- Email: [email protected]
Production of leukotrienes and prostaglandins in the rat uterus during periimplantation period
PROSTAGLANDINS
PRODUCTION OF LEUKOTRIENES AND PROSTAGLANDINS IN THE R A T U T E R U S D U R I N G PERIIMPLANTATION PERIOD P.V. Malathy, H.C. Cheng and S.K. Dey Departments of Obstetrics-Gynecology and Physiology Ralph L. Smith Research Center University of Kansas Medical Center Kansas City, Kansas 66103 (Reprint Requests to SKD) Abstract We have measured by radioimmunoassay the production of leukotrienes (LTC 4 and LTB4) and prostaglandins (PGE 2 and PGF2c 0 in the rat uterus on Days 1 through 6 of pregnancy. The production is defined as the synthesis minus the degradation for a defined period. The production of LTC 4 or LTB 4 remained unaltered on days 1-3, but exhibited a marked increase on Day 4 showing a peak at noon. This was then followed by a sharp decline on Day-5 morning. A small but consistent peak in uterine LT production was also noticed on Day-5 noon prior to implantation and this was followed by a decline on Day-6 morning i.e. after initiation of implantation. The production profile of PGE 2 and PGF2c ~ showed a striking resemblance to that of LTs; one exception being that maximal PG production was noticed on Day-4 morning and preceded the peak production of LTs. These vasoactive arachidonate derivatives reached their peak production rates at around the time when a surge in estrogen level is noticed in the uterus on Day 4. Implantation is a local proinflammatory type of reaction that is associated with increased uterine vascular permeability. Vascular changes in inflammatory reactions are provoked by two kinds of chemical mediators: (1) vasodilators and ( 2 ) a g e n t s that increase vascular permeability. PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. Therefore, an interaction between LTs, PGs, and histamine could be important for uterine preparation for implantation and/or implantation per se. Introduction The biochemical and physiological events leading to blastocyst implantation in the rat depend upon a precise balance between progesterone and estrogen. In the pregnant rat, the uterus becomes receptive on Day 5, while by Day 6 it becomes refractory to the presence of blastocysts (1). A proinflammatory type of reaction accompanied by an increased uterine stromal capillary permeability at the site of blastocyst attachment is considered to be one of the prerequisite events for implantation (1). Prostaglandins (PGs) and histamine, by virtue of their vasoactive properties, have been implicated to participate in the implantation process (2-4). However, they may not be the only agents involved in this process. Involvement of other mediators has recently been suggested, especially with the discovery of a new class of
OCTOBER
1986 VOL.
32 NO.
4
605
PROSTAGLANDINS
mediators, termed leukotrienes (LTs), derived from arachidonic acid molecules from which PGs are synthesized (5,6). LTs are potent vasoactive and chemotactic agents as well as mediators of various kinds of inflammatory reactions (5,6). They are generated from a wide spectrum of inflammatory and resident tissue cells (7-10). Vascular changes in inflammatory reactions are considered to involve two kinds of chemical mediators: (a) vasodilators and (b) agents that increase vascular permeability (11). PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability (5-12). In addition, PGF2e has been reported to induce both deciduogenic (13) and mitotic (14) responses in the rat uterus. The function and modulation of uterine cyclooxygenase pathway have been studied in various species under a variety of conditions (15-18). However, except for a few recent reports on the presence of this pathway in the human (19) and rabbit uterus (17), almost nothing is known about the function and regulation of uterine lipoxygenase pathway. We have, therefore, examined the synthesis of lipoxygenase (LTC 4 and LTB 4) and cyclooxygenase (PGE 2 and PGF2a ) products in the rat uterus on Days 1 through 6 of pregnancy. Materials and Methods For measurements of LTs and PGs, rats (Holtzman strain, weighing 250275 g) were killed by decapitation on Days 1 through 6 of gestation (Day 1 = morning of detecting spermatozoa in the vagina). The uterine horns were cleaned of contaminating blood and adhering fat. They were then homogenized in RPMI-1640 and the homogenates were centrifuged at 750 x g for 15 min. Aliquots of supernatant (500 #1) were incubated at 37°C for 1 h under a gas phase of 95% 02 and 5% C02. After the incubations were terminated by acid precipitation, LTs (LTB 4 and LTC4) and PGs (PGE 2 and PGF2e) were extracted with one volume of isopropanol and four volumes of diethyl ether. The percentages of recoveries were about 80% for both LTs and PGs. Starting levels (0 h) of LTs and PGs were measured in the samples prior to incubation. PGs and LTs were measured by radioimmunoassay (RIA) method (17). Radiolabeled (3H) PGE 2 and PGF2a were procured from New England Nuclear (NEN), Boston, MA, and their specific antibodies were obtained from The Pasteur Institute, Paris, France. LTC 4 and LTB 4 RIA kits were purchased from NEN and Seragen Inc., Boston, MA, respectively. The antisera to PGE 2 or PGF2c ~ react preferentially (100%) with PGE 2 or P G F 2 e , respectively, and negligibly with other PG derivatives. The LTC 4 antiserum cross-reacts with (5S,6 R)-LTC 4 (100%), (5R, 6R)-LTC 4 (100%), l l trans-LTD 4 (60.5%), LTD 4 (55.3%), LTD4-sulfone (10%), LTC4-sulfone (9.5%), LTE 4 (8.6%) and negligibly with other related arachidonate metabolites. The antibodies to LTB 4 cross-reacts 100% with LTB 4 and minimally with other related compounds. The lower limits of sensitivity for PGE2, PGF2e, LTC 4 and LTB 4 were 10-25 pg per tube. Intra and interassay coefficients of variation did not exceed 5% and 10% respectively. Based on the cross-reactivity, LTC 4 assayed should be considered as peptidoleukotrienes. For measurement of estradiol-17/3 (E2-17/3) concentrations, animals were killed by decapitation on Days 1 through 6 of pregnancy. Ovaries and uteri
606
OCTOBER
1986 VOL.
32 NO.
4
PROSTAGLANDINS
were trimmed of fat and homogenized extracts of tissues were dried down concentrations were determined by RIA from NEN and its specific antibody was al.(21).
in 2 ml methanol. The methanolic in a vortex evaporator and E 2-17# (20). Tritiated E2-17fl was obtained prepared and characterized by Exley et
Protein concentrations were determined with bovine serum albumin as the standard by Bradford method (22) using kits from Bio-Rad Laboratory, Richmond, CA. The 0 h levels were subtracted from the incubated values to determine the production rate (ng/mg protein/h). Statistical analyses were performed by Kruskal-Wallis followed by Mann Whitney tests for LTs and oneway ANOVA followed by "post-hoc" t-tests for PGs. Results Because we did not determine the metabolism of PGs and LTs in uterine tissues in vitro, we define production rate of these products as the rate of their synthesis minus their rate of degradation. The temporal patterns of LT and PG production in the rat uterus during the periimplantation period are shown in Figures 1 and 2. The results demonstrate for the first time that, in addition to its PG producing capacity, the rat uterus has the potential to synthesize lipoxygenase-mediated immunoreactive LTC 4 and LTB 4. Although remained unaltered on Days 1-3, the production rate of LTC 4 or LTB 4 started climbing by Day-4 morning reaching a peak by noon of the same day. The peak production rate on Day-4 noon was then followed by a sharp decline by Day-5 morning. A second small but consistent peak in the production rate was observed on Day-5 noon that was followed by a decline again on Day-6 morning (Figure 1). The uterine production rate of PGE 2 or PGF2c~ during early pregnancy follows similar profile as that of LTs, except that peak production rate was observed on Day4 morning instead of Day-4 noon when LT production was at its peak. Another notable difference observed was high rate in PGF2c~ production on Day-I of pregnancy (Fig. 2). This could be attributed to the presence of semen with spermatozoa in the uterine tract on this day. The patterns of ovarian and uterine E2-17fl concentrations are shown in Fig. 3. Both ovarian and uterine tissues showed highest levels of this steroid hormone on Day-4 noon. It is interesting to note that there is a striking similarity between the synthetic pattern of arachidonate metabolites and the E2-17# profile in the uterus (compare Figs. 1 and 2 with Fig. 3). Discussion The results of our study demonstrate that not only cyclooxygenase but lipoxygenase pathways are also operative in rat uterus during the periimplantation stages of gestation. This ability of rat uterine tissue to synthesize LTs and PGs is in line with our earlier observations in the rabbit (17) in which maternal estrogen is not an absolute requirement for implantation (23). The synthetic capacity of LTs and PGs by the rat uterus has been confirmed by in vitro incorporation of [14C]-arachidonic acid into these
OCTOBER 1986 VOL. 32 NO. 4
607
PROSTAGLANDINS
F i g u r e 1. pregnancy.
The uterine production
o- ---o
LTB4
e,
LTC4
o f L T C 4 a n d L T B 4 in the rat d u r i n g early
Ii
-5 •
/ .1=
1.0-
.@
._= 2
Q.
E
"-
o
08-
Q.
//i I
Q6.
2
E v
(10
O4-
'5
0 I-_.1
Q2"
a
I
.I
I
I
I
I
I
I
I
I
I
2
I
2
I
Dayl
Day2 I. 8 AM
Day5
Doy4
Day5
Day6
2. 12 NOON
Vertical lines indicate m e a n _+ S.E.M. E x p e r i m e n t s on d i f f e r e n t days c o m p r i s e d of 7 - 1 0 rats per g r o u p with the e x c e p t i o n of those on D a y s 5 a n d 6 w h i c h consisted o f 4 - 5 a n i m a l s per group. D i f f e r e n c e s in m e a n s with P values less t h a n 0.01 were c o n s i d e r e d significant. T h e p r o d u c t i o n o f L T s was not statistically d i f f e r e n t on Days 1-3. Values on D a y 4 were s i g n i f i c a n t l y h i g h e r f r o m those on o t h e r days. Values on D a y - 5 noon were s i g n i f i c a n t l y h i g h e r t h a n those on D a y - 5 a n d D a y - 6 m o r n i n g s .
608
OCTOBER 1986 VOL. 32 NO. 4
PROSTAGLANDINS
Figure 2. pregnancy.
The uterine production of PGE 2 and PGF2c ~ in the rat during early
o----o A
2.5-
•
•
PGE 2
(7)
PG F2= ~
I
(r)
-15
2.0-
1 2 - *o o.
-.
\\~l /
1.5-
e~ t.OW (.9 0.
'~' \
l#
/ "~/
T \7
1//'
/
9
g
\~"
¢
0.5-
..
.-6 "3
(5) I
I
I
I
I
I
l
I
I
I
I
2
I
2
Doyl
Doy2
Doy5
I. 8AM
Doy4
Day5
I
I
Day6
2. 12 NOON
Vertical lines indicate mean _+ S.E.M. for the n u m b e r of animals shown in parentheses. D i f f e r e n c e s in means with P values less than 0.05 were considered significant. Mean values on Day 4 were significantly higher than those on all the other days with the exception o f PGF2c ~ on Day 1.
OCTOBER 1986 VOL. 32 NO. 4
609
PROSTAGLANDINS
Figure 3. The ovarian and uterine concentrations of E2-17fl on different days of pregnancy.
000.
,40
'\\ A/
000.
-t
\ x
t /-
l
.30
I
~.z'
400.
\\ \
/ \ /.
~'\ x
\, .20
|
r
,-;-.
o o
I
L 200.
.10
1, 6:00 am
,,.t.-
2. 12:00 noon 3. 5 : 0 0 pm 4. 10:00 pm
nAY 1
DAY 2
OAY 3
DAY 4
DAY 5
DAY $
Experiments on different days comprised of 6-9 animals per group. Differences in means with P values less than 0.05 were considered significant, Ovarian E2-17 fl concentrations on Days 3-5 and Day-4 noon were significantly higher than those on Day 1 and all other days, respectively. While uterine concentration of this steroid on Day-4 noon was significantly higher as compared to all other day, concentrations on Days 3-6 were only significantly higher than those on Days 1 and 2.
610
OCTOBER
1986 VOL.
32 N O . 4
PROSTAGLANDINS
products and by the use of esculetin, an inhibitor of lipoxygenase pathway (24) and indomethacin, an inhibitor of cyclooxygenase pathway (25) (to be published). Although, our results demonstrate uterine capacity to synthesize LTs and PGs in vitro during the periimplantation period, it is not clear if similar synthetic patterns occur in vivo. The present study was carried out to examine if the changes in LT and/or PG production patterns were concurrent with parallel alterations in E2-17b levels in a species, such as the rat, in which maternal estrogen is an absolute requirement for implantation. The relatively lower production rate of the immunoreactive arachidonate products during Days 1-3 of pregnancy and a dramatic increment in response on Day 4 (Figs. 1 and 2) are suggestive of a complex hormonal influence required for stimulation of the cyclooxygenase and lipoxygenase pathways. What is more striking is the fact that peak production rates of these vasoactive agents are reached at approximately around the time when a surge in estrogen level occurs in the uterus. This surge of E2-17b is considered to be a prerequisite for triggering blastocyst implantation (26-28). It is, however, not clear whether E 2-17b is one of the predominant hormonal signals modulating uterine PG and LT production during this eariy phase of pregnancy. Furthermore, it is not known if these arachidonate metabolites assume similar importance as estrogen in blastocyst implantation. Because lower production rates of LTs and PGs are observed on Day 1 of pregnancy, proestrous E2-17b surge with accompanied eosinophilia does not appear sufficient to activate this pathway (29). It, therefore, seems reasonable to assume that a surge of E 217b with rising progesterone level may be necessary to stimulate uterine cyclooxygenase and lipoxygenase pathways. Liberation of the fatty acid precursor, arachidonic acid, from tissue phospholipids as a result of phospholipase A 2 (PLA2) activity is widely accepted as the rate-limiting step in the biosynthesis of PGs and LTs (30). The patterns of LT and PG production rates observed in our present study bear a striking similarity to the profile of PLA 2 activity in the uterus during early pregnancy reported earlier (31). However, the role of phospholipase C (PLC) and diacylglyceride lipase in the release of arachidonic acid has been reported (32,33). It should be pointed out that although the PGs and LTs are products of the same precursor, arachidonic acid, generated via PLA 2 or PLC (30,32,33), the maximal activation of the cyclooxygenase and lipoxygenase systems appears to occur at different periods; activation of cyclooxygenase preceding that of lipoxygenase. Such differences in the kinetics of these two enzyme systems have been assigned to the possible involvement of different acylhydrolase pathways active at specific or varied phospholipid pools (33-35) and may account for our present observation. At this juncture, it is premature to speculate if the products of cyclooxygenase exert a stimulatory effect on lipoxygenase system. Because the uterus attains peak synthetic capacity for LTs and PGs a day before and prior to the time of implantation (Day-4 and Day-5 noon), we could speculate that these agents, by virtue of their vasoactive and proinflammatory properties may play an important role both in uterine preparation for implantation and implantation process per se. A synergistic effect among LTs, PGs and histamine that is observed in the generation of inflammatory response
OCTOBER 1986 VOL. 32 NO. 4
611
PROSTAGLANDINS
could also be operative during implantation process. However, specific and potent inhibitors of cyclooxygenase and lipoxygenase pathways will be necessary to examine the hormonal dependence and functional involvement of these potent vasoactive agents in implantation. Acknowledgements This research was supported by an NICHD grant (HD-12304-07). PVM is a Rockefeller Foundation Postdoctoral Fellow. We thank Anne K. Salamon for her technical assistance. References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10)
11) 12) 13) 14)
612
Psychoyos, A. Endocrine control of egg implantation. In: Handbook of Physiology. Section 7, Vol. II (Ed. R.O. Greep) American Physiological Society, Washington, D.C., 1973. p. 187-215. Dey, S.K., and D.C. Johnson. In: The Endometrium (Ed. F. Kimball), SP Medical and Scientific Books, Spectrum Publications, New York, I980. pp. 269-283. Shelesnyak, M.C. Some experimental studies on the mechanism of ovaimplantation in the rat. Recent Prog. Horm. Res. 13:269-322. 1957. Kennedy, T.G. Evidence for a role for prostaglandins in the initiation of blastocyst implantation in the rat. Biol. Reprod. 16:286-291. 1977. Samuelsson, B. Mediators of immediate hypersensitivity reactions and inflammation. Science 22____.00:568-575. 1983. Hammarstrom, S. Leukotrienes. Annu. Rev. Biochem. 5_2:355-377. 1983. Czarnetzki, B.M., and J. Grabbe. Biological and chemical characterization of eosinophil chemotactic factor from human leukocytes. Agents and Actions (Supplements) 12--204-216, 1983. Goetzl, E.J. Mediators of immediate hypersensitivity derived from arachidonic acid. N. Engl. J. Med. 303:822-825. 1980. Goetzl, E.J. Selective feed-back inhibition of the 5-1ipoxygenase of arachidonic acid in human T-lymphocytes. Biochem. Biophys. Res. Commun. 101....___:344-350. 1980. Valone, F.H., M. Franklin, F.F. Sun, and E.J. Goetzl. Alveolar macrophage-lipoxygenase products of arachidonic acid isolation and recognition of the predominant constituents of the neutrophil chemotactic activity elaborated by alveolar macrophages. Cell. Immunol. 54:390-401. 1980. Williams, T.J., and M.J. Peck. Role of prostaglandin-mediated vasodilation in inflammation. Nature 270--530-532. 1977. Ford-Hutchinson, A.W., and A. Rackman. Leukotrienes as mediators of skin inflammation. Br. J. Dermatol. 10....29(Suppl. 25):26-29. 1983. Sonnanes, N., E.E. Banlieu, and C. Legoascogne. Prostaglandins as inductive factor of decidualization in the rat uterus. Mol. Cell. Endocr. 6:153-158, 1976. Peleg, S. The modulation of decidual cell proliferation and differentiation by progesterone and prostaglandins. J. Steroid Biochem. 1_.99:283-289, 1983.
OCTOBER
1986 VOL.
32 N O . 4
PROSTAGLANDINS
15) Pakrasi, P.L., S.K. Dey, and D.C. Johnson. Studies on the temporal pattern of prostaglandin synthesis in the uterus of the delayed implanting rat with or without-implantation inducing stimuli. Prostaglandins, Leukotrienes Med. 14:365-381. 1984. 16) Phillips, C.A., and N.L. Poyser. Studies on the involvement of prostaglandins in implantation in the rat. J. Reprod. Fertil. 6_22:73-81. 1981. 17) Pakrasi, P.L., R. Becka, and S.K. Dey. Cyclooxygenase and lipoxygenase pathways in the preimplantation rabbit uterus and blastocyst. Prostaglandins 2._29:481-495. 1985. 18) Harper, M.J.K., C.J. Norris, and K. Rajkumar. Prostaglandin release by zygotes and endometria of pregnant rabbits. Biol. Reprod. 28:350-362. 1983. 19) Demers, L.M., M.C.P. Rees, and A.C. Turnbull. Arachidonic acid metabolism by the non-pregnant human uterus. Prostaglandins Leukotrienes Med. 1_44:175-180. 1984. 20) Cheng, H.C., and D.C. Johnson. Serum estrogens and gonadotropins in developing androgenized and normal female rats. Neuroendocrinology 13:357-365. 1973. 21) Exley, D., M.W. Johnson, and P.D.G. Dean. Antisera highly specific for 17/%estradiol. Steroids I.~8:605-620. 1971. 22) Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 7_Z2:248-254. 1976. 23) Kwun, J.K., and C.W. Emmens. Hormonal requirements for implantation and pregnancy in the ovariectomized rabbit. Aust. J. Biol. Sci. 2_!:275-283. 1974. 24) Neichi, T., Y. Koshihara, and S.I. Murota. Inhibitory effect of esculetin on 5-1ipoxygenase and leukotriene biosynthesis. Biochim. Biophys. Acta 75__~3:130-132, 1983. 25) Vane, J.R. Inhibition of prostaglandin biosynthesis as a mechanism of action of aspirin-like drugs. Nature, New Biol. 231:232-235, 1971. 26) Shelesnyak, M.C., and P.F. Kraicer. Studies on the mechanism of decidualization. I. The oestrogen surge of pseudopregnancy and progravity and its role in the process of decidualization. Acta Endocrinol. (kbh) 42:225-232. 1963. 27) Yoshinaga, K., R.A. Hawkins, and J.F. Strocker. Estrogen secretion by the rat ovary i~ vivo during estrous cycle and pregnancy. Endocrinology 85:103-112. 1969. 28) Shaikh, A.A. Estrone and estradiol levels in the ovarian venous blood from rats during the estrous cycle and pregnancy. Biol. Reprod. 5_:297-307. 1971. 29) Tchernitchin, A., J. Roorijck, X. Tchernitchin, J. Vandenhende, and P. Galand. Dramatic increase in uterine eosinophils after estrogen administration. Nature 248:142-143. 1974. 30) Samuelsson, B., M. Goldyne, E. Granstrom, M. Hamberg, S. Hammarstrom, and C. Malmsten. Prostaglandins and thromboxanes. Ann. Rev. Biochem. 4"/:997-1029. 1978. 31) Cox, C., H.C. Cheng, and S.K. Dey. Phospholipase A 2 activity in the rat uterus during early pregnancy. Prostaglandins Leukotrienes Med. 8:375-381. 1982.
OCTOBER
1986 V O L . 32 N O . 4
613
PROSTAGLANDINS
32) Okazaki, T., N. Sagawa, J.E. Bleasdale, J.R. Okita, P.C, Macdonald, and J.M. Johnston. Initiation of human parturition: XIII. Phospholipase C, phospholipase A 2 and diglycerol lipase activities in fetal membranes and decidua vera tissues from early and late gestation. Biol. Reprod. 25.'103109. 1980. 33) Bell, R.I., D.A. Kennerly, N. Stanford, and P.N. Majerus. Diglyceride lipase: A pathway for arachidonate release from human platelets. Proc. Natl. Acad. Sci. USA 76:3238-3241. 1979. 34) Humes, J.L., S. Sadowski, M. Galvage, M. Goldenberg, E. Subers, R.J. Bonney, and F.A. Keuhl. Evidence for two sources of arachidonic acid for oxidative metabolism by mouse peritoneal macrophages. J. Biol. Chem. 257:1591-1594. 1982. 35) Billah, M.M., E.G. Lapetina, and P. Cuatrecas. Phospholipase A 2 activity specific for phosphatidic acid: A possible mechanism for the production of arachidonic acid in platelets. J. Biol. Chem. 256:5399-5403. 1981.
Editor:
614
H. R. Behrman
Received:
6-17-86
OCTOBER
Accepted:
9-ii-86
1986 V O L . 32 N O . 4
PRODUCTION OF LEUKOTRIENES AND PROSTAGLANDINS IN THE R A T U T E R U S D U R I N G PERIIMPLANTATION PERIOD P.V. Malathy, H.C. Cheng and S.K. Dey Departments of Obstetrics-Gynecology and Physiology Ralph L. Smith Research Center University of Kansas Medical Center Kansas City, Kansas 66103 (Reprint Requests to SKD) Abstract We have measured by radioimmunoassay the production of leukotrienes (LTC 4 and LTB4) and prostaglandins (PGE 2 and PGF2c 0 in the rat uterus on Days 1 through 6 of pregnancy. The production is defined as the synthesis minus the degradation for a defined period. The production of LTC 4 or LTB 4 remained unaltered on days 1-3, but exhibited a marked increase on Day 4 showing a peak at noon. This was then followed by a sharp decline on Day-5 morning. A small but consistent peak in uterine LT production was also noticed on Day-5 noon prior to implantation and this was followed by a decline on Day-6 morning i.e. after initiation of implantation. The production profile of PGE 2 and PGF2c ~ showed a striking resemblance to that of LTs; one exception being that maximal PG production was noticed on Day-4 morning and preceded the peak production of LTs. These vasoactive arachidonate derivatives reached their peak production rates at around the time when a surge in estrogen level is noticed in the uterus on Day 4. Implantation is a local proinflammatory type of reaction that is associated with increased uterine vascular permeability. Vascular changes in inflammatory reactions are provoked by two kinds of chemical mediators: (1) vasodilators and ( 2 ) a g e n t s that increase vascular permeability. PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. Therefore, an interaction between LTs, PGs, and histamine could be important for uterine preparation for implantation and/or implantation per se. Introduction The biochemical and physiological events leading to blastocyst implantation in the rat depend upon a precise balance between progesterone and estrogen. In the pregnant rat, the uterus becomes receptive on Day 5, while by Day 6 it becomes refractory to the presence of blastocysts (1). A proinflammatory type of reaction accompanied by an increased uterine stromal capillary permeability at the site of blastocyst attachment is considered to be one of the prerequisite events for implantation (1). Prostaglandins (PGs) and histamine, by virtue of their vasoactive properties, have been implicated to participate in the implantation process (2-4). However, they may not be the only agents involved in this process. Involvement of other mediators has recently been suggested, especially with the discovery of a new class of
OCTOBER
1986 VOL.
32 NO.
4
605
PROSTAGLANDINS
mediators, termed leukotrienes (LTs), derived from arachidonic acid molecules from which PGs are synthesized (5,6). LTs are potent vasoactive and chemotactic agents as well as mediators of various kinds of inflammatory reactions (5,6). They are generated from a wide spectrum of inflammatory and resident tissue cells (7-10). Vascular changes in inflammatory reactions are considered to involve two kinds of chemical mediators: (a) vasodilators and (b) agents that increase vascular permeability (11). PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability (5-12). In addition, PGF2e has been reported to induce both deciduogenic (13) and mitotic (14) responses in the rat uterus. The function and modulation of uterine cyclooxygenase pathway have been studied in various species under a variety of conditions (15-18). However, except for a few recent reports on the presence of this pathway in the human (19) and rabbit uterus (17), almost nothing is known about the function and regulation of uterine lipoxygenase pathway. We have, therefore, examined the synthesis of lipoxygenase (LTC 4 and LTB 4) and cyclooxygenase (PGE 2 and PGF2a ) products in the rat uterus on Days 1 through 6 of pregnancy. Materials and Methods For measurements of LTs and PGs, rats (Holtzman strain, weighing 250275 g) were killed by decapitation on Days 1 through 6 of gestation (Day 1 = morning of detecting spermatozoa in the vagina). The uterine horns were cleaned of contaminating blood and adhering fat. They were then homogenized in RPMI-1640 and the homogenates were centrifuged at 750 x g for 15 min. Aliquots of supernatant (500 #1) were incubated at 37°C for 1 h under a gas phase of 95% 02 and 5% C02. After the incubations were terminated by acid precipitation, LTs (LTB 4 and LTC4) and PGs (PGE 2 and PGF2e) were extracted with one volume of isopropanol and four volumes of diethyl ether. The percentages of recoveries were about 80% for both LTs and PGs. Starting levels (0 h) of LTs and PGs were measured in the samples prior to incubation. PGs and LTs were measured by radioimmunoassay (RIA) method (17). Radiolabeled (3H) PGE 2 and PGF2a were procured from New England Nuclear (NEN), Boston, MA, and their specific antibodies were obtained from The Pasteur Institute, Paris, France. LTC 4 and LTB 4 RIA kits were purchased from NEN and Seragen Inc., Boston, MA, respectively. The antisera to PGE 2 or PGF2c ~ react preferentially (100%) with PGE 2 or P G F 2 e , respectively, and negligibly with other PG derivatives. The LTC 4 antiserum cross-reacts with (5S,6 R)-LTC 4 (100%), (5R, 6R)-LTC 4 (100%), l l trans-LTD 4 (60.5%), LTD 4 (55.3%), LTD4-sulfone (10%), LTC4-sulfone (9.5%), LTE 4 (8.6%) and negligibly with other related arachidonate metabolites. The antibodies to LTB 4 cross-reacts 100% with LTB 4 and minimally with other related compounds. The lower limits of sensitivity for PGE2, PGF2e, LTC 4 and LTB 4 were 10-25 pg per tube. Intra and interassay coefficients of variation did not exceed 5% and 10% respectively. Based on the cross-reactivity, LTC 4 assayed should be considered as peptidoleukotrienes. For measurement of estradiol-17/3 (E2-17/3) concentrations, animals were killed by decapitation on Days 1 through 6 of pregnancy. Ovaries and uteri
606
OCTOBER
1986 VOL.
32 NO.
4
PROSTAGLANDINS
were trimmed of fat and homogenized extracts of tissues were dried down concentrations were determined by RIA from NEN and its specific antibody was al.(21).
in 2 ml methanol. The methanolic in a vortex evaporator and E 2-17# (20). Tritiated E2-17fl was obtained prepared and characterized by Exley et
Protein concentrations were determined with bovine serum albumin as the standard by Bradford method (22) using kits from Bio-Rad Laboratory, Richmond, CA. The 0 h levels were subtracted from the incubated values to determine the production rate (ng/mg protein/h). Statistical analyses were performed by Kruskal-Wallis followed by Mann Whitney tests for LTs and oneway ANOVA followed by "post-hoc" t-tests for PGs. Results Because we did not determine the metabolism of PGs and LTs in uterine tissues in vitro, we define production rate of these products as the rate of their synthesis minus their rate of degradation. The temporal patterns of LT and PG production in the rat uterus during the periimplantation period are shown in Figures 1 and 2. The results demonstrate for the first time that, in addition to its PG producing capacity, the rat uterus has the potential to synthesize lipoxygenase-mediated immunoreactive LTC 4 and LTB 4. Although remained unaltered on Days 1-3, the production rate of LTC 4 or LTB 4 started climbing by Day-4 morning reaching a peak by noon of the same day. The peak production rate on Day-4 noon was then followed by a sharp decline by Day-5 morning. A second small but consistent peak in the production rate was observed on Day-5 noon that was followed by a decline again on Day-6 morning (Figure 1). The uterine production rate of PGE 2 or PGF2c~ during early pregnancy follows similar profile as that of LTs, except that peak production rate was observed on Day4 morning instead of Day-4 noon when LT production was at its peak. Another notable difference observed was high rate in PGF2c~ production on Day-I of pregnancy (Fig. 2). This could be attributed to the presence of semen with spermatozoa in the uterine tract on this day. The patterns of ovarian and uterine E2-17fl concentrations are shown in Fig. 3. Both ovarian and uterine tissues showed highest levels of this steroid hormone on Day-4 noon. It is interesting to note that there is a striking similarity between the synthetic pattern of arachidonate metabolites and the E2-17# profile in the uterus (compare Figs. 1 and 2 with Fig. 3). Discussion The results of our study demonstrate that not only cyclooxygenase but lipoxygenase pathways are also operative in rat uterus during the periimplantation stages of gestation. This ability of rat uterine tissue to synthesize LTs and PGs is in line with our earlier observations in the rabbit (17) in which maternal estrogen is not an absolute requirement for implantation (23). The synthetic capacity of LTs and PGs by the rat uterus has been confirmed by in vitro incorporation of [14C]-arachidonic acid into these
OCTOBER 1986 VOL. 32 NO. 4
607
PROSTAGLANDINS
F i g u r e 1. pregnancy.
The uterine production
o- ---o
LTB4
e,
LTC4
o f L T C 4 a n d L T B 4 in the rat d u r i n g early
Ii
-5 •
/ .1=
1.0-
.@
._= 2
Q.
E
"-
o
08-
Q.
//i I
Q6.
2
E v
(10
O4-
'5
0 I-_.1
Q2"
a
I
.I
I
I
I
I
I
I
I
I
I
2
I
2
I
Dayl
Day2 I. 8 AM
Day5
Doy4
Day5
Day6
2. 12 NOON
Vertical lines indicate m e a n _+ S.E.M. E x p e r i m e n t s on d i f f e r e n t days c o m p r i s e d of 7 - 1 0 rats per g r o u p with the e x c e p t i o n of those on D a y s 5 a n d 6 w h i c h consisted o f 4 - 5 a n i m a l s per group. D i f f e r e n c e s in m e a n s with P values less t h a n 0.01 were c o n s i d e r e d significant. T h e p r o d u c t i o n o f L T s was not statistically d i f f e r e n t on Days 1-3. Values on D a y 4 were s i g n i f i c a n t l y h i g h e r f r o m those on o t h e r days. Values on D a y - 5 noon were s i g n i f i c a n t l y h i g h e r t h a n those on D a y - 5 a n d D a y - 6 m o r n i n g s .
608
OCTOBER 1986 VOL. 32 NO. 4
PROSTAGLANDINS
Figure 2. pregnancy.
The uterine production of PGE 2 and PGF2c ~ in the rat during early
o----o A
2.5-
•
•
PGE 2
(7)
PG F2= ~
I
(r)
-15
2.0-
1 2 - *o o.
-.
\\~l /
1.5-
e~ t.OW (.9 0.
'~' \
l#
/ "~/
T \7
1//'
/
9
g
\~"
¢
0.5-
..
.-6 "3
(5) I
I
I
I
I
I
l
I
I
I
I
2
I
2
Doyl
Doy2
Doy5
I. 8AM
Doy4
Day5
I
I
Day6
2. 12 NOON
Vertical lines indicate mean _+ S.E.M. for the n u m b e r of animals shown in parentheses. D i f f e r e n c e s in means with P values less than 0.05 were considered significant. Mean values on Day 4 were significantly higher than those on all the other days with the exception o f PGF2c ~ on Day 1.
OCTOBER 1986 VOL. 32 NO. 4
609
PROSTAGLANDINS
Figure 3. The ovarian and uterine concentrations of E2-17fl on different days of pregnancy.
000.
,40
'\\ A/
000.
-t
\ x
t /-
l
.30
I
~.z'
400.
\\ \
/ \ /.
~'\ x
\, .20
|
r
,-;-.
o o
I
L 200.
.10
1, 6:00 am
,,.t.-
2. 12:00 noon 3. 5 : 0 0 pm 4. 10:00 pm
nAY 1
DAY 2
OAY 3
DAY 4
DAY 5
DAY $
Experiments on different days comprised of 6-9 animals per group. Differences in means with P values less than 0.05 were considered significant, Ovarian E2-17 fl concentrations on Days 3-5 and Day-4 noon were significantly higher than those on Day 1 and all other days, respectively. While uterine concentration of this steroid on Day-4 noon was significantly higher as compared to all other day, concentrations on Days 3-6 were only significantly higher than those on Days 1 and 2.
610
OCTOBER
1986 VOL.
32 N O . 4
PROSTAGLANDINS
products and by the use of esculetin, an inhibitor of lipoxygenase pathway (24) and indomethacin, an inhibitor of cyclooxygenase pathway (25) (to be published). Although, our results demonstrate uterine capacity to synthesize LTs and PGs in vitro during the periimplantation period, it is not clear if similar synthetic patterns occur in vivo. The present study was carried out to examine if the changes in LT and/or PG production patterns were concurrent with parallel alterations in E2-17b levels in a species, such as the rat, in which maternal estrogen is an absolute requirement for implantation. The relatively lower production rate of the immunoreactive arachidonate products during Days 1-3 of pregnancy and a dramatic increment in response on Day 4 (Figs. 1 and 2) are suggestive of a complex hormonal influence required for stimulation of the cyclooxygenase and lipoxygenase pathways. What is more striking is the fact that peak production rates of these vasoactive agents are reached at approximately around the time when a surge in estrogen level occurs in the uterus. This surge of E2-17b is considered to be a prerequisite for triggering blastocyst implantation (26-28). It is, however, not clear whether E 2-17b is one of the predominant hormonal signals modulating uterine PG and LT production during this eariy phase of pregnancy. Furthermore, it is not known if these arachidonate metabolites assume similar importance as estrogen in blastocyst implantation. Because lower production rates of LTs and PGs are observed on Day 1 of pregnancy, proestrous E2-17b surge with accompanied eosinophilia does not appear sufficient to activate this pathway (29). It, therefore, seems reasonable to assume that a surge of E 217b with rising progesterone level may be necessary to stimulate uterine cyclooxygenase and lipoxygenase pathways. Liberation of the fatty acid precursor, arachidonic acid, from tissue phospholipids as a result of phospholipase A 2 (PLA2) activity is widely accepted as the rate-limiting step in the biosynthesis of PGs and LTs (30). The patterns of LT and PG production rates observed in our present study bear a striking similarity to the profile of PLA 2 activity in the uterus during early pregnancy reported earlier (31). However, the role of phospholipase C (PLC) and diacylglyceride lipase in the release of arachidonic acid has been reported (32,33). It should be pointed out that although the PGs and LTs are products of the same precursor, arachidonic acid, generated via PLA 2 or PLC (30,32,33), the maximal activation of the cyclooxygenase and lipoxygenase systems appears to occur at different periods; activation of cyclooxygenase preceding that of lipoxygenase. Such differences in the kinetics of these two enzyme systems have been assigned to the possible involvement of different acylhydrolase pathways active at specific or varied phospholipid pools (33-35) and may account for our present observation. At this juncture, it is premature to speculate if the products of cyclooxygenase exert a stimulatory effect on lipoxygenase system. Because the uterus attains peak synthetic capacity for LTs and PGs a day before and prior to the time of implantation (Day-4 and Day-5 noon), we could speculate that these agents, by virtue of their vasoactive and proinflammatory properties may play an important role both in uterine preparation for implantation and implantation process per se. A synergistic effect among LTs, PGs and histamine that is observed in the generation of inflammatory response
OCTOBER 1986 VOL. 32 NO. 4
611
PROSTAGLANDINS
could also be operative during implantation process. However, specific and potent inhibitors of cyclooxygenase and lipoxygenase pathways will be necessary to examine the hormonal dependence and functional involvement of these potent vasoactive agents in implantation. Acknowledgements This research was supported by an NICHD grant (HD-12304-07). PVM is a Rockefeller Foundation Postdoctoral Fellow. We thank Anne K. Salamon for her technical assistance. References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10)
11) 12) 13) 14)
612
Psychoyos, A. Endocrine control of egg implantation. In: Handbook of Physiology. Section 7, Vol. II (Ed. R.O. Greep) American Physiological Society, Washington, D.C., 1973. p. 187-215. Dey, S.K., and D.C. Johnson. In: The Endometrium (Ed. F. Kimball), SP Medical and Scientific Books, Spectrum Publications, New York, I980. pp. 269-283. Shelesnyak, M.C. Some experimental studies on the mechanism of ovaimplantation in the rat. Recent Prog. Horm. Res. 13:269-322. 1957. Kennedy, T.G. Evidence for a role for prostaglandins in the initiation of blastocyst implantation in the rat. Biol. Reprod. 16:286-291. 1977. Samuelsson, B. Mediators of immediate hypersensitivity reactions and inflammation. Science 22____.00:568-575. 1983. Hammarstrom, S. Leukotrienes. Annu. Rev. Biochem. 5_2:355-377. 1983. Czarnetzki, B.M., and J. Grabbe. Biological and chemical characterization of eosinophil chemotactic factor from human leukocytes. Agents and Actions (Supplements) 12--204-216, 1983. Goetzl, E.J. Mediators of immediate hypersensitivity derived from arachidonic acid. N. Engl. J. Med. 303:822-825. 1980. Goetzl, E.J. Selective feed-back inhibition of the 5-1ipoxygenase of arachidonic acid in human T-lymphocytes. Biochem. Biophys. Res. Commun. 101....___:344-350. 1980. Valone, F.H., M. Franklin, F.F. Sun, and E.J. Goetzl. Alveolar macrophage-lipoxygenase products of arachidonic acid isolation and recognition of the predominant constituents of the neutrophil chemotactic activity elaborated by alveolar macrophages. Cell. Immunol. 54:390-401. 1980. Williams, T.J., and M.J. Peck. Role of prostaglandin-mediated vasodilation in inflammation. Nature 270--530-532. 1977. Ford-Hutchinson, A.W., and A. Rackman. Leukotrienes as mediators of skin inflammation. Br. J. Dermatol. 10....29(Suppl. 25):26-29. 1983. Sonnanes, N., E.E. Banlieu, and C. Legoascogne. Prostaglandins as inductive factor of decidualization in the rat uterus. Mol. Cell. Endocr. 6:153-158, 1976. Peleg, S. The modulation of decidual cell proliferation and differentiation by progesterone and prostaglandins. J. Steroid Biochem. 1_.99:283-289, 1983.
OCTOBER
1986 VOL.
32 N O . 4
PROSTAGLANDINS
15) Pakrasi, P.L., S.K. Dey, and D.C. Johnson. Studies on the temporal pattern of prostaglandin synthesis in the uterus of the delayed implanting rat with or without-implantation inducing stimuli. Prostaglandins, Leukotrienes Med. 14:365-381. 1984. 16) Phillips, C.A., and N.L. Poyser. Studies on the involvement of prostaglandins in implantation in the rat. J. Reprod. Fertil. 6_22:73-81. 1981. 17) Pakrasi, P.L., R. Becka, and S.K. Dey. Cyclooxygenase and lipoxygenase pathways in the preimplantation rabbit uterus and blastocyst. Prostaglandins 2._29:481-495. 1985. 18) Harper, M.J.K., C.J. Norris, and K. Rajkumar. Prostaglandin release by zygotes and endometria of pregnant rabbits. Biol. Reprod. 28:350-362. 1983. 19) Demers, L.M., M.C.P. Rees, and A.C. Turnbull. Arachidonic acid metabolism by the non-pregnant human uterus. Prostaglandins Leukotrienes Med. 1_44:175-180. 1984. 20) Cheng, H.C., and D.C. Johnson. Serum estrogens and gonadotropins in developing androgenized and normal female rats. Neuroendocrinology 13:357-365. 1973. 21) Exley, D., M.W. Johnson, and P.D.G. Dean. Antisera highly specific for 17/%estradiol. Steroids I.~8:605-620. 1971. 22) Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 7_Z2:248-254. 1976. 23) Kwun, J.K., and C.W. Emmens. Hormonal requirements for implantation and pregnancy in the ovariectomized rabbit. Aust. J. Biol. Sci. 2_!:275-283. 1974. 24) Neichi, T., Y. Koshihara, and S.I. Murota. Inhibitory effect of esculetin on 5-1ipoxygenase and leukotriene biosynthesis. Biochim. Biophys. Acta 75__~3:130-132, 1983. 25) Vane, J.R. Inhibition of prostaglandin biosynthesis as a mechanism of action of aspirin-like drugs. Nature, New Biol. 231:232-235, 1971. 26) Shelesnyak, M.C., and P.F. Kraicer. Studies on the mechanism of decidualization. I. The oestrogen surge of pseudopregnancy and progravity and its role in the process of decidualization. Acta Endocrinol. (kbh) 42:225-232. 1963. 27) Yoshinaga, K., R.A. Hawkins, and J.F. Strocker. Estrogen secretion by the rat ovary i~ vivo during estrous cycle and pregnancy. Endocrinology 85:103-112. 1969. 28) Shaikh, A.A. Estrone and estradiol levels in the ovarian venous blood from rats during the estrous cycle and pregnancy. Biol. Reprod. 5_:297-307. 1971. 29) Tchernitchin, A., J. Roorijck, X. Tchernitchin, J. Vandenhende, and P. Galand. Dramatic increase in uterine eosinophils after estrogen administration. Nature 248:142-143. 1974. 30) Samuelsson, B., M. Goldyne, E. Granstrom, M. Hamberg, S. Hammarstrom, and C. Malmsten. Prostaglandins and thromboxanes. Ann. Rev. Biochem. 4"/:997-1029. 1978. 31) Cox, C., H.C. Cheng, and S.K. Dey. Phospholipase A 2 activity in the rat uterus during early pregnancy. Prostaglandins Leukotrienes Med. 8:375-381. 1982.
OCTOBER
1986 V O L . 32 N O . 4
613
PROSTAGLANDINS
32) Okazaki, T., N. Sagawa, J.E. Bleasdale, J.R. Okita, P.C, Macdonald, and J.M. Johnston. Initiation of human parturition: XIII. Phospholipase C, phospholipase A 2 and diglycerol lipase activities in fetal membranes and decidua vera tissues from early and late gestation. Biol. Reprod. 25.'103109. 1980. 33) Bell, R.I., D.A. Kennerly, N. Stanford, and P.N. Majerus. Diglyceride lipase: A pathway for arachidonate release from human platelets. Proc. Natl. Acad. Sci. USA 76:3238-3241. 1979. 34) Humes, J.L., S. Sadowski, M. Galvage, M. Goldenberg, E. Subers, R.J. Bonney, and F.A. Keuhl. Evidence for two sources of arachidonic acid for oxidative metabolism by mouse peritoneal macrophages. J. Biol. Chem. 257:1591-1594. 1982. 35) Billah, M.M., E.G. Lapetina, and P. Cuatrecas. Phospholipase A 2 activity specific for phosphatidic acid: A possible mechanism for the production of arachidonic acid in platelets. J. Biol. Chem. 256:5399-5403. 1981.
Editor:
614
H. R. Behrman
Received:
6-17-86
OCTOBER
Accepted:
9-ii-86
1986 V O L . 32 N O . 4