Molecular Human Reproduction, Vol. 7, No. 1, 35-42,
January 2001
© 2001 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Inhibition of ovulation in the rat by a leukotriene B4 receptor antagonist
1 Department of Obstetrics and Gynecology, Göteborg University, Göteborg, Sweden, and 2 Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, N-15, W-7, Kita-ku, Sapporo, O60, Japan
Abstract
The involvement of leukotriene (LT) B4 in the ovulatory process of the rat was investigated by the use of a LTB4-receptor antagonist (ZK158252 = L-ANT) administered either intrabursally in vivo or to the in-vitro perfused ovary. The in-vivo experiments revealed inhibition of human chorionic gonadotrophin (HCG)-induced ovulation by 500 µmol/l L-ANT (median 5.5, 25–75% range 1.0–6.0) compared with controls (median 9.0, range 6.25–13.5). In vitro, ovulation was induced by LH (0.2 µg/ml) + 3-isobutyl-1-methylxanthine (IBMX; 0.2 mmol/l). The ovary was perfused either for 20 h, to study ovulation rate, or for 10 h to examine ovarian concentrations of the ovulatory mediators matrix metalloproteinase (MMP)-2 and MMP-9, plasminogen activator (PA), prostaglandin (PG)E2 and PGF2
. Addition of LH+IBMX resulted in a marked stimulation of steroid release and ovulations occurred in all ovaries (median 11.0, range 10.0–14.0). The L-ANT inhibited ovulation in a dose-dependent way (median 10.0, range 8.0–13.0 at 1 µmol/l; median 6.0, range 3.5–10.0 at 10µmol/l; median 2.0, range 0.75–5.75 at 100 µmol/l). The intra-ovarian activity of PA was increased 1.5-fold by L-ANT (100 µmol/l), but the concentrations of PGE2 and PGF2 remained unaltered. While no changes in MMP-9 were observed, conversion from pro-MMP-2 to active MMP-2 was inhibited by L-ANT. These results suggest that activation of the LTB4-receptor within the ovary is involved in the ovulatory process and that the effects of LTB4-receptor activation are partly mediated via MMP-2.
leukotriene/matrix metalloproteinase/ovary/ovulation/rat
Introduction
The LH surge causes marked changes in the ovarian vasculature and extracellular matrix (for review, see Brännström et al., 1997) and some of these changes have been proposed to be partly, directly or indirectly, mediated by eicosanoids (LeMaire et al., 1973
; Tsafriri et al., 1973; Abisogun et al., 1988). The precursor of all eicosanoids, arachidonic acid (AA), is enzymatically metabolized either by the actions of cyclo-oxygenases (COX), forming prostaglandins (PGs) and thromboxanes, or by the actions of lipoxygenases, forming leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). Leukotrienes are synthesized by 5-lipoxygenase which catalyses the conversion of AA to LTA4, which is further hydrolysed to LTB4 or conjugated with glutathione to form LTC4 and its metabolites LTD4 and LTE4.
Ovarian lipoxygenase-activity was demonstrated in cultured human granulosa-lutein cells of IVF patients (Feldman et al., 1986) and a stimulatory role of human chorionic gonadotrophin (HCG) on follicular lipoxygenase activity was shown in the rat ovary (Reich et al., 1985a). Leukotrienes, especially LTB and LTC, may have luteolytic effects in the bovine corpus luteum (Blair et al., 1997). Furthermore, HCG has been shown to cause an increase of the 15-lipoxygenase-metabolite, 15-hydroxyeicosatetraenoic (15-HETE) in the rat ovary during ovulation (Tanaka et al., 1989). Indirect evidence for the participation of the lipoxygenase products in ovulation, was that the 5,12-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), dose-dependently suppressed ovulation rate in the rat ovary both in vivo and in vitro (Mikuni et al., 1998). In the latter study, the inhibition was partly reversed by exogenously added LTB4, thus indicating that this specific LT may be operative in the ovulatory cascade of the rat ovary. However, these results have to be interpreted in the light of observations that NDGA also affects synthesis of other ovulatory-mediators such as prostacyclin (Hellberg et al., 1990) and reactive oxygen species (Yocum et al., 1984). A more specific way to block LTB4 action is to use a specific receptor antagonist. The LTB4 receptor is a G-coupled seven transmembrane receptor (Yokomizo et al., 1997), and activation causes a rise in intracellular calcium and inositol trisphosphate concentrations (Crooks and Stockley, 1998).
In this study, we examined the effects of a specific LTB4 receptor antagonist (ZK158252 = L-ANT) on ovulation, both in vivo and in vitro. We also evaluated the time dependency of LTB4 receptor activation and whether L-ANT affects some ovulation-associated mediators, specifically progesterone (Brännström and Janson, 1988), prostaglandins (Armstrong and Grinwich, 1972), plasminogen activator (PA) (Canipari and Strickland, 1985) and matrix metalloproteinases (MMP) (Brännström et al., 1988).
Materials and methods
Animals
Immature Sprague–Dawley rats (B&K Universal AB, Sollentuna, Sweden) were kept under controlled light conditions (lights on between 05.00 and 19.00 h) with food and water ad libitum. The weight-matched rats were randomly allocated to control and experimental groups, and injected s.c. with equine chorionic gonadotrophin (eCG) (10 IU for in-vivo experiments; 20 IU for in-vitro perfusion experiments) at 09.00 h on day 28 of age. This treatment induces follicular growth, with follicles reaching the preovulatory stage 48 h later. All experiments were carried out according to the principles of the NIH Guidelines for the Use of Laboratory Animals and procedures were approved by the Animal Ethics Committee of Göteborg University.
Reagents
ZK158252 (L-ANT) represents a new class of intercyclohexyl LTB4 receptor antagonist, and was generously provided from Schering AG (Berlin, Germany). ZK158252 is a competitive and specific LTB4 receptor antagonist (competition factor = 2.4; Ki 15 nmol/L at [3H]-LTB4 concentration 0.2 nmol/l) with a similar binding affinity as native LTB4 to the LTB4 receptor on isolated human neutrophils. The ZK158252 does not bind to other LT receptors (compound description; Schering AG, Berlin, Germany). Ovine LH (NIADDK-oLH-26) was kindly provided by the National Institutes of Arthritis, Diabetes, Digestive and Kidney diseases (NIADDK) and National Hormone and Pituitary Program (Rockville, MD, USA). eCG and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma Chemical Company (St Louis, MO, USA); ketamine from Park Davis (Barcelona, Spain); bovine serum albumin (BSA; fraction V) from Boehringer Mannheim (Mannheim, Germany); xylazine was from Bayer (Leverkusen, Germany); medium M199 from Gibco (Gaithersburg, MD, USA); gentamicin sulphate from Biological Industries (Kibbutz Beit Haemek, Israel); insulin from Novo Nordisk (Bagsvaerd, Denmark) and heparin from Lövens (Ballerup, Denmark).
Experimental protocols
In-vivo study using intrabursal injection
The compound L-ANT was dissolved in 99.5% ethanol to a concentration of 0.02 mol/l, sonicated on ice for 5 min, and equimolar concentration of NaOH was added to ensure that L-ANT was converted to its sodium salt. This solution was diluted with 0.9% NaCl to concentrations of 50 µmol/l and 500 µmol/l L-ANT. Immature eCG (10 IU)-primed rats were anaesthetized with ketamine:xylazine (67:13 mg/kg bodyweight) given i.p. A small (15 mm) incision was made on the dorsal midline through the skin and the peritoneal cavity was entered through two dorsolateral subrenal incisions. The fat tissue surrounding the ovary, oviduct and the distal end of uterus was exposed. A 30 gauge needle, attached to a 1 ml syringe, was threaded through ~5 mm of the adipose tissue and gently entered into the ovarian bursa. The L-ANT was injected into the ovarian bursal cavity in a volume of 40 µl on the right side, and the left ovarian bursa was injected with an equal volume of the control solution (diluent for L-ANT; 4 µl 99.5% ethanol + 0.05 µl 1 mol/l NaOH + 36 µl saline). Only animals with no visible leakage of the injected fluid and with swelling of the bursa, were included. After the completion of injection procedure, ovaries were repositioned into the abdominal cavity and the muscle and skin were sutured separately. The animals were then injected s.c with 15 IU HCG and killed 20 h later with isolation of both ovaries with connecting oviducts. The ampullary part of the oviduct was isolated and incised. The oocytes were harvested by applying gentle pressure to both ends of the ampulla. It was possible to ascertain that the ampulla was empty due to the transparency of this part, allowing the oocytes to be clearly visible. The oocytes were placed on a slide in PBS with hyaluronidase. The oocytes were counted under a stereomicroscope with the observer being blind to the treatment given.
In-vitro study using ovarian perfusion
On the morning of day 30 of age, the rats were anaesthetized with ketamine:xylazine (67:13 mg/kg bodyweight) i.p. and were then given 300 IU heparin i.v. through a femoral vein. The surgical procedure has been described in detail (Koos et al., 1984). Briefly, a laparotomy was performed and the caudal abdominal parts of the aorta and the vena cava were cannulated in a retrograde direction. All the connecting vessels, except the right ovarian vein and artery, were ligated and severed. At this stage, 2–5 ml of 0.9% NaCl were injected through the aortic cannulae to flush the ovarian tissue from blood. At the end of the surgical procedure, the aorta and the vena cava were ligated and cut cranially to the level of renal vessels and caudally to cannulation. The bursa was surgically opened before the placement of the ovary into the perfusion chamber to enable ovulated oocytes to sediment to the bottom of a beaker, which was placed inside the perfusion chamber. The perfusions were performed as earlier described (Brännström et al., 1987a) with minor modifications to reduce the volume of perfusion medium of 70–30 ml. The perfusion pressure was maintained at 80 mmHg, resulting in an average flow of 0.9 ml/min. The ovaries were initially perfused for at least 30 min before any agents were added, to allow metabolic stabilization of the tissue. To exclude specimens with vascular leakage or partly clotted vascular beds, only ovarian preparations which exhibited flow rates of 0.7–1.3 ml/min at 80 mmHg perfusion pressure were used. The perfusion medium was 30 ml M199 with Earle's salts supplemented with insulin (0.02 IU/ml), gentamicin sulphate (50 µg/ml), 4% BSA and 0.026 mol/l sodium bicarbonate. A pH of 7.4 was maintained when the perfusion medium was continuously gassed with 5% CO2 and 95% O2. Four ovaries were perfused simultaneously in identical perfusion systems. Samples (1 ml) of perfusion medium were withdrawn at specific time points throughout the perfusion period and stored at –70°C for later analysis of steroid concentrations. An equal volume of fresh medium was added to the perfusion medium after each sampling, in order to keep a constant volume in the system.
In the 20 h perfusions, LH (0.2 µg/ml) + IBMX (0.2 mmol/l) was added (at time 0 h) to induce ovulation in vitro. The phosphodiesterase inhibitor, IBMX, was present to achieve optimal ovulation stimulation (Petersen et al., 1993), with an ovulation rate comparable with that observed in vivo (Tanaka et al., 1991). The stock solution (0.02 mol/l) of L-ANT was diluted with M199 medium to obtain final concentrations of 1, 10 and 100 µmol/l in the perfusion media, and added in a 1 ml volume. Diluent solution (1 ml) was added to the perfusion media of LH+IBMX control perfusions. The L-ANT (1, 10, or 100 µmol/l) was added either 30 min prior to LH+IBMX, or (10 µmol/l L-ANT) 3 or 6 h after LH+IBMX. After 20 h of perfusion, the oocytes were collected from the bottom of the beaker and counted under a stereomicroscope with the observer being unaware of the experimental data.
The 10 h perfusions were designed to study the ovarian concentrations of the proposed ovulatory mediators PGE2, PGF2, PA, MMP-2 and MMP-9 after LH+IBMX with or without L-ANT (100 µmol/l). At the end of the perfusion time, each ovary was divided into three pieces, snap-frozen and kept at –70°C.
Extraction of MMPs from ovarian tissue
Soluble and membrane-based MMPs in the ovarian tissue were extracted sequentially with salt and dimethyl sulphoxide (DMSO) buffers respectively (Davis et al., 1998). The ovarian tissues perfused in the presence of LH+IBMX with (n = 5) or without (n = 5) L-ANT were homogenized with Pellet Pestle (Kontes, Vineland, NJ, USA) in TNC buffer (50 mmol/l Tris–HCl pH 7.5, 0.15 mol/l NaCl, 10 mmol/l CaCl2, 10 mol/l E-64, 0.05% Brij 35, 2 mmol/l phenyl methyl sulphonyl fluoride and 0.02% NaN3). The homogenates were centrifuged at 12 000 g for 10 min and the supernatants were removed.
Zymography
Zymography was conducted as previously described (Davis et al., 1998). A set volume of each fraction was diluted with sodium dodecyl sulphate (SDS) buffer before loaded on a 10% acrylamide gel containing 0.1% gelatin as substrate (Novex System, San Diego, CA, USA). As standards, pro-MMP-2 and pro-MMP-9 (Oncogene Research Products, Cambridge, MA, USA) were used with the same treatments as the samples with either TNC-buffer or DMSO-buffer. After electrophoresis, the gels were washed in 2.5% Triton X-100 and incubated in the developing buffer for 24 h at 37°C. Enzymatic activity was visualized by staining with Coomassie Brilliant Blue R-250. The gelatinolytic activities were densitometrically quantified with Flour-S Multimager® and quantity One® software for Windows (Bio-Rad laboratories, Hercules, CA, USA). The treatment of the pro-MMP-2 and pro-MMP-9 standards in the same buffer with gel electrophoresis and incubation in the same manner as the samples did not change their size. This demonstrates that the method per se has no major proteolytic influence on the MMPs.
Assays
Steroid (oestradiol and progesterone) concentrations in perfusion media were analysed by DELFIA assay kits (Wallac Oy, Turku, Finland).
To assay the prostaglandins, the 10 h perfused ovarian tissues were homogenized (glass–glass homogenizer 5000 rpm 30 s), and sonicated (2x15 s) in 1 ml buffer (0.1 mol/l acetate buffer at pH 4.5). After centrifugation at 10 000 g for 20 min, the supernatants were transferred to propylene-tubes and PGE2 and PGF2 concentrations were analysed by enzyme immunoassay kits (RPN222 and TRK900 respectively; Amersham, Buckinghamshire, UK).
The PA activity assay was based on an earlier described method for ovarian PA activity evaluation (Espey et al., 1985) but with minor modifications. Ovarian tissue was homogenized with glass-glass homogenizer (5000 rpm) and sonicated (2x15 s) on ice in a propylene tube followed by centrifugation at 10 000 g for 20 min at 4°C. The PA enzyme reaction consisted of 20 µl ovary supernatant or 100 µl substrate S-2215 (H-D-Val-Leu-Lys-pNA. 2HCl) and plasminogen (both from Chromogenix AB, Mölndal, Sweden). The plate was shaken (900 oscillations/min) for 2–3 min and incubated for 1–2 h at 37°C, followed by the addition of 75 µl of 50% (v/v) acetic acid to stop the enzymatic reaction. The optical density (OD) after the final reaction was measured at 405 nm. Total protein concentrations in each supernatant from the 10 h perfused ovarian sections were assayed by using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). Intra- and inter-assay coefficients of variation were <10% for all assays.
Statistical analysis
The results are presented as the median and interquartile range. Statistical differences regarding ovulation rate and steroid concentrations at each timepoint were calculated by Kruskal–Wallis test followed by Mann–Whitney's U-test. Differences in concentrations of prostaglandins, PA, MMP-2, or MMP-9 between the control and treated groups, were analysed with Mann–Whitney's U-test. P < 0.05 was considered to be statistically significant.
Results
Effects on ovulation numbers
Intrabursal administration in vivo of the higher concentration of L-ANT (500 µmol/l), but not the lower concentration (50 µmol/l) significantly (P < 0.05) inhibited ovulation (median 5.5, 25–75%, range 1.0–6.0), in comparison with the contralateral control ovaries (median 9.0, range 6.25–13.5) (Figure 1).
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The in-vitro perfusion experiments over 20 h demonstrated a dose-dependent inhibition of LH+IBMX-induced ovulation (Figure 2
). The numbers of ovulations at different concentrations of L-ANT were (median 10.0, range 8.0–13.0 at 1 µmol/l; median 6.0, range 3.5–10.0 at 10 µmol/l; median 2.0, range 0.75–5.75 at 100 µmol/l) when L-ANT was given 0.5 h prior to LH+IBMX. Ovulation numbers were not significantly decreased when L-ANT (10 µmol/l) was administered for 3 h (median 9.0, range 3.75–14.25) or 6 h (median 9.0, range 7.5–10.5) after LH+IBMX (Figure 2).
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Effects on ovarian-derived mediators
Progesterone and oestradiol concentrations in the perfusion media were similarly increased in both the LH+IBMX control and the LH+IBMX+L-ANT groups with maximal concentrations reached after 3–8 h perfusion. There were no differences in progesterone or oestradiol concentrations at any timepoint, when comparing the LH+IBMX group and groups with addition of L-ANT at 100 µmol/l (Figures 3 and 4
) or at lower concentrations (data not shown). When L-ANT was given at 3 or 6 h after LH+IBMX, there was no difference in steroid concentrations at any timepoint, when compared with the LH+IBMX group (data not shown).
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Ovarian tissue concentrations of prostaglandins at 10 h were similar in the LH+IBMX and LH+IBMX+L-ANT (100 µmol/l) groups (Figure 5a,b
). The concentrations of PA-activity in the ovary after 10 h perfusion with LH+IBMX were significantly higher in the presence of L-ANT as compared with the LH+IBMX controls (Figure 6).
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In zymography with the salt fractions (soluble proteins), the 72 kDa band (corresponding in size with pro-MMP-2; Nagase and Woessner, 1999) was predominant, but there were also low levels of 62 kDa activity (Figure 7
). There was no significant difference in gelatinolytic activity between LH+IBMX controls and the LH+IBMX+L-ANT (100 µmol/l) group (data not shown). In zymography with the DMSO fraction (membrane-bound proteins), 92, 72 and 62 kDa bands (corresponding to the pro-forms of MMP-9, MMP-2 and the active form of MMP-2 respectively) were observed (Figure 7), but with a very weak 92 kDa signal. No significant differences in gelatinolytic activity of 92 and 72 kDa bands between the groups were observed. Decreased gelatinolytic activity of 62 kDa active form of MMP-2 in relation to total MMP-2 (72+62 kDa) in the group treated with L-ANT was observed (Figures 7 and 8).
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Discussion
It is now recognized that ovulation is a restricted and controlled inflammatory process, with the partipication of several inflammatory mediators such as eicosanoids, kinins and cytokines (Brännström et al., 1997). The LTs, especially LTB4 which is mainly produced by neutrophils and macrophages, induces recruitment and activation of neutrophils, monocytes and eosinophils (Crooks and Stockley, 1998) and the LTB4 receptor has been localized to these types of inflammatory cells and to endothelial cells (Nohgawa et al., 1997). LTB4 is involved in the up-regulation of a number of cytokines, e.g. interleukin (IL)-2, IL-4 and interferon (IFN)- to promote T cells activation (Morita et al., 1999). It also stimulates transcription of immediate response genes in human monocytes (Stankova and Rola-Pleszczynski, 1992) and degranulation from leukocytes (Priddy et al., 1989). Together, these LTB4-induced changes are proposed to augment and prolong tissue inflammation (Hattori et al., 1998). Inflammation-like processes with tissue remodelling within the ovary take place at certain stages of the reproductive cycle and some studies suggest that the LTs, such as LTB4, are of importance for the transition of the preovulatory follicle into a functional corpus luteum (Reich et al., 1985a; Feldman et al., 1986; Priddy et al., 1989; Hattori et al., 1998).
The present study shows that an LTB4-receptor antagonist (L-ANT) inhibits ovulation at the ovarian level in a dose-dependent pattern, as demonstrated both in vivo and in the in vitro perfused rat ovary system. This inhibitory effect in vitro was only seen when L-ANT was present upon gonadotrophin treatment but not when added >3 h after ovulation induction. The inhibitory effects on ovulation by L-ANT did not seem to involve a reduction in the activity of PA or the concentrations of prostaglandins, but a decrease in the active form of MMP-2 was seen.
Since it has been reported that the rat pituitary is a site for local production/action of LTs (Kiesel et al., 1991), with LTB4 having an inhibitory effect on LH release (Rabe et al., 1997), and that LTs also have profound systemic effects in various pathological conditions (Henderson, 1994), we made use of two model systems to study local ovarian influences on ovulation. The intrabursal injection model has been widely used in studies of ovulation mechanisms both in the hamster (Martin et al., 1981) and the rat (Reich et al., 1985c) and the rat ovary perfusion model has also been used extensively (Koos et al., 1984; Brännström et al., 1987a, 1988; Brännström and Janson, 1988).
The LTB4 concentrations in follicular fluid of the natural cycle (Priddy et al., 1989) and the stimulated IVF cycle (Bili et al., 1998) are ~10 nmol/l. Since the binding affinity to the receptor is similar for LTB4 and this L-ANT, an effective blockage of the receptor in the conducted experiments is likely with the higher L-ANT concentration given. The concentration of the L-ANT, which effectively blocked ovulation in the intrabursally injected rats, was fairly high (500 µmol/l) in comparison with the effective concentration (10 µmol/l) in the in-vitro perfused ovary system. A reason for this discrepancy in concentrations needed, may be that in the intrabursally injected ovary model, the active component is initially only in direct contact with the surface epithelium of the ovary and then needs to penetrate into the ovary through this cell layer and the tunica albuginea to reach the theca and granulosa layers, where most of the activity in the regulation of the ovulatory process is presumed to take place. The necessity to use high concentrations of added inhibitors in this intrabursal system is well documented. In a study using non-specific MMP blockers injected intrabursally to the eCG-primed immature rat, inhibition was only demonstrated at a very high (1 mol/l) concentration (Reich et al., 1985a). Furthermore, an even higher concentration (5 mol/l) of serine proteinase inhibitors administered intrabursally was needed to cause inhibition of ovulation numbers in the rat ovary (Reich et al., 1985b). It could be argued that the high concentrations of active substances used in the present study and other studies with this intrabursally injection model, can cause systemic effects after diffusion into the rich vascular system of the theca region of the preovulatory follicle. Some other studies involving intrabursal injection in the rat used the non-operated contralateral ovary as control (Reich et al., 1985b; Simon et al., 1994). To increase the validity of our experiment, we also performed sham operations with diluent injections on the contralateral side, since the increased fluid pressure in the bursa and local trauma may negatively affect the ovulation rate.
To achieve a more general tissue distribution of the active drug by means of the vascular system, we also used the in-vitro perfused rat ovary system, which is well-characterized regarding many aspects of the ovulatory process (Koos et al., 1984; Brännström et al., 1987a, 1997). The LTB4 receptors are present on the vascular endothelial cells (Nohgawa et al., 1997), which would imply that this model is suitable for studies using LTB4 receptor antagonists or blockers of other receptors which are mainly located on the vascular endothelium. It was apparent from the results of the present study that the concentrations needed to produce a significant inhibition in this in-vitro system were considerably lower than that required in the in-vivo model. The dose–response experiments showed that the concentrations of L-ANT, which produced significant inhibition in this in-vitro system, was in the same range as the effective concentrations of other specific inhibitors, such as a cyclo-oxygenase-2 inhibitor (Mikuni et al., 1998) and a nitric oxide synthase inhibitor (Mitsube et al., 1999), also evaluated in the rat ovary perfusion model.
In the present study, we evaluated whether LTB4-receptor activation is of importance at any specific time phase of the ovulatory process. This ovulatory process in the rat, from the start of the gonadotrophin surge until follicular rupture, is ~10–16 h as demonstrated both in vivo (Tanaka et al., 1989) and during in-vitro perfusion conditions (Brännström et al., 1987a). Earlier in-vivo studies in this species have demonstrated that the concentration of LTB4 in the ovary peaks at 4 h (Espey et al., 1989). Another study revealed that intraovarian concentrations of the LTs C4/D4/E4 show bimodal patterns peaking at 2 and 10 h (Higuchi et al., 1995). We could demonstrate, in the perfusion experiments of the present study, that the L-ANT was ineffective when administered 3 or 6 h after gonadotrophin stimulation. This time-dependency was investigated with 10 µmol/l L-ANT and it is possible that the higher L-ANT concentration (100 µmol/l) could have been effective. However, the experiments were done with the lowest effective concentration to increase the sensitivity when time-dependency was studied. Thus, the results of the present study imply that LTB4 receptor activation is of importance only during the hours immediately after HCG/LH. This relatively short window of critical LTB4-receptor activation suggests that some immediate intraovarian events which are of relevance for follicular rupture, such as the prominent vascular changes after the LH-surge are mediated by LTB4. It is known that LH/HCG induces vasodilatation within 30 min in the rabbit ovary (Janson, 1975) and in the mouse ovary increased vascular permeability is seen within seconds, with peak permeability at 1 h (Powers et al., 1995). Similar vascular effects of LTs on the endothelial lining of the post-capillary venule, occuring in vitro after 10 min, have been described in the microvasculature of the hamster cheek pouch (Dahlen et al., 1981).
The results of the present in-vitro perfusion study indicated that L-ANT treatment does not affect steroid output from the ovary, since the steroid concentrations were similar at all timepoints in the control group when compared with the L-ANT groups. Since progesterone is one of the key factors in the ovulatory cascade of the rat (Brännström and Janson, 1988) and the mouse (Lydon et al., 1996), the results of the present study indicate that the ovulation suppression by L-ANT is due to effects on other mediator systems. Studies with human ovarian cells in vitro have shown that LTB4 may enhance progesterone release from granulosa cells (Rabe et al., 1995) and inhibit progesterone production from luteal cells (Yoshimura et al., 1992). Previous studies utilizing the in-vitro perfused rat ovary method to assess the effects of tumour necrosis factor (Brännström et al., 1995) indicated differences in effects on progesterone synthesis compared with the effects observed in other in-vitro systems (Brännström et al., 1993b). Thus, the leukocyte-rich medulla and stroma (Brännström et al., 1993a), which is present in the perfusion model, may contain cells that respond to the L-ANT and could then modify the steroidogenic effects, so that the net effect for this whole system is unchanged progesterone secretion.
In this study, we evaluated the ovarian concentrations of some well-etablished ovulatory mediators in control and L-ANT-treated perfused ovaries 10 h after gonadotrophin stimulation. This timepoint is within 5 h of follicular rupture and the majority of ovulatory mediators show increased tissue levels at this time.
Prostaglandins are important mediators in ovulation (Tsafriri et al., 1973) and the concentrations increase in ovarian tissue after LH-stimulation to reach 4-fold higher concentrations than in the non-stimulated ovaries in this rat ovary perfusion system (Brännström et al., 1987b). In the present study, the concentrations of PGE2 and PGF2 were similar in the LH+IBMX-treated control group and the group also treated with L-ANT. The absolute values were in the same range as previously seen at a similar timepoint during in-vitro perfusion (Brännström et al., 1987a) and in vivo (Bauminger and Lindner, 1975). Taken together the results of the present study show that LTB4 in the ovary may not affect PG production.
The MMP system is presumed to be highly involved in the ovulatory process, by degrading the collagenous extracellular matrix at the apex of the follicle. Several MMPs have been detected in the rat ovary and the expression of some specific MMPs, e.g. MMP-1 and MMP-2, are induced by the gonadotrophin surge (Reich et al., 1991). In the present study we evaluated the activity of MMP-2 (also named 72 kDa gelatinase) and MMP-9 (92 kDa gelatinase), which both are capable of degrading basement membrane structures such as collagen type IV, laminin and fibronectin. The MMP-2 is activated by cleavage of a propeptide domain, and in contrast to most other MMPs, proMMP-2 is not activated by serine proteinases and by other conventional MMPs, but by a membrane-bound MMP-1 (Nagase and Woessner, 1999). The active form of MMP-2 is presumed to be involved in ovulation since its expression is up-regulated by gonadotrophins (Hägglund et al., 1999) and appears exclusively in the theca interstitial layer of the ovulatory follicles (Liu et al., 1998). The results of the present study demonstrate that activation of MMP-2 is inhibited in the presence of L-ANT. This involvement of LTB4 in MMP regulation in the ovary is supported by other studies, where lipoxygenase products were shown to stimulate production of MMP-2 in fibrosarcoma cells (Reich and Martin, 1996) and T-lymphocytes (Leppert et al., 1995). Thus, one important function for LTB4 in ovulation may be to facilitate the degradation of the two basal membranes of the follicle wall by increasing active MMP-2.
PA, both the uro-kinase type PA (u-PA) and tissue-type PA (t-PA) have been suggested to be essential in the ovulatory process (Canipari and Strickland, 1985; Espey et al., 1985; Tsafriri et al., 1989), but later experiments using a double-knockout mice with deficient u-PA and t-PA indicated a functional redundancy since only a very modest decrease in ovulatory rate was seen (Leonardsson et al., 1995). In the present study, the PA activity levels increased ~1.5-fold in the presence of L-ANT. This increased activity of the PA system, was not however able to overcome the L-ANT ovulation-inhibition carried out by effects on other mediator systems.
In conclusion, the present data indicate that LTB4 may play a role in the ovulatory pathways of the rat ovary and that LTB4 may carry out its effects via MMP-2.
Acknowledgments
We would like to thank Schering AG for the generous gift of ZK158252 and the National Hormone and Pituitary Program of the NIDDK for the gift of LH. The study was supported by grants from the Swedish Medical Research Council (11607 to M.B.), the Medical Faculty of Göteborg University, Göteborg Medical Society and Hjalmar Svensson Research Foundation.
Notes
3 To whom corresponendence should be adressed at: Department of Obstetrics and Gynecology, Göteborg University, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden. E-mail: Markus.Matousek{at}sahlgrenska.se 
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Submitted on June 16, 2000; accepted on October 3, 2000.
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