All You Wanted to Know About Spermatogonia but Were Afraid to Ask

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1 All You Wanted to Know About Spermatogonia but Were Afraid to Ask Review DIRK G. DE ROOIJ* AND LONNIE D. RUSSELL From the *Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands; and the Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois. Spermatogonia have entered the limelight in recent years, given the intense interest in stem cells in general (Fuchs and Segre, 2000) and specific interest in cloning (Wilmut, 1998; Wilmut et al, 1997, 1998), transgenesis (Erickson, 1999; Perry et al, 1999; Pintado and Gutiérrez-Adan, 1999; Robl, 1999; Russell and Griswold, 1998), and regulation of spermatogonial numbers (de Rooij and Grootegoed, 1998). Spermatogonial terminology is confusing to many and spermatogonial kinetics is perceived as complex. The field is dominated by only a few individuals whose work is highly specialized and whose techniques are not generally used. Thus, there is a definite need to simplify this little understood topic for others. What follows is information about spermatogonia, in particular the terminology applied to spermatogonia and their function, a description of the kinetics and renewal of spermatogonia, the origin of spermatogonia, the regulation of spermatogonial numbers, and conditions that affect spermatogonia. A question-and-answer format is used in an attempt to simplify a complex area of research for an audience that is generally not familiar with the topic or that has in the past had difficulty with the topic. A variety of other reviews on the topic are embedded in the dialogue. Reviews are suggested for readers who have mastered the basics and wish to examine the details of spermatogonia and their properties. We limit our discussion of spermatogonia to mammalian systems and, unless otherwise specified, direct our comments primarily to rodent systems, from which most of our information about spermatogonia has been derived. Role of Spermatogonia in Gamete Production Question: What specifically do spermatogonia contribute to the process of spermatogenesis? Correspondence to: Lonnie D. Russell, Department of Physiology, Southern Illinois University School of Medicine, Carbondale, IL ( lrussell@siumed.edu). Received for publication May 1, 2000; accepted for publication June 26, Answer: Spermatogonia have three roles. First, spermatogenesis is initiated via spermatogonia. Second, the population of germ cells is greatly increased via the mitotic activity of spermatogonia. One spermatogonium on average goes through 8 to 9 divisions before differentiating into a spermatocyte (Lok and de Rooij, 1983b; Tegelenbosch and de Rooij, 1993). A spermatocyte carries out only 2 meiotic divisions; therefore, on average, ultimately 1 spermatogonium is capable of rendering 2048 or sometimes 4096 spermatozoa (Russell et al, 1990), depending on the number of divisions. Third, regulation of germ cell numbers is accomplished in the spermatogonial population of cells; regulation ensures that the appropriate ratio of germ cells to Sertoli cells is provided (de Rooij and Janssen, 1987; de Rooij and Lok, 1987). Spermatogonial Nomenclature Question: How does one define a spermatogonium? Answer: Spermatogonium is the term given to a relatively unspecialized diploid germ cell present in the seminiferous epithelium after the start of spermatogenesis that is able to carry out mitotic divisions, ultimately giving rise to primary spermatocytes. Question: Is there more than 1 type of spermatogonia? Answer: Yes, there are a number of spermatogonial types. Unfortunately, although the first assigned names were logical to those who conceived of them, the detection of more spermatogonial cell types has made the nomenclature rather confusing. The following questions should help clarify this. Question: How are spermatogonia named? Answer: In the beginning only 2 types of spermatogonia were described. The first, type A spermatogonium, did not display heterochromatin in the nucleus and the second, type B spermatogonium, did display heterochromatin (Figure 1). Shortly thereafter, someone found a type of spermatogonium that had an intermediate amount of heterochromatin, which was termed intermediate (In) spermatogonium. These names are historical, and they are broad categories of cells. When more generations of spermatogonia were discovered, especially among the type A category, new names were devised to categorize them. Question: It was the amount of heterochromatin that cells possessed that allowed them to be initially categorized?

2 de Rooij and Russell Spermatogonia 777 Figure 1. Broad categories of spermatogonia. Answer: Yes, it is a general rule in biology that the more differentiated a cell is in a particular lineage, the more heterochromatin one finds within the nucleus. We can say that type A spermatogonia are the most primitive, followed by type In spermatogonia, followed by type B spermatogonia (Figure 1A and B). Question: When species are compared, is the increase in the amount of chromatin in the nucleus at the various spermatogonial differentiation steps comparable? Answer: Comparisons are only useful within a species. One cannot necessarily expect the same amount of Figure 2. A type A spermatogonium from a mouse testis. heterochromatin in the same cell type of one species to match that of the same cell type of another species. Question: Which is the most primitive type A spermatogonium? Answer: The most primitive is the type A-single (formerly called A-stem cell or A-isolated) or, simply, A s or the stem cell spermatogonium (Figure 2). The cell pictured is a type A spermatogonium and, to the best of our knowledge, it resembles a stem cell. Question: Does a stem cell spermatogonium have all the characteristics of other stem cells of the organism? Answer: Yes, in that it initiates the development of cells that are committed to form terminally differentiated cells, and that it is able to give rise to more stem cells (Figure 3). Question: In what respect is it different? Answer: For all other proliferating systems, the stem cell is a functional definition, meaning it has the activities described earlier (differentiation and self-renewal). In mammalian testes, stem cell spermatogonia (A s ) are also morphologically defined in that they are the only type of spermatogonia without intercellular bridges. Question: What is an intercellular bridge? Answer: When a spermatogonium divides, telophase is incomplete, leaving an open area of cytoplasmic continuity called a cytoplasmic bridge (ie, a bridge ; Faw-

3 andr 21_621 Mp_778 File # 21em 778 Journal of Andrology November/December 2000 Figure 3. Two possible fates of a stem cell spermatogonium. Figure 5. Symmetrical versus asymmetrical divisions of stem cells. cett et al, 1959; Weber and Russell, 1987; Figure 4A and B). Question: What is the function of bridges? Answer: Bridges allow sharing of gene products between cells of a clone. If gene products in adjacent cells are shared, cellular activities become synchronized, resulting in a synchronized development (Lee et al, 1995; Braun et al, 1989). Because bridges are present in cells that are committed to form sperm we can say that single spermatogonia (without bridges) are stem cells. Question: Do we know if the stem cell divisions are symmetrical (dividing into similar cells) or asymmetrical (dividing into dissimilar cells)? Answer: We do not know that as yet. As depicted in Figure 5, when divisions are symmetrical the fate of the daughter cells could directly depend on a regulatory mechanism that decides whether or not a bridge is formed in a subsequent cell division. A stem cell then either divides to form 2 stem cells or to a pair (Apr), which is destined to follow the differentiation path. When divisions are asymmetrical, a stem cell divides into a stem cell and a single cell that at its first division will give rise to a pair. The other daughter cell produces 2 new stem cells. A suggestion has been made that asymmetrical divisions may occur (Huckins, 1971b) but this has not been confirmed (Lok et al, 1984). Until this is confirmed, we assume that there are symmetrical divisions only and that the only single type A spermatogonia are A s cells. Question: Now that we know the beginning cell in the whole process is the type A s spermatogonium, what are the other types of spermatogonia in order of developmental progression? Answer: The A s in most species give rise to pairs and Figure 4. (A) The intercullar bridge connects two spermatogonia. (B) Two type B spermatogonia connected by intercellular bridges.

4 de Rooij and Russell Spermatogonia 779 Figure 6. The so-called undifferentiated spermatogonia. then chains from 4 to 16 cells, called A pr and A al spermatogonia, respectively (Figure 6). The A s,a pr, and A al spermatogonia together have been called undifferentiated spermatogonia (Huckins, 1971b). Whereas the undifferentiated label makes writing about spermatogonia easier because they can be grouped under one label, the term has been confusing. Question: Why is the term undifferentiated confusing? Answer: The term undifferentiated was originally used to describe the morphological appearance of such cells. They show virtually no characteristics of any nuclear or cytoplasmic differentiation. Unfortunately, these cells differ little from subsequent generations of A spermatogonia that we call differentiating, which also show few differentiation characteristics. However, most importantly, A pr and A al spermatogonia in normal seminiferous epithelium are actually differentiating cells in the sense that they are irreversibly committed to take further developmental steps in the direction of spermatocytes. So, although they are called undifferentiated, they are functionally committed. Clearly, the terminology is confusing. Question: So should they be given a name that is less confusing? Answer: Let us agree to eliminate the word undifferentiated and call spermatogonia by their names, A s, A pr,a al, and so on. Question: If the term undifferentiated was once used, was the term differentiated spermatogonia also used? Answer: Yes, A al spermatogonia are able to differentiate into the first of 6 generations of so-called differentiating spermatogonia in mouse and rat, which are subsequently composed of A 1,A 2,A 3,A 4, In, and B spermatogonia. Again, the terminology, in this case differentiating, should be dropped. The progression from A s to B is demonstrated in Figure 7. Question: Why was the terminology inappropriate? Answer: Differentiating should apply to all spermatogonia that are more advanced than stem cells. Because it has been inappropriately named, we should discard the term. Question: What do advanced spermatogonia look like? Answer: Photographs of these cells taken from whole mounts are provided in Figure 8A through L. When looking at these photos, remember that only the nuclei stain, so the bridges that link cells generally cannot be seen, although we have one example of a whole mount in which bridges are visible (Figure 8D). Question: It seems like the A 1 through A 4 spermatogonial cells appear similar. How does one tell them apart? Answer: You are correct, one cannot tell the individual cell types apart. Question: Then if we can t tell them apart, how do we know which is a type A 1,A 2,A 3,orA 4 spermatogonium? Answer: A 1-4 s can be distinguished from each other only by determining in which stage of the cycle of the seminiferous epithelium they are present (Oakberg, 1956; Clermont, 1962). For example, when the area of the rat whole mount is in stage X, A 2 spermatogonia will be present. Although the great majority of A spermatogonia in Stage X will be A 2, some A s,a pr, and A al spermatogonia (which are present throughout the entire epithelial cycle) will also be present. There should be more than 8 16 cells in a clone for cells to be classified as A 2 cells, whereas a maximum of 16 cells would be present in A al. It has been shown that the bridges can be seen in specially prepared whole mounts (Huckins, 1978b), but it is difficult to visualize bridges. In sectioned material, the various types of A spermatogonia cannot be readily distinguished from each other because the intercellular bridges are not always in the plane of section and, as well, the cells that comprise a chain will generally not all be in the same section. Question: Does a division take place from A al to A 1? Answer: No! It is simply a transformation that takes place in cells while they are in G 0 /G 1 phase of the cell cycle, which requires no division (Figure 9). Question: According to a count in Figure 9, there are Figure 7. Progression from A s spermatogonia to B spermatogonia.

5 andr 21_621 Mp_780 File # 21em 780 Journal of Andrology November/December 2000 Figure 8. Photographs of the cells lying on the basal membrane of mouse seminiferous tubules. A s,a pr, and A al spermatogonia can be distinguished from A 1 -A 4 spermatogonia because they do not go through the cell cycle synchronously with the latter cells. Whole mounts of seminiferous tubules are stained with hematoxylin and only the nuclei of the cells can be discerned. (A) A s spermatogonium presumably in G1 phase of the cell cycle (arrow) among A 1 spermatogonia (arrowheads) synchronously in G2 phase just before division into A 2 spermatogonia. Some Sertoli cells (asterisks) and (leptotene) spermatocytes (small arrowheads) are also indicated. 9 or 10 spermatogonial divisions in the rat before spermatocytes are formed. Although there are usually 9 divisions, as many as 16 A al cells have been seen at one time (Huckins, 1978a), indicating that sometimes there are 10 spermatogonial divisions. Answer: Good, you are doing quite well! Question: If we compare cell generations of spermatogonia, which is more constant in terms of what actually happens: that there are more or less 9 to 10 generations, or the uniqueness in the morphology of the nucleus? Answer: We rely on the number of generations and the number of cells connected by bridges rather than the morphology. Figure 8C. A chain of 8 A al spermatogonia, 7 of which are in the photograph (indicated by in-between dashes) among telophasic A 3 spermatogonia (some indicated by arrowheads). Magnification 660. Bar 10 m. Question: How many stem cells are there in a rodent testis? Answer: A mouse testis contains about A s spermatogonia (Tegelenbosch and de Rooij, 1993). In a rat testis weighing about 10 times more, one can speculate that there will be about stem cells per rat testis. Question: How does one determine the number of stem cells? Answer: In whole mounts (see later discussion), by carrying out cell counts, we can determine the ratio between the numbers of A s cells and Sertoli cells. In sections, one can determine the number of Sertoli cells per testis using morphometric methods (eg, the disector method [Sterio, 1984; Russell et al, 1990; Tegelenbosch and de Rooij, 1993]). From there we can use the ratio (Sertoli/ type A s ) to estimate the number of stem cells per testis. Figure 8B. A pr spermatogonia (arrows) among A 3 spermatogonia (arrowheads) in G2 phase. Magnification 660. Bar 10 m. Figure 8D. A pr spermatogonia, the focus of which is on the intercellular bridge, which happens to be visible, among A 1 spermatogonia in G2. Magnification 660. Bar 10 m.

6 de Rooij and Russell Spermatogonia 781 Figure 8G. A 1 spermatogonia (arrowheads). Magnification Bar 10 m. Figure 8E. A chain of 4 A 1 spermatogonia in prophase of mitosis into a chain of 8 (indicated by in-between dashes) among A 4 spermatogonia in G1 phase (some indicated by arrowheads). Magnification 660. Bar 10 m. Spermatogonial Predecessors Question: What cells give rise to spermatogonia? Answer: The lineage leading to spermatogonia is relatively straightforward (Figure 10) but may appear confusing because there are alternative terminologies for precursor cells. The first cell in the lineage is the primordial germ cell (PGC), which in turn, derives from epiblast cells (embryonal ectoderm; Lawson and Pederson, 1992). Later in development, PGCs migrate from the base of the allantois along the hindgut to finally reach the genital ridges at about embryonic day 11.5 in rats and embryonic day 10.5 in mice. They proliferate during migration and after migrating to the gonadal ridge, primordial germ cells become gonocytes as they become enclosed within cords formed by Sertoli precursor cells and are surrounded by peritubular cells (Clermont and Perey, 1957a; Sapsford, 1962). They show a burst of mitotic activity followed by an arrest in the G 0 phase of the cell cycle, and then gonocytes remain mitotically quiescent until after birth, when they give rise to spermatogonia. The terms prospermatogonia (various types) (Hilscher et al, 1974) and prespermatogonia (Byskov, 1986) are other names given to gonocytes. Question: During pubertal development, do gonocytes give rise to A s cells? Answer: How gonocytes divide to form various spermatogonial cell types is still unclear. We know the following: 1) stem cells by definition must be present; 2) bridges that connect some gonocytes have been described (Zamboni and Merchant, 1973); and 3) as discussed later, the kinetics of spermatogenesis at the start of spermatogenesis suggest that gonocytes, aside from giving rise to stem cells, also give rise to A 2 spermatogonia (de Rooij, 1998). Thus, whereas at least some gonocytes give rise to A s spermatogonia at the start of spermatogenesis, other gonocytes give rise to more differentiated types of spermatogonia, some behave like A 1 cells and give rise to A 2 spermatogonia (Figure 11). The latter observation implies that just like A al spermatogonia do in the course of normal spermatogenesis, some gonocytes differentiate into A 1 spermatogonia before division and, after their first division, become A 2 cells. They may not skip several divisions because they are already in clones with intercellular bridges, which suggests some differentiation has already taken place. Question: Are A pr and A al spermatogonia present during the first wave of spermatogenesis? Answer: We do not know whether A pr and A al spermatogonia are also formed directly at the start of spermatogenesis. Figure 8F. An apoptotic clone of A 2 spermatogonia (asterisks) among viable A 2 s (some indicated by arrowheads). Magnification 660. Bar 10 m. Figure 8H. A 2 spermatogonia (arrowheads). Magnification Bar 10 m.

7 andr 21_621 Mp_782 File # 21em 782 Journal of Andrology November/December 2000 Figure 8I. A 3 spermatogonia (arrowheads). Magnification Bar 10 m. Figure 8K. In spermatogonia (arrowheads). Magnification Bar 10 m. Question: It would seem that if gonocytes have the dual responsibility of producing A s, and possibly also A pr and A al spermatogonia and dividing to form A 2 cells, that the yield of spermatogenesis is low during pubertal development. Answer: The first wave is less efficient, possibly for this reason, but surely also because many cells degenerate early in spermatogenic development (Kluin et al, 1982; Russell et al, 1987). Question: When do the first A 2 spermatogonia appear in various rodent species? It appears that this would be important to know because the division of gonocytes to form the first A 2 spermatogonia marks the beginning of spermatogenesis, right? Answer: Yes, the appearance of A 2 spermatogonia is the signal that spermatogenesis has started. The data for the beginning of spermatogenesis in several species/ strains is summarized in Table 1. Question: There is something strange with these data. In the rat, spermatogenesis starts at day 5 and spermatogenesis is complete at day 43. That means that 4 epithelial cycles (from A 2 to spermiation) took place within 38 days, while 1 epithelial cycle takes 12.8 to 12.9 days in the rat. One would expect spermatogenesis not to be complete before day 56. Apparently, in the developing rat, each epithelial cycle lasted only 9.5 days on average instead of 12.8 to 12.9 days. Answer: The epithelial cycle in young animals is much shorter than in adults (Kluin et al, 1982; van Haaster and de Rooij, 1993b). In rats, the epithelial cycle takes about 5 days during the first weeks of life (van Haaster and de Rooij, 1993b) versus 12.8 in adults (Hilscher et al, 1969); in Chinese hamsters, the comparable figures are 9 days (van Haaster and de Rooij, 1993b) versus 17 days in adults (Oud and de Rooij, 1977). Spermatogonial cell cycle times and spermatocyte development are faster in prepubertal animals. It is not known what slows down the process to the adult rate. The switch to a slower spermatogenic process correlates with testicular descent (and a lower testicular temperature), the appearance of stage VII pachytene spermatocytes, and the start of the formation of a tubular lumen (van Haaster and de Rooij, 1993b). Question: A correlation between testicular temperature and the rate of spermatogenesis seems perhaps the most plausible cause for differing rates of spermatogenesis. Is there any evidence for such a correlation? Answer: Yes, one paper has indicated a role for testis temperature in modifying the rate of adult mouse spermatogenesis (Meistrich et al, 1973); however, more studies will be necessary to extend this to the prepubertal situation. Question: Do some PGCs fail to become incorporated into cords and, if so, what happens to PGCs that do not become incorporated into cords? Answer: This has been studied by Byskov (1978; 1986) and it was found that such cells, when they occur, begin the meiotic process. One of us (L.D.R.) has found a condition in a desert hedgehog mouse knockout in which gonocytes residing outside of cords do not enter meiosis (unpublished). Question: If gonocytes do enter meiosis, wouldn t that be like the normal situation in the formation of the ovary in which gonocytes normally never enter cords? Answer: Yes (Byskov, 1986). Figure 8J. A 4 spermatogonia (arrowheads). Magnification Bar 10 m. Figure 8L. B spermatogonia (arrowheads). Magnification Bar 10 m.

8 de Rooij and Russell Spermatogonia 783 Figure 9. Progress of spermatogonia from A s to B. Spermatogonial Kinetics Question: The preceding section described the types of spermatogonia. Can we presume the arrows in Figure 9 imply the pattern of kinetics? Answer: Yes, most investigators in the field now agree on a single stem cell (A s ) theory, which was originally put forward by Huckins (1971b) and Oakberg (1971). Question: Can this theory be simplified? Answer: Yes, A s cells divide to either renew A s cells or to become A pr cells. A pr cells divide to form A al cells that reach clonal sizes of 4, 8, or 16 cells. A s,a pr, and A al spermatogonia divide at random during the cycle of the seminiferous epithelium, but most actively during rat stages X II and only occasionally in stages III IX. During the proliferative activity of these cells the numbers of A s and A pr remain about constant but many A al are formed. At about stages VII VIII nearly all of the A al that were formed transform into A 1 cells (no mitosis), which resume the cell cycle. A 1 cells divide to form A 2 cells (in stage IX); these divide to form A 3 cells (in stage XI in mouse, stage XII in rat) and finally, a division occurs to form A 4 cells (in stage I). A 4 cells divide to form In type spermatogonia (in Stage II) and, in turn, In spermatogonia divide to form type B spermatogonia (in Stage IV). The last spermatogonial division forms preleptotene spermatocytes (in Stage VI). Question: I thought you were trying to make this a simple explanation? Answer: The diagram in Figure 12 simplifies what has been proposed for rats and mice (Huckins, 1971b; Oakberg, 1971). Question: If divisions that lead to A al cells are random during the cycle of the seminiferous epithelium, there must be a temporary arrest for some of them prior to their stage-related commitment to form sperm. Right? Answer: Right, although the relation between cell cycle arrest of A al spermatogonia and their transformation into A 1 spermatogonia is unclear. On the one hand, the arrest of A al spermatogonia can be prolonged in vitamin A-deficient rodents when vitamin A deficiency is induced (van Pelt and de Rooij, 1990a; van Pelt et al, 1995). Cells are arrested in G 1 /G 0 phase of the cell cycle in all tubules and do not progress beyond the A al to A 1 transition; thus, all tubules will contain large numbers of A al cells. Then, when retinoic acid (vitamin A) is administered, all spermatogonia in all tubules go forward at the same time, which leads to a more or less synchronized testis in terms of germ cell development (Morales and Griswold, 1987; van Pelt and de Rooij, 1990b). On the other hand, the arrest can be shortened or may not occur when no In or B spermatogonia are present. A kind of a feedback regulatory mechanism exists due to which the proliferation of A s,a pr, and A al spermatogonia is prolonged when insufficient numbers of A 1 spermatogonia are locally produced during the preceding cycle (de Rooij et al, 1985). Question: What is the cell cycle time of spermatogonial generations? Answer: The cell cycle duration of A 2 through B Figure 10. Predecessors of A s spermatogonia.

9 andr 21_621 Mp_784 File # 21em 784 Journal of Andrology November/December 2000 Figure 12. Some divisions occur anytime during the spermatogenic cycle (random) and others occur at specific stages. Figure 11. Gonocytes apparently have two possible division patterns. spermatogonia has been estimated to be 28.5 hours (Monesi, 1962) in mice, 42 hours in rats (Hilscher et al, 1969; Huckins, 1971a), and 60 hours in Chinese hamsters (Lok and de Rooij, 1983a). For A s,a pr, and A al spermatogonia, cell cycle times have been found to be 56 hours in rats (Huckins, 1971c) and about 90 hours in Chinese hamsters (Lok et al, 1983). Question: Most people believe the scheme originally proposed by Huckins (1971b) and Oakberg (1971); are there other schemes? Answer: Yes, another major stem cell renewal scheme has been advanced by Clermont and Bustos-Obregon (1968), Dym and Clermont (1970), Clermont and Hermo (1975), and Bartmanska and Clermont (1983). In fact, this was the first theory advanced. Question: Can we examine and compare both schemes? Answer: Certainly, it is only through doing so that readers can make up their minds about which scheme is correct and only through knowing both schemes can one or the other scheme be modified or confirmed. Question: What is the name of the other stem cell renewal scheme? Table 1. The age at which spermatogenesis starts and at which it is complete, in various species and strains of rodents (day of birth was taken to be day 1) Species Cpb-N mice CBA Mouse strain not specified Wistar rat Djungarian hamster Chinese hamster Age of Start Day of birth Day of birth Day 3 Day 5 Day 5 Day 9 Age of Completion Day 32 Day 35 Unknown Day 43 Day 32 Day 63 References Kluin et al, 1982 Vergouwen et al, 1991, 1993 Sapsford, 1962 van Haaster and de Rooij, 1993 van Haaster et al, 1993 van Haaster and de Rooij, 1993 Answer: It is the A 0 /A 1 theory. It has also been called the reserve stem cell theory or A 0 theory. Question: What is a reserve stem cell? Answer: It is proposed to be a reserve stem cell, which is normally a quiescent cell, and only divides when needed; thus, it has an indefinite or very slow cell cycle. This has been proposed by Clermont and Bustos-Obregon (1968), Dym and Clermont (1970), Clermont and Hermo (1975), and Bartmanska and Clermont (1983). Question: If A 0 cells are normally nonproliferative, then how do spermatogonia renew according to the A 0 / A 1 scheme? Answer: The idea is that a small number of A 4 cells divide to form A 1 cells, whereas most of the A 4 cells go forward to form Intermediate-type spermatogonia. Question: A 0 cells are around to respond to emergency situations when there is a problem with the A 4 to A 1 transition; for example, after irradiation? On a routine basis, A 1 cells are produced from A 4 cells? Answer: Yes, that is correct (Figure 13). Question: What is the major evidence for each of the schemes? Can we start by reviewing the Huckins-Oakberg scheme (A s )? Answer: In whole mounts of seminiferous tubules (see later discussion of techniques), thanks to the organization provided by the spermatogenic cycle, one can follow the subsequent germ cell stages of the cycle along the length of the tubule. Consequently, one can, for example, see A 1 spermatogonia in G 2 phase; somewhat further on they can be seen in mitosis and, after that, A 2 spermatogonia are present but are small because they are in G 1 phase. Then these A 2 s become larger as they enter S phase, G 2 phase, and then they divide into A 3, etc sort of a logical progression. Spermatogenesis is a highly synchronized process and therefore all spermatogonia in Figure 13. The A 0 /A 1 scheme of spermatogonial renewal.

10 de Rooij and Russell Spermatogonia a particular clone will synchronously enter mitosis and even adjacent clones are fairly synchronous. Huckins (1971b) observed other A spermatogonia topographically arranged as singles, pairs, and chains of up to 16 cells that did not follow the synchrony of the A 1 -B spermatogonia, whose chain size is much larger. Furthermore, in stage IX (rat) these stage-asynchronized spermatogonia were few in number while many more were present in stage II. Cell kinetic analysis of the spermatogonial cell types revealed that the asynchronous A spermatogonia have clearly different cell cycle times from the synchronous ones (see earlier; Huckins, 1971a, 1971c). In mouse testis sections, scrutiny of small morphological differences brought Oakberg to the conclusion that mice also possess a population of A spermatogonia that do not follow the general synchronous developmental pattern of A 1 -B spermatogonia (Oakberg, 1971). Huckins and Oakberg then jointly proposed the scheme of spermatogonial multiplication and stem cell renewal, which was outlined earlier (Huckins, 1971b; Oakberg, 1971). Later on this hypothesis was supported and extended by tubular whole mount studies in mice (de Rooij, 1973) and Chinese hamsters (Lok et al, 1982, 1983; Lok and de Rooij, 1983a, 1983b). In essence, the Huckins-Oakberg theory says that the spermatogonia that give rise to A 1 divide irregularly during the epithelial cycle and are not population-derived from the A 1 or their progeny, the latter dividing in a stagerelated manner. Question: What is the major evidence for the Clermont scheme (A 0 /A 1 theory)? Answer: Clermont and Leblond were pioneers in establishing the cycle of the seminiferous epithelium and its stages using spermatid development as a guide to identify stages (Leblond and Clermont, 1952; Clermont and Perey, 1957b). Spermatogonial multiplication and stem cell renewal were studied by counting cell numbers and mitotic figures in the various epithelial stages. A scheme evolved in which A 1 -A 4, In, and B spermatogonia were distinguished and in which the last generation of type A spermatogonia (A 4 ) gave rise to both A 1 and In spermatogonia (Clermont, 1962), based on the idea that there are no principle differences between the various generations of A spermatogonia. Clermont and Bustos-Obregon (1968) introduced the seminiferous tubular whole-mount technique to study spermatogonial kinetics. Additional A spermatogonia were detected, topographically arranged as singles or pairs. These were present throughout the cycle of the seminiferous epithelium, did not change in numbers much, and were only rarely seen to divide (Clermont and Bustos-Obregon, 1968; Clermont and Hermo, 1975). Accordingly, and also because results of studies after irradiation suggested that these cells were involved in the repopulation of the seminiferous epithelium (Dym and 785 Clermont, 1970), the single or paired cells were termed reserve stem cells or A 0 spermatogonia. The finding that the numbers of the singles and pairs are relatively constant during the epithelial cycle was confirmed in rats (Huckins, 1971b), mice (de Rooij, 1973), and Chinese hamsters (Lok et al, 1982). Question: Can you summarize the main differences between the 2 schemes? Answer: The difference of opinion about the scheme of spermatogonial proliferation and stem cell renewal originates from different results with respect to the proliferative activity of the A s and A pr spermatogonia (A 0 in Clermont s scheme) and a different idea about the nature of the asynchronous chains of A spermatogonia (A al according to Huckins and A 1 -A 4 according to Clermont). Question: So where do you have a problem with the Clermont scheme? Answer: Although Clermont and coworkers did not find much proliferative activity of the singles and pairs of A spermatogonia, Huckins (1971c) and Lok and De Rooij (1983b) did find appreciable 3 H-thymidine incorporation by these cells, indicating active proliferation. It is difficult to reconcile this difference in results with respect to the proliferative activity of A s and A pr spermatogonia. Furthermore, the Clermont group did not distinguish chains of A spermatogonia that we call A al spermatogonia. Chains of asynchronous A spermatogonia were observed but were considered to be delayed or quick A 1 -A 4 spermatogonia, depending on the epithelial stage in which they were encountered. However, in cell kinetic studies in which the labeled mitoses technique was used to study cell cycle times of spermatogonial cell types, it was found that the asynchronous chains of A spermatogonia have a cell cycle time similar to that of A s and A pr spermatogonia (Huckins, 1971c; Lok et al, 1983) and not similar to A 1 to A 4 spermatogonia. Furthermore, because there was a clear second peak in the labeled mitoses curve of the chains of asynchronous A spermatogonia, it could be concluded that these cells consistently have a cell cycle time that is different from that of A 1 -A 4 spermatogonia. Thus, the widely different cell kinetic properties of A al spermatogonia versus A 1 -A 4 clearly indicate that these are 2 different cell populations. Question: This is a huge amount of information to digest. Would you help by simplifying this more? Answer: OK, here is a summary for simplicity. Essential Features of the Two Major Spermatogonial Renewal Schemes A 0 /A 1 Theory A 4 spermatogonia normally give rise to A 1 spermatogonia as well as In spermatogonia. A 0 sper-

11 andr 21_621 Mp_786 File # 21em 786 Journal of Andrology November/December 2000 matogonia divide to form A 1 spermatogonia when spermatogenesis is in need of more cells. A s Stem Cell Theory Only A s cells act as stem cells, they give rise to committed cells that divide irregularly (A pr and A al ) during the spermatogenic cycle and these, in turn, give rise to A 1 spermatogonia. Major Historical Difference in the A s and the A 0 /A 1 Theories In the A s theory, A s,a pr, and A al cells are a separate population of cells that are different from A 1 to A 4 cells. In the A 0 /A 1 theory, A s and A pr cells are considered reserve stem cells and A al are considered A 1 -A 4 cells, and are no different from each other. Question: There are other actively proliferating tissues in the body such as the hemopoetic system, the gastrointestinal tract, and skin. What kind of stem cell renewal do these tissues possess? Are their stem cell renewal schemes more like the A s model or the A 0 model? Answer: The Huckins and Oakberg scheme (A s theory) is similar to other cell-renewing systems in that stem cells divide the least frequently (Lajtha, 1979). It can be argued that this is advantageous because there is only a small chance that stem cells will be damaged by inaccurate DNA duplication. In the Clermont scheme stem cells theoretically divide 4 times each epithelial cycle; in the Huckins-Oakberg scheme, it has been estimated that in mice and Chinese hamsters A s divide 2 to 3 times (Lok and de Rooij, 1983b; Tegelenbosch and de Rooij, 1993). Question: Is there a proposed dedifferentiation system from A 4 to A 1 spermatogonia in the Clermont system? Answer: The Clermont scheme supposes that A 1 -A 4 spermatogonia are at a similar phase of differentiation and that no differentiation takes place; thus, no dedifferentiation is required. At present, no molecular markers have been described that indicate differentiation in A 1 to A 4 spermatogonia. Question: Although no molecular markers have been found, are not intercellular bridges an indication of differentiation? Answer: Some would say so because A s cells do not have bridges. Someday we will know the answer to this question. Question: According to Clermont s theory, A 4 spermatogonia would have to break their bridges to form A 1 cells. By counting the number of cells in bridges, are there examples when this might have occurred? Answer: The point is not easy to explain within the Clermont scheme. It could be established in Chinese hamsters that the A 1 spermatogonia in stage VII are composed of chains of 4, 8, and 16 cells only (Lok et al, 1982) and that chains virtually never posses odd numbers of cells. After division this would render clones of 8 32 A 2 spermatogonia and ultimately A 4 spermatogonia. If the A 0 /A 1 theory is correct, clones of 2, 4, and 8 cells would then have to be pinched off from clones of A 4 spermatogonia to divide into new clones of 4 16 A 1 spermatogonia. One cannot say that this does not occur or is impossible, but it does not seem to be a very likely course of events. Question: Why is there not universal acceptance of the Huckins-Oakberg theory? Answer: It is difficult for most people to critically examine the literature because it requires a detailed knowledge of staging as well as an examination of numerous tables of cell counts at various stages. Most people are unwilling to put in the effort. Moreover, as long as a few people keep advocating the A 0 /A 1 theory, there will always be controversy. Question: I will ask more about a theory of density dependent regulation later, but there is an important point to be brought up now. In this theory, which indicates that the density of advanced germ cells is regulated by spermatogonial apoptosis, you say that the numbers of cells entering meiosis is controlled by apoptotic events, primarily in A 2 through A 4 spermatogonia. If Clermont s scheme is correct would it make good biological sense to eliminate cells prior to stem cell renewal rather than after renewal? Answer: As discussed earlier, cells in every stem-cell system divide as little as possible (Lajtha, 1979). Density regulation among potential stem cells would mean that spermatogonial stem cells divide more than strictly necessary. In this way one would enhance the chance of mutations in the kind of cells one should protect the most. Logically, stem cell renewal should come before elimination of cells as it does in other systems of the body as proposed in the A s theory. Question: I understand that intercellular bridges are not sacred in terms of designating stem cells because they have been described in gonocytes, right? Answer: All gonocytes may not be alike; some may not be stem cells. As discussed earlier, at least some gonocytes give rise to A 1 cells and thus gonocytes may be comparable to the population of A s,a pr, and A al spermatogonia, as a whole, with respect to the occurrence of intercellular bridges. It may well be that only single gonocytes after the start of spermatogenesis will give rise to A s spermatogonia. Do not give up as yet about the sacredness of intercellular bridges in this respect. Future research will surely enlighten us. Techniques to Study Spermatogonia Question: Because spermatogonia are the rarest cell type within the epithelium and are the most difficult to

12 de Rooij and Russell Spermatogonia Figure 14. Spermatogonia as seen using conventionally sectioned tubules and using whole mounted tubules. In the latter the microscope focuses on a plane just under the coverslip. identify in the testis, must it take a special technique to identify them? Answer: Yes, whole mounts of seminiferous tubules have traditionally been the most useful techniques (Clermont and Bustos-Obregon, 1968). Question: How is a whole mount prepared? Answer: Tubule segments of up to several centimeters in length are isolated by teasing apart rodent testis tissue in a Petri dish. The tubules are stained with hematoxylin (a nuclear stain), partially flattened by the pressure of a cover slip, and examined by a microscope that is focused only on the plane of cells on the basal lamina (Clermont and Bustos-Obregon, 1968; Meistrich and van Beek, 1993; Figure 14). Question: How does one identify the types of spermatogonia? Answer: The criteria are as follows: A s : No other similar A spermatogonia are within 25 m of these cells. A pr : Only 2 spermatogonia of the same nuclear morphology are closer than 25 m to each other. A al : More than 2 A spermatogonia can be construed as branched or straight chains of the same morphology with no intervening space of more than 25 m between member nuclei. The chains are generally no greater than 16 cells. Question: What if an A s cell is near, for example, a chain of A 3 spermatogonia? Does that mean that an A s cell could be misidentified? Answer: One could misidentify the A s cell. The easiest way to identify A s,a pr, and A al spermatogonia is to look for them in areas in which A 1 -A 4 spermatogonia are in late G 2 phase or in mitosis. A s -A al spermatogonia then stand out because they are not synchronized in the cell cycle with A 1 -A 4 spermatogonia and their nuclei will appear different. In areas in which A 1 -A 4 spermatogonia are in G 1 or S phase, the differences between these cells and A s -A al are too small to allow identification in each instance and there will be too many doubtful cases to allow reliable counting. Examples appear in Figure 8A through E. 787 Question: With your staining technique, you don t see the intercellular bridges? Answer: No, we generally don t. Figure 8D is an exception; we presume intercellular bridges are present because the cells have the same morphology and, presumably, are all in the same phase of the cell cycle. We can imagine the chain of cells by the pattern of cells and the expected distance between cells. Question: What other techniques do you use to study spermatogonia? Answer: We utilize germ cell-depletion techniques, irradiation, or vitamin A deficiency. Question: How does irradiation affect the testis? Answer: Irradiation kills the proliferating cells (ie, spermatogonia). A 1 through A 4 spermatogonia are the most radiosensitive, followed by A pr and A al spermatogonia. A s are the most radioresistant (van der Meer et al, 1992a, 1992b). Enough irradiation will deplete the entire population of spermatogonia. Spermatocytes and spermatids are much more radioresistant and, after irradiation, develop in an apparently normal way and ultimately leave the testis as spermatozoa. If stem cells are able to survive, they will usually repopulate the seminiferous epithelium (Dym and Clermont, 1970; van den Aardweg et al, 1982), but are found to first replenish their own numbers before again producing differentiating cells (van Beek et al, 1990). Question: What use can be made of vitamin A-deficient testes? Answer: As described earlier, the seminiferous epithelium in a vitamin A-deficient testis contains only A s - A al spermatogonia. Because of this, these testes can be used to purify these cells (van Pelt et al, 1996). Furthermore, because after replacement of vitamin A, virtually all A al spermatogonia throughout the testis synchronously differentiate into A 1 spermatogonia and then start their series of divisions, spermatogenesis in these animals becomes synchronized. Hence, in the testes of vitamin A- deficient rats (Morales and Griswold, 1987; Griswold et al, 1989; van Pelt and de Rooij, 1990a; van Beek and Meistrich, 1991) and mice (van Pelt and de Rooij, 1990b), after replacement of vitamin A, only a few epithelial stages can be seen. Which stages are present depends on the duration of vitamin A replacement and can be calculated from the duration of the epithelial cycle and its stages. These testes can be used to study epithelial stage-dependent processes (Klaij et al 1994). Question: How does one determine if particular spermatogonia have divided or are going to divide? Answer: There are two ways to determine this. One is to examine the epithelial stage and look for mitotic figures. We have also relied heavily on the use of 3 H

13 andr 21_621 Mp_788 File # 21em 788 Journal of Andrology November/December 2000 thymidine or bromodeoxyuridine labeling. Both substances become incorporated into DNA during the S-phase of the cell cycle and may be given as a pulse (Lok and de Rooij, 1983a, 1983b; Lok et al, 1983; van de Kant et al, 1988, 1990; van Pelt et al, 1995), as a series of injections, or continuously (Lok et al, 1984). Question: Has anyone cultured spermatogonia? Answer: Various culture systems for spermatogonia of various species have been described. In a Sertoli cell germ cell coculture from neonatal mouse germ cells, no apparent loss of viability was found after 3 days of culture (Maekawa and Nishimune, 1991). Nagano et al (1998) were able to keep germ cells, isolated from adult mice, alive for more than 4 months in serum containing medium. No attempt was made to quantify the number of viable cells. It is interesting that cells that had been cultured on feeder layers were able to colonize busulphan-emptied recipient testes after spermatogonial stem cell transplantation. Because this was not the case for germ cells cultured without a feeder layer, this suggests that a feeder layer is required for the survival of spermatogonial stem cells. Recently, 50% of cultured, purified spermatogonia from 80-day-old boars were shown to be viable when the cells were cultured in a potassium-rich medium, called KSOM, after 3 days (Dirami et al, 1999). However, in general, a system for the long-term culture of purified spermatogonia is still lacking. Long-term survival of spermatogonial stem cells on a feeder layer has been shown, but this system is still poorly defined in terms of efficiency. Question: Has anyone frozen spermatogonia? Answer: Yes. Brinster s laboratory has now reported spermatogonial stem cell freezing for up to 6 months (Avarbock et al, 1996; Clouthier et al, 1996; Ogawa et al, 1999) and now has done so for 2 years (personal communication). However, no estimate was made of the efficiency of storing spermatogonial stem cells by way of cryopreservation. Question: Has anyone purified spermatogonia? Answer: Yes, several techniques have been published. First, to isolate germ cells, a testis has to be decapsulated, mechanically minced into small pieces, and then enzymatically dissociated (Meistrich et al, 1973; Barcellona and Meistrich, 1977; van Pelt et al, 1996). Various procedures have been developed to further purify spermatogonia from human and rodent testis cell suspensions, which involve sedimentation velocity (Meistrich and Eng, 1972; Bellvé et al, 1977), equilibrium density centrifugation in Percoll gradients (Meistrich and Trostle, 1975), and centrifugal elutriation (Grabske et al, 1975). However, the efficiency of the purification of spermatogonia not only depends on the isolation technique but also Figure 15. Type A spermatogonia isolated and cultured from 9-day-old rat testes using the STAPUT procedure (Dym et al, 1995). The cells display large, spherical nuclei with a thin rim of cytoplasm and perinuclear organelles. The morphology of the cultured spermatogonia is very similar to that seen in vivo. on the relative abundance of these cells in the testis and, consequently, in the testis cell suspension. Starting from young mice, Bellvé et al (1977) succeeded in obtaining greater than 90% pure A spermatogonia and 76% pure B spermatogonia. About 85% pure A spermatogonia could be prepared from young rat testes (Morena et al, 1996). Recently, Dirami et al (1999) described the purification of 95% 98% pure A spermatogonia from 80-day-old pig testes using sedimentation velocity and differential adhesion to eliminate contaminating Sertoli cells, which adhere to culture flasks. Figure 15 shows spermatogonia after they have been isolated. Question: Can the genetic information be modified in stem cell spermatogonia? Answer: There have been numerous claims that the genetics of a small number of spermatogonia can be modified through various manipulations such as electric shock of the testis after introduction of a vector. In a very recent paper from Brinster s laboratory, spermatogonia were isolated, cultured, and transfected with a retrovirus and then transplanted with resulting stable integration of the viral vector (Nagano et al, 2000). Regulation of Spermatogonial Number Question: Can one simply determine what the eventual yield of sperm will be on the basis of the kinetics of spermatogonia, spermatocytes, and spermatids? Answer: It is not that simple. As it turns out, the germ cell population can be regulated. To determine yield of sperm, one must also consider cell degeneration during spermatogenesis. Although there are several sites for possible regulation during spermatogenesis, the regulation at the spermatogonial level appears to have the greatest impact on the eventual yield of cell numbers.

14 de Rooij and Russell Spermatogonia Question: Where does the regulation of spermatogonia take place? Answer: Studies have been performed to examine the cell density of A 1 and other types of spermatogonia and preleptotene spermatocytes in Chinese hamsters (de Rooij and Janssen, 1987; de Rooij and Lok, 1987). Large differences were found in the density of A 1 spermatogonia in different areas of the seminiferous tubular basal membrane. Apparently, there is no mechanism that regulates the proliferation of A s -A al spermatogonia in such a way that A 1 spermatogonia are evenly distributed. However, the density of preleptotene spermatocytes is the same everywhere. Hence, density regulation must take place somewhere in the process from A 2 to B spermatogonia. Question: So, where is the site of regulation under normal conditions? Answer: Regulation under normal conditions takes place among A 2,A 3, and A 4 spermatogonia. Question: What appears to be the raison de être for density-dependent regulation? Answer: The basis is the density of cells; thus, the theory is called density-dependent regulation. The greater the density of A 2 though A 4 spermatogonia in a particular region of the tubule, the greater the amount of cell degeneration is necessary to down-regulate the density to a normal level. Question: Could you tell me more? Answer: It works like other systems of the body (Potten et al, 1997; Conton and Raff, 1999). At first, certain cells are overproduced and are later reduced to physiological numbers by the apoptotic process (Figure 8F). If all the cells produced as spermatogonia were to become sperm, there would be a twofold to fivefold increase in sperm production in the mouse (de Rooij and Lok, 1987). Increasing sperm production sounds nice, but the supporting cells of the testis could not handle such a load. Indeed, this has been found to be the case in some transgenic mice in which spermatogonial apoptosis was inhibited (Knudson et al, 1995; Furuchi et al, 1996; Rodriguez et al, 1997). There was an accumulation in these mice of spermatogonia and early spermatocytes that eventually all entered apoptosis, apparently by way of a backup apoptotic pathway. Question: What would be the mechanism for densitydependent regulation? Answer: We must assume that a mechanism exists that senses too many spermatogonia of more advanced types in a particular area, and which triggers the apoptotic mechanism. Such a mechanism could reside in the Sertoli cells but also in spermatogonia themselves. Possibly, when the clones consisting of at least 16 A 2 to up to 128 A 4 get in each other s way trying to carry out divisions, 789 apoptosis may be induced. This area will need further study. Question: Do cells apoptose individually or as a clone? Answer: Huckins (1978a) found that the entire clone is always lost during density-dependent regulation. In normal epithelium, clones of even-numbered cells are found exclusively. Odd-numbered clones are found under experimental or adverse conditions (van Beek et al, 1984; de Rooij et al, 1999), but this does not necessarily imply single-cell death because there may be forces under abnormal conditions that cause a single cell to separate from a chain. Question: When all is said and done, how finely tuned is the spermatogenic process? Answer: Reviewing the ratio of elongated spermatids per Sertoli cell reveals that for many of the species studied, there is about 20% 40% difference in the number of germ cells each Sertoli cell supports, and the number of spermatids that can be supported by a single Sertoli cell is characteristic of the species (Russell and Peterson, 1984). Question: Why would a biological system be so wasteful? Answer: Stem cells are rare and it may be difficult to regulate their proliferative activity in such a way that they constantly produce a particular number of differentiating cells in all areas. When stem cell renewal and differentiation depends on stochastic events that gives them a 50% chance of self-renewal or differentiation in normal epithelium, there will always be areas in which by chance too many stem cells carried out self-renewal or differentiation, and lead to the production of too few differentiating cells in the same or the next epithelial cycle, respectively. Therefore, it may be better to keep on the safe side and produce so many cells that in all cases, sufficient differentiating cells are produced. In addition, such a system will be better able to cope with situations in which cells are lost by damage or insult to the testis. For example, a toxic insult may result in increased cell loss, but because apoptosis can be cut back, sufficient cells may still be produced to ensure normal fertility. Enough teleological reasoning, let s get back to facts. Question: What factors have been proposed to regulate spermatogonial divisions/apoptosis? Answer: The literature on this topic is voluminous and in keeping with the idea that we should keep this review simple, we present a table (Table 2). Question: Of all the proposed regulators of spermatogonia, which have the strongest physiological evidence to support their proposed role?