Original article

B. WYLOT1,2, K. TWORUS1, S. OKRASA1

THE EFFECTS OF MU-, DELTA- AND KAPPA-OPIOID RECEPTOR ACTIVATION
ON LUTEINIZING AND FOLLICLE-STIMULATING HORMONE SECRETION
FROM PORCINE PITUITARY CELLS

1Department of Animal Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury, Olsztyn, Poland;
2Department of Cell Biology, Nencki Institute of Experimental Biology, Warsaw, Poland
Endogenous opioid peptides (EOP) are involved, among others, in the regulation of endocrine systems, including gonadotropin (LH and FSH) secretion in females during the estrous cycle. EOP preferentially act through three major types of opioid receptors: mu (MOP), delta (DOP) and kappa (KOP). Their influence on gonadotropin secretion at the hypothalamic level was extensively studied in different species, but information pertaining to their modulatory action on LH and FSH secretion at the pituitary level is scarce. Therefore, the aim of the present study was to examine the effects of opioid receptor agonists (mu - DAMGO, delta - DPDPE and kappa - U 50.488) at doses 10–9, 10–8, 10–7 mol/L on both basal and GnRH-stimulated gonadotropin (LH and FSH) secretion in vitro from the anterior pituitary cells of gilts on days 8–10 (luteal phase) and 19–20 (follicular phase) of the estrous cycle. The exposition of pituitary cells in vitro to kappa-receptor agonist (U 50.488; 10–7 mol/L) significantly reduced (p<0.05) basal secretion of LH during both phases of the estrous cycle. The GnRH-stimulated LH secretion was also decreased (p<0.05) by this agonist during the luteal phase (10–7 mol/L) and follicular phase (10–9, 10–8 and 10–7 mol/L). In turn, the FSH secretion was reduced (p<0.05) by kappa-agonist only in the presence of GnRH during the luteal phase (10–8 and 10–7 mol/L) and follicular phase (107 mol/L). The delta-opioid agonist (DPDPE) significantly reduced (p<0.05) the GnRH-affected secretion of LH during the follicular phase (10–8 mol/L) and FSH during the luteal phase (10–7 mol/L). The mu-opioid agonist (DAMGO) affected neither LH nor FSH secretion. These results indicate that opioid peptides, acting mainly through kappa- and delta-opioid receptors, may participate in the modulation of gonadotropin (LH and FSH) secretion at the pituitary level in cyclic gilts.
Key words:
endogenous opioid peptides, opioid receptors, luteinizing hormone, follicle-stimulating hormone, pituitary, cyclic pigs, estrous cycle, luteal phase, follicular phase

INTRODUCTION

Endogenous opioid peptides (EOP) belong to three major families: endorphins (e.g. β-endorphin), enkephalins (e.g. Met- and Leu-enkephalin) and dynorphins (e.g. dynorphin A, dynorphin B, β-neoendorphin). They preferentially act through mu (MOP), delta (DOP) and kappa (KOP) opioid receptors, respectively. Additionally, the affinity of opioid receptors is extended to a wider range of opioid compounds (1, 2).

Endogenous opioids are known to participate in the regulation of reproductive processes in the female by affecting gonadotropin (3-7) and prolactin (8, 9) secretion. Previous studies performed with various species, including the pig, sheep, cow and rat, demonstrated that EOP can mostly act at the level of hypothalamus where they influence GnRH release leading to considerable changes in the amplitude and/or frequency of luteinizing hormone (LH) pulses across the estrous cycle and appearance of its preovulatory surge (6, 7, 10, 11). Relatively few studies raised the possibility that endogenous opioids may directly influence gonadotropin secretion acting at the level of anterior pituitary gland. Such a mechanism of opioid action appears very likely as the presence of opioid receptors and their mRNA was demonstrated in the anterior pituitary of mammals (12, 13) and lower vertebrates (14, 15). Importantly, in rodents and amphibians opioid receptors were found directly on gonadotropic cells (14). Moreover, expression of opioid precursor genes (13, 16, 17) and presence of opioid peptides (18, 19, 20) in the anterior pituitaries of different species as well as changes in synthesis of opioids and their receptors in this gland throughout the estrous cycle (13, 20) suggest a potential role of EOP systems in a local (para- and/or autocrine) regulation of gonadotropin secretion. In addition, it seems possible that the pituitary gland may also be enriched in EOP by their local retrograde transfer from the perihypophyseal vascular complex (21). Consistently, several in vitro studies demonstrated that opioids can directly modulate gonadotropin release from the pituitary cells of gilts (22), cows (23, 24) and rats (25). Altogether, these studies demonstrated that opioids belonging to endorphins and enkephalins reduce secretion of LH from cultured anterior pituitary cells while blocking of opioid receptors with nonspecific opioid antagonist (naloxone) had the opposite effect. Nevertheless, the previous experiments did not precisely distinguish the types of opioid receptors involved in the local modulation of gonadotropin secretion and they did not consider the physiological status of studied females. Moreover, data on the possible role of opioids in a local modulation of gonadotropin release in large animals, including the pig, are rudimentary. Therefore, the aim of the present study was to examine effects of the activation of three major types of opioid receptors mu, delta and kappa by highly specific agonists on the secretion of LH and follicele-stimulating hormone (FSH) from the anterior pituitary cells of gilts representing the luteal and follicular phases of the estrous cycle.

MATERIAL AND METHODS

Animals

All experiments were performed in accordance with the principles and procedures of the Animal Ethics Committee, University of Warmia and Mazury in Olsztyn. Pituitary glands were collected from mature cross-bred gilts (Large White x Polish Landrace) on Days 8–10 (luteal phase) and 19–20 (follicular phase) of the estrous cycle in a local slaughterhouse. The day of the cycle was determined according to the morphology of the ovaries using the tables published by Akins and Morrissette (26). Pituitaries were transported to the laboratory in cooled sterile Dulbecco (Sigma-Aldrich) medium supplemented with 0.1% BSA (Sigma Aldrich), nystatin and gentamycin.

In vitro cell culture

In order to provide sufficient number of cells for in vitro experiments, 6–8 pituitaries from luteal- and follicular-phase gilts were used for every replicate. Anterior pituitary glands were mechanically minced and rinsed with Dulbecco medium. The tissue was further disintegrated with 0.3% trypsin solution (Biomed) at 37°C on a magnetic stirrer. The suspension containing released cells was collected every 10 minutes, rinsed with fresh Dulbecco medium 3 times and centrifuged at 800×g for 10 minutes at 4°C (after each rinsing). Digestion step was repeated until the pituitary tissue was entirely digested. The pituitary cells were filtered through a sterile filter (mesh diameter - 70 µm) and counted in Burker counting chamber. Cell viability, assessed by trypan blue incorporation, ranged between 90–95%. Isolated cells were preincubated in McCoy-5A medium (Sigma-Aldrich) supplemented with 2.5% bovine calf serum (Life Technologies), 10% horse serum (Biomed), MEM vitamins solution (Sigma-Aldrich), MEM non-essential amino acid solution (Sigma-Aldrich), nystatin (240 IU/ml) and gentamycin (100 µg/ml) on 24-well culture plates (Corning) at a density of 3×105 cells/well for 48 hours at 37°C under controlled atmosphere (5% CO2, 95% air). Then, the cell incubation in 1 ml of fresh McCoy-5A medium without serum was continued for additional 24 hours. After the 72-hour pre-incubation, the cells reached high confluence (about 90%). Subsequently, the cells were rinsed with fresh medium and exposed to opioid agonists for 4 hours to test the effects of the treatment on gonadotropin secretion. The effects of opioid receptor agonists (mu - DAMGO, delta - DPDPE and kappa - U 50.488; all from Sigma-Aldrich) at doses 10–9, 10–8, 10–7 mol/L on both basal and GnRH-stimulated gonadotropin (LH and FSH) secretion were tested. The doses of opioid agonists were chosen based on previous studies (22, 24, 27-29). All the experiments were performed in duplicates. After incubation, the culture media were collected and stored at –72°C for further analysis.

Measuring of LH and FSH concentration

Gonadotropin (LH and FSH) concentration in culture media was assessed by radioimmunoassay (RIA) using polyclonal anti-LH (30) and anti-FSH (NIDDK, USA) antibodies, and porcine LH and FSH (both from NIDDK, USA) for labeling with 125I and as standards. Sensitivities of the assay were 0.035 and 0.044 ng/sample for LH for FSH, respectively.

Statistical analysis

The experimental data were analyzed using Statistica 8.0 software (StatSoft). The influence of opioid agonists on LH and FSH secretion was examined with nonparametric Wilcoxon test for independent variables. Values of p<0.05 were considered to be statistically significant. Concentrations of LH and FSH were presented as the means ± S.E.M. and expressed per 105 of seeded cells.

RESULTS

The influence of mu-, delta- and kappa-opioid agonists on the basal and GnRH-stimulated secretion of gonadotropins (LH and FSH) from porcine anterior pituitary cells of cyclic gilts was studied (Fig. 2 and 3). Secretory potential of in vitro cultured pituitary cells used in the study was confirmed by assessing their reaction to GnRH. It was demonstrated that cells obtained from both luteal- and follicular-phase gilts markedly increased secretion of LH and FSH in vitro after 4-hour treatment with 100 ng/ml GnRH (Fig. 1).

Figure 1 Fig. 1. LH and FSH secretion from anterior pituitary cells of gilts in luteal and follicular phases of the estrous cycle treated with 100 ng/ml GnRH. The concentration of LH and FSH was expressed per 105 cells; n=7; mean ± S.E.M., *p<0.05.

The influence of opioid agonists on basal and GnRH-stimulated LH secretion

The activation of mu-opioid receptor did not significantly (p>0.05) influence basal and GnRH-stimulated LH secretion by pituitary cells of gilts in luteal and follicular phase (Fig. 2).

Figure 2
Fig. 2. Basal and GnRH-stimulated secretion of LH from pituitary cells of gilts in luteal (A, B) and follicular (C, D) phases of the estrous cycle incubated with DAMGO, DPDPE and U 50.488 at doses 10–9, 10-8 and 10–7 mol/L. The concentration of LH was expressed per 105 cells; n=7; mean ± S.E.M., * p<0.05.

The delta-opioid agonist (DPDPE) at dose 10–8 mol/L significantly reduced GnRH-affected secretion of LH from the follicular-phase pituitary cells (from 8.59±1.45 to 7.55±1.47 ng/ml; Fig. 2D). The effects of DPDPE on LH secretion in the remaining experimental groups were negligible (Fig. 2A-2C).

The exposition of pituitary cells to kappa-receptor agonist resulted in unequivocal inhibition of LH secretion. U 50.488 at dose 10–7 mol/L markedly (p<0.05) reduced both basal (from 3.18±0.98 to 1.94±0.63 ng/ml) and GnRH-stimulated (from 17.28±7.54 to 11.80±5.12 ng/ml) secretion of LH by cells representing the luteal phase (Fig. 2A and 2B) as well as basal secretion of this gonadotropin from follicular-phase cells (from 1.67±0.26 to 1.29±0.18 ng/ml; Fig. 2C). In the presence of GnRH, pituitary cells collected from follicular-phase gilts decreased secretion of LH in response to all tested doses of kappa agonist (from 8.59±1.45 ng/ml to: 7.60±1.68, 7.05±1.67 and 5.40±1.24 ng/ml for doses: 10–9, 10–8 and 10–7 mol/L, respectively; Fig. 2D).

The influence of opioid agonists on basal and GnRH-stimulated FSH secretion

The activation of mu-opioid receptors by DAMGO did not influence FSH secretion by anterior pituitary cells (Fig. 3); neither the phase of the estrous cycle nor the treatment with GnRH affected this process (p>0.05).

Figure 3
Fig. 3. Basal and GnRH-stimulated secretion of FSH from pituitary cells of gilts in luteal (A, B) and follicular (C, D) phases of the estrous cycle incubated with DAMGO, DPDPE and U 50.488 at doses 10–9, 10–8 and 10–7 mol/L. The concentration of FSH was expressed per 105 cells; n=7; mean ± S.E.M., * p<0.05.

The addition of delta-receptor agonist at dose 10–8 mol/L to the culture media reduced secretion of FSH (from 2.11±1.26 to 1.80±1.07 ng/ml) by GnRH-treated pituitary cells representing luteal phase of the cycle (Fig. 3B). In turn, neither basal nor GnRH-stimulated FSH secretion from cells representing the follicular phase was significantly altered by delta-receptor agonist (Fig. 3C and 3D).

Treatment of the cells with the kappa-agonist significantly (p<0.05) affected FSH secretion only in the presence of GnRH (Fig. 3B and 3D). U 50.488 reduced secretion of FSH by luteal-phase cells (from 2.11±1.26 ng/ml to: 1.74±1.14 and 1.48±0.96 ng/ml for doses 10–8 and 10–7 mol/L, respectively; Fig. 3B) and it lowered this gonadotropin secretion by follicular-phase cells at a dose 10–7 mol/L (from 0.91±0.20 to 0.61±0.21 ng/ml; Fig. 3D).

DISCUSSION

The present study demonstrated that opioid receptor ligands affect gonadotropin secretion depending on the type of receptor involved in the action and physiological status of pituitary cells. The strongest, inhibitory, effects on LH and FSH secretion were observed after treatment of the cells with kappa-opioid agonist. Inhibitory influence of delta-opioid receptor activation was also demonstrated in the presence of GnRH, while specific activation of mu receptors neither significantly altered LH nor FSH secretion.

Activation of kappa-opioid receptors

In our study, the agonist of kappa-opioid receptors, U 50.488, reduced basal and GnRH-stimulated secretion of LH from the cells representing both phases of the estrous cycle. This agonist also reduced FSH secretion by these cells in the presence of GnRH. In previous in vitro studies performed with pituitary cells, the influence of kappa-opioid receptors on gonadotropin secretion has not been studied in detail. However, experiments with the use of the non-specific opioid antagonist (naloxone) to some extent suggested the role of kappa receptors in the modulation of gonadotropin release at the pituitary level (22, 23, 25, 32). It is known that naloxone has the highest affinity to mu-opioid receptors and at higher doses it can efficiently block kappa receptors (31). Barb et al. (22) demonstrated a stimulatory effect of naloxone on basal and GnRH-stimulated LH secretion from isolated pituitary cells of gilts. Similarly, in other studies performed on cows and rats a similar stimulatory effect of the antagonist on LH secretion in vitro was also shown (23, 25, 32). Additionally, naloxone stimulated FSH secretion in vitro from anterior pituitary cells of female rats (32). Collectively, results of these studies suggest that at least part of naloxone effects might have been induced by activation of kappa receptors.

Studies performed in vivo confirm the contribution of dynorphins to the regulation of gonadotropin secretion, however, their results did not specify the site of dynorphin action. Administration of kappa agonists - U 50.488, dynorphin A, dynorphin B or β-neoendorphin to ovariectomized rats reduced LH secretion and blocked preovulatory LH surge (8, 33, 34). In turn, intracerebroventral administration of specific kappa-receptor antagonist, bor-BNI, to proestrus rat accelerated LH surge emergence as well as increased its amplitude (34). In the previous studies, dynorphins were suggested to participate in central (hypothalamic) modulation of gonadotropin secretion, since their presence in GnRH-ergic neurons and influence on GnRH release have been demonstrated (7, 33, 35-37). Our results, in turn, suggest that activation of kappa-opioid receptors may also modulate gonadotropin secretion at the pituitary level.

In the present study the influence of kappa-opioid agonist on LH and FSH secretion from pituitary cells exhibited some differences. At a dose of 10–7 mol/L it decreased both basal and GnRH-stimulated LH secretion but reduced only GnRH-stimulated FSH release (not basal). These results indicate a greater potential of endogenous ligands of kappa receptors for the modulation of LH secretion than FSH throughout the estrous cycle, by affecting the frequency and/or amplitude of its pulses. Moreover, endogenous kappa-receptor ligands (PDYN-derived opioid peptides) seem to participate rather in the modulation of GnRH-induced FSH releases during both phases of the cycle. Effects of the activation of kappa receptors on gonadotropin secretion observed in the present study as well as the fact that dynorphins can be synthesized by gonadotropes (38-40) indicate that these opioid peptides can affect gonadotropin secretion in an autocrine manner. Evident influence of kappa agonist on GnRH-evoked secretion of gonadotropins imply an interaction of intracellular pathways transducing signals from the opioid and GnRH receptors in gonadotropic cells. Previous studies have demonstrated an involvement of phospholipase C-dependent pathway in transduction of kappa- and GnRH-receptor stimulation (41, 42). Nevertheless, the mechanisms of above interaction requires further, more detailed studies.

Activation of delta-opioid receptors

Activation of delta-opioid receptors in our study inhibited GnRH-stimulated secretion of LH and FSH from pituitary cells collected from the follicular- and luteal-phase gilts, respectively. It should be noted that delta receptor agonist affected gonadotropin secretion only at concentration of 10–8 mol/L but not at the highest dose (10–7 mol/L). Similar effect was observed in a previous study (29) and it might result from dose-dependent recruitment of specific subtype of G protein by the ligand.

Our results pertaining to LH are in general agreement with literature data, which demonstrated an inhibitory effect of endogenous delta-agonists, Met- and Leu-enkephalin, on GnRH-stimulated secretion of LH secretion from cultured pituitary cells of cows (23) and rats (28). Moreover, studies with the use of the entire rat pituitaries also showed inhibitory influence of another delta opioid agonist, DSLET, on GnRH-stimulated secretion of this gonadotropin (43). Results of the present study indicate that pituitary enkephalin system can influence LH secretion in gilts only during the late follicular phase. In previous in vivo studies, 6-hour administration of mu- and delta-opioid agonist, FK 33-824, to the late follicular-phase (days 19–20 of the estrous cycle) gilts led to decrease of LH concentration in peripheral blood (4). Authors of that study concluded that FK 33-824 can predominantly act at the level of hypothalamus, because it did not blocked responsiveness of pituitary gland to GnRH. Nevertheless, our present study suggests that effects of delta-opioid receptor activation on LH secretion in the late follicular phase is also possible at the pituitary level. Moreover, enkephalin synthesis was demonstrated in the rat gonado- and somatotropes (44), and this supports the possibility of these peptides to affect gonadotropin secretion in a para- and/or autocrine manner.

Our results demonstrated that delta agonist can reduce GnRH-stimulated FSH secretion from luteal-phase porcine pituitary cells. In the previous experiments performed with the pig or other species, the effects of specific delta receptor activation on FSH secretion has not been studied in detail. However, treatment with naloxone, which also blocks delta receptors, increased FSH secretion from the rat pituitary cells in vitro (32). Moreover, in in vivo studies performed on women, cows and mares, increase of FSH release after treatment with naloxone was demonstrated (5, 45, 46, 47). As opposed to the luteal phase, no influence of DPDPE on FSH secretion by pituitary cells of the follicular-phase gilts was shown in the present study. Similarly, in other studies performed on gilts under in vivo conditions no changes in FSH release were found during the follicular phase after treatment with naloxone (3). Our in vitro study together with previous results suggests that in gilts, EOP participate in the modulation of FSH secretion from the pituitary gland through delta receptors mainly during the luteal phase of the estrous cycle.

Activation of mu-opioid receptors

Results of the present study did not show statistically significant influence of mu-opioid agonist, DAMGO, on gonadotropin secretion from pituitary cells. In previous studies with porcine pituitary cells, Barb et al. (22) observed stimulatory effects of β-endorphin (mainly mu-receptor agonist) on basal secretion of LH after short exposure (4 h). However, in the same study (22) as well as in studies performed with rats (32) longer incubation of pituitary cells (at least 24 h) with β-endorphin inhibited basal LH release. Therefore, the effect of mu-opioid receptor activation on basal LH secretion seems to be dependent on the exposure time to the agonist and it cannot be excluded that longer (e.g. 24 h) stimulation of mu receptors with DAMGO could affect LH secretion in our study. In the present study only short exposure (4 h) of the cells to opioid ligands was considered, as it rather more accurately mimics physiological conditions than longer exposure (4 h), which could reflect chronic stress conditions.

Previous studies performed with porcine and bovine pituitary cells generally showed reduced GnRH-stimulated secretion of LH in vitro after short treatment (4 h) with beta-endorphin (22, 24). In turn, in the present study, DAMGO did not influence GnRH-stimulated LH release from pituitary cells of gilts representing both phases of the estrous cycle. It could be concluded that divergent effects of mu-opioid agonists on GnRH-stimulated LH secretion are, at least partially, due to the use of opioid ligands with differentiated affinity to opioid receptors. DAMGO is highly selective for mu receptors, while β-endorphin, used in the majority of previous studies, can also efficiently bind to delta and kappa receptors (48). Thus, some effects of b-endorphin on LH secretion from pituitary cells noted in the previous studies (22, 24) might have been evoked by the remaining types of opioid receptors, i.e. delta and/or kappa.

In conclusion, results of the present study further support the possibility of EOP participation in a local, para- and/or autocrine, modulation of gonadotropin secretion from anterior pituitary cells of gilts during the estrous cycle. The action of EOP through kappa- and delta-opioid receptors seems to be especially important in this process.

Acknowledgements: The study was supported by the Ministry of Science and Higher Education Grant No: N N303 292934.

REFERENCES

  1. McNally GP, Akil AH. Opioid peptides and their receptors: overview and function in pain modulation. In: Neuropsychopharmacology: The Fifth Generation of Progress, KL Davis, D Charney, JT Coyle, C Nemeroff (eds). Philadelphia, Pennsylvania, Lippincott Williams & Wilkins, 2002, pp. 35-46.
  2. Evans CJ. Secrets of the opium poppy revealed. Neuropharmacology 2004; 47(Suppl. 1): 293-299.
  3. Okrasa S, Weigl R, Mah J, Tilton J. Concentrations of prolactin, LH and FSH after naloxone administration in follicular-phase gilts. Anim Reprod Sci 1990; 22: 39-46.
  4. Okrasa S, Tilton JE. Concentrations of prolactin and LH after administration of Met-enkephalin analogue (FK 33-824) to gilts during the follicular phase. Anim Reprod Sci 1992; 27: 195-207.
  5. Davison LA, McManus CJ, Fitzgerald BP. Gonadotropin response to naloxone in the mare: effect of time of year and reproductive status. Biol Reprod 1998; 59: 1195-1199.
  6. Kadokawa H, Yamada Y. Effect of a long-lasting opioid receptor antagonist (naltrexone) on pulsatile LH release in early postpartum Holstein dairy cows. Theriogenology 2000; 54: 75-81.
  7. Goodman RL, Coolen LM, Anderson GM, et al. Evidence that dynorphin plays a major role in mediating progesterone negative feedback on gonadotropin-releasing hormone neurons in sheep. Endocrinology 2004; 145: 2959-2967.
  8. Leadem CA, Yagenova SV. Effects of specific activation of mu-, delta- and kappa-opioid receptors on the secretion of luteinizing hormone and prolactin in the ovariectomized rat. Neuroendocrinology 1987; 45: 109-117.
  9. Ignacak A, Kasztelnik M, Sliwa T, Korbut RA, Rajda R, Guzik TJ. Prolactin - not only lactotrophin a “new” view of the “old” hormone. J Physiol Pharmacol 2012; 63: 435-443.
  10. Asanovich KM, Johnson B, Chang WJ, Barb CR, Rampacek GB, Kraeling RR. Delay of estradiol-induced surge secretion of LH in gilts by intracerebroventricular injection of morphine. Domest Anim Endocrinol 1998; 15: 45-53.
  11. Ciechanowska MO, Lapot M, Malewski T, Mateusiak K, Misztal T, Przekop F. The central effect of beta-endorphin and naloxone on the expression of GnRH Gene and GnRH receptor (GnRH-R) gene in the hypothalamus, and on GnRH-R gene in the anterior pituitary gland in follicular phase ewes. Exp Clin Endocrinol Diabetes 2008; 116: 40-46.
  12. Carretero J, Bodego P, Rodriguez RE, Rubio M, Blanco E, Burks DJ. Expression of the mu-opioid receptor in the anterior pituitary gland is influenced by age and sex. Neuropeptides 2004; 38: 63-68.
  13. Wylot B, Staszkiewicz J, Okrasa S. The expression of genes coding for opioid precursors, opioid receptors, beta-LH subunit and GnRH receptor in the anterior pituitary of cyclic gilts. J Physiol Pharmacol 2008; 59: 745-758.
  14. Sabbieti MG, Marchetti L, Menghi G, et al. Occurrence of beta-endorphin binding sites in the pituitary of the frog Rana esculenta: effect of beta-endorphin on luteinizing hormone secretion. Gen Comp Endocrinol 2003; 132: 391-398.
  15. Chadzinska M, Hermsen T, Savelkoul HF, Verburg-van Kemenade BM. Cloning of opioid receptors in common carp (Cyprinus carpio L.) and their involvement in regulation of stress and immune response. Brain Behav Immun 2009; 23: 257-266.
  16. Garcia-Garcia L, Llewellyn-Jones V, Fernandez Fernandez I, Fuentes JA, Manzanares J. Acute and repeated ECS treatment increases CRF, POMC and PENK gene expression in selected regions of the rat hypothalamus. Neuroreport 1998; 9: 73-77.
  17. Pierzchala-Koziec K, Zubel J, Rzasa J. Effect of prolonged progesterone treatment on the proenkephalin mRNA gene expression and enkephalins concentration in the sheep brain. Reprod Biol 2006; 6(Suppl. 2): 37-46.
  18. Chao CC, Trout WE, Malven PV. Immunoreactive dynorphin-A in ovine anterior pituitary and effects of estradiol-17 beta administration. Peptides 1987; 8: 367-369.
  19. Yan L, Zhu X, Tseng JL, Desiderio DM. Beta-endorphin-containing proteins in the human pituitary. Peptides 1997; 18: 1399-1409.
  20. Roman E, Ploj K, Gustafsson L, Meyerson BJ, Nylander I. Variations in opioid peptide levels during the estrous cycle in Sprague-Dawley rats. Neuropeptides 2006; 40: 195-206.
  21. Krzymowski T, Stefanczyk-Krzymowska S. Local retrograde and destination transfer of physiological regulators as an important regulatory system and its role. Facts and hypothesis. J Physiol Pharmacol 2012; 63: 3-16.
  22. Barb CR, Barrett JB, Wright JT, Kraeling RR, Rampacek GB. Opioid modulation of LH secretion by pig pituitary cells in vitro. J Reprod Fertil 1990; 90: 213-219.
  23. Chao CC, Moss GE, Malven PV. Direct opioid regulation of pituitary release of bovine luteinizing hormone. Life Sci 1986; 39: 527-534.
  24. Saleri R, Baratta M, Tamanini C. b-endorphin directly influences both basal and GnRH-induced LH release by bovine pituitaries in vitro. Reprod Dom Anim 1998; 33: 27-32.
  25. Sanchez-Franco F, Cacicedo L. Inhibitory effect of beta-endorphin on gonadotropin-releasing hormone and thyrotropin-releasing hormone releasing activity in cultured rat anterior pituitary cells. Horm Res 1986; 24: 55-61.
  26. Akins EL, Morrissette MC. Gross ovarian changes during the estrous cycle of swine. Am J Vet Res 1968; 29: 1953-1957.
  27. Kaminski T, Siawrys G, Bogacka I, Okrasa S, Przala J. The regulation of steroidogenesis by opioid peptides in porcine theca cells. Anim Reprod Sci 2003; 78: 71-84.
  28. Leiva LA, Croxatto HB. Comparison of the effect of hypothalamic neuropeptides upon luteinizing hormone secretion by cultured rat anterior pituitary cells. Biol Res 1994; 27: 113-121.
  29. Krazinski BE, Koziorowski M, Brzuzan P, Okrasa S. The expression of genes encoding opioid precursors and the influence of opioid receptor agonists on steroidogenesis in porcine adrenocortical cells in vitro. J Physiol Pharmacol 2011; 62: 461-468.
  30. Szafranska B, Ziecik A, Okrasa S. Primary antisera against selected steroids or proteins and secondary antisera against gamma-globulins - an available tool for studies of reproductive processes. Reprod Biol 2002; 2: 187-204.
  31. Pasternak GW. Multiple opiate receptors: deja vu all over again. Neuropharmacology 2004; 47(Suppl. 1): 312-323.
  32. Cacicedo L, Sanchez Franco F. Direct action of opioid peptides and naloxone on gonadotropin secretion by cultured rat anterior pituitary cells. Life Sci 1986; 38: 617-625.
  33. Yilmaz B, Gilmore DP. Opioid modulation of hypothalamic catecholaminergic neurotransmission and the pre-ovulatory LH surge in the rat. Neuroendocrinol Lett 1999; 20: 115-121.
  34. Zhang Q, Gallo RV. Presence of kappa-opioid tone at the onset of the ovulatory luteinizing hormone surge in the proestrous rat. Brain Res 2003; 980: 135-139.
  35. Smith MJ, Gallo RV. The effect of blockade of kappa-opioid receptors in the medial preoptic area on the luteinizing hormone surge in the proestrous rat. Brain Res 1997; 768: 111-119.
  36. Foradori CD, Goodman RL, Adams VL, Valent M, Lehman MN. Progesterone increases dynorphin a concentrations in cerebrospinal fluid and preprodynorphin messenger ribonucleic acid levels in a subset of dynorphin neurons in the sheep. Endocrinology 2005; 146: 1835-1842.
  37. Navarro VM, Gottsch ML, Chavkin C, Okamura H, Clifton DK, Steiner RA. Regulation of gonadotropin-releasing hormone secretion by kisspeptin/dynorphin/neurokinin B neurons in the arcuate nucleus of the mouse. J Neurosci 2009; 29: 11859-11866.
  38. Khachaturian H, Sherman TG, Lloyd RV, et al. Pro-dynorphin is endogenous to the anterior pituitary and is co-localized with LH and FSH in the gonadotrophs. Endocrinology 1986; 119: 1409-1411.
  39. Schwaninger M, Knepel W, Dohler KD, Sandow J. Release of dynorphin-like immunoreactivity from rat adenohypophysis in vitro during inhibition of anterior pituitary hormone secretion from individual cell types. Endocrinology 1987; 121: 167-174.
  40. Kaynard AH, Low KG, Melner MH. Differential regulation of anterior pituitary prodynorphin and gonadotropin-subunit gene expression by steroid hormones. Mol Cell Endocrinol 1992; 88: 67-75.
  41. Szafranska B, Tilton JE. Free intracellular calcium [Ca2+] in opioid sensitive cells of the porcine anterior pituitary. J Physiol Pharmacol 2000; 51: 541-554.
  42. Pawson AJ, McNeilly AS. The pituitary effects of GnRH. Anim Reprod Sci 2005; 88: 75-94.
  43. Dragatsis I, Papazafiri P, Zioudrou C, Gerozissis K. Opioids modify the release of LH at the pituitary level: in vitro studies with entire rat pituitaries. J Endocrinol 1995; 145: 263-270.
  44. Slama A, Burg-Poveda D, Tramu G. Colocalized peptides in gonadotrophs: LeuEnkephalin and ACTH interact differently on GnRH induced LH and FSH release. Neuropeptides 1990; 16: 135-140.
  45. Rossmanith WG, Mortola JF, Yen SS. Role of endogenous opioid peptides in the initiation of the midcycle luteinizing hormone surge in normal cycling women. J Clin Endocrinol Metab 1988; 67: 695-700.
  46. Wolfe MW, Roberson MS, Stumpf TT, Kittok RJ, Kinder JE. Modulation of luteinizing hormone and follicle-stimulating hormone in circulation by interactions between endogenous opioids and oestradiol during the peripubertal period of heifers. J Reprod Fertil 1992; 96: 165-174.
  47. Behrens C, Aurich JE, Klug E, Naumann H, Hoppen HO. Inhibition of gonadotrophin release in mares during the luteal phase of the oestrous cycle by endogenous opioids. J Reprod Fertil 1993; 98: 509-514.
  48. Vuong C, Van Uum SH, O’Dell LE, Lutfy K, Friedman TC. The effects of opioids and opioid analogs on animal and human endocrine systems. Endocr Rev 2010; 31: 98-132.
R e c e i v e d : April 4, 2013
A c c e p t e d : August 9, 2013
Author’s address: Prof. Stanislaw Okrasa, Department of Animal Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury, Olsztyn, Poland, 1A Oczapowski Street, 10-719 Olsztyn, Poland e-mail: okrasa@uwm.edu.pl