Spermatogonial Stem Cells Function Research Articles

In Vivo and In Vitro Aging Is Detrimental to Mouse Spermatogonial Stem Cell Function

The development of techniques to maintain the spermatogonial stem cell (SSC) in vivo and in vitro for extended periods essentially allows for the indefinite continuation of an individual germline. Recent evidence indicates that the aging of male reproductive function is due to failure of the SSC niche. SSCs are routinely cultured for 6 mo, and no apparent effect of culture over this period has been observed. To determine the effects of SSC aging, we utilized an in vitro culture system, followed by quantitative transplantation experiments. After culture for 6 mo, SSCs that had been aged in vivo for 1500 days had a slower proliferation rate than SSCs that were aged in vivo to 8 or 300 days. Examination of methylation patterns revealed no apparent difference in DNA methylation between SSCs that were aged 8, 300, or 1500 days before culture. Long-term culture periods resulted in a loss of stem cell potential without an obvious change in the visual appearance of the culture. DNA microarray analysis of in vivo- and in vitro-aged SSCs identified the differential expression of several genes important for SSC function, including B-cell CLL/lymphoma 6, member B (Bcl6b), Lim homeobox protein 1 (Lhx1), and thymus cell antigen 1, theta (Thy1). Collectively, these data indicate that, although both in vitro and in vivo aging are detrimental to SSC function, in vitro aging results in greater loss of function, potentially due to a decrease in core SSC self-renewal gene expression and an increase in germ cell differentiation gene expression.

Expression profile of genes identified in human spermatogonial stem cell-like cells using suppression subtractive hybridization

Spermatogenesis is the process by which testicular spermatogonial stem cells (SSCs) self-renew and differentiate into mature sperm in the testis. Maintaining healthy spermatogenesis requires proper proliferation of SSCs. In this study, we sought to identify factors that regulate the proliferation of SSCs. Human SSC (hSSC)-like cells were isolated from azoospermic patients by a modified culture method and propagated in vitro. After four to five passages, the SSC-like cells spontaneously ceased proliferating in vitro, so we collected proliferating (P)-hSSC-like cells at passage two and senescent (S)-hSSC-like cells at passage five. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed between the P-hSSC-like and S-hSSC-like cells. We selected positive clones up-regulated in P-hSSC-like cells using SSH and functionally characterized them by reference to public databases using NCBI BLAST tools. Expression levels of genes corresponding to subtracted clones were analyzed using RT-PCR. Finally, we confirmed the differential expression of 128 genes in positive clones of P-hSSC-like cells compared with S-hSSC-like cells and selected 23 known and 39 unknown clones for further study. Known genes were associated with diverse functions; 22% were related to metabolism. Fifteen of the known genes and two of the unknown genes were down-regulated after senescence of hSSC-like cells. A comparison with previous reports further suggests that known genes selected, SPP1, may be related to germ cell biogenesis and cellular proliferation. Our findings identify several potential novel candidate biomarkers of proliferating- and senescencet-hSSCs, and they provide potentially important insights into the function and characteristics of human SSCs.

Introductory Overview to the Proceedings of the XXth North American Testis Workshop

Introductory Overview to the Proceedings of the XXth North American Testis Workshop

Inactivation of Numb and Numblike in spermatogonial stem cells by cell-permeant Cre recombinase

Spermatogonial stem cells (SSC) ensure continuous production of mammalian male gametes. In rodents, the SSC are Asingle spermatogonia (As). Gene loss and gain-of-function mutations have provided some clues into SSC function, but genetic dissection of SSC physiology has not yet been accomplished. The adaptor protein Numb is an evolutionarily conserved protein originally implicated in the control of the fate of sibling cells. Mice homozygous for deficient Numb die before embryonic day 11.5, hampering the analysis of its inactivation in postnatal male germline. Here, we have developed an experimental strategy to conditionally inactivate Numb and its homolog Numblike in the postnatal germline by in vitro delivery of cell-permeant Cre recombinase. Cre-transduced SSC isolated from wild-type mice retained their ability to self-renew and to differentiate in vivo, as shown by their ability to give rise to normal spermatogenic colonies when transplanted in recipient testes. Cre-transduced SSC from conditional mutant mouse line were able to colonize recipient testes upon transplantation. Inactivation of either Numb or Numblike in SSC did not impair the development of normal donor-derived spermatogenesis. However, compared to single-null SSC, double-null SSC generated shorter colonies in which the germ cells failed to differentiate beyond the round spermatid stage, underscoring the essential roles of both Numb and Numblike in spermatogenesis. We demonstrate the feasibility of gene inactivation in adult SSC by ex vivo Cre delivery. This provides a means to analyze the function of genes that operate on a cell-autonomous basis or those that are coupled to signals that SSC receive from bystander 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.

FUNCTIONAL DECIPHERING OF THE CORE SIGNALING MECHANISM FOR SPERMATOGONIAL STEM CELL SELF-RENEWAL AND SURVIVAL

The biological fate decisions of self-renewal and differentiation by spermatogonial stem cells (SSCs) provide the foundation for continual spermatogenesis in the mammalian testis. Since SSCs are rare in the testis, it is extremely difficult to study the mechanisms that regulate their biological functions. The SSC self-renewal fate decision has been shown to be dependent on glial cell line-derived neurotrophic factor (GDNF) influence. However, the intracellular mechanisms utilized by GDNF in SSCs are unknown. Recently we identified GDNF-regulated genes in SSCs, which likely encode for molecules that regulate self-renewal. From these data significant up-regulated expression of the transcription factors Bcl6b, Erm, and Lhx1 was identified and Bcl6b was subsequently shown to be important for SSC self-renewal in vitro and in vivo. The objective of this study was to begin deciphering the intracellular signaling mechanisms utilized by GDNF to regulate SSC self-renewal and survival. We utilized a mouse GDNF-dependent culture system that maintains an undifferentiated germ cell population enriched for self-renewing SSCs. Continuous in vitro culture and transplantation analysis are critical elements to define the presence of SSCs in any population of cells subject to examination for factors regulating SSC function. Using siRNA to reduce expression in cultured SSCs and transplantation to quantify stem cell numbers, Bcl6b, Erm, and Lhx1 were all shown to be important for SSC self-renewal in vitro. Next, GDNF was shown to activate AKT in SSCs and this activation could be blocked by inhibition of both PI3K and Src Family Kinases (SFKs). Culture of SSCs with inhibitors to AKT (AKT inhibitor IV), PI3K (LY294002), or SFKs (SU6656) followed by transplantation analysis showed significant impairment of SSC maintenance in vitro compared to control cultures. Apoptosis analysis revealed a significant increase in the percentage of apoptotic cells when both AKT and PI3K inhibitors, but not SFK inhibitor, was added to culture media indicating that multiple signaling pathways are responsible for SSC selfrenewal and survival. Up-regulation of Bcl6b, Erm, and Lhx1 gene expression by GDNF in self-renewing SSCs could be blocked by inhibition of AKT. However, expression of both Plzf and Oct4, which are expressed in SSCs but not GDNF-regulated, were also significantly reduced when AKT was inhibited. GDNF was further shown to phosphorylate SFKs in cultured SSCs and inhibition of SFK function could specifically block GDNF-regulated expression of Bcl6b, Erm, and Lhx1 without effecting either Plzf or Oct4 expression. These data demonstrate that GDNFactivated AKT signaling is essential for SSC survival, but not a specific pathway used for GDNF-regulated gene expression. Importantly, these data also demonstrate that SFK stimulation is an essential intracellular signaling mechanism utilized by GDNF to specifically regulate SSC selfrenewal. (platform)

Glial Cell-Line Derived Neurotrophic Factor-Mediated RET Signaling Regulates Spermatogonial Stem Cell Fate1

Normal spermatogenesis is essential for reproduction and depends on proper spermatogonial stem cell (SSC) function. Genes and signaling pathways that regulate SSC function have not been well defined. We report that glial cell-line-derived neurotrophic factor (GDNF) signaling through the RET tyrosine kinase/GFRA1 receptor complex is required for spermatogonial self-renewal in mice. GFRA1 and RET expression was identified in a subset of gonocytes at birth, was restricted to SSCs during normal spermatogenesis, and RET expressing cells were abundant in a cryptorchid model of SSC self-renewal. We used the whole-testis transplantation technique to overcome the limitation of neonatal lethality of Gdnf-, Gfra1-, and Ret-deficient mice and found that each of these genes is required for postnatal spermatogenesis and not for embryological testes development. Each mutant testis shows severe SSC depletion by Postnatal Day 7 during the first wave of spermatogenesis. These defects were due to lack of SSC proliferation and an inability of SSCs to maintain an undifferentiated state. Our results demonstrate that GDNF-mediated RET signaling is critical for the fate of undifferentiated spermatogonia and that abnormalities in this pathway may contribute to male infertility and testicular germ cell tumors.