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The nature of the metabolic signal that triggers onset of puberty in female rats
Physiology & Behavior 68 (2000) 377–382
The nature of the metabolic signal that triggers onset of puberty in female rats Neal A. Messera, Helen I’Ansonb,* a Brown Science Center, Transylvania University, Lexington, KY, USA Department of Biology, Washington and Lee University, Lexington, VA 24450, USA Received 18 February 1999; received in revised form 31 August 1999; accepted 21 September 1999 b
Abstract These experiments examined the effects of different refeeding stimuli on reproductive activity as measured by the onset of first estrus in prepubertal, food-restricted female rats. Such rats were placed on a restricted food intake until day 50 of age to maintain a weight of 80–90 g, and to suppress onset of puberty (normal time of puberty: 37 ⫾ 1.4 days of age). In Experiment 1, rats were refed at different daily caloric intakes from day 50 through day 62. First estrus was observed in all rats, with highest caloric intake after 5.7 ⫾ 0.6 days of refeeding. Progressively fewer rats achieved first estrus, and the time to first estrus increased as the caloric intake per day decreased. These results suggest that the highest caloric intake most closely resembles an ad lib diet in such realimented rats. The second experiment determined the duration of an ad lib food stimulus needed to initiate first estrus. Similarly growth-restricted rats were refed (on Day 50 of age) ad lib meals of 67.2 ⫾ 0.1 kcal, presented for periods of 12, 24, 48, or 72 h. The majority of rats (6 of 7) achieved first estrus when the ad lib meals were presented for 72 h, but progressively fewer rats achieved first estrus when such meals were presented for less time. These data indicate that an extended ad lib food stimulus (72 h) is necessary to cause onset of cycling in the majority of food-restricted, prepubertal female rats. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Development; Growth-restriction; Puberty; Rat; Metabolism; Food intake
1. Introduction It is well known that a link exists between food intake and reproductive function in female mammals [1–5]. When there is a scarcity of available food resources, anestrus and the cessation of estrous-related behavior often occur. Also, puberty (as determined by the onset of estrous cycling and reproductive fertility) will be delayed in females on a restricted food intake. It is believed that this occurs as a method of preserving valuable energy resources. Reproduction in females is an extremely energetically costly endeavor, as it includes estrous cycling, pregnancy, and lactation. When available energy is low, it is funneled into processes essential for survival such as thermoregulation and cellular maintenance. Drains on energy supply such as cold temperatures [6], exercise [7–9], insulin treatment [10], and food-deprivation [11–14] have all been shown to induce anestrus and delay puberty. The metabolic signal that suppresses reproduction ultimately has its effect by inhibiting the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus [11, * Corresponding author. Tel.: 540-463-8974; Fax: 540-463-8012. E-mail address: [email protected]
15–17]. Inhibition of GnRH secretion in turn decreases luteinizing hormone (LH) secretion and prevents the preovulatory LH surge [18, 19]. The combination of these hormonal effects may inhibit follicular development, ovulation, and estrous cycling. Currently, the exact mechanism used to signal these changes is unknown, although recent evidence points to a glucose sensor in the brain that determines the level of usable energy in the body and switches off reproduction when this level is too low [20–23]. Excess food will rapidly signal metabolic changes in food-restricted, female mammals [11, 16]. Food-restricted, prepubertal female rats resume LH pulsing in as little as 3–4 h after being given access to ad lib food [24]. In addition, they gain considerable body weight in a 24-h period [16]. However, it remains unclear as to the amount of time this excess food stimulus must be present to trigger the onset of puberty and reproductive fertility. We addressed this question in a series of two studies. The first study examined the amount of calories necessary to signal onset of puberty, as determined by first estrus, in growth-restricted, prepubertal female rats with delayed puberty. The second study examined the duration of an ad lib food stimulus needed to induce onset of puberty in such rats. Through these experiments, we hope to develop a model that can be used to study
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the signals that reinstate estrous cycling in nutritionally delayed prepubertal rats. 2. Materials and methods 2.1. General Female Long–Evans rats were purchased from Charles River Breeding Laboratories, Wilmington, MA, and received at 22 days of age. These rats were housed individually at a temperature of approximately 23⬚ C and kept on a 14 L:10 D-h light–dark cycle (lights on at 700 h). They were fed Purina Rat Chow Formula #5008 (4.15 kcal/g) ad lib until they reached 80–90 g. Upon reaching this weight, they received a restricted food intake of approximately 34% of an ad lib diet to maintain a body weight of 80–90 g. While maintained at this weight, all rats remained acyclic despite being beyond the age of normal pubertal onset (37.0 ⫾ 1.4 days [2]). Rats were weighed three times per week. The Formula #5008 Chow is a high energy and high protein diet designed to induce higher reproductive performance. Its composition includes no less than 23% protein, 6.5% fat, and no more than 4% fiber, 8% ash, and 2.5% added minerals. Experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals, and were approved by Transylvania University animal care and use review board. Estrous cyclicity was determined by vaginal smears taken daily throughout the length of the experiment. This method of determining estrous cyclicity has previously been validated in the growth-restricted prepubertal female rat with delayed puberty [16]. Indeed, when such female rats were realimented and subsequently mated on the night of first ovulation, they became pregnant and produced normal-sized litters of normal-sized pups [16]. 2.2. Experimental design 2.2.1. Experiment 1 At 50 days of age, 28 growth-restricted female rats with delayed puberty were divided into four groups so that an equal number of each litter were represented in each experimental group. The groups received the following daily food intake from days 50–62 of age: group 1 ⫽ x ⫹ 0.4 g; group 2 ⫽ x ⫹ 1.2 g; group 3 ⫽ x ⫹ 3.6 g; group 4 ⫽ x ⫹ 10.8 g (where x ⫽ mean daily food intake of individual rat over previous period of food restriction). Daily weights and vaginal smears were taken from Days 50–62 of the experiment to monitor weight gain and cyclicity, respectively. 2.2.2. Experiment 2 At 50 days of age, 28 growth-restricted female rats with delayed puberty were divided into four evenly sized groups in a manner similar to that used in Experiment 1. The four groups were given ad lib access to food (x ⫹ 10.8 g) for periods of 12, 24, 48, and 72 h, respectively. After the period of ad lib feeding, cases were changed to eliminate any remaining excess food, and food intake was dropped to the
mean level of food-restricted daily intake for a period of 3 days. At the end of these 3 days, food intake was manipulated as done previously to stabilize body weights. Again, daily weights and vaginal smears were taken from Days 50– 59 of the experiment to monitor weight gain and cyclicity, respectively. 2.3. Data analyses In each experiment, the number of animals in each group which achieved first estrus was compared using a modification of the Fisher exact test for comparing more than two proportions [25]. Following the exact test, a Tukey-type multiple comparisons test was used to determine which groups were different [25]. Body weights and food intake were compared using analysis of variance (ANOVA, Statview 512⫹, Brainpower, Las Calabas, CA), and the Scheffe test was used in any post hoc comparisons made among means. Growth rates during the refeeding periods were compared using regression analysis (GraphPad version 2.0, San Diego, CA). Linear regression analyses of caloric intake per day versus days to reach first estrus and body weight versus days to reach first estrus were performed post hoc (Statview 512⫹, Brainpower, Las Calabas, CA). In all analyses, p ⬍ 0.05 was considered significant. All data is presented as mean ⫾ standard error of the mean. 3. Results 3.1. Experiment 1 From Day 30 to Day 50 of age, all rats were maintained at a stable body weight of 82.2 ⫾ 0.2 g. There was no significant weight difference among groups at this time. There was also no significant difference in the mean food intake of the different groups before the experimental period. Average food intake before the experimental period was approximately 5.6 ⫾ 0.0 g (23.2 ⫾ 0.1 kcal) per day. On Day 50, daily food intake was raised by 0.4 g, 1.2 g, 3.6 g, and 10.8 g for groups 1–4, respectively. From Day 50 to Day 62, the growth rate of rats was dependent on caloric intake (Fig. 1). In group 1 (24.8 ⫾ 0.2 kcal/day), mean weight of rats increased by 10.7 ⫾ 0.7 g, while in group 2 (28.2 ⫾ 0.5 kcal/day), mean weight increased by 16.3 ⫾ 1.5 g. These increases in body weight were not significantly different from each other. In contrast, the increase in mean body weight observed in group 3 (38.0 ⫾ 0.3 kcal/day: 38.3 ⫾ 1.2 g), and in group 4 (68.1 ⫾ 0.3 kcal/day: 84.0 ⫾ 0.5 g) were different from each other and from the increases observed in groups 1 and 2. The amount of calories provided during this realimentation period determined when and if rats would cycle (Fig. 2). The number of rats, which cycled in response to the increase in caloric intake, was significantly fewer in groups 1 and 2 compared to groups 3 and 4. While the number of animals that cycled in response to the increase in caloric intake was not different between groups 3 and 4 (6 of 7 rats
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Fig. 1. Mean (⫾SEM) body weight of female rats during Experiment 1 (n ⫽ 7 rats per group). The vertical line at Day 50 depicts the onset of increased food intake. Group 1: 24.8 ⫹ 0.2 kcal/day; group 2: 28.2 ⫹ 0.5 kcal/day; group 3: 38.0 ⫹ 0.3 kcal/day; group 4: 68.1 ⫹ 0.3 kcal/day.
versus 7 of 7 rats cycled, respectively), the time required to respond to the smaller increase in caloric intake received by group 3 was longer by 3–4 days compared to the response time of rats in group 4 on the higher caloric intake. Day of first estrus appeared to be more highly correlated with caloric intake per day (Fig. 3, lower panel; p ⫽ 0.0002; r2 value of 0.7) than body weight at first estrus (Fig. 3, upper panel; p ⫽ 0.02; r2 value of 0.4).
Fig. 3. Linear regression analysis of body weight and caloric intake with day of first estrus in all rats that cycled regardless of group in Experiment 1 (15 of 28 rats). Upper panel: body weight relative to day of first estrus. Lower panel: caloric intake per day relative to day of first estrus. The solid line depicts the regression line in both panels. The number next to the circle (open or closed) represents overlapping data points.
3.2. Experiment 2 From Day 30 to Day 50 of age, all rats were maintained at a stable body weight of 86.2 ⫾ 0.2 g. There was no significant weight difference between groups at this time.
Fig. 2. Mean (⫾SEM) time in days from onset of refeeding until first estrus and percentage of rats cycling relative to the size of the refeed meal (kcal/ day) in Experiment 1.
Again, there was no significant difference in the mean food intake of the different groups before the experimental period. Average daily food intake before the experiment was approximately 5.4 ⫾ 0.0 g (22.5 ⫾ 0.1 kcal). On Day 50 of age, rats were given an ad lib meal of approximately 16.2 ⫾ 0.0 g (67.2 ⫾ 0.2 kcal) per day. During the realimentation period, weight gain was proportional to the amount of time spent on an ad lib diet (Fig. 4). Group 4 (72-h ad lib feeding) showed the most substantial weight gain (30.99 ⫾ 1.67g), whereas group 1 (12 h ad lib feeding) showed the least weight gain (14.17 ⫾ 1.09 g). Group 2 (24 h ad lib feeding) and 3 (48 h ad lib feeding) showed intermediate weight gains (18.76 ⫾ 1.29 g and 24.91 ⫾ 1.88 g, respectively). The duration of the ad lib food stimulus determined whether or not they began to cycle (Fig. 5). The number of rats, which cycled in response to the increase in caloric intake, was significantly fewer in groups 1, 2, and 3 compared to group 4. Thus, an ad lib food stimulus of 72 h was required for the majority of rats to achieve first estrus (6 of 7 rats). It was also of interest to note that the time to first estrus was between 3 to 4 days after onset of refeeding in all
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Fig. 4. Mean (⫾SEM) body weight of female rats during Experiment 2 (n ⫽ 7 rats per group). The vertical line at Day 50 depicts the onset of increased food intake. Group 1: 12 h; group 2: 24 h; group 3: 48 h; group 4: 72 h of ad lib food.
rats that cycled regardless of the duration of the ad lib food stimulus (i.e., regardless of group). 4. Discussion The studies reported herein determined if onset of puberty is affected by the size and duration of the realimentation stimulus after puberty was delayed by food restriction. Our results from Experiment 1 show that level of caloric intake has a profound effect on both the number of rats that achieve first estrus and the length of time before cycling begins in such prepubertal female rats. The highest refeed signal at realimentation (68.1 ⫾ 0.3 kcal/day) produced the
Fig. 5. Number and percentage of rats that achieved first estrus relative to the time allowed of ad lib feeding in Experiment 2.
most rapid onset of cycling in all rats, while the lowest refeed signal (24.8 ⫾ 0.2 kcal/day) did not induce onset of cycles. In addition, there was a graded response in terms of the number of rats cycling and the time to onset of cycles in between these two extremes. These findings suggest that the level of increase of food intake appears to determine the amount of time the diet must be sustained to build up sufficient energy reserves to fuel cyclicity and reproduction. Ad lib refeeding is known to increase circulating levels of gonadotropins and trigger onset of puberty and cycling in growth-restricted animals [11, 16]. However, the effect of a lesser refeed signal on pubertal development had not yet been investigated. Because the number of animals in which cycling began was dependent on the size of the refeed meal in Experiment 1, it is possible that levels of gonadotropin secretion after refeeding also increase in a meal size-dependent manner. This phenomenon has been demonstrated in adult male rhesus monkeys in which gonadotropin secretion increased progressively with increasing refeed meal size after a brief period of fasting, indicating that metabolic signals to the GnRH neurosecretory system may be continuous rather than discrete in nature [26]. Thus, in the intact female, GnRH, and therefore LH, release may gradually increase as meal size increases, driving follicular development at a progressively faster pace, and thus, initiating onset of cycling earlier. This possibility remains to be determined. That caloric intake appears to be the crucial signal to the reproductive axis has previously been well established in a variety of models, including the growth-restricted prepubertal female with delayed puberty [18, 27, 28]. In the restricted prepubertal rat and lamb or adult male monkey, an increase in caloric intake only following a period of food restriction causes an increase in gonadotropin secretion regardless of the source of calories. These data also imply that reduced micronutrient and/or protein intake do not limit reproductive function in such food restriction models. In wild mammalian populations, regulation of hormone secretion based on available energy has the effect of linking reproduction to the availability and attainability of food. During periods when food is scarce, the energetically costly female reproductive system is turned off. Available energy resources are channeled towards survival rather than propagation. When food supplies are more plentiful, cycling and reproduction resume and the offspring have a better chance of survival due to better feeding and climactic conditions. This is particularly true in small mammals, whose large surface to volume ratios and minimal fat stores make the demands of reproduction particularly costly [1]. In Whitefooted mice, reproductive activity has been shown to be a better indicator of recent food availability than even body weight and fat content [16]. The duration of an ad lib food stimulus is also critical in determining onset of cycling in growth-restricted, prepubertal female rats. Previous studies have shown that ad lib food stimuli can increase circulating levels of gonadotropin hormones in as little as 3–4 h [24]. In addition, substantial
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weight gain and fat accumulation also occur during the first 24 h after resumption of ad lib feeding (current study; [16]). However, our results show that even an ad lib food stimulus of 48 h duration only initiated cycling in three of seven rats. An extended stimulus of 72 h of ad lib feeding duration was necessary to cause the majority of rats (six of seven) to begin cycling. These data seem to indicate that onset of estrous cycling in growth-restricted, prepubertal female rats requires an extended signal (72 h) of ad lib feeding even though circulating levels of gonadotropins can respond quite quickly (3–4 h) to the same stimulus [24]. The reason for this time lag between resumption of normal hormone secretion and onset of cycling may have its source at the ovarian level. It has been known for some time that degeneration of the ovaries occurs under food-restricted conditions (rats: [29, 30]; hamsters: [31]; guinea pigs: [32]). Larger follicles become atretic and disappear while smaller follicles cease growth. However, no permanent damage to the reproductive system occurs and refeeding will stimulate development of the plentiful smaller follicles and eventually ovulation [29, 31]. Taken together, these data suggest that follicular development is suppressed in food-restricted or food-deprived animals, and that at least 3 days of ovarian stimulation may be required to induce development of follicles to a level where ovulation is possible. Even though many studies have implicated glucose to be the proximate signal in dietary regulation of estrous cycling [20, 33–35], the ultimate signal is associated with the availability of oxidizable metabolic fuels from a variety of sources. Alternate forms of energy, such as fatty acids and proteins, might increase the availability of glucose for use/ detection in the central nervous system, thus indirectly regulating cyclicity. In fattened Syrian hamsters, estrous cyclicity is not depressed by limited food deprivation unless the hamsters also receive doses of MP, an inhibitor of fatty acid oxidation. Similarly, lower doses of MP and 2-DG which have no effect when administered separately, depress cyclicity [36] and estrous behavior [37] when administered together. The results of our experiments lend support to the idea of the ultimate regulatory effects of net caloric intake. Onset of cycling was observed more frequently and more rapidly in those rats receiving higher levels of daily caloric intake. Because we did not measure body fat content, our results do not rule out the possibility that a critical level of body fat is necessary for puberty in rats. However, other studies have shown that rats can reach puberty at vastly different percent body fat contents [38, 39]. Indeed, exogenous administration of GnRH pulses to growth-restricted prepubertal rats will induce estrous cycles with no change in food intake, and by implication, body fat content [16]. Our model will prove useful for testing the metabolic versus adipostatic hypotheses in future studies in which whole body and neuoendocrine parameters can be measured at discrete time intervals during the now well-defined ad lib feeding period. In summary, onset of cycling and the length of time until
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cycling begins in refed female rats varies in a graded manner with daily food intake. Higher levels of food intake lead to higher and quicker incidences of cycling whereas lower levels lead to delayed cycling or none at all. Also, an extended ad lib food stimulus of 72 h is necessary to cause onset of estrous cyclicity in food-restricted, prepubertal, female rats.
Acknowledgments This work was supported by grants from NIH (HD-07433) to HI and CUR undergraduate student summer research fellowship to N.A.M. A preliminary report of the investigations has appeared in the Program of the 29th Annual Meeting of the Society for the Study of Reproduction, London, Ontario (Abstract 489).
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N.A. Messer and H. I’Anson / Physiology & Behavior 68 (2000) 377–382 effects of estrous cycles and metabolic fuels. Endocrinology 1992; 131:2222–8. Bronson FH. Food-restricted, prepubertal female rats; rapid recovery of luteinizing hormone pulsing with excess food, and full recovery of pubertal development with gonadotropin-releasing hormone. Endocrinology 1986;118:2483–7. Li HY, Wade GN, Blaustein JD. Manipulations of metabolic fuel availability alter estrous behavior and neural estrogen-receptors immunoreactivity in Syrian Hamsters. Endocrinology 1994;135:240–7. Foster DL, Ebling FJP, Vannerson LA, Suttie JM, Landefeld TD, Padmanabhan V, Micka AF, Bucholtz DC, Wood RI, Fenner DE. Toward an understanding of interfaces between nutrition and reproduction: the growth-restricted lamb as a model. In: Pirke KM, Wuttke W, Schweiger U, editors. The Menstrual Cycle and Its Disorders: Influences of Nutrition, Exercise, and Neurotransmitters. Heidelberg: Springer Verlag; 1989. pp. 50–65. Schillo KK. Effects of dietary energy on control of luteinizing hormone secretion in cattle and sheep. J Anim Sci 1992;70:1271–82. Bucholtz DC, Vidwans, NM, Herbosa CG, Schillo KK, Foster DL. Metabolic interfaces between growth and reproduction. V. LH secretion is acutely sensitive to glucose availability. Endocrinology 1996;137:601–7. Murahashi K, Bucholtz DC, Nagatani S, Tsukamura H, Foster DL, Maeda K-I. Suppression of luteinizing hormone pulses by restriction of glucose availability is mediated by sensors in the brain stem. Endocrinology 1996;137:1171–6. Schneider JE, Goldman MD, Leo NA, Rosen ME. Central vs. peripheral metabolic control of estrous cycles in Syrian hamsters. II. Glucoprivation. Am J Physiol 1997;272:R406–12. Schneider JE, Hall AJ, Wade GN. Central vs. peripheral metabolic control of estrous cycles in Syrian hamsters. I. Lipoprivation. Am J Physiol 1997;272:R400–5. Bronson FH, Heideman PD. Short-term hormonal responses to food intake in prepubertal female rats. Am J Physiol 1990;259:R25–31. Zar JH. Biostatistical Analysis. Englewood Cliffs, NJ: Prentice-Hall, Inc., 1984. pp. 400–3. Parfitt DB, Church KR, Cameron JL. Restoration of pulsatile luteinizing hormone secretion after fasting in rhesus monkeys (Macaca mulatta): dependence on size of the refeed meal. Endocrinology 1991; 129:749–56.
[27] Bronson FH, Manning J. Food consumption, prolonged exercise, and LH secretion in the peripubertal female rat. In: Pirke KM, Wuttke W, Schweiger U, editors. The Menstrual Cycle and Its Disorders: Influences of Nutrition, Exercise, and Neurotransmitters. Heidelberg: Springer Verlag, 1989; pp. 43–49. [28] Cameron JL, Influence of nutrition on the hypothalamic–pituitary– gonadal axis in primates. In: Pirke KM, Wuttke W, Schweiger U, editors. The Menstrual Cycle and Its Disorders: Influences of Nutrition, Exercise, and Neurotransmitters. Heidelberg: Springer Verlag, 1989; pp. 66–78. [29] Marrian MH, Parkes AS. The effect of anterior pituitary preparations administered during dietary anoestrus. Proc R Soc 1930;105: 248–58. [30] Mulinos MG, Pomerantz J. Pseudohypophysectomy—a condition resembling hypophysectomy produced by malnutrition. J Nutr 1940;19: 493–504. [31] Printz RH, Greenwald GS. Effects of starvation on follicular development in the cyclic hamster. Endocrinology 1970;86:290–5. [32] Loeb, L. Effect of undernourishment on mammalian ovary and the sexual cycle. JAMA 1921;77:1646–8. [33] Nagatani S, Bucholtz DC, Murahashi K, Estacio MAC, Tsukamura H, Foster DL, Maeda K-I. Reduction of glucose availability suppresses pulsatile luteinizing hormone release in female and male rats. Endocrinology 1996;137:1166–70. [34] Schneider JE, Friedenson DG, Hall AJ, Wade GN. Glucoprivation induces anestrus and lipoprivation may induce hibernation in Syrian Hamsters. Am J Physiol 1993;264:R573–7. [35] Schneider JE, Zhu Y. Caudal brain stem plays a role in metabolic control of estrous cycles in Syrian hamsters. Brain Res 1994;661:70–4. [36] Schneider JE, Wade GN. Availability of metabolic fuels controls estrous cyclicity of Syrian hamsters. Science 1989;244:1326–8. [37] Dickerman RW, Li HY, Wade GN. Decreased availability of metabolic fuels suppresses estrous behavior in Syrian hamsters. Am J Physiol 1993;264:R568–72. [38] Bronson FH. Puberty in female rats: relative effects of exercise and food restriction. Am J Physiol 1987;252:R140–4. [39] Glass AR, Dahms WT, Swerdloff RS. Body fat and puberty in rats: alteration by changes in diet. Ped Res 1979;13:7–9.
The nature of the metabolic signal that triggers onset of puberty in female rats Neal A. Messera, Helen I’Ansonb,* a Brown Science Center, Transylvania University, Lexington, KY, USA Department of Biology, Washington and Lee University, Lexington, VA 24450, USA Received 18 February 1999; received in revised form 31 August 1999; accepted 21 September 1999 b
Abstract These experiments examined the effects of different refeeding stimuli on reproductive activity as measured by the onset of first estrus in prepubertal, food-restricted female rats. Such rats were placed on a restricted food intake until day 50 of age to maintain a weight of 80–90 g, and to suppress onset of puberty (normal time of puberty: 37 ⫾ 1.4 days of age). In Experiment 1, rats were refed at different daily caloric intakes from day 50 through day 62. First estrus was observed in all rats, with highest caloric intake after 5.7 ⫾ 0.6 days of refeeding. Progressively fewer rats achieved first estrus, and the time to first estrus increased as the caloric intake per day decreased. These results suggest that the highest caloric intake most closely resembles an ad lib diet in such realimented rats. The second experiment determined the duration of an ad lib food stimulus needed to initiate first estrus. Similarly growth-restricted rats were refed (on Day 50 of age) ad lib meals of 67.2 ⫾ 0.1 kcal, presented for periods of 12, 24, 48, or 72 h. The majority of rats (6 of 7) achieved first estrus when the ad lib meals were presented for 72 h, but progressively fewer rats achieved first estrus when such meals were presented for less time. These data indicate that an extended ad lib food stimulus (72 h) is necessary to cause onset of cycling in the majority of food-restricted, prepubertal female rats. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Development; Growth-restriction; Puberty; Rat; Metabolism; Food intake
1. Introduction It is well known that a link exists between food intake and reproductive function in female mammals [1–5]. When there is a scarcity of available food resources, anestrus and the cessation of estrous-related behavior often occur. Also, puberty (as determined by the onset of estrous cycling and reproductive fertility) will be delayed in females on a restricted food intake. It is believed that this occurs as a method of preserving valuable energy resources. Reproduction in females is an extremely energetically costly endeavor, as it includes estrous cycling, pregnancy, and lactation. When available energy is low, it is funneled into processes essential for survival such as thermoregulation and cellular maintenance. Drains on energy supply such as cold temperatures [6], exercise [7–9], insulin treatment [10], and food-deprivation [11–14] have all been shown to induce anestrus and delay puberty. The metabolic signal that suppresses reproduction ultimately has its effect by inhibiting the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus [11, * Corresponding author. Tel.: 540-463-8974; Fax: 540-463-8012. E-mail address: [email protected]
15–17]. Inhibition of GnRH secretion in turn decreases luteinizing hormone (LH) secretion and prevents the preovulatory LH surge [18, 19]. The combination of these hormonal effects may inhibit follicular development, ovulation, and estrous cycling. Currently, the exact mechanism used to signal these changes is unknown, although recent evidence points to a glucose sensor in the brain that determines the level of usable energy in the body and switches off reproduction when this level is too low [20–23]. Excess food will rapidly signal metabolic changes in food-restricted, female mammals [11, 16]. Food-restricted, prepubertal female rats resume LH pulsing in as little as 3–4 h after being given access to ad lib food [24]. In addition, they gain considerable body weight in a 24-h period [16]. However, it remains unclear as to the amount of time this excess food stimulus must be present to trigger the onset of puberty and reproductive fertility. We addressed this question in a series of two studies. The first study examined the amount of calories necessary to signal onset of puberty, as determined by first estrus, in growth-restricted, prepubertal female rats with delayed puberty. The second study examined the duration of an ad lib food stimulus needed to induce onset of puberty in such rats. Through these experiments, we hope to develop a model that can be used to study
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the signals that reinstate estrous cycling in nutritionally delayed prepubertal rats. 2. Materials and methods 2.1. General Female Long–Evans rats were purchased from Charles River Breeding Laboratories, Wilmington, MA, and received at 22 days of age. These rats were housed individually at a temperature of approximately 23⬚ C and kept on a 14 L:10 D-h light–dark cycle (lights on at 700 h). They were fed Purina Rat Chow Formula #5008 (4.15 kcal/g) ad lib until they reached 80–90 g. Upon reaching this weight, they received a restricted food intake of approximately 34% of an ad lib diet to maintain a body weight of 80–90 g. While maintained at this weight, all rats remained acyclic despite being beyond the age of normal pubertal onset (37.0 ⫾ 1.4 days [2]). Rats were weighed three times per week. The Formula #5008 Chow is a high energy and high protein diet designed to induce higher reproductive performance. Its composition includes no less than 23% protein, 6.5% fat, and no more than 4% fiber, 8% ash, and 2.5% added minerals. Experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals, and were approved by Transylvania University animal care and use review board. Estrous cyclicity was determined by vaginal smears taken daily throughout the length of the experiment. This method of determining estrous cyclicity has previously been validated in the growth-restricted prepubertal female rat with delayed puberty [16]. Indeed, when such female rats were realimented and subsequently mated on the night of first ovulation, they became pregnant and produced normal-sized litters of normal-sized pups [16]. 2.2. Experimental design 2.2.1. Experiment 1 At 50 days of age, 28 growth-restricted female rats with delayed puberty were divided into four groups so that an equal number of each litter were represented in each experimental group. The groups received the following daily food intake from days 50–62 of age: group 1 ⫽ x ⫹ 0.4 g; group 2 ⫽ x ⫹ 1.2 g; group 3 ⫽ x ⫹ 3.6 g; group 4 ⫽ x ⫹ 10.8 g (where x ⫽ mean daily food intake of individual rat over previous period of food restriction). Daily weights and vaginal smears were taken from Days 50–62 of the experiment to monitor weight gain and cyclicity, respectively. 2.2.2. Experiment 2 At 50 days of age, 28 growth-restricted female rats with delayed puberty were divided into four evenly sized groups in a manner similar to that used in Experiment 1. The four groups were given ad lib access to food (x ⫹ 10.8 g) for periods of 12, 24, 48, and 72 h, respectively. After the period of ad lib feeding, cases were changed to eliminate any remaining excess food, and food intake was dropped to the
mean level of food-restricted daily intake for a period of 3 days. At the end of these 3 days, food intake was manipulated as done previously to stabilize body weights. Again, daily weights and vaginal smears were taken from Days 50– 59 of the experiment to monitor weight gain and cyclicity, respectively. 2.3. Data analyses In each experiment, the number of animals in each group which achieved first estrus was compared using a modification of the Fisher exact test for comparing more than two proportions [25]. Following the exact test, a Tukey-type multiple comparisons test was used to determine which groups were different [25]. Body weights and food intake were compared using analysis of variance (ANOVA, Statview 512⫹, Brainpower, Las Calabas, CA), and the Scheffe test was used in any post hoc comparisons made among means. Growth rates during the refeeding periods were compared using regression analysis (GraphPad version 2.0, San Diego, CA). Linear regression analyses of caloric intake per day versus days to reach first estrus and body weight versus days to reach first estrus were performed post hoc (Statview 512⫹, Brainpower, Las Calabas, CA). In all analyses, p ⬍ 0.05 was considered significant. All data is presented as mean ⫾ standard error of the mean. 3. Results 3.1. Experiment 1 From Day 30 to Day 50 of age, all rats were maintained at a stable body weight of 82.2 ⫾ 0.2 g. There was no significant weight difference among groups at this time. There was also no significant difference in the mean food intake of the different groups before the experimental period. Average food intake before the experimental period was approximately 5.6 ⫾ 0.0 g (23.2 ⫾ 0.1 kcal) per day. On Day 50, daily food intake was raised by 0.4 g, 1.2 g, 3.6 g, and 10.8 g for groups 1–4, respectively. From Day 50 to Day 62, the growth rate of rats was dependent on caloric intake (Fig. 1). In group 1 (24.8 ⫾ 0.2 kcal/day), mean weight of rats increased by 10.7 ⫾ 0.7 g, while in group 2 (28.2 ⫾ 0.5 kcal/day), mean weight increased by 16.3 ⫾ 1.5 g. These increases in body weight were not significantly different from each other. In contrast, the increase in mean body weight observed in group 3 (38.0 ⫾ 0.3 kcal/day: 38.3 ⫾ 1.2 g), and in group 4 (68.1 ⫾ 0.3 kcal/day: 84.0 ⫾ 0.5 g) were different from each other and from the increases observed in groups 1 and 2. The amount of calories provided during this realimentation period determined when and if rats would cycle (Fig. 2). The number of rats, which cycled in response to the increase in caloric intake, was significantly fewer in groups 1 and 2 compared to groups 3 and 4. While the number of animals that cycled in response to the increase in caloric intake was not different between groups 3 and 4 (6 of 7 rats
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Fig. 1. Mean (⫾SEM) body weight of female rats during Experiment 1 (n ⫽ 7 rats per group). The vertical line at Day 50 depicts the onset of increased food intake. Group 1: 24.8 ⫹ 0.2 kcal/day; group 2: 28.2 ⫹ 0.5 kcal/day; group 3: 38.0 ⫹ 0.3 kcal/day; group 4: 68.1 ⫹ 0.3 kcal/day.
versus 7 of 7 rats cycled, respectively), the time required to respond to the smaller increase in caloric intake received by group 3 was longer by 3–4 days compared to the response time of rats in group 4 on the higher caloric intake. Day of first estrus appeared to be more highly correlated with caloric intake per day (Fig. 3, lower panel; p ⫽ 0.0002; r2 value of 0.7) than body weight at first estrus (Fig. 3, upper panel; p ⫽ 0.02; r2 value of 0.4).
Fig. 3. Linear regression analysis of body weight and caloric intake with day of first estrus in all rats that cycled regardless of group in Experiment 1 (15 of 28 rats). Upper panel: body weight relative to day of first estrus. Lower panel: caloric intake per day relative to day of first estrus. The solid line depicts the regression line in both panels. The number next to the circle (open or closed) represents overlapping data points.
3.2. Experiment 2 From Day 30 to Day 50 of age, all rats were maintained at a stable body weight of 86.2 ⫾ 0.2 g. There was no significant weight difference between groups at this time.
Fig. 2. Mean (⫾SEM) time in days from onset of refeeding until first estrus and percentage of rats cycling relative to the size of the refeed meal (kcal/ day) in Experiment 1.
Again, there was no significant difference in the mean food intake of the different groups before the experimental period. Average daily food intake before the experiment was approximately 5.4 ⫾ 0.0 g (22.5 ⫾ 0.1 kcal). On Day 50 of age, rats were given an ad lib meal of approximately 16.2 ⫾ 0.0 g (67.2 ⫾ 0.2 kcal) per day. During the realimentation period, weight gain was proportional to the amount of time spent on an ad lib diet (Fig. 4). Group 4 (72-h ad lib feeding) showed the most substantial weight gain (30.99 ⫾ 1.67g), whereas group 1 (12 h ad lib feeding) showed the least weight gain (14.17 ⫾ 1.09 g). Group 2 (24 h ad lib feeding) and 3 (48 h ad lib feeding) showed intermediate weight gains (18.76 ⫾ 1.29 g and 24.91 ⫾ 1.88 g, respectively). The duration of the ad lib food stimulus determined whether or not they began to cycle (Fig. 5). The number of rats, which cycled in response to the increase in caloric intake, was significantly fewer in groups 1, 2, and 3 compared to group 4. Thus, an ad lib food stimulus of 72 h was required for the majority of rats to achieve first estrus (6 of 7 rats). It was also of interest to note that the time to first estrus was between 3 to 4 days after onset of refeeding in all
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Fig. 4. Mean (⫾SEM) body weight of female rats during Experiment 2 (n ⫽ 7 rats per group). The vertical line at Day 50 depicts the onset of increased food intake. Group 1: 12 h; group 2: 24 h; group 3: 48 h; group 4: 72 h of ad lib food.
rats that cycled regardless of the duration of the ad lib food stimulus (i.e., regardless of group). 4. Discussion The studies reported herein determined if onset of puberty is affected by the size and duration of the realimentation stimulus after puberty was delayed by food restriction. Our results from Experiment 1 show that level of caloric intake has a profound effect on both the number of rats that achieve first estrus and the length of time before cycling begins in such prepubertal female rats. The highest refeed signal at realimentation (68.1 ⫾ 0.3 kcal/day) produced the
Fig. 5. Number and percentage of rats that achieved first estrus relative to the time allowed of ad lib feeding in Experiment 2.
most rapid onset of cycling in all rats, while the lowest refeed signal (24.8 ⫾ 0.2 kcal/day) did not induce onset of cycles. In addition, there was a graded response in terms of the number of rats cycling and the time to onset of cycles in between these two extremes. These findings suggest that the level of increase of food intake appears to determine the amount of time the diet must be sustained to build up sufficient energy reserves to fuel cyclicity and reproduction. Ad lib refeeding is known to increase circulating levels of gonadotropins and trigger onset of puberty and cycling in growth-restricted animals [11, 16]. However, the effect of a lesser refeed signal on pubertal development had not yet been investigated. Because the number of animals in which cycling began was dependent on the size of the refeed meal in Experiment 1, it is possible that levels of gonadotropin secretion after refeeding also increase in a meal size-dependent manner. This phenomenon has been demonstrated in adult male rhesus monkeys in which gonadotropin secretion increased progressively with increasing refeed meal size after a brief period of fasting, indicating that metabolic signals to the GnRH neurosecretory system may be continuous rather than discrete in nature [26]. Thus, in the intact female, GnRH, and therefore LH, release may gradually increase as meal size increases, driving follicular development at a progressively faster pace, and thus, initiating onset of cycling earlier. This possibility remains to be determined. That caloric intake appears to be the crucial signal to the reproductive axis has previously been well established in a variety of models, including the growth-restricted prepubertal female with delayed puberty [18, 27, 28]. In the restricted prepubertal rat and lamb or adult male monkey, an increase in caloric intake only following a period of food restriction causes an increase in gonadotropin secretion regardless of the source of calories. These data also imply that reduced micronutrient and/or protein intake do not limit reproductive function in such food restriction models. In wild mammalian populations, regulation of hormone secretion based on available energy has the effect of linking reproduction to the availability and attainability of food. During periods when food is scarce, the energetically costly female reproductive system is turned off. Available energy resources are channeled towards survival rather than propagation. When food supplies are more plentiful, cycling and reproduction resume and the offspring have a better chance of survival due to better feeding and climactic conditions. This is particularly true in small mammals, whose large surface to volume ratios and minimal fat stores make the demands of reproduction particularly costly [1]. In Whitefooted mice, reproductive activity has been shown to be a better indicator of recent food availability than even body weight and fat content [16]. The duration of an ad lib food stimulus is also critical in determining onset of cycling in growth-restricted, prepubertal female rats. Previous studies have shown that ad lib food stimuli can increase circulating levels of gonadotropin hormones in as little as 3–4 h [24]. In addition, substantial
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weight gain and fat accumulation also occur during the first 24 h after resumption of ad lib feeding (current study; [16]). However, our results show that even an ad lib food stimulus of 48 h duration only initiated cycling in three of seven rats. An extended stimulus of 72 h of ad lib feeding duration was necessary to cause the majority of rats (six of seven) to begin cycling. These data seem to indicate that onset of estrous cycling in growth-restricted, prepubertal female rats requires an extended signal (72 h) of ad lib feeding even though circulating levels of gonadotropins can respond quite quickly (3–4 h) to the same stimulus [24]. The reason for this time lag between resumption of normal hormone secretion and onset of cycling may have its source at the ovarian level. It has been known for some time that degeneration of the ovaries occurs under food-restricted conditions (rats: [29, 30]; hamsters: [31]; guinea pigs: [32]). Larger follicles become atretic and disappear while smaller follicles cease growth. However, no permanent damage to the reproductive system occurs and refeeding will stimulate development of the plentiful smaller follicles and eventually ovulation [29, 31]. Taken together, these data suggest that follicular development is suppressed in food-restricted or food-deprived animals, and that at least 3 days of ovarian stimulation may be required to induce development of follicles to a level where ovulation is possible. Even though many studies have implicated glucose to be the proximate signal in dietary regulation of estrous cycling [20, 33–35], the ultimate signal is associated with the availability of oxidizable metabolic fuels from a variety of sources. Alternate forms of energy, such as fatty acids and proteins, might increase the availability of glucose for use/ detection in the central nervous system, thus indirectly regulating cyclicity. In fattened Syrian hamsters, estrous cyclicity is not depressed by limited food deprivation unless the hamsters also receive doses of MP, an inhibitor of fatty acid oxidation. Similarly, lower doses of MP and 2-DG which have no effect when administered separately, depress cyclicity [36] and estrous behavior [37] when administered together. The results of our experiments lend support to the idea of the ultimate regulatory effects of net caloric intake. Onset of cycling was observed more frequently and more rapidly in those rats receiving higher levels of daily caloric intake. Because we did not measure body fat content, our results do not rule out the possibility that a critical level of body fat is necessary for puberty in rats. However, other studies have shown that rats can reach puberty at vastly different percent body fat contents [38, 39]. Indeed, exogenous administration of GnRH pulses to growth-restricted prepubertal rats will induce estrous cycles with no change in food intake, and by implication, body fat content [16]. Our model will prove useful for testing the metabolic versus adipostatic hypotheses in future studies in which whole body and neuoendocrine parameters can be measured at discrete time intervals during the now well-defined ad lib feeding period. In summary, onset of cycling and the length of time until
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cycling begins in refed female rats varies in a graded manner with daily food intake. Higher levels of food intake lead to higher and quicker incidences of cycling whereas lower levels lead to delayed cycling or none at all. Also, an extended ad lib food stimulus of 72 h is necessary to cause onset of estrous cyclicity in food-restricted, prepubertal, female rats.
Acknowledgments This work was supported by grants from NIH (HD-07433) to HI and CUR undergraduate student summer research fellowship to N.A.M. A preliminary report of the investigations has appeared in the Program of the 29th Annual Meeting of the Society for the Study of Reproduction, London, Ontario (Abstract 489).
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