Name: cytological analysis of spermatogenesis: live and fixed preparations of drosophila testes
Uploaded: 2014-01-20T15:00:00
Description: Watch this Scientific Journal Video about Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes at JoVE.com

Forfatterne vil gerne takke Michael Anderson til etablering i Lee lab disse accepterede metoder til at studere spermatogenesen med ekspertrådgivning fra Karen Hales. H. Oda og Y. Akiyama-Oda generøst forudsat at γ-tubulin-GFP flyve lager. Dette arbejde blev støttet af en NIH R01 tilskud til LAL (GM074044).

1.. Testiklerne Dissection <ol><li> Bedøver fluer i en flaske eller hætteglas hjælp af en strøm af CO 2 og overføre til en flue pad. </li><li> Sorter flyver under et dissektionsmikroskop med en lille pensel, og indsamle et passende antal (afhængigt af forsøget) Drosophila hanner af de ønskede genotyper. Unge mænd (0-2 dage gamle) er ideelle til at undersøge celler i hele de tidligere stadier af spermatogenesen (f.eks spermatogonier, spermatocytter og tidlige post-meiotiske sperma...

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Selv testiklerne af vildtype fluer let kan identificeres på grund af deres gule farve (i modsætning de omkringliggende hvide væv), testiklerne af hvide mutant fluer er hvide, og dermed kan lejlighedsvis forveksles med tarmen. Mest transgene stammer, som typisk er på en hvid baggrund, har også hvide testikler fordi mini-hvide gen fundet i P-elementer ikke fremmer pigment akkumulation i testiklerne. Da Drosophila testikler kan ikke skelnes efter farve, andre let genkendeli...

Drosophila testikler er en ideel model for studiet af mange biologiske processer, herunder reguleringen af stamceller, meiose, og sæd udvikling 13-18. De spermatocytter og deres meiotiske spindler er store og dermed praktisk til cytologisk analyse og afslappet cellecykluskontrolpunkter under spermatogenese lette undersøgelsen af ​​mutationer i cellecyklus gener. Forskellige celletyper kan observeres i bestilte progression langs længden af ​​testiklerne, og enhver afbrydelse i spermatogenesen kan føre til ændringer i denne overordnede arrangement. Disse funktioner kombineret med Drosophila genetiske værktøjer har lettet mutationsanalyse af spermatogenesen 21-23. Etaperne i Drosophila spermatogenesen er veldefineret. Germline-celler, der udvikler sig synkront inden cyster fremskridt sekventielt gennem stadier af spermatogenesen langs længden af ​​testiklerne. Under both mitose og meiotiske delinger af de mandlige kønsceller, opstår cytokinese ufuldstændigt sådan, at datteren cellerne forbliver forbundet med cytoplasmatiske broer kendt som ring kanaler (figur 1). Den apikale spids af testis indeholder en population af germlinie stamceller, der giver anledning til spermatogonier, der undergår fire mitotiske divisioner ufuldstændig cytokinese at generere 16-celle cyster af primære spermatocytter. Efter premeiotic S fasen, primære spermatocytter indtaste G2, en længere periode med ~ 90 hr hvorunder cellulære volumen øges ~ 25-fold vækst. Progression gennem meiose I og meiose II resulterer i dannelsen af ​​32-celle cyster af sekundære spermatocytter og 64-celle cyster af haploide spermatider, hhv. De umodne, runde spermatider undergår omfattende cellulær ombygning til at danne modne sædceller. Post-meiotiske celler, især bundter af forlængede og modne spermatider, optager meget af mængden af ​​testiklerne. Than vellykket transport af funktionelle sædceller til kvindelige fluer kræver koordination mellem de forskellige dele af den mandlige reproduktive system, der er sammensat af flere parrede strukturer (testiklerne, sædblærer og tilbehør kirtler) og en enkelt ejakulatorisk kanal (figur 2). Sperm er produceret inden testiklerne og opbevares inden sædblærerne indtil samleje 24. De tilhørende kirtler indeholder sekretoriske celler, der producerer sædvæsken. Sæden migrere fra sædblærerne blandes med sædvæske inden udløsning kanal, der er forbundet til begge sædblærerne og de accessoriske kirtler. Denne blanding af sæd og sædvæske sidste ende pumpes ud af han i skeden af den kvindelige flyver gennem udløsning pære placeret ved den bageste ende af den mandlige maven 25. Proteiner i sædvæsken er afgørende for langvarig opbevaring af sæd inden specialiserede organer, der er kendt som spermathecae i reproductive tarmkanalen af Drosophila hunner 26. Gode ​​metoder til isolering af Drosophila testikler og visualisering af celler på forskellige stadier af spermatogenesen findes i den videnskabelige litteratur 3-12. Vi tilføjer heri til dette organ af viden ved at præsentere eksempler på disse protokoller med en ledsagende video demonstration. Protokollen for forberedelse af levende testikler prøver til fasekontrast-mikroskopi er baseret på en tidligere beskrevet metode 27. Protokollen for formaldehyd fiksering og immunfarvning af testiklerne er også baseret på en tidligere beskrevet metode 28. De heri beskrevne fremgangsmåder er blevet anvendt i mange undersøgelser Drosophila spermatogenese (for eksempel for at vurdere de roller dynein en minus-ende-directed mikrotubulus motor under Drosophila spermatogenese). Ud over de grundlæggende protokoller der forslag fastsat varying dissektion, således at berige for spermatogonier, spermatocytter eller modne sædceller. Forskellige metoder til behandling af testiklerne sådan, at cyster enten forblive intakt eller bliver afbrudt efter behov beskrevet. En fordel ved at anvende Drosophila testikler som en model system er, at sammenlignet med Drosophila oocyter og embryoner, kan antistoffer og farvestoffer let trænge celler efter deres spredning fra testiklerne, og der kræves færre vasketrin, og dermed kan protokoller udføres i et relativt kort tid.

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		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold Antifade Reagent with DAPI</td>
			<td style="width:215px;">Life Technologies</td>
			<td>P36931</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-gamma-tubulin antibody (clone GTU-88)</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td>T6557</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3-AffiniPure Goat Anti-Mouse IgG&#160;</td>
			<td style="width:215px;">Jackson ImmunoResearch</td>
			<td>115-165-003</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Triton X-100</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">BP151-100</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">AC61511-0040</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Methanol</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">A412-4</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">16% Formaldehyde</td>
			<td style="width:215px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">28908</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:351px;">Sigmacote</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:127px;">SL2</td>
			<td style="width:321px;">Use according to manufacturer&#39;s directions to siliconize cover slips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DAPI</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:127px;">D-9542</td>
			<td style="width:321px;">0.5 mg/ml in 75% ethanol; store at -20&#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:351px;">NaCl</td>
			<td style="width:215px;">Research Products International Corp.</td>
			<td style="width:127px;">S23020</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Na2HPO4</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:127px;">S9763</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NaH2PO4</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:127px;">S0751</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipes delicate task wipers</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td>S47299</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:351px;">BSA</td>
			<td style="width:215px;">Research Products International Corp.</td>
			<td style="width:127px;">A30075</td>
			<td style="width:321px;">Molecular biology grade</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Glass Coplin staining jar, screw cap</td>
			<td style="width:215px;">Electron Microscopy Sciences</td>
			<td>70315</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single frosted microscope slides</td>
			<td>Corning</td>
			<td>2948-75X25</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Poly-L-lysine coated microscope slides</td>
			<td>Polysciences, Inc.</td>
			<td>22247-1</td>
			<td style="width:321px;">Optional (to replace untreated microscope slides )</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Square cover glass</td>
			<td>Corning</td>
			<td style="width:127px;">2865-22</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Razor blades</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">12-640</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimax Petri dish</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">S31473</td>
			<td style="width:321px;">Kimble #23060 10015 EMD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Dumont</td>
			<td>52100-51S</td>
			<td>Pattern 5 INOX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;"></td>
			<td style="width:215px;"></td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;"></td>
			<td style="width:215px;"></td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Name of Equipment</td>
			<td style="width:215px;">Company</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Stemi 2000-CS stereoscope</td>
			<td style="width:215px;">Carl Zeiss</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Eclipse 80i</td>
			<td style="width:215px;">Nikon</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Plan-Fluor 40x objective</td>
			<td style="width:215px;">Nikon</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Axiophot</td>
			<td style="width:215px;">Carl Zeiss</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Plan-Neofluar Ph2 40x objective</td>
			<td style="width:215px;">Carl Zeiss</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
		</tr>
	</tbody>
</table>

cytological analysis of spermatogenesis: live and fixed preparations of drosophila testes

Drosophila melanogaster er en kraftfuld model, der har været almindeligt anvendt til at belyse forskellige biologiske processer. For eksempel har studier af både kvindelige og mandlige kimlinjer Drosophila høj grad bidraget til den nuværende forståelse af meiose samt stamcellebiologi. Fremragende protokoller er tilgængelige i litteraturen for isoleringen og billeddannelse af Drosophila æggestokke og testikler 3-12. Heri er metoder til dissektion og udarbejdelse af Drosophila testikler til mikroskopisk analyse beskrevet med en ledsagende video demonstration. En protokol til isolering testikler fra maven af ​​voksne mænd og forberede lysbilleder af levende væv til analyse ved fasekontrast-mikroskopi samt en protokol for fastsættelse og immunfarvning testikler til analyse ved fluorescens mikroskopi præsenteres. Disse teknikker kan anvendes i karakteriseringen af Drosophila mutanter, som udviser defects i spermatogenesen samt i visualisering af subcellulære lokaliseringer af proteiner.

Et eksempel på en korrekt dissekeret par Drosophila mandlige kønsorganer er vist i figur 2A. Testikler fjernet fra maven af den voksne mandlige flue er typisk knyttet til udløsning kanalen (brun, figur 2A) og et par af accessoriske kirtler (grøn, figur 2A &#39;) via et par sædblærer (blå, figur 2A) . For at adskille testiklerne fra de fleste af ledsagende somatiske væv udløsning kanalen og de ​​accessoriske kirtler bør tages af og...

Watch this Scientific Journal Video about Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes at JoVE.com

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Metoder til isolering og forberede Drosophila testikler prøver (leve og faste) til billeddannelse ved fasekontrast-og fluorescensmikroskopi er beskrevet heri.

Cytologiske Analyse af Spermatogenese: Levende og Faste Tilberedt<em&gt; Drosophila</em&gt; Testikler

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

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Biology

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Isolation of Drosophila melanogaster Testes

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Kvantitative levende celler fluorescens-mikroskopi Analyse af Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Preparing Individual Drosophila Egg Chambers for Live Imaging

Forberedelse Individual Drosophila Egg Chambers til Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

Levende Imaging af GFP-mærkede proteiner i<em&gt; Drosophila</em&gt; Oocytter

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Levende Imaging af Apoptotisk Cell Clearance under<em&gt; Drosophila</em&gt; Embryogenese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Levende cellecyklusanalyse af<em&gt; Drosophila</em&gt; Væv bruger Attune Acoustic Fokusering Cytometer og Vybrant DyeCycle Violet DNA Stain

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

Analyse af embryonale og larvestadiet Zebrafisk Skeletal myofibre fra Dissocierede Forberedelser

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...