👤 Ina Dobrinski

Studies on spermatogonial stem cells (SSCs) are of unusual significance because they are the unique stem cells that transmit genetic information to subsequent generations and they can acquire pluripotency to become embryonic stem-like cells that have therapeutic applications in human diseases. MicroRNAs (miRNAs) have recently emerged as critical endogenous regulators in mammalian cells. However, the function and mechanisms of individual miRNAs in regulating SSC fate remain unknown. Here, we report for the first time that miRNA-20 and miRNA-106a are preferentially expressed in mouse SSCs. Functional assays in vitro and in vivo using miRNA mimics and inhibitors reveal that miRNA-20 and miRNA-106a are essential for renewal of SSCs. We further demonstrate that these two miRNAs promote renewal at the post-transcriptional level via targeting STAT3 and Ccnd1 and that knockdown of STAT3, Fos, and Ccnd1 results in renewal of SSCs. This study thus provides novel insights into molecular mechanisms regulating renewal and differentiation of SSCs and may have important implications for regulating male reproduction. Show less

Mammalian spermatogenesis is a process whereby male germ-line stem cells (spermatogonial stem cells) divide and differentiate into sperm. Although a great deal of progress has been made in the isolation and characterization of spermatogonial stem cells (SSCs) in rodents, little is known about human SSCs. We have recently isolated human G protein-coupled receptor 125 (GPR125)-positive spermatogonia and GDNF family receptor alpha 1 (GFRA1)-positive spermatogonia using a 2-step enzymatic digestion and magnetic-activated cell sorting (MACS) from adult human testes. Cell purities of isolated human GPR125- and GFRA1-positive spermatogonia after MACS are greater than 95%, and cell viability is over 96%. The isolated GPR125- and GFRA1-positive spermatogonia coexpress GPR125, integrin, alpha 6 (ITGA6), THY1 (also known as CD90), GFRA1, and ubiquitin carboxyl-terminal esterase L1 (UCHL1), markers for rodent or pig SSCs/progenitors, suggesting that GPR125- and GFRA1-positive spermatogonia are phenotypically the SSCs in human testis. Human GPR125-positive spermatogonia can be cultured for 2 weeks with a remarkable increase in cell number. Immunocytochemistry further reveals that GPR125-positive spermatogonia can be maintained in an undifferentiated state in vitro. Collectively, the methods using enzymatic digestion and MACS can efficiently isolate and purify SSCs from adult human testis with consistent and high quality. The ability of isolating and characterizing human SSCs could provide a population of stem cells with high purity for mechanistic studies on human SSC self-renewal and differentiation as well as potential applications of human SSCs in regenerative medicine. Show less

This study was designed to isolate, characterize, and culture human spermatogonia. Using immunohistochemistry on tubule sections, we localized GPR125 to the plasma membrane of a subset of the spermatogonia. Immunohistochemistry also showed that MAGEA4 was expressed in all spermatogonia (A(dark), A(pale), and type B) and possibly preleptotene spermatocytes. Notably, KIT was expressed in late spermatocytes and round spermatids, but apparently not in human spermatogonia. UCHL1 was found in the cytoplasm of spermatogonia, whereas POU5F1 was not detected in any of the human germ cells. GFRA1 and ITGA6 were localized to the plasma membrane of the spermatogonia. Next, we isolated GPR125-positive spermatogonia from adult human testes using a two-step enzymatic digestion followed by magnetic-activated cell sorting. The isolated GPR125-positive cells coexpressed GPR125, ITGA6, THY1, and GFRA1, and they could be cultured for short periods of time and exhibited a marked increase in cell numbers as shown by a proliferation assay. Immunocytochemistry of putative stem cell genes after 2 wk in culture revealed that the cells were maintained in an undifferentiated state. MAPK1/3 phosphorylation was increased after 2 wk of culture of the GPR125-positive spermatogonia compared to the freshly isolated cells. Taken together, these results indicate that human spermatogonia share some but not all phenotypes with spermatogonial stem cells (SSCs) and progenitors from other species. GPR125-positive spermatogonia are phenotypically putative human SSCs and retain an undifferentiated status in vitro. This study provides novel insights into the molecular characteristics, isolation, and culture of human SSCs and/or progenitors and suggests that the MAPK1/3 pathway is involved in their proliferation. Show less