RESUMO
An experiment was carried out to determine the pattern of follicular expression of mRNAs for aromatase, IGF-I receptor (IGF-IR), IGF-binding protein (IGFBP)-2, -4 and -5, leptin and the long form of the leptin receptor (Ob-Rb) in ten ewes infused with human recombinant leptin (n = 5; 1 microg/h) or saline (n = 5) for 72 h in the luteal phase of the oestrous cycle. At the end of infusion a follicular phase was induced with a luteolytic dose of a prostaglandin F2alpha analogue and the ovaries were collected 32 h later. One ovary from each ewe was serially sectioned at 10 microm using a cryostat at -20 degrees C. All follicles >1 mm in diameter were counted and probed with specific oligoprobes for aromatase, IGF-IR and IGFBP-2, -4 and -5 and specific riboprobes for leptin and Ob-Rb. Leptin mRNA was detected in theca and granulosa cells and Ob-Rb mRNA was detected only in granulosa cells, of some, but not all antral follicles. Leptin doubled the number of follicles with a diameter >/=3.5 mm (1.0 +/- 0.36 (s.e.m.) vs 2.4 +/- 0.24; control vs leptin; P < 0.02) but had no effect on the number of >/=1 < 3.5 mm follicles. Leptin had no effect on the number of follicles expressing aromatase mRNA but it decreased significantly the number of follicles expressing mRNA for IGF-IR (10.7 +/- 0.79 vs 7.4 +/- 0.81; control vs leptin; P < 0.05), IGFBP-2 (10.0 +/- 0.82 vs 5.2 +/- 0.87; control vs leptin; P < 0.05) and IGFBP-5 (5.2 +/- 1.60 vs 1.2 +/- 0.30; control vs leptin; P < 0.05). Leptin increased the diameter of IGFBP-2 mRNA-positive follicles (1.5 +/- 0.15 vs 2.2 +/- 0.31 mm; control vs leptin; P < 0.05) and increased follicular mRNA expression for IGFBP-2 (0.30 +/- 0.021 vs 0.39 +/- 0.027 arbitrary units; control vs leptin; P < 0.05) and IGFBP-5 (0.46 +/- 0.019 vs 0.25 +/- 0.053 arbitary units; control vs leptin; P < 0.05). The mRNA for IGFBP-4 was detected in the theca of only two follicles from the control group. Leptin increased the number of follicles expressing Ob-Rb mRNA (0.25 +/- 0.25 vs 1.40 +/- 1.17; control vs leptin; P < 0.05) but had no effect on the number expressing leptin mRNA. Leptin decreased plasma concentrations of oestradiol (P < 0.05) and increased concentrations of FSH (P < 0.001) and insulin (P < 0.001), with no effect on glucose concentrations. These data show that: (i) ovine granulosa cells express mRNA for Ob-Rb and leptin and (ii) leptin increased the number of follicles >/=3.5 mm. Furthermore, the data suggest that suppression of oestradiol production by leptin is not mediated by inhibition of aromatase gene expression. Finally, the data indicate that the action of leptin in ovarian follicles is mediated by the IGF system, because leptin increased mRNA expression of IGFBP-2 and -5. Leptin also decreased the number of follicles expressing IGF-IR and IGFBP-2 and -5. We suggest that these actions of leptin on the IGF system decrease the bioavailability of IGF-I, resulting in decreased oestradiol production.