Post-ovulatory ageing and egg quality: A proteomic analysis of rainbow trout coelomic fluid
© Rime et al; licensee BioMed Central Ltd. 2004
Received: 15 April 2004
Accepted: 04 June 2004
Published: 04 June 2004
In fish, oocyte post-ovulatory ageing is associated with egg quality decrease. During this period, eggs are held in the body cavity where they bath in a semi-viscous liquid known as coelomic fluid (CF). CF components are suspected to play a role in maintaining oocyte fertility and developmental competence (egg quality). However, CF proteic composition remains poorly studied. Thus rainbow trout CF proteome was studied during the egg quality decrease associated with oocyte post-ovulatory ageing.
High resolution two-dimensional gel electrophoresis was used to analyze the proteome of rainbow trout (Oncorhynchus mykiss) CF in relationship with the egg quality decrease associated with oocyte post-ovulatory ageing. A first experiment was performed using CF pools originating from 17 females sampled at ovulation as well as 7, 14 and 21 days later. These observations were verified using a second set of CF pools originating from 22 females sampled 5 and 16 days following ovulation.
Approximately 200 protein spots of 10–105 kDa molecular mass and 3–10 pI were detected in CF samples. Several protein spots, while undetected at the time of ovulation, exhibited a progressive and strong accumulation in CF during post-ovulatory ageing. After silver-staining and Matrix-Assisted Laser Desorption Time Of Flight (MALDI-TOF) mass spectrometer analysis, some of these protein spots were identified as lipovitellin II fragments.
These observations suggest that egg protein fragments accumulate in the CF during the post-ovulatory period and could therefore be used to detect egg quality defects associated with oocyte post-ovulatory ageing.
In salmonids, ovulated oocytes (eggs) are held in the body cavity where they bath in a semi-viscous fluid known as "ovarian" or "coelomic" fluid (CF). In contrast to most fish species, salmonids can hold their eggs in the body cavity at 10–12°C for at least a week without loss of developmental competence [1, 2]. For longer holding times, post-ovulatory ageing is associated with a significant drop of egg quality characterized by decreased embryonic survival and increased number of morphological and ploidy abnormalities . However, the relative importance of each of these phenomena is highly variable depending on the females . In vitro, trout ovulated oocytes held 3 days at 12°C retain full developmental competence when stored in coelomic fluid but not in trout artificial medium . It is therefore possible that some coelomic fluid components could play a role in maintaining the oocyte ability to be fertilized and allow subsequent normal development.
Trout coelomic fluid pH is around 8 and contains Na+, K+ and Ca++ ions, glucose, fructose, cholesterol, phospholipids, proteins and free amino acids [4, 5]. However, its proteic composition remains poorly documented. Several proteases and anti-proteases of maternal origin were previously identified and an anti-bacterial activity of some of these proteins, the trout ovulatory proteins (TOPs) was also demonstrated [6–9]. In addition, enzyme assays also indicated the presence of phosphate, collagenase, gelatinase and lactate dehydrogenase activities . Although some proteic components are believed to be derived from the blood [11, 12], most of the others probably come from the post-ovulatory ovary which is devoid of ovarian cavity and widely open to the coelomic cavity in salmonids.
The purpose of the present study was to analyze the changes in coelomic fluid proteome during oocyte post-ovulatory ageing and tentatively identify proteins that accumulate or, in contrast, disappear simultaneously with the decrease in egg quality usually observed during post-ovulatory ageing. Such accumulation/degradation patterns could thus become useful markers of oocyte quality.
To do so, we combined a high resolution two-dimensional gel electrophoresis (2-D) that separates proteins according to both isoelectric point and molecular weight, and mass spectrometry. Coelomic fluids (CF) and oocyte samples were taken from females after short and long post-ovulatory times in the body cavity. For each sampling time, egg quality was evaluated by fertilizing an oocyte sample from each female and monitoring early development. The accumulation patterns of identified proteins were described using pooled coelomic fluid samples originating from 17 spring-spawning females. We observed that several proteins, while undetected or poorly detected at the time of ovulation, accumulate in the coelomic fluid during post-ovulatory ageing. Some of these proteins were identified as fragments of lipovitellin II, one of the vitellogenin-derived proteins. These observations were then verified using CF pools sampled from 22 autumn-spawning females 5 and 16 days following ovulation.
Materials and methods
Investigations were conducted according to the guiding principles for the use and care of laboratory animals. Mature male and female rainbow trout were obtained from the INRA-SEDI fish farm (Sizun, France) and held in a recirculated water system at 12°C, under natural photoperiod, in INRA experimental facilities (Rennes, France). Females were checked for ovulation 2 or 3 times a week by manual pressure on the abdomen. For ovulation detection as well as coelomic fluid and gamete collection, fish were lightly anesthetized in 0.05% 2-phenoxy-ethanol.
Coelomic fluid and gamete collection
Coelomic fluid and eggs were collected from the same females after increasing holding times in the body cavity. Coelomic fluid was carefully pipetted out and spun at 3000 g for 15 min to remove red blood cells and debris, and anti-proteases were added (1 mM ethylene-diamine-tetra-acetic-acid (EDTA), 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), 10 μM leupeptin). The protein content of coelomic fluid was determined using a Coomassie protein assay reagent (Interchim, France). A first experiment was performed using 17 females collected at the time of ovulation (OV) and 7 (D7), 14 (D14) and 21 (D21) days following ovulation. A second experiment was performed using 22 females collected 5 (D5) and 16 (D16) days following ovulation.
Fertilization and developmental success monitoring
For each female and each collection time, an egg batch was fertilized as previously described  in order to estimate egg quality. Approximately 250–400 eggs were washed with 10 ml of rinsing saline solution (Ovafish, IMV, France). Sperm originating from 5–10 males was added (10 μl) along with 10 ml of sperm extender (Actifish, IMV), tubes were gently swirled and incubated for 10 minutes. After discarding Actifish, eggs were transferred into plastic incubators and held in the dark at 10°C in a recirculated water system during early development (55 days). The number of embryos reaching eyed-stage, hatching and yolk-sac resorption (YSR) was monitored and the number of alevins exhibiting morphological abnormalities at YSR was measured. The quality of each egg batch was estimated using the number of embryos reaching yolk-sac resorption without exhibiting any obvious morphological abnormality (Living and Normal Embryo Rate, LNER).
Preparation of protein samples
Forty micrograms of each coelomic fluid (CF) sample were pooled for each post-ovulatory ageing time (First experiment: OV, D7, D14 and D21; second experiment: D5 and D16). Coelomic fluids were incubated in a rehydration buffer: 6 M urea, 2 M Thiourea, 4% CHAPS, 1% Dithiothreitol (DTT), 0.5% Immobilized pH Gradients (IPG) buffer (Amersham Biosciences) and bromophenol blue for 1 hr at room temperature.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)
A 2D-PAGE was run for each post-ovulatory ageing time for both experiments. For each gel, forty microgram of CF pools were loaded onto a 18 cm nonlinear immobiline dry strip, pH range 3–10; 180 × 3 × 0,5 mm (Amersham Biosciences) for the first dimension. The second-dimensional run was carried out using a precast ExcelGel XL sodium dodecyl sulphate (SDS)-polyacrylamide 12–14% gradient gel (Amersham Biosciences) with precast anode and cathode buffer strips (Amersham Biosciences). Isoelectric focusing (IEF) was performed using an IPGphor IEF system (Amersham Biosciences) during 18.5 hours with a gradually increasing voltage . Immediately after IEF, the IPG strip was equilibrated for 15 min at room temperature in 20 ml of 50 mM Tris-HCL pH 8.2, 6 M urea, 30% glycerol, 2% SDS and bromophenol blue with 65 mM DTT and later for 10 min with 250 mM iodoacetamide and trace of bromophenol blue. The equilibrated IPG strip was transferred to a horizontal Mutiphor II system at 15°C during 3.5 hours. The electrophoresis conditions were set as previously described .
Gel staining and image analysis
For image analysis, 2-D gels were silver-stained as described by . After staining, gels were scanned and then processed for image analysis using 2-D Elite image master software (Amersham Biosciences). Optic density of protein spots was measured using multigauge software (Fuji Ltd, Tokyo, Japan).
In gel digestion and protein identification by mass spectrometry
Proteins resolved by 2D-PAGE were silver-stained using a method compatible with in-gel digestion of proteins mass spectrometry identification [13, 16]. Silver-stained protein spots of interest were excised from 2-D gels and then processed for trypsin digestion as described previously . The eluted peptides were subsequently analyzed by mass fingerprinting in a Matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) processed using the MoverZ software (ProteoMetrics, NY) and internally calibrated using trypsin auto-digestion peptides.
Protein identification by mass spectrometry was performed as previously described . Monoisotopic masses of tryptic peptides observed in the MALDI-MS spectra were used to query National Centre for Biotechnology Information (NCBl) and SWISS-PROT sequences databases using MASCOT search program http://www.matrixscience.com.
Egg quality differences observed between post-ovulatory ageing times were analyzed using paired Student t-tests.
Results and Discussion
List of proteins identified in coelomic fluid during post ovulation ageing. The spot identification # corresponds to Fig 2. SwissProt accession numbers and corresponding protein names are shown.
Accession No. (SwissProt)
Apolipoprotein A I-1
Apolipoprotein A I-1
Apolipoprotein A I-1
1) Six proteins spots were identified as fragments of O. mykiss vitellogenin (Vtg) and more specifically to the lipovitellin II domain . Whereas these fragments could hardly be detected at the time of ovulation (Figure 3A), they appeared 5–7 days later (Figure 3B), and their abundance progressively increased during the post-ovulatory period (Figure 3C,3D and Figure 4A,4B). Fish vitellogenin is a high molecular weight glyco-phospho-lipoprotein (440–660 kDa in rainbow trout O. mykiss) (according to). It is known to be synthesized by the liver cells of mature females under estrogenic control (see  for review). It is secreted into the blood-stream from where it is taken up by developing oocytes and cleaved into yolk proteins . These are mainly known as lipovitellin I and II, phosvitin and β'-component . Our observations suggest that vitellogenin fragments originating from the ovulated oocytes held in the body cavity accumulate in CF during post-ovulatory ageing. This would suggest that post-ovulatory ageing is associated with a leakage of some of the oocyte components into the CF. In normal living oocytes, yolk degradation is tightly controlled by specific protease inhibitors associated with yolk proteins. In contrast, proteolytic activities can be expected to increase in poor-quality eggs as shown in sea bass; Dicentrachus labrax, in which the amount of cathepsin D enzymatic activity was found to be much higher in poor-quality eggs than in good-quality eggs. Cathepsin D could thus be considered a possible marker for bad-quality eggs in the sea bass as it has been suggested for the sea bream [26, 27]. However, some yolk proteolysis has also been shown to occur in the preovulatory ovary, during the process of oocyte maturation. The resulting increase in the number of smaller proteins fragments has been hypothesized to favor oocyte hydration that can be particularly important before ovulation in some teleost . The hydration process during oocyte maturation is certainly much lower in rainbow trout than in marine teleosts. However, it cannot be excluded that the protein fragments from oocyte origin that we observe in ageing coelomic fluids are already present as such in just ovulated oocytes. It is therefore possible that their progressive appearance in the coelomic fluid during post-ovulatory time is due to the weakening of the cellular membrane, thus allowing some leakage of small protein fragments as a first evidence of oocyte ageing.
2) Two protein spots were identified as C- type mannose-binding lectin of O. mykiss. Lectins are sugar-binding proteins of non-immune origin that agglutinate cells or precipitate glycoconjugates. They are known to be involved in cell defense mechanism . One example is the mannose-binding lectin which can activate the complement system by binding the cell surface glycoproteins of microbes . Lectins inhibit the growth of certain bacterial pathogens of fish . In some fish species, lectins are associated with the content and surrounding membrane of cortical vesicles located within the cytoplasm of maturing oocytes [32, 33]. In the present study, lectin spots are detected in CF. It is not clear however whether lectin accumulates or not during post-ovulatory ageing. Our results could suggest that the presence of lectin in CF is due to a leakage from ovulated oocytes. They could also suggest that lectin is secreted by the female into the body cavity to prevent from bacterial infection.
3) Three of the protein spots analyzed were identified as O. mykiss apolipoprotein A-I-1. Apolipoprotein A-I-1 is the major protein in plasma high-density lipoprotein and is a potent activator of the lecithin/cholesterol acyl transferase which facilitates the removal of free cholesterol to the liver for excretion . Our results show that this protein is very abundant in coelomic fluids at all times post-ovulation. Interestingly, this protein was also observed in bovine ovarian fluid .
Taken together, our observations suggest that cellular leakage occurs from the oocyte to the CF during post-ovulatory ageing. Thus, the higher the proportion of degenerating oocytes present in the body cavity, the higher would be the concentration of some proteins or protein fragments. The accumulation of these proteins or peptides would thus reveal the decreasing overall egg quality of the spawn before it can be detected by direct observations. In addition, the progressive accumulation of some of these proteins in CF would allow their use as markers of oocyte quality defects when the exact post-ovulatory ageing time is unknown. It is also noteworthy that similar observations were made in two different rainbow trout strains (spring spawning and autumn spawning). Indeed the level of these protein fragments in CF could become a useful marker of oocyte quality in rainbow trout and possibly in other salmonids.
In the present study, we observed that the egg quality drop observed during post-ovulatory ageing is associated with the apparition and accumulation of several protein spots in CF. It is believed, however, that CF components are important to maintain ovulated oocyte fertility. Indeed, holding in artificial medium exhibiting pH and osmolarity similar to those of CF leads to a very fast decrease of fertility and developmental competence. Taken together, these observations suggest that the concentration of some CF fluid components could decrease during the egg quality drop observed during oocyte post-ovulatory ageing. However, the proteomic analysis performed here is based on equal loading of total protein quantities on 2-D gels. Therefore, the major increase in the concentration of some protein fragments (e.g., vitellogenin fragments) could have biased the analysis. Specific studies should be designed in the future to carefully monitor the protein levels of some CF components during post-ovulatory ageing, especially proteins or peptides exhibiting low or moderate expression levels.
In conclusion, we identified several previously unknown components of rainbow trout CF (e.g., lectin) some of them exhibiting a progressive and strong accumulation in CF during post-ovulatory ageing. These results suggest a leakage of some oocyte components into the CF during the post-ovulatory period that could allow the use of these protein fragments as markers of oocyte quality during this period.
This work was supported by an INRA-IFREMER grant. We thank S. Aegerter for collection of the spring-spawning coelomic fluid samples and C. Duret for participating in the monitoring of developmental success. Both laboratories belong to the Ouest Genopole® network. The proteomic analysis was performed at the high-throughput proteomic platform of Ouest Genopole®.
- Aegerter S, Jalabert B: Effects of post-ovulatory oocyte ageing and temperature on egg quality and on the occurence of triploid fry in rainbow trout, Oncorhynchus mykiss. Aquaculture. 2004, 231: 59-71. 10.1016/j.aquaculture.2003.08.019.View ArticleGoogle Scholar
- Springate JRC, Bromage NR, Elliott JAK, Hudson DL: The timing of ovulation and stripping and their effects on the rates of fertilization and survival to eying, hatch and swim-up in the rainbow trout (Salmo gairdneri R.). Aquaculture. 1984, 43: 313-322. 10.1016/0044-8486(84)90032-2.View ArticleGoogle Scholar
- Bonnet E, Jalabert B, Bobe J: A 3-day in vitro stage of rainbow trout (Oncorhynchus mykiss) unfertilized eggs in coelomic fluid at 12°C does not affect developmental success. Cybium. 2003, 27: 47-51.Google Scholar
- Lahnsteiner F, Weismann T, Patzer RA: Composition of the ovarian fluid in 4 salmonid species: Oncorhynchus mykiss, Salmo trutta f lacustris, Salvelinus alpinus and Hucho hucho. Reprod Nutr Dev. 1995, 35: 465-474.View ArticlePubMedGoogle Scholar
- Satia BP, Donalson LR, Smith LS, Nightingale JN: Composition of ovarian fluid and eggs of the university of Washington strain of rainbow trout (Salmo gairdneri). Journal of Fisheries Research Board Canada. 1974, 31: 1796-1799.View ArticleGoogle Scholar
- Bobe J, Goetz FW: An ovarian progastricsin is present in the trout coelomic fluid after ovulation. Biol Reprod. 2001, 64: 1048-1055.View ArticlePubMedGoogle Scholar
- Coffman MA, Goetz FW: Trout ovulatory proteins are partially responsible for the anti-proteolytic activity found in trout coelomic fluid. Biol Reprod. 1998, 59: 497-502.View ArticlePubMedGoogle Scholar
- Coffman MA, Pinter JH, Goetz FW: Trout ovulatory proteins: site of synthesis, regulation, and possible biological function. Biol Reprod. 2000, 62: 928-938.View ArticlePubMedGoogle Scholar
- Hajnik CA, Goetz FW, Hsu SY, Sokal N: Characterization of a ribonucleic acid transcript from the brook trout (Salvelinus fontinalis) ovary with structural similarities to mammalian adipsin/complement factor D and tissue kallikrein, and the effects of kallikrein-like serine proteases on follicle contraction. Biol Reprod. 1998, 58: 887-897.View ArticlePubMedGoogle Scholar
- Lahnsteiner F: Morphological and biochemical parameters characterizing the over-ripening of rainbow trout eggs. Fish Physiol Biochem. 2000, 23: 107-118. 10.1023/A:1007839023540.View ArticleGoogle Scholar
- Matsubara T, Hara A, Takano K: Immunochemical identification and purification of coelomic fluid-specific protein in chum salmon (Oncorhynchus keta). Comp Biochem Physiol. 1985, 81: 309-314. 10.1016/0305-0491(85)90318-9.Google Scholar
- Matsubara T, Ito F, Hara H, Takano K: Edema-like protuberances found on the inner surface of coelomic wall of matured masu salmon (Oncorhynchus masu), with special reference to the coelomic fluid production. Bull Fac Fish Hokkaido Univ. 1987, 38: 349-357.Google Scholar
- Com E, Evrard B, Roepstorff P, Aubry F, Pineau C: New insights into the rat spermatogonial proteome: identification of 156 additional proteins. Mol Cell Proteomics. 2003, 2: 248-261.PubMedGoogle Scholar
- Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W: The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis. 2000, 21: 1037-1053. 10.1002/(SICI)1522-2683(20000401)21:6<1037::AID-ELPS1037>3.3.CO;2-M.View ArticlePubMedGoogle Scholar
- Heukeshoven J, Dernick R: Simplified method for silver staining of protein in polyacrylamide gels and the mechanism of the silver staining. Electrophoresis. 1985, 6: 103-112.View ArticleGoogle Scholar
- Mortz E, Krogh TN, Vorum H, Gorg A: Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics. 2001, 1: 1359-1363. 10.1002/1615-9861(200111)1:11<1359::AID-PROT1359>3.3.CO;2-H.View ArticlePubMedGoogle Scholar
- Jensen ON, Larsen MR, Roepstorff P: Mass spectrometric identification and microcharacterization of proteins from electrophoretic gels: strategies and applications. Proteins. 1998, Suppl 2: 74-89. 10.1002/(SICI)1097-0134(1998)33:2+<74::AID-PROT9>3.3.CO;2-2.View ArticlePubMedGoogle Scholar
- Perkins DN, Pappin D, Creasy D, Cottrell J: Probability-based protein identification by searching sequence databases using mass spectrometric peptide mapping information. Electrophoresis. 2004, 20: 3551-3567. 10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2.View ArticleGoogle Scholar
- Nomura M, Sakai K, Takashima F: The over-ripening phenomenon of rainbow trout. (I. Changes in the percentage of eyed eggs, hatching rate and incidence of abnormal alevins during the process of over-ripening. Bull Jap Soc Science Fish. 1974, 41: 855-860.Google Scholar
- Lahnsteiner F, Weismann T, Patzer RA: Physiological and biochemical parameters for eggs quality determination in lake trout, Salmo trutta lacustris. Fish Physiol Biochem. 1999, 20: 375-388. 10.1023/A:1007715621550.View ArticleGoogle Scholar
- Mouchel N, Trichet V, Betz A, Le Pennec JP, Wolff J: Characterization of vitellogenin from rainbow trout (Oncorhynchus mykiss). Gene. 1996, 174: 59-64. 10.1016/0378-1119(96)00336-8.View ArticlePubMedGoogle Scholar
- Hara A, Hirai H: Comparative studies on immunochemical properties of female-specific serum protein and egg yolk proteins in rainbow trout (Salmo gairdneri). Comp Biochem Physiol B. 1978, 59: 339-343. 10.1016/0305-0491(78)90011-1.View ArticlePubMedGoogle Scholar
- Specker J,L, Sullivan C,V: Vitellogenesis in fishes: status and perspectives. Perspectives in Comparative Endocrinology. 1994, 304-315.Google Scholar
- Tyler C: Electrophoretic Patterns of Yolk Proteins Throughout Ovarian Development and Their Relationship to Vitellogenin in the Rainbow-Trout, Oncorhynchus-Mykiss. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. 1993, 106: 321-329. 10.1016/0305-0491(93)90308-R.View ArticleGoogle Scholar
- Matsubara T, Sawano K: Proteolytic cleavage of vitellogenin and yolk protein during vitellogenin uptake and oocyte maturation in Barfin Flounder (verasper moseri). The journal of experimental zoology. 1995, 272: 34-45.View ArticleGoogle Scholar
- Carnevali O, Centonze F, Brooks S, Marota I, Sumpter JP: Molecular cloning and expression of ovarian cathepsin D in seabream, Sparus aurata. Biol Reprod. 1999, 61: 785-91.View ArticlePubMedGoogle Scholar
- Carnevali O, Mosconi G, Cardinali M, Meiri I, Polzonetti-Magni A: Molecular components related to egg viability in the gilthead sea bream, Sparus aurata. Mol Reprod Dev. 2001, 58: 330-335. 10.1002/1098-2795(200103)58:3<330::AID-MRD11>3.0.CO;2-7.View ArticlePubMedGoogle Scholar
- Greeley MS, Calder DR, Wallace RA: Changes in teleost yolk proteins during oocyte maturation: correlation of yolk proteolysis with oocyte hydration. Comp Biochem Physiol B. 1986, 84: 1-9. 10.1016/0305-0491(86)90262-2.View ArticlePubMedGoogle Scholar
- Jensen L, Thiel S, Petersen T, Jensenuis J: A rainbow trout lectin with multimeric structure. Comp Biochem Physiol. 1997, 116B: 385-390.View ArticleGoogle Scholar
- Lu JH, Thiel S, Wiedemann H, Timpl R, Reid KB: Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2C1s2 complex, of the classical pathway of complement, without involvement of C1q. J Immunol. 1990, 144: 2287-2294.PubMedGoogle Scholar
- Voss EW, Fryler JL, Banowetz GM: Isolation, Purifcation, and partial characterization of a Lectin from Chinook Salmon Ova. Archives of Biochemistry and Biophysics. 1978, 186: 25-34.View ArticlePubMedGoogle Scholar
- Dong CH, Yang ST, Yang ZA, Zhang L, Gui JF: A C-type lectin associated and translocated with cortical granules during oocyte maturation and egg fertilization in fish. Dev Biol. 2004, 265: 341-354. 10.1016/j.ydbio.2003.08.028.View ArticlePubMedGoogle Scholar
- Nosek J, Krajhanzl A, Kocourek J: Studies on lectins. LVII. Immunofluorescence localization of lectins present in fish ovaries. Histochemistry. 1983, 79: 131-139.View ArticlePubMedGoogle Scholar
- Brouillette CG, Anantharamaiah GM, Engler JA, Borhani DW: Structural models of human apolipoprotein A-I: a critical analysis and review. Biochim Biophys Acta. 2001, 1531: 4-46. 10.1016/S1388-1981(01)00081-6.View ArticlePubMedGoogle Scholar
- Mortarino M, Vigo D, Maffeo G, Ronchi S: Two-dimensional polyacrylamide gel electrophoresis map of bovine ovarian fluid proteins. Electrophoresis. 1999, 20: 866-869. 10.1002/(SICI)1522-2683(19990101)20:4/5<866::AID-ELPS866>3.0.CO;2-V.View ArticlePubMedGoogle Scholar
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