Abstract: In recent years, embryonic stem (ES) cell-like cells have been obtained from cultured mouse spermatogonial stem cells (SSCs). These advances have shown that SSCs can transition from being the stem cell-producing cells of spermatogenesis to being multipotent cells that can differentiate into derivatives of all three germ layers. As such, they offer new possibilities for studying the mechanisms that regulate stem cell differentiation. The extension of these findings to human SSCs offers a route to obtaining personalized ES-like or differentiated cells for use in regenerative medicine. Here, we compare the different approaches used to derive ES-like cells from SSCs and discuss their importance to clinical and developmental research.

Abstract: Testicular hyperthermia in mice lacking transient receptor potential vanilloid receptor-1 results in a much more rapid and massive germ cell depletion from the seminiferous tubules than in wild-type animals, indicating that this receptor protects germ cells against heat stress.

Abstract: The access to sufficient numbers of spermatogonial stem cells (SSC) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behaviour of bovine type A spermatogonia, a cell population that includes the SSC and can be specifically stained for the lectin DBA (Dolichos bifflorus agglutinin). During short term culture (two weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained A spermatogonia. When LIF, EGF or FGF2 were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of A spermatogonia was significantly higher in cultures to which GDNF was added and highest when GDNF, LIF, EGF and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSC among the cultured cells and in addition strongly suggested a more than 10000-fold increase in the number of SSC after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

Abstract: Phosphoprotein enriched in astrocytes (PEA-15) is a 15 kDa acidic serine-phosphorylated protein expressed in different cell types, especially in the CN. We initially detected the expression of PEA-15 in primary cultures of Sertoli cells. To assess the presence and localization of PEA-15 in the mouse testis, we studied the expression pattern of the PEA-15 protein by immunohistochemistry and mRNA by in situ hybridization. Both the protein and the mRNA of PEA-15 were localized in the cytoplasm of Sertoli cells, all types of spermatogonia, and spermatocytes up till zygotene phase of the meiotic prophase. Subsequently, with ongoing development of the spermatocytes, the expression decreased and was very low in the cytoplasm of diplotene spermatocytes. To analyze the possible role of PEA-15 in the developing testis, null mutants for PEA-15 were examined. As the PEA-15 C terminus contains residues for ERK binding, we studied possible differences between the localization of the ERK2 protein in wild type (WT) and PEA-15(-/-)mice. In the WT testis, ERK2 was localized in the cytoplasm of Sertoli cells, B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes, whereas in the KO testis, ERK2 was primarily localized in the nuclei of these cells and only little staining remained in the cytoplasm. Moreover, in PEA-15-deficient mice, significantly increased numbers of apoptotic spermatocytes were found, indicating an anti-apoptotic role of PEA-15 during the meiotic prophase. The increased numbers of apoptotic spermatocytes were not found at a specific step in the meiotic prophase.

Abstract: Phthalate esters are considered endocrine disruptors that interfere with the endocrine balance and development of the mammalian testis. Mono-2-ethylhexyl phthalate (MEHP), the active metabolite of the ubiquitously used plasticizer di-2-ethylhexyl phthalate (DEHP), acts upon Sertoli cells as initial target. By subtractive cDNA libraries we identified genes deregulated as response to MEHP in primary cultures of mouse Sertoli cells. The expression of mouse stress inducible protein 1 (Stip1) was detected as upregulated as a result of MEHP exposure. Stip1 is a cochaperone protein that is homologous to the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). To assess the presence and localization of Stip1 in mouse testis and its potential role in stress defense, we studied the expression pattern of the Stip1 protein by immunohistochemistry and of the mRNA by in situ hybridization. Both the protein and the mRNA of Stip1 were mainly found in the cytoplasm of all types of spermatogonia and spermatocytes up till zygotene, the expression decreased during late pachytene and was very weak in diplotene spermatocytes and round spermatids. Interestingly, this expression pattern resembled the pattern of stress sensitivity of spermatogenic cells in that the most sensitive cell types show the weakest expression of Stip1. This suggests an important role for Stip1 in the ability of germ cells to survive in stress conditions including high temperatures.