J. Embryo/, exp. Morpli. Vol. 24, 3, pp. 525-533, 1970
Printed in Great Britain
525
Stimulation of cell division
in pronephros of embryonic grafts following
partial nephrectomy in the host (Xenopus laevis)
By D. P. CHOPRA 1 AND J. D. SIMNETT 1
From the Department of Pathology, University of Newcastle upon Tyne
SUMMARY
Following partial nephrectomy in juvenile metamorphosed Xenopus laevis the mitotic
activity in the regenerating kidney reached its maximum on the 6th day and returned to its
normal level by the 16th day.
The mitotic activity was measured in the pronephros and epidermis of prefeeding Xenopus
larvae (stage 38) at different intervals after their implantation into the lymph sacs of partially
nephrectomized and control metamorphosed hosts.
Ten days after partial nephrectomy, during the period of increased mitotic activity in the
regenerating kidney of the host, the mitotic activity in the implant pronephros was twice as
high as that in the implant pronephros of the control hosts.
Eighteen days after partial nephrectomy when the mitotic activity in the regenerating host
kidney had returned to normal, there was no difference between the mitotic activity of
pronephros of implants in nephrectomized and control hosts.
There was no significant difference between the mitotic activity of epidermis of the implants
in nephrectomized and control hosts, nor was there any difference between the epidermal
mitotic activity in implants examined 10 and 18 days after host nephrectomy.
It was concluded that a circulating factor (or factors) responsible for the control of mitotic
activity in the regenerating host kidney enters the implant through its vascular supply and
influences the mitotic activity in the homologous embryonic tissue. It is possible that this
factor is a tissue-specific mitotic inhibitor synthesized by the host kidney.
INTRODUCTION
Embryonic tissues (Willis, 1962) or whole embryos (Kolodziejski, 1933;
Galtsoff & Galtsoff, 1959; Simnett, 1966) grafted into mature hosts of the same
species continue to grow and a range of histologically normal differentiated
tissues can subsequently be identified. The rate of cell proliferation in certain
adult (Goss, 1964; Bullough, 1965) and embryonic tissues (Simnett & Chopra,
1969) is controlled by tissue-specific growth regulatory factors and since the
implanted embryo is linked to its host via the vascular supply it is possible that
the growth rate of the embryonic tissues may be regulated by the growth factors
present in the host circulation. If this assumption is correct the extirpation of a
host organ would be expected to result in an alteration of the level of growth
1
Authors' address: Department of Pathology, the Royal Victoria Infirmary, Newcastle
upon Tyne NE1-4LP, England.
526
D. P. CHOPRA AND J. D. SIMNETT
regulatory factors which in turn would modify the development and differentiation of the homologous organ in the embryonic implant.
This concept was examined in the present work by implanting embryos into
hosts in which the kidney had been partially extirpated and by studying the
subsequent development of the kidney in the embryonic implant. It seemed
probable that the postulated influence of a partially nephrectomized host on
implants would be determined by the time of vascularization of the implant, the
pattern of development of the implant kidney and the time of maximum mitotic
activity in the regenerating host kidney. Separate experiments were therefore
carried out to establish these points.
MATERIALS AND METHODS
Extirpation of the host kidney and its regeneration. Juvenile metamorphosed
Xenopus laevis (weight 0-6-0-8 g) reared in the laboratory were anaesthetized
in 0 1 % MS 222 and a small incision was made in the ventral abdominal cavity
overlying the kidneys. Using fine tungsten needles attached to a crystal-controlled diathermy apparatus one of the exposed kidneys was partially extirpated
by cauterization without any significant loss of blood. The total amount of
kidney tissue thus removed was approximately 40 %. Control animals were
subjected to a similar surgical intervention but without any extirpation (sham
operation). After recovery from the anaesthetic the animals were maintained at
16-17 °C; postoperative mortality was approximately 10 %.
In order to investigate the mitotic activity in the regenerating kidney both
partially nephrectomized and control animals were anaesthetized and injected
into the ventral abdominal cavity with the metaphase-arresting agent colcemid
(0-03 ml of a 100 mg % solution) 4 h before they were sacrificed. To avoid
possible errors due to diurnal variation in mitotic incidence (Bullough, 1948) all
animals were killed at the same time of day. The kidneys were fixed in Worcester's fluid (10 % acetic acid and 10 % formaldehyde in saturated mercuric
chloride), embedded in paraffin wax, sectioned at 5/t and stained in haematoxylin and eosin for histological examination.
Mitotic incidence (MI) was measured by counting the number of arrested
metaphases in a sample of 4000-6000 nuclei of kidney tubule cells per animal.
A value of MI, expressed as the number of metaphases per 105 cells, was thus
obtained for each animal and from these individual values the mean MI and
standard deviation for each experimental group (five animals) were calculated.
Consequently the MI represents the mean proportion of cells entering mitosis
during the 4 h period following the colcemid injection.
Method of implantation. Embryos were obtained by the injection of chorionic
gonadotrophin into adult male and female Xenopus laevis and reared to the required stage identified according to the normal table of Xenopus laevis (Nieuwkoop & Faber, 1967). Juvenile metamorphosed animals (weight 0-6-0-8 g)
Cell division in pronephros
527
were used as hosts. Three embryos were implanted into each host—two in the
dorsal lymph sac and one in the ventral lymph sac—using the method described
earlier (Simnett, 1966). To avoid extrusion of implanted embryos the incisions
were closed with two fine silk sutures.
The mitotic activity in the implants was measured at different intervals by
injecting the hosts with colcemid 4 h before sacrifice. The implants were fixed
in Worcester's fluid, embedded in paraffin wax, sectioned serially at IJLI and
stained in haematoxylin and eosin for histological examination.
The number of arrested metaphases was counted in 1000 nuclei of pronephric
tubule cells from each implant. The MI for each implant and for each experimental group was again expressed as the number of arrested metaphases
per 105 cells.
In order to investigate the question of tissue specificity following partial
nephrectomy of the host similar mitotic counts were made on 1000 nuclei from
epidermal cells of each implant.
RESULTS
Kidney regeneration. Partially nephrectomized metamorphosed animals were
sacrificed in groups of five at intervals of 2, 4, 6, 8, 10, 12 and 16 days after the
operation. Control animals were killed at intervals of 2, 4, 6 and 8 days after
the sham operation. Mitotic counts were made in two sample areas from each
animal and each estimate of MI in a group of five animals thus represents the
mean of ten samples. The data for MI in control and nephrectomized animals
are shown in Fig. 1.
During the period of observation there was no significant change in the MI
of the kidney in the control animals. The mean MI for the whole period was
235± 142/105.
The first change in the MI of the partially nephrectomized animals was noted
after 4 days (MI 344 + 105/105). The maximum value of the MI (1000 ± 352/105
or approximately 4 times the control value) was reached on the 6th day. On the
12th day the MI in nephrectomized animals (306 ± 98/105) was still higher than
in controls but by the 16th day there was no significant difference between the
MI in nephrectomized (239 ± 151/105) and control animals.
These results suggest that following partial nephrectomy changes occur in the
level of circulating factors which may determine the rate of mitosis in the
kidney. It would seem reasonable to suggest that if these changes have any
influence on the renal tissue of implanted embryos the effect would be most
obvious during the period of increased mitotic activity in the regenerating host
kidney.
Implantation of embryos at various stages of development into normal hosts.
Observations were made at three different intervals (4, 8 and 16 days) after
implantation of embryos at four developmental stages (stages 12, 32, 38 and 45).
528
D. P. CHOPRA AND J. D. SIMNETT
Each of the twelve experimental groups contained a maximum of five host
animals which received either two or three implants each.
In agreement with a previous study (Simnett, 1966) the majority of implants
examined after 4 days were found already to contain blood vessels which were
connected to the vascular system of the host. At the end of the experimental
period all implants of stages 32, 38 and 45 were present but only a proportion
of stage 12 (gastrula implants) was recovered: four out of six in the 8-day group
and one out of six in the 16-day group. Since the hosts were checked at regular
intervals during the whole of the experimental period for extruded embryos and
none were found it was concluded that a proportion of implanted gastrulae was
reabsorbed by the host.
• Control
X Partially
nephrectomized
1200
1000
-
<
800
600 -
X
400
-T i
J* 1< .
X
200
T
IX
1i
X
X
1
•I
1
I
4
8
12
Days after partial nephrectomy
16
Fig. 1. Regeneration of kidney in juvenile metamorphosed Xenopus laevis. Mean
mitotic incidence per 105 cells and standard deviation in the residual kidney after
partial nephrectomy, compared with control animals.
Implants of stage 45 were found to contain necrotic areas which were first
observed after 4 days and which became progressively more extensive. This
observation agrees with previous findings (Simnett, 1966). There was no
necrosis at any other stage.
Tissues from stage 32 and 38 implants examined after 4 days were poorly
differentiated and still contained abundant yolk platelets but implants examined
after 8 and 16 days contained a number of well-differentiated tissues, including
pronephric kidney situated at the level of the third somite. The pronephros of
stage 38 implants was more compact and had a better developed tubular structure than that found in the pronephros of stage 32 implants. Mesonephros was
also present in both stages but was more difficult to recognize because of its
Cell division in pronephros
529
dense structural units which resembled gastric glands. In view of this it was
decided to confine subsequent observations to the pronephros of stage 38
implants.
The effect of host partial nephrectomy on the MI of implant pronephros. Partial
nephrectomy is a potentially debilitating operation and it was therefore considered advisable to allow a 2-day recovery period before implantation. Eight
days after implantation (or 10 days after nephrectomy) the host kidney, as we
have shown above, would still have an increased MI while by 16 days after
implantation (18 days after nephrectomy) it would have returned to normal.
From a comparison of 8- and 16-day implants it should therefore be possible
to decide whether there is any correlation between the MI of host and implant
kidney which would be expected if humoral regulatory factors were exchanged
via the common circulation established 4 days after implantation.
Table 1. Mitotic incidence (MI)* in the pronephros and the epidermis of implants
removed from control and partially nephrectomized hosts at 8 and 16 days after
implantation
(Each figure of MI is the mean of ten values)
Pronephros
8 days
16 days
Epidermis
8 days
16 days
Implants from control
hosts
1589 ±676
3051J ± 1188
5641 ±809
5287 ±1658
Implants from nephrectomized hosts
3288f±1409
2792§±819
5591 §±1658 4585§±2103
5
* Ml is the number of arrested metaphases per 10 cells during 4 h ofcolcemid treatment.
t Stimulation of mitosis significant (P < 001) as compared with the control.
t Increase in mitosis significant (P < 005) as compared with the 8-day control.
§ No significant difference as compared with the control.
Twelve metamorphosed Xenopus laevis were partially nephrectomized and,
after a recovery period of 2 days, each host received three implants of stage 38,
two in the dorsal lymph sac and one in the ventral lymph sac underneath the
lower jaw. The implantation procedure was repeated in twelve sham-operated
control hosts. Two animals from each group died during the postoperative
period. Five partially nephrectomized and five control hosts were sacrificed on
the 8th day and the same numbers on the 16th day after implantation. In all
cases colcemid was injected 4 h before the animals were killed. After sacrifice of
the animals the implants were removed and treated for histological examination
in the manner earlier described. In some implants the pronephros was difficult
to identify and mitotic counts were therefore made in only ten from the total of
fifteen implants recovered from each group of five host animals. Each estimate
for MI thus represents the mean of ten samples.
530
D. P. CHOPRA AND J. D. SIMNETT
After 8 days the MI in pronephros of implants from nephrectomized hosts
(3288± 1409/105) was significantly higher (P < 001) than in that from the
control hosts (1589 ± 676/105) but at 16 days no significant difference was found
between the two groups (Table 1).
The MI in epidermis of implants from control and nephrectomized hosts
removed 8 days after implantation were 5641±809/105 and 5591 ± 1658/105
respectively and for those removed after 16 days the corresponding values were
5287 ± 1658/105 and 4585 ±2103/105 respectively. There was no statistically
significant difference between these values.
DISCUSSION
Changes in mitotic activity in the kidney of metamorphosed animals. In adult
mammals the maximum mitotic rate in the compensating kidney usually occurs
2 days after partial nephrectomy (Goss & Rankin, 1960) while in metamorphosed Xenopus maintained at 16-17 °C it was not reached until the 6th postoperative day (Fig. 1). This slower response may be a common feature of
amphibian tissues since in the regenerating liver of Xenopus (at 16-17 °C) the
maximum MI was observed 6 days after partial hepatectomy (D. P. Chopra,
unpublished results) and in Triturus viridescens (at 24 °C) the maximum rate of
DNA synthesis was not attained until 10 days after partial hepatectomy
(MacDonald, Guiney & Tank, 1962). Although the rate of mitosis in amphibian
tissues is temperature-dependent (Simnett & Balls, 1969) the slower mitotic
responses mentioned above cannot be ascribed entirely to the fact that the body
temperature of Amphibia is lower than that of mammals since very rapid
responses have been observed in the compensating tissues of larval Amphibia.
For example, the maximum mitotic activity in the pronephros and mesonephros
of unilaterally pronephrectomized Xenopus tadpoles maintained at 16-18 °C
(Chopra & Simnett, 1969a) and in the injured lens epithelium of Rana catesbiana
tadpoles at 30 °C (Reddan & Rothstein, 1966) was already attained on the 2nd
and 3rd postoperative day respectively.
Mitotic activity in the pronephros of free larvae and of larvae implanted in
non-pronephrectomized hosts. In intact Xenopus larvae maintained at 16-17 °C
the MI in the pronephros after 4 h of colcemid treatment was 550/105 (Chopra
& Simnett, 1969 a) and this decreased to 130/105 over a period of 10 days. In
contrast, the MI of larvae implanted into non-pronephrectomized hosts maintained at 16-17 °C was 1589 and 3051/105 (8 and 16 days after implantation
respectively). A similar increase was observed in the epidermis where the MI in
intact larvae was 1289/105 (authors' unpublished results) compared with the
values of 4000-5000/105 observed in implants. It is not certain whether the
higher MI in implant tissues is due to an increased supply of nutritional or
hormonal factors or whether a specific disturbance of normal growth control
mechanisms is involved.
Cell division in pronephros
531
Mitotic activity in larvae implanted into non-nephrectomized and partially
nephrectomized hosts. Following partial nephrectomy in the host an increase in
the mitotic rate in the kidney of implanted embryos was observed at the time
of increased mitosis in the regenerating host kidney. Similar results were obtained in another series of experiments where the value of MI in the pronephros
of experimental implants at 8 days after implantation (2088 ± 629) was significantly
higher than that of control implants (740 ± 259) (authors' unpublished results).
No such response occurred in the implant epidermis. The tissue specificity of the
response in implants confirms the view that a modification of the growth
control system in one of the host organs can produce a specific alteration in the
growth rate of the homologous embryonic implant organ. Embryonic implantation may be regarded as a form of artificial pregnancy and this type of experiment raises the important question whether the mammalian foetus is similarly
exposed to tissue-specific growth factors present in the maternal circulation or
whether it is shielded from their full effect by the placenta. Such a barrier has
been discussed by Goss (1963) following his finding that no increased mitotic
count was observed in foetal kidney 48 h after maternal unilateral nephrectomy.
However, Goss (1963) did not examine the foetal kidney to see if there was any
increase in weight and since the mitotic response in embryonic and immature
tissues is more rapid than in adult tissues (Chopra & Simnett, 1969 a) it is
possible that the rate of mitosis in the foetal kidney did increase but that it
returned to normal by 48 h. Rollason (1961) observed no increase in foetal
kidney weight after maternal total nephrectomy, but this is not surprising
considering the severe debilitating effects of the operation. He did, however,
report an increase of approximately 50 % in the mitotic count of the foetal
kidney 24 h after operation and it has also been shown that the liver of rat
foetuses responds to partial hepatectomy of the mother (Ballantine, 1965). This
suggests that, if it exists, the placental barrier to growth regulatory substances
is incomplete. Unlike the situation in the non-placental vertebrates, where the
embryo can be regarded as a self-contained unit from the point of view of
tissue-specific growth control, it is possible that in the eutherian foetus growth
regulation may, at least in part, depend on maternal factors transmitted via the
placenta.
The possible nature of the mechanisms which regulate mitotic activity in the
kidney. It has been suggested (Goss, 1964; Johnson & Vera Roman, 1968) that
growth and mitotic homeostasis in the kidney are controlled by the physiological
load placed upon the organ. An alternative hypothesis is that these are regulated
by an organ-specific growth inhibitor or chalone (Bullough, 1965) produced in
the kidney. Evidence in support of the latter hypothesis is provided by our
observations that extracts of kidney from mature Xenopus laevis (Simnett &
Chopra, 1969) and rat (Chopra & Simnett, 19696) contain an organ-specific
factor or chalone which inhibits mitosis in Xenopus laevis embryonic kidney
(pronephros). The increased rate of mitosis in the implant pronephros could
532
D. P. CHOPRA AND J. D. SIMNETT
therefore be due to a decreased concentration of kidney chalone in the host
blood resulting from a reduction in the mass of the tissue which produced the
chalone. In a further series of experiments carried out in vivo (D. P. Chopra,
unpublished results) injection of rat kidney extract caused a tissue-specific
mitotic inhibition both in the regenerating kidney of partially nephrectomized,
metamorphosed Xenopus and in the pronephros of embryos implanted in such
hosts. It would appear that the chalone mechanism is involved not only in the
mitotic homeostasis of mature tissues but also in the regulation of embryonic
growth.
It has been demonstrated (Clayton, 1953) that in amphibian embryos some
organ-specific antigens appear during gastrulation before the morphological
differentiation of the corresponding organs. Similarly, it has been shown
(Flickinger, 1962) that in chick and frog embryos proteins with the immunological characteristics of adult lens antigens appear before the differentiation of
the embryonic lens. Consequently, it is possible that certain of these organspecific antigens may act as growth control factors.
RESUME
Stimulation de la mitose dans le pronephros des greffes embryonnaires apres
nephrectomie partielle de Vhote (Xenopus laevis)
Apres nephrectomie partielle de Xenopus laevis recemment metamorphoses, l'activite
mitotique du rein en regeneration atteint son maximum au 6e jour et retourne a son niveau
normal le 16e jour.
L'activite mitotique a ete mesuree dans le pronephros et l'epiderme de larves de Xenopus
(stade 38) a differents intervalles apres leur implantation dans les sacs lymphatiques d'hotes
metamorphoses temoins et d'hotes partiellement nephrectomises.
Dix jours apres nephrectomie partielle, pendant la periode d'activite mitotique accrue dans
le rein en regeneration de l'hote, l'activite mitotique du pronephros implante est deux fois
plus elevee que dans le pronephros implante dans les hotes temoins.
Dix-huit jours apres nephrectomie partielle, quand l'activite mitotique du rein en regeneration de l'hote est redevenue normale, il n'y a pas de difference entre l'activite mitotique du
pronephros des implantats dans les hotes nephrectomises et dans les hotes temoins.
II n'y a pas de difference significative entre l'activite mitotique de l'epiderme des implantats
dans les hotes temoins ou nephrectomises, ni dans celle de l'epiderme d'implantats examines
10 et 18 jours apres la nephrectomie de l'hote.
On en conclut que le (ou les) facteur circulant responsable du controle de l'activite mitotique
du rein en regeneration de l'hote, penetre dans Pimplantat par ses voies vasculaires et influence
l'activite mitotique du tissu embryonnaire homologue. II est possible que ce facteur soit un
inhibiteur mitotique, tissu-specifique, synthetise par le rein de l'hote.
This work was supported by a grant from the North of England Council of the British
Empire Cancer Campaign for Research. Thanks are due to Professor A. G. Heppleston for
kindly providing research facilities in his Department, to Mr and Mrs H. Elliot for skilled
technical assistance and to Mrs M. Jackson, our Editor of Research Publications, for
valuable editorial help.
Cell division in pronephros
533
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{Manuscript received 6 February 1970)