Name: two types of assays for detecting frog sperm chemoattraction
Uploaded: 2011-12-27T13:00:00
Description: Watch this Scientific Journal Video about Two Types of Assays for Detecting Frog Sperm Chemoattraction at JoVE.com

हम अपने वीडियो माइक्रोस्कोपी काम स्टेशन के उपयोग के लिए WM उबकना Bioimaging प्रयोगशाला धन्यवाद. इस अध्ययन NSF आईबीएन 0615435 अनुदान द्वारा समर्थित किया गया.

1. सामग्री और इस्तेमाल किया बफ़र्स <ol><li> Oocyte घंटी बफर (1.5 x OR2) 124 मिमी NaCl, 3.75 मिमी KCl, 1.5 मिमी 2 CaCl, 1.5 मिमी 2 MgCl, 1.5 मिमी ना 2 4 HPO, 10 मिमी Hepes होते हैं, 7.8 पीएच. निषेचन बफर (एफ 1) 41.25 मिमी NaCl, 1.25 मिमी KCl, .25 मिमी 2 CaCl, .06 मिम?...

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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. 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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>

या तो अमीबीय आंदोलन या flagella संचालित तैराकी से बढ़ कोशिकाओं की chemotaxis कई जैविक संदर्भों में पाया जाता है और इस घटना के अध्ययन के व्यावहारिक और विश्वसनीय assays की उपलब्धता की आवश्यकता है. घटना के कुछ उदाहरणों म?...

<table border="1"><tbody><tr><td> आइटम के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियाँ </td></tr><tr><td> 24 अच्छी तरह प्लेटें </td><td> Becton - Dickenson </td><td> 35/1147 </td><td></td></tr><tr><td> 12 मिमी 12 सुक्ष्ममापी ताकना झिल्ली के साथ बाहरी व्यास आवेषण </td><td> Millipore </td><td> PIXP01250 </td><td> हम पहले transwell costar-Corning थाली +३,४०३ अब # बंद का इस्तेमाल </td></tr><tr><td> Zigmond कक्ष </td><td> Neuroprobe </td><td> Z02 </td><td></td></tr><tr><td> सिलिकॉन तेल </td><td> जनरल इलेक्ट्रिक </td><td> SF1154 </td><td> डॉव Corning 550 द्रव के लिए समतुल्य </td></tr><tr><td> छवि जम्मू सॉफ्टवेयर </td><td> वेन Rasband, अनुसंधान सेवा शाखा, मानसिक स्वास्थ्य के राष्ट्रीय संस्थान </td><td> मुफ्त डाउनलोड पर http://rsbweb.nih.gov/ij </td><td> जावा प्रोग्राम है कि विंडोज, लिन पर चलाता हैUX और मैक </td></tr><tr><td> MtrackJ सॉफ्टवेयर </td><td> एरिक / Meijering / Imagescience <a href="http://www.bigr.nl/">बायोमेडिकल इमेजिंग समूह</a> , इरास्मस एम सी - यूनिवर्सिटी मेडिकल सेंटर रॉटरडैम </td><td> <a href="http://www.imagescience.org/meijering/software/mtrackj/">Http://www.imagescience.org/meijering/software/mtrackj/</a> पर मुफ्त डाउनलोड </td><td> जावा प्रोग्राम है कि Windows, Linux और मैक पर चलाता है </td></tr><tr><td> वर्चुअल डब सॉफ्टवेयर </td><td> <a href="http://www.virtualdub.org/gpl.html">GNU जनरल पब्लिक लाइसेंस</a> सॉफ्टवेयर </td><td> <a href="http://www.virtualdub.org/index.html">Http://www.virtualdub.org/index.html</a> के माध्यम से मुफ्त डाउनलोड </td><td> केवल Windows के लिए, plugins के तहत छवि जम्मू वेबसाइट पर सेटअप निर्देश </td></tr><tr><td> cellSens सॉफ्टवेयर </td><td> ओलिंप </td><td> : वेबसाइट देखें <a href="http://www.olympusamerica.com/seg_section/product.asp?product=1070">http://www.olympusamerica.com/seg_section/p</a>roduct.asp 1070 =? उत्पाद </td><td> नियंत्रण और कैमरों की एक किस्म से छवियों को प्राप्त है. इसके अलावा इमेज प्रोसेसिंग की क्षमता है </td></tr></tbody></table>

two types of assays for detecting frog sperm chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

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

अंडे और अंडे के आसपास कोशिकी कोटिंग्स अक्सर पेप्टाइड्स, प्रोटीन और छोटे अणुओं जारी है कि शुक्राणु के साथ संवाद उन्हें अंडे जिससे निषेचन को बढ़ावा देने का मार्गदर्शन करने के लिए. मेंढक शुक्राणु का उपयोग हम वर्णन और शुक्राणु chemoattraction का पता लगाने के लिए इस्तेमाल किया assays के दो वर्गों की तुलना - शुक्राणु संचय assays और शुक्राणु ट्रैकिंग assays.

पता लगाने मेंढक शुक्राणु Chemoattraction के लिए दो Assays के प्रकार

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

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Biology

Harvesting Sperm and Artificial Insemination of Mice

फसल काटने वाले शुक्राणु और चूहे के कृत्रिम गर्भाधान

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 ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Neurocircuit Assays में मिरगी म्यूटेंट की बरामदगी के लिए ड्रोसोफिला</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

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

Zebrafish शुक्राणु cryopreservation के लिए एक उच्च throughput और विधि इन विट्रो में निषेचन

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

Techniques for Imaging Ca2+ Signaling in Human Sperm

Ca इमेजिंग तकनीक के लिए 2 + मानव शुक्राणु में संकेतन

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

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

श्वान - astrocyte का उपयोग सहभागिता के विश्लेषण इन विट्रो में Assays

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

Extraction of Tissue Antigens for Functional Assays

ऊतक प्रतिजनों के कार्यात्मक assays के लिए निकासी

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

Assessing Differences in Sperm Competitive Ability in Drosophila

शुक्राणु प्रतिस्पर्धी क्षमता में में आकलन मतभेद<em&gt; ड्रोसोफिला</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

संस्कृति में कोशिकाओं के लिए व्यवहार्यता Assays

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

की सेल सेल संपर्क अभाव में दो सेलुलर प्रकार के एक सम्मिलित सह संस्कृति प्रणाली का विकास

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

मछली शुक्राणु मूल्यांकन सॉफ्टवेयर और शीतलक उपकरणों का उपयोग

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