Spermatogonial Stem Cells Activity Research Articles

BLOC1S1 promotes proliferation of goat spermatogonial stem cells

With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.

Glutamine protects mouse spermatogonial stem cells against NOX1-derived ROS for sustaining self-renewal division in vitro.

Reactive oxygen species (ROS) are generated from NADPH oxidases and mitochondria; they are generally harmful for stem cells. Spermatogonial stem cells (SSCs) are unique among tissue-stem cells because they undergo ROS-dependent self-renewal via NOX1 activation. However, the mechanism by which SSCs are protected from ROS remains unknown. Here, we demonstrate a crucial role for Gln in ROS protection using cultured SSCs derived from immature testes. Measurements of amino acids required for SSC cultures revealed the indispensable role of Gln in SSC survival. Gln induced Myc expression to drive SSC self-renewal in vitro, whereas Gln deprivation triggered Trp53-dependent apoptosis and impaired SSC activity. However, apoptosis was attenuated in cultured SSCs that lacked NOX1. In contrast, cultured SSCs lacking Top1mt mitochondria-specific topoisomerase exhibited poor mitochondrial ROS production and underwent apoptosis. Gln deprivation reduced glutathione production; supra-molar Asn supplementation allowed offspring production from SSCs cultured without Gln. Therefore, Gln ensures ROS-dependent SSC-self-renewal by providing protection against NOX1 and inducing Myc.

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.

Distinctive molecular features of regenerative stem cells in the damaged male germline.

Maintenance of male fertility requires spermatogonial stem cells (SSCs) that self-renew and generate differentiating germ cells for production of spermatozoa. Germline cells are sensitive to genotoxic drugs and patients receiving chemotherapy can become infertile. SSCs surviving treatment mediate germline recovery but pathways driving SSC regenerative responses remain poorly understood. Using models of chemotherapy-induced germline damage and recovery, here we identify unique molecular features of regenerative SSCs and characterise changes in composition of the undifferentiated spermatogonial pool during germline recovery by single-cell analysis. Increased mitotic activity of SSCs mediating regeneration is accompanied by alterations in growth factor signalling including PI3K/AKT and mTORC1 pathways. While sustained mTORC1 signalling is detrimental for SSC maintenance, transient mTORC1 activation is critical for the regenerative response. Concerted inhibition of growth factor signalling disrupts core features of the regenerative state and limits germline recovery. We also demonstrate that the FOXM1 transcription factor is a target of growth factor signalling in undifferentiated spermatogonia and provide evidence for a role in regeneration. Our data confirm dynamic changes in SSC functional properties following damage and support an essential role for microenvironmental growth factors in promoting a regenerative state.

Ascorbic acid regulates mouse spermatogonial stem cell proliferation in a Wnt/β-catenin/ROS signaling dependent manner

Spermatogonial stem cells (SSCs) provide a foundation for spermatogenesis, but the mechanism of SSC proliferation is still poorly understood. To investigate whether and how ascorbic acid (AA) regulates the growth of mouse SSCs in vitro, the SSCs were cultured in different concentration AA medium for 14 days. The proliferation, apoptosis and the reactive oxygen species (ROS) levels of SSCs in different AA groups were respectively detected. Moreover, the SSC activity in 40 μg/mL AA group and the control was tested by a transplantation assay. To explore the mechanism of AA regulating mouse SSCs proliferation, the dishevelled homolog 2 (DVL2) and nucleoredoxin (NRX) protein levels, the expression of axis inhibition protein 2 (Axin2), leucine-rich G-protein coupled receptor 5 (Lgr5), B-cell lymphoma-2 (Bcl-2), BCL2-Associated X (Bax), c-myc and cyclin D1 genes in Wnt/β-catenin pathway were respectively confirmed. The results showed that the adding concentration of AA did not affect the main shape of SSCs. A 40 μg/mL AA in culture medium promoted the proliferation, and decreased the ROS production and apoptosis rate of SSCs. Moreover, colonization efficiency in the seminiferous tubules of the recipient testis in 40 μg/mL AA group was higher compared with the control group by a transplantation assay. Finally, the appropriate ROS in the 40 μg/mL AA group further adjust the levels of DVL2 and NRX protein in the Wnt/β-catenin pathway to maintain the nuclear intensity of β-catenin, in turn, the expression of apoptosis gene Bax decreased, while the expression of Bcl2, Axin2, Lgr5, c-myc and cyclin D1 genes increased. The study confirmed that AA adjusts the endogenous ROS level to impact on SSC proliferation in a dose-dependent manner by Wnt/β-catenin signaling pathway.

Dimethyloxaloylglycine promotes spermatogenesis activity of spermatogonial stem cells in Bama minipigs.

BackgroundThe testis has been reported to be a naturally O2-deprived organ, dimethyloxaloylglycine (DMOG) can inhibit hypoxia inducible factor-1alpha (HIF-1α) subject to degradation under normal oxygen condition in cells.ObjectivesThe objective of this study is to detect the effects of DMOG on the proliferation and differentiation of spermatogonial stem cells (SSCs) in Bama minipigs.MethodsGradient concentrations of DMOG were added into the culture medium, HIF-1α protein in SSCs was detected by western blot analysis, the relative transcription levels of the SSC-specific genes were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Six days post-induction, the genes related to spermatogenesis were detected by qRT-PCR, and the DNA content was determined by flow cytometry.ResultsResults revealed that the levels of HIF-1α protein increased in SSCs with the DMOG treatment in a dose-dependent manner. The relative transcription levels of SSC-specific genes were significantly upregulated (p < 0.05) by activating HIF-1α expression. The induction results showed that DMOG significantly increased (p < 0.05) the spermatogenesis capability of SSCs, and the populations of haploid cells significantly increased (p < 0.05) in DMOG-treated SSCs when compared to those in DMOG-untreated SSCs.ConclusionWe demonstrate that DMOG can promote the spermatogenesis activity of SSCs.

TCF3 Regulates the Proliferation and Apoptosis of Human Spermatogonial Stem Cells by Targeting PODXL.

Spermatogonial stem cells (SSCs) are the initial cells for the spermatogenesis. Although much progress has been made on uncovering a number of modulators for the SSC fate decisions in rodents, the genes mediating human SSCs remain largely unclear. Here we report, for the first time, that TCF3, a member of the basic helix-loop-helix family of transcriptional modulator proteins, can stimulate proliferation and suppress the apoptosis of human SSCs through targeting podocalyxin-like protein (PODXL). TCF3 was expressed primarily in GFRA1-positive spermatogonia, and EGF (epidermal growth factor) elevated TCF3 expression level. Notably, TCF3 enhanced the growth and DNA synthesis of human SSCs, whereas it repressed the apoptosis of human SSCs. RNA sequencing and chromatin immunoprecipitation (ChIP) assays revealed that TCF3 protein regulated the transcription of several genes, including WNT2B, TGFB3, CCN4, MEGF6, and PODXL, while PODXL silencing compromised the stem cell activity of SSCs. Moreover, the level of TCF3 protein was remarkably lower in patients with spermatogenesis failure when compared to individuals with obstructive azoospermia with normal spermatogenesis. Collectively, these results implicate that TCF3 modulates human SSC proliferation and apoptosis through PODXL. This study is of great significance since it would provide a novel molecular mechanism underlying the fate determinations of human SSCs and it could offer new targets for gene therapy of male infertility.

Constitutive activation of CTNNB1 results in a loss of spermatogonial stem cell activity in mice.

Spermatogenesis requires that a careful balance be maintained between the self-renewal of spermatogonial stem cells (SSCs) and their commitment to the developmental pathway through which they will differentiate into spermatozoa. Recently, a series of studies employing various in vivo and in vitro models have suggested a role of the wingless-related MMTV integration site gene family/beta-catenin (WNT/CTNNB1) pathway in determining the fate of SSCs. However, conflicting data have suggested that CTNNB1 signaling may either promote SSC self-renewal or differentiation. Here, we studied the effects of sustained CTNNB1 signaling in SSCs using the Ctnnb1tm1Mmt/+; Ddx4-CreTr/+ (ΔCtnnb1) mouse model, in which a stabilized form of CTNNB1 is expressed in all germ cells. ΔCtnnb1 mice were found to have reduced testis weights and partial germ cell loss by 4 months of age. Germ cell transplantation assays showed a 49% reduction in total functional SSC numbers in 8 month-old transgenic mice. In vitro, Thy1-positive undifferentiated spermatogonia from ΔCtnnb1 mice formed 57% fewer clusters, which was associated with decreased cell proliferation. A reduction in mRNA levels of genes associated with SSC maintenance (Bcl6b, Gfra1, Plzf) and increased levels for markers associated with progenitor and differentiating spermatogonia (Kit, Rarg, Sohlh1) were detected in these cluster cells. Furthermore, RNAseq performed on these clusters revealed a network of more than 900 genes regulated by CTNNB1, indicating that CTNNB1 is an important regulator of spermatogonial fate. Together, our data support the notion that CTNNB1 signaling promotes the transition of SSCs to undifferentiated progenitor spermatogonia at the expense of their self-renewal.

Rescue and Conservation of Male Adult Alpacas (Vicugna pacos) Based on Spermatogonial Stem Cell Biotechnology Using Atomized Black Maca as a Supplement of Cryopreservation Medium.

This is the first time that testicular tissue (n = 44) and isolated testicular cells (n = 51) were cryopreserved from alpaca testes 24 h postmortem. For this purpose, internally designed freezing media and cryopreservation protocols were used. Testicular tissue fragments (25 mg) and isolated testicular cells were frozen in MTDB (trehalose and black maca), MTD (trehalose), MSDB (sucrose and black maca), and MSD (sucrose) media. Isolated spermatogonial cells were cryopreserved in two ways, before and after proliferation in vitro. After cryopreservation, the percentage of cell viability in Group 1 (>50% of cell viability) by trypan blue did not show differences within each group (p > 0.05) but showed significant differences when comparing fragments with isolated cells (p < 0.05). Spermatogonial stem cells (SSC) were identified by flow cytometry as strong Dolichos biflorus agglutinin (sDBA) and mitochondrial activity of SSC as strongly positive for MitoSense (sMitoSense+) in intact mitochondria cells, weakly positive for MitoSense (wMitoSense+) in early apoptosis, and necrosis with 7-Aminoactinomycin-D positive (7-AAD). After freezing, in Group 1M (≥30% sMitoSense+), the fragments did not show differences between the media (p > 0.05), but in the isolated cells frozen in MSDB medium, 63.68 ± 8.90% (p < 0.05). In Group 2M (<30% sMitoSense+), necrosis (7AAD+) in MSDB medium was 27.03 ± 5.80%, and necrosis in isolated cells was 14.05 ± 9.3% with significant differences between these groups (p < 0.05); in sMitoSense+, the isolated cells (34.40 ± 23%) had a higher percentage than the fragments (12.4 ± 5.2) (p < 0.05). On the other hand, MSDB and MSD media were significantly higher for isolated cells than for fragments in sDBA+ (p < 0.05). On the other hand, the SSC (sDBA+) had significant differences (p < 0.05) between fresh cells 7.43 ± 1.3% (sDBA+) compared with those cryopreserved in MSDB medium 1.46 ± 0.34% (sDBA+). Additionally, the proliferated and cryopreserved SSC 6.29 ± 1.17% (sDBA+) did not show significant differences concerning the fresh cells (p > 0.05). In conclusion, the black maca showed antioxidant properties when it was included in the freezing medium and, therefore, improved the SSC's conservation of the alpaca. Furthermore, the proliferation of isolated cells in vitro produces a higher amount of SSC after thawing them for further preclinical or clinical work.

Genetic and histopathological analysis of transverse testicular ectopia without persistent M\xfcllerian duct syndrome: two case reports

BackgroundTransverse testicular ectopia (TTE) is a rare anomaly in which both testes descend through a single inguinal canal into the same hemiscrotum. Although almost 20–50% of patients with TTE exhibit persistent Müllerian duct syndrome (PMDS) and many genetic analyses have been performed, no reports have described the genes contributing to TTE without PMDS. Here, we report two cases of TTE without PMDS using immunohistochemical staining and genetic analysis.Case presentationTwo Asian patients with TTE without PMDS were subjected to orchiopexy. We performed testicular biopsies during operation and obtained blood samples before the operation. Testicular tissues were stained for c-kit, placental alkaline phosphatase (PLAP), and undifferentiated embryonic cell transcription factor 1 (UTF1) to evaluate the presence of intratubular malignant germ cells. Additionally, we performed polymerase chain reaction-based direct sequencing to identify single nucleotide polymorphisms in genes associated with regression of the Müllerian duct and testicular descent (that is, anti-Müllerian hormone [AMH], AMH receptor 2 [AMHR2], insulin-like 3 [INSL3], and relaxin family peptide receptor 2 [RXFP2]). The three-dimensional structures of proteins were predicted using SWISS-MODEL. In immunohistochemical analysis, c-kit and UTF1 were positive, whereas PLAP was negative in three testicular tissue samples from the two patients. These features were also detected on the unaffected side. In variant analysis, common missense variants in the AMH gene (g.365G>T; c.165G>T; p.Ser49Ile [rs10407022]) were observed. All variants in INSL3 and RXFP2 genes were intronic or silent.ConclusionsBecause UTF1, a specific marker of spermatogonial stem cell activity, was expressed in both the affected and unaffected sides in the testicular tissues of two patients, the risk of malignancy may be high in these patients. Although the etiology of TTE without PMDS remains unclear, our variant analysis results were consistent with previous reports, and variants in the AMH gene (rs10407022) may contribute to the specific phenotype of TTE without PMDS.

Necrostatin-1 improves the cryopreservation efficiency of murine spermatogonial stem cells via suppression of necroptosis and apoptosis

Cryopreservation of spermatogonial stem cells (SSCs) is important to preserve the lineages of valuable livestock and produce transgenic animals. Although interest in molecular-based cryopreservation methods have been increasing to improve their efficiency, the issue of necroptosis has not yet been considered. Therefore, the purpose of this study was to understand the role of necroptosis using necrostatin-1 (Nec-1), necroptosis inhibitor, in SSC cryopreservation, and to investigate the potential application of Nec-1 as a cryoprotectant. To determine the cryopreservation efficiency of Nec-1, we assessed recovery rate, proliferation potential, cellular membrane damage, RIP1 protein expression, apoptosis, and its mechanism. Stable characterization and functional activity of SSCs was determined via immunofluorescence, RT-qPCR, and in vivo transplantation of SSCs. Our results showed a higher proliferation potential in 50 μM Nec-1 (146.5 ± 16.8%) than in DMSO controls (100.0 ± 3.4%). Furthermore, the cryoprotective effects of Nec-1 were verified by a decrease in RIP1 expression (3.1 ± 0.2-fold vs. 1.3 ± 0.3-fold) and in early apoptosis (4.3 ± 0.8% vs. 2.6 ± 0.1%) compared to DMSO controls. Normal functional activity was observed in the SSCs after cryopreservation with 50 μM Nec-1. In conclusion, necroptosis could be a cause of cryoinjury, and their inhibitor may serve as potential effective cryoprotectant. This study will contribute to establish a molecular-based cryopreservation method, and thereby expanding the use of SSCs into the domestic livestock industry as well as for clinical applications.

Aging- and temperature-related activity of spermatogonial stem cells for germ cell transplantation in medaka

Spermatogonial transplantation can contribute to developing a novel method of producing seedlings for both aquaculture and biotic conservation. This study’s purpose was to investigate aging- and temperature-related changes in the numbers and stem cell functions of type-A spermatogonia (ASG) in the model fish medaka (Oryzias latipes). The ASG numbers in medaka of different ages were quantified via histological observation and enzymatic dissociation of vasa-Gfp medaka testes. The ASG numbers were higher in eight-month-old medaka (maturation) than in four-month-old medaka (the onset of maturation). However, ASG numbers decreased in 18-month-old medaka (senescence). Low water temperature appeared to slow down both testis development and aging processes. To study the effects of aging on ASG stem cell activity, testicular cell suspensions containing GFP-expressed ASG were prepared from vasa-Gfp medaka donors at 4 and 18 months of age and transplanted into recipient hybrid larvae of medaka (O. latipes x O. curvinotus), which provided young stem-cell-niches. The findings revealed no significant differences in ASG colonization rates isolated from medaka of different ages. Each group displayed similar rates of germ-line transmission. Furthermore, water temperature had no significant effects on each ASG’s stem cell activity. Taken together, these results indicated that aging and temperature affect ASG numbers. However, ASG isolated from medaka with different ages were transplanted into gonads with a young niche microenvironment, and there was no evidence of donor aging on stem cell activity.

CD2 is a surface marker for mouse and rat spermatogonial stem cells.

The spermatogonial stem cell (SSC) population in testis is small, and the lack of SSC markers has severely handicapped research on these cells. During our attempt to identify genes involved in SSC aging, we found that CD2 is expressed in cultured SSCs. Flow cytometric analysis and spermatogonial transplantation experiments showed that CD2 is expressed in SSCs from mature adult mouse testes. Cultured SSCs transfected with short hairpin RNAs (shRNAs) against CD2 proliferated poorly and showed an increased frequency of apoptosis. Moreover, functional analysis of transfected cells revealed impairment of SSC activity. Fluorescence activated cell sorting and spermatogonial transplantation experiments showed that CD2 is expressed not only in mouse but also in rat SSCs. The results indicate that CD2 is a novel SSC surface marker conserved between mouse and rat SSCs.

Aging of spermatogonial stem cells by Jnk-mediated glycolysis activation

Because spermatogonial stem cells (SSCs) are immortal by serial transplantation, SSC aging in intact testes is considered to be caused by a deteriorated microenvironment. Here, we report a cell-intrinsic mode of SSC aging by glycolysis activation. Using cultured SSCs, we found that aged SSCs proliferated more actively than young SSCs and showed enhanced glycolytic activity. Moreover, they remained euploid and exhibited stable androgenetic imprinting patterns with robust SSC activity despite having shortened telomeres. Aged SSCs showed increased Wnt7b expression, which was associated with decreased Polycomb complex 2 activity. Our results suggest that aberrant Wnt7b expression activated c-jun N-terminal kinase (JNK), which down-regulated mitochondria numbers by suppressing Ppargc1a Down-regulation of Ppargc1a probably decreased reactive oxygen species and enhanced glycolysis. Analyses of the Klotho-deficient aging mouse model and 2-y-old aged rats confirmed JNK hyperactivation and increased glycolysis. Therefore, not only microenvironment but also intrinsic activation of JNK-mediated glycolysis contributes to SSC aging.

Mechanisms regulating mammalian spermatogenesis and fertility recovery following germcell depletion.

Mammalian spermatogenesis is a highly complex multi-step process sustained by a population of mitotic germ cells with self-renewal potential known as spermatogonial stem cells (SSCs). The maintenance and regulation of SSC function are strictly dependent on a supportive niche that is composed of multiple cell types. A detailed appreciation of the molecular mechanisms underpinning SSC activity and fate is of fundamental importance for spermatogenesis and male fertility. However, different models of SSC identity and spermatogonial hierarchy have been proposed and recent studies indicate that cell populations supporting steady-state germline maintenance and regeneration following damage are distinct. Importantly, dynamic changes in niche properties may underlie the fate plasticity of spermatogonia evident during testis regeneration. While formation of spermatogenic colonies in germ-cell-depleted testis upon transplantation is a standard assay for SSCs, differentiation-primed spermatogonial fractions have transplantation potential and this assay provides readout of regenerative rather than steady-state stemcell capacity. The characterisation of spermatogonial populations with regenerative capacity is essential for the development of clinical applications aimed at restoring fertility in individuals following germline depletion by genotoxic treatments. This review will discuss regulatory mechanisms of SSCs in homeostatic and regenerative testis and the conservation of these mechanisms between rodent models and man.

精子幹細胞の自己複製と維持のメカニズムの解明

精子幹細胞はほぼ個体の一生にわたって毎日莫大な数の精子を産生する。その基盤が幹細胞特有な現象である自己複製分裂である。精子幹細胞にウイルス遺伝子導入により固有の標識を施し精子形成への寄与の動態を調べたところ、その寿命は極めて長いことがわかった。一方精子幹細胞には休眠期がなく全てが分裂していた。これらは精子幹細胞が活発かつ長期の自己複製活性を持つことを示す。精子幹細胞を試験管内で長期に増幅する培養法（Germline Stem; GS細胞）により幹細胞を大量に調製することが可能となり、生化学的解析や遺伝子改変により精子幹細胞の自己複製制御機構などバイオロジーの解明に役立った。またGS細胞は子孫を作成できるため、新しい生殖工学発展の可能性を秘めている。今後この技術が幅広く非齧歯類にも展開し、不妊治療や疾患モデル動物の作成などに繋がることが期待される。

Sendai virus-mediated transduction of mammalian spermatogonial stem cells†.

Spermatogonial stem cells (SSCs) provide the foundation of spermatogenesis. However, because of their small number and slow self-renewal, transfection of SSCs has met with limited success. Although several viral vectors can infect SSCs, genome integration and an inability to maintain long-term gene expression have hampered studies on SSCs. Here we report successful SSC infection by Sendai virus (SV), an RNA virus in the Paramyxoviridae. The SV efficiently transduced germline stem (GS) cells, cultured spermatogonia with enriched SSC activity, and maintained gene expression for at least 5 months. It also infected freshly isolated SSCs from adult testes. The transfected GS cells reinitiated spermatogenesis following spermatogonial transplantation into seminiferous tubules of infertile mice, suggesting that SV transfection does not interfere with spermatogenesis progression. On the other hand, microinjection of SV into the seminiferous tubules of immature mice transduced SSCs and Sertoli cells, but did not transduce Leydig or peritubular cells by interstitial virus injection. SV-infected hamster GS cells, and freshly isolated rabbit or monkey SSC-like cells were identified following xenogeneic spermatogonial transplantation, suggesting that SV transduces SSCs from several mammalian species. Thus, SV is a useful vector that can transduce both SSCs and Sertoli cells and overcome problems associated with other viral vectors.

Cyclical expression of GDNF is required for spermatogonial stem cell homeostasis.

In the murine testis, self-renewal of spermatogonial stem cells (SSCs) requires glial cell line-derived neurotrophic factor (GDNF) secreted from neighboring somatic cells. However, it not clear how GDNF promotes self-renewal in vivo or what downstream signaling pathways are required for SSC maintenance. We found that GDNF is normally expressed cyclically during spermatogenesis. Stage-specific ectopic expression of GDNF caused the accumulation of a GFRA1+ LIN28- Asingle population, which has enhanced SSC activity compared with wild type, suggesting that GDNF normally limits self-renewal to specific stages. Despite the increase in SSC cell number, EdU labeling during steady-stage spermatogenesis, and during recovery after busulfan-mediated spermatogonial depletion, indicated that GDNF promotes self-renewal by blocking differentiation and not by promoting proliferation. Increased GDNF signaling led to increased phosphorylation of AKT3 in undifferentiated spermatogonia, but not of AKT1 or AKT2, and was independent of RPS6 phosphorylation, suggesting that AKT3 functions in SSC self-renewal or progenitor cell expansion.

Co-culture of mouse spermatogonial stem cells with sertoli cell as a feeder layer, stimulates the proliferation and spermatogonial stemness profile

Abstract Objectives Sertoli cells effect the fate map of spermatogonial stem cells (SSCs) to self-renew via providing the special microenvironments. Maintenance of proliferation and self-renewal activity of SSCs may be usable as a therapeutic strategy, leads to increase the recovery of male fertility. This research was aimed to evaluate the effect of mouse sertoli cells on spermatogonia stem cells proliferation and the expression pattern of stemness markers. Methods Spermatogonia stem cells were collected from neonatal mouse testis using a two-step mechanical and enzymatic digestion. SSCs were cultured in three groups: The first group or co-culture group consists of spermatogonia and sertoli cells that were cultured together. The control group, only spermatogonial cells and the group no. 3 included spermatogonial cells in the presence of GDNF. The colony formation of mentioned groups, was monitored during one month in culture. Identification of the colonies, was confirmed using PLZF and Oct4 immunostaining. Spermatogonial stemness genes includes; Stra8, mvh and piwill2 were analyzed by RT-PCR. Results In the co-culture group, cells proliferated rapidly and many colonies were appeared whereas they were rarely formed in the control groups. Colonies were exhibited alkaline phosphatesase activity and were immunopositive to Oct4 and PLZF, strongly. The gene expression of srta8, mvh and piwill2, in SSCs that were cultivated with sertoli cells, were greater significantly than other control groups. Conclusion It is concluded that co-culture of SSCs with sertoli cells prepares conditions which leads to efficient proliferation and maintenance of stemness condition of SSCs, that is usable as a therapeutic approach for treatment of male fertility.

A Phytochemical Approach to Promotion of Self-renewal in Murine Spermatogonial Stem Cell by Using Sedum Sarmentosum Extract

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, which is dependent on the ability to self-renew and differentiation. Controlling self-renewal and differentiation of SSCs could apply to treatment of disease such as male infertility. Recently, in the field of stem cell research, it was demonstrated that effective increase in stem cell activity can be achieved by using growth factors derived from plant extracts. In this study, our aim is to investigate components from natural plant to improve the self-renewal of SSCs. To find the components, germ cells were cultured with comprehensive natural plant extracts, and then the more pure fraction, and finally single compound at different concentrations. As a result, we found 5H-purin-6-amine at 1 µg/mL, originated from Sedum sarmentosum, was a very effective compound induced SSCs proliferation. Our data showed that germ cells cultured with 5H-purin-6-amine could maintain their stable characteristics. Furthermore, transplantation results demonstrated that 5H-purin-6-amine at 1 µg/mL increased the activity of SSCs, indicating the compound could increase true SSC concentration within germ cells to 1.96-fold. These findings would be contributed to improve further reproductive research and treat male infertility by using natural plant extracts.