Peripheral administration of κ-opioid receptor antagonist stimulates gonadotropin-releasing hormone pulse generator activity in ovariectomized, estrogen-treated female goats

https://doi.org/10.1016/j.domaniend.2018.12.011Get rights and content

Highlights

  • Peripheral administration of a KOR antagonist increased the frequency of LH pulses.

  • Peripheral infusion of a KOR antagonist increased multiple unit activity in the ARC.

  • KOR antagonist stimulates LH pulses by acting on the GnRH pulse generator.

Abstract

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is indispensable for reproduction in mammals. Kisspeptin neurons in the hypothalamic arcuate nucleus (ARC), referred to as KNDy neurons because of the coexpression of neurokinin B and dynorphin A, are considered as components of the GnRH pulse generator that produces rhythmic GnRH secretion. The present study aimed to investigate if peripheral administration of PF-4455242, a κ-opioid receptor (KOR, a dynorphin A receptor) antagonist, facilitates pulsatile luteinizing hormone (LH) secretion and GnRH pulse generator activity in estrogen-treated ovariectomized Shiba goats to determine the possibility of using KOR antagonists to artificially control ovarian activities. PF-4455242 was intravenously infused for 4 h (1 or 10 μmol/kg body weight/4 h) or as a single subcutaneous injection (1 or 10 μmol/kg body weight). In a separate experiment, the same KOR antagonist (10 μmol/kg body weight/4 h) was intravenously infused during the recording of multiple unit activity (MUA) in the ARC that reflects the activity of the GnRH pulse generator to test the effects of KOR antagonist administration on GnRH pulse generator activity. Intravenous infusion and single subcutaneous injection of the KOR antagonist significantly increased the frequency of LH pulses compared with controls. Intravenous infusion of KOR antagonist also significantly increased the frequency of episodic bursts in the MUA. The present study demonstrates that peripherally administered KOR antagonist stimulates pulsatile LH secretion by acting on the GnRH pulse generator, and peripheral administration of PF-4455242 can be used to facilitate pulsatile LH secretion, which in turn facilitates ovarian activities in farm animals.

Introduction

The fertility rate of cattle has been reportedly declining in the last several decades in Japan [1], the United States [2], and the United Kingdom [3]. In the livestock industry, ovarian dysfunction, such as ovarian quiescence and cystic ovaries, one of the major reproductive disorders, leads to female infertility and breeding delays [4], [5], resulting in economic loss to the farmer. Ovarian activity is regulated by the hypothalamic-pituitary-gonadal (HPG) axis, and disorders in this axis are associated with ovarian dysfunction. Therefore, pharmacological acceleration of the HPG axis can improve ovarian functions and fertility rates in farm animals such as cattle and sheep.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus and subsequent pulsatile gonadotropin secretion from the pituitary gland are indispensable for ovarian functions in mammals [6], [7]. The neuroendocrine mechanism regulating pulsatile GnRH secretion has been suggested to be located in the hypothalamic arcuate nucleus (ARC) and is referred to as the “GnRH pulse generator” [8]. Accumulating evidence suggests that kisspeptin neurons in the ARC play a critical role in regulating the release of pulsatile GnRH/luteinizing hormone (LH) [9], [10], [11]. The ARC kisspeptin neurons coexpress neurokinin B and dynorphin A in mice [12], goats [13], sheep [14], and cattle [15] and therefore are referred to as KNDy neurons [16]. Our previous electrophysiological studies demonstrated that periodic increases in the multiple unit activity (MUA) volley recorded in the vicinity of KNDy neurons in the caudal ARC are correlated with LH pulses in goats [13], [17]. Moreover, the pulsatile pattern of kisspeptin release and temporal association with pulsatile GnRH secretion were confirmed in the median eminence in rhesus monkeys [18]. Thus, KNDy neurons are considered as components of the GnRH pulse generator [16], [19] as well as a therapeutic target to facilitate reproduction.

Dynorphin A is one of the endogenous opioid peptides and exerts its effect mainly through the κ-opioid receptor (KOR) [20], [21]. Central administration of dynorphin A decreased the frequency of MUA volleys and LH pulses in ovariectomized (OVX) goats either with or without estradiol (E2) treatment [13]. Central administration of the KOR antagonist, norbinaltorphimine (nor-BNI), stimulated GnRH pulse generator activity in OVX, E2-treated goats [13] and pulsatile LH secretion in E2-treated OVX rats [22] and OVX ewes either with or without E2 treatment [23], [24]. Our previous study demonstrated that intraperitoneal infusion of nor-BNI facilitated pulsatile LH secretion in peripubertal female rats [25]. These results suggest that dynorphin A-KOR signaling is a negative regulator of GnRH pulse generator activity in mammals. Thus, KOR antagonists can be used to accelerate GnRH pulses and consequent ovarian activities in domestic animals. However, to the best of our knowledge, no report has tested the effects of peripheral administration of KOR antagonist on pulsatile GnRH/LH secretion or ovarian activity in ruminants. Therefore, we used KOR as a pharmacological target to determine the possibility of using a KOR antagonist to artificially control GnRH pulse generator activity. A nonpeptidic antagonist with high affinity to KOR, PF-4455242 [26], was used in the present study. The antagonist is a relatively small molecule with moderate lipophilicity and penetrates the blood-brain barrier in rats [27]. These characteristics could be advantageous in facilitating GnRH pulse generator activity through peripheral administration in farm animals.

In the present study, the effects of peripheral administration of PF-4455242 via intravenous (i.v.) infusion and subcutaneous (s.c.) injection on pulsatile LH secretion were examined in female goats as models for cattle. We also tested the effects of i.v. PF-4455242 on MUA volleys in the ARC to test if the administration of a KOR antagonist facilitates GnRH pulse generator activity.

Section snippets

Animals

Fifteen adult Shiba goats (Capra hircus) weighing 23.0–32.0 kg each were used. Goats were ovariectomized at ages ranging from 12 to 84 mo. Goats were loosely tied to a stanchion in a condition-controlled room under 12-h light/dark cycle at 23°C and 50% relative humidity. They were fed twice daily with a standard pelleted diet and hay, with free access to water and supplemental minerals. Each goat was subcutaneously implanted with a silicone tube (inner diameter, 3 mm; outer diameter, 5 mm;

Experiment 1: effects of i.v. infusion of the KOR antagonist on pulsatile LH secretion

Profiles of plasma LH concentrations in individual animals receiving i.v. infusion of vehicle or the KOR antagonist (1 or 10 μmol/kg) are shown in Figure 1. After the infusion of both 1 and 10 μmol/kg PF-4455242, interpulse intervals were significantly decreased compared with that of controls (Fig. 2A, P < 0.05), whereas the number of pulses, the mean and baseline plasma LH concentrations, and the area under the curve (AUC) for LH were significantly increased only in the 10 μmol/kg group (Fig. 2

Discussion

The present study demonstrated that peripheral administrations of PF-4455242, a KOR antagonist, can stimulate pulsatile LH release in E2-treated OVX female goats, a model of domestic ruminants. To the best of our knowledge, this is the first report to show that peripheral treatment with a KOR antagonist facilitates pulsatile LH secretion in ruminants, suggesting that peripheral administration of PF-4455242 can be used in farm ruminants to increase pulsatile LH secretion, which in turn promotes

Acknowledgments

The authors thank Ms. K. Yamazaki, Mr. Y. Kono, and Mr. F. Yoshimura for their careful animal care and technical assistance. The radioimmunoassay was performed at the Nagoya University Radioisotope Center. This work was supported in part by Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry from the Ministry of Agriculture, Forestry, and Fisheries of Japan (25022A) and the KAKENHI Grant for the Japan Society for the Promotion of Science

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