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

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

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

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

PGR does not regulate <i>Kiss1</i> gene expression.

<p><i>Kiss1</i> mRNA expression level in 6-month-old WT (n = 9) and Kiss1-PgrKO (n = 4) mice.</p

Kiss1-PgrKO mice have impaired kisspeptin-mediated GnRH release.

<p>A. Serum LH before and after stimulation of kisspeptin neurons by exogenous administration of estradiol in 6-8-month-old ovariectomized WT and Kiss1-PgrKO mice (n = 3); <i>a</i> represent statistical difference between WT, and <i>b</i> represents statistical difference between Kiss1-PgrKO mice (<i>p</i> = 0.037 and <i>p</i> = 0.01, respectively). B. Serum LH concentration 10 min after GnRH stimulation in 5-month-old WT (n = 3) and Kiss1-PgrKO (n = 6) mice (<i>p</i> = 0.47). C. Gross morphology, histology, oocyte morphology, and box plot data representation of oocytes from 5-month-old WT (n = 3) and Kiss1-PgrKO (n = 6) mice after ovarian hyperstimulation with gonadotropins; grossly corpora lutea are small, round, tan, nodular structures that can be seen bulging from the ovarian surface (black arrows), and histologically corpora lutea are characterized as multiple large, round, nodular structures that expand the ovarian cortex (<i>CL</i>–corpora lutea). Gross image scale bar = 1 mm, histology scale bar = 100 μm, oocyte scale bar = 250 μm.</p

Loss of Fertility in the Absence of Progesterone Receptor Expression in Kisspeptin Neurons of Female Mice

<div><p>Ovarian steroids, estradiol and progesterone, play central roles in regulating female reproduction by acting as both positive and negative regulators of gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. Recent studies have identified kisspeptin neurons of the hypothalamus as the target of estrogenic regulation of GnRH secretion. In this study, we aimed to determine the significance of progesterone receptor (PGR) expression in the kisspeptin neurons. To this end, the <i>Pgr</i> gene was selectively ablated in mouse kisspeptin neurons and the reproductive consequence assessed. The hypothalamus of the <i>Pgr</i> deficient female mouse expressed kisspeptin, the pituitary released LH in response to GnRH stimulation, and the ovary ovulated when stimulated with gonadotropins. However, the mutant mouse gradually lost cyclicity, was unable to generate a LH surge in response to rising estradiol, and eventually became infertile. Taken together, these results indicate that the loss of PGR impairs kisspeptin secretory machinery and therefore that PGR plays a critical role in regulating kisspeptin secretion.</p></div

Quantification of kisspeptin positive immunofluorescent cells in the AVPV of WT and Kiss1-PgrKO mice.

<p>A. Immunofluorescence of kisspeptin in the AVPV of 5-month-old WT and Kiss1-PgrKO; arrows indicate kisspeptin positive cells (bar = 25 μ). B. Quantification of the raw cell number of kisspeptin positive cells from the AVPV of 2-3- and 5-month-old WT and Kiss1-PgrKO mice; the raw cell number was calculated from 3 sections per animal. Data represent means ± SD.</p

PGR and kisspeptin are co-localized with ERα in kisspeptin neurons but are not expressed in AVPV kisspeptin neurons of ERαKO mice.

<p>A. Double immunohistochemistry of PGR and kisspeptin in WT and ERαKO mice. Black arrows indicate dark brown nuclear PGR staining. Black arrowheads indicate brown red cytoplasmic kisspeptin staining. Open arrowhead indicates hematoxylin-stained nucleus of a kisspeptin positive and PGR negative neuron. Bar = 25 μm. B. Co-localization of PGR, ERα, and kisspeptin in kisspeptin neuron in the hypothalamic AVPV nucleus from a 2-month-old female C57BL/6 mouse in the estrus stage. From left to right: PGR (blue), kisspeptin (red), ERα (green), and merged image. Yellow arrows indicate nuclear PGR, ERα, and merged PGR/ERα. Green arrows indicate cytoplasmic kisspeptin. White arrows indicate ERα positive nucleus.</p