Gonadotrophin-releasing hormone release in vitro from the stalk median eminence of cyclic and ovariectomized gilts in response to naloxone or morphine

ANIMAL

REPRODUCTION SCIENCE ELSEVIER

Animal ReproductionScience40 (1995) 151-163

Gonadotrophin-releasing hormone release in vitro from the stalk median eminence of cyclic and ovariectomized gilts in response to naloxone or morphine S. Okrasa, H. Kalamarz, A.J. Ziecik * Department of Animal Physiology, Olsztyn University of Agriculture and Technology, 10-718 Olsztyn-Kortowo5, Poland Accepted 12 April 1995

Abstract The effects of naloxone (NAL) and/or morphine on gonadotrophin-(GnRH) release in vitro from the stalk median eminence (SME) of gilts under different conditions were studied. In the first experiment, the SME explants of gilts on Days 10-11 (n --- 10) and Day 19 (n = 10) of the estrous cycle were incubated in Krebs-Ringer bicarbonate buffer (KRB) supplemented with 2 X 10 -5 M bacitracin and 1 mg m l - 1 glucose in an atmosphere of 95% 0 2 and 5% CO 2 at 37°C. KRB was replaced every 30 min during incubation. Following preincubation for determination of the basal GnRH release, the SME explants of each group were incubated in the presence (10 -6 M) of NAL (treatments; n = 2 × 5) or KRB alone (controls; n = 2 × 5) and increased potassium concentration (56 mM). In Experiment 2, ovariectomized (OVX) mature gilts were primed for five subsequent days with estradiol benzoate (EB; 2 mg; n = 4), progesterone (P4; 50 or 120 rag; n = 2 X 4), EB plus P4 (2 mg and 50 mg, respectively; n = 4) or vehicle (n = 4) and their SME explants were submitted to an incubation paradigm like those of treatment groups in Experiment 1. The SME explants of four other OVX gilts receiving vehicle were incubated as a control in KRB alone. In Experiment 3, OVX gilts primed (as in Experiment 2) with steroids (n = 3 × 8) or vehicle (n = 8) were used. The SME explants of these gilts underwent incubation in the presence of either a low (2 X 10 -6 M; n = 4 X 4) or high dose (10 -3 M; n = 4 X 4) of morphine instead of NAL. Gonadotrophin releasing hormone (GnRH) concentrations in media were determined by radioimmunoassay (RIA). Additionally, fl-endorphin-like immunoreactivity (/3-END-LI) was measured by RIA in chosen media in Experiments 1 and 2.

* Corresponding author at: Polish Academy of Sciences, Centre for Agrotechnology and Veterinary Sciences, Division of Reproductive Endocrinologyand Pathophysiology, 10-718 Olsztyn 5, Poland 0378-4320/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved SSDI 0378 -4320(95)01399-7

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NAL stimulated GnRH release from the SME explants of luteal- and follicular-phase gilts (Experiment 1), as well as of OVX gilts primed with steroids or vehicle (Experiment 2). In Experiment 2, GnRH response to NAL was the greatest in gilts primed with EB. Morphine at the lower dose significantly increased spontaneous GnRH release from the SME following priming with P4 or vehicle, but when applied at the high dose GnRH release in gilts primed with P4 was inhibited (Experiment 3). These results suggest that in the pig, nerve terminals releasing GnRH at the SME level are sensitive, to a certain degree, to opioid action independently of steroid hormones which, however, may amplify or modulate the opioid effect on GnRH release. Keywords: Pig endocrinology;GnRH; Naloxone; Morphine; Opioids; Estrous cycle; Steroid hormones

1. Introduction Luteinizing hormone (LH) secretion is dependent upon release of hypothalamic gonadotropin-releasing hormone (GnRH) into the hypothalamo-hypophysal portal blood. Circumstances which cause a specific pattern of GnRH release inducing changes in LH secretion, characteristic of the estrous cycle, are not fully described. Endogenous opioid peptides (EOP) are regarded as one group among many factors affecting GnRH release (reviews: Ellendorff and Parvizi, 1982; Thiery and Martin, 1991; Kraeling et al.i 1992. Distribution of proopiomelanocortin (POMC) and GnRH neurons within the hypothalamus of the pig provides the opportunity for interactions between these systems (Kineman et al., 1988; 1989). A stimulatory effect of NAL on LH secretion was observed in luteal-phase gilts (Barb et al., 1986a) but not in gilts during the early and late follicular phase (Barb et al., 1986a; Okrasa et al., 1990). Barb et al. (1986a) suggested that the ability of NAL to stimulate LH secretion in the pig depends on an elevated 1'4 plasma concentration. Thus, steroid hormones, at least to a certain degree, condition the influence of EOP on LH secretion and thereby its responsiveness to NAL treatment. One of the possible hypothalamic sites of EOP action might be the stalk median eminence (SME), where nerve terminals release GnRH. The porcine SME releases considerable amounts of GnRH in vitro (Sesti and Britt, 1993). The present studies were undertaken to examine in vitro: (1) Whether NAL influences GnRH release from the SME; if so, what is the difference in NAL effects on GnRH release from the SME of luteal- and follicular-phase gilts; (2) What is the effect of NAL on GnRH release from the SME of ovariectomized (OVX) gilts primed with steroid hormones; (3) How priming of OVX gilts with steroid hormones differentiates responsiveness of GnRH release from the SME to morphine. 2. Materials and methods 2.1. Animals and experimental design

The studies were performed in vitro as a series of three experiments using the SME of crossbred gilts.

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2.1.1. Experiment 1: NAL influence on GnRH release from the SME of cyclic gilts In Experiment 1, 20 cycling gilts were selected after they had exhibited two previous estrous periods. Animals were slaughtered on Days 10-11 (group A; n = 10) or on Day 19 (group B; n --- 10) of the estrous cycle, and within 3 - 4 rain the SME tissues were isolated as described by Cox and Britt (1982) and placed in cold KRB. After isolation, as quickly as possible, the entire SME tissues were incubated according to the procedure presented below. In each group five explants were incubated in the presence (10 -6 M) of NAL (treatments; n = 2 x 5), and five others in KRB alone (controls; n = 2 X 5). In incubation media, GnRH concentration and additionally, in chosen media, /3-endorphinlike immunoreactivity (/3-END-LI) were determined by RIA. For confirmation of the cycle stage, during slaughtering ovaries were examined, and peripheral blood samples were collected to establish concentrations of P4 and estradiol (E 2) in plasma. 2.1.2. Experiment 2: NAL influence on GnRH release from the SME of OVX gilts primed with steroid hormones In this experiment 24 gilts ovarectomized at 7 - 8 months of age were used. One month later animals were injected i.m. for five subsequent days with EB (2 mg; n = 4), P4 (50 mg; n = 4 or 120 mg; n = 4), EB plus P4 (2 mg and 50 mg, respectively; n -- 4), or corn oil (1 ml; n = 2 X 4) in which steroid hormones were dissolved before administration. On the day following the last injection of steroid hormones or vehicle, gilts were slaughtered and the SME was immediately separated as in Experiment 1. The SME tissues of all gilts primed with steroids and four gilts receiving vehicle were in vitro treated with NAL (10 -6 M). The SME explants of remaining gilts receiving vehicle (n = 4) served as a control and were incubated in KRB without NAL. In incubation media collected during in vitro studies, concentrations of GnRH and also /3-END-LI (as in Experiment 1) were measured by RIA. 2.1.3. Experiment 3: morphine influence on spontaneous GnRH release from the SME of OVX gilts primed with steroid hormones Experiment 3 involved 40 gilts ovariectomized at 7 - 8 months of age which 1 month later were randomly assigned to one of the following primings: EB (2 mg; n = 2 × 4), P4 (50 mg; n = 2 X 4 or 120 mg; n = 2 X 4), EB plus P4 (2 mg and 50 mg, respectively; n = 2 X 4), or corn oil (n = 2 X 4). As in previous experiments, the SME tissues were obtained and four explants of each pretreatment were incubated in vitro in the presence of either lower (2 X 10 -6 M) or higher (10 -3 M) concentrations of morphine. In the incubation medium, GnRH concentration was determined by RIA. 2.2. In vitro incubation of the SME tissue Incubations of the SME tissue (entire) were performed in glass vials (20 × 50 mm) containing 2 ml KRB (Krebs-Ringer bicarbonate buffer), pH 7.4. KRB comprised (mM) 118.5 NaC1, 4.74 KC1, 2.54 CaC12, 1.19 MgSO 3 - 7 H 2 0 , 1.18 KH2PO4, 25.04 NaHCO3, 2 × 10 .5 M bacitracin, and 1 mg ml -~ glucose. All components of KRB were purchased from Sigma (St Louis, KY). Fresh solutions of NaHCO 3 used for preparation of KRB, as well as KRB before the use, were constantly saturated with a mixture of

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O2/CO 2 (95% and 5%, respectively). Incubation vials were maintained in a water-bath shaker at 37°C with constant shaking (45 cycles min -~) in an atmosphere of 95% 0 2 and 5% CO 2. After a 30 min preliminary equilibration of the tissue under these conditions KRB was replaced and a 30 min preincubation was performed to establish basal release of GnRH. Subsequently, the 30 min main incubation was carried out in the presence of NAL (Naloxone Hydrochloride; Sigma, St Louis, KY), or morphine (Polfa, Poland), or without any drug (control), as specified in the experimental design. Tested drugs were dissolved in KRB and added in a total volume of 50/zl to the incubation vial at the beginning of the incubation after removing the same volume of medium. Thereafter, the SME tissue was incubated (30 rain) in KRB alone and finally exposed for 30 min to an increased concentration of KC1 (56 mM) in KRB. Immediately after each incubation, the liquid contents were transferred into the plastic tubes and centrifuged at 3000 r.p.m, for 20 min at 4°C. The supernatant was decanted and frozen at 20oc. -

2.3. H o r m o n e determinations

GnRH concentration in the incubation medium was determined by RIA procedure using rabbit antiserum from ICN (ImmunoBiological, Lisle, IL). Synthetic GnRH (acetate salt; Sigma, St Louis, KY) was radioiodinated by the iodogen method of Fraker and Speck (1978). The iodinated GnRH was separated from free iodine by ion exchange chromatography using a QAE-Sephadex column (Nett and Adams, 1977). Antiserum diluted in 0.1% BSA-PBS (200 /zl) was added to unknown (200 /zl) and standard samples prepared in PBS supplemented with bacitracin (2 X 10 -5 M), followed by 100 /zl 125I-GnRH in 0.1% BSA-PBS (approximately 20 000 c.p.m.). Incubation was carried for 24 h at 4°C. Free labelled GnRH was separated from bound using a second antibody against rabbit y-globulin (produced in our Institute). Following 2 h incubation with the second antibody (200/zl; at a dilution 1 : 24), 1 ml 6% polyethylene glycol solution was added and 1 h later samples were centrifuged at 3000 r.p.m, for 20 min, and the supernatant was decanted. Bound ~25I-GnRH was estimated by counting the pellet. Sensitivity of the assay was 7.8 pg m1-1 at 93% binding. Intraassay and interassay coefficients of variation for GnRH determinations were 8.4% and 12.3%, respectively. /3-Endorphin-like immunoreactivity (/3-END-LI) present in medium (Experiment 1 and 2; main incubations) was established by the RIA procedure previously described by Ostrowska et al. (1990) with the difference that the second antibody was used to separate free labelled t-END from bound, as in the GnRH assay. Rabbit antiserum against /8-END, which exhibited equimolar cross reactivity (100%) with t-END and fl-lipotropin was obtained from Amersham (Buckinghamshire, UK). Human t-END was used for iodination and standards (Serva, Heidelberg, Germany). Incubation media (unextracted), due to high content of /3-END-LI, were diluted 100 or 200 times before the assay. Sensitivity of the assay and the intraassay and interassay coefficients of variation were 20 pg m1-1 (at 92% binding), 5.6%, and 10.8%, respectively. Plasma P4 and E 2 concentrations were determined with the RIA procedure routinely used in our laboratory (Dusza et al., 1993).

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2.4. Statistical analysis

Data for GnRH release, after logarithmic transformation, were submitted to one-way analysis of variance (ANOVA) using the EPISTAT program. Differences were assessed by paired Student's t-test. /3-END-LI concentrations in media were analysed by one- or two-way ANOVA, as appropriate, and unpaired Student's t-test.

3. Results 3.1. Experiment 1

Examination of the ovaries confirmed that gilts used in this experiment were in the stage of the cycle as indicated. Mean (__+SEM) concentrations of P4 and E 2 in plasma for the luteal phase were 12.3_ 0.5 ng m1-1 and 18.0+ 3.0 pg m1-1 and for the follicular phase 1.4 + 0.2 ng ml-1 and 29.2 _ 3.7 pg ml -~, respectively. Release of GnRH in vitro from the SME of cyclic gilts is depicted in Fig. 1. The SME tissues of gilts during the luteal and follicular phases responded to NAL in vitro treatment with significant increases. In both cases the average increases were approximately 3-fold as compared to the basal release. In control groups (without NAL), GnRH release during main incubations did not differ significantly from the basal release. A high potassium concentration evoked GnRH release above the basal in all groups. /3-END-LI efflux from the SME tissue to incubation medium was variable in both groups, and it was higher for the follicular phase than the luteal phase (29.5 _ 8.7 ng m1-1 vs. 17.9 _ 8.5 ng ml- 1), but the difference was not significant. 3.2. Experiment 2

In all groups of OVX gilts primed with steroids, NAL administration into incubation media evoked some increases of GnRH release from the SME (Fig. 2). However, the increase observed in the group primed with EB plus P4 was not significant. The most evident stimulation of GnRH release was noted in the group primed with EB (1395 _+ 186 pg m1-1 vs. 288 + 117 pg m l - l ) . In the control group, GnRH release remained at the basal level during main incubation. GnRH response to increased potassium content in the medium was highly significant in the control group. Following incubation with NAL, an elevation of GnRH release in response to potassium reached significance in only one group. Overall output of fl-END-LI in vitro during main incubations in groups primed with steroid hormones (n = 12) appeared to be greater than in the group (n = 4) receiving vehicle (87.9 _+ 14.3 ng m1-1 vs. 35.4 + 9.5 ng ml-1; P < 0.01). The highest output of fl-END-LI occurred in the group (n = 4) primed with EB (109.2 ng m1-1 vs. 35.4 + 9.5 ng m l - l ; P < 0.01). 3.3. Experiment 3

GnRH response to morphine treatment in vitro was dependent on the dose of the drug and priming. A lower dose of morphine (2 × 10 -6 M) significantly increased GnRH

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Fig. 1. Basal and in response to treatment with NAL (0 o r 10 - 6 M) and potassium (56 raM) release of GnRH (+SEM) in vitro from the SME explants of gilts on Days 10-11 (A) and Day 19 (B) of the estrous cycle (Experiment 1). Note that data are expressed in a logarithmic scale. Asterisks indicate significant differences when compared with appropiate basal values. * P < 0.05; * * P < 0.01.

release from the S M E tissue o f gilts primed with both doses o f P4 or vehicle (Fig. 3(A)). Conversely, a tendency to decrease was observed in gilts primed with EB. In gilts primed with EB plus P4, G n R H release from the S M E was not affected by morphine at a low dose. In this trial, G n R H responses to the high potassium concentration were significant in gilts primed with P4 and vehicle, but in other groups values were differentiated.

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Fig. 2. Basal and in response to NAL (0 or 10 - 6 M) and potassium(56 mM) release of GnRH (±SEM) in vitro from the SME explants of OVX gilts primed with steroid hormones or vehicle (Experiment 2). Two doses of P4 (l-low, 2-high) were used for priming (for details see Materials and methods). Data are expressed in a logarithmic scale. Asterisks indicate significant differenceswhen comparedwith appropiatebasal values. * P<0.05;

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A higher dose of morphine (10 -3 M) significantly reduced release of GnRH from the SME of P4-primed gilts (Fig. 3(B)). All SME explants of gilts receiving P4 plus EB or vehicle also reacted to the high dose of morphine with some decreases in GnRH release. On the contrary, a tendency for an increase in GnRH release was noted in the group primed with EB. Following a diminution of GnRH release caused by morphine, increased potassium concentration evoked some elevations in GnRH release, but in these cases its concentrations did not return to basal values. Stimulation of GnRH release with potassium was evident only in the group primed with EB.

4. Discussion

NAL and morphine used in this study possess the highest affinity for the /x opioid receptors (Wood et al., 1981). Specific binding of 3H-NAL by ME tissue of heifers was demonstrated by Leshin et al. (1991). Localization o f / z receptors on the membranes of nerve endings in the guinea-pig median eminence was reported by Beauvillain et al. (1992). Implication of /~ receptors in regulation of LH secretion has been multiply confirmed. In the present study, inhibition of opioid receptors with NAL at a dose 1 0 - 6 M, chosen on the basis of the preliminary experiment, augmented GnRH release in vitro from the SME explants of luteal- and follicular-phase gilts. There is a limited number of comparable studies in literature. Generally, this observation is in agreement with those

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Fig. 3. Basal and in response to treatment with morphine (A: 2 × 10 -6 M or B: 10 -3 M) and potassium (56 raM) release of GnRH ( + SEM) in vitro from the SME explants of OVX gilts primed with steroid hormones or vehicle (Experiment 3). Two doses of P4 (l-low, 2-high) were used for priming (for details see Materials and methods). Data ate expressed in a logarithmic scale. Asterisks indicate significant differences when compared with appropiate basal values. * P < 0.05; * * P < 0.01.

published by others. NAL has exerted a stimulatory effect on GnRH release in vitro from the hypothalamic-preoptic area of gilts (Chang et al., 1990). Similar effects of NAL were reported for mid-luteal ewes (Wu et al., 1991), mature beef heifers (Leshin et

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al., 1991), and cycling female rats (Wilkes and Yen, 1981). However, our data are only partially consistent with the results of studies performed in vivo with cyclic gilts. NAL coherently stimulated GnRH release in vitro (in the present study) and LH secretion in vivo (Barb et al., 1986a) in gilts during the luteal phase. In contrast, NAL increased GnRH release in vitro from the SME of follicular-phase gilts, as stated above, whereas its effect on LH secretion has not been observed in vivo in gilts during the early or late follicular phase (Barb et al., 1986a; Okrasa et al., 1990). Nevertheless, an opioid agonist (FK 33-824) infused for 4 h in gilts during the late follicular phase, lowered LH plasma concentrations (Okrasa and Tilton, 1992) in a manner reversible by NAL. It is possible that NAL influence on GnRH release observed in vitro at the SME level in follicularphase gilts is abrogated under physiological conditions in vivo unless an exogenous opioid agonist is administered. To provide more information related to conditions which enable EOP action on LH secretion, OVX gilts primed with steroid hormones or vehicle were studied in Experiments 2 and 3. Generally, the priming regimen of OVX gilts with steroid hormones was based on studies of Flowers et al. (1991). We have changed only pretreatment with P4 using two different doses: 50 and 120 mg per animal. During further in vitro studies NAL concentration in medium was maintained the same (10 -6 M) as in Experiment 1. In the case of morphine, we assumed that testing the effect of distinct levels of opioid receptor activation on the spontaneous (not stimulated) release of GnRH in vitro from the SME tissue might allow observation of a wider range of its action - - from inhibition to stimulation. Morphine concentration 2 × 10 -6 M in medium was regarded as lower and it is comparable to doses used in many other studies (Drouva et al., 1981; Diez-Guerra et al., 1986; Isagarakis et al., 1989; Chang et al., 1990)i High morphine concentration (10 -3 M) was chosen on the basis of studies published by Rassmusen et al. (1988) in which dose 2 × 10 -3 M was used. GnRH release from the SME explants of OVX gilts receiving no steroids increased in response to NAL and the low dose of morphine, and a tendency to decrease was demonstrated in the presence of the high concentration of morphine. These effects confirm a possibility of steroid independent action of opioids. Efficacy of treatment with opioid antagonists or agonists in females at similar status has been reported. NAL challenges in OVX rats in vivo (Karahalios and Levine, 1988) and in OVX ewes in vitro (Wu et al., 1991) stimulated GnRH release. The administration of opioid agonists to different regions of the brain has also imposed the inhibitory effect on LH secretion in 'OVX gilts (Estienne et al., 1990) and OVX miniature pigs (Parvizi and Ellendorff, 1980; Parvizi, 1986). In lactating sows with physiologically low steroid concentrations in plasma, NAL and morphine affect LH secretion (Barb et al., 1986b; Mattioli et al., 1986; Armstrong et al., 1988a, 1988b). However, intravenous administration of NAL to OVX gilts fails to alter plasma LH concentrations (Barb et al., 1986a). Collectively, it seems that under certain conditions opioids may, in part, modify LH secretion in sows independently of steroid hormones. All groups primed with P4 manifested significant in vitro responses of GnRH to NAL and both doses of morphine with directions of changes similar to those observed in OVX gilts receiving no steroids. Priming with P4 mainly enhanced GnRH responsivness to treatments with morphine. A higher efficacy of priming with P4 might be expected

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since in earlier in vivo studies LH responsiveness to NAL was evident in luteal-phase gilts (Barb et al., 1986a), as well as being re-established in OVX gilts by P4 administration (Barb et al., 1988; Chang et al., 1993). It is possible that in gilts P4 has more effect on the relationship between GnRH release and the opioid system at levels other than the SME. The fact that the much-increased dose of P4, did not result in any additional effect rather confirms the sufficiency of P4 pretreatments. In OVX gilts primed with EB, a particularly high stimulation of GnRH release occurred in response to NAL, which would be in accordance with earlier discussed results concerning the follicular phase. In studies on OVX rats primed with E 2 (Rubin, 1993) or EB plus P4 (Leadem et al., 1985), NAL stimulated GnRH release from hypothalamic tissues perfused in vitro. In miniature pigs, E 2 reinstated suppression of LH secretion by Met-enkephalin (Parvizi, 1986). In our studies, GnRH release from the SME of gilts primed with EB demonstrated remarkable tendencies to decrease and increase in the presence of low and high doses of morphine, respectively. It is interesting that the tendencies brought about by morphine in EB primed groups are opposite to its effects following priming with P4. Paradoxical stimulation of GnRH release by morphine (low dose) following priming with P4 or vehicle might be a dose-related effect. The stimulatory influence of morphine on GnRH release (Rasmussen et al., 1988) and LH secretion (Piva et al., 1986) was observed in male rats. In addition, morphine inhibited and stimulated LH secretion in a dose-dependent and NAL-reversible manner in normal and OVX female rats (Van Vugt et al., 1989). The results of studies performed with rats imply a possibility of stimulatory input of opioids to LH secretion. The effects of morphine noted in our experiment may result from the functioning of a similar linkage between the opioid system and GnRH release in the pig. A delay in emergence of the LH surge, induced by EB in OVX gilts, following NAL administration (Asanovich et al., 1992) seems to confirm such a supposition. Multiplicity of GnRH responsiveness to morphine treatments, observed under various steroid environments, might be achieved through steroid influence on the opioid system activity and/or opioid receptors. Following priming with steroids, some increases of /3-END-LI efflux from the SME explants were found during main incubation in Experiment 2. Steroid hormones also stimulated /3-END release in male rats (Nakano et al., 1991) and OVX primates (Wardlaw et al., 1982). Moreover, treatment with steroid hormones affects the density of /x receptors in different brain areas of female rats, including the hypothalamus (Martini et al., 1989; Mateo et al., 1992). In the present study, GnRH responses to potassium in a few cases were reduced or did not occur following pretreatment with steroids. This might happen due to depletion of releasable GnRH from nerve terminals during preceding stimulation (Experiment 2) or as an ensuing effect of earlier inhibition of its release (Experiment 3). Opioid agonists, including morphine, have been found to inhibit potassium-induced GnRH release from rat mediobasal hypothalamus (Drouva et al., 1981). Possibly priming with steroids or the treatments applied during incubations of the SME explants influenced a proportion between releasable and residual amounts of GnRH in the tissue, but we did not assess this parameter. Studies of Sesti and Britt (1993, 1994) have shown that releasable pools of GnRH may considerably change under different physiological

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conditions in sows. Collectively, it seems that an explanation of interaction between steroid hormones, the opioid system, and G n R H release requires a consideration of many aspects and further experimentation. In summary, results of the present study indicate that nerve endings releasing G n R H located in the porcine SME are receptive to signals coming from the opioid system in a manner dependent on, and independent of steroid hormones. Connection of the opioid system with G n R H release at the SME level is potentially very flexible, ranging from inhibition to stimulation, to modulation by steroid hormones. The interactions observed in the SME area seem to be only a part of the complex mechanism through which the opioid system affects G n R H production and release.

Acknowledgement These studies were supported by the State Committee for Scientific Research (Grant 5 5384 91 02 and Project 2030.204).

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