GAMETE BIOLOGY AND GAMETOGENESIS - A
Monday, August 2, 2004
10:30 AM–12:30 PM
(147) ENRICHMENT OF RHESUS MACAQUE SPERMATOGONIAL STEM CELLS.
Pommer, Angela1, Dobrinski, Ina2, Meyers, Stuart1, 1 University of California, Davis, Davis, CA2 University of Pennsylvania, Kennett Square, PA
ABSTRACT- Xenotransplantation of spermatogonial stem cells has been reported in a number of laboratory and domestic livestock species, however, with little development beyond the initial stage of spermatogonial proliferation in non-human primates. The goal of this project is to develop techniques for the preservation of valuable genetic material through the preservation and propagation of spermatogonial stem cells from rhesus macaques. The objective of this study was to develop a successful method to isolate and enrich non-human primate germ line precursor cells for xeno- and autologous transplantation. Testicular tissue was obtained at necropsy from eight rhesus macaques at three (n=4) or six months (n=4) of age. After sampling and fixation for histopathology, the testicular parenchyma was dissected free from the tunica albuginea and 2-3 mm3 minced pieces were processed using sequential incubations in Dulbecco's Modified Eagle Medium (DMEM) containing collagenase, hyaluronidase, DNase, and trypsin/EDTA to dissociate the seminiferous tubules. Viability of cells was determined by trypan blue exclusion. Western blotting was used to screen cell suspensions for presence of c-kit receptor, known to be downregulated on spermatogonial stem cells, 6 integrin and GFR-1, previously shown to be expressed on putative stem cells, and MIH as a marker for Sertoli cells. Cells were labeled with c-kit receptor, 6 integrin, MIH, and GFR-1 polyclonal primary antibodies followed by FITC secondary labeling, and adjusted to 1 x 106/ml for flow cytometric analysis. Stem cell enrichment was performed using c-kit, GFR-1, and 6 integrin polyclonal antibodies and a secondary anti-rabbit IgG conjugated to ferromagnetic microbeads. An anti-rabbit FITC secondary was also added so that cells could be analyzed on the flow cytometer following separation. Sorting was performed using separation columns in a magnetic field and retention of the positive cells in the matrix, followed by elution. The enriched fractions contained 78-93% of the targeted cells as determined by flow cytometry. These results indicate that putative germ line stem cells can be isolated and enriched from non-human primate testes and should prove useful for preservation and transplantation studies.
KEY WORDS: non-human primate, spermatogonia, transplantation