Differentiation Of Spermatogonial Stem Cells Research Articles

Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium.

In vitro gamete derivation has been proposed as an interesting strategy for treatment of infertility, improvement of genetic traits, and conservation of endangered animals. Spermatogonial stem cells (SSCs) are primary candidates for in vitro gamete derivation; however, recently, mesenchymal stem cells (MSCs) have also been proposed as candidates for germ cell (GCs) differentiation mainly due to their transdifferentiating capacity. The objective of the present study was to compare the potential for GC differentiation of bovine peripheral blood-derived MSCs (PB-MSCs) and SSCs under the effect of conditioned medium (CM) derived from Sertoli cells (SCs/CM). Samples were collected every 7 days for 21 days and analyzed for pluripotent, GC, and MSC marker expression. The absence of OCT4 and the increased (p < 0.05) expression of NANOG seems to play a role in SSC differentiation, whereas the absence of NANOG and the increased expression (p < 0.05) of OCT4 may be required for PB-MSC differentiation into GCs. SSCs cultured with SCs/CM increased (p < 0.05) the expression of PIWIL2 and DAZL, while PB-MSCs cultured under the same condition only increased (p < 0.05) the expression of DAZL. Overall, the patterns of markers expression suggest that PB-MSCs and SSCs activate different signaling pathways after exposure to SCs/CM and during differentiation into GCs.

RNA m6A modification regulates L1 retrotransposons in human spermatogonial stem cell differentiation in vitro and in vivo

The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m6A) plays an essential role in regulating various biological processes. However, the function of m6A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m6A methyltransferase METTL3 or the m6A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m6A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m6A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m6A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m6A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m6A modification in restricting L1 retrotransposition.

Regulation of spermatogonial stem cell differentiation by Sertoli cells-derived exosomes through paracrine and autocrine signaling.

In the orchestrated environment of the testicular niche, the equilibrium between self-renewal and differentiation of spermatogonial stem cells (SSCs) is meticulously maintained, ensuring a stable stem cell reserve and robust spermatogenesis. Within this milieu, extracellular vesicles, specifically exosomes, have emerged as critical conveyors of intercellular communication. Despite their recognized significance, the implications of testicular exosomes in modulating SSC fate remain incompletely characterized. Given the fundamental support and regulatory influence of Sertoli cells (SCs) on SSCs, we were compelled to explore the role of SC-derived exosomes (SC-EXOs) in the SSC-testicular niche. Our investigation hinged on the hypothesis that SC-EXOs, secreted by SCs from the testes of 5-day-old mice-a developmental juncture marking the onset of SSC differentiation-participate in the regulation of this process. We discovered that exposure to SC-EXOs resulted in an upsurge of PLZF, MVH, and STRA8 expression in SSC cultures, concomitant with a diminution of ID4 and GFRA1 levels. Intriguingly, obstructing exosomal communication in a SC-SSC coculture system with the exosome inhibitor GW4869 attenuated SSC differentiation, suggesting that SC-EXOs may modulate this process via paracrine signaling. Further scrutiny revealed the presence of miR-493-5p within SC-EXOs, which suppresses Gdnf mRNA in SCs to indirectly restrain SSC differentiation through the modulation of GDNF expression-an indication of autocrine regulation. Collectively, our findings illuminate the complex regulatory schema by which SC-EXOs affect SSC differentiation, offering novel perspectives and laying the groundwork for future preclinical and clinical investigations.

Transcriptome Analysis of Key Genes Involved in the Initiation of Spermatogonial Stem Cell Differentiation.

The purpose of this study was to screen the genes and pathways that are involved in spermatogonia stem cell (SSC) differentiation regulation during the transition from Aundiff to A1. Methods: RNA sequencing was performed to screen differentially expressed genes at 1 d and 2 d after SSC differentiation culture. KEGG pathway enrichment and GO function analysis were performed to reveal the genes and pathways related to the initiation of early SSC differentiation. The GO analysis showed that Rpl21, which regulates cell differentiation initiation, significantly increased after 1 day of SSC differentiation. The expressions of Fn1, Cd9, Fgf2, Itgb1, Epha2, Ctgf, Cttn, Timp2 and Fgfr1, which are related to promoting differentiation, were up-regulated after 2 days of SSC differentiation. The analysis of the KEGG pathway revealed that RNA transport is the most enriched pathway 1 day after SSC differentiation. Hspa2, which promotes the differentiation of male reproductive cells, and Cdkn2a, which participates in the cell cycle, were significantly up-regulated. The p53 pathway and MAPK pathway were the most enriched pathways 2 days after SSC differentiation. Cdkn1a, Hmga2, Thbs1 and Cdkn2a, microRNAs that promote cell differentiation, were also significantly up-regulated. RNA transport, the MAPK pathway and the p53 pathway may play vital roles in early SSC differentiation, and Rpl21, Fn1, Cd9, Fgf2, Itgb1, Epha2, Ctgf, Cttn, Timp2, Fgfr1, Hspa2, Cdkn2a, Cdkn1a, Hmga2 and Thbs1 are involved in the initiation of SSC differentiation. The findings of this study provide a reference for further revelations of the regulatory mechanism of SSC differentiation.

Recent Progress of In Vitro 3D Culture of Male Germ Stem Cells.

Male germline stem cells (mGSCs), also known as spermatogonial stem cells (SSCs), are the fundamental seed cells of male animal reproductive physiology. However, environmental influences, drugs, and harmful substances often pose challenges to SSCs, such as population reduction and quality decline. With advancements in bioengineering technology and biomaterial technology, an increasing number of novel cell culture methods and techniques have been employed for studying the proliferation and differentiation of SSCs in vitro. This paper provides a review on recent progress in 3D culture techniques for SSCs in vitro; we summarize the microenvironment of SSCs and spermatocyte development, with a focus on scaffold-based culture methods and 3D printing cell culture techniques for SSCs. Additionally, decellularized testicular matrix (DTM) and other biological substrates are utilized through various combinations and approaches to construct an in vitro culture microenvironment suitable for SSC growth. Finally, we present some perspectives on current research trends and potential opportunities within three areas: the 3D printing niche environment, alternative options to DTM utilization, and advancement of the in vitro SSC culture technology system.

Recent advances in developing 3D culture systems of spermatogonial stem cell preservation and differentiation: A narrative review.

Male infertility has received vast attention in recent years and has no clear etiology in almost 40% of cases. Several methods have been suggested for preserving sperm and spermatogonial stem cells (SSCs) in both in vivo and in vitro conditions. The efficacy of these methods is related to their abilities, including providing an optimal environment for sperm preservation and long-term SSC culture for in vivo and in vitro differentiation of these cells. In this review article, a full MEDLINE/PubMed search was performed using the following search terms: "Spermatogonial Progenitor Cells, Stem Cells, Fertility Preservations, Sperm Freezing, Cell Differentiations, Tissue Scaffold, 3-Dimensional Cell Culture", which retrieved results from 1973-2022. Related articles were added to the bibliography of selected articles. Exclusion criteria included non-English language, abstract only, and unrelated articles. The production of functioning male germ cells is suggested by introducing modern bioengineered systems as a new hope for the maintenance of male fertility. Till now, few in vitro spermatogenesis investigations have provided appreciable amounts of mature gametes. Each method had benefits and disadvantages, but the 3-dimensional culture method had the greatest impact on the differentiation and preservation of SSCs. One of the critical elements of research is the preservation of sperm and the differentiation of SSCs. Several methods have been employed in this area. Various scaffolds providing an environment similar to an extracellular matrix and conditions for germ cell development and survival have been employed in recent research.

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.

Sox2 Localization During Spermatogenesis and Its Association with other Spermatogenesis Markers Using Protein-Protein Network Analysis.

Sox2 (SRY box2) is an essential transcription factor that plays a vital role in spermatogenesis and regulates the genes in this process. Sox2 is important for pluripotency, self-renewal, and even spermatogonial stem cell differentiation. This gene is found in pluripotent and specialized cells, and it is involved in their biological activities. Protein-protein interaction (PPI) network analysis was performed during spermatogenesis using NCBI, STRING, and Cytoscape databases. Then, after isolating spermatogonial stem cells from 6 C57BL/6 mice, mouse embryonic stem cells and ES-like cells were prepared. In the following, Sox2 expression was examined in differentiated and undifferentiated spermatogonia by immunohistochemistry (IMH), immunocytochemistry (ICC), and Fluidigm PCR (polymerase chain reaction). Finally, the results were compared using the Kruskal-Wallis and Dunn tests at the significance level of p<0.05. The results of this experiment showed that contrary to expectations, Sox2 has cytoplasmic expression in undifferentiated cells and nuclear expression in differentiated cells in in vitro conditions. In addition, the expression of Sox2 increased during differentiation. Fluidigm PCR showed a significantly higher expression of Sox2 (p<0.05) in differentiated compared to undifferentiated spermatogonia. Sox2 has an interaction with other genes during spermatogenesis such as Oct4, Nanog, Klf4, Stra8, Smad1, Tcf3, and Osm. Sox2, which is known as a pluripotency marker, has a vital role in spermatogenesis and could be a differential marker. Sox2 has strong connections with other genes such as Oct4, Nanog, Klf4, Tcf3, Osm, Stra8, Lim2, Smad1, Gdnf, and Kit.

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.

Single-cell transcriptomic dissection of the toxic impact of di(2-ethylhexyl) phthalate on immature testicular development at the neonatal stage.

Di(2-ethylhexyl) phthalate (DEHP) early exposure leads to immature testicular injury, and we aimed to utilize single-cell RNA (scRNA) sequencing to comprehensively assess the toxic effect of DEHP on testicular development. Therefore, we gavaged pregnant C57BL/6 mice with 750mg/kg body weight DEHP from gestational day 13.5 to delivery and performed scRNA sequencing of neonatal testes at postnatal day 5.5. The results revealed the gene expression dynamics in testicular cells. DEHP disrupted the developmental trajectory of germ cells and the balance between the self-renewal and differentiation of spermatogonial stem cells. Additionally, DEHP caused an abnormal developmental trajectory, cytoskeletal damage and cell cycle arrest in Sertoli cells; disrupted the metabolism of testosterone in Leydig cells; and disturbed the developmental trajectory in peritubular myoid cells. Elevated oxidative stress and excessive apoptosis mediated by p53 were observed in almost all testicular cells. The intercellular interactions among four cell types were altered, and biological processes related to glial cell line-derived neurotrophic factor (GDNF), transforming growth factor-β (TGF-β), NOTCH, platelet-derived growth factor (PDGF) and WNT signaling pathways were enriched after DEHP treatment. These findings systematically describe the damaging effects of DEHP on the immature testes and provide substantial novel insights into the reproductive toxicity of DEHP.

Sertoli cells-derived exosomal miR-30a-5p regulates ubiquitin E3 ligase Zeb2 to affect the spermatogonial stem cells proliferation and differentiation.

The role of spermatogonial stem cells (SSCs) is crucial in spermatogenesis, and extracellular vesicles (EVs) have been the focus of research as an important intercellular communication mechanism. Various endogenous regulatory factors secreted by Sertoli cells (SCs) can affect the self-maintenance and regeneration of SSCs, but little is known about the roles of SCs-derived exosomal microRNAs (miRNAs) on SSCs. In this study, we aimed to explore the regulation of the SCs-derived exosomal miR-30a-5p on SSCs proliferation and differentiation. EVs from the SCs were detected by electron microscopy and nanoparticle tracking analysis (NTA). Subsequently, the SSCs were treated with the SCs-derived extracellular vesicles (SCs-EVs). CCK-8 assay and EdU staining was applied to detect the cell proliferation, and the results indicated that SCs-EVs promoted the SSCs proliferation. Western blot detection of the SSCs markers (Gfrα1, Plzf, Stra8, and C-kit) indicated that SCs-EVs promoted the SSCs differentiation. Additionally, we found that SCs-EVs secreted miR-30a-5p to show the promoting effects. Besides, we discovered that miR-30a-5p targeted zinc finger E-box binding homeobox 2 (Zeb2) to regulate the ubiquitination of fibroblast growth factor 9 (Fgf9) in SSCs. miR-30a-3p/Zeb2/Fgf9 promoted the SSCs proliferation and differentiation by activating the mitogen‑activated protein kinase (MAPK) signaling pathway. Taken together, our study showed that SCs-EVs can transport miR-30a-5p to SSCs and affect SSCs proliferation and differentiation by regulating the MAPK signaling pathway via Zeb2/Fgf9. This paper disclosed a novel molecular mechanism that regulates SSCs proliferation and differentiation, which could be valuable for the treatment of male infertility.

MP43-11 EIGHT-YEAR FERTILITY PRESERVATION PROGRAM AT ATRIUM HEALTH WAKE FOREST BAPTIST FOCUSING ON SPERMATOGONIAL STEM CELL AND ROUND SPERMATID TECHNOLOGIES.

You have accessJournal of UrologyCME1 Apr 2023MP43-11 EIGHT-YEAR FERTILITY PRESERVATION PROGRAM AT ATRIUM HEALTH WAKE FOREST BAPTIST FOCUSING ON SPERMATOGONIAL STEM CELL AND ROUND SPERMATID TECHNOLOGIES. Adam Cohen, Janmejay Hingu, Nima Pourhabibi Zarandi, Guillermo Galdon, Omar Abdelaal, Banafsheh Nickmehr, Kimberly Stogner-Underwood, Stanley Kogan, Steve Hodges, Stuart Howards, Thomas McLean, Anthony Atala, and Hooman Sadri-Ardekani Adam CohenAdam Cohen More articles by this author , Janmejay HinguJanmejay Hingu More articles by this author , Nima Pourhabibi ZarandiNima Pourhabibi Zarandi More articles by this author , Guillermo GaldonGuillermo Galdon More articles by this author , Omar AbdelaalOmar Abdelaal More articles by this author , Banafsheh NickmehrBanafsheh Nickmehr More articles by this author , Kimberly Stogner-UnderwoodKimberly Stogner-Underwood More articles by this author , Stanley KoganStanley Kogan More articles by this author , Steve HodgesSteve Hodges More articles by this author , Stuart HowardsStuart Howards More articles by this author , Thomas McLeanThomas McLean More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Hooman Sadri-ArdekaniHooman Sadri-Ardekani More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003289.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Male infertility has been associated with many disease processes, including chemotherapy and radiotherapy for cancer, undescended testis (UDT), and genetic conditions such as Klinefelter Syndrome (KS). Preservation of human spermatogonial stem cells (SSC) may be a suitable option for young male patients at risk of losing fertility. With optimal cryopreservation, cell viability can be retained to reestablish spermatogenesis in the future through in vitro germ cell differentiation or round spermatid injection. METHODS: Urology and hematology-oncology patients at Atrium Health Wake Forest Baptist with malignancy, UDT, or KS were approached for consent and assent (if ≥seven years old) for the fertility preservation program. If participation was accepted, patients were given the option to undergo a fertility-preserving testicular biopsy, preferably during general anesthesia for other procedures such as Port-a-Cath placement, orchidopexy, or lymph node biopsy. They were also allowed to enroll as a registry participant (non-biopsy control). Histological and microbiological analyses were performed on biopsies before being cryopreserved. Further data were obtained retrospectively via chart review. RESULTS: Over eight years of banking, a total of 202 patients were approached for the study, with 125 (61.8%) undergoing a biopsy, 39 (19.3%) enrolling as non-biopsy control, and 38 (18.8%) declining participation. The mean age at which biopsy was performed across all diagnoses was 8.39 years (range 0.61 to 37.39). Of the 125 biopsy patients, 73% opted to donate a portion (up to 20%) of the tissue for basic research with no direct benefit to the donor. Of the biopsied patients, 76 (60.8%) were performed for cancer, 38 (30.4%) for unilateral or bilateral UDT, and 11 (8.8%) for KS. Based on PRM1 immunofluorescence, 48.3% of specimens had round spermatids. Short-term complications for testicular biopsy, primarily pain and swelling, were typically mild to moderate. CONCLUSIONS: A multidisciplinary approach can accommodate patients at risk for reduced future fertility due to various pathologies. The preservation of testicular tissue at present can aid in future fertility restoration through techniques such as in vitro SSC differentiation or round cell spermatid injection (ROSI). Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e605 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Adam Cohen More articles by this author Janmejay Hingu More articles by this author Nima Pourhabibi Zarandi More articles by this author Guillermo Galdon More articles by this author Omar Abdelaal More articles by this author Banafsheh Nickmehr More articles by this author Kimberly Stogner-Underwood More articles by this author Stanley Kogan More articles by this author Steve Hodges More articles by this author Stuart Howards More articles by this author Thomas McLean More articles by this author Anthony Atala More articles by this author Hooman Sadri-Ardekani More articles by this author Expand All Advertisement PDF downloadLoading ...

In vitro complete differentiation of human spermatogonial stem cells to morphologic spermatozoa using a hybrid hydrogel of agarose and laminin

Spermatogenesis refers to the differentiation of the spermatogonial stem cells (SSCs) located in the base seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix (ECM), paracrine factors, and testicular somatic cells which play a supporting role for SSCs. Thus, the present study evaluated the potential of co-culturing Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin, the main components of the ECM. Following the three–week conventional culture of human testicular cells, the cells were cultured in agarose hydrogel or agarose/laminin one (hybrid) for 74 days. Then, immunocytochemistry, real-time PCR, electron microscopy, and morphological staining methods were applied to analyze the presence of SSCs, as well as the other cells of the different stages of spermatogenesis. Based on the results, the colonies with positive spermatogenesis markers were observed in both culture systems. The existence of the cells of all three phases of spermatogenesis (spermatogonia, meiosis, and spermiogenesis) was confirmed in the two groups, while morphological spermatozoa were detected only in the hybrid hydrogel group. Finally, a biologically improved 3D matrix can support all the physiological activities of SSCs such as survival, proliferation, and differentiation.

Differentiation of human spermatogonial stem cells using a human decellularized testicular scaffold supplemented by platelet-rich plasma.

Effective culture systems for attachment, migration, proliferation, and differentiation of spermatogonial stem cells (SSCs) can be a promising therapeutic modality for preserving male fertility. Decellularized extracellular matrix (ECM) from native testis tissue creates a local microenvironment for testicular cell culture. Furthermore, platelet-rich plasma (PRP) contains various growth factors for the proliferation and differentiation of SSCs. In this study, human testicular cells were isolated and cultured for 4 weeks, and SSCs were characterized using immunocytochemistry (ICC) and flow cytometry. Human testicular tissue was decellularized (0.3% SDS, 1% Triton), and the efficiency of the decellularization process was confirmed by histological staining and DNA content analysis. SSCs were cultured on the human decellularized testicular matrix (DTM) for 4 weeks. The viability and the expression of differentiation genes were evaluated by MTT and real-time polymerase chain reaction (PCR), respectively. Histological evaluation and DNA content analysis showed that the components of ECM were preserved during decellularization. Our results showed that after 4 weeks of culture, the expression levels of BAX, BCL-2, PLZF, and SCP3 were unchanged, while the expression of PRM2 significantly increased in the cells cultured on DTM supplemented with PRP (ECM-PRP). In addition, the expression of GFRA1 was significantly decreased in the ECM group compared to the control and PRP groups. Furthermore, the MTT test indicated that viability was significantly enhanced in cells plated on DTM supplemented with PRP. Our study demonstrated that DTM supplemented with PRP can provide an effective culture system for the differentiation and viability of SSCs.

Multi-Omics Approaches for Revealing the Epigenetic Regulation of Histone H3.1 during Spermatogonial Stem Cell Differentiation In Vitro

Epigenetic regulation, particularly post-translational modifications (PTMs) of histones, participates in spermatogonial stem cell (SSCs) differentiation. However, there is a lack of systemic studies of histone PTM regulation during the differentiation of SSCs due to its low number in vivo. Herein, we quantified dynamic changes of 46 different PTMs on histone H3.1 by targeted quantitative proteomics using mass spectrometry during SSCs differentiation in vitro, in combination with our RNA-seq data. We identified seven histone H3.1 modifications to be differentially regulated. In addition, we selected H3K9me2 and H3S10ph for subsequent biotinylated peptide pull-down experiments and identified 38 H3K9me2-binding proteins and 42 H3S10ph-binding proteins, which contain several transcription factors, such as GTF2E2 and SUPT5H, which appear to be crucial for epigenetic regulation of SSC differentiation.

Human in vitro spermatogenesis as a regenerative therapy - where do we stand?

Spermatogenesis involves precise temporal and spatial gene expression and cell signalling to reach a coordinated balance between self-renewal and differentiation of spermatogonial stem cells through various germ cell states including mitosis, and meiosis I and II, which result in the generation of haploid cells with a unique genetic identity. Subsequently, these round spermatids undergo a series of morphological changes to shed excess cytoplast, develop a midpiece and tail, and undergo DNA repackaging to eventually form millions of spermatozoa. The goal of recreating this process in vitro has been pursued since the 1920s as a tool to treat male factor infertility in patients with azoospermia. Continued advances in reproductive bioengineering led to successful generation of mature, functional sperm in mice and, in the past 3 years, in humans. Multiple approaches to study human in vitro spermatogenesis have been proposed, but technical and ethical obstacles have limited the ability to complete spermiogenesis, and further work is needed to establish a robust culture system for clinical application.

The emerging role of extracellular vesicles in the testis.

Extracellular vesicles (EVs) are nano-sized membrane-bounded particles, released by all cells and capable of transporting bioactive cargoes, proteins, lipids, and nucleic acids, to regulate a variety of biological functions. Seminal plasma is enriched in EVs, and extensive evidence has revealed the role of EVs (e.g. prostasomes and epididymosomes) in the male genital tract. Recently, EVs released from testicular cells have been isolated and identified, and some new insights have been generated on their role in maintaining normal spermatogenesis and steroidogenesis in the testis. In the seminiferous tubules, Sertoli cell-derived EVs can promote the differentiation of spermatogonial stem cells (SSCs), and EVs secreted from undifferentiated A spermatogonia can inhibit the proliferation of SSCs. In the testicular interstitium, EVs have been identified in endothelial cells, macrophages, telocytes, and Leydig cells, although their roles are still elusive. Testicular EVs can also pass through the blood-testis barrier and mediate inter-compartment communication between the seminiferous tubules and the interstitium. Immature Sertoli cell-derived EVs can promote survival and suppress the steroidogenesis of Leydig cells. Exosomes isolated from macrophages can protect spermatogonia from radiation-induced injury. In addition to their role in intercellular communication, testicular EVs may also participate in the removal of aberrant proteins and the delivery of antigens for immune tolerance. EVs released from testicular cells can be detected in seminal plasma, which makes them potential biomarkers reflecting testicular function and disease status. The testicular EVs in seminal plasma may also affect the female reproductive tract to facilitate conception and may even affect early embryogenesis through modulating sperm RNA. EVs represent a new type of intercellular messenger in the testis. A detailed understanding of the role of testicular EV may contribute to the discovery of new mechanisms causing male infertility and enable the development of new diagnostic and therapeutic strategies for the treatment of infertile men.

Retinoic acid-induced differentiation of porcine prospermatogonia in vitro

Spermatogenesis is an intricate developmental process occurring in testes by which spermatogonial stem cells (SSCs) self-renew and differentiate into mature sperm. The molecular mechanisms for SSC self-renewal and differentiation, while have been well studied in mice, may differ between mice and domestic animals including pigs. To gain knowledge about the molecular mechanisms for porcine SSC self-renewal and differentiation that have so far been poorly understood, here we isolated and enriched prospermatogonia from neonatal porcine testes, and exposed the cells to retinoic acid, a direct inducer for spermatogonial differentiation. We then identified that retinoic acid could induce porcine prospermatogonial differentiation, which was accompanied by a clear transcriptomic alteration, as revealed by the RNA-sequencing analysis. We also compared retinoic acid-induced in vitro porcine spermatogonial differentiation with the in vivo process, and compared retinoic acid-induced in vitro spermatogonial differentiation between pigs and mice. Furthermore, we analyzed retinoic acid-induced differentially expressed long non-coding RNAs (lncRNAs), and demonstrated that a pig-specific lncRNA, lncRNA-106504875, positively regulated porcine spermatogonial proliferation by targeting the core transcription factor ZBTB16. Taken together, these results would help to elucidate the roles of retinoic acid in porcine spermatogonial differentiation, thereby contributing to further knowledge about the molecular mechanisms underlying porcine SSC development and, in the long run, to optimization of both long-term culture and induced differentiation systems for porcine SSCs.

Application of single-cell RNA sequencing on human testicular samples: a comprehensive review.

So far there has been no comprehensive review using systematic literature search strategies to show the application of single-cell RNA sequencing (scRNA-seq) in the human testis of the whole life cycle (from embryos to aging males). Here, we summarized the application of scRNA-seq analyses on various human testicular biological samples. A systematic search was conducted in PubMed and Gene Expression Omnibus (GEO), focusing on English researches published after 2009. Articles related to GEO data-series were also retrieved in PubMed or BioRxiv. 81 full-length studies were finally included in the review. ScRNA-seq has been widely used on different human testicular samples with various library strategies, and new cell subtypes such as State 0 spermatogonial stem cells (SSC) and stage_a/b/c Sertoli cells (SC) were identified. For the development of normal testes, scRNA-seq-based evidence showed dynamic transcriptional changes of both germ cells and somatic cells from embryos to adults. And dysregulated metabolic signaling or hedgehog signaling were revealed by scRNA-seq in aged SC or Leydig cells (LC), respectively. For infertile males, scRNA-seq studies revealed profound changes of testes, such as the increased proportion of immature SC/LC of Klinefelter syndrome, the somatic immaturity and altered germline autophagy of patients with non-obstructive azoospermia, and the repressed differentiation of SSC in trans-females receiving testosterone inhibition therapy. Besides, the re-analyzing of public scRNA-seq data made further discoveries such as the potential vulnerability of testicular SARS-CoV-2 infection, and both evolutionary conservatism and divergence among species. ScRNA-seq analyses would unveil mechanisms of testes' development and changes so as to help developing novel treatments for male infertility.

Transcriptome Analysis in High Temperature Inhibiting Spermatogonial Stem Cell Differentiation In Vitro.

As one of the factors of male infertility, high temperature induces apoptosis of differentiated spermatogenic cells, sperm DNA oxidative damage, and changes in morphology and function of Sertoli cells. Spermatogonial stem cells (SSCs) are a type of germline stem cells that maintain spermatogenesis through self-renewal and differentiation. At present, however, the effect of high temperature on SSC differentiation remains unknown. In this study, an in vitro SSC differentiation model was used to investigate the effect of heat stress treatment on SSC differentiation, and RNA sequencing (RNA-seq) was used to enrich the key genes and pathways in high temperature inhibiting SSC differentiation. Results show that 2days of 37°C or 43°C (30min per day) heat stress treatment significantly inhibited SSC differentiation. The differentiation-related genes c-kit, stra8, Rec8, Sycp3, and Ovol1 were down-regulated after 2 and 4days of heat stress at 37°C. The transcriptome of SSCs was significantly differentially expressed on days 2 and 4 after heat stress treatment at 37°C. In total, 1660 and 7252 differentially expressed genes (DEGs) were identified by RNA-seq in SSCs treated with heat stress at 37°C for 2 and 4days, respectively. KEGG pathway analysis showed that p53, ribosome, and carbon metabolism signaling pathways promoting stem cell differentiation were significantly enriched after heat stress treatment at 37°C. In conclusion, 37°C significantly inhibited SSC differentiation, and p53, ribosome, and carbon metabolism signaling pathways were involved in this differentiation inhibition process. The results of this study provide a reference for further investigation into the mechanism by which high temperature inhibits SSC differentiation.