To investigate the expression of leptin receptors (Ob-R) in the rat hypothalamus–pituitary–ovarian axis, immature rats were treated with eCG/hCG and Ob-R expression was evaluated by western blot analysis. The Ob-R expression increased 24 h after eCG administration in all the tissues assayed. In the hypothalamus, these levels immediately decreased to those obtained without treatment. In the pituitary, the Ob-R expression continued to be elevated 48 h after eCG administration, whereas the hCG injection did not modify these levels. Similar results were obtained with the ovarian long isoform. To assess the effect of leptin on its receptors, Ob-R was assessed in hypothalamus, pituitary and ovarian explants cultured in the presence or absence of leptin (0.3–500 ng/ml). In the hypothalamus, we found a biphasic effect: the Ob-R expression was either reduced or increased at low or high concentrations of leptin respectively. LH-releasing hormone secretion increased at 1 ng/ml. In the pituitary, Ob-R increased at 10 or 30 ng/ml of leptin for the long and short isoforms respectively. Leptin also induced an increase in LH release at 30 ng/ml. In the ovarian culture, the presence of leptin produced an increase in Ob-R expression at different ranges of concentrations and a dose-dependent biphasic effect on the progesterone production. In conclusion, all these results clearly suggest that leptin is able to modulate the expression of its own receptors in the reproductive axis in a differential way. Moreover, the positive or negative effect that leptin exerts on the ovulatory process may be dependent on this regulation.

Leptin exerts both stimulatory and inhibitory effects on the ovulatory process. In this study, we investigated whether these opposite effects involve changes in the oxidative status in response to different levels of leptin. To this end, we performed both in vivo and in vitro assays using ovaries of immature rats primed with gonadotropins to induce ovulation. Superoxide dismutase (SOD) and catalase (CAT) activity, lipid peroxidation, glutathione (GSH) content, and reactive oxygen species (ROS) were studied as oxidative damage-related parameters. The expression of BCL2, BAX, and caspase 3 were measured by western blot as apoptosis-related biomarkers. The acute treatment with leptin, which inhibits ovulation, decreased SOD activity and increased active caspase 3 expression. No differences were found in CAT activity, lipid peroxidation, or total GSH. In contrast, the daily administration of leptin, which induces ovulation, decreased GSH content, ROS levels, and Bax and active caspase 3 expression, but caused no changes in other parameters. In addition, the daily administration of leptin induced follicular growth, measured by the number of antral follicles in ovarian sections. Using ovarian explant cultures, we found increased BCL2 expression and decreased SOD activity at low and high concentrations of leptin respectively. Thus, leptin can modulate the oxidative status of the ovarian tissue, during the ovulatory process, by acting on different targets according to its circulating levels. At low concentration, leptin seems to play a protective role against the oxidative stress, whereas at high concentrations, this protein seems to be involved in cell death.

Leptin, a protein secreted by different tissues, is able to exert both stimulatory and inhibitory effects on the ovulatory process. Thus, we investigated whether these opposite effects involve changes in the ovarian signalling pathways in response to different levels of leptin. To this end, we performed both in vivo and in vitro assays using immature rats primed with gonadotrophins to induce ovulation. The acute treatment with leptin, which inhibits the ovulatory process, caused a significant decrease in the phosphorylation of both STAT3 and ERK1/2 and a simultaneous increase in suppressors of cytokine signalling 3 (SOCS3) protein. However, daily administration of a low dose of leptin, which induces the ovulatory process, showed increased phosphorylation of both STAT3 and ERK1/2 and a decreased expression of SOCS3 protein. Using ovarian explant cultures, we also found that leptin was able to activate both STAT3 and ERK1/2 at 10 ng/ml but only STAT3 at 300–500 ng/ml. In addition, at 100–300 ng/ml, leptin increased protein but not mRNA expression of SOCS3. The addition of specific inhibitors of JAK/STAT and MAPK signalling pathways suppressed both the increase and the decrease in leptin-induced progesterone secretion. These results indicate that i) different levels of leptin are able to regulate STAT3, ERK1/2 and SOCS3 at both intra- and extra-ovarian level and that ii) the dual action of leptin on steroidogenesis seems to occur, at least in part, through both the ERK and STAT cascades.