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
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Abstract |
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Key words: apoptosis/explant/placenta/release/viability
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Introduction |
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`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, 1990). However, tissue explant incubations often reveal declining hormone secretion, morphological alterations and short-term cell survival (Chung et al., 1969
; Taylor and Hancock, 1973
; Hall et al., 1977
).
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.
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Materials and methods |
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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 (1724 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% CO295% 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 (4896 h) in 10% formaldehydephosphate-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 (3741 weeks; data not shown), with time between delivery and sampling (within 35 min; data not shown), with duration of cold preservation (1724 h; data not shown), or with drug administration during delivery (Meuris et al., 1996).
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., 1990). 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 (4896 h) in 10% formaldehydePBS 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, 1976).
The DNA was extracted from pellets using a published method (Wannemacher et al., 1965), 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, 1965
).
Quantities of HCG and HPL in incubation media and supernatants were determined using homologous radioimmunoassays performed as described previously (Robyn et al., 1971; Polliotti et al., 1990
). 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 DEVDpNA, a positive control obtained with the addition of a known amount of human caspase-3, and a negative control obtained after preincubation with DEVDformaldehyde, a CPP32 inhibitor.
LDH activity was measured spectrophotometrically according to a previously published method (Bergmeyer et al., 1974), modified as follows. The assay was conducted in 1 ml of HEPESNaOH 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 HEPESNaOH 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., 1999). 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., 1980
). 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.
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Results |
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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 I). These differences resulted from a larger release during the equilibration period from freshly delivered explants than from preserved explants (Table I
). Hormone release during the experimental period was not statistically different between freshly delivered and preserved tissues (Table I
).
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 stimulussecretion 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 1). 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 1
). 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 1
). 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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The incidence of TUNEL-positive nuclei remained <1% in both preserved and freshly delivered tissues (Table II). Following a 360 min incubation period, the incidence of positive nuclei did not differ from that observed in the same placenta before incubation (Table II
). 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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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.
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Discussion |
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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, 1975; Lee et al., 1979
; Nolan et al., 1994
).
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., 1990, 1992
; Meuris et al., 1994
; Petit and Belisle, 1995
). Moreover, the responsiveness of the overnight-preserved tissue to the addition of cobaltwhich is a potent competitive inhibitor of ion permeation through calcium channels (Hurwitz, 1986
)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 (0180 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., 1990). Moreover, and as described previously (Atwater et al., 1984
; Sooranna et al., 1999
), 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., 1998) 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., 1998
; Axt et al., 1999
) and after a subsequent incubation at 4°C (Cirelli et al., 1999
). 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, 1996). Indeed, gap junction-mediated cell-to-cell communication has been proposed to be a process whereby cells synchronize calcium-dependent events (Cao et al., 1997
) and modulate their responsiveness to physiological agents (Munari-Silem et al., 1995
). 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, 1990
). 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.
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Acknowledgments |
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Notes |
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References |
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Submitted on September 3, 1999; accepted on December 7, 1999.