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

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