Leptin signaling in GABA neurons, but not glutamate neurons, is required for reproductive function - PubMed (original) (raw)

Leptin signaling in GABA neurons, but not glutamate neurons, is required for reproductive function

Wieteke A Zuure et al. J Neurosci. 2013.

Abstract

The adipocyte-derived hormone leptin acts in the brain to modulate the central driver of fertility: the gonadotropin releasing hormone (GnRH) neuronal system. This effect is indirect, as GnRH neurons do not express leptin receptors (LEPRs). Here we test whether GABAergic or glutamatergic neurons provide the intermediate pathway between the site of leptin action and the GnRH neurons. Leptin receptors were deleted from GABA and glutamate neurons using Cre-Lox transgenics, and the downstream effects on puberty onset and reproduction were examined. Both mouse lines displayed the expected increase in body weight and region-specific loss of leptin signaling in the hypothalamus. The GABA neuron-specific LEPR knock-out females and males showed significantly delayed puberty onset. Adult fertility observations revealed that these knock-out animals have decreased fecundity. In contrast, glutamate neuron-specific LEPR knock-out mice displayed normal fertility. Assessment of the estrogenic hypothalamic-pituitary-gonadal axis regulation in females showed that leptin action on GABA neurons is not necessary for estradiol-mediated suppression of tonic luteinizing hormone secretion (an indirect measure of GnRH neuron activity) but is required for regulation of a full preovulatory-like luteinizing hormone surge. In conclusion, leptin signaling in GABAergic (but not glutamatergic neurons) plays a critical role in the timing of puberty onset and is involved in fertility regulation throughout adulthood in both sexes. These results form an important step in explaining the role of central leptin signaling in the reproductive system. Limiting the leptin-to-GnRH mediators to GABAergic cells will enable future research to focus on a few specific types of neurons.

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Figures

Figure 1.

Figure 1.

Characterization of the knock-out models by labeling leptin-induced phosphorylated STAT3. A–I, Representative photomicrographs of staining in different hypothalamic areas counted. A–C, MnPO and rPOA. D–F, Arc, VMN, LH, and DMN. G–I, Arc and PMV. The first column shows leptin-induced (5 mg/kg) pSTAT3 labeling in control animals. Second and third columns show pSTAT3 labeling in GABA- and glutamate-specific LEPR knock-out animals, respectively. J, Quantification of immunohistochemical staining (number of positive labeled cells per section) in control animals (black bars; n = 11), GABA-specific (gray bars; n = 10), and glutamate-specific (white bars, n = 8; n = 2 for NTS) knock-outs. There was no difference between the pSTAT3 counts of the two control groups (n = 6 and n = 5) that are derived from the two different Cre lines; therefore, they have been pooled in this graph. In all regions, experimental groups are compared with the control animals to show significant differences. MnPO includes the region of the organum vasculosum of the lamina terminalis (OVLT); rPOA, ventral part. Scale bar, 100 μm. *p < 0.05.

Figure 2.

Figure 2.

Effects of LEPR knock-outs on body weight and plasma leptin concentration. A, B, Female and male GABA- and glutamate-specific LEPR knock-out and control animals body weights. Female GABA-specific knock-out animals (gray triangles; n = 10) are significantly heavier than controls (black circles; n = 19) at 4 weeks of age; glutamate-specific LEPR knock-out animals (white squares; n = 9) are significantly heavier than controls (black circles) at 9 weeks of age. Male GABA-specific LEPR knock-out animals (gray triangles; n = 9) had an increase in body weight from 6 weeks onwards and glutamate-specific knock-out males (white squares; n = 6) from 12 weeks onwards compared with controls (black circles; n = 15). We were unable to gather body weight data for the female groups throughout the breeding study because females were at different stages of pregnancy. C, Plasma leptin concentrations of control animals (black bars; female n = 8, male n = 3) are significantly lower than GABA-specific knock-out animals (gray bars; female n = 8, male n = 2). In glutamate-specific LEPR knock-outs, male animals show significantly higher leptin than the controls (white bars; female n = 4, male n = 2). *p < 0.05. **p < 0.01. ***p < 0.001.

Figure 3.

Figure 3.

Puberty onset in GABA- and glutamate-specific LEPR knock-out animals. A, In GABA-specific LEPR knock-out animals (gray bars; n = 7–10), vaginal opening, first estrus, and male puberty onset were all significantly delayed compared with their control littermates (black bars; n = 7–10). B, Vaginal opening, first estrus, and male puberty onset all occurred at the same time in glutamate-specific knock-out animals (white bars; n = 6–9) compared with their control littermates (black bars; n = 8–9). C, Survival profiles showing puberty onset of female GABA-specific LEPR knock-out (gray lines) and control animals (black lines). The percentage of mice showing vaginal opening (intermittent lines) and first estrus (continuous lines) is plotted for each time point. D, Puberty onset (date of first successful mating) profiles of male GABA-specific knock-outs (gray line) and controls (black line) over time is plotted. VO, Vaginal opening. *p < 0.05. **p < 0.01. ***p < 0.001.

Figure 4.

Figure 4.

Adult fertility in knock-out and control groups. A, Frequency of estrous cycle stage was monitored for 28 d in GABA-specific LEPR knock-out females (gray bars; n = 9) and controls (black bars; n = 10). A significant decrease in time spent in proestrus was seen in the knock-out females. B, The time from pairing with a wild-type male to first litter in GABA knock-out females (gray bar; n = 10) was longer than in littermate controls (black bar; n = 10). Number of days between litters was significantly increased in male GABA-specific knock-out animals (n = 7 in both groups). The same trend was seen in surviving females, but low animal number limited statistical comparison (n = 3). C, Frequency of estrous cycle stage was monitored for 10 d in glutamate-specific knock-out mice (white bars; n = 9) and control littermates (black bars; n = 9). No significant differences were seen between the groups. D, Time to first litter and number of days between litters were the same for all glutamate-specific LEPR knock-out females and males (white bars; n = 6 and 9) compared with littermate controls (black bars; n = 8 and 9). *p < 0.05. ***p < 0.001.

Figure 5.

Figure 5.

The effects of estradiol on plasma LH concentration in GABA-specific LEPR knock-outs and controls. A, Estradiol-negative feedback was assessed by measuring plasma LH concentration in serial blood samples. Intact plasma samples were taken on day 0, day 14 (OVX), and day 22 (OVX+implant). The negative feedback actions of estradiol remained intact in GABA-specific LEPR knock-out animals (gray triangles; n = 9) compared with littermate controls (black circles; n = 9). B, Estradiol-positive feedback was assessed based on plasma LH concentration in trunk bloods taken at the time of an estradiol-induced preovulatory-like surge. There was a significant suppression of LH concentration in GABA-specific knock-out female animals (gray bar; n = 9) compared with controls (black bar; n = 9). *p < 0.05.

Figure 6.

Figure 6.

GABAergic (vGAT-positive) appositions on GnRH neurons in male GABA-specific knock-outs and controls. A, Representative image of a GnRH neuron (green) with vGAT (red) appositions on the soma and proximal projection (white arrowheads). White lines indicate 10 μm segments of the neuronal projection. B, Number of vGAT-positive appositions per 10 μm of GnRH soma membrane and the first three 10 μm segments of the projection, in control (black bars; n = 5) and GABA-specific LEPR knock-out animals (gray bars; n = 5). No significant differences were found between groups. Scale bar, 10 μm.

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References

    1. Ahima RS, Dushay J, Flier SN, Prabhakaran D, Flier JS. Leptin accelerates the onset of puberty in normal female mice. J Clin Invest. 1997;99:391–395. doi: 10.1172/JCI119172. - DOI - PMC - PubMed
    1. Bhat GK, Sea TL, Olatinwo MO, Simorangkir D, Ford GD, Ford BD, Mann DR. Influence of a leptin deficiency on testicular morphology, germ cell apoptosis, and expression levels of apoptosis-related genes in the mouse. J Androl. 2006;27:302–310. doi: 10.2164/jandrol.05133. - DOI - PubMed
    1. Bourguignon JP, Gerard A, Alvarez Gonzalez ML, Purnelle G, Franchimont P. Endogenous glutamate involvement in pulsatile secretion of gonadotropin-releasing hormone: evidence from effect of glutamine and developmental changes. Endocrinology. 1995;136:911–916. doi: 10.1210/en.136.3.911. - DOI - PubMed
    1. Chehab FF, Lim ME, Lu R. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet. 1996;12:318–320. doi: 10.1038/ng0396-318. - DOI - PubMed
    1. Clarkson J, Herbison AE. Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons. Endocrinology. 2006;147:5817–5825. doi: 10.1210/en.2006-0787. - DOI - PMC - PubMed

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