Early onset of reproductive function in normal female mice treated with leptin

Science

Volumn 275, Issue 5296, 1997, Pages 88-90

  Chehab, Farid F ^a   Mounzih, Khalid ^a   Lu, Ronghua ^a   Lim, Mary E ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ESTRADIOL; LEPTIN; LUTEINIZING HORMONE;

ADOLESCENT; ANIMAL EXPERIMENT; ANIMAL TISSUE; ARTICLE; BODY FAT; BODY WEIGHT; CONTROLLED STUDY; FEMALE; FEMALE GENITAL SYSTEM; FOOD INTAKE; LIPID STORAGE; MATURATION; MOUSE; NONHUMAN; PRIORITY JOURNAL; PUBERTY; REPRODUCTION;

ANIMALS; BODY WEIGHT; EATING; ESTRADIOL; ESTRUS; FEMALE; GENITALIA, FEMALE; LEPTIN; LUTEINIZING HORMONE; MICE; MICE, INBRED C57BL; ORGAN SIZE; PROTEINS; RECOMBINANT PROTEINS; REPRODUCTION; SEXUAL MATURATION;

EID: 0031014413     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5296.88     Document Type: Article

References (19)

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8. Human recombinant leptin was prepared (7) and quantitated with antibodies specific for human leptin (Linco Research, St. Louis, MO). C57BL/6J adult females (n = 24) weighing 24.1 ± 0.7 g (mean ± SEM) were divided into control (n = 6) and experimental groups (n = 18) and injected intraperitoneally (ip) with PBS and recombinant human leptin (4 μg per gram of body weight), respectively. PBS-treated mice were killed immediately after injection and leptin-treated mice were killed at 1, 2, 3, 4, 5, and 7 hours (three mice per time point). Blood was collected by cardiac puncture and the plasma was separated by centrifugation and frozen at -20°C. Leptin in 5 μl of plasma was quantitated by radioimmunoassay as described above.
9. All procedures were approved by the University of California, San Francisco (UCSF) Committee on Animal Research. Two-week-old lactating prepubertal C57BL/6J female pups were obtained from Jackson Laboratories (Bar Harbor, ME) and allowed to recover for 1 week before initiation of experiments. Male pups were removed from the litters at 2 weeks of age. Mice were housed at the UCSF Animal Care Facility and maintained at 20°C with a 12-hour light/12-hour dark cycle (lights on at 6:00 a.m. and off at 6:00 p.m.). Recombinant human leptin (2 μg/g) was prepared, quantitated (7, 8), and injected ip once daily; control animals received equal amounts of PBS. Prepubertal C57BL/6J mice (n = 25) born on the same day were weaned at 21 days of age and housed in eight cages (three or four mice per cage; littermates were placed in different cages). Leptintreated (n = 13) and PBS-treated (n = 12) mice weighed 10.2 ± 0.4 and 10.3 ± 0.3 g (mean ± SEM), respectively. Mice were monitored continuously for body weight, food intake, and vaginal opening. Unless indicated, P values in this study were calculated by unpaired Student's t test.
10. Young female mice were housed away from males and had no contact with males or their urine until day 27, when C57BL/6J breeder males were housed with the females (1 male per cage) to initiate mating. Plugged females were then housed individually in separate cages to assess whether the copulatory plug was associated with a successful pregnancy. PBS or leptin treatment was continued for 20 days after detection of the plug.
11. C57BL/6J females (n = 24) weighing 9.9 ± 0.2 g (mean ± SEM) were weaned at 21 days and divided equally into PBS and leptin groups. The mice were treated (9) for 8 days and killed in the afternoon of day 29; then blood, ovaries, oviducts, and uteri were collected. Each organ was dissected under a binocular microscope to ensure removal of contaminating tissues. Organs were weighed on a Mettler AE160 high-precision analytical balance.
12. In another experiment (10) not shown in Fig. 2A, 7 of 13 leptin-treated mice and 3 of 12 PBS-treated mice showed vaginal opening at 26 days, whereas the remaining mice in both groups showed vaginal opening at 27 days. The variability between experiments may be due to differences in initial body weights of the mice. Vaginal smears were examined daily after vaginal opening, and the relative abundance of leukocytes, nucleated epithelial cells, and cornified cells was determined independently by two investigators.
13. Plasma samples were assayed for LH (50 μl) and 17β-estradiol (200 μl) with, respectively, a mouserat immunoassay (Peninsula Laboratories, Belmont, CA) and a radioimmunoassay (Diagnostic System Laboratories, Webster, TX).
14. C57BL/6J females (n = 24) were weaned at 24 days of age and divided equally into PBS- and leptin-treated groups. Blood was collected by cardiac puncture at 30, 35, and 39 days of age about 20 hours after the last leptin injection (four mice per time point; one mouse from each cage). Plasma was separated from the cells and frozen at -20°C until use. Endogenous leptin was measured by a mouse-specific radioimmunoassay (Linco Research).
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19. We thank R. Weiner and M. Grumbach for suggestions and comments. Supported by NIH grant HLS 53762 (F.F.C.) and NIH training grant DK 07636 (K.M.).