Evidence that orphanin FQ mediates progesterone negative feedback in the ewe

Abstract

Orphanin FQ (OFQ), a member of the opioid family, is found in many areas of the hypothalamus and, when given centrally OFQ inhibits episodic LH secretion in rodents and sheep. Because GnRH neurons are devoid of the appropriate receptors to mediate steroid negative feedback directly, neurons that release OFQ may be involved. Using immunocytochemistry, we first determined that most OFQ neurons in the arcuate nucleus (ARC) and other hypothalamic regions of luteal phase ewes contained both estrogen receptor α and progesterone (P) receptor. Given a similar high degree of steroid receptor colocalization in other ARC subpopulations, we examined whether OFQ neurons of the ARC contained those other neuropeptides and neurotransmitters. OFQ did not colocalize with kisspeptin, tyrosine hydroxylase, or agouti-related peptide, but all ARC OFQ neurons coexpressed proopiomelanocortin. To test for a role for endogenous OFQ, we examined the effects of an OFQ receptor antagonist, [Nphe1,Arg14,Lys15]Nociceptin-NH2 (UFP-101) (30 nmol intracerebroventricular/h), on LH secretion in steroid-treated ewes in the breeding season and ovary-intact ewes in anestrus. Ovariectomized ewes with luteal phase concentrations of P and estradiol showed a significant increase in LH pulse frequency during infusion of UFP-101 (4.5 ± 0.5 pulses/6 h) compared with saline infusion (2.6 ± 0.4 pulses/6 h), whereas ewes implanted with only estradiol did not. Ovary-intact anestrous ewes displayed no significant differences in LH pulse amplitude or frequency during infusion of UFP-101. Therefore, we conclude that OFQ mediates, at least in part, the negative feedback action of P on GnRH/LH pulse frequency in sheep.

Figure 1.

(A and B) Photomicrographs of OFQ immunoreactive cells (brown cytoplasm) and ERα (blue-black nuclei) in the ovine ARC. Box in A depicts position of neurons shown at larger magnification in B. Magnification bars, 200 μm (A) and 25 μm (B). (C) Mean (±SEM, n = 3) OFQ-positive cell number (top graph), ERα-containing cell number (middle graph), and percentage of OFQ neurons also containing ERα (bottom graph) in the POA, PVN, AHA, VMH, and 3 regions (rost, rostral; mid, middle; caud, caudal) of the ARC (gray bars).

Figure 2.

(A and B) Photomicrographs of OFQ immunoreactive cells (brown cytoplasm) and PR (blue-black nuclei) in the ovine ARC. Box in A depicts position of neurons shown at larger magnification in B. Magnification bars, 200 μm (A) and 25 μm (B). (C) Mean (±SEM, n = 3) OFQ-positive cell number (top graph), PR-containing cell number (middle graph), and percentage of OFQ neurons also containing PR (bottom graph) in the POA, PVN, AHA, VMH, and 3 regions (rost, rostral; mid, middle; caud, caudal) of the ARC (gray bars).

Figure 3.

(A–F) Confocal images (1-μm optical section) through the ovine ARC processed for dual immunofluorescence detection of OFQ (red) and either POMC, kisspeptin, AgRP, or TH (green). (C) Merged image of single-labeled OFQ neurons (A) and POMC neurons (B) to depict yellow dual-labeled OFQ/POMC neurons (eg, arrows). Note presence of a few single-labeled POMC neurons (eg, top-middle portion of B and C). Merged images in bottom row show OFQ and kisspeptin (Kiss) (D), AgRP (E), or TH (F). Magnification bar, 25 μm.

Figure 4.

LH profiles from experiment 3a in ewe number 258 that was OVX and treated sc with implants containing P and E₂. LH profiles during infusion of saline (A), UFP-101 (B), and JTC-801 (C) into the lateral ventricle are shown, with pulses identified using a closed circle.

Figure 5.

Mean (±SEM) LH concentrations (A), LH pulse amplitude (AMPL; B), and LH pulse frequency (FREQ; C) for saline (CONT; open bars), UFP-101 (black bars), and JTC-801 (gray bars) infused into the lateral ventricle of OVX+P+E₂ ewes (n = 8). *Significant difference (P < .05) from saline treatment.

Figure 6.

Mean (±SEM) for LH concentrations (A), LH pulse amplitude (AMPL; B), and LH pulse frequency (FREQ; C) for saline (open bars) and UFP-101 (black bars) infused into the lateral ventricle of OVX+E₂ breeding season (Br Sea) (n = 8) and ovary-intact anestrous (anestrus; n = 6 for mean LH and pulse frequency). LH pulse amplitudes were not statistically analyzed in anestrous ewes due to low animal numbers (n = 3), because many ovary-intact anestrous ewes had no LH pulses during the 4-hour sampling period. Note that for illustrative purposes only, the frequency data for breeding season ewes was converted from pulses/5 hours to pulses/4 hours by multiplying the total number of pulses/5 hours by 80% for each ewe.