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Early Onset of Reproductive Function in Normal Female Mice Treated with Leptin

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Science  03 Jan 1997:
Vol. 275, Issue 5296, pp. 88-90
DOI: 10.1126/science.275.5296.88

Abstract

Numerous studies have revealed an association between nutritional status, adiposity, and reproductive maturity. The role of leptin, a hormone secreted from adipose tissue, in the onset of reproductive function was investigated. Normal prepubertal female mice injected with leptin grew at a slower rate than controls as a result of the hormone's thinning effects, but they reproduced up to 9 days earlier than controls and showed earlier maturation of the reproductive tract. These results suggest that leptin acts as a signal triggering puberty, thus supporting the hypothesis that fat accumulation enhances maturation of the reproductive tract.

A link between body fat content and the onset of puberty in females was first proposed over 30 years ago (1, 2). More recent studies documenting delayed puberty in lean female ballet dancers (3, 4) and accelerated puberty in obese females (5) support the concept that a metabolic signal produced by adipose tissue may control the onset of reproductive function (6). The ability of leptin, a hormone secreted by adipose tissue, to restore fertility to mice that are genetically deficient in leptin (7) suggests that this hormone may be a signal triggering the onset of reproductive function.

To explore this possibility, we injected human recombinant leptin into normal prepubertal female mice and monitored its circulatory levels over time (8). Leptin had a half-life (T1/2) of 60 min and was undetectable 7 hours after injection. Because of its short T1/2, in subsequent experiments we administered leptin daily between 5→00 and 7→00 p.m. near the onset of the dark period, when it was most likely to exert its metabolic effects. Leptin slowed growth of the mice and caused a significant decrease in body weight compared with mice treated with phosphate-buffered saline (PBS) from day 3 (P = 0.003) and throughout the treatment (9) (Fig. 1A). This effect was associated with a significant decrease in food intake (P < 0.001) in the leptin group compared with PBS controls (Fig. 1B).

Fig. 1.

Effect of leptin treatment on body weight (A), food intake (B), and presence of copulatory plugs (C) in prepubertal C57BL/6J mice treated with PBS (•) and leptin (ˆ). Each value represents the mean ± SEM of 12 (PBS) or 13 (leptin) animals. Error bars are too small to be shown on the scale in (B) for PBS and leptin groups (SEM ranges, 0.1 to 1.2 and 0.3 to 0.7, respectively). Pairwise comparison of values from day 22 and onward is statistically significant (P < 0.001) by Student's t test. The age distribution curve of the leptin-treated mice in (C) is shifted to the left, reflecting mating at an earlier age than in controls.

If leptin is involved in signaling puberty and the onset of reproductive function, then leptin-treated mice should attain reproductive maturity earlier than control mice (10). Copulatory plugs were detected in leptin-treated mice at an earlier age than in control mice (Fig. 1C). Between the ages of 30 and 39 days, 85% (11 of 13) of leptin-treated mice and 17% (2 of 12) of PBS-treated mice had a copulatory plug (P = 0.001, Fisher's exact test). Leptin treatment thus appeared to accelerate behavioral estrus and mating. The body weight of the leptin-treated mice at the time the copulatory plug was detected was 13% less than that of controls (15.9 ± 0.2 versus 18.3 ± 0.3 g, respectively; P < 0.0001). Furthermore, successful pregnancies and deliveries occurred in 46 and 42% of leptin- and PBS-treated groups, respectively, showing that leptin treatment did not interfere with successful ovulation, pregnancy, or delivery of pups.

To assess whether leptin affected maturation of the reproductive tract in prepubertal mice, we determined (i) the timing of vaginal opening, (ii) the progress toward the first estrous cycle, and (iii) the weights of uteri, ovaries, and oviducts (11). These parameters are reliable indices for the target actions of reproductive hormones. Vaginal opening was advanced by 1 to 4 days in leptin-treated mice compared with PBS-treated controls (Fig. 2A) (12). As a result, progression toward the first estrus was initiated earlier in the leptin-treated mice. By day 29, 5 of 12 leptin-treated mice had passed through estrus and progressed to metestrus (Fig. 2B), thereby completing their first estrous cycle. In contrast, none of 12 control mice had reached this point at day 29.

Fig. 2.

Reproductive function in mice treated with leptin (□) or PBS (▪). (A) Number of mice with vaginal opening after initiation of treatment; P = 0.014 at day 24 and P < 0.001 at day 25 (Fisher's exact test). (B) Number of mice at different stages of the estrous cycle at day 29. Diestrus, proestrus, estrus, and metestrus are denoted by D2, P, E, and D1, respectively. Metestrus, P = 0.037 (Fisher's exact test). (C) Weights of reproductive organs at day 29. Statistical significance by Student's t test is indicated by an asterisk. Uteri, P < 0.004; ovaries, P < 0.0001; oviducts, P = 0.001.

We evaluated gonadal steroid action by assessing the weights of target reproductive organs. For example, estradiol was assayed by its growth-stimulatory effects on the uterus. In leptin-treated prepubertal mice, weights of the uteri, ovaries, and oviducts were, respectively, 53, 37.5, and 43.8% greater than those of controls (Fig. 2C). We also determined the levels of the gonadotropin luteinizing hormone (LH) and the gonadal steroid 17β-estradiol (13) on day 29 when the mice were killed. LH concentrations were lower in leptin-treated mice than in controls (5.2 ± 0.4 versus 6.9 ± 0.4 ng/ml, respectively; P = 0.007). In addition, there was a trend toward lower concentrations of 17β-estradiol in leptin-treated mice compared with PBS controls, although the difference was not statistically significant (9.5 ± 1.1 versus 10.3 ± 1.1 pg/ml, respectively). The lower concentrations of LH and 17β-estradiol in leptin-treated mice at the time of death are consistent with their earlier progression through estrus compared with PBS-treated mice.

Finally, we assessed the levels of endogenous leptin in PBS- and leptin-treated mice at 30, 35, and 39 days of age (14). In the PBS group, plasma leptin concentrations increased by 61% between 30 and 39 days of age, whereas in the leptin group, endogenous leptin did not increase over time. This result suggests that exogenous leptin interferes with the normal age-related increase in leptin (Table 1).

Table 1.

Endogenous leptin concentrations in PBS- and leptin-treated prepubertal mice during growth. Data are means ± SEM. P value represents statistical significance in mice between 30 and 39 days of age by analysis of variance and Student-Newman-Keuls tests. There are four mice per time point. NS, not significant.

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Our findings suggest that leptin acts as a signal for puberty, as evidenced by its ability to accelerate reproduction, vaginal opening, onset of the first estrous cycle, and maturation of reproductive tissues concomitant with changes in LH and 17β-estradiol levels. The involvement of leptin in initiation of reproductive function supports previous observations that relate extreme leanness with delayed puberty (4) and obesity with acceleration of puberty (5). Thus, leptin may be a factor involved in signaling to neuroendocrine pathways the attainment of a critical fat mass, a determinant for triggering puberty (2, 3, 4). Kennedy first postulated that the hypothalamus receives a puberty-triggering signal related to metabolic rate or food intake (2, 15), and later studies showed that the attainment of a critical percentage of body fat is necessary for initiation of puberty (16, 17). Although the critical fat hypothesis has been challenged (18) and the metabolic signal postulated by Kennedy has remained elusive, our study suggests that leptin may be that signal.

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