2da JORNADA de la SOCIEDAD ARGENTINA DE BIOLOGIA

MECANISMOS DE RECONOCIMIENTO Y

DIFERENCIACIÓN CELULAR

Realizada en Instituto de Biología y Medicina Experimental Ciudad Autónoma de Buenos Aires, ARGENTINA Diciembre 2000

Resúmenes publicados en: BIOCELL (ISSN 0327-9545) 25 (2): 179-193, 2001

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Sesión sobre Oncología Resumen Nº1

Inhibitory effect of Transforming Growth Factor-β2 (tgf-β2) from human breast cyst fluids on human breast cancer MCF-7 cells.

Pablo Enriori1, Stella Maris Vázquez1, Carlos Fischer2, Jorge Gori2, Alberto Etkin3, Ricardo Calandra1,4 Isabel Alicia Lüthy1.

1Instituto de Biología y Medicina Experimental, 2Hospital Alemán, 3Hospital Durand, Buenos Aires, 4Facultad de Ciencias Exactas, Universidad Nacional de la Plata, Argentina. (iluthy@dna.uba.ar).

Gross Cystic Disease is the most common benign disease of the breast in premenopausal women, and is associated with an increased risk of developing a breast carcinoma (Cancer Research 52: 1791, 1992). We have previously found that human cyst fluids (CF) are able to enhance proliferation in human breast cancer MCF-7 cells (Medicina 59(5/2): 593, 1999, abst #155). However, some of them caused inhibition of this parameter. The objective of the present work was to analyze the presence of inhibitory factors which could account for this effect. MCF-7 cells were incubated in the presence of TGF-β2 or CF, and in addition, the concentration of this factor in CF was determined by ELISA (ng/ml). For type I CF, significantly less fluids induced inhibition of MCF-7 proliferation (5%) than for type II, (65%, p<0.0001 by Fisher's exact test). The concentration of TGF-β2 was significantly lower in the 24 type I CF (2,56 ± 0,739, which stimulated cell proliferation 2,3 ± 0,3 fold) than in the 17 type II CF (40,87 ± 5,92 which stimulated 0,9 ± 0,1 fold, p<0,0001 as analyzed by Mann-Whitney's test). The concentration of TGF-β2 was negatively correlated with cell proliferation (Spearman correlation coefficient -0.813, p< 0,0001). The incubation of the cells with 0,8–20 ng/ml TGF-β2 (in the absence of CF) caused a significative inhibition of MCF-7 cell proliferation (0,8 ng/ml: 58.4 ± 1.8 % of 3H-Thymidine incorporation from control values, p<0.001 as analyzed by ANOVA followed by Tukey-Kramer's multiple test). These data suggest that TGF-β2 is likely to be one of the inhibitory factor/s present in CF affecting proliferation of human breast cancer MCF-7 cells.

Resumen Nº2

Description and characterization of α2-adrenergic receptors in tumoral and non-tumoral human breast cell lines.

Stella Maris Vázquez, Alejandro Mladovan, Alberto Baldi and Isabel Lüthy.

Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina. (iluthy@dna.uba.ar).

We have previously described (SAB Workshop, 1998), that in human breast cancer MCF-7, MH-6 and MH-7 cell lines (the last ones developed in our laboratory from primary tumors), the α2-adrenergic compounds were able to significantly stimulate tritiated thymidine incorporation. α-Adrenergic receptors have neither been described in breast cancer tissue (human or experimental) nor in the human normal breast. The objective of the present work was to describe and characterize both at the molecular and biochemical level, the different subtypes of α2-adrenergic receptor (α2-AR) involved in the stimulatory effect found in these tumoral as well as in two non-tumoral cell lines (MCF-10A and HBL-100). Receptor expression was studied by RT-PCR followed by Southern blot and the quantification was done by tritiated rauwolscine binding to whole cells in culture (by Scatchard plot). In tumoral cells (MCF-7, MH-6, MH-7) as well as in non-tumoral cells (MCF-10A and HBL-100) identificatory bands for subtypes α2-C2 and α2-C4 were observed. Subtype α2-C10 was never detected under our assay conditions. Scatchard analysis for the cells found positive in the RT-PCR assay showed (Ka (nM-1), maximal binding (fmol/106 cells)): MCF-7: 0.047, 171.2; MH-6: 0.056, 28.13; MH-7: 0.077, 105.9; MCF-10A: 0.059, 32.06; HBL-100: 0.079, 31.51. Conclusion: We describe here for the first time the expression and binding of functional α2-AR in human tumoral and non-tumoral cells.

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Resumen Nº3

The mechanism of histamine H2 receptor desensitization involves different GRKs.

Natalia Fernández !∗, Carina Shayo ∗ψ, Carlos Davio !, Bibiana Lemos !, Alejandro Mladovan *, Federico Monczor !, and Alberto Baldi ∗.

Instituto de Biología y Medicina Experimental, CONICET∗; Lab. de Radioisótopos, Facultad de Farmacia y Bioquímica, UBA!; Facultad de Ciencias Exactas y Naturales, UBAψ.

(natfer@dna.uba.ar).

We have previously described that U937 cell line express histamine H2 receptors (H2r). Histamine and H2 agonists induced an important cAMP response, but failed to induce differentiation in U937 due to the rapid desensitization of the receptor. The present study focuses on the desensitization mechanism of this receptor. In U937 cell line, we determined that desensitization of H2r involves G protein-coupled receptor kinases (GRKs). By immunoblots, we detected the expression of GRK2, 3 and 6 which were not modified by treatment with H2 agonist. In order to analyse this mechanism, we used transiently transfected COS-7 cells expressing tagged-H2r. Results showed that the receptor was rapidly desensitized with a t _= 0.65 ± 0.15 min. Due to the rapid nature of this process, receptor phosphorylation was examined as a likely mechanism for signal attenuation. H2r desensitization was not affected by PKA and PKC inhibitors, but was remarkable reduced by Zn2+ (GRK inhibitor). Cotransfection experiments with tagged-H2r and different GRKs (2, 3, 5, or 6), demonstrated that GRK2 and GRK3 were the most potent in augmenting desensitization. GRK5 and GRK6 did not alter this signaling program. Receptor phosphorylation correlates with desensitization for each GRK studied. These results indicate that in H2r transfected COS-7 cells, exposure to an H2 agonist caused desensitization controlled by H2r phosphorylation via GRKs.

Resumen Nº4

Reduction in H2 receptor desensitization following antisense-induced suppression of GRK 2.

Natalia Fernández !∗, Federico Monczor !, Bibiana Lemos !, Alberto Baldi*, Carlos Davio ! y Carina Shayo∗ψ.

Instituto de Biología y Medicina Experimental, CONICET∗; Lab. de Radioisótopos, Facultad de Farmacia y Bioquímica, UBA!; Facultad de Ciencias Exactas y Naturales,

UBAψ. (natfer@dna.uba.ar)

In previous works we have described that the promonocytic U937 cell line express histamine H2 receptors (H2r). Through these receptors, histamine and specific agonists, transiently induce an important cAMP response, but fail to induce monocytic differentiation due to a rapid receptor desensitization, mediated by GRKs. In COS-7 transfected cells, we showed that GRK 2 and 3 are involved in this desensitization, through receptor phosphorylation. The aim of the present study was to evaluate, in U937 cells, the effect of the reduction of GRK2 expression, on the H2r desensitization. We attempted to reduce specifically GRK2 expression by stable transfection, with an antisense bovine GRK2 cDNA sequence. This yielded up to a 50 % loss of GRK2 when compared with U937 control cells. On addition of the H2r agonist amthamine, cAMP accumulation, as well as the maximal response, was greater in GRK2 antisense cells (D5) compared with control cells. In contrast, cAMP maximal response by addition of forskolin was not significantly different. Also, the desensitization kinetic of the receptor showed a longer t1/2 for GRK2 antisense transfected cells: 11.7±2.2 min and 66.9±12.8 min for U937 and D5 respectively. These data show a direct correlation between expression of GRK2 and desensitization of natively expressed H2 receptors in intact cells, suggesting that GRK2 plays a major role in the regulation of these receptors.

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Resumen Nº5

Telomerase activity is regulated by heat shock protein-90 (HSP90).

Sara Zurita, María Eugenia Martín, Denise Muñoz y Alberto Baldi.

Instituto de Biología y Medicina Experimental (IBYME), (1428) Buenos Aires, Argentina. (abaldi@dna.uba.ar).

Telomers are essential protein-DNA structures at the end of eukaryotic chromosomes. Telomeric DNA consist in a tandem array of simple 6-bp sequence TTAGGG repeats that in association with the enzyme telomerase, provide a mechanism for maintaining chromosome length. Telomerase activity is modulated both at transcriptional and post-transcriptional levels. This last mechanism was investigated by means of geldanamycin (GD) a benzoquinoid ansamycin that block the ATP-binding domain of HSP90 and other chaperons, inducing its dissociation with several proteins. Quiescent NSO cells were stimulated by the addition of fetal calf serum to the cell culture, simultaneously with GD. Telomerase activity was assessed by the TRAP-PCR assay. Kinetics ant GD titration, was previously established. Cell extracts from control and 1ug/ml GD treated cultures, were immunoprecipitated with a monoclonal antibody against HSP90, and the product concentrated by centrifugation Results indicate that all telomerase activity co-immunoprecipitate in untreated cells, whereas in GD treated cells, the enzymatic activity was absent in both the precipitated and supernatant compartments. In conclusion, we demonstrated that telomerase activity is specifically dependent upon its association with HSP90.

Resumen Nº6

Biodistribution of bpa (boronphenylalanine) in undifferentiated thyroid carcinoma.

1Alejandra Dagrosa, 1Mabel Viaggi, 2 Silvia Farias, 2 Ricardo Garavaglia, 1Rómulo Cabrini, 1Guillermo Juvenal, 1,3 Mario Pisarev.

1Dept. Radiobiology, National Comision of Atomic Energy, 2Dept. Chemistry, National Comision of Atomic Energy. 3Medicine School, Buenos Aires University.

Undifferentiated thyroid carcinoma (UTC) lacks of and effective treatment. Boron neutron capture therapy (BNCT) is based on the selective uptake of a 10B-boronated compounds by tumours, followed by irradiation with a neutron beam. The radioactive boron release alpha particles with destroy the tumour. These studies were performed in order to explore the possibility of applying BNCT to UTC. In vitro studies: the uptake of 10 Borophenylalanine (BPA) by UTC cell line ARO (from Dr. G. Julliard, UCLA) and primary cultures of bovine and human thyroid was studied. Boron was determined by ICP-AES. A linear increase in uptake was observed in cells cultured with increasing amounts of BPA. No difference in uptake (as ng 10B /ug protein + SEM) at 24h was observed between ARO proliferating and non-proliferating cells (2.83 + 0.23 and 2.81 + 0.09. Therefore uptake is independent of the proliferative status. The uptake in ARO was greater than in bovine cells (ratio tumour/normal = 4; p< 0.001) as well as in human follicular adenoma: ARO 2.83 + 0.23 versus adenoma 0.55 + 0.02 (ratio tumour/adenoma = 5; p< 0.001). In vivo studies: ARO cells were transplanted into the scapular region of NIH nude mice and after 2 weeks BPA (350 mg/kg bw) was injected via i.p. The animals were sacrificed between 30 and 150 min. Tumour uptake (as ug 10B/g tissue + SEM) was highest after 60 min: 16.80 + 3.57. At the same time normal thyroid had 4.39+ 0.68; blood has 3.04 + 0.75. The ratio tumour/normal thyroid was 4 while tumour/blood ratio was 5.5. The present experimental results open the possibility of applying BNCT for the treatment of UTC. Supported by grants from the University of Buenos Aires, CNEA, CONICET and CECYT.

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Resumen Nº7

Description of a new undifferentiated carcinoma model in nude mice.

1Mabel Viaggi, 1Alejandra Dagrosa, 1David Gangitano, 2Carolina Belli, 2Irene Larripa, 1Rómulo Cabrini , 1Guillermo Juvenal, 1,3Mario Pisarev. 1

Dept. Radiobiology, National Comision of Atomic Energy, 2 Dept. Genetic, National Medicine Academy. 3Medicine School, Buenos Aires University.

The undifferentiated thyroid carcinoma (UTC) is an aggressive solid tumor that produces early metastasis. The aim of present studies was the development of an animal model with a human cell line of UTC. NIH nude mices were injected into the back with UTC human cell line (ARO, kindly provided by Dr. Juilliard, UCLA, CA, USA). Consecutive passages in mice were performed previous tumor primary culture. The following items were studies: tumoral biokinetics, histology, metastatic capacity, animal survival time, in vitro biokinetics of growth, citogenetics and moleculars analysis were. The tumoral volume reached 17,000 mm3 at 117 days. Early histological specimens showed extensive viability with high mitotic activity. At 117 days lung micrometastasis appeared. The in vitro biokinetics of growth decreased when the number of passages increased. The cariotype exhibited a modal number of 66 chromosomes and 13 markers. No differences on amplification of microsatellites of impaired chromosomes were found in the cell line after numerous passages in the mice when compared to the original cell line. This work provides a new model to study the therapy and natural progression of undifferentiated thyroid cancer.

Resumen Nº8

Cross-Talks between ErbB-2 and IGF-IR signaling pathways in mammary tumor.

Labriola Leticia, Balaña María Eugenia, Salatino Mariana, Mosvsichoff Federico, Peters María Giselle, Charreau Eduardo Hernán, and Elizalde Patricia Virgina.

Instituto de Biología y Medicina Experimental, Buenos Aires. (elizalde@dna.uba.ar).

Interactions between signaling pathways activated by progestins and by type I receptor tyrosine kinases (RTKs) family were addressed in an experimental model in which the progestin medroxyprogesterone acetate (MPA) induced mammary carcinomas in Balb/c mice. MPA induced up-regulation of ErbB-2 and ErbB-3 and tyrosine phosphorylation of both receptors on primary cultures of malignant epithelial cells from a progestin-dependent (HD) tumor line. Blocking ErbB-2 expression with antisense oligodeoxynucleotides (ASODNs) resulted in the complete abolishment of MPA proliferative effects on these cells. The simultaneous blocking of two members of the type I RTKs family, ErbB-2 and ErbB-4, as well as of IGF-IR, a member of the type II RTKs family, by an ASODN directed against a tyrosine kinase consensus sequence, did not result in a significantly higher inhibition of MPA-induced growth than the one found using the ErbB-2 ASODNs. Neither synergistic nor additive effects on the inhibition of MPA-induced proliferation were found using combinations of ErbB-2 ASODNs with ASODNs directed to IGF-IR mRNA, suggesting the existence of a hierarchical interaction between ErbB-2 and IGF-IR in which one of the receptors is essential for the activation of signaling pathways elicited by the other. This was confirmed by the finding that supression of IGF-IR expression by ASONDs resulted in the complete abrogation of MPA-induced phosphorylation of ErbB-2. Blocking ErbB-2 did not affect IGF-IR phosphorylation. Notably, we found herein for the first time that this hierarchical interaction between ErbB-2 and IGF-IR in which IGF-IR directs ErbB-2 phosphorylation involves a physical association resulting in the formation of a binary complex between both receptors.

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Resumen Nº9

Regulation of cell cycle components by TGF-β1 in murine mammary tumors.

Mariana Salatino, Leticia Labriola, Eduardo Charreau, Patricia Elizalde.

Instituto de Biología y Medicina Experimental. Buenos Aires. (elizalde@dna.uba.ar).

TGF-β1 modulation of cell cycle components was assessed in the medroxyprogesterone acetate (MPA)-induced mammary tumor model in BalbB/c mice. TGF-β1 inhibited MPA-induced proliferation of primary cultures of epithelial cells from the MPA-dependent tumor line C4HD (ED50: 0.26+ 0.14ng/ml) and serum-induced growth of the MPA-independent variant C4HI (ED50: 0.27+ 0.12 ng/ml). TGF-β1 treatment of both cell types downregulated cyclins D1 (75±22%) and A ( 90±15%) expression at protein level. Cyclin D2 protein was only reduced when TGF-β1 acted as an antagonist of MPA-induced proliferation. Cyclin D2 associated to both cdk2 and cdk4 in C4HD and HI cells. Significant reduction of cyclin D1/cdk4 (80-90%) complexes abundance was found after TGF-β1 treatment of both cell types. TGF-β1 up-regulated protein expression of p21Cip1 (80±25%) when it acted as antagonist of MPA-induced proliferation of C4HD cells and when it inhibited serum-induced proliferation of C4HI cells. Protein levels of p27kip1 (40±12%) were increased only when TGF-β1 antagonized MPA-stimulated growth. Cdk2 activity, determined using histone H1 as substrate, was markedly decreased (84±8%) after TGF-β1 treatment of both cells types. Our results indicate that there are common targets on TGF-β1 inhibitory action on breast cancer cells. However, regulation of specific targets is required when TGF-β1 antagonizes proliferation exerted through the progesterone receptor.

Resumen Nº10

Antiproliferative and pro-apoptotic effect of 5,6,7-trisubstituted flavones on a multidrug-resistant cell line.

Mariana Beviacqua, Alicia Pomilio and Juan Carlos Calvo.

Instituto de Biología y Medicina Experimental – PROPLAME-CONICET, Departamento de Química Orgánica y Departamento de Química Biológica, Facultad de Ciencias Exactas

y Naturales, Universidad de Buenos Aires (jcalvo@dna.uba.ar).

Certain flavones inhibit oncogene expression and possess anti-proliferative effects. In our laboratory we have verified antitumoral activity in Gomphrena spp on growth and survival of the tumoral cell line HCT-15, originated from a colon adenocarcinoma and mutidrug resistant. Quercetin (3,5,7,3’,4’-pentahydroflavone) (Flavo A) and Flavone (Flavo B), were dissolved in 0.2% Tween-20 and a 90:10 mixture of 5,7-dihydroxy-6-methoxyflavone and 3,6-dimethoxy-5,7-dihidroxyflavone (Flavo C) in propylene glycol. We evaluated the chemiosensitivity using tritiated thymidine and colorimetrically with MTT. The pro-apoptotic activity was determined by cytofluorometry (propidium iodide). As a control, a preadipocytic cell line (3T3-L1) was used. Flavo A and B did not inhibit cell proliferation, being protective from the solvent toxicity. >From a dose-response curve for Flavo C, thymidine incorporation at 10 μg/ml, expressed as percentage + SE% was 81.6 + 14.4 for HCT-15 and 6.8 +8.0 for 3T3-L1, whereas with MTT the differences were smaller (51.0 + 1.0 vs 32.0 + 4.3, respectively). Cytofluorometry would indicate certain induction of apoptosis in HCT-15 at a dose of 15 μg/ml but not in 3T3-L1. Conclusion: Flavo C shows a strong antiproliferative effect in HCT-15 tumoral cells, perhaps inducing apoptosis in the same cell line.

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Resumen Nº11

Effect of conditioned media from adipocytes and tumoral LM3 cells on the migration and proliferation of normal murine mammary gland epithelial

cells.

Carla Molinari, Gabriela Rozenberg, Marcela Sandoval, Gabriel Bertolesi and Juan Carlos Calvo.

Instituto de Biología y Medicina Experimental and Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires

(jcalvo@dna.uba.ar).

Cells in an organism interact with their surroundings. We studied a normal murine mammary epithelial cell line, and evaluated migration and proliferation in environments where they are normally found (adipose tissue, fibroblasts, mammary tumors). NMuMG cells were cultured in DMEM with 10% fetal bovine serum (FBS) and insulin (10 μg/ml). For migration studies, we used the wound-healing technique in a confluent monolayer of NMuMG cells, and evaluated the uncovered area in the presence of conditioned media from fibroblasts 3T3-L1, adipocytes and LM3 cells. Conditioned media (CM) from LM3, 3T3-L1 cells and adipocytes were obtained from 80% confluent cultures after 48h in DMEM with 7% FBS for the 3T3-L1 cells and adipocytes and without serum in the case of the LM3 cells. Proliferation was determined using tritiated thymidine. CM from LM3 and adipocytes inhibited migration of NMuMG (0.131 + 0.03 for control, 0.354 + 0.089 for LM3, 0.314 + 0.062 for adipocytes and 0.193 + 0.053 for fibroblasts 3T3-L1, the higher the value, the broader the uncovered region). When analyzing NMuMG proliferation, CM from LM3 was inhibitory. Conclusions: Tumor cells as well as adipocytes could release migration and proliferation regulatory factors to the culture media acting on the normal epithelial cells, probably involved in tumor development and/or normal organogenesis of the mammary gland.

Resumen Nº12

Gliadin peptides antigens in celiac disease: native sequence versus selectively modified by substitution of glutamine by glutamic acid.

Mabel Aleanzi, Ana Demonte, Cecilia Esper , Silvia Garcilazo.

Cátedra de Bioquímica Básica de Macromoléculas. INTEBIO. Facultad de Bioquímica y Ciencias Biológicas. Universidad Nacional del Litoral. Marta Waggener.Hospital de Niños

Dr Orlando Alasia. Santa Fe. (maleanzi@fbcb.unl.edu.ar).

It was demonstrated that selective enzimatic deamidation of glutamin residues of gliadin peptides by tissue transglutaminase transform them in more potent activators of T cells from gut of celiac disease patients. Our objetive was to determine if also peptides modified by selective substitution of native glutamine residues by glutamic acid ,in a similar pattern as enzymatic deamidation , are better B cell epitopes than natives ones. Antibody reactivity of both isotypes, IgA and IgG were determined by ELISA assay, using as solid phase antigen two native peptides of alfa and omega gliadin and their respective modified sequences. Results: increased reactivity was observed of one or both isotypes antibodies from celiac disease sera against one or both deamidated peptides ( 26 celiac disease without gluten free diet). Interestingly, only two of the 20 healthy control sera, that showed mild reactivity against peptides, did not show difference between native and modified sequence. These results strongly suggest that these selectively deamidated peptides are also celiac disease patients specific B cell epitopes.

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Sesión sobre Inmunología Resumen Nº13

Gliadin peptides antigens in celiac disease: native sequence versus selectively modified by substitution of glutamine by glutamic acid.

Mabel Aleanzi, Ana Demonte, Cecilia Esper , Silvia Garcilazo.

Cátedra de Bioquímica Básica de Macromoléculas. INTEBIO. Facultad de Bioquímica y Ciencias Biológicas. Universidad Nacional del Litoral. Marta Waggener.Hospital de Niños

Dr Orlando Alasia. Santa Fe. (maleanzi@fbcb.unl.edu.ar).

It was demonstrated that selective enzimatic deamidation of glutamin residues of gliadin peptides by tissue transglutaminase transform them in more potent activators of T cells from gut of celiac disease patients. Our objetive was to determine if also peptides modified by selective substitution of native glutamine residues by glutamic acid ,in a similar pattern as enzymatic deamidation , are better B cell epitopes than natives ones. Antibody reactivity of both isotypes, IgA and IgG were determined by ELISA assay, using as solid phase antigen two native peptides of alfa and omega gliadin and their respective modified sequences. Results: increased reactivity was observed of one or both isotypes antibodies from celiac disease sera against one or both deamidated peptides ( 26 celiac disease without gluten free diet). Interestingly, only two of the 20 healthy control sera, that showed mild reactivity against peptides, did not show difference between native and modified sequence. These results strongly suggest that these selectively deamidated peptides are also celiac disease patients specific B cell epitopes.

Resumen Nº14

Cloning and expression of heavy-and light-chain variable region genes of a monoclonal antibody specific against the T. cruzi and mammalian ribosomal

P protein consensus sequence.

Pablo López Bergami, Pablo Mateos, Mariano Levin and Alberto Baldi.

Instituto de Biología y Medicina Experimental (IBYME), Instituto de Ingeniería Genética y Biología Molecular (INGEBI). (1428) Buenos Aires, Argentina. (pbergami@dna.uba.ar).

The P2b ribosomal protein of Trypanosoma cruzi (TcP2b) is a relevant antigen in Chronic Chagasic Miocardiopathy. The antibodies against the TcP2b C-terminal epitope (R13 peptide) have autoimmune properties because they react with lower affinity against the H13 peptide corresponding to the mammalian homologue protein. The aim of this study was to develop a monoclonal antibody against the T. cruzi and mammalian ribosomal P protein C-terminal consensus sequence (peptide R10). Balb/c mice were immunized with MBP-TcP2b fusion protein. Both recombinant proteins GST-TcP2b and GST-MmP and synthetic peptides R13 and H13 were used in Western blot and ELISA to asses the induction of specific antibodies. Spleen cells from a selected mice were fused with myeloma cells and the reactivity of 500 resulting clones were assessed against R13 and H13 by ELISA. The clone B10 showed similar reactivity against both peptides. This antibody profile was similar to those of the anti-P autoantibodies observed in sera from Systemic Lupus Erythematosus patients. The heavy and light chain variable region from monoclonal antibody B10 were PCR-amplified, sequenced and cloned as a single chain variable fragment (scFv). The fine specificity of scFV-B10 resembled the corresponding of the monoclonal antibody B10.

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Resumen Nº15

Insulin-like growth factor-I receptor (IGF-1R) regulation on human T lymphocyte proliferation induced by PHA or IL-2.

María Eugenia Segretin, Adriana Galeano*, Alicia Roldán, Roxana Schillaci.

Instituto de Biología y Medicina Experimental, *Sanatorio Mater Dei. Buenos Aires (Fax 4786-2564)

IGF-1 enhances T lymphocyte proliferation in vitro through endocytosis of its receptor. To know if an autocrine/paracrine mechanism of IGF-1 is involve in this effect, it was studied IGF-1 mRNA levels at 0 time and after 30 min stimulation, as well as the IGF-1R expression and its RNAm during the first 36 h stimulation. Also, was investigated the behavior of IGF-1R in the reentry of a new cell cycle induced by IL-2 in cultures of 7 days activation. With that purpose, human mononuclear cells were stimulated with PHA, RNAms were detected by RT-PCR, while IGF-1R, CD25 and cell cycle were identify by flow cytometry. Results shown that RNAm of IGF-1 decreased 45±1% after 30 min of stimulation, at the same time that IGF-1R reached its minimum (0h:33.6± 4.6; 30 min 14.3± 4.5% IGF-1R+ cells). After 24 h, receptors reappeared arriving at the initial levels after at 36 h stimulation, moreover, cells which were IGF-1R+ expressed higher levels of CD25 than those IGF-1R- (fluorescence intensity:174 ± 18 vs 73±17). Meanwhile, levels of IGF-1R RNAm shown a similar profile than its protein, but with delayed on time. By other hand, IGF-1R remain invariable between the 2º and 8º day of stimulation, including after addition of IL-2 at the 7º day, which produced reentry on the cell cycle (20.1± 5.6% of cells in phase S+G2M). These results suggest that the internalization of IGF-1R is induced only by T cell receptor activation, but not by reactivation with IL-2. Also, the decrease of IGF-1 RNAm after stimulation suggest an autocrine/paracrine

Resumen Nº16

Effect of type 2 Shiga toxin on human and rat colon.

Elsa Zotta, Fernando Martín y Cristina Ibarra.

Departamento de Fisiología. Facultad de Medicina. Universidad de Buenos Aires

Shiga toxin type 2 (Stx2) produced by E. coli (STEC) is responsible of a variety of clinical syndromes such as watery diarrhea, haemorragic colitis and hemolytic uremic syndrome (HUS). The aim of this work was investigate if the morphological changes on human and rat colon were associated with A and/or B Stx 2 subunits. It was decided to clone both subunits in pGEM-T, make a mutation and modified E. coli DH5α. The culture supernatant of recombinant E. coli carrying complete or mutated Stx2 gene was used. Culture supernatant from E. coli carrying only a vector was used as negative control. The degree of the toxin lesion was classified in three levels: level 1(mild): loss of superficial epithelium, infiltration of neutrophils and depletion of globet cells, level 2 (moderate): destruction of the upper third of the colonic mucosa, and level 3 (severe): loss of two thirds of the colonic mucosa. The degree of follicular hyperplasia was classified according the number of linfoid follicles presents in the samples. Human and rat colon was mounted in the Ussing chamber between two saline solution and bubbled with a mix of O2 95 % and CO2 5 % at 378C. Then, they were incubated with culture supernatant from recombinant E. coli during 60 min. After that, they were analyzed by microscopy. The results showed a mild lesion in a superficial human colonic mucosa and a moderated lesion in a rat colonic mucosa when the tissues were incubated with complete Stx2. In both cases a moderated lesion was observed in presence of mutated Stx2. The morphological changes observed in rat colon was significantly higher than those present in the human colon. There results indicated that B subunit present in both experimental condition could produce by itself the mucosa colonic lesion. Moreover a follicular hiperplasia was observed in presence of mutated Stx2 that could indicate a limphocyte up-regulation due to the mutation.

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Sesión sobre Reproducción Resumen Nº17

Study of the possible dual origen (epididymal and testicular) of sperm-associated protein DE.

Da Ros V1, Morgenfeld M1, Busso D1, Ellerman D1, Hayashi M2 and Cuasnicu P.1

1Instituto de Biología y Medicina Experimental, CONICET-UBA, Bs As, Argentina; 2Universidad de Hokkaido, Sapporo, Japón. (vgdaros@ dna.uba.ar)

Rat glycoprotein DE (Mw 32kDa) associates with the sperm surface during epididymal maturation and mediates gamete fusion through complementary sites on the egg surface. Recent results indicated the existence of two populations of DE: a major population loosely associated to sperm and which is released during capacitation, and a tightly associated population, already detectable in caput sperm, which remains after capacitation and participates in gamete fusion. Evidence demonstrating the dual origen (testicular and epididymal) of several proteins that associate with sperm in the epididymis, opened the possibility that this strongly bound protein were in fact of testicular origen. Considering both the high homology between DE and testicular protein TPX-1 (67%), and the similarity of their molecular weights, we first investigated whether the tightly bound population could correspond to TPX-1. Western Blot assays against protein detergent extracts from epididymal and testicular sperm using anti-DE and anti-TPX-1 did not show however, any cross-reaction between the antibodies. To examine whether the DE gen was expressed in the testis, RT-PCR of epididymal and testicular tissue were carried out using specific primers for DE. Results revealed a band of 723pb corresponding to DE mRNA only in the epididymis. Taken together, these results indicate the specific epididymal origen of the DE protein finally involved in gamete interaction.

Resumen Nº18

Relationship between the structure and the biological activity of epididymal protein "DE".

Ellerman D, Cohen D, Morgenfeld M, Da Ros V, Busso D, Cuasnicú P.

Instituto de Biología y Medicina Experimental, Bs. As., Argentina.

Sperm protein DE participates in gamete fusion through complementary sites on the egg surface. DE is a glycoprotein of 227 amino acids (aa) and contains 16 cysteines involved in intramolecular disulfide bonds (S-S). The aim of this study was to analyze the relationship between the structure of DE and its biological activity. Recent evidence indicating that bacterially-expressed DE is capable of inhibiting gamete fusion although less efficiently than native DE, suggested a possible role of carbohydrates in DE activity. To examine this further, native protein DE was deglycosilated enzymatically with PNGase F and used in fusion assays. Results indicated that the presence of the deglycosilated protein during gamete fusion inhibited the penetration of zona-free eggs to levels not significantly different from those corresponding to native DE (control: 85%, deglycDE 14%, DE 16%). The relevance of S-S for DE activity was studied using reduced/alkylated DE (redDE), which inhibited (p<0.05) zona-free egg penetration less efficiently than non-treated DE (51% vs 14%). Finally, in order to identify the region through which DE binds to its complementary sites, zona-free oocytes were exposed to different recombinant fragments coupled to maltose binding protein (MBP): [F1(aa 1-158), F2 (156-227), F3 (62-227), and F4 (62-158)] and subjected to indirect immunofluorescence. Oocytes incubated with F1, F3 and F4, but not with F2 or MBP, showed fluorescent labelling on the egg surface. We conclude that a) the activity of DE resides on the peptidic region of the molecule, b) S-S are important although not essential for DE activity and c) the egg binding site is located on the region spanning aa 62-158.

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Resumen Nº19

IFN-γ modifies in vitro inner cell mass (ICM) and trophoectoderm (TB) lineage morphology in murine blastocysts.

V.Fontana1, F.Trama1, L. Vauthay2, M.Cameo1.

1Biología de la Reproducción; 2Instituto de Oncolgía Roffo. Buenos Aires.

It has been suggested that a TH2-type cytokine response predominates in successful pregnancies; cases in which TH1 cytokines predominate, may lead to intrauterine growth retardation or, even, pregnancy loss. In a previous study we demonstrated that sera from infertile patients and IFN-γ, a TH1-type cytokine, added exogenously, impair mouse embryo development, suggesting that IFN-γ could be one of the embryotoxic factors present in sera from infertile patients (3). The purpose of this study is to better understand whether IFN-γ exerts its action on the ICM and/or TB. Methods: Two cell embryos were recovered from superovulated mice and incubated in vitro for up to 72 h (day 3). Embryos classified as blastocysts were cultured for an additional 96h period to compare their capacity to hatch and implant in vitro in the presence or absence of IFN-γ (10μg/μl). After fixation and staining, morphology was evaluated and embryos were classified as Type A or B according to presence, size and compactness of the ICM, and to the development and differentiation of TB outgrowths. Statistical differences between control and experimental distribution patterns were tested by chi-square analysis. Results: Our experiments showed that the addition of IFN-γ to the culture medium impaired embryo development. Differences between distribution in control (C) and study group were statistically significant for ICM and TB when IFN-γ was added on day 3 of culture. For ICM: Type A 61% C, vs 15% IFN-γ (p<0.0001), for TB: 25%C vs 6% IFN-γ, (p=0.0013). Conclusion: Exogenous IFN-γ decreased growth potential of mouse blastocysts at both levels: ICM and TB lineages. This impairment might be crucial for embryo development: ICM plays a vital rol in organogenesis, and TB in contribution to placental structure.

Resumen Nº20

Binding of human proacrosin to zona pellucida sugar residues.

Laura Furlong1, Romina Barrozo1 Pablo Ghiringhelli2, Mónica Vazquez-Levin1.

1Instituto de Biología y Medicina Experimental (CP1428). 2Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes. (lfurlong@dna.uba.ar).

Proacrosin is a protease localised in the sperm acrosome involved in the fertilisation process. Studies carried out in rabbit and boar suggest that proacrosin would act as an adhesion molecule during sperm penetration through the zona pellucida (ZP). The aim of the present study is to determine whether human proacrosin binds to homologous ZP glycoproteins, and to evaluate the participation of ZP sugar moieties in the interaction. Recombinant human proacrosin (Rec-40: 42-44 kDa), N-terminal fragments (Rec-30: 32-34 kDa, Rec-20: 21 kDa, Rec-10: 18 kDa) (Furlong et al, Biol Reprod (2000). 62, 606-615), 125I-ZP glycoproteins and biotinylated BSA-Mannose were used in the binding studies. Using a Dot blot assay, Rec-40 was found to recognise ZP glycoproteins in a concentration dependent manner. N-terminal truncated products showed ZP binding activity, that decreased in the presence of dextran sulphate (10 μM). BSA-Mannose neoglycoprotein was used to evaluate the participation of mannose residues in the interaction with proacrosin. Rec-40 and the N-terminal truncated products showed specific binding towards BSA-Mannose in a Western blot assay. Studies performed with isolated Rec-40 and Rec-30 revealed that Rec-30 retains 30±5% of Rec-40 binding activity towards BSA-Mannose. A molecular analysis was performed on the human proacrosin sequence aimed at identifying potential ZP binding domains. Putative binding domains were found to be located both in the N-terminal and C-terminal regions of the protein. 3D modelling of acrosin sequence indicated that the putative sites would be on the surface of the molecule, where they could interact with complementary sites on the ZP. These studies describe the interaction of human proacrosin with ZP glycoproteins and their sugar residues, supporting its role in secondary binding during fertilization.

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Resumen Nº21

Effect of antisperm antibodies present in the human follicular fluid upon sperm acrosome reaction and interaction with the zona pellucida.

Clara Marín Briggiler1, Mónica Vazquez-Levin1, Fernanda Gonzalez Echeverría2, Jorge Blaquier2, Patricia Miranda1, Jorge Tezón1.

1Instituto de Biología y Medicina Experimental. CONICET, 2Fertilab, Buenos Aires, Argentina. E-mail: (cmarin@dna.uba.ar).

Previous studies from our laboratory have indicated that human follicular fluid (hFF) contains antibodies (hFFIgG) against human spermatozoa with the ability to induce the acrosome reaction (AR). However, the incidence of this phenomenon in the population of infertile patients is currently unknown. The first objective of the present study was to analyze the AR inducing capacity of IgG retrieved from individual hFF. The hFFIgG were isolated by affinity chromatography (n = 40 hFF) and were incubated (10 μg/ml) with capacitated human spermatozoa (n = 3). The occurrence of AR was evaluated using Pisum sativum-FITC. Results were expressed as AR Stimulation (S = % hFFIgG AR - % spontaneous AR / % hFF AR - % spontaneous AR) and as Induction Frequency (IF = positive assays (S = 8 %) / total assays). According to their IF, the hFFIgG were classified as: Group I (Low IF: 0-0.33, 25 %), Group II (Intermediate IF: 0.34-0.66, 30 %) or Group III (High IF: 0.67-1.00, 45 %). The median S values obtained for these groups were 4 %, 14 % and 21 % (p < 0.05), respectively. Considering that these antibodies could induce the AR by aggregation of zona pellucida (ZP) receptors on sperm plasma membrane, the second objective of this study was to determine whether the hFFIgG were able of inhibiting sperm-ZP interaction. Spermatozoa were preincubated with the hFFIgG in the presence of Sr2+ ions (condition that blocks the AR) and hemizona assays were carried out. While treatment with hFFIgG from Group I (n = 8) did not affect sperm binding to the ZP, preincubation with some hFFIgG from Group III (4 of 6) significantly inhibited sperm-ZP interaction, suggesting that these hFFIgG could be inducing the AR by recognizing ZP receptors. These antibodies constitute an invaluable tool for the isolation and characterization of sperm entities involved in the fertilization process.

Resumen Nº22

The use of genetic immunization in the production of antibodies towards human proacrosin.

Carolina Veaute, Laura Furlong, Juan Biancotti, Mónica Vazquez-Levin.

Instituto de Biología y Medicina Experimental. Buenos Aires (CP1428). (cveaute@dna.uba.ar).

Genetic immunization has proved to be highly advantageous in the production of antibodies when compared to traditional protein immunization protocols. Using this last approach, antibodies towards the sperm serine protease acrosin have been shown to block fertilization in animals. The aim of the present study was to evaluate the humoral response obtained in mice inoculated with the cDNA encoding h-proacrosin, and compare it with that obtained after immunization with a recombinant truncated product of the proenzyme (Rec-30, residues 1 to 300) produced in bacteria. The cDNA encoding h-proacrosin was subcloned in the eukaryotic expression vector pSF2 containing the alpha-1-antitrypsin signal peptide, and the CMV promoter (pSF2-acro). Recombinant proacrosin (Rec-40) and Rec-30 were obtained using preparative electrophoresis of bacterial lysates (Furlong et al., Biol. Reprod., (2000) 62, 606-615). Female BalB/c (7 wo) were inoculated with pSF2-Acro (10, 20, 40 ug; i.m.) or Rec-30 (20 ug; sc/ip) every 3 weeks for a total of 12 weeks. Plasmid pSF2 and buffer were used as controls. Production of antibodies was analyzed starting at week 6 using an ELISA with Rec-40 as antigen. When tested at 1:100 sera dilution, a similar response was obtained using either protein or DNA as immunogen. Maximal response was obtained at week 10 after immunization. Twenty ug of pSF2-acro purified using anionic exchange columns (Qiagen) were sufficient to produce a high specific response. The titer decayed to a 50% of the maximun value 6 months after protocol initiation. Gene immunization with a low amount of cDNA encoding proacrosin resulted in a specific high response, with the production of antibodies against the protein. This experimental model will be utilized in studying proacrosin role(s) in ZP sperm penetration during fertilization.

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Resumen Nº23

Human sperm Hexosaminidase: subcellular localization and possible function.

Silvina Perez Martinez, Jorge Tezón, Patricia Miranda.

Instituto de Biología y Medicina Experimental, Buenos Aires. (pmiranda@dna.uba.ar).

Hexosaminidase (Hex) is the enzyme responsible for the hydrolysis of terminal N-acetylglucosamine residues. Previous studies from our laboratory suggest the participation of a sperm Hex during the interaction with the oocyte's extracellular matrix, the zona pellucida (ZP). The aim of the present study was to determine the subcellular distribution of Hex in human sperm. For this purpose, semen samples from normospermic donors were centrifuged over a Dextran cushion and the sperm pellet was subjected to different extraction procedures. Hex activity was measured in supernatant (SN) and pellet with a specific fluorogenic substrate and the percentage associated to the SN calculated. Extraction in culture medium (BWW) released 61 ± 3 % of total activity (viability 81%). This result was not modified by inclusion of high ionic strength (NaCl 0.5 M) in the medium (68 ± 2 %). Treatment with 0.1% Triton X 100, released 87 ± 6 % of total activity. This distribution did not change significantly when capacitated sperm were extracted. Triton X100 proved to exalt total Hex activity on non-capacitated (100 %, p<0.05) and capacitated sperm (35%, p<0.05). Interestingly, although some enzyme was released to the capacitation medium and sperm associated activity diminished 40 % (p<0.05), a 50 % increase in total activity (medium plus sperm) was noticed (p<0.05). These results indicate that 60 % of Hex is loosely associated to human spermatozoa, probably to plasma membrane. Additionally, a 30% of total the enzyme activity has an acrosomal origin. The subcellular localization of Hex, its permanence in capacitated sperm and the increased in total activity found after capacitation agree with its participation in primary binding and/or penetration of the ZP.

Resumen Nº24

Sperm membrane integrity: correlation with bovine "in vivo" fertility.

C.M. Tartaglione1 and M.N.Ritta1,2 .

1Facultad de Ciencias Agrarias, Universidad Nacional de Lomas de Zamora, Buenos Aires, 2Instituto de Biología y Medicina Experimental, CONICET, Buenos Aires, Argentina.

Standard parameters evaluated in order to assess male fertility yield to controversial correlation to fertility. The integrity of the plasma membrane reflects the viability of spermatozoa and cryopreservation procedures are known to damage it. This damage includes swelling and disruption of plasma and outer acrosome membranes, changes in membrane fluidity, altered influx of calcium and changes in enzyme activity. Several studies have reported that sperm viability in hypoosmotic solution (HOS) provides information concerning the membrane integrity of the sperm tail. However, this parameter is not enough to predict in vivo behavior. The aim of this study was to combine supravital test (ST) and HOS test (STHOS) in order to provide information about integrity of the whole sperm surface (head and tail membrane) and correlate this parameter with field fertility. Frozen semen from 12 bulls (packaged in 0.5 ml straws) was assessed for sperm motility (subjective microscopy examination), undead/dead relation (Eosin-Nigrosin vital stain), morphological abnormalities (Chinese ink stain) and non return rates (NRR). Since the statistical analyses (Anova test) of values obtained for each parameter showed a great dispersion, the samples where divided into two groups according to NRR, named high (68.9±3.5, HF) and low fertility (43.2±1.4, LF). HF straws corresponded to animals showing higher motility (76.3±2.7 vs 65.5±2.1) and STHOS values (57.9±2.5 vs 36.0+1.1). Taking into account the correlation between fertility and membrane integrity assessed by STHOS, as an easy and accessible method, which could be a useful assay to test the fertilizing ability of bovine spermatozoa.

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Resumen Nº25

Interaction between early embryos and uterine cells: development of an in-vitro model for implantation.

Griselda Vallejo, Monica Fazzini, Lino Barañao, Miguel Beato, Patricia Saragueta.

Instituto de Biología y Medicina Experimental (IBYME-CONICET). Institut fur Molekularbiologie und Tumorschung Marburg Germany. Departamento de Química

Biológica. FcyEN-UBA. (sarag@dna.uba.ar).

Embryo implantation is a complex series of events that involves changes in pattern of expression of embryonic as well as uterine cell surface components. In the case of the embryo, these changes have been described as driven mostly by the developmental program. Regarding the uterus, these changes are triggered by both maternal hormonal influences as well as embryo-derived factors. Aspects of the implantation process vary among species; however, interaction between the external surface of the embryonic trophectoderm and the apical surface of the lumenal uterine epithelium is a common event. We are focused on defining a coculture system to study the molecular players in these interactions. We used different cell lines to develop embryonic cell growth in vitro. The coculture on granulosa cells produced outgrowth of the embryos as from the third day of culture, showing an increasing inner cell mass phenotype which was present throughout the 12 days of culture. The coculture with an epithelial uterine cell (RENTROP) failed to develop outgrowth of the embryos. On the other hand, the coculture with a stromal uterine cell line (UIII) showed outgrowths mainly with a trophoblast giant cell phenotype but lost the inner cell mass phenotype. Desmin expression in stromal cells occurs via hormonally regulated, epithelial-mesenchymal interactions and serves as an early marker of uterine receptivity. Trophinin and Bystin form a cell adhesion complex that potentially mediates an initial attachment. Strong expression of these molecules was detected in the stromal cell line used here. Moreover, UIII cocultured with mouse embryos during 12 days showed positive desmin immunostaining. These results suggest that the embryo could modulate directly the uterine differentiation during the attachment process and that the endometrial cells could contribute to differentiate the embryonic cells.

Resumen Nº26

Cryopreservation of llama embryos.

Mariano Lattanzi1, Claudio Santos1, Graciela Chaves2, Marcelo Miragaya2, Enrique Capdevielle2, Judith Egey2, Alicia Agüero2, Lino Barañao1.

1Instituto de Biología y Medicina Experimental, Obligado 2490 Capital. 2Area de Teriogenología, Facultad de Veterinaria , UBA. (lattanzi@dna.uba.ar).

Embryo cryopreservation has not been succesfully developed in camelids. The goal of this study was to determine the feasibility of cryopreserving llama embryos using two methods: gradual freezing (G) and vitrification (V). Embryos were collected non surgically at day 7 after mating from llamas control or superstimulated with eCG and/or pFSH. Vitrification: Embryos were exposed to Equilibration Solution (ES) (TCM 199, 15% Ethylene glycol (EG), 20% Fetal Bovine Serum (FBS), 50 ug/ml gentamicin sulfate) at room temperature until dehydratation and rehydratation. Then embryos were exposed to Vitrification Solution (VS) (TCM 199, 30 % EG, 20% FBS, 1M Sucrose), loaded in 20ul Drummond Microcaps and plunged into liquid nitrogen in less than 1 minute. Gradual freezing: Embryos were exposed to PBS 1,5 M EG, 20% FBS, loaded in 0,25 ml straws and cooled 15 minutes at -7ºC. After seeding, straws were cooled at 0,5ºC /min until –35ºC and then plunged into liquid nitrogen. Straws and Microcaps were thawed directly into Dilution Solution (DS) (TCM 199, 0,5 M sucrose, 20% FBS, 10-15% egg yolk) at 39 oC until dehydratation and rehydratation. Then embryos were cultured in SOFaa + BSA under 5% O2 5% CO2 90% N2 at 39oC. Data were analyzed using chi square test. Embryos cryopreserved by G and V showed similar survival rates (re-expansion 24 hs after thawing 33% n=12 vs. 9.5% n=21, after 48h 57% n=7 vs. V: 54%, n=11). These results demonstrate the feasibility of cryopreserving llama embryos using G or V. Moreover, vitrification is an attractive technique for field application. Survival rates after thawing seems to be increased by longer culture periods in defined medium and low O2 levels. (Supported by UBACyT IV01).

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Resumen Nº27

Effects of leptin on hCG secretion by human term placental trophoblast cells cultured in vitro.

Paula Cameo1, Paul Bischof2 and Juan C. Calvo1.

1Instituto de Biología y Medicina Experimental (IBYME), Buenos Aires, Argentina. 2Dep. of Obstetrics and Gynecology, University Hospital of Geneva, Switzerland

(pcameo@dna.uba.ar)

Several observations suggest a possible role of leptin, a 16 KDa protein transcribed from the obese (OB) gene, in feto-placental physiology: 1) circulating leptin levels are elevated during pregnancy; 2) human placenta expresses both leptin and its receptor. The aim of this work was to study the role of leptin in the regulation of hCG secretion by term Cytotrophoblast (CTB) cells. Term human placentas from caesarean sections or vaginal deliveries following uncomplicated pregnancies were used within 1-2 hs after delivery. CTB cells were purified on a discontinuous Percoll gradient according to a previous report (*). CTB cells were then cultured in triplicate in DMEM in presence or absence of FBS, with different concentrations of rh-leptin (0, 10, 100, 500, 1000 and 2000 ng/ml). At least 3 experiments were run for each culture condition using different cell preparations. Results were corrected per mg of protein content in each individual well and expressed as percentage of the corresponding control (mean ± SEM) or as values per mg of cell protein. Statistical analysis was performed by ANOVA. Results: Rh-leptin stimulated hCG secretion by CTB cells cultured in vitro with or without FBS 10%. hCG released to the medium was higher after incubation with the different concentrations of leptin tested when compared with controls: differences were significant (p<0.05) with 1 and 2 ug/ml of rh-leptin. The same effect was observed after two or four days of culture. Conclusions: Leptin has different effects on human term placental trophoblast cells cultured in vitro. In this study we show that under the present culture conditions rh-leptin has a stimulatory effect on hCG secretion. (supported by WHO HRP no 98166) (*)Bischof P, Haenggeli L, Campana A. Hum Reprod 1995; 10: 734-42.

Resumen Nº28

The use of H-Y antiserum for sexing bovine embryos at different stages of development, obtained by in vitro fertilization.

Juan Carlos Gardón1, Sergio Agüera Carmona2, Francisco Catejón Montijano2.

1Programa de Biotecnologías Aplicadas en Reproducción Animal, Fac. de Cs. Agrarias, Univ. Nac. de Lomas de Zamora. 2Departamento de Biología, Fac. de Veterinaria, Univ. de

Córdoba, España. jcgardon@ciudad.com.ar.

The aim of the present study was to evaluate the sex of bovine embryos obtained by in vitro fertilization at different stages, cultured in presence of H-Y antiserum obtained of immunized rats. For it 758 bovine embryos at 32 cells stage (Group A) and 825 at more than 32 cells stage (Group B), were distributed randomly in 4 subgroups. Control I (n=396) cultured in TCM-199, BOEC cells, SFB and antibiotics; Control II (n=394) cultured in TCM-199, BOEC, SFB, antibiotics and guinea pig serum; Control III (n=396) cultured in TCM-199, BOEC, SFB, antibiotics and H-Y antiserum; and Treated (n=397): cultured in TCM-199, BOEC, SFB, antibiotics, guinea pig serum and H-Y antiserum. After culture, in presence or absence of H-Y antiserum, embryos were evaluated as altered and not altered. Then embryos were sexed. There were not significant differences (p<0.05) for altered and not altered among subgroups Control I, II and III of the Groups A and B. However, there were significant differences (p<0.05) among the subgroups Control (I, II and III) regarding Treated for Groups A and B. The relationship male:female, was significant (p>0.05) regarding the normal one observed in the species 1:1, in the Treated subgroups of the groups A and B regarding the Controls I, II and III. Moreover, a high correlation exists among embryos classified as altered (males), in the Treated subgroups of the Groups A and B. The in vitro culture of the embryos in presence of H-Y antiserum produces alterations in the development at stages of 32 and more than 32 cells. The H-Y antiserum obtained in rats, possesses cross reaction with H-Y antigens bovine embryos obtained by in vitro fertilization.

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Resumen Nº29

An oocyte derived factor mitogenic to granulosa cells is present in the immature oocyte.

Fischman, ML; Santos, C; Barañao, JL.

Facultad de Ciencias Exactas y Naturales, UBA, e Instituto de Biología y Medicina Experimental. (lbaranao@dna.uba.ar).

In previous studies we demonstrated that bovine oocytes stimulate rat granulosa cell proliferation and that this effect decreases as meiotic maturation progresses. In this paper we intend to establish whether the factor responsible for this effect is already present in the immature oocyte (FI) or is synthesized during coculture. Bovine oocytes were obtained from antral follicles and in vitro maturated (IVM). Some of the FI and IVM oocytes were sonicated. Primary granulosa cell cultures were obtained from immature rats and cultured in a definite medium with oFSH added (20 ng/ml) in presence of FI, IVM (15 oocytes/well), soluble fraction (S), precipitated fraction (P) from oocyte homogenate, or combination of both (SP), in a concentration equivalent to 15 oocytes/well. Proliferation was assessed according to the incorporation of tritiated thymidine. Maximum stimulation was obtained with FI followed by IVM (16±1 and 11±0.5 – fold stimulation with respect to a control group). Fraction S of FI and IVM oocytes increased the DNA synthesis ( 7±1.6 and 10±0.9 – fold respectively). Fraction P was less effective (FI: 3.3±0.4, and IVM: 3.2±2 – fold). SP combination produced a stimulation similar to that observed when cells were treated with the soluble fraction (FI: 9.3±0.4, and IVM: 9.2±1.9 – fold). These results indicate that the mitogenic effect is due to a soluble factor present in the immature oocyte. Differences in the mitogenic effect of FI and IVM oocytes could be attributed to a decrease in the secretion of this soluble factor.

Resumen Nº30

Cell proliferation, apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis.

Meresman, GF, Barañao RI, Bilotas M, Vighi, S, Gomez Rueda, N, Buquet, RA, Contreras-Ortiz, O, Tesone, M.

Instituto de Biología y Medicina Experimental (IBYME-CONICET), Hospital de Clínicas José de San Martín, Buenos Aires, Argentina. (meresman@ dna.uba.ar).

Objective: To evaluate and compare cell proliferation, spontaneous apoptosis, and Bcl-2 and Bax expression in eutopic endometrium from women with and without endometriosis (EDT). Design: Cell proliferation, Apoptosis, and Bcl-2 and Bax expression were examined in eutopic endometrium from women with and without EDT. Patients: Women with untreated EDT (n=14) and controls (n=16). Methods: Biopsy specimens of eutopic endometrium were obtained from all subjects by means of a Novack cannula. Apoptotic cells were detected using the dUTP nick-end labeling (TUNEL) assay, Bcl-2 and Bax expressions were assessed using immunohistochemical techniques. As a proliferative marker, Ki-67 expression was also examined immunohistochemically. Results: Spontaneous apoptosis was significantly lower in eutopic endometrium from patients with EDT (2.26±0.53 apoptotic cells/field), than in endometrial tissue from controls (9.37±1.69) (p<0.001), and was independent of cycle phase. An increased expression of Bcl-2 protein was found in proliferative eutopic endometrium from patients with EDT. Bax expression was absent in proliferative endometrium, while there was an increase in its expression in secretory endometrium from both patients and controls. The Ki-67 index was increased in stroma (p<0.03) and in epithelial endometrial cells (p<0.04) from patients with EDT. Conclusions: Women with EDT show decreased number of apoptotic cells and increased cell proliferation index in eutopic endometrium. The abnormal survival of endometrial cells may result in their continuing growth into ectopic locations.

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Resumen Nº31

Apoptosis of early antral follicle granulosa cells obtained from DES treated prepubertal rats is regulated by inhibin.

Alejandra Vitale, Olga Gonzalez, Liliana Dain, Stella Campo, Marta Tesone.

Instituto de Biología y Medicina Experimental- CONICET, Facultad de Ciencias Exactas- Universidad de Buenos Aires y Centro de Investigaciones Endocrinológicas, Buenos Aires,

Argentina. (mtesone@dna.uba.ar).

Inhibins are peptides produced mainly in the gonads, their primary role being the regulation of pituitary FSH synthesis and secretion. Nevertheless, inhibins may exert an autocrine and/or paracrine modulation of ovarian function. The detection of α and β Inh subunits, their messenger RNAs and its receptors in granulosa cells have been documented, showing that the ovary is an important source of Inh production. It was study the effect of intraovarian recombinant Inh A administration (0.5 μg/ovary) to prepubertal DES treated rats. The counterlateral ovary was used as control (C). It was determined by TUNEL the number of apoptotic cells in preantral (PF) and early antral follicles (AF). A significant increase (p< 0.05) in AF from ovaries treated with Inh was observed (C: 0.17±0.01; Inh: 0.50±0.02 apoptotic cells/camp). No change was detected in the number of apoptotic cells in PF. Isolated AF were incubated in serum free medium with FSH and/or different concentrations of Inh during 24 h. Apoptosis was determined by DNA agarose gel electroforesis. It was observed an apoptosis increase in the follicles incubated with Inh (4 ng/ml: 117%, 40 ng/ml: 62%). The in vitro effect of Inh A on the granulosa cell proliferation was measured by thymidine3H incorporation. Immature isolated granulosa cells were cultured and stimulated 48 h with FSH (20 ng/ml), E2 (500 ng/ml) and TGFbeta (5 ng/ml). A proliferation inhibition (p< 0.05) was observed (C: 5543±146; Inh 4 ng/ml: 4767±174; Inh 40 ng/ml: 3480±117 cpm. These results suggest that Inh A may act as a paracrine factor inhibiting the growth of neighbor follicles, modulating by this way the mechanism of follicular selection.

Resumen Nº32

In vivo effect of GnRH on the expression of bcl-2 family members in preovulatory rat ovarian follicles.

Fernanda Parborell, Adalí Pecci, Griselda Irusta, Marta Tesone.

Instituto de Biología y Medicina Experimental- CONICET, Facultad de Ciencias Exactas- UBA, Buenos Aires. (mtesone@dna.uba.ar).

Gonadotropin-releasing hormone (GnRH) and its agonists, have specific effects on extra-pituitary tissues such as placenta, breast, and ovary. A GnRH- like peptide, as well as GnRH receptor and GnRH gene transcription products, have been identified in the ovary. In addition, we have demonstrated the antigonadal effect of GnRH in human and rat granulosa cells. In this work, we have studied the in vivo effect of a GnRH agonist (leuprolide acetate, LA, 1 μg/prepubertal superovulated rat during 2 days) on the apoptosis regulation in preovulatory ovarian follicles (FPO). FPO obtained by microdissection from LA rat ovaries show a significant increase in DNA apoptotic fragmentation measured by TUNEL (Control C: 0.65± 0.19; LA: 6.11± 1.41 apoptotic cells/camp) and by agarose gel electrophoresis (C: 450.5±10.5; LA: 1707.8±16.5 arbitrary units). Gene expression of some members of the bcl-2 family was studied by Western blots. LA did not modify the bcl-2 expression (antiapoptotic protein) after 0, 1 and 2 h of FPO incubation in serum free medium . On the contrary, bax levels (proapoptotic protein) were higher in the FPO obtained from LA treated rats (0 h: 50%; 1 h: 207%; 2 h: 57%). It is concluded that the apoptosis induced by LA in rat preovulatory follicles is mediated by changes in the expression of some bcl-2 family members.

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Resumen Nº33

Expression and localization of ion and water channels in trophoblastic cells from normal and pathological placenta.

Alicia Damiano1,2, Elsa Zotta1, and Cristina Ibarra1,2.

1Departamento de Fisología, Facultad de Medicina, 2 Laboratorio Canales Iónicos, Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires, Argentina.

(ibarra@fmed.uba.ar).

The syncytiotrophoblast of normal human placenta (hST) is a continuous, multi-nucleated structure with minimal tight junctions, which results from fusion of the underlying cytotrophoblast cells. Thus the transport of metabolites, ions and water from mother to fetus should take place primary via transcellular routes. Here, we report the expression and localization of CFTR (cystic fibrosis transmembrane conductance regulator), APXL (human homologue of the apical protein of Xenopus laevis), AQP3 and AQP9 (aquaporin 3 and 9) by RT-PCR, immunoblotting and immunocytochemistry. Total RNA was purified from hST and the RT-PCR technique was applied using primers flanking highly conserved regions of CFTR, APXL and AQPs. PCR amplification bands were 295 bp for CFTR, 360 and 404 bp for APXL, and 398 and 393 bp for AQP3 and AQP9, respectively. Western blot analysis in a plasma membrane fraction from hST using specific antibodies, showed a band of ~170 kDa for CFTR, ~160 kDa for APXL and 27-28 kDa for AQPs. The immunolocalization was restricted to the plasma membrane domains of hST. CFTR and APXL were also found in hydatidiform mole, a placental trophoblastic disease characterized by greatly enlarged avascular ¨grape-like¨ villi. The functional importance of these channels in normal and pathological placenta need to be elucidated.

Sesión sobre Endocrinología Resumen Nº33

Modulation of thyroid radiosensitivity by nicotinamide in normal and Goitrous rats.

Marcos Agote, Mabel Viaggi, Erica Kreimann, Leon Krawiec, Maria A. Dagrosa, Laura V. Bocanera, Guillermo J. Juvenal, Mario A. Pisarev.

Nuclear Biochemistry Division, Dept. of Radiobiology, National Atomic Energy Commission and Dept. of Biochemístry, University of Buenos Aires School of Medicine.

Radioiodine is being used to treat thyroid cancer and hyperthyroidism. Sometimes the doses employed may require hospital admission of the patient to avoid radiation hazards. It is desirable to obtain the same therapeutic effect with lower doses of the isotope, by simultaenous administration of a radiosensitizer. This study was performed to analyze the use of nicotinamide as a radiosensitizer to radioiodine; that would contribute to avoid this problem. Wistar rats were employed. Administration of nicotinamide (NA, 20-40 mg/day, during 30 days) failed to alter thyroid weight. The influence of NA on radiothyroidectomy induced by increasing doses of 131I was examined. NA produced a significant increase in the ablation caused by radioiodine. Goiter was then induced by the administration of methylmercaptoimidazol (MMI) to rats, followed by the treatment with radioiodine with and without simultaneous administration of NA. Thyroid weight/100 g bw (average of 7 rats ± SEM) was: goitrous: 10.56 ± 0.30; goitrous + NA: 10.25 ± 0.50; goitrous + l 31I: 7.90 ± 0.50 (25% decrease); goitrous + 131I + NA: 5.70 ± 0.30 (47% decrease, p<0.01). ADP-ribosilation of nuclear proteins, probably involved in radiation-induced DNA repair, showed no differences between controls and NA-treated rats. Thyroid blood flow (determined by 86Rb uptake) was increased by 84% by NA. Conclusions: nicotinamide has a significant radiosensitizing effect to 131I both in normal and goitrous rats. This action is due to an increase in thyroid blood flow, which enhances tissue oxygenation,and probably free radical production.

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Resumen Nº34

3Isoproterenol regulates the mRNA levels of an Arachidonic Acid-releasing Acyl-CoA thioesterase in cardiac tissue.

Isabel Neuman, Paula Maloberti, Constanza Lisdero, Juan José Poderoso,

Ernesto J. Podestá.

Departamento de Bioquímica Humana y Laboratorio del Oxígeno. Facultad de Medicina, Universidad de Buenos Aires. (ineuman@fmed.uba.ar).

We have recently described an acyl-CoA thioesterase specific for very-long-chain fatty acids, named ARTISt (Arachidonic acid Related Thioesterase Involved in Steroidogenesis). ARTISt is under hormonal regulation and stimulates steroidogenesis through the release of Arachidonic Acid (AA) in adrenal zona fasciculata cells. This protein has been detected in steroidogenic and non steroidogenic tissues, suggesting an alternative pathway for intracelular AA release. We have previously demonstrated that Isoproterenol (ISOP) produces a dose-dependent stimulation of ARTISt´s bioactivity in cardiac tissue, mediated by beta-adrenergic receptors. In the present report we demonstrate the hormonal regulation of the mRNA levels of ARTISt in cardiac tissue. Treatment with ISOP (10-7 M) by cardiac perfusion resulted in a time-dependent increase of the transcript abundance (1,4; 1,6 y 2,6 times at 15, 30 and 60 minutes respectively). The effect of the agonist was blocked by propranolol (10-5 M) perfusion prior to ISOP treatment. Administration of Actinomycin D (500 μg/kg) injection 45 minutes before perfusion abolished the effect of ISOP. Taken together, our results provide evidence of an early control of transcription of ARTISt in heart mediated by beta-adrenergic receptors. Given that in steroidogenic tissues AA released by this pathway regulates genes transcription, the role of ARTIST in cardiac tissue could be also located at transcriptional level.

Resumen Nº35

Serotoninergic and adrenergic receptors in the modulation of steroidogenesis in leydig cells from golden hamster.

Mónica Frungieri1,2, Karina Zitta1, Silvia Gonzalez-Calvar1,2, Ricardo Calandra1,3.

1Instituto de Biología y Medicina Experimental-Conicet; 2Cátedra Bioquímica Humana, Facultad de Medicina, UBA; 3Cátedra de Endocrinología, Facultad de Cs Exactas, UNLP.

(scalvar@dna.uba.ar).

Previous reports have shown the presence of norepinephrine (NE), epinephrine (E) and serotonin (5-HT) in the testis and their involvement in the regulation of testosterone (T) production (Frungieri et al., Neuroendocrinology, 1999). The aim of the present work was to study the heterologous regulation between E/NE and 5-HT on steroidogenic capacity in purified Leydig cells from adult hamsters photoperiod (14 hs light- 10 hs dark). “In vitro” hCG-stimulated (100 mIU/ml) T production was evaluated (29.4 ± 1.3 ng/ 106 .Leydig cell), with or without 10–6 M 5-HT, E, NE and agonists (ag)/antagonists (ant) of these receptors. The present results indicate an inhibitory action of 5-HT and 5-HT1A/2A ags. In contrast, E/NE and α1/β ags are associated with a stimulatory action on T production. Ketanserin (ket) (2A ant) reverte the effect of: 5-HT (5-HT: 21.9±1.1 vs 5-HT+ket: 26.9±1.0*), ag2 (DOI: 21.8±1.3 vs DOI+ket: 30.2±1.4*), ag β (isoproterenol: 36.4±0.8 vs iso+ket: 29.4±2.4*) and α1-ag (phenylephrine: 37.0±0.3 vs phen+ket:31.9±1.1*). However, pMppi (1A ant) reverts the effect of 5HT (5-HT: 21,9±1.1 vs 5-HT+pMppi: 25.5±0.5*) and that of the 1A ag (DPAT: 22.5±1.5* vs DPAT+pMppi: 29.8±0.7*), without alteration in the response to α1/β1 ags (ng/106Leydig cel.; *: p<0.05). In conclusion, the present study demonstrate that, in adult hamster testis, the modulation of T production exerted by α1/β1 adrenergic receptors is mediated by 5-HT2A receptors but not by 5HT1A receptors.

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Resumen Nº36

Possible participation of phospholipase A2 (PLA2) activation on FSH-regulation of lactate production in rat Sertoli cells.

Gabriela Gómez, Silvina Meroni, Fernanda Riera, Eliana Pellizzari, Helena Schteingart, Selva Cigorraga.

Centro de Investigaciones Endocrinológicas. Hospital de Niños R. Gutiérrez. Buenos Aires. (scigorraga@cedie.guti.gov.ar).

FSH plays a pivotal role on Sertoli cell growth and differentiation. PLA2 activation upon FSH binding to its receptor has been demonstrated. The aim of the present study was to determine whether FSH stimulation of 2-deoxiglucose uptake (2-DOG), LDH activity (LDH) and mRNA LDH A levels, biochemical steps that participate in lactate production, involves PLA2 activation and arachidonic acid metabolization via the cyclooxygenase pathway. Sertoli cell cultures from 20-day-old rats were stimulated with PLA2 (10UI/ml), FSH (200ng/ml) and/or the inhibitors of PLA2 and cyclooxygenase, Quinacrine (Q) and Indomethacin (Indo) respectively. The following results were obtained: 2-DOG: Basal: 750±37a, FSH: 2025±55b, PLA2: 1207±131c, FSH+Q: 1402±89c, FSH+Indo: 1245±86c dpm/ μgDNA; LDH: Basal: 31.7±1.8a, FSH: 42.3±3.4b, PLA2: 40.9±2.7b, FSH+Q: 33.5±4a, FSH+Indo: 28.1±1.9a, mUI/μg DNA (mean±SD, n=3, different superscripts indicate statistical significant differences). FSH increased mRNA LDH-A levels and Q treatment partially inhibited FSH-stimulation. These results suggest that PLA2 activation and arachidonic acid metabolization through the cyclooxygenase pathway may participate in FSH regulation of lactate production in Sertoli cells.

Resumen Nº37

Molecular mechanisms involved in the regulation of rat Sertoli cell lactate production by growth factors and cytokines.

Fernanda Riera, Silvina Meroni, Gabriela Gómez, Helena Schteingart, Eliana Pellizzari, Selva Cigorraga.

Centro de Investigaciones Endocrinológicas. Hospital de Niños “R. Gutiérrez”, Buenos Aires (scigorraga@cedie.guti.gov.ar).

Sertoli cells are regulated by FSH, testosterone and locally produced growth factors and cytokines. Sertoli cell lactate production is essential for the normal development of spermatogenesis. In the present study the effect on lactate production of prolonged stimulation of Sertoli cells with the growth factors EGF (50ng/ml), bFGF (10ng/ml), TGFbeta (1ng/ml) and the cytokines IL1beta (50ng/ml), TNFalfa (20ng/ml), interferon gamma (INF 20ng/ml) was analyzed. In addition, LDH activity, 2-deoxiglucose uptake (2-DOG) and the mRNA LDH A levels, biochemical steps that might be altered by these treatments and that are involved in lactate production, were determined (mean±SD, n=3, *p<0.05 vs basal). Lactate production was stimulated by EGF, bFGF and IL1β: basal 8.7±0.85, EGF 12.4±0.2*, bFGF 15.5±1.1*, TGFβ 8.8±0.6, IL1β 19.0±1.5*, TNFα 9.2±1.1, IFNγ 8.6±0.9 μg/μgDNA. The latter increments were accompanied by increases in LDH activity (basal 36.6±2.1, EGF 50.8±5.0*, bFGF 52.7±3.2*, IL1β 55.3±3.3* mUI/μgDNA) and 2-DOG uptake (basal 1111±87, EGF 1503±123*, bFGF 1588±112*, IL1β 1785±127* dpm/μg DNA). No statistical significant increments in LDH activity and 2-DOG uptake were observed with the other growth factors and cytokines tested. Those growth factors and cytokines that stimulated lactate production, also increased mRNA LDH-A levels. These results suggest that peptide regulation of Sertoli cell lactate production is exerted through simultaneous increments in glucose uptake,

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Sesión sobre Neuroendocrinología Resumen Nº39

Effects of Activin A on polyamines and FSH levels in infantile female and male rat anterior pituitaries.

Estela Rey-Roldán, Pablo Hockl and Carlos Libertun.

Instituto de Biología y Medicina Experimental-CONICET. V. de Obligado 2490, 1428. Bs. Aires. (erey@dna.uba.ar).

Activin (Act) is a peptide that specifically stimulates FSH secretion. Polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) are aliphatic amines essential in growth and cell differentiation. We have previously shown that they are elevated in the postnatal period in the pituitary; in 6 day-old female rats these polyamines inhibit FSH and LH secretion. The aim of the present work was to investigate if the endocrine effect of Act on the pituitary was related to polyamine concentrations. Adenohypophyseal fragments of 15 and 7 day-old male and female rats were incubated with Act (50 ng/ml) or medium (control) for 6 hours. FSH was measured by RIA in the culture media, SPD and SPM levels were measured by HPLC (Hockl et al., J.Liq.Chromat. & Rel. Technol. 23:693:2000) in the tissue fragments and DNA was measured by Burton's method. In 15 day-old female rat pituitaries Act significantly lowered SPD (pmol /μg DNA: control:34.8±1.33, Act: 27.58±3.09, p<0.01) and SPM (pmol/μg DNA: control:38.51± 2.64, Act:28.98±3.01, p<0.01) levels; in males no significant differences were observed. Nevertheless, in both sexes Act increased FSH (ng/μg DNA): females: control: 12.85±0.63, Act:16.24±1.27, p<0.05; males: control:5.36±0.37, Act:7.70±0.43, p<0.05. In 7 day-old rats, Act did not modify polyamine levels in the pituitary, even though FSH levels were significantly increased in both sexes. FSH females: control:4.28±0.21, Act:5.29±0.48, p<0.05; males: control: 3.03±0.22, Act: 4.61±0.41, p<0.05. We conclude that a) basal FSH levels are higher in females than in males and increase with age. b) Act induces FSH secretion in 15 and 7 day-old male and female rats. c) Act decreases SPD and SPM titers in 15 day-old females only. The effect of Act on polyamines is observed in females at a critical age, in which the pituitary is highly sensitive to stimulatory and inhibitory factors, and this effect appears not to be related to its gonadotropin- releasing effect. (CONICET-UBA-ANPCyT).

Resumen Nº40

Changes on insuline-like growth factor 1 (IGF-1) expression in the lumbar spinal cord of monosodium glutamate (MSG) treated rats.

Verónica B. Dorfman, M. Cristina Vega, Héctor Coirini*.

Laboratory of Neurobiology, IbyME / CONICET and Department of Human Biochemistry, University of Buenos Aires, Argentina. *e-mail: hcoirini@dna.uba.ar

Animals treated neonatally with MSG show altered sexual behaviour, functional hipogonadism, obesity and growth retardation. IGF-1 has been previously described in the lumbar region of the spinal cord, where it has trophic actions on the cells where it is expressed. Since at this region there are motoneurones related with penil reflexes, erection and ejaculation, we have evaluated the changes in IGF-1 expression by immuno-histochemistry. Male Sprague-Dawley rats were sc. injected with MSG (4mg/g body weight) in 0.9% NaCl solution, days 2, 4, 6, 8, and 10 after birth. Control group rats were equally treated with an NaCl isotonic solution (10%; n=6/group). Four month old animals were anesthetized and perfused through the heart with 0.9% NaCl followed by 4% paraformaldehide (PFA) in phosphate buffer. Spinal cords were removed, post-fixed for 1h (4% PFA), transferred to a 30% sucrose solution overnight, dried, frozen and stored at –70ºC. Presence of IGF-1 was studied on lumbar (L6) coronal sections using a comercial antibody (Peninsula - 1/750 dilution). Specific immunoreativity for IGF-1 was localized on layers IV and V, and in fibers present on the dorsomedial region (DM). MSG treated rats showed a significant 46% reduction of cellular area in layers IV and V associated with an increase in the number of cells smaller than 200µm2. Analysis of the DM region indicated a 48% reduction on the area of immunopositive fibers respect to Controls. The changes observed on the size of IGF-1 expressing neurons but not on its contents, may suggest that MSG treated rats present a deficient development of these neurons that may directly influence the cell in the motor nuclei, leading to an alteration at the muscular response. (PIP 0819/98 and TM-12-UBA).

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Resumen Nº41

Quantitative and ultrastructural immunohistochemical analysis of the somatotrope population in the male golden hamster under short

photoperiod.

Gisela Camihort1, Susana Jurado1, Celia Ferese1, Mónica Carino1, Ricardo Calandra2,3, Karina Zitta3, Gloria Cónsole1.

Cátedra de Histología "B", Facultad de Ciencias Médicas1. Cátedra de Endocrinología, Facultad de Ciencias Exactas2. Universidad Nacional de La Plata. Instituto de Biología y

Medicina Experimental3. (gconsole@atlas.med. unlp.edu.ar).

Exposure of golden hamsters to short photoperiods (<12.5h) determines an early regression of the reproductive organs, followed by spontaneous recrudescence. We studied the effect of light deprivation on the pituitary somatotrope population. Groups of 9 male hamsters were kept under long photoperiods since birth (LP: 14h light, 10h darkness) or exposed to short photoperiods (SP: 6h light, 18h darkness) for 8, 16, 22 and 28 weeks. Pituitaries were processed for both light and electron microscopy. Sections were immunostained by anti-GH- EnVision- DAB. The stereologic parameters were obtained using an image analysis system (Optimas). Volume density (VD x10-2) and cell density (CDx10-4) did not show any significant alterations in SP compared to those of LP. Under SP, the ultrastructure showed a moderate decrease in the number of secretory granules, and images of a dilated RER.

GH LP SP

8w 16w 22w 28w 8w 16w 22w 28w

VD 9.1 ± 0.5 9.5 ± 0.8 12.4 ± 0.4 13.2 ± 0.5 12.4 ± 0.9 8.9 ± 0.4 13.6 ± 0.9 10.6 ± 0.8 CD 10.2 ± 0.9 10.6 ± 0.8 13.8 ± 0.5 14.8 ± 0.6 13.9 ± 0.5 10.0 ± 0.1 15.2 ± 0.4 11.9 ± 0.4 In the SP group, the quantitative immunohistochemical studies detected no significant changes, while at the ultrastrutural level alterations suggestive of a slight increased secretory activity of GH were found.

Resumen Nº42

Effects of light deprivation on the corticotrope population in the male golden hamster.

Cónsole Gloria1, Susana Jurado1, Ricardo Calandra2,3, Gisela Camihort1, Mónica Carino1, César Gómez Dumm1.

Cátedra de Histología "B", Facultad de Ciencias Médicas1. Cátedra de Endocrinología, Facultad de Ciencias Exactas2. Universidad Nacional de La Plata. Instituto de Biología y

Medicina Experimental3. (gconsole@atlas.med.unlp.edu.ar).

The male golden hamster shows variations in the endocrine responses to changes in the photoperiod. In the present study we investigated the effect of light deprivation on the pituitary corticotrope population. Groups of 9 male hamsters were kept under long photoperiods since birth (LP: 14h light, 10h darkness) or exposed to short photoperiods (SP: 6h light, 18h darkness) for 8, 16, 22 and 28 weeks. Pituitaries were processed for both light and electron microscopy. Sections were immunostained by anti-ACTH- EnVision (Dako)-DAB. The stereologic parameters were obtained using an image analysis system (Optimas). Volume density (VD x10-2) and cell density (CDx10-4) showed no significant changes in the SP groups of 8, 16, 22 and 28 weeks when compared to those of LP ones. At the ultrastructural level, an irregular and considerably dilated RER was revealed in the ACTH cells from SP group.

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ACTH LP SP 8w 16w 22w 28w 8w 16w 22w 28w

VD 4.8 ± 0.7 3.9 ± 1.1 4.4 ± 0.4 4.2 ± 0.6 4.3 ± 0.9 4.1 ± 0.8 3.2 ± 0.5 4.0 ± 0.3 CD 5.2 ± 0.9 4.3 ± 0.8 4.8 ± 0.5 4.6 ± 0.6 4.7 ± 0.5 4.5 ± 0.1 3.5 ± 0.4 4.4 ± 0.4 We conclude that in the SP group, the immunohistochemical study did not detect any quantitative alterations in the corticotrope population, while ultrastructural evidence of hypersecretion could be found in a number of ACTH cells.

Resumen Nº43

Influence of photoinhibition on the thyrotrope population in the male golden hamster.

Susana Jurado1, Gloria Cónsole1, Mónica Carino1, Ricardo Calandra2,3, Karina Zitta3, César Gómez Dumm1.

Cátedra de Histología "B", Facultad de Ciencias Médicas1. Cátedra de Endocrinología, Facultad de Ciencias Exactas2. Universidad Nacional de La Plata. Instituto de Biología y

Medicina Experimental3. (gconsole@atlas.med.unlp.edu.ar).

Short photoperiods cause induction of the pineal activity, with atrophy of the reproductive organs. In the present study we investigated the effect of light deprivation on the pituitary thyrotrope population. Groups of 9 male hamsters were kept under long photoperiods since birth (LP: 14h light, 10h darkness) or exposed to short photoperiods (SP: 6h light, 18h darkness) for 8, 16, 22 and 28 weeks. Pituitaries were processed for both light and electron microscopy. Sections were immunostained by anti-TSH-EnVision (Dako)-DAB. The stereologic parameters were obtained using an image analysis system (Optimas). Volume density (VD x10-2) and cell density (CDx10-4) did not show any significant changes in the SP groups of 8, 16, 22 and 28 weeks when compared to those of the LP ones. The ultrastructure revealed a decreased number of secretory granules and an increased RER and Golgi complex in the SP group.

TSH LP SP

H 8w 16w 22w 28w 8w 16w 22w 28w

HVD 0.8 ± 0.1 1.0 ± 0.1 0.7 ± 0.1 0.6 ± 0.1 1.0 ± 0.1 0.9 ± 0.2 0.6 ± 0.2 0.6 ± 0.1 HCD 0.9 ± 0.2 1.1 ± 0.1 0.8 ± 0.3 0.7 ± 0.1 1.1 ± 0.1 1.0 ± 0.1 0.7 ± 0.2 0.7 ± 0.1 HWe conclude that the immunohistochemical studies detected no quantitative alterations in the thyrotrope population, while the ultrastrutural findings showed changes indicative of a higher secretory activity in a number of TSH cells.

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Resumen Nº44

Glucocorticoid regulation of Fos-like immunoreactivity in the normal and injured spinal cord.

Susana González1,2, Claudia González Deniselle1,2, Flavia Saravia1,2, Florencia Labombarda1,2, Paulina Roig1, Rachida Guennoun3, Alejandro De Nicola1,2.

1Instituto de Biología y Medicina Experimental, Argentina, 2Facultad de Medicina, UBA, Argentina, 3INSERM U-488, Kremlin-Bicêtre, Francia. (susanalaura@hotmail.com).

Glucocorticoids (GC) exert beneficial effects in cases of spinal cord injury (SCI). However, the exact mechanism of action still remains unclear. In the present study, we evaluated the time-course of c-fos gene expression to monitor neuronal responses to spinal cord transection in the acute phase of injury and after GC treatment. Rats with sham-operation or SCI at thoracic level received vehicle or 5 mg/kg Dexamethasone (DEX) at 5 min post-lesion and sacrificed 2h or 4h after surgery. A third group of transected rats received vehicle or intensive DEX treatment (5 min, 6, 18, 46h post-lesion) and were sacrificed 48h after surgery. Quantitative analysis of the number of neurons expressing Fos-like immunoreactivity (FLI) was studied using a computer-assisted image analysis system. Neurons were induced to express FLI in Laminae I-III (LI-III) and LX by 2h after SCI (LI-III: sham:4±1 positive nuclei/section, SCI: 15±2, p<0.05; LX: sham: 3±1, SCI:10±2, p<0.05) which reached its maximal levels of expression 4h after injury. DEX treatment significantly enhanced the number of positive nuclei in LI-III by 4h after SCI (SCI:24±2, SCI+DEX:37±2, p<0.05). However, FLI was not maintained by intensive DEX treatment when tested 2 days after injury, suggesting that steroid effects on this parameter may be coupled to early mechanisms arising after trauma. Thus, spinal cord neurons exhibiting nuclear gene products in response to DEX after SCI suggest that GC may play a role in the early activation of this transcriptional cascade in the lesioned tissue (CONICET PIP 4103, CONICET PEI 0328/98, UBA TM13, Agencia PICT-97 0038, Fundación Barceló).

Resumen Nº45

Glucocorticoid and progestin effects on gap-43 mrna and gfap immuno-reactivity in the spinal cord of Wobbler mice.

María Claudia González Deniselle1,2 Gerardo. Piroli1,2, Susana González1,2, Florencia. Labombarda1,2, Daniel Cardinali2 and Alejandro De Nicola1,2.

1Instituto de Biología y Medicina Experimental; 2Facultad de Medicina, UBA. (cldenise@hotmail.com).

The murine mutant Wobbler (wr) presents motoneuron degeneration and astrogliosis in the spinal cord. Expression of the growth-associated protein (GAP-43) and the glial fibrillary acidic protein (GFAP) are increased in the spinal cord of wr mice. In this work, we examined if these markers of neuronal and astrocyte function were sensitive to steroid treatment. A group of wr mice received sc a 20 mg pellet of Progesterone (P4) during 12 days or a 50 mg pellet of Corticosterone (CORT) during 4 days. Basal levels of GAP-43 mRNA were ten-fold elevated in ventral horn motoneurons of untreated wr mice, compared to controls. The high expression of GAP-43 mRNA in wr was reduced by treatment with P4 (18%, p<0.05) and the natural glucocorticoid CORT (41%, p<0.001). Additionaly, the number of GFAP immunoreactive astrocytes were greatly increased in the ventral horn from wr in comparison to controls. The enhanced GFAP protein expression was significantly reduced by CORT treatment in wr (p<0.05) but was unchanged after P4 treatment. These data suggest (1) both CORT and P4 exert a down-regulatory effect upon GAP-43, possibly related to steroid neuroprotection; (2) effects on GAP-43 may be independent of steroid effects on astrogliosis (Supported by CONICET-PIP 4103, UBA TM13, PICT-97 00438, and Peruilh Fellowship).

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Resumen Nº46

Impact of diabetes on the CNS: astrogliosis in hippocampus of nonobese diabetic (NOD) mice, an animal model for insulin-dependent diabetes

mellitus (IDDM).

Flavia Saravia1,2, Yanina Revsin1, Susana Gonzalez1,2, Maria Claudia Gonzalez Deniselle1,2, Paulina Roig1, Veronique Alves3, Josiane Coulaud3, Francoise Homo-Delarche3 and

Alejandro De Nicola1,2

1 Instituto de Biologia y Medicina Experimental, Obligado 2490 (1428) Buenos Aires, 2 Facultad de Medicina, UBA, Argentina, 3Unite CNRS UMR 8603, Paris, France. e-mail:

fsaravia @dna.uba.ar

Diabetes mellitus is one of the most common serious metabolic disorders in man. Manifestations of diabetic cerebral disorders includes increased risk of thrombotic stroke and impairment of cognitive performance. The NOD mouse is a recognized and well-studied spontaneous model of IDDM, sharing many similarities with human disease. In previous work, we observed increased hypothalamic hormones in PVN during diabetes in NOD mice. The PVNis under negative regulation by other brain structures, like hippocampus. Hippocampal damage was associated with diabetes in streptozotocin-treated rats. Accordingly, we investigated possible hippocampal alterations in NOD mice during IDDM. In an immunocytochemistry study, we quantified the number and area of GFAP reactive astrocytes below CA1 region by computerized image analysis. The results showed that the number of GFAP positive cells (in a square of 65x103μm2) increased significantly in diabetic mice (C57Bl/6, control strain (CT) 2.52±0.56, NOD nondiabetic (noDb) 21.38±2.41 p<0.05 , NOD Diabetic (Db) 30.55±6.85 p<0.01. Interestingly, the labeled cell area was also augmented in diabetic animals (CT 35.76±2.51, NOD noDb 154.02±7.58 p<0.01 vs CT, NOD Db 237.01±12.56 p<0.001 vs CT,p<0.05 vs NOD NoDb (Anova one way+Bonferroni post-test). The evidence suggests that the impact of diabetes on the brain is substantial and some aspects of this disease present similarities with neurodegeneration and aging.

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