Proliferation Of Spermatogonial Stem Cells Research Articles

Eif2s3y Promotes the Proliferation of Spermatogonial Stem Cells by Activating ERK Signaling.

The future fertility of males with cancer may be irreversibly compromised by chemotherapy and/or radiotherapy. Spermatogonial stem cell transplantation is believed to be a way to restore fertility in men. However, the survival efficiency of transplanted cells is still low. Eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) located on the Y chromosome of male animals is a coding gene of eIF2γ which mainly functions in translation initiation. Recently, the emerging role of Eif2s3y in spermatogenesis has been emphasized in several studies. However, the underlying mechanism is still unclear. In addition, how Eif2s3y functions in large animals remains largely unknown. In this study, we obtained the CDS sequence of the Eif2s3y gene from the testis of dairy goats and found that this gene was highly expressed in the testis and was evolutionarily conserved among different species. Interestingly, overexpression of Eif2s3y promoted the proliferation of spermatogonial stem cells of dairy goats by activating the ERK signaling pathway. In animal experiments, overexpressing Eif2s3y promoted transplanted goat spermatogonial stem cells and produced more colonies after microinjection into the seminiferous tubules of infertile mice. In conclusion, our study highlights an undiscovered role of Eif2s3y in dairy goat reproduction. This finding may provide an important basis for future works regarding male spermatogenic cell restoration and represent a major advance toward surrogate sires becoming a tool for disseminating and regenerating germplasm in all mammals.

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.

The involvement of bioactive factors in the self-renewal and stemness maintenance of spermatogonial stem cells.

Spermatogenesis is usually accompanied throughout mammalian lifetime, transmitting genetic information to the next generation, which is mainly dependent on the self-renewal and differentiation of spermatogonial stem cells (SSCs). With further investigation on profiles of SSCs, the previous prevailing orthodoxy that SSCs are unipotent stem cells to differentiate into spermatids only, has been challenged. More notably, accumulating evidence has demonstrated that SSCs are capable of giving rise to cell lineages of the three germ layers, highlighting potential important applications of SSCs for regenerative medicine. Nevertheless, it is unknown how the proliferation and stemness maintenance of SSCs are regulated intrinsically and strictly controlled in a special niche microenvironment in the seminiferous tubules. Based on the special niche microenvironment for SSCs, it is of vital interest to summarize the recent knowledge regarding several critical bioactive molecules in the self-renewal and stemness maintenance of SSCs. In this review, we discuss most recent findings about these critical bioactive factors and further address the new advances on the self-renewal and stemness maintenance of SSCs.

Aberrant regulation of RNA methylation during spermatogenesis.

Natural modifications of cellular RNA include various chemical modifications, such as N6-methyladenosine (m6 A), which enable the orderly metabolism and function of RNA structural diversity, thereby affecting gene expression. Spermatogenesis is a complex differentiating developmental process, which includes the proliferation of spermatogonial stem cells, spermatocyte meiosis and sperm maturation. Emerging evidence has shown that RNA methylation can influence RNA splicing, exportation and translation, which are controlled in the male germline in order to ensure coordinated gene expression. In this review, we summarize the typical characteristics of different types of RNA methylation during the process of spermatogenesis. In particular, we emphasize the functions of the RNA methylation effectors during the male germ cell development.

Silk Nanofibrous Electrospun Scaffold Amplifies Proliferation and Stemness Profile of Mouse Spermatogonial Stem Cells

Electrospun nanofibrous scaffolds provide a structural microenvironment similar to the native extracellular matrix, which leads to the maintenance and propagation of the cultured cells under in vitro conditions. Furthermore, we used the sertoli cells to improve SSC proliferation due to its secretory role in growth factors. In this study, a silk electrospun nanofibrous scaffold has been used as a microenvironment for guiding the proliferation of spermatogonial stem cells (SSCs). SSCs were seeded on the prepared scaffolds and then cultured in vitro for 2 weeks. The attachment of SSCs was examined by scanning electron microscopy (SEM). Cell attachment and viability were evaluated via 4′, 6-diamidino-2-phenylindole staining and 3-(4, 5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide assessment, respectively. The expression of the specific SSC genes including stimulated by retinoic acid gene 8 (Stra8), deleted in azoospermia-like (DAZL) and piwil-like RNA-mediated gene silencing 2 (Piwil2), was quantified using real-time-PCR and confirmed by immunofluorescent staining. The data indicated that the prepared scaffold guided more effective SSC attachment and proliferation in comparison to the monolayer control. However, a significant upregulation of DAZL, Stra8, and piwill2 genes was observed in the cells grown on the silk scaffold. Also, the expression of Srta8 and Oct4 in the cultured cells was confirmed by immunofluorescence staining 2 weeks post-culture. Silk scaffold increases the in vitro viability of SSCs and amplifies their proliferation during the culture period. It is concluded that silk scaffold can serve as an appropriate substrate with broad implications for SSC proliferation under in vitro culture medium. We used a silk scaffold for guiding the proliferation of spermatogonial stem cell (SSC) population. Spermatogonia stem cells were seeded and cultured on silk scaffolds for 2 weeks. The attachment of SSCs was examined via SEM and viability was evaluated by DAPI staining and MTT assessments. The expression of specific spermatogonial stem cell markers including Stra8 gene, DAZL, and Piwill2 was quantified via Q-PCR and immunofluorescent staining. Scaffold guides firm attachment of SSCs and proliferates more than a monolayer layer. The significant upregulation of DAZL, Stra8, piwill2, and Oct4 in the experiment was confirmed by the real-time PCR and immunofluorescence staining 2 weeks after culture. Silk scaffold increases the in vitro viability of SSCs and amplifies their proliferation during the culture period. Silk scaffold can serve as an appropriate substrate with wide implications for SSC proliferation under in vitro condition.

FGF9 promotes mouse spermatogonial stem cell proliferation mediated by p38 MAPK signalling.

ObjectivesFibroblast growth factor 9 (FGF9) is expressed by somatic cells in the seminiferous tubules, yet little information exists about its role in regulating spermatogonial stem cells (SSCs).Materials and Methods Fgf9 overexpression lentivirus was injected into mouse testes, and PLZF immunostaining was performed to investigate the effect of FGF9 on spermatogonia in vivo. Effect of FGF9 on SSCs was detected by transplanting cultured germ cells into tubules of testes. RNA‐seq of bulk RNA and single cell was performed to explore FGF9 working mechanisms. SB203580 was used to disrupt p38 MAPK pathway. p38 MAPK protein expression was detected by Western blot and qPCR was performed to determine different gene expression. Small interfering RNA (siRNA) was used to knock down Etv5 gene expression in germ cells.ResultsOverexpression of Fgf9 in vivo resulted in arrested spermatogenesis and accumulation of undifferentiated spermatogonia. Exposure of germ cell cultures to FGF9 resulted in larger numbers of SSCs over time. Inhibition of p38 MAPK phosphorylation negated the SSC growth advantage provided by FGF9. Etv5 and Bcl6b gene expressions were enhanced by FGF9 treatment. Gene knockdown of Etv5 disrupted the growth effect of FGF9 in cultured SSCs along with downstream expression of Bcl6b.ConclusionsTaken together, these data indicate that FGF9 is an important regulator of SSC proliferation, operating through p38 MAPK phosphorylation and upregulating Etv5 and Bcl6b in turn.

Recent advances on identification of spermatogonial stem cells and their niche

Spermatogonial stem cells (SSCs) are the most primitive spermatogonia in the testis. A balance between the self-renewal and differentiation of SSCs, strictly controlled in a special „niche‟ microenvironment in the seminiferous tubules (SSC niche), is essential to maintain normal spermatogenesis. Since the 1950s, many experimental methods, including histology, immunostaining, whole-mount analyses, and pulse-chase labeling, had been used in attempts to identify SSCs. Recent studies demonstrate that Aundiffseem to possess variable levels of stem cell potential to act as SSCs; GFRα1+ population in As has the greatest potential to act as SSCs (can consider as actual SSCs) whereas NGN3+ population in Aundiffhas comparatively much lower potential to act as SSCs (can consider as potential SSCs). The precise identity of SSCs is still being refined. Sertoli cells, directly interact with SSCs, and interstial cells including Leydig cells, testicular macrophages, peritubularmyoid cells and vascular smooth muscle cells, control the proliferation and differentiation of SSCs through the secretion of extrinsic factors, constitute the cellular components of SSC niche which preferentially locates in the region of seminiferous tubules adjacent to the interstitium that always coincide blood vessels.&#x0D; Asian J. Med. Biol. Res. September 2020, 6(3): 375-382

Application of platelet-rich plasma (PRP) improves self-renewal of human spermatogonial stem cells in two-dimensional and three-dimensional culture systems

Spermatogonial stem cells (SSCs) are very sensitive to chemotherapy and radiotherapy, so male infertility is a great challenge for prepubertal cancer survivors. Cryoconservation of testicular cells before cancer treatment can preserve SSCs from treatment side effects. Different two-dimensional (2D) and three-dimensional (3D) culture systems of SSCs have been used in many species as a useful technique to in vitro spermatogenesis. We evaluated the proliferation of SSCs in 2D and 3D culture systems of platelet-rich plasma (PRP). testicular cells of four brain-dead patients cultivated in 2D pre-culture system, characterization of SSCs performed by RT-PCR, flow cytometry, immunocytochemistry and their functionality assessed by xenotransplantation to azoospermia mice. PRP prepared and dosimetry carried out to determine the optimized dose of PRP. After preparation of PRP scaffold, cytotoxic and histological evaluation performed and SSCs cultivated into three groups: control, 2D culture by optimized dose of PRP and PRP scaffold. The diameter and number of colonies measured and relative expression of GFRa1 and c-KIT evaluated by real-time PCR.Results indicated the expression of PLZF, VASA, OCT4, GFRa1 and vimentin in colonies after 2D pre-culture, xenotransplantation demonstrated proliferated SSCs have proper functionality to homing in mouse testes. The relative expression of c-KIT showed a significant increase as compared to the control group (*: p < 0.05) in PRP- 2D group, expression of GFRa1 and c-KIT in PRP scaffold group revealed a significant increase as compared to other groups (***: p < 0.001). The number and diameter of colonies in the PRP-2D group showed a considerable increase (p < 0.01) as compared to the control group. In PRP- scaffold group, a significant increase (p < 0.01) was seen only in the number of colonies related to the control group.Our results suggested that PRP scaffold can reconstruct a suitable structure to the in vitro proliferation of SSCs.

Improvement of in vitro proliferation of cockerel spermatogonial stem cells using different combinations of growth factors

ABSTRACT 1. This study examined whether in vitro proliferation and maintenance of cockerel spermatogonial stem cells (SSCs) could be improved by adding different combinations of growth factors (GFs), including glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF) or leukaemia inhibitory factor (LIF) into the culture medium. 2. The SSCs were isolated from the testes of immature cockerels. For short-term cultures, a medium supplemented with different combinations of GFs for 7 d in 5 replicates was used. The groups were classified as follows: without GF (control group); with GDNF (G group); with GDNF and bFGF (GF group); and with GDNF, bFGF and LIF (GFL group). The number of colonies and cells per colony, as well as the transcript abundance of STRA8 and OCT4 genes, was determined 7 d after the initial culturing. Immunofluorescence staining of SSEA-1, SSEA-3 and VASA protein markers, besides periodic acid-Schiff (PAS) staining, was carried out. 3. The number of colonies and cells per colony increased in the G, GF and GFL groups, compared to the control group (P < 0.01); however, the highest proliferation and colony formation were observed in the GFL group. The positive immunofluorescence staining of SSEA-1, SSEA-3 and VASA protein markers, as well as PAS staining, confirmed the self-renewal and colonisation of cockerel SSCs. The proliferation results were supported by the increased STRA8 and OCT4 transcript abundance in the treated groups (G, GF and GLF), compared to the control group. The SSC proliferation was associated with the higher transcript abundance of STAR8 and OCT4 genes in the GFL group, compared to the G and GF groups (P < 0.01). 4. The results showed that proliferation and colony-forming capacity of cockerel SSCs were positively improved by GDNF, bFGF and LIF. However, the most significant effect was observed when the medium was supplemented with LIF in combination with GDNF and bFGF.

The effect of regulating the Wnt signaling pathway on the proliferation and differentiation of spermatogonial stem cells.

BackgroundSpermatogonial stem cells and organ engineering research has raised new hope in infertility treatment. Spermatogenesis is a complex physiological process. To observe the proliferation ability and differentiation tendency of mice spermatogonial stem cells (SSCs), to study the effect of regulating the Wnt signaling pathway on the proliferation and differentiation of SSCs, and to provide a valuable basis for the clinical application of SSCs.MethodsSSCs were isolated and cultured by immunomagnetic separation. Cell surface markers were identified by flow cytometry. Axin1 was chosen as the target gene to inhibit fibrosis of SSCs by inhibiting the activity of Wnt signaling pathway. Axin-siRNA interference vector was constructed and transfected into spermatogonial stem cells. Cultured SSCs were randomly divided into six groups: control group, SSCs + TGF-β group, SSCs + DKK1 group, SSCs + Axin-RNAi group, SSCs + TGF-β + DKK1 group, SSCs + TGF-β + Axin-RNAi group. Proliferation of SSCs in each group was detected by MTT assay. Immunofluorescence, western blot and real time polymerase chain reaction analysis were used to detect protein expression in the Wnt/β catenin signaling pathways and the molecular markers of fibroblasts in SSCs.ResultsFlow cytometry analysis confirmed that the cultured SSCs were of high purity. MTT assay showed there was no significant difference between Axin-siRNA transfected and non-transfected cells. The proliferation ability was significantly increased in the SSCs + TGF-β group, however, it was retarded in SSCs + Axin-RNAi group. The results of immunofluorescence and western blot analysis showed that the expression levels of the Wnt signaling pathway proteins were relatively inhibited after Axin-siRNA was applied. Real-time polymerase chain reaction showed that the expression levels of the molecular markers of fibroblasts were close to the normal control group.ConclusionsThe Axin-siRNA constructed in this study specifically inhibited Wnt/β-catenin signal pathway activation, then inhibited the differentiation of SSCs into fibroblasts, which provides a valuable basis for the clinical application of SSCs.

An Efficient In Vitro Culture System To Amplify Spermatogonia Stem Cell Markers

Introduction: Proliferation of spermatogonial stem cells (SSCs) can be a treatment for infertile men. Here, we design an efficient method based on culturing in the presence of Sertoli cells to improve the expression level of some specific spermatogonia stem cell genes during two weeks post culture. Materials and Methods: Cells were derived from neonatal (2-6 days old) mice testes and were cultured in DMEM medium with FBS. The colonization of cultured SSCs in days 4, 7, and 14 of culture was counted via phase-contrast microscope and Image J software. Methyl thiazolyl tetrazolium (MTT) test was performed to evaluate the viability of cultured SSCs in days 3, 7, and 14 of culture. The expression level and the alteration pattern of specific spermatogonial markers, i.e., Stra8, DAZL, and Piwill2 was examined via real-time polymerase chain reaction (PCR) during two weeks post culture. Results: The number and the diameters of colonies showed a significant increase in cultured cells. MTT results proved the higher viability of testicular cells during the culture period. The results of ALP staining detected a positive reaction in spermatogonia colonies. Real-time PCR data showed that culturing SSCs in the presence of interstitial cells of the testis, amplified the level and alteration pattern of specific spermatogonia stem cells genes beneficial in the enrichment of SSCs propagation. Conclusion: Providing a similar culture environment to testicular niche increases viability, forms SSCs colonies, and regulates the level and alteration pattern of spermatogonia stem cell genes.

Eif2s3y regulates the proliferation of spermatogonial stem cells via Wnt6/-catenin signaling pathway

Eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) gene, the gene encoding eIF2γ protein, is globally expressed in all tissues and plays important roles in regulating global and gene-specific mRNA translation initiation. It has been noticed that Eif2s3y plays crucial roles in spermatogenesis, however, the mechanism remains unclear. In the current study, transgenic Eif2s3y mice were generated to test our hypothesis that the Eif2s3y promotes the proliferation of spermatogonial stem cells (SSCs). Transgenic Eif2s3y mouse had enhanced SSCs proliferation rate when compared to WT mouse. Interesting, the testes from transgenic Eif2s3y mouse had increased Active-β-catenin protein expression and higher expression pattern of Wnt ligand Wnt6 when compared to testes from WT mouse. This study revealed novel roles of Eif2s3y in the activation Wnt6/β-catenin signal pathway in SSCs. Taken together, we identified Eif2s3y-Wnt6-β-catenin as a critical pathway in the regulation of spermatogenesis, which provides a platform for investigating the molecular mechanisms of male reproduction.

Cytotoxicity of nonylphenol on spermatogonial stem cells via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway.

With continuous advancement of industrial society, environmental pollution has become more and more serious. There has been an increase in infertility caused by environmental factors. Nonylphenol (NP) is a stable degradation product widely used in daily life and production and has been proven to affect male fertility. However, the underlying mechanisms therein are unclear. Thus, it is necessary to study the effect and mechanism of NP on spermatogonial stem cells (SSCs). To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. SSCs were treated with NP at 0, 10, 20 or 30 µmol. MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs. Flow cytometry was conducted to measure SSC apoptosis. The expression of Bad, Bcl-2, cytochrome-c, pro-Caspase 9, SOX-2, OCT-4, Nanog, Nanos3, Stra8, Scp3, GFRα1, CD90, VASA, Nanos2, KIT, PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot, and the mRNA expression of SOX-2, OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction. Compared with untreated cells (0 μmol NP), SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2, Nanog, OCT-4, SOX-2, Nanos3, Stra8, Scp3, GFRα1, CD90, VASA, Nanos2, KIT, and PLZF (P < 0.05), whereas the expression of Bad, cytochrome-c, and pro-Caspase 9 increased significantly (P < 0.05). We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K, AKT, mTORC1, and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs (P < 0.05). NP exerted the greatest effect at 30 μmol among all NP concentrations. NP attenuated the proliferation, differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress. The associated mechanism may be related to the PI3K/AKT/mTOR pathway.

Platelet-derived growth factor-BB and epidermal growth factor promote dairy goat spermatogonial stem cells proliferation via Ras/ERK1/2 signaling pathway

Spermatogonial stem cells (SSCs) have been used for the production of transgenic animals and for the recovery of male fertility. However, the proliferation of SSCs in vitro is still immature, and the mechanisms and pathways involved in the proliferation of SSCs are not clear. Here, the effects of platelet-derived growth factor-BB (PDGF-BB) and epidermal growth factor (EGF) on the proliferation of dairy goat SSCs in vitro were detected. The results showed that 20 ng/ml PDGF-BB or 25 ng/ml EGF was the optimum concentration, and that the BCL2 in the experimental groups was significantly higher than that in the control (P < 0.05), while BAX and BAD were dramatically downregulated (P < 0.05). The pERK1/2 in the experimental groups was about 3–5 times higher than that in the control. After the specific MEK1/2 inhibitor was added, BCL2 was reduced significantly (P < 0.001), while BAX and BAD were upregulated (P < 0.001). The expression of pERK1/2 decreased by 10%–30%. We speculated that these two growth factors may be mediated through the Ras/ERK1/2 signaling pathway to regulate the expression of pERK1/2 protein, and thus enhance the resistance of SSCs to apoptosis. However, further studies are needed to verify this hypothesis.

Three-dimensional co-culture of human spermatogonial stem cells with Sertoli cells in soft agar culture system supplemented by growth factors and Laminin

Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than non-supplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis.

A Testis-Derived Hydrogel as an Efficient Feeder-Free Culture Platform to Promote Mouse Spermatogonial Stem Cell Proliferation and Differentiation.

Fertility preservation and assisted reproductive medicine require effective culture systems for the successful proliferation and differentiation of spermatogonial stem cells (SSCs). Many SSC culture systems require the addition of feeder cells at each subculture, which is tedious and inefficient. Here, we prepared decellularized testicular matrix (DTM) from testicular tissue, which preserved essential structural proteins of testis. The DTM was then solubilized and induced to form a porous hydrogel scaffold with randomly oriented fibrillar structures that exhibited good cytocompatibility. The viability of SSCs inoculated onto DTM hydrogel scaffolds was significantly higher than those inoculated on Matrigel or laminin, and intracellular gene expression and DNA imprinting patterns were similar to that of native SSCs. Additionally, DTM promoted SSC differentiation into round spermatids. More importantly, the DTM hydrogel supported SSC proliferation and differentiation without requiring additional somatic cells. The DTM hydrogel scaffold culture system provided an alternative and simple method for culturing SSCs that eliminates potential variability and contamination caused by feeder cells. It might be a valuable tool for reproductive medicine.

Long-Term Ex Vivo Expansion of Murine Spermatogonial Stem Cells in a Simple Serum-Free Medium.

Spermatogonial stem cells (SSCs) possess both self-renewal and differentiation abilities to sustain lifelong production of enormous numbers of spermatozoa in males. SSCs hold a unique position among tissue-specific stem cells in adults because of their ability to transmit the genetic information to subsequent generations. Ex vivo expansion of SSCs in conjunction with their transplantation is highly invaluable to study SSCs and develop new reproductive technologies for therapeutic applications. In this chapter, we describe a culture system involving a simple serum-free medium for mouse SSCs. Elimination of the serum from the culture is important to enhance the effects of exogenous factors, which are rather masked by the serum, and to avert the serum-induced inflammatory responses of testicular mesenchymal cells, which cause adverse effects on SSC proliferation. Consequently, using this culture system has proven for the first time that glial cell line-derived neurotrophic factor (GDNF) was found to be the key factor to drive the self-renewing proliferation of SSCs, and fibroblast growth factor 2 enhanced the GDNF-dependent proliferation of SSCs. Besides determining these two key cytokines, the simplicity of the system enabled individual modification of its components to develop long-term cultures of rat and rabbit SSCs. The basics of these culture systems will enable development of the culture conditions for human and other mammalian SSCs in the near future.

In Vitro and In Vivo Determinations of The Anti-GDNF Family Receptor Alpha 1 Antibody in Mice by Immunochemistry and RT-PCR.

BackgroundThe glial cell-derived neurotrophic factor (GDNF) family plays essential roles in the maintenance, growth, regulatory and signalling pathways of spermatogonial stem cells (SSCs). In this study, we analysed the ex- pression of anti-GDNF family receptor alpha 1 antibody (GFRa1) by immunohistochemistry (IHC), immunocyto- chemistry (ICC), Fluidigm real-time polymerase chain reaction (RT-PCR) and flow cytometry analyses.Materials and MethodsIn this experiment study, ICC, IHC, Fluidigm RT-PCR and flow cytometry were used to analyse the expression of the germ cell marker GFRa1 in testis tissue and SSC culture.ResultsIHC analysis showed that there were two groups of GFRa1 positive cells in the seminiferous tubules based on their location and expression shape - a small round punctuated shape on the basal compartment donut shape and a C-shaped expression located between the basal and the luminal compartments of the seminiferous tubules. OCT4 and PLZF positive cells may have similar patterns of expression as the first group. Assessment of the seminiferous tubule sections demonstrated that about 27% of the SSCs were positive for GFRa1. Fluidigm RT-PCR confirmed the significant expression (P<0.001) of GFRa1 in the SSCs compared to testicular stromal cells (TSCs). Flow cytometry analysis demonstrated that about 75% of the isolated SSCs colonies were positive for GFRa1.ConclusionThe results indicated that GFRa1 had a specific expression pattern both in vivo and in vitro. This finding could be helpful for understanding the proliferation, maintenance and signalling pathways of SSCs, and differentiation of meiotic and haploid germ cells.

Lrrc34 Is Highly Expressed in SSCs and Is Necessary for SSC Expansion In Vitro.

Objective To discover critical genes contributing to the stemness and maintenance of spermatogonial stem cells (SSCs) and provide new insights into the function of the leucine-rich repeat (LRR) family member Lrrc34 (leucine-rich repeat-containing 34) in SSCs from mice. Methods Bioinformatic methods, including differentially expressed gene (DEG), gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, were used to uncover latent pluripotency-related genes. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence analyses were utilized to verify the mRNA and protein expression levels, respectively. RNA interference of Lrrc34 using siRNA was performed to detect its transient impact on SSCs. Results Eight DEGs between ID4-EGFP+ (G) and ID4-EGFP+/TSPAN8High (TH), eight DEGs between G and ID4-EGFP+/TSPAN8Low (TL) and eleven DEGs between TH and TL were discovered, and eleven protein-protein interaction (PPI) modules were found to be significant in the PPI network of DEGs. One of the DEGs, Lrrc34, was selected as a potential pluripotency-related gene due to its differential expression among ID4-EGFP+ spermatogonia subsets and its interaction with fibroblast growth factor 2 in the fifth module. Immunofluorescence experiments exhibited specific expression of Lrrc34 in a subpopulation of undifferentiated spermatogonia marked by LIN28A, and RT-PCR experiments confirmed the high expression of Lrrc34 in SSCs from P7 and adult mice. The transient knockdown of Lrrc34 in SSCs resulted in reduced colony sizes and significant changes in the transcriptome and apoptotic pathways. Conclusion Lrrc34 is highly expressed in mouse SSCs and is required for SSC proliferation in vitro through effects on transcriptome and signaling transduction pathways.

Moderate hypoxia modulates ABCG2 to promote the proliferation of mouse spermatogonial stem cells by maintaining mild ROS levels

The aim of this study was to investigate the effects of different oxygen (O2) concentrations on the growth of mouse spermatogonial stem cells (SSCs) and the possible mechanisms of cell proliferation in vitro. The SSCs from testicular cells were cultured in various O2 concentrations (1%, 2.5%, 5%, and 20% O2) for 7 days. Colonies of SSCs were identified morphologically and by immunofluorescence. The number of mouse SSC colonies and the area covered by them were measured. Cell cycle progression of the SSCs was analyzed to identify the state of cell proliferation. The effects of O2 concentrations on the levels of intracellular reactive oxygen species (ROS) and expression of ATP binding cassette subfamily G member 2 (ABCG2) were also analyzed in the SSCs. Following culturing for 7 days, the SSCs were treated with Ko143 (a specific inhibitor of ABCG2) for 1 h, and the ROS level and expression of bcl-2, bax, and p53 were analyzed. The results showed that mouse SSCs formed compact colonies and had unclear borders in different O2 concentrations for 7 days, and there were no major morphologic differences between the O2 treatment groups. The expression of the SSC marker, GFR α1 was studied in each O2 treatment group. The number and area of SSC colonies, and the number of GFR α1 positive cells were the highest in the 2.5% O2 treatment group. Compared with other O2 concentrations, the number of cells in G0 cycle was significantly higher, while the level of intracellular ROS was lower at 1% O2. Moreover, the intracellular ROS levels gradually increased with increasing O2 concentration from 1% to 20%. The expression of ABCG2 in the SSCs cultured at 2.5% O2 was higher than in the other O2 groups. Inhibition of ABCG2 increased intracellular ROS generation, and the expression of the pro-apoptotic genes bax and p53, and decreased the expression of the anti-apoptotic gene bcl-2. In conclusion, moderate to low O2 tension increases ABCG2 expression to maintain mild ROS levels, triggers the expression of the anti-apoptotic genes, suppresses the proapoptotic gene pathway, and further promotes the proliferation of mouse SSCs in vitro.