0021-972x/92/7503-0783.$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 75, No. 3 Printed in U.S A.
Rapid Stimulation of Human Chorionic Gonadotropin Secretion by Interleukinl@ from Perifused First Trimester Trophoblast* GILLIAN L. STEELE?, BASIL HO YUEN, AND
W. DAVID CURRIES, EARNEST PETER C. K. LEUNG$
Department of Obstetrics and Gynecology, British Columbia. Canada V6H 3V5
Grace Hospital,
University
ABSTRACT
H. LEUNG, of
British
Columbia,
Vancouver,
stimulated hCG secretion by approximately 80% (P < 0.05; n = 6). The hCG secretory profiles in response to IL-16 and GnRH were similar. Combined treatment with equimolar (10m9 M) IL-10 and GnRH increased hCG secretion by approximately 150% (P < 0.05; n = 5), stimulating hCG secretion as effectively as either hormone alone (P > 0.05). The stimulatory effect of GnRH on hCG secretion was blocked by the concomitant presence of a GnRH antagonist, Nal-Glu-GnRH (P c 0.05; n = 5). However, simultaneous treatment with IL-l@ and Nal-Glu-GnRH did not affect IL-lb-stimulated hCG secretion (100.5 + 3.6 DS. 162.9 & 10.2%; P < 0.05; n = 7). The data suggest that IL-10 and GnRH stimulated a near-maximal physiological hCG secretory response, possibly through different receptor types. Alternatively, these two hormones may share a common signal transduction pathway, or IL-lp may influence a step distal to the coupling of GnRH to its receptor in the placental trophoblast. (J Clin Endocrinol Metab 75: 783-788, 1992)
Placental trophoblast has been implicated as a major source of interleukin-lb (IL-l@), a cytokine that mediates immunological responses in the body. This study evaluated the effect of IL-l/3 on hCG secretion from 8- to 12-week-old placental trophoblast. Physically dissociated trophoblast cells collected from multiple placentae were cultured on carrier beads and loaded into chambers in a perifusion system. Medium was perifused through the chambers, and effluent was collected and assayed for hCG. Basal hCG secretion was not dependent on exogenous IL-lb or GnRH, but varied between mixed placental preparations and increased with duration of culture. Therefore, hCG secretion was expressed as a percentage of mean basal hCG secretion for any given chamber. IL-10 (10e9 M) stimulated a rapid and transient hCG secretory response. hCG release increased by approximately 150% (P < 0.05; n = 5) in response to the cytokine, but lower concentrations (lo-‘” and 10-l’ M) were ineffective (P > 0.05; n = 3 each). GnRH
T
IL-lfi in this tissue. In a study using trophoblasts derived from human choriocarcinoma and first trimester placental cell lines, IL-l@ stimulated hCG secretion in static culture (8). The failure of indomethacin to block this stimulatory effect indicated that it was not mediated by prostaglandin EZ. Molecular studies have revealed that first trimester decidua expressgreater levels of IL-10 and IL-l@ mRNA (9) at a time when hCG levels are maximal. This is in keeping with a regulatory interaction between the two hormones. In this study, 8- to 12-week-old human placental trophoblast cells were cultured for lo-12 days, then treated with recombinant IL-lp in a perifusion system. The possibility of a regulatory interaction with GnRH was also investigated.
HE PLACENTA is an endocrine organ that synthesizes a diversity of hormones, cytokines, and growth factors (l-3). The presenceof receptors for many of these factors in the same tissue raises the possibility of paracrine regulation of hormone secretion. Human placenta synthesizes steroid, protein, and glycoprotein hormones throughout gestation (4). The production of hCG by placenta in early pregnancy is critical for implantation and maintenance of the blastocyst. However, hCG secretory regulation does not appear to have been extensively examined. Locally synthesized GnRH stimulates hCG secretion (5). Interleukin-lp (IL-l@) has been isolated from mononuclear phagocytic cells in human and mouse placenta (6, 7), raising questions as to the function of Received December 13,. 1991. Address all correspondence and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Grace Hospital, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. * Nal-Glu-GnRH was synthesized at The Salk Institute under Contract NOI-HD-0-2906 with the NIH and made available by the Contraceptive Development Branch, Center for Population Research, NICHHD. This work was supported by the Medical Research Council and the British Columbia Health Care Research Foundation. t Recipient of a studentship award from the British Columbia Children’s Hospital. $ Recipient of a postdoctoral fellowship from the Natural Science and Engineering Research Council of Canada. 5 Career Investigator of the British Columbia Children’s Hospital and recipient of a Medical Research Council Scientist Award.
Materials
and Methods
Tissue preparation The experimental protocol involving the use of human placentae was received and approved by the Clinical Screening Committee for Research, University of British Columbia. First trimester placentae were obtained from therapeutic abortion at 8-12 weeks of pregnancy. Tissue preparation and perifusion were performed under sterile conditions in a biosafety cabinet (Forma, Marietta, OH). Tissues were dissociated in medium 199 (M199) with 1% fetal calf serum (vol/vol), 100 U penicillin G sodium/ml, 100 rg streptomycin/ml, 25 mM NaHC03, and 15 rnM HEPES (supplemented M199, Gibco, Burlington, Ontario, Canada) through a 150-pm screen (Sigma, St. Louis, MO). Aggregated cells were removed by filtration through 48-pm nylon mesh (B&SH Thompson, Scarborough, Ontario, Canada). Dissociated cells were centrifuged (1000
783
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STEELE
784
JCE & M. 1992 Vol75.No3
ET AL.
8 for 6 min) and resuspended in supplemented M199. Hematocytes were removed bv centrifuging (1700 X 2 for 20 min) the suspension on Percoll in Hanks’ Buffered Salt Solution (40%, vol/vol; Gibco). The viabilitv i of the urenarations was 85-95%. Cells from three or four placentae were mixed to minimize variation in basal hCG secretion from one tissue to another. Cells were plated for lo-12 days on carrier beads (1 X 1Oh cells/l9 mg beads; Cytodex-3, Sigma) in humidified air with 5% C02. Six such preparations were used.
the culture period and from one placenta pool to another. For this reason, all hCG results are reported with respect to the observed basal secretion in the same chamber. Data are presented as a percentage of the mean hCG concentration during the respective control perifusion period (1 h pretreatment).
Perifusion
Effect of IL-lb
X
and sampling
Beads with cells were loaded into 1.5.mL perifusion chambers (1.5 X 10h cells/chamber; Accusyst microchambers, Endotronics, Minneapolis, MN) in a 36 C bath 24 h before the trials. Perifusion was performed with supplemented Ml99 delivered by a multichannel peristaltic pump (Isco, Lincoln, NE) via silicone tube (id, 0.8 mm; od, 4 mm; Cole-Parmer, Chicago, IL). Beginning 1 h before trials and during trials, chambers were perifused at 15 mL/h. Effluent was collected in 5-min fractions for immediate hCG assay.
Treatments Control and treatment perifusions were performed over two consecutive l-h periods, Chambers were perifused with Ml99 for 1 h, then oerifused with the given hormone treatment (solubilized in M199) for 5 kin, followed by Ml99 without hormone for the remainder of the second hour. At least one chamber was treated with GnRH during each perifusion to confirm the responsiveness of the tissue. Treatments were as follows: 1) recombinant IL-l/3 (lo-” M; Cistron Biotechnology, Pine Brook, NJ; n = 5); 2) IL-1B (lo-‘” and lo-” M; n = 3 for each); 3)GnRH (lo-’ M: Siema: n = 6): 4) Nal-Glu-GnRH (lo-’ M; The Salk Institute, San Diego,“CA; n = 5);“5) iL-lB and Nal-Glu-GnRH (equimolar, 10e9 M; n = 7); 6) GnRH and Nal-Glu-GnRH (equimolar, 10m9 M; n = 5); and 7) IL-lb and GnRH (equimolar, 10m9 M; n = 5).
hCG assay Fractions were analyzed for hCG concentration using reagents from a diagnostic kit (Maioclone, Serono, Allentown, PA). Mouse monoclonal anti-hCG labeled with fluorescein and ‘*‘I was added (100 pL) to duplicate sample aliquots (50 pL) and incubated for 24 h at 4 C. Sheep antifluorescein covalently bound to magnetic particles was added (75 pL), and the assay was incubated for 24 h at 4 C. Supernatant was decanted, and pellets were washed with 10 rnM Tris buffer (500 pL). Supernatant was decanted, pellets were counted for 1 min, and hCG concentrations were determined by spline function (LKB-Wallac 1271 RIAgamma, Turku, Finland). hCG concentrations are expressed in terms of the First International Reference Preparation/Third International Standard 75/537. Approximately 140 cpm/tube of 10,000 cpm [lZ51]antihCG added/tube were detected in the absence of antigen. The mean hCG concentration, dilution factor, and intra- and interassay coefficients of variation follow for pooled human pregnancy reference serum diluted with M199: 1) 7.75 IlJ/L, 1:s x lo”, 7.1%, and 9.9%; 2) 5.26 IU/L, 1:1.6 X 10h, 7.9%, and9.6%, and 3) 3.43 IU/L, 1:3.2 X 106, 12.7%, and 13.3%. The sensitivity of the hCG assay, expressed as the lowest standard different from zero by t test, was 0.5 IU/L medium (P < 0.05).
Statistical
analysis
Results Basal hCG secretion did not depend l/3 or GnRH stimulation, but increased
upon exogenous with the duration
ILof
on hCG secretion
IL-l/3 ( 10m9 M) increased basal hCG secretion in placental trophoblast (99.5 + 0.4% US. 252.3 + 44.6%; P < 0.05; n = 5). The response peaked within 25 min after IL-10 perifusion was initiated, and hCG secretion returned to basal concentrations 10 min later (Fig. 1). Lower doses of IL-10 were tested in the placental perifusion system, but were ineffective in altering hCG output (lo-” M IL-lb, 99.9 + 0.1% VS. 103.6 + 13.7%, control US. treatment, n = 3; and 10-r’ M IL-P, 99.9 2 0.1 VS. 97.8 + 3.1%, n = 3; Fig. 1). Effect of GnRH perifusion
on hCG secretion
Basal hCG secretion was increased by GnRH (99.9 + 0.1 us. 178.4 f 23.7%; P < 0.05; n = 6). GnRH and IL-l/3 (10m9 M) were equally effective in stimulating hCG secretion (P > 0.05). The time course of the hCG response to GnRH was similar to that after IL-l/3 treatment (Fig. 2). A specific GnRH antagonist (Nal-Glu-GnRH; low9 M) completely blocked the GnRH-induced increase in hCG secretion (Fig. 2; n = 5). Specificity
of the IL-l@ effect on hCG
Nal-Glu-GnRH was used in combination with IL-10 (equimolar, 10e9) to determine whether IL-10 stimulation of hCG secretion was via a GnRH-mediated mechanism. The blocker was not effective in inhibiting the IL-1P-stimulated increase in hCG secretion (Fig. 3, middle panel; 100.5 + 3.6 US. 162.9 + 10.2%; n = 7). Furthermore, there was no difference between the IL-l@stimulated hCG increase and that of IL10 in combination with Nal-Glu-GnRH (P > 0.05). The perifusion of placental trophoblast with Nal-GluGnRH alone had no effect on the mean hCG secretion (99.7 f 0.2% us. 99.3 + 3.1%; P > 0.05; n = 5). Basal levels were maintained throughout the treatment period (Fig. 3, lower panel). Combined
Control and treatment means (mean hCG concentration in the first and second hours of perifusion, respectively) were compared by paired t test, The potencies with which treatments affected hCG were compared by analysis of variance of differences between control and treatment means, Scheffe’s test was used to separate means after a significant F test.
perifusion
treatment
with IL-l/?
and GnRH
Perifusion of placental trophoblast with equimolar concentrations of IL-10 and GnRH (1O-9 M) increased basal hCG (99.8 + 0.1% us. 243.6 + 39.4%; P C 0.05; n = 5). The potency of this effect was not different from that observed for either IL-lb or GnRH alone (P > 0.05). The response of hCG stimulation to combined treatment followed a similar time course (Fig. 4). In two of five chambers tested, hCG secretion did not return to baseline during the period of sampling.
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IL-l&STIMULATED
600
-
500
-
400
-
hCG
785
SECRETION
FIG. 1. IL-l@
stimulated hCG secretion from first trimester placental trophoblast. Cells were perifused for 1 h with Ml99 (control perifusion), then perifused with IL-10 for 4 min (0) at the beginning of the following hour (treatment perifusion). Effluent fractions were collected every 5 min, and hCG was calculated as a percentage of the mean control concentration. The agonist was effective in a concentration of 10m9 M (P
IL-ll3 IL-18 IL-la
v -
300 -
(lo-'M) (lo-'%) (lOs"M)
200 -
100
< 0.05; n = 5).
-
-
o!.
0
I.
I.
I.
I.
2
4
6
8
I.
Ii
F
I.
I.
I.
1;
14
16
l-8
Fraction
I.
I.
2-O
I
2;
I
24
(Smin)
400
FIG. 2. GnRH stimulated hCG secretion from first trimester placental trophoblast. Perifusion of cells with lo-’ M GnRH increased basal hCG levels from those observed during the first hour of perifusion with Ml99 (P < 0.05; n = 6). Fractions were collected every 5 min, and hCG is expressed as a percentage of the mean control concentration. Treatment with 10m9 M Nal-Glu-GnRH blocked the effect of GnRH at the same concentration (P < 0.05; n = 5).
300
-
GnFtH (10e9M) GnRH + Nal-Glu-GnFW
--t
( 10-9M)
200
100
0
I
’
2
I
4
-
I
6
-
I
8
-
I
10
-
0 I
12
Fraction
Discussion Cellular perifusion provides a unique technique for the study of hormonal regulation of endocrine tissue. Cells may be exposed transiently to a given factor, and their responses monitored in regular and frequent intervals thereafter. The latency, duration, and characteristic profile of the response can be determined. Further, such information may allow for speculation as to the mechanism of hormone release. That is, an immediate secretory response would be indicative of the release of stored hormone, while a significant delay in the responsemay suggestde novo synthesis of hormone. For these reasons,we have chosen to use the perifusion system
’
I
14
-
1.
16
1
18
-
1
20
”
‘1’
22
24
(5min)
for the study of placental trophoblast hCG secretion and its regulation by various hormonal factors. The results of this study indicate a role for IL-10 in the regulation of hCG secretion from first trimester placental trophoblast. At the concentration used, this cytokine increasedhCG secretion with a potency comparable to that of GnRH. The rapidity of the responsesuggeststhat stored hCG was releasedfrom secretory vesicles, although the effects of IL-l@ on the synthesis of hCG cannot be ruled out. However, it is unlikely that alterations in hCG biosynthesis could be detected over the time course of these experiments. The use of a specific GnRH antagonist, Nal-Glu-GnRH, blocked GnRH-induced hCG secretion, but had no effect on IL-IO-
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STEELE ET AL.
786
600
-
500
-
400
-
300
-
200
-
-
100
IL-1B
JCEtsM.1992 Vol75.No3
(10s9M)
-
o!.,v-,v-I 0
FIG. 3. GnRH antagonist does not attenuate IL-lo-stimulated hCG secretion. Stimulation of hCG from placental trophoblast by IL-l@ (10e9 M) perifusion is illustrated in the upper panel (P < 0.05; n = 5). This effect was not blocked by Nal-Glu-GnRH (10e9 M), as shown in the middle panel (P < 0.05; n = 7). Perifusion with the antagonist alone did not affect hCG secretion (lower panel; P > 0.05; n = 5).
:
.1’1.I.I.‘.‘.” 0 2
4
-
6 5 t 6
6
IL-16
8
10
12
14
16
18
20
22
24
14
16
18
20
22
24
22
24
+ Nal-Glu-GnFW
(10%)
FJ I” z s
50 0
B
2
4
-
100
1
.,.,.,.I’I.I.1.I.I.‘.I”l 0
6
8
Nal-Glu-GnNN
10
12
( 10e9M)
. * L--+--+.
.
.
2
4
6
8
10
12
Fraction
induced hCG secretion. This indicated that the action of ILlp was not mediated by either GnRH or nonspecific interaction with GnRH receptors. IL-lp receptors do not appear to have been isolated from placental tissue to this date, although they have been demonstrated in human endometrial epithelium (10). Further, there is evidence that IL-lstimulated hCG release in static culture may be dependent on IL-6- and IL-6 receptor-mediated signal transduction (11). Despite the apparent GnRH-independent action of IL-l/3 on hCG, combined treatment of perifused trophoblast with the two hormones did not exhibit an additive effect. This observation suggeststhat IL-l@ and GnRH act through the samesignal transduction pathway. That is, if a given second messengeris generated in an all or none fashion, then an additional agonist stimulating the pathway would not gen-
.
. .
CI
o!.,.,.,‘I.I.l.I.I.l.l.‘.‘l 0
.
14
16
18
20
(Smin)
erate a larger response. The mechanism of action of GnRH in the pituitary and ovary is thought to involve inositol phosphate metabolism and mobilization of intracellular free calcium (12, 13). However, the mechanism of action remains to be identified for either hormone in the placenta. The idea of a shared signal transduction mechanism is supported by the observation of a similar time course and degree of increasedhCG secretion in responseto IL-10 and GnRH administered alone. Alternatively, IL-lfl may mediate the stimulatory action of GnRH on hCG secretion, a possibility not ruled out by the experiment with Nal-Glu-GnRH. A somewhat prolonged hCG increase was observed in two of five perifusion chambersin responseto combined treatment. This probably reflects a variability in tissue preparations. However, hCG levels in the remaining chambersreturned to basal
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IL-l&STIMULATED
hCG SECRETION
600 500 -
IL-1A
(10m9M)
400 300 200 100
o!.,.,.,.,., 0
FIG. 4. Combined treatment of placental trophoblast with IL-10 and GnRH. Perifusion of cells with IL-10 and GnRH (equimolar, 10m9 M) stimulated hCG secretion (lower panel; P C 0.05; n = 5) to the same degree as IL-l@ (upper panel; P < 0.05; n = 5) or GnRH (middle panel; P < 0.05; n = 6) alone (P > 0.05). The time course of the response was similar for either agonist alone, but somewhat prolonged in response to combined treatment.
g o5 ? E 6 E g
400
-
300
-
200
-
100
-
.~, 2
4
u
6
8
.,.,.,
.,.,’
10
12
14
16
18
20
22
24
10
12
14
16
18
20
22
24
GnRH (10w9M)
6 5
o!.,.,.,.,.,.~,.,.,.,.,.,, 0
2
4
6
6
H
400
-
300
-
200
-
-
IL-U3
+ GnRH (10m9M)
100 -
o!.,.,.,.,.,.~,.,.,.,.,.,, 0
2
4
6
8
10
12
Fraction
levels over the sametime period as those treated with IL-ID or GnRH alone. Basal hCG output varied between mixed tissue preparations, probably due to variability in the age of the placental samples and the duration of culture. Rather than isolating the cells by enzymatic dispersion, a physical dissociation protocol was followed. This method has been proven to result in high viability and integrity of the cells. A high percentage of cytotrophoblasts is isolated simply due to size exclusion of syncytiotrophoblast, and these cells differentiate rapidly in culture (14). Once differentiated into syncytiotrophoblasts, the cells are capable of secreting larger amounts of hCG as a result of their proliferated peptide synthetic machinery. This may explain the observed increasing basal
14
16
18
20
22
24
(5min)
hCG output with time in culture. Despite this variation in basal hCG, placental preparations appeared responsive to both IL-10 and GnRH between 4-15 days in culture. The progressive increase in basal hCG secretion, independent of exogenous stimuli, may also be subject to regulation by endogenous factors. For instance, GnRH has been shown to be synthesized locally (15, 16), and changes in the concentration of this and/or other hormonal factors may be important for maintaining basal hCG secretion. In these studies IL-lfi was effective in stimulating hCG at a minimum concentration of 10m9M. Although this is within a physiological range, IL-lo-stimulated hCG secretion has been reported in concentrations as low as 10-l’ M (8). However, the reported study used trophoblast cell lines and JAR
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STEELE
188
cells, which may exhibit enhanced responsiveness to hormonal stimuli. Furthermore, the reported protocol used static culture rather than perifusion, and cells were exposed to the agonist for a 24-h period. In the perifusion protocol used in this study, cells were exposed transiently (5 min) to the agonist in a system that better reflects physiological conditions. The biological properties of IL-l/3 include foreign antigen recognition, stimulation of T-cell cytokine production, and T- and B-cell proliferation (17). IL-l@ may be involved in maternal immunological recognition of the fetal semiallograft (9). In the placenta, monocytes may be activated by a number of mechanisms, one being through estradiol and progesterone stimulation (18). The present study suggests that IL-10 may not only itself be under hormonal control, but it may be important in regulating the secretion of one of the critical hormones of pregnancy, hCG.
References 1. Solomon S. 1988 The placenta as an endocrine organ: steroids. In: Knobil E, Neil1 JD, eds. The physiology of reproduction. New York: Raven Press; pp 2085-91. 2. Talamantes F, Ogren L. 1988 The placenta as an endocrine organ: polypeptides. In: Knobil E, Neil1 JD, eds. The physiology of reproduction New York: Raven Press; UD 2093-144. 3. Cunningham FG, MacDonald PC, Grant NF. 1989 The placenta and fetal membranes. In: Patterson L, Williams J, eds. Williams Obstetrics, 18th ed. Norwalk: Appleton and Lange; pp 39-65. 4. Petraglia F, Volpe A, Genazzani AR, Rivier J, Sawchenko PE, Vale W. 1990 Neuroendocrinology of the human placenta. Front Neuroendocrinol. 11:6-37. 5. Khodr GS, Siler-Kodr TM. 1978 The effect of luteinizing hormonereleasing factor on human chorionic gonadotropin secretion, Fertil Steril. 30:301-4. 6. Flynn A, Finke JH, Hilfiker ML. 1982 Placental mononuclear
ET AL.
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phagocytes as a source of interleukin-1. Science. 218:475-7. 7. Flynn A, Finke JH, Loftus MA. 1985 Comparison of interleukin-1 production by adherent cells and tissue pieces from human placenta. Immunopharmacology. 9:19-26. 8. Yagel S, Lala PK, Powell WA, Casper RF. 1989 Interleukin-1 stimulates human chorionic gonadotropin secretion by first trimester human trophoblast. Clin Endocrinol Metab. 68:992-5. 9. Kauma S, Matt D, Strom S, Eierman D, Turner T. 1990 Interleukin-l beta, human leukocyte antigen HLA-DR alpha, and transforming growth factor-beta expression in endometrium, placenta, and placental membranes, Am J Obstet Gynecol. 163:1430-7. 10. Tabibzadeh S, Kaffka KL, Satyaswaroop PG, Kilian PL. 1990 Interleukin-1 (IL-l) regulation of human endometrial function: presence of IL-1 receptor correlates with IL-l-stimulated prostaglandin Er production. J Clin Endocrinol Metab 70:1000-6. 11. Masuhiro K, Matsuzaki N, Nishino E, et al. 1991 Trophoblastderived interleukin-1 (IL-l) stimulates the release of human chorionic gonadotropin by activating IL-6 and IL-6-receptor system in first trimester human trophoblasts. J Clin Endocrinol Metab. 72:594601. 12. Chang JP, McCoy E, Morgan RO, Catt K. 1987 Mechanisms of GnRH action: interactions between GnRH-stimulated calcium-phospholipid pathways mediating gonadotropin secretion. In: Leung PKC, Armstrong DT, Ruf KB, Moger WH, Friesen HG, eds. Endocrinology and physiology of reproduction. New York: Plenum Press; pp 135-53. 13. Leung PCK, Wang J. 1989 Inositol lipids and LHRH action in the rat ovary. J Reprod Fertil. 37(Suppl):287-93. 14. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF. 1986 Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118:156782. 15. Khodr GS, Siler-Khodr TM. 1978 Localization of luteinizing hormone releasing factor (LRF) in the human placenta. Fertil Steril. 29:523-6. 16. Seeburg PH, Adelman JP. 1984 Characterization of cDNA for precursor of human luteinizing hormone releasing hormone. Nature. 311:666-9. 17. Oppenheim JJ, Kovacs EJ, Matsushima K, Durum SK. 1986 There is more than one interleukin-1. Immunol Today. 7:45-56. 18. Flynn A. 1984 Stimulation of interleukin 1 production from placental monocytes. Lymphokine Res. 3:1-5
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Vol. 75, No. 3 Printed in U.S A.
Rapid Stimulation of Human Chorionic Gonadotropin Secretion by Interleukinl@ from Perifused First Trimester Trophoblast* GILLIAN L. STEELE?, BASIL HO YUEN, AND
W. DAVID CURRIES, EARNEST PETER C. K. LEUNG$
Department of Obstetrics and Gynecology, British Columbia. Canada V6H 3V5
Grace Hospital,
University
ABSTRACT
H. LEUNG, of
British
Columbia,
Vancouver,
stimulated hCG secretion by approximately 80% (P < 0.05; n = 6). The hCG secretory profiles in response to IL-16 and GnRH were similar. Combined treatment with equimolar (10m9 M) IL-10 and GnRH increased hCG secretion by approximately 150% (P < 0.05; n = 5), stimulating hCG secretion as effectively as either hormone alone (P > 0.05). The stimulatory effect of GnRH on hCG secretion was blocked by the concomitant presence of a GnRH antagonist, Nal-Glu-GnRH (P c 0.05; n = 5). However, simultaneous treatment with IL-l@ and Nal-Glu-GnRH did not affect IL-lb-stimulated hCG secretion (100.5 + 3.6 DS. 162.9 & 10.2%; P < 0.05; n = 7). The data suggest that IL-10 and GnRH stimulated a near-maximal physiological hCG secretory response, possibly through different receptor types. Alternatively, these two hormones may share a common signal transduction pathway, or IL-lp may influence a step distal to the coupling of GnRH to its receptor in the placental trophoblast. (J Clin Endocrinol Metab 75: 783-788, 1992)
Placental trophoblast has been implicated as a major source of interleukin-lb (IL-l@), a cytokine that mediates immunological responses in the body. This study evaluated the effect of IL-l/3 on hCG secretion from 8- to 12-week-old placental trophoblast. Physically dissociated trophoblast cells collected from multiple placentae were cultured on carrier beads and loaded into chambers in a perifusion system. Medium was perifused through the chambers, and effluent was collected and assayed for hCG. Basal hCG secretion was not dependent on exogenous IL-lb or GnRH, but varied between mixed placental preparations and increased with duration of culture. Therefore, hCG secretion was expressed as a percentage of mean basal hCG secretion for any given chamber. IL-10 (10e9 M) stimulated a rapid and transient hCG secretory response. hCG release increased by approximately 150% (P < 0.05; n = 5) in response to the cytokine, but lower concentrations (lo-‘” and 10-l’ M) were ineffective (P > 0.05; n = 3 each). GnRH
T
IL-lfi in this tissue. In a study using trophoblasts derived from human choriocarcinoma and first trimester placental cell lines, IL-l@ stimulated hCG secretion in static culture (8). The failure of indomethacin to block this stimulatory effect indicated that it was not mediated by prostaglandin EZ. Molecular studies have revealed that first trimester decidua expressgreater levels of IL-10 and IL-l@ mRNA (9) at a time when hCG levels are maximal. This is in keeping with a regulatory interaction between the two hormones. In this study, 8- to 12-week-old human placental trophoblast cells were cultured for lo-12 days, then treated with recombinant IL-lp in a perifusion system. The possibility of a regulatory interaction with GnRH was also investigated.
HE PLACENTA is an endocrine organ that synthesizes a diversity of hormones, cytokines, and growth factors (l-3). The presenceof receptors for many of these factors in the same tissue raises the possibility of paracrine regulation of hormone secretion. Human placenta synthesizes steroid, protein, and glycoprotein hormones throughout gestation (4). The production of hCG by placenta in early pregnancy is critical for implantation and maintenance of the blastocyst. However, hCG secretory regulation does not appear to have been extensively examined. Locally synthesized GnRH stimulates hCG secretion (5). Interleukin-lp (IL-l@) has been isolated from mononuclear phagocytic cells in human and mouse placenta (6, 7), raising questions as to the function of Received December 13,. 1991. Address all correspondence and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Grace Hospital, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. * Nal-Glu-GnRH was synthesized at The Salk Institute under Contract NOI-HD-0-2906 with the NIH and made available by the Contraceptive Development Branch, Center for Population Research, NICHHD. This work was supported by the Medical Research Council and the British Columbia Health Care Research Foundation. t Recipient of a studentship award from the British Columbia Children’s Hospital. $ Recipient of a postdoctoral fellowship from the Natural Science and Engineering Research Council of Canada. 5 Career Investigator of the British Columbia Children’s Hospital and recipient of a Medical Research Council Scientist Award.
Materials
and Methods
Tissue preparation The experimental protocol involving the use of human placentae was received and approved by the Clinical Screening Committee for Research, University of British Columbia. First trimester placentae were obtained from therapeutic abortion at 8-12 weeks of pregnancy. Tissue preparation and perifusion were performed under sterile conditions in a biosafety cabinet (Forma, Marietta, OH). Tissues were dissociated in medium 199 (M199) with 1% fetal calf serum (vol/vol), 100 U penicillin G sodium/ml, 100 rg streptomycin/ml, 25 mM NaHC03, and 15 rnM HEPES (supplemented M199, Gibco, Burlington, Ontario, Canada) through a 150-pm screen (Sigma, St. Louis, MO). Aggregated cells were removed by filtration through 48-pm nylon mesh (B&SH Thompson, Scarborough, Ontario, Canada). Dissociated cells were centrifuged (1000
783
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STEELE
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JCE & M. 1992 Vol75.No3
ET AL.
8 for 6 min) and resuspended in supplemented M199. Hematocytes were removed bv centrifuging (1700 X 2 for 20 min) the suspension on Percoll in Hanks’ Buffered Salt Solution (40%, vol/vol; Gibco). The viabilitv i of the urenarations was 85-95%. Cells from three or four placentae were mixed to minimize variation in basal hCG secretion from one tissue to another. Cells were plated for lo-12 days on carrier beads (1 X 1Oh cells/l9 mg beads; Cytodex-3, Sigma) in humidified air with 5% C02. Six such preparations were used.
the culture period and from one placenta pool to another. For this reason, all hCG results are reported with respect to the observed basal secretion in the same chamber. Data are presented as a percentage of the mean hCG concentration during the respective control perifusion period (1 h pretreatment).
Perifusion
Effect of IL-lb
X
and sampling
Beads with cells were loaded into 1.5.mL perifusion chambers (1.5 X 10h cells/chamber; Accusyst microchambers, Endotronics, Minneapolis, MN) in a 36 C bath 24 h before the trials. Perifusion was performed with supplemented Ml99 delivered by a multichannel peristaltic pump (Isco, Lincoln, NE) via silicone tube (id, 0.8 mm; od, 4 mm; Cole-Parmer, Chicago, IL). Beginning 1 h before trials and during trials, chambers were perifused at 15 mL/h. Effluent was collected in 5-min fractions for immediate hCG assay.
Treatments Control and treatment perifusions were performed over two consecutive l-h periods, Chambers were perifused with Ml99 for 1 h, then oerifused with the given hormone treatment (solubilized in M199) for 5 kin, followed by Ml99 without hormone for the remainder of the second hour. At least one chamber was treated with GnRH during each perifusion to confirm the responsiveness of the tissue. Treatments were as follows: 1) recombinant IL-l/3 (lo-” M; Cistron Biotechnology, Pine Brook, NJ; n = 5); 2) IL-1B (lo-‘” and lo-” M; n = 3 for each); 3)GnRH (lo-’ M: Siema: n = 6): 4) Nal-Glu-GnRH (lo-’ M; The Salk Institute, San Diego,“CA; n = 5);“5) iL-lB and Nal-Glu-GnRH (equimolar, 10e9 M; n = 7); 6) GnRH and Nal-Glu-GnRH (equimolar, 10m9 M; n = 5); and 7) IL-lb and GnRH (equimolar, 10m9 M; n = 5).
hCG assay Fractions were analyzed for hCG concentration using reagents from a diagnostic kit (Maioclone, Serono, Allentown, PA). Mouse monoclonal anti-hCG labeled with fluorescein and ‘*‘I was added (100 pL) to duplicate sample aliquots (50 pL) and incubated for 24 h at 4 C. Sheep antifluorescein covalently bound to magnetic particles was added (75 pL), and the assay was incubated for 24 h at 4 C. Supernatant was decanted, and pellets were washed with 10 rnM Tris buffer (500 pL). Supernatant was decanted, pellets were counted for 1 min, and hCG concentrations were determined by spline function (LKB-Wallac 1271 RIAgamma, Turku, Finland). hCG concentrations are expressed in terms of the First International Reference Preparation/Third International Standard 75/537. Approximately 140 cpm/tube of 10,000 cpm [lZ51]antihCG added/tube were detected in the absence of antigen. The mean hCG concentration, dilution factor, and intra- and interassay coefficients of variation follow for pooled human pregnancy reference serum diluted with M199: 1) 7.75 IlJ/L, 1:s x lo”, 7.1%, and 9.9%; 2) 5.26 IU/L, 1:1.6 X 10h, 7.9%, and9.6%, and 3) 3.43 IU/L, 1:3.2 X 106, 12.7%, and 13.3%. The sensitivity of the hCG assay, expressed as the lowest standard different from zero by t test, was 0.5 IU/L medium (P < 0.05).
Statistical
analysis
Results Basal hCG secretion did not depend l/3 or GnRH stimulation, but increased
upon exogenous with the duration
ILof
on hCG secretion
IL-l/3 ( 10m9 M) increased basal hCG secretion in placental trophoblast (99.5 + 0.4% US. 252.3 + 44.6%; P < 0.05; n = 5). The response peaked within 25 min after IL-10 perifusion was initiated, and hCG secretion returned to basal concentrations 10 min later (Fig. 1). Lower doses of IL-10 were tested in the placental perifusion system, but were ineffective in altering hCG output (lo-” M IL-lb, 99.9 + 0.1% VS. 103.6 + 13.7%, control US. treatment, n = 3; and 10-r’ M IL-P, 99.9 2 0.1 VS. 97.8 + 3.1%, n = 3; Fig. 1). Effect of GnRH perifusion
on hCG secretion
Basal hCG secretion was increased by GnRH (99.9 + 0.1 us. 178.4 f 23.7%; P < 0.05; n = 6). GnRH and IL-l/3 (10m9 M) were equally effective in stimulating hCG secretion (P > 0.05). The time course of the hCG response to GnRH was similar to that after IL-l/3 treatment (Fig. 2). A specific GnRH antagonist (Nal-Glu-GnRH; low9 M) completely blocked the GnRH-induced increase in hCG secretion (Fig. 2; n = 5). Specificity
of the IL-l@ effect on hCG
Nal-Glu-GnRH was used in combination with IL-10 (equimolar, 10e9) to determine whether IL-10 stimulation of hCG secretion was via a GnRH-mediated mechanism. The blocker was not effective in inhibiting the IL-1P-stimulated increase in hCG secretion (Fig. 3, middle panel; 100.5 + 3.6 US. 162.9 + 10.2%; n = 7). Furthermore, there was no difference between the IL-l@stimulated hCG increase and that of IL10 in combination with Nal-Glu-GnRH (P > 0.05). The perifusion of placental trophoblast with Nal-GluGnRH alone had no effect on the mean hCG secretion (99.7 f 0.2% us. 99.3 + 3.1%; P > 0.05; n = 5). Basal levels were maintained throughout the treatment period (Fig. 3, lower panel). Combined
Control and treatment means (mean hCG concentration in the first and second hours of perifusion, respectively) were compared by paired t test, The potencies with which treatments affected hCG were compared by analysis of variance of differences between control and treatment means, Scheffe’s test was used to separate means after a significant F test.
perifusion
treatment
with IL-l/?
and GnRH
Perifusion of placental trophoblast with equimolar concentrations of IL-10 and GnRH (1O-9 M) increased basal hCG (99.8 + 0.1% us. 243.6 + 39.4%; P C 0.05; n = 5). The potency of this effect was not different from that observed for either IL-lb or GnRH alone (P > 0.05). The response of hCG stimulation to combined treatment followed a similar time course (Fig. 4). In two of five chambers tested, hCG secretion did not return to baseline during the period of sampling.
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IL-l&STIMULATED
600
-
500
-
400
-
hCG
785
SECRETION
FIG. 1. IL-l@
stimulated hCG secretion from first trimester placental trophoblast. Cells were perifused for 1 h with Ml99 (control perifusion), then perifused with IL-10 for 4 min (0) at the beginning of the following hour (treatment perifusion). Effluent fractions were collected every 5 min, and hCG was calculated as a percentage of the mean control concentration. The agonist was effective in a concentration of 10m9 M (P
IL-ll3 IL-18 IL-la
v -
300 -
(lo-'M) (lo-'%) (lOs"M)
200 -
100
< 0.05; n = 5).
-
-
o!.
0
I.
I.
I.
I.
2
4
6
8
I.
Ii
F
I.
I.
I.
1;
14
16
l-8
Fraction
I.
I.
2-O
I
2;
I
24
(Smin)
400
FIG. 2. GnRH stimulated hCG secretion from first trimester placental trophoblast. Perifusion of cells with lo-’ M GnRH increased basal hCG levels from those observed during the first hour of perifusion with Ml99 (P < 0.05; n = 6). Fractions were collected every 5 min, and hCG is expressed as a percentage of the mean control concentration. Treatment with 10m9 M Nal-Glu-GnRH blocked the effect of GnRH at the same concentration (P < 0.05; n = 5).
300
-
GnFtH (10e9M) GnRH + Nal-Glu-GnFW
--t
( 10-9M)
200
100
0
I
’
2
I
4
-
I
6
-
I
8
-
I
10
-
0 I
12
Fraction
Discussion Cellular perifusion provides a unique technique for the study of hormonal regulation of endocrine tissue. Cells may be exposed transiently to a given factor, and their responses monitored in regular and frequent intervals thereafter. The latency, duration, and characteristic profile of the response can be determined. Further, such information may allow for speculation as to the mechanism of hormone release. That is, an immediate secretory response would be indicative of the release of stored hormone, while a significant delay in the responsemay suggestde novo synthesis of hormone. For these reasons,we have chosen to use the perifusion system
’
I
14
-
1.
16
1
18
-
1
20
”
‘1’
22
24
(5min)
for the study of placental trophoblast hCG secretion and its regulation by various hormonal factors. The results of this study indicate a role for IL-10 in the regulation of hCG secretion from first trimester placental trophoblast. At the concentration used, this cytokine increasedhCG secretion with a potency comparable to that of GnRH. The rapidity of the responsesuggeststhat stored hCG was releasedfrom secretory vesicles, although the effects of IL-l@ on the synthesis of hCG cannot be ruled out. However, it is unlikely that alterations in hCG biosynthesis could be detected over the time course of these experiments. The use of a specific GnRH antagonist, Nal-Glu-GnRH, blocked GnRH-induced hCG secretion, but had no effect on IL-IO-
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STEELE ET AL.
786
600
-
500
-
400
-
300
-
200
-
-
100
IL-1B
JCEtsM.1992 Vol75.No3
(10s9M)
-
o!.,v-,v-I 0
FIG. 3. GnRH antagonist does not attenuate IL-lo-stimulated hCG secretion. Stimulation of hCG from placental trophoblast by IL-l@ (10e9 M) perifusion is illustrated in the upper panel (P < 0.05; n = 5). This effect was not blocked by Nal-Glu-GnRH (10e9 M), as shown in the middle panel (P < 0.05; n = 7). Perifusion with the antagonist alone did not affect hCG secretion (lower panel; P > 0.05; n = 5).
:
.1’1.I.I.‘.‘.” 0 2
4
-
6 5 t 6
6
IL-16
8
10
12
14
16
18
20
22
24
14
16
18
20
22
24
22
24
+ Nal-Glu-GnFW
(10%)
FJ I” z s
50 0
B
2
4
-
100
1
.,.,.,.I’I.I.1.I.I.‘.I”l 0
6
8
Nal-Glu-GnNN
10
12
( 10e9M)
. * L--+--+.
.
.
2
4
6
8
10
12
Fraction
induced hCG secretion. This indicated that the action of ILlp was not mediated by either GnRH or nonspecific interaction with GnRH receptors. IL-lp receptors do not appear to have been isolated from placental tissue to this date, although they have been demonstrated in human endometrial epithelium (10). Further, there is evidence that IL-lstimulated hCG release in static culture may be dependent on IL-6- and IL-6 receptor-mediated signal transduction (11). Despite the apparent GnRH-independent action of IL-l/3 on hCG, combined treatment of perifused trophoblast with the two hormones did not exhibit an additive effect. This observation suggeststhat IL-l@ and GnRH act through the samesignal transduction pathway. That is, if a given second messengeris generated in an all or none fashion, then an additional agonist stimulating the pathway would not gen-
.
. .
CI
o!.,.,.,‘I.I.l.I.I.l.l.‘.‘l 0
.
14
16
18
20
(Smin)
erate a larger response. The mechanism of action of GnRH in the pituitary and ovary is thought to involve inositol phosphate metabolism and mobilization of intracellular free calcium (12, 13). However, the mechanism of action remains to be identified for either hormone in the placenta. The idea of a shared signal transduction mechanism is supported by the observation of a similar time course and degree of increasedhCG secretion in responseto IL-10 and GnRH administered alone. Alternatively, IL-lfl may mediate the stimulatory action of GnRH on hCG secretion, a possibility not ruled out by the experiment with Nal-Glu-GnRH. A somewhat prolonged hCG increase was observed in two of five perifusion chambersin responseto combined treatment. This probably reflects a variability in tissue preparations. However, hCG levels in the remaining chambersreturned to basal
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IL-l&STIMULATED
hCG SECRETION
600 500 -
IL-1A
(10m9M)
400 300 200 100
o!.,.,.,.,., 0
FIG. 4. Combined treatment of placental trophoblast with IL-10 and GnRH. Perifusion of cells with IL-10 and GnRH (equimolar, 10m9 M) stimulated hCG secretion (lower panel; P C 0.05; n = 5) to the same degree as IL-l@ (upper panel; P < 0.05; n = 5) or GnRH (middle panel; P < 0.05; n = 6) alone (P > 0.05). The time course of the response was similar for either agonist alone, but somewhat prolonged in response to combined treatment.
g o5 ? E 6 E g
400
-
300
-
200
-
100
-
.~, 2
4
u
6
8
.,.,.,
.,.,’
10
12
14
16
18
20
22
24
10
12
14
16
18
20
22
24
GnRH (10w9M)
6 5
o!.,.,.,.,.,.~,.,.,.,.,.,, 0
2
4
6
6
H
400
-
300
-
200
-
-
IL-U3
+ GnRH (10m9M)
100 -
o!.,.,.,.,.,.~,.,.,.,.,.,, 0
2
4
6
8
10
12
Fraction
levels over the sametime period as those treated with IL-ID or GnRH alone. Basal hCG output varied between mixed tissue preparations, probably due to variability in the age of the placental samples and the duration of culture. Rather than isolating the cells by enzymatic dispersion, a physical dissociation protocol was followed. This method has been proven to result in high viability and integrity of the cells. A high percentage of cytotrophoblasts is isolated simply due to size exclusion of syncytiotrophoblast, and these cells differentiate rapidly in culture (14). Once differentiated into syncytiotrophoblasts, the cells are capable of secreting larger amounts of hCG as a result of their proliferated peptide synthetic machinery. This may explain the observed increasing basal
14
16
18
20
22
24
(5min)
hCG output with time in culture. Despite this variation in basal hCG, placental preparations appeared responsive to both IL-10 and GnRH between 4-15 days in culture. The progressive increase in basal hCG secretion, independent of exogenous stimuli, may also be subject to regulation by endogenous factors. For instance, GnRH has been shown to be synthesized locally (15, 16), and changes in the concentration of this and/or other hormonal factors may be important for maintaining basal hCG secretion. In these studies IL-lfi was effective in stimulating hCG at a minimum concentration of 10m9M. Although this is within a physiological range, IL-lo-stimulated hCG secretion has been reported in concentrations as low as 10-l’ M (8). However, the reported study used trophoblast cell lines and JAR
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188
cells, which may exhibit enhanced responsiveness to hormonal stimuli. Furthermore, the reported protocol used static culture rather than perifusion, and cells were exposed to the agonist for a 24-h period. In the perifusion protocol used in this study, cells were exposed transiently (5 min) to the agonist in a system that better reflects physiological conditions. The biological properties of IL-l/3 include foreign antigen recognition, stimulation of T-cell cytokine production, and T- and B-cell proliferation (17). IL-l@ may be involved in maternal immunological recognition of the fetal semiallograft (9). In the placenta, monocytes may be activated by a number of mechanisms, one being through estradiol and progesterone stimulation (18). The present study suggests that IL-10 may not only itself be under hormonal control, but it may be important in regulating the secretion of one of the critical hormones of pregnancy, hCG.
References 1. Solomon S. 1988 The placenta as an endocrine organ: steroids. In: Knobil E, Neil1 JD, eds. The physiology of reproduction. New York: Raven Press; pp 2085-91. 2. Talamantes F, Ogren L. 1988 The placenta as an endocrine organ: polypeptides. In: Knobil E, Neil1 JD, eds. The physiology of reproduction New York: Raven Press; UD 2093-144. 3. Cunningham FG, MacDonald PC, Grant NF. 1989 The placenta and fetal membranes. In: Patterson L, Williams J, eds. Williams Obstetrics, 18th ed. Norwalk: Appleton and Lange; pp 39-65. 4. Petraglia F, Volpe A, Genazzani AR, Rivier J, Sawchenko PE, Vale W. 1990 Neuroendocrinology of the human placenta. Front Neuroendocrinol. 11:6-37. 5. Khodr GS, Siler-Kodr TM. 1978 The effect of luteinizing hormonereleasing factor on human chorionic gonadotropin secretion, Fertil Steril. 30:301-4. 6. Flynn A, Finke JH, Hilfiker ML. 1982 Placental mononuclear
ET AL.
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phagocytes as a source of interleukin-1. Science. 218:475-7. 7. Flynn A, Finke JH, Loftus MA. 1985 Comparison of interleukin-1 production by adherent cells and tissue pieces from human placenta. Immunopharmacology. 9:19-26. 8. Yagel S, Lala PK, Powell WA, Casper RF. 1989 Interleukin-1 stimulates human chorionic gonadotropin secretion by first trimester human trophoblast. Clin Endocrinol Metab. 68:992-5. 9. Kauma S, Matt D, Strom S, Eierman D, Turner T. 1990 Interleukin-l beta, human leukocyte antigen HLA-DR alpha, and transforming growth factor-beta expression in endometrium, placenta, and placental membranes, Am J Obstet Gynecol. 163:1430-7. 10. Tabibzadeh S, Kaffka KL, Satyaswaroop PG, Kilian PL. 1990 Interleukin-1 (IL-l) regulation of human endometrial function: presence of IL-1 receptor correlates with IL-l-stimulated prostaglandin Er production. J Clin Endocrinol Metab 70:1000-6. 11. Masuhiro K, Matsuzaki N, Nishino E, et al. 1991 Trophoblastderived interleukin-1 (IL-l) stimulates the release of human chorionic gonadotropin by activating IL-6 and IL-6-receptor system in first trimester human trophoblasts. J Clin Endocrinol Metab. 72:594601. 12. Chang JP, McCoy E, Morgan RO, Catt K. 1987 Mechanisms of GnRH action: interactions between GnRH-stimulated calcium-phospholipid pathways mediating gonadotropin secretion. In: Leung PKC, Armstrong DT, Ruf KB, Moger WH, Friesen HG, eds. Endocrinology and physiology of reproduction. New York: Plenum Press; pp 135-53. 13. Leung PCK, Wang J. 1989 Inositol lipids and LHRH action in the rat ovary. J Reprod Fertil. 37(Suppl):287-93. 14. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF. 1986 Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118:156782. 15. Khodr GS, Siler-Khodr TM. 1978 Localization of luteinizing hormone releasing factor (LRF) in the human placenta. Fertil Steril. 29:523-6. 16. Seeburg PH, Adelman JP. 1984 Characterization of cDNA for precursor of human luteinizing hormone releasing hormone. Nature. 311:666-9. 17. Oppenheim JJ, Kovacs EJ, Matsushima K, Durum SK. 1986 There is more than one interleukin-1. Immunol Today. 7:45-56. 18. Flynn A. 1984 Stimulation of interleukin 1 production from placental monocytes. Lymphokine Res. 3:1-5
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