Uncovering Biology of Klinefelter Syndrome (47,XXY) Infertility 2019-03-15آ  1Center for Male Reproductive

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  • Background • Finding sperm in rare areas of dilated seminiferous tubules (DSFTs) among

    the mass of collapsed seminiferous tubules (CSFTs) in men with Klinefelter syndrome (KS) is difficult to explain.

    • Two plausible explanations of sperm production in KS are either spontaneous rescue of 47,XXY and loss of the additional X chromosomes in some areas of testis, or existence of low grade mosaicism in tissue levels.

    • Our lab has confirmed that all pre-meiotic cells in testis have two X chromosomes including spermatogonial stem cells (SSCs). Thus, we believe that along SFTs in men with KS there are similar number of SSCs, but due to optimal niche in some regions of testis the rescue of genetic defect is possible

    Objective • The aim of this study was to characterize heterogeneity of SSCs and

    somatic cells along the DSFTs and CSFTs from same patient and same testis using single cell sequencing.

    Methods • Two men with non-mosaic KS and azoospermia underwent microTESE. • The SFTs were divided into DSFTs or CSFTs under the operating

    microscope at the time of surgery. Single cell suspensions were prepared from the DSFTs and CSFTs followed by preparation of libraries for single cell sequencing with 10X Genomics chromium system.

    • Reads were aligned with STAR aligner, normalized and analyzed with Seurat v2.0.

    • Single tubules were stained with antibodies against UTF1 (SSCs) and SOX9 (Sertoli Cells).

    Results • 20,207 reads were mapped for each specimen to GRCh38.

    Uncovering Biology of Klinefelter Syndrome (47,XXY) Infertility Using Novel 10x Genomics Single Cell Sequencing Ryan 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, NY 3Consulting 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 Laks Foundation and Robert Dow Foundation

    Figure 1. Distributed stochastic neighbor embedding (t-SNE) plot demonstrating identifiable cell populations via cell- specific gene localization. Uniquely distinguishing genes to each cluster are reported to identify cell type. Dilated tubules (Left), collapsed tubules (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.

    UTF1 DAPI

    SOX9

    A B

    C6 Leydig Cell HSPA1B Germ Cell HLA-DRA Immune Cell ACTA2 PTMC FATE1 Sertoli Cell

    Figure 2. Discriminative genes were identified by Seurat per cellular cluster. Top 20 genes were visualized for cellular localization and used to assign cell-type to each cluster. Genes coding for proteins as demonstrated in the images above (Violin Plots – top; immunohistochemistry- bottom) are examples of those used to define cellular types. Images are of immunohistochemistry staining, acquired from proteinatlas.org.

    Collapsed Klinefelter Syndrome Testis Tubules Dilated Klinefelter Syndrome Testis Tubules

    Figure 3. Canonical Spermatogonial markers among collapsed tubules. BCL6B appears to be more specifically located to the germ cell cluster.

    Figure 3. Expression of interstitial progenitor cell markers are strongly expressed coinciding with Leydig cell markers, suggesting a possible immature state of Leydig cells.

    Conclusions • Germ cells, predominately spermatogonia are identified

    in both dilated and collapsed tubules. • Among canonical spermatogonial markers, BCL6B

    appears to be most specific to germ cells in our data. • Conventional protein markers of cells in the testis may

    not carry the same specificity as gene expression, reflecting the differential uncoupling of transcription and translation among cells in the testis.

    • Leydig cells may exist in a premature or poorly differentiated state among men with Klinefelter, contributing to hypergonadal hypogonadism.