Secretory characteristics and viability of human term placental tissue after overnight cold preservation

N. Cirelli1,4, P. Lebrun2, C. Gueuning1, A. Moens3, J. Delogne-Desnoeck1, C. Dictus-Vermeulen1, A.-M. Vanbellinghen1 and S. Meuris1

1 Research Laboratory on Reproduction and 2 Laboratory of Pharmacology, Université Libre de Bruxelles (ULB), Brussels, Belgium and 3 Unité Vétérinaire, Université Catholique de Louvain (UCL), Louvain-La-Neuve, Belgium


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collection of human term placentae for research purposes is generally limited during working hours. Preserving placental tissue overnight might help to postpone experiments and, by extent, to increase material availability. In this study, fragments from normal placentae were incubated at 37°C either immediately after delivery or after preservation at 4°C in a HEPES-buffered solution or in a Roswell Park Memorial Institute (RPMI) 1640 culture medium. Protein, human chorionic gonadotrophin (HCG), human placental lactogen (HPL) and lactate dehydrogenase (LDH) contents within preserved explants were similar to those within freshly delivered ones. In contrast, HCG and HPL amounts released during incubation of preserved tissue were lower than with freshly delivered tissue. Differences were significant only during the first 3 h of incubation. Hormone releases were similarly Ca2+-stimulated, and Co2+- and low temperature-inhibited in preserved and freshly delivered tissues. After preservation, LDH leakage was also reduced. Furthermore, before and after 37°C incubation during 6 h, preserved tissue was morphologically indistinguishable from freshly delivered tissue and showed neither higher incidence of DNA fragmentation, nor elevated caspase-3 activity, both of which are markers of apoptosis. This study validates an original, useful and rapid method to preserve placental tissue. Consequently, this preservation model may facilitate the study of physiological processes regulating placental hormone secretion in normal and pathological conditions.

Key words: apoptosis/explant/placenta/release/viability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Throughout pregnancy, human placenta synthesizes and secretes numerous polypeptide hormones into maternal blood. Among them, human chorionic gonadotrophin (HCG) and human placental lactogen (HPL) are structurally and functionally related to hormones produced by the pituitary gland (Chard, 1986Go; Talamantes and Ogren, 1988Go). The releases of both HCG and HPL from term placental explants are stimulated by the influx of calcium ion (Polliotti et al., 1990Go, 1992Go; Meuris et al., 1994Go; Petit and Belisle, 1995Go) in a manner concordant with the `stimulus–secretion coupling' concept (Douglas, 1968Go). However, in contrast to their pituitary homologues whose secretory responses are modulated by defined external stimuli, little is known about the physiological control of the placental HCG and HPL releases (Ringler and Strauss, 1990Go; Handwerger, 1991Go).

`In-vitro' systems represent useful experimental approaches to characterize the influence of physiological and/or pharmacological agents upon the endocrine function. Among the numerous `in-vitro' methods for studying secretion, incubation of placental explants has the potential advantage of maintaining the placental cells in their normal histological environment, thus allowing paracrine and/or autocrine interactions among different cell types (Ringler and Strauss, 1990Go). However, tissue explant incubations often reveal declining hormone secretion, morphological alterations and short-term cell survival (Chung et al., 1969Go; Taylor and Hancock, 1973Go; Hall et al., 1977Go).

Although research on human placenta involves no particular ethical problem, laboratories have to face the limited availability of normal and, more particularly, pathological placentae during working hours. Preserving placentae overnight might help to postpone experiments and, by extent, to increase material availability for research purposes.

In this study, an `in-vitro' preservation methodology was tested in order to increase availability of placental tissue usable for further physiological investigations. Explants from normal-term placentae were incubated either directly after delivery or after a 4°C overnight preservation period, and then compared on the basis of their secretory characteristics and tissue viability.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tissue collection and preservation
Term human placentae were obtained after vaginal delivery following normal pregnancy (37–41 weeks gestation) and immediately transferred from the Obstetric Department of Erasme Hospital (three to four deliveries/day) to the laboratory located on the same campus. As previously shown (Jauniaux et al., 1991Go; Denison et al., 1998Go), for further in-vitro investigations placental tissue needs to be processed as rapidly as possible after delivery. In this study, the mean time between delivery and sampling was 27.5 ± 3.3 min (range 15–35 min; n = 16). Villous tissue free of visible infarct, calcification or haematoma was sampled midway between the chorionic and basal plates, from five to ten cotyledons. These central parts of cotyledons were cut into multiple fragments (~0.5 g) which were thoroughly rinsed in a cold Hanks' medium (pH 7.4), gassed under a 5% CO2–95% O2 atmosphere and containing (in mmol/l): NaCl 137, KCl 5, CaCl2 1, MgSO4 1, Na2HPO4 0.3, KH2PO4 0.4 and NaHCO3 4. All reagents were of analytical grade and purchased from Sigma Chemical Co (St Louis, MO, USA), unless specified.

Sets of fragments (n >=10) from the same placenta were used for immediate incubations or for postponed incubations after an overnight preservation at 4°C.

The preservation procedure consisted of bathing placental fragments overnight (17–24 h) at 4°C in flasks containing 200 ml of medium continuously gassed under an atmosphere of 100% O2. The preservation medium was composed of the incubation medium supplemented with penicillin 50 IU/ml and streptomycin 50 µg/ml (Gibco-BRL, Gaithersburg, MD, USA). The incubation medium was composed of a HEPES-buffered physiological salt solution (pH 7.4) having the following composition (in mmol/l): HEPES 10, NaCl 139, KCl 5, CaCl2 1, MgCl2 1, glucose 4.2 and 0.5% (w/v) dialysed albumin. In comparative experiments (n = 3 placentae), this cold preservation medium was replaced by a Roswell Park Memorial Institute (RPMI) 1640 culture medium (Gibco-BRL) supplemented with 5% (v/v) fetal bovine serum, penicillin 50 IU/ml and streptomycin 50 µg/ml and maintained at 4°C under a 5% CO2–95% O2 atmosphere.

Immediately after delivery, or after the overnight preservation period, fragments (n >=10) were cut into small explants (~20 mg wet weight) and collected in a Petri dish containing cold Hanks' medium. Explants, randomly sampled, were either fixed (48–96 h) in 10% formaldehyde–phosphate-buffered saline (PBS) solution for histological observations (five groups of three explants) or weighed and stored at –20°C for initial content determinations (five groups of three explants). Remaining explants were used for incubation experiments.

Incidentally, it had been noticed previously that no significant variation of placental hormone content was found in relation to gestational age (37–41 weeks; data not shown), with time between delivery and sampling (within 35 min; data not shown), with duration of cold preservation (17–24 h; data not shown), or with drug administration during delivery (Meuris et al., 1996Go).

Experimental design
Freshly delivered and preserved explants were incubated in vials (three per vial) containing the HEPES incubation medium, and placed in a shaking water bath (35 cycles/min) heated at 37°C under a 100% O2 atmosphere. Incubation started with a 3x60 min equilibration period in order to reach a steady basal HCG and HPL release, as described previously (Polliotti et al., 1990Go). Further experimental periods were conducted according to two designs.

First, in order to assess the secretory capacity and the reactivity of the placental tissue, the initial 180 min equilibration period was followed by an 18x5 min experimental period. Explants were transferred, at each time interval, to glass vials containing 1 ml of incubation medium. The increase in Ca2+ or Co2+ concentration and the temperature lowering were performed from the 30th min until the 60th min of this experimental period. When the concentration of divalent cations, Ca2+ 10 mmol/l or Co2+ 0.5 mmol/l, was modified in the incubation medium, the concentration of NaCl was adjusted accordingly to keep osmolarity constant. The media collected at each time interval were stored separately at –20°C until assayed for HCG and HPL. At the end of the incubation, explants from each vial were weighed and stored at –20°C for further determinations of final protein, DNA, HCG and HPL contents. Experiments were repeated with five placentae.

The second experimental design was conducted in order to assess the viability of the placental tissue after longer time incubations. The initial equilibration period was followed by a 3x60 min experimental period. A 1 h time interval was required to assay lactate dehydrogenase (LDH) release which remained undetectable in 5 min incubation media. Explants were transferred, at each time interval, to glass vials containing 5 ml of incubation medium. Media were collected and stored at –20°C in order to further assay hormone and LDH. At the end of incubations, explants were either fixed (48–96 h) in 10% formaldehyde–PBS solution for histological observations or weighed and stored at – 20°C for further determinations of final DNA, HCG, HPL, LDH and caspase-3 (CPP32) contents. Experiments were repeated with three placentae.

Assays
For cellular content determinations, tissue homogenates were prepared as following. Thawed placental samples were sonicated (15 s twice, 50 kHz, 50 W) either in 500 µl ice-cold PBS solution (pH 7.2) containing (in mmol/l) Na2HPO4 40, KH2PO4 10 and NaCl 120 or, for CPP32 determinations, in an ice-cold cell lysis buffer (pH 7.4) comprising (in mmol/l) HEPES 50, EDTA 0.1, dithiothreitol (DTT) 1 and 0.1% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate (CHAPS). Homogenates were then centrifuged for 10 min at 2500 g at 4°C. Pellets were used for DNA determinations whilst supernatants were assayed for protein and hormone contents.

Protein contents were assayed in supernatants using a modification of the method of Lowry et al. (1951) (Bensadoun and Weinstein, 1976Go).

The DNA was extracted from pellets using a published method (Wannemacher et al., 1965Go), with the following modifications. First, pellets were washed twice with 0.2 mol/l perchloric acid instead of 10% trichloroacetic acid before the first 0.3 mol/l KOH extraction step. The final step of DNA extraction involved 0.3 mol/l KOH at 37°C for 15 min instead of 0.5 mol/l perchloric acid at 96°C for 45 min. DNA content was estimated colorimetrically using the Dische's diphenylamine reaction (Giles and Myers, 1965Go).

Quantities of HCG and HPL in incubation media and supernatants were determined using homologous radioimmunoassays performed as described previously (Robyn et al., 1971Go; Polliotti et al., 1990Go). Sensitivities of the assays were 0.6 µg HPL/ml and 1.5 mIU HCG/ml (2nd International Standard distributed by the World Health Organization) respectively. All samples obtained from the same placenta were systematically measured within the same assay. Total hormone amounts released during incubations corresponded to the sum of individual amounts assayed at each time interval. The large placenta-related variations in hormone release led to the expression, for each individual experiment, of changes in hormone release evoked by Ca2+, Co2+ or temperature modifications, with reference to a baseline value (100%) defined as the amount of hormone released by the explants during the first 30 min of the experimental period.

The CPP32-like activity was measured spectrophotochemically using the Caspase-3 Cellular Activity Assay Kit Plus (Biomol, Plymouth, PA, USA). The release of p-nitroaniline (pNA), cleaved by the tissue extract CPP32, from the DEVD tetrapeptide (Asp-Glu-Val-Asp)-pNA substrate was measured at 405 nm after a 60 min incubation at 37°C, using a pNA calibration curve. Results were expressed as pmol pNA released/min/µg DNA. Three control reactions were performed: a blank control using all reagents except substrate DEVD–pNA, a positive control obtained with the addition of a known amount of human caspase-3, and a negative control obtained after preincubation with DEVD–formaldehyde, a CPP32 inhibitor.

LDH activity was measured spectrophotometrically according to a previously published method (Bergmeyer et al., 1974Go), modified as follows. The assay was conducted in 1 ml of HEPES–NaOH buffer pH 7.6 (HEPES 50 mmol/l, EDTA 1 mmol/l, L-lactic acid 50 mmol/l and NAD+ 2 mmol/l). The kinetics of NADH formation was monitored at 30°C by following absorbance changes at 340 nm during 10 min. The blank reading consisted of the HEPES–NaOH buffer without NAD. The results were expressed as mU/µg DNA (1 U = 1 µmol NADH produced/min at 30°C).

Microscopy
For histological observations, 5 µm sections of placental tissue, by groups of three explants, embedded in paraffin were placed onto silanated slides and rehydrated. At least three sections were stained with haematoxylin and eosin for morphological assessment. For three additional tissue sections, nuclei containing fragmented DNA were identified using the TdT-mediated biotinylated dUTP nick end-labelling (TUNEL) method, as previously described (Cirelli et al., 1999Go). Endogenous peroxidase was inactivated by covering the sections for 10 min with 0.6% (v/v) H2O2 containing 0.1% (w/v) NaN3. Peroxidase activity was evidenced by the DAB (3,3'-diaminobenzidine; Fluka Chemica, Buchs, Switzerland) staining (Vacca et al., 1980Go). Slides were counterstained with methyl green, dehydrated, then mounted with a coverslip for histological examination. All tissue sections from the same placenta were processed in the same TUNEL experiment. For each set of experiments, a positive control (testicular tissue from hamster, provided by Dr Nonclercq, Department of Histology, Université de Mons-Hainaut, Mons, Belgium) was systematically included. In the absence of the TdT enzyme, positive nuclei were never observed within testicular and placental tissue.

The incidence of the peroxidase activity staining was microscopically evaluated by two independent observers using a micrometer reticle (Omnilabo, Brussels, Belgium). The observations were processed on five slides for each experimental condition. For each slide, 10 fields (at magnification x200) were taken into account and the number of TUNEL-positive nuclei was expressed as a percentage of the total nuclei counted (approximately 10 000). Only transverse villous sections ranging from 125 to 625 µm2 were considered for this assessment.

Statistical analysis
The statistical significance of differences between mean amounts per placenta of two experimental groups was assessed using paired Student's t-test. A two-tail P-value of < 0.05 was considered significant.


    Results
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 Materials and methods
 Results
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 References
 
The DNA contents measured before or after incubation were similar in freshly delivered and preserved tissues (Table IGo).


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Table I. Measurements of DNA, protein, human chorionic gonadotrophin (HCG) and human placental lactogen (HPL) before, after or during incubation of freshly delivered and preserved placental tissue
 
The initial protein, HCG and HPL contents in relation to DNA were similar after delivery or after cold overnight preservation (Table IGo). The contents were not significantly modified after 37°C incubation. The final protein and HCG contents were similar whichever explants were incubated immediately after placenta delivery or after the overnight preservation. By contrast, the final HPL content in preserved explants was significantly higher (18.3 ± 4.61%) than in explants incubated immediately after placenta delivery (Table IGo).

The total amounts of HCG and HPL secreted into the medium during the entire incubation time were lower when experiments were conducted following overnight preservation (Table IGo). These differences resulted from a larger release during the equilibration period from freshly delivered explants than from preserved explants (Table IGo). Hormone release during the experimental period was not statistically different between freshly delivered and preserved tissues (Table IGo).

Replacement of the HEPES-buffered medium by the RPMI 1640 culture medium during the 4°C preservation period did not modify protein and hormone explant contents, whereas hormone amounts released during incubation were lower than from freshly delivered explants. The total hormone release was reduced by 62.4 ± 3.42% for HCG and by 53.9 ± 2.87% for HPL. These decreases did not differ from those observed when tissue was preserved in the HEPES-buffered medium.

In order to verify the integrity of the stimulus–secretion coupling cascade, extracellular Ca2+ concentration was raised from 1 to 10 mmol/l during 30 min. The Ca2+ increase always elicited a marked stimulation of the HCG and HPL releases (P < 0.001; Figure 1Go). Whether incubations were performed immediately after placenta collection or after an overnight preservation period, the secretory responses were of similar amplitude for both hormones. The amplitude of the HCG and HPL secretory responses to a second rise in calcium concentration to 10 mmol/l (from the 270 to 300th min) was not significantly different from that following the first Ca2+ elevation (data not shown). Moreover, the addition of 0.5 mmol/l Co2+ during 30 min resulted in a marked inhibition of HCG and HPL release (P < 0.001; Figure 1Go). The inhibitory effect of Co2+ observed on freshly delivered and preserved explants was of similar magnitude. Lastly, temperature lowering from 37°C to 4°C during 30 min also resulted in a decrease in hormone release (P < 0.001; Figure 1Go). This inhibition was similar in freshly delivered and preserved explants. The effects of Ca2+, Co2+ and temperature variations on HCG and HPL releases were reversible phenomena (data not shown).



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Figure 1. Effect of raising calcium or cobalt concentrations and of decreasing temperature to 4°C, on human chorionic gonadotrophin (HCG; upper panel) and human placental lactogen (HPL; lower panel) releases from the 210th to the 240th min of incubation, by freshly delivered (open bars) and preserved (stippled bars) tissues. Results are expressed as the percentage of mean hormone release during the previous 30 min period. Values (mean ± SE) refer to 270 min incubations repeated with five groups of three explants from five placentae. All relative releases are significantly different from those observed during the previous 30 min period with P < 0.001 (*). Differences between relative releases observed on freshly delivered and preserved tissues were not significant using the paired Student's t-test (NS).

 
In order to examine further the viability of placental explants, longer incubations (360 min rather than 270 min) were conducted immediately after placenta collection, or after the overnight preservation period at 4°C. In haematoxylin and eosin-stained sections, no histological difference was identified before and after incubation between explants from freshly delivered and preserved tissues. The large majority of nuclei were considered to be morphologically normal, though some (which were randomly distributed in the section) presented evidence of shrinkage and condensation of chromatin. No necrotic area was detected. Morphologically normal red blood cells could be seen in intervillous spaces as well as in fetal vessels, even after incubation.

The incidence of TUNEL-positive nuclei remained <1% in both preserved and freshly delivered tissues (Table IIGo). Following a 360 min incubation period, the incidence of positive nuclei did not differ from that observed in the same placenta before incubation (Table IIGo). These nuclei were distributed randomly within tissue, and could be observed in trophoblastic cells, in Hofbauer cells from the villous stroma, and in endothelial cells from fetal capillaries. The incidence of TUNEL-stained nuclei was similar in the periphery and in the central part of tissue explants.


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Table II. TUNEL, caspase-3 (CPP32) and lactate dehydrogenase (LDH) measurements before, after or during incubation of freshly delivered and preserved placental tissue
 
No difference in CPP32-like activity was found between samples collected either just after the delivery or after the overnight preservation period, or after the incubation (Table IIGo). The LDH contents of explants measured after the delivery were similar to those found following overnight preservation (Table IIGo). Concurrently, LDH content after incubation did not differ from that measured before incubation. The percentage of LDH released into the incubation media, compared with the corresponding LDH final content, was lower for preserved explants than for explants incubated after the delivery (7.66 ± 2.33% and 15.0 ± 4.22% respectively; P = 0.034). Interestingly, >80% of total LDH release was observed during the equilibration period in freshly delivered and preserved tissues (81.6 ± 14.0% and 92.9 ± 15.4% respectively; Table IIGo).

Replacement of the HEPES-buffered medium by the RPMI 1640 culture medium during the 4°C preservation period did not modify the incidence of TUNEL-positive nuclei and the CPP32 and LDH explant contents. The total amount of LDH released during incubations was less than that from freshly delivered explants, but similar to that released from HEPES-preserved tissue. The average percentage of LDH released from RPMI 1640-preserved tissue into the incubation media was 7.18 ± 0.11% of the corresponding LDH final content. The amount of LDH released during the first 3 h amounted to 84.8 ± 2.30% of the total release.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although the easy accessibility of human placenta is of great benefit in overcoming a number of restrictions related to experimental research on human tissue, the limited availability of placental tissue during working hours may lead to difficulties in this respect. Thus, in order to overcome such technical problems, a rapid, simple and efficient experimental model of placental tissue preservation was developed.

Following the cold overnight preservation procedure, total protein, HCG, HPL and LDH contents were similar to those found in samples collected from the same placenta just after the delivery. Moreover, on completion of a subsequent incubation period, those contents remained unchanged. Contents remaining in preserved explants after incubation were equal to (for HCG, total protein and LDH) or even higher than (for HPL) those observed in explants incubated just after delivery. These data indicate that intracellular protein levels were maintained in the placental tissue preserved overnight at 4°C, as well as in placental explants further incubated during periods of up to 360 min. Amounts of total protein, hormone and LDH content, observed here under different experimental conditions, were consistent with those reported previously for normal human term placenta obtained after delivery (Gustke and Kowalewski, 1975Go; Lee et al., 1979Go; Nolan et al., 1994Go).

The amplitude of HCG and HPL responses to extracellular Ca2+ indicates that the preserved tissue maintains its Ca2+ sensitivity at levels similar to those reported previously for freshly delivered tissue (Polliotti et al., 1990Go, 1992Go; Meuris et al., 1994Go; Petit and Belisle, 1995Go). Moreover, the responsiveness of the overnight-preserved tissue to the addition of cobalt—which is a potent competitive inhibitor of ion permeation through calcium channels (Hurwitz, 1986Go)—also supports the integrity of the tissue's plasma membrane protein equipment. The reversibility of the secretory responses to these divalent cations, as well as the similar amounts of hormone released during the experimental period from both freshly delivered and preserved explants, strongly suggest that the overnight treatment did not impair the secretory machinery.

Interestingly, lesser amounts of HCG and HPL were released from preserved explants during the equilibration period (0–180 min). Similar decreases were also observed when the HEPES-buffered solution was replaced by a conventional culture medium such as RPMI 1640 during cold preservation. These features may be related to the lower percentage of LDH tissue content released by explants incubated after an overnight preservation. Besides that, more than 80% of the total LDH released during 360 min was observed during the first 3 h, whether incubated tissue was freshly delivered or preserved. These results indicate that some cellular leakage occurs early during incubation, immediately after preparation of placental fragments into explants. This also confirms the importance of an equilibration period in order to reach a steady state of hormone release before any experiment (Polliotti et al., 1990Go). Moreover, and as described previously (Atwater et al., 1984Go; Sooranna et al., 1999Go), it should be noted that a low percentage of the LDH tissue content released into the incubation media and a reversible decrease in hormone release caused by temperature lowering, further substantiate membrane integrity within placental tissue maintained overnight at 4°C.

Tissue sections from freshly delivered and preserved explants were histologically indistinguishable. Syncytiotrophoblast, cytotrophoblast and stromal cellular components were morphologically preserved, and no nuclear pyknosis was detected.

A low percentage of TUNEL-positive nuclei (<1%) was found in explants fixed just after delivery or after cold preservation. This low incidence of DNA fragmentation was not increased after 6 h of incubation. Moreover, the CPP32-like activity, previously immunolocalized in placental sections (Huppertz et al., 1998Go) and quantified in tissue homogenates for the first time in the present study, was not modified in preserved explants. All these data confirm a low incidence of apoptosis within human term placentae obtained after delivery (Qiao et al., 1998Go; Axt et al., 1999Go) and after a subsequent incubation at 4°C (Cirelli et al., 1999Go). Further incubations of placental tissue at 37°C during 6 h were not associated with higher apoptotic death.

Taken as a whole, the results demonstrate that the preserved tissue remains physiologically and morphologically intact as compared with the freshly delivered tissue. Consequently, the above-described preservation procedure may be used to increase placental tissue availability for research purposes. The incubation of tissue explants allows maintenance of cellular elements in their normal morphological relationships and preserves cell-to-cell communication which is particularly important for endocrine secretory processes (Meda, 1996Go). Indeed, gap junction-mediated cell-to-cell communication has been proposed to be a process whereby cells synchronize calcium-dependent events (Cao et al., 1997Go) and modulate their responsiveness to physiological agents (Munari-Silem et al., 1995Go). The present technical approach is likely to be of major advantage when compared with cell cultures, because cell isolation may damage the plasma membrane and cause degradation of cell surface proteins (Ringler and Strauss, 1990Go). Moreover, our incubation system with short intervals between medium changes makes it possible to observe secretory dynamics and, perhaps, to unmask short-term effects of physiological or pharmacological factors on hormone release. Coupled to the in-vitro preservation model developed here, it might facilitate the study of the physiological control of hormone release while trophoblastic cells are maintained in their histological environment.

In summary, an original and feasible approach to preserve human placental tissue has been developed. The potential use of preserved tissue for experimental purposes has been validated by the assessment of its secretory capacity, its physiological responsiveness to stimuli, and its morphological integrity. This preservation method may be useful in increasing the availability of limited pathological material and early pregnancy trophoblastic tissue.


    Acknowledgments
 
We are very grateful to Professor G.Graff for his helpful comments, as well as to Mrs L.Lammers and the nursing staff from the Erasme Hospital (Brussels, Belgium) for providing placentae. This work was granted by the National Fund for Scientific Research (Belgium, FNRS) from which S.Meuris and P.Lebrun are Research Directors. N.Cirelli was supported by a grant in aid from the `Université Libre de Bruxelles'.


    Notes
 
4 To whom correspondence should be addressed at: Research Laboratory on Reproduction, CPi 626, Faculty of Medicine, Université Libre de Bruxelles, 808, Route de Lennik, B-1070 Brussels, Belgium Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Atwater, I., Goncalves, A., Herchuelz, A. et al. (1984) Cooling dissociates glucose-induced insulin release from electrical activity and cation fluxes in rodent pancreatic islets. J. Physiol., 348, 615–627.[Abstract]

Axt, R., Meyberg, R., Mink, D. et al. (1999) Immunohistochemical detection of apoptosis in the human term and post-term placenta. Clin. Exp. Obstet. Gynecol., XXVI, 56–59.

Bensadoun, A. and Weinstein, D. (1976) Assay of proteins in the presence of interfering materials. Anal. Biochem., 70, 241–250.[ISI][Medline]

Bergmeyer, H.U., Gawhen, K. and Grassl, M. (1974) In Bergmeyer, H.U. in collaboration with Gawhen, K. (eds), Methods of Enzymatic Analysis. Verlag Chemie Weinheim Academic Press, Inc., New York, London. Second English edition translated from the Third German edition. Volume 1, p. 480.

Cao, D., Lin, G., Westphale, E.M. et al. (1997) Mechanisms for the coordination of intercellular calcium signaling in insulin-secreting cells. J. Cell Sci., 110, 497–504.[Abstract/Free Full Text]

Chard, T. (1986) Placental synthesis. Clin. Obstet. Gynaecol., 13, 447–461.[ISI][Medline]

Chung, H.K., McLimans, W.F., Horoszewicz, J. et al. (1969) In vitro studies of human trophoblast. Am. J. Obstet. Gynecol., 104, 945–952.[ISI][Medline]

Cirelli, N., Moens, A., Lebrun, P. et al. (1999) Apoptosis in human term placenta is not increased during labour but can be massively induced in vitro. Biol. Reprod., 61, 458–463.[Abstract/Free Full Text]

Denison, F.C., Kelly, R.W., Calder, A.A. et al. (1998) Cytokine secretion by human fetal membranes, decidua and placenta at term. Hum. Reprod., 13, 3560–3565.[Abstract]

Douglas, W.W. (1968) Stimulus-secretion coupling: the concept and clues from chromaffin and other cells. Br. J. Pharmacol., 34, 451–474.[ISI][Medline]

Giles, K.W. and Myers, A. (1965) An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature, 206, 93.[ISI]

Gustke, H.-H. and Kowalewski, S. (1975) Glycolytic enzymes in the normal human term placenta. Enzyme, 19, 154–164.[ISI][Medline]

Hall, C.S., James, T.E., Goodyer, C. et al. (1977) Short term tissue culture of human midterm and term placenta: parameters of hormonogenesis. Steroids, 30, 569–580.[ISI][Medline]

Handwerger, S. (1991) Clinical counterpoint: the physiology of placental lactogen in human pregnancy. Endocr. Rev., 12, 329–336.[Abstract]

Huppertz, B., Frank, H.G., Kingdom, J.C. et al. (1998) Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta. Histochem. Cell Biol., 110, 495–508.[ISI][Medline]

Hurwitz, L. (1986) Pharmacology of calcium channels and smooth muscle. Annu. Rev. Pharmacol. Toxicol., 26, 225–258.[ISI][Medline]

Jauniaux, E., Moscoso, J.G., Vanesse, M. et al. (1991) Perfusion fixation for placental morphologic investigation. Hum. Pathol., 22, 442–449.[ISI][Medline]

Lee, J.N., Grudzinskas, J.D. and Chard, T. (1979) Circulating levels of pregnancy proteins in early and late pregnancy in relation to tissue concentration. Br. J. Obstet. Gynaecol., 86, 888–890.[ISI][Medline]

Lowry, O.H., Rosebrough, N.J., Farr, A.L. et al. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265–275.[Free Full Text]

Meda, P. (1996) The role of gap-junction membrane channels in secretion and hormonal action. J. Bioenerg. Biomembr., 28, 369–377.[ISI][Medline]

Meuris, S., Polliotti, B., Robyn, C. et al. (1994) Ca2+ entry through L-type voltage-sensitive Ca2+ channels stimulates the release of human chorionic gonadotrophin and placental lactogen by placental explants. Biochim. Biophys. Acta, 1220, 101–106.[ISI][Medline]

Meuris, S., Gavriil, P., Vanbellinghen, A.-M. et al. (1996) In vivo and in vitro assessments of the influence of ritodrine and oxytocin on the placental secretion of human chorionic gonadotrophin and placental lactogen. Hum. Reprod., 11, 664–667.[Abstract]

Munari-Silem, Y., Lebrethon, M.C., Morand, I. et al. (1995) Gap junction-mediated cell-to-cell communication in bovine and human adrenal cells. A process whereby cells increase their responsiveness to physiological corticotropin concentrations. J. Clin. Invest., 95, 1429–1439.[ISI][Medline]

Nolan, G.H., Nahavandi, M., Edwards, C.H. et al. (1994) Deoxyribonucleic acid, ribonucleic acid, and protein in the placentas of normal and selected complicated pregnancies. J. Nutr., 124, 1022S–1027S.[Medline]

Petit, A. and Belisle, S. (1995) Stimulation of intracellular calcium concentration by adenosine triphosphate and uridine 5'-triphosphate in human term placental cells: evidence for purinergic receptors. J. Clin. Endocrinol. Metab., 80, 1809–1815.[Abstract]

Polliotti, B., Meuris, S., Lebrun, P. et al. (1990) Stimulatory effects of extracellular calcium on chorionic gonadotrophin and placental lactogen release by human placental explants. Placenta, 11, 181–190.[ISI][Medline]

Polliotti, B., Meuris, S., Lebrun, P. et al. (1992) Calcium influx similarly stimulates the release of chorionic gonadotrophin and placental lactogen from human placental explants. Trophoblast Res., 6, 189–198.

Qiao, S., Nagasaka, T., Harada, T. et al. (1998) p53, Bax and Bcl-2 expression, and apoptosis in gestational trophoblast of complete hydatiform mole. Placenta, 19, 361–369.[ISI][Medline]

Ringler, G.E. and Strauss, J.F., III (1990) In vitro systems for the study of human placental function. Endocr. Rev., 11, 105–123.[ISI][Medline]

Robyn, C., L'Hermitte, M., Petrusz, P. et al. (1971) Potency estimates of human gonadotrophins by a bioassay and three immunoassay methods. Acta Endocrinol. (Copenh.), 67, 417–433.[Medline]

Sooranna, S.R., Oteng-Ntim, E., Meah, R. et al. (1999) Characterization of human placental explants: morphological, biochemical and physiological studies using first and third trimester placenta. Hum. Reprod., 14, 536–541.[Abstract/Free Full Text]

Talamantes, F. and Ogren, L. (1988) The placenta as an endocrine organ: polypeptides. In Knobil, E. and Neil, J. (eds), The Physiology of Reproduction. Raven Press, New York, pp. 2093–2114.

Taylor, P.V. and Hancock, K.W. (1973) Viability of human trophoblast in vitro. J. Obstet. Gynaecol. Br. Common., 80, 834–838.[ISI][Medline]

Vacca, L.L., Abrahams, S.J. and Naftchi, N.E. (1980) A modified peroxidase-antiperoxidase procedure for improved localization of tissue antigens, localization of substance P in rat spinal cord. J. Histochem. Cytochem., 28, 297–307.[Abstract]

Wannemacher, R.W., Banks, W.L. and Wunner, W.H. (1965) Use of a single tissue extract to determine cellular protein and nucleic acid concentrations and rate of amino acid incorporation. Anal. Biochem., 11, 320–326.[ISI][Medline]

Submitted on September 3, 1999; accepted on December 7, 1999.