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a Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901
b School of Molecular Biosciences, Washington State University, Pullman, Washington 99164
ABSTRACT
The distribution of type A spermatogonia was studied using drawings of cross-sectioned tubules at various stages of the spermatogenic cycle of perfusion-fixed, epoxy-embedded mouse testis. Spermatogonia were classified as either positioned opposite the interstitium or opposite the region where two tubules make contact or in a defined, intermediate region at which the two tubules diverged. At stage V, the population of type A spermatogonia, comprised of As through Aal cells, is randomly positioned around the periphery of the seminiferous tubule. The As through Aal population becomes nonrandomly distributed beginning at stage VI, being located primarily in regions where the tubule opposes the interstitium, and remains nonrandom through stage III of the next cycle. The A1 spermatogonia of stage VII, derived from most Apr and Aal spermatogonia, and the A2 spermatogonia of stage IX, derived from the A1 spermatogonia, are also nonrandomly positioned opposing the interstitium. However, the A3 population of stage XI becomes randomly distributed around the tubule. To our knowledge, these are the first data to show that the more primitive spermatogonial types (As to Aal) move to specific sites within the seminiferous tubule. Division of the regularly spaced, more primitive spermatogonia (As to Aal) leads to the spread of their progeny (A1 to A4) laterally along the base of the seminiferous tubule. The lateral spread from more or less evenly spaced foci ensures that spermatogenesis is conducted uniformly around the entire tubule. The data also suggest that the position of a seminiferous tubule in the mouse is stabilized in relationship to other seminiferous tubules.
mouse, seminiferous tubule, spermatogenesis, spermatogonia
INTRODUCTION
Spermatogenesis is conducted in seminiferous tubules, the ends of which terminate in the rete testis. This process is uniform within seminiferous tubules such that mature spermatids are lined evenly along the lumen and released all around the tubular lumen. Similarly, younger spermatids and spermatocytes are layered circumferentially and distributed uniformly. The organization of spermatogenesis also implies that the youngest cells, the spermatogonia, are also uniformly distributed around the seminiferous tubule.
The pubertal establishment and adult maintenance of spermatogenesis requires that precursor cells not be positioned within the tubule asymmetrically but, rather, uniformly. Transplantation of a single stem cell spermatogonium [1] will result initially in asymmetrical spermatogenesis but, shortly thereafter, in uniform spermatogenesis around the entire tubule [2]. Thus, spermatogonial stem cells are capable of movement. Spermatogenesis is established at the rate of approximately 60 µm/day along the length of the seminiferous tubule after transplantation [3] into an infertile recipient. The capability for stem cells to move and the uniform organization of spermatogenesis around seminiferous tubules suggest that some regulatory influence must govern the even distribution of spermatogenic cells during normal spermatogenesis.
The objective of this study was to examine the distribution of the earliest spermatogonia in the mouse testis. Our results suggest that the distribution of primitive classes of spermatogonia (As to Aal) throughout most of the spermatogenic cycle is not random. These results also suggest that spermatogonia are mobile, and that they demonstrate the ability to cyclically position themselves at periodic intervals along the seminiferous tubule to ensure their progeny are distributed evenly and uniformly around the seminiferous tubule.
MATERIALS AND METHODS
Animals and Tissue Preparation
Seven normal, adult mice (C57BL/6J), weighing 27.71 ± 0.95 g (mean ± SEM) and obtained from Jackson Labs (Bar Harbor, ME) were perfused-fixed with 5% (v/v) glutaraldehyde (biological grade) in cacodylate buffer (0.05 M, pH 7.4) according to the method described by Sprando [4]. Testis tissue with a mean weight of 103 ± 1.57 mg per testis was post-fixed in an osmium-ferrocyanide mixture [5], dehydrated in ethanol, infiltrated with propylene oxide, and embedded in Araldite 502 (Electron Microscopy Sciences, Fort Washington, PA). Tissues were embedded to produce cross-sections of seminiferous tubules on sectioning.
Tissue blocks were cut with an ultramicrotome into sections approximately 1 µm in thickness, stained with toluidine blue, and examined with the light microscope. The four animals with the best fixation were then selected for final study. The criteria for selection were that no visible shrinkage be detected, as judged by the presence of contact of adjacent seminiferous tubules and the presence of protein within the interstitial space. Clear space, without the presence of blue-staining protein, was sufficient to exclude animals from study. More than 20 light-microscope slides from different regions of the testis from each animal were made.
Determining the Position of Spermatogonia
Tubules at stages III, V, VI, VII, IX, and XI were selected for study. The staging criteria were those of Oakberg [6], as modified by Russell et al. [7]. The timing of spermatogenesis was that provided by Oakberg [8]. Tubules were examined at 240x magnification, and a camera lucida was used to draw the boundaries (peritubular tissue) of the tubules at the stages noted above. Nine tubules at each stage were drawn. Three different colors were used to draw the tubular perimeter. One color was used to draw the portions of the tubule in which the peritubular tissue contacted other tubules; contact was defined as no visible space between the peritubular tissue of adjacent seminiferous tubules. Another color was used to draw the interface of the tubule peritubular tissue with the interstitial space. Yet another color was used to define an intermediate region, in which the interstitial region and the tubule-tubule contact region joined. This area was specifically defined as the region between tubule-tubule contact and where two tubules diverged, extending the width of two thicknesses of peritubular tissue. In most cases, approximately four to six regions in each tubule related to the interstitium, although the range was two to eight (Fig. 1). Specifically, the following was noted: 0.85% ± 0.49% (mean ± SEM) of tubules made contact with two other tubules, 6.73% ± 1.25% of the tubules made contact with three tubules, 23.78% ± 4.21% of the tubules made contact with four tubules, 38.28% ± 3.34% of tubules made contact with five tubules, 25.5% ± 1.97% of the tubules made contact with six tubules, 4.03% ± 1.31% of the tubules made contact with seven tubules, and 0.85% ± 0.49% of the tubules made contact with eight tubules. No tubules facing the capsule of the testis were utilized.
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The various kinds of type A spermatogonia were differentiated as reported in a companion paper [9]. Their profiles were outlined using the camera lucida in the same tubules for which the tubule boundary was outlined as described above (Fig. 1). A digitizer was used to determine the total length of each colored region of an outlined seminiferous tubule. The total length of each colored line of each seminiferous tubule was summed for nine seminiferous tubules analyzed from each animal.
The positions of type A spermatogonia were recorded as being opposite the interstitial, intermediate, or tubule contact areas. The total number of cells in each animal was determined for each of these three positions and a mean value obtained (Table 1).
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Type A spermatogonial distribution was initially expressed in line graphs. The line graphs expressed the distribution of spermatogonia as the ratio. In turn, the ratio was determined to be the percentage of type A spermatogonia contacting the interstitium (or another tubule or intermediate regions) divided by the percentage of type A spermatogonia in those regions. For example, if 50% of spermatogonia occupied 50% of a seminiferous tubule, the ratio would be 1.0. In this situation, the topographical distribution of spermatogonia would be considered random. On the other hand, if 80% of the spermatogonia occupied 50% of the tubule, the ratio would be 1.6. Line graphs were used to express results, because the process of adult spermatogenesis is considered to be cyclic, with one cycle being similar to the next.
Statistical Analysis
The ratio values described above were not used in the statistical analysis. Instead, the Strauss linear selectivity index (Li) was chosen to examine region selection by type A spermatogonia in the basal compartment of seminiferous tubules [10]. The Li were calculated for each tubular region as Li = ri - pi, where Li = the linear index value, ri = the proportion of spermatogonia in a specific region, and pi = the proportion of length in the same region. These calculations resulted in an Li value for each region ranging from -1 to +1, with zero representing a random position of type A spermatogonia. A positive number represented positive selection (i.e., preference) of the given region, whereas a negative number represented negative selection (i.e., avoidance) of the given region. The Li values were graphed with their 95% confidence intervals.
The Li values were calculated for each tubular count for four animals used in the present study. It was first necessary to show that no significant differences existed in Li values for any of the four animals. In other words, it was necessary to show homogeneity of the animals with respect to spermatogonial distribution. An ANOVA was performed that made 72 comparisons, 71 of which indicated no differences in Li values. The single positive comparison was considered to be a type II error due to extreme variability in one sampling. For stage comparisons of a single location, Li values were compared using individual t-tests (P < 0.05).
A t-test was also used to determine whether Li values were significantly different from zero and nonrandom (P < 0.05). Generally, the Li index graphs were similar in form to the ratio-derived graphs (the latter graphs are not shown).
RESULTS
Figure 1 shows a micrograph of a seminiferous tubule that was taken at stage VI and utilized in the present study. It depicts the spermatogonia that were analyzed in this single tubule at high magnification. It also shows a computer-graphics drawing made using the camera lucida, detailing both the tubular outline (traced along the peritubular tissue) and the outline of spermatogonia.
Table 1 shows the mean number of type A spermatogonia in specific regions of the seminiferous tubules. Table 1 also depicts the percentage occupancy of seminiferous tubules with other tubules, the percentage occupancy of the intermediate region, and the percentage occupancy of the interstitium.
Distribution of All Types of A Spermatogonia
Ratios of the percentage of spermatogonia in a tubule to the percentage of the tubule periphery that they occupied were determined. These data were graphed but are not shown, because the ratio and Li graphs are similar in form. As an example of the ratios obtained, we present the ratios for stages V and VII, which are stages showing a random and a nonrandom distribution of spermatogonia, respectively. At stage V, interstitial, intermediate, and tubule contact ratios of As to Aal spermatogonia were 1.06 ± 0.22, 1.03 ± 0.18, and 0.94 ± 0.25, respectively. At stage VII, the interstitial, intermediate, and tubule contact ratios of the As to A1 spermatogonia were 1.55 ± 0.13, 0.66 ± 0.17, and 0.57 ± 0.11, respectively.
Figure 2 shows Li values indicating the topographical distribution of all type A spermatogonia at the three defined contact regions of the seminiferous tubule. Nonrandom distribution of type A spermatogonia (As, Apr, and Aal) is shown at stage III, during which significantly more spermatogonia were positioned along the region where the interstitium and tubule make contact.
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The position of As to Aal spermatogonia at stage V was determined statistically to be random at all regions analyzed. The position of the same cell types at stage VI was nonrandom, however, with the As to Aal cells being primarily located at the periphery of the tubule bordering the interstitium. The tubule contact and intermediate region showed significant negative selection. In stage VII, when most of the Aal cells had transitioned to A1 cells, a similar significant, nonrandom distribution was found, with cells showing a significant preference for the region opposing the interstitium and a significant negative selection for spermatogonia in regions termed intermediate and tubule. The cell population composed of As to A2 cells at stage IX showed a significant location preference for the interstitium, but this preference was significantly diminished compared with stage VII. The As to A3 population of type A spermatogonia in stage XI showed no differences compared with stage IX.
Distribution of As, Apr, and Aal Spermatogonia
Figure 3 is plotted based on the identification, separation, and exclusion of the more primitive type A spermatogonia (As, Apr, and Aal) from the more advanced types of spermatogonia (A1 to A3). The Li graph demonstrates the same features shown in Figure 2 up to stage VI, because no A1 to A3 spermatogonia are present in these early stages. When most Apr and Aal spermatogonia transition to A1 spermatogonia at stage VII, a significantly greater positive selection of the As to Aal group is observed for region as opposed to the interstitium compared with the As to Aal cells of stage VI. A nonrandom, negative selection of the As to Aal group of cells at stage VII in the intermediate and tubule regions is also seen. The As to Aal population at stage IX shows a similar distribution as that described for stage VII, except no negative selection for the intermediate or tubule regions is observed. The As to Aal population at stage XI is nonrandom, with the cells preferring the interstitial region. A significant decline in the preference of cells for the interstitial region is seen from their peak at stage VII. Only the tubule contact region shows negative selection for the As to Aal population.
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Distribution of A1 to A3 Spermatogonia
Figure 4 shows that the Li of A1 spermatogonia and Aal to A1 transition cells [9] is strongly nonrandom in stage VII. The A2 cells of stage IX are also nonrandomly positioned along the interstitium, but they are not significantly different in position from the A1 cells of stage VII. The A3 cells of stage XI are randomly positioned, and their position opposing the interstitium is significantly changed compared with their predecessor A2 cells at stage VII, but not compared with those of stage IX.
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Comparison of the As to Aal Population with the A1 to A3 Population
The As to Aal population distribution was compared at stage VII with that of the A1 (or A1 transition cells) population. The As to Aal population had a significantly stronger preference for the interstitial region than did the A1 population. Similar results were obtained for the As to Aal population of cells at stage IX compared with A2 cells of the same stage. No significant difference was found in the preference of As to Aal cells for the interstitium compared with A3 cells of stage IX.
DISCUSSION
Several technical reasons allowed us to show that specific type A spermatogonia were not randomly positioned within the seminiferous tubules at certain stages of the cycle of the seminiferous epithelium. First, perfusion fixation resulted in virtually no shrinkage artifact of the kind that is well known to separate seminiferous tubules from each other. That little or no shrinkage between tubules was evident by the presence of lightly blue-stained interstitial space, a color that is generally known to represent protein normally present in the interstitial space. Had there been shrinkage, a colorless area would have been present to reveal the separation of the interstitial proteinaceous material from the seminiferous tubule. Second, tubule-to-tubule contact in the mouse is large compared with that in many other species, making it possible to define different topographical regions of seminiferous tubules. Finally, the recent identification [9] of more advanced types of spermatogonia (A1 to A4) allowed us to show it was the As, Apr, and Aal cells that maintained the highest degree of tubule-interstitial contact at most stages of the cycle of the seminiferous epithelium.
One interesting finding is that a nonrandom distribution of spermatogonia could only be maintained if seminiferous tubules remained in a relatively constant relationship to one another. One of the author's (L.D.R.) previous concepts regarding the positioning of seminiferous tubules is that they are highly mobile relative to one another. Such a concept is based on the results of nicking the capsule of rat tubules and watching seminiferous tubules ooze out, as noodles might do if enclosed by a pressure-filled capsule. The present study in the mouse, in which slightly more connective tissue is present compared with the rat, strongly suggests that their seminiferous tubules do not move relative to one another or rotate in vivo or change position during fixation.
Seminiferous tubules maintain contact with two to eight other seminiferous tubules, with more tubules making contact with five other tubules than any other situation. The sites around the tubule provide two to eight relatively evenly spaced foci from which their progeny can spread laterally. The spread of their progeny (A1 to A4 cells, intermediate type and type B spermatogonia) occurs progressively as the more differentiated cells divide and are pushed laterally from the interstitial location. When looking under a microscope, it is easy to see that very advanced spermatogonia, such as intermediate and type B spermatogonia, are evenly spread around the seminiferous tubule. Indeed, the graphs (Fig. 4) show that by the end of the cycle, the position of the A3 cells is random.
Sertoli cells of a particular species support a characteristic number of germ cells [11]. To our knowledge, this number has never been shown to increase by experimental manipulation beyond that seen in normal animals. Thus, it appears that the germ cells, up to preleptotene spermatocytes, are rather evenly distributed around the seminiferous tubule. Next, they move centripetally between Sertoli cells [12] and then, as elongating spermatids, form an exclusive association with a single Sertoli cell [13].
Expressed as cells per unit area, approximately threefold as many of the more primitive type A spermatogonia (As to Aal) are present at stage VII in the region of the tubule adjacent to the interstitium than in the tubule contact region. The periodic location of spermatogonia near the interstitium serves to position spermatogonia at evenly spaced foci, from which further cell division occurs that will later spread the progeny of type A spermatogonia evenly around the base of the seminiferous tubule.
It appears that the distribution of the As to Aal spermatogonia is located primarily at the interstitium, but the number of cells is regulated in a different manner. In the mitotic divisions from A2 to A4 spermatogonia, a mechanism limits the number of spermatogonia in particular regions of the tubule, based on their density (i.e., density-dependent regulation). This regulation in mice appears to occur via a prosurvival protein (Bclx-L [14]) and a proapoptotic protein (Bax [15]). Thus, mechanisms are in place to regulate both the numbers and spatial distribution of early germ cells in mice.
To date, the mouse is the only species studied with regard to spermatogonial distribution. As mentioned above, the mouse has a large area of tubule contact; thus, the topography of tubules can easily be mapped. What about other species? We are currently assessing the spermatogonial distribution pattern in the rat, in which tubule-to-tubule contact is minimal compared with the mouse. Because of the species variability in the amount of interstitium that surrounds seminiferous tubules, we do not necessarily propose that the regulation of spermatogonial topography is similar for all species.
This study does not address the factors that regulate spermatogonial topography. We presume that factors from the interstitium, perhaps Leydig cells, or from the vascular system can attract spermatogonia, although the nature of the biological attractant is not known. The basal lamina may be biochemically different in the region at which spermatogonia come to rest.
Testosterone is a possible candidate as an attractant to spermatogonia via stimulation of nearby somatic cells, given that no testosterone receptors are found in spermatogonia. At first examination, this possibility seems unlikely, because considerable information suggests that increased testosterone levels increase spermatogonial apoptotic activity and that lowered levels do the opposite [16, 17]. However, on further examination, the suppression of spermatogonial apoptosis should not be confused with signaling for cells to migrate. Leydig cells are known to produce other factors that may be attractants for spermatogonia [18].
This study demonstrates that spermatogonia migrate within the seminiferous epithelium. Other recent studies have also demonstrated this migratory capability of spermatogonia. For example, a study using radiation depletion of the seminiferous epithelium has shown that when most stem cell spermatogonia are depleted, repopulation of the tubule occurs as the few remaining spermatogonia undergo mitosis and extend along the length of the seminiferous tubule [19]. In a similar way, transplantation of spermatogonia results in spermatogonia moving along the tubule at a rate of more than 50 µm/day [2, 3]. The movements referred to above are accomplished by stem cell spermatogonia. Because the present study shows cyclic, random location followed by nonrandom location, we presume that some portion of the As, Apr, and Aal population must be moving within the seminiferous tubule.
The present study demonstrates overall movement of A1 to A3 spermatogonia. The degree to which A3 cells are associated with the interstitium is less than that of A2 cells, which, in turn, is less than that of A1 cells, suggesting that progressive lateral spread and/or chain elongation with cell division is responsible for movement away from the interstitium. To what degree large, interconnected chains of spermatogonia can move is not known. It is known that the successors of A1 to A3 cells, preleptotene-leptotene spermatocytes, with a theoretical chain length of more than 1000 cells but most probably a chain length of a few hundred cells [20], move centripetally at the time they enter the intermediate compartment of the testis [12]. It has been suggested that Sertoli cells are helpful in relocating preleptotene-leptotene spermatocytes [12], but the data do not indicate if the surrounding Sertoli cells are active in helping spermatogonia move.
It should be stressed that the data presented herein quantify populations of cells and, for the most part, not individual cell types. The As, Apr, and Aal population are not an equal numerical distribution of cell types. In most stages, the Aal population dominates in terms of cell numbers [9]. Thus, the numerical data of their position can make it difficult to ascertain the position of the more primitive As and Aal cells. Some cell types of this population may move and others may not, thus yielding the present summed data regarding their overall topography. When spermatogonia markers are available to distinguish many of the primitive spermatogonial types, the precise cell types and their relative contribution to cell position can be elucidated.
The present study has examined spermatogonia in sectioned tissue. A previous study in the Chinese hamster that examined whole mounts of spermatogonia has shown that the spacing of chains of the same length along the tubule was approximately equal [21]. Thus, evidence from both whole-mount and sectioned-tissue methodologies indicates that the spacing of spermatogonia is regulated—regulated in the longitudinal axis of the tubule or the circumferential axis, respectively. It was also noted in the Chinese hamster study [21] that clones of a particular size dominated certain regions of the tubule, but this feature was not addressed in the present study.
The present study has shown that the more primitive type A (As to Aal) spermatogonial distribution within the seminiferous tubules is not random at most stages of the cycle of the mouse. The As to Aal group is randomly distributed at stage V; however, a significant change in their position occurs at stage VI, when they move to the regions abutting the interstitium and remain at this location at stage VII. As most of the Aal group transform to A1, a significant movement of the A1 population away from the regions adjacent to the interstitium is seen, whereas the remaining As to Aal population remains at the interstitial site. In fact, the As to Aal cells remain preferentially at the interstitium until stage III of the next cycle. As A1 cells develop into A2 (of stage IX) and A3 (of stage XI) spermatogonia, their (i.e., the A3 cells') position along the periphery of the tubule becomes random at stage XI. Thus, the more primitive of the spermatogonia, the As to Aal cells, are preferentially positioned at specific sites along the periphery of the seminiferous tubule. The apparent preferential location of these cells reflects the need for an evenly spaced distribution of selective precursor type A spermatogonia. The data indicate that division of these precursor (As, Apr, and Aal) spermatogonia results in the lateral spread of their progeny (A1 to A3) uniformly along the circumferential axis (i.e., the base) of the seminiferous tubule. This spread ensures that all nurse cells (i.e., Sertoli cells) develop a relationship with relatively immature germ cells that they will maintain throughout spermatogenesis.
ACKNOWLEDGMENTS
Our thanks go to Paul S. Wills for statistical assistance.
FOOTNOTES
1 Supported by the NIH (HD 35494 to M.D.G. and L.D.R.) and a Latin American Fellowship (to H.C.-G.). 
2 Correspondence and current address: Helio Chiarini-Garcia, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil 31270-901. FAX: 55 31 3499 2780; chiarini{at}icb.ufmg.br
Accepted: May 23, 2001.
Received: April 5, 2001.
REFERENCES
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