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Department of Cell Biology, Faculty of Biology, University of Murcia, Campus Universitario de Espinardo, 30100 Murcia, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, gametogenesis, seasonal reproduction, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost that develops asynchronous spermatogenesis during the male phases. The specimens are male during the first 2 yr of life and subsequently change into females [6, 7]. During the male phase, the bisexual gonad has functional testicular and nonfunctional ovarian areas. Therefore, the gonad of this species could be considered as a complex model in which both ovarian and testicular regulatory mechanisms coexist. The morphology of the testicular germinal compartment has been described in some detail in several seasonal breeding teleost species [8, 9]. In contrast, little attention has been paid to the interstitial tissue, even though it probably plays a pivotal role in the reorganization of the testis during postspawning. In some teleost species, a marked increase of interstitial tissue, the vacuolization of interstitial cells, and the presence of macrophages after the shedding of spermatozoa have been observed by conventional microscopy [5, 10, 11]. Moreover, Sertoli cells, alone or together with macrophages, have been observed to be involved in germ cell elimination in teleosts [12, 13]. Of interest, in the gilthead seabream we have previously demonstrated that acidophilic granulocytes infiltrate the testis mostly during postspawning, and they produce interleukin-1ß (IL-1ß), a testicular growth factor for mammalian spermatogonia, rather than being involved in the phagocytosis of degenerative germ cells [14]. In the present study, we extend these observations by in situ immunodetection of proliferative cells labeled with 5-bromo-2'-deoxyuridine (BrdU) and the identification of apoptotic cells by TUNEL. A good correlation between the gonadosomatic index (GSI), the morphological changes of the testis, and cell renewal (proliferation and apoptosis/ phagocytosis) was found in gilthead seabream males. To the best of our knowledge, this is the first paper to provide an overall view of proliferation and apoptosis throughout the reproductive cycle of a seasonal breeding teleost which will, it is hoped, provide a basis for a better understanding of its reproductive physiology.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Healthy specimens of sexually mature male gilthead seabream Sparus aurata (Sparidae, Perciform, Teleostei), with a body weight (BW) of 100 g, were obtained in November 2002, from Predomar, S.L. (Carboneras, Spain). The fish were kept at the Spanish Oceanographic Institute (Mazarrón, Murcia), in 14 m3 running-seawater aquaria (dissolved oxygen 6 ppm, flow rate 20% aquarium volume/hour) with natural temperature and photoperiod, and fed twice a day with a commercial pellet diet (Trouvit, Burgos, Spain). Specimens (n = 9–17 fish/month) were weighed, and the gonads were removed, weighed, and processed for light microscopy as described below. Some specimens were injected i.p. with 50 mg/kg BW of BrdU (Sigma, St. Louis, MO) 2 h before sampling. The studies presented in this manuscript were approved by the Bioethical Committee of the University of Murcia.
Light Microscopy and Immunocytochemical Staining
The gonads were fixed in Bouin solution or 4% paraformaldehyde solution, embedded in paraffin (Paraplast Plus; Sherwood Medical, Athy, Ireland), and sectioned at 5 µm. Some sections were stained with hematoxylin-eosin or with Mallory trichromic to determine the reproductive stage of each fish, and the testicular and total areas of the gonads (see below).
For determining cell proliferation, an indirect immunocytochemical method was performed [15]. The sections were incubated for 40 min in peroxidase-quenching solution [H2O2 (commercial solution at 30%; Panreac, Barcelona, Spain) in methanol, 1:9], for 30 min in 1% periodic acid at 60°C and finally for 30 min in 5% BSA (Sigma) in PBS (pH 7.4). Afterward, they were incubated with a monoclonal antibody anti-BrdU (Becton Dickinson, San Jose, CA) at the optimal dilution of 1:100 in 1% BSA in PBS for 2 h at room temperature. Subsequently, sections were washed in PBS and incubated with a peroxidase-conjugated rabbit anti-mouse immunoglobulin G (whole molecule) at the optimal dilution of 1: 100 in 1% BSA in PBS for 1 h at room temperature. The sections were then washed twice in PBS and in 0.05 M Tris-HCl buffer (pH 7.6) for 5 min each. The peroxidase activity was revealed by incubation with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Fluka, Steinheim, Switzerland) in Tris-HCl buffer (pH 7.6) containing 0.05% H2O2 for 15 min at room temperature. The sections were slightly counterstained with Maller hematoxylin. The specificity of the reactions was determined by omitting the first antiserum and by using tissue sections from fish that had not been injected with BrdU.
In Situ Detection of DNA Fragmentation
TUNEL was performed to identify apoptotic cells (in situ cell death detection kit; Roche, Mannheim, Germany). After hydration, 4% paraformaldehyde-fixed gonad sections were permeabilized for 8 min with 0.1% sodium citrate and 0.1% Triton X-100 at room temperature, washed twice with PBS, and incubated for 1 h at 37°C in a humidified chamber with 50 µl of the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and fluorescein-deoxyuridine triphosphate (f-dUTP), following the supplier's guidelines. Negative controls were processed in an identical manner except that the TdT enzyme was omitted. Positive controls were also performed treating the sections with DNase I (3–3000 U/ ml, Sigma) in 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, and 1 mg/ml BSA for 10 min at room temperature to induce DNA strand breaks before labeling. Slides were examined with an Axiolab (Zeiss) fluorescence microscope.
Electron Microscopy
Samples were fixed with 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 h at 4°C, postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at 4°C, and then embedded in Epon. Toluidine-blue stained semithin sections were obtained. Ultrathin sections were obtained with a Reichert-Jung ultramicrotome, contrasted with uranyl acetate and lead citrate, and examined with a Zeiss EM 10C electron microscope.
Measurement of Testicular and Gonad Areas
Transverse sections (n = 12) stained with hematoxylin-eosin obtained from the middle part of the gonad (n = 6) of each reproductive stage were used to measure the testicular area and the total area of the gonad. The testicular area covers the spermatogenetic tubules and was drawn manually over the digital image. However, the total area covers the spermatogenetic tubules, the efferent duct, and the ovarian area, and was measured using an image analysis thresholding method employed to differentiate borders. The ratio between these two areas was calculated from measurements of gonad tissue images obtained with an Olympus SZ11 overhead projector, a Sony DXC 151 AP video camera, and the software MIP 4.5 Consulting Image Digital (CID, Barcelona).
Calculations and Statistics
As an index of the reproductive stage, we calculated the GSI as 100·[MG/MB] (%), where MG is gonad mass (in grams) and MB is body mass (in grams). The proliferative rates were calculated as the mean number of proliferative cells (spermatogonia stem cells, Sertoli cells, and primary spermatogonia) and proliferative cysts in 50 optical areas at 400x magnification. The areas measured were randomly distributed to cover the whole testis. The testicular ratio was calculated as follows: testicular area (mm2)/gonad area (mm2) x 100. The nuclear and cytoplasmic diameters were drawn manually and measured by image analysis using a Zeiss light microscope, a CoolSNAP digital camera (RS Photometrics), and CoolSNAP software (RS Photometrics). The number of single spermatogonia measured (n = 200) was always higher than the number obtained by the formula (standard deviation·0.83/mean·0.05)2. Data were analyzed by analysis of variance (ANOVA) and a Waller-Duncan multiple range test to determine differences between groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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The reproductive cycle of the gilthead seabream, a protandrous hermaphrodite teleost, can be divided into four stages: spermatogenesis, an asynchronous process that lasts for several months (from September to February), spawning (March), postspawning (from April to May), and resting (from June to September). The gilthead seabream testis was formed by tubules (Fig. 1) consisting of spermatogonia stem cells and cysts (a cohort of synchronically developed germ cells enclosed by a cohort of Sertoli cells) of primary spermatogonia, A and B spermatogonia, spermatocytes, spermatids, and spermatozoa. We distinguished between spermatogonia stem cells and primary spermatogonia according to their size and localization in the germinal compartment (Fig. 1a). Thus, spermatogonial stem cells were the larger spermatogonia with a cytoplasmic diameter of 11.6 ± 0.4 µm and a nuclear diameter of 7.8 ± 0.1 µm, and were mostly located close to the basement membrane of the germinal epithelium. On the other hand, primary spermatogonia were smaller in size with a cytoplasmic diameter of 8.8 ± 0.3 µm and a nuclear diameter of 5.7 ± 0.1 µm. They were surrounded by Sertoli cells, forming a cyst located close to the lumina of the tubules (Fig. 1, a and c, insets).
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As spermatogenesis progressed, the amount of free spermatozoa increased, whereas the cyst variability decreased. Taking this into account, the spermatogenesis stage can be divided into 1) early spermatogenesis, when scarce free spermatozoa are in the lumina; 2) middle spermatogenesis, when a small population of free spermatozoa is present in the lumina of some tubules (Fig. 1a); and 3) late spermatogenesis, when most of the tubules and the efferent duct are full of free spermatozoa (Fig. 1b). During spawning (Fig. 1, c and d), the tubules mainly consisted of spermatogonia stem cells and primary spermatogonia cysts (Fig. 1c). The lumina of the tubules and the efferent duct were enlarged and full of free spermatozoa (Fig. 1d). However, in post-spawning, the spermatozoa were shed and the lumina of the tubules were reduced in size, although some remaining spermatozoa were observed in the tubules closest to the efferent duct (Fig. 1e). During resting, the testis was full of primary spermatogonia cysts and spermatogonia stem cells, forming a dense tissue with no lumina in the tubules (Fig. 1f). Some scattered spermatogonia B cysts were also observed at this stage.
The marked morphological changes observed during the spawning and postspawning stages in the testis were associated with Sertoli cell activity. The Sertoli cells (Fig. 2a) involved in germ cell elimination after the shedding of spermatozoa became more vesiculated due to the presence of numerous lysosomes and some phagocytosed spermatozoa (Fig. 2b). Moreover, they also lost their original organization as the degenerative stage proceeded. At the end of postspawning, the Sertoli cells formed clusters and contained phagosomes and a highly vesiculated cytoplasm (Fig. 2c). However, during the resting stage, the tubules were refilled and showed a narrow lumina with primary spermatogonia enclosed by Sertoli cells.
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The organization of the interstitial tissue also changed markedly during the reproductive cycle. During spermatogenesis, some leukocytes, such an acidophilic granulocytes and macrophage-like cells, were observed between the collagenous fibers of the interstitial tissue, whereas at the end of postspawning they were observed in close association with the basement membrane of the Sertoli cells, which formed cysts containing degenerative germ cells. However, none of these leukocytes were observed to phagocytose testicular cells.
Cell Proliferation and Apoptosis During the Reproductive Cycle
The immunodetection of BrdU (Fig. 3, a–f) and the in situ detection of DNA fragmentation (Fig. 3, g and h) were associated with the reproductive cycle (Fig. 4), although there were some unexpected findings. Notably, spermatogonia stem cells, Sertoli cells, and primary spermatogonia proliferated throughout the year at variable rates (P 
0.05, by ANOVA), which depended on the reproductive stage (Fig. 4). During early spermatogenesis (December), spermatogonia stem cells, Sertoli cells, primary spermatogonia, cysts of A and B spermatogonia, and spermatocytes proliferated. Thus, the number of proliferative (BrdU-positive) cells or cysts per area was 3.9 ± 0.8, 9.9 ± 0.9, 4.9 ± 0.8, and 15.7 ± 2.3, respectively (Fig. 4). As expected, all the germ cell types belonging to the same cyst proliferated at the same time (Fig. 3a), but surprisingly, BrdU-positive Sertoli cells belonged to nonproliferating germ cell cysts (Fig. 3b). In contrast, not all the Sertoli cells belonging to the same cyst proliferated at the same time. Moreover, some cells with fibroblast-shaped nuclei were found to proliferate in the interstitial tissue during spermatogenesis (Fig. 3, a and b). Of interest, during late spermatogenesis (February), the number of proliferative spermatogonia stem cells, Sertoli cells, and primary spermatogonia per area decreased (0.6 ± 0.2, 1.3 ± 0.4, and 1.1 ± 0.3, respectively), whereas the number of A and B spermatogonia and spermatocyte cysts per area was similar (18.2 ± 1.4; Fig. 4). In this stage, some Sertoli cells close to the lumina of the tubules full of free spermatozoa were BrdU-immunostained (Fig. 3c). In contrast to the highly proliferative activity observed during spermatogenesis, scarce proliferative spermatogonia stem cells, Sertoli cells, and primary spermatogonia per area were observed during spawning (0.2 ± 0.2, 1.2 ± 0.4, and 0.4 ± 0.2, respectively; Fig. 4). However, the proliferative activity resumed during postspawning and the resting stage. Thus, during postspawning, the mean number of proliferative spermatogonia stem cells, Sertoli cells, and primary spermatogonia per area was 1.9 ± 0.8, 3.3 ± 0.6, and 4.1 ± 1.2, respectively; whereas during resting, 4.4 ± 1.0 spermatogonia stem cells, 5.7 ± 1.0 Sertoli cells, and 4.3 ± 0.2 primary spermatogonia were found to proliferate per area (Fig. 3, d and e, Fig. 4). Strikingly, some oogonia and somatic cells proliferated in the ovarian area during resting, even when the ovary was not functional and no sex change was under way in any of the fish studied (Fig. 3f).
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Apoptosis is one of the most important mechanisms of cell death involved in different physiological processes. As has recently been described in mammals, apoptosis occurs during spermatogenesis [16], guaranteeing the regular progression of spermatogenesis, or the elimination of germ cells with aberrant chromosomes, or both [17]. Apoptotic cells were observed only during postspawning, when remaining germ cells were being eliminated after the spermatogenetic cycle, although some degenerative nuclei were observed during late spermatogenesis. These cells were settled in the germinal compartment and isolated from each other, and possessed large, round nuclei (Fig. 3g). These observations, together with the cell types observed to form the testis during postspawning, suggest that primary spermatogonia underwent apoptosis during postspawning.
Growth Rate and GSI
Good correlation between the GSI and the different phases of the gilthead seabream reproductive cycle was found (Fig. 5a). The GSI dramatically increased from 0.06 ± 0.01 to 0.26 ± 0.07 at the same time as the number of germ cells observed in the testis during spermatogenesis (see above). The shedding of spermatozoa during spawning resulted in a sharp decrease in GSI from 0.26 ± 0.07 to 0.12 ± 0.03, which continued to decline until it reached 0.06 ± 0.01 at the end of postspawning. Finally, during the resting stage, the growth of the testis was resumed and the GSI reached 0.10 ± 0.01 at the end of the experiment. Of interest, the fish were unable to grow during spermatogenesis or the postspawning stages, despite a considerable temperature increase during the latter (Fig. 5b).
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The ratio between the testicular area without the efferent duct and the total area of the gonad in representative transverse sections was determined to confirm that GSI values correlated with testis development during the reproductive cycle (P 0.05 by ANOVA). Thus, our data showed that the testicular area reached 84% ± 4% of the total gonad at the end of spermatogenesis, whereas it dramatically decreased, together with the GSI, when the spermatozoa had been shed. However, the area of the full gonad remained largely similar because some of the tubules and the empty efferent duct were still swollen. Later, at the end of post-spawning, the total section area decreased, probably due to the reorganization of the tubules and the collapse of the efferent duct. Finally, during resting, both the testicular and the total section areas increased at similar rates (Fig. 5c).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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We were able to differentiate between spermatogonia stem cells and primary spermatogonia according to their size and location in the germinal compartment. As in many teleosts, a reserve stock of spermatogonia is present throughout the year in the gilthead seabream testis. This spermatogonia population has been called spermatogonia A, residual germ cells, dormant spermatogonia, sperm mother cells, residual primary spermatogonia, and primary spermatogonia [18–25]. Moreover, some authors have described two subpopulations of this stable spermatogonia pool, referring to a difference of 2 µm in their nuclear diameters (8.5 ± 0.8 vs. 6.4 ± 0.5) [24]. However, other authors did not distinguish between these two subpopulations, although they described a primary spermatogonia population with nuclear diameters ranging from 5 to 9 µm [26, 27]. Taking into account that the mean decrease in the nuclear diameter during spermatogenesis is approximately 1–2 µm in teleost fish [24, 28], these two subpopulations might represent different spermatogenetic stages at the beginning of the process. Therefore, we defined the spermatogonia stem cells as a stable population of large spermatogonia (7.8 ± 0.1 µm nuclear diameter), which are isolated and located close to the basement membrane of the germinal epithelium, and the primary spermatogonia as being smaller (5.7 ± 0.1 µm nuclear diameter) and isolated, but mainly surrounded by Sertoli cells and dispersed throughout the germinal epithelium.
As the Sertoli cells nurse the developing germ cells, their quantity determines the magnitude of sperm production [29]. In contrast to what happens in mammals, the number of Sertoli cells per cyst in the teleost testis increases as the germ cells develop [30, 31]. Moreover, new cysts are continually formed in mature teleost when Sertoli cells initially encircle a single primary spermatogonium [25]. In this framework, Sertoli cell proliferation has also been previously described in adult teleost testis [25, 32]. However, our data showed that dividing Sertoli cells, which can be identified by their location and nuclear shape, belong to nondividing germ cell cysts, whereas in Thalassoma bifasciatum, both Sertoli cells and spermatogonia were seen to synchronously divide in the same cyst [25].
The origin of the new spermatogonia and Sertoli cells that restart spermatogenesis during the following reproductive cycle has been widely discussed and is still a matter of controversy. Based on morphological data, it has been hypothesized that the somatic cells of the nongerminal compartment might come from the peritoneum [33], while Sertoli cells may arise from mesenchyme-like precursors or fibroblasts [25, 34]. Two hypotheses have also been suggested to explain germ cell renewal using microscopic data [34]. In some species, a small population of spermatogonia stem cells present in the testis brings about germ cell renewal. Other studies have suggested that there is an annual migration of stem cells into the tubules from an interstitial location, although the morphological evidence (light microscopy) of this migration is not convincing [34]. Our data demonstrated that both spermatogonia stem cells and Sertoli cells proliferate not only during spermatogenesis when they permit successive spermatogenetic waves (during several months), but also during the spawning and postspawning stages, thus preventing total depletion of these cell types. Although our data allow us to discard the stem cell migration hypothesis for the recrudescence of spermatogenesis in the gilthead seabream, we cannot exclude the possibility of trans-differentiation of interstitial cells into Sertoli cells. On the other hand, exclusively during spermatogenesis, we observed a high proliferation of an undetermined cell type in some areas of the interstitial tissue that may represent proliferative Leydig cell precursors. The Leydig cells were observed in some areas of the interstitial tissue during spermatogenesis. Although Leydig cells were early described as the main source of testis steroids in teleost fish [35], further studies are needed to determine the steroidogenesis capability of this cell type in the gilthead seabream. Unfortunately, all our attempts to identify these cells by immunocytochemistry using commercial antibodies to testosterone have failed so far.
Although some spermatogonia stem cells and Sertoli cells proliferated in the gilthead seabream testis during spawning and postspawning, this activity is significantly lower than that observed during spermatogenesis and resting stages. In some fish with synchronous spermatogenesis, spermatogonia proliferation is not resumed as long as spermatozoa are present in the lumina of the tubules, not even when hormonal treatment that was supposed to be able to initiate spermatogenesis was used [13, 36, 37]. Our data support this idea, although some differences are evident between both synchronous and asynchronous spermatogenetic processes. Strikingly, in the gilthead seabream, the presence of spermatozoa during spermatogenesis is unable to down-regulate proliferation, whereas it might contribute to doing so during spawning. Moreover, in several sparid species, the nonfunctional ovarian area is able to grow during each resting stage of the male phase [11]. These results are now confirmed in the gilthead seabream, in which both oogonia germ cells and somatic cells proliferate during the resting stage of the male phase. Our data suggest, therefore, that the gilthead seabream shows two different times of gonial stem cell proliferation: one during spermatogenesis, when only spermatogonia stem cells proliferate; and another one during the resting stage, when both spermatogonia and oogonia stem cells proliferate. All these data taken together seem to suggest the existence of different regulatory mechanisms of germ cell proliferation that might be influenced by the hormonal status of the fish.
The specific processes by means of which the testicular cells degenerate are not well known. Several authors have suggested that the remaining germ cells, influenced by Sertoli cells, undergo necrosis and in situ lysis, being phagocytosed by Sertoli cells, macrophages, or both [12, 13, 24, 35]. Our data show that in the gilthead seabream, both apoptosis of spermatogonia and phagocytosis of the remaining spermatozoa by Sertoli cells work together during the degenerative process that takes place during postspawning. It has been described that apoptosis affects nonfunctional germ cells in the cartilaginous fish, Torpedo marmorata, in which apoptotic spermatocytes and spermatids were observed during spermatogenesis [38]. However, that we observed apoptosis only during postspawning suggests that this is not the case in the gilthead seabream and that spermatogonial apoptosis is probably related in some way to the degenerative process.
Unlike the germinal compartment, the testicular interstitial tissue has been relatively unstudied. We have previously described the presence of acidophilic granulocytes in the gilthead seabream testis. It is interesting that the down-regulation during spawning and the resumption during post-spawning of spermatogonial proliferation coincide with increased acidophilic granulocyte infiltration. This fact, together with the capability of the testicular acidophilic granulocytes to produce IL-1ß [14], a positive regulator for spermatogonia proliferation in mammals [39, 40], suggests that these cells may be involved in the regulation of the testicular cell cycle rather than in the elimination of degenerative cells. In Squalus acanthias, the epigonal organ (a lymphomyeloid organ at the mature pole of the testis) and granulocyte secretions inhibited the premeiotic proliferation of testicular germ cells [41]. On the other hand, Sertoli cells show high phagocytic activity in the seabream testis and it is likely that they are the only cell type involved in the phagocytosis of degenerating germ cells in this species, because we did not observe any macrophage-like cells phagocytosing germ cells.
To conclude, our data show that germ cell proliferation correlates with the GSI and with the testicular area changes throughout the reproductive cycle of the gilthead seabream. As far as we know, this is the first time that cell proliferation and elimination has been studied throughout the reproductive cycle in a fish showing asynchronous spermatogenesis. To integrate all these new findings and our previous data [14] we propose a model (Fig. 6) in which all the cellular changes during the reproductive cycle of the gilthead seabream are included. During spermatogenesis, most of the premeiotic and postmeiotic germ cells are destined to finish spermatogenesis, after which the spermatozoa are accumulated in the lumina of the tubules and, then, are shed and eliminated during spawning. In addition, the germinal epithelium decreased in size, while germ cells differentiated into spermatozoa. Thus, at spawning, the germinal epithelium consists of spermatogonia stem cells, Sertoli cells, and some primary spermatogonia. During postspawning, a slight increased proliferative activity is accompanied by an increase in the infiltration of acidophilic granulocytes and apoptosis of germ cells, which means that the testicular area does not grow during this stage. However, the enhancement of proliferation and the absence of apoptosis permit the repopulation of the testis by spermatogonia and Sertoli cells during resting. In contrast to fish with synchronous spermatogenesis [13, 36, 37], the proliferative activity is not blocked by the presence of spermatozoa during spermatogenesis, suggesting regulatory differences in the spermatogenesis process. Moreover, the presence of a nonfunctional ovary, which is able to proliferate but unable to develop, suggests the presence of complex regulatory mechanisms in protandrous teleosts that still remain to be identified.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Alfonsa García Ayala, Department of Cell Biology, Faculty of Biology, University of Murcia, Campus Universitario de Espinardo, 30100 Murcia, Spain. FAX: 34 968 363963; agayala{at}um.es
Received: 13 September 2004.
First decision: 11 October 2004.
Accepted: 1 November 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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