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Stereological analysis of the ultrastructure of liver parenchymal cells during pregnancy and lactation
© 1970 by Academic Press, Inc.
292
J. ULTRASTRUCTURE RESEARCH
33, 292-305 (1970)
Stereological Analysis of the Ultrastructure of Liver Parenchymal Cells During Pregnancy and Lactation J. HOPE
Biology Division, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, England Received February 24, 1970 Alterations in the nuclear volume, cytoplasmic volume, and volume proportions of RER, mitochondria, and microbodies in the cytoplasm were measured by morphometric techniques in the liver parenchymal cells of rats, during gestation and lactation. During gestation the most marked effects were an increased volume proportion of RER and an initial increase, followed by a decrease, of the volume proportion of microbodies. It is suggested that these effects may be associated with hydremia and hyperlipemia of pregnancy, respectively. During lactation the volume proportions of the cell organelles rapidly return to the control values, and the greatest effect is a marked hypertrophy of the parenchymal cells.
This work was undertaken as part of an analytical, biochemical, and morphological investigation of liver enlargement during gestation and lactation aimed at obtaining background information against which toxic liver enlargement could be evaluated (29). Using electron microscopy, it was hoped to identify any ultrastructural changes that might occur and to assess the relative contributions of hyperplasia and hypertrophy to the enlargement of the liver. It was expected that the ultrastructural changes in such a normal physiological condition would be minimal and therefore it was considered that a purely qualitative assessment would not be adequate. As du Boistesselin (9) has emphasized, there are difficulties in the subjective interpretation of subcellular changes in the liver even when such changes are qualitatively pronounced, and Steiner et al. (24) have expressed the need for more facts and less subjective hypothesis. Obviously, a more objective technique is required. In any case a quantitative technique is necessary to distinguish between hypertrophy and hyperplasia and to correlate morphological and biochemical data (27); accordingly, the technique of morphological analysis as described by Weibel et al. (26) and by Loud (15) was employed.
STEREOLOGY OF LIVER
293
MATERIALS AND METHODS Rats were taken at 0, 3, 9, 14, 21 days of gestation and 0, 3, 9, 14, 18, and 49 (recovery) days of lactation. Two test animals and one control animal were taken from each group, a total of 33 animals. Animals were anesthetized with ether and the liver was exposed; a small portion of the median lobe was excised, diced into small pieces, and fixed for 1 hour at 0°C in 1 % osmium tetroxide solution in phosphate buffer at p H 7.4. The tissue was dehydrated through alcohols and propylene oxide and embedded in Araldite epoxy resin. Tissues from the two test animals and control animal of each group were processed together. In most cases ten blocks of tissue from each animal were embedded. Initially, 1 l~ sections were cut from each block, stained with toluidine blue, and scanned with the optical microscope. Loud (15) has shown that there are structural variations between cells in different regions of the liver lobule, but most of the significant differences occur in cells around the central vein and these comprise less than 20 % of the parenchymal ceils. Accordingly, areas containing the central vein and immediately surrounding hepatocytes were trimmed from the block, so that mid-zonal and peripheral cells only of the hepatic lobules were included in the sections. If it was not possible to trim away these areas, the blocks were discarded. In this way it was hoped to reduce the variability of the results, but still retain a representative sample of the liver. There was, therefore, a maximum of 20 blocks for each test group and 10 blocks for each control group available and sections from all the available blocks were examined in the electron microscope. Fifty random electron micrographs were taken from each test and control group for stereological analysis. This number of micrographs was distributed as evenly as possible among the available blocks. R a n d o m micrographs were taken in order to avoid bias in the selection of fields, and care was taken that similar areas of adjacent sections were not photographed. The micrographs were taken at an initial magnification of 3 000 x and were enlarged to 12 000 x for analysis. A probe with 100 points was laid over each micrograph and the number of points overlying certain structures were tabulated. The structures counted were: (1) canaliculi and sinusoids, (2) nuclei, (3) mitochondria, (4) rough endoplasmic reticulum (RER), (5) microbodies, (6) lysosomes, (7) lipid, (8) the remaining cytoplasm. Counts of structures 3 to 8 were totaled to give the total proportion of cytoplasm. It was not possible to obtain counts of glycogen because of vagaries in staining. A t the same time up to 150 electron micrographs were taken of random nuclei in the parenchymal cells for the determination of nuclear volume. Since the accuracy of the magnification is important in this determination, the electron microscope was calibrated with a replica grating before each group of nuclei were photographed. The calibrations were all within 2 % of the nominal magnification.
RESULTS The aggregate counts f r o m all the m i c r o g r a p h s in each g r o u p , given in Tables I a n d II, were used to calculate the n u c l e a r - c y t o p l a s m i c ratio, the v o l u m e p r o p o r t i o n of canaliculi a n d sinusoids to p a r e n c h y m a l cells, a n d the v o l u m e p r o p o r t i o n of m i t o c h o n d r i a , r o u g h e n d o p l a s m i c reticulum, a n d m i c r o b o d i e s in the cytoplasm. T h e r e
294
HOPE TABLE I TOTAL COUNTs--CoNTROL ANIMALS
Day
Number Canaliculi of Microand graphs Sinusoids
Nuclei
Total MitoCytoplasm chondria
RER
Microbodies
Lysosomes
Gestation 0 3 9 14 21
50 50 50 50 47
600 597 468 740 510
200 334 309 301 297
4 198 4 068 4 224 3 960 3 892
1 175 1 067 1 101 l 076 894
135 207 277 191 145
63 65 81 75 72
15 39 17 21 18
Lactation 0 3 9 14 18 49
49 33 48 50 28 49
475 254 488 572 405 519
303 254 155 265 126 196
4 122 2 792 4 158 4 163 2 269 4 185
1 064 711 1 125 1 096 574 1 156
198 192 268 279 144 278
88 79 83 83 40 81
3 23 9 14 15 5
w e r e i n s u f f i c i e n t c o u n t s o f l i p i d t o o b t a i n m e a n i n g f u l r e s u l t s . T h e c o u n t s of l y s o s o m e s a n d of t h e c a n a l i c u l i a n d s i n u s o i d s w e r e a l s o t o o l o w a n d t o o v a r i a b l e b e t w e e n a n i m a l s t o give s t a t i s t i c a l l y u s e f u l r e s u l t s , b u t t h e v o l u m e p r o p o r t i o n
of l y s o s o m e s o v e r all
the control animals could be calculated with reasonable accuracy. The results are given in Tables III and IV. Following the procedure of
Loud (15)
t h e v a l u e f o r t h e R E R h a s b e e n i n c r e a s e d b y 50 % t o a l l o w f o r m e m b r a n e s i n c l i n e d t o t h e p l a n e of t h e s e c t i o n s . I t w a s n o t p o s s i b l e t o a r r i v e a t t h e v o l u m e o f t h e cell d i r e c t l y b y e l e c t r o n m i c r o s c o p y a l o n e , b u t it w a s c a l c u l a t e d f r o m t h e n u c l e a r - c y t o p l a s m i c r a t i o , as m e a s u r e d a b o v e , a n d a m e a s u r e of t h e v o l u m e o f t h e n u c l e i o b t a i n e d as d e s c r i b e d b e l o w . TABLE II TOTAL COUNTS--EXPERIMENTAL ANIMALS
Day
Number Canaliculi of Microand graphs Sinusoids
Nuclei
Total MitoCytoplasm chondrla
RER
Microbodies
Lysosomes
Gestation 0 3 9 14 21
50 50 50 50 50
587 380 671 509 464
266 264 235 233 220
4 146 4 356 4 093 4 256 4 312
1 136 1 089 1 031 1 000 774
133 197 251 298 301
69 95 89 79 57
22 20 20 23 33
Lactation 0 3 9 14 18 49
50 50 50 48 50 50
423 502 494 611 732 483
207 238 97 210 319 304
4 372 4 260 4 412 3 979 3 949 4 212
907 1 030 1 158 1 061 1 080 1 094
228 316 340 282 238 245
38 85 84 92 100 72
18 11 12 11 24 18
295
STEREOLOGY OF LIVER TABLE III VOLUME PROPORTIONS OF STRUCTURES IN THE LIVER OF CONTROL ANIMALSa
Day
NuclearCytoplasmic Ratio (%)
Percent Volume of Mitochondria in Cytoplasm
Percent Volume of RER in Cytoplasm
Percent Volume of Microbodies in Cytoplasm
Percent Volume of Canaliculi and Sinusoids in Total Tissue
4.8 (1.80)
28.0 (1.37)
4.8 (1.10)
1.5 (0.22)
8.2 (1.80)
26.2 (1.37)
7.7 (1.10)
1.6 (0.22)
7.3 (1.80) 7.6 (1.80)
26.1 (1.37) 27.2 (1.37)
9.9 (1.10) 7.2 (1.10)
1.9 (0.22) 1.9 (o.22)
7.6 (1.82)
23.0 (1.38)
5.6 (1.10)
1.8 (0.25)
13.6 13.6 10.3 17.4 12.2
Lactation 0 3 9 14 18 49
7.4 9.1 3.7 6.3 5.5 4.7
25.8 25.5 27.1 26.3 25.3 27.7
2.1 2.8 2.0 2.0 1.8 1.9
(0.22) (0,28) (0.25) (0.22) (0.32) (0.22)
10.7 7.7 10.2 11.4 14.5 10.8
Mean (mean sE)
6.6 (1.84)
8.4 (1.12)
1.9 (0.24)
12.0
SE of mean
0.55
0.34
0.07
Gestation 0 3 9 14 21
(1.80) (1.98) (1.82) (1.80) (2.07) (1.80)
(1.37) (1.49) (1.38) (1.37) (1.55) (1.37)
26.2 (1.40) 0.42
7.2 10.4 9.6 10.1 9.5 10.1
(1.10) (1.17) (1.10) (1.10) (1.21) (1.10)
a Standard error is given in parentheses.
TABLE IV VOLUME PROPORTIONS OF STRUCTURES IN THE LIVER OF EXPERIMENTAL ANIMALSa
NuclearCytoplasmic Ratio (%)
Percent Volume of Mitochondria in Cytoplasm
Percent Volume of RER in Cytoplasm
Percent Volume of Microbodies in Cytoplasm
Percent Volume of Canaliculi and Sinusoids in Total Tissue
Gestation 0 3 9 14 21
6.4 (1.54) 6.1 5.7 5.5 5.1
27.4 (1.08) 25.0 25.2 23.5 17.9
4,8 (0,84) 6.8 9.2 10.5 10.5
1.7 (0.22) 2.2 2.2 1.8 1.3
13.3 8.2 15.5 11.3 10.2
Lactation 0 3 9 14 18 49
4.7 5.6 2.2 5.3 8,1 7,2
20.7 24.2 26.3 26.7 27.3 26.0
7.8 11.1 11.6 10.7 9.0 8.7
0.9 2.0 1.9 2.3 2.5 1.7
9.2 10.0 9.9 12.7 14.6 9.7
Day
SE is given in parentheses. The standard error given next to top entry in each column applies to each entry in column.
296
HOPE
TABLE V VOLUME OF N U C L E U S AND CYTOPLASM IN THE LIVER OF CONTROL ANIMALS~
Day
Number of Measurements
Gestation 0 3 9 14 21 Lactation 0 3 9 14 18 49 Mean (mean SE) SE of mean
141 150 150 143 135 120 144 132 94 115 133 ---
Mean Diameter of Nuclei d (/0
6.62 (0.13) 6.80 (0.13) 6.83 (0.13) 6.69 (0.13) 6.97 (0.14) 7.18 (0.15) 6.43 (0.13) 6.20 (0.14) 6.46 (0.16) 6.23 (0.15) 6.12 (0.14) 6.59 (0.14) 0.04
Correctedb Mean Diameter of Nuclei D (t0
8.43 (0.17) 8.66 (0.17) 8.69 (0.17) 8.52 (0.17) 8.87 (0.18) 9.14 (0.19) 8.19 (0.17) 7.89 (0.18) 8.22 (0.20) 7.93 (0.19) 7.79 (0.18) 8.39 (0.18) 0.05
Volume of Nucleus (#0
Volume of Cytoplasm (/~0
314 (11.8) 340 (12.1) 344 (12.6) 324 (12.0) 365 (13.4) 400 (15.0) 288 (11.1) 257 (10.7) 29l (13.7) 26l (11.6) 248 (10.4) 312 (12.2) 3.7
6540 (1 740) 4150 (710) 4710 (890) 4260 (780) 4800 (890) 5400 (1 010) 3160 (510) 6950 (2 380) 4620 (1 040) 4740 (1 390) 5280 (1 400) 4970 (1 160) 318
a Standard error is given in parentheses. b Using the formula 15 - (4/~) d.
The nuclear volume was calculated f r o m the average nuclear diameter derived f r o m measurements on up to 150 sections t h r o u g h nuclei p h o t o g r a p h e d at a magnification of 3000 ×. A l t h o u g h in histological preparations and in 1 # sections of tissue prepared for electron microscopy, the nuclear sections have circular profiles, in ultrathin sections they m a y be elliptical. It is well k n o w n that considerable compression can occur in ultrathin sections in the direction of sectioning (19), and that measurements in this direction are unreliable. Consequently in the present w o r k measurements were made only in a direction parallel to the knife edge; this was invariably along the major axis of any elliptical profiles. The average measured diameter d is related to the actual mean diameter D by the f o r m u l a /9 = (4/~)d, and this formula was used instead of a Wicksell transformation (28) to arrive at the average nuclear diameter. The disadvantage of using this f o r m u l a is that it gives an average diameter, whereas a Wicksell transformation gives a histog r a m of the distribution of nuclear diameters. However, for the purposes of this paper a comparison of mean diameters is sufficient, and it is unnecessary to go t h r o u g h the rather involved c o m p u t a t i o n of a Wicksell transformation. The results for average nuclear diameter, nuclear volume, and cytoplasmic volume are given in Tables V and VI.
297
S T E R E O L O G Y OF L I V E R
TABLE VI VOLUME OF ~',~UCLEUS AND CYTOPLASM IN THE LIVER OF EXPERIMENTAL ANIMALS a
Day
Gestation 0 3 9 14 21 Lactation 0 3 9 14 18 49
Number of Measurements
Mean Diameter of Nuclei d (~)
150 150 150 150 150 150 141 135 144 95 150
6.42 (0.14) 7.26 (0.14) 6.54 (0.14) 7.05 (0.14) 6.79 (0.15) 7.48 (0.14) 6.85 (0.15) 6.64 (0.15) 6.84 (0.16) 6.99 (0.19) 6.84 (0.15)
Corrected b Mean Diameter of Nuclei D (/~)
8.17 (0.18) 9.24 (0.18) 8.33 (0.18) 8.97 (0.18) 8.64 (0.19) 9.52 (0.18) 8.72 (0.19) 8.45 (0.19) 8.71 (0.20) 8.90 (0.25) 8.71 (0.19)
Volume of Nucleus (/13)
286 (12.1) 413 (14.8) 303 (12.2) 378 (14.4) 338 (14.8) 452 (15.8) 347 (14.0) 316 (13.4) 346 (14.6) 369 (19.1) 346 (14.4)
Volume of Cytoplasm (#2)
4,470 (840) 6,770 (1 300) 5,320 (1 080) 6,870 (1 440) 6,630 (1 510) 9,610 (2 280) 6,200 (1 300) 14,360 (6 810) 6,530 (1 440) 4,560 (760) 4,810 (840)
Standard error is given in parentheses, b Using the formula D - (4In) d.
Treatment of results Although the preparative manipulations of fixation, dehydration, and emlzedding were standardized between groups processed at different times, there still exists the possibility of systematic errors due to processing. However, the experimental and control material from each group was processed side by side and therefore, to minimize any systematic errors, the experimental values have been expressed as a Fercentage of the corresponding control value. The experimental values may then be quoted as this percentage or expressed in absolute terms calculated from the mean control value. The results are given in both forms in Table VII and expressed graphically in Figs. 1-6. The dotted lines on each side of the control value represent two standard errors and give an indication of the statistical significance of the experimental results. Nuclear volume. The nuclear volume (Fig. 1) was elevated above the control values on days 3 and 14 of gestation, but not days 0, 9, and 21. During lactation there was a significant increase at day 3 which was maintained to day 14 after which there was a further increase at days 18 and 49. Nuclear-cytoplasmic ratio. The nuclear-cytoplasmic ratio (Fig. 2) fell within two standard errors either side of the average control value throughout the course of the experiment, but nevertheless, an interesting trend was indicated. In the first 3 days of gestation there was an initial sharp fall in the ratio. Between day 3 of gestation and day 9 of lactation there was a further gradual decrease followed by a sharp increase at days 14 and 18 of lactation. Thereafter there was little change at day 49.
298
HOPE TABLE VII E X P E R I M E N T A L R E S U L T S EXPRESSED I N R E L A T I O N TO THE C O N T R O L V A L U E AS A PERCENTAGE, AND IN ABSOLUTE VALUES
Volume of Nucleus r% of control #~
• % of control
Mean control value Gestation 0 3 9 14 21
100 91 121 88 117 93
312 284 379 275 364 289
Lactation 0 3 9 14 18 49
113 120 123 119 141 140
353 376 384 371 441 435
Day
NuclearCytoplasmic Ratio
Volume of Cytoplasm
,
Percent Volume of Mitechondria
Percent Volume of R E R , ~ ~ % of control %
Percent Volume of Microbodies Percent • " - - - - - - - Volume of % of Canaliculi conand Sinusoids trol % %
/~
% of control
%
"% of control
100 68 163 113 161 138
4970 3 390 8110 5 600 8 010 6 850
100 133 74 78 72 67
6.6 8.8 4.9 5.2 4.8 4.4
100 98 95 97 86 78
26.2 100 8.4 25.6 100 8.4 25.0 88 7.4 25.3 93 7.8 22.6 146 12.2 20.4 187 15.7
100 113 137 116 95 72
1.9 2.2 2.6 2.2 1.8 1.4
12.0 11.7 7.2 18.0 7.8 10.0
178 196 207 141 96 91
8 830 9 720 10270 7020 4 770 4 520
64 62 59 84 147 153
4.2 4.1 3.9 5.6 9.7 10.1
80 95 97 102 108 94
21.0 24.9 25.4 26.6 28.3 24.6
43 0.8 71 1.4 95 1.8 115 2.2 139 2.6 89 1.7
10.3 15.6 11.6 13.4 12.1 10.8
Cytoplasmic volume. A l t h o u g h
%
108 107 121 106 95 86
9.1 9.0 10.1 8.9 8.0 7.2
elevation of the cytoplasmic v o l u m e (Fig. 3) oc-
curred at days 3 and 14 of gestation this was not p r o n o u n c e d . H o w e v e r , it was significantly higher at days 0 to 9 of lactation, and at this time it was double that of the c o n t r o l value.
Cytoplasmic organelles. The
v o l u m e p r o p o r t i o n of r o u g h e n d o p l a s m i c r e t i c u l u m
(Fig. 4) increased rapidly f r o m day 9 of gestation to reach 187 % of the c o n t r o l value -500
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FIG. 1. Nuclear volume of liver parenchymal ceils during gestation and lactation in the rat.
299
STEREOLOGY OF LIVER 225' 200" -12 175" . . . . . . . . . . . . . . . . . . . . .
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FIG. 2. N u c l e a r - c y t o p l a s m i c ratio for liver parenchymal cells during gestation a n d lactation in the rat.
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Fro. 3. V o l u m e of cytoplasm of liver p a r e n c h y m a l cells during gestation a n d lactation in the rat. 20 -- 701831 J . Ultrastructure Researcl~
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Fr6. 4. Volume density of rough endoplasmic reticulum of liver parenchymal cells during gestation and lactation in the rat.
decrease to a significantly low value at day 0 of lactation, followed again by an increase to day 18 of lactation and then a further fall at day 49.
DISCUSSION
Control values The results of the volume proportions of cytoplasmic organelles in the control animals are in general agreement with the results of previous workers (4, 5, 11, 14-16, 23, 27), although there is some variation in results both between workers and for the same author in different papers. This may be due to variations between animal strains or to variations in preparative methods. Loud et al. (16) have shown that the buffering system for the osmium tetroxide fixative, and the embedding medium, may both affect the final results. The volume proportion of 26.2 % for the mitochondria in the present work compares with values of 18.1-27.2 in the literature. The most comprehensive and detailed morphometric studies on the normal liver are those by Loud (15) and Weibel et al. (27). Loud made individual measurements from cells from three different regions of the liver lobule, whereas Weibel et al. (27) considered the hepatocytes as a whole. Since Loud found similar values for peripheral and mid-zonal cells, but significantly different values for the centrilobular cells, the latter were not included in the present study, and the peripheral and mid-zonal cells were treated as a single group. Loud
STEREOLOGY OF LIVER
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Gestation
Lactation Days of G e s t a t i o n a n d L a c t a t i o n
F i t . 5. Volume density of mitochondria of liver parenchymal cells during gestation and lactation in the rat.
(i5) gave values of 19.1% for the volume proportion of mitochondria in the midzonal cells and 19.8 % for peripheral cells. The value of 23 % found by Weibel et al. (27) is closer to the present result of 26.2 % and would probably have been higher had they excluded centrilobular cells from the analysis, since the volume proportion of mitochondria in these cells is only 12.9 % (15). The volume proportion of microbodies of 1.9 % compares with values in the literature of 0.6-2.6 %. This high value was after permanganate fixation and Aquon embedding (14). Loud (15) reported a value of 1.4 % and Weibel et al. (27) reported
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Lactation Days of G e s t a t i o n a n d L a c t a t i o n
F~6. 6. Volume density of microbodies of liver parenchymal cells during gestation and lactation in the rat.
302
HOVE
1.5 %. The volume proportion of lysosomes was given by Loud (15) as 0.3 % and by Weibel et al. as 0.9 %, compared to the present result of 0.4 %. There is a difference in the volume proportion of the rough endoplasmic reticulum, measured as 8.4 % in the present work and reported as 10 % by Weibel et al. (27). This difference is greater than the figures suggest since the results of Weibel et al. were not corrected for the inclination of the membranes to the plane of the section. Previous workers have calculated the nuclear volume from measurements on optical micrographs of 1 # sections (15, 27), but have commented that the section thickness is somewhat too great for optimum accuracy. In the present work, therefore, the nuclear diameter was calculated from measurements made on electron micrographs at a magnification of 3000 ×. The results, after the formula/9 = (4/z0 c7 had been applied, gave a diameter of 8.3 #, compared with 8.1 # (15) and 7.9 # (27). There appears to be very little difference, then, between measurements made on ultrathin and on 1/z sections. The nuclear cytoplasmic ratio measured in the present work (6.6 %) is similar to that calculated from the data of other workers: 5.5 % (15), 6.5 % (27), and 7.9 % (4). Consequently, the cytoplasmic volume calculated from the nuclear volume and nuclear cytoplasmic ratio is similar to the values obtained by other workers by more direct methods, namely 4970 #z in the present work, compared with 5 100/z z (15) and 4 660 #3 (27).
Changes during gestation and lactation These experimental results give a quantitative description of some of the morphological changes that take place within the hepatocyte during gestation and lactation and that can be correlated with histopathological findings and with biochemical results. These results have a bearing on the question of whether the liver enlargement is due to hyperplasia, hypertrophy, or both. The trend of the values for the nuclear cytoplasmic ratio, if taken alone, would indicate an initial sharp hypertrophy of the cells, followed by a more gradual increase in cell size during gestation and lactation, until the end of lactation, when the cells reduce in volume. However, this ignores the variations in nuclear volume that occur, and if these are taken into account to calculate the cytoplasmic volume, then the results given in Fig. 3 are obtained. Apart from a low value at day 9 of gestation, this graph suggests a possible phase of hypertrophy during gestation, since the results fall just outside two standard errors of the control value. A further much greater and statistically more significant phase of hypertrophy occurs during lactation, but the cytoplasmic volume returns to the control value by day 18 of lactation. The liver reaches its maximum size at this time, and therefore the number of cells must necessarily have increased. The results are, there-
STEREOLOGY OF LIVER
303
fore, suggestive of two phases of hypertrophy each followed by hyperplasia, and this is in agreement with parallel histological findings in which phases of hypertrophy in gestation and lactation, as determined by nuclear counts on a standard microscopic field, were followed by waves of mitosis (29). The initiation of mitosis during gestation was thought to be triggered by single cell necrosis, but this was not observed during lactation. However, during lactation the cytoplasm reaches double the control value and it has been suggested that hypertrophy itself is a sufficient trigger for mitosis. The nuclear volume, after some variation during gestation and lactation, increases significantly above the control value at day 3 of lactation and remains elevated even after recovery at day 49. This may be taken as evidence of increased ploidy since the nuclear volume doubles with each increase in ploidy (10). Nadal and Zajdela (17) have shown that an increase in ploidy is preceded by a binucleate stage, but although in the present study the percentage of binucleate cells was noted, there was apparently no difference from the controls and no pattern that could be associated with ploidy. The most marked change in the cytoplasmic organelles was the increase in volume proportion of the rough endoplasmic reticulum during gestation. This correlates with an increase in amount of RNA in the liver determined biochemically and an increased basophilia noted histologically (29). However, an increased basophilia in the first 3 days of lactation was not reflected in an increase in the volume proportion of RER. A similar increase in RER occurs in the postnatal differentiation of hepatocytes (6, 7) and after administration of phenobarbital (23). In the first instance this increase is followed chronologically by a proliferation of smooth endoplasmic reticulum (SER), but in the second instance the SER increase occurs simultaneously with that of the RER. In both cases the SER increase is thought to be derived by the degranulation of the RER at the ends of the cisternae and the budding-off of SER elements (23). In the present study the SER was not measured morphologically, but no proliferation was seen subjectively. This is in agreement with biochemical results (29) which indicate that microsomal drug processing enzymes, apart from azobenzene reductase during gestation, did not increase in activity, but were unchanged or even reduced in activity. This is not unexpected since the liver is responding to an increased normal physiological demand, rather than to an alteration in function. The functions of the RER in the liver are, the manufacture of serum proteins for export, the synthesis of new membranes, and the synthesis of microbodies. The synthesis of cytoplasmic proteins is not a function of the RER since these are synthesized by free polysomes not bound to membranes (3). As we have seen there is no synthesis of new SER, the increase in microbodies precedes the great increase in RER, and at the time of RER increase the volume proportion of microbodies is decreased.
304
HOPE
Therefore it would appear that the increased amount of RER reflects a demand for plasma proteins. During pregnancy there is a vast increase in blood volume, and the blood is diluted with plasma--a condition known as hydremia of pregnancy. There is therefore a physiological demand for plasma proteins, and this demand is such that it cannot be met by the slight amount of hypertrophy and hyperplasia that occurs during gestation and in consequence the RER itself hypertrophies. The volume proportions of the mitochondria and of the microbodies (Figs. 5 and 6) showed similar trends to the biochemical assays for enzymes associated with them, succinate dehydrogenase, glutamate dehydrogenase, and urate oxidase. Studies on the effect on the liver of drugs that reduce serum lipid levels such as ethyl-chlorophenoxy-isobutyrate or biphenylyl methylvaleric acid have shown that these drugs induce a large increase in the numbers of microbodies particularly those types without nucleoids (8, 12, 22, 25). The reduction in serum lipid is associated with increased catalase activity, but reduced urate oxidase activity (12, 22, 25). Catalase activity is associated with the matrix, which increases, whereas urate oxidase is associated with the nucleoid (1, 13). These results give some evidence, albeit circumstantial, that microbodies are associated with lipid metabolism as suggested by Novikoff and Shin (18) and one would expect therefore that the reduction in microbodies toward the end of gestation would be associated with increased serum lipid levels. In fact this does occur. In the rat, blood lipid begins to increase on day 11 of gestation and reaches a maximum on day 20 (21). This increase is mainly in the triglycerides, although phospholipids and cholesterol also increase. Other enzymes contained within microbodies are c~-hydroxy acid oxidase and d-amino acid oxidase (1). Together with urate oxidase and catalase, these enzymes suggest a general catabolic function, and the fall in the volume of microbodies would be associated with the increased emphasis on anabolic functions rather than catabolic functions during gestation that has been suggested by Roberge et al. (20) on the basis of decreased urea production. The variations in the volume proportions of cell organelles that occur during gestation rapidly return to the control values during early lactation, and the hypertrophied cells have the same composition as the controls. The hypertrophy probably represents a functional hypertrophy due to an increased demand associated with the vastly increased food intake during lactation (29). Toward the end of lactation this cellular hypertrophy is reduced after a wave of mitosis, and it has been suggested that hypertrophy itself can induce mitosis although the evidence, reviewed by Bullough (2), is conflicting. After weaning, the liver mass reduces; it would have been interesting to follow the mechanism of this reduction, but it was beyond the scope of this work. The picture that emerges, then, of the liver changes during gestation and lactation is that, during gestation, the liver responds to altered physiological demand by a
STEREOLOGYOF LIVER
305
slight cellular hypertrophy and a variation in the proportion of cell organelles associated with an increased requirement for plasma proteins and an alteration in lipid metabolism. During lactation, however, the proportions of cell organelles rapidly return to control values and the increased functional demand due to hyperphagia is met by an increase in liver mass, initially by cellular hypertrophy, but later by a hyperplastic phase that reduces the cell volume to the control value. The celIular hypertrophy is associated with an increase in ploidy that persists after lactation. REFERENCES 1. BAUDHUIN,P., BEAUFAY, H. and DE DUVE, C., J. Cell Biol. 26, 219 (1965). 2. BULLOUGH,W. S., The Evolution of Differentiation, p. 123. Academic Press, 1965. 3. CAMPBELL,P. N. and LAWEORD, G. R., in GRAN, F. C. (Ed.), Structure and Function of the Endoplasmic Reticulum in Liver, p. 57. Academic Press, New York, 1968. 4. CARPENTER,A.-M., J. Histoehem. Cytochern. 14, 834 (1966). 5. CLAWSON,C., VERNIER, R., CARPENTER,A.-M. and LAZAROW,A., J. Histochem. Cytochem. 6, 393 (1958). 6. DALLNER, G., SIEKEVtTZ,P. and PALADE, G. E., J. Cell Biol. 30, 73 (1966). 7. - - - - ibid. 30, 97 (1966). 8. DE LA IGLESIA,F. A., WALL, C., SOSA-LUCERO, J. C. and LUMB, G., Annu. Proc., 27th, Electron Microsc. Soc. Amer. Claitors Publishing Division, Baton Rouge, 1969. 9. DU BOISTESSEL[N,R., Proc. Eur. Soc. Study Drug Toxicity 5, 119 (1965). 10. EPSTEIN, C. J., Proc. Nat. Acad. Sci. U. S. 57, 327 (1967). 11. GLAS, U. and BAHR, G. F., ./. Cell Biol. 29, 507 (1966). 12. HESS, R., STKUBLI,W. and RIEsS, W., Nature (London) 208, 856 (1965). 13. HRUBAN, Z. and SWIFT, H., Science 146, 1316 (1964). 14. LOUD, A. V., J. Cell Biol. 15, 481 (1962). 15. - ibid. 37, 27 (1968). 16. LOUD, A. V., BARANY, W. C. and PACK, B. A., Lab. Invest. 14, 258 (1965). 17. NADAL, C. and ZAJDELA,F., Exp. Cell Res. 42, 99 (1966). 18. NOVIKOFF,A. B. and SHIN, W. Y., J. Microsc. 3, 187 (1964). 19. PEACHEY,L. D., J. Biophys. Biochem. Cytol. 4, 233 (1958). 20. ROBERGE,A., CHARBONNEAU,R. and BERLINGUET,L., Can. J. Biochem. 45, 1371 (1967). 21. Scow, R. O., CHERNIK,S. S. and BRINLEY, M. S., Amer. J. Physiol. 206, 796 (1964). 22. STEINER,J. W., PHILLIPS,M. J. and MIYAI, K., Int. Rev. Exp. Pathol. 3, 65 (1964). 23. SrJ~UBLI,W. and HESS, R., Proc. Int. Congr. Electron Microscopy, 6th, p. 625. Maruzen, Tokyo, 1966. 24. ST~IUBLI,W., HESS, R. and WEmEL, E. R., J. Cell Biol. 42, 92 (1969). 25. SVOBODA,D. J. and AZARNOFF, D. L., Or. Cell Biol. 30, 442 (1966). 26. WEmEL, E. R., KISTLER,G. S. and SCHERLE,W. F., J. Cell Biol. 30, 23 (1966). 27. WHBEL, E. R., STXUBH, W., GN~.GI, H. R. and HESS, F. A., J. Cell Biol. 42, 68 (1969). 28. WICKSELL,S. D., Biometrika 17, 84 (1925). 29. WILSON, R., DOELL, B. A., GROaER, W., HOPE, J. and GELLATLY,J. B. ~[V[., in ROE, F. J. C. (Ed.), Pro& 2nd NuffieM Food Safety Conference. Blackwell, Oxford, 1970, in press.
292
J. ULTRASTRUCTURE RESEARCH
33, 292-305 (1970)
Stereological Analysis of the Ultrastructure of Liver Parenchymal Cells During Pregnancy and Lactation J. HOPE
Biology Division, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, England Received February 24, 1970 Alterations in the nuclear volume, cytoplasmic volume, and volume proportions of RER, mitochondria, and microbodies in the cytoplasm were measured by morphometric techniques in the liver parenchymal cells of rats, during gestation and lactation. During gestation the most marked effects were an increased volume proportion of RER and an initial increase, followed by a decrease, of the volume proportion of microbodies. It is suggested that these effects may be associated with hydremia and hyperlipemia of pregnancy, respectively. During lactation the volume proportions of the cell organelles rapidly return to the control values, and the greatest effect is a marked hypertrophy of the parenchymal cells.
This work was undertaken as part of an analytical, biochemical, and morphological investigation of liver enlargement during gestation and lactation aimed at obtaining background information against which toxic liver enlargement could be evaluated (29). Using electron microscopy, it was hoped to identify any ultrastructural changes that might occur and to assess the relative contributions of hyperplasia and hypertrophy to the enlargement of the liver. It was expected that the ultrastructural changes in such a normal physiological condition would be minimal and therefore it was considered that a purely qualitative assessment would not be adequate. As du Boistesselin (9) has emphasized, there are difficulties in the subjective interpretation of subcellular changes in the liver even when such changes are qualitatively pronounced, and Steiner et al. (24) have expressed the need for more facts and less subjective hypothesis. Obviously, a more objective technique is required. In any case a quantitative technique is necessary to distinguish between hypertrophy and hyperplasia and to correlate morphological and biochemical data (27); accordingly, the technique of morphological analysis as described by Weibel et al. (26) and by Loud (15) was employed.
STEREOLOGY OF LIVER
293
MATERIALS AND METHODS Rats were taken at 0, 3, 9, 14, 21 days of gestation and 0, 3, 9, 14, 18, and 49 (recovery) days of lactation. Two test animals and one control animal were taken from each group, a total of 33 animals. Animals were anesthetized with ether and the liver was exposed; a small portion of the median lobe was excised, diced into small pieces, and fixed for 1 hour at 0°C in 1 % osmium tetroxide solution in phosphate buffer at p H 7.4. The tissue was dehydrated through alcohols and propylene oxide and embedded in Araldite epoxy resin. Tissues from the two test animals and control animal of each group were processed together. In most cases ten blocks of tissue from each animal were embedded. Initially, 1 l~ sections were cut from each block, stained with toluidine blue, and scanned with the optical microscope. Loud (15) has shown that there are structural variations between cells in different regions of the liver lobule, but most of the significant differences occur in cells around the central vein and these comprise less than 20 % of the parenchymal ceils. Accordingly, areas containing the central vein and immediately surrounding hepatocytes were trimmed from the block, so that mid-zonal and peripheral cells only of the hepatic lobules were included in the sections. If it was not possible to trim away these areas, the blocks were discarded. In this way it was hoped to reduce the variability of the results, but still retain a representative sample of the liver. There was, therefore, a maximum of 20 blocks for each test group and 10 blocks for each control group available and sections from all the available blocks were examined in the electron microscope. Fifty random electron micrographs were taken from each test and control group for stereological analysis. This number of micrographs was distributed as evenly as possible among the available blocks. R a n d o m micrographs were taken in order to avoid bias in the selection of fields, and care was taken that similar areas of adjacent sections were not photographed. The micrographs were taken at an initial magnification of 3 000 x and were enlarged to 12 000 x for analysis. A probe with 100 points was laid over each micrograph and the number of points overlying certain structures were tabulated. The structures counted were: (1) canaliculi and sinusoids, (2) nuclei, (3) mitochondria, (4) rough endoplasmic reticulum (RER), (5) microbodies, (6) lysosomes, (7) lipid, (8) the remaining cytoplasm. Counts of structures 3 to 8 were totaled to give the total proportion of cytoplasm. It was not possible to obtain counts of glycogen because of vagaries in staining. A t the same time up to 150 electron micrographs were taken of random nuclei in the parenchymal cells for the determination of nuclear volume. Since the accuracy of the magnification is important in this determination, the electron microscope was calibrated with a replica grating before each group of nuclei were photographed. The calibrations were all within 2 % of the nominal magnification.
RESULTS The aggregate counts f r o m all the m i c r o g r a p h s in each g r o u p , given in Tables I a n d II, were used to calculate the n u c l e a r - c y t o p l a s m i c ratio, the v o l u m e p r o p o r t i o n of canaliculi a n d sinusoids to p a r e n c h y m a l cells, a n d the v o l u m e p r o p o r t i o n of m i t o c h o n d r i a , r o u g h e n d o p l a s m i c reticulum, a n d m i c r o b o d i e s in the cytoplasm. T h e r e
294
HOPE TABLE I TOTAL COUNTs--CoNTROL ANIMALS
Day
Number Canaliculi of Microand graphs Sinusoids
Nuclei
Total MitoCytoplasm chondria
RER
Microbodies
Lysosomes
Gestation 0 3 9 14 21
50 50 50 50 47
600 597 468 740 510
200 334 309 301 297
4 198 4 068 4 224 3 960 3 892
1 175 1 067 1 101 l 076 894
135 207 277 191 145
63 65 81 75 72
15 39 17 21 18
Lactation 0 3 9 14 18 49
49 33 48 50 28 49
475 254 488 572 405 519
303 254 155 265 126 196
4 122 2 792 4 158 4 163 2 269 4 185
1 064 711 1 125 1 096 574 1 156
198 192 268 279 144 278
88 79 83 83 40 81
3 23 9 14 15 5
w e r e i n s u f f i c i e n t c o u n t s o f l i p i d t o o b t a i n m e a n i n g f u l r e s u l t s . T h e c o u n t s of l y s o s o m e s a n d of t h e c a n a l i c u l i a n d s i n u s o i d s w e r e a l s o t o o l o w a n d t o o v a r i a b l e b e t w e e n a n i m a l s t o give s t a t i s t i c a l l y u s e f u l r e s u l t s , b u t t h e v o l u m e p r o p o r t i o n
of l y s o s o m e s o v e r all
the control animals could be calculated with reasonable accuracy. The results are given in Tables III and IV. Following the procedure of
Loud (15)
t h e v a l u e f o r t h e R E R h a s b e e n i n c r e a s e d b y 50 % t o a l l o w f o r m e m b r a n e s i n c l i n e d t o t h e p l a n e of t h e s e c t i o n s . I t w a s n o t p o s s i b l e t o a r r i v e a t t h e v o l u m e o f t h e cell d i r e c t l y b y e l e c t r o n m i c r o s c o p y a l o n e , b u t it w a s c a l c u l a t e d f r o m t h e n u c l e a r - c y t o p l a s m i c r a t i o , as m e a s u r e d a b o v e , a n d a m e a s u r e of t h e v o l u m e o f t h e n u c l e i o b t a i n e d as d e s c r i b e d b e l o w . TABLE II TOTAL COUNTS--EXPERIMENTAL ANIMALS
Day
Number Canaliculi of Microand graphs Sinusoids
Nuclei
Total MitoCytoplasm chondrla
RER
Microbodies
Lysosomes
Gestation 0 3 9 14 21
50 50 50 50 50
587 380 671 509 464
266 264 235 233 220
4 146 4 356 4 093 4 256 4 312
1 136 1 089 1 031 1 000 774
133 197 251 298 301
69 95 89 79 57
22 20 20 23 33
Lactation 0 3 9 14 18 49
50 50 50 48 50 50
423 502 494 611 732 483
207 238 97 210 319 304
4 372 4 260 4 412 3 979 3 949 4 212
907 1 030 1 158 1 061 1 080 1 094
228 316 340 282 238 245
38 85 84 92 100 72
18 11 12 11 24 18
295
STEREOLOGY OF LIVER TABLE III VOLUME PROPORTIONS OF STRUCTURES IN THE LIVER OF CONTROL ANIMALSa
Day
NuclearCytoplasmic Ratio (%)
Percent Volume of Mitochondria in Cytoplasm
Percent Volume of RER in Cytoplasm
Percent Volume of Microbodies in Cytoplasm
Percent Volume of Canaliculi and Sinusoids in Total Tissue
4.8 (1.80)
28.0 (1.37)
4.8 (1.10)
1.5 (0.22)
8.2 (1.80)
26.2 (1.37)
7.7 (1.10)
1.6 (0.22)
7.3 (1.80) 7.6 (1.80)
26.1 (1.37) 27.2 (1.37)
9.9 (1.10) 7.2 (1.10)
1.9 (0.22) 1.9 (o.22)
7.6 (1.82)
23.0 (1.38)
5.6 (1.10)
1.8 (0.25)
13.6 13.6 10.3 17.4 12.2
Lactation 0 3 9 14 18 49
7.4 9.1 3.7 6.3 5.5 4.7
25.8 25.5 27.1 26.3 25.3 27.7
2.1 2.8 2.0 2.0 1.8 1.9
(0.22) (0,28) (0.25) (0.22) (0.32) (0.22)
10.7 7.7 10.2 11.4 14.5 10.8
Mean (mean sE)
6.6 (1.84)
8.4 (1.12)
1.9 (0.24)
12.0
SE of mean
0.55
0.34
0.07
Gestation 0 3 9 14 21
(1.80) (1.98) (1.82) (1.80) (2.07) (1.80)
(1.37) (1.49) (1.38) (1.37) (1.55) (1.37)
26.2 (1.40) 0.42
7.2 10.4 9.6 10.1 9.5 10.1
(1.10) (1.17) (1.10) (1.10) (1.21) (1.10)
a Standard error is given in parentheses.
TABLE IV VOLUME PROPORTIONS OF STRUCTURES IN THE LIVER OF EXPERIMENTAL ANIMALSa
NuclearCytoplasmic Ratio (%)
Percent Volume of Mitochondria in Cytoplasm
Percent Volume of RER in Cytoplasm
Percent Volume of Microbodies in Cytoplasm
Percent Volume of Canaliculi and Sinusoids in Total Tissue
Gestation 0 3 9 14 21
6.4 (1.54) 6.1 5.7 5.5 5.1
27.4 (1.08) 25.0 25.2 23.5 17.9
4,8 (0,84) 6.8 9.2 10.5 10.5
1.7 (0.22) 2.2 2.2 1.8 1.3
13.3 8.2 15.5 11.3 10.2
Lactation 0 3 9 14 18 49
4.7 5.6 2.2 5.3 8,1 7,2
20.7 24.2 26.3 26.7 27.3 26.0
7.8 11.1 11.6 10.7 9.0 8.7
0.9 2.0 1.9 2.3 2.5 1.7
9.2 10.0 9.9 12.7 14.6 9.7
Day
SE is given in parentheses. The standard error given next to top entry in each column applies to each entry in column.
296
HOPE
TABLE V VOLUME OF N U C L E U S AND CYTOPLASM IN THE LIVER OF CONTROL ANIMALS~
Day
Number of Measurements
Gestation 0 3 9 14 21 Lactation 0 3 9 14 18 49 Mean (mean SE) SE of mean
141 150 150 143 135 120 144 132 94 115 133 ---
Mean Diameter of Nuclei d (/0
6.62 (0.13) 6.80 (0.13) 6.83 (0.13) 6.69 (0.13) 6.97 (0.14) 7.18 (0.15) 6.43 (0.13) 6.20 (0.14) 6.46 (0.16) 6.23 (0.15) 6.12 (0.14) 6.59 (0.14) 0.04
Correctedb Mean Diameter of Nuclei D (t0
8.43 (0.17) 8.66 (0.17) 8.69 (0.17) 8.52 (0.17) 8.87 (0.18) 9.14 (0.19) 8.19 (0.17) 7.89 (0.18) 8.22 (0.20) 7.93 (0.19) 7.79 (0.18) 8.39 (0.18) 0.05
Volume of Nucleus (#0
Volume of Cytoplasm (/~0
314 (11.8) 340 (12.1) 344 (12.6) 324 (12.0) 365 (13.4) 400 (15.0) 288 (11.1) 257 (10.7) 29l (13.7) 26l (11.6) 248 (10.4) 312 (12.2) 3.7
6540 (1 740) 4150 (710) 4710 (890) 4260 (780) 4800 (890) 5400 (1 010) 3160 (510) 6950 (2 380) 4620 (1 040) 4740 (1 390) 5280 (1 400) 4970 (1 160) 318
a Standard error is given in parentheses. b Using the formula 15 - (4/~) d.
The nuclear volume was calculated f r o m the average nuclear diameter derived f r o m measurements on up to 150 sections t h r o u g h nuclei p h o t o g r a p h e d at a magnification of 3000 ×. A l t h o u g h in histological preparations and in 1 # sections of tissue prepared for electron microscopy, the nuclear sections have circular profiles, in ultrathin sections they m a y be elliptical. It is well k n o w n that considerable compression can occur in ultrathin sections in the direction of sectioning (19), and that measurements in this direction are unreliable. Consequently in the present w o r k measurements were made only in a direction parallel to the knife edge; this was invariably along the major axis of any elliptical profiles. The average measured diameter d is related to the actual mean diameter D by the f o r m u l a /9 = (4/~)d, and this formula was used instead of a Wicksell transformation (28) to arrive at the average nuclear diameter. The disadvantage of using this f o r m u l a is that it gives an average diameter, whereas a Wicksell transformation gives a histog r a m of the distribution of nuclear diameters. However, for the purposes of this paper a comparison of mean diameters is sufficient, and it is unnecessary to go t h r o u g h the rather involved c o m p u t a t i o n of a Wicksell transformation. The results for average nuclear diameter, nuclear volume, and cytoplasmic volume are given in Tables V and VI.
297
S T E R E O L O G Y OF L I V E R
TABLE VI VOLUME OF ~',~UCLEUS AND CYTOPLASM IN THE LIVER OF EXPERIMENTAL ANIMALS a
Day
Gestation 0 3 9 14 21 Lactation 0 3 9 14 18 49
Number of Measurements
Mean Diameter of Nuclei d (~)
150 150 150 150 150 150 141 135 144 95 150
6.42 (0.14) 7.26 (0.14) 6.54 (0.14) 7.05 (0.14) 6.79 (0.15) 7.48 (0.14) 6.85 (0.15) 6.64 (0.15) 6.84 (0.16) 6.99 (0.19) 6.84 (0.15)
Corrected b Mean Diameter of Nuclei D (/~)
8.17 (0.18) 9.24 (0.18) 8.33 (0.18) 8.97 (0.18) 8.64 (0.19) 9.52 (0.18) 8.72 (0.19) 8.45 (0.19) 8.71 (0.20) 8.90 (0.25) 8.71 (0.19)
Volume of Nucleus (/13)
286 (12.1) 413 (14.8) 303 (12.2) 378 (14.4) 338 (14.8) 452 (15.8) 347 (14.0) 316 (13.4) 346 (14.6) 369 (19.1) 346 (14.4)
Volume of Cytoplasm (#2)
4,470 (840) 6,770 (1 300) 5,320 (1 080) 6,870 (1 440) 6,630 (1 510) 9,610 (2 280) 6,200 (1 300) 14,360 (6 810) 6,530 (1 440) 4,560 (760) 4,810 (840)
Standard error is given in parentheses, b Using the formula D - (4In) d.
Treatment of results Although the preparative manipulations of fixation, dehydration, and emlzedding were standardized between groups processed at different times, there still exists the possibility of systematic errors due to processing. However, the experimental and control material from each group was processed side by side and therefore, to minimize any systematic errors, the experimental values have been expressed as a Fercentage of the corresponding control value. The experimental values may then be quoted as this percentage or expressed in absolute terms calculated from the mean control value. The results are given in both forms in Table VII and expressed graphically in Figs. 1-6. The dotted lines on each side of the control value represent two standard errors and give an indication of the statistical significance of the experimental results. Nuclear volume. The nuclear volume (Fig. 1) was elevated above the control values on days 3 and 14 of gestation, but not days 0, 9, and 21. During lactation there was a significant increase at day 3 which was maintained to day 14 after which there was a further increase at days 18 and 49. Nuclear-cytoplasmic ratio. The nuclear-cytoplasmic ratio (Fig. 2) fell within two standard errors either side of the average control value throughout the course of the experiment, but nevertheless, an interesting trend was indicated. In the first 3 days of gestation there was an initial sharp fall in the ratio. Between day 3 of gestation and day 9 of lactation there was a further gradual decrease followed by a sharp increase at days 14 and 18 of lactation. Thereafter there was little change at day 49.
298
HOPE TABLE VII E X P E R I M E N T A L R E S U L T S EXPRESSED I N R E L A T I O N TO THE C O N T R O L V A L U E AS A PERCENTAGE, AND IN ABSOLUTE VALUES
Volume of Nucleus r% of control #~
• % of control
Mean control value Gestation 0 3 9 14 21
100 91 121 88 117 93
312 284 379 275 364 289
Lactation 0 3 9 14 18 49
113 120 123 119 141 140
353 376 384 371 441 435
Day
NuclearCytoplasmic Ratio
Volume of Cytoplasm
,
Percent Volume of Mitechondria
Percent Volume of R E R , ~ ~ % of control %
Percent Volume of Microbodies Percent • " - - - - - - - Volume of % of Canaliculi conand Sinusoids trol % %
/~
% of control
%
"% of control
100 68 163 113 161 138
4970 3 390 8110 5 600 8 010 6 850
100 133 74 78 72 67
6.6 8.8 4.9 5.2 4.8 4.4
100 98 95 97 86 78
26.2 100 8.4 25.6 100 8.4 25.0 88 7.4 25.3 93 7.8 22.6 146 12.2 20.4 187 15.7
100 113 137 116 95 72
1.9 2.2 2.6 2.2 1.8 1.4
12.0 11.7 7.2 18.0 7.8 10.0
178 196 207 141 96 91
8 830 9 720 10270 7020 4 770 4 520
64 62 59 84 147 153
4.2 4.1 3.9 5.6 9.7 10.1
80 95 97 102 108 94
21.0 24.9 25.4 26.6 28.3 24.6
43 0.8 71 1.4 95 1.8 115 2.2 139 2.6 89 1.7
10.3 15.6 11.6 13.4 12.1 10.8
Cytoplasmic volume. A l t h o u g h
%
108 107 121 106 95 86
9.1 9.0 10.1 8.9 8.0 7.2
elevation of the cytoplasmic v o l u m e (Fig. 3) oc-
curred at days 3 and 14 of gestation this was not p r o n o u n c e d . H o w e v e r , it was significantly higher at days 0 to 9 of lactation, and at this time it was double that of the c o n t r o l value.
Cytoplasmic organelles. The
v o l u m e p r o p o r t i o n of r o u g h e n d o p l a s m i c r e t i c u l u m
(Fig. 4) increased rapidly f r o m day 9 of gestation to reach 187 % of the c o n t r o l value -500
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FIG. 1. Nuclear volume of liver parenchymal ceils during gestation and lactation in the rat.
299
STEREOLOGY OF LIVER 225' 200" -12 175" . . . . . . . . . . . . . . . . . . . . .
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Lactation Days of Gestation and Lactation
FIG. 2. N u c l e a r - c y t o p l a s m i c ratio for liver parenchymal cells during gestation a n d lactation in the rat.
at day 21 of gestation. Thereafter, it dropped back sharply after parturition and did not differ much from the control value throughout lactation. The volume proportion of mitochondria (Fig. 5) was significantly lower than that of the control value at days 14 and 21 of gestation and day 0 of lactation. The volume proportion of microbodies (Fig. 6) showed a n i n c r e a s e a t day 3 of gestation, followed by a gradual 200
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decrease to a significantly low value at day 0 of lactation, followed again by an increase to day 18 of lactation and then a further fall at day 49.
DISCUSSION
Control values The results of the volume proportions of cytoplasmic organelles in the control animals are in general agreement with the results of previous workers (4, 5, 11, 14-16, 23, 27), although there is some variation in results both between workers and for the same author in different papers. This may be due to variations between animal strains or to variations in preparative methods. Loud et al. (16) have shown that the buffering system for the osmium tetroxide fixative, and the embedding medium, may both affect the final results. The volume proportion of 26.2 % for the mitochondria in the present work compares with values of 18.1-27.2 in the literature. The most comprehensive and detailed morphometric studies on the normal liver are those by Loud (15) and Weibel et al. (27). Loud made individual measurements from cells from three different regions of the liver lobule, whereas Weibel et al. (27) considered the hepatocytes as a whole. Since Loud found similar values for peripheral and mid-zonal cells, but significantly different values for the centrilobular cells, the latter were not included in the present study, and the peripheral and mid-zonal cells were treated as a single group. Loud
STEREOLOGY OF LIVER
301
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Lactation Days of G e s t a t i o n a n d L a c t a t i o n
F i t . 5. Volume density of mitochondria of liver parenchymal cells during gestation and lactation in the rat.
(i5) gave values of 19.1% for the volume proportion of mitochondria in the midzonal cells and 19.8 % for peripheral cells. The value of 23 % found by Weibel et al. (27) is closer to the present result of 26.2 % and would probably have been higher had they excluded centrilobular cells from the analysis, since the volume proportion of mitochondria in these cells is only 12.9 % (15). The volume proportion of microbodies of 1.9 % compares with values in the literature of 0.6-2.6 %. This high value was after permanganate fixation and Aquon embedding (14). Loud (15) reported a value of 1.4 % and Weibel et al. (27) reported
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Lactation Days of G e s t a t i o n a n d L a c t a t i o n
F~6. 6. Volume density of microbodies of liver parenchymal cells during gestation and lactation in the rat.
302
HOVE
1.5 %. The volume proportion of lysosomes was given by Loud (15) as 0.3 % and by Weibel et al. as 0.9 %, compared to the present result of 0.4 %. There is a difference in the volume proportion of the rough endoplasmic reticulum, measured as 8.4 % in the present work and reported as 10 % by Weibel et al. (27). This difference is greater than the figures suggest since the results of Weibel et al. were not corrected for the inclination of the membranes to the plane of the section. Previous workers have calculated the nuclear volume from measurements on optical micrographs of 1 # sections (15, 27), but have commented that the section thickness is somewhat too great for optimum accuracy. In the present work, therefore, the nuclear diameter was calculated from measurements made on electron micrographs at a magnification of 3000 ×. The results, after the formula/9 = (4/z0 c7 had been applied, gave a diameter of 8.3 #, compared with 8.1 # (15) and 7.9 # (27). There appears to be very little difference, then, between measurements made on ultrathin and on 1/z sections. The nuclear cytoplasmic ratio measured in the present work (6.6 %) is similar to that calculated from the data of other workers: 5.5 % (15), 6.5 % (27), and 7.9 % (4). Consequently, the cytoplasmic volume calculated from the nuclear volume and nuclear cytoplasmic ratio is similar to the values obtained by other workers by more direct methods, namely 4970 #z in the present work, compared with 5 100/z z (15) and 4 660 #3 (27).
Changes during gestation and lactation These experimental results give a quantitative description of some of the morphological changes that take place within the hepatocyte during gestation and lactation and that can be correlated with histopathological findings and with biochemical results. These results have a bearing on the question of whether the liver enlargement is due to hyperplasia, hypertrophy, or both. The trend of the values for the nuclear cytoplasmic ratio, if taken alone, would indicate an initial sharp hypertrophy of the cells, followed by a more gradual increase in cell size during gestation and lactation, until the end of lactation, when the cells reduce in volume. However, this ignores the variations in nuclear volume that occur, and if these are taken into account to calculate the cytoplasmic volume, then the results given in Fig. 3 are obtained. Apart from a low value at day 9 of gestation, this graph suggests a possible phase of hypertrophy during gestation, since the results fall just outside two standard errors of the control value. A further much greater and statistically more significant phase of hypertrophy occurs during lactation, but the cytoplasmic volume returns to the control value by day 18 of lactation. The liver reaches its maximum size at this time, and therefore the number of cells must necessarily have increased. The results are, there-
STEREOLOGY OF LIVER
303
fore, suggestive of two phases of hypertrophy each followed by hyperplasia, and this is in agreement with parallel histological findings in which phases of hypertrophy in gestation and lactation, as determined by nuclear counts on a standard microscopic field, were followed by waves of mitosis (29). The initiation of mitosis during gestation was thought to be triggered by single cell necrosis, but this was not observed during lactation. However, during lactation the cytoplasm reaches double the control value and it has been suggested that hypertrophy itself is a sufficient trigger for mitosis. The nuclear volume, after some variation during gestation and lactation, increases significantly above the control value at day 3 of lactation and remains elevated even after recovery at day 49. This may be taken as evidence of increased ploidy since the nuclear volume doubles with each increase in ploidy (10). Nadal and Zajdela (17) have shown that an increase in ploidy is preceded by a binucleate stage, but although in the present study the percentage of binucleate cells was noted, there was apparently no difference from the controls and no pattern that could be associated with ploidy. The most marked change in the cytoplasmic organelles was the increase in volume proportion of the rough endoplasmic reticulum during gestation. This correlates with an increase in amount of RNA in the liver determined biochemically and an increased basophilia noted histologically (29). However, an increased basophilia in the first 3 days of lactation was not reflected in an increase in the volume proportion of RER. A similar increase in RER occurs in the postnatal differentiation of hepatocytes (6, 7) and after administration of phenobarbital (23). In the first instance this increase is followed chronologically by a proliferation of smooth endoplasmic reticulum (SER), but in the second instance the SER increase occurs simultaneously with that of the RER. In both cases the SER increase is thought to be derived by the degranulation of the RER at the ends of the cisternae and the budding-off of SER elements (23). In the present study the SER was not measured morphologically, but no proliferation was seen subjectively. This is in agreement with biochemical results (29) which indicate that microsomal drug processing enzymes, apart from azobenzene reductase during gestation, did not increase in activity, but were unchanged or even reduced in activity. This is not unexpected since the liver is responding to an increased normal physiological demand, rather than to an alteration in function. The functions of the RER in the liver are, the manufacture of serum proteins for export, the synthesis of new membranes, and the synthesis of microbodies. The synthesis of cytoplasmic proteins is not a function of the RER since these are synthesized by free polysomes not bound to membranes (3). As we have seen there is no synthesis of new SER, the increase in microbodies precedes the great increase in RER, and at the time of RER increase the volume proportion of microbodies is decreased.
304
HOPE
Therefore it would appear that the increased amount of RER reflects a demand for plasma proteins. During pregnancy there is a vast increase in blood volume, and the blood is diluted with plasma--a condition known as hydremia of pregnancy. There is therefore a physiological demand for plasma proteins, and this demand is such that it cannot be met by the slight amount of hypertrophy and hyperplasia that occurs during gestation and in consequence the RER itself hypertrophies. The volume proportions of the mitochondria and of the microbodies (Figs. 5 and 6) showed similar trends to the biochemical assays for enzymes associated with them, succinate dehydrogenase, glutamate dehydrogenase, and urate oxidase. Studies on the effect on the liver of drugs that reduce serum lipid levels such as ethyl-chlorophenoxy-isobutyrate or biphenylyl methylvaleric acid have shown that these drugs induce a large increase in the numbers of microbodies particularly those types without nucleoids (8, 12, 22, 25). The reduction in serum lipid is associated with increased catalase activity, but reduced urate oxidase activity (12, 22, 25). Catalase activity is associated with the matrix, which increases, whereas urate oxidase is associated with the nucleoid (1, 13). These results give some evidence, albeit circumstantial, that microbodies are associated with lipid metabolism as suggested by Novikoff and Shin (18) and one would expect therefore that the reduction in microbodies toward the end of gestation would be associated with increased serum lipid levels. In fact this does occur. In the rat, blood lipid begins to increase on day 11 of gestation and reaches a maximum on day 20 (21). This increase is mainly in the triglycerides, although phospholipids and cholesterol also increase. Other enzymes contained within microbodies are c~-hydroxy acid oxidase and d-amino acid oxidase (1). Together with urate oxidase and catalase, these enzymes suggest a general catabolic function, and the fall in the volume of microbodies would be associated with the increased emphasis on anabolic functions rather than catabolic functions during gestation that has been suggested by Roberge et al. (20) on the basis of decreased urea production. The variations in the volume proportions of cell organelles that occur during gestation rapidly return to the control values during early lactation, and the hypertrophied cells have the same composition as the controls. The hypertrophy probably represents a functional hypertrophy due to an increased demand associated with the vastly increased food intake during lactation (29). Toward the end of lactation this cellular hypertrophy is reduced after a wave of mitosis, and it has been suggested that hypertrophy itself can induce mitosis although the evidence, reviewed by Bullough (2), is conflicting. After weaning, the liver mass reduces; it would have been interesting to follow the mechanism of this reduction, but it was beyond the scope of this work. The picture that emerges, then, of the liver changes during gestation and lactation is that, during gestation, the liver responds to altered physiological demand by a
STEREOLOGYOF LIVER
305
slight cellular hypertrophy and a variation in the proportion of cell organelles associated with an increased requirement for plasma proteins and an alteration in lipid metabolism. During lactation, however, the proportions of cell organelles rapidly return to control values and the increased functional demand due to hyperphagia is met by an increase in liver mass, initially by cellular hypertrophy, but later by a hyperplastic phase that reduces the cell volume to the control value. The celIular hypertrophy is associated with an increase in ploidy that persists after lactation. REFERENCES 1. BAUDHUIN,P., BEAUFAY, H. and DE DUVE, C., J. Cell Biol. 26, 219 (1965). 2. BULLOUGH,W. S., The Evolution of Differentiation, p. 123. Academic Press, 1965. 3. CAMPBELL,P. N. and LAWEORD, G. R., in GRAN, F. C. (Ed.), Structure and Function of the Endoplasmic Reticulum in Liver, p. 57. Academic Press, New York, 1968. 4. CARPENTER,A.-M., J. Histoehem. Cytochern. 14, 834 (1966). 5. CLAWSON,C., VERNIER, R., CARPENTER,A.-M. and LAZAROW,A., J. Histochem. Cytochem. 6, 393 (1958). 6. DALLNER, G., SIEKEVtTZ,P. and PALADE, G. E., J. Cell Biol. 30, 73 (1966). 7. - - - - ibid. 30, 97 (1966). 8. DE LA IGLESIA,F. A., WALL, C., SOSA-LUCERO, J. C. and LUMB, G., Annu. Proc., 27th, Electron Microsc. Soc. Amer. Claitors Publishing Division, Baton Rouge, 1969. 9. DU BOISTESSEL[N,R., Proc. Eur. Soc. Study Drug Toxicity 5, 119 (1965). 10. EPSTEIN, C. J., Proc. Nat. Acad. Sci. U. S. 57, 327 (1967). 11. GLAS, U. and BAHR, G. F., ./. Cell Biol. 29, 507 (1966). 12. HESS, R., STKUBLI,W. and RIEsS, W., Nature (London) 208, 856 (1965). 13. HRUBAN, Z. and SWIFT, H., Science 146, 1316 (1964). 14. LOUD, A. V., J. Cell Biol. 15, 481 (1962). 15. - ibid. 37, 27 (1968). 16. LOUD, A. V., BARANY, W. C. and PACK, B. A., Lab. Invest. 14, 258 (1965). 17. NADAL, C. and ZAJDELA,F., Exp. Cell Res. 42, 99 (1966). 18. NOVIKOFF,A. B. and SHIN, W. Y., J. Microsc. 3, 187 (1964). 19. PEACHEY,L. D., J. Biophys. Biochem. Cytol. 4, 233 (1958). 20. ROBERGE,A., CHARBONNEAU,R. and BERLINGUET,L., Can. J. Biochem. 45, 1371 (1967). 21. Scow, R. O., CHERNIK,S. S. and BRINLEY, M. S., Amer. J. Physiol. 206, 796 (1964). 22. STEINER,J. W., PHILLIPS,M. J. and MIYAI, K., Int. Rev. Exp. Pathol. 3, 65 (1964). 23. SrJ~UBLI,W. and HESS, R., Proc. Int. Congr. Electron Microscopy, 6th, p. 625. Maruzen, Tokyo, 1966. 24. ST~IUBLI,W., HESS, R. and WEmEL, E. R., J. Cell Biol. 42, 92 (1969). 25. SVOBODA,D. J. and AZARNOFF, D. L., Or. Cell Biol. 30, 442 (1966). 26. WEmEL, E. R., KISTLER,G. S. and SCHERLE,W. F., J. Cell Biol. 30, 23 (1966). 27. WHBEL, E. R., STXUBH, W., GN~.GI, H. R. and HESS, F. A., J. Cell Biol. 42, 68 (1969). 28. WICKSELL,S. D., Biometrika 17, 84 (1925). 29. WILSON, R., DOELL, B. A., GROaER, W., HOPE, J. and GELLATLY,J. B. ~[V[., in ROE, F. J. C. (Ed.), Pro& 2nd NuffieM Food Safety Conference. Blackwell, Oxford, 1970, in press.