Programming Distinguishes Regenerative Spermatogonial Stem Cells in the Developing Mouse Testis.

Spermatogonial stem cells (SSCs) both self-renew and give rise to progenitors that initiate spermatogenic differentiation in the mammalian testis. Questions remain regarding the extent to which the SSC and progenitor states are functionally distinct. Here we provide the first multiparametric integrative analysis of mammalian germ cell epigenomes comparable with that done for >100 somatic cell types by the ENCODE Project. Differentially expressed genes distinguishing SSC- and progenitor-enriched spermatogonia showed distinct histone modification patterns, particularly for H3K27ac and H3K27me3. Motif analysis predicted transcription factors that may regulate spermatogonial subtype-specific fate, and immunohistochemistry and gene-specific chromatin immunoprecipitation analyses confirmed subtype-specific differences in target gene binding of a subset of these factors. Taken together, these results show that SSCs and progenitors display distinct epigenetic profiling consistent with these spermatogonial subtypes being differentially programmed to either self-renew and maintain regenerative capacity as SSCs or lose regenerative capacity and initiate lineage commitment as progenitors.

Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males.

Spermatogonial stem cell transplantation (SSCT) is an experimental technique for transfer of germline between donor and recipient males that could be used as a tool for biomedical research, preservation of endangered species, and dissemination of desirable genetics in food animal populations. To fully realize these potentials, recipient males must be devoid of endogenous germline but possess normal testicular architecture and somatic cell function capable of supporting allogeneic donor stem cell engraftment and regeneration of spermatogenesis. Here we show that male mice, pigs, goats, and cattle harboring knockout alleles of the NANOS2 gene generated by CRISPR-Cas9 editing have testes that are germline ablated but otherwise structurally normal. In adult pigs and goats, SSCT with allogeneic donor stem cells led to sustained donor-derived spermatogenesis. With prepubertal mice, allogeneic SSCT resulted in attainment of natural fertility. Collectively, these advancements represent a major step toward realizing the enormous potential of surrogate sires as a tool for dissemination and regeneration of germplasm in all mammalian species.

Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

Developmental underpinnings of spermatogonial stem cell establishment.

BACKGROUND: The germline serves as a conduit for transmission of genetic and epigenetic information from one generation to the next. In males, spermatozoa are the final carriers of inheritance and their continual production is supported by a foundational population of spermatogonial stem cells (SSCs) that forms from prospermatogonial precursors during the early stages of neonatal development. In mammals, the timing for which SSCs are specified and the underlying mechanisms guiding this process remain to be completely understood. OBJECTIVES: To propose an evolving concept for how the foundational SSC population is established. MATERIALS AND METHODS: This review summarizes recent and historical findings from peer-reviewed publications made primarily with mouse models while incorporating limited studies from humans and livestock. RESULTS AND CONCLUSION: Establishment of the SSC population appears to follow a biphasic pattern involving a period of fate programming followed by an establishment phase that culminates in formation of the SSC population. This model for establishment of the foundational SSC population from precursors is anticipated to extend across mammalian species and include humans and livestock, albeit on different timescales.

mRBPome capture identifies the RNA-binding protein TRIM71, an essential regulator of spermatogonial differentiation.

Continual spermatogenesis relies on the actions of an undifferentiated spermatogonial population that is composed of stem cells and progenitors. Here, using mouse models, we explored the role of RNA-binding proteins (RBPs) in regulation of the biological activities of this population. Proteins bound to polyadenylated RNAs in primary cultures of undifferentiated spermatogonia were captured with oligo (dT)-conjugated beads after UV-crosslinking and profiled by proteomics (termed mRBPome capture), yielding a putative repertoire of 473 RBPs. From this database, the RBP TRIM71 was identified and found to be expressed by stem and progenitor spermatogonia in prepubertal and adult mouse testes. Tissue-specific deletion of TRIM71 in the male germline led to reduction of the undifferentiated spermatogonial population and a block in transition to the differentiating state. Collectively, these findings demonstrate a key role of the RBP system in regulation of the spermatogenic lineage and may provide clues about the influence of RBPs on the biology of progenitor cell populations in other lineages.

MEIOSIN: A New Watchman of Meiotic Initiation in Mammalian Germ Cells.

The capacity to undergo meiosis defines vertebrate germ cells, yet mechanisms driving initiation of this specialized process are largely undefined. In this issue of Developmental Cell,Ishiguro et al. (2020) identified the transcription factor MEIOSIN as a gatekeeper of meiotic initiation in both male and female germ cells.

Isolation of canine adipose-derived mesenchymal stem cells from falciform tissue obtained via laparoscopic morcellation: A pilot study.

OBJECTIVE: To evaluate the feasibility of stem cell isolation from falciform fat harvested via laparoscopic morcellation. STUDY DESIGN: Pilot study. ANIMALS: Eleven client-owned dogs. METHODS: Falciform was harvested traditionally via laparotomy and laparoscopically via tissue morcellation. Harvested tissue was processed with a commercially available adipose tissue dissociation kit to obtain a stromal vascular fraction (SVF). Cells were subsequently labeled for CD90, CD45, and CD44 cell surface antigens by using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting flow cytometry. CD90+ cells were quantitated, and their viability was assessed with a hemocytometer and a trypan blue exclusion test of cell viability. RESULTS: No perioperative complications occurred in dogs undergoing laparoscopic morcellation. Laparoscopically and traditionally harvested samples yielded an average of 0.39 (±0.1) × 106 and 0.33 (±0.1) × 106 CD90+ cells, respectively, per 10 million SVF cells. CD90+ cell viability after MACS was 89% (±11%) for morcellated and 86% (±7%) for traditionally harvested samples. Neither CD90+ cell quantity nor viability was different between samples obtained via traditional laparotomy vs laparoscopic morcellation (P = .38 and P = .63, respectively). Populations of CD90+ cells isolated with each harvest technique had similar CD44 and CD45 expression profiles. CONCLUSION: Viable populations of CD90+ cells with similar CD44/CD45 expression profiles were isolated from laparoscopically morcellated and traditionally harvested falciform tissue. No appreciable morbidity was associated with laparoscopic falciform morcellation. CLINICAL SIGNIFICANCE: Laparoscopic morcellation is a safe and effective minimally invasive approach to falciform tissue harvest for adipose-derived mesenchymal stem cell isolation.

Developmental kinetics and transcriptome dynamics of stem cell specification in the spermatogenic lineage.

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.

MAGE cancer-testis antigens protect the mammalian germline under environmental stress.

Ensuring robust gamete production even in the face of environmental stress is of utmost importance for species survival, especially in mammals that have low reproductive rates. Here, we describe a family of genes called melanoma antigens (MAGEs) that evolved in eutherian mammals and are normally restricted to expression in the testis (http://MAGE.stjude.org) but are often aberrantly activated in cancer. Depletion of Mage-a genes disrupts spermatogonial stem cell maintenance and impairs repopulation efficiency in vivo. Exposure of Mage-a knockout mice to genotoxic stress or long-term starvation that mimics famine in nature causes defects in spermatogenesis, decreased testis weights, diminished sperm production, and reduced fertility. Last, human MAGE-As are activated in many cancers where they promote fuel switching and growth of cells. These results suggest that mammalian-specific MAGE genes have evolved to protect the male germline against environmental stress, ensure reproductive success under non-optimal conditions, and are hijacked by cancer cells.

Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation†.

Gene editing technologies, such as CRISPR-Cas9, have important applications in mammalian embryos for generating novel animal models in biomedical research and lines of livestock with enhanced production traits. However, the lack of methods for efficient introduction of gene editing reagents into zygotes of various species and the need for surgical embryo transfer in mice have been technical barriers of widespread use. Here, we described methodologies that overcome these limitations for embryos of mice, cattle, and pigs. Using mutation of the Nanos2 gene as a readout, we refined electroporation parameters with preassembled sgRNA-Cas9 RNPs for zygotes of all three species without the need for zona pellucida dissolution that led to high-efficiency INDEL edits. In addition, we optimized culture conditions to support maturation from zygote to the multicellular stage for all three species that generates embryos ready for transfer to produce gene-edited animals. Moreover, for mice, we devised a nonsurgical embryo transfer method that yields offspring at an efficiency comparable to conventional surgical approaches. Collectively, outcomes of these studies provide simplified pipelines for CRISPR-Cas9-based gene editing that are applicable in a variety of mammalian species.