Spermatogonial Stem Cell Niche Research Articles

Fertility Preservation in Children and Adolescents: Where We Are and Where We Are Going.

This review will describe current pediatric and adolescent fertility preservation methodologies and the ethical concerns surrounding these procedures, as well as highlight recent research that may pave the way for the development of new fertility preservation options. Research is ongoing to allow prepubertal patients, particularly those with testes, to be able to have biologic children in the future. Studies on sperm in vitro maturation highlight the importance of supporting the spermatogonial stem cell niche for the development of mature sperm. The live birth of a rhesus macaque from in vitro fertilization using prepubertal testicular tissue and in vivo matured sperm gives hope to future human births. For patients with ovaries, prior work has led to successful fertility but further research is underway to refine these techniques and optimize outcomes. Organoid scaffolds have shown promise when being used for in vitro oocyte maturation. For children and adolescents undergoing gonadotoxic treatment, such as chemotherapy, or hormonal treatment, such as gender-affirming hormone therapy, future fertility potential may be negatively impacted. It is recommended that fertility preservation (FP) be offered to these patients and families prior to undergoing treatment. Fertility preservation for postpubertal patients mimics that in adults. For prepubertal children, however, the options are limited and in some cases still experimental. It is essential that this work continues so that we may offer children and adolescents the right to an open future and preserve their fertility potential.

Fertility Protection, A Novel Concept: Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Protect against Chemotherapy-Induced Testicular Cytotoxicity.

Currently, there is no viable option for fertility preservation in prepubertal boys. Experimentally, controlled vitrification of testicular tissue has been evaluated and found to cause potential structural damage to the spermatogonial stem cell (SSC) niche during cryopreservation. In this report, we leveraged the regenerative effect of human umbilical cord-derived Mesenchymal stem cell exosomes (h-UCMSC-Exo) to protect against testicular damage from the cytotoxic effects of polychemotherapy (CTX). A chemotherapy-induced testicular dysfunctional model was established by CTX treatment with cyclophosphamide and Busulfan in vitro (human Sertoli cells) and in prepubescent mice. We assessed the effects of the exosomes by analyzing cell proliferation assays, molecular analysis, immunohistochemistry, body weight change, serum hormone levels, and fertility rate. Our data indicates the protective effect of h-UCMSC-Exo by preserving the SSC niche and preventing testicular damage in mice. Interestingly, mice that received multiple injections of h-UCMSC-Exo showed significantly higher fertility rates and serum testosterone levels (p < 0.01). Our study demonstrates that h-UCMSC-Exo can potentially be a novel fertility protection approach in prepubertal boys triaged for chemotherapy treatment.

Responses of peritubular macrophages and the testis transcriptome profiles of peripubertal and adult rodents exposed to an acute dose of MEHP.

Exposure of rodents to mono-(2-ethylhexyl) phthalate (MEHP) is known to disrupt the blood-testis barrier and cause testicular germ cell apoptosis. Peritubular macrophages (PTMφ) are a newly identified type of testicular macrophage that aggregates near the spermatogonial stem cell niche. We have previously reported that MEHP exposure increased the numbers of PTMφs by 6-fold within the testis of peripubertal rats. The underlying mechanism(s) accounting for this change in PTMφs and its biological significance is unknown. This study investigates if MEHP-induced alterations in PTMφs occur in rodents (PND 75 adult rats and PND 26 peripubertal mice) that are known to be less sensitive to MEHP-induced testicular toxicity. Results show that adult rats have a 2-fold higher basal level of PTMφ numbers than species-matched peripubertal animals, but there was no significant increase in PTMφ numbers after MEHP exposure. Peripubertal mice have a 5-fold higher basal level of PTMφ compared with peripubertal rats but did not exhibit increases in number after MEHP exposure. Further, the interrogation of the testis transcriptome was profiled from both the MEHP-responsive peripubertal rats and the less sensitive rodents via 3' Tag sequencing. Significant changes in gene expression were observed in peripubertal rats after MEHP exposure. However, adult rats showed lesser changes in gene expression, and peripubertal mice showed only minor changes. Collectively, the data show that PTMφ numbers are associated with the sensitivity of rodents to MEHP in an age- and species-dependent manner.

TLR4 signalling protects Sertoli cells from cell stress via reprogramming inflammasome and autophagy pathways in MAPK1/ERK2 dependent way

Sertoli cells constitutively maintain the spermatogonial stem cell niche and blood-testis barrier via impenetrable tight junctions under hormonal, growth factor and cytokine control. Immature Sertoli cell proliferation and survival benefit from FSH, NF-κB, and IL-1α-dependent signalling effective via the MAPK family of pathways and related to p38 MAPK dependent Ca2+ and K+ fluxes. Little is known about how the NF-κB and MAPK axis interact with pro-inflammatory NOD1/TLR4 and different voltage-gated channels. Silencing MAPK1, we found that MAPK1/ERK2 has a synergistic role with TLR4 in restricting the pro-inflammatory NF-κB/Caspase-1/IL-1β axis while having an antagonistic effect on TLR4 in promoting adult 15 P-1 Sertoli cell line proliferation and migration. In MAPK1/ERK2 absence, there was an unexpected TLR4-NF-κB behaviour, showing a strong NF-κB transactivation, assessed using a pNIFTY-SEAP reporter plasmid, accompanied by significantly activated cell migration and proliferation, as demonstrated by the wound healing assay. Voltage-gated potassium flux, most likely via Kv1.3, was significantly increased after TLR4 and NOD1 challenge in ATP DAMP signal presence, associated with inflammasome activation. TLR4 challenge also activated macroautophagy while inhibiting mitophagy. Thus, TLR4 activation restricted the NF-κB and Caspase-1/IL-1β pro-inflammatory axis, cell migration and proliferation, voltage-gated potassium efflux, and shifted mitophagy to macroautophagy (towards an inflammasome pathway-related cell death), promoting cell survival. MAPK1/ERK2 had a differential effect, as in its absence, TLR4 activation had the opposite effect on NF-κB transactivation and cell migration and proliferation. Thus, TLR4/MAPK1/NF-κB/Kv1.3 interaction provides optimal Sertoli cell maintenance conditions and allows cell death only in extreme circumstances of cumulative DAMP/PAMP signalling stimulation.

Decoding the spermatogonial stem cell niche under physiological and recovery conditions in adult mice and humans.

The intricate interaction between spermatogonial stem cell (SSC) and testicular niche is essential for maintaining SSC homeostasis; however, this interaction remains largely uncharacterized. In this study, to characterize the underlying signaling pathways and related paracrine factors, we delineated the intercellular interactions between SSC and niche cell in both adult mice and humans under physiological conditions and dissected the niche-derived regulation of SSC maintenance under recovery conditions, thus uncovering the essential role of C-C motif chemokine ligand 24 and insulin-like growth factor binding protein 7 in SSC maintenance. We also established the clinical relevance of specific paracrine factors in human fertility. Collectively, our work on decoding the adult SSC niche serves as a valuable reference for future studies on the aetiology, diagnosis, and treatment of male infertility.

Dissecting the spermatogonial stem cell niche using spatial transcriptomics

Spermatogonial stem cells (SSCs) in the testis support the lifelong production of sperm. SSCs reside within specialized microenvironments called "niches," which are essential for SSC self-renewal and differentiation. However, our understanding of the molecular and cellular interactions between SSCs and niches remains incomplete. Here, we combine spatial transcriptomics, computational analyses, and functional assays to systematically dissect the molecular, cellular, and spatial composition of SSC niches. This allows us to spatially map the ligand-receptor (LR) interaction landscape in both mouse and human testes. Our data demonstrate that pleiotrophin regulates mouse SSC functions through syndecan receptors. We also identify ephrin-A1 as a potential niche factor that influences human SSC functions. Furthermore, we show that the spatial re-distribution of inflammation-related LR interactions underlies diabetes-induced testicular injury. Together, our study demonstrates a systems approach to dissect the complex organization of the stem cell microenvironment in health and disease.

Potency Assays for Mesenchymal Stromal Cell Secretome-Based Products for Tissue Regeneration.

Adult stem cells maintaining tissue homeostasis and regeneration are tightly regulated by their specific microenvironments or stem cell niches. The dysfunction of niche components may alter the activity of stem cells and ultimately lead to intractable chronic or acute disorders. To overcome this dysfunction, niche-targeting regenerative medicine treatments such as gene, cell, and tissue therapy are actively investigated. Here, multipotent mesenchymal stromal cells (MSCs), and particularly their secretomes, are of high interest due to their potency to recover and reactivate damaged or lost stem cell niches. However, a workflow for the development of MSC secretome-based products is not fully covered by regulatory authorities, and and this issue significantly complicates their clinical translation and has possibly been expressed in a huge number of failed clinical trials. One of the most critical issues in this regard relates to the development of potency assays. In this review, guidelines for biologicals and cell therapies are considered to be applied for the development of potency assays for the MSC secretome-based products that aim for tissue regeneration. Specific attention is paid to their possible effects on stem cell niches and to a spermatogonial stem cell niche in particular.

Lovastatin promotes the self-renewal of murine and primate spermatogonial stem cells.

The spermatogonial stem cell (SSC) niche is critical for SSC maintenance and subsequent spermatogenesis. Numerous reproductive hazards impair the SSC niche, thereby resulting in aberrant SSC self-renewal and male infertility. However, promising agents targeting the impaired SSC niche to promote SSC self-renewal are still limited. Here, we screen out and assess the effects of Lovastatin on the self-renewal of mouse SSCs (mSSCs). Mechanistically, Lovastatin promotes the self-renewal of mSSCs and inhibits its inflammation and apoptosis through the regulation of isoprenoid intermediates. Remarkably, treatment by Lovastatin could promote the proliferation of undifferentiated spermatogonia in the male gonadotoxicity model generated by busulfan injection. Of note, we demonstrate that Lovastatin could enhance the proliferation of primate undifferentiated spermatogonia. Collectively, our findings uncover that lovastatin could promote the self-renewal of both murine and primate SSCs and have implications for the treatment of certain types of male infertility using small compounds.

Comparing the adult and pre-pubertal testis: Metabolic transitions and the change in the spermatogonial stem cell metabolic microenvironment.

Survivors of childhood cancer often suffer from infertility. While sperm cryopreservation is not feasible before puberty, the patient's own spermatogonial stem cells could serve as a germ cell reservoir, enabling these patients to father their own children in adulthood through the isolation, in vitro expansion, and subsequent transplantation of spermatogonial stem cells. However, this approach requires large numbers of stem cells, and methods for successfully propagating spermatogonial stem cells in the laboratory are yet to be established for higher mammals and humans. The improvement of spermatogonial stem cell culture requires deeper understanding of their metabolic requirements and the mechanisms that regulate metabolic homeostasis. This review gives a summary on our knowledge of spermatogonial stem cell metabolism during maintenance and differentiation and highlights the potential influence of Sertoli cell and stem cell niche maturation on spermatogonial stem cell metabolic requirements during development. Fetal human spermatogonial stem cell precursors, or gonocytes, migrate into the seminiferous cords and supposedly mature to adult stem cells within the first year of human development. However, the spermatogonial stem cell niche does not fully differentiate until puberty, when Sertoli cells dramatically rearrange the architecture and microenvironment within the seminiferous epithelium. Consequently, pre-pubertal and adult spermatogonial stem cells experience two distinct niche environments potentially affecting spermatogonial stem cell metabolism and maturation. Indeed, the metabolic requirements of mouse primordial germ cells and pig gonocytes are distinct from their adult counterparts, and novel single-cell RNA sequencing analysis of human and porcine spermatogonial stem cells during development confirms this metabolic transition. Knowledge of the metabolic requirements and their changes and regulation during spermatogonial stem cell maturation is necessary to implement laboratory-based techniques and enable clinical use of spermatogonial stem cells. Based on the advancement in our understanding of germline metabolism circuits and maturation events of niche cells within the testis, we propose a new definition of spermatogonial stem cell maturation and its amendment in the light of metabolic change.

Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, a complex process supported by a specialized microenvironment, called the SSC niche. Postnatal development of SSCs is characterized by distinct metabolic transitions from prepubertal to adult stages. An understanding of the niche factors that regulate these maturational events is critical for the clinical application of SSCs in fertility preservation. To investigate the niche maturation events that take place during SSC maturation, we combined different '-omics' technologies. Serial single cell RNA sequencing analysis revealed changes in the transcriptomes indicative of niche maturation that was initiated at 11 years of age in humans and at 8 weeks of age in pigs, as evident by Monocle analysis of Sertoli cells and peritubular myoid cell (PMC) development in humans and Sertoli cell analysis in pigs. Morphological niche maturation was associated with lipid droplet accumulation, a characteristic that was conserved between species. Lipidomic profiling revealed an increase in triglycerides and a decrease in sphingolipids with Sertoli cell maturation in the pig model. Quantitative (phospho-) proteomics analysis detected the activation of distinct pathways with porcine Sertoli cell maturation. We show here that the main aspects of niche maturation coincide with the morphological maturation of SSCs, which is followed by their metabolic maturation. The main aspects are also conserved between the species and can be predicted by changes in the niche lipidome. Overall, this knowledge is pivotal to establishing cell/tissue-based biomarkers that could gauge stem cell maturation to facilitate laboratory techniques that allow for SSC transplantation for restoration of fertility.

The Molecular Signature of Human Testicular Peritubular Cells Revealed by Single-Cell Analysis.

Peritubular cells of the human testis form a small compartment surrounding the seminiferous tubules. They are crucial for sperm transport, and they emerge as contributors to the spermatogonial stem cell niche. They are among the least known cell types of the human body. We employed single-cell RNA sequencing of cultured human testicular peritubular cells (HTPCs), which had been isolated from testicular samples of donors with normal spermatogenesis. The significant overlap between our results and recently published ex vivo data indicates that HTPCs are a highly adequate cellular model to define and study these cells. Thus, based on the expression of several markers, HTPCs can be classified as testicular smooth muscle cells. Small differences between the in vivo/in vitro expressed genes may be due to cellular plasticity. Plasticity was also shown upon addition of FCS to the culture medium. Based on transcriptome similarities, four cellular states were identified. Further analyses confirmed the presence of known stem cell niche-relevant factors (e.g., GDNF) and identified unknown functions, e.g., the ability to produce retinoic acid. Therefore, HTPCs allow us to define the signature(s) and delineate the functions of human testicular peritubular cells. The data may also serve as a resource for future studies to better understand male (in)fertility.

Effects of Pgam1-mediated glycolysis pathway in Sertoli cells on Spermatogonial stem cells based on transcriptomics and energy metabolomics.

Spermatogenesis is a complex process involving a variety of intercellular interactions and precise regulation of gene expression. Spermatogenesis is sustained by a foundational Spermatogonial stem cells (SSCs) and in mammalian testis. Sertoli cells (SCs) are the major component of SSC niche. Sertoli cells provide structural support and supply energy substrate for developing germ cells. Phosphoglycerate mutase 1 (Pgam1) is a key enzyme in the glycolytic metabolism and our previous work showed that Pgam1 is expressed in SCs. In the present study, hypothesized that Pgam1-depedent glycolysis in SCs plays a functional role in regulating SSCs fate decisions. A co-culture system of murine SCs and primary spermatogonia was constructed to investigate the effects of Pgam1 knockdown or overexpression on SSCs proliferation and differentiation. Transcriptome results indicated that overexpression and knockdown of Pgam1 in SCs resulted in up-regulation of 458 genes (117 down-regulated, 341 up-regulated) and down-regulation of 409 genes (110 down-regulated, 299 up-regulated), respectively. Further analysis of these DEGs revealed that GDNF, FGF2 and other genes that serve key roles in SSCs niche maintenance were regulated by Pgam1. The metabolome results showed that a total of 11 and 16 differential metabolites were identified in the Pgam1 gene overexpression and knockdown respectively. Further screening of these metabolites indicated that Sertoli cell derived glutamate, glutamine, threonine, leucine, alanine, lysine, serine, succinate, fumarate, phosphoenolpyruvate, ATP, ADP, and AMP have potential roles in regulating SSCs proliferation and differentiation. In summary, this study established a SCs-SSCs co-culture system and identified a list of genes and small metabolic molecules that affect the proliferation and differentiation of SSCs. This study provides additional insights into the regulatory mechanisms underlying interactions between SCs and SSCs during mammalian spermatogenesis.

Sertoli Cell-Germ Cell Interactions Within the Niche: Paracrine and Juxtacrine Molecular Communications.

Male germ cell development depends on multiple biological events that combine epigenetic reprogramming, cell cycle regulation, and cell migration in a spatio-temporal manner. Sertoli cells are a crucial component of the spermatogonial stem cell niche and provide essential growth factors and chemokines to developing germ cells. This review focuses mainly on the activation of master regulators of the niche in Sertoli cells and their targets, as well as on novel molecular mechanisms underlying the regulation of growth and differentiation factors such as GDNF and retinoic acid by NOTCH signaling and other pathways.

Cullin4 E3 Ubiquitin Ligases Regulate Male Gonocyte Migration, Proliferation and Blood-Testis Barrier Homeostasis.

Ubiquitination, an essential posttranslational modification, plays fundamental roles during mammalian spermatogenesis. We previously reported the requirement of two Cullin 4 ubiquitin ligase family genes, Cullin 4a (Cul4a) and Cullin 4b (Cul4b), in murine spermatogenesis. Both genes are required for male fertility despite their distinct functions in different cell populations. Cul4a is required in primary spermatocytes to promote meiosis while Cul4b is required in secondary spermatocytes for spermiogenesis. As the two genes encode proteins that are highly homologous and have overlapping expression in embryonic germ cells, they may compensate for each other during germ cell development. In the present study, we directly address the potential functional redundancy of these two proteins by deleting both Cul4 genes, specifically, in the germ cell lineage during embryonic development, using the germ-cell specific Vasa-Cre line. Conditional double-knockout (dKO) males showed delayed homing and impaired proliferation of gonocytes, and a complete loss of germ cells before the end of the first wave of spermatogenesis. The dKO male germ cell phenotype is much more severe than those observed in either single KO mutant, demonstrating the functional redundancy between the two CUL4 proteins. The dKO mutant also exhibited atypical tight junction structures, suggesting the potential involvement of CUL4 proteins in spermatogonial stem cell (SSC) niche formation and blood–testis-barrier (BTB) maintenance. We also show that deleting Cul4b in both germ and Sertoli cells is sufficient to recapitulate part of this phenotype, causing spermatogenesis defects and drastically reduced number of mature sperms, accompanied by defective tight junctions in the mutant testes. These results indicate the involvement of CUL4B in maintaining BTB integrity.

P-798 Fertility preservation in pre-pubertal boys with cancer: A three-dimensional prepubertal testicular organoid for in vitro spermatogonial stem cell propagation and spermatogenesis

Abstract Study question Can a three-dimensional (3D) prepubertal testicular organoid be formed and provide an in vitro microenvironment for spermatogonial stem cells (SSCs) maintenance and future spermatogenesis? Summary answer Primary cells extracted from immature testicular tissue (ITT) or SSCs can be grown long-term as 3D organoids, providing the potential for in vitro study. What is known already Aggressive cancer treatments, such as chemo- or radiotherapy, can leave young prepubertal boys infertile. Such patients are recommended to undergo the cryopreservation of testicular material to protect future fertility. Within the testes, the specialized 3D structure and direct cell-to-cell interactions play a critical role in the proliferation and development of SSCs. Over recent decades, 3D culture systems and organoids have been used to culture cells in vitro, however, a system that allows investigations into testicular organogenesis in vitro, and its impact on the SSC niche, has yet to be developed. Study design, size, duration This study aims to develop a 3D organoid culture system to support the proliferation of SSCs and spermatogenesis. Primary bovine ITT cells and enriched SSCs were isolated and 3D organoids were generated by in vitro culture for up to 40 days. Organoid formation was observed after using different foundation cells seeded at different densities and cultured in medium containing gonadotropic supplements. Participants/materials, setting, methods Post-thaw bovine ITTs (2 weeks-of-age) were dissociated using two-step enzymatic digestion. Enriched SSCs were selected by Percoll gradients and differential plating. Viability and apoptosis were evaluated by trypan blue staining and TUNEL assays, respectively. SSCs were evaluated immunocytochemically for germ-cell markers (PGP-9.5, PLZF) and Sertoli cell markers (Vimentin, Sox9). Expression levels of SSCs and spermatogenesis-related genes (Plzf, Gfrα-1, Nanog, Oct4, Stra8, Thy1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Main results and the role of chance The viability of digested cells from thawed ITTs was 78.667% ± 2.03. Total testicular cells (&amp;lt;10% SSCs) and enriched SSCs(&amp;gt;50% SSCs) were observed to self-assemble into structurally complex organoids recapitulating the cell type compartmentalization of the testis, in a 3D Matrigel-based culture system with 10% knockout serum replacement (KSR) culture medium, but not with 10% fetal bovine serum(FBS) medium. Testicular organoids were found to exhibit either a grape-like structure and a round-shape structure. Cytoplasmic extensions of spermatogonia/Sertoli cells were in contact with each other within a forming colony. Organoids were formed faster and larger when seeded at a final concentration of 1.5 × 106 cells/ml, compared to 5 × 105 cells/ml and 1.5 × 105 cells/ml. Organoids grew to a diameter of 400 μm within 10 - 15 days and were passaged by mechanical disruption at a ratio of 1:3 every 7 - 10 days. Immunocytochemistry results showed that clusters of PGP9.5 and PLZF-positive cells were present within the organoids. The expression of selected germ cell and spermatogenesis markers in the testicular organoids closely resembled that of primary testicular cells for up to 20 days of culture. Limitations, reasons for caution We used calves (2 weeks-of-age) as an animal model to study testicular organoids. This tissue may act differently than human tissues and may not fully represent prepuberty. Furthermore, we only evaluated gene expression levels for selected markers that may not represent the full functional capability of germ cells. Wider implications of the findings Testicular organoids, as an in vitro bioengineering testicular model, could potentially be used to study testicular tissue development, cellular interactions, endocrinology, and spermatogenesis, in the laboratory but may also be applied for clinical purposes in the future. Trial registration number University of Oxford

P–798 Fertility preservation in pre-pubertal boys with cancer: A three-dimensional prepubertal testicular organoid for in vitro spermatogonial stem cell propagation and spermatogenesis

Abstract Study question Can a three-dimensional (3D) prepubertal testicular organoid be formed and provide an in vitro microenvironment for spermatogonial stem cells (SSCs) maintenance and future spermatogenesis? Summary answer Primary cells extracted from immature testicular tissue (ITT) or SSCs can be grown long-term as 3D organoids, providing the potential for in vitro study. What is known already Aggressive cancer treatments, such as chemo- or radiotherapy, can leave young prepubertal boys infertile. Such patients are recommended to undergo the cryopreservation of testicular material to protect future fertility. Within the testes, the specialized 3D structure and direct cell-to-cell interactions play a critical role in the proliferation and development of SSCs. Over recent decades, 3D culture systems and organoids have been used to culture cells in vitro, however, a system that allows investigations into testicular organogenesis in vitro, and its impact on the SSC niche, has yet to be developed. Study design, size, duration This study aims to develop a 3D organoid culture system to support the proliferation of SSCs and spermatogenesis. Primary bovine ITT cells and enriched SSCs were isolated and 3D organoids were generated by in vitro culture for up to 40 days. Organoid formation was observed after using different foundation cells seeded at different densities and cultured in medium containing gonadotropic supplements. Participants/materials, setting, methods Post-thaw bovine ITTs (2 weeks-of-age) were dissociated using two-step enzymatic digestion. Enriched SSCs were selected by Percoll gradients and differential plating. Viability and apoptosis were evaluated by trypan blue staining and TUNEL assays, respectively. SSCs were evaluated immunocytochemically for germ-cell markers (PGP–9.5, PLZF) and Sertoli cell markers (Vimentin, Sox9). Expression levels of SSCs and spermatogenesis-related genes (Plzf, Gfrα–1, Nanog, Oct4, Stra8, Thy1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Main results and the role of chance The viability of digested cells from thawed ITTs was 78.667% ± 2.03. Total testicular cells (&amp;lt;10% SSCs) and enriched SSCs(&amp;gt;50% SSCs) were observed to self-assemble into structurally complex organoids recapitulating the cell type compartmentalization of the testis, in a 3D Matrigel-based culture system with 10% knockout serum replacement (KSR) culture medium, but not with 10% fetal bovine serum(FBS) medium. Testicular organoids were found to exhibit either a grape-like structure and a round-shape structure. Cytoplasmic extensions of spermatogonia/Sertoli cells were in contact with each other within a forming colony. Organoids were formed faster and larger when seeded at a final concentration of 1.5 × 106 cells/ml, compared to 5 × 105 cells/ml and 1.5 × 105 cells/ml. Organoids grew to a diameter of 400 μm within 10 - 15 days and were passaged by mechanical disruption at a ratio of 1:3 every 7 - 10 days. Immunocytochemistry results showed that clusters of PGP9.5 and PLZF-positive cells were present within the organoids. The expression of selected germ cell and spermatogenesis markers in the testicular organoids closely resembled that of primary testicular cells for up to 20 days of culture. Limitations, reasons for caution We used calves (2 weeks-of-age) as an animal model to study testicular organoids. This tissue may act differently than human tissues and may not fully represent prepuberty. Furthermore, we only evaluated gene expression levels for selected markers that may not represent the full functional capability of germ cells. Wider implications of the findings: Testicular organoids, as an in vitro bioengineering testicular model, could potentially be used to study testicular tissue development, cellular interactions, endocrinology, and spermatogenesis, in the laboratory but may also be applied for clinical purposes in the future. Trial registration number University of Oxford

The transformative impact of extracellular vesicles on developing sperm.

ObjectiveTo review the role of extracellular vesicles (EVs) released from the male reproductive tract and their impact on developing sperm. We discuss how sperm exiting the seminiferous tubules, although developmentally mature, require further modification. Acquisition of various functions including increased motility, transfer of cargoes and ability to undertake the acrosome reaction is mediated through the interaction between sperm and EVs.Methods A review of the literature identified that EVs are released from different portions of the male reproductive tract, notably the epididymis and prostate. These EVs interact with sperm as they pass from the seminiferous tubules to the epididymis and vas deferens prior to ejaculation.ResultsEVs are small lipid-bound particles carrying bespoke RNA, protein and lipid cargoes. These cargoes are loaded based on the state of the parent cell and are used to communicate with recipient cells. In sperm, these cargoes are essential for post-testicular modification.ConclusionsInteractions between developing sperm and EVs are important for the subsequent function of sperm. Prior to ejaculation, these interactions confer important changes for the post-testicular modification and development of sperm. Little is known about the interaction between EVs from the testes and the spermatogonial stem cell niche or developing sperm within the seminiferous tubules. However, the numerous roles of EVs in the post-testicular modification of sperm have led many to suspect that they may also play important roles in developing sperm within the testes.Lay summarySperm are crucial for successful fertility. In order to do this, they must be able to swim a large distance to meet the egg in the female reproductive tract and fertilise it. Once released from the testes, sperm may appear to be fully developed, but this is not the case. Several important modifications are required in order for them to swim and fertilise an egg. These modifications are carried out by sending sperm small packages from other cells which contain messages and cargo. We discuss the release of these small packages along with different parts of the male reproductive tract and how they change the way sperm behave and function. This article reviews the literature and known functions of these packages called extracellular vesicles, which are released by the male reproductive tract and modify sperm, transforming their function, before they are ejaculated.

Peritubular Macrophages Are Recruited to the Testis of Peripubertal Rats After Mono-(2-Ethylhexyl) Phthalate Exposure and Is Associated With Increases in the Numbers of Spermatogonia.

Peripubertal exposure of male rodents to the phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP) causes testicular inflammation, spermatocyte apoptosis, and disruption of the blood-testis barrier. The MEHP-induced inflammatory response in the testis includes an infiltration of macrophages and neutrophils, although the cause and purpose of this response is unknown. Recently, a population of testicular macrophages known as peritubular macrophages that are phenotypically distinct from those resident in interstitium was described in mice. Peritubular macrophages aggregate near the spermatogonial stem cell niche and are believed to stimulate their differentiation. We hypothesized that if testicular peritubular macrophages do indeed stimulate spermatogonial differentiation, MEHP exposure would result in an increase of peritubular macrophages to stimulate the replacement of lost spermatocytes. Male rats were exposed to 700 mg/kg MEHP or corn oil (vehicle control) via oral gavage at postnatal day 28 and euthanized at 48 h, 1 or 2 weeks later. Seminiferous tubules were stained with immunofluorescent markers for macrophages (major histocompatibility complex class II [MHC-II+]) and undifferentiated spermatogonia (PLZF). Peritubular macrophages were observed in rat testis: MHC-II+ cells on the surface of seminiferous tubules with heterogeneous morphology. Quantification of MHC-II+ cells revealed that, unlike in the mouse, their numbers did not increase through puberty (2-week period). MEHP increased macrophage presence by 6-fold 48 h after exposure and remained elevated by 2-fold 2 weeks after exposure. An increase of differentiating spermatogonia occurred 2 weeks after MEHP exposure. Taken together, our results suggest that peritubular macrophages play a crucial role in the testis response to acute injury and the subsequent recovery of spermatogenesis.

Regulation of Cell Types Within Testicular Organoids.

Organoids are 3-dimensional (3D) structures grown in vitro that emulate the cytoarchitecture and functions of true organs. Therefore, testicular organoids arise as an important model for research on male reproductive biology. These organoids can be generated from different sources of testicular cells, but most studies to date have used immature primary cells for this purpose. The complexity of the mammalian testicular cytoarchitecture and regulation poses a challenge for working with testicular organoids, because, ideally, these 3D models should mimic the organization observed in vivo. In this review, we explore the characteristics of the most important cell types present in the testicular organoid models reported to date and discuss how different factors influence the regulation of these cells inside the organoids and their outcomes. Factors such as the developmental or maturational stage of the Sertoli cells, for example, influence organoid generation and structure, which affect the use of these 3D models for research. Spermatogonial stem cells have been a focus recently, especially in regard to male fertility preservation. The regulation of the spermatogonial stem cell niche inside testicular organoids is discussed in the present review, as this research area may be positively affected by recent progress in organoid generation and tissue engineering. Therefore, the testicular organoid approach is a very promising model for male reproductive biology research, but more studies and improvements are necessary to achieve its full potential.

Spermatogonia Loss Correlates with LAMA 1 Expression in Human Prepubertal Testes Stored for Fertility Preservation.

Fertility preservation for male childhood cancer survivors not yet capable of producing mature spermatozoa, relies on experimental approaches such as testicular explant culture. Although the first steps in somatic maturation can be observed in human testicular explant cultures, germ cell depletion is a common obstacle. Hence, understanding the spermatogonial stem cell (SSC) niche environment and in particular, specific components such as the seminiferous basement membrane (BM) will allow progression of testicular explant cultures. Here, we revealed that the seminiferous BM is established from 6 weeks post conception with the expression of laminin alpha 1 (LAMA 1) and type IV collagen, which persist as key components throughout development. With prepubertal testicular explant culture we found that seminiferous LAMA 1 expression is disrupted and depleted with culture time correlating with germ cell loss. These findings highlight the importance of LAMA 1 for the human SSC niche and its sensitivity to culture conditions.