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Page 1: Uncovering Biology of Klinefelter Syndrome (47,XXY) Infertility … · 2019-03-15 · 1Center for Male Reproductive Medicine & Microsurgery, Weill Cornell Medicine, New York, NY 3Consulting

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.

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