Leptin actions through the nitrergic system to modulate the hypothalamic expression of the kiss1 mRNA in the female rat.

Gonadotrophin-releasing hormone (GnRH) is the main controller of the reproductive axis and stimulates the synthesis and secretion of gonadotrophins. Estrogen is the main peripheral factor controlling GnRH secretion, and this action is mainly mediated by the transsynaptic pathway through nitric oxide, kisspeptin, leptin, among other factors. Kisspeptin is the most potent factor known to induce GnRH release. Nitric oxide and leptin also promote GnRH release; however, neurons expressing GnRH do not express the leptin receptor (OB-R). Leptin seems to modulate the expression of genes and proteins involved in the kisspeptin system. However, few kisspeptin-synthesizing cells in the arcuate nucleus (ARC) and few cells, if any, in the preoptic area (POA) express OB-R; this indicates an indirect mechanism of leptin action on kisspeptin. Nitric oxide is an important intermediate in the actions of leptin in the central nervous system. Thus, this work aimed to verify the numbers of nNOS cells were activated by leptin in different hypothalamic areas; the modulatory effects of the nitrergic system on the kisspeptin system; and the indirect regulatory effect of leptin on the kisspeptin system via nitric oxide. Ovariectomized rats were treated with estrogen or a vehicle and received an intracerebroventricular (i.c.v.) injection of a nitric oxide donor, leptin or neuronal nitric oxide synthase (nNOS) enzyme inhibitor. Thirty minutes after the injection, the animals were decapitated. Leptin acts directly on nitrergic neurons in different hypothalamic regions, and the effects on the ventral premammillary nucleus (PMV) and ventral dorsomedial hypothalamus (vDMH) are enhanced. The use of a nitric oxide donor or the administration of leptin stimulates the expression of the kisspeptin mRNA in the ARC of animals with or without estrogenic action; however, these changes are not observed in the POA. In addition, the action of leptin on the expression of the kisspeptin mRNA in the ARC is blocked by a nitric oxide synthesis inhibitor. We concluded that the effects of leptin on the central nervous system are at least partially mediated by the nitrergic system. Also, nitric oxide acts on the kisspeptin system by modulating the expression of the kisspeptin mRNA, and leptin at least partially modulates the kisspeptin system through the nitrergic system, particularly in the ARC.

Estrogen-dependent post-translational change in the nitric oxide system may mediate the leptin action on LH and prolactin secretion.

Gonadotrophin-releasing hormone (GnRH) neurons do not express the leptin receptor (OB-R) in the medial preoptic area (MPOA) and the medial basal hypothalamus (MBH). We assessed whether the effect of leptin on the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in proestrus could be mediated by nitric oxide (NO) under estrogen modulation. Female rats were treated with an estrogen antagonist (tamoxifen s.c. 3mg/rat) or vehicle during metestrus and diestrus. At proestrus, they received leptin (3 or 10 µg/µl) or intracerebroventricular saline at 11:00 am and were decapitated at 5:00 pm. The following were analyzed in this work: plasma LH, FSH and PRL levels (radioimmunoassay); neuronal NO-synthase (nNOS) and OB-R transcription (RT-PCR); nNOS and phosphorylated nNOS (pnNOS) translation levels (western blotting); and pSTAT3 immunoreactivity. Tamoxifen reduced the plasma LH and PRL levels and decreased the nNOS mRNA and pnNOS expression in the MPOA. Three micrograms of leptin increased the LH secretion and pnNOS protein levels in the MPOA and MBH. Ten micrograms of leptin decreased the transcription, translation and phosphorylation of nNOS in the MPOA. In the MBH, 10 µg of leptin increased the protein expression of nNOS but not the mRNA expression neither pnNOS protein. Tamoxifen did not change either the mRNA or protein expression of nNOS or the phosphorylation of nNOS but decreased the number of cells that contained pSTAT3 immunoreactivity in both areas. In conclusion, the stimulatory effect of leptin on the secretion of LH and PRL on the afternoon of proestrus may be mediated by estrogen-dependent post-translational changes in the nNOS in the MPOA and MBH.

Transitory activation of the central and ovarian norepinephrine systems during cold stress-induced polycystic ovary in rats.

Cold stress-induced ovarian sympathetic activation is associated with the development of ovarian cysts in rats. Although we have hypothesised that polycystic ovary (PCO) features induced by cold stress, as prevented by lesion of the noradrenergic nucleus locus coeruleus (LC), were a result of the increased activity of the ovarian norepinephrine (NE) system, this was not evident after 8 weeks of stress. In the present study, we investigated the temporal changes in LC and ovarian NE activities and steroid secretion in rats exposed to single (SS) or repeated (RS) cold stress. SS and 4 week (4W)-RS but not 8 week (8W)-RS increased c-Fos expression in the LC and ovarian NE release. Plasma oestradiol, testosterone and progesterone levels tended to increase in 4W-RS and were elevated in 8W-RS rats, which displayed PCO morphology. ß-adrenergic receptor agonist increased steroid hormone release from the ovary of unstressed (US) but not from 8W-RS rats. To determine whether increased activity of noradrenergic system during the initial 4 weeks of RS would be sufficient to promote PCO, rats were exposed to 4 weeks of cold stress and kept in ambient temperature for the next 4 weeks (4W-RS/4W-US). Accordingly, PCO morphology, increased steroid secretion and decreased ovulation rate were found in 4W-RS/4W-US rats, strengthening the hypothesis that the initial increase in NE release triggers the development of PCO. The correlated activity of LC neurones and ovarian noradrenergic terminals and the induction of PCO in 4W-RS/4W-US rats provide functional evidence for a major role of NE in disrupting follicular development and causing the long-lasting endocrine abnormalities found in stress-induced PCO.

Co-expression of leptin and oestrogen receptors in the preoptic-hypothalamic area.

The interaction between the reproductive axis and energy balance suggests that leptin acts as a possible mediator. This hormone acts in the regulation of metabolism, feeding behaviour and reproduction. Animals homozygous for the gene 'ob' (ob/ob) are obese and infertile, and these effects are reversed after systemic administration of leptin. Thus, the present study aimed to determine: (i) whether cells that express leptin also express oestrogen receptors of type-alpha (ER-alpha) or -beta (ER-beta) in the medial preoptic area (MPOA) and in the arcuate (ARC), dorsomedial (DMH) and ventromedial hypothalamic nucleus and (ii) whether there is change in the gene and protein expression of leptin in these brain areas in ovariectomised (OVX) animals when oestrogen-primed. Wistar female rats with normal oestrous cycles or ovariectomised oestrogen-primed or vehicle (oil)-primed were utilised. To determine whether there was a co-expression, immunofluorescence was utilised for double staining. Confocal microscopy was used to confirm the co-expression. The technique of real-time polymerase chain reaction and western blotting were employed to analyse gene and protein expression, respectively. The results obtained showed co-expression of leptin and ER-alpha in the MPOA and in the DMH, as well as leptin and ER-beta in the MPOA, DMH and ARC. However, we did not detect leptin in the MPOA, ARC and DMH using western blotting and there was no statistical difference in leptin gene expression in the MPOA, DMH, ARC, pituitary or adipose tissue between OVX rats treated with oestrogen or vehicle. In conclusion, the results obtained in the present study confirm that the brain is also a source of leptin and reveal co-expression of oestrogen receptors and leptin in the same cells from areas related to reproductive function and feeding behaviour. Although these data corroborate the previous evidence obtained concerning the interaction between the action of brain leptin and reproductive function, the physiological relevance of this interaction remains uncertain and additional studies are necessary to elucidate the exact role of central leptin.