Ringraziamo il Bioimmagini WM Keck Laboratorio per l&#39;uso della propria postazione di lavoro microscopia video. Questo studio è stato supportato da NSF concedere IBN-0615435.

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	<li>Ward, G. E., Brokaw, C. J., Garbers, D. L., Vacquier, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+of+Arbacia+punctulata+spermatozoa+to+resact,+a+peptide+from+the+egg+jelly+layer.">Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer.</a> J. Cell Biol. 101, 2324-2329 (1985).</li><li>Olivera, R. G., Tomasi, L., Rovasio, R. A., Giojalas, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+velocity+and+induction+of+chemotactic+response+in+mouse+spermatozoa+by+follicular+and+oviductal+fluids.">Increased velocity and induction of chemotactic response in mouse spermatozoa by follicular and oviductal fluids.</a> J. Reprod. Fertil. 115, 23-27 (1999).</li><li>Fabro, G., Rovasio, R. A., Civalero, S., Frenkel, A., Caplan, S. 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Dev. 76, 527-536 (2009).</li><li>Al-Anzi, B., Chandler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+egg+jelly+releases+a+sperm+chemoattractant+during+spawning.">Xenopus laevis egg jelly releases a sperm chemoattractant during spawning.</a> Dev. Biol. 198, 366-375 (1998).</li><li>Ralt, D., Goldenberg, M., Fetterolf, P., Thompson, D., Dor, J., Mashiach, S., Garbers, D. L., Eisenbach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+attraction+to+a+follicular+factor(s)+correlates+with+human+egg+fertilizability.">Sperm attraction to a follicular factor(s) correlates with human egg fertilizability.</a> Proc. Natl. Acad. Sci. U.S.A. 88, 2840-2844 (1991).</li><li>Gakamsky, A., Schechtman, E., Caplan, S. R., Eisenbach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chemotaxis+when+the+fraction+of+responsive+cells+is+small--application+to+mammalian+sperm+guidance.">Analysis of chemotaxis when the fraction of responsive cells is small--application to mammalian sperm guidance.</a> Int. J. Dev. Biol. 52, 481-487 (2008).</li><li>B&#246;hmer, M., Van, Q., Weyand, I., Hagen, V., Beyermann, M., Matsumoto, M., Hoshi, M., Hildebrand, E., Kaupp, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++spikes+in+the+flagellum+control+chemotactic+behavior+of+sperm.">Ca2+ spikes in the flagellum control chemotactic behavior of sperm.</a> EMBO J. 24, 2741-2752 (2005).</li><li>Shiba, K., Baba, S. A., Inoue, T., Yoshida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++bursts+occur+around+a+local+minimal+concentration+of+attractant+and+trigger+sperm+chemotactic+response.">Ca2+ bursts occur around a local minimal concentration of attractant and trigger sperm chemotactic response.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 19312-19317 (2008).</li><li>Burnett, L. A., Sugiyama, H., Bieber, A. L., Chandler, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egg+jelly+proteins+stimulate+directed+motility+in+Xenopus+laevis+sperm.">Egg jelly proteins stimulate directed motility in Xenopus laevis sperm.</a> Mol. Reprod. Dev. 78, 450-462 (2011).</li><li>Abaigar, T., Barbero, J., Holt, W. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trajectory+variance+and+autocorrelations+within+single+sperm+tracks+as+population+level+descriptors+of+sperm+track+complexity,+predictability+and+energy+generating+ability.">Trajectory variance and autocorrelations within single sperm tracks as population level descriptors of sperm track complexity, predictability and energy generating ability.</a> J. Androl. , (2011).</li><li>Mortimer, S. T., Swan, M. A., Mortimer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractal+analysis+of+capacitating+human+spermatozoa.">Fractal analysis of capacitating human spermatozoa.</a> Hum. Reprod. 11, 1049-1054 (1996).</li><li>Guerrero, A., Carneiro, J., Pimentel, A., Wood, C. D., Corkidi, G., Darszon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+locating+the+female+gamete:+the+importance+of+measuring+sperm+trajectories+in+three+spatial+dimensions.">Strategies for locating the female gamete: the importance of measuring sperm trajectories in three spatial dimensions.</a> Mol. Hum. Reprod. 17, 511-523 (2011).</li><li>Yoshida, M., Yoshida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+chemotaxis+and+regulation+of+flagellar+movement+by+Ca2+.">Sperm chemotaxis and regulation of flagellar movement by Ca2+.</a> Mol. Hum. Reprod. 17, 457-465 (2011).</li><li>Spehr, M., Schwane, K., Riffell, J. A., Zimmer, R. K., Hatt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptors+and+olfactory-like+signaling+mechanisms+in+mammalian+sperm.Mol.">Odorant receptors and olfactory-like signaling mechanisms in mammalian sperm.Mol.</a> Cell. Endocrinol. 250, 128-136 (2006).</li><li>Veitinger, T., Riffell, J. R., Veitinger, S., Nascimento, J. M., Triller, A., Chandsawangbhuwana, C., Schwane, K., Geerts, A., Wunder, F., Berns, M. W., Neuhaus, E. M., Zimmer, R. K., Spehr, M., Hatt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+Ca2++dynamics+correlate+with+diverse+behavioral+phenotypes+in+human+sperm.">Chemosensory Ca2+ dynamics correlate with diverse behavioral phenotypes in human sperm.</a> J. Biol. Chem. 286, 17311-17325 (2011).</li><li>Tholl, N., Naqvi, S., McLaughlin, E., Boyles, S., Bieber, A. L., Chandler, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming+of+Xenopus+laevis+sperm+exhibits+multiple+gears+and+its+duration+is+extended+by+egg+jelly+constituents.">Swimming of Xenopus laevis sperm exhibits multiple gears and its duration is extended by egg jelly constituents.</a> Biol. Bull. 220, 174-185 (2011).</li><li>Eisenbach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+chemotaxis.">Sperm chemotaxis.</a> Rev. Reprod. 4, 56-66 (1999).</li><li>Riffell, J. A., Zimmer, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+and+flow:+the+consequences+of+fluid+shear+for+sperm-egg+interactions.">Sex and flow: the consequences of fluid shear for sperm-egg interactions.</a> J. Exp. Biol. 210, 3644-3660 (2007).</li><li>Corkidi, G., Taboada, B., Wood, C. D., Guerrero, A., Darszon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+sperm+in+three-dimensions.">Tracking sperm in three-dimensions.</a> Biochem. Biophys. Res. Commun. 373, 125-129 (2008).</li><li>Himes, J. E., Riffell, J. A., Zimmer, C. A., Zimmer, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+chemotaxis+as+revealed+with+live+and+synthetic+eggs.">Sperm chemotaxis as revealed with live and synthetic eggs.</a> Biol Bull. 220, 1-5 (2011).</li><li>Sun, F., Giojolas, L. C., Rovasio, R. A., Tur-Kaspa, I., Sanchez, R., Eisenbach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+species-specificity+in+mammalian+sperm+chemotaxis.">Lack of species-specificity in mammalian sperm chemotaxis.</a> Dev. Biol. 255, 423-427 (2003).</li><li>Guidobaldi, H. A., Teves, M. E., U&#241;ates, D. R., Anastas&#237;a, A., Giojalas, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+from+the+cumulus+cells+is+the+sperm+chemoattractant+secreted+by+the+rabbit+oocyte+cumulus+complex.">Progesterone from the cumulus cells is the sperm chemoattractant secreted by the rabbit oocyte cumulus complex.</a> PLoS One. 3, e3040-e3040 (2008).</li><li>Oren-Benaroya, R., Orvieto, R., Gakamsky, A., Pinchasov, M., Eisenbach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sperm+chemoattractant+secreted+from+human+cumulus+cells+is+progesterone.">The sperm chemoattractant secreted from human cumulus cells is progesterone.</a> Hum. Reprod. 23, 2339-2345 (2008).</li><li>Teves, M. E., Guidobaldi, H. A., U&#241;ates, D. R., Sanchez, R., Miska, W., Publicover, S. J., Morales Garcia, A. A., Giojalas, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+for+human+sperm+chemotaxis+mediated+by+progesterone.">Molecular mechanism for human sperm chemotaxis mediated by progesterone.</a> PLoS One. 4, 8211-82 (2009).</li><li>Blengini, C. S., Teves, M. E., Uñates, D. R., Guidobaldi, H. A., Gatica, L. V., Giojalas, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+sperm+pattern+of+movement+during+chemotactic+re-orientation+towards+a+progesterone+source.">Human sperm pattern of movement during chemotactic re-orientation towards a progesterone source.</a> Asian J. Androl. 13, 769-773 (2011).</li><li>Strünker, T., Goodwin, N., Brenker, C., Kashikar, N. D., Weyand, I., Seifert, R., Kaupp, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CatSper+channel+mediates+progesterone-induced+Ca2++influx+in+human+sperm.">The CatSper channel mediates progesterone-induced Ca2+ influx in human sperm.</a> Nature. 471, 382-386 (2011).</li><li>Lishko, P. V., Botchkina, I. L., Kirichok, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+activates+the+principal+Ca2++channel+of+human+sperm.">Progesterone activates the principal Ca2+ channel of human sperm.</a> Nature. 471, 387-391 (2011).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> J. Exp. Med. 115, 453-465 (1962).</li><li>Zigmond, S. H., Lauffenburger, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+of+leukocyte+chemotaxis.">Assays of leukocyte chemotaxis.</a> Annu. Rev. Med. 37, 149-155 (1986).</li><li>Villanueva-Diaz, C., Vadillo-Ortega, F., Kably-Ambe, A., Diaz-Pérez, M. A., Krivitzky, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+human+follicular+fluid+contains+a+chemoattractant+for+spermatozoa.">Evidence that human follicular fluid contains a chemoattractant for spermatozoa.</a> Fertil. Steril. 54, 1180-1182 (1990).</li><li>Villanueva-Diaz, C., Arizs-Martinez, J., Bermejo-Martinez, L., Vadillo-Ortega, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+induces+human+sperm+chemotaxis.">Progesterone induces human sperm chemotaxis.</a> Fertil. Steril. 64, 1183-1188 (1995).</li><li>Olson, J., Xiang, X., Ziegert, T., Kittleson, A., Rawls, A., Bieber, A., Chandler, D. E. A. l. l. u. r. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+21+kD+sperm+chemoattractant+from+Xenopus+egg+jelly,+is+homologous+to+mammalian+sperm-binding+proteins.">a 21 kD sperm chemoattractant from Xenopus egg jelly, is homologous to mammalian sperm-binding proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 11205-11210 (2001).</li></ol>

Nessun conflitto di interessi dichiarati.

Chemiotassi di cellule in movimento da entrambe movimento ameboide o flagelli alimentato il nuoto è presente in molti contesti biologici e lo studio di questo fenomeno richiede la disponibilità di test pratici e affidabili. Alcuni esempi del fenomeno, come l&#39;attrazione degli spermatozoi per un uovo di riccio di mare o la raccolta di cellule stampo melma per formare un corpo fruttifero, hanno immediato impatto visivo. La quantificazione di questo fenomeno è stata intrapresa in diversi modi, come descritto da Eisen...

<table border="1"><tbody><tr><td> Nome della voce </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> 24-pozzetti </td><td> Becton, Dickinson </td><td> 35/1147 </td><td></td></tr><tr><td> 12 inserti mm di diametro esterno di 12 micron pori della membrana </td><td> Millipore </td><td> PIXP01250 </td><td> In precedenza avevamo usato Costar-Corning piastra transwell # 3403, ora sospeso </td></tr><tr><td> Zigmond camera </td><td> Neuroprobe </td><td> Z02 </td><td></td></tr><tr><td> Olio di silicone </td><td> General Electric </td><td> SF1154 </td><td> Equivalente a Fluido Dow Corning 550 </td></tr><tr><td> Immagine J software </td><td> Wayne Rasband, servizi Settore di Ricerca, Istituto Nazionale di Salute Mentale </td><td> Download gratuito all&#39;indirizzo http://rsbweb.nih.gov/ij </td><td> Programma Java che gira su Windows, LinUX e Mac </td></tr><tr><td> MtrackJ software </td><td> Erik Meijering / Imagescience / <a href="http://www.bigr.nl/">Biomedical Imaging Group</a> , Erasmus MC - University Medical Center di Rotterdam </td><td> Scaricabile gratuitamente dal <a href="http://www.imagescience.org/meijering/software/mtrackj/">http://www.imagescience.org/meijering/software/mtrackj/</a> </td><td> Programma Java che gira su Windows, Linux e Mac </td></tr><tr><td> Virtual Dub software </td><td> <a href="http://www.virtualdub.org/gpl.html">GNU General Public licenza</a> software </td><td> Download gratuito tramite <a href="http://www.virtualdub.org/index.html">http://www.virtualdub.org/index.html</a> </td><td> Istruzioni di installazione sul sito Immagine J sotto plugins, solo per Windows </td></tr><tr><td> cellSens software </td><td> Olimpo </td><td> Vedere il sito: <a href="http://www.olympusamerica.com/seg_section/product.asp?product=1070">http://www.olympusamerica.com/seg_section/p</a>roduct.asp? prodotto = 1070 </td><td> Controlli e acquisisce immagini da una serie di telecamere. Ha anche capacità di elaborazione immagini </td></tr></tbody></table>

two types of assays for detecting frog sperm chemoattraction

Chemiotassi degli spermatozoi negli invertebrati può essere sufficientemente robusto che si può mettere una pipetta contenente il peptide attraente in una sospensione di spermatozoi e microscopicamente visualizzare accumulo di sperma intorno alla pipetta 1. Chemiotassi degli spermatozoi nei vertebrati come rane, roditori ed esseri umani è più difficile da rilevare e richiede analisi quantitative. Tali dosaggi sono di due tipi principali - saggi che quantificare il movimento degli spermatozoi ad una fonte di chemoattractant, i cosiddetti saggi di accumulo degli spermatozoi, e saggi che in realtà traccia le traiettorie individuali di nuoto degli spermatozoi. Saggi di accumulo degli spermatozoi sono decine permettendo relativamente rapido o centinaia di test da fare in un solo giorno, consentendo in tal modo le curve dose-risposta e corsi di tempo per essere eseguiti in modo relativamente rapido. Questi tipi di test sono stati ampiamente utilizzati per caratterizzare molti sistemi chemiotassi ben consolidato - per esempio, chemiotassi dei neutrofili al bacpeptidi materiali e chemiotassi degli spermatozoi al liquido follicolare. Test di tracciamento degli spermatozoi può essere più laborioso, ma offrono ulteriori dati su come chemoattractancts effettivamente alterare i percorsi di nuoto che lo sperma prendere. Questo tipo di test è necessario per dimostrare l&#39;orientamento del movimento degli spermatozoi rispetto all&#39;asse gradiente chemoattrractant e visualizzare trasforma caratteristica o cambiamenti di orientamento che portano lo sperma più vicino al uovo. Qui si descrivono i metodi utilizzati per ciascuno di questi due tipi di test. Il saggio di accumulazione sperma utilizzato è chiamato un saggio &quot;bicamerale&quot;. Spermatozoi anfibi sono inseriti in un inserto piastra di coltura dei tessuti con un pavimento filtro in policarbonato con pori di diametro 12 micron. Inserti con spermatozoi vengono inseriti in pozzi colture di tessuti piastra contenente tampone e un chemoatttractant attentamente pipettati nella parte inferiore e dove il pavimento incontra il muro (vedi fig. 1). Dopo l&#39;incubazione, l&#39;inserto superiore che contiene il serbatoio degli spermatozoi viene accuratamente removed, e lo sperma nella camera di fondo che sono passati attraverso la membrana vengono rimossi, pellettato e poi contati da emocitometro o citometria a flusso. Il test di tracciamento degli spermatozoi utilizza una camera di Zigmond originariamente sviluppato per l&#39;osservazione chemiotassi dei neutrofili e modificato per l&#39;osservazione di spermatozoi da parte Giojalas e collaboratori 2,3. La camera è costituito da una lastra di vetro di spessore in cui due depressioni verticali sono state lavorate. Questi sono separati da una piattaforma di osservazione 1 mm di larghezza. Dopo l&#39;applicazione di una copertura in vetro, gli spermatozoi vengono caricati in una mangiatoia, l&#39;agente chemiotattico nell&#39;altra e il movimento degli spermatozoi singoli visualizzati al microscopio video. Riprese video viene poi analizzata utilizzando software per identificare bidimensionale movimenti delle cellule nel piano xy in funzione del tempo (XYT set di dati) che formano la traiettoria di ogni sperma.

Watch this Scientific Journal Video about Two Types of Assays for Detecting Frog Sperm Chemoattraction at JoVE.com

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Uova e rivestimenti extracellulare intorno uova di frequente rilascio peptidi, proteine ​​e piccole molecole che comunicano con lo sperma per guidarli verso l&#39;ovulo promuovendo in tal modo la fecondazione. Con sperma rana che descrivere e confrontare due classi di test utilizzato per rilevare chemiotassi degli spermatozoi - analisi di accumulo dello sperma e test di tracciamento degli spermatozoi.

Due tipi di saggi per chemiotassi Frog rilevamento sperma

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

two-types-of-assays-for-detecting-frog-sperm-chemoattraction

Research

JoVE Journal

Biology

19.0K Views.  University of Illinois, Urbana-Champaign. Uova e rivestimenti extracellulare intorno uova di frequente rilascio peptidi, proteine ​​e piccole molecole che comunicano con lo sperma per guidarli verso l'ovulo promuovendo in tal modo la fecondazione. Con sperma rana che descrivere e confrontare due classi di test utilizzato per rilevare chemiotassi degli spermatozoi - analisi di accumulo dello sperma e test di tracciamento degli spermatozoi.

Video: Two Types of Assays for Detecting Frog Sperm Chemoattraction

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

Lo sperma raccolto e l&#39;inseminazione artificiale di topi

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

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Biologia

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

A High-Throughput Metodo per la crioconservazione dello sperma e Zebrafish In Vitro Fecondazione

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Tecniche di Imaging Ca 2 + Segnalazione di sperma umano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Analisi di Schwann-astrociti Interazioni utilizzo In Vitro Saggi

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Isolamento e In vitro Attivazione di Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Estrazione di antigeni dei tessuti per test funzionali

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

Differenze valutando in capacità competitiva sperma in<em&gt; Drosophila</em

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

I saggi di redditività per le cellule in coltura

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Sviluppo di un sistema di inserimento co-coltura di due tipi cellulari in assenza del cellula-cellula di contatto

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

Valutazione di sperma di pesce utilizzando Software e dispositivi di raffreddamento

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...