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Gamete Biology |
a School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, California
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, kinases, signal transduction, sperm, sperm capacitation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism regulating sperm function that is a meaningful indicator of capacitation [7, 11]. Previous studies have demonstrated a key role for cAMP and protein kinase A (PK-A) in the signaling pathways leading to sperm protein tyrosine phosphorylation and capacitation [9, 12, 13]. It has been postulated that the production of cAMP is an event downstream from the site of BSA and NaHCO3 action on spermatozoa [7, 9]. It has been demonstrated that PK-A activity increases during mouse sperm capacitation [10] and that these observed changes reflect elevations of intracellular cAMP. These events have been correlated with an increase in protein tyrosine phosphorylation, which mediates a variety of cellular functions such as growth regulation, cell cycle control, cytoskeleton assembly, ionic current regulation, and receptor regulation [14, 15].
Preserved semen often has decreased spermatozoal motility and fertility that can vary widely between individual stallions [16]. It is plausible that cell longevity is compromised by premature capacitation-related changes associated with storage of sperm. Bovine and equine sperm have been demonstrated to exhibit signs of premature capacitation associated with cryopreservation [17, 18]. There is substantial evidence that cryopreservation promotes the capacitation of sperm [19, 20], and this cryocapacitation is frequently cited as one factor associated with the reduced longevity of cryopreserved sperm in the female reproductive tract [20]. Induction of cryocapacitation has been attributed to an increase in intracellular calcium associated with membrane damage [21]. The reduced longevity of sperm in the female reproductive tract is a major barrier to the application of frozen semen technology in the horse. Preserving sperm function following cooled or frozen storage is essential to maintaining optimum fertility.
This study was designed to determine whether stallion sperm express different levels of tyrosine phosphorylation after in vitro capacitation and whether thawed sperm display similar phosphorylation characteristics in comparison with freshly ejaculated sperm. Experiments were performed to facilitate comparisons in tyrosine phosphorylation, motility, and viability of sperm prior to and following in vitro capacitation in fresh, extended, and frozen-thawed sperm. Fresh and frozen-thawed sperm were incubated in capacitating conditions in order to evaluate baseline and induced levels of tyrosine phosphorylation following incubation of sperm at 37°C in Biggers, Whitten, Whittingham (BWW) capacitation medium. We hypothesized that equine spermatozoa undergo tyrosine phosphorylation during capacitation and that this phosphorylation is modified when sperm have been cryopreserved. We also hypothesized that tyrosine phosphorylation could be enhanced by the use of the activators dibutyryl cAMP (db cAMP) and caffeine as well as methyl ß-cyclodextrin, which causes cholesterol efflux from the spermatozoa and is inhibited by the PK-A inhibitor H-89.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Anti-phosphotyrosine monoclonal antibody (mAb), 4G10, was purchased from Upstate Biotechnology (Lake Placid, NY). Nonreducing sample buffer (5x) was obtained from Pierce (Rockford, IL). ß-Mercaptoethanol was purchased from Bio Rad (Hercules, CA). Dulbecco phosphate-buffered saline (DPBS) was purchased from Gibco BRL (Grand Island, NY). E-Z Mixin-"Basic Formula" equine semen extender was purchased from Animal Reproduction Systems (Chino, CA). LIVE/DEAD Sperm Viability Kit was purchased from Molecular Probes (Eugene, OR). BWW was purchased from Irvine Scientific (Irvine, CA). H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide-dihydrochloride) was obtained from Calbiochem (La Jolla, CA). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
Animals and Semen Collection
Semen was obtained from three fertile stallions individually housed at the Veterinary Medicine Teaching Hospital and the Animal Science Horse Barn located at the University of California, Davis. Stallions were maintained according to Institutional Animal Care and Use Committee protocols at the University of California. This includes a diet consisting of equal proportions of oat and alfalfa hay with fresh water ad libitum and daily exercise. Semen was collected using an artificial vagina and a phantom mare. A nylon mesh filter was used to eliminate the gel fraction and only allow the sperm-rich fraction of the ejaculate to enter into the collection bottle. Approximately 7 ml of the gel-free semen was immediately diluted 1:1 (v:v) into BWW capacitation medium that had been warmed to 37°C prior to semen collection. The diluted semen was transported to the laboratory within 5 min of collection. All ejaculates contained at least 2 billion spermatozoa and had an initial progressive motility >50%.
Experiment 1. Determination of Tyrosine Phosphorylation During Capacitation of Equine Sperm
Experiment 1 was designed to detect changes in phosphorylation of tyrosine residues during a standard process of capacitation of equine sperm using Western blotting and immunofluorescence techniques. Samples were incubated for the standard 3-h in vitro capacitation time [22] and were compared with a 0-h control. Activators (db cAMP and caffeine, C+C) were added to duplicate samples for the 3-h incubation period to stimulate phosphorylation of tyrosine residues. Separate tubes contained either methyl ß-cyclodextrin (Sigma Chemical Company) to determine whether this stimulation could be enhanced or H-89 to ascertain whether this stimulation could be abolished.
Sperm processing for capacitation Upon arrival at the laboratory, the semen sample was centrifuged at 200 x g for 5 min to sediment debris and clumped sperm. The supernatant (3 ml) was layered over two Percoll-BWW gradients, each consisting of an 84% lower layer and a 42% upper layer. After a 20 min centrifugation at 300 x g, the sperm pellets were collected and placed in 4 ml BWW. Post-Percoll sperm viability and total motility were >85%. The sperm suspension was centrifuged for 10 min at 300 x g and the resulting sperm pellet was resuspended in 1 ml BWW. The sample was diluted to 20 x 106/ml following determination of sperm concentration using a hemacytometer. Five hundred microliters of the sperm suspension was aliquoted into each of two siliconized microcentrifuge tubes per treatment. The treatments were 0-h control, 3-h capacitated, and 3-h capacitated with the activators db cAMP (1.2 mM) and caffeine (1 mM). The effects of methyl ß-cyclodextrin and the PK-A inhibitor H-89 on these main treatments were also determined. After incubations, samples were processed for Western blotting and immunofluorescence microscopy as described below. For Western blotting experiments, three concentrations of methyl ß-cyclodextrin and H-89 were used to demonstrate a concentration-dependent effect. For immunofluorescence experiments, only the most effective concentration was chosen. The samples were incubated at 37°C in humidified 95% air and 5% CO2 atmosphere to induce capacitation.
Sperm processing for Western blotting At 0 h and 3 h, protein was extracted from whole sperm as described by Galantino-Homer et al. [13]. Briefly, sperm suspensions were centrifuged at 10 000 x g for 3 min. The supernatant was discarded and 1 ml of 0.2 mM sodium ortho-vanadate in DPBS was added to each microcentrifuge tube to inhibit endogenous phosphatases. The samples were centrifuged again for 3 min at 10 000 x g and the supernatant was removed, leaving 20–30 µl covering the sperm pellet. The tubes were then centrifuged for 1 min at 10 000 x g and the rest of the supernatant was removed. Twenty microliters of water and 7 µl of 5x nonreducing sample buffer were added to each tube. The samples were then vortexed for 5 min, followed by boiling for 5 min. The samples were centrifuged for 3 min and 2 µl of ß-mercaptoethanol (ßME) was added to new 500-µl microcentrifuge tubes. The supernatant was added to the 500-µl tubes containing ßME and the pellets discarded. These small tubes were then vortexed, boiled for 5 min, and centrifuged for 1 min. The samples were stored at -20°C for protein analysis using the DC Protein Assay (Bio Rad, Hercules, CA) according to the manufacturer's instructions.
Prior to SDS-PAGE, the samples were thawed, vortexed, and centrifuged at 10 000 x g for 1 min. Six microliters of See Blue prestained standard (Pierce, Rockford, IL) and 8 µg of total protein from fresh spermatozoa were loaded into each well of a 10% Tris-HCl precast gel (Bio Rad, Hercules, CA). Electrophoresis was performed at 200 volts/40 mAmp for 50 min and the gel was then blotted onto an Immobilon P PVDF membrane (Millipore, Bedford, MA). The membrane was washed in a Tris-buffered saline containing 0.1% Tween 20 (TTBS). After blocking for 2 h at room temperature with 5% gelatin (from cold-water fish skin) in TTBS, the membrane was washed with TTBS and incubated with anti-phosphotyrosine 4G10 mAb (1:1000) in TTBS or without the primary antibody (control) for 1 h on an orbital shaker at room temperature. The blot was washed 4 x 15 min with TTBS and then incubated in goat anti-mouse IgG conjugated to horseradish peroxidase (1:5000) in TTBS on an orbital shaker overnight at 4°C. After washing 4 x 30 min with TTBS, bound peroxidase activity was visualized by the ECL-Plus chemiluminescence system (Amersham, Piscataway, NJ) according to the manufacturer's procedure. The relative molecular weights and densitometry of polypeptides were determined by optical scanning of blots and subsequent analysis using a gel-scanning macro for NIH Image 1.60 on a Macintosh computer (Apple Computer, Cupertino, CA).
Sperm processing for immunofluorescence labeling Samples were processed for immunofluorescence labeling at 0 and 3 h as described by Visconti et al. [10]. Prior to immunolabeling, 100 µl of each sample was aliquoted into a new tube and processed for viability according to the manufacturer's directions (Molecular Probes, Eugene, OR). The remaining sperm suspensions were fixed for 10 min in 2% paraformaldehyde, washed by centrifugation, resuspended with DPBS, and then permeabilized with 0.1% Triton X-100 in DPBS for 10 min. The samples were washed, incubated for 10 min in DPBS containing 5% BSA (blocking solution), and then washed by centrifugation and resuspension with DPBS. The samples were incubated with 4G10 mAb (1:500) or without the primary antibody (control) overnight at 4°C, washed twice with DPBS, and then incubated with fluorescein-conjugated goat anti-mouse (Fab) IgG (1:350) for 1 h at room temperature in the dark. The cells were washed twice with and resuspended in 500 µl DPBS. A drop of a fluorescence enhancer was added (Vectashield; Vector, Burlingame, CA) to enhance and preserve cell fluorescence. Sperm cell samples (5 µl) were placed on glass microscope slides with coverslips and fluorescence was visualized using oil immersion at 1000x magnification with an Olympus BX-60 fluorescence microscope using a fluorescein filter with excitation at wavelength 480/30 nm and emission at wavelength 535/40 nm. One observer determined labeling patterns of at least 100 cells per treatment.
Experiment 2: Determination of Tyrosine Phosphorylation of Cryopreserved Equine Sperm under Capacitating Conditions
Experiment 2 was designed to compare phosphorylation of tyrosine residues between fresh and cryopreserved equine spermatozoa. Sperm were not Percoll washed for this experiment because this is not routinely done for cryopreserved sperm [23]. For this experiment, only immunofluorescence labeling was performed because frozen-thawed sperm samples had been cryopreserved in an egg-yolk-based extender. Therefore, these samples were unable to be electrophoresed and blotted adequately due to the residual yolk proteins from the extender, as determined in preliminary experiments. Baseline tyrosine phosphorylation was determined using fresh and thawed spermatozoa (0 h). Duplicate tubes were incubated for 1 h instead of the normal 3 h capacitation time due to the decrease in viability of frozen-thawed sperm seen in preliminary studies. Samples contained activators (C+C), H-89 (60 µM), or vehicle control for the 1-h incubation period for determination of phosphorylated tyrosine residues. Thawed sperm were diluted with BWW and then centrifuged at 300 x g for 3 min. The pellets were then resuspended in BWW and capacitated and processed for immunofluorescence labeling as described in experiment 1.
Sperm processing for cryopreservation For studies with frozen semen, the raw ejaculate was directly diluted with a commercial skim milk-based semen extender (E-Z Mixin-"Basic Formula"), then centrifuged (350 x g; 20 min) and resuspended in a lactose-EDTA freezing extender (5% glycerol and 20% egg yolk) at a concentration of 400 x 106/ml [23]. The semen was then loaded into 0.5-ml polyvinylchloride straws and frozen using a controlled rate freezer (Planar, Middlesex, U.K.) at a rate of 0.5°C/min from 20 to 5°C, 10°C/min from 5 to -15°C, and 25°C/min from -15 to -150°C before straws were plunged into liquid nitrogen for storage. Cryopreserved sperm were thawed in a waterbath (30 sec at 37°C) prior to use in immunofluorescence experiments.
Statistical Analysis
Mean percentage of sperm tail immunofluorescence of phosphorylated tyrosine residues was analyzed using ANOVA techniques with Minitab statistical software (Minitab, State College, PA) and a PC computer. Comparisons were performed using the Fisher least significant difference procedure with an individual error rate of 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various concentrations of H-89 (10–60 µM) resulted in decreased protein band intensities. At Mr 121 000, H-89 resulted in decreased band intensity from control sperm (without H-89) by 32% at 10 µM and 73% at 30 and 60 µM. The protein band at Mr 65 000 decreased in intensity by 40% at 10 µM and 56% at 30 and 60 µM from control sperm. At Mr 121 000, protein band intensity from sperm incubated with activators plus H-89 decreased 5% at 10 µM and 68% at 30 and 60 µM compared with band intensity from control sperm incubated with activators alone (Fig. 1). At Mr 65 000, protein band intensity was decreased 22% at 10 µM and 76% at 30 and 60 µM relative to control samples.
Four types of immunofluorescent labeling patterns of equine sperm were discerned: a) equatorial band, b) tail, c) equatorial band with tail, and d) none (Fig. 3). For analysis, tail and equatorial band with tail labeling (patterns b and c) were combined and presented as "tail," while equatorial band and none (patterns a and d) were combined and presented as "other." Immunofluorescence labeling indicated that, when fresh sperm were capacitated in vitro for 3 h in the presence of activators, a higher percentage (P < 0.05) of sperm displaying tail-associated tyrosine phosphorylation was observed in all three treatments (control, +CD, and +H89) compared with their respective control cells (0 h) and cells incubated for 3 h without activators (Fig. 4). At 3 h, H-89 decreased sperm tail-associated immunofluorescence by approximately 50% compared with control cells and cells incubated with methyl ß-cyclodextrin in the presence of db cAMP and caffeine (P < 0.05). Initial viability of sperm was 89.5% ± 2.8% and 83.0% ± 2.6% after the 3-h incubation at 37°C.
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The percentage of tail-associated immunofluorescence of fresh and cryopreserved sperm increased after 1 h incubation when activators were present (P < 0.05) compared with their respective samples incubated 0 h and 1 h without activators (Fig. 5). When fresh and thawed sperm were incubated 1 h with activators in the presence of H-89, the increase in protein tyrosine phosphorylation was negated (P < 0.05) (Fig. 5). In fresh sperm, H-89 decreased tail immunofluorescence by 97% from control (no H-89), while H-89 decreased tail labeling by 71% from control in cryopreserved sperm. When frozen sperm were compared with fresh sperm, there was an increase in the percentage of tail labeling at 1 h and 1 h with activators (P < 0.05) (Fig. 5). Viability of sperm did not change in the 1-h incubation period of fresh sperm but was lower after the 1-h incubation of frozen-thawed sperm (P < 0.05) (Table 1). Viability of frozen-thawed sperm was lower than fresh sperm both postthaw and after the 1-h incubation (P < 0.05). Motility did not change in the 1-h incubation period in the fresh or frozen-thawed sperm with or without db cAMP and caffeine (Table 1). However, in the 1-h incubation treatment (without activators), motility was lower in frozen-thawed sperm compared with fresh sperm (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Indirect immunofluorescence images of fresh sperm capacitated for 3 h in the presence of activators showed a localized increase of fluorescence in the midpiece and principal piece of the tail. This tail-associated immunofluorescence distribution was similar to that described in bovine [13], monkey [26], and human [25, 28–30] spermatozoa when incubated under similar conditions. In contrast, a different immunofluorescence pattern localized mainly in the acrosomal domain has been described in boar sperm [31]. These differences in the localization of phosphorylated protein tyrosine residues in sperm have been speculated to be associated with different functions. Tail-associated protein phosphorylation in spermatozoa has been correlated positively with motility and hyperactivation [32, 33]. The localization of tyrosine phosphorylated proteins in the human, bovine, and mouse sperm tail after capacitation in vitro has been related to the presence of A kinase anchoring proteins located in the fibrous sheath of the sperm flagellum [25, 33–35]. These A kinase anchoring proteins anchor PK-A, which is cAMP dependent.
The increase in equine sperm tyrosine phosphorylation induced by activators (db cAMP and caffeine) was negated in a concentration-dependent manner by the addition of H-89, suggesting that PK-A was involved in this signaling mechanism. H-89 is a cell-permeable, selective, and potent inhibitor of PK-A that acts as a competitor for ATP binding to the catalytic subunit of PK-A. The effect observed in equine sperm by the addition of H-89 is in agreement with other studies that have found that both Rp-cAMPs [36], another type of PK-A inhibitor, and H-89 [37, 38] inhibit protein tyrosine phosphorylation and capacitation of sperm [9]. The decrease in tyrosine phosphorylation in the presence of H-89 supports the hypothesis that PK-A has a key role in the signal transduction pathway involved in regulating equine sperm capacitation. In addition, the 20-fold increase in the percentage of cells displaying protein tyrosine phosphorylation tail labeling and the enhancement of protein band intensity of up to 300% observed by Western blot techniques due to the presence of activators, db cAMP and caffeine, provide further evidence for the involvement of PK-A during the process of equine sperm capacitation. As previously mentioned, PK-A activity is dependent on cAMP levels, as reported by Visconti et al. [9]. It is known that methylxanthines such as caffeine, 3-isobutyl-1-methylxanthine, and pentoxifylline inhibit phosphodiesterase activity, causing an increase of intracellular cAMP levels [32, 39–41]. Activators such as caffeine and cAMP appear to be crucial in several species in order to induce sperm in vitro capacitation, and they are a requirement before in vitro fertilization is possible [39, 40].
BSA is thought to be required in capacitation medium due to its ability to bind cholesterol, resulting in the efflux of this sterol from the sperm plasma membrane [42–44]. The removal of cholesterol affects plasma membrane fluidity that can either activate certain signaling pathways involved in fertilization processes [44–46] or affect the diffusion of CO2/HCO3- through the sperm plasma membrane [45, 47]. The presence of high concentrations of HCO3- in the intracellular compartment has been related to the activation of the sperm adenylyl cyclase increasing cAMP levels [48, 49] that activate PK-A activity [50]. As stated by Flesch and Gadella [45], bicarbonate seems to be the key player in triggering tyrosine phosphorylation of proteins in capacitating mammalian sperm. Beta-cyclodextrins are cyclic heptasaccharides that have the ability to efficiently bind cholesterol and remove cholesterol from the cells mimicking even more efficiently the effect of BSA [51–53]. Our study found that the increase of protein tyrosine phosphorylation band intensity of equine sperm during capacitation is amplified by the addition of methyl ß-cyclodextrin. This is in agreement with other studies and suggests that cholesterol release is intimately tied to transmembrane signaling events in the sperm that result in protein tyrosine phosphorylation [50].
All these observations together strongly suggest that equine sperm capacitation is mediated by a signaling pathway that involves cAMP-dependent PK-A and tyrosine kinases. The PK-A activity is enhanced by the direct agonist db cAMP and by indirect agonists such as caffeine and methyl ß-cyclodextrin, and it is inhibited by antagonists such as H-89.
Cryopreserved and capacitated sperm share several characteristics such as plasma membrane reorganization, increased intracellular calcium levels, generation of reactive oxygen species, and acquisition of fertilization capacity [20]. In the second part of our study and after demonstrating an increase of protein tyrosine phosphorylation after in vitro capacitation of equine sperm, we tested whether cryopreservation induced phosphorylation changes in spermatozoa.
It has been widely reported that the freezing-thawing process results in increased sperm death primarily due to plasma membrane damage [19, 54, 55]. In our study, equine sperm viability postthaw and postincubation was approximately 50% and 28%, respectively. Therefore, percentage of tail labeling was normalized to viable cell counts. The observation that there were half as many live cells in 0-h frozen/thawed spermatozoa compared with 0-h fresh spermatozoa and yet there was no difference in percentage of tail-associated immunofluorescence indicates that tyrosine phosphorylation is associated with live cells, as previously reported by Galantino-Homer et al. [13]. Although the process of cryopreservation did not affect the percentage of cells displaying tail-associated immunofluorescence of tyrosine residues at 0 h, a significant increase in tail labeling was observed in postthawed samples after incubation for 1 h, with and without activators, when compared with fresh sperm. Changes in sperm protein tyrosine phosphorylation were not detected after flash freezing bovine sperm [13]. However, it has been recently reported that bovine and ram sperm had a population of cells that displayed tyrosine phosphorylation patterns immediately after thawing, suggesting that cryopreservation induces changes in phosphorylation similar to those observed during capacitation [20, 56]. The increase of protein phosphorylation detected was explained by the occurrence of restructured membranes in surviving sperm and that these conformational changes increase the availability of phosphorylation sites [56] or facilitate calcium influx into the cell [21, 57]. This increased intracellular calcium can stimulate adenylyl cyclase affecting cAMP-mediated tyrosine phosphorylation of sperm proteins as previously mentioned. Our results agree with the idea that the process of cryopreservation itself is not inducing protein tyrosine phosphorylation but that sperm become more susceptible to undergoing tyrosine phosphorylation upon incubation under capacitating conditions, suggesting membrane changes associated with cryopreservation.
As has been seen in the first experiment of the present study, the presence of activators significantly increased the percentage of cells displaying phosphorylation of tyrosine residues in fresh and frozen sperm. Furthermore, the effect of activators was greater in viable cells after freezing compared with fresh because the percentage of cells displaying tail-associated immunofluorescence was approximately 100% versus 70%, respectively. This enhancement of phosphorylation in cryopreserved sperm after 1 h of incubation in the presence of activators further supports the hypothesis of plasma membrane alterations and, in addition, demonstrates that the integrity of the signal transduction pathway involving PK-A is not compromised.
The percentage of cells exhibiting tail tyrosine phosphorylation induced by 1 h of incubation with activators was significantly decreased by H-89 in both fresh and frozen spermatozoa. However, H-89 decreased the percentage of cells displaying phosphorylation 30-fold in fresh sperm but only 3.5-fold in frozen sperm. This discrepancy in the effect of H-89 may reflect involvement of another type of protein kinase (PK) that may be activated in cryopreserved sperm, as has been described in human internal mammary arteries [58]. In addition, this hypothesis of the activation of another PK, besides PK-A, could explain the higher population of cells with phosphorylated tyrosine residues observed after 1 h of incubation in frozen sperm when compared with fresh.
In summary, our results indicate that tail-associated immunofluorescence of phosphorylated tyrosine residues observed after equine sperm in vitro capacitation is a consequence of a pathway that involves cAMP-dependent PK-A and tyrosine kinases. The activity of this PK-A is not affected by the process of cryopreservation itself. However, frozen spermatozoa become more susceptible to undergoing tyrosine phosphorylation upon incubation under capacitating conditions either due to a disrupted plasma membrane and increased intracellular calcium concentrations or activation of another type of PK. The PK-A activity is enhanced by the direct agonist, db cAMP, and by indirect agonists such as caffeine and methyl ß-cyclodextrin, and it is inhibited by antagonists such as H-89. These results suggest that cryopreserved sperm may be more sensitive to stimuli that could explain their limited life span when compared with fresh sperm, and further investigation will allow meaningful improvements in cryopreservation methods for stallion sperm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: S.A. Meyers, Sperm Biology Laboratory, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616. FAX: 530 752 7690; smeyers{at}ucdavis.edu
Received: 6 September 2002.
First decision: 3 October 2002.
Accepted: 16 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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