The Use of Digital Portfolio in Higher Education before and during the COVID-19 Pandemic.

This study is focused on the advantages and disadvantages of using a digital portfolio to improve the learning and evaluation processes in the initial teacher training of 4th-year students in the University of Seville (Spain). One of the interests of this research was to compare the learning capacities perceived by the students to improve their learning process before and during the COVID-19 pandemic. A qualitative, descriptive methodology was applied, identifying the most relevant dimensions, categories and codes for the analysis, management and interpretation of the opinions of the students, with a research triangulation (Cohen's kappa coefficient) and a coding performed using the ATLAS.ti 8.4 software. The results show that the advantages with greater percentage correspond to the following categories: learning, usefulness of OneDrive, autonomy and evaluation. The greatest disadvantages detected were: time, uncertainty, usefulness of OneDrive and autonomy. There are differences in the perceptions of the students, between before and during the COVID-19 pandemic, about the learning capacities developed with the use of digital portfolio, since they consider that they have acquired more significant learning, greater self-regulation of their learning and greater reflection capacity.

Melatonin MT1 and MT2 Receptors in the Ram Reproductive Tract.

Some melatonin functions in mammals are exerted through MT1 and MT2 receptors. However, there are no reports of their presence in the reproductive tract of the ram, a seasonal species. Thus, we have investigated their existence in the ram testis, epididymis, accessory glands and ductus deferens. Real-time polymerase chain reaction (qPCR) revealed higher levels of m-RNA for both receptors in the testis, ampulla, seminal vesicles, and vas deferens, than in the other organs of the reproductive tract (p < 0.05). Western blot analyses showed protein bands compatible with the MT1 in the testis and cauda epididymis, and for the MT2 in the cauda epididymis and deferent duct. Immunohistochemistry analyses revealed the presence of MT1 receptors in spermatogonias, spermatocytes, and spermatids, and MT2 receptors in the newly-formed spermatozoa in the testis, whereas both receptors were located in the epithelial cells of the ampulla, seminal vesicles, and ductus deferens. Indirect immunofluorescence showed significant differences in the immunolocation of both receptors in spermatozoa during their transit in the epididymis. In conclusion, it was demonstrated that melatonin receptors are present in the ram reproductive tract. These results open the way for new studies on the molecular mechanism of melatonin and the biological significance of its receptors.

Induction of protective immunity against experimental Eimeria tenella infection using serum exosomes.

Avian coccidiosis is caused by Eimeria, a unicellular, apicomplexan protist which primarily infects intestinal epithelia resulting in nutrient malabsorption and reduced growth of commercial poultry. Vaccination of chickens with exosomes isolated from antigen presenting cells containing parasite antigens (Ags) represents a promising alternative strategy to control avian coccidiosis, but is restricted in its commercial application due to limitations on production scale-up for mass immunization programs. Here, we report the biochemical and physiologic characteristics of exosomes derived from serum of Eimeria tenella-infected chickens and their feasibility for inducing protective immunity to experimental coccidiosis. Exosomes isolated from the serum of E. tenella-infected chickens contained a subset of protein Ags found in the intact parasite. Serum-derived exosomes containing these E. tenella Ags localized to the intestine and spleen following intramuscular injection into naïve chickens. In vitro ELISPOT assays revealed increased numbers of IL-2-, IL-4-, IL-6-, and IFN-γ-secreting cells in the intestine and spleen of exosome-administered chickens, compared with vehicle controls. Pre-immunization of chickens with serum exosomes from E. tenella-infected chickens increased both body weight gain and feed conversion efficiency, and reduced both fecal parasite shedding and gut lesion scores following parasite infection, compared with vehicle controls. Finally, immunization with CD80(+) serum exosomes stimulated greater numbers of cytokine-producing cells, and higher levels of protective immunity to E. tenella infection, compared with CD80(-) exosomes. These results suggest the possibility of producing an effective, parasite-free vaccine against avian coccidiosis under field conditions using serum-derived CD80(+) exosomes containing parasite Ags.

IL-17A regulates Eimeria tenella schizont maturation and migration in avian coccidiosis.

Although IL17A is associated with the immunological control of various infectious diseases, its role in host response to Eimeria infections is not well understood. In an effort to better dissect the role of IL17A in host-pathogen interactions in avian coccidiosis, a neutralizing antibody (Ab) to chicken IL17A was used to counteract IL17A bioactivity in vivo. Chickens infected with Eimeria tenella and treated intravenously with IL17A Ab, exhibited reduced intracellular schizont and merozoite development, diminished lesion score, compared with untreated controls. Immunohistological evaluation of cecal lesions in the parasitized tissues indicated reduced migration and maturation of second-generation schizonts and reduced lesions in lamina propria and submucosa. In contrast, untreated and infected chickens had epithelial cells harboring second-generation schizonts, which extend into the submucosa through muscularis mucosa disruptions, maturing into second generation merozoites. Furthermore, IL17A Ab treatment was associated with increased parameters of Th1 immunity (IL2- and IFNγ- producing cells), reduced levels of reactive oxygen species (ROS), and diminished levels of serum matrix metalloproteinase-9 (MMP-9). Finally, schizonts from untreated and infected chickens expressed S100, Wiskott-Aldrich syndrome protein family member 3 (WASF3), and heat shock protein-70 (HSP70) proteins as merozoites matured, whereas the expression of these proteins was absent in IL17A Ab-treated chickens. These results provide the first evidence that the administration of an IL17A neutralizing Ab to E. tenella-infected chickens inhibits the migration of parasitized epithelial cells, markedly reduces the production of ROS and MMP-9, and decreases cecal lesions, suggesting that IL17A might be a potential therapeutic target for coccidiosis control.

Tetraspanin-3 regulates protective immunity against Eimeria tenella infection following immunization with dendritic cell-derived exosomes.

The effects of immunization with dendritic cell (DC) exosomes, which had been incubated with a tetraspanin-3 (Tspan-3) blocking antibody (Ab) or with an isotype-matched non-immune IgG, were studied using an experimental model of Eimeria tenella avian coccidiosis. Purified exosomes from cecal tonsil and splenic DCs expressed Tspan-3 protein. Chickens injected with exosomes incubated with the control IgG and derived from cecal tonsil DCs preloaded in vitro with E. tenella Ag had Ag-immunostaining cells in the ceca, but not the spleen. Conversely, Ag-containing cells were found only in the spleen, but not the ceca, of chickens given IgG treated splenic DC exosomes. Interestingly, chickens that received exosomes incubated with Tspan-3 Ab had Ag-containing cells observed in both lymphoid organs following administration of exosomes from either DC population. After injection of exosomes non-incubated with Tspan-3 Ab, greater numbers of cells secreting interleukin-2 (IL-2), IL-16, interferon-γ, and E. tenella-reactive Abs were observed in the cecal tonsils of chickens immunized with cecal DC exosomes compared with the spleen. By contrast, more cytokine-and Ab-producing cells were present in the spleen of chickens given splenic DC exosomes compared with the ceca. Incubation with Tspan-3 Ab gave similar numbers of cytokine- and Ab-producing cells in the cecal tonsils and spleen regardless of the source of exosomes. Immunization with E. tenella Ag-loaded cecal tonsil DC exosomes increased in vivo resistance against subsequent E. tenella infection. Increased protection against infection following cecal DC exosome immunization was partially blocked by incubation of exosomes with Tspan-3 Ab. These results suggest that Tspan-3 is involved in the tissue distribution, as well as cytokine and Ab production, following DC exosome administration, and that Tspan-3 contributes to in vivo protection against experimental E. tenella challenge infection following exosomal immunization.

Identification and immunolocalisation of melatonin MT(1) and MT(2) receptors in Rasa Aragonesa ram spermatozoa.

The reproductive seasonality of sheep suggests that melatonin receptors may be present in ram spermatozoa. The present study confirms the presence of melatonin MT(1) and MT(2) receptors. The MT(1) receptor was detected using immunocytochemistry, with four sperm subpopulations identified based on the following labelling patterns: (1) one small subpopulation with labelling over the entire head and tail; (2) one of two main subpopulations that exhibited reactivity at the equatorial, post-acrosomal, neck and tail regions; (3) another main subpopulation with equatorial and tail labelling only; and (4) a subpopulation in which staining was detected only in the tail. Immunocytochemistry revealed the presence of the melatonin MT(2) receptor, with intense staining on the acrosome, post-acrosomal region and neck and tail regions of all cells, but not in the equatorial region. Western blot identification of ram protein extracts revealed a 39-kDa band compatible with both MT(1) and MT(2) receptors, a 75-kDa band compatible with MT(1)/MT(2) heterodimerisation, a 32-kDa band compatible with MT(1) receptor activation and a double band of 45-55 kDa that is compatible with MT(2) receptor homodimerisation or heterodimerisation with other G-proteins. In conclusion, we provide evidence of the presence of MT(1) and MT(2) receptors in ram spermatozoa, although the biochemical pathway triggered by these receptors and their function in terms of fertility remains to be elucidated.

Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using dendritic cell-derived exosomes.

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting T(h)1 cytokines, T(h)2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the T(h)1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the T(h)2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible.

Induction of protective immunity against Eimeria tenella infection using antigen-loaded dendritic cells (DC) and DC-derived exosomes.

Current methods for sustainable control of avian coccidiosis, whether by prophylactic medication or parasite vaccination, are suboptimal. In this study, we describe an alternative immunization strategy against Eimeria tenella infection using parasite antigen (Ag)-loaded dendritic cells (DCs), or their derived exosomes, in the absence of free Ag. CD45(+) intestinal DCs were isolated from E. tenella-infected chickens and loaded ex vivo with an extract of sporozoites as parasite Ag. Extracellular vesicles purified from the Ag-pulsed DCs expressed surface proteins associated with DC-derived exosomes, including major histocompatibility complex proteins (MHC I and MHC II), CD80, flotillin, and heat shock protein (HSP70). Following intramuscular immunization of chickens with Ag-pulsed DCs or Ag-pulsed DC-derived exosomes, Ag-containing cells were observed diffusely localized in the lymphoid tissue and concentrated in germinal centers of caecal tonsils, and restricted to germinal centers (GC) in the spleen. Chickens immunized with pulsed DCs or exosomes exhibited (a) higher numbers of caecal tonsil and spleen cells expressing IgG and/or IgA antibodies that were reactive with E. tenella Ag, (b) greater numbers of IL-2-, IL-16-, and IFN-γ-producing cells, and (c) higher E. tenella Ag-driven cell proliferation, compared with chickens immunized with Ag in the absence of DCs or exosomes. Chickens immunized with Ag-pulsed DCs or Ag-pulsed DC-derived exosomes and subsequently given a live E. tenella challenge infection at 10d post-immunization displayed (a) increased body weight gains, (b) decreased feed conversion ratios, (c) reduced fecal oocyst shedding, (d) diminished intestinal lesions, and (e) lower mortality, compared with animals given Ag alone. This is the first demonstration of Ag-specific protective immunity against avian coccidiosis using parasite Ag-loaded DCs or DC-derived exosomes.

Location of RAD51-like protein during meiotic prophase in Eimeria tenella.

This study focuses on reporting events in Eimeria tenella oocysts from early to late prophase I in terms of RAD51 protein in association with the synaptonemal complex formed between homologous chromosomes. The aim of the study was the sequential localization of RAD51 protein, which is involved in the repair of double-strand breaks (DSBs) on the eimerian chromosomes as they synapse and desynapse. Structural Maintenance of Chromosome protein SMC3, which plays a role in synaptonemal complex formation, was labeled to identify initiation and progress of chromosome synapsis and desynapsis in parallel with the appearance and disappearance of RAD51 foci. Antibodies directed against RAD51 and cohesin subunit SMC3 proteins were labeled with either fluorescence or colloidal gold to visualize RAD51 protein foci and synaptonemal complexes. RAD51 protein localization during prophase I was studied on meiotic chromosomes spreads obtained from oocysts at different points in time after the start of sporulation. The present findings showed that foci detected with the antibody directed against RAD51 protein first appeared at the pre-leptotene stage before homologous chromosomes began pairing. Subsequently, the foci were detected in association with the lateral elements at the precise sites where synapsis were in progress. These findings lead us to suggest that in E. tenella, homologous chromosome pairing was a DSB-dependent mechanism and reinforced the participation of RAD51 protein in meiotic homology search, alignment and pairing of chromosomes.

Effect of artemisinin on oocyst wall formation and sporulation during Eimeria tenella infection.

The anticoccidial effect of a product extracted from the natural herb Artemisia annua, artemisinin, which has a potential use as a dietary supplement, has been studied. Commercial artemisinin was administered at 10 and 17 ppm in food and tested against infection with Eimeria tenella. A battery trial to quantify the effect of artemisinin on the reproductive and infective capabilities of E. tenella was carried out. For that purpose flow cytometry was combined with electron microscopy and immunofluorescence techniques in order to study the effect of artemisinin on E. tenella gametogenesis. Significantly reduced oocyst output and lesion scores were found in chickens treated with artemisinin. In addition, evidence to support a lower oocyst sporulation rate was obtained. Though the ultrastructural studies showed normal development of gametogenesis in artemisinin-treated chickens, the oocyst wall formation was significantly altered. This resulted in both death of developing oocysts and reduced sporulation rate. Immunofluorescent studies provided evidence that treatment with artemisinin inhibited sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) expression in macrogametes. According to these findings, artemisinin has a deleterious effect on fertilized macrogametes (early zygotes) by inhibiting SERCA. The altered secretion of the wall-forming bodies may be the result of Ca(2+)-dependent ATPase impaired activity which, in turn, is the result of SERCA inhibition.

Meiotic chromosome pairing and bouquet formation during Eimeria tenella sporulation.

In Eimeria tenella, meiotic division occurs exclusively in oocysts within the first 8h of sporulation. Difficulties with the wall-oocyst breakage in gaining access to chromosomes during meiosis have resulted in a scarcity of morphological data on Eimeria chromosomes. This study tracks the general behaviour of telomeres, attachment plaques and synaptonemal complexes in the nucleus of the meiotic oocyst of E. tenella. Fluorescence microscopy methods, in combination with immunoelectron microscopy techniques, were applied to obtain a series of time-lapse images during oocyst sporulation. Antibodies to Structural Maintenance of Chromosome proteins SMC1 and SMC3, and lamin were labelled with either fluorescence or colloidal gold to visualise the telomeres, central elements of the synaptonemal complex (SC) and nuclear periphery, respectively, at both the structural and ultrastructural levels. Using oocyst spreads and ultrathin sections of fixed oocysts it was possible to study telomere dynamics at stages during meiosis. The stages of the meiotic prophase I are delineated on the basis of the telomere position and the SC synapsis and desynapsis. During the leptotene stage, at 4h following the start of sporulation, meiotic chromosomes attached to the nuclear envelope. At that stage, chromosome synapsis was initiated in the telomeric regions but no interstitial synapsis pairing was observed. In the zygotene stage, telomere signals were clustered in a limited area of the nuclear envelope. Bouquet formation occurred at 5h after the start of sporulation, whereas chromosomes did not appear completely synapsed until the pachytene stage at 6h of sporulation. Desynapsis was observed at 8h of sporulation during the diplotene stage. This study provides the first morphological description of both the behaviour of the chromosomes and the timing of the prophase I stages in the meiotic nucleus of E. tenella.

Changes in content and localization of proteins phosphorylated at tyrosine, serine and threonine residues during ram sperm capacitation and acrosome reaction.

Previously, we reported the involvement of tyrosine phosphorylation in events that lead to ram sperm capacitation. In this study, we carried out a comparative analysis of the localization of tyrosine, serine and threonine phosphoproteins in different functional stages of ram spermatozoa (after the swim-up procedure, in vitro capacitation, and ionophore-induced acrosome reaction) by immunofluorescence, immunocytochemistry and confocal microscopy. Capacitation increased protein tyrosine, serine and threonine phosphorylation whereas the induction of the acrosome reaction resulted in significantly decreased phosphorylation, mainly in those proteins that increased following capacitation. Control samples showed tyrosine-phosphorylated proteins restricted to the head, mainly distributed at the equatorial region with some cells also displaying an acrosomal and/or post-acrosomal localization. In vitro capacitation promoted both tail and acrosome phosphorylation, and the acrosome reaction induced the loss of labeling on the acrosome and the subsequent increase in the post-acrosomal region and flagellum. The preferential localization of serine- and threonine-phosphorylated proteins in the equatorial and acrosomal regions found in control samples changed during capacitation, which induced tail phosphorylation in a sequential manner. After the acrosome reaction, the labeling of both phosphoamino acids decreased in the acrosome and increased in the post-acrosome. The obtained results were proved by two immunodetection techniques and strengthened by confocal microscopy, and indicate that changes in phosphorylated proteins during capacitation and acrosome reaction of ram spermatozoa may have physiological significance in consolidating certain phosphorylated proteins to specific sperm regions involved in acrosomal exocytosis and zona pellucida recognition, binding and penetration.

Avian follicular and interdigitating dendritic cells: isolation and morphologic, phenotypic, and functional analyses.

An antiserum against Eimeria tenella sporozoites was used to localize and isolate Ag-binding cells in intestinal cecal tonsils of parasite-infected chickens. Based on their tissue localization, ultrastructural features, and expression of surface markers, two subpopulations of cells were isolated, CD45(+) interdigitating dendritic cells (IDCs) and CD45(-) follicular dendritic cells (FDCs). IDCs expressed MHC class I, MHC class II, and selectin, induced the proliferation of allogeneic naïve CD4(+) T cells, and increased the secretion of IFN-gamma by autologous T cells. FDCs expressed surface IgG, IgM, ICAM-1, and VCAM-1, stimulated the proliferation of LPS-treated allogeneic B cells, and augmented the secretion of IgG by LPS-treated autologous B cells. Final cell yields were 6 x 10(5) to 8 x 10(5) cells per chicken with >95% purity. In summary, this combination of methods using Abs against E. tenella and CD45 made it possible for the first time to obtain a highly enriched IDCs and FDCs which are functionally active in chickens. This novel method will enable the detailed biochemical and immunological characterizations of avian dendritic cells and facilitate the investigation of their role in initiating immune response in normal and disease states.

Malformations of the epididymis, incomplete regression of the mesonephric tubules and hyperplasia of Leydig cells in canine persistence of Müllerian duct syndrome.

Persistence of the Müllerian duct syndrome (PMDS) is a rare form of pseudohermaphroditism characterized by the presence of uterus and oviducts in otherwise normally differentiated SRY-positive 78 XY canine males. Undescended testicles are also common. We report a case of a male PMDS dog with a uterus and bilateral cryptorchidism. The dog had an incomplete regression of the mesonephric tubules. As a consequence of this an abnormally enlarged head of the epididymis was observed. In addition, an extreme reduction in size of both the body and the tail was found. Microscopic examination of both testicles revealed bilateral hyperplasia of Leydig cells. The progesterone blood level was measured by ELISA and was found to be abnormally high (3.18 ng/ml) compared to that of normal male dogs (lower than 1 ng/ml). Three months after surgical removal of the internal genitalia, the serum progesterone, testosterone and oestradiol levels were normal for a castrated male dog.

HSP70 is part of the synaptonemal complex in Eimeria tenella.

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 non-treated 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.

Isolation of chicken follicular dendritic cells.

The aim of the present study was to isolate chicken follicular dendritic cells (FDC). A combination of methods involving panning, iodixanol density gradient centrifugation, and magnetic cell separation technology made it possible to obtain functional FDC from the cecal tonsils from chickens, which had been infected with Eimeria tenella. CD45- dendritic cells were selected using the specific monoclonal antibody against chicken CD45, which is a marker for chicken leukocytes, but is not expressed on chicken FDC. Isolated FDC were characterized morphologically, phenotypically and functionally. The phenotype of the selected cells was consistent with FDC in that they expressed IgG, IgM, complement factors C3 and B, ICAM-1, and VCAM-1, but lacked cell surface markers characteristic of macrophages, T-, and B cells. Transmission electron microscopy confirmed their characteristic dendritic morphology. In addition, the identity of the FDC was further confirmed by their ability to trap chicken immune complexes (ICs) on their surface, whereas they did not trap naive antigen (ovalbumin) or ICs generated with mammalian immunoglobulins. Co-culturing allogeneic or autologous isolated FDC with B cells resulted in enhanced B cell proliferation and immunoglobulin production. The lack of MHC restriction, a functional characteristic feature of FDC, further reinforces the identity of the isolated cells as chicken FDC.

Expression of flotillin-1 on Eimeria tenella sporozoites and its role in host cell invasion.

Lipid rafts are detergent-resistant, liquid-ordered microdomains in plasma membranes that are enriched in cholesterol and sphingolipids and involved in intracellular signal transduction, membrane trafficking, and molecular sorting. In this study, we investigated the possibility that lipid rafts on Eimeria tenella sporozoites may act as platforms for host cell invasion. Flotillin-1, a resident protein of lipid rafts, was identified on E. tenella sporozoites and was prominently expressed at the apex of the cells, a region mediating host cell invasion. Pretreatment of sporozoites with antibody against flotillin-1 blocked parasite invasion. Furthermore, the anticoccidial drug, monensin, disrupted the localization of flotillin-1 within raft structures resulting in loss of invasion. We conclude that Eimeria sporozoites utilize lipid rafts containing flotillin-1 for internalization into host cells.

Immunohistochemical localization of sperm-preserving proteins in the ram reproductive tract.

Previously, we reported that the addition of seminal plasma proteins before cold-shock treatment prevents sperm membrane injury, and that 2 proteins of approximately 14 (P14) and 20 (P20) kDa, the main components of fraction 6 isolated by exclusion chromatography, are responsible for this protective effect. The objective of the present study was to localize P14 and P20 in tissues of the ram reproductive tract to determine their origin. Antiserum generated against purified P14 and P20 reacted with proteins in seminal vesicles and vas deferens by Western blot analyses of protein tissue extracts. However, these antisera failed to detect P14 and P20 in testis, prostate, efferent ductules, bulbourethral glands, and epididymis (caput, corpus, and cauda). Immunohistochemical analyses by both indirect immunofluorescence and the avidin-biotin complex technique confirmed that only seminal vesicles showed reactivity, restricted to the secretory cells, with both antibodies. Obtained results indicate that P14 and P20 are secreted specifically in the seminal vesicles. To further confirm that P14 and P20 are specifically expressed in seminal vesicles, we used Northern blot analyses to investigate the expression of both proteins in seminal vesicles and vas deferens. These assays corroborated again that P14 and P20 were specifically expressed in seminal vesicles. Consequently, we suggest referring to these 2 proteins as RSVP14 and RSVP20, respectively, according to their origin and molecular weight.

Synaptonemal complex karyotype of Eimeria tenella.

In most organisms, biological variability rests on the behaviour of the chromosomes in the meiotic context. Despite the importance of meiosis, very little is known about the meiotic behaviour of the Eimeria chromosomes. The aim of the present study is to describe the standard synaptonemal complex karyotype from Eimeria tenella oocyst spreads by electron microscopy. For that purpose, complete sets of pachytene synaptonemal complexes were obtained and the morphological pachytene karyotype was determined. The authors used a previously reported method that overcomes the difficulty of the extreme resistance of protozoan oocysts to disruption and permits the release of intact meiotic chromosomes. The chromosomes were selected under a light microscope and those selected were stained with phosphotungtic acid and studied by transmission electron microscopy. The authors confirmed 14 chromosomes, which were observed as synaptonemal complexes, and the karyotype was constructed by arranging synaptonemal complexes according to their relative lengths and kinetochore position. Components of the synaptonemal complex, lateral elements, central element, recombination nodules and kinetochore were observed. Measures of the kynetochore, width of the synaptonemal complex, diameter of the recombination nodule and length of the telomeres are given. Minimal and no significant differences were found between measures of chromosomes isolated from different Eimeria tenella strains. To the best of our knowledge, the present investigation for the first time identifies and describes the morphological characteristics of the synaptonemal complex of Eimeria tenella during the meiosis that occurs within the oocysts. In addition, the authors provide evidence of the presence of recombination nodules, suggesting that the recombination process may play an important role in the molecular evolution of this parasite.