Failure of platelet-activating factor (PAF-acether) to induce decidualization in mice and failure of antagonists of. PAF to inhibit implantation. S. R. Milligan and C.
Failure of platelet-activating factor (PAF-acether) to induce decidualization in mice and failure of antagonists of PAF to inhibit implantation S. R.
Milligan and C. A.
Finn
Department of Physiology, Division of Biomedicai Sciences, King's College, London WC2R 2LS, UK; and * Department of Veterinary Preclinical Sciences, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK
Summary. The possible role of platelet-activating factor (PAF) in the uterine responses associated with implantation was investigated. Attempts to trigger a decidual cell response in the uteri of hormonally sensitized, ovariectomized mice by instilling PAF\x=req-\ acether (1\p=n-\1000ng) intraluminally were unsuccessful. The effect of PAF antagonists on implantation was investigated in females ovariectomized on Day 3 of pregnancy and treated with progesterone. Implantation was induced in these females by injection of 10 ng oestradiol-17\g=b\ on Day 8. Hourly intraperitoneal injections of three PAF antagonists (WEB 2086, CV 3988 and BN 52021 at doses of 1\m=.\2\p=n-\1\m=.\4mg/kg) given over a 24-h period starting 1 h before the injection of oestradiol-17\g=b\had no significant effect on the occurrence of implantation sites. Intraluminal injection of WEB 2086 (15 \g=m\g)or BN 52021 (5 pg) either 3 h before or 6 h after the nidatory oestradiol also had no significant inhibitory effect on implantation. SRI 63-441 given once daily over the first 4 days of pregnancy at a dose of 40 \g=m\g/30g body weight had no inhibitory effect on the establishment of pregnancy. These results are not consistent with a critical role for PAF in implantation in mice.
Keywords: PAF; implantation;
mouse;
decidualization; PAF antagonists
Introduction There are still many unsolved problems concerning the various signals and mediators that may be involved in controlling implantation and the associated uterine responses. One suggestion that has arisen in recent years is that platelet-activating factor (PAF) may represent one important signal between the embryo and maternal tissues. This idea stems from the work of O'Neill (1985a) who reported a transient decrease in maternal platelet numbers in the Quackenbush strain of mice in the early stages of pregnancy. It has also been reported that mouse and human embryos produce a platelet-activating factor indistinguishable from those of the well characterized platelet-activating factor PAF-acether ( 1 -o-alkyl-2-acetyl-5«-glycero-3-phosphocholine) (O'Neill, 1985b; Collier et ai, 1988). The idea that PAF might be physiologically important in early pregnancy gained further support from the observation that the production of embryo-derived PAF by human embryos correlated with the potential of embryos produced by in-vitro fertilization procedures (O'Neill et ai, 1987). In addition, Spinks & O'Neill (1987, 1988) reported that the administration of inhibitors of platelet activation (iloprost and SRI 63-441) to mice during early pregnancy inhibited implan¬ tation. Acker et ai (1988a) suggested that another PAF antagonist (BN 52021) could inhibit implantation in rats. In addition to PAF from embryonic sources, the uterus of both the rat (Yasuda et ai, 1986) and rabbit (Angle et ai, 1988) also may produce PAF.
The role(s) that embryo-derived or uterine PAF may play in early pregnancy is (are) uncertain. Embryo-derived PAF would appear to be one of the earliest potential signals for the maternal recognition of pregnancy. PAF may also affect the metabolism of preimplantation embryos (O'Neill, 1987). In addition, in view of its known, potent effects on vascular permeability and vasodilatation and effects on a variety of cell types (Braquet et ai, 1987), a role for PAF at implan¬ tation sites could be envisaged (Adamson et ai, 1987). Increased vascular permeability is one of the earliest obvious uterine responses to an implanting blastocyst (Psychoyos, 1973), preceding decidual transformation of the neighbouring stromal cells (Finn & McLaren, 1967). Acker et ai (1988b) reported that intrauterine administration of PAF in rats could induce decidualization and Angle et ai (1988) observed changes in PAF concentrations in the rabbit uterus at the time of implantation consistent with PAF involvement in implantation. The present study was undertaken to investigate the potential role of PAF in the uterine response associated with implantation in mice. Negative results from initial studies of the potential deciduogenic effects of PAF led to an investigation of the significance of PAF in implantation itself: this involved administering three specific PAF antagonists during an accurately timed period in which implantation was occurring. In addition, the effect on early pregnancy of one of the PAF inhibitors (SRI 63-441) used by Spinks & O'Neill (1988) was investigated. A preliminary report of some of this work has been published (Milligan & Finn, 1988).
Materials and Methods Animals. Swiss albino female mice (A. Tuck & Son Ltd, Battlesbridge, Essex, UK), 2-4 months of age (except in Exp. 1(b), when they were 6-7 months) and weighing 25^10 g were fed on a pelleted diet (41 Oxoid) and housed at 21°C with lights on from 08:00 to 24:00 h. Mated females were obtained by placing 3 adult males in cages containing 10-12 females; the females were checked daily (between 08:30 and 09:30h) for copulatory plugs (Day 1 day of finding plug) and those with plugs were removed and caged individually. Ovariectomies were performed under ether anaesthesia. =
Hormones. Oestradiol-17ß and progesterone (Sigma London Chemical Co. Ltd, Poole, Dorset, UK) were dissolved in arachis oil (British Drug Houses Ltd, Poole, Dorset, UK) and administered by subcutaneous (s.e.) injection (0-1 ml) or by s.e. Silastic implants (Exp. 3; length 1 cm, i.d. 1 -58 mm, o.d. 3-18 mm; Dow Corning, Midland, MI, USA) containing a suspension of progesterone in oil; such implants produce plasma concentrations of pro¬ gesterone similar to those seen in the first half of pregnancy (S. R. Milligan, unpublished). Synthetic PAF-acether (l-o-alkyl-2-acetyl-i«-glycero-3-phosphocholine; Sigma) was stored in absolute alcohol at a concentration of 5 mg/ml at a temperature of 20UC. For administration, dilutions of this stock were made in saline (0-154 M-NaCl) containing 0-25% bovine serum albumin (BSA fraction V powder, Sigma). —
PAF antagonists. WEB 2086 (Boehringer Ingleheim, Ingleheim am Rhein, FRG) and CV-3988 (Takeda Chemical Industries Ltd, Osaka, Japan) were dissolved in saline. BN 52021 (Ginklgolide B, I.H.B. Research Laboratories, Le Plessis-Robinson, France) was dissolved in DMSO at a concentration 165 mg/ml and diluted with saline to give a final dilution of 0-75 mg/ml. SRI 63-441 (Sandoz Pharmaceuticals, Leeds, UK) was dissolved in phosphate-buffered saline (PBS). Solutions of antagonists were freshly made for each experiment and were administered by intraperitoneal injection in a volume of 50 µ (WEB 2086, CV-3988 and BN 52021 ) or 200 µ (SRI 63-441 ) at doses of 1 -4, 1 -3, 1 -2 and 1-3 mg/kg respectively. Control mice received injections of saline or 0-5% DMSO in saline, as appropriate.
Experiment 1. In the initial experiments the possibility that PAF-acether might be an effective deciduogenic stimulus was investigated. Ovariectomized mice were treated with 100 ng oestradiol-17ß for 3 days, followed by 2 days of no treatment and then 3 days of progesterone ( 1 mg) and oestradiol-17ß ( 10 ng): this produces maximal sensitivity to a decidual stimulus on the 3rd day of combined progesterone and oestradiol treatment (Finn & Martin, 1972). Intraluminal instillation (5-20 µ ) of PAF-acether, vehicle or oil into one or both uterine horns was performed using ether anaesthesia. Instillations were made via a 25-gauge needle at the anterior tip of the uterine horn. Daily treatment with 1 mg progesterone continued until the animals were killed for assessment of the uterine decidual cell response by uterine weight and histological examination. Experiment 2. The failure to demonstrate any decidi °^enic effect of PAF-acether in Exp. 1 led to an investigation of the effect of PAF-acether on uterine vascular permeability. Ovariectomized mice were treated with lOOng oestradiol-17ß for 3 days, followed by 2 days of no treatment and then 3 days of progesterone (1 mg). On the 3rd day of progesterone treatment, 5 µ PAF-acether, saline or arachis oil were instilled into one uterine horn. Uterine vascular
permeability was assessed 6 h later by determining the accumulation of radiolabelled albumin 30 min after the injection of 0-5 µ 125I-labelled human serum albumin (Arvidson, 1977; Milligan & Mirembe, 1985). Experiment 3. In this experiment, the ability of PAF antagonists to block implantation was tested by administering antagonists throughout the implantation period. The timing of implantation was controlled by initially delaying implantation by ovariectomy in combination with progesterone treatment and then inducing implantation at a the
time. Mated females were ovariectomized on Day 3 of pregnancy and a Silastic implant containing progesterone was placed subcutaneously. At 09:00 h on Day 8, 10 ng oestradiol-17ß were injected s.e. to induce implantation. Intraperitoneal injections of three PAF antagonists (WEB 2086, BN 52021 or CV 3988) or the vehicle were given at hourly intervals for 24 h from 08:00 h on Day 8. These injections spanned the period of expected implantation and implan¬ tation sites were visualized by the i.v. injection of 0-1 ml 1% Pontamine Sky Blue (Psychoyos, 1961) 24 h after the oestradiol injection.
predetermined
Experiment 4. The effect on delayed implantation of two PAF antagonists (WEB 2086 and BN 52021) administered directly into the uterine lumen was investigated. Implantation was delayed as in Exp. 3. Either 3 h before, or 6 h after, the nidatory oestradiol injection on Day 8, 5 µ WEB 2086 (15 µg), BN 52021 (5 µg) or vehicle were injected into the oviducal tip of one uterine horn. At 24 h after the oestradiol injection, the uteri were inspected for implantation sites following the i.v. injection of 01 ml 1% Pontamine Sky Blue. Experiment 5. The effect of SRI 63-441 on the establishment of pregnancy was investigated using a regimen of administration similar to that employed by Spinks & O'Neill (1988). SRI 63-441 or PBS was administered by i.p. injection to newly mated mice at 16:00 h on Day 1 and 09:00 h on Days 2-4. The uteri were examined for implantation sites on Day 8.
Results The effectiveness of the PAF-acether used in this series of experiments was confirmed by three different methods. Subplantar administration of PAF is known to induce rat paw oedema (Goldenberg & Meurer, 1984); in the present study, 4 rats were given a subplantar injection of 2 pg PAF-acether and all exhibited a marked paw oedema within 60 min. This effect was not seen after injection of the vehicle. In mice, the intravenous injection of PAF will induce death by bronchoconstriction and cardiovascular effects (Myers et ai, 1983; Criscuoli & Subissi, 1987). This was confirmed in the present study when 90 pg PAF-acether/kg injected i.v. in 01 ml caused death within 30 min of 9/10 mice. Finally, the i.p. injection of 2 pg PAF-acether into 4 splenectomized mice caused a 15% decrease in platelet numbers within 60 min. The effectiveness of the PAF antagonists was tested by assessing their ability to block the lethal effect of i.v. administered PAF. Female mice received two i.p. injections of the antagonists 1 h apart and were then injected i.v. with 90 pg PAF-acether/kg. In the absence of any inhibitor, only 3/12 (25%) animals were still surviving 30 min after PAF-acether administration. Pretreatment with the PAF antagonists at the doses used in subsequent experiments significantly improved the survival rate, with the numbers surviving after WEB 2086, BN 52021, CV 3988 and SRI 63-441 being 6/6 (100%), 8/12 (67%), 9/12 (75%) and 8/8 (100%), respectively.
Experiment
1: the
deciduogenic potential of PAF-acether in ovariectomized, steroid-treated mice
PAF-acether was injected intraluminally under a variety of conditions (Table 1). While the instillation of arachis oil produced massive decidual responses along the length of the instilled horn, the instillation of PAF-acether was ineffective regardless of the dose (Exps la and lb). The only signs of decidualization in the horns instilled with PAF were occasionally at the site of instillation. The larger uterine weights in Exp. 1(b) can be explained by the increased age of these animals. The possibility that the negative response to PAF-acether was the result of rapid loss of the instilled material from the cervical end of the horn was tested in Exp. 1(c) by ligating the uterus at the time of instillation but no decidual response was observed.
Table 1. Effect of intrauterine instillation of PAF-acether on decidualization in ovariectomized, steroid-treated mice (Exp. 1) Uterine
Intraluminal
injection
Exp. la: dose-response study PAF-acether PAF-acether PAF-acether PAF-acether Arachis oil 1 ng 10 ng 100 ng 1 µg
Exp. lb: single or double injections of PAF 1 µg PAF-acether 2 1 µg PAF-acether (given 2 h apart)
Exp. lc: ligation of uterine horn after instillation 1 µg PAF-acether Saline Arachis oil
weight (mg)
No. of mice
Days after instillation
7 7 7 7 8
2 2 2 2 2
25-6 ± 30-4+ 29-5+ 27-0 ± 61-0 ±
10 6
3 3
45-4+ 2-6 611 ± 51
51-7+ 4-9 51-7 ± 1-6
6 6 6
2 2 2
20-2 ± 1-4 20-5+ 1-5 26-8 ± 3-2
26-8 ± 1-5 26-5 ± 1-3 172-8 + 26t
Control horn
Values are means + s.e.m. "Values significantly different from corresponding control horns: tTwo control horns also had decidual responses.
Experiment 2: effect of intrauterine instillation progesterone-dominated, ovariectomized mice
of PAF-acether
