Atypical Cristae Morphology of Human Syncytiotrophoblast Mitochondria

Mitochondrial complexes I, III2, and IV from human cytotrophoblast and syncytiotrophoblast associate to form supercomplexes or respirasomes, with the following stoichiometries: I1:(III2)1 and I1:(III2)1–2:IV1–4. The content of respirasomes was similar in both cell types after isolating mitochondria. However, syncytiotrophoblast mitochondria possess low levels of dimeric complex V and do not have orthodox cristae morphology. In contrast, cytotrophoblast mitochondria show normal cristae morphology and a higher content of ATP synthase dimer. Consistent with the dimerizing role of the ATPase inhibitory protein (IF1) (García, J. J., Morales-Ríos, E., Cortés-Hernandez, P., and Rodríguez-Zavala, J. S. (2006) Biochemistry 45, 12695–12703), higher relative amounts of IF1 were observed in cytotrophoblast when compared with syncytiotrophoblast mitochondria. Therefore, there is a correlation between dimerization of complex V, IF1 expression, and the morphology of mitochondrial cristae in human placental mitochondria. The possible relationship between cristae architecture and the physiological function of the syncytiotrophoblast mitochondria is discussed.

cytotrophoblast and the syncytiotrophoblast. The cytotrophoblast shows mitotic activity and is in contact with the endometrium; by contrast, the syncytiotrophoblast is facing the uterus, has no mitotic activity, and displays a high steroidogenic activity, with progesterone as the main product. We have described previously that the morphology of the syncytiotrophoblast mitochondria is far from typical, showing a highly condensed matrix and an irregular folding of the mitochondrial inner membrane (27). The present study represents the first report addressing the presence and analysis of respiratory chain complexes and supercomplexes of human placental mitochondria and their role in cristae morphology. It is shown that the atypical morphology of the syncytiotrophoblast mitochondria correlates with low contents of dimeric F 0 F 1 -ATP synthase and of the inhibitory IF 1 subunit, confirming the key role played by these two factors in determining mitochondrial cristae morphology.

Isolation of Human Cytotrophoblast and Syncytiotrophoblast
Mitochondria-Full term human placenta was collected immediately after normal delivery. Mitochondria were prepared as described previously (27). To purify mitochondria of cytotrophoblast or syncytiotrophoblast, the enriched mitochondrial suspension was loaded on a 35% sucrose solution (25 ml) and centrifuged at 15,000 ϫ g for 45 min, at 4°C. The two mitochondrial fractions were collected and centrifuged at 16,000 times] g for 15 min at 4°C, and the mitochondrial pellet was resuspended in 250 mM sucrose, 10 mM Tris (pH 7.4) and stored at Ϫ70°C until use. Protein concentration was measured as reported (28,29).
ATP synthesis by complex V was measured at 37°C using an assay coupled to the reduction of NADP ϩ (⑀ 340 nm ϭ 6.2 mM Ϫ1 cm Ϫ1 ). The reaction mixture contained 0.5 mM NADP ϩ , 1 mM ADP, 6 units/ml glucose-6-phosphate dehydrogenase, 16 units/ml hexokinase, 10 mM succinate, 100 M P 1 , P 5 -Di(adenosine-5Ј)pentaphosphate penta-ammonium, 10 mM glucose, 150 mM sucrose, 5 mM MgCl 2 , 20 mM Tris/HCl, and 20 mM KH 2 PO 4 , pH 7.5. ATP synthesis was started by the addition of cytotrophoblast or syncytiotrophoblast mitochondria (50 g/ml). The values reported were obtained by subtracting the rate of ATP synthesis in the presence of oligomycin (5 g/mg mitochondrial protein) from the amount of ATP synthesis in the control conditions.
Mitochondrial Progesterone Synthesis-Progesterone synthesis was determined at 37°C as reported previously (30) in 120 mM KCl, 10 mM MOPS, 0.5 mM EGTA, 10 mM isocitrate, 4 g of aprotinin/ml, 1 M leupeptin, 5 mM KH 2 PO 4 , pH 7.4, in a final volume of 500 l with 1 mg/ml cytotrophoblast or syncytiotrophoblast mitochondrial protein. Where indicated, 25 M 22(R)-hydroxy-cholesterol was added. After a 60-min incubation, the reaction was stopped with 50 l of methanol, and progesterone was determined by a radioimmunoassay kit (Diagnostic Systems Laboratories, Inc., Webster, TX) according to the manufacturer's instructions. The concentration of progesterone at time 0 was subtracted from the amount of progesterone at 60 min, and this net value of progesterone synthesis was reported.
Electron Microscopy-Samples for transmission electron microscopy were fixed in 2% glutaraldehyde, post-fixed with osmium tetroxide, and sequentially dehydrated with increasing concentrations of ethanol. Finally, the samples were embedded in an epoxy resin (31). Sections were cut and stained with uranyl acetate and lead citrate and observed under 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 (31).
Sample Preparation for Native Electrophoresis-The respiratory complexes from either cytotrophoblast or syncytiotrophoblast mitochondria were resolved by native PAGE following the general procedures reported previously (32,33) with minor modifications. Briefly, cytotrophoblast (2 mg) or syncytiotrophoblast (2 mg) mitochondria were suspended in 50 mM Bis-Tris and 500 mM 6-aminocaproic acid, pH 7.0, and solubilized by adding dodecyl-␤-D-maltoside or digitonin, at detergent/ protein ratios ranging from 0.5 to 10 (g/g), in a final volume of 200 l. The mixtures were incubated for 30 min at 4°C and centrifuged at 100,000 times] g for 30 min at 4°C. The supernatants were recovered and immediately loaded on a linear polyacrylamide gradient gel (5-10% or 3.25-7.5%) for Blue Native PAGE (BN-PAGE) or Clear Native PAGE (CN-PAGE) (34). The molecular weight of each respiratory complex or supercomplex was estimated by using the bovine mitochondrial complexes as standard.
In-gel Catalytic Activity Assays-The in-gel activity assays were performed as described by Wittig et al. (34). Gel strips were assayed for complex I activity (NADH:methylthiazolyldiphenyl tetrazolium bromide reductase), complex II activity (succinate:methylthiazolyldiphenyl tetrazolium bromide reductase), and complex IV activity (cytochrome c:diaminobenzidine reductase). In all cases, the assays were performed at 20 -25°C and stopped with 50% methanol, 10% acetic acid, after 10 -25 min.
To estimate the in-gel ATP hydrolysis activity of monomeric and dimeric complex V and of the F 1 -ATPase subcomplex, gel strips were preincubated in 50 mM glycine (adjusted to pH 8.0 with triethanolamine) for 2 h at 37°C in the presence or absence of the complex V inhibitor oligomycin (5 g/ml). The equilibration solution was discarded, and the gel strips were then added into the assay buffer (50 mM glycine, adjusted to pH 8.0 with triethanolamine, 10 mM MgCl 2 , 0.2% Pb(NO 3 ) 2 , and 8 mM ATP), with or without 5 g/ml oligomycin. ATP hydrolysis correlated with the development of white lead phosphate precipitates. The reaction was stopped using 50% methanol, and subsequently the gel was transferred to water and scanned against a dark background as described previously (11,35).
Two-dimensional Tricine-SDS Gel Electrophoresis and Western Blot Analysis-Proteins of a gel lane excised from native PAGE were separated by two-dimensional Tricine-SDS-PAGE according to Ref. 32 on a 16% polyacrylamide gel under denaturing conditions. Afterward, the proteins were stained with either Coomassie Brilliant Blue R-125 or silver using a commercial kit (Bio-Rad). Alternatively, the gels were electrotransferred to polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA) in a semidry electroblotting system (Bio-Rad) at 25 V for 50 min or 2 h at 100 mA in a buffer containing 100 mM CAPS, 10% methanol (pH ϭ 11). Membranes were blocked in 500 mM NaCl, 0.05% Tween 20, and 20 mM Tris base, pH 7.5 (TTBS buffer) containing 5% blotting grade blocker nonfat dry milk (Bio-Rad). The antibodies used were anti-␣ subunit polyclonal antibody (1:10,000), anti-␤ subunit antibody (1:250), anti-OSCP subunit antibody (1:1000), and anti-IF 1 monoclonal antibody (1:10,000). Membranes were incubated for 1 h with the primary antibody in TTBS buffer and washed thoroughly, and the immunoreactive bands were visualized with the enhanced chemiluminescence assay (Amersham Biosciences) according to the manufacturer's instructions, using horseradish peroxidase-conjugated goat antimouse IgG (Pierce) at a dilution of 1:35,000, and the densitometric analyses were performed with the Alpha-DigiDoc 1000 software (AlphaEaseFC TM from Alpha Innotech Corp.). The intensities of subunits were measured by peak integration after densitometry analyses. Table 2) were excised from the Coomassie Brilliant Blue R-125stained SDS gels, distained, reduced, carbamidomethylated, and digested with modified porcine trypsin (Promega, Madison, WI). Peptide mass spectrometric analysis was carried out using a 3200 Q TRAP hybrid tandem mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Ontario, Canada), equipped with a nanoelectrospray ion source (NanoSpray II) and a MicroIonSpray II head (36). The instrument was coupled on-line to a nanoAcquity Ultra Performance LC system supplied by Waters (Waters Corp., Milford, MA). Briefly, spectra were acquired in automated mode using information-dependent acquisition. Precursor ions were selected in Q1 using the enhanced MS mode as survey scan. The enhanced MS mode was followed by an enhanced resolution scan of the three most intense ions at the low speed of 250 atomic mass units/s to determine the ion charge states and afterward by an enhanced product ion scan. The precursor ions were fragmented by collisionally activated dissociation in the Q2 collision cell. The fragment ions generated were captured and mass analyzed in the Q3 linear ion trap.

Tandem Mass Spectrometry (LC/ESI-MS/MS)-The protein spots (indicated by numbers in Figs. 2A and 3C and
Database searching and protein identification were performed with the MS/MS spectra data sets using the MASCOT search algorithm (version 1.6b9, Matrix Science, London, UK). Mass tolerances of 0.5 Da for the precursor and 0.3 Da for the fragment ion masses were used. Carbamidomethyl-cysteine was the fixed modification, and one missed cleavage for trypsin was allowed. Searches were conducted using the Human subset of the NCBInr database (www.ncbi.nih.gov). Protein identifications were accepted when at least two MS/MS spectra matched at 95% confidence level (p Ͻ 0.05).
Materials-Analytical grade reagents were purchased from Sigma, Merck (Darmstadt, Germany), and Bio-Rad. Antibodies were purchased from Santa Cruz Biotechnology and MitoSciences.

RESULTS
Human Placental Mitochondria Architecture-Transmission electron micrographs of term placenta sections revealed two morphological types of mitochondria in the trophoblast cells (Fig. 1A). Large mitochondria were observed in the cytotrophoblast cells, whose morphology is similar to that of typical liver mitochondria, containing lamellar (and presumably tubular) cristae in an orthodox configuration. In contrast, the syncytiotrophoblast contained smaller mitochondria with a condensed matrix and cristae composed by vesicular regions connected by narrow tubules. The larger cytotrophoblast mitochondria had a round shape, whereas the syncytiotrophoblast mitochondria display an irregular shape with protuberances of the outer and inner membranes. Isolated cytotrophoblast and syncytiotrophoblast mitochondria only partially retained these structural characteristics (Fig. 1B); in particular, the isolated cytotrophoblast mitochondria had partially lost the original cristae configuration seen in situ (Fig. 1A).
Physiological State of Cytotrophoblast and Syncytiotrophoblast Mitochondria-To determine the functional integrity of isolated cytotrophoblast and syncytiotrophoblast mitochondria, respiratory rates and respiratory controls were calculated from oxygen uptake traces using succinate as substrate ( Table  1). Values of respiratory control ranged between 2.85 and 12, higher than those previously reported for this tissue (37); ATP synthesis by complex V was 151 Ϯ 16 and 153 Ϯ 13 nmol/mg/ min for cytotrophoblast and syncytiotrophoblast mitochondria, respectively, suggesting coupling of mitochondrial respiration and ATP synthesis. In addition, we obtained an activity of 113-110 mol/mg/min for the NADH:DCPIP oxide reductase activity (complex I) and 12-14 mol/mg/min for the succinate:DCPIP oxide reductase activity (complex II) ( Table 1). These results indicate the presence of functional mitochondria in both cell types, retaining the ability to increase the consumption of oxygen and the synthesis of ATP upon the addition of ADP.
Human placental mitochondria are steroidogenic organelles that synthesize progesterone due to the presence of the 3-␤hydroxy steroid dehydrogenase in their inner membrane (27,38,39). Because there are two types of mitochondria, its steroidogenic activity was determined. Table 1 shows that synthesis of progesterone by syncytiotrophoblast mitochondria (35.7 Ϯ 0.9 ng of progesterone/mg/min) was 10-fold higher than that of cytotrophoblast mitochondria (3.6 Ϯ 1.34 ng of progesterone/mg/min). In both cases, 22(R)-hydroxy-cholesterol, a soluble substrate used to assess maximal steroidogenic activity (40), increased steroidogenic activity to 92.2 Ϯ 3.4 and 10.1 Ϯ 3.95 ng of progesterone/mg/h in syncytiotrophoblast and cytotrophoblast mitochondria, respectively. These results are in accordance with the specialized role of each placental tissue (27) and support that isolated mitochondria retain their physiological function.
Identification of Respiratory Supercomplexes in Mitochondria from Cytotrophoblast and Syncytiotrophoblast Cells-To investigate the optimal condition for the separation of mitochondrial complexes and supercomplexes, isolated mitochondria from both cytotrophoblasts and syncytiotrophoblasts were solubilized by varying concentrations of either digitonin or dodecyl-␤-D-maltoside (DDM). Results showed that 1-2 g of DDM/g of mitochondrial protein allowed the solubilization of the respiratory chain complexes in their monomeric state ( Fig.  2A), except for complex III, which exists as a stable dimer. BN-PAGE allowed the resolution of all the complexes of the oxidative phosphorylation system (Fig. 2A). The identity and position of complexes I, II, IV, and V on gels were determined using specific reactions for these complexes (Fig. 2A). The molecular mass estimated for each complex was: complex I, 1000 kDa; complex V, 750 kDa; complex III 2 , 500 kDa; complex IV, 200 kDa; and complex II, 130 kDa. In addition, the identity of each complex was confirmed by their known subunit composition upon resolution on two-dimensional SDS-PAGE and by identification of subunits by mass spectrometry (Fig. 2B and Table  2). Identical results were obtained for cytotrophoblast and syncytiotrophoblast mitochondria ( Table 2).
With digitonin as the solubilizing agent, it was possible to isolate the mitochondrial respiratory chain components as individual entities or as supercomplexes. The optimal digitonin/mitochondrial protein ratio was found between 1.5 and 10. However, the abundance of some supercomplexes decreased slightly at higher detergent/protein ratios; therefore, all further experiments were carried out with a digitonin/protein ratio of 1.5 (g/g). The identity and position of the individual complexes I, V, IV, and II, as well as the composition of supercomplexes named a-e, were determined using specific reactions for these complexes (Fig. 3A). The molecular mass of each individual complex was essentially identical to that obtained using DDM for the solubilization process (Table 2). However, the presence of five different supercomplexes was clearly evident in the gel. To resolve the composition of these supercomplexes, BN-PAGE was carried out in 3.25-7.5% linear polyacrylamide gradient gels. As shown in Fig. 3B, about 70% of the NADH: methylthiazolyldiphenyl tetrazolium bromide oxidoreductase activity was located at the position corresponding to the supercomplexes, whereas the cytochrome c:diaminobenzidine oxidoreductase activity was detected only in three of them (c-e) (Fig. 3B). Occurrence of complex III was determined by twodimensional SDS-PAGE followed by spot identification by mass spectrometry (Fig. 3C). The molecular masses estimated for the supercomplexes and their possible stoichiometry were: a ϭ 1,300 kDa (I 1 :III 2 ); b ϭ 1,500 kDa (I 1 :III 2 ); c ϭ 1,700 kDa (I 1 :III 2 :IV 1 ); d ϭ 2,000 kDa (I 1 :III 2 :IV 2-3 ); and e ϭ 2,300 kDa (I 1 :(III 2 ) 1-2 :IV 1-4 ).
Monomeric and Dimeric F 0 F 1 -ATP Synthase in Mitochondria from Cytotrophoblast and Syncytiotrophoblast-Solubilization of cytotrophoblast and syncytiotrophoblast mitochondria with digitonin (1.5 mg/mg of protein) allowed the visualization of the activity of the monomeric (V) and dimeric Respiratory control ϭ oxygen uptake ratio to state 3 (natoms g of oxygen)/oxygen uptake ratio to state 4 (natoms g of oxygen). c Progesterone synthesis was determined as described in experimental procedure. The 22(R)-hydroxy-cholesterol was used to assess maximal steroidogenic activity. Values shows here are the mean Ϯ S.D. from seven to eight determinations, from eight different placental tissues.
(V 2 ) forms of the F 0 F 1 -ATP synthase complex in BN-PAGE (Fig. 3A). It has been reported that the ATPase activity of the F 0 F 1 complex is affected by the Coomassie Brilliant Blue R-125 used in the BN-PAGE (41); therefore, we used the CN-PAGE to visualize the F 0 F 1 complex activity (Fig. 3A, right panel). In accordance with a previous study (42), the F 0 F 1 -ATP synthase is present in two forms, a monomeric complex of about 750 kDa and a dimeric complex of about 1500 kDa. In cytotrophoblast mitochondria, the in-gel ATPase activity of V, evidenced as a white precipitate, was greater than that of V 2 (Fig. 3A, right  panel), whereas in syncytiotrophoblast mitochondria, this difference in ATPase activity was much larger. Indeed, in some syncytiotrophoblast mitochondria preparations, the ATP synthase activity was represented by the monomeric form exclusively. Thus, the V 2 ATPase activity for the cytotrophoblast mitochondria was higher than that of syncytiotrophoblast mitochondria. Because the differences in intensity of in-gel ATPase bands may result from different specific activities, twodimensional SDS-PAGE was carried out after BN-PAGE to  Table 2).

TABLE 2 Molecular mass and subunit identity of cytotrophoblast and syncytiotrophoblast mitochondrial respiratory complexes
The molecular mass of the complexes was determined by BN-PAGE using mitochondrial respiratory complexes from heart bovine as standard. The subunit molecular mass was determined by two-dimensional SDS-PAGE. The identity of each protein spot was determined. The number into parenthesis in subunit identity column is the same of Figs. 2B and 3C.  (Fig. 3C, spots 2 and 3) confirmed that the ATP synthase extracted from cytotrophoblast mitochondria exists in roughly equal amounts in dimeric and monomeric forms (Fig. 3C, left  panel); in contrast, in syncytiotrophoblast mitochondria, only trace amounts of the F 0 F 1 dimer was observed, and the monomeric ATP synthase was therefore highly enriched ( Fig. 3C and Table 2). These results suggest that in syncytiotrophoblast mitochondria, the ATP synthase exists preferably in the monomeric state. It has been proposed that dimerization of the F 0 F 1 -ATP synthase plays an important role in mitochondrial cristae formation (11, 19 -21). In mammalian mitochondria, the dimer of complex V is stabilized by the IF 1 (12). In this sense, the atypical mitochondrial morphology observed in syncytiotrophoblast cells could be associated with a diminished IF 1 content. To assess this possibility,, Western blot analyses of IF 1 , ␣, ␤, and OSCP subunits were carried out in cytotrophoblast and syncytiotrophoblast mitochondria (Fig. 4A). Densitometry analysis showed that the relative signal of ␣, ␤, and OSCP subunits was similar in both cytotrophoblast and syncytiotrophoblast mitochondria; however, the relative intensity of IF 1 signal is on average twice as high as in cytotrophoblast than in syncytiotropho-blast mitochondria (Fig. 4B). Although the actual IF 1 /complex V stoichiometries cannot be estimated by these densitometry analyses, the results are consistent with the hypothesis that a higher concentration of IF 1 in the cytotrophoblast cells results in larger amounts of F 0 F 1 -ATP synthase dimers, which in turn will promote the formation of mitochondrial cristae.

DISCUSSION
The present work shows that in common with mammalian, plant, and fungi mitochondria, human cytotrophoblast and syncytiotrophoblast mitochondria exhibit association of individual respiratory complexes into supercomplexes. When these organelles were solubilized with digitonin, a low amount of monomeric complex I was obtained, suggesting that in vivo, most of complex I is sequestered into supercomplexes. In addition, complex IV and III 2 associate with complex I into respirasomes; however, more than half of complex IV is in the monomeric free form, suggesting the existence an excess of cytochrome c oxidase. In cytotrophoblast and syncytiotrophoblast mitochondria, there was no evidence of the interaction of complex II with other proteins, although associations disrupted or not resolved by BN-PAGE cannot be ruled out. No significant differences were observed in the formation of respirasomes of cytotrophoblast and syncytiotrophoblast mitochon-dria; however, significant differences were found in complex V dimerization, as discussed below.
The most significant observation of this work shows that the ATP synthase dimer is present in digitonin extracts of cytotrophoblast mitochondria but that it is scarce in syncytiotrophoblast mitochondria. This result is consistent with the presence of orthodox mitochondrial cristae in the former and the lack of cristae in the latter. It is well documented that complex V dimerization is a key element in the formation of tubular mitochondrial cristae (11, 19 -21), and our correlation of a higher content of ATP synthase dimer in cytotrophoblast mitochondria is therefore in accordance with the role of ATP synthase dimer in cristae biogenesis. Furthermore, a critical role in the stability of the dimeric complex V has been proposed for its intrinsic inhibitor, IF 1 . bovine, and rat IF 1 dimerize in solution (43). However, yeast IF 1 is less prone to dimerize (44). This different dimerizing propensity correlates well with the more prominent role of bovine and rat IF 1 in stabilizing the F 1 (45) and F 0 F 1 dimers (12) when compared with the apparent dispensability of yeast IF 1 to dimerize F 0 F 1 (26). Given the known dimerizing role of animal or human IF 1 , we looked for an increase in IF 1 expression in cytotrophoblast over syncytiotrophoblast mitochondria that will correlate with the higher cristae content in the former model. In this line, the relative band intensities as developed by Western blot of IF 1 when compared with F 1 subunits (␣, ␤, and OSCP) from cytotrophoblast and syncytiotrophoblast mitochondria were estimated. The blots showed a similar expression of the F 1 moiety in both cell types. In contrast, a reproducible increase in IF 1 expression relative to F 1 in cytotrophoblast versus syncytiotrophoblast was revealed by the intensity ratios of IF 1 /F 1 subunits (␣, ␤, or OSCP). The IF 1 /␣ intensity ratios were 3.6 and 1.7 for cytotrophoblast and syncytiotrophoblast, respectively (Fig. 4). Although it is not possible to estimate a true IF 1 /F 1 stoichiometry from Western blot data, the increase in IF 1 /F 1 intensity ratios obtained shows an average 2-fold increase in IF 1 expression (relative to F 1 ) in cytotrophoblast mitochondria when compared with the syncytiotrophoblast model. A question therefore emerges as to the extent to which a 2-fold increase in IF 1 expression might affect mitochondrial cristae morphology. Previously, some of us had shown that a 2-fold increase in rat IF 1 /F 1 expression in tumor AS-30D cells led to a higher association of IF 1 with the native F 0 F 1 complex (46). Accordingly, this work shows the actual interaction of IF 1 with both monomeric and dimeric human F 0 F 1 in cytotrophoblast mitochondria by MS identification in two-dimensional SDS-PAGE after BN-PAGE (Fig. 4). Similarly, an average 2.5-fold increase in IF 1 /␤ expression as obtained by transient transfection of HeLa cultured cells with IF 1 led to a major increase in mitochondrial cristae density in situ (47). These results indicate that a 2-fold increase in IF 1 expression may be able to exert significant and evident changes in mitochondrial morphology as those observed here between cytotrophoblast and syncytiotrophoblast mitochondria. Accordingly, excess IF 1 also promotes oligomerization of the ATP synthase in addition to stabilizing the F 0 F 1 dimer (12). Furthermore, recent three-dimensional structure of dimeric yeast ATP synthase suggests that the oligomerizing effect of excess IF 1 may take place at dimer-dimer interfaces of a diagonal ATP synthase oligomer wrapping mitochondrial cristae (35). Ongoing work 3 is assessing the effect of higher IF 1 /F 1 overexpression ratios on cristae morphology. Meanwhile, it is important to note that other factors such as supernumerary F 0 subunits or different lipid composition (including cholesterol or steroid content, see below) besides IF 1 may also contribute to the differences in cristae morphology.
Although many types of mitochondrial cristae structure have been described (45), it is evident from recent electron microscopic tomography studies that there are differences between typical mitochondria and those from steroidogenic tissues. In general, cristae from typical mitochondria are a mixture of tubular and lamellar structures (48,49), whereas in steroidogenic cells, cristae are tubular, vesicular, or tubulovesicular (50,51). It has been suggested that due to this particular morphology of the cristae, mitochondria of Leydig cells should not be able to produce ATP because the narrow gap between lamellae would not allow the location of the F 1 moiety of the ATP synthase (50). However, recent data indicate that mitochondrial membrane potential (⌬⌿m), mitochondrial ATP synthesis, and mitochondrial respiration are all required to support Leydig cell steroidogenesis (52). Furthermore, in steroidogenic syncytiotrophoblast mitochondria, ATP is essential for progesterone synthesis (30). Accordingly, this work shows that both types of isolated mitochondria (steroidogenic and non-steroidogenic) exhibit similar rates of oxygen uptake coupled to ATP synthesis (Table 1). However, to correlate the mitochondrial cristae architecture with the rates of ATP synthesis, further work with intact mitochondria in situ rather than with isolated mitochondria will be needed.
Because the human placenta does not express Steroidogenic Acute Regulatory protein (StAR) (40) and Translocator protein (TSPO) (53) proteins involved in mitochondrial cholesterol flow, it has been suggested that the reduction in the size of syncytiotrophoblast mitochondria and the change in the structure of the cristae may improve the steroidogenic activity of the syncytiotrophoblast cells (27). The translocation of cholesterol to P450scc has long been known to be the rate-limiting step in steroidogenesis; thus, the greater surface to volume ratio could improve the movement of cholesterol to the inner membrane where the P450scc is located. This suggests that the non-orthodox cristae structure in mitochondria from steroidogenic tissue allows the cholesterol flow from the outer to the inner mitochondrial membranes and improves the hormone production. Because placental progesterone synthesis by syncytiotrophoblast mitochondria is required to suppress maternal uterine contractions to maintain pregnancy, the non-orthodox mitochondrial architecture of syncytiotrophoblast mitochondria may play a role in supporting sufficient progesterone synthesis to prevent spontaneous abortion (54). Syncytiotrophoblast mitochondria may lack cristae because these organelles are specialized in progesterone production and may in part dispense with ATP synthase dimerization and oligomerization by reducing IF 1 expression.