Xenogeneic transplantation of equine testicular cells into seminiferous tubules of immunocompetent rats

Abstract

The objectives were to develop a transplantation assay for equine testicular cells using busulfan-treated prepubertal immunocompetent rats as recipients, and to determine if putative equine spermatogonial stem cells (SSCs) could be enriched by flow cytometric cell sorting (based on light scattering properties), thereby improving engraftment efficiency. Four weeks after transplantation of frozen/thawed PKH26-labeled equine testicular cells, 0.029 ± 0.045% (mean ± SD) of viable donor cells transplanted had engrafted. Donor cells were present in seminiferous tubules of all recipient rats forming chains, pairs, mesh structures, or clusters (with two to >30 cells/structure). Cells were localized to the basal compartment by the basement membrane. Although equine cells proliferated within rat seminiferous tubules, no donor-derived spermatogenesis was evident. Furthermore, there was no histologic evidence of acute cellular rejection. No fluorescent cells were present in control testes. When equine testicular cells were sorted based on light scattering properties, the percentage of transplanted donor cells that engrafted was higher after injection of cells from the small, low complexity fraction (II; 0.169 ± 0.099%) than from either the large, high complexity fraction (I; 0.046 ± 0.051%) or unsorted cells (0.009 ± 0.007%; P < 0.05). Seminiferous tubules of busulfan-treated prepubertal immunocompetent rats provided a suitable niche for engraftment and proliferation, but not differentiation, of equine testicular cells. Sorting equine testicular cells based on light scattering properties resulted in a 19-fold improvement in colonization efficiency by cells with high forward scatter and low side scatter, which may represent putative equine SSCs.

Fig. 1

Light scattering properties of equine testicular cells. Each dot represents the forward scatter and side scatter value of a single cell. Cells in Fractions I and II were separated from the total population and transplanted into seminiferous tubules of recipient rats. Fraction III was discarded.

Fig. 2

PKH26-labeled equine testicular cell by the basement membrane (arrows) in a histologic section of a rat seminiferous tubule 4 wk after transplantation (A to C). Whole mount of rat seminiferous tubules with fluorescent equine testicular cells forming a mesh structure and chain of cells (arrow) (D to F). The images correspond to fluorescence (A, D), bright field (B, E) and composite of fluorescence and bright field microscopy (C, F). Please refer to the online version of the manuscript for color images.