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Endocrinology Vol. 142, No. 11 4758-4761
Copyright © 2001 by The Endocrine Society

ARTICLES

Puberty in Female Mice Is Not Associated with Increases in Either Body Fat or Leptin

F. H. Bronson

Section of Integrative Biology, School of Biological Sciences, University of Texas, Austin, Texas 78712

Address all correspondence and requests for reprints to: Dr. F. H. Bronson, Section of Integrative Biology, School of Biological Sciences, University of Texas, Austin, Texas 78712. E-mail: bronson{at}mail.utexas.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been hypothesized that puberty is triggered when body fat and hence circulating levels of leptin exceed critical thresholds. Four kinds of experiments tested that hypothesis in female mice. When age was the independent variable, body fat and circulating levels of leptin decreased rather than increased before the onset of puberty. When stage of reproductive development was the independent variable, neither body fat nor circulating levels of leptin correlated with the onset of puberty. In sharp contrast, reproductive development was well correlated with body weight. A significant nocturnal peak in circulating levels of leptin was seen before and at all stages of reproductive development, but the highest levels were seen after rather than before the first estrous cycle was initiated. Neither acceleration nor deceleration of puberty by varying the female’s social environment had any effect on either body fat or leptin. There is no support in any of these experiments for the hypothesis that an increase in body fat and thus an increase in circulating levels of leptin triggers puberty in female mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PAST 5 yr have seen immense interest in the relationship among leptin, body fat, and puberty in females (1). Mice that are homozygous for the obese (ob) gene are incapable of synthesizing leptin and cannot pass through puberty unless treated with exogenous leptin (2). Likewise, underfeeding suppresses puberty in rats and mice, and unless too severe, this can also be reversed by injecting leptin (3, 4). Indeed, repeated injections of leptin have been reported to accelerate puberty somewhat even in normally fed female mice (5, 6).

As the leptin gene expresses itself primarily in adipocytes, and leptin circulates generally in proportion to the size of a female’s fat stores (7), the findings noted above have suggested a modern version of the body fat hypothesis postulated by Frisch almost 3 decades ago (8). The modern version suggests that puberty is triggered when body fat and thus circulating levels of leptin pass critical thresholds (9).

Support for this hypothesis in rodents is mixed. Although one laboratory has reported a slow rise in circulating concentrations of leptin during peripubertal development in the female rat (10), another laboratory saw no such change (11). Still another laboratory reported a dramatic peak in circulating levels of leptin in female mice during their second week of life, but no change thereafter, including the time when the first estrous cycle would be initiated (12). Other studies have confirmed that exogenous leptin can counter the underfeeding-induced suppression of puberty in rat and mouse females, but they could not confirm the leptin-induced acceleration of puberty in normally fed females (11, 13). The picture has become more clouded by the association of puberty in the female rat with the appearance of a nocturnal peak in circulating leptin, with no marked change during daylight hours (14).

The present experiments address the hypothesis that an increase in body fat and thus an increase in circulating levels of leptin act as a trigger for puberty in female mice. This hypothesis was tested by assessing body fat and circulating levels of leptin when age was the independent variable, when stage of reproductive development was the independent variable, when diurnal rhythms in circulating leptin were of interest, and when puberty was either accelerated or decelerated by manipulating the female’s social environment (15, 16).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Four experiments were conducted with CF-1 female mice born in this laboratory and maintained under standard conditions of 22-23 C, 14 h of light (lights on at 0600 h and off at 2000 h) and a diet of Purina 5008 Formulab chow (Ralston Purina Co., St. Louis, MO). Litter size was adjusted to 10 animals at birth to minimize variation in body weight. All animals were weighed at 15 d of age, and extremely large or small females were culled to yield a still smaller range of variation in body weight. The remaining females were delegated to experimental treatments in a way that produced nearly identical means and ranges in body weight in all groups within an experiment. Females were weaned at 20 d and housed thereafter in isolation, except where noted. All experimental procedures followed the guidelines established by the university’s institutional animal care and use committee.

Exp 1
The object of this experiment was to determine the best age at which to explore the initiation of puberty in detail in CF-1 mice. Seven or eight females were killed at each 5-d interval from 15 to 35 d of age. Uteri were weighed, ovaries were examined microscopically for corpora, and oviducts were searched for eggs. The gastrointestinal tracts were removed, and the carcasses were frozen for later whole body fat extraction using ether in a soxhlet apparatus. Serum, collected by cutting the animals’ throats, was stored for later leptin assay. Part of this first experiment was replicated. Twelve females were killed at each 5-d interval between 15 and 30 d of age, and the same data were collected as in the original experiment, except fat extractions were not performed.

Exp 2
Based on the results of the first experiment (which are presented in Results), 15 animals were killed at 1000 h at each 2-d interval between 18 and 28 d of age. At autopsy, the same data were collected as in the previous experiment. After autopsy, the data were pooled, ignoring age, and were reorganized around four stages of reproductive development, as detailed in Results.

Exp 3
The object of this experiment was to examine circadian variation in circulating concentrations of leptin during peripubertal development. The experiment relied on a 6 (ages) x 6 (times of day) experimental design with 60 females/cell for a total of 360 females. Females were killed at 2-d intervals between 18 and 28 d of age at 1 of 6 clock times, every 4 h starting at noon. After autopsy, age was ignored, and the data were reorganized in relation to stage of reproductive development as in the previous experiment.

Exp 4
Eighteen-day-old females, weighing 13 g or more, were housed in one of three conditions: in isolation vs. with a mature stud male vs. in groups of four with an adult female present. The females were killed 3 d later at 1000 h when they were 21 d old.

Leptin assay
Leptin was assayed in 100-µl samples of serum using the mouse leptin RIA kit purchased from Linco Research, Inc. (St. Charles, MO). The intra- and interassay coefficients of variation were 4.5% and 5.2%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1
Based on uterine weight relative to body weight, some females experienced the onset of first estrous cycle between 20–25 d of age, and most females had experienced first ovulation by the time they were 35 d old (Table 1Go). As shown in Fig. 1Go, however, there was great individual variation in the relationship between age and reproductive development. Percent body fat and circulating levels of leptin declined precipitously between 15 and 20 d of age and then rose slowly only after most females had begun their first cycle. Percent body fat and leptin titers were significantly correlated (r2 = 0.44; P < 0.0001). A replicate experiment tested the repeatability of the marked decrease seen in circulating levels of leptin between 15–20 d of age. As shown in Table 1Go, that observation was confirmed.


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  		Table 1. Relationship between body fat, circulating levels of leptin, and pubertal development at various ages in CF-1 female mice

 


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  		Figure 1. Uterine weight of CF-1 females killed at various ages.

 
Exp 2
To avoid the individual variation seen in the relationship between age and reproductive development in the first experiment (Fig. 1Go), the data obtained in the second experiment were pooled, ignoring age, and then reorganized around four stages of reproductive development: 1) before the onset of puberty, when uteri weighed less than 25 mg; 2) soon after puberty was initiated, as indicated by uteri weighing between 25–75 mg; 3) approaching or during the first proestrus, as indicated by uteri weighing more than 75 mg but with no indication of ovulation; and 4) after the first ovulation had occurred, as indicated by corpora in the ovaries and eggs in the oviducts. Based on these four stages of reproductive development, there was a highly significant correlation between stage of reproductive development and body weight (r2 = 0.80; P < 0.0001), but no correlation between reproductive development and either body fat or circulating levels of leptin (Fig. 2Go).



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  		Figure 2. Correlation between stage of reproductive development (as indicated by uterine weight and the presence or absence of eggs in the oviducts) and body weight, percent body fat, and circulating concentrations of leptin in 18- to 28-d-old females.

 
Exp 3
A nocturnal peak in circulating levels of leptin was seen at all four stages of reproductive development (Fig. 3Go). There was little difference between the two earliest stages of development in this regard, and the highest mean levels and the highest peak were seen in females that had already ovulated for the first time. As tested by a 6 x 6, two-factor ANOVA, the main effect of time of day was significant (P < 0.0001), and the main effect of stage of reproductive development was also significant (P < 0.0001), but the interaction was not significant, indicating that all four experimental groups showed the same pattern of change throughout the 24-h period.



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  		Figure 3. Diurnal rhythm of circulating leptin at each of four stages of reproductive development, as indicated by uterine weight and the presence or absence of eggs in the oviducts.

 
Exp 4
As expected, mean uterine weight was much greater in the male-exposed females than in the isolated controls (Table 2Go). Most of the male-exposed females were either in proestrus or approaching that stage of the estrous cycle when killed after 3 d of cohabiting with an adult male. The uterine weight of the group-housed females was smaller than that of isolated controls, but not significantly so. There were no significant differences in leptin titers or amounts of body fat among the three experimental groups. Indeed, the variation in percent body fat and leptin concentrations approached significance in the opposite direction of that postulated by the hypothesis being tested (Table 2Go).


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  		Table 2. Effect of the social environment on uterine weight, percent body fat, and circulating levels of leptin in young female mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several facts relevant to the present experiments have been well established over the past several years. 1) A female must be in positive energy balance for ovulation to occur, whether the female is adult or peripubertal. 2) Body fat is an important component of energy balance, and circulating leptin is a key signal informing the brain of negative energy balance. 3) Thus, leptin is an important modulator of reproductive development in female mammals (1, 17). None of these facts was of investigative concern in the present studies. The experiments reported here tested the specific hypothesis that puberty is triggered when the size of a female’s fat stores and hence the level of circulating leptin pass critical thresholds.

In the first experiment circulating levels of leptin dropped precipitously between 15 and 20 d of age, before the onset of puberty. This decline was probably the latter half of the dramatic peak in circulating levels of leptin seen by another laboratory during the second week of life in mice (12). The role of this peak in regulating puberty, if any, was not a focus of concern in these experiments.

Body weight proved to be a much better predictor of reproductive development than age in isolated females. However, body weight is not critical for the onset of puberty in female mice, as shown by the experiment involving male exposure. Exposure to male pheromones induced rapid pubertal development at body weights well below those seen in isolated females. Uteri characteristic of proestrus were seen at a mean body weight of 19.6 ± 0.3 g in females exposed to a male (Table 2Go), whereas uteri of this size were not seen in isolated females until they had reached an average body weight of 23.3 ± 0.5 (calculated from the data shown for females with uteri >75 mg in Fig. 2Go).

The procedure of reorganizing data around specific stages of reproductive development post-hoc yielded a much more sensitive experimental design for testing the hypothesis of concern than simply comparing females of different ages. In the experiments in which stage of reproductive development was the independent variable, there was no indication of an increase in body fat or circulating levels of leptin just before the pubertal spurt in development; this was true whether leptin titers were examined only at 1000 h or throughout a 24-h period. Neither was their any indication of changes in body fat or circulating levels of leptin in females in which pheromonal cues were used to accelerate or decelerate the onset of puberty. Indeed, the data generated by that experiment approached significance in the opposite direction of that predicted by the hypothesis; percent body fat and leptin titers were highest in the females in which pubertal development was decelerated by female pheromones and lowest in the females in which reproductive development was accelerated by male pheromones.

The failure to find any evidence that body fat and circulating levels of leptin correlate with the onset of puberty is somewhat at odds with reports that puberty can be accelerated in normally fed female mice by repeated injections of leptin (5, 6). The present results suggest that these reports should be viewed with caution until the fat/leptin/puberty hypothesis can be examined much more thoroughly. In particular, these reports have been cited as evidence that puberty in humans is triggered when body fat and circulating levels of leptin pass critical thresholds (18). The relevance of the rodent puberty model to human and nonhuman primates is unclear, because, unlike primates, rodents do not experience a prolonged period of juvenile quiescence; thus, the central mechanisms controlling puberty may be different in primates and rodents (19, 20). Nevertheless, it seems well established that leptin plays no role in provoking puberty in the male rhesus monkey (21), and although a few clinical observations suggest that it might be important for puberty in humans (reviewed in Ref. 21), several clinical studies have found no relationship between leptin and puberty (22, 23, 24) In this regard it should be noted that the hypothesis that puberty depends on a critical amount of body fat has been rejected repeatedly by experimentalists, and it has also been rejected routinely by researchers working with human athletes (25). In the absence of a key role for body fat, there is no strong conceptual basis for predicting a priori that puberty in girls is triggered by increasing titers of leptin.


   Acknowledgments
 

Received April 5, 2001.

Accepted for publication July 30, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cunningham MJ, Clifton DK, Steiner RA 2001 Leptin’s actions on the reproductive axis: perspectives and mechanisms. Biol Reprod 60:216–222[Abstract/Free Full Text]
  2. Chehab FF, Lim ME, Lu R 1996 Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 12:18–320[CrossRef]
  3. Ahima RS, Prabakaran D, Mantozoros E, Qu D, Lowell B, Maratos-Flier E, Flier JS 1996 Role of leptin in the neuroendocrine response to fasting. Nature 382:250–252[CrossRef][Medline]
  4. Barash IA, Cheung CC, Weigle DS, Ren H, Kramer JM, Fallon M, Kabigting EB, Kuijper JL, Clifton DK, Steiner RA 1996 Leptin is a metabolic signal to the reproductive system. Endocrinology 187:3144–3147
  5. Ahima RS, Dushay J, Flier S, Prabakaran D, Flier JS 1997 Leptin accelerates the onset of puberty in normal female mice. J Clin Invest 99:391–395[Medline]
  6. Chehab FF, Mounzih K, Lu R, Lim ME 1997 Early onset of reproductive function in normal female mice treated with leptin. Science 275:88–90[Abstract/Free Full Text]
  7. Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S, Kern PA, Friedman JM 1995 Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1:1155–1161[CrossRef][Medline]
  8. Frisch R, McArthur J 1974 Menstrual cycles: fatness as a determinant of minimum weight necessary for their maintenance or onset. Science 185:949–951[Abstract/Free Full Text]
  9. Chehab FF 1997 The reproductive side of leptin. Nat Med 3:952–953[CrossRef][Medline]
  10. Gruaz NM, Laloui M, Pierroz DD, Sizonenko PC, Blum WF, Aubert ML 1998 Chronic administration of leptin into the lateral ventricle induces sexual maturation in severely food-restricted female rats. J Neuroendocrinol 10:627–633[CrossRef][Medline]
  11. Cheung CC, Thornton JE, Kuijper KL, Weigle DS, Clifton DK, Steiner RA 1997 Leptin is a metabolic gate for the onset of puberty in the female rat. Endocrinology 138:855–858[Abstract/Free Full Text]
  12. Ahima RS, Prabakaran D, Flier JS 1998 Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding. Implications for energy homeostasis and neuroendocrine function. J Clin Invest 101:1020–1027[Medline]
  13. Cheung CC, Thornton JE, Nurani SD, Clifton DK, Steiner RA 2001 A reassessment of leptin’s role in triggering the onset of puberty in the rat and mouse. Neuroendocrinology 74:12–21[CrossRef][Medline]
  14. Nagatani S, Guthikonda P, Foster DL 2000 Appearance of a nocturnal peak of leptin secretion in the pubertal rat. Horm Behav 37:345–352[CrossRef][Medline]
  15. Vandenbergh JG 1983 Pheromonal regulation of puberty. In: Vandenbergh JG, ed. Pheromones and reproduction in mammals. New York: Academic Press; 95–112
  16. Bronson FH, Rissman EF 1986 The biology of puberty. Biol Rev 61:157–195[Medline]
  17. Schneider JE, Zhou D, Blum RM 2000 Leptin and metabolic control of reproduction. Horm Behav 37:306–326[CrossRef][Medline]
  18. O’Rahilly S 1998 Life without leptin. Nature 392:330–331[CrossRef][Medline]
  19. Ojeda SR, Urbanski HF 1994 Puberty in the rat. In: Knobil E, Neill JD, eds. The physiology of reproduction, 2nd Ed. New York: Raven Press; 363–409
  20. Plant TM 1994 Puberty in primates. In: Knobil E, Neill JD, eds. The physiology of reproduction, 2nd Ed. New York: Raven Press; 453–485
  21. Plant TM 2001 Neurobiological bases underlying the control of the onset of puberty in the rhesus monkey: a representative higher primate. Front Neuroendocrinol 22:107–139[CrossRef][Medline]
  22. Oerter KK, Larmore KA, deLancey E, Brown JM, Considine RV, Hassink SG 1998 Effect of obesity on estradiol level, and its relationship to leptin, bone maturation and bone mineral density in children. J Clin Endocrinol Metab 83:3469–3475[Abstract/Free Full Text]
  23. Heger S, Partsch CJ, Peter M, Blum WF, Kiess W, Sippell WG 1990 Serum leptin levels in patients with progressive central precocious puberty. Pediatr Res 46:71–75[Medline]
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  25. Bronson FH, Manning JM 1991 The energetic regulation of ovulation: a realistic role for body fat. Biol Reprod 44:945–950[Abstract]



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