Genetics of gonadal stem cell renewal.

Stem cells are necessary for the maintenance of many adult tissues. Signals within the stem cell microenvironment, or niche, regulate the self-renewal and differentiation capability of these cells. Misregulation of these signals through mutation or damage can lead to overgrowth or depletion of different stem cell pools. In this review, we focus on the Drosophila testis and ovary, both of which contain well-defined niches, as well as the mouse testis, which has become a more approachable stem cell system with recent technical advances. We discuss the signals that regulate gonadal stem cells in their niches, how these signals mediate self-renewal and differentiation under homeostatic conditions, and how stress, whether from mutations or damage, can cause changes in cell fate and drive stem cell competition.

Niche Tet maintains germline stem cells independently of dioxygenase activity.

Ten-eleven translocation (TET) proteins are dioxygenases that convert 5-methylcytosine (5mC) into 5-hydroxylmethylcytosine (5hmC) in DNA and RNA. However, their involvement in adult stem cell regulation remains unclear. Here, we identify a novel enzymatic activity-independent function of Tet in the Drosophila germline stem cell (GSC) niche. Tet activates the expression of Dpp, the fly homologue of BMP, in the ovary stem cell niche, thereby controlling GSC self-renewal. Depletion of Tet disrupts Dpp production, leading to premature GSC loss. Strikingly, both wild-type and enzyme-dead mutant Tet proteins rescue defective BMP signaling and GSC loss when expressed in the niche. Mechanistically, Tet interacts directly with Bap55 and Stat92E, facilitating recruitment of the Polybromo Brahma associated protein (PBAP) complex to the dpp enhancer and activating Dpp expression. Furthermore, human TET3 can effectively substitute for Drosophila Tet in the niche to support BMP signaling and GSC self-renewal. Our findings highlight a conserved novel catalytic activity-independent role of Tet as a scaffold protein in supporting niche signaling for adult stem cell self-renewal.

brinker levels regulated by a promoter proximal element support germ cell homeostasis.

A limited BMP signaling range in the stem cell niche of the ovary protects against germ cell tumors and promotes germ cell homeostasis. The canonical repressor of BMP signaling in both the Drosophila embryo and wing disc is the transcription factor Brinker (Brk), yet the expression and potential role of Brk in the germarium has not previously been described. Here, we find that brk expression requires a promoter-proximal element (PPE) to support long-distance enhancer action as well as to drive expression in the germarium. Furthermore, PPE subdomains have different activities; in particular, the proximal portion acts as a damper to regulate brk levels precisely. Using PPE mutants as well as tissue-specific RNA interference and overexpression, we show that altering brk expression within either the soma or the germline affects germ cell homeostasis. Remarkably, we find that Decapentaplegic (Dpp), the main BMP ligand and canonical antagonist of Brk, is upregulated by Brk in the escort cells of the germarium, demonstrating that Brk can positively regulate this pathway.

Zebrafish dazl regulates cystogenesis and germline stem cell specification during the primordial germ cell to germline stem cell transition.

Fertility and gamete reserves are maintained by asymmetric divisions of the germline stem cells to produce new stem cells or daughters that differentiate as gametes. Before entering meiosis, differentiating germ cells (GCs) of sexual animals typically undergo cystogenesis. This evolutionarily conserved process involves synchronous and incomplete mitotic divisions of a GC daughter (cystoblast) to generate sister cells connected by intercellular bridges that facilitate the exchange of materials to support rapid expansion of the gamete progenitor population. Here, we investigated cystogenesis in zebrafish and found that early GCs are connected by ring canals, and show that Deleted in azoospermia-like (Dazl), a conserved vertebrate RNA-binding protein (Rbp), is a regulator of this process. Analysis of dazl mutants revealed the essential role of Dazl in regulating incomplete cytokinesis, germline cyst formation and germline stem cell specification before the meiotic transition. Accordingly, dazl mutant GCs form defective ring canals, and ultimately remain as individual cells that fail to differentiate as meiocytes. In addition to promoting cystoblast divisions and meiotic entry, dazl is required for germline stem cell establishment and fertility.

The exocyst complex regulates C. elegans germline stem cell proliferation by controlling membrane Notch levels.

The conserved exocyst complex regulates plasma membrane-directed vesicle fusion in eukaryotes. However, its role in stem cell proliferation has not been reported. Germline stem cell (GSC) proliferation in the nematode Caenorhabditis elegans is regulated by conserved Notch signaling. Here, we reveal that the exocyst complex regulates C. elegans GSC proliferation by modulating Notch signaling cell autonomously. Notch membrane density is asymmetrically maintained on GSCs. Knockdown of exocyst complex subunits or of the exocyst-interacting GTPases Rab5 and Rab11 leads to Notch redistribution from the GSC-niche interface to the cytoplasm, suggesting defects in plasma membrane Notch deposition. The anterior polarity (aPar) protein Par6 is required for GSC proliferation, and for maintaining niche-facing membrane levels of Notch and the exocyst complex. The exocyst complex biochemically interacts with the aPar regulator Par5 (14-3-3ζ) and Notch in C. elegans and human cells. Exocyst components are required for Notch plasma membrane localization and signaling in mammalian cells. Our study uncovers a possibly conserved requirement of the exocyst complex in regulating GSC proliferation and in maintaining optimal membrane Notch levels.

Live imaging of the Drosophila ovarian niche shows spectrosome and centrosome dynamics during asymmetric germline stem cell division.

Drosophila female germline stem cells (GSCs) are found inside the cellular niche at the tip of the ovary. They undergo asymmetric divisions to renew the stem cell lineage and to produce sibling cystoblasts that will in turn enter differentiation. GSCs and cystoblasts contain spectrosomes, membranous structures essential for orientation of the mitotic spindle and that, particularly in GSCs, change shape depending on the cell cycle phase. Using live imaging and a fusion protein of GFP and the spectrosome component Par-1, we follow the complete spectrosome cycle throughout GSC division and quantify the relative duration of the different spectrosome shapes. We also determine that the Par-1 kinase shuttles between the spectrosome and the cytoplasm during mitosis and observe the continuous addition of new material to the GSC and cystoblast spectrosomes. Next, we use the Fly-FUCCI tool to define, in live and fixed tissues, that GSCs have a shorter G1 compared with the G2 phase. The observation of centrosomes in dividing GSCs allowed us to determine that centrosomes separate very early in G1, before centriole duplication. Furthermore, we show that the anterior centrosome associates with the spectrosome only during mitosis and that, upon mitotic spindle assembly, it translocates to the cell cortex, where it remains anchored until centrosome separation. Finally, we demonstrate that the asymmetric division of GSCs is not an intrinsic property of these cells, as the spectrosome of GSC-like cells located outside of the niche can divide symmetrically. Thus, GSCs display unique properties during division, a behaviour influenced by the surrounding niche.

Autophagy accompanying the developmental process of male germline stem cells.

Germline stem cells are a crucial type of stem cell that can stably pass on genetic information to the next generation, providing the necessary foundation for the reproduction and survival of organisms. Male mammalian germline stem cells are unique cell types that include primordial germ cells and spermatogonial stem cells. They can differentiate into germ cells, such as sperm and eggs, thereby facilitating offspring reproduction. In addition, they continuously generate stem cells through self-renewal mechanisms to support the normal function of the reproductive system. Autophagy involves the use of lysosomes to degrade proteins and organelles that are regulated by relevant genes. This process plays an important role in maintaining the homeostasis of germline stem cells and the synthesis, degradation, and recycling of germline stem cell products. Recently, the developmental regulatory mechanism of germline stem cells has been further elucidated, and autophagy has been shown to be involved in the regulation of self-renewal and differentiation of germline stem cells. In this review, we introduce autophagy accompanying the development of germline stem cells, focusing on the autophagy process accompanying the development of male spermatogonial stem cells and the roles of related genes and proteins. We also briefly outline the effects of autophagy dysfunction on germline stem cells and reproduction.

Mad dephosphorylation at the nuclear pore is essential for asymmetric stem cell division.

Stem cells divide asymmetrically to generate a stem cell and a differentiating daughter cell. Yet, it remains poorly understood how a stem cell and a differentiating daughter cell can receive distinct levels of niche signal and thus acquire different cell fates (self-renewal versus differentiation), despite being adjacent to each other and thus seemingly exposed to similar levels of niche signaling. In the Drosophila ovary, germline stem cells (GSCs) are maintained by short range bone morphogenetic protein (BMP) signaling; the BMP ligands activate a receptor that phosphorylates the downstream molecule mothers against decapentaplegic (Mad). Phosphorylated Mad (pMad) accumulates in the GSC nucleus and activates the stem cell transcription program. Here, we demonstrate that pMad is highly concentrated in the nucleus of the GSC, while it quickly decreases in the nucleus of the differentiating daughter cell, the precystoblast (preCB), before the completion of cytokinesis. We show that a known Mad phosphatase, Dullard (Dd), is required for the asymmetric partitioning of pMad. Our mathematical modeling recapitulates the high sensitivity of the ratio of pMad levels to the Mad phosphatase activity and explains how the asymmetry arises in a shared cytoplasm. Together, these studies reveal a mechanism for breaking the symmetry of daughter cells during asymmetric stem cell division.

Different mechanisms of X-ray irradiation-induced male and female sterility in Aedes aegypti.

BACKGROUND: Aedes aegypti (Ae. aegypti) is the major vector that transmits many diseases including dengue, Zika, and filariasis in tropical and subtropical regions. Due to the growing resistance to chemical-based insecticides, biological control methods have become an emerging direction to control mosquito populations. The sterile insect technique (SIT) deploys high doses of ionizing radiation to sterilize male mosquitoes before the release. The Wolbachia-based population suppression method of the incompatible insect technique (IIT) involves the release of Wolbachia-infected males to sterilize uninfected field females. Due to the lack of perfect sex separation tools, a low percentage of female contamination is detected in the male population. To prevent the unintentional release of these Wolbachia-infected females which might result in population replacement, a low dose of X-ray irradiation is deployed to sterilize any female escapees. However, it remains unclear whether these irradiation-induced male and female sterilizations share common mechanisms. RESULTS: In this work, we set out to define the minimum dose of X-ray radiation required for complete female sterilization in Ae. aegypti (NEA-EHI strain). Further results showed that this minimum dose of X-ray irradiation for female sterilization significantly reduced male fertility. Similar results have been reported previously in several operational trials. By addressing the underlying causes of the sterility, our results showed that male sterility is likely due to chromosomal damage in the germ cells induced by irradiation. In contrast, female sterility appears to differ and is likely initiated by the elimination of the somatic supporting cells, which results in the blockage of the ovariole maturation. Building upon these findings, we identified the minimum dose of X-ray irradiation on the Wolbachia-infected NEA-EHI (wAlbB-SG) strain, which is currently being used in the IIT-SIT field trial. Compared to the uninfected parental strain, a lower irradiation dose could fully sterilize wAlbB-SG females. This suggests that Wolbachia-carrying mosquitoes are more sensitive to irradiation, consistent with a previous report showing that a lower irradiation dose fully sterilized Wolbachia-infected Ae. aegypti females (Brazil and Mexican strains) compared to those uninfected controls. CONCLUSIONS: Our findings thus reveal the distinct mechanisms of ionizing X-ray irradiation-induced male or female sterility in Ae. aegypti mosquitoes, which may help the design of X-ray irradiation-based vector control methods.

[Toxic effect and mechanism of Tripterygium glycosides on ovarian germline stem cells of mice in vitro by Notch signaling pathway].

The ovarian germline stem cells(OGSCs) cultured in the optimized culture system were used as the research object to observe the effect of Tripterygium glycosides(TG) on OGSCs and explore the mechanism of reproductive toxicity by the Notch signaling pathway. Cell counting kit-8(CCK-8) was used to observe the viability level of OGSCs in mice cultured in vitro by TG of 3.75, 7.5, and 15 µg·mL~(-1). Immunofluorescence technology and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the protein and gene expression level of OGSCs marker mouse vasa homologue(MVH) and octamer-binding transcription factor 4(Oct4) by TG of 3.75 µg·mL~(-1). RT-PCR detected the gene expression of neurogenic locus Notch homolog protein 1(Notch1), Hes family BHLH transcription factor 1(Hes1), and jagged canonical Notch ligand 1(Jagged1). The RNA was extracted for transcriptome analysis to analyze the mechanism of action of TG intervention on OGSCs. 3.75 µg·mL~(-1) of TG was combined with 40 ng·mL~(-1) Notch signaling pathway γ-secretagocin agonist jagged canonical notch ligand(Jagged) for administration. CCK-8 was used to detect the viability level of OGSCs. Double immunofluorescence technology was used to detect the protein co-expression of MVH with Hes1, Notch1, and Jagged1. The results showed that compared with the blank group, the TG administration group significantly inhibited the activity of OGSCs(P<0.01 or P<0.001). It could reduce the protein and gene expression of OGSC markers, namely MVH and Oct4(P<0.05, P<0.01, or P<0.001). It could significantly inhibit the gene expression of Notch1, Hes1, and Jagged1(P<0.001). Transcriptomic analysis showed that TG affected the growth and proliferation of OGSCs by intervening Jagged1, a ligand associated with the Notch signaling pathway. The experimental results showed that the combination of Notch signaling pathway γ-secretagorein agonist Jagged could significantly alleviate the decrease in OGSC viability induced by TG(P<0.001) and significantly increased the OGSC viability compared with the TG group(P<0.001). It also could significantly increase the co-expression of MVH/Jagged1, MVH/Hes1, and MVH/Notch1 proteins(P<0.01 or P<0.001). It suggested that TG play the role of γ-secretagorease inhibitors by downregulating the OGSC markers including MVH and Oct4 and Notch signaling pathway molecules such as Notch1, Hes1, and Jagged1, participate in the OGSC pathway, and mediate reproductive toxicity caused by the Notch signaling pathway.

RNA Helicase Vasa as a Multifunctional Conservative Regulator of Gametogenesis in Eukaryotes.

Being a conservative marker of germ cells across metazoan species, DEAD box RNA helicase Vasa (DDX4) remains the subject of worldwide investigations thanks to its multiple functional manifestations. Vasa takes part in the preformation of primordial germ cells in a group of organisms and contributes to the maintenance of germline stem cells. Vasa is an essential player in the piRNA-mediated silencing of harmful genomic elements and in the translational regulation of selected mRNAs. Vasa is the top hierarchical protein of germ granules, liquid droplet organelles that compartmentalize RNA processing factors. Here, we survey current advances and problems in the understanding of the multifaceted functions of Vasa proteins in the gametogenesis of different eukaryotic organisms, from nematodes to humans.

Survival of Drosophila germline stem cells requires the chromatin-binding protein Barrier-to-autointegration factor.

The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear envelope. This network includes LAP2-emerin-MAN1 domain (LEM-D) proteins that associate with the chromatin and DNA-binding protein Barrier-to-autointegration factor (BAF). Here, we investigate the partnership between three NL Drosophila LEM-D proteins and BAF. In most tissues, only Emerin/Otefin is required for NL enrichment of BAF, revealing an unexpected dependence on a single LEM-D protein. Prompted by these observations, we studied BAF contributions in the ovary, a tissue where Emerin/Otefin function is essential. We show that germ cell-specific BAF knockdown causes phenotypes that mirror emerin/otefin mutants. Loss of BAF disrupts NL structure, blocks differentiation and promotes germ cell loss, phenotypes that are partially rescued by inactivation of the ATR and Chk2 kinases. These data suggest that, similar to emerin/otefin mutants, BAF depletion activates the NL checkpoint that causes germ cell loss. Taken together, our findings provide evidence for a prominent NL partnership between the LEM-D protein Emerin/Otefin and BAF, revealing that BAF functions with this partner in the maintenance of an adult stem cell population.

Articulating the "stem cell niche" paradigm through the lens of non-model aquatic invertebrates.

Stem cells (SCs) in vertebrates typically reside in "stem cell niches" (SCNs), morphologically restricted tissue microenvironments that are important for SC survival and proliferation. SCNs are broadly defined by properties including physical location, but in contrast to vertebrates and other "model" organisms, aquatic invertebrate SCs do not have clearly documented niche outlines or properties. Life strategies such as regeneration or asexual reproduction may have conditioned the niche architectural variability in aquatic or marine animal groups. By both establishing the invertebrates SCNs as independent types, yet allowing inclusiveness among them, the comparative analysis will allow the future functional characterization of SCNs.

High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of male germline stem cells.

Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans.

Epigenetic regulation of drosophila germline stem cell maintenance and differentiation.

Gametogenesis is one of the most extreme cellular differentiation processes that takes place in Drosophila male and female germlines. This process begins at the germline stem cell, which undergoes asymmetric cell division (ACD) to produce a self-renewed daughter that preserves its stemness and a differentiating daughter cell that undergoes epigenetic and genomic changes to eventually produce haploid gametes. Research in molecular genetics and cellular biology are beginning to take advantage of the continually advancing genomic tools to understand: (1) how germ cells are able to maintain their identity throughout the adult reproductive lifetime, and (2) undergo differentiation in a balanced manner. In this review, we focus on the epigenetic mechanisms that address these two questions through their regulation of germline-soma communication to ensure germline stem cell identity and activity.

Regulation of fatty acid desaturase- and immunity gene-expression by mbk-1/DYRK1A in Caenorhabditis elegans.

BACKGROUND: In the nematode Caenorhabditis elegans, longevity in response to germline ablation, but not in response to reduced insulin/IGF1-like signaling, is strongly dependent on the conserved protein kinase minibrain-related kinase 1 (MBK-1). In humans, the MBK-1 ortholog DYRK1A is associated with a variety of disorders, most prominently with neurological defects observed in Down syndrome. To better understand mbk-1's physiological roles and their dependence on genetic background, we analyzed the influence of mbk-1 loss on the transcriptomes of wildtype and long-lived, germline-deficient or insulin-receptor defective, C. elegans strains by RNA-sequencing. RESULTS: mbk-1 loss elicited global changes in transcription that were less pronounced in insulin-receptor mutant than in germline-deficient or wildtype C. elegans. Irrespective of genetic background, mbk-1 regulated genes were enriched for functions in biological processes related to organic acid metabolism and pathogen defense. qPCR-studies confirmed mbk-1 dependent induction of all three C. elegans Δ9-fatty acid desaturases, fat-5, fat-6 and fat-7, in wildtype, germline-deficient and insulin-receptor mutant strains. Conversely, mbk-1 dependent expression patterns of selected pathogen resistance genes, including asp-12, dod-24 and drd-50, differed across the genetic backgrounds examined. Finally, cth-1 and cysl-2, two genes which connect pathogen resistance to the metabolism of the gaseous messenger and lifespan regulator hydrogen sulfide (H2S), were commonly suppressed by mbk-1 loss only in wildtype and germline-deficient, but not in insulin-receptor mutant C. elegans. CONCLUSION: Our work reveals previously unknown roles of C. elegans mbk-1 in the regulation of fatty acid desaturase- and H2S metabolic-genes. These roles are only partially dependent on genetic background. Considering the particular importance of fatty acid desaturation and H2S for longevity of germline-deficient C. elegans, we propose that these processes at least in part account for the previous observation that mbk-1 preferentially regulates lifespan in these worms.

Unravelling HP1 functions: post-transcriptional regulation of stem cell fate.

Heterochromatin protein 1 (HP1) is a non-histone chromosomal protein first identified in Drosophila as a major component of constitutive heterochromatin, required for stable epigenetic gene silencing in many species including humans. Over the years, several studies have highlighted additional roles of HP1 in different cellular processes including telomere maintenance, DNA replication and repair, chromosome segregation and, surprisingly, positive regulation of gene expression. In this review, we briefly summarize past research and recent results supporting the unexpected and emerging role of HP1 in activating gene expression. In particular, we discuss the role of HP1 in post-transcriptional regulation of mRNA processing because it has proved decisive in the control of germline stem cells homeostasis in Drosophila and has certainly added a new dimension to our understanding on HP1 targeting and functions in epigenetic regulation of stem cell behaviour.

JAK/STAT signaling in stem cells and regeneration: from Drosophila to vertebrates.

The JAK/STAT pathway is a conserved metazoan signaling system that transduces cues from extracellular cytokines into transcriptional changes in the nucleus. JAK/STAT signaling is best known for its roles in immunity. However, recent work has demonstrated that it also regulates critical homeostatic processes in germline and somatic stem cells, as well as regenerative processes in several tissues, including the gonad, intestine and appendages. Here, we provide an overview of JAK/STAT signaling in stem cells and regeneration, focusing on Drosophila and highlighting JAK/STAT pathway functions in proliferation, survival and cell competition that are conserved between Drosophila and vertebrates.

Differentiation Potential of Cultured Extracellular DEAD-Box Helicase 4+ Oogonial Stem Cells from Adult Human Ovaries into Somatic Lineages.

The transdifferentiation potential of human oogonial stem cells (hOSCs) isolated using the antibody against extracellular DEAD-Box Helicase 4 (ecDDX4) remains undetermined. Hence, this study isolated OSCs from ovarian cortical pieces of premenopausal women using ecDDX4 antibody by magnetic activated cell sorting and expanded these cells under embryonic stem cell (ESC)-like culture conditions to inves-tigate their transdifferentiation potential. The number of ecDDX4+ cells obtained was variable in each isolation. When cultured on inactivated mouse embryonic fibroblast feeder layer with human leukemia inhibitory factor (hLIF) and basic fibroblast growth factor (bFGF) in Minimum Essential Medium, the hOSCs aggregated, forming ESC-like colonies. The average size of these cells was around 10 µm. hOSCs in culture were positive for alkaline phosphatase and further formed embryoid bodies (EBs) when grown on low attachment plates containing Essential 6 Medium without hLIF and bFGF. Subsequently, EBs differentiated into 3 germ layers, which were confirmed by staining with beta-III tubulin (TUJ1) for ectoderm, alpha-fetoprotein (AFP) for endoderm, and smooth muscle actin (SMA) for mesoderm. Further, using appropriate induction media, the EBs derived from ecDDX4+ hOSCs were differentiated into somatic lineages such as adipocytes, osteoblasts, cardiomyocytes, and neuronal precursor-like cells, which were confirmed by immunofluorescence using antibodies against specific markers for each cell type. This study corroborated the previous findings that ovaries of adult women possess germ cell progenitors that can be isolated using ecDDX4, and these cells can be manipulated as pluripotent stem cells by culturing them under ESC-like culture conditions akin to their male counterparts, the spermatogonial stem cells. Further, these cells could differentiate into somatic lineages under specific signalling environments.

Isolation and in vitro expansion of porcine spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genetic information to offspring through their ability to both self-renew and differentiate into mature spermatozoa. SSCs can be transplanted to establish donor-derived spermatogenesis in recipient animals, thus offering a novel reproductive tool for multiplication of elite individual animals to benefit livestock production. An optimal SSC culture in vitro can benefit various SSC-based studies and applications, such as mechanistic study of SSC biology, SSC transplantation process and SSC-based transgenesis technique. However, except for some model rodent animals, SSC culture remains an inefficient and unstable process. We here studied a workflow to isolate, purify and in vitro culture porcine SSCs from neonatal pig testes. Pig testicular cells were dissociated by two-step enzymatic digestion with collagenase type IV and trypsin. We enriched the spermatogonia from the testicular cell mix by differential plating for at least 3 times to remove firmly attached non-SSCs. We then tested the optimal culture medium formula by supplementation of different growth factors to the basic medium (DMEM/F12 + 1% FBS) and found that a combination of 20 ng/ml GDNF, 10 ng/ml LIF, 20 ng/ml FGF2 and 20 ng/ml IGF1 had the best effect on SSC growth in our defined experimental system. In the presence of 4 growth factors without specific feeders, the purified SSCs can be cultured in poly-L-lysine- and laminin-coated dishes for 28 days and remain preserving a continuous proliferation without losing the undifferentiated spermatogonial phenotype.