Expansion Of Spermatogonial Stem Cells Research Articles

Propagation of porcine spermatogonial stem cells in serum-free culture conditions using knockout serum replacement.

In vitro culture and expansion of spermatogonial stem cells (SSCs) is an essential prerequisite to enhancing livestock productivity through SSC transplantation. Most of the culture media have been observed to be supplemented with serum. However, the use of serum in culture media may exert detrimental effects on SSC maintenance in vitro. An attempt was made to culture SSCs by replacing serum with 5% 'Knockout Serum Replacement (KSR)' in Doom pig (Sus domesticus), one of the valued indigenous germplasm of North-East India. Testes from 7 to 15 days old piglets were used for isolation, enrichment and in vitro culture of putative SSCs using serum-based and serum-free culture media. The cells were characterized for SSC-specific pluripotent markers expression by immunofluorescence staining and quantitative real-time PCR. The diameter and number of SSC colonies were recorded on days 9, 20 and 30 of culture. Similar morphologies of the SSC colonies were observed in both serum-based and serum-free culture conditions. Colony diameter and colony number were non-significantly higher in serum-free than serum-based media. The cells from both the culture conditions showed high alkaline phosphatase activity. The expression of SSC-specific pluripotent markers was observed in immunofluorescence and quantitative real-time PCR study. The present study revealed that SSCs from porcine species could be maintained in vitro for up to 30 days in serum-free culture using 5% KSR, which is believed to be a promising protein source for improving livestock production, and health care along with their conservation.

Ex-Vivo and In-Vivo ExpansionofSpermatogonial Stem Cells Using Cell-Seeded Microfluidic Testis Scaffolds and Animal Model.

This study was designed to provide both ex-vivo and in-vivo methods for the extraction and expansion of spermatogonial stem cells (SSCs). For in-vivo experiments, azoospermicmousemodel was performed with Busulfan. Isolation, culture, and characterization of neonate mouse SSC were also achieved. We performed an in-vivo injection of labeled SSCs to the testes with azoospermia. In ex-vivo experiments, extracted SSCs were seeded on the fabricated scaffold consisting of hyaluronic acid (HA) and decellularized testis tissues (DTT). Immunofluorescence staining with PLZF, TP1, and Tekt 1 was performed for SSCs differentiation and proliferation. Several studies demonstrated efficient spermatogenic arrest in seminiferous tubules and proved the absence of spermatogenesis. Transplanted SSCs moved and settled in the basement covering the seminiferous tubules. Most of the cells were positive for Dil, after 4weeks. An epithelium containing spermatogonia-like cells with Sertoli-like, and Leydig cells were evident in the seminiferous tubules of biopsies, and the IHC staining was significantly positive, 4weeks after injection of SSCs. The results of the ex-vivo experiments showed positive staining for all markers, which was significantly enhanced in scaffolds of ex-vivo experiments compared with in-vitro seeded scaffolds. Ex-vivo SSC differentiation and proliferation using cell-seeded microfluidic testis scaffolds maybe effective for treatment of the azoospermia.

Effect of poly-D-lysine on proliferation and senescence of caprine spermatogonial stem cells <em>in vitro</em>

Spermatogonial stem cells (SSCs) have the unique ability of both self-renewing and to produce progeny that undergoes differentiation to spermatozoa. As SSCs exist in very low numbers, therefore efficient in vitro expansion of SSCs is important prior to their clinical applications. In this study, we tried to improve the functionality of putative SSCs (pSSCs) during culture using poly-D-lysine (PDL) coating. For this, plates were coated with 0.01% PDL with different coating time interim treatments (5, 30 and 60 min) while control remained uncoated. The adequate amount of pSSCs of the goat was isolated and enriched using two-step enzymatic digestion and differential plating methods. Further, the functionality of pSSCs was evaluated by cell growth analysis, cell proliferation, senescence, and the presence of pluripotency (alkaline phosphatase, OCT-4) and SSC related (PGP-9.5) markers. The number and size of pSSCs colonies in 0.01% PDL coating groups were significantly (P <0.05) higher than in the control group. Similarly, pSSCs on uncoated plates expressed significantly (P <0.05) higher

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

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

Rescue of male infertility through correcting a genetic mutation causing meiotic arrest in spermatogonial stem cells.

Azoospermia patients who carry a monogenetic mutation that causes meiotic arrest may have their biological child through genetic correction in spermatogonial stem cells (SSCs). However, such therapy for infertility has not been experimentally investigated yet. In this study, a mouse model with an X-linked testis-expressed 11 (TEX11) mutation (Tex11PM/Y) identified in azoospermia patients exhibited meiotic arrest due to aberrant chromosome segregation. Tex11PM/Y SSCs could be isolated and expanded in vitro normally, and the mutation was corrected by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated endonuclease 9 (Cas9), leading to the generation of repaired SSC lines. Whole-genome sequencing demonstrated that the mutation rate in repaired SSCs is comparable with that of autonomous mutation in untreated Tex11PM/Y SSCs, and no predicted off-target sites are modified. Repaired SSCs could restore spermatogenesis in infertile males and give rise to fertile offspring at a high efficiency. In summary, our study establishes a paradigm for the treatment of male azoospermia by combining in vitro expansion of SSCs and gene therapy.

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.

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.

Self-Reprogramming of Spermatogonial Stem Cells into Pluripotent Stem Cells without Microenvironment of Feeder Cells.

Spermatogonial stem cells (SSCs) derived from mouse testis are unipotent in regard of spermatogenesis. Our previous study demonstrated that SSCs can be fully reprogrammed into pluripotent stem cells, so called germline-derived pluripotent stem cells (gPS cells), on feeder cells (mouse embryonic fibroblasts), which supports SSC proliferation and induction of pluripotency. Because of an uncontrollable microenvironment caused by interactions with feeder cells, feeder-based SSC reprogramming is not suitable for elucidation of the self-reprogramming mechanism by which SSCs are converted into pluripotent stem cells. Recently, we have established a Matrigel-based SSC expansion culture system that allows long-term SSC proliferation without mouse embryonic fibroblast support. In this study, we developed a new feeder-free SSC self-reprogramming protocol based on the Matrigel-based culture system. The gPS cells generated using a feeder-free reprogramming system showed pluripotency at the molecular and cellular levels. The differentiation potential of gPS cells was confirmed in vitro and in vivo. Our study shows for the first time that the induction of SSC pluripotency can be achieved without feeder cells. The newly developed feeder-free self-reprogramming system could be a useful tool to reveal the mechanism by which unipotent cells are self-reprogrammed into pluripotent stem cells.

Long-Term Propagation of Porcine Undifferentiated Spermatogonia.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis and fertility throughout the adult life of a male. Genetic manipulations of SSCs combined with germ cell transplantation present a novel approach for gene therapy and production of genetically modified animals. However, the rarity of SSCs within mammalian testes remains an impediment to related applications, making in vitro expansion of SSCs a prerequisite. Nevertheless, long-term culture systems of SSCs from large animals have not been established yet. In this study, we developed an optimized in vitro culture condition for porcine undifferentiated spermatogonia. The germ cells were isolated and enriched from 7-day-old porcine testes by an optimized differential planting. We tested different feeder layers and found that neonatal autologous Sertoli cells acted better than the SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cell line and adult Sertoli cells. The effects of several growth factors were also investigated. Using neonatal Sertoli cells as feeder and Dulbecco's modified eagle medium: nutrient mixture F-12 (DMEM/F12) culture medium supplemented with 10% KSR and four cytokines, the undifferentiated spermatogonia can proliferate in vitro for at least 2 months without loss of stemness. The expression of SSC markers indicated that the cultured cells maintained SSC expression profiles. Moreover, xenotransplantation and in vitro induction showed that the long-term cultured cells preserved the capacity to colonize in vivo and differentiate in vitro, respectively, demonstrating the presence of SSCs in the cultured cells. In conclusion, the conditions described in this study can support the normal proliferation of porcine undifferentiated spermatogonia with stemness and normal karyotype for at least 2 months. This culture system will serve as a basic refinement in the future studies and facilitate studies on SSC biology and genetic manipulation of male germ cells.

Human umbilical cord perivascular cells exhibit properties of testicular niche support cells

The ability to expand testicular biopsy-derived spermatogonial stem cells (SSC) ex-vivo may be required for the development male fertility preservation strategies. Our aim was to characterize and assess the potential use of first trimester human umbilical cord perivascular cells (HUCPVC) as a source of human feeder cells for the xeno-free ex-vivo expansion of SSCs.

Testicular Niche Required for Human Spermatogonial Stem Cell Expansion

Prepubertal boys treated with high-dose chemotherapy do not have an established means of fertility preservation because no established in vitro technique exists to expand and mature purified spermatogonial stem cells (SSCs) to functional sperm in humans. In this study, we define and characterize the unique testicular cellular niche required for SSC expansion using testicular tissues from men with normal spermatogenesis. Highly purified SSCs and testicular somatic cells were isolated by fluorescence-activated cell sorting using SSEA-4 and THY1 as markers of SSCs and somatic cells. Cells were cultured on various established niches to assess their role in SSC expansion in a defined somatic cellular niche. Of all the niches examined, cells in the SSEA-4 population exclusively bound to adult testicular stromal cells, established colonies, and expanded. Further characterization of these testicular stromal cells revealed distinct mesenchymal markers and the ability to undergo differentiation along the mesenchymal lineage, supporting a testicular multipotent stromal cell origin. In vitro human SSC expansion requires a unique niche provided exclusively by testicular multipotent stromal cells with mesenchymal properties. These findings provide an important foundation for developing methods of inducing SSC growth and maturation in prepubertal testicular tissue, essential to enabling fertility preservation for these boys.

Phenotypic characterization and in vitro propagation and transplantation of the Nile tilapia (Oreochromis niloticus) spermatogonial stem cells

In association with in vitro culture and transplantation, isolation of spermatogonial stem cells (SSCs) is an excellent approach for investigating spermatogonial physiology in vertebrates. However, in fish, the lack of SSC molecular markers represents a great limitation to identify/purify these cells, rendering it difficult to apply several valuable biotechnologies in fish-farming. Herein, we describe potential molecular markers, which served to phenotypically characterize, cultivate and transplant Nile tilapia SSCs. Immunolocalization revealed that Gfra1 is expressed exclusively in single type A undifferentiated spermatogonia (Aund, presumptive SSCs). Likewise, the expression of Nanos2 protein was observed in Aund cells. However, Nanos2-positive spermatogonia have also been identified in cysts with two to eight germ cells that encompass type A differentiated spermatogonia (Adiff). Moreover, we also established effective primary culture conditions that allowed the Nile tilapia spermatogonia to expand their population for at least one month while conserving their original undifferentiated (stemness) characteristics. The maintenance of Aund spermatogonial phenotype was demonstrated by the expression of early germ cell specific markers and, more convincingly, by their ability to colonize and develop in the busulfan-treated adult Nile tilapia recipient testes after germ cell transplantation. In addition to advancing our knowledge on the identity and physiology of fish SSCs, these findings provide the first step in establishing a system that will allow fish SSCs expansion in vitro, representing an important progress towards the development of new biotechnologies in aquaculture, including the possibility of producing transgenic fish.

GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells

γ-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA is also found in many peripheral tissues, where it has important functions during development. Here, we identified the existence of the GABA system in spermatogonial stem cells (SSCs) and found that GABA negatively regulates SSC proliferation. First, we demonstrated that GABA and its synthesizing enzymes were abundant in the testes 6 days postpartum (dpp), suggesting that GABA signaling regulates SSCs function in vivo. In order to directly examine the effect of GABA on SSC proliferation, we then established an in vitro culture system for long-term expansion of SSCs. We showed that GABAA receptor subunits, including α1, α5, β1, β2, β3 and γ3, the synthesizing enzyme GAD67, and the transporter GAT-1, are expressed in SSCs. Using phosphorylated histone H3 (pH3) staining, we demonstrated that GABA or the GABAAR-specific agonist muscimol reduced the proliferation of SSCs. This GABA regulation of SSC proliferation was shown to be independent of apoptosis using the TUNEL assay. These results suggest that GABA acts as a negative regulator of SSC proliferation to maintain the homeostasis of spermatogenesis in the testes.Electronic Supplementary MaterialSupplementary material is available for this article at 10.2478/s11658-013-0081-4 and is accessible for authorized users.

Of Mice and (wo)Men: Purified Oogonial Stem Cells from Mouse and Human Ovaries

When it comes to reproducing, James Brown summed up the human experience: This is a man’s world. Women produce a limited number of viable eggs (probably considerably fewer than 400) and become reproductively senescent in the forth or fifth decade of life. In contrast, men produce millions of sperm per day, with little evidence of age-related decline, and can father offspring into their seventh decade and beyond—if they can find willing mates. Universal agreement exists that a germline stem cell population in the testis makes continuous sperm production possible. The existence of a corresponding stem germ cell population in the ovary, however, has been the subject of intense debate. The latest chapter in this debate was recently published online in Nature Medicine [1]. What makes this paper especially noteworthy is that it provides the first evidence that isolation techniques used to obtain putative oogonial stem cells (OSCs) from the mouse ovary can be used to isolate similar cells from human ovaries. The findings are the result of collaborative studies between investigators at Harvard and at Saitama Medical Center in Japan. The Harvard group includes Jonathan Tilly, whose laboratory challenged the ‘‘central dogma’’ of reproductive biology—that all eggs produced by the mammalian ovary initiate oogenesis during fetal development—with a paper in 2004 reporting OSCs in the mouse ovary [2]. In males, spermatogonial stem cells (SSCs) at the basement membrane of the seminiferous tubules divide to replenish their numbers and give rise to progenitor spermatogonia that provide the steady supply of new spermatocytes required for continuous sperm production. The SSC population is thought to be small in number, making up 0.03% of the total testicular cell population in adult mice [3]. Techniques for the isolation and in vitro expansion of SSCs have been developed, and the litmus test of the putative SSC cells obtained—their capacity to regenerate continual sperm production following transplantation into a recipient testis—has been firmly established [4, 5]. Despite a flurry of research, the existence of the female germline stem cell counterpart—OSCs—has remained controversial. White et al. [1] expand our understanding of OSCs with the demonstration that a population of mitotically active cells can be isolated from both mouse and human ovarian tissue by cell sorting using an antibody that recognizes the Cterminal portion of DDX4, a germ cell-specific RNA helicase. The isolated cells express genes known to be restricted to germ cells, and like male SSCs, these appear to be a rare type (an estimated 0.014% of the ovarian cell population). Interestingly, in contrast to the male, where isolated SSCs form colonies of actively dividing cells after several days in culture [6–9], from 10 to 12 wk (mouse) and from 4 to 8 wk (human) of culture were required to obtain actively dividing OSC colonies. This smacks of in vitro transformation, raising concern about whether the population of cells that exists in the ovary are

Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro

Spermatogonial stem cells (SSCs) support life-long spermatogenesis by self-renewing and producing spermatogonia committed to differentiation. In vitro, SSCs form three-dimensional spermatogonial aggregates (clusters) when cultured with glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2); serial passaging of clusters results in long-term SSC maintenance and expansion. However, the role of these growth factors in controlling patterns of SSC division and fate decision has not been understood thoroughly. We report here that in a short-term culture, GDNF and FGF2 increase the number of dividing SSCs, but not the total SSC number, compared to a no-growth-factor condition. Since the total germ cell number increases with growth factors, these results suggest that GDNF and FGF2 promote a SSC division pattern that sustains the size of the stem cell pool while generating committed progenitors. Our data also show that SSC numbers increase when the cluster structure is disintegrated and cell–cell interaction in clusters is disrupted. Collectively, these results suggest that in this culture system, GDNF and FGF2 stimulate SSC divisions that promote self-renewal and differentiation in the SSC population, and imply that the destruction of the cluster structure, a potential in vitro niche, may contribute to SSC expansion.

Coculture of Spermatogonia With Somatic Cells in a Novel Three‐Dimensional Soft‐Agar‐Culture‐System

Isolation and culture of spermatogonial stem cells (SSCs) has become an approach to study the milieu and the factors controlling their expansion and differentiation. Traditional conventional cell culture does not mimic the complex situation in the seminiferous epithelium providing a basal, intraepithelial, and adluminal compartment to the developing male germ cells. SSCs are located in specific stem cell niches whose features and functional parameters are thus far poorly understood. It was the aim of this study to isolate SSCs and to explore their expansion and differentiation potential in a novel three-dimensional Soft-Agar-Culture-System (SACS). This system provides three-dimensional structural support and multiple options for manipulations through the addition of factors, cells, or other changes. The system has revolutionized research on blood stem cells by providing a tool for clonal analysis of expanding and differentiating blood cell lineages. In our studies, SSCs are enriched using Gfralpha-1 as a specific surface marker and magnetic-activated cell sorting as a separation approach. At termination of the culture, we determined the type and number of germ cells obtained after the first 24 hours of culture. We also determined cell types and numbers in expanding cell clones of differentiating germ cells during the subsequent 15 days of culture. We analyzed a supportive effect of somatic cell lineages added to the solid part of the culture system. We conclude that our enrichment and culture approach is highly useful for exploration of SSC expansion and have found indications that the system supports differentiation up to the level of postmeiotic germ cells.

Transcriptional Profiling of Mammalian Spermatogonial Stem Cells: Insights into Mechanisms Regulating Their Self-Renewal.

Spermatogenesis is a stem cell dependent process in which the self-renewal and differentiation of spermatogonial stem cells (SSCs) provides the foundation for continual spermatozoa production. Even though crucial for male fertility, knowledge of the mechanisms regulating SSC fate decisions is limited. Rarity in the testis and lack of specific phenotypic or molecular markers to distinguish SSCs from other testis cell populations has posed major challenges in studying their functions. Currently, the only unequivocal means to identify SSCs and examine their biological activity is by functional transplantation. SSC function is controlled by both extrinsic niche stimuli and intrinsic expression of specific genes. The testis somatic cell population is believed to supply extrinsic niche growth factors that influence SSC functions, of which Sertoli cells are thought to be the main contributors but input from other cell populations is likely. The growth factor glial cell line-derived neurotrophic factor (GDNF) is a niche factor produced by Sertoli cells that influences the self-renewal of rodent SSCs. We have identified GDNF-regulated genes in cultures of germ cells proven to be enriched for self-renewing SSCs by functional transplantation. From those studies we discovered that GDNF stimulates expression of the transcription regulators bcl6b, etv5, and lhx1. Our subsequent experiments determined that expression of these molecules is important for SSC self-renewal in vitro. Additionally, disruption of both Bcl6b and Etv5 expression in vivo results in impaired spermatogenesis, with progressive loss of germ cells as a male ages resulting in the appearance of seminiferous tubules with a Sertoli cell only phenotype and male fertility defects. More recently we have examined the transcriptional profile of an SSC-enriched cell fraction isolated form mouse testes and compared gene expression levels to those in an SSC-depleted mixed testis cell population. We isolated the Thy1-expressing cell fraction from mouse pup testes and corresponding Thy1-depeleted cell fractions, and using functional transplantation determined that the Thy1+ fraction was approximately 25-fold enriched for SSCs compared to the Thy1-depelted fraction. Global gene expression profiles were measured in both populations using Affymterix oligonucleotide microarrays, producing a database of gene expression information that serves as a mining tool for identifying potential novel mechanisms regulating SSC functions. From this information we identified abundant expression of colony stimulating factor 1 receptor (CSF-1R) by the SSC-enriched Thy1+ cell fraction. To determine a biological significance of this information, we supplemented culture media with recombinant soluble CSF-1 in addition to GDNF. Using functional transplantation to assay SSC expansion rates, significant enhancement of SSC self-renewal was measured in CSF-1 supplemented cultures compared to GDNF treatment alone. Importantly, examination of testis cross sections using immunofluoresence revealed expression of CSF-1 by clusters of Leydig cells and some peritubular myoid cells. These results implicate Leydig cells and possibly myoid cells as contributors to the SSC niche microenvironment and add CSF-1 to the list of niche factors influencing SSC self-renewal.

Akt mediates self-renewal division of mouse spermatogonial stem cells

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, we assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells.

The Seminiferous Epithelium Cycle Length in the Black Tufted-Ear Marmoset (Callithrix penicillata) Is Similar to Humans1

Marmosets are New World small primates phylogenetically close to humans and are commonly used in biomedical research. Although the reproductive biology of the common marmoset Callithrix jacchus is fairly well investigated, there are few data available for testis function for its close relative, Callithrix penicillata. In this regard, the present study was performed to investigate testis structure, spermatogenic cycle length, and spermatogenic and Sertoli cell efficiencies in eight captive C. penicillata. These animals received (3)H-thymidine injections and had their testes perfused-fixed with glutaraldehyde and embedded in plastic at different time periods after (3)H-thymidine injections, for histomorphometric and autoradiographic evaluation. The analysis of the different germ cell associations showed that two or more stages were observed in about 30% of the seminiferous tubule cross sections investigated. The values found for spermatogenic cycle length and for total duration of spermatogenesis in the marmoset C. penicillata, 15.4 and 69.3 days respectively, were very close to those cited in the literature for humans. However, the results observed for Sertoli cell efficiency (number of round spermatids per Sertoli cell; 8:1) and spermatogenic efficiency (daily sperm production per gram of testis; 18.4 million) were substantially higher than those observed for humans. The results found in the present investigation suggest that the black tufted-ear marmoset C. penicillata might represent an alternative and useful experimental model to perform comparative studies regarding the spermatogenic process, particularly in investigations related to the expansion of spermatogonial stem cells and the establishment of spermatogenic waves.