Agradecemos ao WM Keck Laboratory BioImaging para uso de sua estação de trabalho de vídeo microscopia. Este estudo foi financiado pela NSF conceder IBN-0615435.

<ol>
	<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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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. 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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>

Não há conflitos de interesse declarados.

Quimiotaxia de células de movimento pelo movimento amebóide ou flagelos-powered natação é encontrado em muitos contextos biológicos e estudo deste fenômeno exige a disponibilidade de testes prático e confiável. Alguns exemplos do fenômeno, como a atração de esperma para um ovo de ouriço do mar ou coleta de células do discoideum para formar um corpo de frutificação, têm um impacto visual imediato. Quantificação deste fenômeno tem sido feita em uma variedade de maneiras, como descrito por Eisenbach <su...

<table border="1"><tbody><tr><td> Nome do item </td><td> Companhia </td><td> Número Catálogo </td><td> Comentários </td></tr><tr><td> Placas de 24 poços </td><td> Becton-Dickinson </td><td> 35/1147 </td><td></td></tr><tr><td> 12 milímetros de diâmetro exterior inserções com 12 mM de poros da membrana </td><td> Millipore </td><td> PIXP01250 </td><td> Nós usamos anteriormente Costar-Corning placa transwell # 3403-agora descontinuado </td></tr><tr><td> Zigmond câmara </td><td> Neuroprobe </td><td> Z02 </td><td></td></tr><tr><td> Óleo de silicone </td><td> General Electric </td><td> SF1154 </td><td> Equivalente a Dow Corning 550 Fluid </td></tr><tr><td> Imagem J software </td><td> Wayne Rasband, Research Services Branch, National Institute of Mental Health </td><td> Download gratuito em http://rsbweb.nih.gov/ij </td><td> Java programa que roda em 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 Rotterdam </td><td> Download gratuito em <a href="http://www.imagescience.org/meijering/software/mtrackj/">http://www.imagescience.org/meijering/software/mtrackj/</a> </td><td> Java programa que roda em Windows, Linux e Mac </td></tr><tr><td> Virtual Dub software </td><td> <a href="http://www.virtualdub.org/gpl.html">GNU General Public Licensed</a> software </td><td> Download gratuito via <a href="http://www.virtualdub.org/index.html">http://www.virtualdub.org/index.html</a> </td><td> Instruções de instalação no site da J Imagem em plugins, apenas para Windows </td></tr><tr><td> cellSens software </td><td> Olimpo </td><td> Consulte o site: <a href="http://www.olympusamerica.com/seg_section/product.asp?product=1070">http://www.olympusamerica.com/seg_section/p</a>roduct.asp produto? = 1070 </td><td> Controles e adquire imagens de uma variedade de câmeras. Também possui capacidade de processamento de imagem </td></tr></tbody></table>

two types of assays for detecting frog sperm chemoattraction

Quimiotaxia de esperma em invertebrados podem ser suficientemente robusto que um pode colocar uma pipeta contendo o peptídeo atraente em uma suspensão de espermatozóides e microscopicamente visualizar acúmulo de esperma em torno da pipeta 1. Quimiotaxia esperma em vertebrados, como sapos, roedores e seres humanos é mais difícil de detectar e exige ensaios quantitativos. Tais ensaios são de dois tipos principais - os ensaios que quantificar o movimento de esperma a uma fonte de chemoattractant, os chamados ensaios de acumulação de esperma, e os ensaios que realmente controlar as trajetórias de natação do esperma individual. Ensaios de acumulação de espermatozóides são relativamente rápida permitindo dezenas ou centenas de testes a ser feito em um único dia, permitindo assim que as curvas de dose-resposta e cursos tempo para ser realizada de forma relativamente rápida. Estes tipos de ensaios têm sido amplamente utilizado para caracterizar muitos sistemas quimiotaxia bem estabelecida - por exemplo, quimiotaxia de neutrófilos para bacpeptídeos terial e quimiotaxia esperma para fluido folicular. Ensaios de monitoramento de esperma pode ser mais trabalhoso, mas oferecer dados adicionais sobre como chemoattractancts realmente alterar os caminhos que o esperma nadar tomar. Este tipo de ensaio é necessário para demonstrar a orientação do movimento de esperma em relação ao eixo gradiente chemoattrractant e visualizar curvas características ou mudanças de orientação que trazem os espermatozóides mais próximos do ovo. Aqui descrevemos os métodos utilizados para cada um desses dois tipos de ensaios. O ensaio de acumulação de esperma utilizado é chamado de &quot;duas câmaras&quot; ensaio. Espermatozóides dos anfíbios são colocados em uma inserção de placa de cultura de tecidos com um piso de filtro de policarbonato com poros de diâmetro 12 mM. Pastilhas com os espermatozóides são colocados em poços de cultura de tecidos placa contendo tampão e um chemoatttractant cuidadosamente pipetado para o fundo poço onde o piso encontra a parede (ver Fig. 1.). Após a incubação, a inserção superior contendo o reservatório de esperma é cuidadosamente removed, esperma e na câmara de fundo que passaram através da membrana são removidos, peletizada e depois contadas por hemocitômetro ou citômetro de fluxo. O ensaio de rastreamento de esperma utiliza uma câmara de Zigmond originalmente desenvolvido para a observação de quimiotaxia de neutrófilos e modificados para a observação de espermatozóides por Giojalas e colegas de trabalho 2,3. A câmara consiste de uma lâmina de vidro de espessura em que duas calhas verticais foram usinadas. Estes são separados por uma uma plataforma de observação mm de largura. Após a aplicação de um espermatozóide tampa de vidro, são carregados em uma calha, o agente chemoattractant para o outro eo movimento dos espermatozóides individuais visualizados por microscopia de vídeo. Imagens de vídeo são então analisados ​​usando o software para identificar os movimentos em duas dimensões de células no plano xy como uma função do tempo (conjuntos XYT de dados) que formam a trajetória de cada espermatozóide.

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

Ovos e os revestimentos extracelular em torno de ovos com freqüência liberação de peptídeos, proteínas e pequenas moléculas que se comunicam com o esperma para guiá-los até o óvulo, promovendo assim a fecundação. Usando o esperma de rã que descrever e comparar duas classes de testes usados ​​para detectar quimiotaxia esperma - ensaios de esperma acumulação e ensaios de monitoramento de esperma.

Dois tipos de ensaios para detecção de quimiotaxia Sapo Sperm

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

18.9K visualizações.  University of Illinois, Urbana-Champaign. Ovos e os revestimentos extracelular em torno de ovos com freqüência liberação de peptídeos, proteínas e pequenas moléculas que se comunicam com o esperma para guiá-los até o óvulo, promovendo assim a fecundação. Usando o esperma de rã que descrever e comparar duas classes de testes usados ​​para detectar quimiotaxia esperma - ensaios de esperma acumulação e ensaios de monitoramento de esperma.

Vídeo: 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.

Sperm colheita e Inseminação Artificial de Ratos

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 ...

Biologia

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Ensaios Neurocircuit para Apreensões em mutantes de Epilepsia Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

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.

Um método de alta taxa de transferência para Zebrafish criopreservação de espermatozóides e In Vitro Fertilização

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.

Técnicas de imagem Ca 2 + Sinalização no esperma humano

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.

Análise de Schwann-astrócito Interações Usando In Vitro Ensaios

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 A ativação do 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.

Extracção do Tissue Antigens para Ensaios Funcionais

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 ...

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.

Ensaios de viabilidade para células em cultura

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.

Desenvolvimento de um Sistema Insert Co-cultura de dois tipos celulares na ausência de célula-célula 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 ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Coleção de esperma de qualidade diferencial usando centrifugação gradiente de densidade

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...