Spermatogonial Stem Cell Transplantation Research Articles

In vivo and in vitro sperm production: an overview of the challenges and advances in male fertility restoration.

Male infertility can be caused by genetic anomalies, endocrine disorders, inflammation, and exposure to toxic chemicals or gonadotoxic treatments. Therefore, several recent studies have concentrated on the preservation and restoration of fertility to enhance the quality of life for affected individuals. It is currently recommended to biobank the tissue extracted from testicular biopsies to provide a later source of spermatogonial stem cells (SSCs). Another successful approach has been the in vitro production of haploid male germ cells. The capacity of SSCs to transform into sperm, as in testicular tissue transplantation, SSC therapy, and in vitro or ex vivo spermatogenesis, makes them ideal candidates for in vivo fertility restoration. The transplantation of SSCs or testicular tissue to regenerate spermatogenesis and create embryos has been achieved in nonhuman mammal species. Although the outcomes of human trials have yet to be released, this method may soon be approved for clinical use in humans. Furthermore, regenerative medicine techniques that develop tissue or cells on organic or synthetic scaffolds enriched with bioactive molecules have also gained traction. All of these methods are now in different stages of experimentation and clinical trials. However, thanks to rigorous studies on the safety and effectiveness of SSC-based reproductive treatments, some of these techniques may be clinically available in upcoming decades.

Restoration of fertility in nonablated recipient mice after spermatogonial stem cell transplantation

Spermatogonial stem cell (SSC) transplantation is a valuable tool for studying stem cell-niche interaction. However, the conventional approach requires the removal of endogenous SSCs, causing damage to the niche. Here we introduce WIN18,446, an ALDH1A2 inhibitor, to enhance SSC colonization in nonablated recipients. Pre-transplantation treatment with WIN18,446 induced abnormal claudin protein expression, which comprises the blood-testis barrier and impedes SSC colonization. Consequently, WIN18,446 increased colonization efficiency by 4.6-fold compared with untreated host. WIN18,446-treated testes remained small despite the cessation of WIN18,446, suggesting its irreversible effect. Offspring were born by microinsemination using donor-derived sperm. While WIN18,446 was lethal to busulfan-treated mice, cyclophosphamide- or radiation-treated animals survived after WIN18,446 treatment. Although WIN18,446 is not applicable to humans due to toxicity, similar ALDH1A2 inhibitors may be useful for SSC transplantation into nonablated testes, shedding light on the role of retinoid metabolism on SSC-niche interactions and advancing SSC research in animal models and humans.

Transplantation of Spermatogonial Stem Cells in Stallions

Transplantation of Spermatogonial Stem Cells in Stallions

Favorable culture conditions for spermatogonial propagation in human and non-human primate primary testicular cell cultures: a systematic review and meta-analysis.

Introduction: Autologous transplantation of spermatogonial stem cells (SSCs) isolated from cryopreserved testicular biopsies obtained before oncological treatment could restore fertility in male childhood cancer survivors. There is a clear necessity for in vitro propagation of the limited SSCs from the testicular biopsy prior to transplantation due to limited numbers of spermatogonia in a cryopreserved testicular biopsy. Still, there is no consensus regarding their optimal culture method. Methods: We performed a systematic review and meta-analysis of studies reporting primary testicular cell cultures of human and non-human primate origin through use of Pubmed, EMBASE, and Web of Science core collection databases. Of 760 records, we included 42 articles for qualitative and quantitative analysis. To quantify in vitro spermatogonial propagation, spermatogonial colony doubling time (CDT) was calculated, which measures the increase in the number of spermatogonial colonies over time. A generalized linear mixed model analysis was used to assess the statistical effect of various culture conditions on CDT. Results: Our analysis indicates decreased CDTs, indicating faster spermatogonial propagation in cultures with a low culture temperature (32°C); with use of non-cellular matrices; use of StemPro-34 medium instead of DMEM; use of Knockout Serum Replacement; and when omitting additional growth factors in the culture medium. Discussion: The use of various methods and markers to detect the presence of spermatogonia within the reported cultures could result in detection bias, thereby potentially influencing comparability between studies. However, through use of CDT in the quantitative analysis this bias was reduced. Our results provide insight into critical culture conditions to further optimize human spermatogonial propagation in vitro, and effectively propagate and utilize these cells in a future fertility restoration therapy and restore hope of biological fatherhood for childhood cancer survivors.

DAZL Knockout Pigs as Recipients for Spermatogonial Stem Cell Transplantation.

Spermatogonial stem cell (SSC) transplantation into the testis of a germ cell (GC)-depleted surrogate allows transmission of donor genotype via donor-derived sperm produced by the recipient. Transplantation of gene-edited SSCs provides an approach to propagate gene-edited large animal models. DAZL is a conserved RNA-binding protein important for GC development, and DAZL knockout (KO) causes defects in GC commitment and differentiation. We characterized DAZL-KO pigs as SSC transplantation recipients. While there were GCs in 1-week-old (wko) KO, complete GC depletion was observed by 10 wko. Donor GCs were transplanted into 18 DAZL-KO recipients at 10-13 wko. At sexual maturity, semen and testes were evaluated for transplantation efficiency and spermatogenesis. Approximately 22% of recipient seminiferous tubules contained GCs, including elongated spermatids and proliferating spermatogonia. The ejaculate of 89% of recipients contained sperm, exclusively from donor origin. However, sperm concentration was lower than the wild-type range. Testicular protein expression and serum hormonal levels were comparable between DAZL-KO and wild-type. Intratesticular testosterone and Leydig cell volume were increased, and Leydig cell number decreased in transplanted DAZL-KO testis compared to wild-type. In summary, DAZL-KO pigs support donor-derived spermatogenesis following SSC transplantation, but low spermatogenic efficiency currently limits their use for the production of offspring.

Optimizing Immature Testicular Tissue and Cell Transplantation Results: Comparing Transplantation Sites and Scaffolds.

For patients who had testicular tissue cryopreserved before receiving gonadotoxic therapies, transplantation of testicular tissues and cells has been recommended as a potential therapeutic option. There are no studies that indicate the generation of sperm after human immature testicular tissue (ITT) or spermatogonial stem cells (SSCs) transplantation. The use of releasing scaffolds and localized drug delivery systems as well as the optimizing transplantation site can play an effective role in increasing the efficiency and improving the quality of testicular tissue and cell transplantation in animal models. Current research is focused on optimizing ITT and cell transplantation, the use of releasing scaffolds, and the selection of the right transplantation site that might restore sperm production or male infertility treatment. By searching the PubMed and Google Scholar databases, original and review papers were collected. Search terms were relevant for SSCs and tissue transplantation. In this review, we'll focus on the potential advantages of using scaffolds and choosing the right transplantation site to improve transplantation outcomes.

Successful Cryopreservation of Spermatogonia Stem Cells of Neotropical Catfish (Rhamdia quelen) and Enriched Germ Cell Transplantation into Common Carp (Cyprinus carpio) Testes

Cryopreservation and transplantation of spermatogonial stem cells (SSCs) offer new possibilities in the conservation of valuable genetic resources. Therefore, the present study developed a cryopreservation method for whole testicular tissue and for spermatogonial stem cells of jundia catfish (Rhamdia quelen) and developed an enriched germ cell transplantation of jundia catfish into depleted common carp (Cyprinus carpio) testes. Our findings from whole testes indicate that the cryoprotectants MeOH (1.3 M), DMSO (1.4 M), and EG (1.4 M) resulted in high cell viability rates of 67%, 62%, and 51.5%, respectively. Notably, in the case of enriched post-thaw SSCs, DMSO exhibited the highest cell viability at 27%, followed by EG at 16% and MeOH at 7%. Additionally, we observed the successful colonization and proliferation of jundia germ cells within the recipient gonads of common carp following transplantation. Notably, Sertoli cells were identified in the recipient gonads, providing support to the stained donor germ cells and indicated the formation of cysts. Our data suggest that cryopreserving entire testicular tissue presents a viable alternative to cryopreserving isolated testicular cells, and the spermatogonial cells isolated from testes of jundia retained transplantability characteristics. Nonetheless, more investigations are required to reach the goal of functional gamete and to assess the effectiveness of transplantation using these cryopreserved tissues. Taken together, proper cryopreservation methodology and transplantation technology could aid the preservation practice of fish genetic resources.

Recovering Spermatogenesis By Protected Cryopreservation Using Metformin and Transplanting Spermatogonial Stem Cells Into Testis in an Azoospermia Mouse Model.

Cryopreservation and transplantation of spermatogonial stem cells (SSCs) may serve as a new method to restore male fertility in patients undergoing chemotherapy or radiotherapy. However, SSCs may be damaged during cryopreservation due to the production of reactive oxygen species (ROS). Therefore, different antioxidants have been used as protective agents. Studies have shown that metformin (MET) has antioxidant activity. The aim of this study was to assess the antioxidant and antiapoptotic effects of MET in frozen-thawed SSCs. In addition, the effect of MET on the proliferation and differentiation of SSCs was evaluated. To this end, SSCs were isolated from mouse pups aged 3-6 days old, cultured, identified by flow cytometry (ID4, INTEGRIN β1+), and finally evaluated for survival and ROS rate. SSCs were transplanted after busulfan and cadmium treatment. Cryopreserved SSCs with and without MET were transplanted after 1 month of cryopreservation. Eight weeks after transplantation, the recipient testes were evaluated for the expression of apoptosis (BAX, BCL2), proliferation (PLZF), and differentiation (SCP3, TP1, TP2, PRM1) markers using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction. The findings revealed that the survival rate of SSCs was higher in the 500 μm/mL MET group compared to the other groups (50 and 5000 μm/mL). MET significantly decreased the intracellular ROS production. Transplantation of SSCs increased the expression level of proliferation (PLZF) and differentiation (SCP3, TP1, TP2, PRM1) markers compared to azoospermia group, and their levels were significantly higher in the MET group compared to the cryopreservation group containing basic freezing medium (p < 0.05). MET increased the survival rate of SSCs, proliferation, and differentiation and decreased the ROS production and the apoptosis rate. Cryopreservation by MET seems to be effective in treating infertility.

Allogeneic offspring produced by induction of PD-L1 in spermatogonial stem cells via self-renewal stimulation.

The testis is an immune-privileged organ. It is considered that the testis somatic microenvironment is responsible for immune suppression. However, immunological properties of spermatogonial stem cells (SSCs) have remained unknown. Here, we report the birth of allogeneic offspring by enhanced expression of immunosuppressive PD-L1 in SSCs. Invitro supplementation of GDNF and FGF2 increased expression of PD-L1 in SSCs. Cultured SSCs maintained allogeneic spermatogenesis that persisted for >1 year. However, depletion or gene editing of Pd-l1 family genes in SSCs prevented allogeneic spermatogenesis, which suggested that germ cells are responsible for suppression of the allogeneic response. PD-L1 was induced by activation of the MAPK14-BCL6B pathway, which drives self-renewal by reactive oxygen species (ROS) generation. By contrast, reduced ROS or Mapk14 deficiency downregulated PD-L1. Allogeneic offspring were born after SSC transplantation into congenitally infertile and chemically castrated mice. Thus, SSCs have unique immunological properties, which make allogeneic recipients into "surrogate fathers."

Conserved Transcriptome Features Define Prepubertal Primate Spermatogonial Stem Cells as Adark Spermatogonia and Identify Unique Regulators.

Antineoplastic treatments for cancer and other non-malignant disorders can result in long-term or permanent male infertility by ablating spermatogonial stem cells (SSCs). SSC transplantation using testicular tissue harvested before a sterilizing treatment is a promising approach for restoring male fertility in these cases, but a lack of exclusive biomarkers to unequivocally identify prepubertal SSCs limits their therapeutic potential. To address this, we performed single-cell RNA-seq on testis cells from immature baboons and macaques and compared these cells with published data from prepubertal human testis cells and functionally-defined mouse SSCs. While we found discrete groups of human spermatogonia, baboon and rhesus spermatogonia appeared less heterogenous. A cross-species analysis revealed cell types analogous to human SSCs in baboon and rhesus germ cells, but a comparison with mouse SSCs revealed significant differences with primate SSCs. Primate-specific SSC genes were enriched for components and regulators of the actin cytoskeleton and participate in cell-adhesion, which may explain why the culture conditions for rodent SSCs are not appropriate for primate SSCs. Furthermore, correlating the molecular definitions of human SSC, progenitor and differentiating spermatogonia with the histological definitions of Adark/Apale spermatogonia indicates that both SSCs and progenitor spermatogonia are Adark, while Apale spermatogonia appear biased towards differentiation. These results resolve the molecular identity of prepubertal human SSCs, define novel pathways that could be leveraged for advancing their selection and propagation in vitro, and confirm that the human SSC pool resides entirely within Adark spermatogonia.

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.

Isolation of Undifferentiated Spermatogonia from Adult and Developing Mouse Testes.

In the mammalian testis, the mitotic complements of spermatogenic cells are spermatogonia, including spermatogonial stem cells (SSCs) which form the basis of life-long spermatogenesis and male fertility. Thus, investigating spermatogonia and subdivisions thereof is essential to increase our understanding of male germline development and infertility. This protocol describes the isolation of spermatogonia from both adult and developing [postnatal day 6 (P6)] mouse testes. Cell suspensions of the adult mouse testis from the Id4-Egfp transgenic mouse line are obtained through a two-step enzymatic digestion and are subjected to Percoll pre-enrichment before spermatogonia are isolated by selecting testis cells that are CD9bright and ID4-EGFP+ through FACS. For P6 mice, the testis is digested using trypsin-DNase, and spermatogonia are isolated by FACS selection of ID4-EGFP+ testis cells. In both cases, nearly pure populations of undifferentiated spermatogonia are obtained that can be further subdivided using additional parameters (e.g., EGFP intensity, cell surface protein immunostaining), and recovered for use in various downstream applications, such as biochemical analyses (e.g., transcriptome/epigenome), functional analyses by SSC transplantation or propagation in vitro.

WIN18,446 enhances spermatogonial stem cell homing and fertility after germ cell transplantation by increasing blood-testis barrier permeability.

Spermatogonial stem cells (SSCs) possess a unique ability to recolonize the seminiferous tubules. Upon microinjection into the adluminal compartment of the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) to the basal compartment of the tubule and reinitiate spermatogenesis. It was recently discovered that inhibiting retinoic acid signaling with WIN18,446 enhances SSC colonization by transiently suppressing spermatogonia differentiation, thereby promoting fertility restoration. In this study, we report that WIN18,446 increases SSC colonization by disrupting the BTB. WIN18,446 altered the expression patterns of tight junction proteins (TJPs) and disrupted the BTB in busulfan-treated mice. WIN18,446 upregulated the expression of FGF2, one of the self-renewal factors for SSCs. While WIN18,446 enhanced SSC colonization in busulfan-treated wild-type mice, it did not increase colonization levels in busulfan-treated Cldn11-deficient mice, which lack the BTB, indicating that the enhancement of SSC colonization in wild-type testes depended on the loss of the BTB. Serial transplantation analysis revealed impaired self-renewal caused by WIN18,446, indicating that WIN18,446-mediated inhibition of retinoic acid signaling impaired SSC self-renewal. Strikingly, WIN18,446 administration resulted in the death of 45% of busulfan-treated recipient mice. These findings suggest that TJP modulation is the primary mechanism behind enhanced SSC homing by WIN18,446 and raise concerns regarding the use of WIN18,446 for human SSC transplantation.

Propagation of porcine spermatogonial stem cells in serum-free culture conditions using knockout serum replacement.

In vitro culture and expansion of spermatogonial stem cells (SSCs) is an essential prerequisite to enhancing livestock productivity through SSC transplantation. Most of the culture media have been observed to be supplemented with serum. However, the use of serum in culture media may exert detrimental effects on SSC maintenance in vitro. An attempt was made to culture SSCs by replacing serum with 5% 'Knockout Serum Replacement (KSR)' in Doom pig (Sus domesticus), one of the valued indigenous germplasm of North-East India. Testes from 7 to 15 days old piglets were used for isolation, enrichment and in vitro culture of putative SSCs using serum-based and serum-free culture media. The cells were characterized for SSC-specific pluripotent markers expression by immunofluorescence staining and quantitative real-time PCR. The diameter and number of SSC colonies were recorded on days 9, 20 and 30 of culture. Similar morphologies of the SSC colonies were observed in both serum-based and serum-free culture conditions. Colony diameter and colony number were non-significantly higher in serum-free than serum-based media. The cells from both the culture conditions showed high alkaline phosphatase activity. The expression of SSC-specific pluripotent markers was observed in immunofluorescence and quantitative real-time PCR study. The present study revealed that SSCs from porcine species could be maintained in vitro for up to 30 days in serum-free culture using 5% KSR, which is believed to be a promising protein source for improving livestock production, and health care along with their conservation.

Localization and characterization of SSCs from pre-pubertal bovine testes

Self renewal and proliferation ability of spermatogonial stem cells (SSCs) support spermatogenesis during adult life. Theoretically, these stem cells can be utilized for transmission of genetic information to descendants via testicular transplantation. However, lack of knowledge in methodologies for identification of SSCs limits the application of SSCs transplantation in domestic animals. Accumulated studies have shown that SSCs specific markers (DBA, UCHL1) and stem cell marker (Sox2, Oct4) are useful to screen SSCs that able to be used for transplantation. However, in cattle, less information is available on the expression status of these markers till date. Therefore, a study was carried out in 2019 at Tsukuba University, Japan where testes from 3, 5 and 7 months old calves were utilized to examine testicular localization and in vitro propogation of stem cell markers. SSCs were isolated by enzymatic digestion combined with centrifugal separation on discontinuous Percoll density gradient. Cell propagation and SSCs marker expression were determined at 5, 10 and 15 days post-culture. Immunostaining in conjunction with Western Blot analysis of cultured cells showed that stem cell markers (UCHL1, Oct4 and Sox2) were expressed in SSCs suggesting that differentiation of gonocyte started by 3 months and SSCs differentiation begins after 5 months of age. Taken together, these results demonstrated marker expression and localization of bull SSCs and showed that in vitro culturing of bull SSCs is implementable.

RNASEQ ANALYSIS OF FACS SORTED SPERMATOGONIAL CELLS USED TO IDENTIFY MELANOCORTIN RECEPTOR 2 AS A NOVEL AND FUNCTIONAL MEMBRANE-ASSOCIATED SPERMATOGONIAL STEM CELL MARKER

Spermatogonial stem cell (SSC) transplantation is successful to restore fertility in sterile animal models. It is not safe in human since there is no precise methodology to isolate pure SSCs from cancer cells. Therefore, the purpose of this study is to identify a functional membrane-associate specific SSC markers.

In vitro propagation of XXY human Klinefelter spermatogonial stem cells: A step towards new fertility opportunities.

Klinefelter Syndrome (KS) is characterized by a masculine phenotype, supernumerary sex chromosomes (47, XXY), and impaired fertility due to loss of spermatogonial stem cells (SSCs). Early testicular cryopreservation could be an option for future fertility treatments in these patients, including SSCs transplantation or in vitro spermatogenesis. It is critically essential to adapt current in vitro SSCs propagation systems as a fertility option for KS patients. KS human testicular samples (13,15- and 17-year-old non-mosaic KS boys) were donated by patients enrolled in an experimental testicular tissue banking program. Testicular cells were isolated from cryopreserved tissue and propagated in long-term culture for 110 days. Cell-specific gene expression confirmed the presence of all four main cell types found in testes: Spermatogonia, Sertoli, Leydig, and Peritubular cells. A population of ZBTB16+ undifferentiated spermatogonia was identified throughout the culture using digital PCR. Flow cytometric analysis also detected an HLA-/CD9+/CD49f+ population, indicating maintenance of a stem cell subpopulation among the spermatogonial cells. FISH staining for chromosomes X and Y showed most cells containing an XXY karyotype with a smaller number containing either XY or XX. Both XY and XX populations were able to be enriched by magnetic sorting for CD9 as a spermatogonia marker. Molecular karyotyping demonstrated genomic stability of the cultured cells, over time. Finally, single-cell RNAseq analysis confirmed transcription of ID4, TCN2, and NANOS 3 within a population of putative SSCs population. This is the first study showing successful isolation and long-term in vitro propagation of human KS testicular cells. These findings could inform the development of therapeutic fertility options for KS patients, either through in vitro spermatogenesis or transplantation of SSC, in vivo.

Metabolic transitions define spermatogonial stem cell maturation.

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

Fertility Preservation and Restoration Options for Pre-Pubertal Male Cancer Patients: Current Approaches.

Fertility preservation for prepubertal male patients undergoing gonadotoxic therapies, potentially depleting spermatogonial cells, is an expanding necessity, yet most of the feasible options are still in the experimental phase. We present our experience and a summary of current and novel possibilities regarding the different strategies to protect or restore fertility in young male patients, before proceeding with chemotherapy or radiotherapy for malignances or other diseases. Adult oncological patients should always be counselled to cryopreserve the semen before starting treatment, however this approach is not suitable for prepubertal boys, who aren’t capable to produce sperm yet. Fortunately, since the survival rate of pediatric cancer patients has skyrocketed in the last decade and it’s over 84%, safeguarding their future fertility is becoming a major concern for reproductive medicine. Surgical and medical approaches to personalize treatment or protect the gonads could be a valid first step to take. Testicular tissue autologous grafting or xenografting, and spermatogonial stem cells (SSCs) transplantation, are the main experimental options available, but spermatogenesis in vitro is becoming an intriguing alternative. All of these methods feature both strong and weak prospects. There is also relevant controversy regarding the type of testicular material to preserve and the cryopreservation methods. Since transplanted cells are bound to survive based on SSCs number, many ways to enrich their population in cultures have been proposed, as well as different sites of injection inside the testis. Testicular tissue graft has been experimented on mice, rabbits, rhesus macaques and porcine, allowing the birth of live offspring after performing intracytoplasmic sperm injection (ICSI), however it has never been performed on human males yet. In vitro spermatogenesis remains a mirage, although many steps in the right direction have been performed. The manufacturing of 3D scaffolds and artificial spermatogenetic niche, providing support to stem cells in cultures, seems like the best way to further advance in this field.

Spermatogonial Stem Cell-Based Therapies: Taking Preclinical Research to the Next Level.

Fertility preservation via biobanking of testicular tissue retrieved from testicular biopsies is now generally recommended for boys who need to undergo gonadotoxic treatment prior to the onset of puberty, as a source of spermatogonial stem cells (SSCs). SSCs have the potential of forming spermatids and may be used for therapeutic fertility approaches later in life. Although in the past 30 years many milestones have been reached to work towards SSC-based fertility restoration therapies, including transplantation of SSCs, grafting of testicular tissue and various in vitro and ex vivo spermatogenesis approaches, unfortunately, all these fertility therapies are still in a preclinical phase and not yet available for patients who have become infertile because of their treatment during childhood. Therefore, it is now time to take the preclinical research towards SSC-based therapy to the next level to resolve major issues that impede clinical implementation. This review gives an outline of the state of the art of the effectiveness and safety of fertility preservation and SSC-based therapies and addresses the hurdles that need to be taken for optimal progression towards actual clinical implementation of safe and effective SSC-based fertility treatments in the near future.