CHARACTERIZATION, ENRICHMENT, AND IN VITRO CULTURE OF SPERMATOGONIAL STEM CELLS IN THE DOMESTIC CAT: A MODEL FOR RARE AND ENDANGERED FELIDS
Vansandt, Lindsey Marie
Keefer, Carol L
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Spermatogenesis is a highly prolific process in which millions of spermatozoa are produced daily. Spermatogonial stem cells (SSCs), the adult stem cell population of the testis, sustain this process by providing a constant source of new progenitor cells. The ability of this stem cell population to self-renew makes it a promising alternative to spermatozoa for genetic preservation of rare and endangered animals. While innovative advances in SSC technologies have been made in the mouse, there is a paucity of information concerning felid SSCs. Therefore, the overall objective of the dissertation was to develop SSC technology in the domestic cat (<italic>Felis catus</italic>) as a model for rare and endangered felids. In the first study, mRNA transcripts for six SSC marker genes (<italic>THY1</italic>, <italic>GPR125</italic>, <italic>GFRalpha1</italic>, <italic>PLZF</italic>, <italic>UCHL1</italic>, and <italic>OCT4</italic>) were identified in cat testes. Localization within the appropriate <italic>in situ</italic> niche was confirmed by immunohistochemistry for three of the markers (PLZF, UCHL1, and OCT4). The expression pattern of these markers was conserved in the cheetah (<italic>Acinonyx jubatus</italic>) and Amur leopard (<italic>Panthera pardus orientalis</italic>), validating the cat as an appropriate felid model. In Study 2, we explored two techniques to enrich cat testis cells for SSCs. We found that the efficiency of enrichment depends on age of the donor and that prepubertal testes are the preferred source for differential plating. Magnetic-activated cell sorting did not achieve any level of enrichment for cat SSCs, likely due to unsuitability of the antibody. The final study modified the traditional mouse SSC culture system for use in the cat. A clear effect of feeder cell type was demonstrated, with mouse endothelial C166 cells supporting a significantly higher number of germ cell colonies as compared to STO cells or primary cat fetal fibroblasts. Identity of germ cell colonies was confirmed by co-expression of UCHL1, PLZF, and OCT4. During subculture, colonies maintained SSC marker co-expression and displayed alkaline phosphatase activity. At the time of writing, cells had been maintained for 78 days <italic>in vitro</italic>. Together, these studies provide the groundwork towards application of SSC technology in management of rare and endangered felid populations.