Introduction Spermatogenesis is a complex, highly organized process that originates from stem cell spermatogonia. In non- primate mammals, the A s (A single) spermatogonia are considered to be the stem cells of spermatogenesis (Huckins, 1971; Oakberg, 1971; de Rooij, 1973). Upon division of the A s spermatogonia, the daughter cells either migrate away from each other and become two new stem cells, or stay together through an intercellular bridge and become A-paired (A pr ) spermatogonia. The A pr sper- matogonia develop further into chains of four, eight or 16 A-aligned (A al ) spermatogonia. The A al spermatogonia differentiate into A1 spermatogonia and after six mitotic divisions result in A2, A3, A4 and, finally, B spermatogonia, which give rise to spermatocytes at the last mitotic division. Irrespective of species-specific differences, in the bovine testis, a comparable classification with another terminology has been reported (Wrobel et al., 1995a). In this species, spermatogonial precursor cells have been divided into basal stem cells (BSC), aggregated spermatogonial precursor cells (ASPC) and committed spermatogonial precursor cells (CSPC), which, according to this classification, represent A s –A pr spermatogonia, A al spermatogonia and A 1 –A 4 differ- entiating spermatogonia, respectively. Hence, in bulls, A pr spermatogonia are also thought to have stem cell properties (Wrobel, et al., 1995a). As there are relatively few stem cells that can be defined only by their function, the identification and isolation of these cells has been very difficult. To date, the most effective way to enrich germ cell populations for stem cells is to purify all forms of type A spermatogonia. In the adult mammalian testis, owing to the presence of multiple generations of germinal cells, purification of spermatogonia is more diffi- cult than it is before puberty. Bellve et al. (1977) obtained a 90% pure fraction of type A spermatogonia from immature mice. Similarly, highly purified spermatogonia have been obtained from immature rat (Morena et al., 1996) and pig (Dirami et al., 1999) testes. In addition, vitamin A-deficient animals can be used as a source of spermatogonia as, in these animals, spermatogenesis stops at a spermatogonial Isolation and purification of type A spermatogonia from the bovine testis F. Izadyar 1,2 , G. T. Spierenberg 3 , L. B. Creemers 1,2 , K. den Ouden 1,2 and D. G. de Rooij 1,2 1 Department of Endocrinology, Faculty of Biology, University Medical Center Utrecht, Utrecht, The Netherlands; and Departments of 2 Cell Biology and 3 Immunology, University Medical Center Utrecht, Utrecht, The Netherlands Reproduction (2002) 124, 85–94 Research 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 isola- tion and purification was determined under a light micro- scope 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 colon- ization 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 discon- tinuous 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 under- gone 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 sper- matogonial 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 cells. © 2002 Society for Reproduction and Fertility 1470-1626/2002 Email: fizadyar@lab.azu.nl Downloaded from Bioscientifica.com at 02/17/2023 02:44:42PM via free access