Post on 11-Aug-2020
Background• Findingsperminrareareasofdilatedseminiferoustubules(DSFTs)among
themassofcollapsedseminiferoustubules(CSFTs)inmenwithKlinefeltersyndrome(KS)isdifficulttoexplain.
• TwoplausibleexplanationsofspermproductioninKSareeitherspontaneousrescueof47,XXYandlossoftheadditionalXchromosomesinsomeareasoftestis,orexistenceoflowgrademosaicismintissuelevels.
• Ourlabhasconfirmedthatallpre-meioticcellsintestishavetwoXchromosomesincludingspermatogonial stemcells(SSCs).Thus,webelievethatalongSFTsinmenwithKStherearesimilarnumberofSSCs,butduetooptimalnicheinsomeregionsoftestistherescueofgeneticdefectispossible
Objective• TheaimofthisstudywastocharacterizeheterogeneityofSSCsand
somaticcellsalongtheDSFTsandCSFTsfromsamepatientandsametestisusingsinglecellsequencing.
Methods• Twomenwithnon-mosaicKSandazoospermiaunderwentmicroTESE.• TheSFTsweredividedintoDSFTsorCSFTsundertheoperating
microscopeatthetimeofsurgery.SinglecellsuspensionswerepreparedfromtheDSFTsandCSFTsfollowedbypreparationoflibrariesforsinglecellsequencingwith10XGenomicschromiumsystem.
• ReadswerealignedwithSTARaligner,normalizedandanalyzedwithSeuratv2.0.
• SingletubuleswerestainedwithantibodiesagainstUTF1(SSCs)andSOX9(Sertoli Cells).
Results• 20,207readsweremappedforeachspecimentoGRCh38.
UncoveringBiologyofKlinefelter Syndrome(47,XXY)InfertilityUsingNovel10xGenomicsSingleCellSequencingRyan Flannigan1, Ishaan Gupta, Ana-Maria Sutii, Fabien Campagne, Anna Mielnik1, Jackson Hobgood, Russell Hayden1, Alex Bolyakov1, Peter N. Schlegel1, Darius A. Paduch1,3
1Center for Male Reproductive Medicine & Microsurgery, Weill Cornell Medicine, New York, NY3Consulting Research Services Inc., Bergen, New Jersey
FUNDING SOURCE: Urology Care Foundation Research Scholar Award, New York Section. NIH grants P50HD076210 (Project II and outreach core) with additional funds from Howard and Irena LaksFoundation and Robert Dow Foundation
Figure1.Distributedstochasticneighborembedding(t-SNE)plotdemonstratingidentifiablecellpopulationsviacell-specificgenelocalization.Uniquelydistinguishinggenestoeachclusterarereportedtoidentifycelltype.Dilatedtubules(Left),collapsedtubules(right).
Figure 2. Confocal microscopy & immunofluorescence (400x magnification) of a seminiferous tubule from a man with Klinefelter Syndrome and Sertoli Cell Only (A) UTF1 positive SSCs (B) SOX9 positive Sertoli cells.
UTF1DAPI
SOX9
A B
C6Leydig Cell HSPA1BGermCell HLA-DRAImmuneCell ACTA2PTMC FATE1Sertoli Cell
Figure2.DiscriminativegeneswereidentifiedbySeuratpercellularcluster.Top20geneswerevisualizedforcellularlocalizationandusedtoassigncell-typetoeachcluster.Genescodingforproteinsasdemonstratedintheimagesabove(ViolinPlots– top;immunohistochemistry- bottom)areexamplesofthoseusedtodefinecellulartypes.Imagesareofimmunohistochemistrystaining,acquiredfromproteinatlas.org.
CollapsedKlinefelter SyndromeTestisTubulesDilatedKlinefelter SyndromeTestisTubules
Figure 3. Canonical Spermatogonial markers among collapsed tubules. BCL6B appears to be more specifically located to the germ cell cluster.
Figure3. ExpressionofinterstitialprogenitorcellmarkersarestronglyexpressedcoincidingwithLeydig cellmarkers,suggestingapossibleimmaturestateofLeydig cells.
Conclusions• Germcells,predominatelyspermatogonia areidentified
inbothdilatedandcollapsedtubules.• Amongcanonicalspermatogonial markers,BCL6B
appearstobemostspecifictogermcellsinourdata.• Conventionalproteinmarkersofcellsinthetestismay
notcarrythesamespecificityasgeneexpression,reflectingthedifferentialuncouplingoftranscriptionandtranslationamongcellsinthetestis.
• Leydig cellsmayexistinaprematureorpoorlydifferentiatedstateamongmenwithKlinefelter,contributingtohypergonadal hypogonadism.