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The regulation of growth in the mesonephric kidney of adult Xenopus laevis by an endogenous inhibitor of proliferation
DEVELOPMENTAL
BIOLOGY
66,529~538 (1978)
The Regulation of Growth in the Mesonephric Kidney of Adult Xenopus laevis by an Endogenous inhibitor of Proliferation’ GEOFFREYGOLDINANDBARRYFABIAN Department
of Zoology,
University Received
of the Witwatersrand, March
16, 1978; accepted
Johannesburg June
2001, South
Africa
5, 1978
The effect of dorsal lymph sac implanted tissue fragments, of a 100,000g kidney supernatant, and of various kidney-derived ultrafiltrate fractions on the percentage of DNA synthesizing cells in the mesonephric kidney of Xenopus Zaeuis following partial unilateral nephrectomy was investigated autoradiographically. Using Amicon falters with cut-off values of MW 50,000 and 10,000, three ultrafiltrate fractions were obtained: a fraction containing molecules of MW 50,000 and less, a fraction containing molecules of MW 10,000 and less, and one containing molecules in the range of MW 10,000 to 50,000. The ultrafiltrates containing molecules of less than 10,000 MW were found to depress DNA synthetic activity on the sixth postoperative day by 30 to 40%, while the fraction containing molecules between MW 10,000 and 50,000 showed no significant effect. It has been concluded that an endogenous inhibitor of proliferation, with the attributes of a chalone, is present in the fraction of less than 10,000 MW. The loss ,of inhibitor action following Pronase treatment of the ultrafiltrate suggests that the inhibitor substance may be a protein or polypeptide, or that such constituent may be the carrier for the active agent. Since a depression in DNA synthetic activity of 60% was obtained in normal adult mesonephric kidneys following the injection of the ultrafdtrate, it is concluded that both compensatory growth and reparative growth in the kidney of Xenopus Laeois are regulated by a G, kidney chalone of less than 10,000 MW. INTRODUCTION
The maintenance of a steady-state cell population size (reparative growth) in both expanding organs and renewing tissues (Messier and Leblond, 1960) is dependent upon the balance between cell loss and cell production. The preservation of such an equilibrium requires some homeostatic mechanism for regulating proliferative activity (Goss, 1967; Bullough, 1968). Similarly, the progressive decline and eventual cessation of postembryonic growth in expanding organs necessitate the regulation of cell proliferation. It is conceivable, therefore, that both postembryonic and replacement growth in the various expanding organs and renewing tissues are subject to similar, if not the same, regulatory mecha’ This study comprises part of a thesis by G. V. Goldin submitted for the degree of Doctor of Philosophy at the University of the Witwatersrand, Johannesburg.
nisms (Bullough, 1968,1975; Leblond, 1972; Lozzio et al., 1975; Rytomaa, 1976). The view that proliferative growth is regulated by a negative feedback system involving tissue-specific inhibitors has been proposed by several authors (Saetren, 1956; Weiss and Kavanau, 1957; Bullough and Laurence, 1960; Bullough, 1962, 1965), and in this direction experimental support for the regulation of growth by chalones (Bullough, 1962) has been accumulating. This type of endogenous mitotic inhibitor produced by tissue cells and which, by a negative feedback mechanism, regulates the mitotic activity in the same tissue, has been extensively examined in the epidermis (Bullough and Laurence, 1960, 1964; Hondius-Boldingh and Laurence, 1968; Elgjo, 1973; Marks, 1973; Iversen et aZ., 1974). Two types of epidermal chalone have been identified and, on the basis of their specificity of action during the cell cycle (Thornley
529
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DEVELOPMENTALBIOLOGY
and Laurence, 1975, 1976), have been termed GI and Gz chalones, respectively (Elgjo, 1973). Chalones which have similar characteristics to the epidermal chalones have been reported for other renewing tissues, for example, erythrocyte chalones (Kivilaakso and Rytiimaa, 1971), granulocyte chalones (Rytiimaa & Kiviniemi, 1968), and intestinal chalones (Brugal, 1973; Tutton, 1973; Brugal and Pehnont, 1975). The role of tissue-specific growth inhibitors in the maintenance of organ mass and the control of regenerative growth in organs such as the kidney and the liver has been less rigorously studied. While several experiments suggest the involvement of humoral substances in the control of proliferative growth in the kidney, there has been little progress in identifying either a stimulant or an inhibitor involved in this regulation. The existence of a circulating mitotic inhibitor, specific for liver, was first suggested by Glinos and Gey (1952) on the basis of their finding that mitotic stimulation in normal liver could be achieved by the mere dilution of the plasma. In recent years this notion has received considerable experimental support (Scaife, 1970; Verly et al., 1971; Aujard et al., 1973; Simard et al., 1974; Verly, 1976). In particular, Verly et al. (1971) have purified a liver chalone, reported to be trypsin labile and of MW < 4000, which inhibited proliferation in cultured liver slices. In a subsequent study involving both in viva and in vitro assays, these authors (Simard et al., 1974) concluded that the liver chalone exerted its inhibition on hepatocytes during the G1 phase of the cell cycle, blocking the transition of these cells into the S phase. Following the initial postulation of an organ-specific inhibitor of proliferation in the kidney (Saetren, 1956), little progress toward the isolation and characterization of a kidney chalone has been made. The existence of a kidney chalone is implied by the experiments of Chopra and Simnett
VOLUME 66,1978
(1971), who reported that the mitotic activity in the mesonephros of juvenille Xenopus laeuis, following partial unilateral nephrectomy, was depressed by 88% 4 hr after the injection of a crude kidney extract (NO,OOOgsupernatant) . In an earlier study (Chopra and Simnett, 1969) it had been demonstrated that mitotic activity in cultured pronephric kidneys was depressed by the addition to the medium of a lOO,OOOg supernatant. These authors (Chopra and Simnett, 1971) concluded that, since mitotic inhibition was obtained in a 4-hr period following the administration of the extract, the inhibitory factor had acted on the cells in the GBphase of the cell cycle. While these data suggest the existence of a kidney chalone in crude extract, their assay does not in itself preclude S-phase inhibition or cytotoxicity (see Rytomaa, 1976). Evidence for the existence of kidney chalones has also been provided by Dicker (1972), who found that the injection of either kidney homogenates or the microsomal fraction of such homogenates inhibited compensatory renal growth in rata. More recently, Dicker and Morris (1974) found that the 2000g supernatant from homogenates of the renal cortex of either mouse, rat, guinea pig, rabbit, or pig inhibited growth of neonatal rat kidney explants through cortex-specific inhibition of mitosis. Finally, Hall and Seibert (1976) have reported in abstract form the existence of two “bovine-kidney-derived chalones” that inhibit an established cell line at the Gi and G2 phases of the cell cycle, respectively. In the present study, attempts have been made to ascertain whether a kidney-specific inhibitor of proliferation exists, within crude homogenates and various ultrafiltrates, which meets the criteria required to be considered a chalone. Chalones, according to Bullough (1975) and Rytomaa (1976), are cell line-specific, species-nonspecific inhibitors of cell proliferation, which are produced by the same cell line upon which they act and whose action is reversible and
GOLDIN
AND
FABIAN
Growth Regulation
noncytotoxic. Since it had been previously established that partial unilateral nephrectomy in adult Xenopus laevis is followed by a compensatory growth response in which 19% of the residual kidney cells synthesize DNA on the sixth postoperative day (Goldin and Fabian, 1975), the problem was tackled by treating the nephrectomized animals with the various tissue extracts and then scoring the percentage of thymidine-labeled cells. Accordingly, the presence of inhibitor(s) of proliferation in the extracts would result in a depression of the percentage of DNA synthesizing cells. To further test the hypothesis that compensatory renal growth is a response to tissue mass depletion, which results in a decrease in the concentration of specific inhibitor(s) of proliferation, fragments of renal tissue were implanted into the dorsal lymph sac of nephrectomized animals, and the effect of such implants on DNA synthesis in the residual kidney was studied. MATERIALS
AND
METHODS
Partial
Unilateral Nephrectomy Adult Xenopus laevis males that had
been acclimatized to laboratory conditions for several months prior to experimentation were anesthetized with 0.25% MS 222 (Sandoz, Basel, Switzerland) and opened ventrolaterally under aseptic conditions. The right kidney was exteriorized, and the posterior 30 to 40% of tissue destroyed by electrocauterization. Antibiotics (streptomycin and penicillin) were introduced into the abdominal cavity and the wound was closed with two or three sutures of fine silk. The animals were allowed to recover and heal in dechlorinated tap water to which no antibiotics were added.
Implantation
of Tissue Fragments
Following partial unilateral nephrectomy, fragments of either renal, hepatic, or cardiac tissue, obtained from normal adult donor Xenopus laevis, were implanted into
in X. laevis Kidney
531
the dorsal lymph sac of experimental animals. The tissue fragments (1 to 2 mm3) were excised from donor animals in which the blood was replaced by perfusing Steinberg solution (Hamburger, 1960) through the systemic arch. Prior to implantation, the tissue fragments were stored at 4°C in amphibian culture medium (Wolf and Quimby; GIBCO, Grand Island, New York). The implantation procedure was performed on completion of the nephrectomy before the animal had recovered from anesthesia. Several tissue fragments (either renal, hepatic, or cardiac tissue fragments), roughly equivalent in mass to the amount of renal tissue destroyed by cauterization, were then inserted through a small cutaneous incision into the dorsal lymph sac. The wound was closed with a single suture and the nephrectomized host animals were left to recover for 6 days. At the end of this period of time, tritiated thymidine (5 &i/g wt) was administered. The animals were killed 12 hr after the injection, and autoradiographs of host mesonephric kidneys were prepared. The implants were recovered and were prepared for routine histological examination and for autoradiography.
Preparation of Tissue Extracts Crude extract. Kidney pairs from 10 adult Xenopus laevis were perfused in situ with cold (4°C) Steinberg solution to deplete the endogenous blood reserves and to remove any circulating stress hormones. The kidneys were then excised, cleared of superfluous tissues, and weighed. The pooled mesonephric kidneys (combined weight, 3 to 4 g) were then homogenized in 30 to 40 ml of Steinberg solution for 10 set using an Ultra-Turrax homogenizer, and for 5 min using a Potter-S hand homogenizer. The homogenization and subsequent centrifugation steps were carried out at 4°C. The homogenate was subjected to ultracentrifugation at 100,OOOgand the supernatant solution (hereafter referred to as “crude extract”) was collected.
532
DEVELOPMENTAL BIOLOGY VOLUME66.1978
The crude extract injected into the experimental animals was either freshly prepared or that stored for a maximum period of 2 weeks at -20°C. UZtrufiZtrates. Crude extract, obtained by the procedure outlined above, was subjected to Amicon ultrafiltration under pure nitrogen at 40 psi. Two ultrafiltration steps were employed, the first using an Amicon XM 50 Diaflo membrane (cutoff MW 50,000) and the second using a PM 10 Diaflo membrane (cutoff MW 10,000). The ultrafiltrate obtained after the filtration of crude extract through the XM 50 membrane was collected and divided. Half of the ultrafiltrate solution was used for injection into experimental animals without further modification, and the remaining XM 50 ultrafiltrate was then subjected to a second ultrafiltration step through a PM 10 membrane. The PM 10 ultrafiltrate was then collected and used, without further modification, for injection into experimental animals. A third fraction was obtained by resuspending in Steinberg solution those constituents of the XM 50 ultrafiltrate that had been excluded from the PM 10 ultrafiltrate and which had accumulated as a residue on the PM 10 membrane. A portion of the PM 10 ultrafiltrate was subjected to 12-l-n digestion in 0.1% Pronase (predigested) at 30°C (MilesSeravac, Cape Town). [The protein content of each of the ultrafiltrate fractions was determined using the method of Lowry et al. outlined by Rutter (1967).] Autoradiographic Activity
Assay of DNA Synthetic
In order to test the tissue extracts for the presence of a kidney-specific inhibitor of proliferation, nephrectomized animals were injected, subcutaneously, above the dorsal lymph sac, with [ methyZ-3H]thymidine ([3H]TdR) (The Radiochemical Centre, Amersham, England; specific activity, 19 Ci/mmole), together with either crude extract or ultrafiltrate, on the sixth postoperative day. The animals were killed after
12 hr, and the kidneys, liver, small intestine, and testis excised and prepared for autoradiography as described previously (Goldin and Fabian, 1975). The amount of isotope administered corresponded to 5 &i/g body weight of frog, and the dosage of crude extract or ultrafiltrate (0.75 ml) was estimated to represent the volume that would have been derived from 30 to 40% of the tissue from one kidney. As controls, a few animals were injected with an equivalent volume of Steinberg solution together with tritiated thymidine, while others were injected only with the isotope to verify the previously recorded DNA synthetic activity in the residual mesonephric tissue on the sixth postoperative day (Goldin and Fabian, 1975). To minimize the influence of diurnal variation in proliferative activity, all experiments were carried out according to the same time schedule, that is, injections of tritiated thymidine being administered at 0700. In addition to the above treatments, four of the animals were administered a second injection of 0.25 ml of PM 10 ultrafiltrate 6 hr after the initial dosage of 0.75 ml. DNA synthetic activity in the normal untreated adult mesonephros as well as the normal adult injected with PM 10 ultrafiltrate 12 hr before killing was studied in a few animals by the autoradiographic procedure. All sections were coated with Ilford G5 nuclear emulsion [diluted 1:2 (v/v) with 2% glycerol in deionized water] by the dipping technique. After 10 to 14 days’ exposure in the dark at 4°C the slides were developed with D 19 for 3 min at 18°C (or 2 min at 2O”C), fixed with 26% sodium thiosulfate for 5 min, and washed in running water for 30 min. The sections were then stained through the emulsion with alcian blue (pH 2.5) and nuclear fast red. The sections were examined and scored for percentage of labeled cells. Every 20th section through the length of the mesonephric kidney pairs was examined and scored
GOLDIN AND FABIAN
Growth Regulation
for percentage of labeled cells. In the liver, random lobules were scored for labeled cells, and in the intestine, randomly selected crypts were similarly treated. RESULTS
The Effect Synthetic following tomy
of Tissue Implants on DNA Activity in the Mesonephros Partial Unilateral Nephrec-
The percentage of mesonephric cells synthesizing DNA on the sixth postoperative day was examined in 14 animals whose recovery from partial unilateral nephrectomy occurred with renal, hepatic, or cardiac fragments present in the dorsal lymph sac. The level of DNA synthetic activity wa;5 not significantly depressed by the presence of either the liver implants or the cardiac implants (see Table 1). However, in the renal implant-bearing nephrectomized host animals, the percentage of DNA synthesizing cells in the mesonephros was almost totally depressed to a level approaching that which normally occurs in adult mesonephric kidneys (0.2%) (see Table 1).
533
in X. laevis Kidney TABLE
1
THE PERCENTAGE OF DNA SYNTHESIZING CELLS IN THE MESONEPHROS ON THE SIXTH POSTOPERATIVE DAY FOLLOWING RECOVERY FROM PARTIAL UNILATERAL NEPHRECTOMY IN THE PRESENCE OF DORSAL LYMPH SAC IMPLANTED TISSUE FRAGMENTS DepresNynr DNA synthesrzmg sion (W) mals cells (mean % +- SD) Nephrectomized (controls) Renal tissue implant Liver tissue implant Cardiac tissue implant
3
19 f 0.57
-
6
0.2 + 0.075
98.9
4
17 + 2.36
10.5*
4
18 k 1.89
5.8**
* P = 0.3-0.4 (not significant). ** P = 0.6 (not significant).
The Effect of “Crude Extra& on DNA Synthetic Activity in the Mesonephros following Partial Unilateral Nephrectomy The injection of 0.75 ml of 100,OOOgsupernatant obtained from Xenopus mesonephric kidney homogenate into 10 partially nephrectomized animals on the sixth day of recovery was found to markedly depress the percentage of DNA synthesizing cells in the residual mesonephric tissue. In the autoradiographs prepared from the excised kidneys the percentage of thymidine-labeled cells was found to be 0.8% (Fig. 1). In comparison to the 19% of labeled cells (Fig. 2) found in the mesonephros on the sixth postoperative day, this represents a depression in DNA synthetic activity of the order of 96%. Since these experiments were considered to be pilot ones, the effect of crude extract on DNA synthetic activity in other tissues was not systematically studied.
FIG. 1. Autoradiograph of a portion of one mesonephros following the injection of 100,OOOg kidney homogenate supernatant and [1H]TdR on the sixth day of recovery from partial unilateral nephrectomy. The percentage of DNA synthesizing cells is of the order of 0.8%. which represents a depression of 96% from that usually recorded on the sixth postoperative day. Distal tubules containing labeled cells are indicated by arrows. Dt = distal tubules. Px = proximal tubules. x 90.
534
DEVELOPMENTAL BIOLOGY
VOLUME 66,1978
inhibitory action following the treatment of the PM 10 ultrafiltrate with Pronase points to the inhibitor being either a protein.or a polypeptide, or to such substances being vehicles for some other type of agent. Since the treatment involving a second injection of PM 10 ultrafiltrate, 6 hr after the first, did not enhance the depression of labeled cells obtained following the administration of a single dose, it would appear that the inhibitor(s) within the extract was effective for at least 12 hr. The target tissue specificity of the inhibitory effect of both the XM 50 and the PM 10 ultrafiltrates was tested by examining DNA synthetic activity in intestine, liver, and testis. In the liver lobules, intestinal crypts, and seminiferous tubules of the testis, sampled randomly, DNA synthetic activity was found to be unaffected by either of the ultrafiltrates.
FIG. 2. Autoradiograph of a portion of one mesonephros on the sixth postoperative day following partial unilateral nephrectomy. Labeling after 12-hr exposure to tritiated thymidine is maximal at this stage of recovery when 19% of cells are synthesizing DNA. The extensive labeling occurs in the distal tubules (Dt) and not in the proximal tubules (Px). x 90.
The Effect of Ultrafiltrates on DNA Synthetic Activity in the Mesonephros following Partial Unilateral Nephrectomy The data obtained following the administration of the various ultrafiltrates into partially nephrectomized animals are detailed in Table 2. Both the XM 50 ultrafiltrate (MW < 50,000) and the PM 10 ultrafiltrate (MW < 10,000) were found to depress the percentage of DNA synthesizing cells in the mesonephros by 35 to 40%, while the fraction containing molecules between MW 10,000 and 50,000 was not found to exhibit any significant inhibitory action. These results indicate that the depression in the percentage of DNA synthesizing cells is due to the presence of one or more species of molecule of MW < 10,000. The loss of such
The Effect of Ultrafiltrate Adult Kidney
on Normal
The percentage of labeled cells in the normal adult mesonephros following the injection of
The finding of this investigation that the various mesonephric extracts depress the percentage of DNA synthesizing cells usually associated with the compensatory growth response provides support for the view that renal growth is regulated by specific inhibitors of cell proliferation (Saetren, 1956; Chopra and Simnett, 1969, 1971; Dicker, 1972; Dicker and Morris, 1974). Despite the relative crudeness of the ultrafiltrate fractions used in this study, the conclusion that a G1 kidney chalone does exist may be justifiable. While the reversi-
GOLDIN AND FABIAN
Growth Regulation TABLE
535
in X. laeuis Kidney
2
THE PERCENTAGE OF DNA SYNTHESIZING CELLS IN THE MESONEPHROS FOLLOWING THE INJECTION OF ULTRAFILTRATES AND [aH]TdR ON THE SIXTH DAY OF RECOVERY FROM PARTIAL UNILATERAL NEPHRECTOMY Ultrafiltrate
injected
Number of animals
Steinberg solution (control) MW < 50,000 (XM 50) MW < 10,000 (PM 10) (single injection)* MW < 10,000 (PM 10) (two injections)* PM 10 (Pronase treated) MW lO,OOO-50,000 (PM lo-XM 50) * P = 0.6 (not significantly
3 5 7 4 3 3
DNA synthesizing cells (mean % + SD) 18.7 +- 1.15 11.8 + 1.64 11.7 f 1.97 11.0 f 1.8 17.0 -c 1.7 17.6 + 2.1
Depression 6) 36.8 37.4 41.2 9.1 5.9
P value
0.3 (ns) >0.5 (ns)
different).
bility and species nonspecificity of the inhibitory effect of the ultrafiltrates has not been tested, our results suggest that a substance in kidney extract fulfills the criteria listed as characteristics of chalones, namely, that they are tissue-specific inhibitors of cell proliferation that inhibit proliferation in the same tissue whence they are produced (Bullough, 1975; Rytomaa, 1976). Since the injection of either the 40,000 or the <10,000 MW ultrafiltrates was not found to achieve depressions in the percentage of DNA synthesizing cells in excess of 45% when administered concomitantly with tritiated thymidine, it appears that the resultant inhibition was due to a blockade of the cells in the G1 phase of the cell cycle, and not to some direct influence on S-phase events. Accordingly, those cells that were unaffected by the inhibitor(s) had presumably entered into the S phase prior to the injection of the extract and isotope, and consequently would continue to incorporate the tritiated thymidine. That the inhibition was noncytotoxic and tissue specific in its action was supported by the nondepression of the percentage of DNA synthesizing cells recorded in the intestine and liver. In addition, a cytotoxic effect is regarded as a less serious problem in an in viuo system where the organism has effective mechanisms of detoxification (Rytomaa, 1976). Since the depression of the percentage of DNA synthesizing cells was considerably
less extensive in the extracts of MW < 50,000 compared to that recorded after the injection of crude extract, it seems likely that additional inhibitors (MW > 50,000), which presumably acted on cells in the S phase, were present in the crude extract. A similar conclusion was made by Simard et al. (1974) with respect to the inhibition of hepatocyte proliferation by crude liver homogenates. The near-total depression of the percentage of DNA synthesizing cells obtained after implanting renal fragments into the dorsal lymph sac of partially nephrectomized animals clearly supports the hypothesis that compensatory growth is a response to tissue mass depletion, rather than the result of the release of a mitotic stimulator (wound hormone) or the consequence of an increased physiological load. The finding that mesonephric kidney extracts inhibit the proliferative response usually associated with compensatory renal growth in Xenopus further suggests that this response to tissue mass depletion is a consequence of a decrease in the concentration of specific inhibitor(s) of proliferation (chalone) normally produced by the tissue cells. Accordingly, the presence of viable, albeit nonfunctional, renal tissue (roughly equivalent in mass to the amount of tissue depleted) in the dorsal lymph sac of nephrectomized animal would result in a more or less unaltered production of the tissuespecific inhibitor(s) of proliferation. De-
DEVELOPMENTALBIOLOGY
536
spite the topographical separation of the renal tissue masses (residual mesonephros and mesonephric implant), the level of circulating kidney chalone(s) would be expetted to compare with that concentration maintained by an intact mass of kidney
VOLUME 66,1978
(normal mesonephros) . Consequently, no proliferative response by the residual kidney mass would be expected, a result reported in this study. The basis of the preceding discussion is indicated diagrammatically in Fig. 3.
Norma I ki dney ma55 Inhibits pfol i feration I II 1
Normal concentration circulating chalone(s)
I
Residual kidney mass fol lowing S nephrectomy I pro1 i feratlbn
t Decreased circulating
,
I
concentration thaIone
Restored inhibition
of
pro1 i feration
I
chilone(sl
chalonefs
)
normal concentration circulating chalones
FIG. 3. Model to explain the depression of the percentage of DNA synthesizing cells in the mesonephros found on the sixth postoperative day following recovery from partial unilateral nephrectomy in the presence of dorsal lymph sac implanted fragments of renal tissue.
GOLDIN AND FABIAN
Growth Regulation
Although our data indicate a chalone system in the regulation of compensatory growth in Xenopus laeuis, it is important to consider the relevance of these findings with respect to (a) the regulation of postembryonic kidney growth and (b) the maintenance of a steady-state cell population in the normal adult mesonephros. The view that compensatory growth merely represents an amplification of reparative growth has long been subscribed to (Saetren, 1956; Goss, 1964, 1967; Bullough, 1968; Malt, 1969; Bucher and Malt, 1971; Leblond, 1972), and, accordingly, the various hypotheses presented to explain the control of compensatory growth have been extended to include the regulation of replacement growth. There is little experimental justification for this extrapolation except for the evidence obtained in the studies involving liver regeneration where it was found that the destruction of less than 10% of the liver tissue evokes a localized proliferative response, while tissue ablation of a greater magnitude leads to a widespread hyperplastic response (see Bucher and Malt, 1971). Since normal adult mesonephric cells were found to be responsive to the inhibitory effect of the 40,000 MW ultrafiltrate (chalone) which was derived from normal adult mesonephros, the role of a chalone system in the maintenance of organ size remains a distinct possibility. Thus, in terms of the maintenance of a steady-state cell population size within the mesonephros, a chalone system could serve to regulate proliferative activity in the organ so that a balance between cell death and cell production is preserved. The present investigation does not provide data that would enable a discussion on the possible role of chalones in bringing about a decline in and ultimate cessation of post-embryonic growth. There is clearly a need to test the inhibitory ultrafiltrate fractions on mesonephric kidneys at various times from metamorphosis through to their attaining socalled adult size.
in X. laevis Kidney
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The authors express their sincere thanks to Dr. Alan ThornIey for helpful discussions and to Dr. Norman WesseIIs for his criticism of the manuscript. This research was supported by grants from the Atomic Energy Board and the Council for Scientific and Industrial Research and by a George Elkin Bequest. REFERENCES AUJARD, C., CHANY, E., AND FRAYSSINET, C. (1973). Inhibition of DNA synthesis of synchronized cells by liver extracts acting in vitro. Exp. Cell Res. 78, 476-478. BRUGAL, G. (1973). Effects of adult intestine and liver extracts on the mitotic activity of corresponding embryonic tissues of Pleurodeles waltlii Michah. Cell Tissue Kinet. 6,519-524. BRUGAL, G., AND PELMONT, J. (1975). Existence of two chalone-like substances in intestinal extract from the adult newt, inhibiting embryonic intestinal cell proliferation. Cell Tissue Kinet. 8,171-187. BUCHER, N. L. R., AND MALT, R. A. (1971). “Regeneration of Liver and Kidney.” Little, Brown, Boston. BULLOUGH, W. S. (1962). The control of mitotic activity in adult mammalian tissues. Biol. Rev. 37, 307-342. BULLOUGH, W. S. (1965). Mitotic and functional homeostasis: A speculative review. Cancer Res. 25, 1683-1727. BULLOUGH, W. S. (1968). The control of tissue growth. Zn “Biological Basis of Medicine” (E. E. Bittar and N. Bittar, eds.), Vol. 1, pp. 311-333. Academic Press, New York. BULLOUGH, W. S. (1975). Mitotic control in adult mammalian tissues. Biol. Rev. 50,99-127. BULLOUGH, W. S., AND LAURENCE, E. B. (1960). The control of epidermal mitotic activity in the mouse. Proc. Roy. Sot., London Ser. B 151, 517-536. BULLOUGH, W. S., AND LAURENCE, E. B. (1964). Mitotic control by internal secretion: The role of the chalone-adrenalin complex. Exp. Cell Res. 33, 176-194. CHOPRA, D. P., AND SIMNETT, J. D. (1969). Demonstration of an organ-specific mitotic inhibitor in amphibian kidney. The effects of adult Xenopus tissue extracts on the mitotic rate of embryonic tissue in vitro. Exp. Cell Res. 58, 319-322. CHOPRA, D. P., AND SIMNETT, J. D. (1971). Tissuespecific mitotic inhibition in the kidneys of embryonic grafts and partially nephrectomized host Xenopus laevis. J. Embryol. Exp. Morphol. 25,321-329. DICKER, S. E. (1972). Inhibition of compensatory renal growth. J. Physiol. London 225,577-588. DICKER, S. E., AND MORRIS, C. A. (1974). Investigation of a substance of renal origin which inhibits the growth of renal cortex explants in vitro. J. Embryol. Exp. Morphol. 31, 655-665. ELGJO, K. (1973). Epidermal chalone: Ceil cycle specificity of two epidermal growth inhibitors. Nat. Can-
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cer Znst. Monogr. 38, 71-76. GLINOS, A. D., AND GEY, G. 0. (1952). Humoral factors involved in the induction of liver regeneration in the rat. Proc. Sot. Exp. Biol. Med. 80,421-425. GOLDIN, G., AND FABIAN, B. (1975). DNA synthesis during larval development and compensatory growth in the mesonephros of Xenopus laevis. Develop. Biol. 46, 202-210. GOSS, R. J. (1964). “Adaptive Growth.” Academic Press, New York. Goss, R. J. (1967). The strategy of growth. In “Control of Cellular Growth in Adult Organisms” (H. Teir and T. Rytomaa, eds.), pp. 3-27. Academic Press, New York. HALL, R. G., AND SEIBERT, H. (1976). A cell culture study of G, and G2 chalones from kidney tissue. J. Cell Biol. 70, 54a. HAMBURGER, V. (1960). “A Manual of Experimental Embryology.” University of Chicago Press, Chicago. HONDIUS-BOLDINGH, W., AND LAURENCE, E. B. (1968). Extraction, purification and preliminary characterization of epidermal chalone. Eur. J. Biothem. 5, 191-198. IVERSEN, 0. H., BHANGOO, K. S., AND HANSEN, K. (1974). Control of epidermal cell renewal in the bat web. Virchows Arch. B Cell Pathol. 16, 157-179. KIVILAAKSO, E., AND RYT~MAA, T. (1971). Erythrocytic chalone, a tissue-specific inhibitor of cell proliferation in the erythron. Cell Tissue Kinet. 4, 1-9. LEBLOND, C. P. (1972). Growth and renewal. In “Regulation of Organ and Tissue Growth” (R. J. GOSS, ed.), pp. 13-39. Academic Press, New York. LOZZIO, B. B., LOZZIO, C. B., BAMBERGER, E. G., AND LAIR, S. V. (1975). Regulators of cell division: Endogenous mitotic inhibitors of mammalian cells. Znt. Rev. Cytol. 42, 1-47. MALT, R. A. (1969). Compensatory growth of the kidney. N. Engl. J. Med. 280, 1446-1459. MARKS, F. S. (1973). A tissue-specific factor inhibiting DNA synthesis in mouse epidermis. Nat. Cancer Inst. Monogr. 38, 79-96. MESSIER, B., AND LEBLOND, C. P. (1960). Cell proliferation and migration as revealed by autoradiogra-
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phy after injection of thymidine-H3 into male rats and mice. Amer. J. Anat. 106, 247-285. RUTTER, W. J. (1967). Protein determination in embryos. Zn “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), pp. 671-683. Thomas Y. Crowell, New York. RYT~MAA, T. (1976). The chalone concept. Znt. Rev Exp. Pathol. 16, 155-206. RYTBMAA, T., AND KIVINIEMI, K. (1968). Control of granulocyte production. Cell Tissue Kinet. 1, 329-350. SAETREN, H. (1956). A principle of auto-regulation of growth. Production of organ-specific mitose inhibitors in kidney and liver. Exp. Cell Res. 11,229-232. SCAIFE, J. F. (1970). Liver homeostasis: An in vitro evaluation of a possible specific chalone. Experientia 26,1071-1072. SIMARD, A., CORNEILLE, L., DESCHAMPS, Y., AND VERLY, W. G. (1974). Inhibition of cell proliferation in the livers of hepatectomized rats by a rabbit hepatic chalone. Proc. Nat. Acad. Sci. USA 71, 1763-1766. THORNLEY, A. L., AND LAURENCE, E. B. (1975). Chalone regulation of the epidermal cell cycle. Experientia 31, 1024-1026. THORNLEY, A. L., AND LAURENCE, E. B. (1976). The specificity of epidermal chalone action: The results of in vivo experimentation with two purified skin extracts. Develop. Biol. 61, 10-22. TUTTON, P. J. M. (1973). Control of epithelial cell proliferation in the small intestinal crypt. CelZ Tissue Kinet. 6, 211-216. VERLY, W. G. (1976). The control of liver growth. Zn “Chalones” (J. C. Houck, ed.), pp. 401-428. NorthHolland, Amsterdam. VERLY, W. G., DESCHAMPS, Y., PUSHPATHADAM, J., AND DESROSIERS, M. (1971). The hepatic chalone. I. Assay method for the hormone and purification of the rabbit liver chalone. Canad. J. Biochem. 49, 1376-1383. WEISS, P., AND KAVANAU, J. L. (1957). A model of growth and growth control in mathematical terms. J. Gen. Physiol. 41, I-47.
BIOLOGY
66,529~538 (1978)
The Regulation of Growth in the Mesonephric Kidney of Adult Xenopus laevis by an Endogenous inhibitor of Proliferation’ GEOFFREYGOLDINANDBARRYFABIAN Department
of Zoology,
University Received
of the Witwatersrand, March
16, 1978; accepted
Johannesburg June
2001, South
Africa
5, 1978
The effect of dorsal lymph sac implanted tissue fragments, of a 100,000g kidney supernatant, and of various kidney-derived ultrafiltrate fractions on the percentage of DNA synthesizing cells in the mesonephric kidney of Xenopus Zaeuis following partial unilateral nephrectomy was investigated autoradiographically. Using Amicon falters with cut-off values of MW 50,000 and 10,000, three ultrafiltrate fractions were obtained: a fraction containing molecules of MW 50,000 and less, a fraction containing molecules of MW 10,000 and less, and one containing molecules in the range of MW 10,000 to 50,000. The ultrafiltrates containing molecules of less than 10,000 MW were found to depress DNA synthetic activity on the sixth postoperative day by 30 to 40%, while the fraction containing molecules between MW 10,000 and 50,000 showed no significant effect. It has been concluded that an endogenous inhibitor of proliferation, with the attributes of a chalone, is present in the fraction of less than 10,000 MW. The loss ,of inhibitor action following Pronase treatment of the ultrafiltrate suggests that the inhibitor substance may be a protein or polypeptide, or that such constituent may be the carrier for the active agent. Since a depression in DNA synthetic activity of 60% was obtained in normal adult mesonephric kidneys following the injection of the ultrafdtrate, it is concluded that both compensatory growth and reparative growth in the kidney of Xenopus Laeois are regulated by a G, kidney chalone of less than 10,000 MW. INTRODUCTION
The maintenance of a steady-state cell population size (reparative growth) in both expanding organs and renewing tissues (Messier and Leblond, 1960) is dependent upon the balance between cell loss and cell production. The preservation of such an equilibrium requires some homeostatic mechanism for regulating proliferative activity (Goss, 1967; Bullough, 1968). Similarly, the progressive decline and eventual cessation of postembryonic growth in expanding organs necessitate the regulation of cell proliferation. It is conceivable, therefore, that both postembryonic and replacement growth in the various expanding organs and renewing tissues are subject to similar, if not the same, regulatory mecha’ This study comprises part of a thesis by G. V. Goldin submitted for the degree of Doctor of Philosophy at the University of the Witwatersrand, Johannesburg.
nisms (Bullough, 1968,1975; Leblond, 1972; Lozzio et al., 1975; Rytomaa, 1976). The view that proliferative growth is regulated by a negative feedback system involving tissue-specific inhibitors has been proposed by several authors (Saetren, 1956; Weiss and Kavanau, 1957; Bullough and Laurence, 1960; Bullough, 1962, 1965), and in this direction experimental support for the regulation of growth by chalones (Bullough, 1962) has been accumulating. This type of endogenous mitotic inhibitor produced by tissue cells and which, by a negative feedback mechanism, regulates the mitotic activity in the same tissue, has been extensively examined in the epidermis (Bullough and Laurence, 1960, 1964; Hondius-Boldingh and Laurence, 1968; Elgjo, 1973; Marks, 1973; Iversen et aZ., 1974). Two types of epidermal chalone have been identified and, on the basis of their specificity of action during the cell cycle (Thornley
529
0012-1606/78/0662-0529$02.00/0 Copyright All rights
0 1978 by Academic of reproduction in any
Press,Inc. form
reserved.
530
DEVELOPMENTALBIOLOGY
and Laurence, 1975, 1976), have been termed GI and Gz chalones, respectively (Elgjo, 1973). Chalones which have similar characteristics to the epidermal chalones have been reported for other renewing tissues, for example, erythrocyte chalones (Kivilaakso and Rytiimaa, 1971), granulocyte chalones (Rytiimaa & Kiviniemi, 1968), and intestinal chalones (Brugal, 1973; Tutton, 1973; Brugal and Pehnont, 1975). The role of tissue-specific growth inhibitors in the maintenance of organ mass and the control of regenerative growth in organs such as the kidney and the liver has been less rigorously studied. While several experiments suggest the involvement of humoral substances in the control of proliferative growth in the kidney, there has been little progress in identifying either a stimulant or an inhibitor involved in this regulation. The existence of a circulating mitotic inhibitor, specific for liver, was first suggested by Glinos and Gey (1952) on the basis of their finding that mitotic stimulation in normal liver could be achieved by the mere dilution of the plasma. In recent years this notion has received considerable experimental support (Scaife, 1970; Verly et al., 1971; Aujard et al., 1973; Simard et al., 1974; Verly, 1976). In particular, Verly et al. (1971) have purified a liver chalone, reported to be trypsin labile and of MW < 4000, which inhibited proliferation in cultured liver slices. In a subsequent study involving both in viva and in vitro assays, these authors (Simard et al., 1974) concluded that the liver chalone exerted its inhibition on hepatocytes during the G1 phase of the cell cycle, blocking the transition of these cells into the S phase. Following the initial postulation of an organ-specific inhibitor of proliferation in the kidney (Saetren, 1956), little progress toward the isolation and characterization of a kidney chalone has been made. The existence of a kidney chalone is implied by the experiments of Chopra and Simnett
VOLUME 66,1978
(1971), who reported that the mitotic activity in the mesonephros of juvenille Xenopus laeuis, following partial unilateral nephrectomy, was depressed by 88% 4 hr after the injection of a crude kidney extract (NO,OOOgsupernatant) . In an earlier study (Chopra and Simnett, 1969) it had been demonstrated that mitotic activity in cultured pronephric kidneys was depressed by the addition to the medium of a lOO,OOOg supernatant. These authors (Chopra and Simnett, 1971) concluded that, since mitotic inhibition was obtained in a 4-hr period following the administration of the extract, the inhibitory factor had acted on the cells in the GBphase of the cell cycle. While these data suggest the existence of a kidney chalone in crude extract, their assay does not in itself preclude S-phase inhibition or cytotoxicity (see Rytomaa, 1976). Evidence for the existence of kidney chalones has also been provided by Dicker (1972), who found that the injection of either kidney homogenates or the microsomal fraction of such homogenates inhibited compensatory renal growth in rata. More recently, Dicker and Morris (1974) found that the 2000g supernatant from homogenates of the renal cortex of either mouse, rat, guinea pig, rabbit, or pig inhibited growth of neonatal rat kidney explants through cortex-specific inhibition of mitosis. Finally, Hall and Seibert (1976) have reported in abstract form the existence of two “bovine-kidney-derived chalones” that inhibit an established cell line at the Gi and G2 phases of the cell cycle, respectively. In the present study, attempts have been made to ascertain whether a kidney-specific inhibitor of proliferation exists, within crude homogenates and various ultrafiltrates, which meets the criteria required to be considered a chalone. Chalones, according to Bullough (1975) and Rytomaa (1976), are cell line-specific, species-nonspecific inhibitors of cell proliferation, which are produced by the same cell line upon which they act and whose action is reversible and
GOLDIN
AND
FABIAN
Growth Regulation
noncytotoxic. Since it had been previously established that partial unilateral nephrectomy in adult Xenopus laevis is followed by a compensatory growth response in which 19% of the residual kidney cells synthesize DNA on the sixth postoperative day (Goldin and Fabian, 1975), the problem was tackled by treating the nephrectomized animals with the various tissue extracts and then scoring the percentage of thymidine-labeled cells. Accordingly, the presence of inhibitor(s) of proliferation in the extracts would result in a depression of the percentage of DNA synthesizing cells. To further test the hypothesis that compensatory renal growth is a response to tissue mass depletion, which results in a decrease in the concentration of specific inhibitor(s) of proliferation, fragments of renal tissue were implanted into the dorsal lymph sac of nephrectomized animals, and the effect of such implants on DNA synthesis in the residual kidney was studied. MATERIALS
AND
METHODS
Partial
Unilateral Nephrectomy Adult Xenopus laevis males that had
been acclimatized to laboratory conditions for several months prior to experimentation were anesthetized with 0.25% MS 222 (Sandoz, Basel, Switzerland) and opened ventrolaterally under aseptic conditions. The right kidney was exteriorized, and the posterior 30 to 40% of tissue destroyed by electrocauterization. Antibiotics (streptomycin and penicillin) were introduced into the abdominal cavity and the wound was closed with two or three sutures of fine silk. The animals were allowed to recover and heal in dechlorinated tap water to which no antibiotics were added.
Implantation
of Tissue Fragments
Following partial unilateral nephrectomy, fragments of either renal, hepatic, or cardiac tissue, obtained from normal adult donor Xenopus laevis, were implanted into
in X. laevis Kidney
531
the dorsal lymph sac of experimental animals. The tissue fragments (1 to 2 mm3) were excised from donor animals in which the blood was replaced by perfusing Steinberg solution (Hamburger, 1960) through the systemic arch. Prior to implantation, the tissue fragments were stored at 4°C in amphibian culture medium (Wolf and Quimby; GIBCO, Grand Island, New York). The implantation procedure was performed on completion of the nephrectomy before the animal had recovered from anesthesia. Several tissue fragments (either renal, hepatic, or cardiac tissue fragments), roughly equivalent in mass to the amount of renal tissue destroyed by cauterization, were then inserted through a small cutaneous incision into the dorsal lymph sac. The wound was closed with a single suture and the nephrectomized host animals were left to recover for 6 days. At the end of this period of time, tritiated thymidine (5 &i/g wt) was administered. The animals were killed 12 hr after the injection, and autoradiographs of host mesonephric kidneys were prepared. The implants were recovered and were prepared for routine histological examination and for autoradiography.
Preparation of Tissue Extracts Crude extract. Kidney pairs from 10 adult Xenopus laevis were perfused in situ with cold (4°C) Steinberg solution to deplete the endogenous blood reserves and to remove any circulating stress hormones. The kidneys were then excised, cleared of superfluous tissues, and weighed. The pooled mesonephric kidneys (combined weight, 3 to 4 g) were then homogenized in 30 to 40 ml of Steinberg solution for 10 set using an Ultra-Turrax homogenizer, and for 5 min using a Potter-S hand homogenizer. The homogenization and subsequent centrifugation steps were carried out at 4°C. The homogenate was subjected to ultracentrifugation at 100,OOOgand the supernatant solution (hereafter referred to as “crude extract”) was collected.
532
DEVELOPMENTAL BIOLOGY VOLUME66.1978
The crude extract injected into the experimental animals was either freshly prepared or that stored for a maximum period of 2 weeks at -20°C. UZtrufiZtrates. Crude extract, obtained by the procedure outlined above, was subjected to Amicon ultrafiltration under pure nitrogen at 40 psi. Two ultrafiltration steps were employed, the first using an Amicon XM 50 Diaflo membrane (cutoff MW 50,000) and the second using a PM 10 Diaflo membrane (cutoff MW 10,000). The ultrafiltrate obtained after the filtration of crude extract through the XM 50 membrane was collected and divided. Half of the ultrafiltrate solution was used for injection into experimental animals without further modification, and the remaining XM 50 ultrafiltrate was then subjected to a second ultrafiltration step through a PM 10 membrane. The PM 10 ultrafiltrate was then collected and used, without further modification, for injection into experimental animals. A third fraction was obtained by resuspending in Steinberg solution those constituents of the XM 50 ultrafiltrate that had been excluded from the PM 10 ultrafiltrate and which had accumulated as a residue on the PM 10 membrane. A portion of the PM 10 ultrafiltrate was subjected to 12-l-n digestion in 0.1% Pronase (predigested) at 30°C (MilesSeravac, Cape Town). [The protein content of each of the ultrafiltrate fractions was determined using the method of Lowry et al. outlined by Rutter (1967).] Autoradiographic Activity
Assay of DNA Synthetic
In order to test the tissue extracts for the presence of a kidney-specific inhibitor of proliferation, nephrectomized animals were injected, subcutaneously, above the dorsal lymph sac, with [ methyZ-3H]thymidine ([3H]TdR) (The Radiochemical Centre, Amersham, England; specific activity, 19 Ci/mmole), together with either crude extract or ultrafiltrate, on the sixth postoperative day. The animals were killed after
12 hr, and the kidneys, liver, small intestine, and testis excised and prepared for autoradiography as described previously (Goldin and Fabian, 1975). The amount of isotope administered corresponded to 5 &i/g body weight of frog, and the dosage of crude extract or ultrafiltrate (0.75 ml) was estimated to represent the volume that would have been derived from 30 to 40% of the tissue from one kidney. As controls, a few animals were injected with an equivalent volume of Steinberg solution together with tritiated thymidine, while others were injected only with the isotope to verify the previously recorded DNA synthetic activity in the residual mesonephric tissue on the sixth postoperative day (Goldin and Fabian, 1975). To minimize the influence of diurnal variation in proliferative activity, all experiments were carried out according to the same time schedule, that is, injections of tritiated thymidine being administered at 0700. In addition to the above treatments, four of the animals were administered a second injection of 0.25 ml of PM 10 ultrafiltrate 6 hr after the initial dosage of 0.75 ml. DNA synthetic activity in the normal untreated adult mesonephros as well as the normal adult injected with PM 10 ultrafiltrate 12 hr before killing was studied in a few animals by the autoradiographic procedure. All sections were coated with Ilford G5 nuclear emulsion [diluted 1:2 (v/v) with 2% glycerol in deionized water] by the dipping technique. After 10 to 14 days’ exposure in the dark at 4°C the slides were developed with D 19 for 3 min at 18°C (or 2 min at 2O”C), fixed with 26% sodium thiosulfate for 5 min, and washed in running water for 30 min. The sections were then stained through the emulsion with alcian blue (pH 2.5) and nuclear fast red. The sections were examined and scored for percentage of labeled cells. Every 20th section through the length of the mesonephric kidney pairs was examined and scored
GOLDIN AND FABIAN
Growth Regulation
for percentage of labeled cells. In the liver, random lobules were scored for labeled cells, and in the intestine, randomly selected crypts were similarly treated. RESULTS
The Effect Synthetic following tomy
of Tissue Implants on DNA Activity in the Mesonephros Partial Unilateral Nephrec-
The percentage of mesonephric cells synthesizing DNA on the sixth postoperative day was examined in 14 animals whose recovery from partial unilateral nephrectomy occurred with renal, hepatic, or cardiac fragments present in the dorsal lymph sac. The level of DNA synthetic activity wa;5 not significantly depressed by the presence of either the liver implants or the cardiac implants (see Table 1). However, in the renal implant-bearing nephrectomized host animals, the percentage of DNA synthesizing cells in the mesonephros was almost totally depressed to a level approaching that which normally occurs in adult mesonephric kidneys (0.2%) (see Table 1).
533
in X. laevis Kidney TABLE
1
THE PERCENTAGE OF DNA SYNTHESIZING CELLS IN THE MESONEPHROS ON THE SIXTH POSTOPERATIVE DAY FOLLOWING RECOVERY FROM PARTIAL UNILATERAL NEPHRECTOMY IN THE PRESENCE OF DORSAL LYMPH SAC IMPLANTED TISSUE FRAGMENTS DepresNynr DNA synthesrzmg sion (W) mals cells (mean % +- SD) Nephrectomized (controls) Renal tissue implant Liver tissue implant Cardiac tissue implant
3
19 f 0.57
-
6
0.2 + 0.075
98.9
4
17 + 2.36
10.5*
4
18 k 1.89
5.8**
* P = 0.3-0.4 (not significant). ** P = 0.6 (not significant).
The Effect of “Crude Extra& on DNA Synthetic Activity in the Mesonephros following Partial Unilateral Nephrectomy The injection of 0.75 ml of 100,OOOgsupernatant obtained from Xenopus mesonephric kidney homogenate into 10 partially nephrectomized animals on the sixth day of recovery was found to markedly depress the percentage of DNA synthesizing cells in the residual mesonephric tissue. In the autoradiographs prepared from the excised kidneys the percentage of thymidine-labeled cells was found to be 0.8% (Fig. 1). In comparison to the 19% of labeled cells (Fig. 2) found in the mesonephros on the sixth postoperative day, this represents a depression in DNA synthetic activity of the order of 96%. Since these experiments were considered to be pilot ones, the effect of crude extract on DNA synthetic activity in other tissues was not systematically studied.
FIG. 1. Autoradiograph of a portion of one mesonephros following the injection of 100,OOOg kidney homogenate supernatant and [1H]TdR on the sixth day of recovery from partial unilateral nephrectomy. The percentage of DNA synthesizing cells is of the order of 0.8%. which represents a depression of 96% from that usually recorded on the sixth postoperative day. Distal tubules containing labeled cells are indicated by arrows. Dt = distal tubules. Px = proximal tubules. x 90.
534
DEVELOPMENTAL BIOLOGY
VOLUME 66,1978
inhibitory action following the treatment of the PM 10 ultrafiltrate with Pronase points to the inhibitor being either a protein.or a polypeptide, or to such substances being vehicles for some other type of agent. Since the treatment involving a second injection of PM 10 ultrafiltrate, 6 hr after the first, did not enhance the depression of labeled cells obtained following the administration of a single dose, it would appear that the inhibitor(s) within the extract was effective for at least 12 hr. The target tissue specificity of the inhibitory effect of both the XM 50 and the PM 10 ultrafiltrates was tested by examining DNA synthetic activity in intestine, liver, and testis. In the liver lobules, intestinal crypts, and seminiferous tubules of the testis, sampled randomly, DNA synthetic activity was found to be unaffected by either of the ultrafiltrates.
FIG. 2. Autoradiograph of a portion of one mesonephros on the sixth postoperative day following partial unilateral nephrectomy. Labeling after 12-hr exposure to tritiated thymidine is maximal at this stage of recovery when 19% of cells are synthesizing DNA. The extensive labeling occurs in the distal tubules (Dt) and not in the proximal tubules (Px). x 90.
The Effect of Ultrafiltrates on DNA Synthetic Activity in the Mesonephros following Partial Unilateral Nephrectomy The data obtained following the administration of the various ultrafiltrates into partially nephrectomized animals are detailed in Table 2. Both the XM 50 ultrafiltrate (MW < 50,000) and the PM 10 ultrafiltrate (MW < 10,000) were found to depress the percentage of DNA synthesizing cells in the mesonephros by 35 to 40%, while the fraction containing molecules between MW 10,000 and 50,000 was not found to exhibit any significant inhibitory action. These results indicate that the depression in the percentage of DNA synthesizing cells is due to the presence of one or more species of molecule of MW < 10,000. The loss of such
The Effect of Ultrafiltrate Adult Kidney
on Normal
The percentage of labeled cells in the normal adult mesonephros following the injection of
The finding of this investigation that the various mesonephric extracts depress the percentage of DNA synthesizing cells usually associated with the compensatory growth response provides support for the view that renal growth is regulated by specific inhibitors of cell proliferation (Saetren, 1956; Chopra and Simnett, 1969, 1971; Dicker, 1972; Dicker and Morris, 1974). Despite the relative crudeness of the ultrafiltrate fractions used in this study, the conclusion that a G1 kidney chalone does exist may be justifiable. While the reversi-
GOLDIN AND FABIAN
Growth Regulation TABLE
535
in X. laeuis Kidney
2
THE PERCENTAGE OF DNA SYNTHESIZING CELLS IN THE MESONEPHROS FOLLOWING THE INJECTION OF ULTRAFILTRATES AND [aH]TdR ON THE SIXTH DAY OF RECOVERY FROM PARTIAL UNILATERAL NEPHRECTOMY Ultrafiltrate
injected
Number of animals
Steinberg solution (control) MW < 50,000 (XM 50) MW < 10,000 (PM 10) (single injection)* MW < 10,000 (PM 10) (two injections)* PM 10 (Pronase treated) MW lO,OOO-50,000 (PM lo-XM 50) * P = 0.6 (not significantly
3 5 7 4 3 3
DNA synthesizing cells (mean % + SD) 18.7 +- 1.15 11.8 + 1.64 11.7 f 1.97 11.0 f 1.8 17.0 -c 1.7 17.6 + 2.1
Depression 6) 36.8 37.4 41.2 9.1 5.9
P value
different).
bility and species nonspecificity of the inhibitory effect of the ultrafiltrates has not been tested, our results suggest that a substance in kidney extract fulfills the criteria listed as characteristics of chalones, namely, that they are tissue-specific inhibitors of cell proliferation that inhibit proliferation in the same tissue whence they are produced (Bullough, 1975; Rytomaa, 1976). Since the injection of either the 40,000 or the <10,000 MW ultrafiltrates was not found to achieve depressions in the percentage of DNA synthesizing cells in excess of 45% when administered concomitantly with tritiated thymidine, it appears that the resultant inhibition was due to a blockade of the cells in the G1 phase of the cell cycle, and not to some direct influence on S-phase events. Accordingly, those cells that were unaffected by the inhibitor(s) had presumably entered into the S phase prior to the injection of the extract and isotope, and consequently would continue to incorporate the tritiated thymidine. That the inhibition was noncytotoxic and tissue specific in its action was supported by the nondepression of the percentage of DNA synthesizing cells recorded in the intestine and liver. In addition, a cytotoxic effect is regarded as a less serious problem in an in viuo system where the organism has effective mechanisms of detoxification (Rytomaa, 1976). Since the depression of the percentage of DNA synthesizing cells was considerably
less extensive in the extracts of MW < 50,000 compared to that recorded after the injection of crude extract, it seems likely that additional inhibitors (MW > 50,000), which presumably acted on cells in the S phase, were present in the crude extract. A similar conclusion was made by Simard et al. (1974) with respect to the inhibition of hepatocyte proliferation by crude liver homogenates. The near-total depression of the percentage of DNA synthesizing cells obtained after implanting renal fragments into the dorsal lymph sac of partially nephrectomized animals clearly supports the hypothesis that compensatory growth is a response to tissue mass depletion, rather than the result of the release of a mitotic stimulator (wound hormone) or the consequence of an increased physiological load. The finding that mesonephric kidney extracts inhibit the proliferative response usually associated with compensatory renal growth in Xenopus further suggests that this response to tissue mass depletion is a consequence of a decrease in the concentration of specific inhibitor(s) of proliferation (chalone) normally produced by the tissue cells. Accordingly, the presence of viable, albeit nonfunctional, renal tissue (roughly equivalent in mass to the amount of tissue depleted) in the dorsal lymph sac of nephrectomized animal would result in a more or less unaltered production of the tissuespecific inhibitor(s) of proliferation. De-
DEVELOPMENTALBIOLOGY
536
spite the topographical separation of the renal tissue masses (residual mesonephros and mesonephric implant), the level of circulating kidney chalone(s) would be expetted to compare with that concentration maintained by an intact mass of kidney
VOLUME 66,1978
(normal mesonephros) . Consequently, no proliferative response by the residual kidney mass would be expected, a result reported in this study. The basis of the preceding discussion is indicated diagrammatically in Fig. 3.
Norma I ki dney ma55 Inhibits pfol i feration I II 1
Normal concentration circulating chalone(s)
I
Residual kidney mass fol lowing S nephrectomy I pro1 i feratlbn
t Decreased circulating
,
I
concentration thaIone
Restored inhibition
of
pro1 i feration
I
chilone(sl
chalonefs
)
normal concentration circulating chalones
FIG. 3. Model to explain the depression of the percentage of DNA synthesizing cells in the mesonephros found on the sixth postoperative day following recovery from partial unilateral nephrectomy in the presence of dorsal lymph sac implanted fragments of renal tissue.
GOLDIN AND FABIAN
Growth Regulation
Although our data indicate a chalone system in the regulation of compensatory growth in Xenopus laeuis, it is important to consider the relevance of these findings with respect to (a) the regulation of postembryonic kidney growth and (b) the maintenance of a steady-state cell population in the normal adult mesonephros. The view that compensatory growth merely represents an amplification of reparative growth has long been subscribed to (Saetren, 1956; Goss, 1964, 1967; Bullough, 1968; Malt, 1969; Bucher and Malt, 1971; Leblond, 1972), and, accordingly, the various hypotheses presented to explain the control of compensatory growth have been extended to include the regulation of replacement growth. There is little experimental justification for this extrapolation except for the evidence obtained in the studies involving liver regeneration where it was found that the destruction of less than 10% of the liver tissue evokes a localized proliferative response, while tissue ablation of a greater magnitude leads to a widespread hyperplastic response (see Bucher and Malt, 1971). Since normal adult mesonephric cells were found to be responsive to the inhibitory effect of the 40,000 MW ultrafiltrate (chalone) which was derived from normal adult mesonephros, the role of a chalone system in the maintenance of organ size remains a distinct possibility. Thus, in terms of the maintenance of a steady-state cell population size within the mesonephros, a chalone system could serve to regulate proliferative activity in the organ so that a balance between cell death and cell production is preserved. The present investigation does not provide data that would enable a discussion on the possible role of chalones in bringing about a decline in and ultimate cessation of post-embryonic growth. There is clearly a need to test the inhibitory ultrafiltrate fractions on mesonephric kidneys at various times from metamorphosis through to their attaining socalled adult size.
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