Genes involved in spermatogonial stem cell self-renewal and differentiation

Abstract

Spermatozoa are produced by the process of spermatogenesis. At the base of this process is a pool of spermatogonial stem cells that generate new stem cells as well as committed spermatogonia that will eventually differentiate in spermatozoa. A tight balance between self-renewal and differentiation must be maintained to prevent accumulation of stem cells or depletion of the seminiferous tubules. The mechanisms by which self-renewal and differentiation are regulated are largely unknown. Therefore we aimed at identifying genes involved in spermatogonial stem cell self-renewal and/or differentiation. Genes have been identified using both a candidate and a genomics approach. The gene Ly6a/e, also known as Sca-1, is frequently used for the isolation of hematopoietic stem cells and plays a role in the repopulating capacity of these cells and the development of committed progenitor cells. Furthermore, LY6a/e was reported to be expressed by the testicular side population. In this thesis, expression of SCA-1 has been localized to mesenchymal and peritubular cells and histological examination of Ly6a/e null mice testes revealed no abnormal phenotype. Together these observations indicate that LY6A/E is not expressed by spermatogonial stem cells and that LY6A/E does not play a role in spermatogonial stem cells self-renewal or differentiation. Expression of Utf1 is rapidly down-regulated upon differentiation of embryonic stem cells. In chapter 3, expression of UTF1 in the rat testis is shown to be restricted to the population of cells that contains the stem cells, i.e. gonocytes in embryonic and neonatal rats and to a subpopulation of early A spermatogonia in adult rat testis. This result together with the recently proposed notion that UTF1 may be involved in the maintenance of a specific epigenetic profile suggests that UTF1 plays a role in maintaining the spermatogonial stem cells in an undifferentiated state. Two protocols of spermatogonial stem cell differentiation have been established. Both, BMP4 and activin A induce differentiation of the GC-6spg spermatogonial stem cell line. Expression of c-KIT, a marker of differentiated spermatogonia, is up-regulated and BMP4 and activin A receptors are expressed. Using BMP4 induced differentiation of the GC-6spg cells as a model in a time course micro-array experiment revealed numerous transcripts that are differentially regulated during the differentiation process. Among the differentially regulated genes are several TGF? family members and downstream targets. Furthermore, a number of genes more generally known to play a role in stem cell maintenance or differentiation are identified. Quantitative RT-PCR confirmed the expression pattern of six differentially regulated genes, indicating that the list of genes identified can be used as candidate genes for further research. Furthermore, our experiments indicate that activin A and BMP4 act via distinct and identical downstream target genes. Our results form a strong foundation for further research as multiple genes have been identified that likely play a role in spermatogonial stem cell self-renewal and/or differentiation. In addition, two distinct methods for in vitro differentiation of a spermatogonial stem cell line have been established, which can be used as a unique model to study spermatogonial stem cell fate and differentiation.