Targeted inhibition of kisspeptin neurons reverses hyperandrogenemia and abnormal hyperactive LH secretion in a preclinical mouse model of polycystic ovary syndrome

Abstract

Study question: Do hyperactive kisspeptin neurons contribute to abnormally high LH secretion and downstream hyperandrogenemia in polycystic ovary syndrome (PCOS)-like conditions and can inhibition of kisspeptin neurons rescue such endocrine impairments?

Summary answer: Targeted inhibition of endogenous kisspeptin neuron activity in a mouse model of PCOS reduced the abnormally hyperactive LH pulse secretion and hyperandrogenemia to healthy control levels.

What is known already: PCOS is a reproductive disorder characterized by hyperandrogenemia, anovulation, and/or polycystic ovaries, along with a hallmark feature of abnormal LH hyper-pulsatility, but the mechanisms underlying the endocrine impairments remain unclear. A chronic letrozole (LET; aromatase inhibitor) mouse model recapitulates PCOS phenotypes, including polycystic ovaries, anovulation, high testosterone, and hyperactive LH pulses. LET PCOS-like females also have increased hypothalamic kisspeptin neuronal activation which may drive their hyperactive LH secretion and hyperandrogenemia, but this has not been tested.

Study design, size, duration: Transgenic KissCRE+/hM4Di female mice or littermates Cre- controls were treated with placebo, or chronic LET (50 µg/day) to induce a PCOS-like phenotype, followed by acute (once) or chronic (2 weeks) clozapine-N-oxide (CNO) exposure to chemogenetically inhibit kisspeptin cells (n = 6 to 10 mice/group).

Participants/materials, setting, methods: Key endocrine measures, including in vivo LH pulse secretion patterns and circulating testosterone levels, were assessed before and after selective kisspeptin neuron inhibition and compared between PCOS groups and healthy controls. Alterations in body weights were measured and pituitary and ovarian gene expression was determined by qRT-PCR.

Main results and the role of chance: Acute targeted inhibition of kisspeptin neurons in PCOS mice successfully lowered the abnormally hyperactive LH pulse secretion (P < 0.05). Likewise, chronic selective suppression of kisspeptin neuron activity reversed the previously high LH and testosterone levels (P < 0.05) down to healthy control levels and rescued reproductive gene expression (P < 0. 05).

Large scale data: N/A.

Limitations, reasons for caution: Ovarian morphology was not assessed in this study. Additionally, mouse models can offer mechanistic insights into neuroendocrine processes in PCOS-like conditions but may not perfectly mirror PCOS in women.

Wider implications of the findings: These data support the hypothesis that overactive kisspeptin neurons can drive neuroendocrine PCOS-like impairments, and this may occur in PCOS women. Our findings complement recent clinical investigations using NKB receptor antagonists to lower LH in PCOS women and suggest that pharmacological dose-dependent modulation of kisspeptin neuron activity may be a valuable future therapeutic target to clinically treat hyperandrogenism and lower elevated LH in PCOS women.

Study funding/competing interest(s): This research was supported by NIH grants R01 HD111650, R01 HD090161, R01 HD100580, P50 HD012303, R01 AG078185, and NIH R24 HD102061, and a pilot project award from the British Society for Neuroendocrinology. There are no competing interests.

Keywords: GnRH; Kiss1; LH pulses; PCOS; androgen; hyperandrogenism; infertility; kisspeptin; reproduction; testosterone.

Figure 1.

Chemogenetic intervention in a preclinical mouse model of polycystic ovary syndrome (PCOS). (A) Schematic summary of crossing a validated Kiss^CRE mouse line with ‘floxed’ hM4Di (inhibitory DREADD) mice to ultimately obtain Kiss^CRE+/hM4Di^fl/fl mice (Kiss^Di) and Cre−/hM4Di^fl/fl control (CON) females that were then treated with letrozole (LET) in Experiments 1 and 2 to induce PCOS-like phenotypes. (B and C) Experimental paradigm for LET induction of PCOS-like phenotype in female Kiss^Di and CON mice and timeline for acute CNO (Experiment 1) and chronic CNO (Experiment 2) effects on reproductive hormones. CNO, clozapine-N-oxide.

Figure 2.

In vivo LH pulse secretion is decreased in LET Kiss^Di but not LET CON mice following acute CNO treatment. (A) Representative profiles of in vivo LH secretion before and after acute CNO treatment in 2 LET-treated Kiss^Di mice, 2 LET-treated Cre− control females, and 2 placebo females. LH was measured in serial tail-tip bleeds from awake animals every 6 min before and after i.p. CNO injection. Pulses were identified by the PULSAR Otago software program and are indicated by *. (B and C) Mean blood LH values across the entire baseline and CNO sampling periods. ***, significantly different between baseline and post-CNO period (P < 0.001). LET, letrozole; CON, control; CNO, clozapine-N-oxide.

Figure 3.

Mean LH pulse parameters of LET Kiss^Di, LET CON, and placebo Cre− female mice from in vivo serial blood sampling, before and after acute CNO treatment. Mean LH pulse parameters analyzed include (A) pulse frequency (pulses/h), (B) basal LH level, (C) pulse peak (zenith value of a pulse), and (D) pulse amplitude. Significant difference between baseline and post-CNO period indicated by * (P < 0.05), ** (P < 0.01), or *** (P < 0.001). LET, letrozole; CON, control; CNO, clozapine-N-oxide.

Figure 4.

Mean body weights (BWs) of LET female mice before and after chronic CNO treatment. (A) BW (grams) over time and % gain in BW over time in LET Kiss^Di, LET CON, and placebo female mice, with chronic 2-week CNO treatment beginning at the end of Week 3 and lasting until the end of Week 5. (B) Mean BW at sacrifice at the end of Week 5. Different letters above bars indicate significantly differences (P < 0.05) from each other. LET, letrozole; CON, control; CNO, clozapine-N-oxide.

Figure 5.

Improved reproductive neuroendocrine measures after chronic kisspeptin neuron inhibition in LET Kiss^Di mice. (A and B) Blood levels of testosterone (T) and LH of LET or placebo mice before and after chronic CNO treatment (from the end of Week 3 to the end of Week 5 of LET treatment, respectively). Two weeks of CNO treatment significantly lowered the high LH and T levels in LET Kiss^Di mice but not in LET CON. (C–E) Mean blood levels of T, LH, and FSH of LET and placebo mice at sacrifice (end of Week 5) following 2 weeks of chronic CNO treatment. (F, G, H) Mean serum LH:FSH ratio and pituitary gonadotropin gene expression, measured via qRT-PCR, in LET and placebo mice at the end of Week 5 following chronic CNO treatment. Different letters above bars indicate significantly differences (P < 0.05) from each other. LET, letrozole; CON, control; CNO, clozapine-N-oxide.

Figure 6.

Improved ovarian measures after chronic kisspeptin neuron inhibition in LET Kiss^Di females. (A) Mean ovary weights of LET and placebo females at the end of Week 5, following 2 weeks of chronic CNO. (B–D) Ovarian gene expression of Cyp17a1, Fshr, and Amh in LET and placebo mice at the end of Week 5 following chronic CNO treatment. Different letters above bars indicate significantly differences (P < 0.05) from each other. LET, letrozole; CON, control; CNO, clozapine-N-oxide.

Figure 7.

Schematic of neuroendocrine abnormalities in the preclinical LET PCOS-like mouse model and effects of targeted inhibition of kisspeptin neurons on reproductive neuroendocrine features. Letrozole (LET) females have elevated hypothalamic arcuate nucleus (ARC) kisspeptin synthesis, more detectable kisspeptin neurons, and markedly increased kisspeptin neuron activation relative to healthy control females. This overactive kisspeptin neuron phenotype correlates with abnormally rapid, elevated LH pulse secretion, as also occurs in PCOS women. Hyperactive LH secretion provides an excessive stimulatory drive to the ovary, causing elevated testosterone synthesis and hyperandrogenemia which contributes to reproductive and metabolic impairments in both LET-PCOS mice and PCOS women. Chemogenetic inhibition of kisspeptin neurons in LET females reduces the hyperactive LH pulse secretion and, correspondingly, reverses the hyperandrogenemia. This suggests that future novel therapeutics selectively targeting kisspeptin neurons could be beneficial for ameliorating similar endocrine impairments in PCOS women.