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Gamete Biology |
Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, gamete biology, in vitro fertilization, sperm, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We exploited this observation as a unique opportunity to study the effects on embryogenesis of manipulating the paternal genome. We hypothesized that the impairment of embryo development is associated with chromosomal abnormalities and that these abnormalities are induced by foreign DNA itself or by conditions used to enhance integration of the DNA.
Sperm chromosome damage can be prevented, suggesting another approach for this study. Previous reports have indicated that the presence of EDTA and the absence of Ca2+ and Mg2+ from medium for sperm head isolation could improve chromosome stability [6, 7]. EGTA is important for retaining chromosome integrity in freeze-dried mouse spermatozoa [8]. In our recent study on sperm DNA structure, we observed that chelating agents could partially inhibit chromosome damage induced by detergent and dithiothreitol (DTT) [9]. We hypothesized that EDTA and EGTA might prevent foreign DNA-dependent chromosomal damage.
Lavitrano et al. [3] suggested that endonucleases might play a role in the incorporation of transgene DNA into sperm genome. Several other investigators reported evidence for nuclease activity in fully mature spermatozoa [10, 11]. We speculated that ion chelators might also affect transgene integration in ICSI-mediated transgenesis.
The objectives of this study were to determine whether the incubation of spermatozoa with foreign DNA resulted in paternal chromosome breaks and whether this association was related to transgene expression, and to determine the effect of EDTA/EGTA on these processes. We examined the chromosomal status of zygotes produced with ICSI-mediated transgenesis and its relationship to embryo development in vitro and to the efficiency of transgene expression. By exposing spermatozoa to foreign DNA, we induced breakage of the paternal chromosomes. The addition of ion chelators completely suppressed this chromosomal damage. We also observed an inverse relationship between paternal chromosome stability and integration of exogenous DNA into the embryonic genome in ICSI-mediated transgenesis. Ion chelators that were able to suppress chromosome damage also decreased transgene expression. The data suggest that spermatozoa respond to exogenous DNA with a mechanism that results in paternal chromosome damage in the embryo. Furthermore, because transgene expression persisting through the blastocyst stage is often associated with transgene integration [12], the mechanism responsible for paternal chromosome damage may be related to that for transgenic integration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B6D2F1 (C57BL/6 x DBA/2) mice, 8–16 wk of age, were used as oocyte and sperm donors. All animals used in this study were maintained in accordance with the guidelines of the Laboratory Animal Services at the University and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources National Research Council (DHEF publication 80-23, revised in 1985). The protocol for animal handling and treatment procedures was reviewed and approved by the Animal Care and Use Committee at the University of Hawaii.
Reagents
All inorganic and organic compounds were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. As an exogenous DNA, we used the 3.1-kilobase (kb) SalI-BamHI fragment of plasmid pCX-EGFP (a gift from Dr. Masaru Okabe [Osaka University]) that contains a GFP gene expressed from the cytomegalovirus-IE-chicken ß-actin enhancer-promoter combination but lacks a eukaryotic origin of replication.
Sperm preparation
Sperm samples were pretreated in three different ways: 1) freeze-thawing, 2) 0.5 % Triton X-100, or 3) 0.5 % Triton X-100 with a sucrose wash. Live motile swim-up spermatozoa were used as controls. Within each pretreatment method, four treatments were examined: 1) exposure to exogenous DNA (EGFP; [13]), 2) exposure to ion chelators EGTA/EDTA, 3) exposure to exogenous DNA in the presence of EGTA/EDTA, and 4) no DNA or ion chelators. Exogenous DNA was used at a concentration of 5 ng DNA/µl of sperm suspension (50 ng DNA
0.5 x 106 spermatozoa). EGTA and EDTA were used at a concentration of 50 mM each. The incubation time with DNA was 2 min for all treatments, except for swim-up, where spermatozoa were incubated with DNA for 10 min. Spermatozoa were used for ICSI immediately after treatment. Before injection into the oocytes, spermatozoa were washed by sequential transfer from one polyvinylpyrrolidone (PVP)-enriched Hepes-CZB drop to another. The details of sperm preparation are as follows.
Freeze-thawing Cauda epididymides were removed from one male mouse and placed in 300 µl of Hepes-CZB [14] solution, with EGTA/EDTA if appropriate, in an organ tissue culture dish (cat. no. 3513037; Falcon, Bedford, MA). The epididymal contents were expressed from the cauda epididymides with needles, and the tissue was discarded. The spermatozoa were allowed to disperse for 2–5 min at room temperature (25°C), and then 20 µl of the sperm suspension was transferred to a 250-µl tube. The tube was then plunged into liquid nitrogen (1 min), thawed at room temperature (1 min), and frozen in liquid nitrogen again (1 min). After a second thawing, 10 µl of sperm suspension was transferred to another tube, and the DNA was added.
Triton X-100 Cauda epididymides were removed and dispersed as above. The sperm suspension was transferred to a tube. Triton X-100, and EGTA/EDTA if appropriate, was added, and the mixture was pipetted twice and left for 10 min at room temperature. Subsequently, 10 µl of the sperm suspension was transferred to another tube, and the DNA was added.
Triton X-100 with sucrose wash The epididymal fluid was squeezed out and placed in a tube with chilled Hepes-CZB and Triton X-100, and EGTA/EDTA when appropriate. Spermatozoa were vigorously pipetted to disperse them evenly in the solution and then incubated for 15 min at 25°C, and 1 ml of this suspension was carefully layered over a 0.5-ml cushion of 1 M sucrose, 25 mM Tris, pH 7.4, in a microfuge test tube. This step gradient was centrifuged at 3000 x g in a swing bucket rotor for 10 min at 4°C. The pellet was resuspended in 100 µl of Hepes-CZB, with EGTA/EDTA when appropriate, and then 10 µl of the sperm suspension was transferred to another tube, and the DNA was added.
Swim-up Swim-up spermatozoa were prepared by gently placing a drop of epididymal fluid on the bottom of a 1.5-ml microfuge tube containing 500 µl of Hepes-CZB and incubating the suspension for 15 min at 37°C. Next, 10 µl was gently taken off the top of the suspension and transferred to another tube, and DNA and/or EGTA/EDTA were added.
Preparation of Oocytes
Female mice were induced to superovulate by consecutive injections of 5 IU eCG and 5 IU hCG, 48 h apart. Oocytes collected from oviducts 12–14 h after hCG injection were freed from cumulus cells by treatment with 0.1% bovine testicular hyaluronidase (300 USP units/ng) in Hepes-CZB. The oocytes were washed thoroughly and used immediately for ICSI.
Intracytoplasmic Sperm Injection
ICSI was carried out according to the method of Kimura and Yanagimachi [15] with some modifications. A small drop of sperm suspension was mixed thoroughly with an equal volume of Hepes-CZB containing 12% (w/v) PVP (360 kDa) immediately before ICSI. ICSI was performed using Eppendorf micromanipulators (Micromanipulator TransferMan; Eppendorf, Hamburg, Germany) with a Piezo-electric actuator (PMM Controller, model PMAS-CT150; Prima Tech, Tsukuba, Japan). Before tail removal, spermatozoa were washed by sequential transfer from one PVP-Hepes-CZB drop to another. A single spermatozoon was drawn, tail first, into the injection pipette and moved back and forth until the head-midpiece junction (the neck) was at the opening of the injection pipette. The head was separated from the midpiece by applying one or more piezo pulses. After discarding the midpiece and tail, the head was redrawn into the pipette and injected immediately into an oocyte. ICSI was performed in Hepes-CZB within 1 h after oocyte collection and sperm pretreatment. Sperm-injected oocytes were transferred into CZB medium [16] and cultured at 37°C. The oocytes were examined 6 h after ICSI for survival and activation.
Chromosome Analysis
After 6–8 h of culture, injected oocytes were transferred into CZB containing 0.006 µg/ml vinblastine, which was added to inhibit spindle formation and syngamy. Between 19 and 21 h after ICSI, oocytes were treated with 1% pronase (1000 tyrosine units/mg; Kaken Pharmaceuticals, Tokyo, Japan) for 5 min at room temperature to soften the zonae pellucidae. Oocytes were then treated with hypotonic solution (1:1 mixture of 1% sodium citrate:30% fetal bovine serum) for 5 min at 37°C or 10 min at 25°C. Chromosomes were spread on glass slides by the gradual fixation/air-drying method [17]. The preparations were stained with 2% Giemsa (Merck, Darmstadt, Germany) in PBS (pH 6.8) for 10 min for conventional chromosome analysis. The percentages of zygotes with normal and abnormal chromosomes were determined. The paternal chromosomes were considered normal when an egg contained 40 structurally normal metaphase chromosomes. It was not always possible to distinguish between chromosomes of paternal and maternal origin. However, because oocyte chromosomes rarely show structural aberrations at the first cleavage metaphase after parthenogenetic activation (unpublished observations), abnormal chromosomes (chromosome and chromatid breaks and exchanges) within fertilized oocytes were considered of sperm origin. When >9 aberrations per karyoplate were observed, we called them multiple and scored them as 10. To express the intensity of the chromosomal damage, aberrations-per-spermatozoon rate was calculated by dividing the total number of aberrations by the total number of karyoplates examined in one treatment.
Embryo Culture
After ICSI, the oocytes were placed in 50-µl drops of CZB medium preequilibrated overnight with humidified 5% CO2 in air. The culture drops were contained in plastic culture dishes (cat. no. 351007; Falcon) and overlaid with mineral oil (Squibb and Sons, Princeton, NJ). The number of two-cell embryos with a second polar body (fertilized) was recorded after 24 h in culture. Progress through preimplantation to the blastocyst stage was subsequently examined up to 96 h after the commencement of culture.
Transgene Expression
Three to 3.5 days after injection, embryos were examined for GFP expression by epifluorescence microscopy with an ultraviolet light source (480 nm) with fluorescein isothiocyanate filters. Fluorescent and nonfluorescent embryos were scored, and the proportion of "green" embryos at this developmental stage was recorded.
Statistics
In the examination of chromosome stability, the percentage of paternal pronuclei with normal chromosomes (from karyoplates examined) in all subgroups was analyzed and compared. Experiments within each treatment were repeated at least three times. The variation between experiments within the same treatment was determined by calculating the percentage of normal karyoplates in each experiment, taking the mean, and calculating the SD. For comparing the different treatment groups, chi-square analysis was performed. Significance was determined at P < 0.05. The computations were done using KyPlot version 2.0-beta 13 software (Kyence, Tokyo, Japan). Linear regressions were performed using Microsoft Excel 2000 (Microsoft, Redmond, WA). For comparing the relationship between chromosomal breakage and embryo development, values within each pretreatment group were analyzed. The correlation coefficient, r, was calculated in the program using standard formulas. For comparing the relationship between chromosome damage and transgene expression, all treatments in all pretreatment groups were compared together.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, spermatozoa were pretreated with three methods causing membrane disruption: freeze-thawing (FT), Triton X-100 (TX), and Triton X-100 followed by sucrose wash (TX-Suc). Live motile spermatozoa were used as a control. The spermatozoa were injected into oocytes, and the percentage of embryos with normal chromosomes was calculated (Table 1). The FT and TX groups had significantly lower percentages of normal chromosomes compared with swim-up controls (P < 0.001), but the TX-Suc group was not significantly different. These results demonstrated that differences in sperm preparation affected sperm chromatin structure. Therefore, in all subsequent experiments the changes in chromatin structure noted were compared with a control (no DNA, no EGTA/EDTA) within the same pretreatment.
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Treatment of Spermatozoa with Exogenous DNA Leads to Paternal Chromosome Damage
Spermatozoa from each pretreatment group were incubated with or without 5 ng/µl of EGFP plasmid DNA and were injected into oocytes using the ICSI method. Oocytes fertilized by ICSI were divided, and approximately half of them were cultured in vitro to the morula/blastocyst stage, whereas the other half were used for chromosome analysis. Most of the zygotes used for chromosome examination produced chromosome spreads suitable for analysis. There were no differences in the survival and fertilization rates among all pretreatments/treatments. Approximately 80–90% of oocytes used for ICSI survived manipulations, and 95–100% of them were fertilized (as indicated by two pronuclei and a second polar body). For each treatment, the percentages of karyoplates with normal paternal chromosomes, of two-cell embryos that developed to blastocysts, and of EGFP-expressing embryos were analyzed (Table 1). Figure 1 shows an example of one normal and one abnormal paternal karyoplate. In all groups, a decrease in the frequency of normal karyoplates was found when spermatozoa were incubated with foreign DNA (Fig. 2). The decrease was approximately 2-fold, with the biggest difference in the TX group. In the swim-up sample, a decrease in the percentage of normal karyoplates was observed when spermatozoa were incubated with DNA, but the difference was much less than that observed with membrane-disrupted spermatozoa. Aberrations-per-sperm ratios, expressing the intensity of chromosome damage, were elevated in all treatments that included incubation with exogenous DNA.
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EGTA and EDTA Prevent Sperm Chromosome Breakage
One of the causes of chromosome breakage could be the release of endogenous nucleases from plasma membrane-damaged spermatozoa. Most endonucleases are dependent on calcium and/or magnesium ions. We tested whether the presence of chelating agents EGTA and EDTA in the sperm isolation medium could contribute to the improvement in chromosome integrity. Addition of EGTA and EDTA to the sperm-handling medium prevented chromosome breakage and reduced the aberrations-per-sperm ratios (Table 1 and Fig. 2). This phenomenon was observed in all groups examined. In the TX group, the addition of chelating agents resulted in a higher ratio of normal karyoplates than that found in the no-DNA group. This finding suggests that there is a procedure-related effect that can be prevented by the addition of EGTA/EDTA.
Transgene Expression Is Related to Sperm Chromosome Damage
In ICSI-mediated transgenesis, spermatozoa are incubated with foreign DNA and then injected into oocytes [5]. Some of the resulting embryos are transgenic. When EGFP is used as a marker gene, it is possible to easily recognize transgenic embryos with a fluorescence microscope and appropriate filters; transgenic embryos fluoresce green. When spermatozoa subjected to different membrane-damaging pretreatments were exposed to EGFP plasmids, green fluorescence was observed in the produced embryos (Fig. 3). We then compared the percentage of embryos expressing the EGFP protein in all treatments. Transgene expression observed at the morula/blastocyst stage of embryos produced with ICSI-mediated transgenesis reached the highest level in those treatments that included some form of sperm membrane disruption pretreatment (Fig. 4 and Table 1). The addition of chelating agents to these spermatozoa significantly decreased the percentage of embryos expressing EGFP in every case. Regression analyses of the percentage of EGFP-expressing embryos and either the percentage of embryos with normal chromosomes or the aberrations-per-sperm ratios produced correlation coefficients of -0.6321 and 0.5708, respectively. We were also able to produce green embryos when membrane-intact motile spermatozoa were used for ICSI. In this case, however, the level of transgene expression was significantly lower than that in all other pretreatments. In these swim-up controls, EGTA/EDTA did not decrease the level of transgene expression, suggesting that the sperm membrane may prevent access of the chelators to the sperm nucleus or that some other mechanism prevents EGTA/EDTA from acting.
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Blastocyst Development Is Correlated with Normal Chromosomes
Embryo development in vitro was evaluated in all experimental groups (Table 1). Two different types of results were obtained. First, the proportion of blastocysts differed for the different pretreatments without DNA or EDTA/EGTA. The development was most severely impaired in the FT and TX groups but was also impaired in the TX-Suc group when compared with ICSI with swim-up spermatozoa. These data suggest that different sperm pretreatments used in this study can affect blastocyst development. Second, for all four treatments (with or without DNA and/or EGTA/EDTA) within each pretreatment group, there was a direct correlation between the percentages of embryos that developed to blastocysts in culture and the percentages of embryos that had normal paternal chromosomes (Fig. 5). Under each of the conditions, linear regression analysis resulted in very high correlation coefficients (0.8736–0.9586). The slopes and y-intercepts were different for each of the conditions, probably reflecting the fact that factors that other than chromosome integrity affect embryo development. However, there was still a strong correlation (r = 0.7519) between the presence of normal paternal chromosomes and the development to blastocyst when all conditions were compared in one regression analysis, indicating that DNA integrity is an important factor in all treatments.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The FT and TX pretreatments caused significant chromosome breakage even without the presence of exogenous DNA (Fig. 2), suggesting that factors other than exogenous DNA can lead to paternal chromosome damage. Mouse spermatozoa are sensitive to mechanical [18] and chemical- and radiation-based [19] insults. All membrane disruption methods used were relatively harsh, yet in only two of three pretreatments were aberrations observed as a result of the procedure-only (no DNA) treatment. One possible explanation for why TX-pretreated spermatozoa led to a higher percentage of abnormal karyoplates and less successful embryo development than did the TX-Suc group is that in the TX group traces of TX might have been introduced into the oocytes, somehow affecting the embryos. Another possibility is that when TX was washed out by centrifugation through sucrose in the TX-Suc group some other factors present in spermatozoa and possibly detrimental to sperm chromatin, such as acrosin or other sperm enzymes freed by cell lysis, were also removed.
As a control for the three membrane-disruption pretreatments we used motile swim-up spermatozoa. When swim-up spermatozoa were used for ICSI, almost all karyoplates were normal and the ability of embryos to develop was high. However, even those membrane-intact spermatozoa expressed the same pattern of changes when exposed to exogenous DNA. When motile spermatozoa were exposed to EGFP plasmid, a decrease was observed in both chromosome integrity and development in vitro, but this decrease was not as marked as in all other pretreatment groups.
The most important finding of our study was the relationship between paternal chromosome breakage and transgene expression. The one treatment that always resulted in chromosome breaks, regardless of pretreatment, was the addition of exogenous DNA. We obtained green embryos in all pretreatments where the sperm membrane was disrupted, with the highest percentage of EGFP-expressing embryos in the TX-Suc group. We also obtained green embryos with membrane-intact spermatozoa, but the proportion was small. Our results revealed a relationship between the frequency of chromosome breaks and transgene expression. When EDTA/EGTA was included, the chromosome breaks were inhibited and transgene expression decreased. However, there was a direct correlation between the proportion of normal chromosomes and the potential of the embryo to develop in all examined groups. Thus, those embryos that were most likely to incorporate the EGFP transgene, e.g., the ones with broken paternal chromosomes, were also the most likely not to develop to the morula/blastocyst stage.
In all pretreatment groups, the same or higher percentage of normal karyoplates was obtained when EGTA/EDTA was included in sperm handling medium with EGFP compared with when only EGFP plasmid was added. The ability of EDTA and EGTA to inhibit the DNA-mediated paternal chromosome damage points to a possible involvement of Ca2+- and Mg2+-dependent enzymes in this process. Most endonucleases require calcium and/or magnesium ions, and their activity can be inhibited by the presence of chelating agents. Thus, the mechanism responsible for chromosome degradation associated with the presence of exogenous DNA may be based on the activity of endonucleases. These nucleases may also be involved in DNA integration in ICSI-mediated transgenesis. Eukaryotic DNA ligases are ATP-dependent strand-joining enzymes and require Mg2+ for their activity [20]. The reduction in transgene integration with the addition of EGTA/EDTA may be due to ligase inhibition. Integration requires both ligases and endonucleases, and because both require divalent cations, EGTA/EDTA may inhibit both.
We did not assay directly for transgene integration because blastocysts do not provide enough DNA for Southern blot analysis. The other method of reliably assaying for integration is to produce transgenic offspring. However, we were interested in studying the effects of exogenous DNA on embryo developmental potential. By focusing on the early stages of embryo development, we were able to demonstrate correlations between chromosome damage, embryo development, and transgene expression. The persistence of transgene expression through the number of divisions required to form blastocysts suggests that integration is necessary to form green blastocysts. There is a strong correlation between the persistence of the transgene through preimplantation development and integration into the genome [12]. In the presence of EGTA and EDTA, the efficiency of the production of transgene-expressing blastocysts was much lower than when no chelating agents were included in sperm handling medium.
Our control experiments with membrane-intact swim-up spermatozoa indicate an important role for the sperm plasma membrane in both chromosomal breakage and DNA integration. For swim-up spermatozoa, there was no protection against exogenous DNA-induced paternal chromosome breakage and no decrease in the number of EGFP-expressing embryos after addition of EDTA and EGTA. It is unclear what role the plasma membrane plays in paternal chromosome breakage or ICSI-mediated transgenesis. The membrane may have prevented EDTA/EGTA from entering the sperm nucleus, which is why we did not observe the same protection from DNA-mediated chromosome damage as observed in the other three pretreatment groups. However, we noted much less DNA-mediated damage and much lower transgene integration when swim-up spermatozoa were used. Thus, the plasma membrane may interfere with DNA association with spermatozoa, in agreement with the previous suggestion that exogenous DNA may interact with demembranated spermatozoa more efficiently [5].
There may be two different mechanisms for paternal chromosome damage, one that is inhibited by EDTA/EGTA and presumably is nuclease related and another that is not. The chromosome breaks induced by pretreatment with FT alone, without DNA, could not be prevented by EDTA/EGTA, but those induced by TX pretreatment alone and all those induced by exogenous DNA could be prevented by EDTA/EGTA treatment (Fig. 2). We recently observed a similar phenomenon when treating spermatozoa with a combination of detergent and DTT [9]. When these spermatozoa were injected into oocytes, the paternal chromosomes were broken in most resulting embryos. The damage induced by Triton X-100 + DTT was only partially inhibited by chelating agents. Thus, the chromosome damage caused by detergent and DTT and that caused by exogenous DNA may have different origins.
It is unclear whether the events causing paternal chromosome damage occur in the sperm cell itself or whether they evoke some changes in the sperm chromatin that signal the oocyte to initiate paternal chromosome breakage. If the paternal chromosome breakage occurs within the sperm cell before entry into the oocyte, it would mean that the sperm nucleus contains the necessary enzymes to cause this DNA breakage. If the chromosomes become broken in the oocyte, it would mean that the oocyte can recognize some change in the sperm chromatin that signals it to disrupt the paternal DNA. Experiments are currently underway to explore this very important mechanism.
Whether the mechanism is active within the spermatozoon or shortly after it enters the oocyte, we encounter a paradox: how can the chromatin in the sperm nucleus, which is much more condensed than that in somatic cells and is packaged in a way that makes the DNA relatively inaccessible to proteins [21], also be functionally dynamic? We proposed an extended model of sperm structure (Fig. 6) emphasizing its functional implications for spermatozoa [22, 23], and a similar model was suggested by Spadafora [24]. Approximately 2% of rodent sperm DNA [11] (R. Balhorn, personal communication) is bound to histones, not to protamines. In our donut-loop model, each protamine-DNA toroid is a single DNA loop domain, and the two ends of the 50 kb that make up one toroid are each attached to the nuclear matrix at sites called matrix attachment regions (MARs). The essence of the model is that the DNA near the MARs is associated with histones rather than protamines (Fig. 6). The results presented here suggest that even though sperm DNA is very highly condensed, it may be vulnerable to endonuclease-induced chromosome breakage in these linker regions. It is also possible that oocyte nucleases act after the protamines have been removed from the sperm nucleus. Our model provides a possible explanation for paternal DNA degradation before sperm chromatin decondensation.
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Incubation of exogenous DNA with demembranated spermatozoa increased paternal chromosome breakage when these spermatozoa were injected into oocytes, and the expression of this exogenous DNA at the morula/blastocyst stage was directly proportional to the level of paternal chromosome damage, suggesting that the two mechanisms are related. Both paternal chromosome damage and transgene integration were prevented by EGTA/EDTA, indicating that some enzymes requiring Mg2+ and Ca2+, such as endonucleases and/or ligases, may be involved in the mechanisms regulating these two processes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Monika A. Szczygiel, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822. FAX: 808 956 7316; szczygie{at}hawaii.edu
Received: 14 October 2002.
First decision: 8 November 2002.
Accepted: 16 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, without DNA or EDTA/EGTA; 
, with DNA; 
, with EDTA/EGTA; and 
, with DNA and EDTA/EGTA