- Email: [email protected]
HSP70 is part of the synaptonemal complex in Eimeria tenella
Parasitology International 57 (2008) 454–459
Contents lists available at ScienceDirect
Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
HSP70 is part of the synaptonemal complex in Eimeria tenella Emilio del Cacho a,⁎, Margarita Gallego a, Marc Pages b, Luis Monteagudo c, Caridad Sánchez-Acedo a a b c
Department of Animal Pathology, Faculty of Veterinary Sciences, Miguel Servet 177, University of Zaragoza, 50013 Zaragoza, Spain HIPRA Laboratories, Gerona, Spain Department of Anatomy, Embryology and Genetics, Faculty of Veterinary Sciences, Miguel Servet 177, 50013 Zaragoza, Spain
A R T I C L E
I N F O
Article history: Received 25 February 2008 Received in revised form 16 May 2008 Accepted 24 May 2008 Available online 5 June 2008 Keywords: Heat shock proteins Synaptonemal complex Quercetin Eimeria tenella
A B S T R A C T In the current study the expression and ultrastructural localization of heat shock protein 70 (HSP70) was analyzed by immunogold labelling of surface spreads of meiotic chromosomes from Eimeria tenella oocysts. The authors used a previously reported method that overcomes the difficulties of the resistance of Eimeria oocysts to disruption and permits the release of intact meiotic chromosomes. HSP70 was localized at the ultrastructural level using an anti-HSP70 monoclonal antibody in combination with a secondary antibody coupled to colloidal gold. Synaptonemal complexes (SCs) were visualized by means of the surface spreading technique to study both HSP70 expression and the consequences of the lack of HSP70 in the behaviour of the eimerian chromosomes during meiosis. For that purpose E. tenella oocysts were treated with quercetin, a flavonoid that is known to inhibit the synthesis of HSP70. The results showed a close association of HSP70 with the lateral elements (LEs) of the SCs. That association began at the time that SCs were formed and persisted until disassemble. Comparison between distribution of immunogold label over the SCs from nontreated and treated oocysts revealed a decreasing number of gold particles as the concentration of quercetin increased. The current results demonstrated three dose-dependent effects of the quercetin treatment of Eimeria oocysts: a reduction in the HSP70 synthesis; defects in SC formation or desynapsis, and inhibition of sporulation. HSP70, as a structural component of the SCs, may be involved in SC functions such as chromosomal pairing, recombination, or disjunction. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Comparison between heat shock proteins (HSPs) from different organisms, from bacteria to human, has revealed that they have been highly conserved in evolution. They play different roles in the homeostatic mechanisms that preserve cellular functions from the deleterious effects of stressful conditions [1]. These proteins have been identified in a wide variety of protozoan parasites, such as Leishmania [2], Trypanosoma spp. [3], Plasmodium spp. [4], Toxoplasma spp. [5], and Eimeria spp. [6,7]. Furthermore, heat shock protein 70 (HSP70) has been shown to play important roles in stage conversion, infectivity, and virulence of several protozoan parasites [8,9]. The finding of HSP70 in Eimeria tenella oocysts during sporulation [10] is particularly important as significant cellular and molecular events occur during the process that forms sporocysts and sporozoites. A major process that takes place within the first hours of sporulation is meiosis [11]. In meiosis, chromosome synapsis ensures the faithful segregation of chromosomes by establishing physical connections between homologs. The synaptonemal complex (SC), a ⁎ Corresponding author. Present address: Parasitología y Enfermedades Parasitarias, Facultad de Veterinaria, Miguel Servet 177, 50013 Zaragoza, Spain. E-mail address: [email protected] (E. del Cacho). 1383-5769/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2008.05.005
specialized structure that develops at initiation of the chromosome synapsis, holds the homologous chromosomes together along their entire lengths, stabilizes their pairing and provides the structural framework necessary for genetic recombination events [12]. The structural elements of the SC (lateral and central elements, recombination nodules, kinetochores and telomeres) have remained highly conserved throughout the evolution of protozoa, fungi, plants, insects and mammals [13]. Evolutionary conservation of SC function must be matched by conservation of at least some common molecular components. The origin of the SC and the assembly of its proteinaceous elements have not been well established. However, in mammals, HSP70-2 is thought to be associated with SC [14] and, what is more, to have a key roll in chromosome rearrangement [15]. Del Cacho et al. [16] reported a modified version of the air-dry technique, which permits visualisation of the SC by transmission electron microscopy (TEM) when applied to eimerian oocysts in pachytene. Recently, the normal rearrangement of the E. tenella chromosomes and the 14 SCs that mediate chromosome pairing have been described during meiosis [17]. The study of the SCs by means of the surface spreading technique provides the opportunity to apply immunohistochemical techniques to the eimerian CS in order to study their molecular composition. The aim of this present work was to study both HSP70 expression in the CS of E. tenella and the
E. del Cacho et al. / Parasitology International 57 (2008) 454–459
consequences of the lack of HSP70 in the behaviour of the eimerian chromosomes during meiosis. E. tenella oocysts were treated with quercetin, a flavonoid that is known to inhibit the synthesis of HSP70. 2. Materials and methods 2.1. Animals A total of 50 one-day-old White Leghorn chickens were hatched and reared coccidia-free under routine laboratory conditions with free access to feed and water. All experiments were performed in accordance with the guidelines approved by the Animal Ethics Committee of our institution. 2.2. Parasite An E. tenella strain was originally obtained from Merck Sharp and Dohme (Madrid, Spain). Oocysts were propagated, isolated and sporulated using standard procedures [18]. Chickens were infected with sporulated oocysts (stored for less than four weeks) by oral inoculation into the crop. 2.3. HSP70 inhibition Unsporulated oocysts were harvested and purified using standard procedures [18]. To overcome the difficulty of the impervious nature of the oocyst wall, oocysts were treated with hypoclorite and dimethyl sulfoxide (DMSO) according to the procedure by Wang and Stotish [19] to facilitate quercetin penetration into the oocyst. The hypocloritetreated oocysts were divided into 4 groups, consisting of 10 × 106 oocysts each, to be incubated under 4 experimental conditions as follows. Oοcysts from groups 1, 2, and 3 were incubated in quercetin (Sigma) at a concentration of 25, 50 and 100 μM, respectively, in the presence of 5% (v/v) DMSO from the outset of sporulation. Oocysts from group 4 (the control group) were incubated without quercetin, with the remaining procedure being the same. Aliquots of 3 × 106 oocysts were taken at 6 and 8 h following the outset of sporulation for synaptonemal complex study and blot analyses. 2.4. Synaptonemal complex study Sporulating oocysts were treated according to the method described by del Cacho et al. [16] in order to permit isolation of meiotic chromosomes and their subsequent study by TEM. Briefly, oocysts were incubated in an ethanol: HCl solution for 15 min to partially digest and lower the resistance of the oocyst-wall. After washing in phosphate buffered saline (PBS) (pH 7.4), the oocysts were re-suspended in PBS at a concentration of 5 × 103 oocysts/ml. Six to eight drops of the oocyst suspension were placed on a coat slide with colodion in amyl acetate. The slides were then dried at 37 °C, frozen at − 20 °C for 30 min and subsequently thawed. The process of freezing and thawing was repeated three times to disrupt the oocyst-wall and permit the release and spread of intact meiotic chromosomes. Selected areas showing a high density of chromosomes were marked and then placed on a grid. Grids were then incubated in normal horse serum (blocking reagent) (Vector Lab.) for 10 min, followed by incubation with a mouse anti-HSP70 monoclonal antibody (Clone 7, Pharmingen, San Diego, California) at a 1:100 dilution for 90 min. After washing in PBS, grids were incubated with the secondary antibody conjugated with 5 nm colloidal gold. Gold-conjugated goat antimouse IgG (Sigma, St. Louis, Missouri) was applied (dilution 1:10) at room temperature, for 45 min. Grids were fixed in 4% paraformaldehyde and subsequently stained for 3 min with phosphotungstic acid (PTA) (one part 4% aqueous solution PTA and three parts 95% ethanol) following the method described by Switonski et al. [20]. Finally, chromosomes were observed by TEM. Negative control girds, to which
455
the primary antibody was not applied with all other conditions remaining the same, were prepared concurrently. To determine whether HSP70 was bound non-specifically to SCs, surface-spread preparations were treated with high-salt lysis buffer [14] consisting of 300 mM NaCl, 5 mM MgCl2, 0.5% nonylphenylpolyethylene glycol, 0.1% sodium deoxycholate, and 20 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid buffered (HEPES), pH 7.4. The grids were placed in lysis buffer for 10 min at 37 °C immediately before the paraformaldehyde treatments. 2.5. Blot analyses Blot analyses were applied to oocysts treated and non-treated with quercetin. Extraction of proteins for HSP70 measurements was performed by sonication to disrupt the oocysts, followed by centrifugation at 100,000 ×g for 20 min. The supernatant was stored in liquid nitrogen until use. Equal amounts of protein samples from the oocysts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a discontinuous gel system based on a standard protocol [21]. An SDS-reducing sample buffer containing SDS and 2-mercaptoethanol was used for SDS-PAGE. Electrophoresis was performed at 40 mA. After electrophoresis, the proteins in the gel were electrophoretically transferred to nitrocellulose paper in a TransBlot cell (Bio Rad, Richmond, California). Electrophoresis was performed with a transfer buffer at room temperature for 18 h at 40 mV. After the transfer of the proteins, excess binding sites on the nitrocellulose paper were blocked by washing the paper in PBS containing 4% blocking reagent. The blots were then treated with the mouse anti-HSP70 monoclonal antibody for 1 h at room temperature. The blots were rinsed 3 times in PBS for 5 min, and then exposed to peroxidase-conjugated goat anti-mouse IgG. They were then rinsed in PBS and treated for peroxidase activity with diaminobenzidine (DAB) and hydrogen peroxide solution (20 mg DAB in 100 ml of 0.05 M Tris– HCl buffer, pH 7.6, containing 0.005% H2O2) for 5 min. After immunoblotting, the immunoreactivity of the samples was quantified using an image analyzer (Program MIP 4.5, Microm, Heidelberg, Germany). The images on the immunoblots were analyzed for both image area and mean optical density (OD). The integrated optical density (IOD) (image area x mean OD) was used for quantitation of immunoreactivity. Comparisons between blots were made possible by using an internal control included on every blot. The control lines used in the blots were loaded with 1μg of human HSP70. Prior to the current studies, experiments were carried out following the procedure described by Martinez et al. [22] to demonstrate the linearity of the immunoblot results under the conditions to be used in the present study. Concentrations of human HSP70 running from 0.1 μg to 3 μg were used. Evaluation of the test results by regression analysis
Fig. 1. Complete set of Eimeria tenella bivalents represented by 14 synaptonemal complexes from pachytene oocyst spread obtained 6 h after the start of sporulation. Kinetochores (arrows) are visible on each synaptonel complex. Telomeres (arrowhead). Bar = 1 μm.
456
E. del Cacho et al. / Parasitology International 57 (2008) 454–459
Fig. 2. Immunoelectron micrograph of tightly synapsed Eimeria tenella homologous chromosomes showing labelled lateral elements. Gold grains are distributed along the total length of the lateral elements and appear concentrated at the telomeric region (arrowsheads), whereas de centromeric region (arrows) has no gold grains. Note the decreasing number of grains as the oocysts are incubated with increasing concentrations of quercetin. A. Oocysts not treated with quercetin (control). B. Oocysts incubated with 25 μM of quercetin. C. Oocysts incubated with 50 μM of quercetin. Pachytene oocyst spread obtained 6 h after the start of sporulation. Bar = 0.2 μm.
revealed, in all cases, a very high correlation (r N 0.95) between the observed immunoreactivity and the amount of protein applied in the test. 2.6. Oocyst sporulation Quercetin treated and non-treated oocysts were sporulated following the standard procedures reported by Raether et al. [18]. Sporulation rates were determined by counting 400 oocysts under ×400 magnification (five replications) at 72 h of sporulation. 3. Results 3.1. Synaptonemal complexes study At 6 h after the start of the sporulation, complete sets of 14 pachytene SCs were observed by TEM in surface spreads of meiotic chromosomes from non-treated oocysts and oocysts treated with either 25 or 50 μM of quercetin (Fig. 1). Each of the 14 SCs was formed by the parallel association of the lateral elements (LEs), which were tightly synapsed (Fig. 1). In addition, a strongly stained centromere was observed in most of the 14 SCs (Fig. 1). However, SCs were not
seen in the surface spreads of meiotic chromosomes from oocysts treated with 100 μM of quercetin. SCs spreads were labelled to study the expression of HSP70. HSP70 was detected at the ultrastructural level with an anti-HSP70 monoclonal antibody, whose distribution was revealed using colloidal gold. Gold label was located over the LEs showing a homogeneous distribution along their entire length. Accumulation of particles was detected at the telomeres, where clusters of gold grains were observed (Fig. 2A). The central element and kinetochore in each SC were devoid of gold particles (Fig. 2A). SCs from oocysts treated with 25 or 50 μM of quercetin showed the same pattern of labelling as that found over the SCs from non-treated oocysts (Fig. 2B, C). Gold label was associated with LEs and concentrate at the telomere in the 14 SCs (Fig. 2B, C). However, comparison between distribution of immunogold label over the SCs from non-treated and treated oocysts revealed a decreasing number of gold particles as the concentration of quercetin increased (Fig. 2). The scant number of particles found in SCs from oocysts treated with 50 μM of quercetin (Fig. 2C) is worth noting. Meiotic chromosomes from oocysts treated with 100 μM of quercetin did not form SCs and the LEs were not observed. Consequently, no HSP70 expression was detected in surface spreads of those meiotic chromosomes.
Fig. 3. Electron micrograph showing desynapsis of paired homologous chromosomes seen at 8 h of the start of sporulation. A. Oocysts not treated with quercetin (control). B. Oocysts incubated with 50 μM of quercetin. Breakage of one of the homologous chromosomes forming the bivalent is observed (arrows). Note that the homologous remained associated at both ends of the bivalent. Oocyst spread obtained 8 h after the start of sporulation. Bar = 1 μm.
E. del Cacho et al. / Parasitology International 57 (2008) 454–459
457
Fig. 4. Immunoelectron micrograph showing separation of the two homologous chromosomes in bivalents from control oocysts (A) and oocysts incubated with 25 μM (B) or 50 μM of quercetin (C). Note both the decreasing number of gold grains as the oocysts are incubated with increasing concentrations of quercetin and the breakage of a homolog in most bivalents from the oocysts incubated with 50 μM of quercetin. Oocyst spreads obtained 8 h after the start of sporulation. Bar = 0.3 μm.
At 8 h from the start of sporulation, desynapsis of bivalents was observed showing identical features in non-treated oocysts and oocysts treated with 25 μM of quercetin (Fig. 3A). At that point of sporulation, homologous chromosomes in each bivalent pulled away from each other to some extend, marking the end of synapsis. The terminal arms of the homologous chromosomes were held together by persisting segments of SC (Fig. 3A). However, in the surface spread of chromosomes from 50 μM quercetin-treated oocysts, breakage of one of the homologs forming the bivalent was observed (Fig. 3B) as paired chromosomes opened out and separated. Breaks were found in most of the bivalents (Fig. 3B). The expression of HSP70 on meiotic chromosomes from both treated and non-treated oocysts showed the same distribution at 8 h from the start of sporulation as that found at 6 h (Fig. 4). Likewise, at that point of sporulation and during separation of paired chromosomes, decreasing numbers of gold particles were detected as the concentration of quercetin increased (Fig. 4). 3.2. Blot analysis and quantification of HSP70 expression Immunoblot analysis of the total proteins from the oocysts treated with 25 or 50 μM of quercetin and the non-treated oocysts showed a band of approximately 70 kDa (Fig. 5). Therefore, the anti-HSP70 antibody reacted with a protein with a similar molecular weight in both the E. tenella oocysts and the control (human HSP70). Significantly, the density of the bands diminished as the amount of quercetin increased (Fig. 5). Densitometric studies showed that the control exhibited slightly higher IOD values than those shown by the non-treated oocysts (Table 1). The statistical analysis demonstrated
Fig. 5. Expression of HSP70 in non-treated (lanes 3–4) and treated (lanes 5–10) E. tenella oocysts. Lanes 1–2: human HSP70 (control). Lanes 3–4: oocysts incubated without quercetin. Lanes 5–6: oocysts treated with 25 μM of quercetin. Lanes 7–8: oocysts treated with 50 μM of quercetin. Lanes 9–10: oocysts treated with 100 μM of quercetin. Molecular mass (kDa) is indicated.
that the variation of IOD between the control (human HSP70) and the non-treated oocysts was not significant, whereas IOD values were significantly higher in non-treated oocysts when compared to quercetin-treated oocysts. The greatest decrease in IOD was observed in oocysts treated with 50 μM of quercetin, whose IOD values fell dramatically to 2.5 (Table 1). However, the IOD values found in the oocysts treated with 25 μM of quercetin showed a decrease in IOD to 80.5 (Table 1). Therefore, treated oocysts showed a progressive decrease in the expression of HSP70 as the concentration of quercetin increased. Decreases of 52.2% and 98.6% in IOD were found in the oocysts treated with 25 and 50 μM of quercetin, respectively (Table 1). It should be emphasised that oocysts treated with 100 μM of quercetin lacked HSP70 expression. 3.3. Oocyst sporulation Sporulation rates of the quercetin-treated and non-treated oocysts are given in Fig. 6. The rates found in 25 μM quercetin-treated oocysts were slightly lower than those found in non-treated oocysts. However, the difference in the number of oocysts that sporulated was not statistically significant. In contrast, the oocysts treated with 50 and 100 μM quercetin failed to sporulate. In addition, our results demonstrate that in non-treated and 25 μM quercetin-treated oocysts, sporozoites were formed within each sporulated oocyst and the
Table 1 Densitometric analysis of HSP70 immunoreactivity Optical density Integrated optical % Integrated optical density (IOD) (mean ± SD) density (IOD) (mean ± SD) (mean ± SD)
Sample
Area
Human HSP70 No quercetin 25 μM quercetin 50 μM quercetin 100 μM quercetin
220.5 ± 13.2a 0.80 ± 0.04a
176.4 ± 7.7a
104.9%a
218.4 ± 10.6a 167.8 ± 10.9b
0.77 ± 0.03a 0.48 ± 0.04b
168.1 ± 6.8a 80.5 ± 6.9b
100%a 47.8%b
84.3 ± 12.5c
0.03 ± 0.007c
2.5 ± 0.6c
1.4%c
0
0
0
0
Integrated optical density: area x optical density. The % of integrated optical density has been calculated taking the IOD found in the oocysts incubated without quercetin as representing 100%. Data marked with different superscript sign differ significantly (P b 0.05).
458
E. del Cacho et al. / Parasitology International 57 (2008) 454–459
Fig. 6. Sporulation rates of the quercetin-treated and non-treated oocysts. Significant differences between untreated (solid area) and 25 μM quercetin-treated (dotted area) oocysts were not found. Note the oocysts treated with either 50 μM or 100 μM of quercetin did not sporulate. Each bar represents the mean ± SD.
appearance of the Stieda bodies was observed (Fig. 7A). However, in oocysts treated with 50 and 100 μM quercetin, neither cleavage of the sporoplasm nor sporocyst formation was seen (Fig. 7B). Furthermore, the oocysts showed a granular cytoplasm which was enclosed in a distinct cytoplasmic membrane instead of the standard sporocysts (Fig. 7B). 4. Discussion To increase our understanding of how the structural components of SCs accomplish their many functions in chromatin arrangement, chromosome pairing, recombination, and disjunction at meiosis it is necessary to know the components that combine to form the SCs [23]. Antibodies directed against SC components provide a first approach to the identification of SC substructures and their functions. In the current study, the expression and ultrastructural localization of HSP70 was analyzed by immunogold labelling of surface spreads of meiotic chromosomes from E. tenella oocysts. HSP70 was detected with a specific monoclonal antibody whose distribution was revealed using colloidal gold, and examined by TEM. Previous studies on HSPs during Eimeria sporogony have demonstrated that HSP70 is regulated developmentally and highly expressed during sporulation [10]. In the present study, HSP70 was found to be a component of the SC. From the beginning of pachytene until SCs disassembly in diplotene, the authors found a constant pattern of staining, which demonstrated a higher concentration of HSP70 on the telomeres. During SC formation, the telomere attaches to the inner nuclear envelope to form a structure named attachment plaque,
which functions as a kind of platform for SC movement [24]. Several reports have shown the ability of HSP70 to assembly and disassembly cytoskeletal proteins playing a role in cellular movements [25,26,27]. Therefore, the continued presence of HSP70 on the SCs suggests that the HSP70 has a continuous function in the chromosomal behaviour playing a role in the SCs movements that take place during chromosomal pairing, recombination, or disjunction. Quercetin is a specific inhibitor of HSP70 synthesis with a limited effect on the synthesis of other HSPs [28]. The mechanism of this inhibitory effect is not thoroughly understood. Hosokawa et al. [29] have found that quercetin inhibits HSP70 synthesis by changing the tertiary structure of heat shock factor which is required for the transcription of the HSP70 gene. The current results demonstrated three dose-dependent effects of the quercetin treatment: a reduction in the HSP70 synthesis; defects in SC formation or desynapsis; and inhibition of sporulation. Treatment with 25 μM quercetin inhibited HSP70 synthesis to 47.8%; nevertheless, it did not affect chromosome behaviour or the sporulation process. Treatment with 50 or 100 μM quercetin dramatically inhibited HSP70 synthesis which showed a decrease ranging from 98.6% to 100%. The consequences were failure to form SCs or to complete desynapsis and failure to develop haploid sporozoites. Although the significance of HSP70 in eimerian SCs is unknown, the present results suggest that HSP70 supports fundamental meiotic mechanisms. Furthermore, the role of HSP70 proteins as chaperones that assist the folding and the assembly and disassembly of other proteins is well known [25]. Accordingly, HSP70 in eimerian SCs may help to stabilize structures that are important in achieving chromosomal paring and segregation as has been proposed to occur in mouse SCs [15]. Recently, in mammals and in the protozoan parasite Leishmania, quercetin has been demonstrated to inhibit DNA topoisomerases I and II [30,31]. As a consequence, DNA religation is inhibited, chromosome instability occurs and chromosome breakage may result [32]. However, DNA topoisomerases have not been reported during sporulation of apicomplexan protozoa. In the light of our findings one may assume that the effect of HSP70 on SC formation and desynapsis is essential in order to complete the sporulation process. The current results provide an important basis for the development of new lines of research aimed at clarifying the mechanisms that play a role in the sporulation of E. tenella. Acknowledgements This work was supported by grant A46 from the Research Council of Aragón, Spain.
Fig. 7. Eimeria tenella oocysts after 72 h of sporulation. A) Sporulated oocysts showing the sporozoites and the Stieda body. B) Oocyst treated with 50 μM of quercetin. Note the condensation of the sporoplasm. The cytoplasm appeared granular and enclosed in a distinct cytoplasmic membrane.
E. del Cacho et al. / Parasitology International 57 (2008) 454–459
References [1] Maresca B, Carratú L. The biology of the heat shock response in parasites. Parasitol Today 1992;8:260–6. [2] Coulson RMR, Smith DF. Isolation of genes showing increased or unique expression in the infective promastigotes of Leishmania major. Mol Biochem Parasitol 1990;40: 63–76. [3] Hausler T, Clayton C. Post-transcriptional control of hsp70 mRNA in Trypanosoma brucei. Mol Biochem Parasitol 1996;76:57–71. [4] Sharma YD. Structure and possible function of heat-shock proteins of falciparum malaria (mini-review). Comp Biochem Physiol 1992;102B:437–44. [5] Lyons RE, Johnson AM. Heat shock proteins of Toxoplasma gondii. Parasite Immunol 1995;17:353–9. [6] Robertson NP, Reese RT, Speer CA. Heat shock-like polypeptides of the sporozoites and merozoites of Eimeria bovis. J Parasitol 1988;74:1004–8. [7] Dunn PPJ, Billington K, Bumstead JM, Tomley FM. Isolation and sequences of cDNA clones for cytosolic and organellar hsp70 species in Eimeria spp. Mol Biochem Parasitol 1995;70:211–5. [8] Lyons RE, Johnson AM. Gene sequence and transcription differences in 70 kDa heat shock protein correlate with murine virulence of Toxoplasma gondii. Int J Parasitol 1998;28:1041–51. [9] Del Cacho E, Gallego M, Lopez-Bernad F, Quilez J, Sanchez-Acedo C. Differences in HSP70 expression in the sporozoites of the original strain and precocious lines of Eimeria tenella. J Parasitol 2005;91:1127–31. [10] Del Cacho E, Gallego M, Pereboom D, Lopez-Bernad F, Quilez J, Sanchez-Acedo C. Eimeria tenella: HSP70 expression during spoogony. J Parasitol 2001;87:946–50. [11] Canning EU, Anwar M. Studies on meiotic division in coccidial and malarial parasites. J Protozool 1968;15:290–8. [12] Roeder GS. Chromosome synapsis and genetic recombination: their roles in meiotic chromosome segregation. Trends Genet 1990;6:385–9. [13] Westergaard M, Wettstein D. The synaptonemal complex. Ann Rev Genet 1972;6: 71–110. [14] Allen JW, Dix DJ, Collins BW, Merrick BA, He C, Selkrik JK, et al. HSP70-2 is part of the synaptonemal complex in mouse and hamster spermatocytes. Chromosoma 1996;104:414–21. [15] Dix DJ, Allen JW, Collins BW, Poorman-Allen P, Mori C, Blizard DR, et al. HSP70-2 is required for desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes. Development 1997;124:4595–603. [16] Del Cacho E, Gallego M, Monteagudo L, López-Bernad F, Quílez J, Sánchez-Acedo C. A method for the sequential study of eimerian chromosomes by light and electron microscopy. Vet Parasitol 2001;94:221–6. [17] Del Cacho E, Pages M, Gallego M, Monteagudo F, Quílez J, Sánchez-Acedo C. Synaptonemal complex karyotype of Eimeria tenella. Intl J Parasitol 2005;35: 1445–51.
459
[18] Raether W, Hofmann J, Uphoff M. Biotechnology. In: Eckert J, Brown R, Shirley MW, Coudert P, editors. Guidelines on Techniques in Coccidiosis Research. Brussels: European Commission; 1995. p. 79–84. [19] Wang CC, Stotish RL. Changes of nucleic acids and proteins in the oocysts of Eimeria tenella during sporulation. J Protozool 1975;22:438–43. [20] Switonski M, Gustavsson I, Höjer K, Plöen L. Synaptonemal complex analysis of the B-chromosomes in spermatocytes of the silver fox (Vulpes fulvus Desm.). Cytogenet Cell Genet 1987;45:84–92. [21] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [22] Martínez J, Pérez-Serrano J, Bernadina WE, Rodríguez-Caabeiro F. Detection of heat shock protein-70 from Trichinella spiralis larvae using a modification of the routine western blotting procedure. J Parasitol 2000;86:637–9. [23] Moens PB, Heyting C, Dietrich AJJ, van Raamsdonk W, Chen Q. Synaptonemal complex antigen location and conservation. J Cell Biol 1987;105:93–103. [24] Alsheimer M, von Glasenapp E, Hock R, Benavente R. Architecture of the nuclear periphery of rat pachytene spermatocytes: distribution of nuclear complex attachment sites. Mol Biol 1999;10:1235–45. [25] Kiang JG, Tsokos GC. Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol Ther 1998;80:183–201. [26] Agueli C, Geraci F, Guidice G, Chimenti L, Cascino D, Sconzo G. A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed. Biochem J 2001;360:413–9. [27] Botha M, Pesce ER, Blatch GL. The Hsp40 proteins of Plasmodium falciparum and other apicomplexa: Regulating chaperone power in the parasite and the host. Int J Biochem Cell Biol 2007;39:1781–803. [28] Elia G, Santoro G. Regulation of heat shock protein synthesis by quercetin in human erythroleukaemia cells. Biochem J 1994;300:201–9. [29] Hosokawa N, Hirayoshi K, Kudo H, Takechi H, Aoike A, Kawai K, et al. Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids. Mol Cell Biol 1992;12:3490–8. [30] Cantero G, Campanella C, Mateos S, Cortés F. Topoisomerase II inhibition and high yield of endoreduplication induced by the flavonoids luteolin and quercetin. Mutagenesis 2006;21:321–5. [31] Cortázar TM, Coombs GH, Walker J. Leishmania panamensis: comparative inhibition of nuclear DNA topoisomerase II enzymes from promastigotes and human macrophages reveals anti-parasite selectivity of fluoroquinolones, flavonoids and pentamidine. Exp Parasitol 2007;116:475–82. [32] Das BB, Sen N, Roy A, Dasgupta SB, Ganguly A, Mohanta BC, et al. Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I. Nucleic Acids Res 2006;34:1121–32.
Contents lists available at ScienceDirect
Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
HSP70 is part of the synaptonemal complex in Eimeria tenella Emilio del Cacho a,⁎, Margarita Gallego a, Marc Pages b, Luis Monteagudo c, Caridad Sánchez-Acedo a a b c
Department of Animal Pathology, Faculty of Veterinary Sciences, Miguel Servet 177, University of Zaragoza, 50013 Zaragoza, Spain HIPRA Laboratories, Gerona, Spain Department of Anatomy, Embryology and Genetics, Faculty of Veterinary Sciences, Miguel Servet 177, 50013 Zaragoza, Spain
A R T I C L E
I N F O
Article history: Received 25 February 2008 Received in revised form 16 May 2008 Accepted 24 May 2008 Available online 5 June 2008 Keywords: Heat shock proteins Synaptonemal complex Quercetin Eimeria tenella
A B S T R A C T In the current study the expression and ultrastructural localization of heat shock protein 70 (HSP70) was analyzed by immunogold labelling of surface spreads of meiotic chromosomes from Eimeria tenella oocysts. The authors used a previously reported method that overcomes the difficulties of the resistance of Eimeria oocysts to disruption and permits the release of intact meiotic chromosomes. HSP70 was localized at the ultrastructural level using an anti-HSP70 monoclonal antibody in combination with a secondary antibody coupled to colloidal gold. Synaptonemal complexes (SCs) were visualized by means of the surface spreading technique to study both HSP70 expression and the consequences of the lack of HSP70 in the behaviour of the eimerian chromosomes during meiosis. For that purpose E. tenella oocysts were treated with quercetin, a flavonoid that is known to inhibit the synthesis of HSP70. The results showed a close association of HSP70 with the lateral elements (LEs) of the SCs. That association began at the time that SCs were formed and persisted until disassemble. Comparison between distribution of immunogold label over the SCs from nontreated and treated oocysts revealed a decreasing number of gold particles as the concentration of quercetin increased. The current results demonstrated three dose-dependent effects of the quercetin treatment of Eimeria oocysts: a reduction in the HSP70 synthesis; defects in SC formation or desynapsis, and inhibition of sporulation. HSP70, as a structural component of the SCs, may be involved in SC functions such as chromosomal pairing, recombination, or disjunction. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Comparison between heat shock proteins (HSPs) from different organisms, from bacteria to human, has revealed that they have been highly conserved in evolution. They play different roles in the homeostatic mechanisms that preserve cellular functions from the deleterious effects of stressful conditions [1]. These proteins have been identified in a wide variety of protozoan parasites, such as Leishmania [2], Trypanosoma spp. [3], Plasmodium spp. [4], Toxoplasma spp. [5], and Eimeria spp. [6,7]. Furthermore, heat shock protein 70 (HSP70) has been shown to play important roles in stage conversion, infectivity, and virulence of several protozoan parasites [8,9]. The finding of HSP70 in Eimeria tenella oocysts during sporulation [10] is particularly important as significant cellular and molecular events occur during the process that forms sporocysts and sporozoites. A major process that takes place within the first hours of sporulation is meiosis [11]. In meiosis, chromosome synapsis ensures the faithful segregation of chromosomes by establishing physical connections between homologs. The synaptonemal complex (SC), a ⁎ Corresponding author. Present address: Parasitología y Enfermedades Parasitarias, Facultad de Veterinaria, Miguel Servet 177, 50013 Zaragoza, Spain. E-mail address: [email protected] (E. del Cacho). 1383-5769/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2008.05.005
specialized structure that develops at initiation of the chromosome synapsis, holds the homologous chromosomes together along their entire lengths, stabilizes their pairing and provides the structural framework necessary for genetic recombination events [12]. The structural elements of the SC (lateral and central elements, recombination nodules, kinetochores and telomeres) have remained highly conserved throughout the evolution of protozoa, fungi, plants, insects and mammals [13]. Evolutionary conservation of SC function must be matched by conservation of at least some common molecular components. The origin of the SC and the assembly of its proteinaceous elements have not been well established. However, in mammals, HSP70-2 is thought to be associated with SC [14] and, what is more, to have a key roll in chromosome rearrangement [15]. Del Cacho et al. [16] reported a modified version of the air-dry technique, which permits visualisation of the SC by transmission electron microscopy (TEM) when applied to eimerian oocysts in pachytene. Recently, the normal rearrangement of the E. tenella chromosomes and the 14 SCs that mediate chromosome pairing have been described during meiosis [17]. The study of the SCs by means of the surface spreading technique provides the opportunity to apply immunohistochemical techniques to the eimerian CS in order to study their molecular composition. The aim of this present work was to study both HSP70 expression in the CS of E. tenella and the
E. del Cacho et al. / Parasitology International 57 (2008) 454–459
consequences of the lack of HSP70 in the behaviour of the eimerian chromosomes during meiosis. E. tenella oocysts were treated with quercetin, a flavonoid that is known to inhibit the synthesis of HSP70. 2. Materials and methods 2.1. Animals A total of 50 one-day-old White Leghorn chickens were hatched and reared coccidia-free under routine laboratory conditions with free access to feed and water. All experiments were performed in accordance with the guidelines approved by the Animal Ethics Committee of our institution. 2.2. Parasite An E. tenella strain was originally obtained from Merck Sharp and Dohme (Madrid, Spain). Oocysts were propagated, isolated and sporulated using standard procedures [18]. Chickens were infected with sporulated oocysts (stored for less than four weeks) by oral inoculation into the crop. 2.3. HSP70 inhibition Unsporulated oocysts were harvested and purified using standard procedures [18]. To overcome the difficulty of the impervious nature of the oocyst wall, oocysts were treated with hypoclorite and dimethyl sulfoxide (DMSO) according to the procedure by Wang and Stotish [19] to facilitate quercetin penetration into the oocyst. The hypocloritetreated oocysts were divided into 4 groups, consisting of 10 × 106 oocysts each, to be incubated under 4 experimental conditions as follows. Oοcysts from groups 1, 2, and 3 were incubated in quercetin (Sigma) at a concentration of 25, 50 and 100 μM, respectively, in the presence of 5% (v/v) DMSO from the outset of sporulation. Oocysts from group 4 (the control group) were incubated without quercetin, with the remaining procedure being the same. Aliquots of 3 × 106 oocysts were taken at 6 and 8 h following the outset of sporulation for synaptonemal complex study and blot analyses. 2.4. Synaptonemal complex study Sporulating oocysts were treated according to the method described by del Cacho et al. [16] in order to permit isolation of meiotic chromosomes and their subsequent study by TEM. Briefly, oocysts were incubated in an ethanol: HCl solution for 15 min to partially digest and lower the resistance of the oocyst-wall. After washing in phosphate buffered saline (PBS) (pH 7.4), the oocysts were re-suspended in PBS at a concentration of 5 × 103 oocysts/ml. Six to eight drops of the oocyst suspension were placed on a coat slide with colodion in amyl acetate. The slides were then dried at 37 °C, frozen at − 20 °C for 30 min and subsequently thawed. The process of freezing and thawing was repeated three times to disrupt the oocyst-wall and permit the release and spread of intact meiotic chromosomes. Selected areas showing a high density of chromosomes were marked and then placed on a grid. Grids were then incubated in normal horse serum (blocking reagent) (Vector Lab.) for 10 min, followed by incubation with a mouse anti-HSP70 monoclonal antibody (Clone 7, Pharmingen, San Diego, California) at a 1:100 dilution for 90 min. After washing in PBS, grids were incubated with the secondary antibody conjugated with 5 nm colloidal gold. Gold-conjugated goat antimouse IgG (Sigma, St. Louis, Missouri) was applied (dilution 1:10) at room temperature, for 45 min. Grids were fixed in 4% paraformaldehyde and subsequently stained for 3 min with phosphotungstic acid (PTA) (one part 4% aqueous solution PTA and three parts 95% ethanol) following the method described by Switonski et al. [20]. Finally, chromosomes were observed by TEM. Negative control girds, to which
455
the primary antibody was not applied with all other conditions remaining the same, were prepared concurrently. To determine whether HSP70 was bound non-specifically to SCs, surface-spread preparations were treated with high-salt lysis buffer [14] consisting of 300 mM NaCl, 5 mM MgCl2, 0.5% nonylphenylpolyethylene glycol, 0.1% sodium deoxycholate, and 20 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid buffered (HEPES), pH 7.4. The grids were placed in lysis buffer for 10 min at 37 °C immediately before the paraformaldehyde treatments. 2.5. Blot analyses Blot analyses were applied to oocysts treated and non-treated with quercetin. Extraction of proteins for HSP70 measurements was performed by sonication to disrupt the oocysts, followed by centrifugation at 100,000 ×g for 20 min. The supernatant was stored in liquid nitrogen until use. Equal amounts of protein samples from the oocysts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a discontinuous gel system based on a standard protocol [21]. An SDS-reducing sample buffer containing SDS and 2-mercaptoethanol was used for SDS-PAGE. Electrophoresis was performed at 40 mA. After electrophoresis, the proteins in the gel were electrophoretically transferred to nitrocellulose paper in a TransBlot cell (Bio Rad, Richmond, California). Electrophoresis was performed with a transfer buffer at room temperature for 18 h at 40 mV. After the transfer of the proteins, excess binding sites on the nitrocellulose paper were blocked by washing the paper in PBS containing 4% blocking reagent. The blots were then treated with the mouse anti-HSP70 monoclonal antibody for 1 h at room temperature. The blots were rinsed 3 times in PBS for 5 min, and then exposed to peroxidase-conjugated goat anti-mouse IgG. They were then rinsed in PBS and treated for peroxidase activity with diaminobenzidine (DAB) and hydrogen peroxide solution (20 mg DAB in 100 ml of 0.05 M Tris– HCl buffer, pH 7.6, containing 0.005% H2O2) for 5 min. After immunoblotting, the immunoreactivity of the samples was quantified using an image analyzer (Program MIP 4.5, Microm, Heidelberg, Germany). The images on the immunoblots were analyzed for both image area and mean optical density (OD). The integrated optical density (IOD) (image area x mean OD) was used for quantitation of immunoreactivity. Comparisons between blots were made possible by using an internal control included on every blot. The control lines used in the blots were loaded with 1μg of human HSP70. Prior to the current studies, experiments were carried out following the procedure described by Martinez et al. [22] to demonstrate the linearity of the immunoblot results under the conditions to be used in the present study. Concentrations of human HSP70 running from 0.1 μg to 3 μg were used. Evaluation of the test results by regression analysis
Fig. 1. Complete set of Eimeria tenella bivalents represented by 14 synaptonemal complexes from pachytene oocyst spread obtained 6 h after the start of sporulation. Kinetochores (arrows) are visible on each synaptonel complex. Telomeres (arrowhead). Bar = 1 μm.
456
E. del Cacho et al. / Parasitology International 57 (2008) 454–459
Fig. 2. Immunoelectron micrograph of tightly synapsed Eimeria tenella homologous chromosomes showing labelled lateral elements. Gold grains are distributed along the total length of the lateral elements and appear concentrated at the telomeric region (arrowsheads), whereas de centromeric region (arrows) has no gold grains. Note the decreasing number of grains as the oocysts are incubated with increasing concentrations of quercetin. A. Oocysts not treated with quercetin (control). B. Oocysts incubated with 25 μM of quercetin. C. Oocysts incubated with 50 μM of quercetin. Pachytene oocyst spread obtained 6 h after the start of sporulation. Bar = 0.2 μm.
revealed, in all cases, a very high correlation (r N 0.95) between the observed immunoreactivity and the amount of protein applied in the test. 2.6. Oocyst sporulation Quercetin treated and non-treated oocysts were sporulated following the standard procedures reported by Raether et al. [18]. Sporulation rates were determined by counting 400 oocysts under ×400 magnification (five replications) at 72 h of sporulation. 3. Results 3.1. Synaptonemal complexes study At 6 h after the start of the sporulation, complete sets of 14 pachytene SCs were observed by TEM in surface spreads of meiotic chromosomes from non-treated oocysts and oocysts treated with either 25 or 50 μM of quercetin (Fig. 1). Each of the 14 SCs was formed by the parallel association of the lateral elements (LEs), which were tightly synapsed (Fig. 1). In addition, a strongly stained centromere was observed in most of the 14 SCs (Fig. 1). However, SCs were not
seen in the surface spreads of meiotic chromosomes from oocysts treated with 100 μM of quercetin. SCs spreads were labelled to study the expression of HSP70. HSP70 was detected at the ultrastructural level with an anti-HSP70 monoclonal antibody, whose distribution was revealed using colloidal gold. Gold label was located over the LEs showing a homogeneous distribution along their entire length. Accumulation of particles was detected at the telomeres, where clusters of gold grains were observed (Fig. 2A). The central element and kinetochore in each SC were devoid of gold particles (Fig. 2A). SCs from oocysts treated with 25 or 50 μM of quercetin showed the same pattern of labelling as that found over the SCs from non-treated oocysts (Fig. 2B, C). Gold label was associated with LEs and concentrate at the telomere in the 14 SCs (Fig. 2B, C). However, comparison between distribution of immunogold label over the SCs from non-treated and treated oocysts revealed a decreasing number of gold particles as the concentration of quercetin increased (Fig. 2). The scant number of particles found in SCs from oocysts treated with 50 μM of quercetin (Fig. 2C) is worth noting. Meiotic chromosomes from oocysts treated with 100 μM of quercetin did not form SCs and the LEs were not observed. Consequently, no HSP70 expression was detected in surface spreads of those meiotic chromosomes.
Fig. 3. Electron micrograph showing desynapsis of paired homologous chromosomes seen at 8 h of the start of sporulation. A. Oocysts not treated with quercetin (control). B. Oocysts incubated with 50 μM of quercetin. Breakage of one of the homologous chromosomes forming the bivalent is observed (arrows). Note that the homologous remained associated at both ends of the bivalent. Oocyst spread obtained 8 h after the start of sporulation. Bar = 1 μm.
E. del Cacho et al. / Parasitology International 57 (2008) 454–459
457
Fig. 4. Immunoelectron micrograph showing separation of the two homologous chromosomes in bivalents from control oocysts (A) and oocysts incubated with 25 μM (B) or 50 μM of quercetin (C). Note both the decreasing number of gold grains as the oocysts are incubated with increasing concentrations of quercetin and the breakage of a homolog in most bivalents from the oocysts incubated with 50 μM of quercetin. Oocyst spreads obtained 8 h after the start of sporulation. Bar = 0.3 μm.
At 8 h from the start of sporulation, desynapsis of bivalents was observed showing identical features in non-treated oocysts and oocysts treated with 25 μM of quercetin (Fig. 3A). At that point of sporulation, homologous chromosomes in each bivalent pulled away from each other to some extend, marking the end of synapsis. The terminal arms of the homologous chromosomes were held together by persisting segments of SC (Fig. 3A). However, in the surface spread of chromosomes from 50 μM quercetin-treated oocysts, breakage of one of the homologs forming the bivalent was observed (Fig. 3B) as paired chromosomes opened out and separated. Breaks were found in most of the bivalents (Fig. 3B). The expression of HSP70 on meiotic chromosomes from both treated and non-treated oocysts showed the same distribution at 8 h from the start of sporulation as that found at 6 h (Fig. 4). Likewise, at that point of sporulation and during separation of paired chromosomes, decreasing numbers of gold particles were detected as the concentration of quercetin increased (Fig. 4). 3.2. Blot analysis and quantification of HSP70 expression Immunoblot analysis of the total proteins from the oocysts treated with 25 or 50 μM of quercetin and the non-treated oocysts showed a band of approximately 70 kDa (Fig. 5). Therefore, the anti-HSP70 antibody reacted with a protein with a similar molecular weight in both the E. tenella oocysts and the control (human HSP70). Significantly, the density of the bands diminished as the amount of quercetin increased (Fig. 5). Densitometric studies showed that the control exhibited slightly higher IOD values than those shown by the non-treated oocysts (Table 1). The statistical analysis demonstrated
Fig. 5. Expression of HSP70 in non-treated (lanes 3–4) and treated (lanes 5–10) E. tenella oocysts. Lanes 1–2: human HSP70 (control). Lanes 3–4: oocysts incubated without quercetin. Lanes 5–6: oocysts treated with 25 μM of quercetin. Lanes 7–8: oocysts treated with 50 μM of quercetin. Lanes 9–10: oocysts treated with 100 μM of quercetin. Molecular mass (kDa) is indicated.
that the variation of IOD between the control (human HSP70) and the non-treated oocysts was not significant, whereas IOD values were significantly higher in non-treated oocysts when compared to quercetin-treated oocysts. The greatest decrease in IOD was observed in oocysts treated with 50 μM of quercetin, whose IOD values fell dramatically to 2.5 (Table 1). However, the IOD values found in the oocysts treated with 25 μM of quercetin showed a decrease in IOD to 80.5 (Table 1). Therefore, treated oocysts showed a progressive decrease in the expression of HSP70 as the concentration of quercetin increased. Decreases of 52.2% and 98.6% in IOD were found in the oocysts treated with 25 and 50 μM of quercetin, respectively (Table 1). It should be emphasised that oocysts treated with 100 μM of quercetin lacked HSP70 expression. 3.3. Oocyst sporulation Sporulation rates of the quercetin-treated and non-treated oocysts are given in Fig. 6. The rates found in 25 μM quercetin-treated oocysts were slightly lower than those found in non-treated oocysts. However, the difference in the number of oocysts that sporulated was not statistically significant. In contrast, the oocysts treated with 50 and 100 μM quercetin failed to sporulate. In addition, our results demonstrate that in non-treated and 25 μM quercetin-treated oocysts, sporozoites were formed within each sporulated oocyst and the
Table 1 Densitometric analysis of HSP70 immunoreactivity Optical density Integrated optical % Integrated optical density (IOD) (mean ± SD) density (IOD) (mean ± SD) (mean ± SD)
Sample
Area
Human HSP70 No quercetin 25 μM quercetin 50 μM quercetin 100 μM quercetin
220.5 ± 13.2a 0.80 ± 0.04a
176.4 ± 7.7a
104.9%a
218.4 ± 10.6a 167.8 ± 10.9b
0.77 ± 0.03a 0.48 ± 0.04b
168.1 ± 6.8a 80.5 ± 6.9b
100%a 47.8%b
84.3 ± 12.5c
0.03 ± 0.007c
2.5 ± 0.6c
1.4%c
0
0
0
0
Integrated optical density: area x optical density. The % of integrated optical density has been calculated taking the IOD found in the oocysts incubated without quercetin as representing 100%. Data marked with different superscript sign differ significantly (P b 0.05).
458
E. del Cacho et al. / Parasitology International 57 (2008) 454–459
Fig. 6. Sporulation rates of the quercetin-treated and non-treated oocysts. Significant differences between untreated (solid area) and 25 μM quercetin-treated (dotted area) oocysts were not found. Note the oocysts treated with either 50 μM or 100 μM of quercetin did not sporulate. Each bar represents the mean ± SD.
appearance of the Stieda bodies was observed (Fig. 7A). However, in oocysts treated with 50 and 100 μM quercetin, neither cleavage of the sporoplasm nor sporocyst formation was seen (Fig. 7B). Furthermore, the oocysts showed a granular cytoplasm which was enclosed in a distinct cytoplasmic membrane instead of the standard sporocysts (Fig. 7B). 4. Discussion To increase our understanding of how the structural components of SCs accomplish their many functions in chromatin arrangement, chromosome pairing, recombination, and disjunction at meiosis it is necessary to know the components that combine to form the SCs [23]. Antibodies directed against SC components provide a first approach to the identification of SC substructures and their functions. In the current study, the expression and ultrastructural localization of HSP70 was analyzed by immunogold labelling of surface spreads of meiotic chromosomes from E. tenella oocysts. HSP70 was detected with a specific monoclonal antibody whose distribution was revealed using colloidal gold, and examined by TEM. Previous studies on HSPs during Eimeria sporogony have demonstrated that HSP70 is regulated developmentally and highly expressed during sporulation [10]. In the present study, HSP70 was found to be a component of the SC. From the beginning of pachytene until SCs disassembly in diplotene, the authors found a constant pattern of staining, which demonstrated a higher concentration of HSP70 on the telomeres. During SC formation, the telomere attaches to the inner nuclear envelope to form a structure named attachment plaque,
which functions as a kind of platform for SC movement [24]. Several reports have shown the ability of HSP70 to assembly and disassembly cytoskeletal proteins playing a role in cellular movements [25,26,27]. Therefore, the continued presence of HSP70 on the SCs suggests that the HSP70 has a continuous function in the chromosomal behaviour playing a role in the SCs movements that take place during chromosomal pairing, recombination, or disjunction. Quercetin is a specific inhibitor of HSP70 synthesis with a limited effect on the synthesis of other HSPs [28]. The mechanism of this inhibitory effect is not thoroughly understood. Hosokawa et al. [29] have found that quercetin inhibits HSP70 synthesis by changing the tertiary structure of heat shock factor which is required for the transcription of the HSP70 gene. The current results demonstrated three dose-dependent effects of the quercetin treatment: a reduction in the HSP70 synthesis; defects in SC formation or desynapsis; and inhibition of sporulation. Treatment with 25 μM quercetin inhibited HSP70 synthesis to 47.8%; nevertheless, it did not affect chromosome behaviour or the sporulation process. Treatment with 50 or 100 μM quercetin dramatically inhibited HSP70 synthesis which showed a decrease ranging from 98.6% to 100%. The consequences were failure to form SCs or to complete desynapsis and failure to develop haploid sporozoites. Although the significance of HSP70 in eimerian SCs is unknown, the present results suggest that HSP70 supports fundamental meiotic mechanisms. Furthermore, the role of HSP70 proteins as chaperones that assist the folding and the assembly and disassembly of other proteins is well known [25]. Accordingly, HSP70 in eimerian SCs may help to stabilize structures that are important in achieving chromosomal paring and segregation as has been proposed to occur in mouse SCs [15]. Recently, in mammals and in the protozoan parasite Leishmania, quercetin has been demonstrated to inhibit DNA topoisomerases I and II [30,31]. As a consequence, DNA religation is inhibited, chromosome instability occurs and chromosome breakage may result [32]. However, DNA topoisomerases have not been reported during sporulation of apicomplexan protozoa. In the light of our findings one may assume that the effect of HSP70 on SC formation and desynapsis is essential in order to complete the sporulation process. The current results provide an important basis for the development of new lines of research aimed at clarifying the mechanisms that play a role in the sporulation of E. tenella. Acknowledgements This work was supported by grant A46 from the Research Council of Aragón, Spain.
Fig. 7. Eimeria tenella oocysts after 72 h of sporulation. A) Sporulated oocysts showing the sporozoites and the Stieda body. B) Oocyst treated with 50 μM of quercetin. Note the condensation of the sporoplasm. The cytoplasm appeared granular and enclosed in a distinct cytoplasmic membrane.
E. del Cacho et al. / Parasitology International 57 (2008) 454–459
References [1] Maresca B, Carratú L. The biology of the heat shock response in parasites. Parasitol Today 1992;8:260–6. [2] Coulson RMR, Smith DF. Isolation of genes showing increased or unique expression in the infective promastigotes of Leishmania major. Mol Biochem Parasitol 1990;40: 63–76. [3] Hausler T, Clayton C. Post-transcriptional control of hsp70 mRNA in Trypanosoma brucei. Mol Biochem Parasitol 1996;76:57–71. [4] Sharma YD. Structure and possible function of heat-shock proteins of falciparum malaria (mini-review). Comp Biochem Physiol 1992;102B:437–44. [5] Lyons RE, Johnson AM. Heat shock proteins of Toxoplasma gondii. Parasite Immunol 1995;17:353–9. [6] Robertson NP, Reese RT, Speer CA. Heat shock-like polypeptides of the sporozoites and merozoites of Eimeria bovis. J Parasitol 1988;74:1004–8. [7] Dunn PPJ, Billington K, Bumstead JM, Tomley FM. Isolation and sequences of cDNA clones for cytosolic and organellar hsp70 species in Eimeria spp. Mol Biochem Parasitol 1995;70:211–5. [8] Lyons RE, Johnson AM. Gene sequence and transcription differences in 70 kDa heat shock protein correlate with murine virulence of Toxoplasma gondii. Int J Parasitol 1998;28:1041–51. [9] Del Cacho E, Gallego M, Lopez-Bernad F, Quilez J, Sanchez-Acedo C. Differences in HSP70 expression in the sporozoites of the original strain and precocious lines of Eimeria tenella. J Parasitol 2005;91:1127–31. [10] Del Cacho E, Gallego M, Pereboom D, Lopez-Bernad F, Quilez J, Sanchez-Acedo C. Eimeria tenella: HSP70 expression during spoogony. J Parasitol 2001;87:946–50. [11] Canning EU, Anwar M. Studies on meiotic division in coccidial and malarial parasites. J Protozool 1968;15:290–8. [12] Roeder GS. Chromosome synapsis and genetic recombination: their roles in meiotic chromosome segregation. Trends Genet 1990;6:385–9. [13] Westergaard M, Wettstein D. The synaptonemal complex. Ann Rev Genet 1972;6: 71–110. [14] Allen JW, Dix DJ, Collins BW, Merrick BA, He C, Selkrik JK, et al. HSP70-2 is part of the synaptonemal complex in mouse and hamster spermatocytes. Chromosoma 1996;104:414–21. [15] Dix DJ, Allen JW, Collins BW, Poorman-Allen P, Mori C, Blizard DR, et al. HSP70-2 is required for desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes. Development 1997;124:4595–603. [16] Del Cacho E, Gallego M, Monteagudo L, López-Bernad F, Quílez J, Sánchez-Acedo C. A method for the sequential study of eimerian chromosomes by light and electron microscopy. Vet Parasitol 2001;94:221–6. [17] Del Cacho E, Pages M, Gallego M, Monteagudo F, Quílez J, Sánchez-Acedo C. Synaptonemal complex karyotype of Eimeria tenella. Intl J Parasitol 2005;35: 1445–51.
459
[18] Raether W, Hofmann J, Uphoff M. Biotechnology. In: Eckert J, Brown R, Shirley MW, Coudert P, editors. Guidelines on Techniques in Coccidiosis Research. Brussels: European Commission; 1995. p. 79–84. [19] Wang CC, Stotish RL. Changes of nucleic acids and proteins in the oocysts of Eimeria tenella during sporulation. J Protozool 1975;22:438–43. [20] Switonski M, Gustavsson I, Höjer K, Plöen L. Synaptonemal complex analysis of the B-chromosomes in spermatocytes of the silver fox (Vulpes fulvus Desm.). Cytogenet Cell Genet 1987;45:84–92. [21] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [22] Martínez J, Pérez-Serrano J, Bernadina WE, Rodríguez-Caabeiro F. Detection of heat shock protein-70 from Trichinella spiralis larvae using a modification of the routine western blotting procedure. J Parasitol 2000;86:637–9. [23] Moens PB, Heyting C, Dietrich AJJ, van Raamsdonk W, Chen Q. Synaptonemal complex antigen location and conservation. J Cell Biol 1987;105:93–103. [24] Alsheimer M, von Glasenapp E, Hock R, Benavente R. Architecture of the nuclear periphery of rat pachytene spermatocytes: distribution of nuclear complex attachment sites. Mol Biol 1999;10:1235–45. [25] Kiang JG, Tsokos GC. Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol Ther 1998;80:183–201. [26] Agueli C, Geraci F, Guidice G, Chimenti L, Cascino D, Sconzo G. A constitutive 70 kDa heat-shock protein is localized on the fibres of spindles and asters at metaphase in an ATP-dependent manner: a new chaperone role is proposed. Biochem J 2001;360:413–9. [27] Botha M, Pesce ER, Blatch GL. The Hsp40 proteins of Plasmodium falciparum and other apicomplexa: Regulating chaperone power in the parasite and the host. Int J Biochem Cell Biol 2007;39:1781–803. [28] Elia G, Santoro G. Regulation of heat shock protein synthesis by quercetin in human erythroleukaemia cells. Biochem J 1994;300:201–9. [29] Hosokawa N, Hirayoshi K, Kudo H, Takechi H, Aoike A, Kawai K, et al. Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids. Mol Cell Biol 1992;12:3490–8. [30] Cantero G, Campanella C, Mateos S, Cortés F. Topoisomerase II inhibition and high yield of endoreduplication induced by the flavonoids luteolin and quercetin. Mutagenesis 2006;21:321–5. [31] Cortázar TM, Coombs GH, Walker J. Leishmania panamensis: comparative inhibition of nuclear DNA topoisomerase II enzymes from promastigotes and human macrophages reveals anti-parasite selectivity of fluoroquinolones, flavonoids and pentamidine. Exp Parasitol 2007;116:475–82. [32] Das BB, Sen N, Roy A, Dasgupta SB, Ganguly A, Mohanta BC, et al. Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I. Nucleic Acids Res 2006;34:1121–32.