Genes Involved in Post-transcriptional Regulation are Over Represented in Stem/progenitor Spermatogonia of Cryptorchid Mouse Testes

¹ Departments of Obstetrics, Gynecology Reproductive Sciences and Molecular Genetics Biochemistry, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
² Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104
³ Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
⁴ University of Regensburg, Regensburg, Germany 93042
⁵ Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

^* To whom correspondence should be addressed. E-mail: cpope{at}vet.upenn.edu.

Correspondence may also be addressed to Kyle Orwig at korwig@pdc.magee.edu.

	Abstract

Gene expression and consequent biological activity of adult tissue stem cells are regulated by signals emanating from the local microenvironment (niche). To gain insights into the molecular regulation of spermatogonial stem cells (SSCs), gene expression was characterized from SSCs isolated from their cognate niches of cryptorchid (stem cell enriched), wild type, and busulfan-treated (stem cell depleted) mouse testes. Quantitative assessment of stem cell activity in each testis model was determined using an in vivo functional assay and correlated with gene expression using Affymetrix MGU74Av2 microarrays and the ChipStat algorithm optimized to detect gene expression from rare cells in complex tissues. We identified 389 stem/progenitor spermatogonia candidate genes, which exhibited significant overlap with genes expressed by embryonic, hematopoietic, and neural stem cells, enriched spermatogonia and cultured SSCs identified in previous studies. Candidate cell surface markers identified by the microarray may facilitate the isolation and enrichment of stem and/or progenitor spermatogonia. Flow cytometric analyses confirmed the expression of chemokine receptor 2 (Ccr2) and Cd14, on a subpopulation cryptorchid testis cells (6-integrin⁺, side scatter^lo) enriched for SSCs. These cell surface molecules may mark progenitor spermatogonia, but not SSCs because Ccr2⁺ and Cd14⁺ fractions failed to produce spermatogenesis upon transplantation to recipient testes. Functional annotation of candidate genes and subsequent immunohistochemistry revealed proteins involved in post-transcriptional regulation are overrepresented in cryptorchid testes that are enriched for SSCs. Comparative analyses indicated that this is a recurrent biological theme among stem cells.