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Embryo |
Department of Cell Biology and Physiology,3 Instituto de Investigaciones Biomédicas, UNAM. México, D.F. México 04510
Prince Henry's Institute of Medical Research,4 Monash Medical Centre, Clayton, Victoria 3168, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gene regulation, ovary, Sertoli cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been proposed that B6-Ydom sex reversal results from an improper interaction between the Ydom testis-determining gene (Sry) and dominant autosomal genes in the genome of the C57BL/6J strain [6, 9, 10], but the identity of these genes remains unknown. On the other hand, it was found that transcription of Sry in B6-Ytir gonads starts at the same time as in normal XY gonads, but its expression persists longer in B6-Ytir gonads [11]. The finding that levels of Sry were lower in sex-reversed B6.Ytir gonads than in non-sex-reversed B6-Ydom strains suggests that low levels and/or mistiming of Sry may cause failure of testis differentiation [12]. However, Lee and Taketo [13] observed that levels of Sry in normal male gonads of the C57BL/6J strain are as low as in the B6-Ytir gonad, suggesting that low levels of Sry transcripts cannot be the sole cause of B6-Ytir sex reversal. Thus the molecular events underlying sex reversal in the B6-Ytir mouse still remain to be elucidated.
SOX9, a SRY-related gene, was originally identified as responsible for campomelic dysplasia, a severe form of dwarfism often associated with XY male-to-female sex reversal [14, 15]. Like SRY, dosage may be important for proper function of SOX9, since mutations in humans appear to be present in only one allele, suggesting that sex reversal and campomelic dysplasia could be due to haploinsufficiency of this gene product [16]. Sox9 has been detected in the mouse urogenital complex of both sexes at 10.5 dpc. Beginning at 11.5 dpc, just after Sry is expressed in the male gonad, Sox9 is up-regulated in male gonads and down-regulated in female gonads. After seminiferous cord formation, Sox9 expression is specific to Sertoli cells [17], and Sox9 is probably one of the first genes induced by Sry in male gonads [18]. The DNA-binding domains of both Sry and Sox9 proteins are highly conserved and can functionally substitute for each other [19]. A mouse line with a transgene inserted upstream of Sox9 showed that the development of XX males is consistent with Sry being an indirect repressor downstream target of Sox9 [20, 21]. Recently, female to male sex-reversal induced by ectopically expressing Sox9 in XX gonads indicated that it can substitute for Sry in the events necessary to form a testis and is likely to act downstream [22].
It is assumed that Sry and Sox9 are expressed in pre-Sertoli cells from which an orchestrated series of intercellular signals leads the undifferentiated genital ridge to form a testis [23]. Since it is known that Sry is expressed in B6-Ytir fetal gonads, it seems likely that formation of ovotestis or ovaries in B6-Ytir gonads may be due to a mismatch of intercellular signals before and/or after Sox9 expression in pre-Sertoli cells. The aim of the present study was to correlate the timing and distribution of pre-Sertoli cells that express Sox9 by immunofluorescence with the profile levels of Sox9 transcripts in fetal and postnatal B6-Ytir gonads. It was found that pre-Sertoli cells appear in all undifferentiated B6-Ytir gonads as in wild-type B6 gonads at 11.5–12.5 dpc. From 13.5 dpc onward, however, pre-Sertoli cells expressing the Sox9 protein were absent in about 50% of the gonads. This corresponds to the number of B6-Ytir gonads that develop as ovaries and in the cranial and caudal regions in fetal ovotestis. In contrast, reverse transcriptase polymerase chain reaction (RT-PCR) revealed the presence of Sox9 transcripts in all fetal and postnatal B6-Ytir gonads, although levels were lower in B6-Ytir ovaries. Thus it is tempting to speculate that abnormal development of B6.Ytir gonads may be due to partial failure of Sox9 function as a consequence of reduced dosage in pre-Sertoli cells, which leads to ovotestis or the development of follicular cells in B6.Ytir ovaries. In turn, the reduced dosage could be a consequence of the reduced ability of Sry to activate Sox9 transcription. Furthermore, the abnormal presence of Sox9 transcripts in B6-Ytir ovaries found here may cause infertility in B6-Ytir females.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals from the Institute for Laboratory Animal Research of the National Academy of Sciences [24]. B6.Ytir mice kept in our colony (backcross generations N 30–35) that possess the B6 genetic background and the Y chromosome from M. musculus domesticus (from Tirano, Italy) were produced as previously described [7]. B6.Ytir males were mated with B6 females, and the day of vaginal plug was taken to be 0.5 days postcoitum (dpc). Bilateral gonads were dissected from embryos at 11.5 up to 18.5 dpc and 30 and 60 days postpartum (dpp).
Zfy PCR Analysis
The chromosomal sex of the B6-Ytir embryos was determined by PCR amplification of the 600 base pair (bp) fragment of the Y-encoded gene Zfy. The PCR was performed using DNA isolated from the fetal tissues or from the tails at 30 and 60 dpp. The tissue was homogenized and incubated in lysis buffer (100 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 0.5% Tween 20, 0.5% NP-40, and 0.4 mg/ml proteinase K) [13] at 55°C overnight, and proteinase K was inactivated by heating at 85°C for 10 minutes. Fifteen microliters of the lysate were diluted in 85 µl of ddH2O. Two microliters of each DNA sample was processed for PCR amplification in a total volume of 20 µl with 0.25 µM of each of the following primers: Zfy1, 5'-AAG ATA AGC TTA CAT AAT CAC ATG GA-3'; Zfy2, 5'-CCT ATG AAA TCC TTT GCT GCA CAT GT-3' for Zfy; Actin 1, GGG TCA GAA GGA TTC CTA TG; and Actin 2, GGT CTC AAA CAT GAT CTG GG for ß-actin used as an internal control [26]. The primers were in 10 mM Tris-HCl pH 8.3, 50 mM KCl, 0.01% gelatin, 2.0 mM MgCl2, 0.2 mM of each dNTP and 1.0 U Taq DNA polymerase.
The amplification program was 94°C/10 min (once), 94°C/15 sec, 60°C/15 sec, 72°C/30 sec (35 cycles), and 72°C/10 min (once) [26]. The PCR reactions were electrophoresed on a 1% agarose gel in TAE 1x buffer and visualized with ethidium bromide fluorescence in UV light.
Immunocytochemical Detection of SOX9
Antigen-affinity antibody raised to the human SOX9 C-terminal 24-amino-acid epitope (VPSIPQTHSPQHWEQPVYTQLTRP) was prepared according to the previously described protocol [17]. For immunocytochemical detection of Sox9, B6-Ytir gonads were dissected from embryos from 11.5 to 18.5 dpc and fixed in 4% paraformaldehyde in PBS for 10 min, washed in PBS, and incubated in 30% sucrose overnight. The gonads were embedded in OCT medium (Tissue-Tek, Sakura Finetek, Torrance, CA) and frozen at -70°C. Serial sections of 10-µm thickness were used. After air drying, sections were treated with 1% Triton X-100 in water, rinsed with PBS, and blocked using 1% bovine serum albumin. The sections were then incubated overnight in Sox9 polyclonal antibody diluted 1:500. After washing in PBS, the sections were treated with fluorescein-goat anti-rabbit IgG (Sigma, St. Louis, MO) diluted 1:200 in blocking solution.
RNA Isolation and RT-PCR
Gonads were dissected from B6 and B6-Ytir embryos of 18.5 dpc and from animals of 30 and 60 dpp. The tissues were immediately frozen in dry ice and stored at -70° C. Detection was carried out in the two fetal gonads or single gonads of postnatal animals. Unilateral fetal gonads from hermaphrodite mice were pooled as two ovaries or two testes. A DNA probe containing a 243 bp fragment of ß-actin was used as the internal expression control. Total RNA was isolated from gonads using Trizol reagent (Life Technologies Invitrogen, Carlsbad, CA); based on the method of Chomczynski and Sacchi [27], a few modifications were introduced to improve the RNA isolation, the volume of chloroform was increased to double, and the time of precipitation in isopropanol was increased from 10 minutes to overnight. Total RNA was resuspended in 12 µl of DEPC-treated ddH2O. Reverse transcriptase polymerase chain reaction (RT-PCR) amplifications were performed using primers designed on the basis of the mouse Sox9 as previously reported [18]. The sense primer was Sox9-1, 5'-GTG GCA AGT ATT GGT CAA-3'; the antisense primer was Sox9-2, 5'-GAA CAG ACT CAC ATC TCT-3'. For expression control, amplification of oligonucleotides used as internal controls were Actin 1 and Actin 2 as previously described [25]. The amplification reactions were carried out by means of the One Step RT-PCR kit (Life Technologies Invitrogen) using 3 µl of total RNA per reaction, 1x buffer reaction mix containing 0.2 mM of each dNTP, 1.2 mM MgSO4, and 200 ng of ß-actin primers or 300 ng of Sox9 primers in separate tubes. The enzyme used was a mixture of Superscript II RT and Taq DNA polymerase Platinum (Life Technologies Invitrogen) in a volume of 20µl. The amplification program was 50°C for 30 min (once); 94°C/5 min (once), 94°C/30 sec, 56°C/60 sec, 72°C/60 sec (25 cycles), 72°C/10 min (once) within the linear range previously described [18]. In order to confirm that the amplified products were not genomic DNA in origin, we used an RT-free control (Taq DNA polymerase but not template DNA added) for each RT-PCR [28].
Ten microliters of each RT-PCR reaction were electrophoresed on a 2.5% agarose gel with 0.1 µg/ml ethidium bromide in TAE 1x buffer [29]. Gels were visualized by UV transillumination and photographed with a DS-34 Polaroid camera (Polaroid, Waltham, MA). The size of the amplified products was 310 bp for Sox9 and 243 for ß-actin. The intensity of each band was quantified by densitometry using the Scion Image program. Results were expressed as arbitrary units of the ratio between Sox9 and ß-actin mRNA levels.
In Situ Hybridization
In embryos at 12.5 and 13.5 dpp, the gonads were processed in situ attached to the dorsal body wall. At 18.5 dpc and 30 dpp, the gonads were dissected out and processed. A total of 11, 15, 7, and 11 gonads at 12.5 dpc, 13.5 dpc, 18.5 dpc, and 30 dpp, respectively, were processed. Whole-mount in situ hybridization for detection of Sox9 transcripts was carried out with probes kindly provided by Dr. Ramón Merino (Cantabria University, Spain). Reactions were performed in vitro using T3 or T7 RNA polymerase to generate sense and antisense RNA probes labeled with digoxigenin-UTP. The complete procedure was as previously described by Wilkinson and Nieto [30]. Reactions were developed with BCIP/NBT or with purple AP substrate (Boehringer-Mannheim, Roche, Indianapolis, IN).
Statistical Analysis
Profiles of Sox9 transcripts were quantified as mean ± standard deviation of at least three independent determinations. Variance between experiments was assessed by two-way ANOVA. The mean was used to evaluate the significant differences by paired Tukey test (P < 0.001).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pre-Sertoli cells were identified by immunofluorescent staining with an antibody against Sox9 protein and were observed in all 12 B6.Ytir gonads at 11.5 and 12.5 dpc (Table 1). Longitudinal and cross-serial sections of urogenital ridges revealed a distribution pattern of pre-Sertoli cells within the still undifferentiated genital ridges. Cross serial sections revealed that pre-Sertoli cells were located among the innermost cells of the thickening genital ridges and rarely formed part of the coelomic epithelium (Fig. 1A). Longitudinal sections showed that pre-Sertoli cells were located in the medial region, whereas they were absent from both the cranial and caudal regions of the genital ridges (Fig. 1B).
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Interestingly, whereas at 13.5 dpc Sox9-positive cells were present in both gonads of six out of 12 serially sectioned B6.Ytir embryos, they were considerably diminished in one gonad of three of these embryos. In three other embryos both gonads lacked Sox9-positive cells, and in the other three embryos Sox9-positive cells were absent in one of the two gonads (Table 1). Sox9-positive cells were located in the medial region of the gonad where they organize the incipient seminiferous cords together with the germ cells (Fig. 1C). In the B6.Ytir genital ridges showing Sox9-positive cells at 13.5 dpc, their number and the area of distribution along the medial region showed individual variations. Some gonads had scant randomly distributed Sox9-positive cells, whereas others revealed only a few at the mesonephric-gonad boundary (Fig. 1, D and E). In contrast to normal male gonads of B6 where seminiferous cords formed by Sox9-positive cells segregated from the coelomic epithelium at 12.5 dpc, in B6.Ytir gonads such segregation was delayed approximately for 1 day and was evident at 13.5 dpc. Furthermore, although well-formed seminiferous cords were evident in the middle region of the B6.Ytir gonads, at the cranial and caudal regions Sox9-positive cells were scant or absent (Fig. 1F).
At 15.5–17.5 dpc, seminiferous cords formed by Sox9-positive cells are organized in the middle region of all B6.Ytir gonads that form ovotestis. They expand toward the cranial and caudal regions where ovarian tissues are undergoing regression as suggested by the presence of abundant pyknotic cells (Fig. 2). In addition to the immunolocalization of Sox9 in Sertoli cells, the presence of testicular and ovarian tissues in B6.Ytir ovotestis is supported by the different meiotic behavior of germ cells in each of them (Fig. 2). Whereas in the well-organized seminiferous cords the germ cells entered the mitotic resting stage (Fig. 3A), in the ovarian regions they entered the prophase of the first meiotic division as evidenced by the synaptonemal complexes clearly detected with the electron microscope (Fig. 3B).
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At 18.5 dpc, B6.Ytir gonads that differentiated as ovotestes or as ovaries were distinguishable by their location within the abdominal cavity and the presence or absence of Sox9-positive Sertoli cells. Although ovotestes showed intra-abdominal descent and were located 2–3 mm below the kidneys, ovaries devoid of Sox9-positive cells remained in the upper position attached to the kidneys. Although several B6.Ytir embryos had either bilateral ovotestes or ovaries, some embryos had an ovotestis and a contralateral ovary (Fig. 4, A and B). The extension of well-organized seminiferous cords containing Sox9-positive Sertoli cells varies in individual gonads. In some gonads Sox9-positive cells forming seminiferous cords occupied almost the whole gonad (Fig. 4C). In others, however, the seminiferous cords were located mainly in the middle region, whereas at the cranial and caudal gonadal regions the Sox9-positive cells appeared scattered and failed to form seminiferous cords (Fig. 4, A and D).
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Table 1 summarizes our observations of Sox9-positive cells in B6.Ytir embryos at various developmental stages. Although in 50% both gonads and in 25% one gonad showed pre-Sertoli cells at 13.5 dpc, in 25% both gonads and in 25% one gonad showed Sox9-positive Sertoli cells by 18.5 dpc. Taken together, Sox9-positive cells were absent in about half of the gonads studied between 13.5 and 18.5 dpc. This corresponds to the reported proportion of ovotestis and ovaries that form in B6.Ytir mice [31].
Profiles of Sox9 Transcripts in B6.Ytir Ovaries and Ovotestis
In contrast to the immunocytochemical detection of the Sox9 protein, RT-PCR detection of Sox9 transcripts showed that they were present at all stages and in all B6.Ytir fetal gonads (Fig. 5). A semiquantitative densitometric analysis revealed that the levels of Sox9 transcripts increased from 13.5 to 17.5 dpc in both B6 testes and B6.Ytir ovotestes. Nevertheless, statistical analysis indicated that B6.Ytir ovotestis had lower Sox9 than B6 testis at 13.5–17.5 dpc, which was significantly lower (P < 0.001) at 17.5 dpc. In contrast, B6.Ytir ovaries showed significantly lower Sox9 earlier at 15.5 and 17.5 dpc (P < 0.001; Fig. 5). On the other hand, Sox9 transcripts were not detected in all B6 XX fetal ovaries at 13.5–17.5 dpc stages (not shown).
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Representative gels and semiquantitative densitometric analyses of Sox9 transcripts in fetal (18.5 dpc) and postnatal gonads (30 and 60 dpp) of two normal strains (CD1 and B6) and B6.Ytir gonads are shown in Figure 6. Although at 18.5 dpc and 30 dpp, Sox9 mRNA was detected in all fetal gonads with a Y chromosome from CD1, B6, or Ytir, the expression levels varied according to the strain and the type of gonad formed (ovotestis, testis, or ovary). Sox9 levels were not significantly different between testes of CD1 and B6 at all three stages. Comparing fetal gonads of B6 testes with B6.Ytir ovotestis at 18.5 dpc, no significant differences were found. In contrast, B6.Ytir ovaries had significantly lower levels than B6 testis at 18.5 dpc (Fig. 6A).
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At 30 dpp as at 18.5 dpc, the levels of Sox9 appeared similar in testes of CD1 and B6 strains. In contrast to fetal testes, B6.Ytir gonads that developed as testes or bilateral ovaries did not show significant differences in Sox9 transcript levels compared with those in B6 testes. However, B6.Ytir ovaries of hermaphrodite B6.Ytir mice showed significantly lower levels than B6.Ytir females with bilateral ovaries (Fig. 6B). Finally, at 60 dpp the levels of Sox9 were not significantly different between B6 and B6.Ytir testes. In contrast, in B6.Ytir ovaries, Sox9 transcripts were no longer detected (Fig. 6C).
Whole Mount In Situ Hybridization
In situ hybridization with a Sox9 probe performed in whole mount samples confirmed the absence of Sox9 transcripts in B6 XX ovaries and their presence in all gonads with genotype B6.Ytir, regardless of whether they developed into testes or ovaries. The extent and distribution of tissue expressing Sox9 within the gonads varied both between contralateral gonads and between gonads of individuals of the same B6.Ytir litter (Fig. 7). At 13.5 dpc, Sox9-expressing cells were not detected in any B6 XX gonads (Fig. 7A). In contrast, Sox9 transcripts were evident in the middle region of all B6.Ytir gonads. Although in some gonads Sox9-positive tissue extends toward the cranial and caudal regions, in others only the middle region was Sox9-positive (Fig. 7, B and C). At 30 dpp, B6 XX ovaries remained Sox9-negative. In contrast, in B6 and B6.Ytir testis Sox9-positive tissue was strongly detected in the seminiferous tubules, whereas in B6.Ytir ovary clusters of randomly distributed Sox9-positive tissue were evident (Fig. 7D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The polyclonal antibody used here was raised against the human SOX9 C-terminal 24 amino-acid epitope. This region forms part of the transcriptional activation domain and is highly conserved between species. The specificity of the antibody to Sox9 protein was previously shown to correlate with Sox9 mRNA by in situ hybridization in developing mice gonads [17]. At 11.5 dpc, Sox9 was immunolocalized in the cytoplasm of several cells, suggesting that functional activity of Sox9 may depend on its nuclear translocation in pre-Sertoli cells. In the present study, however, Sox9 was always located within the nuclei of pre-Sertoli cells at 11.5 and 12.5 dpc, making it unlikely that the inhibition of differentiation of pre-Sertoli to Sertoli cells in B6.Ytir gonads is due to failure of nuclear translocation of Sox9.
In B6.Ytir gonads that take the ovarian pathway, Sertoli cells expressing Sox9 protein disappeared beyond 13.5 dpc; we did detect Sox9 transcripts in B6.Ytir ovaries, but at significantly lower levels than in gonads that form seminiferous cords (at 17.5 dpc). Since Sox9 transcripts in B6.Ytir ovaries are lower than in B6 or CD1 testes, our results indicate that maintenance of Sox9 transcripts at threshold levels may be necessary for pre-Sertoli cells to undergo further differentiation. Although Sry can weakly initiate Sox9 expression in the B6.Ytir gonads at 12.5 dpc, its low activity or amount may lead to failure of Sox9-positive cells to further differentiate into Sertoli cells. Since Sertoli cells are necessary for testis tissue differentiation, it is tempting to speculate that lower levels of Sox9 transcripts in pre-Sertoli cells lead to B6.Ytir gonads developing as phenotypic ovaries.
As in normal males, Sox9-positive cells appear in the genital ridges of B6.Ytir males in cells considered to be the pre-Sertoli cell lineage [17]. Although this lineage appears in 100% of 11.5 and 12.5 dpc embryos, from 13.5 dpc onward the number of Sox9-positive cells diminishes and disappears in about half of B6.Ytir embryos. This observed loss of Sox9 protein suggests that unknown factors in B6.Ytir gonads prevent further differentiation of pre-Sertoli cells and lead them down the female pathway. The factors could be genetic modifiers that are manifested when Sry and/or Sox9 function is at threshold levels.
A regional distribution of cells that express Sox9 in undifferentiated B6.Ytir gonads was observed. Sox9-positive cells appeared in the middle region, whereas they were absent from the cranial and caudal regions. Furthermore, cross sections of genital ridges at 11.5 and 12.5 dpc showed Sox9-positive cells among the innermost cells and rarely among coelomic epithelial cells. This distribution was similar in all embryos at 11.5 and 12.5 dpc, suggesting that regional location of pre-Sertoli cells in early genital ridges does not preclude their fate to differentiate as ovotestes or ovaries. The observed distribution of Sox9-positive pre-Sertoli cells in B6.Ytir genital ridges is similar to that of Sry-positive cells in morphologically undifferentiated genital ridges of normal XY mice [32]. Thus the pattern of Sry expression followed by the location of Sox9-positive cells and the organization of the seminiferous cords [33, together with current results] suggests that the presence of short-range extracellular cues acting along the genital ridge during normal testis differentiation may be disturbed in B6.Ytir gonads. Alternatively, a cell autonomous effect due to weak Sry expression causing weak Sox9 transcription may occur. Thus normal entrance of cells from the mesonephros into the normal developing testes [34, 35] may be partially or totally prevented in B6.Ytir gonads [36].
Furthermore, in the present study Sox9 transcripts were detected by RT-PCR in both ovotestis and ovaries of B6.Ytir embryos at all developmental stages. This finding suggests that failure to up-regulate Sox9 in pre-Sertoli cells may be posttranscriptional of either Sry, Sox9, or both. Assuming that cells immunopositive to Sox9 detected at 11.5 and 12.5 dpc in B6.Ytir genital ridges are pre-Sertoli cells, current results indicate that 13.5 dpc represents a critical stage for the developmental fate of these gonads.
In B6.Ytir ovotestis, seminiferous cords comprising Sox9-positive cells gradually develop toward the cranial and caudal regions of the gonad after 14.5 dpc. Although scattered Sox9-positive cells remain among the sex cords in the ovarian regions, the great abundance of pyknotic cells indicates that in most fetal ovotestes the ovarian tissue regresses. Besides lacking clear epithelial cord organization, the ovarian regions of B6.Ytir ovotestis were identified by the onset of meiosis of the germ cells in these areas. Thus current results support previous observations suggesting that postnatal ovotestes in litters of B6.Ytir mice are rare because most or all ovarian tissue regresses before birth [9, 37].
The levels of Sox9 transcripts at 30 dpp are lower in unilateral (ovary and testis in each side) than bilateral B6.Ytir ovaries (two ovaries), but at 60 dpp Sox9 transcripts are undetected in both. At 30 dpp, unilateral ovaries had more growing follicles than bilateral B6.Ytir ovaries. By 60 dpp, most follicles disappeared in all B6. Ytir ovaries (unpublished results). Sox9 transcripts are absent in normal ovaries. Correlating the levels of Sox9 transcripts with the ovarian morphology suggested that the lower levels of Sox9 transcripts observed in unilateral ovaries at 30 dpp may favor follicular development. Considering that infertility in B6.Ytir females occurs at the ovarian level [31, 37, 38], current results suggest that the abnormal presence of Sox9 transcripts may disturb oocyte development in follicles of B6.Ytir ovaries.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Horacio Merchant-Larios, Instituto de Investigaciones Biomédicas, UNAM. Apartado Postal 70228, México, D.F. México 04510. FAX: 52 55 5662 3897; merchant{at}servidor.unam.mx
Received: 3 March 2003.
First decision: 10 April 2003.
Accepted: 2 September 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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