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. 2014 Feb;28(2):225-38.
doi: 10.1210/me.2013-1319. Epub 2014 Jan 1.

Disrupted kisspeptin signaling in GnRH neurons leads to hypogonadotrophic hypogonadism

Horacio J Novaira  1 , Momodou L Sonko, Gloria Hoffman, Yongbum Koo, Chemyong Ko, Andrew Wolfe, Sally Radovick
Affiliations

Disrupted kisspeptin signaling in GnRH neurons leads to hypogonadotrophic hypogonadism

Horacio J Novaira et al. Mol Endocrinol. 2014 Feb.

Abstract

Landmark studies have shown that mutations in kisspeptin and the kisspeptin receptor (Kiss1r) result in reproductive dysfunction in humans and genetically altered mouse models. However, because kisspeptin and its receptor are present in target cells of the central and peripheral reproductive axis, the precise location(s) for the pathogenic signal is unknown. The study described herein shows that the kisspeptin-Kiss1r signaling pathway in the GnRH neuron is singularly critical for both the onset of puberty as well as the attainment of normal reproductive function. In this study, we directly test the hypothesis that kisspeptin neurons regulate GnRH secretion through the activation of Kiss1r on the plasma membrane of GnRH neurons. A GnRH neuron-specific Kiss1r knockout mouse model (GKirKO) was generated, and reproductive development and phenotype were assessed. Both female and male GKirKO mice were infertile, having low serum LH and FSH levels. External abnormalities such as microphallus and decreased anogenital distance associated with failure of preputial gland separation were present in GKirKO males. A delay in pubertal onset and abnormal estrous cyclicity were observed in female GKirKO mice. Taken together, these data provide in vivo evidence that Kiss1r in GnRH neurons is critical for reproductive development and fertility.

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Figures

Figure 1.
Figure 1.
Development of GnRH-specific Kiss1r knockout mouse. A, Schematic diagrams of constructs used to generate GKirKO mice. Mice bearing LoxP sites flanking exon 2 of the Kiss1r were crossed with transgenic mice expressing Cre recombinase specifically in GnRH neurons. Primers used in PCR genotyping are labeled P1 (exon 1) and P3 (exon 3). X, XbaI; A, Asp718I; S, SpeI restriction enzyme sites. B, Genotyping by PCR analysis of the genomic DNA produced a band migrating at 2096 bp in the mice bearing a floxed allele and a band at 1882 bp in WT mice. These primers were designed to produce a 1120-bp band after excision of the sequence between the LoxP sites in DNA extracted from the hypothalamus (that is, tissue that express Cre recombinase). C, Southern blot analysis was performed by digesting the DNA with Asp718I and detected a band of 9.2 kb in the floxed allele and 12.2 kb in the WT allele with probe 1. D, qPCR analysis of Kiss1r mRNA extracted from male and female mouse tissues (P ≤ .001, n = 3). WT, wild-type; Kiss1rWT/fl, heterozygous floxed; Kiss1rfl/fl, homozygous floxed; ND, not detected.
Figure 2.
Figure 2.
External anatomical abnormalities in GKirKO mice and puberty assessment. A, External anatomic abnormalities were not observed in GKirKO female mice. B, GKirKO males exhibited microphallus and decreased anogenital distance. Delayed puberty in GKirKO mice. C, Graphic representation of the time course of the day of VO in females (n = 5–12). D, Time course of the day of PPS in males (n = 7–15). E, Body weight change over time in female mice (n = 4–7). F, Body weight change over time in male mice (n = 9).
Figure 3.
Figure 3.
GKirKO mice have an abnormal estrous cycle. A, Graphic representation of the estrous cycle in WT and GKirKO mice determined by vaginal cytology followed for 12 days (n = 5). B–E, Baseline serum LH and FSH levels in female (left, n = 6–8) and male (right, n = 3–10) mice. Assay detection limit = 0.048 ng/mL. F and H, GnRH stimulation test. Evaluation of serum LH levels 20 minutes after injection of GnRH agonist (0.1 ng/g via ip). Increased serum LH levels in mice treated with GnRH agonist (indicated as GnRH +) was observed in both genders of WT and GKirKO (n = 7–9). G and I, Kisspeptin stimulation test. Evaluation of serum LH levels 10 minutes after injection of kisspeptin-10 (1 nmol ip). No LH response to kisspeptin (indicated as Kiss +) was observed in female (n = 7–8) or male (n = 3–10) GKirKO mice. Significant differences compared with saline-treated control groups (indicated as GnRH − or Kiss −): *, P ≤ .05; **, P ≤ .01; ***, P ≤ .001. Two-way ANOVA showed an interaction between genotype and treatment in F (P ≤ .05), G (P ≤ .01), and I (P ≤ .001), an effect of treatment in F (P ≤ .01), G (P ≤ .05), H (P ≤ .001), and I (P ≤ .001) and an effect of genotype in G (P ≤ .001) and I (P ≤ .001).
Figure 4.
Figure 4.
Matrix of the representative breeding study. Six WT and GKirKO female mice were paired with 3 WT and GKirKO males for 14 days and then returned to their own cages for 3 weeks to allow for birth of pups, indicating a successful pairing. Each row represents 1 individual female, and each box represents one of her pairings. Black boxes represent a successful pregnancy and white boxes a lack of pregnancy noted.
Figure 5.
Figure 5.
Gross gonadal anatomy and gonadal histology of GKirKO mice. A, Small ovaries (size and weight) and uterus found in GKirKO mice compared with WT. B, Smaller size and weight of the testes in GKirKO male mice compared with WT mice. C, Representative sections from a WT ovary (left image) showing follicles at all stages of development, including primary, preantral, antral, preovulatory follicles as well as corpora lutea (CL); GKirKO mice (right image), in contrast, do not contain follicles past the antral stage and have no corpora lutea formation. Scale bars correspond to 100 μm. The most representative microphotographs were chosen. D, Representative sections of testes. Seminiferous tubules from a WT mouse (left, top image) show all stages of spermatogenesis with numerous sperm. In contrast, seminiferous tubules from GKirKO mice (right, top image) lack spermatogenesis. Representative sections of the epididymis: the lumen from a WT mouse is filled with sperm (left, bottom image), whereas that of GKirKO mice have fewer sperm (right, bottom image). Scale bars represent 50 μm.
Figure 6.
Figure 6.
Reduced estradiol negative feedback in GKirKO mice. A and B, Serum LH and FSH levels in intact WT control mice in metestrus and intact GKirKO mice (noncycling) compared with OVX WT control mice and OVX GKirKO mice, respectively (n = 4–11). C and D, Kiss1 mRNA in AVPV nucleus and ARC nucleus of intact WT control mice in metestrus and intact GKirKO mice compared with OVX WT control mice and OVX GKirKO mice, respectively (n = 4–5). Significant differences compared with intact WT and GKirKO control groups: *, P ≤ .05; **, P ≤ .01; ***, P ≤ .001. Two-way ANOVA showed an interaction between genotype and treatment in A (P ≤ .001) and B (P ≤ .001), an effect of treatment in A, B, C, and D (P ≤ .001) and an effect of genotype in A and B (P ≤ .001).
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