Name: a seminiferous tubule squash technique for the cytological analysis of spermatogenesis using the mouse model
Uploaded: 2018-02-06T13:00:00
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Dette arbejde blev støttet af NIGMS (R01GM11755) til P.W.J. og af en uddannelse grant stipendium fra National Cancer Institute (NIH) (CA009110) til S.R.W. og J.H.

Brugen af mus blev godkendt af institutionelle Animal Care og bruge Udvalget af Johns Hopkins University. Forsøgene blev udført på ungdomskriminalitet (20-26 dage post-partum, dpp) C57BL/6J mus, at drage fordel af den semisynkron første bølge af spermatogenesen. Dog, denne teknik kan også udføres ved hjælp af voksen mus.
1. dissektion og isolation af musen seminiferous tubules
<ol><li>Forberede den rettelse/løsning og antistof fortynding lysisbuffer (ADB) er beskrevet i tabe...

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Centrosome-autonomous+signal+that+involves+centriole+disengagement+permits+centrosome+duplication+in+G2+phase+after+DNA+damage.">A Centrosome-autonomous signal that involves centriole disengagement permits centrosome duplication in G2 phase after DNA damage.</a> Mol Biol Cell. 21, 3866-3877 (2010).</li><li>Firat-Karalar, E. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+a+spermatogenic+wave+into+a+tsunami:+synchronizing+murine+spermatogenesis+using+WIN+18,446.">Turning a spermatogenic wave into a tsunami: synchronizing murine spermatogenesis using WIN 18,446.</a> Biol Reprod. 88 (2), 40 (2013).</li><li>Yoshida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+round+of+mouse+spermatogenesis+is+a+distinctive+program+that+lacks+the+self-renewing+spermatogonia+stage.">The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage.</a> Development. 133 (8), 1495-1505 (2006).</li><li>Kluin, P. M., Kramer, M. F., Rooij, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis+in+the+immature+mouse+proceeds+faster+than+in+the+adult.">Spermatogenesis in the immature mouse proceeds faster than in the adult.</a> Int J Androl. 5 (3), 282-294 (1982).</li><li>Rodriguez, I., Ody, C., Araki, K., Garcia, I., Vassalli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+early+and+massive+wave+of+germinal+cell+apoptosis+is+required+for+the+development+of+functional+spermatogenesis.">An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.</a> EMBO J. 16 (9), 2262-2270 (1997).</li><li>Kangasniemi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+basal+and+FSH+dependent+cyclic+AMP+production+in+rat+seminiferous+tubules+staged+by+an+improved+transillumination+technique.">Modulation of basal and FSH dependent cyclic AMP production in rat seminiferous tubules staged by an improved transillumination technique.</a> Anat Rec. 227 (1), 62-76 (1990).</li><li>Martianov, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+arrest+of+spermiogenesis+and+germ+cell+apoptosis+in+mice+lacking+the+TBP-like+TLF/TRF2+gene.">Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene.</a> Mol Cell. 7 (3), 509-515 (2001).</li><li>Henriks&#233;n, K., Parvinen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+apoptosis+of+male+germ+cells+in+the+rat:+Mechanisms+of+cell+death+studied+by+supravital+squash+preparations.">Stage-specific apoptosis of male germ cells in the rat: Mechanisms of cell death studied by supravital squash preparations.</a> Tissue Cell. 30 (6), 692-701 (1998).</li></ol>

Mus har vist sig for at være en nyttig model organisme for at studere de cellulære begivenheder, der styrer meiotiske progression i spermatogenesen. Yderligere, det er nødvendigt at udvikle værktøjer dækket til studiet af spermatogenesen, fordi mange begivenheder, såsom exit fra meiotiske prophase jeg, er seksuelt dimorfe. Denne protokol beskriver en seminiferous tubulus squash metoden for visualisering og undersøgelse af forskellige stadier af musen spermatogenesen. Denne metode bevarer cellulære integritet og ...

Meiose er en kompleks cellulære begivenhed hvor en enkelt runde af DNA-replikation er efterfulgt af to successive runder af celledeling. Flere meiose-specifikke begivenheder skal koordineres i de indledende faser af meiose at sikre nøjagtig kromosom adskillelse. Disse arrangementer omfatter gennemførelsen af homologe rekombination, co orientering af søster kinetochores under den første meiotiske deling og trinvis tabet af cohesin komplekser til at løse chiasmata mellem homologs. Præcis regulering af disse processer er nødvendige for at opretholde frugtbarhed og forebygge kromosom missegregation begivenheder, der kan føre til genetiske udviklingsforstyrrelser og spontan abort1.
Mens de vigtigste begivenheder af meiose finder sted i både hanner og hunner, findes væsentlige timeligt og mekanistiske forskelle mellem spermatogenesen og oogenesen2. For eksempel, under kvindelige meiose, prophase jeg opstår under fosterudviklingen og anholdelser i dictyate fase indtil puberteten. Derimod begynder spermatogenesen ved puberteten og skrider frem i bølger i hele voksenlivet uden anholdelse. Forskelle mellem mandlige og kvindelige meiose understreger behovet for at udvikle metoder, der er specifikt dækket mod vurdering af disse processer i både spermatocytes og oocyter. I øjeblikket bygger vurderer meiotiske progression i vid udstrækning på anvendelse af kromatin opslag3,4,5. Mens kromatin spreads er nyttigt for at studere meiotiske kromosomer, undlader de at bevare cellulære integritet, forhindrer evaluering af cellulære strukturer som spindel mikrotubuli, centrosomes, nuklear kuvert og telomere vedhæftede filer. Live imaging og langsigtede dyrkningsbaserede teknikker er meget avancerede vores forståelse af kvindelige meiose; lignende metoder til at visualisere hele intakt cellen, dog gennemføres mindre hyppigt for studiet af spermatogenesen6,7. For at visualisere dynamiske begivenheder i hele mandlige meiose, har vi tilpasset etablerede tubulus squash teknikker til hurtigt at vurdere de cytologiske funktioner for at udvikle mus spermatocytes8,9. Metoden beskrevet her opretholder integriteten af cellen, giver studiet af flere cellulære strukturer i forskellige faser af spermatogenesen.
Denne tubulus squash teknik er en hele cellen tilgang, som giver mulighed for vurdering af cellulære strukturer via immunofluorescens mikroskopi. Fælles histologiske tilgange til at visualisere meiotiske progression i mandlige mus, såsom haematoxylin og eosin pletter af paraffin indlejret testiklerne, og immunfluorescent mærkning af snit frosset organmateriale giver mulighed for et bredt overblik over meiotiske progression. Men disse teknikker ikke løse enkelt celler i det omfang, der er nødvendige for detaljeret analyse af hændelser i hele meiose10,11. Alternative teknikker til at visualisere meiotiske processer er afhængige af betydelig kemiosmotisk afbrydelse af spermatocyte til at isolere og løse nukleare materialer3,4,5. Disse kemiske behandlinger hindre observation af celletyper end primære spermatocytes. En nylig beskrevne metode af Namekawa har aktiveret forskersamfundet til at bevare den nukleare arkitektur af isolerede spermatocytes, men kræver brug af en cytospin og tilbehør, der ikke kan være let tilgængelige for nogle laboratorier4. Tubulus squash teknik kræver derimod kun udstyr, der er generelt standard i de fleste celle biologi laboratorier.
Tubulus squash metode beskrevet her kan bruges til at visualisere de forskellige celletyper findes inden for den seminiferous tubulus, herunder sertoli celler, spermatogonia, primære og sekundære spermatocytes og spermatider. Ved at koble denne teknik med den nær-synkron første bølge af spermatogenesen i juvenile mus, er det muligt at opnå beriget populationer af spermatogenic celler, som de fremskridt gennem meiose12. Denne proces tillader den detaljerede analyse af processer i hele spermatogenesen, som tidlig prophase begivenheder, G2/MI og metafase anaphase overgange og spermiogenesis. Tubulus squash præparater kan desuden bruges til at visualisere cytologiske funktioner af kromosomer (f.eks. interchromatid domæner (ICDs) og kinetochores) og centrosomes (centrioler og pericentriolar materiale/matricer). Metoden squash kan udføres let parallelt med andre eksperimenterende tilgange, såsom kromatin spreads og protein udvinding. Desuden, er denne teknik ændret med held til at deponere levende spermatogenic celler på dias for direkte visualisering13.
Den metode beskrevet her indebærer en hel celle seminiferous tubulus squash teknik til at analysere G2/MI overgangen i wild-type C57BL/6J mus. De cytologiske træk ved primær spermatocytes ind i den første meiotiske deling blev visualiseret med immunofluorescens mikroskopi til at observere meiotiske spindlen. Denne alsidige teknik kan let modificeret til at visualisere anden meiotiske faser og forskellige celletyper. Teknikken er også åbne over for alternative visualisering strategier, såsom DNA og RNA fisk tilgange.

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			<td height="21" style="height:21px;width:251px;">16% Paraformaldehyde Aqueous</td>
			<td style="width:247px;">Electron Microscopy Sciences (EMS)</td>
			<td style="width:137px;">15710</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">10x PBS</td>
			<td style="width:247px;">Quality Biological</td>
			<td style="width:137px;">119-069-161</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Triton X-100</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:137px;">T8787</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">BSA</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:137px;">A1470</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Horse Serum</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:137px;">H-1270</td>
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			<td style="width:167px;">1 in 1000</td>
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			<td height="21" style="height:21px;width:251px;">Goat anti-SCP3</td>
			<td style="width:247px;">Santa Cruz</td>
			<td style="width:137px;">sc-20845</td>
			<td style="width:167px;">1 in 50</td>
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			<td height="21" style="height:21px;width:251px;">Human anti-Centromere Protein</td>
			<td style="width:247px;">Antibodies Incorporated</td>
			<td style="width:137px;">15-235</td>
			<td style="width:167px;">1 in 100</td>
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			<td height="21" style="height:21px;width:251px;">Mouse anti-alpha tubulin</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:137px;">T9026</td>
			<td style="width:167px;">1 in 1000</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mouse anti-AIM1</td>
			<td style="width:247px;">BD Biosciences</td>
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			<td style="width:167px;">1 in 200</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mouse anti-&#947;H2AX</td>
			<td style="width:247px;">Thermo Fisher</td>
			<td style="width:137px;">MA1-2022</td>
			<td style="width:167px;">1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mouse anti-CENT3</td>
			<td style="width:247px;">Abnova</td>
			<td style="width:137px;">H00001070-M01</td>
			<td style="width:167px;">1 in 200</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Rabbit anti-pericentrin</td>
			<td style="width:247px;">Abcam</td>
			<td style="width:137px;">ab4448</td>
			<td style="width:167px;">1 in 200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Rabbit anti-REC8</td>
			<td style="width:247px;">Courtesy of&#160; Dr. Karen Schindler</td>
			<td style="width:137px;">N/A</td>
			<td style="width:167px;">1 in 1000</td>
		</tr>
	</tbody>
</table>

a seminiferous tubule squash technique for the cytological analysis of spermatogenesis using the mouse model

Meiotiske progression i hanner er en proces, der kræver en samordnet indsats af en række stærkt regulerede cellulære begivenheder. Fejl, der opstår under meiose kan føre til barnløshed, graviditet tab eller genetiske defekter. Begyndende ved indtræden af puberteten og fortsætter gennem voksenalderen, kontinuerlig semisynkron bølger af spermatocytes gennemgå spermatogenesen og i sidste ende udgør haploide sædceller. Den første bølge af mus spermatocytes gennemgår meiotiske indledning vises på dag 10 post-partum (10 dpp) og er udgivet i lumen af seminiferous tubules som modne sæd på 35 dpp. Derfor er det fordelagtigt at bruge mus i dette udviklingsmæssige tidsvindue for at få højt beriget populationer af interesse. Analyse af sjældne celle faser er vanskeligere i ældre mus af successive spermatogenic bølger, der øger mangfoldigheden af de cellulære befolkninger i tubuli bidrag. Metoden beskrevet her er en let gennemføres teknik for cytologiske evaluering af de celler, der findes inden for mus, herunder spermatogonia, spermatocytes og spermatider seminiferous tubules. Tubulus squash teknik opretholder integriteten af isolerede mandlige kønsceller og giver mulighed for undersøgelse af cellulære strukturer, der ikke er let visualiseres med andre teknikker. For at vise de mulige anvendelser af denne tubulus squash teknik, blev spindel forsamling overvåget i spermatocytes forløber gennem prophase til metafase jeg overgang (G2/MI overgang). Derudover blev centrosome dobbeltarbejde, meiotiske sex kromosom Inaktiveringen (MSCI) og kromosom buket dannelse vurderet som eksempler på de cytologiske strukturer, som kan observeres ved hjælp af denne tubulus squash metode. Denne teknik kan bruges til at indkredse specifikke defekter under spermatogenesen, der er forårsaget af mutation eller eksogen undertrykkelse af netbårne, og således bidrager til vores Molekylær forståelse af spermatogenesen.

Her, vi har brugt metoden tubulus squash for at visualisere forbigående cellepopulationer gennemgår prophase til metafase jeg (G2/MI) overgang, som var beriget af høst testiklerne fra juvenile wild-type mus gennemgår den første bølge af spermatogenese (24 DPP). Figur 1 viser repræsentative billeder af forskellige celle faser, der kan visualiseres ved hjælp af metoden tubulus squash. Beriget populationer af metafase jeg spermatocytes blev visualiseret ...

Watch this Scientific Journal Video about A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model at JoVE.com

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Målet med denne tubulus squash teknik er til hurtigt at vurdere cytologiske funktioner for at udvikle mus spermatocytes samtidig bevare cellulære integritet. Denne metode giver mulighed for undersøgelse af alle faser af spermatogenesen, og nemt kan gennemføres sammen med andre biokemiske og molekylær biologiske metoder til undersøgelse af mus meiose.

En Seminiferous tubulus Squash teknik til cytologiske analyse af spermatogenesen ved hjælp af musen Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

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