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Division of Reproductive Endocrinology and Infertility, Departments of Obstetrics and Gynecology and Physiology, University of Toronto, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5
ABSTRACT
Cytoplasmic transfer is an assisted reproductive technique that involves the infusion of ooplasm from a donor oocyte into a recipient oocyte of inferior developmental competence. Although this technique has shown some success for couples with recurrent in vitro fertilization failure, it results in mitochondrial heteroplasmy in the offspring, defined as the presence of two different mitochondrial genomes in the same individual. Because the long-term health consequences of mitochondrial heteroplasmy are unknown, there is a need for appropriate animal models to evaluate any physiological changes of dual mtDNA genotypes. This longitudinal study was designed as a preliminary screen of basic physiological functions for heteroplasmic mice (NZB mtDNA on a BALB/cByJ background). The mice were tested for cardiovascular and metabolic function, hematological parameters, body mass analysis, ovarian reserve, and tissue histologic abnormalities over a period of 15 mo. Heteroplasmic mice developed systemic hypertension that corrected over time and was accompanied by cardiac changes consistent with pulmonary hypertension. In addition, heteroplasmic animals had increased body mass and fat mass compared with controls at all ages. Finally, these animals had abnormalities in electrolytes and hematological parameters. Our findings suggest that there are significant physiological differences between heteroplasmic and control mice. Because ooplasm transfer appears to be consistently associated with mitochondrial heteroplasmy, children conceived through ooplasm transfer should be closely followed to determine if they are at risk for any health problems.
assisted reproductive technology, cytoplasm transfer, heteroplasmy, mitochondria, ooplasm transfer, ovary
The presence of different mtDNA mutations has been studied intensively in the human because of the prevalence of heritable mtDNA diseases occurring in at least 1 of 8000 adults, making mtDNA disorders among the most frequently inherited metabolic disorders [1]. The physiological effects of heteroplasmy are generally not seen until the ratio of mutant to wild-type mtDNA exceeds a specific threshold [2]. It appears that a small number of mitochondria with normal mtDNA are sufficient to protect individuals from defects in oxidative phosphorylation [3], with disease onset primarily occurring during the later stages of adulthood [4]. The natural occurrence of heteroplasmy may, therefore, be underestimated in the human population both because of the lack of symptomatic phenotypes and inherent inaccuracies in identifying the presence of sequence polymorphisms in the mtDNA genome [5].
Clinically, mitochondrial diseases often present as oxidative phosphorylation disorders affecting the heart, muscle, and brain—highly aerobic and postmitotic tissues [6]. Interestingly, the same mtDNA mutation can result in different pathologies in different patients, perhaps because of a variation in tissue distribution and mutational load among individuals. Point mutations within the coding region of mitochondrial genes, usually in combination with the previously described common polymorphisms, have also been associated with hypertension and cardiovascular and renal pathologies [7]. Additionally, because of the dependence of mitochondria on nuclear-encoded genes, genomic mutations can result in mitochondrial depletion syndromes that are inherited in mendelian ratios (for a review, see Suomalainen and Kaukonen [8]).
Mitochondrial heteroplasmy became an important topic in the reproductive sciences with the development of cytoplasmic transfer as a fertility treatment. This treatment was based on results of earlier animal experiments involving mouse embryos from strains that experience a developmental block. Injection of cytoplasm from an oocyte of a nonblocking strain into a blocking strain increased cleavage rates of the recipient embryos compared with noninjected controls, suggesting the presence of an ooplasmic factor capable of rescuing the developmental block [9]. Cohen et al. [10], Barritt et al. [11], and Lanzendorf et al. [12] have performed ooplasm transfers from healthy fertile donors into oocytes of patients with repeated embryonic developmental failure, leading to the birth of at least 30 children worldwide [13]. There have been several concerns surrounding both the scientific methodology and ethics of performing this technique in the clinical setting [14–17], as the underlying mechanism and the long-term safety of cytoplasmic transfer remain unknown.
A number of ooplasm components are transferred from the donor to the recipient oocyte, including mRNA, proteins, ribosomes, and other organelles, including mitochondria. The injection of multiple factors has, therefore, hampered the identification of the cytoplasmic factor responsible for improved developmental potential. We believe it is likely that mitochondria play a significant role in the embryonic rescue, as the injection of an enriched mitochondrial fraction, isolated from granulosa or embryonic stem cells, is capable of reducing the incidence of in vitro fragmentation of oocytes [18]. In addition, transfer of mitochondrial fractions isolated from developmentally competent pig oocytes into developmentally compromised oocytes has been shown to improve fertilization rates [19]. Although it is presently unknown whether mitochondria alone are responsible for the embryonic rescue seen with clinical ooplasm transfer, children born following this procedure have demonstrated mitochondrial heteroplasmy. Mitochondrial heteroplasmy created as a consequence of ooplasm transfer results from the persistence of donor mitochondria in the recipient child [13]. The health impact of induced mitochondrial heteroplasmy in these children is unknown and has created the need for a comprehensive study of the physiological effects of induced mitochondrial heteroplasmy [16, 17]. Although an animal model is not an ideal comparison for clinical ooplasm transfer, it does represent the most accessible source for study of long-term physiological consequences because of the relatively short life span of the mouse.
Animal models of heteroplasmy have been created through various means in the mouse [20–25], and natural heteroplasmy has been studied in the bovine [26, 27]. The resulting animals were primarily used to investigate mtDNA segregation patterns [20, 21, 23] and the effect of a specific mtDNA mutation on the organism [24, 25]. Heteroplasmic mice have been described as normal and healthy, as no overt phenotype was observed [25]. However, none of these studies has focused on the general effects of neutral heteroplasmy on physiological development. We believe it is essential to study the genetic consequences of mitochondrial heteroplasmy in greater detail in order to determine the long-term physiological consequences of ooplasm transfer.
Nine female mice of a BALB/cByJ (Mus musculus domesticus) genomic background carrying varying heteroplasmic levels of NZB (Mus musculus spretus) mitochondria (range, 19%–56% NZB mtDNA [20]) and 12 age-matched controls of a BALB/cByJ genomic background (Jackson Labs) were used in this study. The heteroplasmic mice used in this study were created by electrofusing BALB/cByJ embryos with cytoplasts from NZB oocytes, resulting in the creation of heteroplasmic embryos, which were then transferred into pseudopregnant females [20]. Since the creation of this heteroplasmic strain, mitochondrial heteroplasmy has been stably maintained through the generations over the last decade. NZB mtDNA contains 108 neutral base substitutions, resulting in 15 conservative amino acid differences between the BALB/cByJ and NZB mtDNA genomes [20, 28]. Previous in vitro hepatocyte studies have shown that NZB mtDNA in this nuclear background has neither a decrease in respiratory or replication efficiency [28]. The control and heteroplasmic mice were screened at 4, 8, and 12 mo of age by the physiological tests outlined below. Two heteroplasmic and one control mouse contracted an eye infection and were not run through the final screen at 12 mo. At the end of the study, the mice were subjected to a thorough necropsy, in which a subset of tissues was isolated for histological evaluation and determination of tissue-specific mitochondrial heteroplasmy levels. A second group of heteroplasmic (19%–58%) and age-matched control mice were killed at 4 mo of age, and tissues were obtained to assess histological defects in the organs. The Mount Sinai Hospital Animal Care Committee approved all animal protocols, and protocols were in compliance with standards for the ethical treatment of animals.
Determination of mtDNA Heteroplasmy
The level of mtDNA heteroplasmy in isolated organs was determined according to the method previously described [20, 28]. Briefly, isolated mtDNA between positions 3571 and 4059 of the mtDNA genome was amplified and radioactively labeled by PCR. The product was then digested with the restriction enzyme RsaI (Invitrogen), and the digested fragments were separated on a 10% nondenaturing polyacrylamide gel. The optical density of each band was determined, and the proportion of heteroplasmy was calculated as previously described [20]. There was no positive correlation between the degree of mitochondrial heteroplasmy in any of the tissues analyzed at the time of necropsy and the outcomes of the physiological screens.
Mouse blood pressure and heart rate were measured by a tail cuff system (Visitech BP-2000 Blood Pressure Analysis System) [29]. The final report from the analysis included the average arterial pressure (millimeters of mercury), average pulse rate (beats per minute), and number of successful measurement cycles. The mice were tested at 4, 8, and 12 mo of age. This monitoring system has been shown to be particularly effective for screening and monitoring changes in blood pressure and heart rate between different groups over time [30, 31]. Saphenous vein blood (
30 µl) was collected and analyzed with a COULTER AC•T diff Analyzer (Coulter). The system output consisted of erythrocyte count (x1012/L), hemoglobin concentration (HGB; grams per liter), hematocrit (HCT; liters per liter), mean corpuscular volume (femtoliters), mean corpuscular hemoglobin (MCH; picograms per cell), mean corpuscular hemoglobin concentration (MCHC; grams per liter), platelet (x109/L), and leukocyte count (x109/L).
A diabetes screen was performed by the glucose tolerance test. The mice had unlimited access to water but were fasted overnight prior to the test. The body weight and a baseline glucose level were determined for each mouse prior to administering the glucose challenge. Blood glucose concentration was determined before and 20 min after i.p. injection of 1.5 mg of glucose per gram of body weight.
Blood biochemistry was measured with the Nova Biomedical Stat Profile M7 (Nova Biomedical). Whole blood (120 µl) was collected from the saphenous vein of the mouse and analyzed for levels of Na+, K+, Cl–, iCa2+, glucose, lactate, and blood urea nitrogen (all measured in millimoles per liter).
The renal system was further assessed through urinalysis, testing for the presence of glucose, protein, and blood in the urine. Urine was collected from conscious restrained mice, and 2 µl was placed onto a Chemstrip 4MD urinalysis test strip (Roche). This method was capable of monitoring glucose concentrations in the range of 2.8–55 mmol/L, protein in the range of 0–500 mg/dl, and blood in the range of 0–250 erythrocytes/µl.
The body composition was determined through use of the PIXI-mus II Small Animal Densitometer (version 1.43.020; Lunar Corp.) [32]. The analysis was performed on anesthetized live mice, and obtained bone variables included bone mineral density (BMD; grams per square centimeter), bone mineral content (BMC; grams), and total bone area (square centimeters). The reported tissue variables included lean mass (grams), fat mass (grams), total body mass (grams), and the percentage of fat mass.
The heteroplasmic mice are known to be fertile, as the original transgenic mice have been able to propagate and maintain the heteroplasmic line [20]. At 15 mo of age, the mice were stimulated with exogenous gonadotropins (10 U of eCG and hCG, respectively; Sigma). Sixteen hours following the hCG injection, the mice were killed, and their oviducts were flushed to determine ovulation rate. One ovary from each of the young (4 mo) and old (15 mo) control and heteroplasmic mice was fixed and subjected to histomorphometric analysis as previously described [33].
At 15 mo of age, the mice were killed, and the kidneys and heart were fixed in 10% PBS. Additionally, small pieces of tissue were taken from kidneys, ovaries, pancreas, heart, lungs, tail, skeletal muscle, spleen, liver, and blood in order to perform mtDNA heteroplasmy analysis on those tissues. DNA was extracted and analyzed for mitochondrial heteroplasmy levels in both heteroplasmic and control mice as previously described [20].
All longitudinal data were analyzed by Repeated Measures Greenhouse-Geisser Analysis, while follicle counts were measured by t-tests (SPSS Release 11.0.1, Standard Version; LEAD Technologies). Data were analyzed for effects of heteroplasmy and aging and further to determine whether heteroplasmy had a direct effect on age-related changes in physiology. Most results obtained were consistent with those previously reported for female mice of the BALB/cByJ strain [34–37] (http://www.jax.org/phenome).
The heteroplasmic mice were found to have a highly significant increase in blood pressure compared with controls at the ages of 4 and 8 mo (P = 0.0001, F(1.0, 12.0) = 27.8). The blood pressure was the same as controls by the age of 12 mo. The change in blood pressure with increasing age (P = 0.017, F(1.45, 17.4) = 5.95) was found to be dependent on the heteroplasmic state (P = 0.002, F(1.45, 17.4) = 11.2) (Fig. 1). There was a significant increase in heart rate with increasing age (P = 0.007, F(1.97, 23.7) = 6.23), irrespective of mitochondrial status (data not shown).
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Histological analysis of the heart demonstrated mild physiological adaptations to the increased blood pressure. The majority of the hearts from the heteroplasmic animals had a left:right ventricular ratio of less than 3:1, the standard normal range for most mammalian species. This slight increase in size of the right ventricle is a common adaptation to pulmonary hypertension and is also indicative of a potential increase in afterload volume, which may decrease cardiac output from the left ventricle [38]. There were no myocardial or endocardial changes in the heteroplasmic hearts compared with controls. Some deeply basophilic stippled material was found in the epicardium of both ventricles in both control and heteroplasmic mice. There were varying degrees of moderate increases in fibrous tissue proliferation but no evidence of myocardiocyte degeneration or necrosis in these affected areas. These lesions are common in various strains of mice, including BALB/c, in which epicardial mineralization occurs spontaneously with an incidence of 11% in males and 4% in females [39]. The lack of abnormal serum calcium levels in either of the groups indicates that the pathology is unrelated to mitochondrial status and is a variant related to mouse strain. An internal control was performed to verify this finding, in which separate cohorts of control and heteroplasmic mice were necropsied at the age of 4 mo. Similar changes to the epicardial tissue were apparent in this cohort, supporting the fact that the lesions are an artifact of mouse strain and are not related to the heteroplasmic state.
A number of hematological parameters were found to be significantly different (Table 1). Heteroplasmic mice had a significant decrease in (P = 0.032, F(1.00, 16.0) = 5.52) and MCHC (P = 0.0001, F(1.00, 16.0) = 21.0) compared with control mice, and both parameters were significantly affected by the interaction between age and heteroplasmic state (MCH: P = 0.001, F(1.00, 16.0) = 14.9; MCHC: P = 0.038, F(1.00, 16.0) = 0.502). The alterations in MCH and MCHC suggest a change in either the amount or structure of HGB. Additionally, the heteroplasmic mice were observed to have an increased erythrocyte count (P = 0.03, F(1.00, 16.0) = 5.86) and a significant decrease in mean corpuscular volume dependent on age (MCV, P = 0.0001, F(1.00, 16.0) = 68.0) compared with controls. The heteroplasmic genotype also increased both HCT (P = 0.023, F(1.00, 16.0) = 6.29) and HGB (P = 0.037, F(1.00, 16.0) = 5.19), likely a reflection of the greater change in erythrocyte volume over time in the heteroplasmic group.
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Small adaptations were also noted in the kidney, as all heteroplasmic mice displayed a slight thickening of the mesangium, a layer of cells surrounding the capillaries in the juxtaglomerular apparatus that play a role in controlling the surface area of the capillaries for filtration. However, this increase in mesangium was not extensive enough to negatively affect glomerular filtration. Additionally, the majority of both control and heteroplasmic mice demonstrated an increase in fibrous tissue between the tubules of the outer cortex and medulla at the age of 15 mo. The interstitial changes in the kidneys are an incidental finding that is frequently encountered in aging mice and is not thought to contribute to impaired renal function.
There were no significant differences between control and heteroplasmic mice noted in either the glucose tolerance test or the urinalysis (data not shown). There were, however, significant differences in some aspects of the blood biochemistry assays, including potassium, chloride, and urea content (Table 2). Most striking was an initial decrease in blood urea nitrogen content in the heteroplasmic mice compared with controls (P = 0.011, F(1.00, 16.0) = 8.24). This change disappeared by the age of 12 mo, but the age-related changes were both significant (P = 0.038, F(1.00, 16.0) = 3.78) and dependent on the mitochondrial genotype of the mice (P = 0.015, F(1.00, 16.0) = 29.5). Potassium content was increased in heteroplasmic mice at the age of 8 mo (P = 0.015, F(1.00, 16.0) = 7.40), and the heteroplasmic state of the mice influenced the potassium balance as a function of age (P = 0.011, F(1.00, 16.0) = 10.9). Changes in chloride concentration occurred as the mice aged, with heteroplasmic mice having a significantly lower level of serum chloride at the ages of 8 and 12 mo (P = 0.029, F(1.00, 16.0) = 5.75).
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One of the most striking differences occurred in body mass in the heteroplasmic and control mice, both initially and with age (Fig. 2 and Table 3). Heteroplasmic mice were consistently heavier than control mice, with a significantly higher total body mass at both 4 and 12 mo (P = 0.0001, F(1.00, 16.0) = 21.2). Although aging caused a significant increase in total mass in both groups (P = 0.0001, F(1.00, 16.0) = 117), this increase was not affected by genotype (P = 0.714, F(1.00, 16.0) = 0.139). However, there was a difference in the type of mass gained, with heteroplasmic mice gaining significantly more fat mass (P = 0.016, F(1.00, 16.0) = 10.2) than controls. This imbalance in fat mass gained with age was directly affected by the heteroplasmic state (P = 0.0018, F(1.00, 16.0) = 6.91).
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Differences between the two genotypes for bone variables were not found to be significant. However, the genotype of the mice significantly affected how the variables changed with age. The heteroplasmic mice displayed a decreased bone area with age (P = 0.0001, F(1.00, 16.0) = 13.2) and slightly increased BMD (P = 0.0460, F(1.00, 16.00) = 4.67). The increase in BMD resulted from the more dramatic decrease in bone area with age in the heteroplasmic mice rather than any significant changes in BMC.
The heteroplasmic females typically have four to six pups per litter (B. Battersby, personal communication), whereas BALB/cByJ control females are reported to have approximately seven pups per litter (http://www.jax.org/phenome). Because the heteroplasmic mice were fertile, we decided to investigate whether differences in ovarian reserve existed and whether these differences were maintained with aging. Ovaries of heteroplasmic (n = 6) and age-matched control mice (n = 5) were subjected to histomorphometric analysis of follicles at the age of 4 mo. Heteroplasmic mice had a lower number of primordial and primary follicles, accompanied by a significant increase in secondary follicles, suggesting altered follicular dynamics. Surprisingly, this difference was not sustained with aging, as there were no differences in follicular reserve at the age of 15 mo in the mice run through the physiology screens (Fig. 3). Moreover, ovulation rate triggered by administration of exogenous gonadotropins was not significantly altered between the heteroplasmic (range, 0–4; mean, 1.18 ± 2.14; n = 11) and control mice (range, 0–7; mean 2.0 ± 1.63; n = 7) at the age of 15 mo.
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The analysis of mtDNA content in the control and heteroplasmic mice determined that there were no detectable levels of NZB mtDNA in any of the control mice and that the levels of NZB mtDNA remained relatively constant over the course of the study in the heteroplasmic mice. We compared the level of mtDNA heteroplasmy from the original tail analysis to the levels found at the time of necropsy. There were no statistically significant tissue-specific changes in the distribution of mtDNA within this cohort of mice (average change, 0.94 ± 0.2, n = 7; Fig. 4). The lack of preferential selection for mtDNA genotypes is in contrast to previous reports [28, 40] that have shown tissue-specific selection for either the NZB (liver/kidney) or BALB/cByJ (blood/spleen) mitochondria. Although our results do not parallel previous reports, it is possible that the small sample size contributed to the lack of significant tissue-specific selection.
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The most striking results of the study were the incidence of significantly elevated blood pressure and the increased body and fat mass in the heteroplasmic mice. Although the elevated blood pressure decreased to normal over the course of the study, its occurrence does cause concern. Pathological adaptations to hypertension were noted in both the heart and kidney. In addition, there were changes in right ventricular volume consistent with possible pulmonary hypertension. Pulmonary hypertension is associated with increased afterload volume, resulting in decreased cardiac output, often leading to hypotension [41], and is a potential source of the decline in blood pressure evidenced in our hypertensive heteroplasmic mice. The mice in this study were observed up to the age of 15 mo, whereas a longer-term study of these mice could have provided more information concerning the potential for associated pathological changes to the heart, vasculature, and kidney.
There were significant differences noted in the total weight and proportion of fat mass in the heteroplasmic and control mice. The heteroplasmic mice had a higher starting total mass, fat mass, and lean mass than controls, although they were within the range of reported weights and fat percentages for BALB/c mice of similar age [37]. However, the heteroplasmic mice demonstrated a disproportionate weight gain, leading to an increased percentage of fat mass. The increased weight and increased fat mass, together with the cardiovascular system changes, is reminiscent of some of the changes associated with the metabolic syndrome [42]. The glucose tolerance test results were normal in the heteroplasmic mice, although this test did not directly measure insulin levels, and the results cannot be used to comment on the possible incidence of insulin resistance in the heteroplasmic mice, which is known to be tissue/cell type-specific.
There were a number of hematological changes associated with both increased biological age and heteroplasmy. The decreased levels of MCH and MCHC in heteroplasmic mice suggest a mild iron deficiency in these mice similar to the age-related decline in MCHC in the laboratory rat [43]. Decreased blood volume and MCH with increased age is usually indicative of a decrease in average red blood cell size, clinically diagnosed as hypochromic microcytic anemia in the human population. The results from our study indicate that although there are similar hematological changes with age between the control and heteroplasmic mice, the heteroplasmic mice adapt less favorably to these changes, demonstrating a decreased oxygen-carrying capacity of the erythrocytes.
Differences between the heteroplasmic and control mice were noted in potassium, chloride, and urea content. The small differences do not suggest difficulties in acid-base balance or filtration in the kidney, supported by the normal results of the urinalysis testing. The cause and effect of these imbalances cannot be conclusively determined from the data set collected.
One possible cause of these physiological abnormalities was inefficient cross-talk between the nuclear and mitochondrial genomes. Success rates of nuclear cloning are exceptionally low, thereby restricting the ability to accurately assess the incidence of defective mitochondrial-nuclear interactions that may lead to embryo demise, fetal distress, or physiological abnormalities in offspring [44]. Experiments involving maternal pronuclear exchange between different strains of mice led to heritable abnormalities in gene expression, morphology, and physiology, perhaps due to errors in cross-talk between the mismatched nuclear and mitochondrial genomes [45]. These cross-talk issues could also be a factor in the success of therapeutic cloning, as the resultant embryonic stem cells and their differentiated daughter cells would also suffer from the same genetic burden of mitochondrial heteroplasmy. Although the heteroplasmic mice used in this study were created from two different inbred strains, previous studies on hepatic cells showed no deficiency in mitochondrial respiratory chain function or replication deficiency of NZB mtDNA on the BALB/c genetic background in these animals [28]. Thus, the physiological differences that we observed are not likely due to cross-talk issues between the mtDNA genotypes and the nuclear mtDNA replication machinery.
Although the electrofusion technique and the cross-species nature of the founder heteroplasmic mice differ significantly from the techniques and donor source used in ooplasm transfer in the clinical setting, the final outcome with respect to heritable coexistence of mitochondrial heteroplasmy is the same. The results of this study clearly indicate that although the heteroplasmic mice appeared to be healthy, there were several underlying abnormalities in standard physiological and metabolic parameters. We believe that the physiological relevance of these abnormalities should be followed up in a targeted longitudinal study of a larger cohort of control and first generation heteroplasmic mice created by the same techniques as ooplasm transfer performed in the clinical setting. The phenotypes caused by mitochondrial heteroplasmy in animal models raise concerns that may have possible clinical relevance and thus should be adequately assessed in the long-term follow-up of children born with mitochondrial heteroplasmy.
ACKNOWLEDGMENTS
We would like to thank Dr. E.A. Shoubridge and Dr. B. Battersby, Montreal Neurological Institute, McGill University, Montreal, QB, Canada, for their generosity with heteroplasmic mice. The authors would like to thank the staff and faculty of the Center for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada. Additionally, thanks are extended to Shathiyah Kulandavelu and Dr. Peter Liu for helpful discussions and to Dr. Theodore J. Brown for assistance with statistical analysis.
FOOTNOTES
3Correspondence: A. Jurisicova, 600 University Avenue, Samuel Lunenfeld Research Institute, Room 876, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5. FAX: 416 586 8588; e-mail: jurisicova{at}mshri.on.ca 
4Current address: SUNY Canton, Department of Health, Science and Professional Studies, Canton, NY 13617.
1Supported by Canadian Institute of Health Research (operating grant MOP14048). B.M.A. was supported by a Canadian Institute of Health Research Doctoral Research Award, and A.J. was supported by a Canadian Institute of Health Research New Investigator Award.
Correspondence: 2R.F. Casper, 600 University Ave., Samuel Lunenfeld Research Institute, Room 876, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5. FAX: 416 586 8588; e-mail: rfcasper{at}aol.com
Received: 11 February 2007.
First decision: 12 March 2007.
Accepted: 5 June 2007.
REFERENCES
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