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

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.

A developmental study of the Desert hedgehog-null mouse testis.

Desert hedgehog (Dhh) is a cell-signaling molecule that was first discovered in Drosophila. A unique testicular phenotype has been described in neonatal and adult Dhh-null animals that includes anastomotic seminiferous tubules, pertitubular cell abnormalities, and absence of adult-type Leydig cells. In the present study, we addressed the developmental basis for the abnormalities previously described for the adult Dhh-null phenotype. The source of Dhh is the Sertoli cell, and receptors are localized on peritubular cells and possibly Leydig cells. The development of testes from Dhh-null mouse embryos was studied using light and electron microscopy at 11.5, 12.5, 13.5, and 16.5 days postcoitum (dpc) and was compared with that in control Dhh heterozygous and wild-type embryos. Dhh-null and control testes were generally similar during the period of early cord formation (11.5-12.5 dpc). By 13.5 dpc, the basal lamina delimiting the cords was lacking in some regions and disorganized in Dhh-null testes, and occasional germ cells were seen outside cords. At 16.5 dpc, these defects were more prominent and cord organization was less well defined than in controls. In addition, there were numerous extracordal germ cells, some of which were partially enclosed by a somatic cell of unknown identity. Numerous fibroblast-like cells, apparently secreting collagen and basal lamina, characterized the interstitium of the Dhh-null testis. These defects likely stem from abnormal peritubular stimulation due to the lack of Dhh, leading to the abnormalities seen in the developmental stages studied here and in the adult testis.