A study of young rats, weighing 55 to 59 g, after being for 10 days in conditions of limited mobility, shows a retardation of body growth as well as that of liver growth. The decrease in the rate of growth is accompanied by a reduction of cell proliferation and by delay polyploidization of hepatocytes in the liver of experimental rats. The materials, methods, and results of research are discussed

Brodskiy, V. Y.

Faktor, V. M.

Li, S. Y.

Malyutin, V. F.

English

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NASA TECHNICAL MEMORANDUM
	 NASA TM-76515
POLYPLOIDIZATION!DELAY IN RAT HEPATOCYTES UNDER LIVER
GROWTH INHIBITION PRODUCED BY HYPOKINESIA
V. M. Faktor, V. F. Malyutin, S. Ye. Li and V. Ya. Brodskiy
Translation of °Zaderzhka poliploidizatsii gepatotsitov krysy
v usloviyakh tormozheniya rosta pecheni pri gipcakinezii,
Tsitologiya, Vol. 21, No. 4, 1979, pp 397-400.
(NASI-Td- 76515) POIYPLOIDI,ZATION DELAY :TN
	 N82-14794
BIT HEPATOCYTES UNDER LIVER GROWTH
INHIBITION BY HYPOKINESIA (National
Aeronautics and Space. Admin.istratiom) 10 p
	 Unclas
HC 402/!!F A01
	 CSCL 06C G3/51 08666
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STANDAPD TITLE PAGE
k Report No.
,.NASA Tk1-7 G515 2. Gowmmant Accaasion No. T:	 Recipient's Catalog No,
d. Title end Subtlti.POLYPLOIDIZATION DLtAY IN- RAT S.	 Report Date
EI EPATOCYTES UNDER LIVER GROWTff INHIBITIO Jul	 1981
♦. Performing Organisation codeP RODUCED BY HYPOKINESIA
7. Authorfs) V. M.	 Faktor,
	
V.	 F.	 Ma lyutin , 1. Performing Organisati on Report No.
S . Ye. Li and V. Y•a. Hrodskiy
10, wont unit No,•
9. Performing Organisation Name and Address
Leo.Kanner Associates
11. Contract or Grant fie,
NASW-319,
R edwood City, California 94063 13. Type of Report end Period Covered
Translation
1Z. Sponsoring Agency Name and Address' 	 +
N ational Aeronautics and Space Admin.
Washington , D . C .
	
20 54 G 14. Sponsoring Agency rod.
IS. Supplementary Notes
T ranslation of "ZadeYzhka poliploidizatsii gepatotsitov krysy.
V usloviyakh :tormozhen,ya rosta pecheni pri gipokinezii",
T sitologiya, Vol.	 21, No.	 4,	 1979, pp 397-400.
18,	 Absttoct
'Young rats, weighing 55-59 g, after being for 10 days
in conditions of limited mobility, show a retardation of body
g rowth as well as that of liver•growth.
	 The decrease in. the
rate of growth is zicconlpanien by a reduction of cell prolifera
Lion and by delay in ^olyploidization of hepatocytes in the
li.ver of experimental rats.
•
`
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this report) 20.	 Security Ciotti#. (of Ohi a Pago) 21. No. of Pogo 22.
Unclassified Unclassif ied
POLYPLOIDIZATION DELAY IN RAT HEPATOCYTES UNDER LIVER
GROWTH INHIBITION PRODUCED BY HYPOKINESIA
V. M. Faktor, V. F. Malyutin, S. Ye. Li and V. Ya. Brodskiy
USSR Academy of Sciences Institute of Developmental Biology, Moscow and
Institute of Marine Biology, Far East Scientific Center, Vladivostok
According to current concepts there is at the base of polyploi- 397*
dy development in the liver a polyploidizing mitosis that induces the
formation of cells with an enhanced number of chromosomes (.see in the
bibliography: Brodski.y and Urivayeva, 1977). Mitosis of the diploid
hepatocyte, in the course of which there is no cell wall formation,
starts off the first polyploid cells -- binuclear with diploid nuceli.
The division of binuclear cells, which proceeds with the joining of
;aired metaphase plates, results in the formation of tetraploid hepa-
tocytes, etc. We may consider it established, that all cell transfor-
mations occurring in the liver in the course of histogenesis (,2n+2nx2}
4n+4nx2+8n+8nx2 ... ) result from sequential transition on the part of
cells of different ploidity in the mitotic cycle. At the same time
the transformations of cell types are stable and directed toward in-
creasing cellular ploidity (Faktor, Urynayeva,, 1975).
j
However these ideas are out of line with the data in a number
of reports of observed reduction in cell ploidity under conditions
where mitotic activity was repressed (Zaletayeva, 1953, 1965; Li,
Kirillov, 1972, 1974; Kirillov, 1977). To explain the reduced num-
ber of polypload cells and the increased amount of :low-ploid cells
there was hypothesized an amitotic division of nuclei and division
of binuclear cells with the formation of mononuclear ones. Since
these conclusions are important for assessing the mechanisms of'hepa-
tocyte polyploidization, we decided to study once more the effect of
growth inhibition, using oae of the experimental models of the au-
thors. We studied the effect of growth inhibition, in animals subjec
*Numbers in the margin indicate pagination in the foreign text.
1	 ^.
ted to hypokinesia on the ratio of cells of different ploidity in
the liver cells of the rat.
Material and Method
Wistar male rats were used that weighed 56-59 g at the beginning
of the experiment. To restrict their mobility they were placed in
narrow plastic cases which allowed relative freedom of movement to the
head, tail and extremities. Control and experimental animals were fed
to excess and simultaneously.
16 young rats were divided into 3 groups: 7 as initial control,/398
5 put in the boxes, 4 as controls of growth. The experiment ended 10
days following the beginning of hypokinesia. Before hypokinesia began
a part of each group were impregnated with 3H-thymidin administered
every 5-9 hr over 3 days. The 3H-thymidin (specific activity 12 curies
per millimole) was given IP in the amount of 0.7 microcuries per gram
of weight. The animals were decapitated during the morning hours from
9 to 11 a.m. Body weight was noted and that of the perfused liver.
Preparations of isolated cells were made for study (for methodology see
Faktor, Uryvayeva, 1975). Following 30 min fixation in 96% alcohol the
smears were stained by the Feulgen method: 15 min hydrolysis in 5 n
HC1 at 37°C and treatment with Schiff's reagent for l hr at room tem-
perature. To obtain radi.oautographs we covered the smears with an M
type emulsion (Gosniikhimfotoproyekt) and exposed them in darkness at
4 0 for a month. When the smears developed we identified nuclei of va-
rious ploidities on the basis of combined criteria: nuclei dimensions
and intensity of stain in the Feulgen reaction (see Faktor, Uryvayeva,
1975). For this purpose we examined 500-1000 cells in the smear of 	
3
each animal. The index for labeled cells was determined on the basis
of a 1000-3000 cell count and expressed in percent.
Results and Discussion
Under conditions of hypokinesia there is a considerable slow-down
in body and liver growth (Table I). We know of the decrease in the
2
ORIGINAL PAGE IS
OF POOR QUALITY
TABLE I. EFFECT OF HYPOKINESIA ON RAT BODY AND LIVER WEIGHT
Experimental variant 	 Number
of
rats
wt of rat in g, x ± sx Liver wt, g
x ± sx
start of end of
experiment experiment
F.
t	 Initial control	 7 59.0±0.7 --- 3.8±4.2
Hypokinesia, 10 days 	 5 56....0±2.0 61.0}1.2 4.4±0.3
Growing control	 4 58.0±2.5 84. 0±3.3 5.9±0.4
TABLE II RATIO OF CELLS OF DIFET REW PLOIDITY. IN
NORMAL AND HYPOKINWIC RAT LIVER
4
Relative number of cells in respect toExperimental variant ploidity and different number of nuclei,
in percent
tin I	 2n X 2	
I
4n	 I 4n X 2 I	 $n
Initial control ._^^
04.1 41.2 4.7 0 0
61.2 33.5 5.1 0.2 0
41.6 47.7 9.1 1.6 0
44.1 4 4. 2 10.5 1.2 0
18.8 50.3 29.3 1.6 0
58.1 38.8 2.9 0.2 0
x + s x 4,119 51.5 4.0 0.6 0
46.0±5.8 43.9±2.7 9,4±3.8 0.8±0.2 0	 8
Hypokinesia, 10 days 27..t 62.7 9.5 0.7 0
19.6 59.9 18.8 1.7 0
19.9 60.7 16.8 2.6 0
27.4 64.6 6.9 1.1 0
x + s 23.5 55.8 17.1 3.3 0.2x 23.5±1.9 60.7±1.7 13,8±2.6 1.9±0.5 0.04
Growing control 26.6 47.0 21.5 2.7 0.2
14.7 40.8 38.9 5.4 0.3
30.1 44.7 23.4 1.6 0.2
x + s - 30.5 48.4 19.4 1.7 01
x
x2.6.0+4.4 45.2	 1.2	 25.7±	 +5.0 2.9±1.0 0.18±0.04
in absolute weight for the liver and a number of other internal organs in longtenr.
hypokinesia (Kirillov, 19771_. 	 Animal growth lag was matched by a lag in liver cell
pcyploidization for experimental rats (Table II).	 This is particularly evident
3
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in the case of cells with tetraploid nuclei - the 4n and the
	 /399
4nx2 type. The number of diploid cells in hypokinesia dropped as
sharply as in the growing control, while the number of binuclear
hepatocytes with diploid nuclei increased. Similar relationships
between cells of varying ploidity are typical of young growing ani-
mals (Carriere, 1969). During this period of ontogenesis the drop
!lli
	
	
in the number of diploid cells goes hand in hand with tissue accumu-
lation of binuclear cells with diploid nuclei and their subsequent
entry into mitosis results in the appearance of tetraploid cells.
In, our experiments reduced intensity in liver growth showed itself
in the accumulation, in tissue, of high-ploid cells of types 4n and
4nx2. This agrees well with literature data. Interruption or re-
tardation of liver growth, as provoked by a whole range of experi-
mental factors -- hypophysectomy, thyroidectomy (see the literature:
Carriere, 1969), nonprotein diet (Nadal, Zajdela, 1966), prolonga-
tion of the lactation period (Wheatley, 1.972) -- results in delayed
polyploid development in the tissue.
e
Prior impregnation of young growing rats with 3H-thymidin made
it possible for us to accumulate in the tissue a group of labeled
cells and to follow their transformation under conditions of normalcy
and of retarded growth due to hypokinesia. Among cells so labeled
at the start of the experiment there was a_notable increase in the
proportion of tetraploid hepatocytes (Table III, initial control)
indicating an intensive process_of_polydiploidization typical of
the ontogenesis segment under study. At the 'close of the experiment
all animals showed an increase in labeled cells, evidence of the
continued growth of the organ during the experimental period. How-
ever, the control's showed a higher index of labeled cells (Table III,
growing control). in both cases not only was there no decrease in
the number of labeled tetraploid cells in t-he course of the experi-
ment, but there was an actual increase (Table. III)
	
This_indi-	 /400
cates that in hypokinesia, despite decreased intensity of prolifer-
ation, a polyploidization process goes on although`, in a smaller
number of cells. The important point is that, 'both under conditions
of restricted growth and under normal circumstances, cell transforma-
tion follows the scheme 2n42nx2-*4n->4nx2-}... , i. e. it is oriented
_	
5
toward increased cell ploidity (Tables II, III). We have previously
pointed out (Brodskiy et al., 1969; Faktor, 1972; Faktor,•Uryvayeva,
1975) the stability of cell transformation and the absence of divi-
Sion of high-diploid cells as the source of low-diploid cell forma-
tion.
in the assessment of a change in cell distribution on the basis
of ploidity under different experimental conditions an important fea-
ture is comparison with the control, particularly if the experiments
last a long time. For example, in experiments with chronic stress,
lasting usually 10-20 days, the liver of control animals may show a
real growth-related polyploidization of hepatocytes. only when we
compare the ratio between cells of varying ploidity in the liver of
experimental animals with both the initial and the growing controls
does it become clear that the development level of polyploidy in the
experimental rats reflects not a reduction of the polyploid cells al-
ready formed but a lag in the growth-related polyploidization of the
hepatocytes (Table II): At the present time there is no reason to
assume that amitosis processes, as means of dividing high-ploid cells,
have a role in any transformations of cellular types whatever that
take place in the normal liver or under experimental conditions.
v
r
REFERENCES
1. Brodskiy, V. Ya., V. M. Faktor, N. A. Milyutina and I. V. Ury-
vayeva, Issledovaniye sinteza DNK i mitozov v regeneriruyush-
chey pecheni myshi dlya • vyyavleniya G 2 -populyatsii gepatotsi-
tov [Study of DNA Synthesis and Mitoses in Regeneration of the
Mouse Liver for Revealing G 2
 Populations of Hepatocytes],
Proceedings of the Soviet Academy of Sciences 189, 3, 639-642(1969) .
2. Brodskiy, V. Ya. and I. V. Uryvayeva, Cell Polyploidy: its Re-
lation to Tissue Growth and Function, Intern. Rev. Cytol. 50,
275-332 (1977).
3. Carriere, R., The Growth of Liver Parenchymal Nuclei and its En-
docrine Regulation, Intern. Rev. Cytol. 25, 201-278 (1969).
4. Faktor, V. M.., Izucheniye mekhanizmov proliferatsii i poliploi-
di g. v pecheni myshi [Study of the Mechanisms of Proliferation
and Polyploidy in the Mouse Liver], Author's abstract of doc-
toral dissertation, Moscow, 1972.
5. Faktor, V. M. and I. V. Uryvayeva, Progressirovaniye poliploidii-
v pecheni myshi pri povtornykh gepatektomiyakh [Progress of
Polyploidy  in the Mouse Liver with Repeated Hepatectomies],
Tsitologiya 17, 8, 909-915 (1975).
6. Kirillov, 0. 1., Protsessy kletochno2o obnovleniya i rosta v u-
sloviyakh stressa [Processes of Cell Renewal and Growth under
Stress Conditions], Nauka Press, Moscow, 1977.
7. Li, S. Ye. and 0. L. Kirillov, Kletochnyye izmeneniya v pecheni
krys pri povtornoy dvigatel'noy nagruzke [Cellular Changes in
the Rat. Liver with Repeated Mobility Loading], Byul. eksper.
biol. med. 74, 12, 89-91 (1972).
B. Li, S. Ye. and O. I. Kirillov, Kle-,-ochnyye izmeneniya v pecheni
krys pri dlitel'noy gipokinezii [Cellular Changes in the Rat
Liver with Prolonged Hypokinesia], Kosm. biol. med.8, 2,
13-17 (1974) .
9. Nadal, C. and F. Zajdela, Polyploidie dans le foie de rat. II.
Le role de 1 hypophyse et de la corrence proteique JPolyploi-
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Flow], Exper. Cell Res. 42, 117-129 (1966).
10. Wheatley, D. N. Binucleation in Mammalian Liver. Studies on
th C t 1 f C t k'	 E	 11	 74e on ro o y oinesis in vivo, xe=.L Ce Res. 
455-465 (1972).
a
11. Zaletayeva, T. A., O-sootnoshenii kolichestva poliploidykh i
dvuyadernykh kletok pecheni v techeniye sutok IRatio'between
Ngmher o„ roiyploid and Binuclear Liver Cells in 24 hr] ,
Byul. eks ^ er. biol. med. 56, 10, 93-95 (;1963).
7
a	 ^
12. Zaletayeva, T. A., O nekotorykh destruktivnykh i vosstanovitel'-
nykh protsessakh v pecheni pri golodanii [Some Destructive
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Copyright Holder "Izdatel'stvo "Nauka", "Tsitologiya", 1979


Polyploidization delay in rat hepatocytes under liver growth inhibition by hypokinesia

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