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Funktionel vurdering af kinesin-7 CENP-E i spermatocytter ved anvendelse af in vivo-hæmning , immunofluorescens og flowcytometri

Published: December 28, 2021
doi:
10.3791/63271
, , , , , , ,
1Department of Cell Biology and Genetics, The School of Basic Medical Sciences,Fujian Medical University, 2Key Laboratory of Stem Cell Engineering and Regenerative Medicine,Fujian Province University, 3Fujian Obstetrics and Gynecology Hospital, 4Medical Research Center, Fujian Maternity and Child Health Hospital,Affiliated Hospital of Fujian Medical University

Summary

Denne artikel rapporterer en in vivo hæmning af CENP-E gennem abdominal kirurgi og testikel injektion af GSK923295, en værdifuld model for mandlig meiotisk deling. Ved hjælp af immunofluorescens-, flowcytometri- og transmissionselektronmikroskopi-assays viser vi, at CENP-E-hæmning resulterer i kromosomforskydning og genomustabilitet i musespermatocytter.

Abstract

I eukaryoter er meiose afgørende for genomstabilitet og genetisk mangfoldighed i seksuel reproduktion. Eksperimentelle analyser af spermatocytter i testikler er afgørende for undersøgelserne af spindelsamling og kromosomadskillelse i mandlig meiotisk division. Musens spermatocyt er en ideel model for mekanistiske undersøgelser af meiose, men de effektive metoder til analyse af spermatocytter mangler. I denne artikel rapporteres en praktisk og effektiv metode til in vivo-hæmning af kinesin-7 CENP-E i musespermatocytter. En detaljeret procedure for testikelinjektion af en specifik hæmmer GSK923295 gennem abdominal kirurgi hos 3 uger gamle mus præsenteres. Desuden er beskrevet her en række protokoller til vævsindsamling og fiksering, hæmatoxylin-eosinfarvning, immunofluorescens, flowcytometri og transmissionselektronmikroskopi. Her præsenterer vi en in vivo hæmningsmodel via abdominal kirurgi og testikelinjektion, der kunne være en kraftfuld teknik til at studere mandlig meiose. Vi demonstrerer også, at CENP-E-hæmning resulterer i kromosomforskydning og metafasestop i primære spermatocytter under meiose I. Vores in vivo-hæmningsmetode vil lette mekanistiske undersøgelser af meiose, tjene som en nyttig metode til genetiske modifikationer af mandlige kønsceller og kaste lys over fremtidige kliniske anvendelser.

Introduction

Meiose er en af de vigtigste, meget stive, evolutionære bevarede begivenheder i eukaryote organismer og er afgørende for gametogenese, seksuel reproduktion, genomintegritet og genetisk mangfoldighed 1,2,3. Hos pattedyr gennemgår kimcellerne to på hinanden følgende celledelinger, meiose I og II, efter en enkelt runde DNA-replikation. I modsætning til søsterkromatider i mitose parres duplikerede homologe kromosomer og adskilles i to datterceller under meiose I 4,5. I meiose II trækker søsterkromatider sig fra hinanden og adskilles for at danne haploide gameter uden DNA-replikation6. Fejl i en af de to meiotiske divisioner, herunder spindelsamlingsfejl og kromosomfejlegregering, kan resultere i tab af kønsceller, sterilitet eller aneuploidisyndromer 7,8,9.

Akkumulerende undersøgelser har vist, at kinesinfamiliemotorer spiller en afgørende rolle i reguleringen af kromosomjustering og adskillelse, spindelsamling, cytokinese og cellecyklusprogression i både mitotiske og meiotiske celler10,11,12. Kinesin-7 CENP-E (Centromere protein E) er en plus-end-rettet kinetochore motor, der kræves til kromosomkongres, kromosomtransport og justering og regulering af spindelsamlingskontrolpunkt i mitose 13,14,15,16,17,18. Under meiose fører CENP-E-hæmning af den specifikke hæmmer GSK923295 til cellecyklusstop, kromosomforskydning, spindeldisorganisering og genomstabilitet i spermatogene celler19. Lokaliseringsmønstrene og dynamikken i CENP-E ved centromererne af delende spermatocytter indikerer, at CENP-E interagerer med kinetochore-proteiner til sekventiel samling af centromerer under meiose I20,21. I oocytter kræves CENP-E til kromosomjustering og afslutning af meiose I13,22,23. Antistoffer eller morpholino injektion af CENP-E resulterer i forkert justerede kromosomer, unormal kinetochore orientering, og meiose jeg arresterer i både mus og Drosophila oocytter23. Sammenlignet med CENP-E’s væsentlige roller i mitose forbliver CENP-E’s funktioner og mekanismer i meiose stort set ukendte. Detaljerede mekanismer for CENP-E i kromosomkongresion og genomstabilitet i mandlige meiotiske celler mangler stadig at blive afklaret.

Spermatogenese er en kompleks og langvarig fysiologiproces, der involverer sekventiel spermatogonia-proliferation, meiose og spermiogenese. Derfor er hele processen ekstraordinært vanskelig at reproducere in vitro hos pattedyr og andre arter24,25. Det er umuligt at inducere spermatocytter differentiering efter pachytenstadiet in vitro. Undersøgelser af mandlige meiotiske divisioner har generelt været begrænset til eksperimentelle analyser af tidlig meiotisk profase25,26. På trods af mange teknologiske bestræbelser, herunder kortvarig kultur af spermatocytter27,28 og organkulturmetoder25, er der få effektive metoder til at studere mandlig meiotisk opdeling. Desuden resulterer genetisk sletning af essentielle gener normalt i udviklingsstop og embryonal dødelighed. F.eks. undlader museembryoner, der mangler CENP-E, at implantere og kan ikke udvikle sig efter implantation29, hvilket er en hindring i mekanistiske undersøgelser af CENP-E ved meiose. Samlet set kan etablering af et praktisk og gennemførligt system til undersøgelse af mandlig meiotisk opdeling i høj grad fremme forskningsområdet meiose.

Den lille cellegennemtrængelige hæmmer er et kraftfuldt værktøj til at studere kinesinmotorer i celledeling og udviklingsprocesser. Den allosteriske inhibitor, GSK923295, binder specifikt til CENP-E-motordomænet, blokerer frigivelsen af ADP (adenosindiphosphat) og stabiliserer endelig interaktionerne mellem CENP-E og mikrotubuli30. I denne undersøgelse præsenteres en in vivo hæmningsmusemodel gennem abdominal kirurgi og testikelinjektion af GSK923295. CENP-E-hæmning resulterer i kromosomforskydning i metafase I af primære spermatocytter. Desuden fører CENP-E-hæmning til meiotisk anholdelse af spermatocytter og forstyrrelse af spermatogenese. En række protokoller er beskrevet til analyse af spermatocytter og kan anvendes til at observere meiotiske spindelmikrotubuli, homologe kromosomer og subcellulære organeller i spermatocytter. Vores in vivo hæmningsmetode er en effektiv metode til studier af meiotisk deling og spermatogenese.

Protocol

Alle dyreforsøg blev gennemgået og godkendt af Animal Care and Use Committee ved Fujian Medical University (protokolnummer SYXK 2016-0007). Alle museforsøg blev udført i overensstemmelse med de relevante retningslinjer for pleje og brug af forsøgsdyr fra National Institutes of Health (NIH-publikationer nummer 8023, revideret 1978). 1. Konstruktion af GSK923295-medierede CENP-E hæmningsmusemodeller De kirurgiske instrumenter steriliseres ved 121 °C i 30 min. Be…

Representative Results

Vi har med succes konstrueret en in vivo CENP-E hæmningsmodel af musetestikler gennem abdominal kirurgi og testikelinjektion afGSK923295 19. De vigtigste tekniske trin i denne metode blev vist i figur 1. Efter testikelinjektion af GSK923295 i 4 dage blev testiklerne høstet til yderligere analyser. I kontrolgruppen var den spermatogene bølge i de seminiferøse tubuli regelmæssig og organiseret (figur 2A). I GSK923295-gruppen b…

Discussion

I dette studie har vi etableret en in vivo CENP-E hæmningsmodel af musetestikler ved hjælp af abdominal kirurgi og mikroinjektion af GSK923295. Den abdominale kirurgi og testikelinjektionsmetode, der anvendes i denne undersøgelse, har følgende fordele. For det første er det ikke begrænset til musens alder. Eksperimenter kan udføre testikelinjektion på et tidligt stadium, for eksempel hos 3 uger gamle eller yngre mus. For det andet har GSK923295 en specifik og fremragende hæmmende virkning på CENP-E. Fo…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi takker alle medlemmer af cytoskeletlaboratoriet ved Fujian Medical University for nyttige diskussioner. Vi takker Jun-Jin Lin på Public Technology Service Center, Fujian Medical University for teknisk assistance inden for flowcytometri. Vi takker Ming-Xia Wu og Lin-Ying Zhou på Electron Microscopy Lab of Public Technology Service Center, Fujian Medical University for teknisk assistance i elektronmikroskopi. Vi takker Si-Yi Zheng, Ying Lin, Qi Ke, og Jun Song på Experimental Teaching Center of Basic Medical Sciences ved Fujian Medical University for deres støtter. Denne undersøgelse blev støttet af følgende bevillinger: National Natural Science Foundation of China (bevillingsnummer 82001608), Natural Science Foundation of Fujian-provinsen, Kina (bevillingsnummer 2019J05071), Fujian Provincial Health Technology Project (bevillingsnummer 2018-1-69), Startup Fund for Scientific Research, Fujian Medical University (bevillingsnummer 2017XQ1001), Fujian Medical University talenter på højt niveau videnskabeligt forskningsstartfinansieringsprojekt (bevillingsnummer XRCZX2017025) og Forskningsprojekt af online uddannelse og undervisning af kinesiske medicin kandidatstuderende (bevilling nummer B-YXC20200202-06).

Materials

0.25% Trypsin-EDTA Gibco 25200056
1 ml Syringe Several commercial brands available Sterile.
1.5 mL Centrifuge tube Axygen MCT-150-C
50 mL Centrifuge Tube Corning 430828
6 cm Petri dish Corning 430166
95% Ethanol Sinopharm Chemical Reagent Co.,Ltd 10009164
tubulin rabbit polyclonal antibody Beyotime AF0001 For immunofluorescence assays. Use at 1:100.
rabbit anti-Histone H3 (phospho S10) monoclonal antibody Abcam ab267372 For immunofluorescence assays. Use at 1:100.
rabbit anti-TUBA4A polyclonal antibody Sangon Biotech D110022 For immunofluorescence assays. Use at 1:100.
Anti-SYCP3 rabbit monoclonal antibody Abcam ab175191 For immunofluorescence assays. Use at 1:100.
Adhesion microscope slides CITOTEST 188105
Alexa fluor 488-labeled goat anti-rabbit antibody Beyotime A0423 Sencodary antibody. Use at 1:500.
Aluminium potassium sulphate Sinopharm Chemical Reagent Co.,Ltd 10001060
Anhydrous ethanol Sinopharm Chemical Reagent Co.,Ltd 100092690
Anti-fade mounting medium Beyotime P0131 Prevent photobleching of flourescent signals.
BD FACS Canto II BD Biosciences FACS Canto II
Bovine Serum Albumin Sinopharm Chemical Reagent Co.,Ltd 69003435
Centrifuge Eppendorf 5424BK745380
Chloral hydrate Sinopharm Chemical Reagent Co.,Ltd 80037516
Citric acid Shanghai Experiment Reagent Co., Ltd 122670
Collagenase Sangon Biotech A004194-0100
Coverslips CITOTEST 10212020C 20 × 20 mm. Thickness 0.13-0.16 mm.
DAPI Beyotime C1006
Dye vat Several commercial brands available 91347802
Eosin Y, alcohol soluble Sinopharm Chemical Reagent Co.,Ltd 71014460
Ether Sinopharm Chemical Reagent Co.,Ltd 10009318
Formaldehyde – aqueous solution Sinopharm Chemical Reagent Co.,Ltd 10010018
GSK923295 MedChemExpress HY-10299
Hematoxylin, anhydrous Sinopharm Chemical Reagent Co.,Ltd 71020784
ICR mouse Shanghai SLAC Laboratory Animal Co., Ltd
Image J software National Institutes of Health https://imagej.nih.gov/ij/ Fluorescent image analysis.
Leica ultramicrotome Leica
Leica EM UC-7 ultramicrotome Leica EM UC7
Modfit MFLT32 Verity Software House For analysis of flow cytometry results.
Nail polish Several commercial brands available
Neutral gum Sinopharm Chemical Reagent Co.,Ltd 10004160
Nikon Ti-S2 microscope Nikon Ti-S2
Picric acid Sinopharm Chemical Reagent Co.,Ltd J60807
Rheodyne Sangon Biotech F519160-0001 10 μl rheodyne
Sliced paraffin Sinopharm Chemical Reagent Co.,Ltd 69019461
Sodium iodate Sinopharm Chemical Reagent Co.,Ltd 80117214
Surgical instruments Several commercial brands available For abdominal surgery. Sterilize at 121 °C, 20 min.
Transmission electron microscope FEI Tecnai G2
Trisodium citrate dihydrate Shanghai Experiment Reagent Co., Ltd 173970
Triton X-100 Sinopharm Chemical Reagent Co.,Ltd 30188928 Dilute in sterile PBS to make a 0.25% working solution.
Tween 20 Sinopharm Chemical Reagent Co.,Ltd 30189328 Dilute in sterile PBS to make a 0.1% working solution.
Paraformaldehyde Sinopharm Chemical Reagent Co.,Ltd 80096618
Xylene Sinopharm Chemical Reagent Co.,Ltd 10023418
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Tags

Keywords: MeiosisSpermatogenesisSpermatocytesCENP-EGSK923295ImmunofluorescenceFlow CytometryGene EditingMouse ModelMicrotome
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Xu, M., Yang, Y., Wei, Y., Zhang, J., Lin, X., Lin, X., Chen, H., She, Z. Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry. J. Vis. Exp. (178), e63271, doi:10.3791/63271 (2021).
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