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Original Studies |
Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229
Address correspondence and requests for reprints to: Theresa M. Siler-Khodr, Ph.D., Professor, Department Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, Room 416E, San Antonio, Texas 78229. E-mail: Silerkhodr{at}UTHSCSA.edu
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Abstract |
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These studies are the first to demonstrate bioactivity of a second form of GnRH in a human tissue and to identify the pulsatile release of chicken II GnRH from a human tissue. These data led us to propose that chicken II GnRH and its synthetic analogs may be potent ligands for hormone regulation during pregnancy.
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Introduction |
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In the case of the human placenta and paracrine regulation of pregnancy, multiple paracrine/autocrine axes regulating the maintenance of pregnancy and the initiation of labor and parturition have now been defined (6). However, complex and sometimes contradictory findings have been reported. Very high concentrations of mammalian GnRH are needed to bind to the receptor or to effect a hCG response (7, 8). Although the limited activity of mammalian GnRH can be partially explained by the presence of a highly active postproline peptidase in the human placenta (9, 10), even mammalian GnRH analogs having increased resistance to this enzymatic degradation are still relatively inactive at the placental receptor (11). In addition, we and other investigators have observed that placental GnRH exhibits differing reactivity with certain antisera to mammalian GnRH (12, 13), indicating nonidentity with mammalian GnRH. Also, mammalian GnRH seems to act as a partial agonist on placental hormonal releases (14, 15, 16). These findings have led us to hypothesize that placenta produces a GnRH that is not identical to mammalian GnRH.
The existence of multiple forms of GnRH in nonmammalian vertebrates has been recognized for many years, and they are thought to have various evolutionary functions in different cells (17, 18, 19). Not until chicken II GnRH in the brain of the tree and musk shrew and mole (20, 21, 22, 23) was described was it realized that multiple forms of GnRH existed in mammalian species. Recently, the expression of the messenger RNA (mRNA) for chicken II GnRH in human (24, 25) and primate brain (26) has been reported. In addition, the chicken II GnRH receptor has been described in human tissues (27). However, it was speculated that the function of the chicken II GnRH receptor should be vestigial (27) because GnRH isoforms, other than the mammalian isoform, have limited to no ability to stimulate gonadotropin release from the mammalian pituitary (28).
We proposed that a different isoform of GnRH is expressed in the human placenta and this isoform of GnRH has enhanced activity for the human placental GnRH receptor. In these studies, we have investigated the possibility that chicken II GnRH is a more active ligand for the placental receptor than mammalian GnRH. The studies described herein provide the first direct evidence for the production and action of a potent second isoform of GnRH in the human placenta and a specific GnRH receptor for this isoform. The receptor binding and bioactivity of enzymatically resistant analogs of GnRH, both mammalian and chicken II isoforms, were compared using human term placental tissues.
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Materials and Methods |
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Mammalian and chicken II GnRH were purchased from Sigma (St. Louis, MO) and Peninsula Laboratories, Inc. (Belmont, CA). Analogs of chicken II GnRH were synthesized by Peninsula Laboratories, Inc.
Human tissues
Human placental tissues were obtained immediately following vaginal delivery or suction curettage. The tissues were obtained from "apparently" normal pregnancies, however, no history or identifiers were obtained from the patients for confidentiality reasons. The study was approved by the Institutional Review Board for investigation of human subjects as an exempt protocol with the use of patient identifiers and was performed in accordance with the Helsinki Declaration of 1975, as revised in 1983.
Enzyme assay
Competition for the enzymatic degradation of mammalian GnRH by a postproline peptidase was studied by determining the remaining GnRH after incubation of varying concentrations of mammalian GnRH with a highly active postproline peptidase, C-ase-1, isolated from human term placentas (9), in the presence or absence of varying concentrations of other GnRH isoforms or analogs. The remaining GnRH was measured using a RIA specific for mammalian GnRH (9) having less than 0.1% cross-reactivity for any of the analogs or isoforms tested. The concentration of the product of the degraded GnRH was quantified by subtracting the remaining mammalian GnRH from the starting concentration of mammalian GnRH. Analogs and isoforms of GnRH studied were Buserelin, chicken II GnRH, and its D-Arg (6), Des-Gly (10) GnRH-ethylamide, and its D-Arg (6), aza-Gly (10)-amide analogs. The Ks for the degradation of mammalian GnRH was calculated from the x-axis intercept a using Lineweaver-Burke double reciprocal plot of the concentration of the product formed vs. the concentration of the substrate used. The inhibitor constant (Ki) was also calculated from the point of converging lines formed from the plot of the concentration of the product formed using a given concentration of mammalian GnRH in the presence of different concentrations of competing analogs or isoform.
GnRH receptor-binding assay
Placental GnRH receptors were purified from human term placentas after homogenization in 40 mM Tris buffer (pH 7.4) and filtered through cheesecloth, followed by an initial centrifugation at 1,000 x g for 10 min. The resulting supernatant was, again, centrifuged at 35,000 x g for 30 min, and the membrane pellet was collected and resuspended in Tris buffer with 0.3 M sucrose. The protein concentration was determined by the Bio-Rad Laboratories, Inc. Protein Assay (Hercules, CA). Membranes were stored frozen (-20 C) until used. Before use, placental membranes were diluted to 5,000 µg/mL with Tris buffer containing 0.5% BSA and 50 U/mL bacitracin. Placental membranes (100 µL) were mixed with varying concentrations of mammalian GnRH, Buserelin, chicken II GnRH, D-Arg (6)-chicken II GnRH-des-Gly (10)-ethylamide, or D-Arg (6)-chicken II GnRH-aza-Gly (10)-NH2 (100 µL), and either radio-labeled Buserelin or radiolabeled D-Arg (6)-chicken II GnRH-aza-Gly (10)-amide (100 µL/tube, iodinated by the method of Hunter and Greenwood (29) to 100,000 cpm/100 µL). Following incubation at room temperature for 4 h, the bound and the free hormones were separated using polyethylene glycol precipitation, followed by centrifugation. The binding affinity for each GnRH isoform or analog was calculated using the double reciprocal plot of bound vs. the free ligand. Each study was done using three different human term placental tissues.
Biopotency studies
An explant culture system (8) was used to determine
the effect of mammalian GnRH, chicken II GnRH, or the D-Arg
(6)-chicken II GnRH-aza-Gly (10)-NH2 analog on
the release of hCG, progesterone, and prostaglandin
E2 (PGE2). Human
term placentas were dissected free of membranes, minced into fragments
of 5 mm3, rinsed in medium 199, and a total
weight of 
100 mg (20 explants) was placed on a sterile filter paper
resting on an organ culture grid such that they touched the surface of
the culture medium but were not immersed in it. Medium 199 (2 mL)
containing penicillin, streptomycin, and fungizone (100 U/mL, 100
µg/mL, and 2.5 µg/mL, respectively) with and without varying doses
of GnRH isoforms or analogs was added to each Petri dish. Triplicate
chambers for each media were made and incubated at 37 C in a humidified
chamber with an atmosphere of 5% CO2 and 95%
air. Spent media were collected and replaced after 2 h, 24 h
and 48 h of culture and stored frozen at -20 C until assayed for
hormones. hCG, progesterone, and PGE2 were
measured using specific double antibody procedures as described
previously (8, 30). The chicken II GnRH analog was studied
using four different human term placentas, and the native chicken II
GnRH isoform was also studied using one human term placenta.
Perifusion of early human placentas
Fresh early placental tissues (7–8 weeks LMP) were
dissected free of decidua, membranes, and visible large vessels, and
20–50 mg tissue was placed in the 3-mL chamber surrounded by glass
wool and placed in a water bath at 37 C. Replicate chambers were
perifused with medium 199 containing 0.05% BSA, 100 U/mL penicillin,
100 µg/mL streptomycin, 10 ng/mL estradiol, and 200 ng/mL
progesterone. Steroid concentrations were chosen to emulate the early
intrauterine milieu. The influx medium flow rate was 2 mL/h and was
collected at half-hourly intervals for 4.5 h into glass test tubes
containing bacitracin (final concentration, 20 U/mL). Samples were
stored frozen at -20 C until assayed. Four early human term placenta
tissues were studied using this system.
RIA for chicken II GnRH
Using a polyclonal antibody generated in rabbits to chicken II
GnRH at a final dilution of 1:30,000, a specific and sensitivity RIA
for chicken II GnRH was developed. Standard chicken II GnRH was
purchased from Peninsula Laboratories, Inc. Chicken II
GnRH was radio-iodinated by the method of Hunter and Greenwood
(29), and 100 fmole were added to each tube. The bound
hormone was precipitated using magnetic beads coated with antirabbit
globulin (PolySciences, Inc., Warrington, PA). The assay
sensitivity was 1 fmol/tube, and the intra- and interassay coefficients
of variations were 4.5% and 5.9%, respectively. Crossreactivity
with mammalian GnRH was less than 0.5%.
Statistical analyses
The comparison of receptor binding for different analogs or isoforms was done using Student’s t test. Significant difference in response to the various isoforms or analogs of GnRH was determined using two-way ANOVA. The Student’s-Newman-Keul’s test was used to determine the points of significant difference compared with untreated tissues. Linear-line regression analysis was used to determine dose-response relationships. A P value less than 0.05 was considered significantly different.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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30 nM. Using the reciprocal
plot of the product vs. the concentration of the GnRH
isoform or analog to determine the Ki, it was
determined that Buserelin was degraded by C-ase-1, although at one
fourth the rate of its native mammalian GnRH isoform (i.e.
it exhibited a Ki of 110
nM). Chicken II GnRH competed for the degradation
of mammalian GnRH with a Ki of 200
nM (i.e. only one sixth that of
mammalian GnRH; Fig. 1A
). The
D-Arg-chicken II GnRH-ethyamide had a
Ki of more than 200 nM, and
D-Arg, aza-Gly (10)-NH2
analog of chicken II GnRH was essentially undegraded
(Ki, >400 nM; Fig. 1B)
(i.e. >6- and >150-fold, respectively, that of mammalian
GnRH).
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compares the average
Kd observed for the three different placental
membrane preparations for mammalian GnRH, Buserelin,
D-Arg (6)-chicken II GnRH-Des-Gly
(10)-ethylamide, or D-Arg (6)-chicken II
GnRH-aza-Gly (10)-NH2 using the
D-Arg (6)-chicken II
GnRH-aza-Gly-NH2 radiolabeled analog. The most
potent affinity constant was observed for the
D-Arg (6)-chicken II GnRH-aza-Gly
(10)-NH2 analog, having a
Ks of 68 nM when using the
placenta 2 membrane preparation (Fig. 2).
The average binding affinity observed for this analog was 93 ± 23
nM [i.e. 25-fold that observed for
mammalian GnRH (2421 ± 596 nM)].
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). However, the response to
D-Arg-chicken II
GnRH-aza-Gly-NH2 was biphasic [i.e.
an inhibition of hCG was observed using the higher concentrations (1–9
µM) of this chicken II GnRH analog]. After 24
and 48 h of incubation with this analog, a similar pattern of
response was observed, even though the basal hCG release had fallen 10-
to 20-fold during the 2 days in vitro. A significant
dose-related inhibition of hCG release (P < 0.05) was
observed after 2, 24, and 48 h of treatment with the
D-Arg-chicken II
GnRH-aza-Gly-NH2 (Fig. 4 and Table 2). The progesterone release was also
inhibited when incubated with the higher concentrations of this analog,
but not as markedly as hCG (Table 3).
PGE2 was not significantly changed by exposure to
this analog (Table 4, A and
B).
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The pulsatile release of chicken II GnRH from early human placental
tissues was demonstrated using a perifusion system. As shown in Fig. 5, pulses were observed approximately
every 90 min. Chicken II GnRH production over the 4 h of
perifusion was relatively stable, ranging from 15–60 fmol/h from the
20–50 mg placenta tissue. Similar data were found in four different
placental perifusion studies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When mammalian GnRH was first defined nearly 3 decades ago (1), it was believed that releasing and inhibiting activities were synthesized only in the hypothalamus, and it was thought that only one form of GnRH was expressed in mammals. Our demonstration of a GnRH-like activity in the human placenta (2, 3, 4), and the presence of a paracrine axis in the human placenta (5), was the first definition of the first extra-hypothalamic-pituitary paracrine axis, negating the teaching that such activity was limited to the hypothalamus. Since that time, multiple paracrine axes have been defined in other species as well as various human tissues, leading to an expanded understanding of local regulation of tissue functions. In the case of the placental GnRH-hCG-steroid axis, a gene for mammalian GnRH, which is identical to that in the hypothalamus (31) has been defined. Steroid regulatory sites in the promoter region of this gene have also been identified (32), and effects of steroids on the expression of GnRH mRNA have been demonstrated as well (33, 34, 35). GnRH receptor activity and the mRNA for GnRH receptor vary throughout gestation in the human placenta (36, 37).
However, other data negates the hypothesis that mammalian GnRH plays a significant role in the human placenta. First, very high concentrations of mammalian GnRH are needed to bind to the receptor or to effect a hCG response (7, 8). In addition, we and other investigators have observed that placental GnRH exhibits differing reactivity with certain antisera to mammalian GnRH (12, 13), indicating nonidentity with mammalian GnRH. Other studies have demonstrated that mammalian GnRH acts as a partial agonist on placental hormonal releases (14, 15, 16). Although the limited activity of mammalian GnRH can be partially explained by the presence of a highly active postproline peptidase in the human placenta (9, 10), even mammalian GnRH analogs that are more resistance to enzymatic degradation are still relatively inactive at the placental receptor (11). In light of these conflicting data, we proposed that more than one isoform of GnRH, which is not identical to mammalian GnRH, is active in the human placenta. This hypothesis is also supported by the finding that multiple isoforms for the GnRH molecule exist in nonvertebrate species, which may have various evolutionary functions in different cells (17, 18, 19). GnRH evolved more than 500 million years ago, before the time of the evolution of vertebrates. The mammalian isoform evolved 350 million years ago and was thought to be the only form expressed in mammals. It was not until the mid-1990s, when Dellovade et al. (21) and King et al. (22) described chicken II GnRH in the brain of the tree and musk shrew and mole, was it realized that two different forms of GnRH existed in a mammalian species. Recently, the expression of the gene for chicken II GnRH in the shrew (20) and guinea pig (38) has been described. Chicken II GnRH in the rhesus monkey brain (26, 39) and in the human brain (24, 25) was demonstrated using specific immunoassays and mRNA expression. Subsequently, the chicken II receptor was identified in a variety of human tissues (27), although these investigators speculated that their function should be vestigial.
The studies described herein provide the first direct evidence of a pulsatile release of a potent second isoform of GnRH from the human placenta and a specific high-affinity receptor for chicken II GnRH isoform. Our data demonstrate both potent receptor binding and bioactivity of enzymatically resistant analogs of chicken II GnRH and, taken together with the existence of the chicken II GnRH receptor (27) in the human placenta, provide support for the hypothesis that this isoform of GnRH is active in regulating human placental functions. The biphasic response of hCG to this chicken II GnRH analog may reflect its stimulatory activity at the placental receptor with low, acute exposure to this analog. With continued exposure or higher concentrations of this chicken II GnRH analog, a down-regulation of the receptor may lead to inhibition of hCG production. Supporting this hypothesis are these and previous studies, which demonstrate the pulsatile release of a GnRH-like activity from the human placenta, as in the hypothalamus (40), as well as the down-regulation of the placental GnRH receptor with chronic exposure to mammalian GnRH agonist (41). We propose that hCG inhibition occurs via down-regulation of the placental GnRH receptor, as at the pituitary when exposed to high and chronic levels of GnRH agonist. We also propose that the hormonal effect of chicken II GnRH or its analogs will depend on the frequency and duration of treatment. Thus, such analogs may be used to stimulate or inhibit placental function.
The relative ineffectiveness of mammalian GnRH on hCG in these cultures of human term placentas may reflect both its limited affinity for the placental receptor and/or its rapid degradation in the placenta. The enhanced receptor binding and bioactivity of chicken II GnRH supports our proposal that the chicken II GnRH isoform is a potent and active form of GnRH in the placenta, although susceptible to degradation.
In summary, we have demonstrated the pulsatile production of chicken II GnRH from human placenta and a specific analog to the chicken II GnRH isoform was designed. This analog is stable in the presence of placental extracts and the highly active human placental C-ase-1 postproline peptidase. These studies demonstrate not only the presence, but also the action of a second isoform of GnRH in human tissues. The recognition that multiple forms of GnRH are expressed and regulate physiologic functions in nonmammalian species is not novel, but the knowledge of their bioactivity in a human tissue is. These findings have led us to speculate that chicken II GnRH may be the true paracrine GnRH-like activity at extra-hypothalamic tissues, such as the placenta. In addition, our ongoing studies indicate that chicken II GnRH is active in a number of other extra-hypothalamic tissues, which is consistent with the widespread expression of the chicken II GnRH receptor (42). We have observed that chicken II GnRH and its analogs are potent regulators of cell growth and can have direct action on tumor growth (Siler-Khodr, T. M., unpublished results). Specific chicken II GnRH analogs may be useful for the site-specific regulation of placental function in pregnancy, or other extra-pituitary sites, and may have limited interference with pituitary function.
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Footnotes |
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Received July 21, 2000.
Revised October 17, 2000.
Accepted October 29, 2000.
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References |
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-Human chorionic gonadotropin, human chorionic
gonadotropin and human chorionic somatomammotropin. Biol Reprod. 34:245–254.[Abstract]
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