- Email: [email protected]
Polyploidization of liver after partial hepatectomy in the dwarf mouse and hypophysectomized rat: Effect of extended regenerative periods
0 1967 by Academic Press Inc. Experimental
Cell Research 48, 557-568 (1967)
557
POLYPLOIDIZATION OF LIVER AFTER PARTIAL HEPATECTOMY IN THE DWARF MOUSE AND HYPOPHYSECTOMIZED RAT: EFFECT OF EXTENDED REGENERATIVE PERIODS F. J. SWARTZ Department
of Anatomy, University Louisville, Ky Received
of Louisville
School of Medicine,
40202,U.S.A.
February
17, 1967s
ALTHOUGH it is generally
known that the livers of most mammals become predominantly polyploid during postnatal development [l, 13, 181, various attempts to understand the significance of this phenomenon have led to certain discrepancies concerning its control. For example, the failure of nuclear classes to develop in the hereditary pituitary dwarf mouse [9], the arrest of polyploidization in the rat by hypophysectomy [6] and restoration of polyploidy in the dwarf mouse by growth hormone replacement [9, 111 have all implied the requirement of pituitary growth hormone. Other studies have implicated growth hormone indirectly [4, 151. Recently, it has been suggested that any factor which prevents liver growth will also inhibit the formation of polyploid cells [12]. Conversely, it has been reported that large number of polyploid cells can be formed during liver regloneration in the hypophysectomized rat [8]. In fact, more polyploid nuclei were observed in the liver during and following regeneration than in livers of non-hepatectomized animals. Increased polyploidy in normal animals, which persists indefinitely following hepatectomy, has also been reported frequently ([3, lo] for reviews). That this excess “polyploidy” is due in part to delay of the liver parenchyma to equilibrate post-operatively is suggested by the work of James et al. [lo], who extended the post-hepatectomy period in rats to two months and observed a steady depression in the percentage of polyploid cells formed during regeneration. At the conclusion of the experiment, however, the polyploid population remained elevated. Thus, two problems are presented. The first concerns the role of the 1 Supported by USPHS Research Grant HD-01245. s Revised version received June 11, 1967. Experimental
Cell Research 48
558
F. J. Swartz
pituitary in polyploidization and the second the question of “over-polyploidization” of liver following partial hepatectomy. The current study was designed to approach both problems by [ 1 ] insuring a sufficiently long period free of pituitary influence before hepatectomy, and [2] a posthepatectomy period sufficiently long to permit equilibration of the parenchyma. At the same time, the regenerative period was hastened by removing only a relatively small amount of hepatic tissue, while the use of adult, relatively stabilized, animals permitted interpretation of the cytological events of regeneration in the absence of those events accompanying normal polyploidization. Two separate studies are reported. In the first, “adult” pituitary dwarf mice, selected because of presumed postnatal absence of growth hormone [7], were hepatectomized and maintained for one month. The somewhat equivocal results, arising from difficulty in maintaining the post-hepatectomy dwarfs and subsequent loss of desirable controls, necessitated the second study. In this, hypophysectomized adult rats were maintained for two months, partially hepatectomized and killed two months later. Equilibration of the hepatic parenchyma was apparently assured by the longer post-hypophysectomy and post-hepatectomy periods, and the minimal hepatectomy. It will be shown that the events interpreted as excess polypioidization in both the intact and hypophysectomized rat are observed only temporarily and probably represent residual regenerative activity. In the normal mouse, the percentage of polyploid nuclei is much higher than in the rat, and response to partial hepatectomy more prolonged. However, a tendency to return to control levels of polyploidy following hepatectomy is demonstrated. In the dwarf liver, equilibration is apparently further delayed.
MATERIALS
AND
METHODS
Hereditary pituitary dwarf mice.-Male and female dwarf mice and their normal littermates, from Jackson Memorial Laboratory stock, were maintained until 70 days of age and subdivided into experimental and control groups (Fig. 1). Ten normal mice (group A) were partially hepatectomized by ligating and removing the left lateral lobe, which was used for determining percentages of polyploid nuclei. Of the 10 mice, 6 survived until 100 days of age at which time they were killed by cervical dislocation and their livers analyzed for polyploidy (group B). Eight normal, unoperated animals, killed at 100 days of age, served as normal controls (group Cl. Eighteen dwarf mice were treated in a manner parallel to that of the normal mice. Eleven were partially hepatectomized at 70 days (group D) and the 8 survivors killed at 100 days (group E). Seven unoperated dwarf controls were killed at 100 Experimental
Cell Research 48
Polyploidy after partial hepafecfomy
559
days (group F). Livers were analyzed for polyploidy both at time of bepatectomy and at the termination of the experiment. Hypophysectomized rats.-The experimental procedure is diagrammed in Fig. 2. Fifty-one male Sprague-Dawley rats were selected for homogeneity of age (108-112 days) and size (353-393 g). Of 31 non-hypopbysectomized animals, ten were killed PARTIAL
I (6)-KILLE,,
(6)
AGE
(DAYS)
70
(B)
KILLED
(0
100
Fig. l.-Experimental procedure for dwarf mouse study. Letters in parentheses indicate experimental groups and numbers in parentheses, number of animals.
on day 110 (group A) to determine percentages of hepatic polyploid nuclei on day 0 of the study. Ten normal animals were killed at 167 days (group B) and, of the remaining 11 non-hypophysectomized animals, 6 were partially hepatectomized (left lateral lobe) on day 219 (group C). The four surviving hepatectomized animals of group C were killed on day 273 (group D) as were the remaining 5 unoperated controls (group E). This progression yielded the polyploid percentages of a series of normal livers from 110 to 273 days of age as well as the effect of partial hepatectomy on hepatic polyploid composition two months after the operation. Twenty of the animals were hypophysectomized (Hormone Assay Laboratories, Chicago) on day 110. Their hepatic polyploid state at time of operation is reflected by that of group A. Of the 20, 10 were killed on day 167 (group F), and, of the ten remaining animals, 5 were partially hepatectomized on day 219 (group G) and killed on day 273 (group H). The 5 remaining non-hepatectomized animals were also killed on day 273 (group J). A progression parallel to that of the non-hypophysectomized animals was thus provided for the hypophysectomized group. The relatively large number of control groups was necessitated by the extended time of the experiment, during which the control, or normal, polyploid populations, although relatively stabilized, nevertheless continued to increase gradually. Changes in polyploidy following hepatectomy were, therefore, measured against a continually changing control population. General procedures Percentages of polyploid nuclei.-Two mm cubes of liver were removed from the left lateral lobes of animals with intact livers and from lobes removed at hepatectomy. Experimental Cell Research 48
560
F. J. Swartz
Cubes from the right lobe were removed from previously hepatectomized animals. Tissues were fixed overnight in Lavdowsky’s fluid (formalin, 5 parts; 95 per cent ethyl alcohol, IO parts; glacial acetic acid, 1 part; distilled water, 40 parts) and processed routinely. Ten and 15 ,U sections were Feulgen stained under controlled conditions. DAYS
AFTER
AGE
(DAYS)
HYPOPHYSECTOMY
0
57
109
163
I IO
167
219
273
KILLED NON-HYPOPHYSECTOMIZED (31)
t
(A)
.9-c \ -141-KILLED
-I
‘KILLED
KILLED HYPOPHYHYPOPHYSECTOMIZED (20)
(D)
!E)
(F)
7
SECToMY Ml
yTRT--, %KlLLED SKILLED
(HI (J)
Fig. Z.-Experimental procedure for rat liypophysectomy study. Letters in parenthesesindicate experimental groups and numbers in parentheses,number of animals. In this laboratory percentages of polyploid nuclei are determined by first establishing a DNA-volume relationship. DNA per nucleus, in arbitrary units, is measured by cytophotometry on at least 50 nuclei per IO ,u section, using a Barr and Stroud Integrating Microdensitometer [5]. Nuclei are measured selectively rather than randomly with the intent of finding all existing combinations of DNA-volume relationships. If the results indicate a strict DNA-volume relationship (e.g. Fig. 3), photographs are made of the 15 y sections, enlarged appropriately and analyzed randomly for volume distributions of parenchymal nuclei. For this purpose at least 1000 nuclei are classified as diploid, tetraploid or octaploid with the aid of a Zeiss Particle Size Analyzer. For reproducibility in classifying nuclei the necessity of measuring at least 1000 nuclei has been determined empirically in this laboratory. Use of the Particle Size Analyzer, after establishment of a DNA-volume relationship, greatly facilitates the large number of measurements required. In the current study, a strict DNA-volume relationship was observed consistently, as is usual in adult, relatively stabilized liver. Experimental
Cell Research 48
Polyploidy
after partial
561
hepatectomy
Growth measurements.--Body weights and tail lengths were measured weekly on all animals. Wet liver weights and relative liver weights were determined as each group was killed. Statistics.-p values were calculated according to the method for small samples by Bancroft [2].
l--Lr Ii
J-y-,,,, 40
50
60
70
I
8090100
VOLUME
.
200
(CUBIC
L?
I
300
I 400
.
1 !5OC
MICRONS)
Fig. 3.-Procedure for determining percentages of hepatic polyploid nuclei. A DNA-volume relationship is first established by cytophotometry on 50 selected nuclei (points) and then the volume distribution of 1000 randomly measured nuclei is determined by semi-automatic particle size analysis (histogram). Cytophotometric points, which are superimposed on the volume histogram, aid in the analysis of nuclear classes.
RESULTS
Hereditary
pituitary
dwarf mice
Growth.-The normal mice, weighing 25-33 g at 70 days of age, did not gain significantly in body weight or tail length during the 30 day period studied. The dwarfs, however, increased their body weight by 18 +5.7 per cent and tail length by 10.0 k3.3 per cent during the 30 day period, showing a small but significant growth. At the termination of the study (100 days of age) the normals outweighed the dwarfs by a factor of almost 3 (Table I), while tail lengths differed by a factor of about 2. Experimental Cell Research 48
562
F. J. Swartz
Liver and relative liver weights of partially hepatectomized and unoperated mice, in both groups, were not different at 100 days, indicating that regeneration of liver mass was complete. Within each group, partially hepatectomized and unoperated animals were essentially identical in all of the TABLE
partially
I. Body and liver weights, relative liver weights and tail lengths of hepatectomized dwarf and normal mice and unoperated normal littermates at 100 days of age.
Group Normal (C)
No. of mice
Body weight 63)
8
28.1 k 1.47 p > 0.9
Hepatectomized normal (B)
6
28.4 + 1.71
Dwarf (F)
7
9.4 + 0.44 0.4 > p > 0.3 lO.OrtO.47
Hepatectomized dwarf (E)
8
Relative Liver weight liver weight (g/100 g body wt) (B) l.SkO.12 0.6 B p > 0.5 1.7kO.14
5.8 + 0.30 0.7 > p > 0.6 6.0 & 0.35
0.5 + 0.03
5.6 IL 0.21 0.6 z p z 0.5 5.2 f 0.23
p > 0.9
0.5 + 0.03
Tail length (mm) 8.8 f 0.04 0.01 > p > 0.001
8.6 f 0.04
4.7kO.11 p > 0.9
4.7kO.12
parameters determined; only questionable significance can be attached to the small difference between tail lengths of operated and unoperated dwarfs. Polyploidy.-The polyploid percentages of livers of the six groups are shown in Fig. 4. From 70 to 100 days the normal unoperated mice (groups A and C) show no significant increases in either tetraploid or octaploid nuclear classes (p >0.2 in both classes). The previously hepatectomized normal group (B) also failed to add significantly to its octaploid population when compared with either group A or C (p>O.3). Within group B, however, the individual response to partial hepatectomy was highly varied, and there is little doubt that at least two of the animals had greatly increased percentages of higher DNA classes. The dwarfs, whose livers were essentially diploid at 70 days (group D) increased their tetraploid nuclear population slightly but significantly (p ~0.05) at 100 days (group F). Octaploid and higher nuclei, virtually absent at 70 days, failed to increase significantly during the next 30 days. Following partial hepatectomy (group E), however, both tetraploid and octaploid populations were significantly elevated (p cO.05). Experimental
Cell Research 48
Polyploidy
after partial NORMAL 0
A
100 -
563
hepatectomy C
90 80 - 70 w = 60 i’
g
-
-;:i:i:i: :;:i:i:i .. .. .. .. .. .. .. .. i;$i;i; .:::::::
.:::::::: .:.:.:.:. .. .. .. .. .. .. .. .. :iii$ii :::::::::
100 DAYS HEPATECTOMIZED
100 DAYS NON-HEPATEC. TOMIZED
2 5040 t
$30
20 IO-
m :::::::: :::::::: $i$ii i:;:;:;:
Of 70 DAYS PARTIAL HEPATECTOMY 100 :;:i:i:i :.:::::: :.:::::: :;:::::: : : : :
ii~
i:i;:i:‘ :;;i:;:; ;::I:;;; ..
~
. . . . ..i. :.:;;...
o;
::::::::
.:......
E
F
DWARF
Fig. 4.-Percentages of polyploid nuclei in the 6 experimental groups of the dwarf mouse study. Normals, above; dwarfs, below. Octaploid and higher classes are grouped together. Letter designations of experimental groups are given at top or bottom of each bar. W, Octaploid and higher; 0, tetraploid; m, diploid.
II. Mean DNA values and percent distribution of nuclear classes in livers of unoperated and partially hepatectomized normal and dwarf mice.
TABLE
Mean DNA in arbitrary units
% Distribution Ifrstandard error 4n
8n
5.8
56.5 +3.7
6.8 * 2.2
0.3
9.4 14.9/28.2 8.6 13.4/17.2 8.1 -
32.3k14.0 28.9& 3.7 96.4& 1.0
45.6k8.7 60.7 +2.3 3.6kl.O
16.7k10.2 9.8* 2.1
5.4 0.6 -
8.8 8.2
63.1k12.6 92.6* 1.7
30.7 +9.1 7.3 + 1.6
Group
2n
4n
8n
Higher
A. 70 Day normal B. 100 Day hepatectomized normal C. 100 Day normal D. 70 Day dwarf E. 100 Day hepatectomized dwarf F. 100 Day dwarf
2.9
5.0
9.0
13.3
3.0 2.9 2.8
5.3 4.9 5.0
2.9 2.9
5.0 5.0
14.3 14.4
2n 36.4f
Experimental
Higher
5.9* 4.5 0.1 i: 0.04
0.3 -
Cell Research 48
564
F. J. Swartz
It is of interest that normal mice, both at 70 and 100 days, have small but distinct populations of nuclei which fall approximately in the 12-ploid range (Table II). Following partial hepatectomy the livers of the dwarf mice add significantly to the class. Hypophysectomized
rats
Growth (Fig. 5).-Normal rats (group D) increased in body weight from 365 kS.9 g to 564 + 10.4 g during the course of the study, while the hypophysectomized animals dropped to 295 k34.9 g (group J). Tail lengths between the controls of the two groups (E and J) were not significantly
BODY WEIGHT
300
250 F
TAIL ;E $L
22w I
20-
--.,G ----
LENGTH /E 2-H
,.-.crrd,J
-----
20 % <
z
LIVER
15 -
IO -
.
5 m 40gi 0% 7 J5“$ m
A-
----.
---d------,J
RELATIVE
LIVER
k--
----
a-H
WEIGHT
t --.
3.0
I I
. . -
8J
I
167
Fig. 5.-Growth curves for normal and hypophysectomized mental groups. See text for p values. Cell Research 48
---Ii
t PARTIAL HEPATECT
t HVPOX I
AGE iDAYS)IIO
Experimental
WEIGHT
219 rats. Letters
at right indicate
experi-
Polyploidy
after partial
565
h’epatectomy
different (p> 0.9). Liver weights maintained a significant differential as did relative liver weights, both substantially lowered in the hypophysectomized group (p ~0.001). The effect of hypophysectomy at this age appears to be on organ and body weight rather than on growth in length. NON-HYPOPHYSECTOMIZED
Q
OlPLOlD
(DAYS)
110
TREATMENT 1 DAYS
AFTER
c
0
:::::::: . .:.:.:.: I:;:;::: .:.:.:.: :.:.:.:. .:.:.:.:
:::::::: ;i;;;i;i ..:.:.:.: .. ... . :.:.:.:. .:.:.:.: . .. ... .. ;,L
:::::::: :.:.:.:. :i:;:i:; :.:.:.:.
E
; so-
I AGE
B
HYPOX HYPOX
0
167
219 PARTIAL HEPATECT
-
57
109
I;$::; i:i;i$ ::::::i: $$$:i i:i;i;.; :::::::: F
273 PREVIOUSLY HEPATECT 163
.._.. ..:. .:::::::: :ii;;$i ::::::::: ::::::::. ~~~~~~~~ H
:.:.:.:. ._._._._. . ...._. .:.:.:_: :.:.:.;. .:.:.:.: 273 NONHEPATECT 163
:::::::: :::::::: :::::::: ::::I:;: J
HYPOPHYS&TOMlZED
Fig. 6.-Percentages of polyploid nuclei in the 10 experimental groups of the rat hypophysectomy study. Non-hypophysectomized rats, above; hypophysectomized, below. Letter designations of experimental groups are given at top or bottom of each bar.
From the lack of significant differences (p > 0.8 in all cases) between the partially hepatectomized and non-hepatectomized animals in both hypophysectomized and non-hypophysectomized groups, it is apparent that the gross regenerative process had been completed. Polyploidy (Fig. 6).-Since the percentage of octaploid nuclei was quite low, and changes minimal, only the percentages of tetraploid nuclei were considered indicative of change in polyploidy. In the non-hypophysectomized control group, the tetraploid class increased significantly (p ~0.05) from 54.6 k5.1 per cent (group A) to 72.8 t-5.0 per cent (group E). During the same period the tetraploid class in the hypophysectomized animals increased comparably (from 54.6 k5.1 per cent in group A to 70.6 k2.9 per cent in Experimental
Cell Research 48
566
F. J. Swartz
group J, p <0.05), despite the decrease in liver and body weight (Fig. 5). In the previously hepatectomized normal group (D), the tetraploid class was identical (72.4 & 8.0 per cent, p>O.9) to that of its non-hepatectomized control group (E); however, the percentage of tetraploid nuclei in previously hepatectomized hypophysectomized animals (group H) remained at the same level as that observed at time of hepatectomy (group G), and, at the termination of the experiment, was significantly less (p ~0.05) than that of its non-hepatectomized control (group J). DISCUSSION
Two questions can be considered here. The first relates to the problem of of the liver after partial hepatectomy and the “excess” polyploidization second to the general hormonal control of hepatic polyploid levels. Regarding the first, it has been shown that, following partial hepatectomy, the liver exhibits a tendency to lose its excess polyploid nuclei with time [lo]. The rat study reported here indicates that all excess polyploid nuclei are eventually lost in animals with intact pituitaries and that even fewer polyploid nuclei are seen ultimately in the regenerated livers of hypophysectomized rats. In the normal mouse, the parenchyma shows a strong tendency for return to control levels following hepatectomy while, in the dwarf, the percentage of apparently polyploid nuclei remains elevated longer. The reason for this difference is unclear but may be related to difference in size and developmental potential between the two groups of mice. Thus the dwarfs, in contrast to the normals of the same age, continue to grow slowly and retain a latent capacity for extensive growth. In this respect the dwarfs are unlike the other three experimental groups, all of which have attained a more or less stabilized adult condition. A more suitable control for the dwarfs, perhaps, would have been a group of normal mice of comparable weight. Such controls, however, would have presented additional difficulties in that they, unlike the dwarfs, would have been in an active growth and polyploidization phase during the extended study. If it is assumed that the basic cytological phenomena of liver regeneration differ only quantitatively among mammals, then it is probable that the regenerating dwarf liver would also have exhibited a return to control levels or lower. Nevertheless, in the rat, it is clear that excess polyploid classes are not permitted either in the presence or absence of the pituitary and, in the mouse, in the presence of the pituitary. Those phenomena which have been interpreted as excess polyploidization are probably temporary and may, as already suggested [lo], represent residual signs of earlier vigorous regenerative activity. Experimental
Cell Research 48
Polyploidy
after partial
hepatectomy
567
With respect to the second question, it seems that the pituitary regulates the general level of polyploidization by its control over the growth of the body as a whole. Thus polyploid classes largely fail to develop in the pituitary dwarf [9] and their development is arrested by early hypophysectomy in the rat [6]. In both, body and organ growth are also inhibited. In this context it has also been shown that growth inhibition by protein deprivation, polyploidization [ 121. even in the presence of growth hormone, inhibits Therefore, the general level of liver polyploidization seems to be limited by the growth of the organ which, in turn, is under the influence of the pituitary. Growth hormone is apparently not specific in this role. But other factors must also come into play. It has been shown, for example, that both testosterone and estrogen support the development of higher polyploid classes in rat liver, although estrogen at the same time inhibits liver growth [16, 171. In the rat a small but significant increase in polyploid nuclei, comparable to that in the intact animal, is seen after hypophysectomy despite the gradual decrease of both liver and relative liver weight. A similar observation has been reported previously [4]. And finally, a direct effect of the pituitary itself cannot be completely discounted for it has been shown in this study that the eventual return of polyploidy to control levels after hepatectomy is inhibited or retarded in hypophysectomized adult animals. The contradictory results may be due to different control mechanisms operating in normally growing and regenerating livers, but the final interpretation of “polyploidy” in the liver must await clarification of exactly what the DNA classes represent. In the normal, relatively equilibrated liver parenchyma there is little doubt that real polyploid classes exist [18] but there is also evidence that temporary phenomena, such as those reported here, may linger and that more permanent ones, such as a substantial G2 population, may confuse the picture [14]. In the various attempts to elucidate the controlling factors in polyploidization, it should be remembered that the basic problem to be resolved is the significance of the large populations of polyploid cells in mammalian tissues. Studies such as these are pursued, of course, with the expectation that they will provide insight into that significance.
SUMMARY
Elevated levels of hepatic polyploid nuclei following partial hepatectomy largely disappear if minimal amounts of liver are removed and extended regenerative periods are permitted. In the normal rat all excess polyploid Experimental
Cell Research 48
F. J. Swartz
568
classes disappear while in the hypophysectomized rat the percentage of polyploid nuclei remains below normal. The liver of the normal mouse shows a strong tendency for equilibration while that of the dwarf mouse remains in a state of “excess” polyploidization for at least a month after partial hepatectomy. It is concluded that excess polyploid classes in the rat are not permitted permanently either in the presence or absence of the pituitary. A species difference is noted in the mouse, in that initial polyploid levels are higher and response to partial hepatectomy greater. The increased response to hepatectomy in the dwarf mouse liver is attributed to its greater potential for growth than the livers of the other experimental groups. It is assumed that equilibration in the dwarf mouse liver would have also occurred with time. The pituitary is not required for polyploidization during hepatic regeneration but indirectly controls the general level of polyploidy through its control over organ growth. Other factors also come into play. The interpretation of polyploidy in terms of DNA classes is considered. The author expresses appreciation to Mr Erno Nagy and Mrs J. Smith for capable technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
ALFERT, M. and GESCHWIND, I. I., Expptl Cell Res. 15, 230 (1958). BANCROFT, H., Introduction to biostatistics, p. 172. Hoeber-Harper, New York, 1957. BUCHER, N. L., Int. Rev. Cyt. 15, 245 (1963). CARRIERE, R., J. Endocrinol. 70, 761 (1962). DEELEY, E. M., J. Sci. Znstr. 32, 263 (1955). DI STEFANO, H. S., DIERMEIER, H. F. and TEPPERMAN, J., J. Endocrinol. 57, 158 (1955). FRANCIS, T., The Development of the Pituitary in Hereditary Anterior Pituitary Dwarfism in Mice. Munksgaard, Copenhagen, 1944. GESCHWIND, I. I.. ALFERT, M. and SCHOOLEY, C., Expil Cell Res. 15, 232 (1958). HELWEG-LARSEN; H. FR., Acta Pafhol. Microbial. S&d. 26, 609 (1949). JAMES, J., SCHOPMAN, M. and DELFGAAUW, P., Exptl Cell Res. 42, 375 (1966). LEUCHTENBERG-ER,C.,HELWEG-LARSEN, H. F~.and MURMANIS, L., Lnb. Invest.3,245 (1954). NADAL, C. and ZAJDELA, F., Exptl Cell Res. 42, 117 (1966). NAORA, H., J. Biophys. Biochem. Cyfol. 3, 949 (1957). PERRY, L. D. and SWARTZ, F. J. Expptl Cell Res., 48, 155 (1967). SWARTZ, F. J. and FORD, J. D., Proc. Sot. Expfl Biol. Med. 104, 756 (1960). SWARTZ, F. and SAMS, B. F., Anat. Rec. 141, 219 (1961). SWARTZ, F., SAMS, B. F. and BARTON, A. G., Expfl Cell Res. 20,438 (1960). SWIFT, H., Znt. Rev. Cytol. 2, 1 (1953).
Experimental
Cell Research 48
Cell Research 48, 557-568 (1967)
557
POLYPLOIDIZATION OF LIVER AFTER PARTIAL HEPATECTOMY IN THE DWARF MOUSE AND HYPOPHYSECTOMIZED RAT: EFFECT OF EXTENDED REGENERATIVE PERIODS F. J. SWARTZ Department
of Anatomy, University Louisville, Ky Received
of Louisville
School of Medicine,
40202,U.S.A.
February
17, 1967s
ALTHOUGH it is generally
known that the livers of most mammals become predominantly polyploid during postnatal development [l, 13, 181, various attempts to understand the significance of this phenomenon have led to certain discrepancies concerning its control. For example, the failure of nuclear classes to develop in the hereditary pituitary dwarf mouse [9], the arrest of polyploidization in the rat by hypophysectomy [6] and restoration of polyploidy in the dwarf mouse by growth hormone replacement [9, 111 have all implied the requirement of pituitary growth hormone. Other studies have implicated growth hormone indirectly [4, 151. Recently, it has been suggested that any factor which prevents liver growth will also inhibit the formation of polyploid cells [12]. Conversely, it has been reported that large number of polyploid cells can be formed during liver regloneration in the hypophysectomized rat [8]. In fact, more polyploid nuclei were observed in the liver during and following regeneration than in livers of non-hepatectomized animals. Increased polyploidy in normal animals, which persists indefinitely following hepatectomy, has also been reported frequently ([3, lo] for reviews). That this excess “polyploidy” is due in part to delay of the liver parenchyma to equilibrate post-operatively is suggested by the work of James et al. [lo], who extended the post-hepatectomy period in rats to two months and observed a steady depression in the percentage of polyploid cells formed during regeneration. At the conclusion of the experiment, however, the polyploid population remained elevated. Thus, two problems are presented. The first concerns the role of the 1 Supported by USPHS Research Grant HD-01245. s Revised version received June 11, 1967. Experimental
Cell Research 48
558
F. J. Swartz
pituitary in polyploidization and the second the question of “over-polyploidization” of liver following partial hepatectomy. The current study was designed to approach both problems by [ 1 ] insuring a sufficiently long period free of pituitary influence before hepatectomy, and [2] a posthepatectomy period sufficiently long to permit equilibration of the parenchyma. At the same time, the regenerative period was hastened by removing only a relatively small amount of hepatic tissue, while the use of adult, relatively stabilized, animals permitted interpretation of the cytological events of regeneration in the absence of those events accompanying normal polyploidization. Two separate studies are reported. In the first, “adult” pituitary dwarf mice, selected because of presumed postnatal absence of growth hormone [7], were hepatectomized and maintained for one month. The somewhat equivocal results, arising from difficulty in maintaining the post-hepatectomy dwarfs and subsequent loss of desirable controls, necessitated the second study. In this, hypophysectomized adult rats were maintained for two months, partially hepatectomized and killed two months later. Equilibration of the hepatic parenchyma was apparently assured by the longer post-hypophysectomy and post-hepatectomy periods, and the minimal hepatectomy. It will be shown that the events interpreted as excess polypioidization in both the intact and hypophysectomized rat are observed only temporarily and probably represent residual regenerative activity. In the normal mouse, the percentage of polyploid nuclei is much higher than in the rat, and response to partial hepatectomy more prolonged. However, a tendency to return to control levels of polyploidy following hepatectomy is demonstrated. In the dwarf liver, equilibration is apparently further delayed.
MATERIALS
AND
METHODS
Hereditary pituitary dwarf mice.-Male and female dwarf mice and their normal littermates, from Jackson Memorial Laboratory stock, were maintained until 70 days of age and subdivided into experimental and control groups (Fig. 1). Ten normal mice (group A) were partially hepatectomized by ligating and removing the left lateral lobe, which was used for determining percentages of polyploid nuclei. Of the 10 mice, 6 survived until 100 days of age at which time they were killed by cervical dislocation and their livers analyzed for polyploidy (group B). Eight normal, unoperated animals, killed at 100 days of age, served as normal controls (group Cl. Eighteen dwarf mice were treated in a manner parallel to that of the normal mice. Eleven were partially hepatectomized at 70 days (group D) and the 8 survivors killed at 100 days (group E). Seven unoperated dwarf controls were killed at 100 Experimental
Cell Research 48
Polyploidy after partial hepafecfomy
559
days (group F). Livers were analyzed for polyploidy both at time of bepatectomy and at the termination of the experiment. Hypophysectomized rats.-The experimental procedure is diagrammed in Fig. 2. Fifty-one male Sprague-Dawley rats were selected for homogeneity of age (108-112 days) and size (353-393 g). Of 31 non-hypopbysectomized animals, ten were killed PARTIAL
I (6)-KILLE,,
(6)
AGE
(DAYS)
70
(B)
KILLED
(0
100
Fig. l.-Experimental procedure for dwarf mouse study. Letters in parentheses indicate experimental groups and numbers in parentheses, number of animals.
on day 110 (group A) to determine percentages of hepatic polyploid nuclei on day 0 of the study. Ten normal animals were killed at 167 days (group B) and, of the remaining 11 non-hypophysectomized animals, 6 were partially hepatectomized (left lateral lobe) on day 219 (group C). The four surviving hepatectomized animals of group C were killed on day 273 (group D) as were the remaining 5 unoperated controls (group E). This progression yielded the polyploid percentages of a series of normal livers from 110 to 273 days of age as well as the effect of partial hepatectomy on hepatic polyploid composition two months after the operation. Twenty of the animals were hypophysectomized (Hormone Assay Laboratories, Chicago) on day 110. Their hepatic polyploid state at time of operation is reflected by that of group A. Of the 20, 10 were killed on day 167 (group F), and, of the ten remaining animals, 5 were partially hepatectomized on day 219 (group G) and killed on day 273 (group H). The 5 remaining non-hepatectomized animals were also killed on day 273 (group J). A progression parallel to that of the non-hypophysectomized animals was thus provided for the hypophysectomized group. The relatively large number of control groups was necessitated by the extended time of the experiment, during which the control, or normal, polyploid populations, although relatively stabilized, nevertheless continued to increase gradually. Changes in polyploidy following hepatectomy were, therefore, measured against a continually changing control population. General procedures Percentages of polyploid nuclei.-Two mm cubes of liver were removed from the left lateral lobes of animals with intact livers and from lobes removed at hepatectomy. Experimental Cell Research 48
560
F. J. Swartz
Cubes from the right lobe were removed from previously hepatectomized animals. Tissues were fixed overnight in Lavdowsky’s fluid (formalin, 5 parts; 95 per cent ethyl alcohol, IO parts; glacial acetic acid, 1 part; distilled water, 40 parts) and processed routinely. Ten and 15 ,U sections were Feulgen stained under controlled conditions. DAYS
AFTER
AGE
(DAYS)
HYPOPHYSECTOMY
0
57
109
163
I IO
167
219
273
KILLED NON-HYPOPHYSECTOMIZED (31)
t
(A)
.9-c \ -141-KILLED
-I
‘KILLED
KILLED HYPOPHYHYPOPHYSECTOMIZED (20)
(D)
!E)
(F)
7
SECToMY Ml
yTRT--, %KlLLED SKILLED
(HI (J)
Fig. Z.-Experimental procedure for rat liypophysectomy study. Letters in parenthesesindicate experimental groups and numbers in parentheses,number of animals. In this laboratory percentages of polyploid nuclei are determined by first establishing a DNA-volume relationship. DNA per nucleus, in arbitrary units, is measured by cytophotometry on at least 50 nuclei per IO ,u section, using a Barr and Stroud Integrating Microdensitometer [5]. Nuclei are measured selectively rather than randomly with the intent of finding all existing combinations of DNA-volume relationships. If the results indicate a strict DNA-volume relationship (e.g. Fig. 3), photographs are made of the 15 y sections, enlarged appropriately and analyzed randomly for volume distributions of parenchymal nuclei. For this purpose at least 1000 nuclei are classified as diploid, tetraploid or octaploid with the aid of a Zeiss Particle Size Analyzer. For reproducibility in classifying nuclei the necessity of measuring at least 1000 nuclei has been determined empirically in this laboratory. Use of the Particle Size Analyzer, after establishment of a DNA-volume relationship, greatly facilitates the large number of measurements required. In the current study, a strict DNA-volume relationship was observed consistently, as is usual in adult, relatively stabilized liver. Experimental
Cell Research 48
Polyploidy
after partial
561
hepatectomy
Growth measurements.--Body weights and tail lengths were measured weekly on all animals. Wet liver weights and relative liver weights were determined as each group was killed. Statistics.-p values were calculated according to the method for small samples by Bancroft [2].
l--Lr Ii
J-y-,,,, 40
50
60
70
I
8090100
VOLUME
.
200
(CUBIC
L?
I
300
I 400
.
1 !5OC
MICRONS)
Fig. 3.-Procedure for determining percentages of hepatic polyploid nuclei. A DNA-volume relationship is first established by cytophotometry on 50 selected nuclei (points) and then the volume distribution of 1000 randomly measured nuclei is determined by semi-automatic particle size analysis (histogram). Cytophotometric points, which are superimposed on the volume histogram, aid in the analysis of nuclear classes.
RESULTS
Hereditary
pituitary
dwarf mice
Growth.-The normal mice, weighing 25-33 g at 70 days of age, did not gain significantly in body weight or tail length during the 30 day period studied. The dwarfs, however, increased their body weight by 18 +5.7 per cent and tail length by 10.0 k3.3 per cent during the 30 day period, showing a small but significant growth. At the termination of the study (100 days of age) the normals outweighed the dwarfs by a factor of almost 3 (Table I), while tail lengths differed by a factor of about 2. Experimental Cell Research 48
562
F. J. Swartz
Liver and relative liver weights of partially hepatectomized and unoperated mice, in both groups, were not different at 100 days, indicating that regeneration of liver mass was complete. Within each group, partially hepatectomized and unoperated animals were essentially identical in all of the TABLE
partially
I. Body and liver weights, relative liver weights and tail lengths of hepatectomized dwarf and normal mice and unoperated normal littermates at 100 days of age.
Group Normal (C)
No. of mice
Body weight 63)
8
28.1 k 1.47 p > 0.9
Hepatectomized normal (B)
6
28.4 + 1.71
Dwarf (F)
7
9.4 + 0.44 0.4 > p > 0.3 lO.OrtO.47
Hepatectomized dwarf (E)
8
Relative Liver weight liver weight (g/100 g body wt) (B) l.SkO.12 0.6 B p > 0.5 1.7kO.14
5.8 + 0.30 0.7 > p > 0.6 6.0 & 0.35
0.5 + 0.03
5.6 IL 0.21 0.6 z p z 0.5 5.2 f 0.23
p > 0.9
0.5 + 0.03
Tail length (mm) 8.8 f 0.04 0.01 > p > 0.001
8.6 f 0.04
4.7kO.11 p > 0.9
4.7kO.12
parameters determined; only questionable significance can be attached to the small difference between tail lengths of operated and unoperated dwarfs. Polyploidy.-The polyploid percentages of livers of the six groups are shown in Fig. 4. From 70 to 100 days the normal unoperated mice (groups A and C) show no significant increases in either tetraploid or octaploid nuclear classes (p >0.2 in both classes). The previously hepatectomized normal group (B) also failed to add significantly to its octaploid population when compared with either group A or C (p>O.3). Within group B, however, the individual response to partial hepatectomy was highly varied, and there is little doubt that at least two of the animals had greatly increased percentages of higher DNA classes. The dwarfs, whose livers were essentially diploid at 70 days (group D) increased their tetraploid nuclear population slightly but significantly (p ~0.05) at 100 days (group F). Octaploid and higher nuclei, virtually absent at 70 days, failed to increase significantly during the next 30 days. Following partial hepatectomy (group E), however, both tetraploid and octaploid populations were significantly elevated (p cO.05). Experimental
Cell Research 48
Polyploidy
after partial NORMAL 0
A
100 -
563
hepatectomy C
90 80 - 70 w = 60 i’
g
-
-;:i:i:i: :;:i:i:i .. .. .. .. .. .. .. .. i;$i;i; .:::::::
.:::::::: .:.:.:.:. .. .. .. .. .. .. .. .. :iii$ii :::::::::
100 DAYS HEPATECTOMIZED
100 DAYS NON-HEPATEC. TOMIZED
2 5040 t
$30
20 IO-
m :::::::: :::::::: $i$ii i:;:;:;:
Of 70 DAYS PARTIAL HEPATECTOMY 100 :;:i:i:i :.:::::: :.:::::: :;:::::: : : : :
ii~
i:i;:i:‘ :;;i:;:; ;::I:;;; ..
~
. . . . ..i. :.:;;...
o;
::::::::
.:......
E
F
DWARF
Fig. 4.-Percentages of polyploid nuclei in the 6 experimental groups of the dwarf mouse study. Normals, above; dwarfs, below. Octaploid and higher classes are grouped together. Letter designations of experimental groups are given at top or bottom of each bar. W, Octaploid and higher; 0, tetraploid; m, diploid.
II. Mean DNA values and percent distribution of nuclear classes in livers of unoperated and partially hepatectomized normal and dwarf mice.
TABLE
Mean DNA in arbitrary units
% Distribution Ifrstandard error 4n
8n
5.8
56.5 +3.7
6.8 * 2.2
0.3
9.4 14.9/28.2 8.6 13.4/17.2 8.1 -
32.3k14.0 28.9& 3.7 96.4& 1.0
45.6k8.7 60.7 +2.3 3.6kl.O
16.7k10.2 9.8* 2.1
5.4 0.6 -
8.8 8.2
63.1k12.6 92.6* 1.7
30.7 +9.1 7.3 + 1.6
Group
2n
4n
8n
Higher
A. 70 Day normal B. 100 Day hepatectomized normal C. 100 Day normal D. 70 Day dwarf E. 100 Day hepatectomized dwarf F. 100 Day dwarf
2.9
5.0
9.0
13.3
3.0 2.9 2.8
5.3 4.9 5.0
2.9 2.9
5.0 5.0
14.3 14.4
2n 36.4f
Experimental
Higher
5.9* 4.5 0.1 i: 0.04
0.3 -
Cell Research 48
564
F. J. Swartz
It is of interest that normal mice, both at 70 and 100 days, have small but distinct populations of nuclei which fall approximately in the 12-ploid range (Table II). Following partial hepatectomy the livers of the dwarf mice add significantly to the class. Hypophysectomized
rats
Growth (Fig. 5).-Normal rats (group D) increased in body weight from 365 kS.9 g to 564 + 10.4 g during the course of the study, while the hypophysectomized animals dropped to 295 k34.9 g (group J). Tail lengths between the controls of the two groups (E and J) were not significantly
BODY WEIGHT
300
250 F
TAIL ;E $L
22w I
20-
--.,G ----
LENGTH /E 2-H
,.-.crrd,J
-----
20 % <
z
LIVER
15 -
IO -
.
5 m 40gi 0% 7 J5“$ m
A-
----.
---d------,J
RELATIVE
LIVER
k--
----
a-H
WEIGHT
t --.
3.0
I I
. . -
8J
I
167
Fig. 5.-Growth curves for normal and hypophysectomized mental groups. See text for p values. Cell Research 48
---Ii
t PARTIAL HEPATECT
t HVPOX I
AGE iDAYS)IIO
Experimental
WEIGHT
219 rats. Letters
at right indicate
experi-
Polyploidy
after partial
565
h’epatectomy
different (p> 0.9). Liver weights maintained a significant differential as did relative liver weights, both substantially lowered in the hypophysectomized group (p ~0.001). The effect of hypophysectomy at this age appears to be on organ and body weight rather than on growth in length. NON-HYPOPHYSECTOMIZED
Q
OlPLOlD
(DAYS)
110
TREATMENT 1 DAYS
AFTER
c
0
:::::::: . .:.:.:.: I:;:;::: .:.:.:.: :.:.:.:. .:.:.:.:
:::::::: ;i;;;i;i ..:.:.:.: .. ... . :.:.:.:. .:.:.:.: . .. ... .. ;,L
:::::::: :.:.:.:. :i:;:i:; :.:.:.:.
E
; so-
I AGE
B
HYPOX HYPOX
0
167
219 PARTIAL HEPATECT
-
57
109
I;$::; i:i;i$ ::::::i: $$$:i i:i;i;.; :::::::: F
273 PREVIOUSLY HEPATECT 163
.._.. ..:. .:::::::: :ii;;$i ::::::::: ::::::::. ~~~~~~~~ H
:.:.:.:. ._._._._. . ...._. .:.:.:_: :.:.:.;. .:.:.:.: 273 NONHEPATECT 163
:::::::: :::::::: :::::::: ::::I:;: J
HYPOPHYS&TOMlZED
Fig. 6.-Percentages of polyploid nuclei in the 10 experimental groups of the rat hypophysectomy study. Non-hypophysectomized rats, above; hypophysectomized, below. Letter designations of experimental groups are given at top or bottom of each bar.
From the lack of significant differences (p > 0.8 in all cases) between the partially hepatectomized and non-hepatectomized animals in both hypophysectomized and non-hypophysectomized groups, it is apparent that the gross regenerative process had been completed. Polyploidy (Fig. 6).-Since the percentage of octaploid nuclei was quite low, and changes minimal, only the percentages of tetraploid nuclei were considered indicative of change in polyploidy. In the non-hypophysectomized control group, the tetraploid class increased significantly (p ~0.05) from 54.6 k5.1 per cent (group A) to 72.8 t-5.0 per cent (group E). During the same period the tetraploid class in the hypophysectomized animals increased comparably (from 54.6 k5.1 per cent in group A to 70.6 k2.9 per cent in Experimental
Cell Research 48
566
F. J. Swartz
group J, p <0.05), despite the decrease in liver and body weight (Fig. 5). In the previously hepatectomized normal group (D), the tetraploid class was identical (72.4 & 8.0 per cent, p>O.9) to that of its non-hepatectomized control group (E); however, the percentage of tetraploid nuclei in previously hepatectomized hypophysectomized animals (group H) remained at the same level as that observed at time of hepatectomy (group G), and, at the termination of the experiment, was significantly less (p ~0.05) than that of its non-hepatectomized control (group J). DISCUSSION
Two questions can be considered here. The first relates to the problem of of the liver after partial hepatectomy and the “excess” polyploidization second to the general hormonal control of hepatic polyploid levels. Regarding the first, it has been shown that, following partial hepatectomy, the liver exhibits a tendency to lose its excess polyploid nuclei with time [lo]. The rat study reported here indicates that all excess polyploid nuclei are eventually lost in animals with intact pituitaries and that even fewer polyploid nuclei are seen ultimately in the regenerated livers of hypophysectomized rats. In the normal mouse, the parenchyma shows a strong tendency for return to control levels following hepatectomy while, in the dwarf, the percentage of apparently polyploid nuclei remains elevated longer. The reason for this difference is unclear but may be related to difference in size and developmental potential between the two groups of mice. Thus the dwarfs, in contrast to the normals of the same age, continue to grow slowly and retain a latent capacity for extensive growth. In this respect the dwarfs are unlike the other three experimental groups, all of which have attained a more or less stabilized adult condition. A more suitable control for the dwarfs, perhaps, would have been a group of normal mice of comparable weight. Such controls, however, would have presented additional difficulties in that they, unlike the dwarfs, would have been in an active growth and polyploidization phase during the extended study. If it is assumed that the basic cytological phenomena of liver regeneration differ only quantitatively among mammals, then it is probable that the regenerating dwarf liver would also have exhibited a return to control levels or lower. Nevertheless, in the rat, it is clear that excess polyploid classes are not permitted either in the presence or absence of the pituitary and, in the mouse, in the presence of the pituitary. Those phenomena which have been interpreted as excess polyploidization are probably temporary and may, as already suggested [lo], represent residual signs of earlier vigorous regenerative activity. Experimental
Cell Research 48
Polyploidy
after partial
hepatectomy
567
With respect to the second question, it seems that the pituitary regulates the general level of polyploidization by its control over the growth of the body as a whole. Thus polyploid classes largely fail to develop in the pituitary dwarf [9] and their development is arrested by early hypophysectomy in the rat [6]. In both, body and organ growth are also inhibited. In this context it has also been shown that growth inhibition by protein deprivation, polyploidization [ 121. even in the presence of growth hormone, inhibits Therefore, the general level of liver polyploidization seems to be limited by the growth of the organ which, in turn, is under the influence of the pituitary. Growth hormone is apparently not specific in this role. But other factors must also come into play. It has been shown, for example, that both testosterone and estrogen support the development of higher polyploid classes in rat liver, although estrogen at the same time inhibits liver growth [16, 171. In the rat a small but significant increase in polyploid nuclei, comparable to that in the intact animal, is seen after hypophysectomy despite the gradual decrease of both liver and relative liver weight. A similar observation has been reported previously [4]. And finally, a direct effect of the pituitary itself cannot be completely discounted for it has been shown in this study that the eventual return of polyploidy to control levels after hepatectomy is inhibited or retarded in hypophysectomized adult animals. The contradictory results may be due to different control mechanisms operating in normally growing and regenerating livers, but the final interpretation of “polyploidy” in the liver must await clarification of exactly what the DNA classes represent. In the normal, relatively equilibrated liver parenchyma there is little doubt that real polyploid classes exist [18] but there is also evidence that temporary phenomena, such as those reported here, may linger and that more permanent ones, such as a substantial G2 population, may confuse the picture [14]. In the various attempts to elucidate the controlling factors in polyploidization, it should be remembered that the basic problem to be resolved is the significance of the large populations of polyploid cells in mammalian tissues. Studies such as these are pursued, of course, with the expectation that they will provide insight into that significance.
SUMMARY
Elevated levels of hepatic polyploid nuclei following partial hepatectomy largely disappear if minimal amounts of liver are removed and extended regenerative periods are permitted. In the normal rat all excess polyploid Experimental
Cell Research 48
F. J. Swartz
568
classes disappear while in the hypophysectomized rat the percentage of polyploid nuclei remains below normal. The liver of the normal mouse shows a strong tendency for equilibration while that of the dwarf mouse remains in a state of “excess” polyploidization for at least a month after partial hepatectomy. It is concluded that excess polyploid classes in the rat are not permitted permanently either in the presence or absence of the pituitary. A species difference is noted in the mouse, in that initial polyploid levels are higher and response to partial hepatectomy greater. The increased response to hepatectomy in the dwarf mouse liver is attributed to its greater potential for growth than the livers of the other experimental groups. It is assumed that equilibration in the dwarf mouse liver would have also occurred with time. The pituitary is not required for polyploidization during hepatic regeneration but indirectly controls the general level of polyploidy through its control over organ growth. Other factors also come into play. The interpretation of polyploidy in terms of DNA classes is considered. The author expresses appreciation to Mr Erno Nagy and Mrs J. Smith for capable technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
ALFERT, M. and GESCHWIND, I. I., Expptl Cell Res. 15, 230 (1958). BANCROFT, H., Introduction to biostatistics, p. 172. Hoeber-Harper, New York, 1957. BUCHER, N. L., Int. Rev. Cyt. 15, 245 (1963). CARRIERE, R., J. Endocrinol. 70, 761 (1962). DEELEY, E. M., J. Sci. Znstr. 32, 263 (1955). DI STEFANO, H. S., DIERMEIER, H. F. and TEPPERMAN, J., J. Endocrinol. 57, 158 (1955). FRANCIS, T., The Development of the Pituitary in Hereditary Anterior Pituitary Dwarfism in Mice. Munksgaard, Copenhagen, 1944. GESCHWIND, I. I.. ALFERT, M. and SCHOOLEY, C., Expil Cell Res. 15, 232 (1958). HELWEG-LARSEN; H. FR., Acta Pafhol. Microbial. S&d. 26, 609 (1949). JAMES, J., SCHOPMAN, M. and DELFGAAUW, P., Exptl Cell Res. 42, 375 (1966). LEUCHTENBERG-ER,C.,HELWEG-LARSEN, H. F~.and MURMANIS, L., Lnb. Invest.3,245 (1954). NADAL, C. and ZAJDELA, F., Exptl Cell Res. 42, 117 (1966). NAORA, H., J. Biophys. Biochem. Cyfol. 3, 949 (1957). PERRY, L. D. and SWARTZ, F. J. Expptl Cell Res., 48, 155 (1967). SWARTZ, F. J. and FORD, J. D., Proc. Sot. Expfl Biol. Med. 104, 756 (1960). SWARTZ, F. and SAMS, B. F., Anat. Rec. 141, 219 (1961). SWARTZ, F., SAMS, B. F. and BARTON, A. G., Expfl Cell Res. 20,438 (1960). SWIFT, H., Znt. Rev. Cytol. 2, 1 (1953).
Experimental
Cell Research 48