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Influence of hormone concentration and time factor on development of receptor memory in a unicellular (Tetrahymena) model system
Comp. Biochem. Physiol. Vol. 73B, No. 2, pp. 357 to 360, 1982
0305-0491/82/100357-04503.00./'0 © 1982 Pergamon Press Ltd
Printed in Great Britain.
INFLUENCE OF HORMONE CONCENTRATION AND TIME F A C T O R O N D E V E L O P M E N T O F R E C E P T O R M E M O R Y IN A U N I C E L L U L A R (TETRAHYMENA) M O D E L SYSTEM G. CSABA, G. NI~METHt a n d P. VARGHA* Department of Biology, Biometry and Clinical Epidemiology Unit,* Semmelweis University of Medicine and Department of Medical Biology,t University Medical School, Szeged, Hungary
(Received 17 November 1981) 1. Treatment of Tetrahymena pyriformis cells with diiodotyrosine (T2) gave rise to a considerable, concentration-dependent increase of the growth rate within the range of 10-~5 and 10 _9 M, but did not influence it at the level of 10 ~8 M. 2. Re-exposure of the cells 1, 2 and 4 weeks later to the hormone concentrations originally used accounted for a marked increase of growth rate at all h o r m o n e levels tested, indicating that the extremely low concentration of l0 ~8 M, which failed to stimulate growth on first exposure, did nevertheless give rise to hormonal imprinting, which caused the cells to "'remember" the hormone, as judged from their increased responsiveness to it on re-exposure. 3. The degree of growth response was concentration-dependent on both first and second exposure: higher levels of treatment gave rise to firmer imprinting, and to greater response on re-exposure. 4. The length of exposure time proved to be more decisive than the level of treatment in respect of the development of hormonal imprinting. 5. Short-term exposures up to 60 rain, although they stimulated cell growth by direct effect, gave rise to lasting inhibition of cellular response to re-exposure(s) rather than to hormonal imprinting. Abstract
INTRODUCTION T h e first i n t e r a c t i o n b e t w e e n target cell a n d h o r m o n e is o f decisive i m p o r t a n c e at all levels o f p h y l o g e n e s i s , f r o m unicellular o r g a n i s m s to n e o n a t a l m a m m a l s , for it gives rise to t h e so-called h o r m o n a l i m p r i n t i n g ( C s a b a , 1980, 1981), w h i c h a c c o u n t s for cellular " r e c o g n i t i o n " of, a n d c o n s e q u e n t g r e a t e r cellular res p o n s i v e n e s s to, t h e h o r m o n e , o n s u b s e q u e n t exposure(s). It h a s been s h o w n e x p e r i m e n t a l l y t h a t 24-hr e x p o s u r e o f t h e Tetrahymena to a h o r m o n e is sufficient to e n d o w t h e u n i c e l l u l a r with a " m e m o r y " over m o r e t h a n 500 g e n e r a t i o n s ( C s a b a et al., 1982); this h a s clearly i n d i c a t e d t h e h e r e d i t a r y t r a n s m i s s i o n of hormonal imprinting. Since, h o w e v e r , t h e Tetrahymena m a y divide 4 - 5 t i m e s w i t h i n 24 h r (Hill, 1972), t h a t period is too l o n g for the s t u d y of e v e n t s p e r t a i n i n g to its life cycle. In t h e p r e s e n t e x p e r i m e n t s we a t t e m p t e d to identify the s h o r t e s t e x p o s u r e t i m e a n d lowest h o r m o n e c o n c e n t r a t i o n r e s p o n s i b l e for h o r m o n a l i m p r i n t i n g at t h e u n i c e l l u l a r level, to o b t a i n m o r e i n f o r m a t i o n o n the underlying mechanism. MATERIAL AND METHODS
Tetrahymena pyr![brmis GL cells, maintained in l°o yeast extract containing 1°~, Bacto trypton (Difco, Michigan) medium, were used in the experiments. A. Mass cultures of Tetrahymena were, in the logarithmic phase of growth, transferred for 24 hr to a medium containing 10 9 M diiodotyrosine (T2; Fluka, Buchs, Switzerland), were washed thoroughly after incubation, and were returned to the basal medium again. Twenty cells were subcultured immediately after 24-hr treatment with ~..p 73,2, L
T 2, and the clonal cell counts were read after 24hr. Further 20-cell subcultures were derived from the mass cultures 1, 2 and 4 weeks after the first exposure in a medium containing 10 -18 , 10 15, 10 ~2 or 1 0 - g M T 2 , and were read after 24hr. Not pretreated Tetrahymena cultures were exposed to T 2 treatment at all levels (10 is, 10-15 10- 12 10-9 M) as controls to the second exposure. The results were evaluated by two-factor variance analysis, taking as one factor the time (weeks) elapsed after the first exposure, and as the other the h o r m o n e concentration applied for re-exposure. B. Mass cultures of Tetrahymena were, in the logarithmic phase of growth, exposed to 10 -18, 10 15 10 12 and 10 9 M diiodothyrosine for 24 hr, washed thoroughly, and returned for 1 week to normal medium. After 1 week all cultures were treated again with T2, at 10 .9 or 1 0 - ~ 8 M level. The control cultures set up with the test were, naturally, not treated. Twenty cells were subcultured in each group, and were examined for growth after 24 hr. The results were evaluated by two-factor variance analysis, taking as one factor the level of pretreatment, and as the other that of re-exposure. C. Tetrahymena cultures were exposed to 10 .9 M T z in the logarithmic phase of growth, for periods of 10, 30, 60rain, 4, 8 and 24hr. After treatment the cells were washed, 20 cells were subcultured in each lot and were read after 24 hr. The control cultures set up with the test were not treated. One, 2, 3 and 4 weeks after the first exposure, the mass cultures were treated again with 10 9 M T 2, cubcultured as above and read after 24 hr. The results were evaluated by variance analysis and regression analysis. RESULTS AND DISCUSSION
A m o n g t h e h o r m o n e s a n d p r e c u r s o r s b e l o n g i n g to t h e t h y r o x i n e series, d i i o d o t y r o s i n e (T2) h a s been f o u n d to s t i m u l a t e t h e g r o w t h of t h e Tetrahymena to 357
358
G. CSABA et al. T a b l e 1. G r o w t h r e s p o n s e of Tetrahymena to a single t r e a t m e n t with T 2
a. Mean growth rates at different levels of treatment Hormone conc.(M) Mean growth rate in 24 hr
Source of variation Regression Deviation from linearity Error
10 ~ 21.0
10 12 17.6
10 15 14.6
10 ta 12.2
b. Variance table Degrees of Sum of squares freedom Mean square 870.25 6.30 358.20
1 2 76
870.25 3.15 4.71
K 12.4
F
P
184 0.67
>0.05
~0.001
Equation of regression: y = 29.7 + 0.98x where y = growth and x = log concentration.
the greatest degree (Csaba & Ndmeth, 1980). Experimental observations suggest that at the low phylogenetic level represented by the unicellular, T2 is more hormone-like in action than the thyronine derivatives. In view of this we used T2 as a h o r m o n e in our experimental study of cellular (receptor) memory at the unicellular level. Since the untreated control cultures set up with the tests did not notably differ from one another ( F [ 1 0 ; 2 0 9 ] = 0 . 7 8 : P < 0 . 0 5 ) in respect of growth rate a direct comparison of mean values has been possible, at a mean variance of 4.36. The mean growth rate for controls was assessed as 12.3, A single treatment with T2 gave rise to an abrupt increase of the growth rate, whose degree depended on the applied concentration of h o r m o n e : the correlation between the logarithm of concentration and growth rate was practically linear in the given range (Table 1). This correlation seems to apply in a very narrow interval outside the given concentration range, since the growth of cultures exposed to 10 18 M T2 was similar to the control (the difference was not significant statistically), while the m a x i m u m stimulatory concentration was around 10 9 M. Thus only a minimal increase, resp. decrease can be expected over the limits of the critical range. The results of re-exposure at different levels of horm o n e (experiment A, Table 2) were to a certain degree surprising: the length of the time elapsed after pre-
treatment had no significant influence on the growth rate; e.g. the mean clonal cell counts were higher at 2 weeks than at 1 week after the first exposure. O n re-exposure the concentration dependence of h o r m o n e effect proved to be statistically significant, but not unequivocal: at lO 9 M the growth rate increased significantly over that observed at 10- 12 and 10 15 M (P < 0.01: P < 0.05), but did not differ significantly from that observed at 1018 M, which was greater, although not significantly, than the rates observed at 10-12 and 1 0 - i s M. While on first exposure 1 0 - i s M had been practically inactive (to judge from similar growth rate to the control in this and all preceding experimental series), on re-exposure even that low concentration developed a notable stimulatory effect in cultures pretreated at the same level. It follows that a rery low hormone concentration, which does not in itself b!fluence celhdar growth in any way, can also give rise to hormonal imprintin.q. Naturally, as indicated by the results of experiment B, the levels o1 first exposure and re-exposure equally influence cell growth, and higher concentrations develop a greater stimulatory action (Table 3). It should be noted that, although 1 0 - 1 S M T 2 stimulated the growth of the Tetr~thymena to a lesser degree than 10 -9 M, the difference was only significant statistically. As to the role of the length of first exposure, 10-rain treatment with T2 gave already rise to considerable growth stimulation (Table 4). The growth rate tended
Table 2. Growth response of Tetrahymena to re-exposure at different concentrations of T 2 a. Mean growth rates Interval between 1st and 2nd treatment (weeks)
10 ~
1 2 4
20.7 21.4 19.8
Source of variation Time interval Concentration Interaction Error
Level of re-exposure {M) 10 - i s I0 lz 18.6 19.3 19.9
19.6 20.2 19.1
b. Variance table Degrees of Sum of squares freedom Mean square 36.86 64.72 45.3l 1583.1
2 3 6 228
10 lu
18.43 21.57 7.55 6.94
19.7 21.0 19.8
F
P
2.65 3.11 1.09
>0.05 <0.05 >0.05
Development of receptor memory Table 3. Growth response of
359
Tetrahymenato application of different hormone levels for first and second exposure a. Mean growth rates
First exposed at Re-exposed at 10 9 M 10
t8 M
10 - 9 M
10 -~2 M
10 -15 M
10 -~8 M
21.2 19.9
19.7 18.8
19.2 17.6
18.1 16.3
b. Variance table Degrees of Sum of squares freedom Mean square
Source of variation At first exposure At re-exposure Interaction Error
242.32 79.81 5.27 768.40
to increase further with the prolongation of exposure, and showed a practically linear relationship with the length of exposure. The growth values determined one week later were considerably lower, but exhibited a similar linearity. The two regression lines showed a parallelism, i.e. 1-week decrease was identical after different lengths of exposure. Re-exposure for 24 hr 1 week after pretreatment did not further stimulate the growth of cultures originally exposed for short 10-rain, 30-rain periods, but accounted for a statistically significant (P < 0.01), although biologically not notable, increase in the growth rate of those pretreated for 60 rain. Stimu-
3 l 3 152
80.8 79.81 1.76 5.05
F
P
15.9 15.8 0.35
<0.001 < 0.001 > 0.05
lation of growth by re-exposure was considerable in the cultures originally exposed for 4 hr or longer; the mean cell counts were practically identical in subcultures prepared after first exposure for 4, 8 and 24 hr. Two weeks after pretreatment, 24-hr re-exposure to the concentration originally applied had similar effects to re-exposure after 1 week. In cultures treated for 60 min or a shorter time the growth rates were markedly inferior to those observed on first exposure for identical periods, and did not differ significantly from the control. In cultures originally exposed for 4 h r or longer, re-exposure-stimulated growth rates were greater than in the short-term series, but still
Table 4. Influence of the length of exposure to 10-9 M T2 on growth response of the a. Mean growth rates Period of first exposure 10 min
Sampling times Immediately after firstexposure One week after first exposure After 24-hr re-exposure for identical periods At 2 weeks At 2 + 3 weeks At 2 + 3 + 4 weeks
30 min
1 hr
4 hr
17.8 13.3
18.9 14.2
20.8 13.8
2 1 . 1 2 1 . 0 22.3 15.1 16.5 17.9
13.4 12.4 14.2 16.0
14.1 13.2 13.5 17.7
15.8 13.2 16.0 18.8
21.8 19.0 20.0 20.5
b. Variance table 1. Values assessed immediately after treatment Degrees of Source of variation Sum of squares freedom Mean square Regression Deviation from linearity Error
236.5 34.4 494.3
Tetrahymena
1 4 114
236 8.6 4.34
8 hr
21.6 20.0 20.8 20.5
24 hr
21.5 20.0 21.2 20.8
F
P
55 2.0
,~ 0.001 >0.05
F
P
66 1.8
40.001 > 0.05
Equation of regression: y = 20 + 0.8x where y = growth and x = log time.
Source of variation Regression Deviation from linearity Error
2. Values assessed 1 week after treatment Degrees of Sum of squares freedom Mean square 284 31.5 488.8
1 4 114
284 7.9 4.29
Equation of regression: y = 14 + 0.9x where y = growth and x = log time.
360
G. CSABA et ol.
inferior to those observed on first exposure for 4, 8 and 24 hr. Two weeks after pretreatment, 24-hr re-exposure to the concentration originally applied had similar effects to re-exposure after 1 week. In cultures treated for 6 0 m i n or a shorter time, the growth rates were markedly inferior to those observed on first exposure for identical periods, and did not differ significantly from the control. In cultures originally exposed for 4 hr or longer, re-exposure-stimulated growth rates were greater than in the short-term series, but still inferior to those observed on first exposure. Twenty-four hour long re-exposure after 3 weeks was followed by a not significant increase of the growth rate over the 2-week values and over the baseline as well. The growth decrease observed on re-exposure after 4 weeks was negligible in cultures originally exposed for 4, 8 and 24 hr; the maximum growth rates did not differ between the three series exposed for different times. (It should be noted that the cultures originally exposed for 4 hr showed maximum increase in growth rate after re-exposure at 4 weeks.) In cultures originally exposed for 1 hr or shorter periods, re-exposure after 3 and 4 weeks was followed by a considerable increase of the growth rate over that observed on first exposure; the increase showed a parallelism with the length of exposure in all cases except one (the mean growth rate was higher in cultures pretreated for 10 min than in those pretreated for 30 min, but this may have been a chance phenomenon). It follows that, for first exposure, 10-min treatment with the h o r m o n e already accounts for a considerable stimulation of cell growth, which increases further with the prolongation of exposure. At the same time, short-term {10, 30 or 60-min) pretreatments, although causing growth stimulation by direct effect, inhibit the development of such an effect on later prolonged reexposure. The inhibitory influence tends to decrease with progressing time. but seems to be of extraordinarily long duration. Summarizing the information emerging from experiments A, B and C, the conclusion lies close at hand that h o r m o n e concentrations too low (10 ~s M) for direct stimulation of cell growth can nevertheless induce h o r m o n a l imprinting, while short-term exposures sufficient for direct growth stimulation by the h o r m o n e inhibit rather than promote the development of h o r m o n a l imprinting, to judge from suppression of cellular response to later re-exposure{s) in cultures so treated.
Remarkably, at the extremely low concentration of 10 ~8 M, T2 did not in any way influence the growth of the Tetrahymena, but did give rise to hormonal imprinting, as indicated by the increased responsivehess of the cells so treated to re-exposure to the same low level of the hormone. Mention should also be made of the fact thai hmgterm first exposure for 4, 8 and 24 hr accounted m itself for a considerable increase of the growth rate over the control (15.1: 16.5:17.9 opposed to 12.41 for as long as a week, while short-term first exposure for 10, 30 and 6 0 m i n gave rise only to a minor increase thereof ( 13.3 : 14.2:13.8 against 12.4). This supports our earlier hypothesis that the first interaction with the h o r m o n e practically "'resets" the growth rate of the target cells, and the altered mechanism persists over the progeny generations if the process of imprinting is long enough. It follows from the experimental facts that the concentration of the h o r m o n e is less decisive for a succesful imprinting than the length of exposure. Supposing that Koch's (1979) dynamic receptor pattern generation theory is valid, and the cell is "screening" its environment for information carrier molecules through presenting on its surface wlrious configurations of sub-patterns (receptors), it might as well be postulated that the presentation (chance arisalt of an adequate configuration requires a certain time. If that time is not available e.g. at short-term tirst exposures in the present experiment no imprinting can take place. Long-term inhibition of cellular response to re-exposure(s) after non-imprinting short exposures is, however, not explicable by the above theory.
REFERENCES
CSABA G. (1980) Phylogeny and ontogeny of hormone receptors: the selection theory of receptor formation and hormonal imprinting. Biol. Rer. (Cambridge) 551,47 63. CSABA G. (1981) Ontogeny mid Phylogef U, of ftormone Receptors. Karger, Basel, New York. CSABA G. & N/:MEXH G. (1980) Effect of hormones and their precursors on protozoa the selective responsiveness of Tetrahymena. Comp. Bioehem. Physiol. 65B, 387 390. CSABA G., N~MVrH G. & VAR(~HA P. (1982) Development and persistence of receptor "memory" in a unicellular model system. Exp. Cell. Biol. In press. HILL D. L. (1972) The Biochemistry and Phvsiologty ¢!1 Tetrahymena. Academic Press, New York. KOCH A. S., F~:Hf:RJ. & LUKOVITS I. (1979) A simple model of dynamic receptor pattern generation. Biol. Cyhernet, 32, 125 138.
0305-0491/82/100357-04503.00./'0 © 1982 Pergamon Press Ltd
Printed in Great Britain.
INFLUENCE OF HORMONE CONCENTRATION AND TIME F A C T O R O N D E V E L O P M E N T O F R E C E P T O R M E M O R Y IN A U N I C E L L U L A R (TETRAHYMENA) M O D E L SYSTEM G. CSABA, G. NI~METHt a n d P. VARGHA* Department of Biology, Biometry and Clinical Epidemiology Unit,* Semmelweis University of Medicine and Department of Medical Biology,t University Medical School, Szeged, Hungary
(Received 17 November 1981) 1. Treatment of Tetrahymena pyriformis cells with diiodotyrosine (T2) gave rise to a considerable, concentration-dependent increase of the growth rate within the range of 10-~5 and 10 _9 M, but did not influence it at the level of 10 ~8 M. 2. Re-exposure of the cells 1, 2 and 4 weeks later to the hormone concentrations originally used accounted for a marked increase of growth rate at all h o r m o n e levels tested, indicating that the extremely low concentration of l0 ~8 M, which failed to stimulate growth on first exposure, did nevertheless give rise to hormonal imprinting, which caused the cells to "'remember" the hormone, as judged from their increased responsiveness to it on re-exposure. 3. The degree of growth response was concentration-dependent on both first and second exposure: higher levels of treatment gave rise to firmer imprinting, and to greater response on re-exposure. 4. The length of exposure time proved to be more decisive than the level of treatment in respect of the development of hormonal imprinting. 5. Short-term exposures up to 60 rain, although they stimulated cell growth by direct effect, gave rise to lasting inhibition of cellular response to re-exposure(s) rather than to hormonal imprinting. Abstract
INTRODUCTION T h e first i n t e r a c t i o n b e t w e e n target cell a n d h o r m o n e is o f decisive i m p o r t a n c e at all levels o f p h y l o g e n e s i s , f r o m unicellular o r g a n i s m s to n e o n a t a l m a m m a l s , for it gives rise to t h e so-called h o r m o n a l i m p r i n t i n g ( C s a b a , 1980, 1981), w h i c h a c c o u n t s for cellular " r e c o g n i t i o n " of, a n d c o n s e q u e n t g r e a t e r cellular res p o n s i v e n e s s to, t h e h o r m o n e , o n s u b s e q u e n t exposure(s). It h a s been s h o w n e x p e r i m e n t a l l y t h a t 24-hr e x p o s u r e o f t h e Tetrahymena to a h o r m o n e is sufficient to e n d o w t h e u n i c e l l u l a r with a " m e m o r y " over m o r e t h a n 500 g e n e r a t i o n s ( C s a b a et al., 1982); this h a s clearly i n d i c a t e d t h e h e r e d i t a r y t r a n s m i s s i o n of hormonal imprinting. Since, h o w e v e r , t h e Tetrahymena m a y divide 4 - 5 t i m e s w i t h i n 24 h r (Hill, 1972), t h a t period is too l o n g for the s t u d y of e v e n t s p e r t a i n i n g to its life cycle. In t h e p r e s e n t e x p e r i m e n t s we a t t e m p t e d to identify the s h o r t e s t e x p o s u r e t i m e a n d lowest h o r m o n e c o n c e n t r a t i o n r e s p o n s i b l e for h o r m o n a l i m p r i n t i n g at t h e u n i c e l l u l a r level, to o b t a i n m o r e i n f o r m a t i o n o n the underlying mechanism. MATERIAL AND METHODS
Tetrahymena pyr![brmis GL cells, maintained in l°o yeast extract containing 1°~, Bacto trypton (Difco, Michigan) medium, were used in the experiments. A. Mass cultures of Tetrahymena were, in the logarithmic phase of growth, transferred for 24 hr to a medium containing 10 9 M diiodotyrosine (T2; Fluka, Buchs, Switzerland), were washed thoroughly after incubation, and were returned to the basal medium again. Twenty cells were subcultured immediately after 24-hr treatment with ~..p 73,2, L
T 2, and the clonal cell counts were read after 24hr. Further 20-cell subcultures were derived from the mass cultures 1, 2 and 4 weeks after the first exposure in a medium containing 10 -18 , 10 15, 10 ~2 or 1 0 - g M T 2 , and were read after 24hr. Not pretreated Tetrahymena cultures were exposed to T 2 treatment at all levels (10 is, 10-15 10- 12 10-9 M) as controls to the second exposure. The results were evaluated by two-factor variance analysis, taking as one factor the time (weeks) elapsed after the first exposure, and as the other the h o r m o n e concentration applied for re-exposure. B. Mass cultures of Tetrahymena were, in the logarithmic phase of growth, exposed to 10 -18, 10 15 10 12 and 10 9 M diiodothyrosine for 24 hr, washed thoroughly, and returned for 1 week to normal medium. After 1 week all cultures were treated again with T2, at 10 .9 or 1 0 - ~ 8 M level. The control cultures set up with the test were, naturally, not treated. Twenty cells were subcultured in each group, and were examined for growth after 24 hr. The results were evaluated by two-factor variance analysis, taking as one factor the level of pretreatment, and as the other that of re-exposure. C. Tetrahymena cultures were exposed to 10 .9 M T z in the logarithmic phase of growth, for periods of 10, 30, 60rain, 4, 8 and 24hr. After treatment the cells were washed, 20 cells were subcultured in each lot and were read after 24 hr. The control cultures set up with the test were not treated. One, 2, 3 and 4 weeks after the first exposure, the mass cultures were treated again with 10 9 M T 2, cubcultured as above and read after 24 hr. The results were evaluated by variance analysis and regression analysis. RESULTS AND DISCUSSION
A m o n g t h e h o r m o n e s a n d p r e c u r s o r s b e l o n g i n g to t h e t h y r o x i n e series, d i i o d o t y r o s i n e (T2) h a s been f o u n d to s t i m u l a t e t h e g r o w t h of t h e Tetrahymena to 357
358
G. CSABA et al. T a b l e 1. G r o w t h r e s p o n s e of Tetrahymena to a single t r e a t m e n t with T 2
a. Mean growth rates at different levels of treatment Hormone conc.(M) Mean growth rate in 24 hr
Source of variation Regression Deviation from linearity Error
10 ~ 21.0
10 12 17.6
10 15 14.6
10 ta 12.2
b. Variance table Degrees of Sum of squares freedom Mean square 870.25 6.30 358.20
1 2 76
870.25 3.15 4.71
K 12.4
F
P
184 0.67
>0.05
~0.001
Equation of regression: y = 29.7 + 0.98x where y = growth and x = log concentration.
the greatest degree (Csaba & Ndmeth, 1980). Experimental observations suggest that at the low phylogenetic level represented by the unicellular, T2 is more hormone-like in action than the thyronine derivatives. In view of this we used T2 as a h o r m o n e in our experimental study of cellular (receptor) memory at the unicellular level. Since the untreated control cultures set up with the tests did not notably differ from one another ( F [ 1 0 ; 2 0 9 ] = 0 . 7 8 : P < 0 . 0 5 ) in respect of growth rate a direct comparison of mean values has been possible, at a mean variance of 4.36. The mean growth rate for controls was assessed as 12.3, A single treatment with T2 gave rise to an abrupt increase of the growth rate, whose degree depended on the applied concentration of h o r m o n e : the correlation between the logarithm of concentration and growth rate was practically linear in the given range (Table 1). This correlation seems to apply in a very narrow interval outside the given concentration range, since the growth of cultures exposed to 10 18 M T2 was similar to the control (the difference was not significant statistically), while the m a x i m u m stimulatory concentration was around 10 9 M. Thus only a minimal increase, resp. decrease can be expected over the limits of the critical range. The results of re-exposure at different levels of horm o n e (experiment A, Table 2) were to a certain degree surprising: the length of the time elapsed after pre-
treatment had no significant influence on the growth rate; e.g. the mean clonal cell counts were higher at 2 weeks than at 1 week after the first exposure. O n re-exposure the concentration dependence of h o r m o n e effect proved to be statistically significant, but not unequivocal: at lO 9 M the growth rate increased significantly over that observed at 10- 12 and 10 15 M (P < 0.01: P < 0.05), but did not differ significantly from that observed at 1018 M, which was greater, although not significantly, than the rates observed at 10-12 and 1 0 - i s M. While on first exposure 1 0 - i s M had been practically inactive (to judge from similar growth rate to the control in this and all preceding experimental series), on re-exposure even that low concentration developed a notable stimulatory effect in cultures pretreated at the same level. It follows that a rery low hormone concentration, which does not in itself b!fluence celhdar growth in any way, can also give rise to hormonal imprintin.q. Naturally, as indicated by the results of experiment B, the levels o1 first exposure and re-exposure equally influence cell growth, and higher concentrations develop a greater stimulatory action (Table 3). It should be noted that, although 1 0 - 1 S M T 2 stimulated the growth of the Tetr~thymena to a lesser degree than 10 -9 M, the difference was only significant statistically. As to the role of the length of first exposure, 10-rain treatment with T2 gave already rise to considerable growth stimulation (Table 4). The growth rate tended
Table 2. Growth response of Tetrahymena to re-exposure at different concentrations of T 2 a. Mean growth rates Interval between 1st and 2nd treatment (weeks)
10 ~
1 2 4
20.7 21.4 19.8
Source of variation Time interval Concentration Interaction Error
Level of re-exposure {M) 10 - i s I0 lz 18.6 19.3 19.9
19.6 20.2 19.1
b. Variance table Degrees of Sum of squares freedom Mean square 36.86 64.72 45.3l 1583.1
2 3 6 228
10 lu
18.43 21.57 7.55 6.94
19.7 21.0 19.8
F
P
2.65 3.11 1.09
>0.05 <0.05 >0.05
Development of receptor memory Table 3. Growth response of
359
Tetrahymenato application of different hormone levels for first and second exposure a. Mean growth rates
First exposed at Re-exposed at 10 9 M 10
t8 M
10 - 9 M
10 -~2 M
10 -15 M
10 -~8 M
21.2 19.9
19.7 18.8
19.2 17.6
18.1 16.3
b. Variance table Degrees of Sum of squares freedom Mean square
Source of variation At first exposure At re-exposure Interaction Error
242.32 79.81 5.27 768.40
to increase further with the prolongation of exposure, and showed a practically linear relationship with the length of exposure. The growth values determined one week later were considerably lower, but exhibited a similar linearity. The two regression lines showed a parallelism, i.e. 1-week decrease was identical after different lengths of exposure. Re-exposure for 24 hr 1 week after pretreatment did not further stimulate the growth of cultures originally exposed for short 10-rain, 30-rain periods, but accounted for a statistically significant (P < 0.01), although biologically not notable, increase in the growth rate of those pretreated for 60 rain. Stimu-
3 l 3 152
80.8 79.81 1.76 5.05
F
P
15.9 15.8 0.35
<0.001 < 0.001 > 0.05
lation of growth by re-exposure was considerable in the cultures originally exposed for 4 hr or longer; the mean cell counts were practically identical in subcultures prepared after first exposure for 4, 8 and 24 hr. Two weeks after pretreatment, 24-hr re-exposure to the concentration originally applied had similar effects to re-exposure after 1 week. In cultures treated for 60 min or a shorter time the growth rates were markedly inferior to those observed on first exposure for identical periods, and did not differ significantly from the control. In cultures originally exposed for 4 h r or longer, re-exposure-stimulated growth rates were greater than in the short-term series, but still
Table 4. Influence of the length of exposure to 10-9 M T2 on growth response of the a. Mean growth rates Period of first exposure 10 min
Sampling times Immediately after firstexposure One week after first exposure After 24-hr re-exposure for identical periods At 2 weeks At 2 + 3 weeks At 2 + 3 + 4 weeks
30 min
1 hr
4 hr
17.8 13.3
18.9 14.2
20.8 13.8
2 1 . 1 2 1 . 0 22.3 15.1 16.5 17.9
13.4 12.4 14.2 16.0
14.1 13.2 13.5 17.7
15.8 13.2 16.0 18.8
21.8 19.0 20.0 20.5
b. Variance table 1. Values assessed immediately after treatment Degrees of Source of variation Sum of squares freedom Mean square Regression Deviation from linearity Error
236.5 34.4 494.3
Tetrahymena
1 4 114
236 8.6 4.34
8 hr
21.6 20.0 20.8 20.5
24 hr
21.5 20.0 21.2 20.8
F
P
55 2.0
,~ 0.001 >0.05
F
P
66 1.8
40.001 > 0.05
Equation of regression: y = 20 + 0.8x where y = growth and x = log time.
Source of variation Regression Deviation from linearity Error
2. Values assessed 1 week after treatment Degrees of Sum of squares freedom Mean square 284 31.5 488.8
1 4 114
284 7.9 4.29
Equation of regression: y = 14 + 0.9x where y = growth and x = log time.
360
G. CSABA et ol.
inferior to those observed on first exposure for 4, 8 and 24 hr. Two weeks after pretreatment, 24-hr re-exposure to the concentration originally applied had similar effects to re-exposure after 1 week. In cultures treated for 6 0 m i n or a shorter time, the growth rates were markedly inferior to those observed on first exposure for identical periods, and did not differ significantly from the control. In cultures originally exposed for 4 hr or longer, re-exposure-stimulated growth rates were greater than in the short-term series, but still inferior to those observed on first exposure. Twenty-four hour long re-exposure after 3 weeks was followed by a not significant increase of the growth rate over the 2-week values and over the baseline as well. The growth decrease observed on re-exposure after 4 weeks was negligible in cultures originally exposed for 4, 8 and 24 hr; the maximum growth rates did not differ between the three series exposed for different times. (It should be noted that the cultures originally exposed for 4 hr showed maximum increase in growth rate after re-exposure at 4 weeks.) In cultures originally exposed for 1 hr or shorter periods, re-exposure after 3 and 4 weeks was followed by a considerable increase of the growth rate over that observed on first exposure; the increase showed a parallelism with the length of exposure in all cases except one (the mean growth rate was higher in cultures pretreated for 10 min than in those pretreated for 30 min, but this may have been a chance phenomenon). It follows that, for first exposure, 10-min treatment with the h o r m o n e already accounts for a considerable stimulation of cell growth, which increases further with the prolongation of exposure. At the same time, short-term {10, 30 or 60-min) pretreatments, although causing growth stimulation by direct effect, inhibit the development of such an effect on later prolonged reexposure. The inhibitory influence tends to decrease with progressing time. but seems to be of extraordinarily long duration. Summarizing the information emerging from experiments A, B and C, the conclusion lies close at hand that h o r m o n e concentrations too low (10 ~s M) for direct stimulation of cell growth can nevertheless induce h o r m o n a l imprinting, while short-term exposures sufficient for direct growth stimulation by the h o r m o n e inhibit rather than promote the development of h o r m o n a l imprinting, to judge from suppression of cellular response to later re-exposure{s) in cultures so treated.
Remarkably, at the extremely low concentration of 10 ~8 M, T2 did not in any way influence the growth of the Tetrahymena, but did give rise to hormonal imprinting, as indicated by the increased responsivehess of the cells so treated to re-exposure to the same low level of the hormone. Mention should also be made of the fact thai hmgterm first exposure for 4, 8 and 24 hr accounted m itself for a considerable increase of the growth rate over the control (15.1: 16.5:17.9 opposed to 12.41 for as long as a week, while short-term first exposure for 10, 30 and 6 0 m i n gave rise only to a minor increase thereof ( 13.3 : 14.2:13.8 against 12.4). This supports our earlier hypothesis that the first interaction with the h o r m o n e practically "'resets" the growth rate of the target cells, and the altered mechanism persists over the progeny generations if the process of imprinting is long enough. It follows from the experimental facts that the concentration of the h o r m o n e is less decisive for a succesful imprinting than the length of exposure. Supposing that Koch's (1979) dynamic receptor pattern generation theory is valid, and the cell is "screening" its environment for information carrier molecules through presenting on its surface wlrious configurations of sub-patterns (receptors), it might as well be postulated that the presentation (chance arisalt of an adequate configuration requires a certain time. If that time is not available e.g. at short-term tirst exposures in the present experiment no imprinting can take place. Long-term inhibition of cellular response to re-exposure(s) after non-imprinting short exposures is, however, not explicable by the above theory.
REFERENCES
CSABA G. (1980) Phylogeny and ontogeny of hormone receptors: the selection theory of receptor formation and hormonal imprinting. Biol. Rer. (Cambridge) 551,47 63. CSABA G. (1981) Ontogeny mid Phylogef U, of ftormone Receptors. Karger, Basel, New York. CSABA G. & N/:MEXH G. (1980) Effect of hormones and their precursors on protozoa the selective responsiveness of Tetrahymena. Comp. Bioehem. Physiol. 65B, 387 390. CSABA G., N~MVrH G. & VAR(~HA P. (1982) Development and persistence of receptor "memory" in a unicellular model system. Exp. Cell. Biol. In press. HILL D. L. (1972) The Biochemistry and Phvsiologty ¢!1 Tetrahymena. Academic Press, New York. KOCH A. S., F~:Hf:RJ. & LUKOVITS I. (1979) A simple model of dynamic receptor pattern generation. Biol. Cyhernet, 32, 125 138.