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 cells.
Izadyar
F
F
Department of Endocrinology, Faculty of Biology, University Medical Center Utrecht, Utrecht, The Netherlands. fizadyar@lab.azu.nl
Spierenberg
G T
GT
Creemers
L B
LB
den Ouden
K
K
de Rooij
D G
DG
D000818:Animals; D002417:Cattle; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D005434:Flow Cytometry; D007150:Immunohistochemistry / Q000379:methods; D037102:Lectins; D008297:Male; D037121:Plant Lectins; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis; D013093:Spermatogonia / Q000166:cytology / Q000637:transplantation; D013737:Testis
D037102:Lectins; D037121:Plant Lectins; C036554:dolichos biflorus agglutinin; D019009:Proto-Oncogene Proteins c-kit