Effect of neonatal androgenization on the circadian rhythm of feeding behavior in rats

Physiology&Behavior.Vol. 53, pp. 329-335, 1993

0031-9384/93 $6.00 + .00 Copyright© 1993 PergamonPressLtd.

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Effect of Neonatal Androgenization on the Circadian Rhythm of Feeding Behavior in Rats J U A N A. M A D R I D , *1 C L E M E N T E

LOPEZ-BOTEt

AND EUGENIO MARTIN:~

*Department of Physiology and Pharmacology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain, i'Department of Zootechny, Faculty of Veterinary Science, University of Extremadura, Cdceres, Spain, and ~cDepartment of Animal Biology, Faculty of Biology, University of Granada, Granada, Spain Received 29 July 1991 MADRID, J. A., C. LOPEZ-BOTE AND E. MARTfN. Effect of neonatalandrogenizationon the circadianrhythm offeeding behaviorin rats.PHYSIOL BEHAV 53(2) 329-335, 1993.--The effect of neonatal androgenization with testosterone propionate (TP) on growth, food intake, and feeding circadian and ultradian rhythms was studied in male and female rats. TP-female rats but not TP-male rats weighed more and ate more in adulthood than control animals injected with oil. TP administration to male rats induced few changes in the circadian organization of food intake and meal size. However, neonatal androgenization of female rats produced an approach of feeding parameters to those showed by male rats: diminished meal number and increased meal size and food intake. Moreover, the spectral analysis of meal frequency revealed that TP induced in female rats an enlargement in the 4.8 h ultradian component. Although hormone secretions produced in adult animals contribute to sexual dimorphism, it is noticeable that androgen presence during the organizational period is largely responsible for differences between sexes. Sex differentiation Sex behavior Meal pattern Testosterone

Androgenization

Circadian rhythms

IT is a well-known phenomenon that males of many mammalian species are larger than their female counterparts and perform differently in terms of overall growth rate and carcass leanness. These sexual differences are determined mainly by the hormones secreted by the male and female gonads, which are controlled by brain substrate and circuits that respond to endogenous hormones in sexually different dimorphic ways (7-9,26). In assessing sexual differences it is useful to distinguish two separate actions of gonadal hormones. The first is an organizational or inductive effect of the undifferentiated neuroendocrine system of the immature organism, related to those differences displayed in adulthood (13,25). The second is to activate or suppress those metabolic processes in target tissues affecting the physiological responsiveness, especially the growth of organs and tissues (3). Androgens, estrogens, and progestins affect circadian motor activity rhythm in characteristically different ways (3,18). These hormones have been reported to modulate the synchrony, amplitude, phase, and period of adult mammalian circadian rhythms (14,24). In male rodents, testosterone increases the amplitude and shortens the period of the circadian activity rhythm (5). In females, the onset, amplitude, and period of motor activity are systematically altered during the various stages of the 4- to 5-day estrous cycle (l, 15,27). Changing levels of estrogen and progesterone during the estrous cycle appear t Requests for reprints should be addressed to J. A. Madrid.

329

Ultradian rhythms

Critical period

to mediate these day-to-day variations in circadian periodicity. Also, the neuronal structure of the suprachiasmatic nuclei, essential for the generation and entrainment of many physiological and behaviorial variables, appear to be sexually differentiated (8,16). Although several papers have been published showing sex differences in circadian rhythms of motor activity (2,6,29), in corticosterone secretion (4), and in $35 methionine uptake by brain tissue (23), so far the published literature fails to examine the circadian organization of feeding behavior in relation to sexual status. The purpose of the present study was to examine the effect of neonatal androgenization on circadian rhythms of eating patterns in male and female rats. METHOD

Animals The experiments were carried out using equally sized litters of female and male Wistar rats. Two litters of eight female and two litters of eight male were obtained by rearranging the original litters on the day of birth. On this same day, eight females (TP-females) and eight males (TP-males) were subcutaneously injected with 2.5 mg testosterone propionate (Sigma, 1875) in 0.25 ml sesame oil (Sigma, 7131). Eight control females (Cfemales) and eight control males (C-males) were injected with

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only 0.25 ml sesame oil. The cages were placed in the same light-, temperature-, and humidity-controlled r o o m (LD 12: 12, light 0800-2000 h; temperature 22 _+ I°C; relative humidity 60 ___ 10%). Animals were weaned at 23 days of age and kept individually in polycarbonate cages (Makrolom Type 28 X 28 X 14 cm). A

pelleted diet (Letica, Barcelona) and tap water were available ad lib. From 32 to 71 days of age animals were weighed daily at 0900 h, and food consumption was measured over periods of approximately 12 h, immediately after lights on (0800-0900 h) and before lights off (1900-2000 h). Before sacrifice, at 71 days

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ANDROGENIZATION AND FEEDING RHYTHM

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of age, blood samples for testosterone determination were obtained from male animals by means of a retro-orbital sinus puncture at 0800 h and 2000 h. Then, all male animals were killed under ether anaesthesia and the seminal vesicle glands were collected and weighed.

331

Apparatus and Data Analysis Eating activity was recorded using a computerized system. Rats took food pellets from a food hopper provided with a lightweight perspex flap, which had to be pushed up to reach the pellets. The flap was connected to a detector, the output of which was fed into a microcomputer (VIC-20, Commodore) and was cumulatively recorded at 10-min intervals. All further calculations are based on these 10-min totals. For visual inspection and meal frequency calculations, event records were printed at 10min intervals over 48-h scale with each day being repeated (double plot). Feeding events with no interruption for 20 rain were defined as a meal. This criterion are similar to those used by others to discriminate within-meal from between-meal pauses (10,12,20). Total intake in each light or dark period was determined by weighing the amount of food offered and the amount remaining in the cage after 12 h. Meal size was obtained by division of total intake at each period for frequency values. To determine the shape of the circadian functions of the feeding behavior under LD, the meal frequency (based on the time of onset of each meal) was calculated every 30 rain. This value was then averaged across 10 days for each subject to yield circadian functions for frequency of meal occurrence. Group meal parameter rhythms were obtained by averaging individual functions across subjects. These data were analyzed for circadian rhythm detection by a computerized inferential statistical method, the cosinor (17), involving the fit of a 24h cosine curve to averaged data series by the method of least squares. The chi-square periodogram (22) was used to determine the exact period of circadian rhythms in rats exposed to LL conditions. A spectral analysis based in the fit of different cosine curves to the original data was used to compare the power content of the different harmonics. The period of the cosine function that best fitted the data produced the highest amplitude. Thus, the amplitudes of the first 12 harmonics were obtained, and the power content of each harmonic was expressed as a percentage of the total power of the feeding activity function. A two-way ANOVA, with factors being treatment and age, was used to determine differences in daily body weight and food intake between groups. If there were significant effects, individual comparisons were made by means of the Scheffe's test. The overall mean values of the daily food intakes, meal frequencies, meal sizes, cosinor parameters, testosterone levels, and weight of seminal vesicles were statistically processed by one-way ANOVA. Differences between groups in the A.E.I. and spectral analysis were assessed with the nonparametric Mann-Whitney U-test. RESULTS

Testosterone Concentration After blood collection, serum was obtained by letting it clot at room temperature for 45 min. After centrifugation, the serum was frozen and stored at - 2 0 ° C until hormone analysis. Serum testosterone concentration was determined by radioimmunoassay (Serono Diagnostic Kit) after extraction with diethyl ether. The antiserum had a very high specificity for testosterone and relatively low crossreactivity for dihydrotestosterone (22.5%). The sensitivity of the radioimmunoassay was 0.16 ng/ml, and the linear range of the standard curve was 0.2-24 ng/ml. Intraand interassay coefficients of variation were 12.3 and 15.5, respectively.

Figure 1 shows the evolution of body weight in the four groups of animals. Neonatal administration of testosterone to males did not produce significant variations in body weight during the experimental period when compared to C-males, F(1,546) = 1.89; p > 0.1. On the contrary, in female rats both the treatment, F(I, 546) = 232.4; p < 0.0001, age, F(38, 546) = 61.4, p < 0.0001, and treatment × age, F(38, 546) = 2.22, p < 0.01, effects were statistically reliable. Post hoc contrast analysis indicated that the differences between control males and TP-females were not significant until day 46 but became greater from day 47 to day 70. Changes both in food intake and in the alimentary efficiency index (AEI) (100 X g body weight gained/g food consumed) were also observed. Androg-

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enized females showed a higher food intake, from 35 to 71 days of age, than control females, F(1, 112) = 77.5, p < 0.0001 (Fig. 2). Males showed a higher consumption than all the other groups, but no significant differences were detected between C-males and TP-males, F(1, 112) = 3.11, p > 0.05. TP-females also showed higher AEI than C-females (20.21 + 0.48% vs. 17.22 _ 0.39%, p < 0.05). For this variable, no statistically

significant differences between male groups of rats were obtained (24.26 + 0.62% in C-males vs. 23.66 -+ 0.51% in TPmales, p > 0.5).

Circadian Organization Markedly nocturnal rhythms in food intake were evident in all animals. Under conditions of L D alternance only a 15.63

ANDROGENIZATION AND FEEDING RHYTHM

and a 14.12 percent of food were consumed during the light period in C-males and C-females, respectively. Neonatal testosterone administration significantly increased the food ingested during the light phase (Fig. 3). The number of meals in 24 h was higher in C-females than in C-males, considering both the dark and light period. Neonatal testosterone administration caused a nonsignificant increase in the number of meals in males and a significant decrease in females (Fig. 3). Meal size also exhibited a clear sexual differentiation. This parameter was higher in C-males than in C-females. Neonatal testosterone administration decreased both overall (24 h) and nocturnal meal size but increased diurnal meal size in TP-males. On the other hand, diurnal and nocturnal meal size was increased in testosterone-treated females (Fig. 3). The daily pattern of meal frequency was characterized in all animals by a sharp drop of the feeding activity at or around the light onset, followed by a gradual increase during the rest of the light segment (Fig. 4). During darkness, the animals showed hior trimodal feeding patterns with an evening peak in the first or second hour, and a morning peak in the last or next-to-last hour. Cosinor analysis revealed important differences related to sex. C-males displayed a lower amplitude in circadian oscillation (60% respect the mesor in C-males vs. 73.5% in C-females) and a phase advance in circadian rhythm (Table 1). Meal frequency started to increase in C-males slightly earlier than in C-females. This difference was reflected in the phase relations between these groups, as determined by the sine-wave curve-fitting procedure. The acrophase of the meal frequency rhythm occurred at 0000 h in C-males, whereas in C-females it occurred 70 min later. Testosterone treatment induced slightly, but nonsignificant, F(l, 14) = 1.75, p > 0.05, phase advance of 29 min in TP-males and a nonsignificant advance of 6 min in TP-females, F(1, 14) = 0.8, p > 0.1. To determine differences in ultradian rhythms in meal frequency under LD conditions, the spectral analysis was performed. The spectral analysis, involving the first 12 harmonic components, is shown in Fig. 5. Female rats showed a higher amplitude in the 24-h spectral estimates than male rats (p < 0.01). Administration of testosterone propionate at day 1 to female rats elicited a decrease in the amplitude of 8-h spectral estimates (17< 0.05) and a significant increase in the 5th harmonic component (4.8 h) (p < 0.05). This hormonal treatment did not appear to affect the male ultradian rhythms. When female rats were exposed to LL, tau (r) of free-running rhythms for meal frequency ranged from 24.33 to 25.5 h for both groups. The mean free running of androgenized females

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24 12 8 6 4.8 4 3.4 3 2.6 2.4 2.2 2 Period (h) FIG. 5. Spectral analysis of the meal frequency patterns. Each value represents the mean + SEM of the percentage of amplitude for distinct spectral estimates. This parameter is a measure of the extent to which oscillationsof various periods lengths contribute to the total variability of a time series. Stars indicate significantdifference (p < 0.05) with reference to the respective control group. were not statistically different from C-females (1486 min in TPfemales vs. 1481 min in C-females, F(1, 14) = 0.6, p > 0.01. Testosterone concentration showed a marked light-dark difference in the C-male group. Serum levels at the onset of light was 4.78 + 0.725 ng/ml whereas at 2000 h, when lights were turned off, the concentration of this steroid reached 11.46 + 3.790 ng/ml. Neonatal testosterone administration decreased

TABLE 1 COSINORSUMMARYOF THE EFFECTSOF NEONATALANDROGENIZATION ON CIRCADIANRHYTHMIN MEALFREQUENCY Mesor

Amplitude

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A

95% CI

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36.7 26.9 48.4 52.9

0.30 0.28 0.34 0.36

0.25-0.36 0.23-0.34 0.29-0.40 0.30-0.41

0.18 0.15 0.25 0.27

0.11-0.26 0.08-0.23 0.18-0.33 0.19-0.34

0000 0029 0110 0116

2215-0142 2355-0251 2351-0229 0005-0227

PR = percentage rhythm; CI = confidence intervals. Acrophase is referenced to local midnight.

334

M A D R I D , LOPEZ-BOI[-£ A N D MAR IiN

serum testosterone of adult male rats in both lighting conditions, 1.95 _+ 0.604 ng/ml at 0800 h, F(I, 14) - 10.5, p < 0.01, and 2.16 _+ 0.243 ng/ml at 2000 h, F(I, 14) = 9.78, p < 0.01. The mean of the seminal vesicle weights in control males at the time of slaughter { 1.03 +_ 0.127 g) was much higher than in the neonatally treated group (0.30 _+ 0.049 g) F(I, 14) 21.5, p < 0.001. DISCUSSION It has been already reported that neonatal supplementation of androgen to males during the critical period of sexual differentiation produce a decrease of LH production by the hypophysis and lower testicular activity in mice (11). In our present experiment it can be seen that males neonatally treated with testosterone had lower testosterone circulating levels in adulthood and also lower seminal vesicle weight. This general low amount of androgens in adulthood might produce certain patterns of behavior characteristic of females. On the other hand, if the neonatal androgenization occurs in a genetic female, her hormonal status will be modified in such a manner that her estrogen levels will be lower than in the genetic phenotypie females, this leading to an artificial masculinization (androgenization) of the behavior, and other morphological and physiological manifestations (3,19,21,25). So far it has not been clearly demonstrated the process by which sex differences in the circadian system of adult animals take place. They might be derived solely from the genetic structure of the animal, or they might be highly influenced by the developmental process of sexual differentiation which, as already stated, depends on the steroid concentration during the critical period of sexual differentiation (1,29). From our results it can observed that in all the parameters studied (body weight, food intake, AEI, and circadian organization) there are physiological differences between the two control groups. Neonatal administration of testosterone propionate to male rats induced no significant differences in the circadian and ultradian patterns of meal frequency, total food intake, and growth rate. However, food intake and meal size showed a statistically significant decrease in light-dark differences in TP-males vs. Cmales. Also, the amplitude of circadian rhythm in meal frequency was slightly greater in C-males than in TP-males. This result agrees with that reported by Daan et al. (5), according to which, in mice, testosterone induces in adult male animals an increase

in the amplitude of the circadian motor activity rhythm. In this sense, our results show that serum testosterone in TP-males was strongly reduced with respect to C-males. Alternatively, the reduced light-dark differences observed in testosterone levels in the TP-male group could explain the decrease obtained in the amplitude of circadian rhythm in feeding behavior variables. The effects of neonatal androgenization in female rats arc more dramatic, in this sex, testosterone treatment increases food intake: however, this parameter did not reach the level of Cmales. The effect of treatment on body weight gain was even more marked than the effect on food intake; consequently, TPfemales had a significantly higher AEI than C-females. The above-mentioned effects were accompanied by significant changes in the circadian organization of feeding behavior; thus, neonatal androgenization of females showed four significant effects on the circadian feeding rhythm in LD: I. 2. 3. 4.

a differential increase in light and dark food intake, a reduction in the meal number during light and 24 h, an increase in light, dark, and 24-h meal size, an increase in the fifth spectral harmonic (4.8 h) and a reduction in the third harmonic (8 h).

In addition, control female rats difli~r from control male rats in all the variables characterizing feeding behavior. In general, neonatal androgenization of female rats causes feeding parameters to approach those showed by male rats, i.e., decreases meal number, and increases meal size, and food intake. However, neonatal androgenization also elicits some specific effects, the most significant is the increase in the fifth harmonic component, with a 4.8-h period, that occurs in TP-females. This effect is similar to the increase in the ultradian activity reported by Wollink et al. (28) in laborator3' LEW/Ztn female rats. In these rats, perinatal treatment with testosterone propionate resulted in a significant increase of the 4.8-h and 4-h spectral stimates with respect to control females. The results of our study demonstrate that neonatal injection of testosterone propionate can permanently alter the neonatal pattern of growth and feeding behavior in female rats. Although hormone secretions produced in adult animals contribute undoubtedly to sexual dimorphism, it is noticeable that androgen presence during the organizational period is largely responsible for differences between sexes, not only in body weight and food intake, but also in the circadian and ultradian feeding rhythm behavior.

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ANDROGENIZATION AND FEEDING RHYTHM

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