Morphological and Quantitative Analysis of Spermatogonia in Mouse Testes Using Whole Mounted Seminiferous Tubules I. THE NORMAL TESTES
'
C. HUCKINS ' AND E. F. OAKBERG Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 and Biology Diuision, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
ABSTRACT
The spermatogonial populations in ten normal adult mice were analyzed using whole mounted seminiferous tubules. The undifferentiated A spermatogonia as well as the six generations of differentiating spermatogonia were clearly identifiable on whole mounts. Description plus quantitation of these cell types revealed t h a t they behaved in essentially the same manner as their counterparts in the rat. Single undifferentiated A cells were classified as type A, stem cell spermatogonia. They were distributed throughout the seminiferous epithelium, and by periodic mitoses, maintained their stock and furnished cells which would eventually differentiate. Although initially resembling the A, spermatogonia, the progeny which were destined to differentiate were classified as type Aal spermatogonia because they were linked by cytoplasmic bridges, and because they usually underwent one or more synchronous mitotic divisions t o form short chains of aligned cells. Ultimately, division of Aal cells were no longer seen, and the cells appeared to gradually acquire the typical morphological characteristics of A , spermatogonia; these continued to differentiate according to the well-established pattern. It was concluded that the cyclic production of cohorts of A, cells in this manner would ensure a continual supply of spermatogonia for differentiation.
Over the years, the rodent model has been and continues t o be widely used t o study spermatogenesis under both normal and experimental conditions. The mouse has provided much of the important information on irradiation effects on the testis (Oakberg, '55a,b, '56, '75; Withers et al., '741, while the rat in particular has furnished information on the qualitative and quantitative aspects of normal spermatogenesis (Leblond and Clermont, '52; Clermont, '62; Clermont and Bustos-Obregon, '68; Huckins, '71a,b; Clermont and Hermo, '76). In both r a t and mouse, a new concept of spermatogonial stem cell renewal and differentiation has recently been evolved (Oakberg, '71; Huckins, '71b,c; de Rooij, '73). However, the experimental pathways leading to the formulation of this theory have been widely divergent. In the case of the rat, analysis of spermatogonia in normal whole mounted ANAT. REC. (1978)192: 519-528.
seminiferous tubules plus kinetic studies after labeling with tritiated thymidine generated new data on their morphology, quantitation and cell cycle parameters (Huckins, '7la,b,c,d), and culminated in a stem cell theory t h a t differed substantially from other models (Clermont, '72). Concurrent labeling studies in the mouse on cross-sections of normal and irradiated tubules (Oakberg, '71) have provided data t h a t resulted in a similar theory. The present investigation was designed to both confirm and complement the rat studies by analyzing the spermatogonial population in whole mounts of normal and irradiated Received April 25. '78. Accepted July 19, '78. ' By acceptance of this article, the publisher or recipient acknowledges the right of the US. Government to retain a nonexclusive. royalty-free license in and to any copyright covering the article. Send reprint requests to: Dr. C. Huckins, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030. 30perated by the Union Carbide Corporation for the Department of Energy.
519
520
C. HUCKINS A N D E. F. OAKBERG
mouse seminiferous tubules. In this paper, the spermatogonia in normal tubules will be characterized and quantitated. The essential features and behavior of the rat spermatogonial population are confirmed in the mouse. MATERIALS AND METHODS
Ten 12-week-old F, hybrid (101 x C3H) male mice were used in these studies. To prepare whole mounts, segments of seminiferous tubules were dissected free from each testis and fixed for two hours in Bouin's fluid. Following removal of picric acid in successive washes of 70% alcohol, tubules were stained for two minutes in a 1:5 dilution of Harris hematoxylin. After dehydration through a graded series of alcohol, and clearing in xylene, tubules were mounted in toto. Undifferentiated and differentiating spermatogonia were described at each stage of the cycle, using a classification scheme based on the six successive generations of differentiating spermatogonia. Stage l began with the first generation of A , spermatogonia, and progressed to stage 6, which contained the sixth and last generation of B spermatogonia (Huckins, '78a; Oakberg and Huckins, '76). Spermatogonia were quantitated at each stage, using the same methodology that had been applied to the rat (Huckins, '71b). Essentially, an ocular grid (area of 7,225 p z, was placed over the domed center of the tubule a t the beginning of a stage. All spermatogonial nuclei with their geometric center within the frame of the grid were mapped on matching paper grids. Successive frames were mapped along the length of the tubule until the end of the stage was reached. Counts were expressed as number of cells per frame. In addition, Sertoli cell nucleoli were counted at every fifth frame. Since the Sertoli cell population is numerically constant, all spermatogonial counts were corrected to 20 Sertoli cells per frame to overcome any variability due t o shrinkage or stretching of tubules during preparation. RESULTS
Differentiating spermatogonia The six generations of differentiating spermatogonia occupied discrete and usually spatially consecutive areas along the tubular wall. Morphologically, each type of spermatogonia bore the same features that had been described earlier in cross-sections of mouse testes (Oakberg, '56) and on whole mounts of rat tubules (Clermont and Bustos-
Obregon, '68; Huckins, '71b). In general, with each successive generation of spermatogonia, the nuclei became progressively smaller and more rounded, and displayed increasing amounts of heterochromatin, both free in the nucleoplasm and adherent t o the nuclear membrane (figs. 2-7). Degeneration was common among groups of A, and A, and, to a lesser extent, A, spermatogonia. Therefore, quantitation revealed only a modest expansion in the size of the population from A, to A, spermatogonia despite the three intervening mitotic divisions (table 1).There were only about 3.4 times the number of A, as A, cells where, theoretically, there should have been an 8-fold increase. Subsequently, however, the population doubled with each successive division of A,, In, and B spermatogonia (table 1). Undifferentiated spermatogonia
The undifferentiated A population could be topographically classified as consisting of single stem cells (A,) as well as pairs and short chains (A.J of spermatogonia which were linked by cytoplasmic bridges (figs. 2-71. The latter differentiated into A , spermatogonia (fig. 11).Undifferentiated A cells were scattered among all classes of differentiating spermatogonia. Although they were hard to recognize definitively among the A2 and young A, cohorts, they were easy to delineate among mature A, cells (figs. 3, 4). Regardless of their topographical location, they all had similar morphological features. In Bouin fixed whole mounts, they usually displayed a slightly oval homogeneously granular nucleus. The nuclear membrane was free of adherent chromatin particles. A spherical nucleolus was usually conspicuous (figs. 2, 7). Both single and chains of nuclei were seen to enlarge and enter mitosis synchronously (figs. 2, 7-10). Mitotic activity was particularly noticeable in stages 4 and 5 , and was unrelated to the behavior of the A, and In spermatogonia in the same respective areas. Occasionally, mitotic figures were observed in stages 6 and 1 (figs. 7, 9, 10). The undifferentiated A cells were counted in all stages except stage 2, where it was impossible to accurately distinguish them from A, spermatogonia. An effort was made t o separately enumerate undifferentiated A s and A3cells in stage 3, but this should only be regarded as a tentative value. As can be seen from table 2, the undifferentiated A popula-
52 1
SPERMATOGONIA IN NORMAL MOUSE TESTES TABLE 1
Number of spermatogonia in testes of adult mice: whole mount tubule analysis Stage
Spermatogonial type
1
No.iframe2S.E.'
A, + A A3 A,+A A A3+A A A4 A In A
2
3 4 5
2.75-3.72
-
8.35*0.26 1 . 1 6 2 0.24 9.66-tO.26 1 . 6 9 2 0.19 17.5520.34 2.7730.31 36.39-t 0.37 3.35-t 0.17
B
6
Animal range
3.3620.14 0.52 5 . 7 4 2 0.33
A
15.7-18.8 1.94-3.77 34.7-38.1 3.05-4.33
' Corrected to 20 Sertoli cells/frarne. 'Range of values for the ten animals quantitated Undifferentiated type A cells. TABLE 2
Number of undifferentiated A spermatogonia in mouse testes: whole mount tubule analysis Stage
I
No. of frames
ZA
591
2,171
-
-
309 222 177 325 591
231 391 512 1,272 2,171
Mitotic index I V )
XA+S.E. 0.52
'
-
-
-
1.16 k0.24 1.69 3 0 . 1 9 2.77 2 0 . 3 1 3.35 2 0 . 1 7 3.36 '20.14
3.03 3.32 4.10 1.17 0.36
As. As + A , .
TABLE 3
Large us. small A spermatogonia in stage 1-2 Total A
LargeA Small A
As
Apr
Aal
Frames
No.
x
No.
x
No.
x
No.
%,
100 100
387 71
84.4 15.6
3 29
0.8 40.8
16 31
4.1 43.7
368 9
95.0 12.7
tion was minimal in stage 1, since most of them had morphologically transformed into A, cells which differentiated. Mitotic activity led to a gradual expansion in the size of the undifferentiated A population from 0.52 cellslframe in stage 1 to 3.50 cells/frame in stage 6. During stage 6, divisions became infrequent, and the population plateaued a t its maximal size. During stages 6 and 1, most of the undifferentiated A s acquired the characteristics of A, spermatogonia and another cycle of the seminiferous epithelium began. Just before the onset of the A, division,
most chains of spermatogonia were clearly enlarged, while most single As cells remained small (fig. 11).Counts revealed that 85%of all spermatogonia were enlarged; of these, 95% were chains of A , cells and only 5% were enlarged single or paired cells (table 3). Of the remaining 15% small resting A spermatogonia, there were equal numbers of singles and pairs with an occasional chain of four cells. In another analysis, the percent distribution of the various types of undifferentiated A spermatogonia was determined (table 4). The numbers of As and, t o a lesser extent, pairs
522
C . HUCKINS AND E. F. OAKBERG TABLE 4
Distribution of undifferentiated A sperrnatogonia Stage
1 4 5 6
No. of frames
Total A uncorrected no./frame
No.
As
x,
No.
Apr %,
No.
L
100 100 100 100
4.08 1.57 2.96 4.08
0.46 0.48 0.53 0.48
11.3 30.6 17.9 11.8
0.51 0.38 0.56 0.60
12.5 24.2 18.9 14.7
3.11 0.71 1.84 3.00
76.2 45.2 62.2 73.5
were fairly constant from stage to stage. The Aal were fewest just after t h e formation of A , cells, but gradually increased in numbers until stage 6, at which time they made up 75% of t h e total population. There was considerable animal t o animal variability in t h e average number of A spermatogonia found in each stage (table 1). For example, in stage 1,there could be as many as 40% more A cells in one animal as in t h e next. This variation was not sustained through differentiation, so t h a t t h e numbers of In and B spermatogonia for individual animals fell within a narrow range. DISCUSSION
Previous studies in both r a t and mouse have led to a plausible mechanism by which spermatogonia renew and differentiate themselves (Huckins, '71b,c; Oakberg, '71). The single A, cells in t h e undifferentiated spermatogonial population have been identified as stem cells which sporadically replenish their own stock, as well as give rise to conjoined pairs, most of which continue to proliferate and form chains of A , ] cells (Huckins, '71b,c). As the present study confirms, t h e A a l cells appear to morphologically transform into A , spermatogonia which will go on to differentiate. Evidence from normal and experimental studies which depleted t h e spermatogonial population clearly fit well with this model (Oakberg, '71; de Rooij, '73; Withers e t al., '74; vanKeulen and de Rooij, '75; Oakberg and Huckins, '76; Erickson, '76; Huckins, '78b). Unlike t h e cohorts of differentiating spermatogonia which divide synchronously in peaks of mitoses, i t could be seen in whole mounts t h a t t h e stem cells appear in all stages and t h a t isolated mitoses occur in all stages in patterns unrelated t o t h e activities of t h e differentiating cells. Moreover, quantitation revealed t h a t t h e number of A, remains remarkably constant from stage to stage. Simi-
Aa1 or A1
lar findings have been reported in t h e rat (Huckins, '71b). All of t h e undifferentiated cells have similar morphological features, t h u s posing t h e question of whether spermatogonia in pairs or chains do retain stem cell potential. Counts made a t t h e time of t h e stage-1 division clearly showed t h a t 88%of t h e residual stem cell population is either single or paired. Whether t h e latter are simply two single cells close together or truly linked pairs could not be ascertained. Moreover, t h e members of chains invariably number 4, 8 or 16, indicating t h a t single cells do not usually break off from chains t o re-enter t h e stem cell compartment. Thus, while not finally resolving t h e issue, indications from this study a r e t h a t in normal testes, stem cell capability is restricted to the single elements. As in t h e r a t , t h e rebuilding of the undifferentiated population from its minimal size at t h e beginning of stage 2 is initiated by the periodic and random divisions of A, cells. Subsequent mitoses produce chains of Aal cells so t h a t , by stage 3, short chains a r e clearly recognizable among cohorts of A3 spermatogonia. Chains tend to remain shorter than in the rat, with 4- and 8-cell groups predominating. Proliferative activity is greatest during stages 4 and 5 , and tapers off in stage 6 as t h e cells transform into type A , cells. There is considerable animal to animal variability in terms of t h e number of undifferentiated A's finally produced (table 1). For ten animals, there was a range from 2.75-3.72 in cell number in stage 1. Nonetheless, there was only a narrow range of 34.7-38.1 in t h e number of B spermatogonia for these same animals. Based on t h e range in A , spermatogonia, and t h e number of subsequent divisions, one would anticipate t h a t t h e number of B cells should extend over a 32-cell range rather than just a 3.4-cell range. Presumably, this gap is narrowed through a variable degree of degen-
SPERMATOGONIA IN NORMAL MOUSE TESTES
523
Time
1
Sptd 16
1 Splr Release
Fig. 1 Model for t h e Renewal and Differentiation of Spermatogonia in Mouse.
eration of A, and A3 spermatogonia. This may suggest t h a t degeneration plays a critical role in ensuring t h a t optimal numbers of cells differentiate. Similar observations have been made in the rat (Huckins, ’78a). In summary, then, morphological and quantitative analysis of the undifferentiated A population in whole mounts of seminiferous tubules in the mouse reveal a pattern of behavior which is consistent with that described for the rat. Although labeling studies comparable t o those in r a t were not undertaken, ancillary data suggest that the cell cycle kinetics of mouse germ cells would likewise be complementary (Monesi, ‘62; Oakberg, ’75; Oakberg and Huckins, ’76). As summarized in figure 1, i t is proposed t h a t through periodic divisions, A, cells both renew themselves and contribute pairs of cells t o the proliferating compartment. Within the proliferating compartment, the pairs may undergo one or several additional divisions to form chains of aligned spermatogonia (A,,). Eventually, however, mitosis ceases, and all of the spermatogonia in the proliferating compartment transform without further division into A, cells. This cohort of A , spermatogonia synchronously enters the differentiating com-
partment t o initiate maturation, and the whole process begins again. Thus, the stem cell compartment maintains a constant size while the proliferating compartment is regularly depleted and rebuilt again. By this mechanism, generations of A, spermatogonia cyclically begin differentiation. ACKNOWLEDGMENTS
We thank Greg Duncan and Lois Layton for their technical help and expertise in these experiments, and Larry Swain for his assistance in preparing the photographs. These studies were supported by HD 07655 and the U S . Energy Research and Development Administration under contract with the Union Carbide Corporation. LITERATURE CITED Clermont, Y. 1962 Quantitative analysis of spermatogenesis of the rat: A revised model for the renewal of spermatogonia. Am. J. Anat., 2 2 2 : 111-129. 1972 Kinetics of spermatogenesis in mammals: Seminiferous epithelial cycle and spermatogonial renewal. Physiol. Rev., 52: 198-236. Clermont, Y., and E. Bustos-Obregon 1968 Re-examination of spermatogonial renewal in t h e rat by means of seminiferous tubules mounted “in toto.” Am. J. Anat., 222: 231-248. Clermont, Y., and L. Hermo 1976 Spermatogonial stem cells in the albino rat. Am. J. Anat., 242: 159-176.
524
C. HUCKINS AND E. F. OAKBERG
Erickson, B. H. 1976 Effect of ““Co-radiation in the stem and differentiating spermatogonia of the post puberal rat. Rad. Res., 68: 433-448. Huckins, C. 1971a Cell cycle properties of differentiating spermatogonia in adult Sprague-Dawley rats. Cell Tissue Kinet., 4: 139.154. 1971h The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat. Rec., 169: 533-558. 1971c The spermatogonial stem cell population in adult rats. 11. A radioautographic analysis of their cell cycle properties. Cell Tissue Kinet., 4: 313-334. 1971d The spermatogonial stem cell population in adult rats. 111. Evidence for a long-cycling spermatogonial population. Cell Tissue Kinet., 4: 335-349. 1978a The morphology and kinetics of spermatogonial degeneration in adult rats: An analysis using a simplified classification of the germinal epithelium. Anat. Rec., 190: 905-926. 1978b Behavior of stem cell spermatogonia in the adult rat irradiated testis. Biol. Reprod. 19: in press. Leblond, C. P., and Y. Clermont 1952 Definition of the stages of the cycle of the seminiferous epithelium of the rat. Ann. N.Y. Acad. Sci., 55: 548-573. Monesi, V. 1962 Relation between X-ray sensitivity and stages of the cell cycle on spermatogonia of the mouse. Rad. Res.. 17: 809-838. Oakberg, E. F. 1955a Sensitivity and time of degeneration of spermatogenic cells irradiated in various stages of maturation in the mouse. Radiat. Res.. 2: 369-391.
1955h Degeneration of spermatogonia of the mouse following exposure to X-rays and stages in the mitotic cycle a t which cell death appears. J. Morph., 97: 39-54. Oakberg, E. 1956 A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat., 99: 391-413. 1971 Spermatogonial stem-cell renewal in t h e mouse. Anat. Rec., 169: 515-532. 1975 Effects of radiation on the testis. In: Handbook of Physiology. Vol. 5. Section 7, The Male Reproductive System. E. B. Astwood and R. 0. Greep, eds. Am. Physiological Society, pp. 233-243. Oakberg, E. F., and C. Huckins 1976 Spermatogonial stem cell renewal in t h e mouse as revealed by “H-thymidine labeling and irradiation. In: Stem Cells of Renewing Cell Populations. A. B. Cairnie, P. Lala and D. G. Osmond, eds. Academic Press, New York, pp. 287-302. de Rooij, D. G. 1973 Spermatogonial stem cell renewal in the mouse. I. Normal situation. Cell Tissue Kinet., 6: 281-287. vanKeulen, C. J. G., and D. G. de Rooij 1975 Spermatogenic clones developing from repopulating stem cells surviving a high dose of an alkylating agent. I. First 15 days after injury. Cell Tissue Kinet., 8: 543-551. Withers, H. R., N. Hunter, H. T. Barkley and B. 0. Reid 1974 Radiation survival and regeneration characteristics of spermatogenic stem cells of mouse testis. Rad. Res., 57: 88-103.
All photographs are of whole mounted seminiferous tubules from normal mouse. Fixed in Bouin’s fluid and stained by Harris hematoxylin. PLATE 1 EXPLANATION O F FIGURES
2
Area of tubule in stage 6 showing cluster of B spermatogonia. Among them is a resting A, cell and an enlarged As cell (arrows). Large Sertoli cell nuclei (S)are conspicuous.
3 Area of tubule in stage 3. Undifferentiated type Aai cells (arrows) are easily discernible among t h e differentiating A3 cohort. The dark shadows are zygotene spermatocytes (Z). 4
Late stage 3 showing Aai spermatogonia (arrows) among mature A, cells. Zygotene spermatocytes (Z).
5 Aai spermatogonia (arrows) among type A, cells in stage 4. Dark shadows are young pachytene spermatocytes (P). 6
Part of a chain of Aai cells (arrows) in stage 6 among type B spermatogonia. Note the similarity in morphology of A cells in figures 3-6. Sertoli cells (S).
7
Part of a chain of eight enlarged Aai cells (arrows) in stage 6 among young type B spermatogonia. The undifferentiated A in the lower left corner is just entering early prophase. Comparison of figures 6 and 7 reveals a doubling of nuclear size, hut retention of similar morphology
SPERMATOGONIA IN NORMAL MOUSE TESTES C Huckins and E. F. Oakherg
PLATE 1
525
PLATE 2 EXPLANATION OF FIGURES
8
Single AS stem cells in prophase of mitosis (arrow). Stage 1 among pre-leptotene spermatocytes (P-L). Sertoli cells (S).
9 Pair of dividing undifferentiated A cells (arrows) in stage 1 among pre-leptotene spermatocytes (P-L). Note the cytoplasmic bridge joining t h e cells (large arrow). 10 Part of a chain of eight dividing Aal spermatogonia (arrows) in stage 6 among young type B cells (B). Sertoli cells 6 ) .
1 1 In late stage 1, typically enlarged A cells are present (arrows). In the same field, two small A spermatogonia are seen (As). Pre-leptotene spermatocytes are maturing into leptotene cells (L).
526
SPERMATOGONIA IN NORMAL MOUSE TESTES C. Huckins and E. F. Oakberg
PLATE 2
527
'
C. HUCKINS ' AND E. F. OAKBERG Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 and Biology Diuision, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
ABSTRACT
The spermatogonial populations in ten normal adult mice were analyzed using whole mounted seminiferous tubules. The undifferentiated A spermatogonia as well as the six generations of differentiating spermatogonia were clearly identifiable on whole mounts. Description plus quantitation of these cell types revealed t h a t they behaved in essentially the same manner as their counterparts in the rat. Single undifferentiated A cells were classified as type A, stem cell spermatogonia. They were distributed throughout the seminiferous epithelium, and by periodic mitoses, maintained their stock and furnished cells which would eventually differentiate. Although initially resembling the A, spermatogonia, the progeny which were destined to differentiate were classified as type Aal spermatogonia because they were linked by cytoplasmic bridges, and because they usually underwent one or more synchronous mitotic divisions t o form short chains of aligned cells. Ultimately, division of Aal cells were no longer seen, and the cells appeared to gradually acquire the typical morphological characteristics of A , spermatogonia; these continued to differentiate according to the well-established pattern. It was concluded that the cyclic production of cohorts of A, cells in this manner would ensure a continual supply of spermatogonia for differentiation.
Over the years, the rodent model has been and continues t o be widely used t o study spermatogenesis under both normal and experimental conditions. The mouse has provided much of the important information on irradiation effects on the testis (Oakberg, '55a,b, '56, '75; Withers et al., '741, while the rat in particular has furnished information on the qualitative and quantitative aspects of normal spermatogenesis (Leblond and Clermont, '52; Clermont, '62; Clermont and Bustos-Obregon, '68; Huckins, '71a,b; Clermont and Hermo, '76). In both r a t and mouse, a new concept of spermatogonial stem cell renewal and differentiation has recently been evolved (Oakberg, '71; Huckins, '71b,c; de Rooij, '73). However, the experimental pathways leading to the formulation of this theory have been widely divergent. In the case of the rat, analysis of spermatogonia in normal whole mounted ANAT. REC. (1978)192: 519-528.
seminiferous tubules plus kinetic studies after labeling with tritiated thymidine generated new data on their morphology, quantitation and cell cycle parameters (Huckins, '7la,b,c,d), and culminated in a stem cell theory t h a t differed substantially from other models (Clermont, '72). Concurrent labeling studies in the mouse on cross-sections of normal and irradiated tubules (Oakberg, '71) have provided data t h a t resulted in a similar theory. The present investigation was designed to both confirm and complement the rat studies by analyzing the spermatogonial population in whole mounts of normal and irradiated Received April 25. '78. Accepted July 19, '78. ' By acceptance of this article, the publisher or recipient acknowledges the right of the US. Government to retain a nonexclusive. royalty-free license in and to any copyright covering the article. Send reprint requests to: Dr. C. Huckins, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030. 30perated by the Union Carbide Corporation for the Department of Energy.
519
520
C. HUCKINS A N D E. F. OAKBERG
mouse seminiferous tubules. In this paper, the spermatogonia in normal tubules will be characterized and quantitated. The essential features and behavior of the rat spermatogonial population are confirmed in the mouse. MATERIALS AND METHODS
Ten 12-week-old F, hybrid (101 x C3H) male mice were used in these studies. To prepare whole mounts, segments of seminiferous tubules were dissected free from each testis and fixed for two hours in Bouin's fluid. Following removal of picric acid in successive washes of 70% alcohol, tubules were stained for two minutes in a 1:5 dilution of Harris hematoxylin. After dehydration through a graded series of alcohol, and clearing in xylene, tubules were mounted in toto. Undifferentiated and differentiating spermatogonia were described at each stage of the cycle, using a classification scheme based on the six successive generations of differentiating spermatogonia. Stage l began with the first generation of A , spermatogonia, and progressed to stage 6, which contained the sixth and last generation of B spermatogonia (Huckins, '78a; Oakberg and Huckins, '76). Spermatogonia were quantitated at each stage, using the same methodology that had been applied to the rat (Huckins, '71b). Essentially, an ocular grid (area of 7,225 p z, was placed over the domed center of the tubule a t the beginning of a stage. All spermatogonial nuclei with their geometric center within the frame of the grid were mapped on matching paper grids. Successive frames were mapped along the length of the tubule until the end of the stage was reached. Counts were expressed as number of cells per frame. In addition, Sertoli cell nucleoli were counted at every fifth frame. Since the Sertoli cell population is numerically constant, all spermatogonial counts were corrected to 20 Sertoli cells per frame to overcome any variability due t o shrinkage or stretching of tubules during preparation. RESULTS
Differentiating spermatogonia The six generations of differentiating spermatogonia occupied discrete and usually spatially consecutive areas along the tubular wall. Morphologically, each type of spermatogonia bore the same features that had been described earlier in cross-sections of mouse testes (Oakberg, '56) and on whole mounts of rat tubules (Clermont and Bustos-
Obregon, '68; Huckins, '71b). In general, with each successive generation of spermatogonia, the nuclei became progressively smaller and more rounded, and displayed increasing amounts of heterochromatin, both free in the nucleoplasm and adherent t o the nuclear membrane (figs. 2-7). Degeneration was common among groups of A, and A, and, to a lesser extent, A, spermatogonia. Therefore, quantitation revealed only a modest expansion in the size of the population from A, to A, spermatogonia despite the three intervening mitotic divisions (table 1).There were only about 3.4 times the number of A, as A, cells where, theoretically, there should have been an 8-fold increase. Subsequently, however, the population doubled with each successive division of A,, In, and B spermatogonia (table 1). Undifferentiated spermatogonia
The undifferentiated A population could be topographically classified as consisting of single stem cells (A,) as well as pairs and short chains (A.J of spermatogonia which were linked by cytoplasmic bridges (figs. 2-71. The latter differentiated into A , spermatogonia (fig. 11).Undifferentiated A cells were scattered among all classes of differentiating spermatogonia. Although they were hard to recognize definitively among the A2 and young A, cohorts, they were easy to delineate among mature A, cells (figs. 3, 4). Regardless of their topographical location, they all had similar morphological features. In Bouin fixed whole mounts, they usually displayed a slightly oval homogeneously granular nucleus. The nuclear membrane was free of adherent chromatin particles. A spherical nucleolus was usually conspicuous (figs. 2, 7). Both single and chains of nuclei were seen to enlarge and enter mitosis synchronously (figs. 2, 7-10). Mitotic activity was particularly noticeable in stages 4 and 5 , and was unrelated to the behavior of the A, and In spermatogonia in the same respective areas. Occasionally, mitotic figures were observed in stages 6 and 1 (figs. 7, 9, 10). The undifferentiated A cells were counted in all stages except stage 2, where it was impossible to accurately distinguish them from A, spermatogonia. An effort was made t o separately enumerate undifferentiated A s and A3cells in stage 3, but this should only be regarded as a tentative value. As can be seen from table 2, the undifferentiated A popula-
52 1
SPERMATOGONIA IN NORMAL MOUSE TESTES TABLE 1
Number of spermatogonia in testes of adult mice: whole mount tubule analysis Stage
Spermatogonial type
1
No.iframe2S.E.'
A, + A A3 A,+A A A3+A A A4 A In A
2
3 4 5
2.75-3.72
-
8.35*0.26 1 . 1 6 2 0.24 9.66-tO.26 1 . 6 9 2 0.19 17.5520.34 2.7730.31 36.39-t 0.37 3.35-t 0.17
B
6
Animal range
3.3620.14 0.52 5 . 7 4 2 0.33
A
15.7-18.8 1.94-3.77 34.7-38.1 3.05-4.33
' Corrected to 20 Sertoli cells/frarne. 'Range of values for the ten animals quantitated Undifferentiated type A cells. TABLE 2
Number of undifferentiated A spermatogonia in mouse testes: whole mount tubule analysis Stage
I
No. of frames
ZA
591
2,171
-
-
309 222 177 325 591
231 391 512 1,272 2,171
Mitotic index I V )
XA+S.E. 0.52
'
-
-
-
1.16 k0.24 1.69 3 0 . 1 9 2.77 2 0 . 3 1 3.35 2 0 . 1 7 3.36 '20.14
3.03 3.32 4.10 1.17 0.36
As. As + A , .
TABLE 3
Large us. small A spermatogonia in stage 1-2 Total A
LargeA Small A
As
Apr
Aal
Frames
No.
x
No.
x
No.
x
No.
%,
100 100
387 71
84.4 15.6
3 29
0.8 40.8
16 31
4.1 43.7
368 9
95.0 12.7
tion was minimal in stage 1, since most of them had morphologically transformed into A, cells which differentiated. Mitotic activity led to a gradual expansion in the size of the undifferentiated A population from 0.52 cellslframe in stage 1 to 3.50 cells/frame in stage 6. During stage 6, divisions became infrequent, and the population plateaued a t its maximal size. During stages 6 and 1, most of the undifferentiated A s acquired the characteristics of A, spermatogonia and another cycle of the seminiferous epithelium began. Just before the onset of the A, division,
most chains of spermatogonia were clearly enlarged, while most single As cells remained small (fig. 11).Counts revealed that 85%of all spermatogonia were enlarged; of these, 95% were chains of A , cells and only 5% were enlarged single or paired cells (table 3). Of the remaining 15% small resting A spermatogonia, there were equal numbers of singles and pairs with an occasional chain of four cells. In another analysis, the percent distribution of the various types of undifferentiated A spermatogonia was determined (table 4). The numbers of As and, t o a lesser extent, pairs
522
C . HUCKINS AND E. F. OAKBERG TABLE 4
Distribution of undifferentiated A sperrnatogonia Stage
1 4 5 6
No. of frames
Total A uncorrected no./frame
No.
As
x,
No.
Apr %,
No.
L
100 100 100 100
4.08 1.57 2.96 4.08
0.46 0.48 0.53 0.48
11.3 30.6 17.9 11.8
0.51 0.38 0.56 0.60
12.5 24.2 18.9 14.7
3.11 0.71 1.84 3.00
76.2 45.2 62.2 73.5
were fairly constant from stage to stage. The Aal were fewest just after t h e formation of A , cells, but gradually increased in numbers until stage 6, at which time they made up 75% of t h e total population. There was considerable animal t o animal variability in t h e average number of A spermatogonia found in each stage (table 1). For example, in stage 1,there could be as many as 40% more A cells in one animal as in t h e next. This variation was not sustained through differentiation, so t h a t t h e numbers of In and B spermatogonia for individual animals fell within a narrow range. DISCUSSION
Previous studies in both r a t and mouse have led to a plausible mechanism by which spermatogonia renew and differentiate themselves (Huckins, '71b,c; Oakberg, '71). The single A, cells in t h e undifferentiated spermatogonial population have been identified as stem cells which sporadically replenish their own stock, as well as give rise to conjoined pairs, most of which continue to proliferate and form chains of A , ] cells (Huckins, '71b,c). As the present study confirms, t h e A a l cells appear to morphologically transform into A , spermatogonia which will go on to differentiate. Evidence from normal and experimental studies which depleted t h e spermatogonial population clearly fit well with this model (Oakberg, '71; de Rooij, '73; Withers e t al., '74; vanKeulen and de Rooij, '75; Oakberg and Huckins, '76; Erickson, '76; Huckins, '78b). Unlike t h e cohorts of differentiating spermatogonia which divide synchronously in peaks of mitoses, i t could be seen in whole mounts t h a t t h e stem cells appear in all stages and t h a t isolated mitoses occur in all stages in patterns unrelated t o t h e activities of t h e differentiating cells. Moreover, quantitation revealed t h a t t h e number of A, remains remarkably constant from stage to stage. Simi-
Aa1 or A1
lar findings have been reported in t h e rat (Huckins, '71b). All of t h e undifferentiated cells have similar morphological features, t h u s posing t h e question of whether spermatogonia in pairs or chains do retain stem cell potential. Counts made a t t h e time of t h e stage-1 division clearly showed t h a t 88%of t h e residual stem cell population is either single or paired. Whether t h e latter are simply two single cells close together or truly linked pairs could not be ascertained. Moreover, t h e members of chains invariably number 4, 8 or 16, indicating t h a t single cells do not usually break off from chains t o re-enter t h e stem cell compartment. Thus, while not finally resolving t h e issue, indications from this study a r e t h a t in normal testes, stem cell capability is restricted to the single elements. As in t h e r a t , t h e rebuilding of the undifferentiated population from its minimal size at t h e beginning of stage 2 is initiated by the periodic and random divisions of A, cells. Subsequent mitoses produce chains of Aal cells so t h a t , by stage 3, short chains a r e clearly recognizable among cohorts of A3 spermatogonia. Chains tend to remain shorter than in the rat, with 4- and 8-cell groups predominating. Proliferative activity is greatest during stages 4 and 5 , and tapers off in stage 6 as t h e cells transform into type A , cells. There is considerable animal to animal variability in terms of t h e number of undifferentiated A's finally produced (table 1). For ten animals, there was a range from 2.75-3.72 in cell number in stage 1. Nonetheless, there was only a narrow range of 34.7-38.1 in t h e number of B spermatogonia for these same animals. Based on t h e range in A , spermatogonia, and t h e number of subsequent divisions, one would anticipate t h a t t h e number of B cells should extend over a 32-cell range rather than just a 3.4-cell range. Presumably, this gap is narrowed through a variable degree of degen-
SPERMATOGONIA IN NORMAL MOUSE TESTES
523
Time
1
Sptd 16
1 Splr Release
Fig. 1 Model for t h e Renewal and Differentiation of Spermatogonia in Mouse.
eration of A, and A3 spermatogonia. This may suggest t h a t degeneration plays a critical role in ensuring t h a t optimal numbers of cells differentiate. Similar observations have been made in the rat (Huckins, ’78a). In summary, then, morphological and quantitative analysis of the undifferentiated A population in whole mounts of seminiferous tubules in the mouse reveal a pattern of behavior which is consistent with that described for the rat. Although labeling studies comparable t o those in r a t were not undertaken, ancillary data suggest that the cell cycle kinetics of mouse germ cells would likewise be complementary (Monesi, ‘62; Oakberg, ’75; Oakberg and Huckins, ’76). As summarized in figure 1, i t is proposed t h a t through periodic divisions, A, cells both renew themselves and contribute pairs of cells t o the proliferating compartment. Within the proliferating compartment, the pairs may undergo one or several additional divisions to form chains of aligned spermatogonia (A,,). Eventually, however, mitosis ceases, and all of the spermatogonia in the proliferating compartment transform without further division into A, cells. This cohort of A , spermatogonia synchronously enters the differentiating com-
partment t o initiate maturation, and the whole process begins again. Thus, the stem cell compartment maintains a constant size while the proliferating compartment is regularly depleted and rebuilt again. By this mechanism, generations of A, spermatogonia cyclically begin differentiation. ACKNOWLEDGMENTS
We thank Greg Duncan and Lois Layton for their technical help and expertise in these experiments, and Larry Swain for his assistance in preparing the photographs. These studies were supported by HD 07655 and the U S . Energy Research and Development Administration under contract with the Union Carbide Corporation. LITERATURE CITED Clermont, Y. 1962 Quantitative analysis of spermatogenesis of the rat: A revised model for the renewal of spermatogonia. Am. J. Anat., 2 2 2 : 111-129. 1972 Kinetics of spermatogenesis in mammals: Seminiferous epithelial cycle and spermatogonial renewal. Physiol. Rev., 52: 198-236. Clermont, Y., and E. Bustos-Obregon 1968 Re-examination of spermatogonial renewal in t h e rat by means of seminiferous tubules mounted “in toto.” Am. J. Anat., 222: 231-248. Clermont, Y., and L. Hermo 1976 Spermatogonial stem cells in the albino rat. Am. J. Anat., 242: 159-176.
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C. HUCKINS AND E. F. OAKBERG
Erickson, B. H. 1976 Effect of ““Co-radiation in the stem and differentiating spermatogonia of the post puberal rat. Rad. Res., 68: 433-448. Huckins, C. 1971a Cell cycle properties of differentiating spermatogonia in adult Sprague-Dawley rats. Cell Tissue Kinet., 4: 139.154. 1971h The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat. Rec., 169: 533-558. 1971c The spermatogonial stem cell population in adult rats. 11. A radioautographic analysis of their cell cycle properties. Cell Tissue Kinet., 4: 313-334. 1971d The spermatogonial stem cell population in adult rats. 111. Evidence for a long-cycling spermatogonial population. Cell Tissue Kinet., 4: 335-349. 1978a The morphology and kinetics of spermatogonial degeneration in adult rats: An analysis using a simplified classification of the germinal epithelium. Anat. Rec., 190: 905-926. 1978b Behavior of stem cell spermatogonia in the adult rat irradiated testis. Biol. Reprod. 19: in press. Leblond, C. P., and Y. Clermont 1952 Definition of the stages of the cycle of the seminiferous epithelium of the rat. Ann. N.Y. Acad. Sci., 55: 548-573. Monesi, V. 1962 Relation between X-ray sensitivity and stages of the cell cycle on spermatogonia of the mouse. Rad. Res.. 17: 809-838. Oakberg, E. F. 1955a Sensitivity and time of degeneration of spermatogenic cells irradiated in various stages of maturation in the mouse. Radiat. Res.. 2: 369-391.
1955h Degeneration of spermatogonia of the mouse following exposure to X-rays and stages in the mitotic cycle a t which cell death appears. J. Morph., 97: 39-54. Oakberg, E. 1956 A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am. J. Anat., 99: 391-413. 1971 Spermatogonial stem-cell renewal in t h e mouse. Anat. Rec., 169: 515-532. 1975 Effects of radiation on the testis. In: Handbook of Physiology. Vol. 5. Section 7, The Male Reproductive System. E. B. Astwood and R. 0. Greep, eds. Am. Physiological Society, pp. 233-243. Oakberg, E. F., and C. Huckins 1976 Spermatogonial stem cell renewal in t h e mouse as revealed by “H-thymidine labeling and irradiation. In: Stem Cells of Renewing Cell Populations. A. B. Cairnie, P. Lala and D. G. Osmond, eds. Academic Press, New York, pp. 287-302. de Rooij, D. G. 1973 Spermatogonial stem cell renewal in the mouse. I. Normal situation. Cell Tissue Kinet., 6: 281-287. vanKeulen, C. J. G., and D. G. de Rooij 1975 Spermatogenic clones developing from repopulating stem cells surviving a high dose of an alkylating agent. I. First 15 days after injury. Cell Tissue Kinet., 8: 543-551. Withers, H. R., N. Hunter, H. T. Barkley and B. 0. Reid 1974 Radiation survival and regeneration characteristics of spermatogenic stem cells of mouse testis. Rad. Res., 57: 88-103.
All photographs are of whole mounted seminiferous tubules from normal mouse. Fixed in Bouin’s fluid and stained by Harris hematoxylin. PLATE 1 EXPLANATION O F FIGURES
2
Area of tubule in stage 6 showing cluster of B spermatogonia. Among them is a resting A, cell and an enlarged As cell (arrows). Large Sertoli cell nuclei (S)are conspicuous.
3 Area of tubule in stage 3. Undifferentiated type Aai cells (arrows) are easily discernible among t h e differentiating A3 cohort. The dark shadows are zygotene spermatocytes (Z). 4
Late stage 3 showing Aai spermatogonia (arrows) among mature A, cells. Zygotene spermatocytes (Z).
5 Aai spermatogonia (arrows) among type A, cells in stage 4. Dark shadows are young pachytene spermatocytes (P). 6
Part of a chain of Aai cells (arrows) in stage 6 among type B spermatogonia. Note the similarity in morphology of A cells in figures 3-6. Sertoli cells (S).
7
Part of a chain of eight enlarged Aai cells (arrows) in stage 6 among young type B spermatogonia. The undifferentiated A in the lower left corner is just entering early prophase. Comparison of figures 6 and 7 reveals a doubling of nuclear size, hut retention of similar morphology
SPERMATOGONIA IN NORMAL MOUSE TESTES C Huckins and E. F. Oakherg
PLATE 1
525
PLATE 2 EXPLANATION OF FIGURES
8
Single AS stem cells in prophase of mitosis (arrow). Stage 1 among pre-leptotene spermatocytes (P-L). Sertoli cells (S).
9 Pair of dividing undifferentiated A cells (arrows) in stage 1 among pre-leptotene spermatocytes (P-L). Note the cytoplasmic bridge joining t h e cells (large arrow). 10 Part of a chain of eight dividing Aal spermatogonia (arrows) in stage 6 among young type B cells (B). Sertoli cells 6 ) .
1 1 In late stage 1, typically enlarged A cells are present (arrows). In the same field, two small A spermatogonia are seen (As). Pre-leptotene spermatocytes are maturing into leptotene cells (L).
526
SPERMATOGONIA IN NORMAL MOUSE TESTES C. Huckins and E. F. Oakberg
PLATE 2
527
