5.1. Morphological and Cell Cycle Characteristics of Spermatogonial Stem Cells
Spermatogonial stem cells lie amid the other types of spermatogonia, newly formed spermatocytes, and Sertoli cells on the basal membrane of the tubules. These stem cells can be recognized morphologically using a special technique. Pieces of seminiferous tubules are prepared in their entirety to produce whole mounts of these structures (19). These whole mounts of seminiferous tubules enable study of the topography of the spermatogonia lying on the basal membrane and to distinguish singles, pairs, and chains of these cells (Fig. 3). Hence, differential cell counts of stem cells and Apr and Aal spermatogonia can be carried out. Furthermore, a method was developed to perform autoradiography on these whole mounts (20). Using 3H-thymidine and the labeled mitoses technique, it was found that spermatogonial stem cells have a relatively long cell cycle time of at least 56 h in the rat (11) and 90 h in the Chinese hamster (14). These cell cycle times resemble those of Apr and Aal spermatogonia, but are longer than in subsequent types of spermatogonia (12,13).
5.2. Purification of Spermatogonial Stem Cells
In one adult mouse testis, there are about 35,000 stem cells, which is only 0.03% of all germ cells (18).Various techniques have been developed to purify the total population of A spermatogonia, achieving a purity varying between 85 and 98% (21-23). Unfortunately, in the mouse only about 3% of the A spermatogonia were calculated to be stem cells (18), and it will not likely be much different in other animals. Hence, although a 100-fold enrichment of stem cells can be achieved by purifying A spermatogonia, the purity is still very low. To increase the purity, a method has been developed to isolate spermatogonia from rats deficient in vitamin A (24). In these rats and mice, spermatogenesis is arrested at the differentiation step of Aal into A1 spermatogonia, and the testes of these animals only contain As, Apr, and Aal spermatogonia (25). A cell population containing roughly 10% stem cells can be obtained using animals deficient in vitamin A (18,24).
Certain biochemical markers have been used to enrich spermatogonial stem cells (26,27). Using anti-P(1)- and anti-a(6)-integrin and negatively selecting for the c-kit receptor, which is not present on spermatogonial stem cells (28), a 40-fold enrichment of spermatogonial stem cells from testicular germ cells could be accomplished (26).
Taken together, the state of the art in the purification of spermatogonial stem cells has not yet reached any further than purity of about 10% at most. More specific membrane markers for these cells will have to be found for further progress in this field.
5.3. Functional Test for Spermatogonial Stem Cells: Spermatogonial Stem Cell Transplantation
The presence of spermatogonial stem cells or their functionality can be checked by the spermatogonial transplantation technique developed by Brinster and coworkers (29,30). In this technique, germ cells of one mouse are transplanted into the testes of a recipient mouse, the endogenous sper-matogenesis of which is depleted by treatment with the alkylating agent busulfan. Also, mice carrying the Wv/Wv mutation can be used since their testes do not contain germ cells. The donor stem cells repopulate the seminiferous epithelium of the recipient mice.
Interestingly, rat spermatogonial stem cells also are able to repopulate the mouse testes and produce normal rat spermatogenesis in the mouse (31,32). However, stem cells from other species transplanted into mouse testes either produce defective spermatogenesis (for hamster, see ref. 33) or initiate repopulation by spermatogonia only (for rabbit and dog, see ref. 34; for the bull, see ref. 35). Spermatogonial cells from rabbit, dog, and bull form pairs and chains of A spermatogonia, indicating that these stem cells do produce differentiating Apr spermatogonia, but these cells fail to develop further into A1.
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