Uncovering Novel Reproductive Defects in Neurokinin B Receptor Null Mice: Closing the Gap Between Mice and Men

Patients bearing mutations in TAC3 and TACR3 (which encode neurokinin B and its receptor, respectively) have sexual infantilism and infertility due to GnRH deficiency. In contrast, Tacr3−/− mice have previously been reported to be fertile. Because of this apparent phenotypic discordance between mice and men bearing disabling mutations in Tacr3/TACR3, Tacr3 null mice were phenotyped with close attention to pubertal development, estrous cyclicity, and fertility. Tacr3−/− mice demonstrated normal timing of preputial separation and day of first estrus, markers of sexual maturation. However, at postnatal d 60, Tacr3−/− males had significantly smaller testes and lower FSH levels than their wild-type littermates. Tacr3−/− females had lower uterine weights and abnormal estrous cyclicity. Approximately half of Tacr3−/− females had no detectable corpora lutea on ovarian histology at postnatal d 60. Despite this apparent ovulatory defect, all Tacr3−/− females achieved fertility when mated. However, Tacr3−/− females were subfertile, having both reduced numbers of litters and pups per litter. The subfertility of these animals was not due to a primary ovarian defect, because they demonstrated a robust response to exogenous gonadotropins. Thus, although capable of fertility, Tacr3-deficient mice have central reproductive defects. The remarkable ability of acyclic female Tacr3 null mice to achieve fertility is reminiscent of the reversal of hypogonadotropic hypogonadism seen in a high proportion of human patients bearing mutations in TACR3. Tacr3 mice are a useful model to examine the mechanisms by which neurokinin B signaling modulates GnRH release.

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Neurokinin B Is Critical for Normal Timing of Sexual Maturation but Dispensable for Adult Reproductive Function in Female Mice

Endocrinology ◽

10.1210/en.2014-1862 ◽

2015 ◽

Vol 156 (4) ◽

pp. 1386-1397 ◽

Cited By ~ 30

Author(s):

Cadence True ◽

Sayeda Nasrin Alam ◽

Kimberly Cox ◽

Yee-Ming Chan ◽

Stephanie B. Seminara

Keyword(s):

Sexual Maturation ◽

Pubertal Development ◽

Critical Role ◽

Reproductive Function ◽

Wild Type ◽

Neurokinin B ◽

Gene Encoding ◽

Estrous Cycles ◽

Pubertal Delay ◽

Deficient Mice

Abstract Humans carrying mutations in neurokinin B (NKB) or the NKB receptor fail to undergo puberty due to decreased secretion of GnRH. Despite this pubertal delay, many of these patients go on to achieve activation of their hypothalamic-pituitary-gonadal axis in adulthood, a phenomenon termed reversal, indicating that NKB signaling may play a more critical role for the timing of pubertal development than adult reproductive function. NKB receptor-deficient mice are hypogonadotropic but have no defects in the timing of sexual maturation. The current study has performed the first phenotypic evaluation of mice bearing mutations in Tac2, the gene encoding the NKB ligand, to determine whether they have impaired sexual development similar to their human counterparts. Male Tac2−/− mice showed no difference in the timing of sexual maturation or fertility compared with wild-type littermates and were fertile. In contrast, Tac2−/− females had profound delays in sexual maturation, with time to vaginal opening and first estrus occurring significantly later than controls, and initial abnormalities in estrous cycles. However, cycling recovered in adulthood and Tac2−/− females were fertile, although they produced fewer pups per litter. Thus, female Tac2−/− mice parallel humans harboring NKB pathway mutations, with delayed sexual maturation and activation of the reproductive cascade later in life. Moreover, direct comparison of NKB ligand and receptor-deficient females confirmed that only NKB ligand-deficient animals have delayed sexual maturation, suggesting that in the absence of the NKB receptor, NKB may regulate the timing of sexual maturation through other tachykinin receptors.

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Exogenous Kisspeptin Administration as a Probe of GnRH Neuronal Function in Patients With Idiopathic Hypogonadotropic Hypogonadism

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2014-2233 ◽

2014 ◽

Vol 99 (12) ◽

pp. E2762-E2771 ◽

Cited By ~ 15

Author(s):

Yee-Ming Chan ◽

Margaret F. Lippincott ◽

James P. Butler ◽

Valerie F. Sidhoum ◽

Cindy X. Li ◽

...

Keyword(s):

Neuronal Network ◽

Functional Capacity ◽

Pubertal Development ◽

Hypogonadotropic Hypogonadism ◽

Fundamental Property ◽

Neuronal Function ◽

Idiopathic Hypogonadotropic Hypogonadism ◽

The Subject ◽

Lh Secretion ◽

Robust Response

Context: Idiopathic hypogonadotropic hypogonadism (IHH) results from defective synthesis, secretion, or action of GnRH. Kisspeptin is a potent stimulus for GnRH secretion. Objective: We probed the functional capacity of the GnRH neuronal network in patients with IHH. Participants: Eleven subjects with congenital IHH (9 men and 2 women) and one male subject who underwent reversal of IHH were studied. Six of the twelve subjects had an identified genetic cause of their IHH: KAL1 (n = 1), FGFR1 (n = 3), PROKR2 (n = 1), GNRHR (n = 1). Intervention: Subjects underwent q10 min blood sampling to measure GnRH-induced LH secretion at baseline and in response to intravenous boluses of kisspeptin (0.24 nmol/kg) and GnRH (75 ng/kg) both pre- and post-six days of treatment with exogenous GnRH (25 ng/kg sc every 2 h). Results: All subjects with abiding IHH failed to demonstrate a GnRH-induced LH response to exogenous kisspeptin. In contrast, the subject who achieved reversal of his hypogonadotropism demonstrated a robust response to kisspeptin. Conclusions: The functional capacity of the GnRH neuronal network in IHH patients is impaired, as evidenced by their inability to respond to the same dose of kisspeptin that effects a robust GnRH-induced LH response in healthy men and luteal-phase women. This impairment is observed across a range of genotypes, suggesting that it reflects a fundamental property of GnRH neuronal networks that have not been properly engaged during pubertal development. In contrast, a patient who had experienced reversal of his hypogonadotropism responded to exogenous kisspeptin.

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Kisspeptin cell-specific PI3K signaling regulates hypothalamic kisspeptin expression and participates in the regulation of female fertility

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00385.2014 ◽

2014 ◽

Vol 307 (11) ◽

pp. E969-E982 ◽

Cited By ~ 5

Author(s):

Matthew Beymer ◽

Ariel L. Negrón ◽

Guiqin Yu ◽

Samuel Wu ◽

Christian Mayer ◽

...

Keyword(s):

Intracellular Signaling ◽

Pubertal Development ◽

Cell Number ◽

Pi3k Signaling ◽

Catalytic Subunits ◽

Gnrh Release ◽

Estrous Cyclicity ◽

Males And Females ◽

Specific Deletion

Hypothalamic kisspeptin neurons integrate and translate cues from the internal and external environments that regulate gonadotropin-releasing hormone (GnRH) secretion and maintain fertility in mammals. However, the intracellular signaling pathways utilized to translate such information into changes in kisspeptin expression, release, and ultimately activation of the kisspeptin-receptive GnRH network have not yet been identified. PI3K is an important signaling node common to many peripheral factors known to regulate kisspeptin expression and GnRH release. We investigated whether PI3K signaling regulates hypothalamic kisspeptin expression, pubertal development, and adult fertility in mice. We generated mice with a kisspeptin cell-specific deletion of the PI3K catalytic subunits p110α and p110β (kiss-p110α/β-KO). Using in situ hybridization, we examined Kiss1 mRNA expression in gonad-intact, gonadectomized (Gdx), and Gdx + steroid-replaced mice. Kiss1 cell number in the anteroventral periventricular hypothalamus (AVPV) was significantly reduced in intact females but not in males. In contrast, compared with WT and regardless of steroid hormone status, Kiss1 cell number was lower in the arcuate (ARC) of kiss-p110α/β-KO males, but it was unaffected in females. Both intact Kiss-p110α/β-KO males and females had reduced ARC kisspeptin-immunoreactive (IR) fibers compared with WT animals. Adult kiss-p110α/β-KO males had significantly lower circulating luteinizing hormone (LH) levels, whereas pubertal development and fertility were unaffected in males. Kiss-p110α/β-KO females exhibited a reduction in fertility despite normal pubertal development, LH levels, and estrous cyclicity. Our data show that PI3K signaling is important for the regulation of hypothalamic kisspeptin expression and contributes to normal fertility in females.

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TAC3/TACR3 Mutations Reveal Preferential Activation of GnRH Release by Neurokinin B in Neonatal Life Followed by Reversal in Adulthood

Endocrine Reviews ◽

10.1210/edrv.31.2.9979 ◽

2010 ◽

Vol 31 (2) ◽

pp. 254-255

Author(s):

Elena Gianetti ◽

Cintia Tusset ◽

Sekoni D. Noel ◽

Margaret G. Au ◽

Andrew A. Dwyer ◽

...

Keyword(s):

Hypogonadotropic Hypogonadism ◽

Tertiary Care ◽

Sequence Variants ◽

Neurokinin B ◽

Coding Sequence ◽

Base Pair Deletion ◽

Gnrh Release ◽

Reproductive Life ◽

Tertiary Care Centers

ABSTRACT Context Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established. Objective A broad cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships. Design Sequencing of TAC3/TACR3; in vitro functional assays, neuroendocrine phenotyping. Setting Tertiary care centers world-wide. Patients or other participants 345 probands, 18 family members, 292 controls. Intervention Examination of reproductive phenotypes throughout reproductive life and pre/post therapy. Main Outcome Measure Rare sequence variants in TAC3/TACR3. Results In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants (3 nonsense mutations, 6 non-synonymous, 4 synonymous [one predicted to affect splicing]). In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and 7 females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and 5/7 females were assessed after discontinuation of therapy and 6/7 males and 4/5 females demonstrated evidence for reversibility of their hypogonadotropism. Conclusions Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. While the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time.

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Increased Neurokinin B (Tac2) Expression in the Mouse Arcuate Nucleus Is an Early Marker of Pubertal Onset with Differential Sensitivity to Sex Steroid-Negative Feedback than Kiss1

Endocrinology ◽

10.1210/en.2012-1529 ◽

2012 ◽

Vol 153 (10) ◽

pp. 4883-4893 ◽

Cited By ~ 41

Author(s):

John C. Gill ◽

Víctor M. Navarro ◽

Cecilia Kwong ◽

Sekoni D. Noel ◽

Cecilia Martin ◽

...

Keyword(s):

Negative Feedback ◽

Arcuate Nucleus ◽

Pubertal Development ◽

Differential Sensitivity ◽

Sex Steroid ◽

Wild Type ◽

Neurokinin B ◽

Pubertal Onset ◽

Steroid Dependent

Abstract At puberty, neurokinin B (NKB) and kisspeptin (Kiss1) may help to amplify GnRH secretion, but their precise roles remain ambiguous. We tested the hypothesis that NKB and Kiss1 are induced as a function of pubertal development, independently of the prevailing sex steroid milieu. We found that levels of Kiss1 mRNA in the arcuate nucleus (ARC) are increased prior to the age of puberty in GnRH/sex steroid-deficient hpg mice, yet levels of Kiss1 mRNA in wild-type mice remained constant, suggesting that sex steroids exert a negative feedback effect on Kiss1 expression early in development and across puberty. In contrast, levels of Tac2 mRNA, encoding NKB, and its receptor (NK3R; encoded by Tacr3) increased as a function of puberty in both wild-type and hpg mice, suggesting that during development Tac2 is less sensitive to sex steroid-dependent negative feedback than Kiss1. To compare the relative responsiveness of Tac2 and Kiss1 to the negative feedback effects of gonadal steroids, we examined the effect of estradiol (E2) on Tac2 and Kiss1 mRNA and found that Kiss1 gene expression was more sensitive than Tac2 to E2-induced inhibition at both juvenile and adult ages. This differential estrogen sensitivity was tested in vivo by the administration of E2. Low levels of E2 significantly suppressed Kiss1 expression in the ARC, whereas Tac2 suppression required higher E2 levels, supporting differential sensitivity to E2. Finally, to determine whether inhibition of NKB/NK3R signaling would block the onset of puberty, we administered an NK3R antagonist to prepubertal (before postnatal d 30) females and found no effect on markers of pubertal onset in either WT or hpg mice. These results indicate that the expression of Tac2 and Tacr3 in the ARC are markers of pubertal activation but that increased NKB/NK3R signaling alone is insufficient to trigger the onset of puberty in the mouse.

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Reversal and Relapse of Hypogonadotropic Hypogonadism: Resilience and Fragility of the Reproductive Neuroendocrine System

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2013-2809 ◽

2014 ◽

Vol 99 (3) ◽

pp. 861-870 ◽

Cited By ~ 80

Author(s):

Valerie F. Sidhoum ◽

Yee-Ming Chan ◽

Margaret F. Lippincott ◽

Ravikumar Balasubramanian ◽

Richard Quinton ◽

...

Keyword(s):

Medical Center ◽

Pubertal Development ◽

Hypogonadotropic Hypogonadism ◽

Academic Medical Center ◽

Brain Magnetic Resonance Imaging ◽

Magnetic Resonance Imaging Scan ◽

Event Analysis ◽

Neurokinin B ◽

Olfactory Bulbs ◽

History Of

Context: A subset of patients diagnosed with idiopathic hypogonadotropic hypogonadism (IHH) later achieves activation of their hypothalamic-pituitary-gonadal axis with normalization of steroidogenesis and/or gametogenesis, a phenomenon termed reversal. Objective: The objective of this study was to determine the natural history of reversal and to identify associated phenotypes and genotypes. Design, Setting, and Subjects: This was a retrospective review of clinical, biochemical, and genetic features of patients with IHH evaluated at an academic medical center. Main Outcome Measures: History of spontaneous fertility, regular menses, testicular growth, or normalization of serum sex steroids, LH secretory profiles, brain imaging findings, and sequences of 14 genes associated with IHH were reviewed. Results: Of 308 patients with IHH, 44 underwent reversal. Time-to-event analysis estimated a lifetime incidence of reversal of 22%. There were no differences in the rates of cryptorchidism, micropenis, or partial pubertal development in patients with reversal vs IHH patients without reversal. Fifteen patients with reversal (30%) had Kallmann syndrome (IHH and anosmia); one had undetectable olfactory bulbs on a brain magnetic resonance imaging scan. Subjects with reversal were enriched for mutations affecting neurokinin B signaling compared with a cohort of IHH patients without reversal (10% vs 3%, P = .044), had comparable frequencies of mutations in FGFR1, PROKR2, and GNRHR, and had no mutations in KAL1. Five men did not sustain their reversal and again developed hypogonadotropism. Conclusions: Reversal of IHH may be more widespread than previously appreciated and occurs across a broad range of genotypes and phenotypes. Enrichment for mutations that disrupt neurokinin B signaling in patients who reversed indicates that, despite the importance of this signaling pathway for normal pubertal timing, its function is dispensable later in life. The occurrence of reversal in a patient with no olfactory bulbs demonstrates that these structures are not essential for normal reproductive function. Patients with IHH require lifelong monitoring for reversal and, if reversal occurs, subsequent relapse also may occur.

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Functional Corpora Lutea Are Formed in Matrix Metalloproteinase Inhibitor-Treated Plasminogen-Deficient Mice

Endocrinology ◽

10.1210/en.2006-0669 ◽

2007 ◽

Vol 148 (3) ◽

pp. 1226-1234 ◽

Cited By ~ 9

Author(s):

Patrik Wahlberg ◽

Ida Bodén ◽

Josefin Paulsson ◽

Leif R. Lund ◽

Kui Liu ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Plasminogen Activator ◽

Tissue Remodeling ◽

Corpora Lutea ◽

Wild Type ◽

Serum Progesterone ◽

Paracrine Factors ◽

Proteolytic Activation ◽

Luteal Function ◽

Deficient Mice

Corpus luteum (CL) formation involves dramatic tissue remodeling and angiogenesis. To determine the functional roles of the plasminogen activator and matrix metalloproteinase (MMP) systems in these processes, we have studied CL formation and function in plasminogen (plg)-deficient mice, with or without treatment with the broad-spectrum synthetic MMP inhibitor galardin. Both the adult pseudopregnant CL model and the gonadotropin-primed immature mouse model were used. We found that CL formed normally not only in plasminogen-deficient mice and in galardin-treated wild-type mice, but also in galardin-treated plg-deficient mice, suggesting that neither of the plasminogen activator and MMP systems is essential for CL formation. Nevertheless, in plg-deficient mice, serum progesterone levels were reduced by approximately 50%, and the progesterone levels were not reduced further by galardin treatment. When CL from plg-deficient mice were stained for several molecular markers for CL development and regression, they appeared healthy and vascularized, and were indistinguishable from CL from wild-type mice. This implies that the reduced progesterone levels were not caused by impaired CL formation. Taken together, our data suggest that neither plasmin nor MMPs, alone or in combination, are required for CL formation. Therefore, the tissue remodeling and angiogenesis processes during CL formation may be mediated by redundant protease systems. However, the reduced serum progesterone levels in plg-deficient mice suggest that plasmin, but not MMPs, plays a role in maintenance of luteal function. This role may be performed through proteolytic activation of growth factors and other paracrine factors.

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Mutations of the KISS1 Gene in Disorders of Puberty

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2009-2421 ◽

2010 ◽

Vol 95 (5) ◽

pp. 2276-2280 ◽

Cited By ~ 169

Author(s):

L. G. Silveira ◽

S. D. Noel ◽

A. P. Silveira-Neto ◽

A. P. Abreu ◽

V. N. Brito ◽

...

Keyword(s):

Human Serum ◽

Precocious Puberty ◽

Inositol Phosphate ◽

Pubertal Development ◽

Hypogonadotropic Hypogonadism ◽

Central Precocious Puberty ◽

Control Group ◽

Missense Mutations ◽

Wild Type

Abstract Context: Kisspeptin, encoded by the KISS1 gene, is a key stimulatory factor of GnRH secretion and puberty onset. Inactivating mutations of its receptor (KISS1R) cause isolated hypogonadotropic hypogonadism (IHH). A unique KISS1R-activating mutation was described in central precocious puberty (CPP). Objective: Our objective was to investigate KISS1 mutations in patients with idiopathic CPP and normosmic IHH. Patients: Eighty-three children with CPP (77 girls) and 61 patients with IHH (40 men) were studied. The control group consisted of 200 individuals with normal pubertal development. Methods: The promoter region and the three exons of KISS1 were amplified and sequenced. Cells expressing KISS1R were stimulated with synthetic human wild-type or mutant kisspeptin-54 (kp54), and inositol phosphate accumulation was measured. In a second set of experiments, kp54 was preincubated in human serum before stimulation of the cells. Results: Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in three unrelated children with idiopathic CPP. Both mutations were absent in 400 control alleles. The p.P74S mutation was identified in the heterozygous state in a boy who developed CPP at 1 yr of age. The p.H90D mutation was identified in the homozygous state in two unrelated girls with CPP. In vitro studies revealed that the capacity of the P74S and H90D mutants to stimulate IP production was similar to the wild type. After preincubation of wild-type and mutant kp54 in human serum, the capacity to stimulate signal transduction was significantly greater for P74S compared with the wild type, suggesting that the p.P74S variant is more stable. Only polymorphisms were found in the IHH group. Conclusion: Two KISS1 mutations were identified in unrelated patients with idiopathic CPP. The p.P74S variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype.

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