Morphological and Quantitative Analysis of Spermatogonia in Mouse Testes Using Whole Mounted Seminiferous Tubules II. THE IRRADIATED 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 In adult male mice exposed to 300 R X-irradiation, the spermatogonial population was selectively killed except for the radioresistant type As stem cells. Type A spermatogonia were minimal two days after irradiation, when only 20% of the control population was present in stages 5-6; these were predominately single and paired undifferentiated cells. When multiple injections of 3HTdR were given between 2 and 3.5 days post-irradiation, 90-95%of these survivors in stages 4-6 became labeled. Enhanced proliferation of these stem cells, and a t times when they were normally quiescent, led to restoration of all classes of spermatogonia by 11 days after irradiation. Several autoradiographic studies were undertaken to better characterize the radioresistant cells. In mice given single or multiple injections of 3HTdR prior t o irradiation, there was appreciable retention of label by those type A, spermatogonia that had originally incorporated 3HTdR in stages 2-4. This labeling pattern was identical t o that of the long-cycling A, stem cells in nonirradiated testes. Since the long-cycling As stem cells are thought to be characterized by a prolonged GI or "A-phase'' which is known to be a highly radioresistant portion of the cell cycle, i t was clear why these cells could preferentially survive irradiation doses that killed other spermatogonial types. It was proposed t h a t following germ cell depletion, as after irradiation injury, the long-cycling A, survivors could be prematurely triggered from A-phase into DNA synthesis, thereby, initiating restoration of the germ cell population. The preceding article provided a detailed analysis of the undifferentiated type A spermatogonia in normal mouse testes (Huckins and Oakberg, '78). It was shown t h a t the mode of spermatogonial renewal and differentiation was identical to that described earlier in the rat (Huckins, '71b,c). The present paper will focus on the behavior of this population, particularly the class of type A, stem cells, following irradiation. It is already firmly established in mouse (Oakberg, '71, '75; Withers et al., '74; Oakberg and Huckins, '76) and in r a t (Huckins, '78c; Erickson, '76) that type A, stem cells are the radioresistant elements which survive doses of irradiation t h a t are lethal to all other spermatogonial types. In the current study, ANAT. REC. (1978)192: 529-542
analyses of whole mounted seminiferous tubules have led to elucidation of the behavior of these cells during repopulation of the irradiated epithelium. Furthermore, data from experiments in which these spermatogonia were labeled with radioactive thymidine either before or after irradiation allowed us to more fully understand their kinetic activity. As a result, an hypothesis for the interrelationship of long and short cycling stem cells will be proposed. Received Apr. 25, '78. Accepted July 19, '78. ' By acceptance of this article, the publisher or recipient acknowledges the right of the U.S.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. Operated by the Union Carbide Corporation for the Department of Energy.
529
530
C. HUCKINS AND E. F. OAKBERG MATERIALS AND METHODS
Adult male mice of t h e F , hybrid .(lo1 x C3H) strain were used in all of these experiments. Animals were exposed t o 150 R whole body radiation using a Norelco 300 kv X-ray machine operated a t 200 kv, with inherent filtration of 0.2 mmCu, 90 R/min and a targettestis distance of 53.5 cm. Animals were killed a t several times up to 11 days after irradiation. Non-irradiated control mice were also sacrificed a t each interval. Three mice were killed a t each point in each group. For the radioactive experiments, 3HTdR with a specific activity of 2-5 Ci/mmol was injected intraperitoneally at a dose of 17.5 pCi/0.25 ml/animal. Detailed protocol for each of these experiments will be given with t h e results. For all studies, testicular sections and tubular whole mounts were prepared from each animal. For histological sections, testes were fixed in Zenker formol, and embedded in paraffin; 5-c( sections were stained with PASchiff-hematoxylin. Sections for autoradiography were processed by t h e technique of Kopriwa and Huckins ('72). To prepare tubular whole mounts, segments of seminiferous t u bules were fixed in Bouin's fluid, stained in Harris hematoxylin and mounted in toto. The seminiferous epithelium was staged using a classification scheme based on t h e 6 generations of differentiating spermatogonia (Oakberg and Huckins, '76; Huckins, '78a). To analyze t h e autoradiographed sections, labeled and unlabeled undifferentiated A spermatogonia were counted in each stage in both control and irradiated groups. A total of 28 cross-sections were counted for stages 2 through 5, and 60 cross sections were counted for stage 1, giving a total of 88 tubules scored per mouse. Counts of undifferentiated A spermatogonia were made on tubular whole mounts according t o t h e procedure given in t h e preceding paper. Although boundaries between successive stages were less apparent on these irradiated whole mounts, this was not a critical handicap in counting because of t h e paucity of differentiating spermatogonia. RESULTS
Morphological analysis Spermatogonial degeneration was widespread in t h e first two days after 1 5 0 R Xirradiation. During t h a t period, almost all of
t h e undifferentiated and differentiating spermatogonia, except for t h e A, and some refractory A, cells, were eliminated from the epithelium (figs. 8, 9). Degeneration occurred in large clusters of cells, and involved cells which initially were linked by cytoplasmic bridges into a common syncytium (figs. 8,10,11). The bridges ruptured as degeneration became pronounced. The radioresistant survivors were primarily single type A, stem cells. Some of these divided as early as two days post-irradiation, and by three days, almost t h e entire A, population was enlarged preparatory to mitosis, or actually in division (figs. 12, 13). A few pairs of undifferentiated cells in late telophase or early interphase appeared by three days. Chains of A,, cells, as well as generations of A, t o B spermatogonia, were found only very rarely at these times. However, some chains of A, cells, originally in stages 6 and 1 a t the time of irradiation, persisted in the epithelium for up to four days, dying only as they a t tempted t o enter division. A few chains appeared refractory to this dose of irradiation, and these successfully completed division t o form some A, spermatogonia between two and three days post-irradiation. By five days after irradiation, single and paired undifferentiated spermatogonia were seen in all stages; many of them were enlarged, and some were dividing. Chains of undifferentiated A cells were seen infrequently in stages 6 and 1, but appeared more often in stages 4 and 5. The epithelium had begun to be replenished with differentiating spermatogonia. Dividing A, cells were few and usually paired; rarely, a dividing chain of 4 was seen. Although only occasional short chains of A, cells were observed, there was a particular abundance of chains of developing and dividing A, spermatogonia which had been A, cells at t h e time of irradiation (fig. 14). Groups of A,, In and B spermatogonia were still infrequent, while pre-leptotene through zygotene spermatocytes were extremely rare. At 8.5 days, or one cycle of t h e seminiferous epithelium after irradiation, active spermatogonial repopulation continued (fig. 15). Except for stage 1, where A cells remained infrequent, there were abundant undifferentiated spermatogonia in all stages. Dividing A, cells, and type A, spermatogonia remained sparse, and were in fact less abundant than at five days. Some A, and In spermatogonia had reap-
53 1
SPERMATOGONIA IN IRRADIATED MOUSE TESTES TABLE 1
Number of A sperrnatogonia following irradiation -
X
Interval
Stage
Frames
LA
2 days
1 2-3 4 5-6 1 2-3 4 5-6 1 2-3 4 5-6 1 2-3 4 5-6 1 3 4 5 6
408 366 382 385 617 239 402 308 171
1,080 635 482 852 1,160 342 409 215 133
3 days
5 days
8.5 days
Control
I
'
-
f
S.E.?
3.14 -t 1.72 ? 1.41 2 1.26 5 1.71 ? 1.26 k 0.95 ? 0.64 ? 0.89 ?
0.20 0.16 0.18 0.14 0.20 0.09 0.06 0.05 0.20
0.28 1.73 1.45 1.30 0.60 3.22 1.71 1.90 7.53
0.18 0.09 0.16 0.26
1.46 4.73 1.82 3.50
0.21 0.14 0.24 0.19 0.31 0.17
1.44 0.36 3.03 3.32 4.10 1.17
-
-
139 391 588 129
137 317 935 257
0.97 It 0.82 ? 1.69 1.96 -t
287 59 1 309 222 177 325
696 2,171 231 391 512 1,272
2.54 k 3.36 5 1.16 2 1.69 -t 2.77 ? 3.35 &
-
*
-
Mit. Ind. ( $ 1
-
-
-
From Huckins and Oakberg, '78. Corrected to 20 Sertoli cellslfrarne
peared, and there was a particularly noticeable increase in type B cells. Preleptotene through young pachytene spermatocytes were absent from t h e epithelium. Finally, by 11 days, all spermatogonial types had reappeared in good numbers (fig. 16). There were many enlarged and dividing undifferentiated A spermatogonia in stages 6 and 1, areas in which these cells did not normally divide. A few pre-leptotene and leptotene spermatocytes had reappeared, but more m a t u r e spermatocytes were now lacking.
Quantitative analysis Whole mounted tubules were used t o quant i t a t e surviving and repopulating A spermatogonia at 2 , 3 , 5 and 8.5 days (table 1). In a separate study, distribution of these cells as single, paired or aligned elements was made in stages 5-6 and 1 (figs. 1, 2). Minimal spermatogonial values were reached three days after irradiation in stages 5-6 where only 0.64 celldframe were recorded compared t o control levels of 3.35 celldframe (table 1). This was some 20% of t h e control population, and t h e cells were almost entirely single and paired A's which had been in stages 3-4 at t h e time of irradiation. Already by three days, many of these spermatogonia were in division. Proliferative activity reached its maximum five
-a
100
b-
00
0
I
n
CONTROL
TIME
AFTER
3d
0.5d
IRRADIATION
Fig. 1 Distribution of undifferentiated A spermatogonia as single, paired or aligned cells in stages 5-6 a t two intervals after irradiation.
days after irradiation when a mitotic index of 4.7% was recorded (table 1). Minimal numbers of A spermatogonia were not seen in t h e long stage 1 until five days post-irradiation when 0.89 cells/frame were recorded compared t o control values of 3.36 cells/frame (table 1). Counts made here were less valuable since they were made up, in
532
C. HUCKINS AND E. F. OAKBERG CONTROL
a -
0
Z 0
(c)
I R R A D I A T E D (R)
el
0 100 IQ
' z
a
CONTROL
36
5d
85d
75
-
TIME
A F T E R IRRADIATION
Ild
TIME
AFTER IRRADIATION Fig. 2 Distribution of undifferentiated A spermatogonia as single, paired or aligned cells in stage 1 a t several intervals after irradiation.
varying proportion depending on the time after irradiation, of (1) viable chains of A, cells, (2) lethally damaged A, cells which had not yet degenerated, and (3) newly formed cells. This was reflected in the persistence of chains a t five days where 24% of the population was aligned (fig. 2). Counts in other stages followed similar patterns, reaching their lowest levels between three and five days. Again, however, these counts represented a mixture of undifferentiated A and reappearing cohorts of differentiating cells. By 8.5 days after irradiation, the numbers of A spermatogonia in all stages had begun to be restored toward control values. The mitotic index in stages 5, 6 and 1, while still greater than that seen in normal testes, was less than that found a t three and five days. Counts of B spermatogonia a t the 8.5-day interval had returned t o half that found in normal testes (18.8 vs. 36.4).
Kinetic analysis of radioresistant A spermatogonia Expt. 1. Control and irradiated mice, three per group, were given single injections of "TdR 2.5 days after irradiation (fig. 3). As expected, there was incorporation of label by spermatogonia in all stages in control testes. A similar pattern emerged in irradiated testes where there was appreciable uptake of label by surviving Type A cells in all stages (fig. 3). This was particularly striking in stages 5 and 6 where single and paired A's had been shown on whole mounts to be the predominant members of the population at this time after irradiation (fig. I). Similar and confirmatory findings were obtained in mice injected with
STAGE Fig. 3 Irradiated (R) and control (C) mice were injected with 3HTdR(*) 2.5 days after irradiation and killed one hour later. The comparative distribution of labeled A spermatogonia in various stages of the cycle is shown.
3HTdR three days after irradiation (data not shown). When exposure t o the radioactive tag was increased to four injections of 3HTdR a t 12hour intervals between 2 and 3.5 days after irradiation, almost the entire radioresistant A population became labeled (fig. 4). This was most apparent in stages 4-6 where 89-97%of all surviving cells were tagged. Compared to controls where there was little change from the pattern with a single injection, there was a conspicuous augmentation in labeled cells in stage 6 and 1 of the irradiated testes. Expt. 2. Four groups of control and irradiated mice (3 per group) were given a single injection of 3HTdR 24 hours prior to irradiation (fig. 5). Two groups (C, and R,) were killed 2.5 days later. There was appreciable retention of label by the radioresistant population (Rl), especially in stages 5, 6 and 1 (fig. 5). These spermatogonia would have been in stages 3, 4 and 5-6 respectively at the time of labeling. A comparable labeling pattern was seen in the nonirradiated controls (Group Cl). Groups of irradiated and control animals were also killed two days post-irradiation with almost identical results (data not shown). In a modification of the above experiment, two groups (C, and Rz) were given a second injection of 3HTdR 2.5 days after irradiation and killed one hour later (fig. 5). The labeling percentages for groups C, and R, were su-
533
SPERMATOGONIA IN IRRADIATED MOUSE TESTES
l a
CONTROL
CONTROL (C)
GROUP C, CONTROL
L
GROUP Ce
0 IRRADIATED
RI m IRRADIATED GROUP R 2 GROUP
0 I 2 3 4 TIME A F T E R 1RRADIAT)ON
E
1 TIME
A F T E R IRRADIAT1ON
a n 50 W
J
w m
a J
s
25
0
1
2
STAGE Fig. 4 Irradiated (R) and control (C) mice were given four injections of 3HTdR (*I a t 12-hour intervals between 2 and 3.5 days after irradiation, and killed one hour after t h e last injection. The comparative distribution of labeled A spermatogonia in various stages of cycle is shown.
perimposed on those for groups C, and R, in order to visualize the amount of additional label resulting from the second injection. As expected, the second injection led t o increased labeling in both control and irradiated groups. This was particularly striking in stages 5, 6 and 1 of the irradiated groups where increments of 45%,35%and 45%respectively were recorded; in comparable control stages, there was supplemental labeling of 20%, 10% and 25% respectively. In an ancillary study, animals were given a single injection of 3HTdR 24 hours before irradiation a t a dose of 300R. Irradiated and control groups were killed a t 2 and 2.5 days thereafter. The labeling patterns following this higher dose of irradiation were almost identical t o those shown in figure 5 for Groups C1an R,. Expt. 3. In a third set of experiments, mice in Groups c6 and R6 were given six injections of 3HTdRa t 12-hour intervals; mice in Groups C, and R1 were given a single injection which coincided with the sixth (fig. 6). Mice in groups R6 and R1 were irradiated 6.5 days after the last injection of 3HTdR. All mice were killed two days later, or 8.5 days (1 cycle of the seminiferous epithelium) after the last injection of 3HTdR. Labeled spermatogonia were retained in all stages. In the case of the irradiated animals receiving a single injection of 3HTdR, most of the labeled cells were
STAGE Fig. 5 Two groups of control (C) and two groups of irradiated (R) mice were given a single injection of 3HTdR(*) one day before irradiation. At 2.5 days after irradiation, one control (C2) group and one irradiated (R,) received a second injection of 3HTdR(*). All groups were killed one hour later. The distribution of labeled spermatogonia in various stages is depicted. The percent labeled bars for groups c, and RZare superimposed on those for groups C, and R , so t h a t the amount of additional labeling caused by the second injection of 3HTdR can be visualized.
retained in stages 2-4 (fig. 6). For irradiated mice receiving multiple injections, all stages were well labeled, the maximum occurring in stage 4. Analysis of the single injection studies indicated that a t the time of the original labeling 8.5 days earlier, i t was cells in stages 2, 3 and 4 which retained label and survived irradiation. DISCUSSION
General response to irradiation Analyses of irradiated whole mounted seminiferous tubules confirm previous studies on mouse testicular sections (Oakberg, '55, '71; Oakberg and Huckins, '76) that irradiation doses of 150 R selectively eliminate the mitotically active spermatogonia from the germinal epithelium while allowing spermatocytes and spermatids to continue development. The whole mount study has permitted unequivocal identification of the spermatogonia which survive irradiation and, as well, has allowed more extensive depiction of spermatogonial behavior during repair. Within two to three days after irradiation, the germinal epithelium is devoid of most spermatogonia except for some lingering A, cells and the radioresistant A, stem cells. At subse-
534
C. HUCKINS AND E. F. OAKBERG
0".
--
-I
6
= =$"-
2
Y
7 2
-9 -8 -7 -6
-5 -4 -3 -2 -I 0 I
2 3 4 5
TIME FROM IRRADIATION CONTROL
5100 z
0
[7 IRRADIATED
a
9e
75
LL
r
W
a v)
U
r
50
O
W
dm
25
a -t
$ INJ.
1
STAGE
6
1
I
6
2
1
6
3
1
6
4
1
6
5
1
6
6
Fig. 6 Control and irradiated groups were given six injections of 3H-TdR(*)a t 12-hour intervals prior to irradiation (Cs, RJ. Other mice (G, R,) were given single injections at the time of the last multiple injection. Two groups (R, and RJ were irradiated and all mice were killed two days after irradiation and 8.5 days (1 cycle) after the last injection of 3HTdR.
quent intervals, proliferative activity of the stem cells leads t o gradual reappearance of successive spermatogonial generations.
The radiosurvivors By three days post-irradiation, minimal numbers of A cells are recorded in stages 5-6, a t which time only 20% of the control A population is present. Some 95%of these survivors are single or paired cells, while only 5%are in chains as compared to control testes where single and paired cells make up only 26% of the population. These resistant A's had been in stages 3 and 4 a t the time of irradiation. A different picture emerges in stage 1 where minimal numbers of A cells are not recorded until five days post-irradiation; these values never do drop to the nadir observed in stages 5-6, primarily due to the persistence of some A, chains. Thus, a t five days, 24%of all A s in stage 1are aligned. Several factors contribute to this. Firstly, stage 1is approximately two times longer in duration than each of the other five stages, and chains of lethally irradiated A, cells will not degenerate until they attempt to divide a t its end; thus, there is gradual loss of this population over a period of
several days. Secondly, during this same time, repopulation begins s o t h a t some newly formed chains become intermingled with those destined t o die. Thirdly, it is clear from whole mount observations and quantitation that, in addition to the A, stem cells, some A, spermatogonia survive 150 R X-irradiation and undergo normal divisions t o form A, cells (table 1, fig. 2). The most likely explanation for this is t h a t a fraction of the A, cells had been moderately radioresistant at the time of the irradiation insult. In the rat, i t has been shown that the undifferentiated A,, spermatogonia start to leave the active growth fraction in the proliferating compartment a t the beginning of stage 5 and enter the prolonged GI phase for A, cells (Huckins, ' 7 1 ~ )In . these mice, the viable A, cells would have been in a similar position in stage 5 a t the time of irradiation. Since in somatic lines, i t is well established t h a t one of the most radioresistant parts of a cell cycle occurs during G, (Little, '68; Prescott, '76; Chaffey, '711, we conclude that this could explain the persistence and continuing development of a few A, spermatogonia. Increasing the irradiation dose, however,
SPERMATOGONIA IN IRRADIATED MOUSE TESTES
does eliminate these A, spermatogonia. In experiments where the irradiation dose was increased to 300 R (Oakberg and Huckins, '761, minimal numbers of A spermatogonia- 10%of control values-were reported five days after irradiation. Moreover, they were entirely single and paired cells; no chains persisted. Likewise, in the rat testis, it has been found that 300-400 R X-ray or cobalt irradiation is necessary to eliminate most spermatogonia (Dym and Clermont, '70; Erickson, '76; Huckins, ' 7 8 ~ )Minimal . numbers of radioresistant spermatogonia are not found until between 11and 13 days, when some 11-12%of control values are present; these are for the most part A, stem cells (Huckins, ' 7 8 ~ ) .It may be concluded then that in both rats and mice this higher dose of irradiation is required to eradicate all except the A, stem cell spermatogonia. Interestingly, the sizes of the surviving radioresistant populations a t doses less than 400 R are the same as the control stem cell populations.
535
though a long-cycling stem cell component has yet t o be unequivocally identified in mouse epithelium, the available data suggests that such a population should indeed exist.
Repopulation As Oakberg ('71) has pointed out, in mice exposed to 150 R, repopulation by viable stem
cells begins before all the lethally irradiated spermatogonia have degenerated and disappeared. This is confirmed by the whole mount observations of A spermatogonia in mitosis as early as two to three days following irradiation, as well as by the presence of labeled A spermatogonia in sections taken a t this time. Moreover, as previously noted in both mouse (Oakberg, '71, '75) and rat (Dym and Clermont, '70; Huckins, '78c) enhanced mitotic activity is seen in stages in which it is normally rare or absent, namely during stages 5-6 and 1. This additional proliferation is vividly confirmed by radioisotope studies. In mice given an acute injection of 3HTdR 2.5 or 3 days after irradiation, approximately 50% of the survivIdentity and kinetics of the radiosurvivors ing A's in stage 5, and 30% of those in both It has already been shown that the A sper- stages 6 and 1 incorporate label. This is furmatogonia which survive irradiation injury ther dramatized by the fact that multiple can be labeled with 3HTdR prior to irradia- injections administered between 2 and 3.5 tion, thus confirming that they are cells in days post-irradiation label 90%or more of the active mitotic cycle (mouse, 100-1,000 R, Oak- A s in stages 4, 5 and 6, and 80% of those in berg, '64, '71; Oakberg and Huckins, '76; rat, stage 1. (The high percentage of labeled cells 330 R, Huckins, ' 7 8 ~ )Since . it is primarily A, in stages 2 through 4 in control testes, and t o a cells which survive irradiation, the inference lesser extent in irradiated testes, is likely due has been that i t is this population which is la- in part to contamination by differentiating beled and initiates repopulation (Oakberg, '7 1, spermatogonia which are difficult t o distin'75). In recent radioisotope experiments in guish from undifferentiated spermatogonia on normal and irradiated rat testis, i t has been histological sections). Clearly, then, the A, demonstrated t h a t these radioresistant A's spermatogonia which comprise most of the preferentially incorporate label during stages post-irradiation population are induced by 2-4 of the cycle of the seminiferous epithe- some factor, to divide a t times when they are lium, survive in the epithelium for periods up normally quiescent. This activity very effecto one cycle or longer, and are in all respects tively and rapidly restores the undifferentithe same as the long cycling A, spermatogonia ated A population to normal size and a t the same time allows some A s t o begin differentifound in normal testes (Huckins, ' 7 8 ~ ) The . current experiments in mice confirm a similar ation. It should be emphasized that production pattern of label uptake and retention by the of differentiating spermatogonia begins concells which will survive irradiation. Moreover, siderably before the complete rebuilding of the fact t h a t matching whole mount quantita- the undifferentiated population. Lastly, these experiments provide some tive studies show that most of the surviving spermatogonia 2.5 days post-irradiation in data on the behavior of the putative long-cystages 5-6 are single and paired A's, provides cling stem cells during repopulation. Firstly, compelling evidence that the labeled cells i t is clear from comparison of figures 4 and 6 shown in figure 6 a t the same interval are in- that a t least some of the spermatogonia which deed type A, stem cells. Thus, the cell cycle ki- incorporate label and divide after irradiation netics of the radioresistant A spermatogonia must be the same cells which had picked up in both mice and rats appears similar. Al- 3HTdR before irradiation and retained i t for
536
C. HUCKINS AND E. F. OAKBERG
one cycle. For example, in stage 6, 30%of the cells have incorporated label six days prior to irradiation and retained i t two days thereafter, while 98% of spermatogonia in stage 6 pick up label given between two and three days post irradiation. This gives support to the proposal developed in rat (Huckins, '78c) that the long-cycling A, spermatogonia are not blocked in G,, but divide and enter a prolonged GI phase. Otherwise, they would have been unable t o incorporate the post-irradiation label in the current experiments. More importantly, since GI is a highly radioresistant phase, this would explain the ability of the stem cells t o survive a radiation insult of 150 R or greater which so quickly eliminates other spermatogonial types (Oakberg and Huckins, '76; Oakberg, '78). The data of Oakberg ('78) in which labeling preceded irradiation by 24 hours, showed that the frequency of labeled cells 207 hours after irradiation was the same for controls and doses of 100, 300, 500, and 660 R. This pointed to the stability of the radioresistant population, and its likely location in G, phase. Secondly, it is clear from comparison of the irradiation data from figures 3 and 6 (fig. 7) that whereas most of the pre-irradiation labeling of the long cycling survivors occurs between late stage 2 and early stage 4, most of the post-irradiation labeling occurs in stages 5, 6 and 1. The implication is that some yet to be identified factor (possibly the paucity of differentiating spermatogonia) induces these spermatogonia to begin D N A synthesis a t times when they would normally be in a prolonged G,. The role of the long-cycling stem cells in spermatogenesis and their relationship to the short-cycling elements as well as to other spermatogonia poses an elusive and difficult problem to address experimentally. In normal steady state kinetics, two possibilities exist. The long-cycling A, could be the true stem cells in spermatogenesis, maintaining themselves and giving rise to short-cycling A, spermatogonia which go on to differentiate. Alternatively, there could be a single stem cell compartment in which spermatogonia are usually short -cycling, but periodically and randomly enter a longer cycle as suggested by the model of Smith and Martin ('73). Conceivably, the trigger t o enter a long cycle might be synthesis of D N A in stages 2-4; certainly it is well established that the long-cycling cells synthesize D N A exclusively in that period, while
a
U W
a
v,
50-
a
2
3 4 STAGE
5
6
Fig. 7 The irradiated data from figures 3 and 6 have been placed together for comparative purpses. In the case of figure 3, label was injected 2.5 days after irradiation, with sacrifice one hour later. In the case of figure 6, label was given 6.5 days pre-irradiation and mice were killed two days after irradiation, or 8.5 days after tagging. Thus, the two curves are separated by one complete cycle.
the A, spermatogonia which incorporate label a t any other time are always in short cycle. Another interesting correlate from rat data shows that this period of DNA synthesis by long-cycling cells coincides with the period of maximum degeneration of differentiating spermatogonia (Huckins, '78a,c). Regardless of which possibility is correct, an important biological mechanism exists t o conserve the stem cell line for a lifetime by sequestering some portion of it under the protective umbrella of GI where cells are relatively impervious to damage. This, moreover, insures a mechanism for rapid response to injury. Ample experimental data have documented that when the spermatogonial populations are smaller than normal adult ones, as in growth or following injury, steady state kinetics are disrupted and additional (extra) spermatogonial proliferation occurs. We propose that in such instances, most stem cells would be in short cycle, but t h a t once restoration of the epithelial population is completed, some would again be sequestered in longer cycle. There is ancillary data to support this notion. It is noteworthy, as the data in figures 4 and 6 reveal, that a t least 30%of the spermatogonia which incorporate label after irradiation in stages 4-6 must belong to the long-cycling population. Moreover, in the forming germinal epithelium of young rats, long cycling stem cells are not identifiable (Huckins, unpublished). Furthermore, the irradiation response
SPERMATOGONIA IN IRRADIATED MOUSE TESTES
in young testes is different from the adult (Erickson, '76; Selby, '731, and might be a t tributable t o the absence of a long-cycling population, as Cattanach ('77) has suggested. Lastly, recent data from split dose irradiation studies in mouse has indicated the population of radioresistant spermatogonia may become radiosensitive after exposure to the first dose of radiation (de Ruiter-Bootsma et al., '77). The implications of such a proposal would suggest a particular vulnerability of the stem cell line a t particular intervals during repair, a vulnerability that could lead t o lasting sterility through elimination of the stem cells themselves. ACKNOWLEDGMENTS
We thank Lois Layton and Pat Tyrell 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 Cattanach, B. M., I. Murray and J. M. Tracey 1977 Translocation yield from the immature mouse testes and the nature of spermatogonial stem cell sensitivity. Mut. Res., 44: 105-117. Chaffey, J. T., and S. Hellman 1971 Differing responses to radiation of murine bone marrow stem cells in relation to t h e cell cycle. Can. Res., 31: 1613-1615. Dym, M., and Y. Clermont 1970 Role of spermatogonia in t h e repair of t h e seminiferous epithelium following xirradiation of the rat testes. Am. J. Anat., 128: 265-282. Erickson, B. H. 1976 Effect of 6oCo-radiationin the stem and differentiating spermatogonia of the post pubertal rat. Rad. Res., 68: 433-448. Huckins, C. 1971a The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat. Rec., 169: 533-558. 1971b The spermatogonial stem cell population in adult rats. 11. A radioautographic analysis of their cell cycle properties. Cell Tissue Kinet., 4: 313-334. 1978a The morphology and kinetics of spermatogonial degeneration in adult rats: An analysis using
537
a simplified classification of t h e germinal epithelium. Anat. Rec., 190: 905-926. 1978b Spermatogonial intercellular bridges in whole mounted seminiferous tubules from normal and irradiated rodent tests. Am. J. Anat., 153: 97-122. 1978c Behavior of stem cell spermatogonia in t h e adult rat irradiated testis. Biol. Reprod., 19. In press. Huckins, C., and E. F. Oakberg 1978 Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules. I. The normal testes. Anat. Rec., 192: 519-528. Kopriwa, B., and C. Huckins 1972 A method for the use of Zenker-Formol fixation and the periodic acid Schiff staini n g technique in light microscope radioautography. Histochem., 32: 231-244. Little, J. B. 1978 Cellular effects of ionizing radiation. New Engl. J. Med., 278: 308-315, 369-376. Oakberg, E. F. 1955 Degeneration of spermatogonia of the mouse following exposure to X-rays and stages in the mitotic cycle at which cell death appears. J.Morph., 97: 39-54. 1964 The effects of dose, dose-rate and quality of radiation on t h e dynamics and survival of the spermatogonial population of the mouse. Japan J. Genetics (Suppl.), 40: 119-127. 1971 Spermatogonial stem-cell renewal in the mouse. Anat. Rec., 169: 515-532. 1975 Effects of radiation on the testis. In: Handbook of Physiology. Vol. 5. Section 7. The Male Reprcductive System. E. B. Astwood and R. 0. Greep, eds. Am. Physiological Society, pp. 233-243. - 1978 Differential spermatogonial stem cell survival and mutation frequency. Mut. Res., 50: 327-340. Oakberg, E. F., and C. Huckins 1976 Spermatogonial stem cell renewal in t h e mouse as revealed by 3H-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. Prescott, D. M. 1976 The cell cycle and the control of cellular reproduction. Adv. in Gen., 99-117. de Ruiter-Bootsma, A. L., M. F. Kramer and D. G. de Rooij 1977 Survival of spermatogonial stem cells in the mouse after split-dose irradiation with fission neutrons of 1MeV mean energy or 300-kV X-rays. Rad. Res., 71: 579-592. Selby, P. B. 1973 X-ray-induced specific-locus mutation rates in young male mice. Mut. Res., 18: 77-88. Smith, J. A,, and L. Martin 1973 Do cells cycle? Proc. Nat. Acad. Sci. (U.S.A.), 70: 1263-1267. 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 fixed in Bouin's fluid and stained by Harris hematoxylin. PLATE 1 EXPLANATION O F FlGURES
8
Degeneration (enclosed area) during the division of intermediate (In) spermatogonia to form type B cells. Six hours after 150 R X-irradiation.
9 Degeneration (enclosed area) during the division of type B spermatogonia. Twelve hours after 150 R X-irradiation. 10 Vertical chains of normal (right) and degenerating (left) A, spermatogonia. With the disappearance of spermatogonia, the deeper layer of pachytene (PI spermatocytes becomes conspicuous. Six hours post-irradiation. 11 Enlargement of the degenerating cells (arrows) in figure 10 reveals that intercellular bridges (*) persist until a late stage in deterioration. The Sertoli cell nuclei (S) retain a normal morphology.
538
SPERMATOGONIA IN IRRADIATED MOUSE TESTES C . Huckins and E. F. Oakberg
PLATE 1
PLATE 2 EXPLANATION OF FIGURES 12
By two days after irradiation, only occasional A, spermatogonia (arrow) are scattered in t h e germinal epithelium. Most of these are enlarged in preparation for division.
13 Two newly formed daughter cells (arrows) which have become widely separated. Two days after 1 5 0 R X-irradiation. 1 4 Chains of mature and dividing A2 cells. Some intercellular bridges Five days after 1 5 0 R X-irradiation. 15
(*I are in focus.
Branching chain of conjoined A, spermatogonia which has been regenerated. Some bridges are visible (*I. Eight and a half days post-irradiation.
16 By 1 1 days after irradiation, the epithelium is being extensively repopulated. This area depicts t h e carpet of type B cells. Sertoli cell nuclei (S) are conspicuous in the absence of pachytene spermatocytes.
540
SPERMATOGONIA IN IRRADIATED MOUSE TESTES C . Huckins and E. F. Oakberg
PLATE 2
541
'
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 In adult male mice exposed to 300 R X-irradiation, the spermatogonial population was selectively killed except for the radioresistant type As stem cells. Type A spermatogonia were minimal two days after irradiation, when only 20% of the control population was present in stages 5-6; these were predominately single and paired undifferentiated cells. When multiple injections of 3HTdR were given between 2 and 3.5 days post-irradiation, 90-95%of these survivors in stages 4-6 became labeled. Enhanced proliferation of these stem cells, and a t times when they were normally quiescent, led to restoration of all classes of spermatogonia by 11 days after irradiation. Several autoradiographic studies were undertaken to better characterize the radioresistant cells. In mice given single or multiple injections of 3HTdR prior t o irradiation, there was appreciable retention of label by those type A, spermatogonia that had originally incorporated 3HTdR in stages 2-4. This labeling pattern was identical t o that of the long-cycling A, stem cells in nonirradiated testes. Since the long-cycling As stem cells are thought to be characterized by a prolonged GI or "A-phase'' which is known to be a highly radioresistant portion of the cell cycle, i t was clear why these cells could preferentially survive irradiation doses that killed other spermatogonial types. It was proposed t h a t following germ cell depletion, as after irradiation injury, the long-cycling A, survivors could be prematurely triggered from A-phase into DNA synthesis, thereby, initiating restoration of the germ cell population. The preceding article provided a detailed analysis of the undifferentiated type A spermatogonia in normal mouse testes (Huckins and Oakberg, '78). It was shown t h a t the mode of spermatogonial renewal and differentiation was identical to that described earlier in the rat (Huckins, '71b,c). The present paper will focus on the behavior of this population, particularly the class of type A, stem cells, following irradiation. It is already firmly established in mouse (Oakberg, '71, '75; Withers et al., '74; Oakberg and Huckins, '76) and in r a t (Huckins, '78c; Erickson, '76) that type A, stem cells are the radioresistant elements which survive doses of irradiation t h a t are lethal to all other spermatogonial types. In the current study, ANAT. REC. (1978)192: 529-542
analyses of whole mounted seminiferous tubules have led to elucidation of the behavior of these cells during repopulation of the irradiated epithelium. Furthermore, data from experiments in which these spermatogonia were labeled with radioactive thymidine either before or after irradiation allowed us to more fully understand their kinetic activity. As a result, an hypothesis for the interrelationship of long and short cycling stem cells will be proposed. Received Apr. 25, '78. Accepted July 19, '78. ' By acceptance of this article, the publisher or recipient acknowledges the right of the U.S.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. Operated by the Union Carbide Corporation for the Department of Energy.
529
530
C. HUCKINS AND E. F. OAKBERG MATERIALS AND METHODS
Adult male mice of t h e F , hybrid .(lo1 x C3H) strain were used in all of these experiments. Animals were exposed t o 150 R whole body radiation using a Norelco 300 kv X-ray machine operated a t 200 kv, with inherent filtration of 0.2 mmCu, 90 R/min and a targettestis distance of 53.5 cm. Animals were killed a t several times up to 11 days after irradiation. Non-irradiated control mice were also sacrificed a t each interval. Three mice were killed a t each point in each group. For the radioactive experiments, 3HTdR with a specific activity of 2-5 Ci/mmol was injected intraperitoneally at a dose of 17.5 pCi/0.25 ml/animal. Detailed protocol for each of these experiments will be given with t h e results. For all studies, testicular sections and tubular whole mounts were prepared from each animal. For histological sections, testes were fixed in Zenker formol, and embedded in paraffin; 5-c( sections were stained with PASchiff-hematoxylin. Sections for autoradiography were processed by t h e technique of Kopriwa and Huckins ('72). To prepare tubular whole mounts, segments of seminiferous t u bules were fixed in Bouin's fluid, stained in Harris hematoxylin and mounted in toto. The seminiferous epithelium was staged using a classification scheme based on t h e 6 generations of differentiating spermatogonia (Oakberg and Huckins, '76; Huckins, '78a). To analyze t h e autoradiographed sections, labeled and unlabeled undifferentiated A spermatogonia were counted in each stage in both control and irradiated groups. A total of 28 cross-sections were counted for stages 2 through 5, and 60 cross sections were counted for stage 1, giving a total of 88 tubules scored per mouse. Counts of undifferentiated A spermatogonia were made on tubular whole mounts according t o t h e procedure given in t h e preceding paper. Although boundaries between successive stages were less apparent on these irradiated whole mounts, this was not a critical handicap in counting because of t h e paucity of differentiating spermatogonia. RESULTS
Morphological analysis Spermatogonial degeneration was widespread in t h e first two days after 1 5 0 R Xirradiation. During t h a t period, almost all of
t h e undifferentiated and differentiating spermatogonia, except for t h e A, and some refractory A, cells, were eliminated from the epithelium (figs. 8, 9). Degeneration occurred in large clusters of cells, and involved cells which initially were linked by cytoplasmic bridges into a common syncytium (figs. 8,10,11). The bridges ruptured as degeneration became pronounced. The radioresistant survivors were primarily single type A, stem cells. Some of these divided as early as two days post-irradiation, and by three days, almost t h e entire A, population was enlarged preparatory to mitosis, or actually in division (figs. 12, 13). A few pairs of undifferentiated cells in late telophase or early interphase appeared by three days. Chains of A,, cells, as well as generations of A, t o B spermatogonia, were found only very rarely at these times. However, some chains of A, cells, originally in stages 6 and 1 a t the time of irradiation, persisted in the epithelium for up to four days, dying only as they a t tempted t o enter division. A few chains appeared refractory to this dose of irradiation, and these successfully completed division t o form some A, spermatogonia between two and three days post-irradiation. By five days after irradiation, single and paired undifferentiated spermatogonia were seen in all stages; many of them were enlarged, and some were dividing. Chains of undifferentiated A cells were seen infrequently in stages 6 and 1, but appeared more often in stages 4 and 5. The epithelium had begun to be replenished with differentiating spermatogonia. Dividing A, cells were few and usually paired; rarely, a dividing chain of 4 was seen. Although only occasional short chains of A, cells were observed, there was a particular abundance of chains of developing and dividing A, spermatogonia which had been A, cells at t h e time of irradiation (fig. 14). Groups of A,, In and B spermatogonia were still infrequent, while pre-leptotene through zygotene spermatocytes were extremely rare. At 8.5 days, or one cycle of t h e seminiferous epithelium after irradiation, active spermatogonial repopulation continued (fig. 15). Except for stage 1, where A cells remained infrequent, there were abundant undifferentiated spermatogonia in all stages. Dividing A, cells, and type A, spermatogonia remained sparse, and were in fact less abundant than at five days. Some A, and In spermatogonia had reap-
53 1
SPERMATOGONIA IN IRRADIATED MOUSE TESTES TABLE 1
Number of A sperrnatogonia following irradiation -
X
Interval
Stage
Frames
LA
2 days
1 2-3 4 5-6 1 2-3 4 5-6 1 2-3 4 5-6 1 2-3 4 5-6 1 3 4 5 6
408 366 382 385 617 239 402 308 171
1,080 635 482 852 1,160 342 409 215 133
3 days
5 days
8.5 days
Control
I
'
-
f
S.E.?
3.14 -t 1.72 ? 1.41 2 1.26 5 1.71 ? 1.26 k 0.95 ? 0.64 ? 0.89 ?
0.20 0.16 0.18 0.14 0.20 0.09 0.06 0.05 0.20
0.28 1.73 1.45 1.30 0.60 3.22 1.71 1.90 7.53
0.18 0.09 0.16 0.26
1.46 4.73 1.82 3.50
0.21 0.14 0.24 0.19 0.31 0.17
1.44 0.36 3.03 3.32 4.10 1.17
-
-
139 391 588 129
137 317 935 257
0.97 It 0.82 ? 1.69 1.96 -t
287 59 1 309 222 177 325
696 2,171 231 391 512 1,272
2.54 k 3.36 5 1.16 2 1.69 -t 2.77 ? 3.35 &
-
*
-
Mit. Ind. ( $ 1
-
-
-
From Huckins and Oakberg, '78. Corrected to 20 Sertoli cellslfrarne
peared, and there was a particularly noticeable increase in type B cells. Preleptotene through young pachytene spermatocytes were absent from t h e epithelium. Finally, by 11 days, all spermatogonial types had reappeared in good numbers (fig. 16). There were many enlarged and dividing undifferentiated A spermatogonia in stages 6 and 1, areas in which these cells did not normally divide. A few pre-leptotene and leptotene spermatocytes had reappeared, but more m a t u r e spermatocytes were now lacking.
Quantitative analysis Whole mounted tubules were used t o quant i t a t e surviving and repopulating A spermatogonia at 2 , 3 , 5 and 8.5 days (table 1). In a separate study, distribution of these cells as single, paired or aligned elements was made in stages 5-6 and 1 (figs. 1, 2). Minimal spermatogonial values were reached three days after irradiation in stages 5-6 where only 0.64 celldframe were recorded compared t o control levels of 3.35 celldframe (table 1). This was some 20% of t h e control population, and t h e cells were almost entirely single and paired A's which had been in stages 3-4 at t h e time of irradiation. Already by three days, many of these spermatogonia were in division. Proliferative activity reached its maximum five
-a
100
b-
00
0
I
n
CONTROL
TIME
AFTER
3d
0.5d
IRRADIATION
Fig. 1 Distribution of undifferentiated A spermatogonia as single, paired or aligned cells in stages 5-6 a t two intervals after irradiation.
days after irradiation when a mitotic index of 4.7% was recorded (table 1). Minimal numbers of A spermatogonia were not seen in t h e long stage 1 until five days post-irradiation when 0.89 cells/frame were recorded compared t o control values of 3.36 cells/frame (table 1). Counts made here were less valuable since they were made up, in
532
C. HUCKINS AND E. F. OAKBERG CONTROL
a -
0
Z 0
(c)
I R R A D I A T E D (R)
el
0 100 IQ
' z
a
CONTROL
36
5d
85d
75
-
TIME
A F T E R IRRADIATION
Ild
TIME
AFTER IRRADIATION Fig. 2 Distribution of undifferentiated A spermatogonia as single, paired or aligned cells in stage 1 a t several intervals after irradiation.
varying proportion depending on the time after irradiation, of (1) viable chains of A, cells, (2) lethally damaged A, cells which had not yet degenerated, and (3) newly formed cells. This was reflected in the persistence of chains a t five days where 24% of the population was aligned (fig. 2). Counts in other stages followed similar patterns, reaching their lowest levels between three and five days. Again, however, these counts represented a mixture of undifferentiated A and reappearing cohorts of differentiating cells. By 8.5 days after irradiation, the numbers of A spermatogonia in all stages had begun to be restored toward control values. The mitotic index in stages 5, 6 and 1, while still greater than that seen in normal testes, was less than that found a t three and five days. Counts of B spermatogonia a t the 8.5-day interval had returned t o half that found in normal testes (18.8 vs. 36.4).
Kinetic analysis of radioresistant A spermatogonia Expt. 1. Control and irradiated mice, three per group, were given single injections of "TdR 2.5 days after irradiation (fig. 3). As expected, there was incorporation of label by spermatogonia in all stages in control testes. A similar pattern emerged in irradiated testes where there was appreciable uptake of label by surviving Type A cells in all stages (fig. 3). This was particularly striking in stages 5 and 6 where single and paired A's had been shown on whole mounts to be the predominant members of the population at this time after irradiation (fig. I). Similar and confirmatory findings were obtained in mice injected with
STAGE Fig. 3 Irradiated (R) and control (C) mice were injected with 3HTdR(*) 2.5 days after irradiation and killed one hour later. The comparative distribution of labeled A spermatogonia in various stages of the cycle is shown.
3HTdR three days after irradiation (data not shown). When exposure t o the radioactive tag was increased to four injections of 3HTdR a t 12hour intervals between 2 and 3.5 days after irradiation, almost the entire radioresistant A population became labeled (fig. 4). This was most apparent in stages 4-6 where 89-97%of all surviving cells were tagged. Compared to controls where there was little change from the pattern with a single injection, there was a conspicuous augmentation in labeled cells in stage 6 and 1 of the irradiated testes. Expt. 2. Four groups of control and irradiated mice (3 per group) were given a single injection of 3HTdR 24 hours prior to irradiation (fig. 5). Two groups (C, and R,) were killed 2.5 days later. There was appreciable retention of label by the radioresistant population (Rl), especially in stages 5, 6 and 1 (fig. 5). These spermatogonia would have been in stages 3, 4 and 5-6 respectively at the time of labeling. A comparable labeling pattern was seen in the nonirradiated controls (Group Cl). Groups of irradiated and control animals were also killed two days post-irradiation with almost identical results (data not shown). In a modification of the above experiment, two groups (C, and Rz) were given a second injection of 3HTdR 2.5 days after irradiation and killed one hour later (fig. 5). The labeling percentages for groups C, and R, were su-
533
SPERMATOGONIA IN IRRADIATED MOUSE TESTES
l a
CONTROL
CONTROL (C)
GROUP C, CONTROL
L
GROUP Ce
0 IRRADIATED
RI m IRRADIATED GROUP R 2 GROUP
0 I 2 3 4 TIME A F T E R 1RRADIAT)ON
E
1 TIME
A F T E R IRRADIAT1ON
a n 50 W
J
w m
a J
s
25
0
1
2
STAGE Fig. 4 Irradiated (R) and control (C) mice were given four injections of 3HTdR (*I a t 12-hour intervals between 2 and 3.5 days after irradiation, and killed one hour after t h e last injection. The comparative distribution of labeled A spermatogonia in various stages of cycle is shown.
perimposed on those for groups C, and R, in order to visualize the amount of additional label resulting from the second injection. As expected, the second injection led t o increased labeling in both control and irradiated groups. This was particularly striking in stages 5, 6 and 1 of the irradiated groups where increments of 45%,35%and 45%respectively were recorded; in comparable control stages, there was supplemental labeling of 20%, 10% and 25% respectively. In an ancillary study, animals were given a single injection of 3HTdR 24 hours before irradiation a t a dose of 300R. Irradiated and control groups were killed a t 2 and 2.5 days thereafter. The labeling patterns following this higher dose of irradiation were almost identical t o those shown in figure 5 for Groups C1an R,. Expt. 3. In a third set of experiments, mice in Groups c6 and R6 were given six injections of 3HTdRa t 12-hour intervals; mice in Groups C, and R1 were given a single injection which coincided with the sixth (fig. 6). Mice in groups R6 and R1 were irradiated 6.5 days after the last injection of 3HTdR. All mice were killed two days later, or 8.5 days (1 cycle of the seminiferous epithelium) after the last injection of 3HTdR. Labeled spermatogonia were retained in all stages. In the case of the irradiated animals receiving a single injection of 3HTdR, most of the labeled cells were
STAGE Fig. 5 Two groups of control (C) and two groups of irradiated (R) mice were given a single injection of 3HTdR(*) one day before irradiation. At 2.5 days after irradiation, one control (C2) group and one irradiated (R,) received a second injection of 3HTdR(*). All groups were killed one hour later. The distribution of labeled spermatogonia in various stages is depicted. The percent labeled bars for groups c, and RZare superimposed on those for groups C, and R , so t h a t the amount of additional labeling caused by the second injection of 3HTdR can be visualized.
retained in stages 2-4 (fig. 6). For irradiated mice receiving multiple injections, all stages were well labeled, the maximum occurring in stage 4. Analysis of the single injection studies indicated that a t the time of the original labeling 8.5 days earlier, i t was cells in stages 2, 3 and 4 which retained label and survived irradiation. DISCUSSION
General response to irradiation Analyses of irradiated whole mounted seminiferous tubules confirm previous studies on mouse testicular sections (Oakberg, '55, '71; Oakberg and Huckins, '76) that irradiation doses of 150 R selectively eliminate the mitotically active spermatogonia from the germinal epithelium while allowing spermatocytes and spermatids to continue development. The whole mount study has permitted unequivocal identification of the spermatogonia which survive irradiation and, as well, has allowed more extensive depiction of spermatogonial behavior during repair. Within two to three days after irradiation, the germinal epithelium is devoid of most spermatogonia except for some lingering A, cells and the radioresistant A, stem cells. At subse-
534
C. HUCKINS AND E. F. OAKBERG
0".
--
-I
6
= =$"-
2
Y
7 2
-9 -8 -7 -6
-5 -4 -3 -2 -I 0 I
2 3 4 5
TIME FROM IRRADIATION CONTROL
5100 z
0
[7 IRRADIATED
a
9e
75
LL
r
W
a v)
U
r
50
O
W
dm
25
a -t
$ INJ.
1
STAGE
6
1
I
6
2
1
6
3
1
6
4
1
6
5
1
6
6
Fig. 6 Control and irradiated groups were given six injections of 3H-TdR(*)a t 12-hour intervals prior to irradiation (Cs, RJ. Other mice (G, R,) were given single injections at the time of the last multiple injection. Two groups (R, and RJ were irradiated and all mice were killed two days after irradiation and 8.5 days (1 cycle) after the last injection of 3HTdR.
quent intervals, proliferative activity of the stem cells leads t o gradual reappearance of successive spermatogonial generations.
The radiosurvivors By three days post-irradiation, minimal numbers of A cells are recorded in stages 5-6, a t which time only 20% of the control A population is present. Some 95%of these survivors are single or paired cells, while only 5%are in chains as compared to control testes where single and paired cells make up only 26% of the population. These resistant A's had been in stages 3 and 4 a t the time of irradiation. A different picture emerges in stage 1 where minimal numbers of A cells are not recorded until five days post-irradiation; these values never do drop to the nadir observed in stages 5-6, primarily due to the persistence of some A, chains. Thus, a t five days, 24%of all A s in stage 1are aligned. Several factors contribute to this. Firstly, stage 1is approximately two times longer in duration than each of the other five stages, and chains of lethally irradiated A, cells will not degenerate until they attempt to divide a t its end; thus, there is gradual loss of this population over a period of
several days. Secondly, during this same time, repopulation begins s o t h a t some newly formed chains become intermingled with those destined t o die. Thirdly, it is clear from whole mount observations and quantitation that, in addition to the A, stem cells, some A, spermatogonia survive 150 R X-irradiation and undergo normal divisions t o form A, cells (table 1, fig. 2). The most likely explanation for this is t h a t a fraction of the A, cells had been moderately radioresistant at the time of the irradiation insult. In the rat, i t has been shown that the undifferentiated A,, spermatogonia start to leave the active growth fraction in the proliferating compartment a t the beginning of stage 5 and enter the prolonged GI phase for A, cells (Huckins, ' 7 1 ~ )In . these mice, the viable A, cells would have been in a similar position in stage 5 a t the time of irradiation. Since in somatic lines, i t is well established t h a t one of the most radioresistant parts of a cell cycle occurs during G, (Little, '68; Prescott, '76; Chaffey, '711, we conclude that this could explain the persistence and continuing development of a few A, spermatogonia. Increasing the irradiation dose, however,
SPERMATOGONIA IN IRRADIATED MOUSE TESTES
does eliminate these A, spermatogonia. In experiments where the irradiation dose was increased to 300 R (Oakberg and Huckins, '761, minimal numbers of A spermatogonia- 10%of control values-were reported five days after irradiation. Moreover, they were entirely single and paired cells; no chains persisted. Likewise, in the rat testis, it has been found that 300-400 R X-ray or cobalt irradiation is necessary to eliminate most spermatogonia (Dym and Clermont, '70; Erickson, '76; Huckins, ' 7 8 ~ )Minimal . numbers of radioresistant spermatogonia are not found until between 11and 13 days, when some 11-12%of control values are present; these are for the most part A, stem cells (Huckins, ' 7 8 ~ ) .It may be concluded then that in both rats and mice this higher dose of irradiation is required to eradicate all except the A, stem cell spermatogonia. Interestingly, the sizes of the surviving radioresistant populations a t doses less than 400 R are the same as the control stem cell populations.
535
though a long-cycling stem cell component has yet t o be unequivocally identified in mouse epithelium, the available data suggests that such a population should indeed exist.
Repopulation As Oakberg ('71) has pointed out, in mice exposed to 150 R, repopulation by viable stem
cells begins before all the lethally irradiated spermatogonia have degenerated and disappeared. This is confirmed by the whole mount observations of A spermatogonia in mitosis as early as two to three days following irradiation, as well as by the presence of labeled A spermatogonia in sections taken a t this time. Moreover, as previously noted in both mouse (Oakberg, '71, '75) and rat (Dym and Clermont, '70; Huckins, '78c) enhanced mitotic activity is seen in stages in which it is normally rare or absent, namely during stages 5-6 and 1. This additional proliferation is vividly confirmed by radioisotope studies. In mice given an acute injection of 3HTdR 2.5 or 3 days after irradiation, approximately 50% of the survivIdentity and kinetics of the radiosurvivors ing A's in stage 5, and 30% of those in both It has already been shown that the A sper- stages 6 and 1 incorporate label. This is furmatogonia which survive irradiation injury ther dramatized by the fact that multiple can be labeled with 3HTdR prior to irradia- injections administered between 2 and 3.5 tion, thus confirming that they are cells in days post-irradiation label 90%or more of the active mitotic cycle (mouse, 100-1,000 R, Oak- A s in stages 4, 5 and 6, and 80% of those in berg, '64, '71; Oakberg and Huckins, '76; rat, stage 1. (The high percentage of labeled cells 330 R, Huckins, ' 7 8 ~ )Since . it is primarily A, in stages 2 through 4 in control testes, and t o a cells which survive irradiation, the inference lesser extent in irradiated testes, is likely due has been that i t is this population which is la- in part to contamination by differentiating beled and initiates repopulation (Oakberg, '7 1, spermatogonia which are difficult t o distin'75). In recent radioisotope experiments in guish from undifferentiated spermatogonia on normal and irradiated rat testis, i t has been histological sections). Clearly, then, the A, demonstrated t h a t these radioresistant A's spermatogonia which comprise most of the preferentially incorporate label during stages post-irradiation population are induced by 2-4 of the cycle of the seminiferous epithe- some factor, to divide a t times when they are lium, survive in the epithelium for periods up normally quiescent. This activity very effecto one cycle or longer, and are in all respects tively and rapidly restores the undifferentithe same as the long cycling A, spermatogonia ated A population to normal size and a t the same time allows some A s t o begin differentifound in normal testes (Huckins, ' 7 8 ~ ) The . current experiments in mice confirm a similar ation. It should be emphasized that production pattern of label uptake and retention by the of differentiating spermatogonia begins concells which will survive irradiation. Moreover, siderably before the complete rebuilding of the fact t h a t matching whole mount quantita- the undifferentiated population. Lastly, these experiments provide some tive studies show that most of the surviving spermatogonia 2.5 days post-irradiation in data on the behavior of the putative long-cystages 5-6 are single and paired A's, provides cling stem cells during repopulation. Firstly, compelling evidence that the labeled cells i t is clear from comparison of figures 4 and 6 shown in figure 6 a t the same interval are in- that a t least some of the spermatogonia which deed type A, stem cells. Thus, the cell cycle ki- incorporate label and divide after irradiation netics of the radioresistant A spermatogonia must be the same cells which had picked up in both mice and rats appears similar. Al- 3HTdR before irradiation and retained i t for
536
C. HUCKINS AND E. F. OAKBERG
one cycle. For example, in stage 6, 30%of the cells have incorporated label six days prior to irradiation and retained i t two days thereafter, while 98% of spermatogonia in stage 6 pick up label given between two and three days post irradiation. This gives support to the proposal developed in rat (Huckins, '78c) that the long-cycling A, spermatogonia are not blocked in G,, but divide and enter a prolonged GI phase. Otherwise, they would have been unable t o incorporate the post-irradiation label in the current experiments. More importantly, since GI is a highly radioresistant phase, this would explain the ability of the stem cells t o survive a radiation insult of 150 R or greater which so quickly eliminates other spermatogonial types (Oakberg and Huckins, '76; Oakberg, '78). The data of Oakberg ('78) in which labeling preceded irradiation by 24 hours, showed that the frequency of labeled cells 207 hours after irradiation was the same for controls and doses of 100, 300, 500, and 660 R. This pointed to the stability of the radioresistant population, and its likely location in G, phase. Secondly, it is clear from comparison of the irradiation data from figures 3 and 6 (fig. 7) that whereas most of the pre-irradiation labeling of the long cycling survivors occurs between late stage 2 and early stage 4, most of the post-irradiation labeling occurs in stages 5, 6 and 1. The implication is that some yet to be identified factor (possibly the paucity of differentiating spermatogonia) induces these spermatogonia to begin D N A synthesis a t times when they would normally be in a prolonged G,. The role of the long-cycling stem cells in spermatogenesis and their relationship to the short-cycling elements as well as to other spermatogonia poses an elusive and difficult problem to address experimentally. In normal steady state kinetics, two possibilities exist. The long-cycling A, could be the true stem cells in spermatogenesis, maintaining themselves and giving rise to short-cycling A, spermatogonia which go on to differentiate. Alternatively, there could be a single stem cell compartment in which spermatogonia are usually short -cycling, but periodically and randomly enter a longer cycle as suggested by the model of Smith and Martin ('73). Conceivably, the trigger t o enter a long cycle might be synthesis of D N A in stages 2-4; certainly it is well established that the long-cycling cells synthesize D N A exclusively in that period, while
a
U W
a
v,
50-
a
2
3 4 STAGE
5
6
Fig. 7 The irradiated data from figures 3 and 6 have been placed together for comparative purpses. In the case of figure 3, label was injected 2.5 days after irradiation, with sacrifice one hour later. In the case of figure 6, label was given 6.5 days pre-irradiation and mice were killed two days after irradiation, or 8.5 days after tagging. Thus, the two curves are separated by one complete cycle.
the A, spermatogonia which incorporate label a t any other time are always in short cycle. Another interesting correlate from rat data shows that this period of DNA synthesis by long-cycling cells coincides with the period of maximum degeneration of differentiating spermatogonia (Huckins, '78a,c). Regardless of which possibility is correct, an important biological mechanism exists t o conserve the stem cell line for a lifetime by sequestering some portion of it under the protective umbrella of GI where cells are relatively impervious to damage. This, moreover, insures a mechanism for rapid response to injury. Ample experimental data have documented that when the spermatogonial populations are smaller than normal adult ones, as in growth or following injury, steady state kinetics are disrupted and additional (extra) spermatogonial proliferation occurs. We propose that in such instances, most stem cells would be in short cycle, but t h a t once restoration of the epithelial population is completed, some would again be sequestered in longer cycle. There is ancillary data to support this notion. It is noteworthy, as the data in figures 4 and 6 reveal, that a t least 30%of the spermatogonia which incorporate label after irradiation in stages 4-6 must belong to the long-cycling population. Moreover, in the forming germinal epithelium of young rats, long cycling stem cells are not identifiable (Huckins, unpublished). Furthermore, the irradiation response
SPERMATOGONIA IN IRRADIATED MOUSE TESTES
in young testes is different from the adult (Erickson, '76; Selby, '731, and might be a t tributable t o the absence of a long-cycling population, as Cattanach ('77) has suggested. Lastly, recent data from split dose irradiation studies in mouse has indicated the population of radioresistant spermatogonia may become radiosensitive after exposure to the first dose of radiation (de Ruiter-Bootsma et al., '77). The implications of such a proposal would suggest a particular vulnerability of the stem cell line a t particular intervals during repair, a vulnerability that could lead t o lasting sterility through elimination of the stem cells themselves. ACKNOWLEDGMENTS
We thank Lois Layton and Pat Tyrell 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 Cattanach, B. M., I. Murray and J. M. Tracey 1977 Translocation yield from the immature mouse testes and the nature of spermatogonial stem cell sensitivity. Mut. Res., 44: 105-117. Chaffey, J. T., and S. Hellman 1971 Differing responses to radiation of murine bone marrow stem cells in relation to t h e cell cycle. Can. Res., 31: 1613-1615. Dym, M., and Y. Clermont 1970 Role of spermatogonia in t h e repair of t h e seminiferous epithelium following xirradiation of the rat testes. Am. J. Anat., 128: 265-282. Erickson, B. H. 1976 Effect of 6oCo-radiationin the stem and differentiating spermatogonia of the post pubertal rat. Rad. Res., 68: 433-448. Huckins, C. 1971a The spermatogonial stem cell population in adult rats. I. Their morphology, proliferation and maturation. Anat. Rec., 169: 533-558. 1971b The spermatogonial stem cell population in adult rats. 11. A radioautographic analysis of their cell cycle properties. Cell Tissue Kinet., 4: 313-334. 1978a The morphology and kinetics of spermatogonial degeneration in adult rats: An analysis using
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a simplified classification of t h e germinal epithelium. Anat. Rec., 190: 905-926. 1978b Spermatogonial intercellular bridges in whole mounted seminiferous tubules from normal and irradiated rodent tests. Am. J. Anat., 153: 97-122. 1978c Behavior of stem cell spermatogonia in t h e adult rat irradiated testis. Biol. Reprod., 19. In press. Huckins, C., and E. F. Oakberg 1978 Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules. I. The normal testes. Anat. Rec., 192: 519-528. Kopriwa, B., and C. Huckins 1972 A method for the use of Zenker-Formol fixation and the periodic acid Schiff staini n g technique in light microscope radioautography. Histochem., 32: 231-244. Little, J. B. 1978 Cellular effects of ionizing radiation. New Engl. J. Med., 278: 308-315, 369-376. Oakberg, E. F. 1955 Degeneration of spermatogonia of the mouse following exposure to X-rays and stages in the mitotic cycle at which cell death appears. J.Morph., 97: 39-54. 1964 The effects of dose, dose-rate and quality of radiation on t h e dynamics and survival of the spermatogonial population of the mouse. Japan J. Genetics (Suppl.), 40: 119-127. 1971 Spermatogonial stem-cell renewal in the mouse. Anat. Rec., 169: 515-532. 1975 Effects of radiation on the testis. In: Handbook of Physiology. Vol. 5. Section 7. The Male Reprcductive System. E. B. Astwood and R. 0. Greep, eds. Am. Physiological Society, pp. 233-243. - 1978 Differential spermatogonial stem cell survival and mutation frequency. Mut. Res., 50: 327-340. Oakberg, E. F., and C. Huckins 1976 Spermatogonial stem cell renewal in t h e mouse as revealed by 3H-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. Prescott, D. M. 1976 The cell cycle and the control of cellular reproduction. Adv. in Gen., 99-117. de Ruiter-Bootsma, A. L., M. F. Kramer and D. G. de Rooij 1977 Survival of spermatogonial stem cells in the mouse after split-dose irradiation with fission neutrons of 1MeV mean energy or 300-kV X-rays. Rad. Res., 71: 579-592. Selby, P. B. 1973 X-ray-induced specific-locus mutation rates in young male mice. Mut. Res., 18: 77-88. Smith, J. A,, and L. Martin 1973 Do cells cycle? Proc. Nat. Acad. Sci. (U.S.A.), 70: 1263-1267. 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 fixed in Bouin's fluid and stained by Harris hematoxylin. PLATE 1 EXPLANATION O F FlGURES
8
Degeneration (enclosed area) during the division of intermediate (In) spermatogonia to form type B cells. Six hours after 150 R X-irradiation.
9 Degeneration (enclosed area) during the division of type B spermatogonia. Twelve hours after 150 R X-irradiation. 10 Vertical chains of normal (right) and degenerating (left) A, spermatogonia. With the disappearance of spermatogonia, the deeper layer of pachytene (PI spermatocytes becomes conspicuous. Six hours post-irradiation. 11 Enlargement of the degenerating cells (arrows) in figure 10 reveals that intercellular bridges (*) persist until a late stage in deterioration. The Sertoli cell nuclei (S) retain a normal morphology.
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SPERMATOGONIA IN IRRADIATED MOUSE TESTES C . Huckins and E. F. Oakberg
PLATE 1
PLATE 2 EXPLANATION OF FIGURES 12
By two days after irradiation, only occasional A, spermatogonia (arrow) are scattered in t h e germinal epithelium. Most of these are enlarged in preparation for division.
13 Two newly formed daughter cells (arrows) which have become widely separated. Two days after 1 5 0 R X-irradiation. 1 4 Chains of mature and dividing A2 cells. Some intercellular bridges Five days after 1 5 0 R X-irradiation. 15
(*I are in focus.
Branching chain of conjoined A, spermatogonia which has been regenerated. Some bridges are visible (*I. Eight and a half days post-irradiation.
16 By 1 1 days after irradiation, the epithelium is being extensively repopulated. This area depicts t h e carpet of type B cells. Sertoli cell nuclei (S) are conspicuous in the absence of pachytene spermatocytes.
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SPERMATOGONIA IN IRRADIATED MOUSE TESTES C . Huckins and E. F. Oakberg
PLATE 2
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