Ablation of KNDy Neurons Results in Hypogonadotropic Hypogonadism and Amplifies the Steroid-Induced LH Surge in Female Rats

Abstract

In the human infundibular (arcuate) nucleus, a subpopulation of neurons coexpress kisspeptin and neurokinin B (NKB), 2 peptides required for normal reproductive function. A homologous group of neurons exists in the arcuate nucleus of rodents, termed KNDy neurons based on the coexpression of kisspeptin, NKB, and dynorphin. To study their function, we recently developed a method to selectively ablate KNDy neurons using NK3-SAP, a neurokinin 3 receptor agonist conjugated to saporin (SAP). Here, we ablated KNDy neurons in female rats to determine whether these neurons are required for estrous cyclicity and the steroid induced LH surge. NK3-SAP or Blank-SAP (control) was microinjected into the arcuate nucleus using stereotaxic surgery. After monitoring vaginal smears for 3-4 weeks, rats were ovariectomized and given 17β-estradiol and progesterone in a regimen that induced an afternoon LH surge. Rats were killed at the time of peak LH levels, and brains were harvested for NKB and dual labeled GnRH/Fos immunohistochemistry. In ovary-intact rats, ablation of KNDy neurons resulted in hypogonadotropic hypogonadism, characterized by low levels of serum LH, constant diestrus, ovarian atrophy with increased follicular atresia, and uterine atrophy. Surprisingly, the 17β-estradiol and progesterone-induced LH surge was 3 times higher in KNDy-ablated rats. Despite the marked increase in the magnitude of the LH surge, the number of GnRH or anterior ventral periventricular nucleus neurons expressing Fos was not significantly different between groups. Our studies show that KNDy neurons are essential for tonic levels of serum LH and estrous cyclicity and may play a role in limiting the magnitude of the LH surge.

Figure 1.

Schematic diagrams of experimental protocols. In experiment 1, rats were injected with Blank-SAP or NK₃-SAP (d 0), and daily vaginal smears were started on experimental day 7 and conducted until ovariectomy (shown at d 30 in this example). In experiment 2, rats were implanted with an E₂ capsule 7 days after ovariectomy, and on the morning of day 9, they were implanted with a P capsule to induce an afternoon LH surge. Rats were killed at the expected time of peak LH levels (shown here at 4 pm), and the brains were harvested for immunohistochemistry.

Figure 2.

Photomicrographs of pro-NKB immunohistochemistry from a representative Blank-SAP and NK₃-SAP rat. A, The Blank-SAP control shows intense staining of NKB neuronal elements in the arcuate nucleus. There is jet-black staining of scattered cell bodies with a dense plexus of NKB axons. B, In contrast, rats with accurate targeting of NK₃-SAP injections did not show the intense jet-black staining of neurons but only a faint cellular blush consistent with background staining. In addition, the dense plexus of NKB axons within the arcuate neuropil was absent. 3V, third ventricle; ARC, arcuate nucleus. Scale bar, 100 μm (applies to A and B).

Figure 3.

Serum LH (A) and FSH (B) immediately before (d 0) and approximately 30 days after stereotaxic injections of Blank-SAP or NK₃-SAP (mean ± SEM). At day 30, serum LH was significantly reduced in KNDy-ablated rats compared with Blank-SAP controls. Serum FSH was not significantly different between Blank-SAP and NK₃-SAP rats. n = 7 Blank-SAP rats, n = 3 (d 0) or 4 (d 30) NK₃-SAP rats. +, significantly different, Blank-SAP vs NK₃-SAP, P = .003.

Figure 4.

Effects of KNDy neuron ablation on estrous cycles (A–C), ovary (D–F), and uterus (G–I). Representative graphs show 4- to 5-day estrous cycles in 4 Blank-SAP rats (A) and constant diestrus in 3 out of 4 NK₃-SAP rats (B). One NK₃-SAP rat showed a long period of constant diestrous, followed by a 7-day cycle and the beginning of the next cycle. C, Vaginal smears from NK₃-SAP rats showed a marked increase in the percentage of time spent in diestrous compared with control rats. D and E, Representative photographs of the ovaries from a Blank-SAP and NK₃-SAP rat. F, The ovaries weighed significantly less in NK₃-SAP rats. G and H, Representative photomicrographs of paraffin-embedded, hematoxylin and eosin-stained uterine sections from a Blank-SAP and NK₃-SAP rat. The arrow in G marks endometrial glands, which were not present in 3 out of 4 NK₃-SAP rats. I, The endometrial area was significantly reduced in KNDy-ablated rats. Scale bars, 5 mm (in D; applies to D and E) and 200 μm (in G; applies to G and H). n = 7 Blank-SAP and n = 4 NK₃-SAP rats. All data represents mean ± SEM. +, significantly different, Blank-SAP vs NK₃-SAP (P < .001 for C and F; P < .05 for I).

Figure 5.

A–D, Photomicrographs of hematoxylin and eosin-stained ovarian sections from Blank-SAP (A) and NK₃-SAP rats (B–D). The arrowheads (A, B, and D) show developing (secondary or antral) follicles and the arrows show type III follicular atresia. C, Photomicrograph of a large antral follicle with numerous apoptotic cells indicative of type II atresia. D, Type III follicular atresia (arrow) is characterized by collapse of the follicle and fragmentation of the basal lamina. An adjacent viable antral follicle is also seen (arrowhead). E–G, Quantitative morphology showing the number of viable follicles (E) atretic follicles (F) and corpora lutea per section from Blank-SAP and NK₃-SAP rats. In KNDy-ablated rats, the number of follicles exhibiting type III atresia is increased, with decreased numbers of corpora lutea. CL, corpora lutea. Scale bars, 200 μm (in A; applies to A and B) and 50 μm (in C; applies to C and D). n = 7 Blank-SAP and n = 4 NK₃-SAP rats. +, significantly different, Blank-SAP vs NK₃-SAP (P < .01 for type III atresia; P < .05 for corpora lutea).

Figure 6.

Serum LH (A) and FSH (B) in Blank-SAP and NK₃-SAP rats receiving an E₂P regimen to induce an LH surge in the afternoon (mean ± SEM). The level of LH during the surge was 3-fold higher in KNDy-ablated rats relative to controls. The FSH surge was also higher in KNDy-ablated rats. The am blood sample was taken at 8:30 am, and the pm blood sample was taken between 3:30 and 4:30 pm. n = 7 Blank-SAP and n = 4 NK₃-SAP rats. +, significantly different, Blank-SAP vs NK₃-SAP, P < .001; #, significantly different, Blank-SAP vs NK₃-SAP, P < .01; *, significantly different than am, within NK₃-SAP, P < .001.

Figure 7.

A–D, Computer-assisted maps of GnRH (green squares) and GnRH/Fos (red circles) neurons in a Blank-SAP rat. The distance from bregma is indicated in upper right corners. E–H, Fluorescent photomicrographs of GnRH (green) and Fos (red) immunoreactivity. E and F show single-labeled GnRH neurons (arrowheads), and G and H show green GnRH neurons with red nuclear staining for Fos neurons (arrows). Although the LH surge was markedly amplified in KNDy-ablated rats, there was no change in number of neurons expressing GnRH or GnRH/Fos between Blank-SAP and NK₃-SAP rats (Table 2). 3V, third ventricle; ac, anterior commissure; oc, optic chiasm. Scale bars, 250 μm (in A; applies to A–D) and 20 μm (in E; applies to E–H).

Figure 8.

Computer-assisted maps of Fos-positive nuclei in the AVPV of a Blank-SAP (A) and NK₃-SAP rat (B) killed at the peak of the LH surge. There was no significant difference in the number of AVPV Fos cells between groups. 3V, third ventricle; oc, optic chiasm. Scale bar, 100 μm (in A; applies to A and B).