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RESEARCH |
Grup de Metabolisme Energètic i Nutrició, Departament de Biologia Fonamental i Ciències de la Salut, Institut Universitari d’Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears i CIBER Fisiopatologia Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, Spain
Correspondence should be addressed to M Gianotti; Email: magdalena.gianotti{at}uib.es
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Abstract |
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coactivator-1
, nuclear respiratory factors 1 and 2, mitochondrial single-strand DNA-binding protein, mitochondrial transcription factor A), and mitochondrial function (cytochrome c oxidase subunit IV, cytochrome c oxidase subunit I and ß-ATP phosphohydrolase) in rat embryo throughout the placentation period (gestational days 11, 12 and 13). Our results reflect that embryo mitochondria were enhancing their OXPHOS potential capacities, pointing out that embryo mitochondria become more differentiated during the placentation period. Besides, the current findings show that the mRNAs of the nuclear genes involved in mitochondrial biogenesis were downregulated, whereas their protein content together with the mitochondrial DNA expression were upregulated throughout the period studied. These data indicate that the molecular regulation of the mitochondrial differentiation process during placentation involves a post-transcriptional activation of the nuclear-encoded genes that would lead to an increase in both the nuclear- and mitochondrial-encoded proteins responsible for the mitochondrial biogenic process. As a result, embryo mitochondria would reach a more differentiated stage with a more efficient oxidative metabolism that would facilitate the important embryo growth during the second half of the pregnancy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Alterations in mitochondrial function and biogenesis have been involved in several human degenerative diseases (Wallace 1999). Thus, research on mitochondrial biogenesis is of great interest, since it would provide the knowledge to understand the pathophysiology of mitochondrial diseases. Numerous protein factors have been reported to be involved in mitochondrial biogenesis in mammals so far, contributing insight into the molecular pathways taking part in this biological process. In short, the family of peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) coactivates different transcription factors in response to energy requirements (Scarpulla 2006). Among these transcription factors, nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) regulate the expression of most nuclear genes involved in mitochondrial biogenesis, such as genes encoding subunits of respiratory complexes, mitochondrial transcription factors A, B1 and B2 (TFAM, TFB1M and TFB2M respectively), mitochondrial replication factors like mitochondrial single-strand DNA-binding protein (mtSSB) and mitochondrial RNA-processing endoribonuclease (RNase MRP), as well as other proteins necessary for mitochondrial function (Virbasius & Scarpulla 1994, Gleyzer et al. 2005, Scarpulla 2006).
Mitochondriogenesis is a complex event, including both mitochondrial proliferation and differentiation processes (Izquierdo et al. 1995, Ostronoff et al. 1996), which takes place according to specific energy demands in response to several physiological conditions (Williams et al. 1987, Pillar & Seitz 1997), including prenatal (May-Panloup et al. 2005, Thundathil et al. 2005) and postnatal developmental stages (Luis et al. 1993, Izquierdo et al. 1995, Ostronoff et al. 1996). In middle pregnancy, rat embryo mitochondria undergo considerable morphofunctional changes (Mackler et al. 1973, Shepard et al. 1998, Yang et al. 1998) coinciding with the switch from glycolytic to oxidative metabolism that takes place on gestational day 12 (Akazawa et al. 1994, Shepard et al. 1997), just when the placentary circulation is established and oxygen becomes more available to the embryo (Jollie 1986, Akazawa 2005). In addition, a previous study in our laboratory reported the activation of mitochondrial gene expression throughout the placentation period, suggesting that mitochondrial biogenesis is an active process in rat embryo during this developmental stage (Alcolea et al. 2006). Therefore, embryo development during the placentation period is a suitable model to further understand the mitochondrial proliferation and differentiation processes (Justo et al. 2002).
In light of this background, the aim of the present work was to study the oxidative phosphorylation system (OXPHOS) enzymatic activities in rat embryo throughout gestational days 11, 12 and 13, when placentation takes place (Jollie 1986, Akazawa 2005), to determine whether the changes seen in mitochondrial gene expression were translated into an activation of the mitochondrial respiratory and phosphorylative capacities. Furthermore, we investigated the expression of genes involved in the coordinated transcriptional regulation of both nuclear and mitochondrial genomes (PGC-1, NRF-1, NRF-2 and TFAM; Gaspari et al. 2004, Scarpulla 2006), as well as genes related to mitochondrial replication (mtSSB; Hoke et al. 1990), and function, such as cytochrome c oxidase subunit IV, cytochrome c oxidase subunit I and ß-ATP phosphohydrolase (COX IV, COX I and ß-ATPase; Hatefi 1985) to go further into the knowledge of the molecular regulation of rat embryo mitochondrial biogenesis during the placentation period.
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Materials and Methods |
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Oligonucleotide primer sequences were from Roche Diagnostics. Chemicals for mRNA RT were obtained from Applied Biosystems (Foster City, CA, USA). Lightcycler-FastStart DNA Master SYBR Green I for real-time PCR and oligonucleotide primer sequences were supplied by Roche Diagnostics.
Antibodies for COX IV were obtained from Mito-Sciences (MS407; Eugene, Oregon, USA), antibodies for ß-ATPase were from Santa Cruz Biotechnology Inc. (sc-16690; Santa Cruz, CA, USA) and rabbit antisera against TFAM was kindly provided by Inagaki et al.(2000). Chemicals for immunoblot development were provided by Amersham.
Animals
Animal experiments were performed in accordance with general guidelines approved by our institutional ethics committee and the EU (86/609/EEC) regulations. Three-month-old virgin female Wistar rats, purchased from Charles River (Barcelona, Spain), were housed at 22 °C with a ratio of 12 h light:12 h darkness cycle (lights on at 0800 h) with free access to tap water and pelleted standard diet (Panlab, Barcelona, Spain). The females were mated overnight with males of the same strain and, when sperm-positive vaginal smears were found the following morning (reference point for gestational day 0), they were placed in individual cages.
Sacrifice and isolation of samples
Pregnant rats were killed by decapitation on gestational days 11, 12 or 13. Uteri were removed and between one and three conceptuses (embryos with intact amniotic and yolk sacs) per mother were dissected into a sterile culture dish with physiological saline solution which contained 0.1% (v/v) diethyl pyrocarbonate (DEPC) to inactivate RNase activity. The embryos obtained were immediately frozen in liquid nitrogen and stored at –70 °C for their subsequent RNA extraction.
The remaining conceptuses were dissected without DEPC added to saline solution. The embryos were rapidly pooled into preweight Eppendorf tubes, which were spun down to remove the remaining saline solution and weighed to determine the average embryo weight. Then, the embryos were homogenised in isolation buffer (300 mM sucrose, 5 mM 4-morpholine propanesulfonic acid (MOPS), 5 mM KH2PO4, 1 mM EDTA and 0.01% BSA (pH 7.4)) and used for mitochondrial isolation. Seven independent experiments were performed, in each of which three to four rats were used on day 11, two to three on day 12 and one to two on day 13 in order to pool large enough samples to be able to perform all measurements. The total number of rats used was 22 on day 11, 15 on day 12 and 10 on day 13, which contributed a total of 219 embryos on day 11, 163 embryos on day 12 and 117 embryos on day 13.
Isolation of mitochondria
The mitochondrial fraction was obtained according to Justo et al.(2002). In brief, nuclei and cell debris were first removed using a refrigerated centrifuge (Sigma-3K30) at 700 g for 10 min at 4 °C. The resulting supernatant was subjected to 10-min centrifugation at 1000 g rendering the mitochondrial fraction to be studied. The pellets were resuspended in the isolation buffer. An aliquot of both homogenate and isolated mitochondria were used to determine cytochrome c oxidase activity and the recovery percentage of the isolated mitochondria was calculated. The remaining samples were frozen at –70 °C for subsequent analysis.
Total protein content was measured in both homogenate and mitochondrial fractions (Lowry et al. 1951). Total DNA content was tested in homogenate samples (Thomas & Farquhar 1978).
OXPHOS enzymatic activities
OXPHOS enzymatic activities were measured in the mitochondrial fraction using a microtitre plate spectrophotometer (BioTek Instruments, Winooski, VT, USA).
NADH dehydrogenase (NADH:ubiquinone oxidoreductase, complex I, EC 1.6.5.3 [EC] ) and ATPase (complex V, EC 3.6.1.3 [EC] ) activities were assessed using an adaptation based on a spectrophotometric method (Ragan et al. 1987). The activities were measured at 37 °C and were calculated using an extinction coefficient of 6.81 mM/cm for complex I and of 6.22 mM/cm for ATPase.
Ubiquinol cytochrome c reductase (ubiquinol:ferri-cytochrome c oxidoreductase, complex III, EC 1.10.2.2 [EC] ) activity was determined using a spectrophotometric assay (Krahenbuhl et al. 1991). In brief, isolated mitochondria were incubated with 0.1 mM EDTA, 0.1% BSA (w/v), 150 µM cytochrome c, 3 mM sodium azide and 50 mM potassium phosphate (pH 7.4) in a final volume of 210 µl. After a 5-min equilibration period, the reactions were started by adding 100 µM reduced coenzyme Q2, and the increase in absorbance was monitored at 550 nm. Activities were calculated using an extinction coefficient of 19.1 mM/cm for reduced cytochrome c. Antimycin A-sensitive activities were obtained by subtraction of the rate determined in incubations containing 10 µg antimycin A from the rate in parallel with antimycin A-free incubations. Reduced coenzyme Q2 was synthesised according to Ragan et al.(1987).
Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, COX, EC 1.9.3.1 [EC] ) activity was determined by a spectrophotometric method (Chrzanowska-Lightowlers et al. 1993) following the oxidation of 3,3'diaminobenzidine-tetrahydrochloride at 450 nm and 37 °C.
mtDNA extraction and semi-quantification
mtDNA extraction and semi-quantification were carried out in isolated mitochondria as previously described (Justo et al. 2002). The extracted mtDNA was quantified by real-time PCR using SYBR Green technology on a LightCycler rapid thermal cycler (Roche Diagnostics). PCR was performed to amplify a 162 nt region of the mitochondrial NADH dehydrogenase subunit 4 gene. The primer sequences were 5'-TACACGATGAGGCAACCAAA-3' and 5'-GGTAGGGGGTGTGTTGTGAG-3'.
Real-time RT-PCR analysis
Total RNA was isolated from frozen embryo samples using TriPure isolation reagent and quantified using a spectrophotometer set at 260 nm.
Total RNA (1 µg) was reverse transcribed to cDNA at 42 °C for 60 min with 25 U MuLV reverse transcriptase in a 10 µl retrotranscription reaction mixture containing 10 mM Tris–HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl2, 2.5 µM random hexamers, 10 U RNase inhibitor and 500 µM of each dNTP. Each cDNA was diluted 1/10 and aliquots were frozen (–70 °C) until the PCRs were carried out.
Real-time PCR was carried out for seven target genes: PGC-1; NRF-1 and NRF-2 DNA-binding subunit, mtSSB, TFAM, COX IV and COX I; and one housekeeping gene: 18S rRNA (18S). All oligonucleotide primer sequences were obtained from primer 3 and tested with IDT OligoAnalyzer 3.0 (NCBI; Table 1
). Finally, a basic local alignment search tool (NCBI BLAST) revealed that the primer sequence homology was obtained only for the target genes.
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and 62 °C for 18S, 10 s at 58 °C for mtSSB and COX IV at 60 °C for TFAM and at 64 °C for NRF-2, 8 s at 55 °C for COX I, 5 s at 56 °C for NRF-1) and an extension step (12 s at 72 °C for all, except for NRF-1 and COX I, which were 8 s at 72 °C and 26 s at 72 °C respectively). After each cycle, fluorescence was measured at 72 °C. A negative control without cDNA template was run in each assay. The real-time PCR efficiencies (e) were estimated on average of all sample efficiencies, which were calculated by the following formula
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where n and n0 were the crossing point values of F and F0 respectively, fluorescences that belong to the linear section of the logarithmic quantification curve.
Western blot analysis
Equal amounts of mitochondrial protein (40 µg) were electrophoresed on SDS–PAGE gels containing 15% (v/v) polyacrylamide. Proteins were electrotransferred onto a nitrocellulose filter and Ponceau S staining was performed systematically to check the correct loading and electrophoretic transfer. Blots with mitochondrial proteins were incubated with mouse polyclonal antibody for COX IV, goat polyclonal antibody for ß-ATPase and rabbit antisera against TFAM. Immunoblot development was performed using an ECL Western blotting analysis system. Film bands were quantified by photodensitometric analysis (Kodak 1D Image Analysis Software). The apparent molecular weights of the proteins detected were 14, 51 and 25 kDa for COX IV, ß-ATPase and TFAM respectively.
Statistical analysis
All data are expressed as mean ± S.E.M. Differences between gestational days were assessed by one-way ANOVA followed by least significant difference (LSD) post-hoc test using the statistical software package SPSS 13.0 for Windows (SPSS, Chicago, IL, USA).
The statistical PCR data analysis was performed using the Relative Expression Software Tool (REST 2005 BETA V1.9.12). The statistical model used was a pair-wise fixed reallocation randomisation test (Pfaffl et al. 2002, 2004). Differences in expression between groups were assessed using the means for statistical significance by randomisation tests. Results were considered statistically significant at the P < 0.05 level.
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Results |
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). These results reflect that the important growth of the embryo during the placentation period entailed both cellular hypertrophy and hyperplasia processes.
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). As mtDNA content can be used as an indicator of mitochondrial content (Djouadi et al. 1994, Koekemoer & Oelofsen 2001), these data are in agreement with a previous work (Alcolea et al. 2006), suggesting a reduction in the number of mitochondria per embryo cell accompanied by an enrichment in the protein content of these mitochondria during the current developmental stage.
Mitochondrial function
Table 3 shows the enzymatic activities of complex I, III, IV and ATPase measured on gestational days 11, 12 and 13 in isolated mitochondria. All OXPHOS enzymatic activities presented a similar profile, i.e. a rise during the placentation period. Although all OXPHOS activities showed an increasing trend when they were expressed per tDNA and per mitochondrial protein (1.3- to 2.3-fold and 1.0- to 3.9-fold respectively), this profile was statistically significant only for complex III and ATPase. The ratio between OXPHOS activities and mtDNA, however, showed a significant increase in all cases and the rise was higher than those observed per both tDNA and mitochondrial protein (3.6- to 7.1-fold).
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, NRF-1, NRF-2 and TFAM), mitochondrial replication (mtSSB) and function (COX IV and COX I) is shown in Table 4. The mRNA levels of most of the nuclear-encoded genes studied (NRF-1, NRF-2, TFAM, mtSSB and COX IV) showed a profile of decrease throughout the placentation period which was statistically significant in all cases except for TFAM, in which a downward trend was observed. Nevertheless, mRNA levels of PGC-1, which is also a nuclear-encoded gene, did not show significant differences.
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Western blot analysis of nuclear-encoded proteins
The levels of the nuclear-encoded proteins COX IV, ß-ATPase and TFAM are given in Fig. 1. These data showed an increasing profile throughout the period studied, which was statistically significant on gestational day 13.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Rat embryo mitochondria during placentation are a suitable model to further understand mitochondrial biogenesis due to the important oxidative metabolism activation that takes place (Mackler et al. 1971, Akazawa et al. 1994). However, this developmental stage has not been further characterised. Our results show that although embryo cells were decreasing their mitochondria content throughout the placentation period, as suggested by the mtDNA content, they were actually increasing their OXPHOS capacities. Furthermore, the rise seen in the OXPHOS enzymatic activities when expressed per mtDNA reflect that each embryo mitochondrion was increasing its potential respiratory capacity. All these results suggest that a mitochondrial differentiation process would predominate over mitochondrial proliferation in rat embryo throughout the placentation period, increasing the mitochondrial efficiency in order to face the important energy demands of this developmental stage.
For the purpose of going further into the knowledge of the molecular regulation of rat embryo mitochondrial differentiation during the placentation period, we studied the factors involved in the coordinated regulation of the nuclear and mitochondrial genomes since they both take part in mitochondriogenesis (Shadel & Clayton 1997, Fernandez-Silva et al. 2003). This orchestrated process is a complex event carried out by nuclear transcriptional activators and coactivators that modulate the expression of nuclear genes encoding the necessary proteins for maintenance, replication and expression of mtDNA, as well as for most OXPHOS subunits and other mitochondrial proteins (Kelly & Scarpulla 2004). The downward trend shown by most genes encoded at a nuclear level (NRF-1, NRF-2, TFAM, mtSSB and COX IV) reflected that the PGC-1 pathway, by which this coactivator together with NRFs regulate the expression of several nuclear genes involved in mitochondrial biogenesis, among which TFAM, mtSSB and COX IV are found (Virbasius & Scarpulla 1994, Kelly & Scarpulla 2004), would be reduced. Nevertheless, the increasing profile observed in the protein content of some nuclear-encoded proteins, such as COX IV, ß-ATPase as well as TFAM, indicates that the regulation of the expression of the nuclear-encoded proteins related to the mitochondriogenic process was taking place at a post-transcriptional level, in such a way that despite the reduction of the transcription their protein content would increase leading to a mitochondrial differentiation process.
The lack of correlation observed between the mRNA levels of the nuclear genes encoding proteins involved in mitochondrial biogenesis and the protein content of some of them reflects that the mitochondrial biogenic process together with the concomitant activation of the respective molecular pathways would have started very early during development, maybe during the preimplantation period as previously described in mouse and bovine embryos (May-Panloup et al. 2005, Thundathil et al. 2005). As a result, during the gestational days studied in the current work, the nuclear transcriptional signals involved in mitochondrial biogenesis could already be reduced and other post-transcriptional signals, such as increased mRNA stability and/or translational efficiency, would become available to keep the mitochondrial differentiation process active. Therefore, such regulation would be similar to what has previously been reported during the rat perinatal period (Luis et al. 1993, Izquierdo et al. 1995, Ostronoff et al. 1996).
Besides, during the placentation period, embryo mitochondria showed a significant increase in TFAM content, which as one of the regulatory factors necessary for proper mtDNA transcription (Montoya et al. 1997, Larsson et al. 1998, Garstka et al. 2003, Gaspari et al. 2004) could cause the significant rise seen in the mRNA expression of COX I, a protein encoded by the mtDNA heavy strand. Taking into account that the transcription of all the mRNAs encoded by the mtDNA heavy strand originates a polycistronic molecule (Garesse & Vallejo 2001, Fernandez-Silva et al. 2003), the current results reflect an increase in the gene expression of the mitochondrial proteins encoded by this strand, i.e. the majority of the mitochondrial-encoded proteins. Thus, the aforementioned increase in the nuclear-encoded proteins, such as TFAM, would lead to an activation of the mtDNA transcription and all together would help the embryo mitochondria to reach a more differentiated stage through a coordinate regulation of both nuclear and mitochondrial-encoded proteins. This idea corroborates the activation of the mitochondrial differentiation process in rat embryo during the placentation period that we have previously suggested (Alcolea et al. 2006).
In conclusion, throughout the placentation period, rat embryo mitochondria would be undergoing a mitochondrial differentiation process involving an increase in OXPHOS potential enzymatic activities. The molecular regulation of this mitochondrial process throughout this developmental stage would be carried out through a post-transcriptional activation of nuclear-encoded proteins involved in the mitochondriogenic pathway. This activation would, among others, provide most OXPHOS subunits as well as regulatory proteins that would activate the expression of the mtDNA, which encodes the catalytic core subunits of these enzymes. This, all in all, would lead to an increased OXPHOS capacity and would, thus, prepare the embryo to use the oxidisable metabolites now available from the newly established chorioallantoic circulation.
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Acknowledgements |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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