Lack of coexpression of leptin receptor (LepR) and Kiss1 before puberty.

<p>A-F. Fluorescence photomicrographs showing the distribution of Kiss1 (hrGFP) and LepR (tdTomato) in prepubertal (21 days of age, P21) (A, C, E) and ovariectomized adult female mice (B, D, F). Note the lack of colocalization of Kiss1 and LepR in prepubertal mice and the higher colocalization rate in ovariectomized adult female mice (arrows indicate dual labeled neurons). Scale bar: A–F  =  200 µm. 3V, third ventricle; ME, median eminence.</p

Re-expression of LepR selectively in Kiss1 neurons causes no amelioration of the reproductive or metabolic phenotype of LepR null mice.

<p>A. Agarose gel showing Cre-induced DNA recombination (higher band) of LepR<i>^Lox</i>^TB in the hypothalamus and testis (but not in the white adipose tissue and tail) of Kiss1-Cre LepR null mice. B. Survival graphs showing the progression of vaginal opening and pregnancy in wild type, LepR null and Kiss1-Cre LepR null mice; C. Image comparing the size of the uterus of a wild type female on diestrus and adult Kiss1-Cre LepR null mice; D. Image showing sections of the ovary of a female on diestrus and of an adult Kiss1-Cre LepR null female. Note the presence of corpora lutea (CL) only in the ovary of the wild type female mice. E. Graph showing the progression of body weight of wild type, LepR null and Kiss1-Cre LepR null female mice. F. Bar graphs showing body composition (percentage of fat and lean mass) of LepR null (black) and Kiss1-Cre LepR null (red) mice at 3 different ages: 20 weeks, 35 weeks (males) and 28 weeks (females).</p

Lack of re-expression of functional LepR in Kiss1-Cre LepR null mice.

<p>A-C. Brightfield photomicrographs showing the distribution of leptin-induced phosphorylation of STAT3 immunoreactivity (pSTAT3-ir) in the arcuate nucleus (Arc) of wild type female mice on diestrus (A) and lack of pSTAT3-ir in the Arc of LepR null (B) and of Kiss1-Cre LepR null (C) adult female mice; <b>D-G.</b> Identification of Kiss1-Cre/GFP cells for whole-cell patch-clamp recordings. (D) Brightfield illumination showing a targeted neuron; (E) the same neuron under fluorescent (FITC) illumination; (F) complete dialysis of AlexaFluor 594 from the intracellular pipette at the end of the recording; (G) colocalization of AlexaFluor 594 and GFP. <b>H</b>. A current-clamp recording demonstrates that leptin (100 nM) depolarizes Kiss1-Cre/GFP neurons. The dashed line indicates the resting membrane potential. Scale bar: A–C  =  400 µm.</p

Kiss1 neurons are not responsive to leptin before completion of sexual maturation.

<p>A-B. Brightfield and fluorescence photomicrographs showing lack of colocalization of Kiss1-Cre GFP and LepR mRNA (A) or leptin-induced phosphorylation of STAT3 (B) in prepubertal mice. <b>C-H.</b> Validation of Kiss1 human renilla GFP (Kiss1-hrGFP) mouse model. To optimize the detection of Kiss1 mRNA, we performed colocalization studies (Kiss1 mRNA and hrGFP) in ovariectomized (OVX) and OVX estrogen primed (OVX+E2) mice. Virtually all Kiss1 neurons in the anteroventral periventricular nucleus, anterior periventricular nucleus (AVPV/PeN) and arcuate nucleus (Arc) of OVX+E2 and OVX mice respectively coexpressed hrGFP immunoreactivity. C-D, F-G. Brightfield photomicrographs showing distribution of hrGFP immunoreactivity in the AVPV and Arc of OVX (C, F) and OVX+E2 (D, G) mice. Note changes in hrGFP expression due to sex steroids manipulation. E, H. Higher magnification of D and F, respectively (arrows indicate same cells), showing coexpression of hrGFP-ir and Kiss1 mRNA in the AVPV of OVX+E2 mice (E) and in the Arc of OVX mice (H). Scale bar: A-B  =  200 µm; C-D, F-G  =  400 µm; E, H  =  80 µm. 3V, third ventricle.</p

Prepubertal and leptin signaling-deficient mice display decreased numbers of Kiss1 neurons.

<p>A-F. Fluorescence photomicrographs showing the distribution of Cre activity (Kiss1 reporter gene) in adult (WT on diestrus and Kiss1-Cre LepR null) and prepubertal (WT) female mice. <b>G-I.</b> Bar graphs showing quantification of Kiss1-Cre tdTomato neurons in prepubertal and adult WT female mice and in adult Kiss1-Cre LepR null female mice. Note higher numbers of Kiss1-Cre tdTomato neurons in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (Arc) of adult WT mice on diestrus compared to adult Kiss1-Cre LepR null and prepubertal WT mice. Scale bar: A–F  =  400 µm. 3V, third ventricle.</p