| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Obstetrics and Gynecology (D.M.), Faculty of Medicine, University of Torino, 10127 Torino, Italy; Institute of Normal Human Morphology (D.M., A.G., F.V., M.C.), Faculty of Medicine, Polytechnic University of Marche, I-60020 Ancona, Italy; Chair of Obstetrics and Gynaecology (G.F., P.F., F.P.), Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, 53100 Siena, Italy; and Dipartimento di Medicina Sperimentale e Sanità Pubblica (M.N.), University of Camerino, 62032 Camerino, Italy
Address all correspondence and requests for reprints to: Mario Castellucci, M.D., Institute of Normal Human Morphology-Anatomy, Polytechnic University of Marche, Via Tronto, 10/a, I-60020 Ancona, Italy. E-mail: anatomia2{at}univpm.it.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Because little information is available on the role of VIP and/or SP on the secretion of placental hormones, we investigated the effect of these neuropeptides on human chorionic gonadotropin (hCG) and progesterone release from primary cultured human trophoblastic and JEG-3 cells. The addition of increasing doses of VIP resulted in a dose-dependent stimulation of hCG release from cultured human trophoblast and JEG-3 cells. Increasing doses of VIP also dose-dependently stimulated progesterone secretion from primary cultured trophoblastic cells at all time points evaluated and from JEG-3 cells only after 3 h. SP did not affect hCG and progesterone secretion either in cultured human trophoblast or in JEG-3 cells.
In conclusion, the present study demonstrates that VIP and SP are mainly expressed in human trophoblasts, and that VIP modulates the in vitro secretion of hCG and progesterone, suggesting a different role in trophoblastic function of the two peptides.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It has been shown that vasoactive intestinal peptide (VIP), a neurotransmitter and neuromodulator with neurotrophic properties, regulates embryonic growth in rodents (2), promotes neural growth and survival, and modulates immune functions (3). A specific high-affinity receptor gene for VIP is found in placental tissues (3), and specific binding sites for VIP have been found on JEG-3 cells (4). In these cells, VIP activates adenylcyclase and modulates human chorionic gonadotropin (hCG) gene expression via the cAMP pathway (4). The potential for VIP to act as a pregnancy hormone has been suggested by the observation that pregnant sheep have higher plasma VIP levels than nonpregnant ewes (5).
Substance P (SP) is another tachykinin neuropeptide that is localized in a population of sensory fibers that innervate the mammalian female reproductive tract (6). SP exhibits a wide variety of biological effects, including regulation of blood flow, smooth muscle activity, transmission of pain signals, and stimulatory action on the gut immune system (7). SP interacts with the receptor NK1R and activates it (6). Candenas et al. (8) have shown that NK1R mRNA uterine levels are low in early pregnant rats, increase in late gestation, and decline on d 1 postpartum with a return to levels similar to those at early pregnancy. In addition, a complex relation between tachykinin NK1R mRNA levels and the ratio of estrogen and progesterone serum levels has been observed (8), suggesting that steroid hormones regulate the expression of the NK1R during the course of pregnancy. Little information is available on the correlation between neuropeptides and hormone production during human gestation. An autocrine mechanism for VIP and SP actions on the trophoblast is a theoretical possibility. In the current study, the expressions of VIP and SP in first- and third-trimester human placentas were investigated by immunohistochemistry and by quantitative slot blot analysis. In addition, we analyzed the effect of VIP and SP on hCG and progesterone release from primary cultures of human cytotrophoblastic cells and from JEG-3 cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Twenty human placentas aged 7 (n = 3), 8 (n = 3), 10 (n = 1), 12 (n = 5), 38 (n = 5), and 40 (n = 3) wk postmenstruation were collected from clinically normal pregnancies interrupted by curettage (aspiration technique) for psycho-social and medical reasons that were unlikely to affect placental structure and function or terminated by cesarean sections and normal vaginal deliveries. Written informed consent was obtained from each pregnant woman, and the permission of the Human Investigation Committee was granted for the study.
Tissue preparation for immunohistochemistry
Placental tissue was cut into small blocks and immediately fixed in 4% neutral buffered formaldehyde solution for 8–12 h at 4 C. The specimens were then routinely processed for paraffin embedding at temperatures not exceeding 56 C as previously described (9). Sections (4 µm) were cut and stretched at 45 C, allowed to dry, and stored at 4 C until use.
Immunohistochemistry
Paraffin sections were deparaffinized and rehydrated via xylene and a graded series of ethyl alcohols. Sections were incubated for 30 min with 1% hydrogen peroxide in methanol to inhibit endogenous peroxidase and then for 1 h at room temperature with 10% PBS/BSA.
Afterward the sections were incubated overnight with one of the following primary antibodies diluted in PBS containing 1% normal serum: 1) a rabbit polyclonal antibody anti-SP (Peninsula Laboratories, Belmont, CA), dilution 1:200 vol/vol; 2) a rabbit polyclonal antibody anti-VIP (Peninsula), dilution 1:100 vol/vol; 3) a mouse monoclonal antibody antihuman pan-cytockeratin (Dako, Glostrup, Denmark), dilution 1:500 vol/vol; and 4) a mouse monoclonal antibody anticytokeratin 7 (Dako), dilution 1:200 vol/vol.
After washing with PBS, the bound antibody was visualized by the peroxidase avidin biotin complex method (10) using the following biotinylated secondary antibodies: for the primary polyclonal antibodies, a goat antirabbit (Vector Laboratories, Burlingame, CA), dilution 1:200 vol/vol; for the primary monoclonal antibodies, a horse antimouse (Vector Laboratories) dilution 1:200 vol/vol.
3',3'-Diaminobenzidine hydrochloride (Sigma, St. Louis, MO) was used as chromogen. The sections were rinsed with distilled water, counterstained with hematoxylin, dehydrated, and mounted in Eukitt (Kindler GmbH & Co., Freiburg, Germany).
For the above-described immunohistochemical procedures, controls were performed replacing primary antibodies by PBS or nonimmune rabbit or mouse serum. Additional controls were performed omitting the secondary antibody. The controls were always negative.
Tissue preparation for RNA extraction
Placental specimens were collected and immediately frozen and stored in liquid nitrogen until used for total RNA extraction. Total RNA was extracted from 2 µg for each sample of placenta using TRIzol reagent (Life Technologies, Inc. Brl, Grand Island, NY), based on the acid guanidinium thiocyanate-phenol-chloroform extraction method (11). Final total RNA concentration was determined by OD reading at 260 nm, and integrity was verified by ethidium bromide staining of 28 S and 18 S RNA bands on a denaturing 1.2% agarose gel.
Oligonucleotide primer sequences
VIP antisense primer was designed based on the sequence of the primary structure of human pre-pro VIP/peptide histidine-methionine-27 mRNA (1) and was purchased from Life Technologies, Inc. Brl. The 26-mer VIP primer was complementary to the nucleotide region 385–411 of the pre-pro VIP/peptide histidine-methionine-27 mRNA. Preprotachykinin-A (PPTA) (SP) antisense was designed based on the sequence of Homo sapiens delta preprotachykinin mRNA (AF050658) and purchased by Life Technologies, Inc. Brl. The 32-mer was complementary to the nucleotide region 68–99 of the human delta preprotachykinin mRNA (sequence ID, AF 0500658). VIP primer, 5'-CTAGGCGGGTATAGTTGTCAGTGAAG-3'; SP primer, 5'-CATTAATCCAAAGAACTGCTGAGGCTTGGGTC-3'.
Slot blot analysis
One hundred picomoles of VIP and SP oligonucleotides were 3' end-labeled using digoxigenin (DIG)-oligonucleotide 3' end labeling kit (Roche Diagnostic S.p.A., Milan, Italy). The labeling efficiency was estimated by a spot test with a DIG-labeled control following kit protocol (Roche).
For slot blot hybridization, 15 µg of total RNA from each sample was diluted in RNA dilution buffer [H2O: 20x NaCl 3 M, sodium citrate 0.3 M, pH 7.0 (SSC): formaldehyde; 5:3:2] and spotted onto a nylon membrane (Nytran; Schleicher & Schuell, Dassel, Germany) using a slot blot vacuum system. The RNA was fixed to the membrane by backing in an oven at 120 C for 30 min. The blot was placed in a hybridization tube containing 25 ml of DIG Easy Hyb solution (Roche) for 2 h at 42 C. Before hybridization, the appropriate amount of DIG-VIP and DIG-SP oligonucleotide probes (50 pmol) were heat-denaturated at 70 C for 10 min, immediately chilled on ice, and added to 5 ml DIG-Easy Hyb solution. The membrane was hybridized at 42 C overnight in a hybridization oven (Stuart Scientific-Bibby Sterilin, Stone, UK). The posthybridization washes were performed: 1) 2x SSC/0.1% sodium dodecyl sulfate for 5 min at room temperature (two times), 2) 0.5x SSC/0.1% sodium dodecyl sulfate for 15 min at 44 C (two times).
The detection was as described by the manufacturer’s protocol (Roche), using CSPD as chemioluminescent substrate. Membrane was exposed to film for 30 min at 37 C.
Cell cultures
Cell culture reagents and fetal bovine serum (FBS) were purchased from Celbio S.p.a. (Milan, Italy) and Sigma (Milan, Italy), respectively. All cell culture plastic material was purchased from Sarstedt (Verona, Italy).
Primary trophoblast cell cultures
Placentas were obtained under sterile conditions from normal pregnancies undergoing elective cesarean section at term. Maternal consent was obtained according to the guidelines of the local ethics committee. Trophoblast-enriched cell cultures were purified according to the method of Kliman et al. (12). Briefly, chunks of placental cotyledons weighing approximately 30 g were thoroughly rinsed in calcium- and magnesium-free Hanks’ solution; villous tissue was identified and isolated from membranes, large vessels, decidua, and connective tissue under a dissection microscope and was then coarsely minced with scissors and transferred to 150 ml Hanks’ solution containing 0.25% trypsin (from porcine pancreas T4799; Sigma), 25 mmol/liter HEPES, and 0.4 mg/ml (0.4 g/liter) type I DNase. The tissue was then incubated in a water shaking bath at 37 C for 30 min. The resultant cell suspensions were layered over FBS and centrifuged at 1000 x g for 5 min at room temperature, while the remaining placental tissue was digested two more times with the addition of fresh digestion solution.
The pellets collected after the three digestions were resuspended in DMEM containing 25 mmol/liter HEPES, 25 mmol/liter glucose, and 2 mmol/liter glutamine, pooled, centrifuged at 1000 x g for 10 min, and resuspended in 4 ml DMEM. This suspension was placed on a discontinuous Percoll gradient (5–70% Percoll in a 5% steps) and centrifuged at 1200 x g for 30 min. A population of mononuclear cells resulting as a band at a density of 1.051–1.065 g/ml (1051–1065 g/liter) was removed, washed with DMEM, and resuspended in cold PBS/BSA 0.1%. The nontrophoblast cells expressing common leukocyte antigen were removed by immunodepletion using a monoclonal antibody against CD45RB (clone PD7/26; Dako). After depletion the cells were resuspended in DMEM with 4 mmol/liter glutamine and 50 µg/ml (50 mg/liter) gentamicin. This process of digestion, isolation, and depletion yields a trophoblast-enriched preparation containing approximately 95% trophoblast and 5% stromal contaminants, as ascertained by immunocytochemical analyses with cytokeratin 7 and vimentin (Dako).
Purified trophoblast was plated in six-well plastic plates at a density of 6x105 cells/well and cultured for 48 h at 37 C under humidified 5% CO2, with the culture medium refreshed after 24 h. The experiments were performed 48 h after plating, and before the stimulation by VIP and SP at doses addressed below the cells were washed twice with Hanks’ solution. Conditioned medium with VIP and SP at concentrations of 100 pg, 10 ng, and 1 µg was then used to replace the culture medium. Control cells were present in each experiment. After 3, 6, 24, 48, and 72 h of incubation, the medium was harvested and stored at –20 C until the progesterone and ß-hCG assays. The experiments were performed without FBS.
VIP and SP were kindly donated by Dr. W. Vale from The Salk Institute (La Jolla, CA).
JEG-3 choriocarcinoma cells
JEG-3 cells were obtained from the American Type Culture Collection (Rockville, MD). JEG-3 cells were maintained routinely in DMEM supplemented with 10% FBS, 2 mmol/liter glutamine, 100 IU/ml (105 IU/liter) penicillin, and 100 µg/ml (100 mg/liter) streptomycin at 37 C under 5% CO2-95% air.
Cells were plated at a density of 4x105 cells/well in six-well plastic plates containing culture medium. JEG-3 cells were cultured for 2 d. When the cells had reached a subconfluent state, the culture medium was removed and the cells were treated with VIP and SP at concentrations of 100 pg, 10 ng, and 1 µg. Control cells were present in each experiment. After 6, 12, and 24 h of incubation, the medium was harvested and stored at –20 C until the progesterone and hCG assays. The experiments were performed without FBS.
Determination of vitality
At the end of the exposure period with and without VIP and SP, the vitality rate was determined with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromides (MTT) assay.
Cells were plated in 96-well plastic plates and were treated as described above in the experimental protocol. MTT stock solution (5 g/liter) was added to each well being assayed to equal one tenth the original culture volume and incubated for 4 h. At the end of the incubation period, converted dye was solubilized with acidic isopropanol [0.04–0.1 N (0.04–0.1 mol/liter) HCl in absolute isopropanol]. Absorbance of converted dye was measured at a wavelength of 570 nm with background subtraction at 630 nm.
Measurement of ß-hCG and progesterone
The concentrations of ß-hCG and progesterone in the culture medium were measured with commercially available enzyme immunoassay kits (RADIM Co., Pomezia, Italy; and DRG International, Inc., Mountainside, NJ, respectively). Each measurement was performed in triplicate according to the manufacturer’s recommendations.
The ß-hCG assay detection limit was 2 mIU/ml (2 x 103 mIU/liter), and the precision was less than 3.4% for intraassay and less than 5% for the interassay. The assay does not recognize FSH, TSH, prolactin, and human placental lactogen and showed 0.2% cross-reactivity with LH.
The progesterone assay detection limit was 0.05 ng/ml (0.05 µg/liter), the precision was less than 4.6% for intraassay and less than 5.5% for the interassay, and the cross-reaction with 17-OH progesterone, estriol, 17ß-estradiol, testosterone, dehydroepiandrosterone sulfate, cortisol, and pregnenolone was less than 0.3%.
Statistical analysis
Results were expressed as the mean and SEM, and statistical significance of hormone concentrations was assessed by using the one-way ANOVA test. Results were considered statistically significant whenever P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the tissues collected during the first trimester of pregnancy, immunoreactive VIP and SP were present in the syncytiotrophoblast (Fig. 1
, A and B); whereas VIP was mainly expressed in the cytoplasm of the syncytiotrophoblast (Fig. 1A), SP showed a more intense staining on the apical plasma membrane of the syncytium (Fig. 1B). Villous cytotrophoblastic cells and numerous stromal cells of the chorionic villi were found to express VIP as well as SP (Fig. 1, A and B). VIP showed a positive reaction product in the wall of the fetal vessels (Fig. 1A). The small fetal vessels and the majority of the large vessels showed a negative staining pattern for SP. VIP and SP immunostaining was present in extravillous cytotrophoblast of cell columns and cell islands.
|
), whereas SP showed an intense staining pattern at the apical surface of the syncytiotrophoblast (Fig. 1D). Only a few villous cytotrophoblastic cells showed weak immunostaining for VIP (Fig. 1C), whereas SP was negative in these cells (Fig. 1D). The extravillous cytotrophoblastic cells (positive for cytokeratin) of the basal plate as well as the decidual cells (negative for cytokeratin) were weakly stained for both VIP and SP. In the villous stroma few stromal cells were positive for VIP (Fig. 1C), whereas the villous stroma was mainly negative for SP (Fig. 1D). Immunostaining was absent from control sections (Fig. 1E). Slot blot analyses
To establish a simplified, nonradioactive approach to identify VIP and PPTA mRNA levels, specific VIP and PPTA antisense oligonucleotides were designed and used as probes in combination with chemiluminescence-based detection. Moreover, ß-actin antisense oligonucleotide was used as probe for the normalization of the detection signals. The signals obtained with VIP and PPTA probes were normalized against ß-actin signal giving a value of VIP and PPTA mRNA expressions (quantitative slot blot analysis). VIP and PPTA mRNA levels slightly increased in the third trimester of gestation in comparison with the first trimester (Fig. 2).
|
The addition of increasing doses of VIP to trophoblast cells resulted in a dose-dependent significant (P < 0.001) stimulation of hCG release from cultured human trophoblast cells (but not after 72 h of incubation), as well as from JEG-3 cells (Fig. 3). With respect to progesterone, VIP significantly (P < 0.001) and dose-dependently stimulated its secretion from primary cultured trophoblastic cells at all time points evaluated with the exception of 72 h, and from JEG-3 cells only after 3 h (Fig. 4).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene expression (4). Thus, the present data and the finding that VIP is able to stimulate the release of placental GnRH (1, 16) lead us to suggest that this neuropeptide is able to modulate the placental hypothalamus-pituitary axis, and this suggestion is further reinforced by our data showing that VIP also stimulates progesterone secretion in vitro. Aside from the local control of placental hormonogenesis, the stimulation of hCG secretion also suggests a paracrine modulation for VIP on trophoblast differentiation, so that the lack of VIP action on the expression of hCG could cause insufficient trophoblast differentiation and may play a role in placental dysfunctions. The role of VIP in human placenta is also reinforced by our findings showing the stimulatory activity on progesterone release, because progesterone is known to be important for successful maintenance of pregnancy and normal parturition. It has been previously demonstrated that VIP stimulates progesterone production in cultured rat granulosa cells by enhancement of cAMP and protein kinase A pathway (17). Previous data showed that VIP also exerts other important biological functions including the control of the resistance in the vascular system (5). VIP binding sites are located on fetal vascularization (18). Although the role of VIP as vasoregulator in the human placenta is still far from being elucidated, the evidence of the expression of VIP on villous fetal endothelium is intriguing and suggests the possibility that VIP may play a role in the modulation of placental blood flow.
In the present study we also found that human placenta expresses SP; however, SP does not stimulate placental hCG and progesterone secretion. Unfortunately, no data are available on the immunolocalization of SP receptor (NK1R) in the human placenta. However, recent data show that NK1R mRNA is detectable in human term placental tissues (19). In addition, in fetal placental circulation it has been shown that the vasodilatory effect of neurokinin B, a kind of tachykinin which is similar to SP, is predominantly due to direct activation of NK1R (19, 20, 21). Thus, we can speculate that the SP may play a role in the maintenance of high placental blood flow in normal pregnancy. So it could be interesting to investigate the role of SP in preeclampsia, which is a pathology with reduced placental blood flow.
In conclusion, our data suggest that human placenta expresses VIP and SP, and that VIP may be responsible for the control of hCG and progesterone secretion and, consequently, the physiological development of the placental tissues. In addition, these peptides could represent two factors involved in the modulation of placental vascular reactivity.
|
Footnotes |
|---|
First Published Online December 28, 2004
Abbreviations: DIG, Digoxigenin; hCG, human chorionic gonadotropin; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromides; PPTA, preprotachykinin A; SP, substance P; SSC, NaCl 3 M, sodium citrate 0.3 M, pH 7.0; VIP, vasoactive intestinal peptide
Received August 2, 2004.
Accepted December 16, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene activity in JEG-3 syncytial trophoblasts. J Biol Chem 265:10274–10281This article has been cited by other articles:
 |
|
 |
|
  V. Roca, M. Calafat, L. Larocca, R. Ramhorst, M. Farina, A. M. Franchi, and C. Perez Leiros Potential immunomodulatory role of VIP in the implantation sites of prediabetic nonobese diabetic mice Reproduction, October 1, 2009; 138(4): 733 - 742. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.E. Green, C. Thota, G.D.V. Hankins, C. Yallampalli, and Y.-L. Dong Calcitonin gene-related peptide stimulates human villous trophoblast cell differentiation in vitro Mol. Hum. Reprod., July 1, 2006; 12(7): 443 - 450. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
