Animal models of Soft Tissue Sarcoma for alternative anticancer therapy studies: characterization of the A-72 Canine Cell Line.

Canine Soft Tissue Sarcoma (STS) cell line A-72 has been largely employed for antiviral and antiproliferative studies. However, there are few information on their characteristics. Our aim was to evaluate A-72 expression level of genes and proteins involved in the innate immune response and cell cycle, their ability to respond to infective stressors and their possible use as a cellular model for anti-cancer studies in human and animal medicine. For this purpose, we evaluated the basal expression of immune-related, cell cycle and DNA repair genes on this cell line and tumoral tissues. A-72 ability to respond to a wild-type strain of Salmonella typhimurium was assessed. S. typhimurium showed ability to penetrate A-72 causing pro-inflammatory response accompanied by a decrease of cell viability. IL10 and IL18 genes were not expressed in A-72 while CXCL8, NOS2, CXCR4 and PTEN were highly expressed in all samples and TP53 was slightly expressed, as shown in human STS. Our results outline the ability of A-72 to respond to a bacterial agent by modifying the expression of important genes involved in innate immune response and provide a useful model for in vitro evaluation of new therapeutic approaches that could be translated into the human oncology.

Investigation and Follow-Up of a Staphylococcal Food Poisoning Outbreak Linked to the Consumption of Traditional Hand-Crafted Alm Cheese.

Staphylococcal food poisoning (SFP) is one of the most important foodborne diseases. This work describes a SFP event linked to the consumption of alm cheese and involved three people belonging to the same family. Leftovers of the consumed cheese, samples from the grocery store and the producing alm were collected and tested for Coagulase positive staphylococci (CPS) enumeration and for the presence of staphylococcal enterotoxins (SEs). Isolates were typed with MLST, spa typing, and tested for SEs and methicillin resistance genes. An in vitro test evaluated SEs production in relation to bacterial growth. The presence of CPS and SEs was detected in all cheese samples and all isolates belonged to the same methicillin sensitive ST8/t13296 strain harbouring sed, ser and sej genes. The in vitro test showed the production of enterotoxins started from 105 CFU/mL. The farmer was prescribed with corrective actions that led to eradication of the contaminating strain.

In vitro and in vivo characterization of stem-like cells from canine osteosarcoma and assessment of drug sensitivity.

Cancer stem cell (CSC) self-renewing and drug resistance cause treatment failure and tumor recurrence. Osteosarcoma is an aggressive bone tumor characterized by biological and molecular heterogeneity, possibly dependent on CSCs. CSC identification in osteosarcoma and their efficient targeting are still open questions. Spontaneous canine osteosarcoma shares clinical and biological features with the human tumors, representing a model for translational studies. We characterized three CSC-enriched canine osteosarcoma cultures. In serum-free conditions, these CSC cultures grow as anchorage-independent spheroids, show mesenchymal-like properties and in vivo tumorigenicity, recapitulating the heterogeneity of the original osteosarcoma. Osteosarcoma CSCs express stem-related factors (Sox2, Oct4, CD133) and chemokine receptors and ligands (CXCR4, CXCL12) involved in tumor proliferation and self-renewal. Standard drugs for osteosarcoma treatment (doxorubicin and cisplatin) affected CSC-enriched and parental primary cultures, showing different efficacy within tumors. Moreover, metformin, a type-2 diabetes drug, significantly inhibits osteosarcoma CSC viability, migration and self-renewal and, in co-treatment with doxorubicin and cisplatin, enhances drug cytotoxicity. Collectively, we demonstrate that canine osteosarcoma primary cultures contain CSCs exhibiting distinctive sensitivity to anticancer agents, as a reliable experimental model to assay drug efficacy. We also provide proof-of-principle of metformin efficacy, alone or in combination, as pharmacological strategy to target osteosarcoma CSCs.

Canine osteosarcoma cell lines contain stem-like cancer cells: biological and pharmacological characterization.

Cancer stem cells (CSCs) represent a small subpopulation of cells responsible for tumor formation and progression, drug resistance, tumor recurrence and metastasization. CSCs have been identified in many human tumors including osteosarcoma (OSA). CSC distinctive properties are the expression of stem cell markers, sustained growth, self-renewal and tumorigenicity. Here we report the isolation of stem-like cells from two canine OSA cultures, characterized by self-renewal, evaluated by sphere formation ability, differential marker expression, and in vitro proliferation when cultured in a medium containing EGF and bFGF. Current therapies for OSA increased survival time, but prognosis remains poor, due to the development of drug resistance and metastases. Chemotherapy shrinks the tumor mass but CSCs remain unaffected, leading to tumor recurrence. Metformin, a drug for type 2 diabetes, has been shown to possess antitumor properties affecting CSC survival in different human and animal cancers. Here we show that metformin has a significant antiproliferative effect on canine OSA stem-like cells, validating this in vitro model for further pre-clinical drug evaluations. In conclusion, our results demonstrate the feasibility of obtaining CSC-enriched cultures from primary canine OSA cells as a promising model for biological and pharmacological studies of canine and human OSAs.

In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors.

BACKGROUND: Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. METHODS: Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. RESULTS: We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. CONCLUSIONS: Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals.

Cytoskeletal proteins associate with components of the ribosomal maturation and translation apparatus in Xenopus stage I oocytes.

Actin-based cytoskeleton (CSK) and microtubules may bind to RNAs and related molecules implicated in translation. However, many questions remain to be answered regarding the role of cytoskeletal components in supporting the proteins involved in steps in the maturation and translation processes. Here, we performed co-immunoprecipitation and immunofluorescence to examine the association between spectrins, keratins and tubulin and proteins involved in 60S ribosomal maturation and translation in Xenopus stage I oocytes, including ribosomal rpl10, eukaryotic initiation factor 6 (Eif6), thesaurins A/B, homologs of the eEF1α elongation factor, and P0, the ribosomal stalk protein. We found that rpl10 and eif6 cross-reacted with the actin-based CSK and with tubulin. rpl10 co-localizes with spectrin, particularly in the perinuclear region. eif6 is similarly localized. Given that upon ribosomal maturation, the insertion of rpl10 into the 60S subunit occurs simultaneously with the release of eif6, one can hypothesise that actin-based CSK and microtubules provide the necessary scaffold for the insertion/release of these two molecules and, subsequently, for eif6 transport and binding to the mature 60S subunit. P0 and thesaurins cross-reacted with only spectrin and cytokeratins. Thesaurins aggregated at the oocyte periphery, rendering this a territory favourable site for protein synthesis; the CSK may support the interaction between thesaurins and sites of the translating ribosome. Moreover, given that the assembly of the ribosome stalk, where P0 is located, to the 60S subunit is essential for the release of eif6, it can be hypothesised that the CSK can facilitate the binding of the stalk to the 60S.

Spatial and temporal expressions of prune reveal a role in Müller gliogenesis during Xenopus retinal development.

The development of stratified retinal cell architecture is highly conserved in all vertebrates, implying that a common fundamental molecular mechanism is involved in the generation of the organized retina. However, the detailed molecular mechanisms of retinal development are not fully understood. Here we have identified the Xenopus ortholog of prune and show that it is expressed in both differentiating and differentiated retinal domains during development. Interestingly, these spatial and temporal expression patterns coincide with the expression of prune binding partners, the NM23 family members. Overexpression of prune in retinal precursor cells significantly increases the ratio of Müller glial cells as observed by modulation of NM23 activity (Mochizuki et al., 2009). However, a mutated form of prune that has replacement of four aspartate (D) residues (D'Angelo et al., 2004), essential for phosphodiesterase activity, does not exhibit gliogenic activity. Our observations suggest that Xenopus prune may regulate Müller gliogenesis through phosphodiesterase-mediated regulation of NM23 family members.

Sperm motility initiation by egg jelly of the anuran, Discoglossus pictus may be mediated by sperm motility-initiating substance of the internally-fertilizing newt, Cynops pyrrhogaster.

The egg jelly of Discoglossus pictus contains sperm motility-activating activity, the molecular basis of which has not been studied. Discoglossus pictus sperm initiated motility immediately after immersion in egg-jelly extract, as well as after immersion in hyposmotic solution, which initiates sperm motility in the external fertilization of anuran amphibians. Sequential treatment of the D. pictus sperm with these two solutions revealed the predominant effect of hyposmolality in initiation of motility. The motility initiation induced by jelly extract was suppressed by a monoclonal antibody (mAb) that is specific for the 34 kDa sperm motility-initiating substance (SMIS) in the egg jelly of the newt, Cynops pyrrhogaster. Immunoblotting using the anti-SMIS mAb revealed several antigenic proteins that included major ones with sizes of 18- and 34-kDa in D. pictus jelly extract. Scanning electron microscopic observation revealed that granules of jelly matrix, in which SMIS localizes and which have a critical role in the internal fertilization of C. pyrrhogaster, were not observed near the surface of the D. pictus egg jelly. These results suggest that sperm motility-activating activity in egg jelly of D. pictus may be mediated by SMIS homologous proteins that act through a mechanism that is partially different from that of C. pyrrhogaster.

Involvement of the eukaryotic initiation factor 6 and kermit2/gipc2 in Xenopus laevis pronephros formation.

The translation initiation factor Eif6 has been implicated as a regulator of ribosome assembly, selective mRNA translation and apoptosis. Many of these activities depend upon the phosphorylation of eif6 Serine 235 by protein kinase C (PKC). Eif6-60S is probably part of the RNA-induced silencing complex (RISC). eif6 over-expression in Xenopus embryos causes aberrant eye development. kermit2/gipc2 morphants have an eye phenotype similar to that of the eif6 overexpressors. Eye formation is regulated by insulin growth factor (IGF) signalling. eif6 interacts with the IGF receptor (IGFR) and kermit2/gipc2, which also binds to igfr. eif6 over-expression in Xenopus causes also the formation of antero-ventral oedema, suggesting a malfunction of the excretory system. Here we evaluated the pronephros phenotype. The oedema grows into the nephrocoel, expanding its boundary and is accompanied by a strong reduction of the pronephros. The three main components of the pronephros are severely impaired in eif6 over-expressors, while are not affected in eif6 morphants. Conversely, gipc2 depletion induces the oedema phenotype and reduction of the pronephros, while gipc2 overexpression does not. p110*, a constitutively active p110 subunit of the PI3 kinase partially recovers the oedema phenotype. We also determined that PKC-dependent phosphorylation of Ser235 in eif6 is not required to produce defective pronephroi. These results indicate that the levels of eif6 are highly regulated during development and instrumental for proper morphogenesis of the pronephros. Moreover, it appears that for proper pronephros development the gipc2 level should be kept within or over the physiological range and that the oedema phenotype is partly due to the inhibition of IGF signalling.

CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis.

BACKGROUND: Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation. RESULTS: A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours. CONCLUSIONS: These results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis.

Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential.

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs.

Evaluation of internal reference genes for quantitative expression analysis by real-time PCR in ovine whole blood.

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells.

Detection of a morphogenetically novel Sarcocystis hominis-like in the context of a prevalence study in semi-intensively bred cattle in Italy.

The aim of this study was to determine the prevalence of sarcosporidiosis in semi-intensively bred cattle in northwestern Italy. A diagnostic protocol was setup in which infected animals were identified by rapid histological examination of the esophagus, diaphragm, and heart and the detected Sarcocystis spp. were subsequently typed using conventional electron microscopy in combination with molecular techniques. Sarcosporidia cysts were detected in 78.1% of the animals and were seen most often in the esophagus. The cattle is intermediate host for Sarcocystis hominis (final host, humans and some primates), Sarcocystis cruzi (final host, domestic and wild canids), and Sarcocystis hirsuta (final host, wild and domestic cats).All these three species of Sarcocystis were identified, variously associated, with the following prevalence: S. cruzi (74.2%), S. hirsuta (1.8%), and S. hominis (42.7%). Furthermore, a new S. hominis-like (prevalence 18.5%), characterized by hook-like structures of villar protrusion and a different sequence of the 18S rRNA gene, was identified. The cattle sheds testing positive for zoonotic Sarcocystis were assessed for risk factors contributing to the maintenance of the parasite's life cycle. Significant associations emerged between consumption of raw meat by the farm owner, mountain pasturing, and absence of a sewerage system on the farm and cattle breed. Our study demonstrates that sarcosporidiosis may constitute a public health problem in Italy and indicates several issues to be addressed when planning surveillance and prevention actions. The applied diagnostic approach revealed that cattle can harbor a further type of Sarcocystis, of which life cycle and zoonotic potential should be investigated.

Multiple-Particle-Tracking to investigate viscoelastic properties in living cells.

Cell mechanical properties play an important role in determining many cellular activities. Passive microrheology techniques, such as Multiple-Particle-Tracking (MPT) give an insight into the structural rearrangements and viscoelastic response of a wide range of materials, in particular soft materials and complex fluids like cell cytoplasm in living cells. The technique finds an important field of application in large cells such as oocytes where, during their growth, several organelles and molecules are displaced in specific territories of the cell instrumental for later embryonic development. To measure cell mechanics, cells are usually deformed by many techniques that are slow and often invasive. To overcome these limits, the MPT technique is applied. Probe particles are embedded in the viscoelastic sample and their properties are extracted from the thermal fluctuation spectra measured using digital video-microscopy. The Brownian motion of a probe particle immersed in a network is directly related to the network's mechanical properties. Particles exhibit larger motions when their local environments are less rigid or less viscous. The mean-square-displacement (MSD) of the particle's trajectory is used to quantify its amplitude of motions over different time scales.

Organization of the vitelline envelope in ovarian follicles of Torpedo marmorata Risso, 1810 (Elasmobranchii: Torpediniformes).

In Torpedo marmorata, the vitelline envelope (VE), an extracellular envelope surrounding the growing oocyte, consists of fibrils and amorphous materials that are deposited in the perivitelline space starting from the initial steps of oocyte growth. SDS-PAGE analysis of the isolated and purified VE reveals that it consists of different glycoproteins. Furthermore, our investigations showed that the 120 and 66 kDa glycoproteins are positive to an antibody directed against gp69/64 of the Xenopus laevis VE and are synthesized under the control of 17beta-estradiol in the liver, that, together follicle cells and the oocyte, is the biosynthetic site of VE components.

Expression of p27BBP/eIF6 is highly modulated during Xenopus laevis embryogenesis.

Protein p27BBP/eIF6 is necessary for ribosomal function of all cells. Previous data showed that from mammals to yeast p27BBP/eIF6 is involved in the biogenesis of ribosomal subunit 60S and its association with the 60S prevents premature 80S formation regulated by PKC signaling, indicating that phosphorylation of p27BBP/eIF6 is needed for translation to occur. While in vitro p27BBP/eIF6 is constitutively expressed, and it has a high level of expression in cycling cells, in vivo its expression varies according to tissues and appears regulated by factors up to now unknown. p27BBP/eIF6 has never been investigated in developing organisms where its upregulation can be correlated with tissue growth and differentiation. In this study we have sequenced p27BBP/eIF6 cDNA and studied its expression during development of Xenopus laevis, as the first step for studying its regulation. The amino acid sequence is highly conserved with two putative PKC phosphorylation sites in serine, one site being typical of Xenopus. At the end of gastrulation, the p27BBP/eIF6 riboprobe localizes in the neural plate and in the paraxial mesoderm. In particular, from stage 24, a clear-cut localization occurs in the perspective head. In embryos exposed to teratogens, the localization of p27BBP/eIF6 riboprobe varies according to the change of head size caused by the treatment. p27BBP/eIF6 expression is particularly evident in differentiating olfactory pits, the lens, otic vesicles, and in branchial arches. Features of particular interest are p27BBP/eIF6 high level of expression in the eye field, and in the mid-hindbrain-boundary, two regions with high proliferative activity. Altogether, data indicate that a modulated expression of p27BBP/eIF6 occurs in developing anlagens in addition to a basal level of expression, and may suggest a correlation between p27BBP/eIF6 and proliferative activity. Moreover, the X. laevis cDNA isolation and characterization offer new hints for further studies in relation to potential p27BBP/eIF6 phosphorylation.

Sulphated glycoconjugates are powerful inhibitors of spermatozoa binding to the vitelline envelope in amphibian eggs.

BACKGROUND INFORMATION: In amphibians, the role of sulphated glycans has not been determined in spermatozoa-egg interaction, although they are known to be involved in other systems. In previous studies, it was found that, in Discoglossus pictus, a VE (vitelline envelope) glycoprotein of 63 kDa exhibits high homology to Xenopus laevis gp69/gp64 and to ZP2 of mammals. gp63 and a glycoprotein of 75 kDa are both capable of binding the spermatozoa in in vitro assays and, having similar peptide maps and different glycosylation, are probably two glycoforms of the same protein. RESULTS: In the present study, binding assays performed by treating dejellied eggs with metaperiodate suggest that hydroxy groups of sugars are not directly involved in spermatozoa-vitelline envelope binding. Competition assays between dejellied eggs and spermatozoa preincubated with dextran, dextran sulphate or fucoidan indicated that sulphated oligosaccharides have an inhibitory effect on spermatozoa binding. In similar competition assays, Le(x) (Lewis(x)) trisaccharide 3'-sulphate inhibited spermatozoa binding to VE in contrast with 3'-sialyl-Le(x) tetrasaccharide. Assays performed with gp75- or gp63-coated beads and spermatozoa treated with fucoidan or dextran sulphate indicated that sulphated oligosaccharides competitively inhibit spermatozoa binding to gp75-coated beads, yet not to gp63-coated beads. Finally, solubilized VE digested with N-glycosidase F retains the inhibitory activity in spermatozoa-VE binding assays in contrast with VE treated with alpha-N-acetylgalactosaminidase. CONCLUSION: It was concluded that VE sulphate groups are involved in spermatozoa binding. These groups are present in gp75 glycoconjugates and are probably located in O-linked glycoconjugates.

Vitelline envelope gps 63 and 75 specifically bind sperm in "in vitro" assays in Discoglossus pictus.

In Xenopus, conflicting data related to sperm-vitelline envelope (VE) binding suggest that further experiments should be performed to study the role of VE glycoproteins in sperm binding. In this article, we studied the VE of Discoglossus pictus, where gp63, the product of the Dp ZP2 gene, has high molecular identity to Xenopus gp69/64 and to mouse ZP2 and only A23187-treated sperm bind to VE. Sperm bind to VE all over the egg, yet a sperm tuft was found only in the animal half of the egg, where the dimple, the site of fertilization, is located and an intense immunostain was detected in VE by an antiserum directed against gp69/64. The same antiserum inhibited sperm binding to VE. Sperm binding to beads coated with gp63, gp40, or gp75 was in the range of 62-70% for gp63-beads, 67-75% for 75 beads, and about 20% for BSA beads and gp40-coated beads. Soluble purified gp63 and gp75 competitively inhibited binding of sperm to gp63-coated beads. Similarly, the same glycoproteins inhibited sperm binding to gp75-coated beads. SDS-polyacrylamide gels (PAGE) of FE and comparison of VE and FE peptide maps showed that gp63 undergoes a minor shift to about 62 kDa in FE. In sperm binding assays with beads coated with FEs gp62, there was no binding. Following fertilization, in the region of the dimple, an F-layer is formed as well as an alteration of the VE structure. Lectin blots of the FE showed that the FE and in particular gp62 acquires a stronger affinity to Maackia amurensis agglutinin (MAA) with respect to VEs gp63. These results indicate that gps 63 and 75 are the sperm binding glycoproteins of D. pictus VE, where major post-fertilization changes occur as in other anuran species.

Spectrin and Ankyrin-like Proteins in the Egg of Discoglossus pictus (Anura): Their Identification and Localization in the Site of Sperm Entrance versus the Rest of the Egg: (spectrin/ankyrin/anuran eggs/fertilization).

The Discoglossus pictus egg has a specific site of sperm-egg interaction, the dimple, which has a well-defined cytoskeleton. We studied whether there are cytoskeletal and cytoskeleton-related proteins typically involved in the polarization of plasma membrane proteins. The identity and the localization of the molecules cross-reacting with antispectrin, antifodrin and antiankyrin antiobodies were investigated by immunofluoresecence and immunoblotting of the proteins of the dimple (D) and of the rest of the egg (dimple-less-egg; DLE). Two polypeptides of about 254-and 246-kD were detected in the D and DLE, and localized in the egg cortex. A third molecule, weakly cross-reacting with antispectrin and antifodrin, was found in the subcortical cytoplasm. The 246-kD polypeptide was labile in samples prepared for SDS-PAGE; a mild prefixation of eggs prevented its dispersion. Mild fixation was also needed to retain antispectrin reactivity in cryostat sections of the DLE cortex, while this is not necessary in D. A molecule of about 204-kD, cross-reacting with antiankyrin, was detected in the cortex of the whole egg. These data and the finding that the concentrations of both the 254-kD polypeptide and ankyrin are about 12-fold higher in D than in the DLE, suggest that, in D, spectrin has a specific organization.

Cytochemical Ca2+ Distribution in Activated Discoglossus pictus Eggs: A Gradient in the Predetermined Site of Fertilization.

The K-pyroantimonate/OsO4 (PA) cytochemical method coupled with EGTA and X-ray microanalytical controls has been used to localize Ca2+ at egg activation in Discoglossus pictus eggs. The results show that: 1) the PA method is able to selectively localize Ca2+ pools mobilized by activating stimuli; 2) the smooth endoplasmic reticulum (SER) elements located in the animal dimple region, i.e. in the predetermined site of fertilization, are the first egg components labeled by precipitates; 3) a decreasing gradient of precipitates is present from the center beyond the boundaries of the dimple region; 4) precipitates are lacking in the remainder of the egg even at late times after activation. The possibilities are discussed that a) SER is the major Ca2+ -releasing store at activation in Discoglossus, and b) the observed gradient of pyroantimonate-detected Ca2+ reflects an ionic Ca2+ gradient.