Decidualization in the bat: Role of leukotrienes and prostaglandins

Prostaghdins, (il Longman

Leukotriencs and Medicine Group UK Ltd 1987

(1987) 29, Z-227

DECIDUALIZATION IN TEE RAT: ROLE OF LEUKOTBIENKSAND PROSTAGIANDINS O.W. Tawfik, Cathleen Sagrillo, D.C. Johnson and S.K.Dey Departments of Gynecology h Obstetrics and Physiology R.L. Smith Research Center, University of Kansas Medical Center Kansas City, Kansas 66103 (reprint requests to SKD) AESTKACT The role of prostaglandins (PGs) and leukotrienes (LTs) in the induction of decidualization in the rat uterus was investigated. In the hypophysectomized progesterone (P4) primed rat, intraluminal infusion for four days by osmotic minipump, of PGE2 (lug/h), LTC4 (long/h) or 0.15M saline (lul/h) significantly elevated uterine weight when compared to the noninfused horn: all were equally effective. In contrast, simultaneous infusion of PGE2 and LTC4 produced an increase in uterine weight which was markedly higher than any other conditions and the reaction was elicited along the entire length of the uterine horn. Infusion of PGE2, LTC4, a combination of the two or vehicle, into one uterine horn of day-5 pseudopregnant rats elicited a huge decidual response. Infusion of indomethacin, an inhibitor of PG synthesis, FPL 55712 (FPL), an antagonist of LTs, or a combination of these inhibitors evoked a minimal decidual response. In addition, FPL infused along with PGE2 or LTC4 markedly reduced the response that could be induced by these arachidonate metabolites alone. Furthermore, infusion of indomethacin along with LTC4 resulted in a far smaller response than that obtained with LTC4 alone. These results are interpreted to indicate that there is an interaction between LTs and PGs in the induction of the uterine decidual response. INTKODUCTION Implantation of the blastocyst in the rat uterus, normally depends upon a precise balance between P4 and estrogen. P4 in the absence of estrogen results in blastocyst dormancy and delayed implantation (1). initiates implantation in a However, the mechanism by which estrogen P4-primed uterus is still an open question. One of the earliest discernible prerequisite events in blastocyst implantation is the increased uterine stromal capillary permeability at the site of the blastocyst (2). During early pregnancy, the endometrial stromal cells undergo

221

growth and differentiation in response to the implanting blastocyst, resulting in the formation of the cells of the decidua of pregnancy (2,3). The decidual cell reaction also can be elicited in the uterus by artificial stimuli but this can be achieved only during a narrow window of receptivity in a pregnant, pseudopregnant or hormonally primed uterus (2). Prostaglandins (PGs), especially PGE2, have been proposed not only as the mediators of endometrial vascular permeability changes induced by artificial stimuli (4), but also as a required constituent for the progression of decidual cell reactions(5,6). Confusing the picture however, is the finding that administration of indomethacin, for inhibition of PG synthesis, only delays, but does not completely inhibit implantation (7). A critical analysis of the previous observations, along with current developments regarding the interaction of various mediators in inflammatory reactions, warrants a reevaluation of our present interpretation on the role of the mediators involved in decidualization. Evidence of involvement of other mediators is gradually accumulating especially with the discovery of a new class of mediators, the leukotrienes (LTs). Leukotrienes, like the PGs, are derivatives of arachidonic acid (8,9) and are potent vasoactive agents and mediators of various kinds of inflammatory reactions (8,9). A possible interplay between PGs and LTs in implantation and decidualization has added a new dimension to the problem and given us the opportunity to address the issue with a new insight. Although both the cyclooxygenase and the lipoxygenase pathways are operative in the rabbit and rat uterus during the preimplantation period (lo-12), little is known about their interaction in uterine The present study was undertaken in an attempt to reveal the functions. role of LTs and PGs in the induction of decldualization in the rat. MATERIALS AND MRTHODS Adult (275-350 g) virgin female rats (Holtzman strain) exhibiting at least two successive estrous cycles were used. In order to induce pseudopregnancy, females, on the day of proestrus, were caged overnight with vasectomized males. The finding of vaginal plugs on the following For simulating the condition morning indicated day-l of pseudopregnancy. of delayed implantation, rats were hypophysectomized (13) on day-3 of pseudopregnancy and maintained thereafter by daily injections of progess.c.) (Sigma Chemical Co., St. Louis, MO.) disterone (P4) (2 mg/rat, solved in 0.1 ml sesame seed oil. The daily P4 treatment was continued until the animals were killed. PGE2 (Sigma), LTC4 (a gift from Dr. J. or their combination was Rokach of Merck Frosst Labs, Quebec, Canada), delivered (lul/hr dissolved in saline) by osmotic minipump (Model 2001, Palo Alto, Ca.). The pumps were attached to the uterine Alza Corp., lumen at the ovarian end so that the effluent could be instilled from the tubal end of one uterine horn (5). Treatment was begun on day-8 of on day-6 of P4 treatment. Immediatly following pseudopregnancy, i.e. placement of the pumps animals received an i.v. injection of 50ng of a daily subcutaneous injection of estradiol-17 (E2) in saline (Sigma): 100 ng of E2 in oil was given until they were killed on day 12. To determine the effect of inhibiting PG synthesis and function of an osmotic minipump containing these agents, LTs on decidualization, or a combination of both, was attached to one uterine their antagonists, horn of intact pseudopregnant rats at 1200 h on day 5, the period of 222

maximum sensitivity for induction of decidualization (2). Animals were killed on day 9. Indomethacin (Sigma) dissolved in a vehicle consisting of 10% ethanol, 50% propylene glycol and 40% saline, was used as an inhibitor of PG synthesis (14). FPL 55712 (FPL) dissolved in distilled water (a gift from Sterling-Winthrop Research Institute) was used as an antagonist of LTC4 (15). Wet weights of both infused and noninfused uterine horns were recorded immediately after killing the animals, and a portion of the uterine horn was processed for histological examination. Statistical analyses were calculated on the square root of weight of infused horn minus the weight of the noninfused horn, followed by two-way analysis of variance and Scheffe post hoc comparisons between treatment groups. The square root transformation was used to stabilize the within group variances. A P value of less than 0.05 was considered statistically significant. RESULTS Table 1: EFFECT OF UNILATERAL INFUSION OF PGE2 AND/OR LTC4 ON THE DECIDUAL REACTION IN HYPOPHYSECTOMIZED PSEUDOPREGNANT RATS. Intraluminal treatment

No of animals

Uterine weighttmg) Infused horn Noninfused horn

Percent * increase

Saline

6

264t24

109*9

143

PGE2

6

334&28

116&7

187

LTC4

6

326~42

13629

139

PGEZ+LTC4 6 629266 132_+20 376 -_------__----___---~--~~~--~---~~~------------------~~~~~~~~~~~~~~~~~+ S.E.M.; Values of all noninfused horns were statistically different from those of the infused horns. Values with the same superscript are not significantly different. *Calculated as wttinfused horn)-wtcnoninfused) divided by wtcnoninfused horn) X 100. EFFECT OF PGE2 AND/OR LTC4 INFUSION ON DECIDUALIZATION IN NYPOPEYSECTOMIZED RATS: As shown in table 1, infusion of PGE2 (1 ug/h), LTC4 (10 ng/h), or 0.15M saline (1 ul/h) increased significantly the wet weight of the uterine horns as compared to the noninfused horns. However, neither PGE2 nor LTC4 was significantly better than saline in increasing the wet weight of the infused horn. In contrast, a combination of PGE2 (lug/h) and LTC4 (long/h) significantly increased weight of the infused horns as compared with any other treatment and the reaction was evoked along the entire length of the uterine horn in most of the animals. EFFECT OF INFUSING ARACBIDONATE NBTABOLITBS AND THEIR INNIBITOES ON DECIDUALIZATION IN PSEUDOPRGNANT INTACT RATS: Uterine wet weights of the infused horns were higher than those of the noninfused horns in all groups but the degree of response varied between the different treatment groups. Degeneration of the uterine luminal epithelium, as determined by histological examination, indicated a decid-

223

ual cell reaction in all infused uteri. As shown in table 2, infusion of the vehicle (1 ul/h; Group l), PGE2 (1 ug/h; Group 2), LTC4 (10 rig/h;; Group 31, or PGEP (lug/h) + LTC4 (long/h) (Group 4) produced a large decidual response. Because there were no differnces in responses induced by saline or the vehicle used for indomethacin and FPL, the results with these were pooled and cited under the group for vehicle. Infusion of

Table 2: EFFECT OF UNILATERAL INFUSION OF PGE2, LTC4, AND THEIR INHIBITORS ON DECIDUALIZATION OF THE UTERUS IN PSEUDOPREGNANT RATS Group

No of

Intraluminal

Uterine wt(mg)

Percent *

NO:____~~Z.S___tfeqtmeIft______lnflls""_9fifr_____"""~"f""'~_~"f"__~"f"_"""__ 1

9

Vehicle

1038+104

128*8

712

2

6

PGE2

1555+184

163&14

864

3

6

LTC4

1232+54

130*9

848

4

6

PGEZ+LTC4

1485270

144fll

923

5

6

FPL+PGEZ

481241

133210

262

6

6

FPL+LTC4

491286

134f8

266

7

6

4092148

140flO

193

8

6

Indomethacin+ LTC4 Indomethacin

234k23

1601t27

9

6

FPL

334238

143*9

134

10

5

Indomethacin+ FPL

170*15

134f2

27

71

All values for the noninfused horn, except group 8, were statiS.E.M.; istically significantly different from the corresponding infused horn. *Calculated as indicated in table 1. Groups with the same superscript are not significantly different from each other. indomethacin (1 ug/h; Group 81, FPL (1 ug/h; Group 91, or their combination (Group IO), evoked a minimal decidual response. Furthermore, FPL infused with PGE2 or LTC4 markedly blunted the response induced by PGE2 or LTC4 alone; compare groups 5 and 6 with 2 and 3. Similarly, indomethacin infused with LTC4 (Group 7) reduced the response induced by LTC4 alone (Group 3). Infusion of either the vehicle, PGE2, or LTC4 induced decidualization along the entire length of the uterine horn, whereas in the presence of inhibitors, the decidual reaction was restricted to the area of installation of the pump. DISCUSSIOB Mechanisms that operate at the cellular level to modulate uterine sensitivity to implantation and subsequent decidualization are ill de-

224

fined. Because the decidual cell reaction involves interactions between the uterine luminal epithelial and stromal cells, changes in the stromal vascular system, growth and differentiation of the endometrium, as well it has been difficult to determine the as other biochemical changes, causative factors associated with this process. Kennedy and his associates have proposed that changes in the endometrial vascular permeability, which precede and are a prerequisite for decidualization, are mediated by PGs of the E and I series (16). However, Kennedy has also shown that differential vascular permeability at various phases of uterine sensitivity are not related to the uterine concentration of PGs. He postulated that either (a) the timing of uterine sensitivity is closely related to the capacity of the uterus to respond to PGs for increased vascular permeability, or (b) the latter response may need other mediators in addition to PGs (17). Vascular changes in inflammatory reactions are considered to involve two kinds of chemical mediators: (i> vasodilators and (ii) agents that increase vascular permeability (18). PGs (especially of the E and I series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. However, LTs are much more potent (5000 times) than histamine in this regard (8,lq). Coadministration of LTs with PGs potentiates capillary leakage of plasma in the tissues of various species (8). That there is a synergistic effect between LTs and PGs in the generation of inflammatory edema has been proposed (20). Because the increases in uterine vascular permeability preceeding the decidual cell reaction are of the proinflammatory type, it could be postulated that an interaction exists between PGs and LTs in the generation of the decidual reaction. In the present investigation, we used two experimental protocols that produced rats with different sensitivities for decidualization. Because the hypophysectomixed delayed-implanting rat treated with P4 is extremely sensitive to the implantation initiating action of estrogen that this model should be quite sensitive to the arach(211, we reasoned idonate metabolites in the decidualization reaction. This did not prove to be true because much smaller responses were obtained than with intact animals (Table 1 vs 2). Although PGE2 and LTC4 induced a decidual response in the hypophysectomized animals, it was no larger than that induced by saline alone. When PGEP and LTC4 were combined in the infusate a significant additive effect was produced. We interpret this to indicate that both lipoxygenase and cyclooxygenase products are involved in the decidual response. The second model consisted of intact females on day-5 of pseudopregperiod for decidualization (2). Although both nancy , the most sensitive PGE2 and LTC4 increased the weight of the infused horn over that seen with the vehicle they did not produce a significant additive effect when combined. On the other hand, blockade of the PG pathway for arachidonic acid metabolism by use of indomethacin nearly completely inhibited the response. FPL had a similar effect and a combination of the two nearly abolished the response. The combination of an inhibitor of one arachidonate metabolite and infusion of another metabolite produced most interesting results. PGE2 in the presence of an inhibitor of LTC4 action or LTC4 in the presence of a cyclooxgyenase inhibitor produced a decidual response about 60% of that obtained with vehicle alone. An important aspect that emerges from this study is that PGs and LTs act synergistically and are interdependent on each other in the in-

225

duction of decidualization. This is evident from the observation that FPL interfered with PGEZ-induced decidualization, while indomethacin interfered with LTC4-induced decidualization. This was probably the consequence of the interference of the functions of endogenous LTs or PGs by these inhibitors. Because FPL is a specific antagonist to LTs (15), even infusion of LTC4 with FPL did not overcome the inhibitory effect of FPL on decidualization. It is possible that by increasing the dose of LTCC the inhibitory effect of FPL may disappear. Also we do not know whether LTs other than LTC4 have a similar role in decidualization. Although PGF2a has a moderate effect on the induction of decidualization in the rat (51, it will be interesting to see whether other PGs, especially in combination with various LTs, augement the decidual cell reaction. Although our study clearly demonstrates the interdependence of PGE2 and LTC4 for a decidual cell reaction, we do not know yet whether these two mediators act on the same site or whether they have site-specific actions in this process. Nonetheless, our present study, for the first time, provides evidence of involvement of LTs and their interaction with PGs in the induction of decidualization. ACKNOWLEDGEMENT This study was supported by a grant from the National Institute of Child Health and Human Development (NIH) (HD-12304). Special thanks are given to Dr. Janet Levy , of the R.L. Smith Center Computer Center, for statistical analyses of results. REFEBENCES 1. Mayer, G. Delayed nidation in rats: A method of exploring the mechanisms of ovo-implantation. In: Delayed Implantation (Ed. A.C. Enders). University of Chicago Press Chicago, pp. 213-231, 1963. 2. Psychoyos, A. Endocrine control of egg implantation. In: Handbook of Physiology, Section 7, Vol. II, (Ed. R.O. Greep). Am. Physiol sot., Washington, D.C., pp. 187-215, 1973. 3. DeFeo, V.J. Decidualization. In: Cellular Biology of the uterus (Ed. R.M. Wynn). Appleton-Century-Crofts, New York, pp. 192-290, 1967. 4. Kennedy, T.G. Prostaglandins and increased endometrial vascular permeability resulting from the application of an artificial stimulus to the uterus of the rat sensitized for the decidual cell reaction. Biol. Reprod. 20:560-566, 1979. 5. Kennedy, T.G., and Lukash, L.A. Induction of decidualization in rats by the intraluminal infusion of prostaglandins. Biol. Reprod. 27:253-260, 1982. 6. Kennedy, T.G. Evidence for the involvement of prostaglandins throughout the decidual cell reaction in the rat. Biol. Reprod. 33:140-146, 1985. 7. Kennedy, T.G. Evidence for a role for prostaglandins in the initiation of blastocyst implantation in the rat. Biol. Reprod. 16:286-291, 1977. 8. Samuelsson, B. Leukotrienes: Mediators of immediate hypersensitivity reactions and inflammation. Science 220:568-575, 1983. 9. Hammarstrom, S. Leukotrienes. Ann. Rev. Biochem. 52~355-377, 1983.

226

lO.Pakrasi, P.L., Becka, R., and Dey, S.K. Cyclooxygenase and lipoxygenase pathways in the preimplantation rabbit uterus and blastocyst. Prostaglandins 29:481-495, 1985. Il.Pakrasi, P.L. and Dey, S.K. Evidence for an inverse relationship between cyclooxygenase and lipoxygenase pathways in the pregnant rabbit endometrium. Prostaglandins, Leukotrienes and Medicine 18~347-352, 1985. 12.Malathy, P.V., Cheng, H.C., and Dey, S.K. Production of leukotrienes and prostaglandins in the rat uterus during periimplantation period. Prostaglandins 32:605-614, 1986. 13.Dey, S.K., Hoversland, R.C., and Johnson, D.C. Phospholipase A2 activity in the rat uterus: Modulation by steroid hormones. Prostaglandins 32:619-630, 1982. 14.Vane, J.R. Inhibition of prostaglandin synthesis as a mechanism of aspirin-like drugs. Nature (London) New Biol. 231:232-235,1971 lS.Casey, F.B., Appleby, B.J., and Buck, D.C. Selective inhibition of lipoxygenase metabolic pathway of arachidonic acid by the SRS-A antagonist FPL 55712. Prostaglandins 25:1-11, 1983. 16.Kennedy, T.G., and Armstrong, D.T. The role of prostaglandins in endometrial vascular changes at implantation. In cellular and Molecular Aspects of Implantation (Eds. S.R. Galsser, and D.W. Bullock), Plenum Press, New York, pp. 349-363, 1981. 17.Kennedy, T.G. Timing of uterine sensitivity for the decidual cell reaction: Role of prostaglandins. Biol. Reprod. 22:519-525, 1980 18.Williams, T.J., and Peck, M.J. Role of prostaglandin-mediated vasodilatation in inflammation. Nature 270:530-532, 1977. 19.Ford-Hutchinson, A.W., and Rackman, A. Leukotrienes as mediators of skin inflammation. Br. J. Dermatol. 109 (Suppl. 25):26-29, 1983. ZO.Williams, T.J., Jose, P.J., Wedmore, C.V., Peck, M.J., and Forest, M.J. Mechanisms underlying inflammatory edema: The importance of synergism between prostaglandins, leukotrienes, and complementderived peptides. Ln Advances in Prostaglandin, Thromboxane and Leukotriene Research (Eds. B. Samuelsson, R. Raoletti, and Ramwell) Vol 11, Raven press, New York, pp. 33-37, 1983. 21 .Johnson, D.C., and Dey, S.K. Role of histamine in implantation: Dexamethasone inhibits estradiol-induced implantation in the rat. Biol. Reprod. 22:1136-1141, 1980.

227