Spermatogonial Stem Cells Markers Research Articles

Characterization of potential spermatogonia biomarker genes in the European eel (Anguilla anguilla).

Identification of specific molecular markers for spermatogonial stem cells in teleost is crucial for enhancing the efficacy of reproductive biotechnologies in aquaculture, such as transplantation and surrogate production in fishes. Since it is not yet possible to distinguish spermatogonial stem cells of European eel (Anguilla anguilla) using specific molecular markers, we isolated spermatogonial cells from immature European eels to find these potential markers. We attempted this by studying three candidate genes: vasa, nanos2, and dnd1. Two vasa (vasa1 and vasa2) genes, nanos2, and dnd1 were identified, characterized, and studied in the muscle, testis, and isolated spermatogonia. Our results showed that vasa1 and vasa2 had the highest levels of expression when measured by qPCR. In situ hybridization and immunochemistry assays showed that the four genes were localized explicitly in type A spermatogonia. However, vasa1 and vasa2 exhibited stronger signals in the immature testicular tissue than the other two potential markers. According to this, vasa1 and vasa2 were found to be the most effective markers for spermatogonial cells in the European eel.

High incidence of imperforate vagina in ADGRA3-deficient mice

BackgroundTen percent of the female population suffers from congenital abnormalities of the vagina, uterus, or oviducts, with severe consequences for reproductive and psychological health. Yet, the underlying causes of most of these malformations remain largely unknown. ADGRA3 (GPR125) is involved in WNT signaling and planar cell polarity, mechanisms vital to female reproductive tract development. Although ADGRA3 is a well-established spermatogonial stem cell marker, its role within the female urogenital system remains unclear.ResultsIn this study, we found Adgra3 to be expressed throughout the murine female urogenital system, with higher expression pre-puberty than after sexual maturation. We generated a global Adgra3−/− mouse line and observed imperforate vagina in 44% of Adgra3−/− females, resulting in distension of the reproductive tract and infertility. Ovarian morphology, plasma estradiol, ovarian Cyp19a1, and vaginal estrogen receptor α (Esr1) expression were unaffected. However, compared to controls, a significantly lower bone mineral density was found in Adgra3−/− mice. Whereas vaginal opening in mice is an estrogen-dependent process, 17β-estradiol treatment failed to induce vaginal canalization in Adgra3−/− mice. Furthermore, a marked reduction in vaginal and ovarian progesterone receptor expression was observed concomitant with an upregulation of apoptotic regulators Bcl2, Bid, and Bmf in adult Adgra3−/− females with a closed vagina.ConclusionsOur collective results shed new insights into the complex mechanisms by which the adhesion receptor ADGRA3 regulates distal vaginal tissue remodeling during vaginal canalization via altered sex hormone responsiveness and balance in apoptotic regulators. This highlights the potential of ADGRA3 as a target in diagnostic screening and/or therapy for obstructive vaginal malformations in humans.

Analysis of OCT4 and PGP9.5 gene expression in prenatal and postnatal buffalo (Bubalus bubalis) testes

This study aimed to investigate and characterize the spermatogonial stem cells (SSCs) in buffaloes at different stages of development, including prenatal, neonatal, prepubertal, and adult testes. We sought a comprehensive understanding of these cells through a combination of histological, immunohistochemical, and ultrastructural analyses. Specifically, we examined changes in the expression of two potential SSC markers, OCT4 and PGP9.5, using immunohistochemistry. Additionally, we conducted a real-time quantitative polymerase chain reaction (RT-qPCR) to assess the relative gene expression of OCT4 and PGP9.5. The relative expression of the OCT4 gene was down-regulated in the adult testes compared to its expression during prepubertal and neonatal life. The relative expression of the PGP9.5 gene was up-regulated in the neonatal testes and down-regulated in the prepubertal and adult testes. The spermatogonia were round, oval-to-ellipsoidal cells lying over the basement membrane (BM) with a round-to-oval nucleus. Based on the immunoexpression of the putative SSC markers, OCT4 and PGP9.5, we concluded that the proportion of stem cells was highest during the neonatal stage, followed by the prepubertal and prenatal stages. This finding sheds light on the dynamics of spermatogonial stem cells in buffalo testes at different developmental stages, providing valuable insights into these cells’ regulation and potential applications.

Characterization and enrichment of spermatogonial stem cells of common carp (Cyprinus carpio)

Spermatogenesis is a systematically organized process that ensures uninterrupted sperm production in which the spermatogonial stem cells (SSCs) play a crucial role. However, the existing absence of teleost-specific molecular markers for SSCs presents a notable challenge. Herein we characterized phenotypically the spermatogonial stem cells using specific molecular markers and transmission electron microscopy. Moreover, we also describe a simple method to suppress common carp spermatogenesis using the combination of Busulfan and thermo-chemical treatment, and finally, we isolate and enrich the undifferentiated spermatogonial fraction. Our results showed that C-kit, GFRα1, and POU2 proteins were expressed by germ cells, meanwhile, undifferentiated spermatogonial populations preferentially expressed GFRα1 and POU2. Moreover, the combination of high temperature (35 °C) and Busulfan (40 mg/kg/BW) effectively suppressed the spermatogenesis of common carp males. Additionally, the amh expression analysis showed differences between the control (26 °C) when compared to 35 °C with a single or two Busulfan doses, confirming that the testes were depleted by the association of Busulfan at high temperatures. In an attempt to isolate the undifferentiated spermatogonial fraction, we used the Percoll discontinuous density gradient. Thus, we successfully dissociated the carp whole testes in different cellular fractions; subsequently, we isolated and enriched the undifferentiated spermatogonial population. Therefore, our results suggest that probably both GFRα-1 and POU2 are highly conserved factors expressed in common carp germinative epithelium and that these molecules were well conserved along the evolutionary process. Furthermore, the enriched undifferentiated spermatogonial population developed here can be used in further germ cell transplantation experiments to preserve and propagate valued and endangered fish species.

Expression of Phospholipase D Family Member 6 in Bovine Testes and Its Molecular Characteristics.

Spermatogonial stem cells (SSCs) are the only primitive spermatogonial cells in males that can naturally transmit genetic information to their offspring and replicate throughout their lives. Phospholipase D family member 6 (PLD6) has recently been found to be a surface marker for SSCs in mice and boars; however, it has not been validated in cattle. The results of reversed transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) found that the relative expression of the PLD6 gene in the testicular tissues of two-year-old Simmental calves was significantly higher than that of six-month-old calves. Immunofluorescent staining further verified the expression of PLD6 protein in bovine spermatogenic cells like germ cell marker DEAD box helicase 4 (DDX4, also known as VASA). Based on multiple bioinformatic databases, PLD6 is a conservative protein which has high homology with mouse Q5SWZ9 protein. It is closely involved in the normal functioning of the reproductive system. Molecular dynamics simulation analyzed the binding of PLD6 as a phospholipase to cardiolipin (CL), and the PLD6-CL complex showed high stability. The protein interaction network analysis showed that there is a significant relationship between PLD6 and piwi-interacting RNA (piRNA) binding protein. PLD6 acts as an endonuclease and participates in piRNA production. In addition, PLD6 in bovine and mouse testes has a similar expression pattern with the spermatogonium-related genes VASA and piwi like RNA-mediated gene silencing 2 (PIWIL2). In conclusion, these analyses imply that PLD6 has a relatively high expression in bovine testes and could be used as a biomarker for spermatogenic cells including SSCs.

BMI1 fine-tunes gene repression and activation to safeguard undifferentiated spermatogonia fate.

Introduction: Spermatogenesis is sustained by the homeostasis of self-renewal and differentiation of undifferentiated spermatogonia throughout life, which is regulated by transcriptional and posttranscriptional mechanisms. B cell-specific Moloney murine leukemia virus integration site 1 (BMI1), one of spermatogonial stem cell markers, is a member of Polycomb repressive complex 1 (PRC1) and important to spermatogenesis. However, the mechanistic underpinnings of how BMI1 regulates spermatogonia fate remain elusive. Methods: We knocked down BMI1 by siRNA to investigate the role of BMI1 in undifferentiated spermatogonia. Differentially expressed genes were identified by RNA-seq and used for KEGG pathway analysis. We performed ChIP-seq analysis in wild type and BMI1 knockdown cells to explore the underlying molecular mechanisms exerted by BMI1. BMI1-associated alterations in repressive histone modifications were detected via Western blotting and ChIP-seq. Furthermore, we performed mass spectrometry and Co-immunoprecipitation assays to investigate BMI1 co-factors. Finally, we demonstrated the genomic regions occupied by both BMI1 and its co-factor. Results: BMI1 is required for undifferentiated spermatogonia maintenance by both repressing and activating target genes. BMI1 preserves PI3K-Akt signaling pathway for spermatogonia proliferation. Decrease of BMI1 affects the deposition of repressive histone modifications H2AK119ub1 and H3K27me3. BMI also positively regulates H3K27ac deposited genes which are associated with proliferation. Moreover, we demonstrate that BMI1 interacts with Sal-like 4 (SALL4), the transcription factor critical for spermatogonia function, to co-regulate gene expression. Discussion: Overall, our study reveals that BMI1 safeguards undifferentiated spermatogonia fate through multi-functional roles in regulating gene expression programs of undifferentiated spermatogonia.

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.

A Fundamental Research in In Vitro Spermatogonial Stem Cell Culturing: What Are Clump Cells?

Spermatogonial stem cells (SSCs) are a small group of testicular cells located in the basement membrane of seminiferous tubules and can balance self-renewal and differentiation during spermatogenesis. Our in vitro culture experiments of mouse SSCs indicated heterogeneity of cultured cells. Highly compact colonies were observed next to SSC colonies, which we call clump cells. We used immunocytochemical staining to identify SSCs and somatic cells with VASA and Vimentin antibodies. Subsequently, we compared mRNA expression levels of VASA, DAZL, PLZF, GFRA1, Lin28, Kit, Myc and Vimentin genes using Fluidigm real-time RT-polymerase chain reaction in clump cells, SSCs, and testicular stromal cells. To better understand the functions of selected genes, we created a protein-protein interaction network and performed an enrichment analysis using different databases. Based on the data collected, we state that clump cells do not express the molecular markers of SSCs, so we cannot consider them as SSCs; however, we claim that these cells are altered SSCs. The molecular mechanism of this conversion is still obscure. Therefore, this study can support the analysis of germ cell development both in vitro and in vivo. In addition, it can be effective in finding new and more efficient treatments for male infertility.

Generation of Transgenic Sperm Expressing GFP by Lentivirus Transduction of Spermatogonial Stem Cells In Vivo in Cynomolgus Monkeys.

Nonhuman primates (NHPs) have been considered as the best models for biomedical research due to their high similarities in genomic, metabolomic, physiological and pathological features to humans. However, generation of genetically modified NHPs through traditional methods, such as microinjection into the pronuclei of one-cell embryos, is prohibitive due to the targeting efficiency and the number of NHPs needed as oocyte/zygote donors. Using spermatogonial stem cells (SSCs) as the target of gene editing, producing gene-edited sperm for fertilization, is proven to be an effective way to establish gene editing animal disease models. In this experiment, we used ultrasound to guide the echo dense injection needle into the rete testis space, allowing the EGFP lentivirus to be slowly injected at positive pressure from the rete testis into seminiferous tubules. We found Thy1 can be used as a surface marker of cynomolgus monkey SSCs, confirming that SSCs carry the GFP gene. Finally, we successfully obtained transgenic sperm, with a similar freezing and recovery rate to that of WT animals.

Loss of Adgra3 causes obstructive azoospermia with high penetrance in male mice.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3-/- ), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

Signal regulatory protein alpha is a conserved marker for mouse and rat spermatogonial stem cells†.

Characterization of spermatogonial stem cells (SSCs) has been hampered by their low frequency and lack of features that distinguish them from committed spermatogonia. Few conserved SSC markers have been discovered. To identify a new SSC marker, we evaluated SIRPA expression in mouse and rat SSCs. SIRPA was expressed in a small population of undifferentiated spermatogonia. SIRPA, and its ligand CD47 were expressed in cultured SSCs. Expression of both SIRPA and CD47 was upregulated by supplementation of GDNF and FGF2, which promoted SSC self-renewal. Sirpa depletion by short hairpin RNA impaired the proliferation of cultured SSCs, and these cells showed decreased MAP2K1 activation and PTPN11 phosphorylation. Immunoprecipitation experiments showed that SIRPA associates with PTPN11. Ptpn11 depletion impaired SSC activity in a manner similar to Sirpa depletion. SIRPA was expressed in undifferentiated spermatogonia in rat and monkey testes. Xenogenic transplantation experiments demonstrated that SIRPA is expressed in rat SSCs. These results suggest that SIRPA is a conserved SSC marker that promotes SSC self-renewal division by activating the MAP2K1 pathway via PTPN11.

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.

P-803 DNA-FISH analysis in testicular tissue cells of prepuberal patients with Klinefelter Syndrome

Abstract Study question Can we accurately assess sex-chromosomes number in testicular cells (germ and somatic) of Klinefelter Syndrome (KS) prepuberal patients by DNA-FISH in paraffin embedded sections? Summary answer We have evaluated the sex-chromosomes content of testicular cells in KS prepuberal patients and the presence of XY lines evidenced a gonadal mosaicism status. What is known already Prepuberal boys with Klinefelter Syndrome usually have fertility problems in adulthood, mainly azoospermia. Due to the symptoms cause by the disease, Spermatogonial Stem Cells (SSCs) are less abundant and spermatogenesis does not occur. These patients do not have any alternative to restore the fertility in the future. When a boy has both XY and XXY cell lines, this patient is mosaic (46,XY/47,XXY). Meaning some cells could undergo spermatogenesis and generate gametes with normal sex-chromosomes number. The aim of this work is to test if KS patients diagnosed as pure can have testicular cell lines with XY chromosome number (mosaic). Study design, size, duration We obtained samples from human prepuberal patients diagnosed with pure Klinefelter Syndrome (47,XXY) for the last 5 years. Testicular biopsy fragments are fixed for histological studies and other fragments are cryopreserved. Some fragments are used for immunofluorescence and subsequently processed by DNA-FISH to determine the sex-chromosomes content of testicular cells. Participants/materials, setting, methods In this study, we used 10 prepuberal patients with KS and 5 prepuberal patients with other fertility problems with normal chromosome set as controls. We performed immunofluorescence to determine expression of germ cells (VASA) and SSCs (MAGEA4) markers and somatic cells markers such as Leydig cells (StAR) and Sertoli cells (SOX9). Afterwards, we perform DNA-FISH, with probes specific for chromosomes X and Y and chromosome 18 as a control. Main results and the role of chance The methodology used allows cytogenetic characterization of testicular tissue in paraffin embedded sections. Testicular mosaicism has been observed in all patients diagnosed as pure KS. We have observed a degree of mosaicism of 66-80% in SSCs, of 20-50% in Sertoli cells and of 30-50% in Leydig cells. We pursued a protocol with a good FISH efficiency that allows colocalization of previous immunocharacterized testicular cells. Limitations, reasons for caution The DNA-FISH technique is made just in a single paraffin section of each KS patient testicular sample. Due to the limited and difficulty to obtain KS testicular tissue, we use a limited number of samples. Wider implications of the findings We succeed to demonstrate the mosaicism of testicular cells in prepuberal patients that are diagnosed with KS. Thus, the better understanding of the SSCs with normal chromosome set (XY) could be useful for future in vitro expansion and stem cell therapies. Trial registration number not applicable

Models and Molecular Markers of Spermatogonial Stem Cells in Vertebrates: To Find Models in Nonmammals

Spermatogonial stem cells (SSCs) are the germline stem cells that are essential for the maintenance of spermatogenesis in the testis. However, it has not been sufficiently understood in amphibians, reptiles, and fish because numerous studies have been focused mainly on mammals. The aim of this review is to discuss scientific ways to elucidate SSC models of nonmammals in the context of the evolution of testicular organization since rodent SSC models. To further understand the SSC models in nonmammals, we point out common markers of an SSC pool (undifferentiated spermatogonia) in various types of testes where the kinetics of the SSC pool appears. This review includes the knowledge of (1) common molecular markers of vertebrate type A spermatogonia including putative SSC markers, (2) localization of the markers on the spermatogonia that have been reported in previous studies, (3) highlighting the most common markers in vertebrates, and (4) suggesting ways of finding SSC models in nonmammals.

Retinoic acid promotes formation of chicken (Gallus gallus) spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. Systematically exploring the critical factors associated with the formation of SSCs will provide new insight to improve the formation efficiency, and their practical application. Here we explore the regulatory mechanism of the ECM-receptor interaction signaling pathway and related genes during differentiation of SSCs in chicken. Firstly, the positive cell rate of SSCs protein marker was detected by immunofluorescence and flow cytometry and qRT-PCR was used to identify, the expression of related marker genes after 10days of RA-induction. Secondly, the ESCs on 0d/ 4d /10d after RA- induction/self-differentiation were collected, and the total RNA was then extracted from cells. Finally, high-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. After PCA analysis of the RNA-seq data, Venny analysis, GO and KEGG enrichment were further used to find the key signaling pathways and genes in the RA-induction process. The results showed that on day 10 of RA-induction, grape cluster growth cells expressed integrinβ1, the specific marker protein of SSCs cells, and the integrinβ1 positive rate was 35.1%. Also, SSCs marker genes CVH, Integrinβ1, Integrinα6 were significantly up-regulated during RA-induction. Moreover, the significantly enriched pathway, ECM-receptor interaction signaling, in current study may play a crucial role in RA-induction. Then, JASPAR was used to predict the differential gene transcription factors in the signaling pathway, finding that RA receptor was a transcription factor of COL5A1, COL5A2 and COL3A1. The qRT-PCR results showed that the expression levels of RA receptors (RXRA, RARA and RXRG) and the predicted genes (COL5A1, COL5A2 and COL3A1) were both significantly increased during RA-induction. Also, dual-luciferase reporter assay showed that RA could affect the luciferin activities of COL5A1, COL5A2 and COL3A1. These results suggest that RA plays a crucial role in the formation of chicken spermatogonial stem cells via the transcription levels of COL5A1, COL5A2 and COL3A1 to regulate the ECM-receptor interaction signaling pathway. Additionally, knockdown of COL5A1/COL5A2/COL3A1 could effectively reduce the formation efficiency of SSCs. This indicated that the interference of RA receptor binding genes in the ECM-receptor interaction signaling pathway could decrease the efficiency of RA induced SSCs formation. Therefore, this study concludes that RA promotes formation of chicken spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.

MiR-22-5p regulates the self-renewal of spermatogonial stem cells by targeting EZH2.

MiRNAs play an important role in spermatogonial stem cells (SSCs). The purpose of this study was to investigate the basic function of miR-22-5p in cryptorchidism. The results of RT-PCR, western blot, and immunohistochemistry showed that miR-22-5p was increased while EZH2 decreased in the testicular tissues of patients with cryptorchidism. Overexpression of miR-22-5p inhibited the proliferation of SSCs, increased cell apoptosis rate, and reduced expression of SSC marker proteins (GDNF and DAZL); however, knockout of miR-22-5p has the opposite effect. The Luciferase reporter gene assays demonstrated that EZH2 is a direct target of miR-22-5p. Moreover, EZH2 overexpression could reverse the effect of miR-22-5p mimic on SSCs’ proliferation, apoptosis, and expression of SSC marker proteins. Our results demonstrated that miR-22-5p regulates SSCs’ self-renewal by targeting EZH2, which indicated that miR-22-5p may serve as a biological marker for the treatment of infertility caused by cryptorchidism.

PD39-10 A XENO-FREE CULTURE METHOD FOR THE IN VITRO EXPANSION OF HUMAN SPERMATOGONIAL STEM CELLS

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (PD39)1 Sep 2021PD39-10 A XENO-FREE CULTURE METHOD FOR THE IN VITRO EXPANSION OF HUMAN SPERMATOGONIAL STEM CELLS Meghan Robinson, Ryan Flannigan, and Luke Witherspoon Meghan RobinsonMeghan Robinson More articles by this author , Ryan FlanniganRyan Flannigan More articles by this author , and Luke WitherspoonLuke Witherspoon More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002049.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In vitro expansion of spermatogonial stem cells (SSCs) has been established using animal-derived fetal bovine serum (FBS), however, animal components introduce the risk of contaminating with pathogens, making them unsuitable for medical use. This study set out to develop xeno-free culture conditions for the expansion of human SSCs. METHODS: SSCs were derived from human induced pluripotent stem cells (hiPSCs) and tested in various xeno-free media conditions in combination with growth factors Prostaglandin D2 (PDG2) and Insulin-Like Growth Factor 1 (IGF1). Primary SSCs then underwent 3 passages in the best condition, were cryopreserved and thawed, and then analysed for changes in gene and protein expression by real time polymerase chain reaction (RT-qPCR) and immunocytochemistry. RESULTS: 10 ng/mL PDG2 with 10 ng/mL IGF1 was found to replace FBS and BSA without loss of viability or growth. ROCK inhibitor Y-27632 was determined to be necessary for viability upon thawing. Compared to established conditions, the SSCs shared identical protein expression profiles for the SSC markers Glial Cell-Derived Neurotrophic Factor Family Receptor Alpha 1 (GFRA1), G-Protein Coupled Receptor 125 (GPR125), Thy-1 Cell Surface Antigen (CD90), and Stage-Specific Embryonic Antigen 4 (SSEA4) (Figure 1A-B). RT-qPCR analyses revealed no significant variation in gene expression of undifferentiated germ cell markers and Inhibitor Of Differentiation 4 (ID4), and Fibroblast Growth Factor Receptor 3 (FGFR3). The greatest change seen was an increase in Deleted In Azoospermia Like (DAZL), a regulator of both SSC proliferation and differentiation (23-fold) (Figure 1C). CONCLUSIONS: This study identified a combination of growth factors capable of replacing FBS and BSA components in established SSC expansion cell culture. This xeno-free defined formulation allows standardized SSC culture and removes the risk of animal pathogens. Source of Funding: VCHRI, CUASF, CIHR, UBC © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e671-e672 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Meghan Robinson More articles by this author Ryan Flannigan More articles by this author Luke Witherspoon More articles by this author Expand All Advertisement Loading ...

MicroRNA-30a-5p promotes differentiation in neonatal mouse spermatogonial stem cells (SSCs)

BackgroundThe importance of spermatogonial stem cells (SSCs) in spermatogenesis is crucial and intrinsic factors and extrinsic signals mediate fate decisions of SSCs. Among endogenous regulators, microRNAs (miRNAs) play critical role in spermatogenesis. However, the mechanisms which individual miRNAs regulate self- renewal and differentiation of SSCs are unknown. The aim of this study was to investigate effects of miRNA-30a-5p inhibitor on fate determinations of SSCs.MethodsSSCs were isolated from testes of neonate mice (3–6 days old) and their purities were performed by flow cytometry with ID4 and Thy1 markers. Cultured cells were transfected with miRNA- 30a-5p inhibitor. Evaluation of the proliferation (GFRA1, PLZF and ID4) and differentiation (C-Kit & STRA8) markers of SSCs were accomplished by immunocytochemistry and western blot 48 h after transfection.ResultsBased on the results of flow cytometry with ID4 and Thy1 markers, percentage of purity of SSCs was about 84.3 and 97.4 % respectively. It was found that expression of differentiation markers after transfection was significantly higher in miRNA-30a- 5p inhibitor group compared to other groups. The results of proliferation markers evaluation also showed decrease of GFRA1, PLZF and ID4 protein in SSCs transfected with miRNA-30a-5p inhibitor compared to the other groups.ConclusionsIt can be concluded that inhibition of miRNA-30a-5p by overexpression of differentiation markers promotes differentiation of Spermatogonial Stem Cells.

Effect of a Freezing Medium Containing Melatonin on Markers of Pre-meiotic and Post-meiotic Spermatogonial Stem Cells (SSCs) After Transplantation in an Azoospermia Mouse Model Due to Testicular Torsion.

Spermatogonial stem cells (SSCs) are essential to the initiation of spermatogenesis. Cryopreservation, long-term maintenance, and auto-transplantation of SSCs could be a new treatment for infertility. The aim of this study was to add melatonin to the basic freezing medium and to evaluate its effect on the efficiency of the thawed SSCs after transplantation into the testicles of azoospermic mice. SSCs were isolated from newborn NMRI mice, and the cells were enriched to assess morphological features. The thawed SSCs were evaluated for survival, apoptosis, and ROS level before transplantation, and the proliferation (MVH and ID4) and differentiation (c-Kit, SCP3, TP1, TP2, and Prm1) markers of SSCs were examined using immunofluorescence, western blot, and quantitative real-time polymerase chain reaction (PCR) after transplantation. It was found that the survival rate of SSCs after thawing was significantly higher in the melatonin group compared with the cryopreservation group containing basic freezing medium, and the rate of apoptosis and level of ROS production also decreased significantly in the cryopreservation group with melatonin (p < 0.05). The expression of proliferation and differentiation markers after transplantation was significantly higher in the cryopreservation group with melatonin compared to the cryopreservation group (p< 0.05). The results suggest that adding melatonin to the basic freezing medium can effectively protect the SSCs by increasing the viability and reducing the ROS production and apoptosis and improve the transplantation efficiency of SSCs after cryopreservation, which will provide a significant suggestion for fertility protection in the clinic.