Marcadores moleculares, nicho e criopreservação de células-tronco espermatogoniais em equídeos

Abstract

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and are located in a highly dynamic microenvironment called niche that influences all aspects of stem cell function, including homing, self-renewal and differentiation. Several studies have recently identified specific proteins that regulate the fate of SSCs. These studies also aimed at identifying surface markers that would facilitate theisolation of these cells in different vertebrate species. The present study is the first to investigate SSC physiology and niche in stallions and to offer a comparative evaluation of undifferentiated type A spermatogonia (Aund) markers (GFRA1, PLZF and CSF1R) in three different domestic equid animals (stallions, donkeys, and mules). Aund were first characterized according to their morphology and expression of the GFRA1 receptor.Our findings strongly suggest that in stallions these cells were preferentially located in the areas facing the interstitium, particularly those nearby blood vessels. This distribution is similar to what has been observed in other vertebrate species. In addition, all three Aund markers were expressed in the equid animals evaluated in this study.These markers have been well characterized in other mammalian species, which suggests that the molecular mechanisms that maintain the niche and Aund/SSCs physiology may be conserved among mammals. Regarding spermatogonial cryopreservation, proper conditions for germ cells storage represent important biotechnological procedures for studies involving germ cells transplantation and the preservation of genetic stock of valuable animals. In order toevaluate the effects of different cryopreservation protocols on the survival rates of spermatogonial stem cells (SSCs) in horse, we enzymatically isolated SSCs from testis of 8 adult horses. After Percoll gradient enrichment of cell suspension, immunolabeling and western blotting for GFRA1 receptor (undifferentiated spermatogonial marker)demonstrated that most germ cells present in the suspension were GFRA1+. Also, three cryomedia [DMSO+DMEM+BFS (1); ethylene glycol (2); DMSO+sucrose (3)], associated with different methods (vitrification, slow and fast-freezing) were tested. The cell viability was evaluated before and post-thawing by trypan blue assay. Annexin V and propidium iodide assays were used to estimate the rate of apoptotic and necroticcells post-thawing by flow cytometry. The cell metabolic activity and stemness potential were also evaluated post-thawing, using respectively, MTT assay and immunolabeling for GFRA1 and VASA after 24 days in culture. Based on the rates of viable SSCs before and post-thawing, as well as the number of recovered cells after cryopreservation, the best results were obtained utilizing the DMSO-based cryomedia atfast (Medium 3) and slow freezing methods (Media 1 and 3). In addition, the MTT data have indicated that cryopreserved cells were as metabolically active as fresh cells, and also they were expressing typical stem cell proteins (GFRA1 and VASA). Thus, the results have indicated that equine SSCs could be cryopreserved without impairment oftheir metabolic activity and stemness. We hope that our findings will help future studies aiming the isolation and cryopreservation of equids SSCs. In addition, our data will be very useful for studies related to the preservation of the germplasm of valuable animals, and involving germcell transplantation or xenografts of equids testicular cells suspensions.