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24. Alzheimer's disease-related amyloid-β peptide induces the loss of human sperm function.

Author

Tavares, R., Martins, S., Almeida-Santos, T., Sousa, A., Ramalho-Santos, J., and Cruz e Silva, O.

Subjects
ALZHEIMER'S disease, SPERMATOZOA, AMYLOID beta-protein precursor, IMMUNOCYTOCHEMISTRY, PHOSPHORYLATION
Abstract

Characteristically identified as the main component of senile plaques present in patients suffering from Alzheimer's disease, Aβ has been detected in human testis and reproductive fluids, but its effect on spermatozoa has not been addressed. The present study evaluated whether the most toxic and aggregant amyloid precursor protein (APP)-proteolytic product, amyloid-β (Aβ), was capable of affecting sperm functionality. Normozoospermic samples were either exposed to different Aβ doses or to the untreated and scrambled controls for a maximum of 48 h at 37 °C and 5%CO, and motility, viability and mitochondrial status were evaluated. Additionally, tyrosine phosphorylation was analyzed by immunocytochemistry and acrosomal integrity through PSA-FITC. A shorter treatment period was used to monitor prompt Ca responses. Aβ peptide decreased motility before inducing mitochondrial impairment ( p < 0.05; n = 6). Both outcomes became more pronounced with time, reaching their maximal decrease at 48 h, where even 1 μM produced undesirable effects ( p < 0.05; n = 6). Aβ peptide also decreased cell survival ( p < 0.05; n = 6). Furthermore, although no effects on tyrosine phosphorylation were observed ( p > 0.05; n = 6), reduced acrosomal integrity was detected ( p < 0.05; n = 7), which was not correlated with viability loss ( p > 0.05). In parallel, all Aβ concentrations elicited a [Ca] rise but a significant difference was only observed at 20 μM ( p < 0.05; n = 7) and a tendency was obtained with 10 μM ( p = 0.053; n = 7). In conclusion, Aβ peptide oligomers impair sperm function in vitro, although further studies are required to determine the clinical relevance of these findings. [ABSTRACT FROM AUTHOR]

Published
2017
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27. Golgi apparatus dynamics during mouse oocyte in vitro maturation: effect of the membrane trafficking inhibitor brefeldin A

Author

Ricardo Moreno, Schatten, G., and Ramalho-Santos, J.

Subjects
Meiosis, Gamete biology, Gametogenesis, Oocyte development
Abstract

We have studied Golgi apparatus dynamics during mouse oocyte in vitro maturation, employing both live imaging with the fluorescent lipid BODIPY-ceramide and immunocytochemistry using several specific markers (beta-COP, giantin, and TGN38). In germinal vesicle oocytes the Golgi consisted of a series of structures, possibly cisternal stacks, dispersed in the ooplasm, but slightly more concentrated in the interior than at the cortex. A similar pattern was detected in rhesus monkey germinal vesicle oocytes. These "mini-Golgis" were functionally active because they were reversibly disrupted by the membrane trafficking inhibitor brefeldin A. However, the drug had no visible effect if the oocytes had been previously microinjected with GTP-gamma-S. During in vitro maturation the large Golgi apparatus structures fragmented at germinal vesicle breakdown, and dispersed homogenously throughout the ooplasm, remaining in a fragmented state in metaphase-II oocytes. Similarly to what has been reported using protein synthesis inhibitors, the presence of brefeldin A blocked maturation at the germinal vesicle breakdown stage before the assembly of the metaphase-I spindle. These results suggest that progression of murine oocyte maturation may require functional membrane trafficking

Published
2002

28. Energy metabolism in human pluripotent stem cells and their differentiated counterparts

Author

Varum, S, Rodrigues, AS, Moura, MB, Momcilovic, O, Easley IV, CA, Ramalho-Santos, J, van Houten, B, Schatten, G, Varum, S, Rodrigues, AS, Moura, MB, Momcilovic, O, Easley IV, CA, Ramalho-Santos, J, van Houten, B, and Schatten, G

Abstract

Background: Human pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells. Methodology/Principal Findings: We compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism. Conclusions/Findings: Our results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human p

Published
2011

29. Female (in)fertility

Author

Saad, H., primary, Khalil, E., additional, Bora, S. A., additional, Parikh, J., additional, Abdalla, H., additional, Thum, M. Y., additional, Bina, V., additional, Roopa, P., additional, Shyamala, S., additional, Anupama, A., additional, Tournaye, H., additional, Polyzos, N. P., additional, Guzman, L., additional, Nelson, S. M., additional, Lourenco, B., additional, Sousa, A. P., additional, Almeida-Santos, T., additional, Ramalho-Santos, J., additional, Okhowat, J., additional, Wirleitner, B., additional, Neyer, T., additional, Bach, M., additional, Murtinger, M., additional, Zech, N. H., additional, Nwoye, M., additional, Corona, R., additional, Blockeel, C., additional, Stoop, D., additional, Camus, M., additional, Rajikin, M. H., additional, Kamsani, Y. S., additional, Chatterjee, A., additional, Nor-Ashikin, M. N. K., additional, Nuraliza, A. S., additional, Scaravelli, G., additional, D'Aloja, P., additional, Bolli, S., additional, De Luca, R., additional, Spoletini, R., additional, Fiaccavento, S., additional, Speziale, L., additional, Vigiliano, V., additional, Farquhar, C., additional, Brown, J., additional, Arroll, N., additional, Gupta, D., additional, Boothroyd, C., additional, Al Bassam, M., additional, Moir, J., additional, Johnson, N., additional, Pantasri, T., additional, Robker, R. L., additional, Wu, L. L., additional, Norman, R. J., additional, Buzaglo, K., additional, Velez, M., additional, Shaulov, T., additional, Sylvestre, C., additional, Kadoch, I. J., additional, Krog, M., additional, Prior, M., additional, Carlsen, E., additional, Loft, A., additional, Pinborg, A., additional, Andersen, A. N., additional, Dolleman, M., additional, Verschuren, W. M. M., additional, Eijkemans, M. J. C., additional, Dolle, M. E. T., additional, Jansen, E. H. J. M., additional, Broekmans, F. J. M., additional, Van der Schouw, Y. T., additional, Fainaru, O., additional, Pencovich, N., additional, Hantisteanu, S., additional, Barzilay, I., additional, Ellenbogen, A., additional, Hallak, M., additional, Cavagna, M., additional, Baruffi, R. L. R., additional, Petersen, C. G., additional, Mauri, A. L., additional, Massaro, F. C., additional, Ricci, J., additional, Nascimento, A. M., additional, Vagnini, L. D., additional, Pontes, A., additional, Oliveira, J. B. A., additional, Franco, J. G., additional, Canas, M. C. T., additional, Nicoletti, A., additional, Martins, A. M. V. C., additional, Lichtblau, I., additional, Olivennes, F., additional, Aubriot, F. A., additional, Junca, A. M., additional, Belloc, S., additional, Cohen-Bacrie, M., additional, Cohen-Bacrie, P., additional, de Mouzon, J., additional, Nandy, T., additional, Caragia, A., additional, Balestrini, S., additional, Zosmer, A., additional, Sabatini, L., additional, Al-Shawaf, T., additional, Seshadri, S., additional, Khalaf, Y., additional, Sunkara, S. K., additional, Joy, J., additional, Lambe, M., additional, Lutton, D., additional, Nicopoullos, J., additional, Faris, R., additional, Behre, H. M., additional, Howles, C. M., additional, Longobardi, S., additional, Chimote, N., additional, Mehta, B., additional, Nath, N., additional, Chimote, N. M., additional, Mine, K., additional, Yoshida, A., additional, Yonezawa, M., additional, Ono, S., additional, Abe, T., additional, Ichikawa, T., additional, Tomiyama, R., additional, Nishi, Y., additional, Kuwabara, Y., additional, Akira, S., additional, Takeshita, T., additional, Shin, H., additional, Song, H. S., additional, Lim, H. J., additional, Hauzman, E., additional, Kohls, G., additional, Barrio, A., additional, Martinez-Salazar, J., additional, Iglesias, C., additional, Velasco, J. A. G., additional, Tejada, M. I., additional, Maortua, H., additional, Mendoza, R., additional, Prieto, B., additional, Martinez-Bouzas, C., additional, Diez-Zapirain, M., additional, Martinez-Zilloniz, N., additional, Matorras, R., additional, Amaro, A., additional, Bianco, B., additional, Christofolini, J., additional, Mafra, F. A., additional, Barbosa, C. P., additional, Christofolini, D. M., additional, Pesce, R., additional, Gogorza, S., additional, Ochoa, C., additional, Gil, S., additional, Saavedra, A., additional, Ciarmatori, S., additional, Perman, G., additional, Pagliardini, L., additional, Papaleo, E., additional, Corti, L., additional, Vanni, V. S., additional, Ottolina, J., additional, de Michele, F., additional, Marca, A. L., additional, Vigano, P., additional, Candiani, M., additional, Li, L., additional, Yin, Q., additional, Huang, L., additional, Huang, J., additional, He, Z., additional, Yang, D., additional, Tiplady, S., additional, Ledger, W., additional, Godbert, S., additional, Hart, S., additional, Johnson, S., additional, Wong, A. W. Y., additional, Kong, G. W. S., additional, Haines, C. J., additional, Franik, S., additional, Nelen, W., additional, Kremer, J., additional, Gillett, W. R., additional, Lamont, J. M., additional, Peek, J. C., additional, Herbison, G. P., additional, Sung, N. Y., additional, Hwang, Y. I., additional, Choi, M. H., additional, Song, I. O., additional, Kang, I. S., additional, Koong, M. K., additional, Lee, J. S., additional, Yang, K. M., additional, Celtemen, M. B., additional, Telli, P., additional, Karakaya, C., additional, Bozkurt, N., additional, Gursoy, R. H., additional, Younis, J. S., additional, Ben-Ami, M., additional, Pundir, J., additional, Pundir, V., additional, Omanwa, K., additional, and El-Toukhy, T., additional

Published
2013
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31. ANDROLOGY

Author

Hu, J. C. Y., primary, Seo, B. K., additional, Neri, Q. V., additional, Rozenwaks, Z., additional, Palermo, G. D., additional, Fields, T., additional, Monahan, D., additional, Rosenwaks, Z., additional, Szkodziak, P., additional, Plewka, K., additional, Wozniak, S., additional, Czuczwar, P., additional, Mroczkowski, A., additional, Lorenzo Leon, C., additional, Hernandez, J., additional, Chinea Mendez, E., additional, Concepcion Lorenzo, C., additional, Sanabria Perez, V., additional, Puopolo, M., additional, Palumbo, A., additional, Toth, B., additional, Franz, C., additional, Montag, M., additional, Boing, A., additional, Strowitzki, T., additional, Nieuwland, R., additional, Griesinger, G., additional, Schultze-Mosgau, A., additional, Cordes, T., additional, Depenbusch, M., additional, Diedrich, K., additional, Vloeberghs, V., additional, Verheyen, G., additional, Camus, M., additional, Van de Velde, H., additional, Goossens, A., additional, Tournaye, H., additional, Coppola, G., additional, Di Caprio, G., additional, Wilding, M., additional, Ferraro, P., additional, Esposito, G., additional, Di Matteo, L., additional, Dale, R., additional, Dale, B., additional, Daoud, S., additional, Auger, J., additional, Wolf, J. P., additional, Dulioust, E., additional, Lafuente, R., additional, Lopez, G., additional, Brassesco, M., additional, Hamad, M., additional, Montenarh, M., additional, Hammadeh, M., additional, Robles, F., additional, Magli, M. C., additional, Crippa, A., additional, Pescatori, E., additional, Ferraretti, A. P., additional, Gianaroli, L., additional, Zahiri, M., additional, Movahedin, M., additional, Mowla, S. J., additional, Noruzinia, M., additional, Crivello, A. M., additional, Sermondade, N., additional, Dupont, C., additional, Hafhouf, E., additional, Cedrin-Durnerin, I., additional, Poncelet, C., additional, Benzacken, B., additional, Levy, R., additional, Sifer, C., additional, Ferfouri, F., additional, Boitrelle, F., additional, Clement, P., additional, Molina Gomes, D., additional, Bailly, M., additional, Selva, J., additional, Vialard, F., additional, Yaprak, E., additional, Basar, M., additional, Guzel, E., additional, Arda, O., additional, Irez, T., additional, Norambuena, P., additional, Krenkova, P., additional, Tuettelmann, F., additional, Kliesch, S., additional, Paulasova, P., additional, Stambergova, A., additional, Macek, M., additional, Rivera, R., additional, Garrido-Gomez, T., additional, Galletero, S., additional, Meseguer, M., additional, Dominguez, F., additional, Garrido, N., additional, Mallidis, C., additional, Sanchez, V., additional, Weigeng, L., additional, Redmann, K., additional, Wistuba, J., additional, Gross, P., additional, Wuebbelling, F., additional, Fallnich, C., additional, Burger, M., additional, Schlatt, S., additional, San Celestino Carchenilla, M., additional, Pacheco Castro, A., additional, Simon Sanjurjo, P., additional, Molinero Ballesteros, A., additional, Rubio Garcia, S., additional, Garcia Velasco, J. A., additional, Macanovic, B., additional, Otasevic, V., additional, Korac, A., additional, Vucetic, M., additional, Garalejic, E., additional, Ivanovic Burmazovic, I., additional, Filipovic, M. R., additional, Buzadzic, B., additional, Stancic, A., additional, Jankovic, A., additional, Velickovic, K., additional, Golic, I., additional, Markelic, M., additional, Korac, B., additional, Gosalvez, J., additional, Ruiz-Jorro, M., additional, Garcia-Ochoa, C., additional, Sachez-Martin, P., additional, Martinez-Moya, M., additional, Caballero, P., additional, Hasegawa, N., additional, Fukunaga, N., additional, Nagai, R., additional, Kitasaka, H., additional, Yoshimura, T., additional, Tamura, F., additional, Kato, M., additional, Nakayama, K., additional, Oono, H., additional, Kojima, E., additional, Yasue, K., additional, Watanabe, H., additional, Asano, E., additional, Hashiba, Y., additional, Asada, Y., additional, Das, M., additional, Al-Hathal, N., additional, San-Gabriel, M., additional, Phillips, S., additional, Kadoch, I. J., additional, Bissonnette, F., additional, Holzer, H., additional, Zini, A., additional, Zebitay, A. G., additional, Ocal, P., additional, Sahmay, S., additional, Karahuseyinoglu, S., additional, Usta, T., additional, Repping, S., additional, Silber, S., additional, Van Wely, M., additional, Datta, A., additional, Nayini, K., additional, Eapen, A., additional, Barlow, S., additional, Lockwood, G., additional, Tavares, R., additional, Baptista, M., additional, Publicover, S. J., additional, Ramalho-Santos, J., additional, Vaamonde, D., additional, Rodriguez, I., additional, Diaz, A., additional, Darr, C., additional, Chow, V., additional, Ma, S., additional, Smith, R., additional, Jeria, F., additional, Rivera, J., additional, Gabler, F., additional, Nicolai, H., additional, Cunha, M., additional, Viana, P., additional, Goncalves, A., additional, Silva, J., additional, Oliveira, C., additional, Teixeira da Silva, J., additional, Ferraz, L., additional, Madureira, C., additional, Doria, S., additional, Sousa, M., additional, Barros, A., additional, Herrero, M. B., additional, Delbes, G., additional, Troueng, E., additional, Chan, P. T. K., additional, Vingris, L., additional, Setti, A. S., additional, Braga, D. P. A. F., additional, Figueira, R. C. S., additional, Iaconelli, A., additional, Borges, E., additional, Sargin Oruc, A., additional, Gulerman, C., additional, Zeyrek, T., additional, Yilmaz, N., additional, Tuzcuoglu, D., additional, Cicek, N., additional, Scarselli, F., additional, Terribile, M., additional, Franco, G., additional, Zavaglia, D., additional, Dente, D., additional, Zazzaro, V., additional, Riccio, T., additional, Minasi, M. G., additional, Greco, E., additional, Cejudo-Roman, A., additional, Ravina, C. G., additional, Candenas, L., additional, Gallardo-Castro, M., additional, Martin-Lozano, D., additional, Fernandez-Sanchez, M., additional, Pinto, F. M., additional, Balasuriya, A., additional, Serhal, P., additional, Doshi, A., additional, Harper, J., additional, Romany, L., additional, Fernandez, J. L., additional, Pellicer, A., additional, Ribas-Maynou, J., additional, Garcia-Peiro, A., additional, Fernandez-Encinas, A., additional, Prada, E., additional, Jorda, I., additional, Cortes, P., additional, Llagostera, M., additional, Navarro, J., additional, Benet, J., additional, Kesici, H., additional, Cayli, S., additional, Erdemir, F., additional, Karaca, Z., additional, Aslan, H., additional, Ocakli, S., additional, Tas, U., additional, Ozdemir, A. A., additional, Aktas, R. G., additional, Tok, O. E., additional, Li, S., additional, Lu, C., additional, Hwu, Y., additional, Lee, R. K., additional, Landaburu, I., additional, Gonzalvo, M. C., additional, Clavero, A., additional, Ramirez, J. P., additional, Pedrinaci, S., additional, Serrano, M., additional, Montero, L., additional, Carrillo, S., additional, Weiss, J., additional, Ortiz, A. P., additional, Castilla, J. A., additional, Sahin, O., additional, Bakircioglu, E., additional, Serdarogullari, M., additional, Bayram, A., additional, Yayla, S., additional, Ulug, U., additional, Tosun, S. B., additional, Bahceci, M., additional, Yoon, S. Y., additional, Shin, D. H., additional, Shin, T. E., additional, Park, E. A., additional, Won, H. J., additional, Kim, Y. S., additional, Lee, W. S., additional, Yoon, T. K., additional, Lee, D. R., additional, Hattori, H., additional, Nakajo, Y., additional, Kyoya, T., additional, Kuchiki, M., additional, Kanto, S., additional, Kyono, K., additional, Park, M., additional, Park, M. R., additional, Lim, E. J., additional, Choi, Y., additional, Mitra, A., additional, Bhattacharya, J., additional, Kundu, A., additional, Mukhopadhaya, D., additional, Pal, M., additional, Enciso, M., additional, Alfarawati, S., additional, Wells, D., additional, Abad, C., additional, Amengual, M. J., additional, Esmaeili, V., additional, Safiri, M., additional, Shahverdi, A. H., additional, Alizadeh, A. R., additional, Ebrahimi, B., additional, Brucculeri, A. M., additional, Ruvolo, G., additional, Giovannelli, L., additional, Schillaci, R., additional, Cittadini, E., additional, Scaravelli, G., additional, Perino, A., additional, Cortes Gallego, S., additional, Gabriel Segovia, A., additional, Nunez Calonge, R., additional, Guijarro Ponce, A., additional, Ortega Lopez, L., additional, Caballero Peregrin, P., additional, Heindryckx, B., additional, Kashir, J., additional, Jones, C., additional, Mounce, G., additional, Ramadan, W. M., additional, Lemmon, B., additional, De Sutter, P., additional, Parrington, J., additional, Turner, K., additional, Child, T., additional, McVeigh, E., additional, Coward, K., additional, Tosun, S., additional, Ciray, N., additional, Saeidi, S., additional, Shapouri, F., additional, Hoseinifar, H., additional, Sabbaghian, M., additional, Pacey, A., additional, Aflatoonian, R., additional, Bosco, L., additional, Carrillo, L., additional, Pane, A., additional, Manno, M., additional, Roccheri, M. C., additional, Selles, E., additional, Garcia-Herrero, S., additional, Martinez, J. A., additional, Munoz, M., additional, Durmaz, A., additional, Dikmen, N., additional, Gunduz, C., additional, Tavmergen Goker, E., additional, Tavmergen, E., additional, Gozuacik, D., additional, Vatansever, H. S., additional, Kara, B., additional, Calimlioglu, N., additional, Yasar, P., additional, Semerci, B., additional, Baka, M., additional, Ozbilgin, K., additional, Karabulut, A., additional, Tekin, A., additional, Sabah, B., additional, Cottin, V., additional, Kottelat, D., additional, Fellmann, M., additional, Halm, S., additional, Rosenthaler, E., additional, Kisida, T., additional, Kojima, F., additional, Sakamoto, T., additional, Makutina, V. A., additional, Balezin, S. L., additional, Rosly, O. F., additional, Slishkina, T. V., additional, Hatzi, E., additional, Lazaros, L., additional, Xita, N., additional, Makrydimas, G., additional, Sofikitis, N., additional, Kaponis, A., additional, Stefos, T., additional, Zikopoulos, K., additional, Georgiou, I., additional, Hibi, H., additional, Ohori, T., additional, Sumitomo, M., additional, Anarte, C., additional, Calvo, I., additional, Domingo, A., additional, Presilla, N., additional, Aleman, M., additional, Bou, R., additional, Guardiola, F., additional, Agirregoikoa, J. A., additional, De Pablo, J. L., additional, Barrenetxea, G., additional, Zhylkova, I., additional, Feskov, O., additional, Feskova, I., additional, Zozulina, O., additional, Somova, O., additional, Nabi, A., additional, Khalili, M. A., additional, Roudbari, F., additional, Parmegiani, L., additional, Cognigni, G. E., additional, Bernardi, S., additional, Taraborrelli, S., additional, Troilo, E., additional, Ciampaglia, W., additional, Pocognoli, P., additional, Infante, F. E., additional, Tabarelli de fatis, C., additional, Arnone, A., additional, Maccarini, A. M., additional, Filicori, M., additional, Silva, L., additional, Oliveira, J. B. A., additional, Petersen, C. G., additional, Mauri, A. L., additional, Massaro, F. C., additional, Cavagna, M., additional, Baruffi, R. L. R., additional, Franco, J. G., additional, Fujii, Y., additional, Endou, Y., additional, Mtoyama, H., additional, Shokri, S., additional, and Aitken, R. J., additional

Published
2012
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38. Identification of endogenous metabolites in human sperm cells using proton nuclear magnetic resonance (1H-NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS).

Author

Paiva, C., Amaral, A., Rodriguez, M., Canyellas, N., Correig, X., Ballescà, J. L., Ramalho‐Santos, J., and Oliva, R.

Subjects
METABOLITE analysis, METABOLITE synthesis, SPERMATOZOA, GAS chromatography/Mass spectrometry (GC-MS), LABORATORY techniques
Abstract

The objective of this study was to contribute to the first comprehensive metabolomic characterization of the human sperm cell through the application of two untargeted platforms based on proton nuclear magnetic resonance (1H-NMR) spectroscopy and gas chromatography coupled to mass spectrometry (GC-MS). Using these two complementary strategies, we were able to identify a total of 69 metabolites, of which 42 were identified using NMR, 27 using GC-MS and 4 by both techniques. The identity of some of these metabolites was further confirmed by two-dimensional 1H-1H homonuclear correlation spectroscopy (COSY) and 1H-13C heteronuclear single-quantum correlation (HSQC) spectroscopy. Most of the metabolites identified are reported here for the first time in mature human spermatozoa. The relationship between the metabolites identified and the previously reported sperm proteome was also explored. Interestingly, overrepresented pathways included not only the metabolism of carbohydrates, but also of lipids and lipoproteins. Of note, a large number of the metabolites identified belonged to the amino acids, peptides and analogues super class. The identification of this initial set of metabolites represents an important first step to further study their function in male gamete physiology and to explore potential reasons for dysfunction in future studies. We also demonstrate that the application of NMR and MS provides complementary results, thus constituting a promising strategy towards the completion of the human sperm cell metabolome. [ABSTRACT FROM AUTHOR]

Published
2015
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43. ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.

Author

Ramalho-Santos, João, Sutovsky, Peter, Simerly, Calvin, Oko, Richard, Wessel, Gary M., Hewitson, Laura, Schatten, Gerald, Ramalho-Santos, J, Sutovsky, P, Simerly, C, Oko, R, Wessel, G M, Hewitson, L, and Schatten, G

Abstract

We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle. [ABSTRACT FROM AUTHOR]

Published
2000
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44. On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTracker(TM) and high resolution differential interference contrast microscopy.

Author

Sutovsky, Peter, Ramalho-Santos, João, Moreno, Ricardo D., Oko, Richard, Hewitson, Laura, Schatten, Gerald, Sutovsky, P, Ramalho-Santos, J, Moreno, R D, Oko, R, Hewitson, L, and Schatten, G

Subjects
ANIMAL experimentation, CATTLE, CELL separation, COMPARATIVE studies, ELECTRON microscopy, FLUORESCENT antibody technique, RESEARCH methodology, MEDICAL cooperation, MITOCHONDRIA, PRIMATES, RESEARCH, SPERMATOZOA, EVALUATION research, FLUORESCENT dyes
Abstract

The selection of individual round spermatids for round spermatid injection (ROSI), a prerequisite for the successful application of this infertility treatment, has been hampered by the ambiguous definition of a round spermatid and the lack of specific vital and non-vital markers. Using cells from rhesus monkey and bull, we describe a non-invasive method for the on-stage selection of individual round spermatids for ROSI, based on the polarized patterns of mitochondria, visualized in live round spermatid cells by epifluorescence microscopy after incubation with MitoTracker(TM), a vital, mitochondrion-specific fluorescent probe. The correct identification of live round spermatid was confirmed by the presence of the acrosomal granule or acrosomal cap in parallel observations by Nomarski differential interference contrast microscopy. The existence of mitochondrial polarization was first established by the labelling of MitoTracker-tagged round spermatids with spermatid-specific antibodies against proteins of nascent sperm accessory structures combined with antibodies against a nuclear pore complex component, known to disappear at the round spermatid stage. Using an inverted microscope equipped with epifluorescence, the round spermatids can be individually selected from a heterogeneous population of testicular cells labelled with MitoTracker dyes. A major advantage of this approach is that the dyes are incorporated into the paternal mitochondria, destined for rapid elimination after fertilization. In addition, the relatively high excitation and emission wavelengths of MitoTracker dyes are less harmful to DNA after their photon excitation. Before the appropriate clinical testing is conducted, the MitoTracker-based round spermatid selection may be instrumental in the training of clinical staff. [ABSTRACT FROM AUTHOR]

Published
1999
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45. LOCALIZATION OF SNARES, NSF AND CAVEOLIN 1 IN HUMAN SPERMATOZOA: RELATIONSHIP WITH SEMINAL PARAMETERS

Author

Sousa, A. P. M., Gomes-Santos, C. S. S., and Ramalho-Santos, J.

Abstract

Membrane fusion is a very important process in gametes. The mechanism of membrane fusion during the AR has been proposed to involve SNAREs. Our aim is to quantify patterns of localization of Caveolin 1, SNAREs (Syntaxin 1A, Syntaxin 2 and VAMP 1) and NSF on human sperm, to determine how the differential distribution of these proteins might be interdependent and to evaluate if this distribution is related with seminal parameters. These proteins are present in different regions of the head of human sperm: anterior, equatorial and posterior regions and that Syntaxin 2 and Syntaxin 1A had a slightly different pattern of labelling. The presence and localization of SNAREs, NSF and Caveolin 1 do not correlate with seminal parameters. There is significant correlation between NSF and SNAREs, which may indicate a cooperation of these proteins in membrane fusion mechanisms of human sperm.

Published
2006
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46. PRESENCE OF N-ETHYL MALEIMIDE SENSITIVE FACTOR (NSF) ON THE ACROSOME OF MAMMALIAN SPERM

Author

Ramalho-Santos, J. and Schatten, G.

Abstract

We have extended previous observations to show that the ATPase N-ethyl maleimide sensitive factor (NSF) an important regulator of membrane trafficking and fusion in somatic cells, is present on bovine, murine and rhesus macaque sperm. However, NSFs main effectors, alfa- and beta-SNAP, although present in the developing acrosome, could not be detected in the mature organelle. The fact that NSF localizes mainly to the acrosome suggests that this protein, together with other factors such as rabs and SNAREs, may be a common feature in the triggering/regulation of membrane merging during the mammalian acrosome reaction.

Published
2004
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