Leptin-Induced CART (Cocaine- and Amphetamine-Regulated Transcript) Is a Novel Intraovarian Mediator of Obesity-Related Infertility in Females

Abstract

Obesity is considered detrimental to women's reproductive health. Although most of the attention has been focused on the effects of obesity on hypothalamic function, studies suggest a multifactorial impact. In fact, obesity is associated with reduced fecundity even in women with regular cycles, indicating that there may be local ovarian effects modulating fertility. Here we describe a novel mechanism for leptin actions directly in the ovary that may account for some of the negative effects of obesity on ovarian function. We find that normal cycling, obese, hyperleptinemic mice fed with a high-fat diet are subfertile and ovulate fewer oocytes compared with animals fed with a normal diet. Importantly, we show that leptin induces expression of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in the granulosa cells (GCs) of ovarian follicles both in vitro and in vivo. CART then negatively affects intracellular cAMP levels, MAPK signaling, and aromatase mRNA expression, which leads to lower estradiol synthesis in GCs and altered ovarian folliculogenesis. Finally, in human samples from patients undergoing in vitro fertilization, we show a significant positive correlation between patient body mass index, CART mRNA expression in GCs, and CART peptide levels in follicular fluid. These observations suggest that, under obese conditions, CART acts as a local mediator of leptin in the ovary to cause ovarian dysfunction and reduced fertility.

Figure 1.

Granulosa cells from ovaries of obese mice have inherent defects with respect to follicular physiology. A, Five-week-old mice (n = 18 animals/diet group) were fed with ND or HFD (60% kcal fat diet) for 12 weeks, and their weights were measured every other week. B, Serum leptin levels (n = 10 animals/diet group) were determined after the 12-week diet; *, P ≤ .05. C, Histological analysis of ovarian morphology of HFD and ND mice (n = 3 ovaries from 3 different mice/diet). P, primordial; Pr, primary; PA, preantral; A, antral; Atr, atretic. D, Aromatase mRNA levels in GCs isolated from ND and HFD mouse ovaries determined by real-time PCR (n = 10 animals/diet group). E, Estradiol levels extracted from ovaries of ND and HFD mice (n = 10 animals/diet group); *, P ≤ .05.

Figure 2.

Leptin inhibits aromatase mRNA levels and induces CART expression in the ovaries. Aromatase expression in (A) primary culture of mouse GCs (n = 3) isolated from ND mice treated with leptin for 18 hours and in (B) GCs isolated from ovaries of ND mice (n = 5 animals/treatment) injected (ip) with vehicle or 30 mg of leptin/d for 1 week. CART mRNA levels in primary culture of mouse GCs (n = 3) isolated from ND mice treated with different concentrations of leptin for 18 hours (C) and CART (55–102) peptide levels in the media of mouse primary culture GCs (n = 3) treated with/without leptin (30 ng/mL) (D). E, CART mRNA levels in GCs isolated from ovaries of ND mice (n = 5 animals/treatment) injected (ip) with vehicle or 30 mg of leptin/d for 1 week. Serum leptin levels (n = 10 animals/diet group) (F) and CART mRNA expression in GCs (n = 14 animals/diet group) (G) of ND and HFD mice compared with leptin receptor-deficient mice (db/db mice, n = 3 animals). Data are normalized to GAPDH; *, P ≤ .05.

Figure 3.

Leptin- or HFD-induced inhibition of aromatase expression is mediated through CART. A, Primary GCs from normal mice were cultured in vitro and transfected with CART or non-specific (Nsp) siRNA and knockdown of CART mRNA was confirmed by real-time PCR as shown in Supplemental Figure 1. GC cultures were then treated for 18 hours with/without leptin (30 ng/mL), and aromatase expression was measured by real-time PCR. B, siRNA-mediated knockdown of CART expression rescues aromatase expression in GCs isolated from HFD-treated obese mice. Data are normalized to GAPDH and represented as mean of 3 separate experiments; *, P ≤ .05 vs Nsp-control.

Figure 4.

In vitro and in vivo CART treatment disrupts follicular physiology. A, Primary mouse GC cultures from ND mice were treated with 0.1μM CART (55–102) peptide for 18 hours, and aromatase mRNA level was determined by real-time PCR. Data are normalized to GAPDH and represented as mean of 3 separate experiments; *, P ≤ .05. CART (55–102) peptide (50 ng/mouse) or vehicle (0.2 mL/mouse) was injected (ip) for 1 week in ND-fed mice (n = 8 animals); and GC aromatase mRNA expression (B), estradiol levels (C), and number of ovulated oocytes (D) were determined; *, P ≤ .05.

Figure 5.

CART inhibits cAMP and pMAPK3/1 signaling in GCs. Primary GCs isolated from ND mice were pretreated with CART (55–102) or an inactive CART (negative control) peptide for 15 minutes followed by FSH or IGF stimulation (30 min). Intracellular cAMP (A) and total and phosphorylated (B and C) MAPK3/1 levels were determined by ELISA and Western blot analysis, respectively. Western blottings were quantified by densitometric analyses where phospho-MAPK3/1 levels were normalized to corresponding total-MAPK3/1 levels, and data are displayed as mean ± SEM of fold increase relative to media (M). Data are represented as mean of 3 separate experiments; P ≤ .05.

Figure 6.

Correlation between body mass index (BMI) and follicular CART levels in humans. GCs and follicular fluids were isolated during oocyte retrieval in IVF patients and (A) CART mRNA in GCs and (B) CART peptide in follicular fluids were determined by real-time PCR and ELISA, respectively; and correlated with corresponding patient BMI. Each circle or triangle represents 1 patient. C, Proposed model of CART-mediated obesity-related infertility.