Stem Cell Renewal

Stem cells are common to all epithelial systems because they have two fundamental properties (30). First, stem cells have the ability to produce more stem cells (to maintain a constant number of these cells). Second, stem cells can produce differentiated progeny; this property of spermatogenetic stem cells was described in the Spermatogonia and Mitosis section. The former capability represents stem cell renewal.

Stem cell renewal is the mechanism that ensures that remarkably large numbers of spermatozoa are produced by a relatively small number of stem cells throughout the adult primate's life. Whether each stem cell produces one stem cell and one differentiated cell, asymmetrical mitosis, or one stem cell produces two stem cells while another adjacent stem cell produces two daughter cells, symmetrical mitosis, is not known for most epithelia (31,32). As discussed later, evidence relating to this intriguing issue is presented that suggests that the renewing stem cells of some primates, if not all, divide symmetrically.

Historically, two fundamentally different models of stem cell renewal during mammalian spermatogenesis were postulated from the results of studies performed in rodents (32). A review of the evidence supporting each model in rodents is necessary, because the original scheme of stem cell renewal has been generally accepted to occur in primates and the original scheme postulated from results in rodents has been replaced by the newer scheme. Moreover, the mechanism of stem cell renewal that is widely accepted in primates has recently been challenged by the notion that the newer model may be applicable to primates (32).

Clermont and colleagues (33), using mophometric analysis of histological sections and incorporation of tritiated thymidine during the DNA synthetic phase of the cell cycle, identified four morphologically distinct type A spermatogonia. These spermatogonia, called A1-A4, were distinguishable from each other by the increase of heterochromatin in the nuclei of spermatogonia of each succeeding generation. These workers determined that each type Ai spermatogonium divided mitotically and theoretically produced two type A2 spermatogonia. In turn, each type A2 spermatogonium divided and potentially produced two type A3 spermatogonia. Each type A3 spermatogonium, in turn, divided, and produced two type A4 spermatogonia. With the eventual appearance of type A4 a departure from this straightforward system of spermatogonial proliferation was postulated. The type A4 spermatogonia divided and produced either the differentiated intermediate type spermatogonia or the rather undifferentiated type Ai spermato-gonia. In other words, the most differentiated of the four generations of type A spermatogonia, A4 cells, produced progeny that were either more differentiated or the least differentiated type A spermatogonia. Because of this potential to produce undifferentiated spermatogonia, all four generations of type A spermatogonia were considered renewing stem cells. This singular state of affairs was further complicated.

Indeed, another type A spermatogonia population was also described (33). These type A spermatogonia were morphologically less differentiated than any of the other four type A spermatogonia and did not incorporate the labeled nucleotide that indicated a cell was preparing for mitosis. Furthermore, the number of these cells in the seminiferous epithelium did not change. Therefore, these rather undifferentiated type A sper-matogonia were not mitotically active but were quiescent. These stem spermatogonia were designated type A0 spermatogonia and postulated to represent stem cells held in reserve that would repopulate the seminiferous epithelium only when all more mature germ cells were destroyed by some catastrophe. The A0 spermatogonia were postulated to divide mitotically and produce type Ai spermatogonia until the seminiferous epithelium was reestablished. It was posited that one or all type A1-4 spermatogonia produced a factor, or factors, that normally held the A0 stem cells in a quiescent state (34). However, the search for this factor was not fruitful (35-38).

The second scheme of stem cell renewal was posited by Huckins (39,40), based on studies of rodent testes. A technique for isolating, fixing, and mounting long segments of seminiferous tubules for observation with the light microscope was developed. This technique permitted examination of the outer layer of the seminiferous tubule; that is, the layer containing the spermatogonia. The spermatogonia, both stem and differentiated, could be identified easily, and long strings of spermatogonial nuclei exhibiting the same morphology were readily apparent. Because the segments of seminiferous tubule were intact, the nuclei of each string were enumerated. In addition, the spacing of the nuclei was rather constant from one string to the other. Furthermore, when tritiated thymidine was administered to the rodent before removing the testes, all the nuclei in a string were labeled. The conclusion was that as the spermatogonial nuclei divided, cytokinesis was not completed and cytoplasmic bridges connected the spermatogonia. After the strings with identical morphology were examined, three kinds of nuclear strings were identified. Pairs of type A spermatogonia were identified but were too far from the next nuclei of similar morphology to expect that they were part of the same string. Longer strings, consisting of 4, 8, or 16 nuclei, were readily distinguished. The type A spermatogonia, with a nucleus that was clearly separated from the next nucleus with the same morphology, were called A isolated or A single (As). When two nuclei of similar morphology were adjacent to one another, they were called A paired (Apr). The last class were strings of nuclei containing 4, 8, or 16 nuclei. These latter strings were called A aligned (Aai). These observations led to the conclusion that the type As were the renewing stem cells, whereas the remaining categories of nuclei strings were simply the result of mitosis without complete cytokinesis. Long strings of nuclei of type Ai_4 were also recognized, but the results suggested that the Type Aa nuclei, whether strings of 4, 8, or 16, would, at the proper time in spermatogenesis, transform directly into type A1 spermatogonia. This transformation of nuclei from strings of cells of different lengths is the greatest weakness of this model.

The latter scheme of stem cell renewal overcomes the problem of the former scheme, namely the dedifferentiation of type A4 cells producing the undifferentiated type A1 spermatogonia. This scheme, however, cannot explain easily how the transformation of nuclei strings with differing numbers can all transform simultaneously at the appropriate time to produce type A1 spermatogonia. The greatest weakness of this model is that the cells can transform into A1 whether they are in strings of 4, 8, or 16 nuclei. This poses a substantial problem, because the timing of the seminiferous epithelial cycle is clearly determined by the germ cells and not by external factors (41). It is difficult to conceive a mechanism whereby the chains of 4, 8, or 16 nuclei can detect the appropriate environment and directly transform.

In summary, the two schemes of stem cell renewal are distinctive in two respects. The first scheme has two types of stem cells, namely reserve stem cells and renewing stem cells. The reserve stem cells, type A0 spermatogonia, are quiescent and only become active when the renewing stem cells, types A1-4 spermatogonia, are destroyed. The four generations of renewing stem cells require that a more differentiated cell, A4 spermatogonium, produces less differentiated daughter cells, type A1 spermatogonia. A mechanism that produces a pattern of differentiation immediately followed by dedifferentiation is difficult for the author to conceptualize. The second scheme of stem cell renewal is simpler than the first because no reserve stem cell is postulated, however, it has difficulty accounting for coordination of the strings of spermatogonial nuclei that have divided a different number of times, so that they all transform at the same time into the type A1 spermatogonia. This coordination is required because, as described in the Wave of the Cycle section, the various types of germ cells and the processes of spermatoogonial proliferation, meiosis, and spermiogenesis are ordered and highly synchronized. Neither scheme is entirely satisfactory.

For stem cell renewal in higher primates, the mechanism is less complicated compared to that of rodents. Only two types of A spermatogonia are found in the seminiferous epithelia of these species, types Ad and Ap spermatogonia (see Spermatogonia and Mitosis section).

Type Ad spermatogonia are rather mitotically inactive.4 This conclusion is based on two lines of evidence. First, the number of Ad spermatogonia in the higher primate testis is constant, and these cells rarely incorporate tritiated thyminidine, a marker of DNA synthesis preparatory to mitosis (18-20). Second, X-irradiation of the testes of adult rhesus monkeys destroyed all germ cells except the Ad spermatogonia (42). After documenting the decline and subsequent disappearance of all germ cells except Ad

4 It should be noted that the initial descriptions of spermatogenesis in man (16) suggested that the Ad spermatogonia were the renewing stem cells. Later studies in monkey and man showed that few Ad spermatogonia incorporate radiolabeled nucleotides, suggesting that these cells were mitotically quiescent (20).

spermatogonia, all irradiation was stopped, and the seminiferous epithelia of the monkeys were allowed to recover. The type Ad spermatogonia transformed without mitosis directly into the type Ap spermatogonia. Once the population of Ap spermatogonia was reestablished, some of the Ap spermatogonia transformed back to Ad spermatogonia. These results unequivocally demonstrate that the Type Ad spermatogonia are reserve stem cells.

The case for Ap spermatogonia being the renewing stem cells is based on morphome-tric analyses and the pattern of incorporation of tritiated thymidine. Clermont and Antar (20) injected the testes of adult stump-tailed macaques (M. arctoides) with the radiolabeled nucleotide. Testes were removed either 3 h or 12 d and 3 h after the injection. In addition, the number of each cell type was enumerated. The number of Ad spermatogo-nia was unchanged at both 3 h and 12 d 3 h after injection. Few Ad spermatogonia were labeled with the tritiated thymidine, and 12 d later, none was identified. These results led to the notion that the Ad spermatogonia were reserve stem cells. By contrast, the Ap spermatogonia were increased in number, and incorporated the radiolabeled nucleotide at 3 h after injection. After the increase in Ap spermatogonia, the number of these cell types returned to the number before the increase. This transient increase in cell number was observed in the testes removed 14 d 3 h after the administration of tritiated thymidine. Moreover, Ap spermatogonia were labeled with tritiated thymidine in the testes removed 12 d 3 h after the single injection with labeled nucleotide.

Two schemata for the mechanism of stem cell renewal have been postulated (20). The first scheme, which occurs in the rhesus monkey and African green monkey, and, perhaps, in man, is distinguished by a single peak of mitosis of the Ap spermatogonia. During that peak, all type Ap spermatogonia divide and yield either more Ap spermatogonia, thus renewing the population, or type B spermatogonia, beginning spermatogenesis. Whether this cell division is symmetrical or asymmetrical has not been determined, although cell counts and labeling patterns suggest that the mitosis of Ap spermatogonia mitosis is symmetrical; that is, half of the population of Ap spermatogonia divides and produces two daughter cells, both of which are Ap spermatogonia. The remaining half of the Ap spermatogonia divide, and produce two identical type B spermatogonia.

The other scheme, which occurs in the stump-tailed macaque and the crab-eating macaque, is distinguished by two peaks of Ap spermatogonia mitosis during every cycle of the seminiferous epithelium. The first peak results in a transitory doubling of the number of Ap spermatogonia and is followed in the same cycle by a second peak of mitosis. Only half of these newly formed type Ap spermatogonia divide during the second peak, resulting only in the production of the first generation of B spermatogonia. This scheme seems more likely to support the notion that the mitotic events are symmetrical.

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