189 Isolation and purification of rhinoceros and horse spermatogonial stem cells

The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is critically endangered. Testicular tissue (TT) of an NWR that died at 46 years was cryopreserved and stored. Spermatogonial stem cells (SSC) have been isolated from TT and, following transplantation or in vitro culture, differentiated into mature spermatozoa. These SSC have been phenotypically characterised by the expression of markers specific for SSC, and in vitro culture methods optimized to isolate, purify, and enrich populations of SSC from mixed germ cells. The reproductive age may affect the percent of SSC in TT, affecting recovery from older animals. We previously identified in TT of NWR the surface markers GFRa1 and GPR125, both of which were expressed at various stages of spermatogenesis. However, characterisation, isolation, and purification of rhinoceros SSC have not been studied. Because of the limited availability of rhinoceros TT, we used the horse as a model for rhinoceros (both species are in the same order: Perissodactyla). In this study, we (1) identified and compared the expression of markers specific for SSC (GFRa1, GPR125, PLZF) and pluripotent markers (SSEA-1, SSEA-4, OCT-4) in rhinoceros and horse mixed germ cells; (2) evaluated whether rhinoceros and horse SSC could be purified and enriched by sequential culture with collagen (2 days) and laminin (3 weeks); and (3) determined whether age may affect the percentages of SSC by comparing the expression of SSC and pluripotent markers in horse mixed germ cells at different reproductive stages: pubertal (PU=1-1.5 years) v. post-pubertal (PP=2-3 years) v. adult (AD=>5 years). Mixed germ cells were isolated from testes of one male of each of 3 rhinoceros species: NWR, southern white rhinoceros (SWR; Ceratotherium simum simum, 49 years), and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis, 5 years), and horses at different reproductive stages (PU=2, PP=2, AD=7). The SWR and GOHR testes were refrigerated overnight, whereas NWR mixed germ cells were isolated from frozen-thawed TT. Flow cytometry analysis showed expression of SSC and pluripotent markers but not for transcription factor PLZF. The marker expression was similarly distributed between the 3 species of rhinoceros, with a larger portion of cells positive for SSEA-4 (mean%±s.e.m.: 6.3±1.1%) and smaller proportion for GDNFa1 (0.4±0.2%), GRP125 (0.1±0.1%), OCT-4 (0.4±0.2%), and SSEA-1 (0.4±0.2%; P<0.05). Similarly, horse expressed the markers GDNFa1 (0.9±0.3%), GRP125 (0.9±0.4%), OCT-4 (1.7±0.5%), and SSEA-1 (0.12±0.1%), but the abundance of SSEA-4 (1.8±0.8%) was less than that of rhinoceros (P<0.05). In rhinoceros, sequential culture enhanced the numbers of cells expressing all markers compared with that before culture, whereas in horses, the increase was observed only for cells expressing GDNFa1, GPR125, and SSEA-1. Reproductive stage did not affect the percentages of horse cells expressing germ cell and pluripotent markers. Overall, these results showed that rhinoceros SSC can be isolated from TT and expressed the same SSC markers as horses, and that differential culture enriched a population of SSC.