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Short period rhythms of liver glycogen content
Camp. Biochem. Physiof., 1971, Vol. 39A,
SHORT
SHERWIN
PERIOD
MIZELL,
pp. 219 to 226. Pergamon Press. Printed in Great Britain
RHYTHMS OF LIVER GLYCOGEN CONTENT* HERBERT
THOMAS
MOSKOW,
SALLY
A. WILLINGHAM,
WARREN
and
III
Department of Anatomy and Physiology, Indiana University, Bloomington, Indiana; and Department of Anatomy, Medical College of South Carolina, Charleston, South Carolina (Received 7 September 1970)
Abstract-l.
Statistically significant short period rhythms of glycogen content were observed in livers of Rana pip&m. 2. Male animals exhibit rhythms with a period length of S-10 hr. 3. Female animals exhibit rhythms with a period length of 13-15 hr. 4. These rhythms do not appear to be completely synchronized by a controlled lighting environment. INTRODUCTION A RHYTHM
in liver glycogen content was first observed in the rabbit by Forsgren
(1927). A recent review by Sollberger reports rhythms of this type in other species investigated since that date. Light has been demonstrated as being the dominant synchronizer of these rhythms (Sollberger, 1964). Although food was also thought to have an effect, studies utilizing starved animals have shown that these rhythms persist in the absence of food (Sollberger, 1964; Chaikulus, X966; Haus, 1966). We have demonstrated an annual rhythm of liver glycogen content in &zaa pi&ens (Mizell, 1965). I n order to more fully appreciate the seasonal aspects of this rhythm we wished to examine this species for the presence of short period (circadian) rhythms. Therefore, using controlled light as a synchronizer, we undertook to determine the characteristics of the daily rhythm of liver glycogen content in R.pipiens. MATERIALS
AND
METHODS
To insure a uniform population, all animals were obtained from the same geographica location. After arriving in the laboratory the animals were checked for external signs of disease and placed in individual, numbered, opaque plastic containers. Males and females were identified and placed in alternate containers filied with tap water. Seven groups consisting of four males and four females each were placed in a constant temperature room (21& 1’C) and were exposed to a 12 : 12 LD light regime for 14 undisturbed days. After the 14 day acchmation period the animals were sacrificed in groups of eight at 4-hr intervals over a 24-hr period. The frogs were double pithed and the liver was immediately excised for chemical analysis. Glycogen content was determined using the anthrone * Supported by Grant No. GB-671 Foundation.
and Grant No. GB-6937 219
from the National Science
220
SHORT
PERIOD
RHYTHMS
OF LIVER
GLYCOGEN
CONTENT
method (Seifter, 1950). Complete details of this procedure are found in an earlier report (Mizell, 196.5). Statistical tests, analysis of variance and power spectrum analysis (Dixon, 196.5), were performed using a Control Data Corporation 3600/8090 computer. These experiments were repeated, under identical controlled conditions, on l-2 July, 22-23 July and S-6 August of two consecutive years. A total of 336 animals were utilized. RESULTS
Following the per cent deviation, glycogen content in Figs. 1 and 2, stant throughout fashion, glycogen
method of Halberg et al, (1960), all results are expressed as the of glycogen content at a particular time period, from the mean of al1 animals tested during a 24-hr period. The results, as seen show that the concentration of glycogen in frog liver is not cona 24-hr period. Even though all animals were treated in the same content fluctuated throughout each of the test periods. Lwer glycogen content . Run
I
o Run
2 A Run3
-40 -50
I
/
0900
1300
1700
2100
0100
0500
0900
Hour of day FIG. 1. Liver glycogen content of R. pipiens during year one. Biack bar indicates the interval during which the animals were in complete darkness. Each point represents the average of eight animals tested at that time.
The data was also analyzed statistically for a time effect (analysis of variance). Demonstration of significant variation from one time point to the next is essential before any statements can be made on the presence or absence of a rhythm. With the demonstration of a significant variation the data was then tested for an estimate of the period length of any repeatable cycles. The results of these analyses are found in Table 1. When plotted graphically (Fig. 1, Run 1) the first experimental run appeared to have rhythmic characteristics with a peak in glycogen content occurring at 21:O0. However, preliminary statistical analysis of the total population, testing
S.
MIZELL, H.
S. WARREXAND ‘I’. A. W~LI‘INGHAM,III
MOSKOW,
Liver glycogen
content
. RunI o Run2 A Run3
I
-80’
ocioo
1200
221
.
I 1600
2000
2400
A 0400
I 0800
Hour of day
FIG. 2, Liver glycogen content of fz. pipiens during year two. Black bar indicates the interval during which the animals were in complete darkness. Each point represents the average of eight animals tested at that time. TABLE ~-STATISTICAL ANALYSIS OF DATA No. of animals
Group
Year
1,
F ratio Runl*
Run2
Run3
Year
2,
Analysis of variance
Run 1
Run2
Run3
C.t F. M. C. F. M. C. F. M.
55 27 28 54 27 27 55 28 27
C. F. M. C. F. AM. C. F. M.
53 26 27 55 31 24 55 27 28
1.4293 4.0146 2.3003 46472 2.3158 2.5863 13.3680 5.2119 7.5514 9.0055 4.0360 7.4255 1.3310 1~7051 0.3373 2.6040 15301 2.4130
P n.s. 0.01 n.s. 0.01 n.s. O*OS 0.01 0.01 0.01 0.01 0.01 O-01 n.5. n.s. n.s, 0.05 n.s. n.s.
Power spectrum analysis period length (hr)
13-15 8-10 8-10 22-24 22-24 22-24 8-10 13-1s 8-10
S-10
* Run 1, performed on l-2 July; run 2, performed on 22-23 July; run 3, performed on S-6 August. t C. = combined; F. = females; M. = males.
222
SHORT PERIOD RHYTHMS
OF LIVER GLYCOGEN CONTENT
for a time effect (analysis of variance), indicated that there was no statistical significance to this variation (Table 1). When the population was divided on the basis of sex and then tested statistically, the female population showed a significant time effect with a period of 13-15 hr. The results of the analysis of variance using data obtained from male frogs indicated that this fluctuation was not statistically significant. The results of this first experiment indicated that there could be a sexual difference in the frogs’ response to a light stimulus. Experiments of a different nature, utilizing the same animals, suggested that light could synchronize physiological rhythms in this species (Mizeil, 1970). This work had also suggested that there could be a sexual difference in response to a light regime stimulus. Because of this we repeated the glycogen experiment three weeks later under the same controlled conditions. The results of this second experiment are seen in Fig. 1, Run 2 and Table 1. In this case a prominent peak was observed at 13:00 (8 hr prior to the peak observed in the earlier experiment). Smaller peaks were observed at 21:OO(similar to the earlier experiment), 01:OOand 09:OO(8-12 hr after the peak in the earlier experiment). Statistical analysis indicated the total population exhibited a significant variation in liver glycogen content with a period of 8-10 hr. The male group from this population showed a significant variation with a period of 8-10 hr. The females exhibited fluctuations in liver glycogen content which, however, were not statistically significant. Due to the discrepancy in peak times, statistical significance and period lengths of the rhythms observed in the first two experimental runs, this phenomenon was investigated a third time (Fig. 1, Run 3). Jn this case a peak in glycogen content was observed at 17:00 (4 hr after the major peak of the second experiment). This observed variation in glycogen content was highly significant statistically, and the period length was determined as 22-24 hr. Both the female and male segments of the test population also exhibited significant variations with periods of 22-24 hr. These highly unusual results prompted us to perform the same experiments under identical conditions on the same calendar days one year later (Fig. 2). Again it was impossible to superimpose the results of one experiment on another. The results of the fourth experimental run (Fig. 2, Run 1) indicated peaks of glycogen content at 1200, 2O:OOand 0800. Statistical analysis showed a highly significant time effect in the males, females and total population. The total population exhibited a period of 8-10 hr, the females one of 13-15 hr and the males a period which was 8-10 hr in length (Table 1). These results are similar to those observed earlier. When the results of the fifth experiment were graphed (Fig. 2, Run 2) a prominent peak was observed at 24:O0. However, analysis of variance for a time effect indicated that these variations were not statistically significant. The graphical representation of the results obtained during the sixth experimental run (Fig. 2, Run 3) indicated peaks of glycogen content at ZOO, 2O:OO and 04:OO. Examination, using the technique of analysis of variance testing for
S. MIZELL, H. MOSKOW,S. WARRENANDT. A. WILLINGHAM,III
223
a time effect, showed a statistically significant variation for the total group. The period length for this significant variation was eight hours. The male and female segments of this population did not exhibit significant variations. DISCUSSION
The results listed above leave no doubt that statistically significant short period rhythms of liver glycogen content do occur in the frog, R.pi&ens. Those experimental runs which exhibited significant variations in glycogen content from one time point to the next also had rhythms whose periods were similar in length from one run to the next. That we did not observe statistically significant rhythms during every experimental run is probably due to a small sample size accentuating normal biological variation. Four of the experimental runs showed significant rhythms for the total test population. Th ree of these had period lengths of 8-10 hr while the fourth had a period length of 22-24 hr. When the populations are segregated on the basis of sex the period length of the females is 13-15 hr while that of the males is 8-10 hr. This was true of all significant runs with one exception when period length for both sexes was 22-24 hr. This period length could be a harmonic of the other periods. Although these rhythms have been demonstrated, we are still left with two pertinent questions : 1. What is the synchronizer of these rhythms ? 2. Why is there a sexual difference in the observed rhythms ? Light has been most successful as a synchronizer of a wide range of rhythmic processes (Sollberger, 1965). Liver glycogen rhythms in particular have been demonstrated as being affected by light synchronizations. Halberg (1960) has reported the inversion of the rhythm of liver glycogen content in mice by inverting the light regime. After synchronizing the glycogen rhythm to a specific LD light regime, inversion of this light regime caused complete inversion of the glycogen rhythm. Although the light regime used in our experiments was very close to the ambient light conditions for that time of the year, we allowed a 14 day acclimation period before any animals were sacrificed. Even with this precaution we did not observe any synchronization of the liver glycogen rhythms by the controlled light stimulus. One might believe that the frog is in general refractive to a light stimulus. This, however, is not the case. Some of the same frogs subjected to the environmental conditions above were tested for rhythms of mitotic division rate in cornea1 epithelium. In this case the frogs responded as we had expected. The rhythms observed in mitotic division rates were synchronized by a light stimulus (Mizell, 1971), and graphs of results of successive experiments were superimposable. This is also true for DNA synthesis in frog cornea1 epithelium (Morgan & Mizell, 1971) and for DNA content and synthesis in frog epidermis (Morgan & Mizell, 1971). Apparently the rhythms of liver glycogen content that we have observed in the frog are free running and may be synchronized not by a light stimulus but rather by a parameter we have not yet controlled. That this is the case is further indicated
224
SHORT PERIOD RHYTHMS
OF LIVER GLYCOGFN
CONTENT
by the results of a long term experiment. In this experiment liver glycogen content was determined over a 37-day period and indicated an ultradian rhythm of 110 hr. Again a controlled light environment based on a 24-hr pattern did not synchronize this rhythm of glycogen content (Mizell, 1971). What the synchronizer of this rhythm could be is an interesting question. During the acclimation period these animals were not disturbed, were not fed, and were exposed to an environment which consisted of a constant temperature and a 12 : 12 LD light regime. It is entirely possible that the glycogen rhythm in these animals is more responsive to a particular wave length of light or perhaps light of a different intensity than that to which they were exposed. Slight variations in temperature or humidity may have some effect, although we have already demonstrated a seasonal rhythm in the frog which is independent of temperature (Mizell, 1955). Temperature independence is also one of the characteristics of biological rhythms (Biinning, 1964; Sollberger, 1965). Perhaps changes in the neuroendocrine system are more responsible for the rhythm in liver glycogen that we have observed in R. pipiens, than changes in the external environment. Glycogen content in the frog may truly be endogenous with no external correlates. Another problem which presents itself is that of a sexual difference in liver glycogen rhythms. The statistical analysis of this phenomenon was somewhat hampered by the small sample size of each sex. However, there is an indication of a difference in the rhythms of glycogen content of males and females. The female segment of the population appears to exhibit a glycogen rhythm with a longer period than the males (13-15 hr as opposed to 8-10 hr). This result was also obtained in the mitotic division study (Mizell, 1971) and the studies on DNA (Morgan & Mizell, 1971). The rhythm of mitotic division rate of female frogs has a longer period than that of males (24 hr as opposed to 8 hr). The males also have the same period length in both liver glycogen content and mitotic division rate. In the long-term study of liver glycogen rhythms (Mizell, 1971) a sexual However, we have also reported a sexual difference difference was not apparent. in the rhythm of gonadogenesis in frogs (Mizell, 1964) It is quite possible that the different sexes respond differently to the same stimulus. Although apparent in R. pipiens, we have not seen this reported in the literature for other species. Aside from the information obtained on the peculiarities of rhythmic phehomena in R, pipiens, another important point was raised by this data. That is the importance of repeating studies when looking for a time effect. That one appears to have a rhythm when the data of a single experiment is plotted on a That the high and low points on a graph is not necessarily proof of a rhythm. graph are different from one another is also not necessarily proof of a time effect. That an apparent rhythm occurred while a biological system is exposed to a particular light regime and tested for a single 24-hr period is not necessarily proof of synchronization of a rhythm by light. These should be established by 1.
Repeating
the experiments
several
2.
Statistical
tests for time effects.
times,
and
S. MIZELL, H. MOSKOW,S. WARRENAND T. A. WILLINGHAM,III
225
In view of the fact that the frog is so commonly used for laboratory demonstrations and as an experimental animal, it is imperative that we obtain a thorough knowledge of its physiology. As a poikilothermic hibernator, the frog undergoes changes from an active to an inactive stage during each year. During these times of the year, the frog’s physiological responses are different. Not only are there long period seasonal and annual changes in the physiology of this vertebrate but short period rhythms as well. It appears that in addition to this there is a sexual Moreover, not all systems in this difference in response to a controlled stimulus. animal respond to what can be called the classical synchronizer of short period rhythmic phenomena, light. Apparently we have a great deal to learn about the common laboratory animal, R. pipiens. SUMMARY
Male and female R. pipiens were stored in individual containers in a constant unfed and undistemperature, and a 12 : 12 LD light rCgime. After remaining turbed for 14 days in this controlled environment, groups were sacrificed at 4-hr intervals over a 24-hr period. This was performed a total of six times, on the same three calendar periods of two consecutive years. Statistical analysis indicated that significant short period rhythms of glycogen content are measurable in this species. However, a controlled light stimulus did not completely synchronize these rhythms. Furthermore, male and female frogs exhibit statistically significant rhythms of glycogen content which are of different period length. Acknowledgement-The authors wish to thank Mr. John Thomas Tielking for his help in the statistical analysis of the data. REFERENCES B~~NNING E. (1964) The Physiological Clock. Academic Press, New York. CHIAKULAS J. J. & SCHEVINC L. E. (1966) Periodicity in liver glycogen of urodele larvae, Comp. Biochem. Physiol. 17, 87-91. DIXON W. J. (1965) Biomedical Computer Programs, pp. 459-494. Health Sciences computing Facility, University of California, Los Angeles. FORSCRENE. (1927) Mikroskopiska och experimentella leverundersckningar. Dissertation, University of Stockholm, Sweden. HALBERCF., ALBRECHTP. G. & BARNUMC. P. JR. (1960) Phase shifting of liver glycogen rhythm in intact mice, Am.J. Physiol. 199,400-402. HAUS E. & HALBERGF. (1966) Persisting circadian rhythms in hepatic glycogen of mice during inanition and dehydration. Experientia 22, 113. MIZELL S. (1955) Seasonal variation in gastric hydrochloric acid production in Rana pipiens. Am.J. Physiol. 180, 650-654. MIZELL S. (1964) Seasonal differences in spermatogenesis and oogenesis, Nature, Land. 202, 875-876. MIZELL S. (1965) Seasonal changes in energy reserves in the common frog, Rana pipiens. J. Cell Comp. Physiol. 66, 251-258. MIZELL S. & HUTTOA. (1971) Mitotic division rates in cornea1 epithelium. Comp. Biochem. Physiol. 39, 227. MIZELL S., MOSKOWA. & WARRENS. (1971) A long period rhythm of liver glycogen content. Comp. Biochem. Physiol. (In press).
226
SHORTPERIODRHYTHMSOF LIVER GLYCOGENCONTENT
MORGANW. W. & MIZELL S. (1971) Diurnal fluctuation in DNA content and DNA synthesis in the dorsal epidermis of Rana pipiens. Camp. Biochem. Physiol., 39, 591. MORGANW. W. & MIZELL S. (1971) Daily fluctuations in cornea1 DNA synthesis of Rana pipiens. Submitted to journal. SEIFTER S., DAYTON S., NOVIC B. & MUNTWYLERE. (19.50) The estimation of glycogen with the anthrone reagent, Archs Biochem. 25, 191-200. SOLLBERGERA. (1964) The control of circadian glycogen rhythms. Ann. IV. Y. Acad. Sci. 117, 519-554. SOLLBERGERA. (1965) Biological Rhythm Research. Elsevier, New York. Key Word Index-Rhythms in liver glycogen ; rhythms in Rana pipiens; biological rhythms; sexual differences in rhythms; non-synchronization of rhythms by light.
SHORT
SHERWIN
PERIOD
MIZELL,
pp. 219 to 226. Pergamon Press. Printed in Great Britain
RHYTHMS OF LIVER GLYCOGEN CONTENT* HERBERT
THOMAS
MOSKOW,
SALLY
A. WILLINGHAM,
WARREN
and
III
Department of Anatomy and Physiology, Indiana University, Bloomington, Indiana; and Department of Anatomy, Medical College of South Carolina, Charleston, South Carolina (Received 7 September 1970)
Abstract-l.
Statistically significant short period rhythms of glycogen content were observed in livers of Rana pip&m. 2. Male animals exhibit rhythms with a period length of S-10 hr. 3. Female animals exhibit rhythms with a period length of 13-15 hr. 4. These rhythms do not appear to be completely synchronized by a controlled lighting environment. INTRODUCTION A RHYTHM
in liver glycogen content was first observed in the rabbit by Forsgren
(1927). A recent review by Sollberger reports rhythms of this type in other species investigated since that date. Light has been demonstrated as being the dominant synchronizer of these rhythms (Sollberger, 1964). Although food was also thought to have an effect, studies utilizing starved animals have shown that these rhythms persist in the absence of food (Sollberger, 1964; Chaikulus, X966; Haus, 1966). We have demonstrated an annual rhythm of liver glycogen content in &zaa pi&ens (Mizell, 1965). I n order to more fully appreciate the seasonal aspects of this rhythm we wished to examine this species for the presence of short period (circadian) rhythms. Therefore, using controlled light as a synchronizer, we undertook to determine the characteristics of the daily rhythm of liver glycogen content in R.pipiens. MATERIALS
AND
METHODS
To insure a uniform population, all animals were obtained from the same geographica location. After arriving in the laboratory the animals were checked for external signs of disease and placed in individual, numbered, opaque plastic containers. Males and females were identified and placed in alternate containers filied with tap water. Seven groups consisting of four males and four females each were placed in a constant temperature room (21& 1’C) and were exposed to a 12 : 12 LD light regime for 14 undisturbed days. After the 14 day acchmation period the animals were sacrificed in groups of eight at 4-hr intervals over a 24-hr period. The frogs were double pithed and the liver was immediately excised for chemical analysis. Glycogen content was determined using the anthrone * Supported by Grant No. GB-671 Foundation.
and Grant No. GB-6937 219
from the National Science
220
SHORT
PERIOD
RHYTHMS
OF LIVER
GLYCOGEN
CONTENT
method (Seifter, 1950). Complete details of this procedure are found in an earlier report (Mizell, 196.5). Statistical tests, analysis of variance and power spectrum analysis (Dixon, 196.5), were performed using a Control Data Corporation 3600/8090 computer. These experiments were repeated, under identical controlled conditions, on l-2 July, 22-23 July and S-6 August of two consecutive years. A total of 336 animals were utilized. RESULTS
Following the per cent deviation, glycogen content in Figs. 1 and 2, stant throughout fashion, glycogen
method of Halberg et al, (1960), all results are expressed as the of glycogen content at a particular time period, from the mean of al1 animals tested during a 24-hr period. The results, as seen show that the concentration of glycogen in frog liver is not cona 24-hr period. Even though all animals were treated in the same content fluctuated throughout each of the test periods. Lwer glycogen content . Run
I
o Run
2 A Run3
-40 -50
I
/
0900
1300
1700
2100
0100
0500
0900
Hour of day FIG. 1. Liver glycogen content of R. pipiens during year one. Biack bar indicates the interval during which the animals were in complete darkness. Each point represents the average of eight animals tested at that time.
The data was also analyzed statistically for a time effect (analysis of variance). Demonstration of significant variation from one time point to the next is essential before any statements can be made on the presence or absence of a rhythm. With the demonstration of a significant variation the data was then tested for an estimate of the period length of any repeatable cycles. The results of these analyses are found in Table 1. When plotted graphically (Fig. 1, Run 1) the first experimental run appeared to have rhythmic characteristics with a peak in glycogen content occurring at 21:O0. However, preliminary statistical analysis of the total population, testing
S.
MIZELL, H.
S. WARREXAND ‘I’. A. W~LI‘INGHAM,III
MOSKOW,
Liver glycogen
content
. RunI o Run2 A Run3
I
-80’
ocioo
1200
221
.
I 1600
2000
2400
A 0400
I 0800
Hour of day
FIG. 2, Liver glycogen content of fz. pipiens during year two. Black bar indicates the interval during which the animals were in complete darkness. Each point represents the average of eight animals tested at that time. TABLE ~-STATISTICAL ANALYSIS OF DATA No. of animals
Group
Year
1,
F ratio Runl*
Run2
Run3
Year
2,
Analysis of variance
Run 1
Run2
Run3
C.t F. M. C. F. M. C. F. M.
55 27 28 54 27 27 55 28 27
C. F. M. C. F. AM. C. F. M.
53 26 27 55 31 24 55 27 28
1.4293 4.0146 2.3003 46472 2.3158 2.5863 13.3680 5.2119 7.5514 9.0055 4.0360 7.4255 1.3310 1~7051 0.3373 2.6040 15301 2.4130
P n.s. 0.01 n.s. 0.01 n.s. O*OS 0.01 0.01 0.01 0.01 0.01 O-01 n.5. n.s. n.s, 0.05 n.s. n.s.
Power spectrum analysis period length (hr)
13-15 8-10 8-10 22-24 22-24 22-24 8-10 13-1s 8-10
S-10
* Run 1, performed on l-2 July; run 2, performed on 22-23 July; run 3, performed on S-6 August. t C. = combined; F. = females; M. = males.
222
SHORT PERIOD RHYTHMS
OF LIVER GLYCOGEN CONTENT
for a time effect (analysis of variance), indicated that there was no statistical significance to this variation (Table 1). When the population was divided on the basis of sex and then tested statistically, the female population showed a significant time effect with a period of 13-15 hr. The results of the analysis of variance using data obtained from male frogs indicated that this fluctuation was not statistically significant. The results of this first experiment indicated that there could be a sexual difference in the frogs’ response to a light stimulus. Experiments of a different nature, utilizing the same animals, suggested that light could synchronize physiological rhythms in this species (Mizeil, 1970). This work had also suggested that there could be a sexual difference in response to a light regime stimulus. Because of this we repeated the glycogen experiment three weeks later under the same controlled conditions. The results of this second experiment are seen in Fig. 1, Run 2 and Table 1. In this case a prominent peak was observed at 13:00 (8 hr prior to the peak observed in the earlier experiment). Smaller peaks were observed at 21:OO(similar to the earlier experiment), 01:OOand 09:OO(8-12 hr after the peak in the earlier experiment). Statistical analysis indicated the total population exhibited a significant variation in liver glycogen content with a period of 8-10 hr. The male group from this population showed a significant variation with a period of 8-10 hr. The females exhibited fluctuations in liver glycogen content which, however, were not statistically significant. Due to the discrepancy in peak times, statistical significance and period lengths of the rhythms observed in the first two experimental runs, this phenomenon was investigated a third time (Fig. 1, Run 3). Jn this case a peak in glycogen content was observed at 17:00 (4 hr after the major peak of the second experiment). This observed variation in glycogen content was highly significant statistically, and the period length was determined as 22-24 hr. Both the female and male segments of the test population also exhibited significant variations with periods of 22-24 hr. These highly unusual results prompted us to perform the same experiments under identical conditions on the same calendar days one year later (Fig. 2). Again it was impossible to superimpose the results of one experiment on another. The results of the fourth experimental run (Fig. 2, Run 1) indicated peaks of glycogen content at 1200, 2O:OOand 0800. Statistical analysis showed a highly significant time effect in the males, females and total population. The total population exhibited a period of 8-10 hr, the females one of 13-15 hr and the males a period which was 8-10 hr in length (Table 1). These results are similar to those observed earlier. When the results of the fifth experiment were graphed (Fig. 2, Run 2) a prominent peak was observed at 24:O0. However, analysis of variance for a time effect indicated that these variations were not statistically significant. The graphical representation of the results obtained during the sixth experimental run (Fig. 2, Run 3) indicated peaks of glycogen content at ZOO, 2O:OO and 04:OO. Examination, using the technique of analysis of variance testing for
S. MIZELL, H. MOSKOW,S. WARRENANDT. A. WILLINGHAM,III
223
a time effect, showed a statistically significant variation for the total group. The period length for this significant variation was eight hours. The male and female segments of this population did not exhibit significant variations. DISCUSSION
The results listed above leave no doubt that statistically significant short period rhythms of liver glycogen content do occur in the frog, R.pi&ens. Those experimental runs which exhibited significant variations in glycogen content from one time point to the next also had rhythms whose periods were similar in length from one run to the next. That we did not observe statistically significant rhythms during every experimental run is probably due to a small sample size accentuating normal biological variation. Four of the experimental runs showed significant rhythms for the total test population. Th ree of these had period lengths of 8-10 hr while the fourth had a period length of 22-24 hr. When the populations are segregated on the basis of sex the period length of the females is 13-15 hr while that of the males is 8-10 hr. This was true of all significant runs with one exception when period length for both sexes was 22-24 hr. This period length could be a harmonic of the other periods. Although these rhythms have been demonstrated, we are still left with two pertinent questions : 1. What is the synchronizer of these rhythms ? 2. Why is there a sexual difference in the observed rhythms ? Light has been most successful as a synchronizer of a wide range of rhythmic processes (Sollberger, 1965). Liver glycogen rhythms in particular have been demonstrated as being affected by light synchronizations. Halberg (1960) has reported the inversion of the rhythm of liver glycogen content in mice by inverting the light regime. After synchronizing the glycogen rhythm to a specific LD light regime, inversion of this light regime caused complete inversion of the glycogen rhythm. Although the light regime used in our experiments was very close to the ambient light conditions for that time of the year, we allowed a 14 day acclimation period before any animals were sacrificed. Even with this precaution we did not observe any synchronization of the liver glycogen rhythms by the controlled light stimulus. One might believe that the frog is in general refractive to a light stimulus. This, however, is not the case. Some of the same frogs subjected to the environmental conditions above were tested for rhythms of mitotic division rate in cornea1 epithelium. In this case the frogs responded as we had expected. The rhythms observed in mitotic division rates were synchronized by a light stimulus (Mizell, 1971), and graphs of results of successive experiments were superimposable. This is also true for DNA synthesis in frog cornea1 epithelium (Morgan & Mizell, 1971) and for DNA content and synthesis in frog epidermis (Morgan & Mizell, 1971). Apparently the rhythms of liver glycogen content that we have observed in the frog are free running and may be synchronized not by a light stimulus but rather by a parameter we have not yet controlled. That this is the case is further indicated
224
SHORT PERIOD RHYTHMS
OF LIVER GLYCOGFN
CONTENT
by the results of a long term experiment. In this experiment liver glycogen content was determined over a 37-day period and indicated an ultradian rhythm of 110 hr. Again a controlled light environment based on a 24-hr pattern did not synchronize this rhythm of glycogen content (Mizell, 1971). What the synchronizer of this rhythm could be is an interesting question. During the acclimation period these animals were not disturbed, were not fed, and were exposed to an environment which consisted of a constant temperature and a 12 : 12 LD light regime. It is entirely possible that the glycogen rhythm in these animals is more responsive to a particular wave length of light or perhaps light of a different intensity than that to which they were exposed. Slight variations in temperature or humidity may have some effect, although we have already demonstrated a seasonal rhythm in the frog which is independent of temperature (Mizell, 1955). Temperature independence is also one of the characteristics of biological rhythms (Biinning, 1964; Sollberger, 1965). Perhaps changes in the neuroendocrine system are more responsible for the rhythm in liver glycogen that we have observed in R. pipiens, than changes in the external environment. Glycogen content in the frog may truly be endogenous with no external correlates. Another problem which presents itself is that of a sexual difference in liver glycogen rhythms. The statistical analysis of this phenomenon was somewhat hampered by the small sample size of each sex. However, there is an indication of a difference in the rhythms of glycogen content of males and females. The female segment of the population appears to exhibit a glycogen rhythm with a longer period than the males (13-15 hr as opposed to 8-10 hr). This result was also obtained in the mitotic division study (Mizell, 1971) and the studies on DNA (Morgan & Mizell, 1971). The rhythm of mitotic division rate of female frogs has a longer period than that of males (24 hr as opposed to 8 hr). The males also have the same period length in both liver glycogen content and mitotic division rate. In the long-term study of liver glycogen rhythms (Mizell, 1971) a sexual However, we have also reported a sexual difference difference was not apparent. in the rhythm of gonadogenesis in frogs (Mizell, 1964) It is quite possible that the different sexes respond differently to the same stimulus. Although apparent in R. pipiens, we have not seen this reported in the literature for other species. Aside from the information obtained on the peculiarities of rhythmic phehomena in R, pipiens, another important point was raised by this data. That is the importance of repeating studies when looking for a time effect. That one appears to have a rhythm when the data of a single experiment is plotted on a That the high and low points on a graph is not necessarily proof of a rhythm. graph are different from one another is also not necessarily proof of a time effect. That an apparent rhythm occurred while a biological system is exposed to a particular light regime and tested for a single 24-hr period is not necessarily proof of synchronization of a rhythm by light. These should be established by 1.
Repeating
the experiments
several
2.
Statistical
tests for time effects.
times,
and
S. MIZELL, H. MOSKOW,S. WARRENAND T. A. WILLINGHAM,III
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In view of the fact that the frog is so commonly used for laboratory demonstrations and as an experimental animal, it is imperative that we obtain a thorough knowledge of its physiology. As a poikilothermic hibernator, the frog undergoes changes from an active to an inactive stage during each year. During these times of the year, the frog’s physiological responses are different. Not only are there long period seasonal and annual changes in the physiology of this vertebrate but short period rhythms as well. It appears that in addition to this there is a sexual Moreover, not all systems in this difference in response to a controlled stimulus. animal respond to what can be called the classical synchronizer of short period rhythmic phenomena, light. Apparently we have a great deal to learn about the common laboratory animal, R. pipiens. SUMMARY
Male and female R. pipiens were stored in individual containers in a constant unfed and undistemperature, and a 12 : 12 LD light rCgime. After remaining turbed for 14 days in this controlled environment, groups were sacrificed at 4-hr intervals over a 24-hr period. This was performed a total of six times, on the same three calendar periods of two consecutive years. Statistical analysis indicated that significant short period rhythms of glycogen content are measurable in this species. However, a controlled light stimulus did not completely synchronize these rhythms. Furthermore, male and female frogs exhibit statistically significant rhythms of glycogen content which are of different period length. Acknowledgement-The authors wish to thank Mr. John Thomas Tielking for his help in the statistical analysis of the data. REFERENCES B~~NNING E. (1964) The Physiological Clock. Academic Press, New York. CHIAKULAS J. J. & SCHEVINC L. E. (1966) Periodicity in liver glycogen of urodele larvae, Comp. Biochem. Physiol. 17, 87-91. DIXON W. J. (1965) Biomedical Computer Programs, pp. 459-494. Health Sciences computing Facility, University of California, Los Angeles. FORSCRENE. (1927) Mikroskopiska och experimentella leverundersckningar. Dissertation, University of Stockholm, Sweden. HALBERCF., ALBRECHTP. G. & BARNUMC. P. JR. (1960) Phase shifting of liver glycogen rhythm in intact mice, Am.J. Physiol. 199,400-402. HAUS E. & HALBERGF. (1966) Persisting circadian rhythms in hepatic glycogen of mice during inanition and dehydration. Experientia 22, 113. MIZELL S. (1955) Seasonal variation in gastric hydrochloric acid production in Rana pipiens. Am.J. Physiol. 180, 650-654. MIZELL S. (1964) Seasonal differences in spermatogenesis and oogenesis, Nature, Land. 202, 875-876. MIZELL S. (1965) Seasonal changes in energy reserves in the common frog, Rana pipiens. J. Cell Comp. Physiol. 66, 251-258. MIZELL S. & HUTTOA. (1971) Mitotic division rates in cornea1 epithelium. Comp. Biochem. Physiol. 39, 227. MIZELL S., MOSKOWA. & WARRENS. (1971) A long period rhythm of liver glycogen content. Comp. Biochem. Physiol. (In press).
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MORGANW. W. & MIZELL S. (1971) Diurnal fluctuation in DNA content and DNA synthesis in the dorsal epidermis of Rana pipiens. Camp. Biochem. Physiol., 39, 591. MORGANW. W. & MIZELL S. (1971) Daily fluctuations in cornea1 DNA synthesis of Rana pipiens. Submitted to journal. SEIFTER S., DAYTON S., NOVIC B. & MUNTWYLERE. (19.50) The estimation of glycogen with the anthrone reagent, Archs Biochem. 25, 191-200. SOLLBERGERA. (1964) The control of circadian glycogen rhythms. Ann. IV. Y. Acad. Sci. 117, 519-554. SOLLBERGERA. (1965) Biological Rhythm Research. Elsevier, New York. Key Word Index-Rhythms in liver glycogen ; rhythms in Rana pipiens; biological rhythms; sexual differences in rhythms; non-synchronization of rhythms by light.