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. 2014 Jun;124(6):2550-9.
doi: 10.1172/JCI65928. Epub 2014 May 8.

Leptin-dependent neuronal NO signaling in the preoptic hypothalamus facilitates reproduction

Nicole Bellefontaine, Konstantina Chachlaki, Jyoti Parkash, Charlotte Vanacker, William Colledge, Xavier d'Anglemont de Tassigny, John Garthwaite, Sebastien G Bouret, Vincent Prevot

Leptin-dependent neuronal NO signaling in the preoptic hypothalamus facilitates reproduction

Nicole Bellefontaine et al. J Clin Invest. 2014 Jun.

Erratum in

  • J Clin Invest. 2014 Aug 1;124(8):3678

Abstract

The transition to puberty and adult fertility both require a minimum level of energy availability. The adipocyte-derived hormone leptin signals the long-term status of peripheral energy stores and serves as a key metabolic messenger to the neuroendocrine reproductive axis. Humans and mice lacking leptin or its receptor fail to complete puberty and are infertile. Restoration of leptin levels in these individuals promotes sexual maturation, which requires the pulsatile, coordinated delivery of gonadotropin-releasing hormone to the pituitary and the resulting surge of luteinizing hormone (LH); however, the neural circuits that control the leptin-mediated induction of the reproductive axis are not fully understood. Here, we found that leptin coordinated fertility by acting on neurons in the preoptic region of the hypothalamus and inducing the synthesis of the freely diffusible volume-based transmitter NO, through the activation of neuronal NO synthase (nNOS) in these neurons. The deletion of the gene encoding nNOS or its pharmacological inhibition in the preoptic region blunted the stimulatory action of exogenous leptin on LH secretion and prevented the restoration of fertility in leptin-deficient female mice by leptin treatment. Together, these data indicate that leptin plays a central role in regulating the hypothalamo-pituitary-gonadal axis in vivo through the activation of nNOS in neurons of the preoptic region.

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Figures

Figure 1
Figure 1. Leptin activates nNOS in the preoptic region and increases circulating LH levels.
(A) Representative Western blots for phosphorylated and total nNOS at the times indicated (in minutes) following leptin treatment (see Supplemental Figure 4 for full-length photographs of the Western blots). Leptin promotes the phosphorylation of nNOS acutely at 15 minutes. (B) Coronal sections of the OVLT, showing an increase in the percentage of nNOS cells expressing P-nNOS immunoreactivity (ir) 15 minutes after leptin stimulation. 3V, third ventricle; oc, optic chiasm. Scale bar: 100 μm. (C) Quantification of immunolabeling shown in B. (D) Circulating LH levels surge 15 minutes after leptin administration. *P < 0.05.
Figure 2
Figure 2. The blockade of nNOS blunts leptin’s ability to induce LH release.
(A) nNOS activity is required for leptin-induced LH release, independently of kisspeptin/GPR54 signaling. (B) The pharmacological inhibition of nNOS, specifically within the preoptic region (POA), is sufficient to block leptin-induced LH release. The diagram and the corresponding photomicrograph show the target site into which l-NAME was stereotaxically infused in the preoptic region, in which GnRH (green dots) and nNOS (red dots) neuronal cell bodies are intermingled. ac, anterior commissure. The asterisk in the image indicates the trajectory of the implanted needle. Scale bar: 500 μm. (C) Schematic representation of the hypothetical mechanisms of regulation of LH secretion in the absence (top panel) or presence (bottom panel) of exogenous leptin treatment in diestrus mice (see Supplemental Figure 2 for mathematical modeling). *P < 0.05, **P < 0.01.
Figure 3
Figure 3. The pharmacological inhibition of nNOS with l-NAME prevents the rescue of the estrous cycle and LH levels by leptin in fasted mice.
(A) Representative 15-day estrous cycles of animals subjected or not to 24-hour fasting in diestrus 1 three days before death (gray shadow). Mice were subjected to leptin (red arrows), l-NAME (blue arrows), or vehicle (white arrows) injection twice daily on diestrus 1 three days before death. Each circle represents 1 day. Di, diestrus; P, proestrus; E, estrus. (B) Leptin treatment in fasting mice rescued proestrous-like uterine weight (UW), whereas concomitant l-NAME injection blunted this effect. (C) Leptin treatment restores surge levels of LH in mice subjected to 24-hour fasting in diestrus 1 and killed on the expected day of proestrus, i.e., 2 days after. l-NAME treatment impedes this leptin-rescuing effect in fasting mice. *P < 0.05, ***P < 0.001, leptin vs. vehicle.
Figure 4
Figure 4. A deficiency in NO signaling renders leptin unable to induce puberty in Lepob/ob mice.
(A) Nos1–/– Lepob/ob mice displayed a lower body weight than Nos1+/+ Lepob/ob littermates prior to the leptin regimen, although both groups lost weight with leptin treatment. (B) Nos1–/– Lepob/ob mice never underwent first vaginal estrus. ND, not detected. (C) No difference in body weight was observed between Lepob/ob mice infused with either vehicle or l-NAME, although both groups responded to leptin with a decrease in body weight. (D) The Lepob/ob mice treated chronically with l-NAME never demonstrated first vaginal estrus. (E) Blockade of nNOS in Lepob/ob mice, either by genetic or pharmacologic means, resulted in a lack of estrous cyclicity, while this was corrected in control mice. C, cornified (estrus); N, nucleated (proestrus); L, lymphocytic (diestrus). (F) Blockade of nNOS in Lepob/ob mice leads to nondetectable levels of LH. Nos1+/+ Lepob/ob and Nos1–/– Lep+/+ mice showed partial to full surge-like LH levels following exposure to male odor. (G) Ovarian sections from Nos1+/+ Lepob/ob mice with no leptin treatment, following 28 days of leptin treatment, and Nos1–/– Lepob/ob mice, following 28 days of leptin treatment. Note the presence of corpora lutea (CL) in Nos1+/+ Lepob/ob mice treated with leptin. Scale bar: 100 μm. AF, atretic follicles; i.c., intracranial.
Figure 5
Figure 5. nNOS deficiency does not alter GnRH expression in Lep+/+ and Lepob/ob mice.
(A) The number of GnRH neurons in the preoptic region and (B and C) the density of GnRH-immunoreactive (ir, green) fibers in the median eminence. Interestingly, the Nos1+/+ Lepob/ob mice showed an increased GnRH immunoreactivity, while that of Nos1–/– Lepob/ob and Nos1+/+ Lepob/ob female mice treated with leptin was equivalent to that of wild-type and Nos1–/– Lep+/+ mutant mice. Scale bar: 200 μm. *P < 0.05, **P < 0.01.
Figure 6
Figure 6. Site-specific deletion of LepR disrupts basal LH levels in female mice.
(A) Bilateral injections of TAT-Cre protein into the OVLT/MEPO prevented P-STAT3 45 minutes following peripheral leptin injection in the preoptic region (POA) of Leprfl/fl, but not in Lepr+/+, littermates. However, leptin was still able to induce P-STAT3 in caudal areas of the hypothalamus, such as the ARH and PMv, in TAT-CrePOA Leprfl/fl mice. Scale bar: 200 μm. (B) Body weight did not differ between TAT-CrePOA Lepr+/+ and TAT-CrePOA Leprfl/fl mice. (C) Lack of leptin signaling in the OVLT/MEPO resulted in higher uterine weight in TAT-CrePOA Leprfl/fl mice when compared with that of TAT-Cre–injected wild-type littermates. (D) Strikingly, basal levels of LH were increased in females lacking LepR signaling in the preoptic region when compared with those of wild-type mice injected with TAT-Cre. When injected with leptin, TAT-CrePOA Leprfl/fl mice were unable to respond further with a rise in LH levels. Scale bar: 50 μm. *P < 0.05.
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