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. 2012 Jun;153(6):2800-12.
doi: 10.1210/en.2012-1045. Epub 2012 Apr 16.

Arcuate kisspeptin/neurokinin B/dynorphin (KNDy) neurons mediate the estrogen suppression of gonadotropin secretion and body weight

Melinda A Mittelman-Smith  1 , Hemalini Williams, Sally J Krajewski-Hall, Josephine Lai, Philippe Ciofi, Nathaniel T McMullen, Naomi E Rance
Affiliations

Arcuate kisspeptin/neurokinin B/dynorphin (KNDy) neurons mediate the estrogen suppression of gonadotropin secretion and body weight

Melinda A Mittelman-Smith et al. Endocrinology. 2012 Jun.

Abstract

Estrogen withdrawal increases gonadotropin secretion and body weight, but the critical cell populations mediating these effects are not well understood. Recent studies have focused on a subpopulation of hypothalamic arcuate neurons that coexpress estrogen receptor α, neurokinin 3 receptor (NK(3)R), kisspeptin, neurokinin B, and dynorphin for the regulation of reproduction. To investigate the function of kisspeptin/neurokinin B/dynorphin (KNDy) neurons, a novel method was developed to ablate these cells using a selective NK(3)R agonist conjugated to the ribosome-inactivating toxin, saporin (NK(3)-SAP). Stereotaxic injections of NK(3)-SAP in the arcuate nucleus ablated KNDy neurons, as demonstrated by the near-complete loss of NK(3)R, NKB, and kisspeptin-immunoreactive (ir) neurons and depletion of the majority of arcuate dynorphin-ir neurons. Selectivity was demonstrated by the preservation of proopiomelanocortin, neuropeptide Y, and GnRH-ir elements in the arcuate nucleus and median eminence. In control rats, ovariectomy (OVX) markedly increased serum LH, FSH, and body weight, and these parameters were subsequently decreased by treatment with 17β-estradiol. KNDy neuron ablation prevented the rise in serum LH after OVX and attenuated the rise in serum FSH. KNDy neuron ablation did not completely block the suppressive effects of E(2) on gonadotropin secretion, a finding consistent with redundant pathways for estrogen negative feedback. However, regardless of estrogen status, KNDy-ablated rats had lower levels of serum gonadotropins compared with controls. Surprisingly, KNDy neuron ablation prevented the dramatic effects of OVX and 17β-estradiol (E(2)) replacement on body weight and abdominal girth. These data provide evidence that arcuate KNDy neurons are essential for tonic gonadotropin secretion, the rise in LH after removal of E(2), and the E(2) modulation of body weight.

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Figures

Fig. 1.
Fig. 1.
Representative photomicrographs of sections from rats with injections of Blank-SAP (left) or NK3-SAP (right) in the arcuate nucleus, stained with Cresyl-violet (A) or immunohistochemistry (B–H). A, Cresyl violet-stained sections show preservation of the Nissl architecture in NK3-SAP rats. B–D, NK3-SAP rats exhibited near total depletion of NK3R, NKB, and kisspeptin-ir neurons and fibers in the arcuate nucleus and median eminence. E, The majority of dynorphin-ir neurons and fibers in the arcuate nucleus and median eminence were ablated in NK3-SAP rats. F and G, There was no difference in the number of POMC neurons or NPY fiber density between Blank-SAP and NK3-SAP rats. H, GnRH fibers in the median eminence were preserved in NK3-SAP rats. 3V, Third ventricle; ARC, arcuate nucleus; ME, median eminence; pro-DYN, prodynorphin; VMN, ventromedial nucleus. Scale bar, 100 μm.
Fig. 2.
Fig. 2.
Effects of KNDy neuron ablation on serum LH (A) and FSH (B). Rats were ovariectomized and injected with NK3-SAP or Blank-SAP in the arcuate nucleus on d 0, given E2 capsules 20–23 d later (shown as d 22), and killed after 11 d of E2. Serum LH and FSH were markedly increased by OVX and reduced by E2 treatment in Blank-SAP animals. In contrast, serum LH after OVX was not significantly different from intact values in NK3-SAP rats, and the rise in FSH was attenuated (mean ± sem, n = 6–11 rats/group for both A and B). Serum LH and FSH were significantly lower in KNDy-ablated rats than Blank-SAP controls in both OVX and OVX + E2-treated groups. #, Significantly different, NK3-SAP vs. Blank-SAP, P ≤ 0.05; +, significantly different from intact values on d 0, P ≤ 0.01; *, significantly different after E2 (vs. OVX), P ≤ 0.05.
Fig. 3.
Fig. 3.
Effects of KNDy neuron ablation on body weight and abdominal girth. Rats were ovariectomized and injected with NK3-SAP or Blank-SAP in the arcuate nucleus on d 0, given E2 capsules 20–23 d later (shown as d 22), and killed after 11 d of E2. A, After OVX, Blank-SAP animals were 25% heavier relative to their initial body weight and lost weight after E2 treatment. Rats with arcuate NK3-SAP injections gained small amounts of weight throughout the study, regardless of E2 status (mean ± sem, n = 9–11 rats/group). B, After OVX, the girth of NK3-SAP rats was significantly smaller than Blank-SAP rats. Girth was significantly decreased by E2 in Blank-SAP but not NK3-SAP rats (mean ± sem, n = 3–4 rats/group). #, Significantly different, Blank-SAP vs. NK3-SAP, P < 0.01; +, significantly different from intact values on d 0, P < 0.05; *, significantly different after E2 (vs. OVX), P < 0.01.
Fig. 4.
Fig. 4.
Representative maps of NK3R neurons from a Blank-SAP-injected (A), arcuate NK3-SAP-injected (B) or lateral hypothalamic NK3-SAP-injected (C) rat. A, Map from Blank-SAP rat showing the location of NK3R neurons in the arcuate nucleus, lateral hypothalamus, supraoptic nucleus, and zona incerta. B, Map from a rat with an arcuate NK3-SAP injection with KNDy neuron ablation and incomplete loss of NK3R neurons in the lateral hypothalamus and zona incerta. C, Map from rat with a lateral hypothalamic NK3-SAP injection showing NK3R neurons in the arcuate nucleus and incomplete loss of NK3R cell bodies in the lateral hypothalamus and zona incerta. Each dot represents one labeled neuron. 3V, Third ventricle; ARC, arcuate nucleus; LHA, lateral hypothalamic area; ot, optic tract; SON, supraoptic nucleus; VMN, ventromedial nucleus; ZI, zona incerta. Scale bar, 250 μm.
Fig. 5.
Fig. 5.
Effects of NK3-SAP injections in the lateral hypothalamus on serum LH (A), FSH (B), and body weight (C). Rats were ovariectomized and injected with NK3-SAP or Blank-SAP on d 0, given E2 capsules 20–23 d later (shown as d 22), and killed after 11 d of E2. A–C, There were no significant differences between rats with lateral hypothalamic NK3-SAP and Blank-SAP injections (mean ± sem, n = 4 values/group, except n = 2 for Blank-SAP gonadotropin levels). +, Significantly different from intact values on d 0, P < 0.05; *, significantly different after E2 (vs. OVX), P < 0.05.
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