Variations in opioid peptide levels during the estrous cycle in Sprague–Dawley rats☆
Introduction
Female gonadal hormones exert potent effects on mental state and behavior. This implies modulation of brain neurotransmission and selectivity for certain brain regions, either by a direct effect at membrane-bound sites or by a more indirect influence on genomic mechanisms. Converging evidence from behavioral and biochemical studies has indicated that the functional consequences of this neuroendocrine action are associated not only with classical transmitters such as serotonin, dopamine, γ-aminobutyric acid and acetylcholine, but also with neuropeptides (Argiolas, 1999, Etgen, 2002, McEwen, 2002, McEwen and Parsons, 1982). These investigations have involved both intact and ovariectomized (OVX) hormone-substituted animals. However, the data available on female sex steroid hormones and opioid peptide interaction are mainly derived from the latter model. The present investigation concerns possible fluctuations in endogenous peptides in the brain and pituitary gland in untreated, freely cycling rats.
In female rats that show a 4- or 5-day estrous cycle, the cycle is usually divided into diestrus, proestrus and estrus. Diestrus is characterized by low levels of progesterone, while estradiol levels start to increase gradually during diestrus. During proestrus, estradiol levels increase rapidly, followed by a surge of luteinizing hormone (LH) and increasing concentrations of progesterone, which together set into action a number of events that result in ovulation and a condition of sexual motivation and receptivity (Smith et al., 1975).
The endogenous opioid peptides, β-endorphin, enkephalins and dynorphins, act on μ-, δ- and κ-opioid receptors (Akil et al., 1984, Mansour et al., 1988, Terenius, 2000) to mediate a variety of physiological effects such as pain modulation (Przewlocki and Przewlocka, 2001, Terenius, 1992), neuroendocrine secretion (Pfeiffer and Herz, 1984), motivation and reward (Gianoulakis, 2004, Nylander and Silberring, 1998, Van Ree et al., 2000), and sexual behavior (Meyerson, 1988). The dynorphin peptides dynorphin A (DYNA) and dynorphin B (DYNB) are derived from the prodynorphin precursor protein (Civelli et al., 1985). Larger fragments of prodynorphin are also present in the brain, for example dynorphin 32 (which includes the sequences of DYNA and DYNB) and dynorphin B 29 (the C-terminal end of the precursor protein with one copy of DYNB within its sequence) (Civelli et al., 1985, Day et al., 1998, Seizinger et al., 1984a). DYNA and DYNB can be metabolized further into biologically active peptide fragments. One common cleavage product of this tissue- and peptide-specific enzymatic conversion is the N-terminal peptide Leu-enkephalin-Arg6 (Leu-Arg) (Nyberg et al., 1985, Nyberg and Silberring, 1990, Nylander et al., 1995, Sandin et al., 1997). DYNA can also be cleaved to DYNA (1–8) (Seizinger et al., 1984b, Zamir et al., 1984) and, in addition, to des-Tyr-fragments that are devoid of opioid receptor activity (Young et al., 1987). Nociceptin/orphanin FQ (N/OFQ) is a peptide with a structure similar to that of endogenous opioid peptides, especially that of DYNA. It does not bind to opioid receptors, however, and opioid peptides do not bind to the N/OFQ receptor, ORL1 (opioid receptor-like 1) (Mogil and Pasternak, 2001). In spite of this, N/OFQ has been shown to be involved in pain, feeding, anxiety, learning and memory, reward, and also in endocrine effects (Harrison and Grandy, 2000).
Opioids play an important role in the regulation of sexual receptivity in female rats, and they have been studied extensively in this respect (Meyerson, 1988). Most studies have concerned μ opioid receptors (e.g. Hammer, 1990, Piva et al., 1995, Quinones-Jenab et al., 1997) and β-endorphin levels (e.g. Ge et al., 1993, Hammer et al., 1994, Hulse et al., 1984) in the hypothalamus and the pituitary gland. For example, β-endorphin has been shown to inhibit lordosis in OVX, steroid-primed rats and this effect was found to be reversed by administration of the opioid antagonist naloxone. Furthermore, infusions of naloxone facilitate lordosis behavior in OVX, steroid-primed females (Pfaus and Gorzalka, 1987). Changes in gonadal hormone activity also affect opioid peptide systems. In a recent study, it was shown that ovariectomy induced an estradiol-reversible decrease in preproenkephalin mRNA levels in the striatum and the nucleus accumbens, while no effect was found in the cortex (Le Saux and Di Paolo, 2005). In the anterior pituitary gland, alterations in prodynorphin mRNA levels were detected after estradiol injections (Spampinato et al., 1990). Furthermore, it was found that levels of prodynorphin mRNA doubled in OVX rats, in parallel with an increase in immunoreactive (ir) DYNA levels, and they were restored after estradiol replacement (Spampinato et al., 1995). Consistent with these findings, it was shown that ovariectomy resulted in an estradiol-reversible increase in the anterior pituitary lobe ir DYN 32 levels. Neither ovariectomy nor estrogen treatment altered ir levels of DYNA (1–8), DYNA or DYN 32 in the hypothalamus and the neurointermediate pituitary lobe (Fullerton et al., 1988). Less is known about the involvement of N/OFQ in sexual behavior, but it has been shown that administration of N/OFQ facilitates lordosis in a dose-dependent manner in estrogen-primed female rats (Sinchak et al., 1997). Moreover, OVX rats treated with estradiol had increased ORL-1 mRNA expression in the hypothalamic ventromedial nucleus which was not increased further by treatment with estrogen in combination with progesterone (Sinchak et al., 2001).
There have been very few studies on the influence of gonadal hormones on brain opioid peptides and N/OFQ under physiological conditions in freely cycling females. Cyclic fluctuations in hormonal secretion in relation to the female estrous cycle may provide us with the means of understanding neurochemical variations in relation to the complex endocrine conditions during the various phases of the estrous cycle. Because of an increasing interest in sex differences in the neurobiological mechanisms underlying vulnerability for drug dependence, we have included female rats in our recent studies (Gustafsson et al., 2005, Ploj et al., 2001, Roman et al., 2004, Roman et al., 2005). During the course of these studies, it became apparent that information about opioid peptides in various areas of the brain in freely cycling rats was difficult to find in the literature. To our knowledge, a quantitative analysis has not been performed; thus, the general objective of the present investigation has been evaluation of possible fluctuations in opioid peptides in female rats. Basal ir levels of endogenous opioid peptides and also N/OFQ were measured at different phases of the estrous cycle in Sprague–Dawley rats. The prodynorphin-derived peptides DYNA, DYNB and Leu-Arg, the proenkephalin-derived peptide Met-enkephalin-Arg6Phe7 (MEAP), and also N/OFQ were measured using specific radioimmunoassays (RIAs) during diestrus, proestrus and estrus in the pituitary gland, the hypothalamus, and various areas of the brain. Furthermore, correlations between peptides and brain areas were investigated.
Section snippets
Animals and housing
Thirty 10-week-old female Sprague–Dawley rats (Scanbur BK AB, Sollentuna, Sweden) were used. Upon arrival, the rats were housed in groups of five in standard macrolon cages (59 × 38 × 20 cm) containing woodchip bedding material. The animals were maintained on standard pellet food (R36 Labfor; Lactamin, Vadstena, Sweden) and water ad libitum. All animals were housed in a temperature-controlled (22 ± 1 °C) and humidity-controlled (50 ± 10%) animal room on a 12-h light/dark cycle, with lights on at 6:00
Results
The estrous cycles were of the 4-day type and no trend of synchronization was seen. According to our definition (see Section 2.2), the animals were divided in three groups, i.e. diestrus, proestrus and estrus, respectively, with the majority of animals in the diestrus group being of the second day of diestrus. A summary of ir peptide levels in the pituitary gland, hypothalamus and various areas of the brain during diestrus, proestrus and estrus is given in Table 1. With the cation exchange
Discussion
The current study, which involves evaluations of four opioid peptides and N/OFQ in the pituitary gland and the brain, shows alterations associated with the estrous cycle with regard to (1) ir opioid peptide levels in several areas of the brain, (2) the ratio between dynorphins and their conversion product Leu-Arg, and (3) correlations within as well as between certain areas of the brain. The most frequent finding, in terms of significant differences, was between the diestrus condition and the
Acknowledgements
The authors wish to thank Ms. Marita Berg for technical assistance in determination of the vaginal smears and Ms. Jenny Lantz and Ms. Hanna Lindgren for valuable help with the radioimmunoassays.
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Sources of support: Grants from the Swedish Research Council (K2002-04X-12588-05A), the Alcohol Research Council of the Swedish Alcohol Retailing Monopoly (98-21) and AFA supported this study.