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Effect of exogenous melatonin on in vivo and in vitro prostaglandin secretion in Rasa Aragonesa ewes
Theriogenology 60 (2003) 1345–1355
Effect of exogenous melatonin on in vivo and in vitro prostaglandin secretion in Rasa Aragonesa ewes Jose´-Alfonso Abeciaa,*, Fernando Forcadaa, Jose´-Antonio Valaresa, Olga Zu´n˜igaa, Hans Kindahlb a
Department of Animal Production and Food Science, Faculty of Veterinary Medicine, University of Zaragoza, Miguel Servet, 177, 50013 Zaragoza, Spain b Department of Obstetrics and Gynaecology, SLU, Box 7039, SE-75007 Uppsala, Sweden
Received 21 October 2002; received in revised form 7 April 2003; accepted 8 April 2003
Abstract The effect of exogenous melatonin on prostaglandin secretion was measured on Rasa Aragonesa ewes. Fourteen ewes received an 18 mg melatonin implant (Mþ) on 10 April and were compared with 13 control animals (without implants M). Twenty days later, intravaginal pessaries were inserted in all animals to induce a synchronized oestrus (day 0). On day 14, ewes were injected, i.v., with 0.5 IU oxytocin. Plasma 15-ketodihydro-PGF2a (PGFM) concentrations were measured to assess uterine secretory responsiveness to oxytocin. After euthanasia, pieces of endometrium were collected to determine progesterone content and PGE2 and PGF2a secretion in vitro, in the presence or absence of either 20 mg/ml recombinant ovine interferon-tau (roIFNt) or 1 nmol/l oxytocin in the medium. Endometrial progesterone content was similar in the two treatments (Mþ: 50:25 17:34 ng/mg tissue, M: 43:08 11:21 ng/mg tissue). Mþ ewes that responded to oxytocin had significantly higher plasma PGFM concentrations between 10 and 80 min after oxytocin administration, a higher mean PGFM peak (P < 0:001), higher plasma PGFM levels after the challenge (P < 0:05) and higher plasma progesterone concentrations (P < 0:01) than control ewes. In the in vitro experiment, Mþ and M control samples secreted similar amounts of PGE2. The presence of roIFNt and oxytocin only stimulated PGE2 production (P < 0:05) in M tissues. Control Mþ tissues secreted higher amounts of PGF2a (P ¼ 0:07) and PGF2a secretion was significantly (P < 0:01) stimulated by roIFNt. Oxytocin produced this effect only in M samples (P < 0:01). In conclusion, although previous studies have demonstrated a positive effect of melatonin on lamb production, PGF2a secretion is higher in vitro and the PGE2:PGF2a ratio is unfavourable in response to
* Corresponding author. Tel.: þ34-976761000; fax: þ34-976761612. E-mail address: [email protected] (J. Abecia).
0093-691X/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0093-691X(03)00168-7
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IFNt, which could affect embryo survival. Whether or not these mechanisms are similar in pregnant ewes remains to be elucidated. # 2003 Elsevier Science Inc. All rights reserved. Keywords: Sheep; Melatonin; Prostaglandins
1. Introduction Sexual activity in sheep is mainly affected by photoperiod. Changes in day length are conveyed to the reproductive neuroendocrine axis by melatonin [1]. Subcutaneous implants with this hormone are commonly used to advance the breeding season in sheep [2–4]. The implants increase melatonin concentration over a 24-h period, causing a short day-like response without suppressing the endogenous rhythm of secretion [5,6]. About 40–60 days after treatment the pulse frequency of LH and GnRH are increased [7]. Melatonin treatment affects the reproductive physiology of the ewe at the central level as well as other physiological processes, including an increased ovulation rate [8,9], an annual rhythm of luteal secretion of progesterone and improved luteal function [10]. In fact, melatonin receptors have been found in human granulosa cells [11]. Melatonin also stimulates progesterone secretion by luteal tissue both in vitro and in vivo [12–14]. In a previous study, our group has shown that plasma progesterone concentration increases following a melatonin challenge [15]. Melatonin could also affect fertility rate by provoking changes in embryo development or maternal pregnancy recognition (MPR) mechanisms. We observed that in vitro embryo development improved in the presence of melatonin [15]. However, in an embryo transfer program, McEvoy et al. [16] did not find more embryos in melatonin-treated donor ewes. It is unclear whether melatonin has a direct effect on the MPR system in sheep. The main modulators of these mechanisms are prostaglandin (PG) secretion from the uterus and interferon-tau (IFNt) from the embryo. In rats, melatonin appears to regulate prostaglandins produced by the uterus and hypothalamus [17] and inhibit spontaneous and oxytocininduced contractions of the myometrium [18]. In ruminants, oestrus is brought about by the luteolytic action of PGF2a, after it is released by the endometrium at the end of the cycle [19]. This mechanism can be blocked by the presence of an embryo (10–21 days old), which inhibits the endometrial oestrogen receptor by the production of IFNt, prevents an increase in oxytocin receptors and suppresses the pulsatile release of PGF2a by oxytocin [20]. In contrast to PGF2a, PGE2 has a luteoprotective action that is either luteotrophic or antiluteolytic [21]. The aim of this study was to determine the effect of melatonin on prostaglandin secretion and how this may alter the luteolytic mechanisms.
2. Material and methods The study was carried out at the experimental farm of the University of Zaragoza, Spain (latitude 418 400 N), which meets the requirements of the European Community
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Commission (1986) for Scientific Procedure Establishments. All protocols were approved by the Animal Experimentation Ethics Committee of University of Zaragoza. 2.1. Animals We used 27 non-pregnant Rasa Aragonesa ewes with a mean (S.E.M.) live weight of 68:2 1:7 kg and a mean body condition score [22] of 3:38 0:06. On 10 April, 14 ewes (group Mþ) received a single implant at the base of the left ear containing 18 mg melatonin (Melovine, CEVA Salud Animal, Spain). The remaining animals (group M, n ¼ 13) were not implanted. Animals were housed in communal yards with an uncovered area and fed a concentrate ration and barley straw at rates designed to provide their maintenance requirements. Fresh, clean water was available at all times. 2.2. Experimental procedures To determine ovarian activity at the time of implantation, blood samples were collected into heparin coated tubes on 3 and 7 April, and assayed for progesterone. Ewes were deemed cyclic if their plasma progesterone concentrations were greater than 0.5 ng/ml, which indicates the presence of a corpus luteum in this breed [23]. Twenty days after melatonin implantation, intravaginal pessaries containing 30 mg of FGA (Chronogest, Intervet, Spain) were inserted in all animals. The pessaries were withdrawn 12 days after insertion, after which 300 UI eCG (Intervet, Spain) were injected intramuscularly. Estrus (day 0) was detected using vasectomized rams. Thereafter, blood samples were collected daily and assayed for progesterone. On day 14, ewes were challenged with 0.5 IU oxytocin (Sigma, St. Louis, USA) in 1 ml saline. Plasma concentrations of 15-ketodihydro-PGF2a (PGFM, the main metabolite of PGF2a) were measured in blood samples collected at 15 min intervals for 1 h before the injection of oxytocin and then at 10 min intervals for 2 h after the challenge. Four hours after the end of the bleeding period, ewes were euthanized (Euta-Lener, Normon, Spain), and pieces of endometrium were collected from the body of the uterus ipsilateral to the ovulating ovary. Samples were either stored at 20 8C until the determination of progesterone content or cultured to determine prostaglandin secretion in vitro. The numbers of corpora lutea on each ovary were also recorded. 2.3. Endometrial progesterone content Uterine progesterone concentrations were determined using the technique described by Abecia et al. [24]. Endometrial tissue samples (500 mg) were homogenized in 10 ml isotonic buffer (102.5 g/l sucrose, 0.375 g/l EDTA, 1.825 g/l Tris, pH 7.4), and the supernatant was removed after centrifugation at 1000 g for 10 min. Progesterone was extracted four times from 1 ml supernatant, using 2 ml diethyl ether each time. The pooled extracts were then assayed to determine progesterone concentration. The proportion of progesterone recovered was estimated by measuring the recovery of 125 I-progesterone added to the homogenate. The appropriate correction factor was applied to the measured concentrations.
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2.4. Endometrial tissue incubation Pieces of endometrial tissue (500 mg) were placed in individual wells of a 24-well tissue culture plate containing 500 ml of cell culture medium, and incubated in duplicate for 24 h at 37 8C in an atmosphere of 5% CO2 in either control medium or medium containing either 20 mg/ml recombinant ovine interferon-tau (roIFNt) or 1 nmol/l oxytocin. The cell culture medium consisted of Ham’s F-12 medium supplemented with antibiotics, L-glutamine (0.29 mg/ml), insulin (5 mg/ml), transferrin (5 mg/ml) and selenium (5 ng/ml) [25]. The roIFNt was provided by Dr. Nicole Cheˆ ne (INRA Jouy-en-Josas, France), produced and purified as described in Ref. [26]. Its antiviral activity was 0:8 108 IU/ml, using MDBK cells and vesicular stomatitis virus. After culture, the medium was collected and stored at 20 8C until analysis to determine prostaglandin concentrations. 2.5. PGFM determination 15-Ketodihydro-PGF2a concentrations were determined in duplicate by radioimmunoassay of plasma samples using the antibody and the technique described by Granstro¨ m and Kindahl [27]. The sensitivity of the method was 60 pmol/l. The intra-assay coefficients of variation ranged between 6.6 and 11.7% for the different ranges of the standard curve. The inter-assay coefficient of variation was 14%. 2.6. Prostaglandin determination Culture media from wells were analysed using prostaglandin enzyme immunoassay kits (Cayman Chemical Co., Ann Arbor, USA), to determine PGE2 and PGF2a concentrations. The intra- and inter-assay coefficients of variation were 2.5 and 2.3% and 2.4 and 10%, for PGE2 and PGF2a, respectively. 2.7. Progesterone determination Progesterone was determined using solid-phase RIA kits based on antibody coated tubes, 125 I-labelled progesterone and rabbit antiserum (CIS bio international, Gif-surYvette, France). The assay sensitivity was 0.05 ng/ml. The intra- and inter-assay coefficients of variation were 13.6 and 15.7%, respectively. All material employed for tissue cultures was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.8. Statistical analysis A PGFM response was defined as an increase at two successive points during the posttreatment period above the mean þ 2 S.D. values of the pre-treatment period. Peak height (PGFM peak) was defined as the greatest increase recorded during the post-treatment period after subtracting the initial baseline to account for basal differences among ewes. An analysis of variance was applied to compare PGFM peak, and pre- and post-treatment PGFM concentrations between groups, excluding data from ewes which did not respond to oxytocin.
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The main effects of melatonin treatment on prostaglandin secretion in vitro and plasma and endometrial progesterone concentrations were compared by an analysis of variance. Ovulation rate was compared using a Chi-square test. A paired t-test was used to compare prostaglandin concentrations in the treated and control media after incubation with roIFNt or 1 nmol/l oxytocin.
3. Results According to the concentrations of plasma progesterone before melatonin treatment, a mean of 26% of the ewes exhibited ovarian cyclicity, with no differences between groups. The mean ovulation rate was similar in both groups (Mþ: 2:29 0:22, M: 2:15 0:32). During the luteal phase (days 5–14), plasma progesterone was higher in Mþ ewes but not significantly different than controls (Fig. 1). However, the overall mean level throughout the experiment was significantly higher in treated ewes (Mþ: 1:06 0:13 ng/ml, M: 0:75 0:09 ng/ml, P ¼ 0:05). There were no significant differences in the progesterone content of endometrial tissue (Mþ: 50:25 17:34 ng/mg tissue, M: 43:08 11:21 ng/mg tissue, NS). The number of ewes with a PGFM response to oxytocin challenge was similar in both groups (Mþ: 7/13, M: 6/14). Plasma concentrations of PGFM were similar between groups prior to oxytocin challenge (Table 1) but increased rapidly in Mþ approximately 10–80 min after oxytocin administration (P < 0:05, Fig. 2). Mþ ewes had significantly higher mean PGFM peak height (P < 0:001) and mean plasma PGFM (P < 0:05) after the challenge (Table 1). Mþ ewes had significantly higher (P < 0:01) concentrations of plasma progesterone between day 10 and 14, among the ewes in both groups that responded to the oxytocin challenge throughout the cycle (Fig. 3). PGE2 secretion by the control tissue was similar in incubates from Mþ and M samples. However, IFNt and oxytocin stimulated the production of PGE2 relative to controls only in tissues collected from M ewes (P < 0:05, Fig. 4). PGF2a concentrations were higher in
Fig. 1. Plasma progesterone (P4) concentration throughout the oestrous cycle of ewes with (Mþ) or without (M) exogenous melatonin implants.
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Table 1 Mean (S.E.M.) plasma PGFM concentrations (pmol/l) before and after oxytocin challenge on day 14 of the oestrous cycle, mean PGFM peak and time (min) of PGFM peak of ewes treated (Mþ) or not (M) with exogenous melatonin (ewes responding to the challenge) Mþ Before After Peak Peak time
453 748 553 28
M
82 (456 118) 150 (552 54) 54 7
360 329 130 53
Significance
60 (246 42) 53 (130 30) 30 14
NS P < 0.05 (P < 0.0001) P < 0.001 NS
Fig. 2. Mean (S.E.M) plasma concentration of PGFM before and after oxytocin challenge on day 14 of the oestrous cycle in ewes that responded to oxytocin administration, with (Mþ) or without (M) exogenous melatonin (P < 0:05, P < 0:01, P < 0:001).
Fig. 3. Plasma progesterone (P4) concentration throughout the oestrous cycle in ewes that responded to an oxytocin challenge on day 14 (with (Mþ) or without (M) exogenous melatonin, P < 0:01).
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Fig. 4. Mean (S.E.M) PGE2 and PGF2a concentration and PGE2:PGF2a ratio in the culture medium after incubation of endometrial samples in ewes with (Mþ) or without (M) exogenous melatonin, collected on day 14 of the oestrous cycle, after incubation in medium (control), medium with 20 mg/ml roIFNt or medium with 1 nmol oxytocin/l. Different letters indicate differences within groups of at least P < 0:05, (P < 0:05 and P < 0:01).
control tissues Mþ ewes (P ¼ 0:07) and PGF2a secretion was significantly (P < 0:01) stimulated by roIFNt. Oxytocin produced the same effect in M samples (P < 0:01). As a result of the different patterns of secretion the PGE2:PGF2a ratio was significantly higher in the M ewes after incubation with IFNt (P < 0:05).
4. Discussion The melatonin-treated ewes (Mþ) appeared to produce more PGF2a than control ewes both in vivo (after an oxytocin challenge) and in vitro (with or without IFNt in the culture medium). Since the oxytocin-induced PGFM response in OVX ewes is under the
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quantitative control of progesterone and estradiol, ewes that do not respond to the challenge probably had an abnormal secretion of these hormones [28]. In early pregnancy, ewes with a high estradiol:progesterone ratio may generate larger PGF2a episodes, which increases the risk of failure of the maternal recognition of pregnancy. Moreover, progesterone treatment in OVX ewes substantially increases the concentration of PGF2a in uterine venous plasma and the PGFM response [29]. In this experiment, melatonin-treated ewes which responded to oxytocin had higher plasma progesterone than the controls that also responded to oxytocin. The progesterone concentrations were considerably higher compared with normal levels in this breed at the same time in the estrous cycle [30,31]. Recently, we have shown that intravenous injection of melatonin has an effect on progesterone secretion [15]. Wallace et al. [32] observed higher peripheral progesterone concentrations on days 7 and 12 of the cycle in melatonin-treated ewes compared to naturally ovulating control animals. Further evidence supporting a direct effect of melatonin on progesterone production has been obtained with studies involving sheep [33], cattle [13] and humans [13,14], and melatonin receptors have been found in human granulosa cells [11]. Thus, the higher secretion of PGFM by Mþ ewes after the oxytocin challenge could be mediated indirectly by a higher production of progesterone instead of through a direct effect of melatonin on endometrial cells. Progesterone probably induces responsiveness to oxytocin by up regulating the post-receptor signalling pathways and/or enzyme involved in prostaglandin synthesis [29]. In fact, progesterone can increase the number of oxytocin receptors [34] and up-regulate COX-2 expression, which in turn increases the capacity of prostaglandin synthesis [35]. Thus, the elevated plasma progesterone concentrations caused by melatonin could be sufficient to strongly modulate oxytocin receptors and prostaglandin enzymes. The regulation of receptors in endometrial tissue could also be similar since their progesterone content was homogeneous. Estradiol concentration has also been correlated with the mean response of PGFM to oxytocin, via the estrogenic stimulation of uterine oxytocin receptors [36]. We could not confirm the role of estradiol in the response to oxytocin in Mþ ewes since plasma estradiol concentrations were not determined. On the other hand, a faster increase in PGFM may reflect a faster release of PGF2a or a greater rate of metabolism of PGF2a to PGFM [29]. In ruminants, photoperiod regulates sexual activity as well as other physiological processes, including basal metabolism. Short days induce a higher rate of tissue metabolism and increased lipolytic activity [37]. The photoperiodic signal provided by melatonin implants causes a short-day-like response [5,6]. Therefore, perhaps the higher and faster response to oxytocin in Mþ ewes in terms of plasma PGFM concentration reflects differences in PGF2a metabolism rather than PGF2a secretion. However, the in vitro results seem to confirm the effects on secretion in vivo, indicating that other mechanisms should be considered. Most of the information on in vitro prostaglandin production in the presence of both roIFNt and oxytocin is based on bovine tissue. The results depend on the origin of the cultured tissue and the culture techniques. Since we used endometrial explants, we could not distinguish between epithelial or stromal endometrial cells. Samples from Mþ ewes produced more PGF2a (P ¼ 0:07) than controls (M), especially with roIFNt in the culture medium (P < 0:01). However, basal secretion of PGE2 was similar between groups. Adding roIFNt only increased PGE2 in the endometrium in control ewes. In
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cultured ovine endometrial cells, oIFNt attenuates release of both PGE2 and PGF2a, suggesting a direct or indirect action of interferons on the prostaglandin synthase enzyme which converts arachidonic acid to the endoperoxide intermediate (PGH2) [38]. Moreover, bovine IFNt suppresses secretion of PGE2 and PGF2a from the bovine endometrium in vitro [39]. However, Asselin et al. [40] demonstrated that IFNt triggers an important increase of PGE2 by bovine endometrial cells via upregulation of COX-2 gene expression. These authors [41] also observed that roIFNt increases the PGE2:PGF2a ratio, preferentially in the caruncular zone of the bovine endometrium. Thus, at the cellular level, the relative production of PGE2 and PGF2a in vitro may give an indication of the direction of the response at pregnancy recognition in vivo. An increase may indicate a luteoprotective response whereas a reduction may indicate a luteolytic response. We only observed a greater PGE2:PGF2a ratio in the presence of roIFNt in explants from control ewes (significantly higher than the Mþ group). In the presence of oxytocin, there were no differences between groups but PGE2 and PGF2a secretion increased significantly in cultures from untreated ewes compared to control cultures. Oxytocin seems to increase PGE2 and PGF2a secretion in vitro by epithelial cells [39,41,42], with COX-2 being involved in the oxytocin regulation of PGF2a production in the endometrium [42]. However, as concluded by Charpigny et al. [43], the physiological significance of these in vitro experiments could be questionable since the tissues probably do not maintain their normal cellular function in culture. In conclusion, the increase in litter size brought about by melatonin implants occurs despite higher PGF2a secretion in vitro and an unfavourable PGE2:PGF2a ratio in response to IFNt, which could be detrimental for embryo survival. First, it is possible that the positive effect of melatonin on ovulation rate [8,9] or on embryo survival [15] could compensate for the negative effect on prostaglandin secretion, resulting in a higher number of lambs born per treated ewe. Second, the prostaglandin secretion in treated ewes could be lower than the necessary threshold to induce luteolysis. Whether or not these mechanisms are similar in pregnant ewes and the effect of melatonin on IFNt secretion remains to be elucidated.
Acknowledgements This study was supported by grant AGF98-0575 and AGF2001-1817 from CICYT (Spain). J.A. Valares is grateful to DGA (Spain) for a pre-doctoral grant. The authors would like to thank Dr. Alex Martino (CEVA Salud Animal, Spain) and Dr. Nicole Cheˆ ne (INRA, France) for generously donating melatonin implants and roIFNt, respectively. References [1] Bittman EL, Karsch FJ, Hopkins JW. Role of the pineal gland in ovine photoperiodism: regulation of seasonal breeding and negative feedback effects of estradiol upon luteinizing hormone secretion. Endocrinology 1983;113:329–36. [2] Koumitzis SA, Belibasaki S, Doney JM. Melatonin advances and condenses the onset of seasonal breeding in Greek dairy ewes. Anim Prod 1989;48:399–405.
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Effect of exogenous melatonin on in vivo and in vitro prostaglandin secretion in Rasa Aragonesa ewes Jose´-Alfonso Abeciaa,*, Fernando Forcadaa, Jose´-Antonio Valaresa, Olga Zu´n˜igaa, Hans Kindahlb a
Department of Animal Production and Food Science, Faculty of Veterinary Medicine, University of Zaragoza, Miguel Servet, 177, 50013 Zaragoza, Spain b Department of Obstetrics and Gynaecology, SLU, Box 7039, SE-75007 Uppsala, Sweden
Received 21 October 2002; received in revised form 7 April 2003; accepted 8 April 2003
Abstract The effect of exogenous melatonin on prostaglandin secretion was measured on Rasa Aragonesa ewes. Fourteen ewes received an 18 mg melatonin implant (Mþ) on 10 April and were compared with 13 control animals (without implants M). Twenty days later, intravaginal pessaries were inserted in all animals to induce a synchronized oestrus (day 0). On day 14, ewes were injected, i.v., with 0.5 IU oxytocin. Plasma 15-ketodihydro-PGF2a (PGFM) concentrations were measured to assess uterine secretory responsiveness to oxytocin. After euthanasia, pieces of endometrium were collected to determine progesterone content and PGE2 and PGF2a secretion in vitro, in the presence or absence of either 20 mg/ml recombinant ovine interferon-tau (roIFNt) or 1 nmol/l oxytocin in the medium. Endometrial progesterone content was similar in the two treatments (Mþ: 50:25 17:34 ng/mg tissue, M: 43:08 11:21 ng/mg tissue). Mþ ewes that responded to oxytocin had significantly higher plasma PGFM concentrations between 10 and 80 min after oxytocin administration, a higher mean PGFM peak (P < 0:001), higher plasma PGFM levels after the challenge (P < 0:05) and higher plasma progesterone concentrations (P < 0:01) than control ewes. In the in vitro experiment, Mþ and M control samples secreted similar amounts of PGE2. The presence of roIFNt and oxytocin only stimulated PGE2 production (P < 0:05) in M tissues. Control Mþ tissues secreted higher amounts of PGF2a (P ¼ 0:07) and PGF2a secretion was significantly (P < 0:01) stimulated by roIFNt. Oxytocin produced this effect only in M samples (P < 0:01). In conclusion, although previous studies have demonstrated a positive effect of melatonin on lamb production, PGF2a secretion is higher in vitro and the PGE2:PGF2a ratio is unfavourable in response to
* Corresponding author. Tel.: þ34-976761000; fax: þ34-976761612. E-mail address: [email protected] (J. Abecia).
0093-691X/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0093-691X(03)00168-7
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IFNt, which could affect embryo survival. Whether or not these mechanisms are similar in pregnant ewes remains to be elucidated. # 2003 Elsevier Science Inc. All rights reserved. Keywords: Sheep; Melatonin; Prostaglandins
1. Introduction Sexual activity in sheep is mainly affected by photoperiod. Changes in day length are conveyed to the reproductive neuroendocrine axis by melatonin [1]. Subcutaneous implants with this hormone are commonly used to advance the breeding season in sheep [2–4]. The implants increase melatonin concentration over a 24-h period, causing a short day-like response without suppressing the endogenous rhythm of secretion [5,6]. About 40–60 days after treatment the pulse frequency of LH and GnRH are increased [7]. Melatonin treatment affects the reproductive physiology of the ewe at the central level as well as other physiological processes, including an increased ovulation rate [8,9], an annual rhythm of luteal secretion of progesterone and improved luteal function [10]. In fact, melatonin receptors have been found in human granulosa cells [11]. Melatonin also stimulates progesterone secretion by luteal tissue both in vitro and in vivo [12–14]. In a previous study, our group has shown that plasma progesterone concentration increases following a melatonin challenge [15]. Melatonin could also affect fertility rate by provoking changes in embryo development or maternal pregnancy recognition (MPR) mechanisms. We observed that in vitro embryo development improved in the presence of melatonin [15]. However, in an embryo transfer program, McEvoy et al. [16] did not find more embryos in melatonin-treated donor ewes. It is unclear whether melatonin has a direct effect on the MPR system in sheep. The main modulators of these mechanisms are prostaglandin (PG) secretion from the uterus and interferon-tau (IFNt) from the embryo. In rats, melatonin appears to regulate prostaglandins produced by the uterus and hypothalamus [17] and inhibit spontaneous and oxytocininduced contractions of the myometrium [18]. In ruminants, oestrus is brought about by the luteolytic action of PGF2a, after it is released by the endometrium at the end of the cycle [19]. This mechanism can be blocked by the presence of an embryo (10–21 days old), which inhibits the endometrial oestrogen receptor by the production of IFNt, prevents an increase in oxytocin receptors and suppresses the pulsatile release of PGF2a by oxytocin [20]. In contrast to PGF2a, PGE2 has a luteoprotective action that is either luteotrophic or antiluteolytic [21]. The aim of this study was to determine the effect of melatonin on prostaglandin secretion and how this may alter the luteolytic mechanisms.
2. Material and methods The study was carried out at the experimental farm of the University of Zaragoza, Spain (latitude 418 400 N), which meets the requirements of the European Community
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Commission (1986) for Scientific Procedure Establishments. All protocols were approved by the Animal Experimentation Ethics Committee of University of Zaragoza. 2.1. Animals We used 27 non-pregnant Rasa Aragonesa ewes with a mean (S.E.M.) live weight of 68:2 1:7 kg and a mean body condition score [22] of 3:38 0:06. On 10 April, 14 ewes (group Mþ) received a single implant at the base of the left ear containing 18 mg melatonin (Melovine, CEVA Salud Animal, Spain). The remaining animals (group M, n ¼ 13) were not implanted. Animals were housed in communal yards with an uncovered area and fed a concentrate ration and barley straw at rates designed to provide their maintenance requirements. Fresh, clean water was available at all times. 2.2. Experimental procedures To determine ovarian activity at the time of implantation, blood samples were collected into heparin coated tubes on 3 and 7 April, and assayed for progesterone. Ewes were deemed cyclic if their plasma progesterone concentrations were greater than 0.5 ng/ml, which indicates the presence of a corpus luteum in this breed [23]. Twenty days after melatonin implantation, intravaginal pessaries containing 30 mg of FGA (Chronogest, Intervet, Spain) were inserted in all animals. The pessaries were withdrawn 12 days after insertion, after which 300 UI eCG (Intervet, Spain) were injected intramuscularly. Estrus (day 0) was detected using vasectomized rams. Thereafter, blood samples were collected daily and assayed for progesterone. On day 14, ewes were challenged with 0.5 IU oxytocin (Sigma, St. Louis, USA) in 1 ml saline. Plasma concentrations of 15-ketodihydro-PGF2a (PGFM, the main metabolite of PGF2a) were measured in blood samples collected at 15 min intervals for 1 h before the injection of oxytocin and then at 10 min intervals for 2 h after the challenge. Four hours after the end of the bleeding period, ewes were euthanized (Euta-Lener, Normon, Spain), and pieces of endometrium were collected from the body of the uterus ipsilateral to the ovulating ovary. Samples were either stored at 20 8C until the determination of progesterone content or cultured to determine prostaglandin secretion in vitro. The numbers of corpora lutea on each ovary were also recorded. 2.3. Endometrial progesterone content Uterine progesterone concentrations were determined using the technique described by Abecia et al. [24]. Endometrial tissue samples (500 mg) were homogenized in 10 ml isotonic buffer (102.5 g/l sucrose, 0.375 g/l EDTA, 1.825 g/l Tris, pH 7.4), and the supernatant was removed after centrifugation at 1000 g for 10 min. Progesterone was extracted four times from 1 ml supernatant, using 2 ml diethyl ether each time. The pooled extracts were then assayed to determine progesterone concentration. The proportion of progesterone recovered was estimated by measuring the recovery of 125 I-progesterone added to the homogenate. The appropriate correction factor was applied to the measured concentrations.
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2.4. Endometrial tissue incubation Pieces of endometrial tissue (500 mg) were placed in individual wells of a 24-well tissue culture plate containing 500 ml of cell culture medium, and incubated in duplicate for 24 h at 37 8C in an atmosphere of 5% CO2 in either control medium or medium containing either 20 mg/ml recombinant ovine interferon-tau (roIFNt) or 1 nmol/l oxytocin. The cell culture medium consisted of Ham’s F-12 medium supplemented with antibiotics, L-glutamine (0.29 mg/ml), insulin (5 mg/ml), transferrin (5 mg/ml) and selenium (5 ng/ml) [25]. The roIFNt was provided by Dr. Nicole Cheˆ ne (INRA Jouy-en-Josas, France), produced and purified as described in Ref. [26]. Its antiviral activity was 0:8 108 IU/ml, using MDBK cells and vesicular stomatitis virus. After culture, the medium was collected and stored at 20 8C until analysis to determine prostaglandin concentrations. 2.5. PGFM determination 15-Ketodihydro-PGF2a concentrations were determined in duplicate by radioimmunoassay of plasma samples using the antibody and the technique described by Granstro¨ m and Kindahl [27]. The sensitivity of the method was 60 pmol/l. The intra-assay coefficients of variation ranged between 6.6 and 11.7% for the different ranges of the standard curve. The inter-assay coefficient of variation was 14%. 2.6. Prostaglandin determination Culture media from wells were analysed using prostaglandin enzyme immunoassay kits (Cayman Chemical Co., Ann Arbor, USA), to determine PGE2 and PGF2a concentrations. The intra- and inter-assay coefficients of variation were 2.5 and 2.3% and 2.4 and 10%, for PGE2 and PGF2a, respectively. 2.7. Progesterone determination Progesterone was determined using solid-phase RIA kits based on antibody coated tubes, 125 I-labelled progesterone and rabbit antiserum (CIS bio international, Gif-surYvette, France). The assay sensitivity was 0.05 ng/ml. The intra- and inter-assay coefficients of variation were 13.6 and 15.7%, respectively. All material employed for tissue cultures was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.8. Statistical analysis A PGFM response was defined as an increase at two successive points during the posttreatment period above the mean þ 2 S.D. values of the pre-treatment period. Peak height (PGFM peak) was defined as the greatest increase recorded during the post-treatment period after subtracting the initial baseline to account for basal differences among ewes. An analysis of variance was applied to compare PGFM peak, and pre- and post-treatment PGFM concentrations between groups, excluding data from ewes which did not respond to oxytocin.
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The main effects of melatonin treatment on prostaglandin secretion in vitro and plasma and endometrial progesterone concentrations were compared by an analysis of variance. Ovulation rate was compared using a Chi-square test. A paired t-test was used to compare prostaglandin concentrations in the treated and control media after incubation with roIFNt or 1 nmol/l oxytocin.
3. Results According to the concentrations of plasma progesterone before melatonin treatment, a mean of 26% of the ewes exhibited ovarian cyclicity, with no differences between groups. The mean ovulation rate was similar in both groups (Mþ: 2:29 0:22, M: 2:15 0:32). During the luteal phase (days 5–14), plasma progesterone was higher in Mþ ewes but not significantly different than controls (Fig. 1). However, the overall mean level throughout the experiment was significantly higher in treated ewes (Mþ: 1:06 0:13 ng/ml, M: 0:75 0:09 ng/ml, P ¼ 0:05). There were no significant differences in the progesterone content of endometrial tissue (Mþ: 50:25 17:34 ng/mg tissue, M: 43:08 11:21 ng/mg tissue, NS). The number of ewes with a PGFM response to oxytocin challenge was similar in both groups (Mþ: 7/13, M: 6/14). Plasma concentrations of PGFM were similar between groups prior to oxytocin challenge (Table 1) but increased rapidly in Mþ approximately 10–80 min after oxytocin administration (P < 0:05, Fig. 2). Mþ ewes had significantly higher mean PGFM peak height (P < 0:001) and mean plasma PGFM (P < 0:05) after the challenge (Table 1). Mþ ewes had significantly higher (P < 0:01) concentrations of plasma progesterone between day 10 and 14, among the ewes in both groups that responded to the oxytocin challenge throughout the cycle (Fig. 3). PGE2 secretion by the control tissue was similar in incubates from Mþ and M samples. However, IFNt and oxytocin stimulated the production of PGE2 relative to controls only in tissues collected from M ewes (P < 0:05, Fig. 4). PGF2a concentrations were higher in
Fig. 1. Plasma progesterone (P4) concentration throughout the oestrous cycle of ewes with (Mþ) or without (M) exogenous melatonin implants.
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Table 1 Mean (S.E.M.) plasma PGFM concentrations (pmol/l) before and after oxytocin challenge on day 14 of the oestrous cycle, mean PGFM peak and time (min) of PGFM peak of ewes treated (Mþ) or not (M) with exogenous melatonin (ewes responding to the challenge) Mþ Before After Peak Peak time
453 748 553 28
M
82 (456 118) 150 (552 54) 54 7
360 329 130 53
Significance
60 (246 42) 53 (130 30) 30 14
NS P < 0.05 (P < 0.0001) P < 0.001 NS
Fig. 2. Mean (S.E.M) plasma concentration of PGFM before and after oxytocin challenge on day 14 of the oestrous cycle in ewes that responded to oxytocin administration, with (Mþ) or without (M) exogenous melatonin (P < 0:05, P < 0:01, P < 0:001).
Fig. 3. Plasma progesterone (P4) concentration throughout the oestrous cycle in ewes that responded to an oxytocin challenge on day 14 (with (Mþ) or without (M) exogenous melatonin, P < 0:01).
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Fig. 4. Mean (S.E.M) PGE2 and PGF2a concentration and PGE2:PGF2a ratio in the culture medium after incubation of endometrial samples in ewes with (Mþ) or without (M) exogenous melatonin, collected on day 14 of the oestrous cycle, after incubation in medium (control), medium with 20 mg/ml roIFNt or medium with 1 nmol oxytocin/l. Different letters indicate differences within groups of at least P < 0:05, (P < 0:05 and P < 0:01).
control tissues Mþ ewes (P ¼ 0:07) and PGF2a secretion was significantly (P < 0:01) stimulated by roIFNt. Oxytocin produced the same effect in M samples (P < 0:01). As a result of the different patterns of secretion the PGE2:PGF2a ratio was significantly higher in the M ewes after incubation with IFNt (P < 0:05).
4. Discussion The melatonin-treated ewes (Mþ) appeared to produce more PGF2a than control ewes both in vivo (after an oxytocin challenge) and in vitro (with or without IFNt in the culture medium). Since the oxytocin-induced PGFM response in OVX ewes is under the
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quantitative control of progesterone and estradiol, ewes that do not respond to the challenge probably had an abnormal secretion of these hormones [28]. In early pregnancy, ewes with a high estradiol:progesterone ratio may generate larger PGF2a episodes, which increases the risk of failure of the maternal recognition of pregnancy. Moreover, progesterone treatment in OVX ewes substantially increases the concentration of PGF2a in uterine venous plasma and the PGFM response [29]. In this experiment, melatonin-treated ewes which responded to oxytocin had higher plasma progesterone than the controls that also responded to oxytocin. The progesterone concentrations were considerably higher compared with normal levels in this breed at the same time in the estrous cycle [30,31]. Recently, we have shown that intravenous injection of melatonin has an effect on progesterone secretion [15]. Wallace et al. [32] observed higher peripheral progesterone concentrations on days 7 and 12 of the cycle in melatonin-treated ewes compared to naturally ovulating control animals. Further evidence supporting a direct effect of melatonin on progesterone production has been obtained with studies involving sheep [33], cattle [13] and humans [13,14], and melatonin receptors have been found in human granulosa cells [11]. Thus, the higher secretion of PGFM by Mþ ewes after the oxytocin challenge could be mediated indirectly by a higher production of progesterone instead of through a direct effect of melatonin on endometrial cells. Progesterone probably induces responsiveness to oxytocin by up regulating the post-receptor signalling pathways and/or enzyme involved in prostaglandin synthesis [29]. In fact, progesterone can increase the number of oxytocin receptors [34] and up-regulate COX-2 expression, which in turn increases the capacity of prostaglandin synthesis [35]. Thus, the elevated plasma progesterone concentrations caused by melatonin could be sufficient to strongly modulate oxytocin receptors and prostaglandin enzymes. The regulation of receptors in endometrial tissue could also be similar since their progesterone content was homogeneous. Estradiol concentration has also been correlated with the mean response of PGFM to oxytocin, via the estrogenic stimulation of uterine oxytocin receptors [36]. We could not confirm the role of estradiol in the response to oxytocin in Mþ ewes since plasma estradiol concentrations were not determined. On the other hand, a faster increase in PGFM may reflect a faster release of PGF2a or a greater rate of metabolism of PGF2a to PGFM [29]. In ruminants, photoperiod regulates sexual activity as well as other physiological processes, including basal metabolism. Short days induce a higher rate of tissue metabolism and increased lipolytic activity [37]. The photoperiodic signal provided by melatonin implants causes a short-day-like response [5,6]. Therefore, perhaps the higher and faster response to oxytocin in Mþ ewes in terms of plasma PGFM concentration reflects differences in PGF2a metabolism rather than PGF2a secretion. However, the in vitro results seem to confirm the effects on secretion in vivo, indicating that other mechanisms should be considered. Most of the information on in vitro prostaglandin production in the presence of both roIFNt and oxytocin is based on bovine tissue. The results depend on the origin of the cultured tissue and the culture techniques. Since we used endometrial explants, we could not distinguish between epithelial or stromal endometrial cells. Samples from Mþ ewes produced more PGF2a (P ¼ 0:07) than controls (M), especially with roIFNt in the culture medium (P < 0:01). However, basal secretion of PGE2 was similar between groups. Adding roIFNt only increased PGE2 in the endometrium in control ewes. In
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cultured ovine endometrial cells, oIFNt attenuates release of both PGE2 and PGF2a, suggesting a direct or indirect action of interferons on the prostaglandin synthase enzyme which converts arachidonic acid to the endoperoxide intermediate (PGH2) [38]. Moreover, bovine IFNt suppresses secretion of PGE2 and PGF2a from the bovine endometrium in vitro [39]. However, Asselin et al. [40] demonstrated that IFNt triggers an important increase of PGE2 by bovine endometrial cells via upregulation of COX-2 gene expression. These authors [41] also observed that roIFNt increases the PGE2:PGF2a ratio, preferentially in the caruncular zone of the bovine endometrium. Thus, at the cellular level, the relative production of PGE2 and PGF2a in vitro may give an indication of the direction of the response at pregnancy recognition in vivo. An increase may indicate a luteoprotective response whereas a reduction may indicate a luteolytic response. We only observed a greater PGE2:PGF2a ratio in the presence of roIFNt in explants from control ewes (significantly higher than the Mþ group). In the presence of oxytocin, there were no differences between groups but PGE2 and PGF2a secretion increased significantly in cultures from untreated ewes compared to control cultures. Oxytocin seems to increase PGE2 and PGF2a secretion in vitro by epithelial cells [39,41,42], with COX-2 being involved in the oxytocin regulation of PGF2a production in the endometrium [42]. However, as concluded by Charpigny et al. [43], the physiological significance of these in vitro experiments could be questionable since the tissues probably do not maintain their normal cellular function in culture. In conclusion, the increase in litter size brought about by melatonin implants occurs despite higher PGF2a secretion in vitro and an unfavourable PGE2:PGF2a ratio in response to IFNt, which could be detrimental for embryo survival. First, it is possible that the positive effect of melatonin on ovulation rate [8,9] or on embryo survival [15] could compensate for the negative effect on prostaglandin secretion, resulting in a higher number of lambs born per treated ewe. Second, the prostaglandin secretion in treated ewes could be lower than the necessary threshold to induce luteolysis. Whether or not these mechanisms are similar in pregnant ewes and the effect of melatonin on IFNt secretion remains to be elucidated.
Acknowledgements This study was supported by grant AGF98-0575 and AGF2001-1817 from CICYT (Spain). J.A. Valares is grateful to DGA (Spain) for a pre-doctoral grant. The authors would like to thank Dr. Alex Martino (CEVA Salud Animal, Spain) and Dr. Nicole Cheˆ ne (INRA, France) for generously donating melatonin implants and roIFNt, respectively. References [1] Bittman EL, Karsch FJ, Hopkins JW. Role of the pineal gland in ovine photoperiodism: regulation of seasonal breeding and negative feedback effects of estradiol upon luteinizing hormone secretion. Endocrinology 1983;113:329–36. [2] Koumitzis SA, Belibasaki S, Doney JM. Melatonin advances and condenses the onset of seasonal breeding in Greek dairy ewes. Anim Prod 1989;48:399–405.
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