Cell-cell communication between Mouse mammary epithelial cells and 3T3-L1 preadipocytes: effect on triglyceride accumulation and cell proliferation

Interaction between parenchyma and stroma is essential for organogenesis, morphogenesis, and differentiation. Mammary gland has being the chosen model for developmental biologist because the most striking changes in morphology and function take place after birth. We have demonstrated a regulation of triglyceride accumulation by protein factors synthesized by normal mouse mammary gland epithelial cells (NMMG), acting on a cell line, 3T3-L1, long used as a model for adipogenesis. In this paper, we demonstrate that this inhibitory effect seems to be shared by other cells of epithelial origin but not by other cell types. We found a regulation of cell proliferation when NMMG cells are cultured in the presence of conditioned media from Swiss 3T3 or 3T3-L1 cells. We found a possible point of regulation for the mammary factor on a key enzyme of the lipid metabolic pathway, the glycerol-3-phosphate dehydrogenase. The inhibitory factor seems to have an effect on this enzyme´s activity and reduces it. The results presented herein contribute to the understanding of cell-cell communication in a model of a normal mammary gland.Fil: Julianelli, Vanina Laura. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guerra, Liliana Noemi. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Calvo, Juan Carlos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

O papel dos glicosaminoglicanos no processo de descondensação cromatínica do espermatozoide humano

En el núcleo del espermatozoide, la cromatina se encuentra 6 a 8 veces más condensada que en los núcleos de las células somáticas. esta condensación es el resultado del intercambio de histonas por protaminas durante el proceso de formación de los espermatozoides, otorgando al material genético una resistencia única al ataque por especies reactivas de oxígeno como también al estrés que significa el trayecto hasta su encuentro con el ovocito en el oviducto. Esta condensación tan alta debe ser relajada, una vez producida la fecundación del ovocito, para permitir la formación del pronúcleo masculino y la posterior singamia. Para que ocurra la descondensación es necesaria la reducción de los puentes S-S de las protaminas, reacción que lleva a cabo el glutatión y, además, la remoción de las protaminas con intercambio posterior o concomitante por las histonas ovocitarias. En este punto, todavía no hay consenso sobre la molécula involucrada. En nuestro laboratorio postulamos que sería el heparán sulfato, basados en análisis de descondensación in vitro, en las características estructurales de la molécula y en su presencia en el ovocito murino, con evidencias preliminares de su presencia también en el ovocito humano (datos no mostrados)In the sperm nucleus, chromatin is 6 to 8 times more condensed than in the nuclei of somatic cells. This high condensation is the result of the exchange between histones and protamines during the process of spermatogenesis, and provides the genetic material with unique resistance to the attack of reactive oxygen species, as well as to the stress of the voyage towards the encounter with the oocyte in the oviduct. This high condensation must be relaxed, once the fertilization of the oocyte has taken place, in order to allow for the formation of male pronucleus and subsequent syngamy. For the decondensation to occur, it is necessary to reduce the disulfide bonds in the protamines, reaction that takes place with the assistance of glutathione, and also to remove the pro-tamines with the subsequent or concomitant exchange with oocyte histones. At this point, there is no consensus regarding the molecule involved in this process. In these studies, it has been postulated that this molecule is heparan sulfate, based on in vitro decondensation studies, on the structural characteristics of the molecule and the fact that it is present in the murine oocyte, with preliminary evidence of its presence also in the human oocyte (data not shown).Fil: Romanato, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Julianelli, Vanina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Farrando, Bárbara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Calvo, Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Calvo, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Heterogeneous distribution of histone methylation in mature human sperm

Purpose To analyze the presence of various histone modifications in ejaculated human spermatozoa Methods In this prospective study, seminal ejaculates from 39 normozoospermic individuals were evaluated for semen analysis and the presence of histone modifications in isolated nuclei. Results We observed heterogeneous presence of histone methylation in normal mature human sperm. We observed that 12 to 30 % of the nuclei of normal sperm contain a heterogeneous distribution of the marks H3K4Me1, H3K9Me2, H3K4Me3, H3K79Me2, and H3K36Me3. Moreover, the presence of these marks is higher in the poor motile fraction of the ejaculate, which is associated with poor morphology and functional quality. In contrast, we did not observe histone acetylation (H3K4Ac and H4K5Ac) in normal or abnormal mature human sperm Conclusions Defects in the process of spermatogenesis may alter the correct epigenetic programing in mature sperm. Further studies are required to evaluate the impact of these findings in human infertilityFil: la Spina, Florenza Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Romanato, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Brugo Olmedo, Santiago. Centro Médico Seremas; ArgentinaFil: de Vicentiis, Sabrina. Centro Médico Seremas; ArgentinaFil: Julianelli, Vanina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Rivera, Rocío M.. University Of Missouri; Estados UnidosFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Heparan sulfate and nuclear sperm decondensation in humans: study of its specific role in this process and molecular characterization of heparan sulfate-DNA-Protamines interaction

A diferencia de las células somáticas, el espermatozoide posee su cromatina empaquetada principalmente por protaminas en lugar de histonas. Las protaminas son proteínas pequeñas y básicas, debido a su alto contenido de residuos lisina, arginina y cisteína. Las cisteínas forman puentes disulfuro intra e intercatenarios, lo que le proporciona al núcleo espermático gran estabilidad y resistencia para poder atravesar el tracto masculino, el femenino y llegar con su ADN intacto hasta el sitio de fecundación. Una vez que el espermatozoide penetra en el ovocito, es imprescindible que se produzca la descondensación de su cromatina, es decir el desempaquetamiento del ADN para poder reemplazar las protaminas por histonas ovocitarias. La descondensación de la cromatina consiste en dos eventos, deben reducirse los puentes disulfuro (tiorreducción) y removerse las protaminas. Las moléculas involucradas en estos procesos son el glutatión reducido (GSH) y el heparán sulfato (HS) ovocitarios. Hasta el momento se consideraban eventos independientes y sucesivos: primero el GSH tiorreduce las protaminas, y luego el HS las remueve. Sin embargo, nuestros resultados indican que estos procesos podrían ser simultáneos y cooperativos. El objetivo de esta tesis fue avanzar en el estudio del mecanismo de la descondensación de la cromatina del espermatozoide humano in vitro en presencia de heparina (análogo estructural del HS) y GSH y su extrapolación al mecanismo in vivo durante la fertilización. Al coincubar espermatozoides humanos o núcleos espermáticos aislados con heparina y GSH, se produce un mayor porcentaje de descondensación de la cromatina, con respecto a la incubación en forma secuencial con los mismos agentes, independientemente del orden de los mismos. Utilizando naranja de acridina como técnica para evaluar en forma indirecta el estado de tiorreducción de los núcleos espermáticos, se observa que la heparina colabora con el GSH en la tiorreducción, durante la descondensación. Estos resultados fueron confirmados utilizando monobromobimane como método directo para evaluar tiorreducción. Los resultados aportados por esta tesis indican que la tiorreducción producida por el GSH, y la remoción de las protaminas producida por la heparina, durante la descondensación de la cromatina de espermatozoides humanos in vitro, ocurriría en forma simultánea, y no secuencial, ya que ambos factores cooperarían entre sí.Unlike somatic cells, sperm have a highly packed chromatin due to the presence of protamines instead of histones. Protamines are small basic proteins, very rich in cysteine, lysine and arginine. Cysteine residues form disulfide bonds, both intra and interchain, rendering the sperm nucleus high stability and resistance during transit along the male and female reproductive tracts, thus enabling the spermatozoon to arrive at the fertilization site intact. Once the sperm penetrates the oocyte, chromatin decondensation must occur, implying the unpacking of DNA through the replacement of protamines by oocyte histones. Decondensation comprises two steps: disulfide bonds must be reduced (thioreduction) and protamines must be removed. The molecules involved in these steps are reduced glutathione (GSH) and heparan sulfate (HS) from the oocyte. Until now, these events were considered independent and consecutive: first GSH thioreduced protamines and, then HS removed them. Our results indicate otherwise, suggesting that both processes could be simultaneous and cooperative. The aim of this thesis was to advance in the study of the mechanism of nuclear decondensation of human sperm in vitro, using heparin (HS structural analog) and GSH and the extrapolation to the in vivo mechanism during fertilization. A higher degree of decondensation was observed upon coincubating of human sperm or isolated nuclei with heparin and GSH, compared to sequential incubation with the same agents, regardless of the order in which they were added. Use of an acridine orange staining technique as a means to indirectly evaluate the thioreduced status of sperm chromatin, revealed that heparin enhanced GSH thiol reducing activity, suggesting the existence of a cooperative effect between both reagents during decondensation. These results were confirmed by direct thiol reduced status evaluation uthrough labeling of free thiols in sperm chromatin with monobromobimane. The results presented herein indicate that protamine thiol reduction by GSH and removal of protamines by heparin during chromatin decondensation of human sperm in vitro, occur simultaneously rather than sequentially, since a cooperative effect could be observed between both decondensing agents.Fil:Julianelli, Vanina Laura. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

The presence of heparan sulfate in the mammalian oocyte provides a clue to human sperm nuclear decondensation in vivo

BACKGROUND: Previous results from our laboratory have led us to propose heparan sulfate (HS) as a putative protamine acceptor during human sperm decondensation in vivo. The aim of this paper was to investigate the presence of glycosaminoglycans in the mammalian oocyte in an effort to better support this contention. METHODS: Two experimental approaches are used: oocyte labeling to identify the presence of HS and analysis of sperm decondensing ability of fresh oocytes in the presence or absence of specific glycosidases. RESULTS: Staining of mouse zona-intact oocytes with the fluorescent cationic dye, Rubipy, at pH 1.5 allowed for the detection of sulfate residues in the ooplasm by confocal microscopy. HS was detected in the ooplasm by immunocytochemistry. A sperm decondensation microassay using heparin and glutathione was successfully developed. The same level of sperm decondensation could be attained when heparin was replaced by mouse zona-free oocytes. Addition of heparinase to the oocyte/glutathione mixture significantly reduced sperm decondensation (P = 0.0159), while there was no effect following addition of either chondroitinase ABC or hyaluronidase. CONCLUSIONS: The results presented in this paper demonstrate for the first time that HS is present in the mammalian oocyte and show that HS is necessary for fresh oocytes to express their sperm decondensing ability in vitro. © The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.Fil: Romanato, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Julianelli, Vanina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Zappi, M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Piñeiro, Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Calvo, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Role of versican and hyaluronan in the differentiation of 3T3-L1 cells into preadipocytes and mature adipocytes

We have previously shown that during the adipose conversion of these cells the culture medium changed its viscoelastic properties due to the presence of hyaluronan and a chondroitin sulfate proteoglycan [Calvo, J.C., Rodbard, D., Katki, A., Chernick, S., and Yanagishita, M., 1991. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans. J. Biol. Chem. 266, 11237-11244., Calvo, J.C., Gandjbakhche, A.H., Nossal, R., Hascall, V.C., and Yanagishita, M., 1993. Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures. Arch. Biochem. Biophys. 302, 468-475]. Here, we analyze the time course for the appearance of these molecules during drug-induced cell differentiation. The synthesis of both hyaluronan and the proteoglycan, was maximal at 48 h in the presence of isobutylmethylxanthine and dexamethasone, but while hyaluronan remained high after changing the culture medium, the proteoglycan dropped to almost basal levels after a few days. Northern analysis revealed the presence of message for a "versican-like" molecule as well as the possibility of alternative splicing. Three major bands of 9.39, 8.48, and 7.69 kb appeared in the analysis. These bands showed a dramatic increase in intensity when RNA from non-differentiated cells was compared to differentiating 3T3-L1 cells. In addition, when the time course of appearance for this message was analyzed, it perfectly correlated the metabolic labeling of the glycosaminoglycans during cell culture. The nucleotide sequencing of two exons revealed between a 100-94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation.Fil: Zizola, Cynthia F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Julianelli, Vanina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Bertolesi, Gabriel. University of Calgary; CanadáFil: Yanagishita, Masaki. Tokyo Medical and Dental University; JapónFil: Calvo, Juan Carlos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Dermatan sulfate synergizes with heparin in murine sperm chromatin decondensation

El núcleo del espermatozoide de mamíferos contiene la cromatina inusualmente condensada, gracias al reemplazo de la mayoría de las histonas por otras proteínas básicas llamadas protaminas. Sin embargo, apenas el espermatozoide penetra el ooplasma durante la fertilización, la descondensación de esta cromatina densamente compactada, debe ocurrir para permitir la formación del pronúcleo masculino y singamia. La descondensación se logra con la reducción de los puentes disulfuro por el glutatión ovocitario y el reemplazo de las protaminas por histonas ovocitarias, con la ayuda de una molécula aceptora. Resultados previos de nuestro laboratorio demostraron que el heparán sulfato presente en el ooplasma funciona como aceptor de protaminas durante la descondensación del espermatozoide humano in vivo. En el presente trabajo, analizamos el rol de la heparina, análogo estructural del heparán sulfato, y dermatán sulfato en la descondensación de la cromatina espermática murina, incluyendo la posibilidad de un efecto sinérgico entre ambos glicosaminoglicanos. La descondensación fue corroborada bajo microscopio de contraste de fases luego de la incubación de espermatozoides murinos con glutatión y ya sea heparina, dermatán sulfato, o la combinación de ambos. Cambios ultraestructurales que tienen lugar durante la descondensación fueron analizados por microscopia electrónica de transmisión. Ambos glicosaminoglicanos fueron capaces de promover la descondensación de espermatozoides murinos in vitro pero la habilidad de la heparina fue significativamente mayor. El uso de ambos glicosaminoglicanos juntos, revelo la existencia de un efecto sinérgico. El análisis de microscopia electrónica de transmisión de espermatozoides descondensados apoyó estos hallazgos. El sinergismo entre heparina y dermatán sulfato fue observado tanto en espermatozoides capacitados como en no capacitados, pero la cinética de descondensación fue mas rápida en los primeros. Estos resultados obtenidos indican un nuevo rol potencial para el dermatán sulfato en espermatozoides murinos en la descondensación durante la fertilización y proveen evidencia de diferencias en el grado de condensación cromatínica en el núcleo de espermatozoides murinos.The mammalian sperm nucleus contains an unusually condensed chromatin, due to replacement of the majority of histones by protamines. However, soon after the spermatozoon penetrates the ooplasm at fertilization, decondensation of this densely packed chromatin must occur to allow formation of the male pronucleus and syngamy. Decondensation is accomplished by protamine disulfide bond reduction by oocyte glutathione and replacement of protamines by oocyte histones with the aid of an acceptor molecule. Previous results from our laboratory have demonstrated that heparan sulfate (HS) present in the ooplasm functions as protamine acceptor during human sperm decondensation in vivo. In the present paper, we analyze the role of heparin, structural analogue of HS, and dermatan sulfate (DS) in murine sperm chromatin decondensation in vitro, including the possibility of a synergistic effect between both glycosaminoglycans. Decondensation was assessed under phase contrast microscopy following incubation of murine spermatozoa with glutathione and either heparin, DS, or a combination of both. Ultrastructural changes taking place during decondensation were analyzed by transmission electron microscopy. Both glycosaminoglycans were able to promote the decondensation of murine spermatozoa in vitro but the decondensing ability of heparin was significantly higher. Use of both glycosaminoglycans together revealed the existence of a synergistic effect. Transmission electron microscopy analysis of decondensing spermatozoa supported these findings. Synergism between heparin and DS was observed both in capacitated and non-capacitated spermatozoa but decondensation kinetics was faster in the former. The results obtained indicate a new potential role for dermatan sulfate in murine sperm decondensation at fertilization and provide evidence of differences in the degree of chromatin condensation throughout the murine sperm nucleus.Fil: Sanchez, Melisa Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Alverez Sedo, Cristian. Centro de Estudios En Ginecologia y Reproduccion; ArgentinaFil: Julianelli, Vanina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Romanato, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Calvo, Lucrecia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Calvo, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Fontana, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa

BACKGROUND: Human sperm nuclear decondensation in vivo involves protamine disulfide bond reduction by glutathione (GSH) and protamine/histone exchange, presumably with heparan sulfate (HS) as the protamine acceptor. The aim of the present study was to test the hypothesis that these two events occur simultaneously rather than sequentially, as has been hitherto accepted, and to test for the presence of HS in the human oocyte. METHODS: Spermatozoa and isolated sperm nuclei obtained from normal volunteers were exposed in vitro to heparin, the functional analogue of HS and either GSH or dithiothreitol (DTT) as the disulfide reducing agent. Decondensing reagents were added either simultaneously or sequentially. Percentage sperm nuclear decondensation was assayed by phase contrast microscopy. Thiol reduced status of isolated sperm nuclei was evaluated both indirectly [acridine orange (AO) staining of acid-denatured DNA] and directly [monobromobimane (mBBr) staining of protamine-free thiols]. The presence of HS in mature metaphase II (MII) human oocytes was analyzed by immunocytochemistry. RESULTS: Sequential addition of reagents always resulted in significantly lower decondensation if GSH was used as the disulfide bond reducer (P < 0.05 for sperm and P < 0.001 for nuclei), but only when heparin was used first, when DTT was the disulfide reducing agent (P < 0.05 for sperm and P < 0.01 for nuclei). Both AO staining of DNA and mBBr staining of protamines revealed that the addition of heparin to GSH but not to DTT significantly increased the thiol reduced status of sperm chromatin. HS was detected in the ooplasm of zona-free MII human oocytes. CONCLUSIONS: The results presented in this paper clearly show that heparin enhances the sperm chromatin thiol reducing activity of GSH in vitro, suggesting that in vivo thiol reduction and protamine/histone exchange could occur as simultaneous, rather than sequential, events. We also demonstrate for the first time the presence of HS in the human oocyte.Fil: Julianelli, Vanina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Farrando, Bárbara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Alvarez Sedó, C.. CEGYR Medicina Reproductiva; ArgentinaFil: Piñeiro, Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Romanato, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Calvo, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Human sperm decondensation in vitro is related to cleavage rate and embryo quality in IVF

Purpose: To investigate whether the ability of human spermatozoa to decondense in vitro in the presence of heparin (Hep) and glutathione (GSH) is related to assisted reproduction (ART) success. Methods: Cross-sectional pilot study involving male partners of 129 infertile couples undergoing ICSI with (45) or without (84) donor oocytes at two infertility clinics in CABA, Argentina, between October 2012 and December 2013. In vitro decondensation kinetics with Hep and GSH and DNA fragmentation (TUNEL) were determined on the same sample used for ICSI. The possible relationship of decondensation parameters (maximum decondensation and decondensation velocity) and TUNEL values with ART success was evaluated. Results: Embryo quality correlated positively with decondensation velocity (D60/D30) (Spearman?s correlation, p < 0.05). According to D60/D30 values, patients were classified as slow decondensers (SlowD) (n = 68) or fast decondensers (FastD) (n = 61). Embryo quality was better in FastD (unpaired t test, p < 0.05). FastD and SlowD were subdivided according to use of donor oocytes. Among SlowD, biochemical and clinical pregnancy rates per transfer were significantly higher in donor (n = 19) vs. in non-donor (n = 31) cycles (Fisher?s exact test, p < 0.05). TUNEL values were not related to embryo quality, but no clinical pregnancies or live births were achieved in TUNEL+ SlowD (n = 7). Conclusion: Decondensation kinetics of human spermatozoa in vitro with Hep and GSH could be related to embryo quality and ART success.Fil: Galotto, Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cambiasso, Maite Yael. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Julianelli, Vanina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Procrearte; ArgentinaFil: Rey Valzacchi, Gaston Javier. Procrearte; Argentina. Hospital Italiano; ArgentinaFil: Rolando, R. N.. Hospital Italiano; ArgentinaFil: Rodriguez, M. L.. Hospital Italiano; ArgentinaFil: Calvo, L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Calvo, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires; ArgentinaFil: Romanato, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin