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Original Studies |
Department of Anatomy, University of Cambridge (A.L.W., J.N.S., G.J.B.), Cambridge, United Kingdom CB2 3DY; and the Academic Department of Obstetrics and Gynecology, University College London Medical School (E.J.), London, United Kingdom WC1E 6HX
Address all correspondence and requests for reprints to: Dr. Adrian L. Watson, Department of Anatomy, University of Cambridge, Downing Street, Cambridge, United Kingdom CB2 3DY. E-mail: aw10016{at}pop.hermes.cam.ac.uk
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Abstract |
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Introduction |
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Recent anatomical and physiological findings indicate that the oxygen tension within the feto-placental unit may be surprisingly low during early pregnancy (3, 4). Direct polarographic electrode measurements in vivo have demonstrated that the mean oxygen (O2) tension measured within the feto-placental unit is only 17.9 mm Hg (SD = 6.9) at 8–10 weeks gestation, but rises to 60.7 mm Hg (SD = 8.5) at 12–13 weeks (5). It is thought that this is due to accumulations of trophoblast cells from the cytotrophoblastic shell blocking the mouths of many of the maternal spiral arteries during the first trimester, preventing significant continuous flow of maternal blood cells into the intervillous space (6, 7). As pregnancy proceeds, the accumulations progressively loosen, allowing increased circulation of maternal blood through the intervillous space. These findings have been challenged strongly (8, 9, 10), and the issue is clearly not resolved. However, if the prevailing oxygen tension is low in vivo during the first trimester, then placental tissues will be exposed to considerable oxidative stress when delivered into ambient oxygen concentrations.
Evidence has accumulated to suggest that hyperoxic tissue damage is mediated through increased intracellular production of superoxide anions (O2.-). If the level of this molecule, and the species into which it is converted, exceeds the capacity of the cellular defense systems, then destructive oxidations will result. There are numerous potential sources of O2.- within the cell, for example in the cytoplasm, endoplasmic reticulum, peroxisomes, and mitochondria, with generation resulting from both enzymatic and nonenzymatic activities (11). Of the various sources, mitochondrial production seems to be most significant in causing the damage observed to lung tissue when exposed to 85% oxygen (12).
The predominant cause of superoxide anion generation within mitochondria is thought to be the leakage of electrons from enzymes of the respiratory chain to molecular oxygen (13). Such leakage occurs in a nonenzymatic fashion, so O2.- generation will continue to increase with oxygen concentration even after respiratory enzymes become saturated with this substrate. The main protection is provided by the enzyme manganese superoxide dismutase (MnSOD) located within the inner mitochondrial matrix (14). This catalyzes the dismutation of O2.- to hydrogen peroxide, which is, in turn, converted to oxygen and water by the enzyme catalase. As might be expected, higher concentrations of MnSOD are induced by exposure to hyperoxic conditions (15), although the exact mechanism by which the oxygen level controls expression is not yet fully understood.
To test the hypothesis that rapid degeneration of the first trimester syncytiotrophoblast after delivery is due to oxygen toxicity, this study examined mitochondrial activity and ultrastructure in villi maintained under contrasting oxygen tensions. Short term cultures (up to 6 h) were performed because of the rapidity of the degenerative process. The localization and activity of the mitochondrial SOD in villi of different gestational ages were also monitored.
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Materials and Methods |
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Culture materials, 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), nitro blue tetrazolium (NBT), riboflavin, N,N,N',N'-tetramethylethylene diamine (TEMED), and BSA were all obtained from Sigma Chemical Co. (St. Louis, MO). Polyfreeze embedding medium was purchased from Polysciences (Warrington, PA). Fixatives and araldite were obtained from TAAB (Reading, UK). Sheep polyclonal antiserum to MnSOD was obtained from Calbiochem Novabiochem (Nottingham, UK). Biotinylated antibodies and avidin-biotin-peroxidase complex were obtained from Vectorlabs (Peterborough, UK). Histotec aqueous mountant was purchased from Serotec (Oxford, UK). Gas mixtures were provided by BOC (Guildford, UK).
Tissue collection and maintenance in culture
Placental villous tissue was obtained from terminations of
pregnancy carried out at University College Hospital (London, UK) with
the informed consent of the patients and approval of the local ethics
committee. Before the termination procedure, pregnancies were dated by
ultrasound scan. Initially, villous tissue, obtained by dilatation and
vacuum aspiration, was washed free of the aspirated products of
conception after transportation to the laboratory. Later, the majority
of experiments were conducted using villous tissue obtained in
situ using a chorionic villous sampling procedure performed under
ultrasound guidance before dilatation and curettage or aspiration.
Tissue was then immediately placed into the appropriate culture medium.
Low oxygen concentration conditions were achieved by equilibrating 120
mL culture medium (medium 199, 5% heat-inactivated FBS, 1 mmol/L
L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin,
and 250 µg/mL amphotericin) with a 2.5% O2-5%
CO2-92.5% N2 gas mixture. Ambient oxygen
conditions were as described above, with medium equilibrated to 21%
oxygen-5% CO2-74% N2. Glass bottles were used
rather than traditional culture plastics because of the inherent gas
permeability of the latter. Once tissue (
100 mg) had been placed
into culture medium, the bottles were quickly sealed and transported to
the laboratory, where they were placed into a controlled atmosphere
incubator at 37 C for up to 6 h (total). Lids on bottles used for
high oxygen incubations were loosened at this point to allow regassing
of the medium at 21% oxygen-5% CO2.
To control for regional variation, sampling of the larger volume, second trimester placenta was performed randomly from four different areas of the bulk tissue. These samples were then pooled for assays.
Transmission electron microscopy
Tissue to be used for transmission electron microscopy was fixed by immersion in 3% glutaraldehyde-0.3% hydrogen peroxide in 0.1 mol/L 1,4-piperazine diethane sulfonic acid (PIPES) buffer (pH 7). After 2-h fixation at room temperature, tissue was washed for 30 min in 0.1 mol/L PIPES buffer. Secondary fixation was achieved by immersing the tissue specimen in 1% osmium tetroxide in PIPES buffer for 1 h at room temperature. After three washes in buffer, specimens were dehydrated in graded ethanol and embedded in Araldite epoxy resin. Ultrathin sections (50 nm) were cut on a Reichert-Jung Ultracut S (Reichert-Jung, Vienna, Austria). Sections were counterstained with uranyl acetate, followed by lead citrate, before viewing in a Philips CM100 electron microscope (Philips Electronics, Mahway, NJ).
Stereological assessment of mitochondria
Stereological assessment of the mitochondria was performed on a Philips CM 100 transmission electron microscope equipped with the Quantimet 500 image analysis system. Tissue samples were coded so that the observer was blinded to the incubation protocol, and fields of view were selected on a systematic random basis by taking areas of the syncytiotrophoblast adjacent to vertical grid bars (10–15/placenta). Magnification to the screen was x125,000. The profiles of mitochondria in each area were traced, providing estimates of the area of each profile, and of its aspect ratio or "roundness."
The surface density of the cristae was then measured according to the method of Mattfeldt et al. (16). A quadratic test grid was superimposed on the profiles, with the intersects of the lines acting as test points. Each test point was associated with a test line length of U nanometers on the screen. To correct for the apparent loss of membranes due to tangential sectioning, the number of intersections the test lines made with the outer boundary of the mitochondria, whether this could be clearly resolved or not, was counted I(tot;out). The number of these intersections at which the layers of the membrane could be clearly resolved was also counted I(app;out). This provided the correction factor R, represented by I(app;out)/I(tot;out). The number of intersections that could be identified with the inner mitochondrial membranes I(app;inn) was also counted, along with the number of test points, P, falling on the mitochondria. The surface density SV of an object is given by the formula SV = 2 x IL. The surface density of the inner mitochondrial membranes corrected for apparent loss of membranes was then derived from: SV(inn) = 2 x I(app;inn)/P x U x R. The volume fraction of the mitochondria represented by intracristal space was estimated by counting the number of test points falling on the intracristal space and expressing this as a fraction of the total number of points falling on the mitochondria.
Mitochondrial dehydrogenase activity
Activity within villous tissue was assessed immediately on receipt and after a period of culture using an adaptation of the MTT cleavage assay originally described by Mossman (17). Briefly, tissue (10–15 mg) was placed into a 0.5 mg/mL MTT solution (in culture medium) and incubated for 20 min at 37 C. MTT solution equilibrated at 2.5% oxygen was used for low oxygen tissue and at 21% oxygen solution for ambient oxygen tissue. Tissue was then briefly washed in medium 199 and frozen in Polyfreeze aqueous embedding medium in an isopentane slush. Cryosections (8 µm) were cut and covered with an aqueous mountant for viewing. Mitochondrial activity, as evidenced by incorporation of the blue-black formazan reaction product, was viewed and photographed via interference light microscopy (Axiophot, Zeiss, New York, NY).
Assessment of MTT staining
The pattern of MTT staining was assessed in a semiquantitative way as follows. Samples were again coded so that they were examined blind by the observer. Sections were viewed with a x40 phase contrast objective, enabling the syncytiotrophoblastic and cytotrophoblastic layers to be differentiated. Fields of view were selected in a systematic fashion with a random starting point. A test line was superimposed over the villous profiles. Where this intersected the trophoblast layer, it was recorded regardless of whether there was staining in the syncytiotrophoblast. This was repeated 50 times/placenta, and the frequency of syncytiotrophoblastic staining was expressed as a percentage. No instances were found when there was syncytiotrophoblastic staining without reaction product also being present in the underlying cytotrophoblast cells.
Immunostaining of cryosections
Fresh tissue of different gestational ages was washed briefly in medium 199 and immediately frozen in Polyfreeze tissue freezing medium by submersion in isopentane slush. Serial cryostat sections (7 µm) were cut using a Leica Jung CM3000 (Leica, Vienna, Austria) and mounted on gelatin-subbed slides. Before staining, sections were washed and rehydrated for 10 min in Tris-buffered saline (TBS; 20 mmol/L Tris-HCl and 0.15 mol/L NaCl, pH 7.4), fixed for 5 min in acetone at 4 C, and blocked for 15 min with 0.3% hydrogen peroxide in TBS and then 30 min with 5% BSA also in TBS. Immunoreactivity was determined via indirect immunohistochemistry using a sheep polyclonal antiserum to MnSOD (final IgG concentration, 30 µg/mL). Controls for nonspecific immunoreactivity were conducted using a purified sheep IgG at the same concentration. A second layer of biotinylated antisheep antiserum and a third of a avidin-biotin-peroxidase complex were then added to amplify the signal (Vectorstain ABC kit). Immunolabeling was developed using a Vector VIP chromogenic substrate kit. Sections were counterstained with hemotoxylin and mounted in Histotec aqueous mountant (Serotec, Ltd., Oxford, UK).
MnSOD activity
MnSOD activity was assessed according to a modification of the method described by Salin and McCord (18). Prewashed, frozen-thawed placental villous tissue ([aim]100 mg wet weight) was homogenized in 50 mmol/L potassium phosphate buffer (pH 7.4) containing 0.1 mmol/L ethylenediamine tetraacetate using a tight-fitting Potter homogenizer (Glas-col, Terre Haute, IN) at 4 C. The tissue debris was then pelleted at 13,000 rpm for 10 min in a bench-top microfuge, and the supernatant was used for protein concentration assessment (Sigma Total Protein Reagent).
Equivalent amounts of supernatant protein (200 µg) were loaded onto a native acrylamide gel for electrophoretic separation. The resolving gel comprised 7.5% acrylamide and a final Tris-acetate (pH 8.0) concentration of 0.1 mol/L. A stacking gel was used containing 5% acrylamide and a final Tris-acetate (pH 8.0) concentration of 0.1 mol/L. The gel was run in 0.1 mol/L Tris-acetate buffer, and samples were loaded in 0.02 mol/L Tris-acetate (pH 8.0). The gel was run for 4 h at 100 V (4 C).
After electrophoresis, gels were soaked in the dark for 45 min in a freshly prepared solution containing 50 mmol/L potassium phosphate (pH 7.8), 1 mmol/L ethylenediamine tetraacetate, 0.25 mmol/L NBT, 20 mmol/L tetramethylethylenediamine, and 30 µmol/L riboflavin. Gels were then washed briefly with two changes of distilled water and exposed for 30 min to an incandescent lamp. The photogenerated superoxide reduced the dye to an insoluble purple formazan, except for those areas where SOD was present, forming achromatic bands on the gel.
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Results |
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Figure 1A
is an electron micrograph
demonstrating the typical syncytiotrophoblastic ultrastructure at 9
weeks gestation. The nuclei are condensed, and there is an abundance of
organelles, particularly mitochondria, and a rich covering of
microvilli. The syncytiotrophoblastic morphology illustrated in Fig. 1B, which shows tissue from the same placenta after 4 h of culture
at 21% oxygen, is somewhat different. Now the syncytiotrophoblast is
extensively vacuolated, the microvillous cover has deteriorated, and it
is difficult, at this magnification, to identify mitochondria or other
organelles. When 9-week tissue is cultured for a similar period at
2.5% oxygen (Fig. 1C), it is immediately clear that the morphology
most closely approximates that of the tissue at time zero (Fig. 1A).
There is little or no vacuolation, and the brush border is still
largely intact. Under all circumstances described, the cytotrophoblasts
and stromal cells appeared healthy and unaffected.
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Figure 2A shows 9-week
syncytiotrophoblastic mitochondria at time zero. These mitochondria
have clearly defined cristae and an electron-dense matrix. Figure 2, B
and C, shows high magnification images of mitochondria from the
syncytiotrophoblast of 9-week tissue after 4 h of culture in 21%
oxygen and 2.5% oxygen, respectively. Under ambient oxygen
concentrations, the mitochondria became swollen, and their matrix less
electron dense. They had irregular shapes and displayed extensive
degeneration of their cristae, as is clear from the three remnants
shown in Fig. 2B. In many cases loss of structural integrity was so
extensive that recognition of organelles such as mitochondria was
difficult even at this higher magnification. Conversely,
syncytiotrophoblastic mitochondria cultured under low oxygen conditions
generally retained their structural integrity (Fig. 2C). Most were
regular in shape, were in a condensed state, and had clearly defined
cristae. Overall, there seemed to be a marked improvement in
mitochondrial appearance within tissue that had been maintained under
low oxygen con-ditions.
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). Thus, the cristae were distended and
distorted, rather than necessarily being fewer in number.
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Activity was virtually absent from syncytiotrophoblast of first
trimester placenta within 90 min if tissue was first transported to the
laboratory in the presence of aspirated products of conception (Fig. 3). When 8-week 4-day tissue was obtained
by chorionic villous sampling, mitochondrial activity declined less
dramatically, although still significantly, after 3 h of exposure
to ambient culture conditions (culture medium, pH 7.3, 21% oxygen;
Fig. 4A). This occurred despite a lag of
less than 45 s between tissue removal from the uterus and
immersion in culture medium. This deterioration continued and was
virtually complete after 6 h (Fig. 4B). However, we found that if
tissue was put into similar medium that had been equilibrated to 2.5%
oxygen, again within 45 s of removal, mitochondrial dehydrogenase
activity could be maintained at time zero levels for 6 h or more
(Fig. 4, C and D).
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). The same tissue
cultured at low oxygen retained full mitochondrial function, as
determined by the MTT assay (Fig. 5B).
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).
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Immunolocalization and activity of MnSOD
Antiserum to the human form of MnSOD was used for immunohistochemistry to establish patterns of expression within villous tissue. Staining was punctate on all occasions, with no general staining of the cytoplasm observed. This pattern of immunoreactivity was assumed to be due to the mitochondrial localization of MnSOD, although no confirmation of this specificity was obtained.
MnSOD immunoreactivity before 11 weeks was very weak, with only
occasional cells giving a signal. By 11 weeks, detection was more
widespread. The majority of the trophoblastic immunoreactivity at this
age was cytotrophoblastic (Fig. 6, A and
B), although there were a few regions where MnSOD could also be
detected in syncytiotrophoblastic mitochondria (Fig. 6B). Detection was
also seen within stromal cells. Immunostaining of tissue at 16 weeks
gestational age is shown in Fig. 6C. At this age the same punctate
pattern was found more regularly within the syncytiotrophoblast as well
as in cytotrophoblast and stromal cells. However, some areas of
syncytiotrophoblast still did not contain detectable levels of MnSOD.
The patterns of immunoreactivity shown here were consistently
reproduced on a number of occasions. No detection was seen when
nonimmune sheep IgG was used instead of the primary antibody.
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). The top band on the gel
shows MnSOD activity; it is about 52 kDa larger in the native state
than in the copper/zinc (Cu/Zn) form of the enzyme (lower
band, 85 vs. 33 kDa). The MnSOD band clearly shows a
significant increase in activity between the ages of 8 and 11 weeks 5
days gestational age. The barely detectable activity at 6 and 8 weeks
may explain the difficulty experienced in detecting the enzyme
immunohistochemically before 11 weeks 5 days. There was also a slight
increase in the detection of activity between 11-week 5-day and 14-week
tissue.
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Discussion |
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We have shown previously that the syncytiotrophoblast of placental villi from early pregnancy (<12 weeks) contains concentrations of the antioxidant enzymes Cu/ZnSOD (20) and catalase (21) that are barely detectable via immunohistochemistry. In contrast, the cytotrophoblast cells and the cells of the stromal core demonstrate strong immunoreactivity for these two enzymes. The reason for this apparently profound differential between cells of the same organ may lie in the reported lack of transcriptional activity within the syncytiotrophoblast (22), which will inevitably limit the synthetic capabilities of this tissue. Perhaps the more intriguing question, however, is how the syncytiotrophoblast is able to cope with so little antioxidant defenses, particularly considering that its location on the villous surface must ensure that it is the best oxygenated of all the fetal tissues. Interestingly, there is now a sizable body of opinion, based on physiological and anatomical evidence, that the amount of oxygen reaching the placenta during early pregnancy is relatively low (for review, see Ref.4). It is possible, therefore, that the early syncytiotrophoblast survives perfectly well in vivo without enzymatic defense, perhaps employing only nonenzymatic defenses such as vitamins A and C and reduced glutathione at this time. Thus, although the tissue may be adequately protected for a low oxygen environment in vivo, sudden exposure to ambient oxygen levels could render the syncytiotrophoblast acutely vulnerable to oxidative stress. It is possible that oxidative metabolic processes that have previously been limited by the restricted supply of oxygen may suddenly become more active as oxygen becomes readily available. It is widely held that such metabolic activities in the mitochondria are responsible for generating a significant proportion of cellular reactive oxygen species (ROS), in particular the superoxide anion (O2.-) (13, 23, 24). This theory is given extra credence by the observation that mitochondria contain their own enzyme for detoxifying superoxide, namely the manganese ion containing isoform of superoxide dismutase (MnSOD). Therefore, the observation made by ourselves as well as others (1) that the syncytiotrophoblast degenerates rapidly under conventional culture conditions could be explained in terms of oxygen sensitivity.
Oxidative stress is often described as a situation in which the cellular capacity for dealing with oxygen and derivative ROS is overwhelmed, resulting in damage to various cellular components and thus cell homeostasis (25). Therefore, the barely detectable tissue activity and absence of syncytiotrophoblastic immunoreactivity for MnSOD in pre-12-week placental villi suggest that the generation of superoxide within mitochondria during this period of gestation occurs at a very low rate in vivo. If appreciable quantities of free radicals were to be produced and then allowed to react with mitochondrial components, a likely outcome would be damage to membrane ion transporters, resulting in swelling of the intercristal space and inhibition of various dehydrogenases of the type detected here by the MTT assay. Our results are clearly consistent with such a sequence of events when first trimester syncytiotrophoblast is exposed to ambient oxygen concentrations.
A further inference of our findings is that if early tissue were cultured in a low oxygen environment, then prevention or at least retardation of the degenerative process should result. We found this to be the case; both mitochondrial morphology and dehydrogenase activity were largely retained, although only if introduction to low oxygen conditions was ensured within a very short time after termination of the pregnancy. The latter requirement exposes the rapidity with which the destabilizing effects of greater oxygen availability affect the syncytiotrophoblast and suggests that the ROS produced are capable of causing irreparable damage within a very short space of time.
As well as an elevated survival rate for the syncytiotrophoblast at lower oxygen tensions, there was also a trend for improved viability at ambient (21%) oxygen tension in tissue of increasing gestational age. Concomitantly, the syncytiotrophoblast of this tissue, as well as having more Cu/ZnSOD and catalase (20, 21), is shown here to contain greater concentrations of MnSOD. All of these enzymes provide the cell with protection against ROSs. Significantly, at all ages the cytotrophoblast and stromal cells contained Cu/ZnSOD, catalase, and to lesser extent MnSOD. Therefore, although stromal cells may well experience an oxidative stress at the same time as the syncytiotrophoblast, they have the requisite defenses for survival, as was demonstrated for all tissue studied. What may also be important, if not critical, here is the fact that cytotrophoblast and stromal cells contain full transcriptional machinery, thereby permitting them to respond to the changing environment by synthesizing new antioxidants.
By contrast, due to the general transcriptional inactivity of the syncytiotrophoblast, the response in this tissue will presumably be elicited in the cytotrophoblast cells. This would explain why areas of MnSOD immunoreactivity are restricted to cytotrophoblast cells when first detected immunochemically. As a result one would predict a considerable lag while a sufficient number of these cells fuse to existing syncytiotrophoblast and "feed" in their contents. The important contents here may be MnSOD-containing mitochondria or MnSOD protein alone, which is competent for transfer across the membranes of existing syncytiotrophoblastic mitochondria. It is also possible that a form of posttranscriptional regulation is in operation. Redox-sensitive binding proteins influencing the stability of the messenger ribonucleic acids for the main antioxidant enzymes have been identified in the lung (26, 27). Such a mechanism would provide for a more rapid response within the syncytiotrophoblast to changing conditions, but further work is required to establish whether it operates in the placenta.
The low concentrations of antioxidant enzymes in the syncytiotrophoblast are consistent with the theory that the oxygen tension in the feto-placental unit is low during the first trimester. In general, tissues are not overendowed with antioxidant enzymes, as there is now strong evidence that physiological concentrations of oxygen-derived free radicals play an important role in signaling pathways, enabling cells to respond quickly to changing oxygen availability (28, 29). It is possible that syncytiotrophoblastic vulnerability to ROS is a by-product of the need to transfer information regarding oxygen tension to deeper cells of the placenta and ultimately the fetus itself.
There are clearly many more questions to be addressed with respect to oxygen supply and placental physiology during the first trimester. It is also clear that given the nature of the syncytiotrophoblastic degeneration observed, there is a strong likelihood of clinical implications of oxygen sensitivity to placental operation and the success of an ongoing pregnancy. For example, the syncytiotrophoblast is the source of a number of hormones that are critical to maintaining the relationship between mother and fetus. Loss or even perturbation of this synthetic activity may have very serious implications for the conceptus.
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Acknowledgments |
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Footnotes |
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Received November 17, 1997.
Revised January 15, 1998.
Accepted February 13, 1998.
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