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a Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One approach to studying sperm capacitation is to use mutant mice with sperm that are unable to complete fertilization. The t haplotypes are the best known of such systems. The t haplotypes are variant alleles of genes in the proximal one-third of mouse chromosome 17, called the t complex. The variant alleles are linked together by a series of inversions (relative to the wild type). Genes within the t complex control sperm flagellar assembly and movement [4, 5], and zona-free oocyte penetration [6]. Males carrying two t haplotypes are invariably sterile. They have extremely poor motility and lower zona-binding ability, and are unable to penetrate zona pellucida-free oocytes. However, solubilized zona pellucida proteins are able to induce the AR among capacitated sperm from t/t mice [7].
An earlier study of capacitation found no differences between sperm from t/t mice and sperm from +/+ mice until 4 h of incubation [8]. Thus, it was not clear whether this difference was relevant to any of the other defects in fertilization exhibited by these sperm. The present study was undertaken to re-examine capacitation in wild-type (+/+) and congenic (genetically identical except in the region of the t haplotype) t/+ and t/t mice, and to determine whether differences in capacitation could affect their ability to bind to the zona pellucida.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wild-type male mice were produced by crossing 129-+/+ and C57BL/6-+/+ mice. All tw5/+, tw32/+, and tw32/tw5 mice were congenic to the +/+ mice [8]. Since there were no significant differences in the acrosome reaction (AR) and sperm-zona binding between the sperm from tw5/+ and tw32/+ mice, both are referred to as t/+ mice. Consequently, tw32/tw5 mice are referred to as t/t mice. Males used in this study were 3–9 mo old.
The proximal portion of mouse chromosome 17 was genotyped by restriction fragment length polymorphism (RFLP) analysis of genomic DNA extracted from tail-tip biopsies [9]. Briefly, digested DNAs were separated by electrophoresis in agarose gels and blotted to nylon membrane. DNAs were bound to the membrane by UV light and hybridized with radiolabeled probes that were informative for the chromosome 17 genotype. The presence of complete t haplotypes were detected by using the marker DNA clones Tu48 [10] and Tu89 [11]. Pim 1A [12] was used to distinguish between tw5 and tw32 haplotypes.
Isolation and Incubation of Sperm
Media used were as follows: a modified Krebs-Ringer-bicarbonate buffer containing 2% BSA, 1.7 mM CaCl2, and 50 mM Hepes instead of 10 mM TAPSO (3-[N-Tris-(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid) in vitro fertilization (IVF) medium [13]; IVF medium without HCO3- (NB); IVF medium without Ca2+ (NC), and IVF medium without HCO3- and Ca2+ (NCB). The cauda epididymides were minced and placed in either IVF or NCB medium for 10 min; then epididymal tissue was removed, and aliquots of the sperm suspension were diluted 1:1 with fresh media of the same or different composition. The sperm suspensions were incubated at 37°C in 5% CO2 in air, except for those in media without HCO3-, which were incubated at 37°C in air. All media were maintained at pH 7.43 ± 0.01 by the presence of 50 mM HEPES.
Assessment of Capacitation
Sperm were assayed for capacitation by two methods. The first method was a modification of the chlortetracycline (CTC) assay [14]. At various times (5 min, 1, 2, and 3 h) of incubation, 5 µl of each sperm suspension was mixed on a warmed slide with 5 µl of a solution (kept on ice) containing 20 mM TAPSO, 130 mM NaCl, 5 mM cysteine, and 500 µM CTC HCl (Sigma, St. Louis, MO). After 30 sec a coverslip was applied, and then a piece of filter paper was pressed upon the coverslip to absorb excess fluid. The sperm were viewed with a Leitz Ortholux II epifluorescent microscope equipped with an excitation filter BP 355–425 and suppression filter LP 460 (Leitz filter block D; New York, NY).
For each time and treatment, more than 100 sperm were classified as having one of the following five staining patterns: 1) diffuse fluorescence over the entire head (F pattern), characteristic of noncapacitated sperm; 2) a dark band over the posterior head (B pattern), characteristic of intact sperm able to undergo the zona pellucida-induced AR; 3) a dim apical and dark posterior head (S pattern), characteristic of sperm in an early stage of the AR [15] (the S pattern percentage was less than 10% of the total); 4) the entire head nonfluorescent (AR pattern), characteristic of acrosome-reacted sperm; 5) none of the above patterns (nonidentified sperm; this class was never more than 7% of the total).
The second method of assaying capacitation was by induction of the AR with L-
-lysophosphatidylcholine (LPC). LPC (type I from egg yolk; Sigma) was prepared as a stock solution (2 mg/ml) on the day of use. To induce the AR with LPC, sperm were preincubated for 1 h in the appropriate medium (IVF, NBC, NC, or NB). Sperm were then incubated for 15 min in the presence of LPC and, if not already present, 1.7 mM CaCl2. The percentage of AR sperm among these LPC-treated sperm was determined immediately after CTC staining.
Sperm-Zona Binding Assay
CF-1 female mice (Charles River Laboratory) were induced to superovulate by i.p. injection of 5 IU of eCG (Sigma), followed by 5 IU of hCG (Sigma) 48 h later. The females were killed 16 h after hCG injection. The cumulus/oocyte clumps were obtained by puncturing the distended ampulla as described previously [13]. To disperse cumulus cells, cumulus/oocyte clumps were transferred to IVF medium containing 0.0025% bovine testis hyaluronidase (Calbiochem, San Diego, CA) and 0.01% soybean trypsin inhibitor (Sigma) for approximately 5 min. Completely denuded eggs were washed twice and placed in IVF medium containing 0.3% BSA. Two-cell embryos used as a control [16] were obtained by flushing the oviducts of superovulated CF-1 females having a vaginal plug after overnight mating with a fertile male. The embryos were also kept in IVF medium containing 0.0025% bovine testis hyaluronidase and 0.01% soybean trypsin inhibitor for approximately 5 min, then washed twice with IVF medium. Eight to twelve oocytes and 3–4 two-cell embryos were dispensed into 0.75 µl IVF medium containing 0.3% BSA under silicone oil or mineral oil in an atmosphere of 90% N2:5% CO2:5% O2.
For each male, one cauda epididymidis was minced and placed in IVF medium and the other in NCB medium as described above (right and left epididymides were alternated between treatments). One hundred microliters of each sperm suspension was diluted 1:1 with the same medium and incubated for 1.5–2 h at 37°C either with 5% CO2:95% air (IVF medium) or with air (NCB medium). Sperm concentrations were determined by counting motile and immotile sperm in a hemocytometer 10 min after dilution. The capacitated sperm concentration (incubated in IVF) was adjusted to 5 x 106/ml with IVF medium containing 0.3% BSA. The noncapacitated sperm concentration (incubated in NCB) was first adjusted to 1 x 107/ml with NCB medium containing 0.3% BSA, then diluted 1:1 with IVF medium containing 3.4 mM CaCl2, 50 mM NaHCO3, and 0.3% BSA. Ten microliters of capacitated and noncapacitated sperm at a concentration of 5 x 106/ml were coincubated for 15 min with 8–12 zona-intact oocytes and 3–6 two-cell embryos kept in 0.75 µl of IVF medium for each experiment. To measure sperm-egg attachment, oocytes and embryos were washed either 1) once using a micropipette with a bore size of ~110 µm, or 2) 5 times using a micropipette with a bore size of ~90 µm. The pipette bore size was determined by examination under the light microscope using an ocular micrometer. The oocytes and embryos were then transferred to a siliconized slide and air-dried. They were fixed with 2.5% glutaraldehyde (Sigma; grade I, 25% solution) for 30 sec, rinsed twice with 2.3% sodium-citrate:ethanol (3:1, rinsing buffer), stained with 10 µg/ml Hoechst 33342 dye in rinsing buffer, and mounted with 5 µl of mounting medium. Oocytes and sperm were viewed with a Leitz Ortholux II epifluorescent microscope equipped with an excitation filter BP 355–425 and suppression filter LP 460 (Leitz filter block D).
Data Analysis
Each experiment was replicated at least 3 times for each treatment and genotype (see figure legends). The overall mean was determined from the individual means. Significant differences among the four treatments or among the three genotypes were determined by ANOVA, followed when appropriate by the Neuman-Keuls test. Significant differences between NCB and IVF media (within a genotype) were determined by the Mann-Whitney test. Results were considered significantly different when p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cauda epididymal sperm were isolated in NCB, then diluted 1:1 into IVF, NB, NC, or NCB medium, incubated 5 min to 3 h, and assayed by the B pattern of fluorescence after CTC staining. Sperm from +/+ mice incubated in IVF medium reached a maximal percentage of the B pattern after 1 h of incubation, while sperm from t/+ mice reached a similar maximum after 2 h of incubation (Fig. 1). Minimal capacitation was observed for sperm of these genotypes incubated in NC or NCB medium. In NB medium, the level of B pattern appeared slightly higher than in NC or NCB medium, but this was not significant except for t/+ mice at 3 h. In contrast, sperm from t/t mice incubated in any of the four media showed no increase in the percentage of B pattern with time (Fig. 1). The lack of capacitation was not due to death of the sperm, since at least 40% of those incubated in NBC medium exhibited moving flagella after 90 min (data not shown).
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The observation that capacitation did not occur in sperm from t/t mice conflicted with an earlier study of sperm of the same genotypes [8]. However, in the earlier study sperm were isolated in IVF medium. Therefore, additional experiments were done in which sperm were isolated from one epididymis in IVF medium and from the other epididymis in NCB medium; then both suspensions were diluted 1:1 into IVF medium and incubated for 3 h. Sperm from t/t mice isolated in IVF and then incubated in IVF medium did exhibit a significant increase in the percentage of B pattern after 1–3 h of incubation, but the level of capacitation was significantly lower than that of sperm from +/+ and t/+ mice (Fig. 2). Thus, while some sperm from t/t mice isolated and incubated in IVF medium were able to become capacitated, the level of capacitation was significantly lower than among sperm from congenic +/+ or t/+ mice at 1, 2, and 3 h of incubation.
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The medium used for sperm isolation, IVF or NCB, made no difference in the percentage of B pattern among sperm from +/+ and t/+ mice. In contrast, sperm from t/t mice were significantly more capacitated when isolated in IVF medium than when isolated in NCB medium (Fig. 2). Since the isolation medium in these experiments lacked both Ca2+ and HCO3-, it was not clear whether the lack of either or both ion species subsequently affected the level of capacitation of sperm from t/t mice. To distinguish among these possibilities, sperm from t/t mice were isolated in NC or NB medium, then diluted 1:1 into IVF medium. In three experiments, after 3 h of incubation, the percentage of B pattern among sperm isolated in NC was 27 ± 4 (mean ± SD), not significantly different from that of sperm isolated in NCB, whereas that of sperm isolated in NB was 33 ± 4, significantly higher than that of sperm isolated in NCB, but significantly less than that of sperm isolated in IVF medium. Thus, the concentration of both Ca2+ and HCO3- during isolation of sperm from t/t mice appeared to influence the level of capacitation after transfer to IVF medium. However, the lack of Ca2+ had a greater effect than the lack of HCO3-.
To confirm that capacitation was abnormal in sperm from t/t mice after 1 h of incubation, capacitation was also assayed by another, independent procedure. Capacitated sperm of several mammalian species can be induced to undergo the AR by exposure to the fusogenic lipid LPC [17, 18]. However, the use of LPC with mouse sperm has not yet been reported. Therefore, we first determined whether induction of the AR by LPC in mouse sperm was capacitation-dependent. Cauda epididymidal sperm from wild-type mice were incubated in IVF for 1 h to allow capacitation and then exposed to various concentrations of LPC for 15 min, and the percentage of acrosome-reacted sperm was determined. LPC induced capacitated sperm to undergo the AR in a concentration-dependent manner (Fig. 3). A concentration of 300 µg/ml was arbitrarily chosen as the concentration to be used for all subsequent experiments. To ensure that exposure to LPC was not inducing general sperm membrane damage leading to a "false" AR, sperm were incubated in 300 µg/ml LPC for 75 min, then assayed for motility (standard motion analysis) [8] and for their ability to penetrate zona-free oocytes [6]. There were no significant differences between LPC-treated and control sperm in either motility or egg-penetration ability (data not shown). Likewise, noncapacitated sperm populations freshly isolated from the epididymides and incubated for 15 min with 300 µg/ml LPC had less than 6% AR (three +/+, six t/+ and three t/t mice tested). This suggested that the LPC-induced AR occurred only in capacitated mouse sperm.
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Sperm isolated in NCB were diluted 1:1 into IVF, NB, NC, or NCB and incubated 60 min, then exposed to 300 µg/ml LPC for 15 min and examined for the AR pattern after CTC staining. For sperm incubated in NCB and NC, a final concentration of 1.7 mM Ca2+ was added with the LPC (since Ca2+ is required for the AR). About one-third of the sperm from +/+ and +/t mice incubated in IVF medium were acrosome-reacted after treatment with LPC, while sperm from the same males incubated in NCB or NC medium had significantly lower levels of LPC-induced AR (Fig. 4), confirming that LPC induced the AR only in capacitated sperm. Sperm from these males incubated in NB had an LPC-induced AR level significantly higher than that in NC or NCB but significantly lower than that of sperm incubated in IVF medium (Fig. 4). Thus, this assay suggested that some capacitation could occur even when sperm were incubated in medium with a very low level of bicarbonate ions.
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The level of LPC-induced AR of sperm from t/t mice incubated in either IVF or in NB medium was significantly lower than that of sperm from +/+ or t/+ mice (Fig. 4). Thus this assay also suggested that the sperm from t/t mice were deficient in capacitation. However, sperm from t/t mice exhibited a significantly higher level of LPC-induced AR in IVF or NB medium than in NC or NCB medium, suggesting that capacitation was not entirely absent in these populations.
Sperm-Zona Pellucida Binding
A previous study from this laboratory found that sperm from t/t mice were deficient in binding to the zona pellucida [19]. It seemed possible that this defect could be due to their lower level of capacitation, since some studies have suggested that capacitation enhances sperm-zona binding [1]. However, some studies suggested that this might not be true of mouse sperm [20, 21]. Therefore, we tested the zona-binding ability of capacitated and noncapacitated sperm from +/+, t/+, and t/t mice. Sperm were incubated in either capacitating (IVF) medium or noncapacitating (NCB) medium for 90–120 min, then coincubated with cumulus-free oocytes and two-cell embryos in IVF medium. A sperm concentration of 5 x 106 cells/ml was used because coincubation at this concentration for longer periods of time had shown that the majority of oocytes became fertilized (data not shown). This indicated that under these conditions, sperm were capable of zona penetration as well as binding.
After a 15-min coincubation with sperm, oocytes and embryos were washed according to two different protocols: 1) one wash with a ~110-µm-bore pipette or 2) five washes with a ~90-µm-bore pipette. Using the latter procedure, there were very few attached sperm per embryo and no significant differences among sperm from mice of different genotypes (Fig. 5, embryos). Therefore, sperm-zona binding was defined as the sperm remaining on the zona of oocytes after five washes with a ~90-µm-bore pipette. There were no differences in sperm binding between +/+ and t/+ mice, but more of these genotypes of sperm bound to the zona of oocytes than did sperm from t/t mice (Fig. 5). For sperm of all genotypes, the level of sperm binding was significantly lower for sperm incubated in NCB (before the binding assay) than for sperm incubated in IVF medium (Fig. 5).
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The less stringent washing procedure (procedure 1) was expected to serve as an indication of the number of sperm that were capable of reaching the oocyte, including sperm adhering to the zona nonspecifically. However, for fertile mice (with normal capacitation levels), significantly more sperm adhered to the oocytes than to two-celled embryos in the same group (Table 1). This suggested that some of the sperm (those in excess of the number bound to the embryo zonae) could be specifically bound to the oocytes. Indeed, when the mean number of sperm/embryo was subtracted from the mean number of sperm/oocyte in each experiment, the mean remainder was significantly less for sperm from t/t mice than for sperm from the other two genotypes. This was true whether sperm were incubated in IVF or NCB medium (data not shown). Furthermore, significantly fewer sperm incubated in NCB (and thus not capacitated) attached to oocytes than sperm incubated in IVF medium (Table 1), suggesting that more capacitated than noncapacitated sperm could attach to the zona pellucida.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When sperm from t/t mice were examined after CTC staining, the percentage with a B pattern at 1, 2, and 3 h was significantly lower than the percentage of sperm from congenic t/+ and +/+ mice, even when sperm were isolated and incubated in complete medium (Fig. 2). This suggests that fewer sperm from t/t mice are capable of capacitation. An earlier study from this lab, however, found no differences in the percentage of B pattern between sperm from +/+ and t/t mice until 4 h of incubation [8]. This discrepancy was not due to a difference between the two studies in the levels of B pattern among sperm from t/t mice. Rather, sperm populations from +/+ mice in the earlier study became capacitated more slowly, so that maximal capacitation was not reached until after 2 h [8]. We have no reasonable explanation for this, except that in the previous experiments the medium contained 10 mM TAPSO, while in the present experiments the medium contained 50 mM HEPES (in addition to bicarbonate). Since the latter buffer is much better at maintaining the pH (data not shown), it is possible that this difference could have affected the rate of capacitation among fertile sperm.
To confirm that the mutant sperm were defective in capacitation, another assay of capacitation was developed. Sperm were incubated for 60 min, then exposed to LPC for 15 min, and examined for the AR. It has been shown that LPC induces AR in capacitated, but not noncapacitated, bovine and human sperm [17, 18]. LPC did not induce an AR in noncapacitated mouse sperm (freshly isolated from the epididymis, or incubated 60 min in calcium-deficient medium), but did induce the AR in capacitated sperm of the three genotypes (Fig. 4). However, the level of LPC-induced AR was significantly lower for sperm from t/t mice than for sperm from t/+ or +/+ mice. Thus, the LPC-induced AR assay confirmed that capacitation is reduced in sperm populations from t/t mice.
These data suggest that a gene (or genes) within the t complex plays a role in capacitation. The significant reduction in capacitation of sperm from t/t mice suggests that mutations within the t haplotype affect one or more components in the signal transduction pathway leading to capacitation. Identification of the mutated gene(s) could uncover an as yet unknown step in the capacitation pathway. Since the t haplotypes contain inversions, the standard methods of determining the location of mutations cannot be used [7]. However, a novel approach, called hybrid sterility analysis, has been developed to locate and identify sterility genes within the t haplotypes. This method has already been used to locate genes important for sperm motility and penetration of the zona-free egg [4–6]. Thus, it should also be possible to use hybrid sterility analysis to identify the gene (or genes) affecting capacitation. We have begun this process by locating at least one such gene in the distal end of the most distal inversion of the t haplotype (preliminary observations).
What could be the biochemical function of the mutated gene product(s) that are responsible for the reduction in capacitation? That these sperm were totally unable to become capacitated when isolated in medium with very low levels of Ca2+ and HCO3- (NCB medium) might be a clue. Isolation of sperm from t/t mice in NCB medium caused an irreversible change in these sperm, such that when subsequently transferred to IVF medium, no increase in capacitation was observed (Fig. 2). This irreversible change is unlikely to be morbidity, since nearly half of the sperm from t/t mice were observed to have motile flagella after a 90-min incubation in NCB medium. Thus, this irreversible change most likely involves some Ca2+ and/or HCO3--dependent event. Both HCO3- and Ca2+ can exert their functions through the cAMP-dependent protein kinase A pathway [2]. Alternatively, HCO3- could facilitate the influx of Ca2+ [22], and in the cell Ca2+ can bind to calmodulin (CaM). The Ca2+-CaM complex activates several enzymes that could play a crucial role in capacitation [23]. Thus, it appears that when sperm from t/t mice are isolated in medium deficient in Ca2+ and HCO3-, they become incapable of proceeding along a pathway necessary for capacitation in vitro. Identification of the gene responsible, and an understanding of the protein's function in the cell, could reveal a previously unknown step in the uptake or use of these ions in the pathway leading to normal capacitation.
Consequences of the Reduced Capacitation for Fertilization In Vivo
The primary reason for sterility of t/t mice is that in vivo virtually no sperm ever reach the site of fertilization ([24] and references therein). Although capacitation is thought to be important in retaining sperm within the oviduct [1], the significant reduction in capacitation of sperm populations from t/t mice is unlikely to be responsible for their defective transport through the female genital tract. Because few sperm can be recovered from the oviducts of females mated to t/t males even as soon as 0.5 h after coitus, it seems likely that the large majority of these sperm never enter the oviduct. Since progressive motility is thought to be important for rodent sperm to move through the uterotubal junction [25], it is probable that the poor motility of these sperm, rather than the reduced capacitation, causes the great deficiency in oviductal sperm. However, for those few sperm that do reach the oviduct, their lower level of capacitation could further decrease the chances that any will reach the site of fertilization in vivo.
Capacitation Influences Sperm-Zona Binding
The relationship of capacitation to zona pellucida binding is controversial. While several reports suggest that capacitation in other mammalian species can either reduce the time needed for zona binding [1] or enhance zona binding directly [26], studies of mouse sperm have suggested that capacitation is not necessary for zona binding [20, 21, 27]. However, in the studies with mouse sperm, capacitation was not directly determined. To resolve this issue, we have determined the zona-binding ability of noncapacitated sperm (incubated in NCB) and compared it to that of capacitated sperm (incubated in IVF medium). Regardless of genotype, the zona-binding ability of minimally capacitated sperm was significantly reduced, relative to that of fully capacitated sperm from the same males (compare Fig. 2 with Fig. 5). Although the data from the less stringent washing procedure included both nonspecifically and specifically attached sperm, they also suggest that more capacitated than noncapacitated sperm can attach to the zona of oocytes (Table 1).
The small amount of binding that occurred in NCB medium could have been due to capacitation during the 15-min gamete coincubation in IVF medium (see Materials and Methods). Thus, regardless of whether or not sperm-zona binding occurs in the absence of capacitation, it is clear that capacitation significantly increases the probability that a mouse sperm can bind to the zona pellucida.
Sperm from t/t mice are deficient in their ability to bind to the zona pellucida, relative to sperm from congenic t/+ and +/+ mice ([19] and this report). The cause of the reduced zona-binding ability of sperm from t/t mice is not known. It is unlikely that the reduction in zona binding was a result of less sperm-egg contact, since after 1 wash with a ~110-µm-bore pipette, similar numbers of sperm of all three genotypes preincubated in IVF medium were associated with oocytes and with embryos (Table 1). It has been suggested that the reduction in zona binding occurs because sperm from t/t mice have four times the activity level of sperm surface ß-1,4-galactosyltransferase (GalTase), relative to wild-type siblings [28], and overexpression of GalTase has been shown to reduce sperm-zona binding [29]. However, the data presented here suggest another possible reason for poor zona binding.
Since capacitation greatly facilitates sperm-zona binding, sperm populations with reduced capacitation levels would be expected to bind less well to zonae pellucidae. Thus, the reduced level of capacitation of sperm from t/t mice could explain their decreased zona-binding ability. Although the level of binding of sperm from t/t mice was significantly lower than that of sperm from fertile mice after incubation in NCB medium (Fig. 5, eggs), this could have been caused by a small amount of capacitation occurring in fertile sperm during the 15-min gamete coincubation. Whether the reduction in zona-binding ability of sperm from t/t mice is due to their higher levels of GalTase, or to their lower level of capacitation, remains to be determined by mapping the genetic factors involved.
In summary, the data presented here suggest that sperm from t/t mice are deficient in capacitation. This defect appears to be separate from the defects in motility and zona-free egg penetration. However, since evidence presented here demonstrates that zona binding is greatly enhanced by capacitation, it is possible that the deficiency in capacitation could be, at least in part, responsible for the decreased zona binding of sperm from t/t mice. Isolation and characterization of the gene(s) responsible for this deficiency could contribute to our understanding of pathways important for normal capacitation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Patricia Olds-Clarke, Department of Anatomy and Cell Biology, Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140. FAX: 215 707 2966; polds-cl{at}vm.temple.edu
Accepted: February 19, 1999.
Received: November 9, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5). Each concentration is significantly different from all other concentrations.
