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Regular Article |
a Department of Animal Science and Production, University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland
b Dpto. de Mejora Genética y Biotecnologíaand
c Dpto. de Reproducción Animal y Conservación de Recursos Zoogenéticos, INIA, Madrid 28040, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, environment
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro-produced bovine embryos differ from their in vivo counterparts in many respects, including having darker cytoplasm and lower buoyant density [4] as a consequence of their higher lipid content [5], a more fragile zona pellucida [6], reduced expression of intercellular communicative devices [7], and a higher incidence of chromosome abnormalities [8, 9], rendering them less tolerant of cryopreservation. Studies employing reverse-transcriptase polymerase chain reaction (RT-PCR) methods have revealed differences between the relative abundance of some developmentally important gene transcripts between in vivo-produced and in vitro-produced bovine embryos [10, 11]. In addition, it is known that the conditions of culture in vitro can alter gene expression in the embryo [12–17], and indeed, such changes have been implicated in the occurrence of the large offspring syndrome in ruminants [12]. The analysis of such differences in mRNA expression may explain the observed differences in cryotolerance between in vivo-produced and in vitro-produced embryos, and allow the opportunity to modify gene expression through modification of culture systems to overcome the reduced postthaw viability of the latter.
In the present study we used reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) differential display to analyze differential mRNA expression between bovine blastocysts produced under various conditions of culture (in vitro or in vivo) known to produce blastocysts of divergent quality [2, 3]. We also examined quantitative differences in the expression of 7 selected genes that are known to be important during the preimplantation stages of development. Our observations indicate that whereas mRNA expression patterns are highly conserved, differences in mRNA expression profiles do exist between such embryos. In addition, the differences between the relative abundance of some developmentally important gene transcripts at the blastocyst stage are dramatic and may well explain the differential quality of such embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation [2, 3], were used in this study: 1) in vitro culture in synthetic oviduct fluid (SOF) of in vitro matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) [2] of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery.
In vitro maturation Cumulus oocyte complexes (COCs) were obtained by aspirating follicles from the ovaries at slaughter. After 4 washes in PBS supplemented with 36 µg/ml pyruvate, 50 µg/ml gentamycin, and 0.5 mg/ml BSA (Sigma Chemical Company, St. Louis, MO [catalog number A-9647]), groups of up to 50 COCs were placed in 500 µl of maturation medium in 4-well dishes (Nunc, Roskilde, Denmark) and cultured for 24 h at 39°C in an atmosphere of 5% CO2 in air with maximum humidity. The maturation medium was TCM-199 supplemented with 10% (v/v) fetal calf serum (FCS) and 10 ng/ml epidermal growth factor.
In vitro fertilization For IVF, COCs were washed 4 times in PBS and then in fertilization medium before being transferred in groups of up to 50 into 4-well dishes containing 250 µl of fertilization medium (Tyrode medium with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg/ml fatty acid-free BSA, and 10 µg/ml heparin-sodium salt [184 units/mg heparin] per well; Calbiochem, San Diego, CA). Motile spermatozoa were obtained by centrifugation of frozen-thawed semen (Dairygold A.I. Station, Mallow, Ireland) on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient (2.5 ml 45% [v/v] Percoll over 2.5 ml 90% [v/v] Percoll) for 8 min at 700 x g at room temperature. Viable spermatozoa, collected at the bottom of the 90% fraction, were washed in Hepes-buffered Tyrode and pelleted by centrifugation at 100 x g for 5 min. Spermatozoa were counted in a hemocytometer and diluted in the appropriate volume of fertilization medium to give a concentration of 2 x 106 spermatozoa/ml. A 250-µl aliquot of this suspension was added to each fertilization well to obtain a final concentration of 1 x 106 spermatozoa/ml. Plates were incubated for 24 h at 39°C in an atmosphere of 5% CO2 in air with maximum humidity. Semen from the same bull was used for all experiments.
In vitro culture At approximately 20 h after insemination, presumptive zygotes were denuded by gentle vortexing, washed 4 times in PBS and twice in culture medium, before being transferred to 25-µl culture droplets (1 embryo per microliter) under mineral oil. Culture took place either in 1) SOF, to which FCS (10%, v/v) was added 24 h after placement in culture, or 2) TCM-199 + 10% FCS containing a granulosa cell monolayer [2].
Culture of IVM/IVF zygotes in the ewe oviduct Presumptive zygotes produced following IVM/IVF, as described above, were surgically transferred to the ligated ewe oviduct (approximately 100 per oviduct) at 20 h after insemination. Embryos were recovered from the ewe 6 days later (i.e., Day 7 after insemination) by flushing the oviduct with 20 ml of PBS.
In vivo embryo production
Cross-bred beef heifers were synchronized using a CIDR device (InterAg, Hamilton, New Zealand) for 8 days. Three days before CIDR removal, heifers received 2 ml (15 mg) of prostaglandin F2
analogue (Prosolvin, Intervet, Dublin, Ireland). Heifers were checked for standing estrus (= Day 0). The dominant follicle was ablated by transvaginal aspiration on Day 8 of the estrous cycle. Beginning on Day 10, animals were superovulated with a total of 180 mg FSH (Folltropin, Vetrepharm Canada Inc., London, ON) given as twice-daily injections over 4 days on a decreasing-dose schedule. Luteolysis was induced with 15 mg prostaglandin given on Day 12. Heifers were inseminated with frozen-thawed semen at 48 h and 60 h after prostaglandin injection. The same semen batch as used in IVF was used for artificial insemination. Day 7 embryos were recovered by nonsurgical flushing 9 days after prostaglandin injection.
All animal experiments were performed in accordance with Institutional Animal Care and USE Committee guidelines and in adherence with guidelines established in the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the Society for the Study of Reproduction.
RNA Extraction
Poly(A) RNA from 3 pools of 10–25 embryos per treatment was isolated as previously described [18] using QuickPrep Micro RNAm Purification Kit (Amersham Pharmacia, Uppsala, Sweden). The concentration of RNA was determined by measurement of absorbance at 260 nm.
RT-AFLP
First-strand and second-strand cDNA synthesis was carried out according to standard protocols with 2-base anchored oligo(dT) primers. Double-stranded cDNA was used in the AFLP analysis, performed according to Bachem et al. [19] with the modification of Gutiérrez-Adán et al. [18]. Complementary DNA was digested with MseI (New England Biolabs, Taunus, Germany) and EcoRI (Pharmacia, Barcelona, Spain). After ligation adapter, preamplification of purified DNA templates was performed with primers complementary to the adapter sequences with an additional selective 2' nucleotide. PCRs were performed in a 20 µl volume with 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM of each dNTP, 30 ng of each primer EcoRI+1, MseI+1 (Table 1), 0.4 units of Taq DNA polymerase (Boehringer, Barcelona, Spain), and 5 µl of diluted digested-ligated DNA fragments. Selective amplifications were performed using combinations of primers EcoRI+2 and MseI+2 (Table 1) with 33P-labeled EcoRI primers. We used 5 µl of the preamplification template for each PCR reaction. Three replicates of the PCR reactions from 3 independent RNA extractions were performed to determine the accuracy of the analysis. Samples amplified with different primer combinations were loaded onto 4.5% denaturing polyacrylamide gels and electrophoresed for 2 h. Gels were later dried onto chromatography paper, and exposed to autoradiographic film.
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Reamplification of DNA Fragments
Selected bands, which were present in the 3 replicates, were excised from the gel and transferred into a centrifuge tube. Elution of DNA was done by a 1-h incubation of gel pieces with 25 µl of Tris/EDTA buffer at room temperature. Samples were centrifuged in a microfuge, and 1 µl of supernatant was subjected to PCR under standard conditions using 60°C of annealing temperature and the primers used in the second PCR of the AFLP assays.
Cloning and Sequencing of Reamplified DNA Fragments
Reamplified PCR products that matched the size of the original DNA fragment were cloned into pGEM T (Promega, Madison, WI) according to the manufacturer's protocol. Selected positive white colonies were cultured overnight in Luria-Bertani medium and plasmids were isolated using a Miniprep Kit (Promega). Plasmids were sequenced and were checked for similarities using the web-based basic local alignment search tool algorithm to other sequences available in nonredundant nucleotides sequences database at the National Center for Biotechnology Information.
RT-PCR of Selected Genes
The RT reaction was carried out following the manufacturer's instructions (Gibco-BRL, Grand Island, NY) using a mix of a reverse primer (Table 2) plus ß-actin primer [20], and Superscript RT enzyme in a volume of 20 µl to prime the RT reaction and to produce specific cDNAs of the respective locus plus ß-actin locus, respectively. Tubes were heated in a thermal cycling block to 70°C for 5 min to denature the secondary RNA structure, the RT mix was then completed with the addition of 5 units of Superscript RT enzyme, and then incubated at 58°C for 20 min to allow the reverse transcription of RNA. PCR was performed by adding 5-µl aliquots of each sample to the PCR mix containing the specific forward primers (Table 2). ß-Actin amplification was used to normalize the amount of mRNA in each preparation of blastocysts.
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Primer sequences, annealing temperature, and the approximate sizes of the amplified fragments are listed in Table 2. Because sequences of the bovine leukemia inhibitory (BLI) factor-receptor-ß (LR-ß), sarcosine oxidase (SOX), and connexin 31 (Cx31) genes were not available, primer sequences were chosen on the basis of conserved regions in available gene sequences (human, mouse, pig, and rat). The rest of the primers (BLI factor, mitochondrial manganese-superoxide dismutase, MnSOD; Bos taurus apoptosis regulator box-, Bax, and connexin 43) were selected based on the bovine sequence of the respective genes (sequences available in the gene database at the National Center for Biotechnology Information).
PCRs were carried out in 25-µl volumes in a PHC-3 Thermal Cycler (Techne, Cambridge, U.K.). The reaction mixture contained 2.5 µl of 10x buffer (Promega), 1 unit of Taq polymerase (Promega), 100 µM of each dNTP, 0.2 µM of each reverse primer, and 2 mM MgCl2. Samples were loaded from ice directly into the heating block at 93°C to minimize the time required to reach denaturation temperatures. The PCR protocol was an initial step of 93°C (2 min), followed by 33 cycles at 93°C (30 sec), 55°C (30 sec), and 72°C (30 sec). The final cycle of extension was at 72°C for 10 min. PCR products were resolved in 2% TBE agarose gels, followed by staining with ethidium bromide, and visualized using ultraviolet light. Semiquantitation of the RT-PCR was performed by carrying out a 5-fold dilution series in the RT-PCR reactions. Each preparation of blastocysts (3 pools of blastocysts per treatment) was first normalized for an equal amount of ß-actin mRNA in the RT-PCR. Each analysis of pools of blastocyst was repeated 3 times. As negative controls, tubes were always prepared in which RNA or RT was omitted during the RT reaction. Generation of the expected fragments was strictly dependent on the presence of RNA in the RT reaction, because omitting reverse transcriptase enzyme from the RT did not generate any fragment. In addition, amplicon identities were confirmed by appropriate restriction digests or sequencing of the products. Relative abundance was determined as appearance or nonappearance of RT-PCR amplification after 3 serial dilutions in 5-fold steps.
Statistical Analysis
Data of mRNA expression were analyzed using the SigmaStat (Jandel Scientific, San Rafael, CA) software package. One-way repeated-measures ANOVA (followed by multiple pair-wise comparisons using the Student-Newman-Kleus method) was used for the analysis of differences in mRNA expression assayed by semiquantitative RT-PCR.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The differential display method described here is more sensitive that those in which the cDNA is subjected to PCR amplification using poly(dT) primer (3') and arbitrary oligonucleotides for the 5'-primer or when both arbitrary primers are used for cDNA synthesis and PCR amplification. Both variants require a low annealing temperature, and as a consequence, the product quality is not only a function of initial concentration of cDNA species, but also is dependent on the quality of a particular match between primer and template. Our system is based on the use of highly stringent PCR conditions. Our RNA fingerprinting, based on AFLP, allows a detailed characterization of mRNA expression. To obtain an optimum number of scorable polymorphic bands per primer combination, we tested different numbers of selective nucleotides on a few selected bovine blastocyst samples. The best results were produced by the combination of 2 selective nucleotides in both the EcoRI and the MseI primers. These combinations yielded an average of 180 bands per amplification reaction (Fig. 1). The combinations EcoRI+AC/T/G/A and MseI+CT, were selected for the analysis of the samples yielding a total of 720 amplified products. Figure 1 shows a fingerprint of some of the samples after PCR amplification with primers EcoRI+AC MseI+CT, and autoradiography.
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Although cDNA banding between the different groups of blastocysts was highly conserved, our analysis has also indicated a small number of putative mRNAs that were uniquely associated with embryos produced under specific conditions (Fig. 1, Table 3). Analysis of more than 720 bands generated by using AFLP-differential display PCR indicated a difference of only 7% in bands that are either missing or expressed when blastocysts from different culture systems are compared. A total of 50 markers were sequenced from the differential bands found in the gel. GenBank matches (known cDNA or genes) were found for 60% of the markers. Of those, 16 matched described cDNA (7% of total bands), 14 corresponded to ribosomal and mitochondrial nonspecific RNA, and 20 are entirely unknown to date. The significance of this observation is yet to be determined.
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Expression of mRNA for Seven Selected Genes
In order to analyze quantitative differential expression of 7 selected genes, gene-specific primers were designed and used for RT-PCR. Each analysis of the 3 pools of blastocysts was repeated 3 times with similar results. As illustrated in Figures 2 and 3, differences in mRNA expression among the different blastocysts were found for genes related to apoptosis and oxidative stress such as Bax, SOD, and SOX, in genes related to communication through gap junctions such as Cx31 and Cx43, and in genes related to differentiation and implantation such as LIF and LR-ß.
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Bax was strongly expressed in blastocysts produced in vitro in SOF, but was far less prevalent in all other groups. MnSOD was strongly expressed in in vivo-produced blastocysts and those from the ewe oviduct, being expressed at a lower level in both in vitro-cultured groups. The pattern of SOX expression was inversely related to that of MnSOD, with a low level of expression in in vivo-produced blastocysts, the level increasing as culture was carried out in the ewe oviduct, coculture, and SOF, respectively. LIF and LR-beta had a low level of expression in both in vivo cultured groups compared with those cultured in vitro. There was a very low level of expression of Cx43 in SOF-cultured blastocysts compared with all other groups. In contrast, Cx31 mRNA was expressed strongly in SOF-derived blastocysts, was intermediate in coculture and ewe oviduct-derived blastocysts, with very low expression in in vivo-derived blastocysts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is perhaps not surprising that there was such a high degree of conservation in the cDNA banding patterns between the groups of blastocysts, because it has been demonstrated recently that cDNA banding patterns are largely conserved from the 8- to 16-cell stage through to the blastocyst stage in in vitro-produced bovine embryos [21]. It may be the case, however, that although the percentage of differences is small, these culture environment-specific cDNAs represent critical genes involved in the determination of embryo quality and may help to explain the variation in terms of cryotolerance observed between these blastocysts [3]. In addition, a number of bands did not display any significant sequence similarity to known gene products, and therefore, may represent novel transcripts. Genes expressed only in in vitro-produced blastocysts (SOF or coculture) may have an important role in supporting development under artificial in vitro culture (IVC) conditions (for example, the expression of the transient receptor potential channel-related mRNA could be due to modification in calcium stores) or may be due to nonspecific expression (for instance, demethylation of Bos taurus cyp17A2 has been observed during IVC [22]). Among the genes that are expressed in blastocysts produced following in vivo culture (ewe oviduct or heifer oviduct), are genes related to growth, immune differentiation, transcription, and translation. It is a challenge to change the culture conditions in vitro in order to induce the expression of these genes, and in that way, improve the quality of the embryos. These genes could well prove useful as markers for identification of embryos of high quality. Many of these gene products represent new targets for future research.
We also demonstrated clear differences in the level of expression of several developmentally important gene transcripts. The expression of Bax was higher in blastocysts produced in SOF than those developed in coculture or in vivo. In agreement, Gjorret et al. [23] reported that apoptosis is more frequent in in vitro-produced than in vivo-produced blastocysts. Consistent with these observations, a higher incidence of apoptosis has been reported in in vitro-produced blastocysts derived from late-cleaving zygotes than those that cleave earlier [24]. The same authors reported that the incidence of apoptosis increases in bovine embryos produced in the presence of serum [24]. Distortions of apoptosis in the blastocyst may lead to either early embryonic death or the formation of anomalies in the fetus that produce early abortions [25].
Oxidative stress is also involved in the etiology of defective embryo development. Conditions of culture can enhance the production of reactive oxygen species (ROS), which can alter most types of cellular molecules, and can also induce development retardation. An increased production of ROS has been measured in mouse embryos produced in vitro compared with those derived in vivo [26]. In the present study, MnSOD expression was detected in blastocysts from each of the 4 culture environments. However, expression was highest in in vivo-produced blastocysts and those cultured in the ewe oviduct and lower in those produced by culture in vitro. This would be consistent with our observations on the cryotolerance of these embryos [2, 3]. In addition, our observations are consistent with those of Lequarre et al. [14] who demonstrated a culture environment-dependent expression of MnSOD. In that study, no expression was detected at the 5- to 8-cell, 9- to 16-cell and morula stages when culture took place in the absence of serum, while it was detected in almost 80% of blastocysts. In contrast, in the presence of 5% serum, or in vivo-produced embryo mRNA expression was detectable in 58% of morulae and 74% of blastocysts. The low level of expression of MnSOD in in vitro-produced blastocysts may be indicative of low mitochondrial activity, because Farin et al. [27] have reported that the mitochondrial population in embryos produced using in vitro culture systems is compromised.
In contrast to MnSOD, the SOX enzyme is expressed at higher levels in embryos produced in vitro or in coculture than the in those produced in vivo. SOX is a member of a recently recognized family of enzymes that contain covalently bound flavin and catalyze oxidation reactions [28]. SOX is a peroxisomal enzyme that may be associated with the peroxisomal membrane. Peroxisomas act in detoxification and also in the first 2 steps of the synthesis of some lipids, and in the oxidation of some lipid of more than 18-carbon-long chains. Alteration of this transcription could be related to the high level of H2O2 measured in bovine embryos produced in vitro, with high production of glycine and glucose, with lipid peroxidations, and in general with lipid metabolism [26]. The expression of many genes can be up-regulated or down-regulated by ROS (i.e., ROS may activate the antioxidant defense genes) [26].
At least 4 connexins contribute to gap junctions in preimplantation development [29]. These junctions are essential for the transport of cryoprotectant and fluids during freezing and thawing. We have analyzed 2 of these, Cx31 and Cx43. In mice, Cx31 and Cx43 transcripts are abundant in the zygote and they are degraded at the 4-cell stage to low levels of Cx31 and undetectable levels of Cx43. Reexpression of Cx43 and Cx31 mRNA occurs from the compacted morula stage onward. At the blastocyst stage, both connexins are coexpressed in the trophectoderm as well as in the inner cell mass. After implantation, compartmentalization of both connexins is observed. This compartmentalization in connexin expression between the derivatives of the inner cell mass and the trophectoderm may maintain the different developmental programs. Apparently, Cx31 is not related to the first step in trophoblast lineage development and could serve as a compensatory channel during preimplantation development [30].
Wrenzycki et al. [31, 32] reported that Cx43 mRNA was detectable in in vitro-produced bovine embryos from the oocyte to the morula stage, but was not detectable in blastocysts or hatched blastocysts, in contrast to its detection in in vivo-derived blastocysts. The same authors subsequently observed that the expression pattern for Cx43 in vitro was altered in the presence of serum, disappearing at the 8- to 16-cell stage, and reappearing at the hatched blastocyst stage [16]. In the present study, Cx43 was strongly expressed in blastocysts derived from coculture, culture in the ewe oviduct, or in vivo. In contrast, Cx31 mRNA was expressed strongly in SOF-derived blastocysts, was intermediate in coculture and ewe oviduct-derived blastocysts, with very low expression in in vivo-derived blastocysts. This pattern of expression is entirely consistent with the previously reported quality of these blastocysts from our group in terms of ultrastructure [33] and survival following vitrification [2, 3].
LIF plays an essential role during early differentiation and implantation in embryos. Oviductal cells synthesize LIF to promote and condition the embryo for implantation [34]. LIF has been reported to bind to blastocyst-stage embryos and affect their development in culture [35]. The receptor consists of the 2 dimerizing subunits, glycoprotein 130 and LR-ß. Human and murine embryos produced in vitro express LIF and LR-ß mRNA transcripts throughout the preimplantaion period [36]. In cattle, Eckert and Niemann [37] reported differences in the expression of LIF and LR-ß between in vitro-derived and in vivo-derived embryos. We have detected LIF and LR-ß mRNAs at higher levels in in vitro-produced bovine blastocysts, irrespective of culture system, than in in vivo-derived blastocysts; blastocysts derived from culture in the ewe oviduct were somewhat intermediate. As suggested by Eckert and Niemann [37], some perturbation of the mRNA expression patterns of the specific LIF/LIF-receptor system occur during the development of bovine embryos in vitro. This may lead to abnormal development of the inner cell mass and trophectoderm in the blastocyst.
It is perhaps not surprising that this period of postfertilization culture is the period having the greatest effect on blastocyst quality [3] when one considers that during that 6-day window in the bovine embryo, several major developmental events take place. These include 1) the first cleavage division, the timing of which is known to be of critical importance in determining the subsequent development of the embryo [38]; 2) the switching on of the embryonic genome [39]; 3) compaction of the morula, which involves the establishment of the first intimate cell-to-cell contacts in the embryo; and 4) blastocyst formation, involving the differentiation of 2 cell types, the trophectoderm and the inner cell mass. Clearly, any modifications of the culture environment, which could affect any or all of these processes could have a major effect on the quality of the embryo.
In conclusion, our data indicate that conditions of postfertilization culture, the period of development in vitro having the greatest effect on blastocyst quality [3], influence mRNA expression in the resulting blastocyst. The observed alterations in mRNA expression may be directly linked with the quality of these blastocysts. The challenge for the future is to modify the conditions of in vitro culture during this critical window of development in order to try and mimic the pattern of mRNA expression as it occurs in vivo, and in that way, produce embryos of higher quality in vitro.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was financed by the European Union Commission (QLK3-CT-1999-00104) and by Ministerio de Ciencia y Tecnología (Spain) (RTA01-064 and RTA01-063-C2-1). D.R. was supported by a grant from the Greek State Scholarships Foundation. 
2 Correspondence: Alfonso Gutiérrez-Adán, Dpto. de Reproducción Animal y Conservación de Recursos Zoogenéticos, INIA, Ctra de la Coruña Km 5.9, Madrid 28040, Spain. FAX: 91 347 4014; agutierr{at}inia.es
Accepted: October 9, 2001.
Received: July 30, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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D. Rizos, A. Gutierrez-Adan, S. Perez-Garnelo, J. de la Fuente, M.P. Boland, and P. Lonergan Bovine Embryo Culture in the Presence or Absence of Serum: Implications for Blastocyst Development, Cryotolerance, and Messenger RNA Expression Biol Reprod, January 1, 2003; 68(1): 236 - 243. [Abstract] [Full Text] [PDF]
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