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

Abstract

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.