Isolation and purification of type A spermatogonia from the bovine testis

The aim of this study was to isolate and purify bovine type A spermatogonia. Testes from 5-7-month-old calves were used to isolate germ cells using a two-step enzymatic digestion. During the isolation and purification steps, the viability of cells was determined using live/dead staining. The identity of type A spermatogonia during isolation and purification was determined under a light microscope equipped with a Nomarski lens. Isolated cells were characterized further by using specific markers for type A spermatogonia, including Dolichos biflorus agglutinin (DBA) and c-kit. The cell suspension was transplanted into immunodeficient recipient mouse testes and the colonization was assessed 1-3 months after transplantation, to assess the stem cell population among the isolated cells. After isolation, a cell suspension was obtained containing about 25% type A spermatogonia, which was enriched further by differential plating and separation on a discontinuous Percoll gradient. Finally, fractions containing 65-87% pure type A spermatogonia were obtained. Large and small type A spermatogonia with different numbers and sizes of nucleoli were found. DBA stained both large and small type A spermatogonia and its application in fluorescence-activated cell sorting (FACS) resulted in comparable percentages of type A spermatogonia to those determined by morphological examination under a light microscope equipped with a Nomarski lens. Nearly all of the large type A spermatogonia showed strong c-kit immunoreactivity, indicating that these cells had undergone at least an initial differentiation step. In contrast, approximately half of the small type A spermatogonia were negative for c-kit, indicating the presence of the spermatogonial stem cells in this population. At 3 months after transplantation, groups of bovine type A spermatogonia were found in most tubule cross-sections of the recipient mouse testes, showing the presence of spermatogonial stem cells among the isolated cell