Liver regeneration and hepatic polyploidy in the hypophysectomized rat

I. I. Geschwind, M. Alfert and Caroline Schooley

232

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

BEAMS, H. W. and KING, R. L., Anat. Record 83, 281 (1942). DI STEFANO, H. S., DIERMEIER, H. F. and TEPPERMAN, J., Endocrinology

57, 158

(1955).

HARRISON, M. F., Nafure 171, 611 (1953). HEIZER, P., Chromosoma 7, 281 (1955). HELWEG-LARSEN, H. FR., Acta Pathol. Microbial. Stand. 26, 609 (1949). HIMES, M., HOFFMAN, J., POLLISTER, A. W. and POST, J., J. ,k’t. Sinai Hosp. 24, 935 (1957). SWARTZ, F. J., Chromosoma 8, 53 (1956). SWIFT, H., Physiol. Zool. 23, 169 (1950). WILSON, J. W. and L~riuc, E. H., Am. J. Anat. 82, 353 (1948).

LIVER

REGENERATION

AND

HEPATIC

HYPOPHYSECTOMIZED I. I. GESCHWIND, Hormone

M. ALFERT

and

POLYPLOIDY

IN THE

RAT’ CAROLINE

SCHOOLEYe

Research Laboratory and Department of Zoology and its Cancer Research Genetics Laboratory, University of California, Berkeley, Calif., U.S.A. Received

May

13, 1958

1-r has b een known for some time that the cells of many adult mammalian livers show polyploidy. In the present paper an examination is made of some factors involved in the hormonal control of polyploidy in rat liver (cf. preceding note [I]). As shown by Di Stefano et al. [2] in rats the progression towards higher levels of ploidy in the liver can be arrested by hypophysectomy; furthermore, the polysomatic pattern is essentially absent in livers,of hereditary pituitary dwarf mice [3, 51 but its appearance can be induced by treatment with pituitary preparations more or less pure with respect to growth hormone. Similarly, the progression toward higher levels of ploidy can be reinstated in the hypophysectomized rat by means of such preparations [2]. The present experiments were undertaken to determine whether growth hormone is a sine qua non for the progressive development of the polysomatic growth pattern. As mentioned above, hypophysectomy is known to arrest the further increase of polyploidy with age; it is also known that liver restoration following hepatectomy [6] and Ccl, poisoning [4] in otherwise intact animals is accompanied by an accelerated shift towards higher levels of ploidy. We have now determined the cell population in the regenerated livers of rats subjected first to hypophysectomy and subsequently to partial hepatectomy. In two preliminary experiments male rats of the Long-Evans strain were hypo1 Supported by Cancer ResearchFunds of the University of California. * With

the expert

Experimental

technical

Cell Research 15

assistance

of Charles

Jordan

and

Norma

0. Goldstein.

Polyploidy TABLE

in regenerated liver

233

of binucleate

I. Distribution

and polyploid cells in regenerating the hypophysectomized rat. Percentage

No. of animals

Lobeb

H at 21 d, S at 28d H at 21 d, Hepat at 28 d, S at 42 d

4

R

3

R

H at 42 d, S at 49 d H at 42 d, Hepat at 49 d, S at 63d

5

R

8

R

H at 21 d, S at 28 or 42 d H at 21 d, Hepat at 28 d, S 42d

7 9

Esperiment

1

2

3

Groupa

Normals, S at 42 d a ’ ’ d e f

6

H, hypophysectomized; R, right lateral; L, left Range. Mean i- standard error. “p” values for R at 42 “p” values for Normals

physectomized animals were

2n

2n Binucleate

liuer of

of cell types

4n

4n Binucleate

8n

0.0

72.2 (63.1-77.6)’ 54.8 (53.4-57.0)

21.9 (16.3-30.5) 10.7 (9.6-11.6)

4.5 (3.1-5.6) 31.5 (28.8-34.7)

0.3 (0.1-0.5) 1.2 (0.8-1.5)

0.4 (0.3-0.5)

36.6 (28.1-47.0) 18.4 (11.2-25.0)

21.9 (18.5-25.7) 2.7 (0.5-4.8)

37.4 (24.7-48.5) 69.8 (63.1-77.9)

2.2 (1.5-2.9) 1.7 (0.7-2.9)

0.2 (0.1-0.4) 4.7 (1.8-10.2)

R

81.2 i 2.2d

13.5k1.9

3.8 i: 0.6

0.1

0.0

L at 28 d

77.5 & 1.4

15.5+ 1.6

4.6kO.7

0.2

0.0

R at 42 d

59.7 ir 2.3 p
9.6 + 1.2 p = 0.01 14.8+ 1.8 pso.05

28.4 i 1.9 p~o.601 25.4 k 4.2 p
0.8 310.2 pi 0.02 0.7 + 0.1 p
R

Hepat, lateral.

partially

hepatectomized;

0.4 * 0.1 p < 0.001 0.2 I!I 0.1 p z 0.05

S, sacrificed.

d vs. L at 28 d (line 7 vs. line 6). vs. H at 21 d, S at 28 or 42 d (line 8 vs. line 5).

either at 21 or 42 days of age; one week later a number of these hepatectomized and after two more weeks they were sacrificed. Right

lateral lobes were removed, fixed, sectioned and stained together; the nuclei of at least 1000 cells per animal were then scored by previously described methods [l]. The results of these experiments, incomplete due to the loss of tissues from terminal control animals, are given in the upper half of Table I. It may be seen that the ploidy distribution arrested by hypophysectomy at 42 days (line 3, Table I) is much more advanced (fewer 2n, and more 4n and 8n) than that arrested at. the al-day age level (line 1, Table I). Furthermore, the regenerated livers of both groups (lines 2 and 4, Table I) exhibit a marked shift towards higher levels of ploidy. Although an unExperimental

Ceft Research 15

234

I. I. Geschwind, M. Alferl and Caroline Schooley

ambiguous evaluation of these results, considered by themselves, cannot be made due to the loss of the appropriate controls, accumulated evidence suggests that the data for such a control group would not have differed markedly from the reported control values. Thus, Di Stefano et al. [2] have shown that hypophysectomy halts the development of ploidy, and in unpublished experiments we have found that in animals 100 days posthypophysectomy, the ploidy pattern had progressed an equivalent of at most one week’s further growth after the time of hypophysectomy. This is also borne out by the results of Experiment 3, reported below. More extensive results of a third experiment are given in the lower half of Table I. Two groups of animals were used, one intact control group sacrificed at 42 days and a group hypophysectomized at 21 days. From the latter, two animals were sacrificed at 28 and five at 42 days; the remainder of the hypophysectomized group was partially hepatectomized (approximately 65 per cent of the liver removed) one week after hypophysectomy and sections of the removed left lateral lobe were fixed; these same animals were sacrificed 2 weeks later (at 42 days). At sacrifice the right lateral lobes from all the animals were fixed. All tissues were then sectioned at 15 ,u and stained together. At least 1000 cells from each liver sample were scored. The distributions of cell types in hypophysectomized control animals sacrificed at 28 and 42 days were very similar and the results from these two groups have been combined in Table I, line 5 (the average percentage distributions of 2n, 2n binucleate and 4n cells were 81.6, 14.4, 2.6 and 81.0, 13.1, 4.5 for livers from animals sacrificed at 28 and 42 days, respectively). These data reveal several points of interest: (1) There are no significant differences between right and left lateral lobes with respect to the ploidy distribution patterns (lines 5 and 6). (2) Hypophysectomized and normal controls (lines 5 and 8) exhibit highly significant differences in the categories 2n, 4n and 4n binucleate. In these two groups, the percentages of binucleate diploid cells present are not significantly different, probably reflecting similar levels on the ascending and descending parts of their frequency curve, shown in Fig. 1 of the preceding note [l]. (3) Finally, a comparison between lines 6 and 7 shows that a highly significant increase in polyploidy has taken place in the livers of hypophysectomized animals during regeneration. In fact, the polysomatic pattern in these animals is very similar to that of the normal controls listed in line 8. In a discussion of his observations on polyploidy in human livers Swartz [7], following a suggestion of Leuchtenberger et al. [5], mentioned the possibility that growth hormone may be specifically responsible for polyploidization, but not for the general growth of the liver. The present results lead rather to the opposite conclusion since they show unequivocally that neither growth hormone nor any other pituitary hormone is indispensable for the process of polyploidization, per se. In intact animals growth hormone may well play a role in normal liver growth and concomitant development of polysomaty. The mechanism responsible for polyploidy, and its control have not been definitively disclosed by any of these experiments. Nuclear fusion or endomitosis or both may be involved, while selective proliferation of higher polyploid cells is a less likely possibility. Unless it is controlled by a specific, still unknown factor, polyploidization may simply be a natural consequence of postnatal liver proliferation, perhaps resulting from a constant discrepancy between the rates of mitosis and cytokinesis. Experimental

Cell Research 15

Polyploidy

in regenerafed liver

235

Summary.-Although the progressive development of hepatic polysomaty is arrested in the hypophysectomized rat, liver regeneration following partial hepatectomy in hypophysectomized rats is accompanied by an increase in the degree of polyploidy. Thus, no pituitary hormone is indispensible for the development of polyploidy. REFERENCES ALFERT, M. and GESCHWIND, I. I., Exptl. Cell Research 15, 230 (1958). DI STEFAKO, H. S., DIERMEIER, H. F. and TEPPERMAN, J., Endocrinology 57, 158 (1955). HELWEG-LARSEN, H. FR., Acta Pathol. Microbial. Stand. 26, 609 (1949). HEXES, M., HOFFMAN, J., POLLISTER, A. W. and POST, J., J. Mt. Sinai Hosp. 24, 935 (1957). 5. LEUCHTENBERGER, C., HELWEG-LARSEN, H. FR. and MURMAKIS, L., Lab. Investigations 3,

1. 2. 3. 4.

6. SULKIN, 7. SWARTZ,

245 (1954). N. M., Am. J. Anat. 73, 107 (1943). F. J., Chromosoma 8, 53 (1956).

INCORPORATION

OF 32P INTO THE

DNA

OF RAT LYMPHOCYTES

E. H. COOPER’ Department

of Biochemistry,

University

of Oxford, Eqland

Received May 17, 1958

THEstudy

of the life span of human lymphocytes by means of radioactive isotopic labelling of the deoxyribonucleic acid (DNA) have been made in patients with chronic lymphatic leukaemia, Hamilton [5] and Osgood, Tivey, Davison, Seaman and Li [7] and in two patients with normal white cell counts, Ottesen [8]. Since it is probable that the isotope is only incorporated into the DNA of these cells at the time of their synthesis, these observations indicate that the human lymphocyte may have a life span of several months and this may be prolonged in chronic lymphatic leukaemia. This isotopic technique has been used by Cossain and Kline [2] who found that in the rabbit lymphocytes labelled with szP in the DNA could be recovered from the circulation 15 days after giving the isotope by injection. The thoracic duct lymph of rats contains an almost pure population of lymphocytes; this lymph can be obtained by an experimental fistula, Bollman, Cain and Grindlay [l]. Hence it seemed that this preparation was most suitable for the study of the survival of lymphocytes labelled with SaPin the DNA. The initial experiment was made on female Wistar strain rats weighing 145-200 g which were given two doses of 0.5 PC “*P/g body weight intramuscularly at six hour intervals. Pairs of 1 Clinical

Research Fellow,

Medical

Research

Council. Experimental

Cell Research 15