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Departamento de Bioquímica (F.M.), Facultad de Medicina, Universidad Nacional Autónoma de México, México, and Center for Research on Reproduction and Women’s Health and the Department of Obstetrics and Gynecology (M.K., J.F.S.), University of Pennsylvania, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: Dr. Federico Martínez, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado. Postal 70–159, 04510, Coyoacan, México, Distrito Federal, México.
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Introduction |
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Mitochondria are involved in the synthesis of progesterone; the first step in the formation of this steroid hormone is catalyzed by the cholesterol side-chain cleavage enzyme, P450scc, which is located on the inner mitochondrial membranes (7, 8, 9). Mitochondria have been isolated by differential centrifugation from human term placenta in dense, less dense, and heavy forms (10, 11, 12). The respiratory properties and progesterone synthesizing activities of these different mitochondrial fractions have been reported to be similar. In contrast, ultrastructural studies suggest that the mitochondria of the syncytiotrophoblast are significantly smaller than those found in the cytotrophoblast cells (1, 13, 14), indicating that there are differences among these organelles in the two main trophoblast phenotypes. The goals of the present work were to document that the mitochondria of syncytiotrophoblast cells have properties that are distinct from those of cytotrophoblast cells and to establish methods to prepare enriched preparations of syncytiotrophoblast mitochondria from human term placenta.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Electron microscopy and immunogold localization of
P450scc
Specimens for transmission electron microscopy were fixed in 2%
glutaraldehyde and postfixed with osmium tetroxide; sequentially
dehydrated with increasing concentrations of ethanol, and embedded in
an epoxy resin (14). Sections were cut and stained with uranyl acetate
and lead citrate, and observed in a Jeol electron microscope, operated
at 60 kV.
Placental tissue was fixed for immunogold studies with 4% paraformaldehyde, 0.2% glutaraldehyde and phosphate buffered saline (17). Immunostaining was performed on thin sections on nickel grids as described by Smith and Jarret (17). Sections were incubated with anti-P450scc antibody overnight at 4 C and the antibody was detected with 10 nm gold particles complexed to antirabbit Ig. Control sections were incubated with preimmune serum. Sections were stained with 2% uranyl acetate.
Isolation of mitochondria from human placenta
Placentas were collected immediately following normal delivery
at term. Mitochondria were prepared as previously reported (18).
Briefly, placental cotyledons were isolated and placed into ice-cold
250 mM sucrose, 1 mM EDTA, pH 7.4. The tissue
was washed three times with the same solution, and then minced into
small pieces. The tissue suspension was homogenized with a Polytron at
3,000 rpm for 1 min for two cycles at intervals of 1 min in a cold
room. The pH of the homogenate was adjusted to 7.4 with Tris and then
centrifuged at 1,500 x g for 15 min in a refrigerated
centrifuge. The supernatant was recovered and centrifuged at 4000
x g to pellet the large mitochondria and then the
supernatant was centrifuged again at 16,000 x g for 15
min. The pellet containing the mitochondria was suspended in the same
solution and centrifuged at 1,500 x g for 10 min to
remove remaining erythrocytes. Finally, mitochondria were pelleted at
12,000 x g for 10 min.
Culture of the BeWo choriocarcinoma cells and isolation of
mitochondria
The b30 clone of BeWo cells, kindly provided by Dr. Arnold
Schwartz (Washington University, St. Louis, MO) were cultured in DMEM
with 25 mM glucose, supplemented with 2 mM
L-glutamine, 10% heat inactivated FBS, 25 mM
HEPES, and 50 µg gentamicin per ml (19). When the cells attained 80
or 90% confluence, they were treated with 1 mM 8-Br-cAMP
for 3 days.
To isolate mitochondria, the BeWo cells were released from the culture dishes with 0.05% trypsin and 0.50 mM EDTA in calcium and magnesium-free HBSS solution during a 5–8 min incubation at room temperature. The released cells were collected by centrifugation at 1,000 x g for 10 min. The cell pellets were suspended in 10 ml of a solution containing 250 mM sucrose, 1 mM EDTA, and 5 mM HEPES, pH 7.2. After 5 min of continuous stirring in an ice bath, digitonin was added until 80% of the cells were lysed. The suspension was then diluted with 2 vol of the isolation medium and centrifuged at 3,000 x g for 10 min. The pellet was suspended in 3 ml of fresh isolation medium and homogenized with three to six strokes in a glass homogenizer. The homogenate was diluted to 35 ml, and centrifuged at 6,000 x g for 10 min. This last step was repeated three times and the supernatants were pooled, and centrifuged at 25,000 x g for 20 min. The pellet containing the mitochondria was suspended in the isolation media.
Sucrose and Percoll gradients
Discontinuous sucrose and Percoll gradients were used to purify
mitochondria. The sucrose gradients were made from 35–50% sucrose, 10
mM Tris-HCl, pH 7.4, at 5% intervals. The formation of
Percoll gradients was accomplished using a procedure modified from a
previously reported technique (20) by using 20, 25, 30, and 40% of
Percoll, maintaining the concentration of sucrose at 250
mM, 1 mM EDTA, pH 7.4, throughout the gradient.
The final vol for both gradients was 8 ml plus 1 ml of mitochondrial
protein (10 mg). Gradients were centrifuged at 104,000 x
g for 30 min at 4 C in an SW 40.1 rotor in a Beckman
refrigerated ultracentrifuge. Fractions were collected with a Pasteur
pipette, diluted with 250 mM sucrose, 1 mM
EDTA, pH 7.4, centrifuged at 16,000 x g for 15 min,
and the protein content was then determined by the dye-binding method
(21). Comparisons of the sucrose and Percoll (Sigma Chemical Co., St.
Louis, MO) gradient procedures for preparation of placental
mitochondria were performed with five different placenta to establish
the reproducibility of the methods.
Metrizamide gradients
A continuous gradient of 10–40% metrizamide (Nyegaard & Co.
To/S, Oslo, Norway) was prepared in a solution containing 1
mM EDTA and 5 mM HEPES, pH 7.3. An aliquot of
0.5 ml of mitochondria was layered over the gradient, with no more than
5 mg of protein/ml, and centrifuged at 106,000 x g,
for 75 min at 4 C in a SW41 rotor in a Beckman (Palo Alto, CA)
ultracentrifuge, as reported previously (22).
Aliquots of 0.5 ml, from the bottom to the top, were collected using a peristaltic pump. The fractions were diluted with 0.5 ml of the isolation medium and centrifuged at 14,000 rpm for 25 min in a cold room in an Eppendorf (Westbury, NY) centrifuge. The supernatants were discarded and 50 µl of sucrose-EDTA-HEPES solution was added. The concentration of protein was then determined.
Enzyme activities
Succinate dehydrogenase (SDH) activity was assayed at room
temperature in a dual-beam Aminco (Urbana, IL) DW-2a spectrophotometer,
using the difference between the absorbance at 550 nm and 575 nm. The
reaction medium contained 50 mM phosphate buffer, pH 7.6,
100 mM KCN, 25 mM cytochrome c, and
50 µg of sample protein in a final volume of 1 ml. After the baseline
was obtained, 6 mM sodium succinate, pH 7.6, was added to
start the reaction. SDH activity was followed for 1–2 min, and then 20
mM malonate was added to stop the reaction (23). SDH
activity was determined in mitochondria from BeWo cells using 10 or 20
µg of protein in a mixture containing: 50 mM phosphate
buffer, pH 7.6, 20 µM cytochrome c, and 100
µM KCN. SDH activity was calculated using an extinction
coefficient for cytochrome c of 22.7 mmol-1
cm-1. Activity is reported as µmol of cytochrome c
reduced mg-1 min-1.
Citrate synthase (Cit S) was assayed at room temperature as reported by Srere (24) using 50 µg of protein in a final volume of 1 ml.
Western blotting of cytochrome P450scc, adrenodoxin,
and cytochrome oxidase
SDS-PAGE was performed as described by Laemmli (25). For
cytochrome P450scc, 10% polyacrylamide gels were used,
whereas for adrenodoxin and cytochrome oxidase, 15% polyacrylamide
gels were employed. Eight micrograms of mitochondrial protein was
loaded in each lane. After the electrophoresis, the gels were placed on
a Immobilon membranes (Millipore, Bedford, MA) and proteins were
transferred overnight at 60 mV (26). Polyclonal antibodies raised
against bovine P450scc and adrenodoxin have been described
previously (15). The antibody against cytochrome oxidase was a gift of
Dr. Diego González-Halphen, Instituto de Fisiología
Celular, UNAM, México, which was prepared with the technical
assistance of Q. F. B. Miriam Vázquez-Acevedo (27). The membranes
were incubated overnight in at 4 C with the primary antibodies and then
washed with a saline solution containing 0.1% of Tween 20 and
incubated with the second antibody (antirabbit IgG peroxidase) at a
dilution of 1:2,000 at room temperature for 2 h. The membranes
were then washed and incubated with chemiluminescence detection
reagents (ECL, Amersham) and exposed to film.
Determination of respiratory chain cytochromes
Samples (0.5 mg) were added to one ml of a medium containing 50
mM phosphate buffer, pH 7.6, and 100 mM KCN. A
baseline from 400–620 nm was taken and subtracted from each scan. The
cytochromes were revealed after adding 6 mM sodium
succinate and dithionite to the sample cuvette as reported previously
(28).
Pregnenolone synthesis
Transformation of 22(R)-OH-cholesterol into pregnenolone by
isolated mitochondria was measured in the following media: 250
mM sucrose, 5 mM MgSO4, 20
mM KH2PO4, 25 mM
Tris-HCl, 0.2 mM EDTA, 1 mg BSA/ml, 10 mM
trilostane, 15 µM 22(R)-OH-cholesterol, 20 mM
glucose-6-phosphate, and 3 U glucose-6-phosphate dehydrogenase.
Mitochondrial protein (0.25 mg) in a total volume of 0.5 ml was
incubated at 37 C. The reaction was started by the addition of 5
mM isocitrate and 0.5 mM NADP+. At
the indicated times, an aliquot of 0.1 ml was withdrawn and mixed with
0.5 ml petroleum ether to stop the reaction. Pregnenolone was extracted
twice with petroleum ether. The evaporated samples were used to measure
pregnenolone by RIA, as described by (29) using an antibody kindly
provided by Dr. Charles Strott (NIH, Bethesda, MD).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The occasional
cytotrophoblast cells in the chorionic villi were not stained.
Identical results were obtained with the two different
anti-P450scc used. Ultrastructural examination revealed two
forms of mitochondria in the trophoblast cells (Fig. 2).
Large mitochondria were found in the cytotrophoblast cells. These
organelles had a structure similar to the morphology of liver
mitochondria, with lamellar cristae in an orthodox configuration. The
syncytiotrophoblast contained smaller mitochondria with a condensed
matrix and vesicle-like cristae. The larger cytotrophoblast
mitochondria had a round shape, whereas the syncytiotrophoblast
mitochondria were irregular in shape with protuberances of the outer
and inner membranes. P450scc was localized in the small
syncytiotrophoblast mitochondria by immunogold electron microscopy
(Fig. 3). Approximately 75% of the mitochondrial
profiles in the syncytiotrophoblast contained from 1–5
particles/profile. Gold particles were rarely found in the
cytotrophoblast mitochondria (<7% of profile contained a gold
particle), confirming the distribution of P450scc observed
at the light microscope level. When the sections were stained with
preimmune serum, gold particles were infrequently seen (<1% of
mitochondrial profiles contained a gold particle).
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Mitochondria are generally isolated by differential centrifugation at 2,500–2,800 x g followed by a second centrifugation of the supernatant at 9,800–16,000 x g, in which mitochondria are pelleted. To separate large from small mitochondria, we included another centrifugation at 4,000 x g. The pellets obtained at 4,000 and 16,000 x g contained SDH activity, an enzyme exclusively found in mitochondria.
Mitochondria sedimented at 16,000 x g were first
loaded onto a sucrose gradient (from 25–50%). Four well-defined bands
at the 30–35, 35–40, 40–45, and 45–50% interfaces were observed
(Fig. 4). A gradient from 35–50% sucrose resulted in
better resolution of these bands. The bands containing mitochondria
were localized by measurements of SDH and citrate synthase (Cit S)
activities, and pregnenolone synthesis. In addition, cytochrome
P450scc and adrenodoxin were identified by Western
blotting, and cytochromes from the electron transport chain were
determined by spectrophotometry. Electron microscopic examination of
each band was performed.
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shows the distribution of the mitochondrial fractions in the
sucrose gradient, and the activities of SDH and Cit S in the four
bands. Band 2 had four times higher SDH activity and two times greater
Cit S activity compared with the starting material, suggesting that
band two contains intact mitochondria, since enzymes of the matrix
(e.g. Cit S) were retained.
P450scc and adrenodoxin distribution among the different
bands is shown in Fig. 4B. P450scc in band 2 was enriched
approximately 4- to 6-fold, while adrenodoxin was enriched around 3- to
4-fold compared with the starting material. Electron microscopy
demonstrated that band 1 contains a mixture of vesicles with some
swollen mitochondria (Fig. 5). Band 2 contained mostly
mitochondria, with some broken outer membranes. Bands 3 and 4 contained
a mixture of mitochondria, vesicles, and other unidentified structures.
As noted above, low SDH and Cit S activities were observed in these two
bands as well as a low concentration of P450scc and
adrenodoxin.
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). The characteristic
Soret 
, ß- and 
-bands of cytochrome c are at
415–420 nm, 521–524 nm and 550–556 nm, respectively. Cytochrome
b is detected at 427–432 nm, 528–531 nm and 559–566 nm.
The band at 444 nm and the -band at 601–605 nm reflect
cytochrome oxidase (cytochrome aa3). Band 2 of
the sucrose gradient contained the cytochromes of the respiratory chain
in higher concentrations than the other bands.
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shows the time course of pregnenolone
synthesis for each of the bands. Band 2 synthesized four to five times
greater amounts of pregnenolone than the starting material. The
relative cholesterol side-chain cleavage activities paralleled the
distribution of P450scc and adrenodoxin in the different
bands as determined by Western blotting.
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).
A gradient of sucrose was always run in parallel with the Percoll
gradient for comparison. SDH and Cit S activities from the Percoll
gradient are shown in Fig. 4A. Only three bands were obtained, and band
2 contained the highest activities of SDH and Cit S. The other
functional properties of the mitochondria studied including contents of
P450scc and adrenodoxin and ability to convert
22(R)-0H-cholesterol into pregnenolone were generally similar to those
obtained with mitochondria isolated on sucrose gradients (Figs. 4B, 6, and 7), suggesting that band 2 from the Percoll gradient was enriched
with syncytiotrophoblast mitochondria. Electron microscopic analysis
confirmed that band 2 contained intact mitochondria (data not shown).
Some differences between mitochondria isolated on sucrose and Percoll
gradients were noted including a lower rate of pregnenolone synthesis
during the initial 5 min of incubation by mitochondria isolated on
Percoll gradients in association with a different spectrum of reduced
cytochromes.
Differentiation of mitochondria in BeWo choriocarcinoma cells
induced by 8-Br-cAMP
The ultrastructural and biochemical observations discussed above
imply that cytotrophoblast mitochondria undergo a process of structural
and functional differentiation as they form syncytiotrophoblast cells.
It has been reported that a clonal line of BeWo choriocarcinoma cells,
which display features of normal cytotrophoblast cells (31), can be
induced to differentiate into syncytial structures by cAMP (19). cAMP
analogs also increase the abundance of mRNAs encoding steroidogenic
enzymes, including P450scc, in choriocarcinoma cells and
substantially stimulate their secretion of progesterone (32). To
determine if cAMP-induced differentiation of BeWo cells is associated
with changes in mitochondrial structure, we analyzed cells after 3 days
of treatment with control medium or 1 mM 8-Br cAMP. This
treatment protocol with 8-Br-cAMP converts >70% of the cells into
syncytia (19). Compared with the untreated BeWo cells, the cells
exposed to the cAMP analog had smaller mitochondria which contained
fewer cristae, often with a vesicular configuration (Fig. 8). The mean of the largest diameter of the
mitochondrial profiles of the control cells (N = 22) was 1.4 times
greater (P < 0.005 by Student’s t test)
than that of the mitochondrial profiles in photomicrographs of the
cells treated with 8-Br-cAMP (N = 16).
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). Also,
the highest amount of cytochrome oxidase was observed in band 3, and
the SDH activity paralleled this distribution (Fig. 9).
A substantial increment of cytochrome P450scc was observed
in mitochondria isolated from BeWo cells treated with 8-Br-cAMP
compared with control cells, whereas the amount of cytochrome oxidase
was equivalent in the control cells and in cells treated with
8-Br-cAMP.
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). Cytochrome oxidase was
most abundant in fraction 4. In control cells, fraction 3 contained the
highest content of cytochrome oxidase, suggesting a shift in density of
the organelles.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported that the cytoplasmic differentiation of primary cultures of human cytotrophoblast cells isolated from term placenta treated with 8-Br-cAMP included the accumulation of numerous small mitochondria (14). These observations suggest that mitochondrial changes can be induced in cultured trophoblast cells. However, these primary cultures contain heterogeneous populations of cells at different stages of differentiation and the cells have limited replicative activity (5). The inability to fully synchronize differentiation and the limited amount of material make it difficult to isolate subcellular organelles from primary cultures (5, 33). To examine the structural and functional differentiation of trophoblast mitochondria in vitro, we studied a clonal line of BeWo choriocarcinoma cells that are transformed from cytotrophoblast-like cells into multinucleated syncytia by cAMP in association with a marked increase in steroidogenic activity. The ability to initiate the differentiation process with a homogeneous cell population and the rapid growth of the BeWo cells offer distinct advantages over the primary cultures. Three days of 8-Br-cAMP treatment produced significant morphological changes in the BeWo cell mitochondria including a reduction in size and a marked change in the configuration of the cristae accompanied by a substantial increase in P450scc content. The structural changes, although showing trends towards those implied from the analysis of cytotrophoblast and syncytiotrophoblast cells in situ, did not achieve the striking size and matrix alterations seen in the syncytiotrophoblast mitochondria or in primary cultures of term cytotrophoblast cells treated with 8-Br-cAMP (14). This probably reflects the recognized inability of the BeWo cell lines to undergo the complete program of trophoblast terminal differentiation (6, 19).
The reduction in size of the mitochondria and the change in the structure of the cristae may improve the steroidogenic activity of the syncytiotrophoblast mitochondria. The smaller surface to volume ratio could enhance the movement of cholesterol, the P450scc substrate, to the inner membranes where the enzyme is located. The translocation of cholesterol to P450scc has long been known to be a rate-limiting step in steroidogenesis that is facilitated in gonadal and adrenal steroid producing cells by steroidogenic acute regulatory protein (StAR). However, the StAR gene is not expressed in the human placenta or choriocarcinoma cells (34). Thus, other mechanisms must subserve the role of StAR in the human placenta. It is attractive to speculate that structural changes in the mitochondria, such as those described in this report, allow the syncytiotrophoblast mitochondria to efficiently transfer cholesterol from the outer to inner membranes in the absence of StAR.
What causes the change in mitochondrial structure as cytotrophoblasts differentiate into syncytiotrophoblast? Our observations on the BeWo choriocarcinoma cells suggest that cAMP could be one factor that triggers the mitochondrial differentiation process. This could be the result of an effect of the cyclic nucleotide on the phosphorylation status of mitochondrial proteins or the consequence of activation of a generalized differentiation program at the genomic level. For example, cAMP increases the levels of proteins that are imported into mitochondria, including P450scc, and this change in mitochondrial protein composition could influence organelle structure. The mitochondrial changes may take place as syncytiotrophoblasts form or they may occur over a longer time span, perhaps as the syncytiotrophoblast undergoes maturation. The concept of progressive syncytiotrophoblast maturation was recently proposed by Babischkin et al. (35), who reported increasing accumulation of P450scc mRNA as pregnancy advances in syncytiotrophoblast-enriched fractions obtained from baboon placentas, whereas relative adrenodoxin mRNA abundance was constant.
In addition to cAMP, drugs that act on the peripheral benzodiazepine receptor induce morphological changes in mitochondria and mitochondrial proliferation. Shiraishi et al. (36) demonstrated that these agents cause changes in mitochondrial structure and increase the number of mitochondria and the percentage of dividing mitochondria in glioma cells. Others have suggested that drugs that act on the peripheral benzodiazepine receptor modulate mitochondrial function, causing inhibition of mitochondrial respiratory control (37) and decreasing O2 consumption in mouse neuroblastoma cells. It is notable that human term placental mitochondria display a low respiratory capacity, with respiratory control rations of 2.5–5 (38). Moreover, peripheral benzodiazepine receptor ligands stimulate steroid secretion by human placental explants (39). Thus, the possibility that the endogenous ligand for the peripheral benzodiazepine receptor promotes changes in mitochondrial structure that alter respiratory activity and enhance steroidogenesis is attractive, but at this juncture still speculative.
Human term placental mitochondria have been isolated by others using sucrose gradients and differential centrifugation (40, 41, 42, 43). In these reports, electron microscopic observations revealed size heterogeneity with some contamination by nonmitochondrial membranes. Gasnier et al. (30), using a Percoll gradient, isolated placental mitochondria. However, only two steps of centrifugation were employed and the electron microscopic images showed large mitochondria, similar to those observed in the cytotrophoblast. In placenta obtained early in gestation, isolated mitochondria had a broad range of sizes with significant contamination (43). Here we demonstrate that sucrose and Percoll gradients are useful tools that can be used to obtain highly enriched preparations of steroidogenic mitochondria from human placenta and BeWo choriocarcinoma cells. In contrast to previously reported methods, we employed a sequential centrifugation protocol to obtain the small mitochondria from human placenta that correspond to the P450scc-containing mitochondria of the syncytiotrophoblast. The mitochondrial fractions enriched with P450scc displayed 2- to 5-fold greater cholesterol side-chain cleavage activity with 22(R)-0H-cholesterol as substrate than reported by others using human placental mitochondria isolated by differential centrifugation alone (44). Although highly purified mitochondria were obtained using both sucrose and Percoll gradients, there were some differences in the characteristics of the organelles isolated on the two different gradients, probably reflecting the effects of the separation media (40).
The human placenta at term weighs approximately 500 g and 100 g of this mass is syncytiotrophoblast (45). The placenta produces about 250 mg of progesterone/day. Assuming that 10% of the wet weight of syncytiotrophoblast is protein and 10% of the cellular protein is mitochondrial protein, the syncytiotrophoblast mitochondria in vivo produce 250 mg of progestin/g of protein. The placental mitochondria that we isolated on sucrose or Percoll density gradients produce approximately 270 mg of pregnenolone from 22(R)-0H-cholesterol/day·g of protein. Thus, the method we have developed appears to yield organelles that reflect the synthetic capacity of the syncytiotrophoblast mitochondria in situ. It is important to note that the BeWo cell mitochondria displayed a slightly different pattern of distribution on density gradient centrifugation compared with placental mitochondria. These differences could be related to differences in the lipid composition and the protein content of the organelles.
In summary, this report documents structural and functional changes in trophoblast mitochondria with the formation of syncytial structures. Methods to isolate highly enriched preparations of steroidogenic mitochondria from human trophoblast have been established and an in vitro system that can be used to explore the mechanisms underlying the alterations in trophoblast mitochondrial morphology and function associated with terminal differentiation in the trophoblast lineage has been characterized.
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Acknowledgments |
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Footnotes |
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Received December 12, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-3ß-hydroxysteroid dehydrogenase/isomerase messenger
ribonucleic acids during baboon pregnancy. Placenta 17:595–602[CrossRef][Medline]
-glycerophosphate oxidation by human placental
mitochondria. Biochim Biophys Acta 429:46–54[Medline]
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