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Prostaglandin production by human term placentas in vitro
Prostaglandins Leukotrienes and Medicine 11: lZ!l-l29,1983
PROSTAGlA.NDJN PROQUCTION BY HUMAN TERM PLACENTAS -IN VITRO M.J.K.
Harper,
G.S.
Khodr
and G. Valenzuela
Department of Obstetrics and Gynecology, The University of Texas Health Science Canter at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284, USA. [Reprints requests to MJKH] ABSTRACT The release of PGs increases with increasing incubation time and the main PG produced is different in different tissuaa. This could PGE release is implicate the presence of an inhibitory factor 5 a. the same in patients in labor and not in labor, and this PG is mainly Villi, membranes and deck&a in tissue produced by the villi. cultures from patients not in labor release more PGF than pa@8nb in labor, the difference for the decidual tissue being strikingly Iscga. Conversion of labelled arachidonic acid by pieces of term placenta from women in labor during short-term incubations was mainly to PGE, and its metabolites and PGD,. INTRODUCTION Prostaglandins (PGs) are increased in plasma and amniotic fluid PG synthesis inhibitors have been used during labor (1,2). successfully in the treatment of premature labor (31, and m&emal ingestion of aspirin prolongs the normal duration. of pregnancy (4). The human placenta and attached fetal membranes contain high concentrations of enzymes capable of catabolizing PGs to much lass The precursor of most PGs, arachidonic acid active metabolites (5). (AA), comprises 20% of the lipids in fetal membranes and dachdua compared to only 0.4% in the mesenteric adipose tissue ($1, and the decidua also contains phospholipase A the enzyme respansWe for cleaving AA from the phosphollpid m&&es where it is sWW (7). One of the major theories on initiation of labor postuta%us thee a decrease in the total intracellular concentratEon of p-e is caused by the appearance of a specific progesterone reoq%ar (8). This produces a relative estrqenic dominance, and a lysis of the in resulting r&aria of lysosomes their qrtzymes, the tissue cot~en$r&ion of AA is ant However, phosphollpase. slightly decreased in women who un&qp spantan@@us W#W to those not in labor who are delivered by cesarean section (6).
121
Furthermore, recently been
some questions raised (9).
about
the
lysis
of
the
lysosomes
have
We performed short term -in vitro incubations and tissue cultures of placentas and membranes, in an attempt to define the role of endogenous AA in PG production, and the effect on this system of deprivation of hormonal support. MATERIALS .LQ Vitro
AND
METHODS
incubations
Placentas (n = 8) obtained from term were rinsed with normal saline became clear and were then frozen work has shown that there is no activity between “fresh” and frozen
women in spontaneous labor at at 4% until the rinse solution at -60°C until used. Previous significant decrease in enzyme placentas over the short term.
Once the placentas were defrosted, pieces of about I g were obtained and rinsed several times with 50 mM Tris HCI buffer containing I mM EDTA (pH 7.7). The tissues were weighed, and the pieces then were placed into polypropylene tubes (Falcon, Oxnard, Ca #2059; I7 x 100 mm) with 2 ml (final volume) of Tris-HCI buffer. To each tube was added approximately 200,000 cpm of [5,6,8,9,11,12,14, l5-sH(N)]-arachidonic acid (New England Nuclear: Sp. act. 72 One half of the tubes received, in addition, 100 mM of [Ci/mmolel). non-labelled arachidonic acid (Sigma Chemical Co., St. Louis, MO). The purity of the arachidonic acid was checked by thin-layer chromaThe incubations were run for I hr at tography prior to use (IO). 37OC and the medium was constantly bubbled with 95% 0,:5% CO,. The incubations were stopped by adding an equal volume of cold A 2 ml aliquot was extracted with acetate buffer (0.6 M, pH 4.5). redistilled) by gentle shaking 4 ml of ethyl acetate (analytical grade, and the samples were then centrifuged in the cold for for 20 min, The ethyl acetate fractions were pooled and 30 min at 1400 x g. They were then temperature. dried under nitrogen at room resuspended in I ml of ethanol to which IO ug of each prostaglandin the following In most experiments, standard had been added. 13,14-dihydro-IS-keto I5-keto-PGF,o, standards were used : PGF2,, TxB,, PGE,, 15-keto PGE,, 13,14-dihydro6- keto PGF The sample was again dried under nitrogen, I%$!&PGE and P&’ in ethyl acetate, and then resuspended2 in 50 ul 2of I% acetic acid activated and pre-soaked onto plastic-baked, spotted quantitatively acetic acid 99:l v/v) silica gel thin-layer plates (in ethyl acetate: The standards were (Eastman-Kodak, No. 13181, Rochester, NY). The plates spotted on the plates separately as reference markers. of ethyl were developed to a distance of 15 cm in the same system They were then re-run in acetate:acetic acid (99: I v/v) and dried. acetate: water: isooctane:acetic acid ethyl organic the phase of This is an adaptation of the methods described (110:100:50:20 v/v>. by Flower -et al. and Sun et al. (IO, II). The average R values that 6-keto- b GF,e, we observed with this system-were as follows: 00-q:; PGE,, . ; l5-keto-PGF2 and 0.37; 0.26; TxB,, PGF,,, and l5- ketoPGE, PGD,, 0.59; 0.54; 13,14-dihydro-15keto-PGF2,, 122
The unchanged arachidonic and 13,14-dihydro-Wketo-PGEs, 0.69. wel I ahead of the PGE metabolites. The zones acid runs corresponding to the authentic markers were visualized by brief with a iodine and then scanned exposure to vapor also Radiochromatographic scanner (Packard model no. 7201) to identify the In this manner, we detected a peak of major peaks of radioactivity. radioactivity corresponding to an unknown compound with an average RF value of 0.17. The peaks corresponding to each compound were cut out for quantitative determination of radioactivity by liquid scintillation counting. The dried extracts were placed in Poly-Q vials (Beckman Instrument Co. , Fullerton, CA) in IO ml scintillation $cktail composed of POPOP, PPO, 1.2 g 400 ml Bio-Solv BBS-3 26 g (Beckman) and 3600 ml of toluene, and counted in a Beckman LS330 ambient temperature scintillation counter with an average efficiency of 3H. The counts per minute found in each relevant zone 40% for were corrected for loss of tritium, and expressed as a percentage of the total number of counts on the plate. Regression analysis was performed to determine whether weight of tissue was correlated with percent conversion of arachidonic acid (12). Since none of these correlations reach the level of significance (P <0.05), the results were not corrected for weight of tissue incubated, but are shown in the tables as percent conversion of labelled arachidonic acid. Statistical comparisons between means were effected by analysis of variance followed by a Student-Newman-Kuels test (13). Tissue
culture
Placentas were obtained from patients in spontaneous labor at term (n = 6) and from patients undergoing elective cesarean section (not in labor) (n = 4). Decidua was obtained by curettage of a portion of the uterus, and was rinsed with normal saline solution at 4Y until the rinse solution was clear. It was then transported at the same temperature under sterile conditions to the laboratory. Pieces of membrane, decidua and villi (approximately 100 mg each) were dissected out, weighed and placed on grids in organ culture dishes just touching the culture medium. The total volume of tissue culture medium was 0.8 ml of McCoy’s medium con ‘ning penicillin % (100 u/ml), streptomycin (100 Pg/ml) and Fungitone (2.5 P&ml). The tissues were incubated in a humidity controlled incubator at 37V, gassed with 9S% air:S% CO,. The incubation medium was collected daily for 4 days and placed in polypropylene tubes (#2059; Falcon, Oxnard, CA) containing a final concentration of IO Pg indomethacin/ml, and then frozen at -20°C until assayed. To some of the culture dishes an excess of arachidonic acid (3.3 PM final concentration) was added (Sigma Chemical Co., St. Louis, MO). At the end of the incubation period, the pieces of tissues were collected, dissolved in I ml 4N NaOH (9O’C for I5 min), and the protein concentration was measured by the BioRad procedure (BioRad Laboratories, Richmond, CA). PGF and PGE were measured by radioimmunoassay by Harper et al. (14,lS). The antisera used (obtained -Inc., Boston, MA) cross-reacted with the one and two so the results have been expressed as immunoreactive
123
as described from Seragen series of PGs, PGE and PGF.
The prostaglandin measured by RIA The produced three-way
F metabolite (13,14-dihydro-l5-keto as described by the method of Cornette
results were expressed as ng/mg by each tissue every 24 hr, and of Analysis of Variance.
PGF, ) was et af? (16). --
protein of PGF and PGE they were analyzed by a
R ESU LTS The short term incubations of placenta did not show any correlation between the weight of tissue and total conversion of labelled arachidonic acid. The placenta converted the labelled arachidonic acid mainly to PGE, metabolites and to PGD, (Table I). Lesser amounts, not differing from each other, of all other PGs were produced. Addition of non-labelled arachidonic acid (100 nM) to the incubation reduced the production of PGE, metabolites significantly, but the total amount of all other prostaglandins formed was unchanged. The main objective of these experiments was to evaluate the different products resulting from conversion of the labelled arachidonic acid. This gives a better perspective of the importance of the various PGs produced. As shown in Table I, there is a production of all major prostaglandins, i.e. the prostacyclin metabolite (6-keto-PGF1 >, PGF,o and its metabolites, and thromboxane B,, PGD, and PeE, and tts metabolites. There was production of an unknown substance that migrated close to 6-keto PGFl . The main product seems to be PGE, and its metabolites, because #e know from previous experiments that, although l5-keto PGF, and PGE, run together in this TLC system, most of the IS-keto %GF,o is rapidly converted to the 13,14-dihydro-IS-keto metabolite. Table Prostaglandin Production by (Mean 2 SE % conversion Wet weight (g), 6-Keto PGF,o
1
Human Term Placenta in Vitro of labelled arachidonic-acm
mean 2 SE
TxB2 pGF2c, IS-Keto PGF, and PGE, 13,14-Dihydr&15-keto PGD, PGF,o IS-Keto and 13,14-dihydro-IS-keto
PGE,
0.93 0.48 0.49 0.50 0.76 0.79 1.20 2.77
2 + 2 f +t f ?
0.60 0.07 0.09 0.09 0.14 0.11 0.16 0.43
Pieces of arachidonic quantitated
placenta were incubated for 1 hour at 37OC with labelled acid and prostaglandins in the medium extracted and by TLC.
The dift’arences difference
showed marked however, tissue culture experiments, between the production of PGF and PGE. There was no of PGE between tissues obtained from in the release 124
patients in labor or not in labor, for villi, decidua or membranes (P >0.05). Therefore, the data were pooled for further analysis. The villi release significantly greater amounts of PGE than the decidua or membranes (Figure I), and this selective production increases throughout the incubation period. Membranes and d%cidus release only moderate amounts of PGE, which did not incrwse with the time of incubation (P <.0002). The addition of arachidonic acid did not alter the amount of prostaglandins released by these latter tissues, which suggests that production not limited by was insufficient substrate. In addition, the concentrations of the major F metabolite, 13,14-dihydro-15-keto PGF, , in selected samples of tissue culture medium were measured. Th&e was no indication of any change with more days of incubation of the tissue. The actual concentrations were low, ranging from 0.2 - 0.8 ng/ml for all samples except for the villi from patients not in labor, where values ranging from 14 - 17 ng/ml were recorded. Although the concentrations of this F metabolite did not change, it is possible that changes could have occurred in the concentrations of other PG metabolites that were not measured in this experiment.
OATASOM
PwLm fvxNTs INIABORANDNOTHIABCH
100,
mo-
Soob i L
-
zoo-
om
Figure
,
___-a------_A-__-------* ----___-___--e l., I 3 Gs
35-* em&LOvrLvo 1.8
4
1. Release of PGE (ng/mg of protein) into the culture of human placental tissue in vitro.
medium during
By contrast with PGE, PGF release was different in tissues derived from patients in labor compared to those not in labor (Figure 2). There were no significant differences between any of the tissues, except for decidua. Contrary to our expectation, we found a significantly higher release of PGF by decidua from patients not in labor than by any other tissue (P <0.004). All the tissues showed an increase in the release of PGF with increase of time of incubation (P <0.0012). 125
DISCUSSION Prostaglandins of their synthesis been suggestions
are involved and its relation of a steroid
in to
parturition labor are
hormonal
(17), but unclear.
control
of
the
control
There labor in
have lower
T Figure
2.
Release culture
of PGF (ng/mg of protein) into the of human placental tissue -in vitro.
medium
during
animals, but in humans at term, the peripheral levels of progesterone studies by and estrogens seem to remain stable (18,19). --In vitro Lackritz et al. (20) showed that addition of progesterone or estradiol to human-p&ental cultures produced a decrease in the synthesis of the appearance of a PGF. Schwartz c al. (8) demonstrated and progesterone-binding in fetal membranes at term, protein Kinoshita e al. (21) described the presence of a heat-unstable, PG production inhibitory substance in the microsomal non-dialyrable, supernanant prepared from placentas. The present studies show that the villi from the placentas of women not in labor produce more PGE, and that the decidua from similar women produces more PGF than the same tissues from women These differences increase with increasing time of in labor. (a) Tissues incubation. There are several probeble expiandions. from women in labor may contain fewer prosteglendin precursors than in the difference those from Howaver, women not in labor. arachidonic acid content of fetal tissues derived from women in I&or to those from women not in labor is minimal (8). compared arachidonic acid did not non-labelled Furthermore, addition of (b) Activity of the increase the amounts of prosE&glmdins measured. major enzymes metabolizing prostuglandins, IShydroxyprostegkndin 126
dehydrogenase and A13-prostaglandin reductase, may be lower in the tissues of women who are not in labor compared to those who are. However, no obvious changes in the amount of the major PGF metabolite were observed in this study, and we have previously found no differences in levels of PGF its metabol i te (13,14-dihydro-IS-keto PGF) in the uterine veiy blood of women in The fact that the levels of one metabolite labor or not in labor (22). did not change does not, of course, exclude the possibility that those of other metabolites might have done so. (c) The present results however, consistent with the presence of an inhibitory factor are, that disappears as the incubation time increases. This finding is reinforced by the fact that the major increase observed was in PGF release by the decidua from patients not in labor (which would be expected to be more inhibited). However, the inhibitory substance seems to be acting differently in the different tissue compartments of the products of conception, because the major release from the villi was PGE. Theoretically, an increase in all the prostaglandins from all the tissues, especially from those from women not in labor, might have been expected. Short-term incubations with labelled arachidonic acid permit an analysis of the types of prostaglandins formed without the constraints imposed by availability of specific radioimmunoassays. However, thin layer is sensitive chromatography much less than the and does not permit such precise quantitation. radioimmunoassay Nonetheless, in the present experiments there was a good qualitative correlation between the two systems, more PGE than PGF being measured. We conclude that the present results indicate the presence of an inhibitory substance acting in vivo that gradually disappears with thuspermitting production of increased time in in vitro culture, The nature of this putative inhibitor is amounts of prostaglandin. not known. ACKNOWLEDGEMENTS This work was supported in part by an NIH Institutional Research Grant, by the Radioimmunoassay Core of the NIH-supported Center for Research in Reproductive Biology (HDl0202) and by NIH grant HDl4046. Thanks are due to G. Cadena and C. J. Norris for technical assistance, and to Dr. K.T. Kirton, Upjohn Co., for the gift of antibody to the PGF metabolite. REFERENCES 1.
Primary F. and Frydman, R. Dray, fluid in pregnancy and spontaneous Gynecol. 126:13, 1961.
2.
Gr&en, K., Bydgeman, M., Toppozada, M. and Wiqvist, N. The role of prostegiendln F,,, in human parturition. Am. J. Obstet. Gynecol. 120:25, 1974.
127
prostaglandins labor, Am.
in J.
amniotic Obstet.
3.
Schwartz, A., Brook, I., Insler, V., Kohen, F., Zor, U. and Lindner, H. R. Effect of flufenamic acid on uterine contractions and plasma levels of 15-keto-13,14-dihydroprostaglandin F20 in preterm labor. Gynecol. Obstet. Invest. 9:139, 1978.
4.
Collins, salicylate
5.
Kinoshita, K., Satoh, K., Yasumizu, T., Sakamoto, S. and Gr6en, K. Bioconversion of arachidonic acid in human amnion during pregnancy and labor, Adv. Prostaglandin Thromboxane Res. 8:1419, 1980.
6.
Schwarz, B.E., Schultz, F.M., MacDonald, P.C. and J.M. Initiation I I I. of human parturition Fetal content of prostaglandin E, and F,o precursor, Obstet. 46~564, 1975.
7.
Schultz, F.M., Schwarz, B. E., MacDonald, P.C. and Johnston, J.M. Initiation of human parturition II. Identification of phospholipase A, in fetal chorioamnion and uterine decidua, Am. J. Obstet. Gynecol. 123:650, 1975.
8.
Schwarz, B. E., Milewich, L., Johnston, J.M., Porter, J.C. and Initiation of human parturition V. MacDonald, P.C. Progesterone binding substance in fetal membranes, Obstet. Gynecol. 48:685, 1976.
9.
Nehemiah, J.L., Schnitzer, J.A., Schulman, H. and Novikoff, cells, and decidual A.B. Human chorionic trophoblasts, macrophages: A histochemical and electron microscopic study, Am. J. Obstet. Gynecol. 140:261, 1981.
10.
Quantitative Flower, R. J., Cheung, H .S. and Cushman, D.W. determination of prostaglandins and malondialdehyde formed by the arachidonate oxygenase (prostaglandin synthetase) system of bovine seminal vesicle, Prostaglandins 4:325, 1973.
11.
Sun, F.F., Chapman, J.P. prostaglandin endoperoxide 14:1055, 1977.
12.
Texas
13.
Sokal, R.R. San Francisco,
14.
Harper, M.J.K., Valenzuela, G. and Hodgson, B. J. Accelerated Changes in ovum transport in rabbits induced by endotoxin. I. effect, endotoxin and reversal of levels prostaglandin Prostaglandins 15:43, 1978.
E. and ingestion
Instruments
Turner, G. in pregnancy,
Statistics
Maternal effects of Lancet ii:335, 1975.
and Rohlf, F.J. 1969, p. 239.
In:
128
Program Biometry.
Johnston, membrane Gynecol.
Metabolism of Prostaglandins
and McGuire, J.C. in animal tissues,
Library
regular
ST108. W. H .
Freeman,
15.
16.
Harper, M.J.K., Norris, C.J., Friedrichs, W.E. and Moreno, A. Poly I :C accelerates ovum transport in the rabbit by a prostaglandin-mediated mechanism, ,J. Reprod. Fertil . 63: 81, -)_961. Cornette, J.C., Harrison, prostaglandin F, Prostaglandins 5:155, l&4.
Of
Measurement K.T. radioimmunoassay,
K. L. and Kirton, metabolites by
17.
and Liggins, G.C. Role of Novy, M.J. prostacyclin, and thromboxanes in the physiologic uterus and in parturition, Semin. Perinatol. 4:45,
18.
Challis, Endocrinology of I ate and J.R.G. pregnancy Review of Physiology. parturition. In : International (R.O. Greep, ed.) University Park Press, Baltimore, 1980, vol. 22, p. 277.
19.
Tulchinsky, D., Hobel, Plasma estrone, estradiol progesterone in human 112:1095, 1972.
20.
Lackritz, production Gynaecol.
21.
Kinoshita, K., Satoh, K. and Sakamoto, prostaglandin in human decidual amnion, Endocrinol . Jpn . 24: 343, 1977.
22.
Valenzuela, G., Harper, M.J.K. and Hayashi, R.H. Uterine venous, peripheral venous and radial arterial levels of prostaglandins E and F in women with pregnancy induced hypertension (P. I .H), Am. J. Obstet. Gynecol., in press.
prostaglandins, control of the 1980.
C.J., Yeager, E. and Marshall, J.R. , estriol, progesterone and 17-hydroxy pregnancy, Am. J. Obstet. Gynecol.
R., Cohen, W.R. and Tulchinsky, by tissue explants from term Obstet. 17:585, 1980.
129
D. Prostaglandin pregnancies, Int.
S.
F J.
Byosynthesis of chorion and villi,
PROSTAGlA.NDJN PROQUCTION BY HUMAN TERM PLACENTAS -IN VITRO M.J.K.
Harper,
G.S.
Khodr
and G. Valenzuela
Department of Obstetrics and Gynecology, The University of Texas Health Science Canter at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284, USA. [Reprints requests to MJKH] ABSTRACT The release of PGs increases with increasing incubation time and the main PG produced is different in different tissuaa. This could PGE release is implicate the presence of an inhibitory factor 5 a. the same in patients in labor and not in labor, and this PG is mainly Villi, membranes and deck&a in tissue produced by the villi. cultures from patients not in labor release more PGF than pa@8nb in labor, the difference for the decidual tissue being strikingly Iscga. Conversion of labelled arachidonic acid by pieces of term placenta from women in labor during short-term incubations was mainly to PGE, and its metabolites and PGD,. INTRODUCTION Prostaglandins (PGs) are increased in plasma and amniotic fluid PG synthesis inhibitors have been used during labor (1,2). successfully in the treatment of premature labor (31, and m&emal ingestion of aspirin prolongs the normal duration. of pregnancy (4). The human placenta and attached fetal membranes contain high concentrations of enzymes capable of catabolizing PGs to much lass The precursor of most PGs, arachidonic acid active metabolites (5). (AA), comprises 20% of the lipids in fetal membranes and dachdua compared to only 0.4% in the mesenteric adipose tissue ($1, and the decidua also contains phospholipase A the enzyme respansWe for cleaving AA from the phosphollpid m&&es where it is sWW (7). One of the major theories on initiation of labor postuta%us thee a decrease in the total intracellular concentratEon of p-e is caused by the appearance of a specific progesterone reoq%ar (8). This produces a relative estrqenic dominance, and a lysis of the in resulting r&aria of lysosomes their qrtzymes, the tissue cot~en$r&ion of AA is ant However, phosphollpase. slightly decreased in women who un&qp spantan@@us W#W to those not in labor who are delivered by cesarean section (6).
121
Furthermore, recently been
some questions raised (9).
about
the
lysis
of
the
lysosomes
have
We performed short term -in vitro incubations and tissue cultures of placentas and membranes, in an attempt to define the role of endogenous AA in PG production, and the effect on this system of deprivation of hormonal support. MATERIALS .LQ Vitro
AND
METHODS
incubations
Placentas (n = 8) obtained from term were rinsed with normal saline became clear and were then frozen work has shown that there is no activity between “fresh” and frozen
women in spontaneous labor at at 4% until the rinse solution at -60°C until used. Previous significant decrease in enzyme placentas over the short term.
Once the placentas were defrosted, pieces of about I g were obtained and rinsed several times with 50 mM Tris HCI buffer containing I mM EDTA (pH 7.7). The tissues were weighed, and the pieces then were placed into polypropylene tubes (Falcon, Oxnard, Ca #2059; I7 x 100 mm) with 2 ml (final volume) of Tris-HCI buffer. To each tube was added approximately 200,000 cpm of [5,6,8,9,11,12,14, l5-sH(N)]-arachidonic acid (New England Nuclear: Sp. act. 72 One half of the tubes received, in addition, 100 mM of [Ci/mmolel). non-labelled arachidonic acid (Sigma Chemical Co., St. Louis, MO). The purity of the arachidonic acid was checked by thin-layer chromaThe incubations were run for I hr at tography prior to use (IO). 37OC and the medium was constantly bubbled with 95% 0,:5% CO,. The incubations were stopped by adding an equal volume of cold A 2 ml aliquot was extracted with acetate buffer (0.6 M, pH 4.5). redistilled) by gentle shaking 4 ml of ethyl acetate (analytical grade, and the samples were then centrifuged in the cold for for 20 min, The ethyl acetate fractions were pooled and 30 min at 1400 x g. They were then temperature. dried under nitrogen at room resuspended in I ml of ethanol to which IO ug of each prostaglandin the following In most experiments, standard had been added. 13,14-dihydro-IS-keto I5-keto-PGF,o, standards were used : PGF2,, TxB,, PGE,, 15-keto PGE,, 13,14-dihydro6- keto PGF The sample was again dried under nitrogen, I%$!&PGE and P&’ in ethyl acetate, and then resuspended2 in 50 ul 2of I% acetic acid activated and pre-soaked onto plastic-baked, spotted quantitatively acetic acid 99:l v/v) silica gel thin-layer plates (in ethyl acetate: The standards were (Eastman-Kodak, No. 13181, Rochester, NY). The plates spotted on the plates separately as reference markers. of ethyl were developed to a distance of 15 cm in the same system They were then re-run in acetate:acetic acid (99: I v/v) and dried. acetate: water: isooctane:acetic acid ethyl organic the phase of This is an adaptation of the methods described (110:100:50:20 v/v>. by Flower -et al. and Sun et al. (IO, II). The average R values that 6-keto- b GF,e, we observed with this system-were as follows: 00-q:; PGE,, . ; l5-keto-PGF2 and 0.37; 0.26; TxB,, PGF,,, and l5- ketoPGE, PGD,, 0.59; 0.54; 13,14-dihydro-15keto-PGF2,, 122
The unchanged arachidonic and 13,14-dihydro-Wketo-PGEs, 0.69. wel I ahead of the PGE metabolites. The zones acid runs corresponding to the authentic markers were visualized by brief with a iodine and then scanned exposure to vapor also Radiochromatographic scanner (Packard model no. 7201) to identify the In this manner, we detected a peak of major peaks of radioactivity. radioactivity corresponding to an unknown compound with an average RF value of 0.17. The peaks corresponding to each compound were cut out for quantitative determination of radioactivity by liquid scintillation counting. The dried extracts were placed in Poly-Q vials (Beckman Instrument Co. , Fullerton, CA) in IO ml scintillation $cktail composed of POPOP, PPO, 1.2 g 400 ml Bio-Solv BBS-3 26 g (Beckman) and 3600 ml of toluene, and counted in a Beckman LS330 ambient temperature scintillation counter with an average efficiency of 3H. The counts per minute found in each relevant zone 40% for were corrected for loss of tritium, and expressed as a percentage of the total number of counts on the plate. Regression analysis was performed to determine whether weight of tissue was correlated with percent conversion of arachidonic acid (12). Since none of these correlations reach the level of significance (P <0.05), the results were not corrected for weight of tissue incubated, but are shown in the tables as percent conversion of labelled arachidonic acid. Statistical comparisons between means were effected by analysis of variance followed by a Student-Newman-Kuels test (13). Tissue
culture
Placentas were obtained from patients in spontaneous labor at term (n = 6) and from patients undergoing elective cesarean section (not in labor) (n = 4). Decidua was obtained by curettage of a portion of the uterus, and was rinsed with normal saline solution at 4Y until the rinse solution was clear. It was then transported at the same temperature under sterile conditions to the laboratory. Pieces of membrane, decidua and villi (approximately 100 mg each) were dissected out, weighed and placed on grids in organ culture dishes just touching the culture medium. The total volume of tissue culture medium was 0.8 ml of McCoy’s medium con ‘ning penicillin % (100 u/ml), streptomycin (100 Pg/ml) and Fungitone (2.5 P&ml). The tissues were incubated in a humidity controlled incubator at 37V, gassed with 9S% air:S% CO,. The incubation medium was collected daily for 4 days and placed in polypropylene tubes (#2059; Falcon, Oxnard, CA) containing a final concentration of IO Pg indomethacin/ml, and then frozen at -20°C until assayed. To some of the culture dishes an excess of arachidonic acid (3.3 PM final concentration) was added (Sigma Chemical Co., St. Louis, MO). At the end of the incubation period, the pieces of tissues were collected, dissolved in I ml 4N NaOH (9O’C for I5 min), and the protein concentration was measured by the BioRad procedure (BioRad Laboratories, Richmond, CA). PGF and PGE were measured by radioimmunoassay by Harper et al. (14,lS). The antisera used (obtained -Inc., Boston, MA) cross-reacted with the one and two so the results have been expressed as immunoreactive
123
as described from Seragen series of PGs, PGE and PGF.
The prostaglandin measured by RIA The produced three-way
F metabolite (13,14-dihydro-l5-keto as described by the method of Cornette
results were expressed as ng/mg by each tissue every 24 hr, and of Analysis of Variance.
PGF, ) was et af? (16). --
protein of PGF and PGE they were analyzed by a
R ESU LTS The short term incubations of placenta did not show any correlation between the weight of tissue and total conversion of labelled arachidonic acid. The placenta converted the labelled arachidonic acid mainly to PGE, metabolites and to PGD, (Table I). Lesser amounts, not differing from each other, of all other PGs were produced. Addition of non-labelled arachidonic acid (100 nM) to the incubation reduced the production of PGE, metabolites significantly, but the total amount of all other prostaglandins formed was unchanged. The main objective of these experiments was to evaluate the different products resulting from conversion of the labelled arachidonic acid. This gives a better perspective of the importance of the various PGs produced. As shown in Table I, there is a production of all major prostaglandins, i.e. the prostacyclin metabolite (6-keto-PGF1 >, PGF,o and its metabolites, and thromboxane B,, PGD, and PeE, and tts metabolites. There was production of an unknown substance that migrated close to 6-keto PGFl . The main product seems to be PGE, and its metabolites, because #e know from previous experiments that, although l5-keto PGF, and PGE, run together in this TLC system, most of the IS-keto %GF,o is rapidly converted to the 13,14-dihydro-IS-keto metabolite. Table Prostaglandin Production by (Mean 2 SE % conversion Wet weight (g), 6-Keto PGF,o
1
Human Term Placenta in Vitro of labelled arachidonic-acm
mean 2 SE
TxB2 pGF2c, IS-Keto PGF, and PGE, 13,14-Dihydr&15-keto PGD, PGF,o IS-Keto and 13,14-dihydro-IS-keto
PGE,
0.93 0.48 0.49 0.50 0.76 0.79 1.20 2.77
2 + 2 f +t f ?
0.60 0.07 0.09 0.09 0.14 0.11 0.16 0.43
Pieces of arachidonic quantitated
placenta were incubated for 1 hour at 37OC with labelled acid and prostaglandins in the medium extracted and by TLC.
The dift’arences difference
showed marked however, tissue culture experiments, between the production of PGF and PGE. There was no of PGE between tissues obtained from in the release 124
patients in labor or not in labor, for villi, decidua or membranes (P >0.05). Therefore, the data were pooled for further analysis. The villi release significantly greater amounts of PGE than the decidua or membranes (Figure I), and this selective production increases throughout the incubation period. Membranes and d%cidus release only moderate amounts of PGE, which did not incrwse with the time of incubation (P <.0002). The addition of arachidonic acid did not alter the amount of prostaglandins released by these latter tissues, which suggests that production not limited by was insufficient substrate. In addition, the concentrations of the major F metabolite, 13,14-dihydro-15-keto PGF, , in selected samples of tissue culture medium were measured. Th&e was no indication of any change with more days of incubation of the tissue. The actual concentrations were low, ranging from 0.2 - 0.8 ng/ml for all samples except for the villi from patients not in labor, where values ranging from 14 - 17 ng/ml were recorded. Although the concentrations of this F metabolite did not change, it is possible that changes could have occurred in the concentrations of other PG metabolites that were not measured in this experiment.
OATASOM
PwLm fvxNTs INIABORANDNOTHIABCH
100,
mo-
Soob i L
-
zoo-
om
Figure
,
___-a------_A-__-------* ----___-___--e l., I 3 Gs
35-* em&LOvrLvo 1.8
4
1. Release of PGE (ng/mg of protein) into the culture of human placental tissue in vitro.
medium during
By contrast with PGE, PGF release was different in tissues derived from patients in labor compared to those not in labor (Figure 2). There were no significant differences between any of the tissues, except for decidua. Contrary to our expectation, we found a significantly higher release of PGF by decidua from patients not in labor than by any other tissue (P <0.004). All the tissues showed an increase in the release of PGF with increase of time of incubation (P <0.0012). 125
DISCUSSION Prostaglandins of their synthesis been suggestions
are involved and its relation of a steroid
in to
parturition labor are
hormonal
(17), but unclear.
control
of
the
control
There labor in
have lower
T Figure
2.
Release culture
of PGF (ng/mg of protein) into the of human placental tissue -in vitro.
medium
during
animals, but in humans at term, the peripheral levels of progesterone studies by and estrogens seem to remain stable (18,19). --In vitro Lackritz et al. (20) showed that addition of progesterone or estradiol to human-p&ental cultures produced a decrease in the synthesis of the appearance of a PGF. Schwartz c al. (8) demonstrated and progesterone-binding in fetal membranes at term, protein Kinoshita e al. (21) described the presence of a heat-unstable, PG production inhibitory substance in the microsomal non-dialyrable, supernanant prepared from placentas. The present studies show that the villi from the placentas of women not in labor produce more PGE, and that the decidua from similar women produces more PGF than the same tissues from women These differences increase with increasing time of in labor. (a) Tissues incubation. There are several probeble expiandions. from women in labor may contain fewer prosteglendin precursors than in the difference those from Howaver, women not in labor. arachidonic acid content of fetal tissues derived from women in I&or to those from women not in labor is minimal (8). compared arachidonic acid did not non-labelled Furthermore, addition of (b) Activity of the increase the amounts of prosE&glmdins measured. major enzymes metabolizing prostuglandins, IShydroxyprostegkndin 126
dehydrogenase and A13-prostaglandin reductase, may be lower in the tissues of women who are not in labor compared to those who are. However, no obvious changes in the amount of the major PGF metabolite were observed in this study, and we have previously found no differences in levels of PGF its metabol i te (13,14-dihydro-IS-keto PGF) in the uterine veiy blood of women in The fact that the levels of one metabolite labor or not in labor (22). did not change does not, of course, exclude the possibility that those of other metabolites might have done so. (c) The present results however, consistent with the presence of an inhibitory factor are, that disappears as the incubation time increases. This finding is reinforced by the fact that the major increase observed was in PGF release by the decidua from patients not in labor (which would be expected to be more inhibited). However, the inhibitory substance seems to be acting differently in the different tissue compartments of the products of conception, because the major release from the villi was PGE. Theoretically, an increase in all the prostaglandins from all the tissues, especially from those from women not in labor, might have been expected. Short-term incubations with labelled arachidonic acid permit an analysis of the types of prostaglandins formed without the constraints imposed by availability of specific radioimmunoassays. However, thin layer is sensitive chromatography much less than the and does not permit such precise quantitation. radioimmunoassay Nonetheless, in the present experiments there was a good qualitative correlation between the two systems, more PGE than PGF being measured. We conclude that the present results indicate the presence of an inhibitory substance acting in vivo that gradually disappears with thuspermitting production of increased time in in vitro culture, The nature of this putative inhibitor is amounts of prostaglandin. not known. ACKNOWLEDGEMENTS This work was supported in part by an NIH Institutional Research Grant, by the Radioimmunoassay Core of the NIH-supported Center for Research in Reproductive Biology (HDl0202) and by NIH grant HDl4046. Thanks are due to G. Cadena and C. J. Norris for technical assistance, and to Dr. K.T. Kirton, Upjohn Co., for the gift of antibody to the PGF metabolite. REFERENCES 1.
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