Department of Urology
Vancouver Prostate Centre, University of British Columbia
A novel cell-sorting strategy for the isolation of germ cells transitioning into meiosis
Meghan Robinson1, Ryan Flannigan1,3,4, Luke Witherspoon1,2,3.
1Urology, Vancouver Prostate Centre, Vancouver, BC, Canada; 2Urology, Ottawa Hospital, Ottawa, ON, Canada; 3Urology, University of British Columbia, Vancouver, BC, Canada; 4Urology, Weill Cornell Medicine, New York, NY, United States
Vancouver Coastal Health Research Institute (VCHRI). Canadian Urological Association Scholarship Foundation (CUASF). Canadian Institute of Health Research (CIHR). University of British Columbia Department of Urological Sciences.
Introduction: Spermatogenesis is driven by meiotic division, wherein a single cell divides twice to produce four cells containing half the original amount of genetic information. Entry into meiosis is regulated by Stimulated by Retinoic Acid Gene 8 (STRA8), and its dysfunction leads to infertility. In this study we set out to isolate STRA8-expressing germ cells using fluorescent-activated cell sorting (FACs).
Methods: 10X Genomics Single Cell Sequencing of a human testis biopsy identified surface proteins associated with pre-meiotic and early meiotic germ cells, which were used to sort cells via FACS. Sorted cells were analyzed for gene expression using real time polymerase chain reaction (RT-qPCR) assays. Results were validated by immunostaining of tissue sections.
Results: Screening for a surface marker to isolate a pre to early meiotic stage of germ cell resulted in the identification of Serine Protease 50 (PRSS50/TSP50). TSP50-sorted cells had enhanced expression of pre-meiotic and early meiotic stage germ cells (Figure 1B). The highest fold change in gene expression was STRA8 (56-fold), followed by the early meiotic marker Doublesex- And Mab-3-Related Transcription Factor B1 (DMRTB1), the meiotic transcription factor Deleted In Azoospermia Like (DAZL), the pre-meiotic/mitotic spermatagonia marker Melanoma-Associated Antigen 4 (MAGEA4), the meiotic marker Synaptonemal Complex Protein 3 (SYCP3), and the pre-meiotic marker Mast/Stem Cell Growth Factor Receptor Kit (KIT). Immunostaining confirmed the localization of TSP50 to germ cells, and co-expression of TSP50 and SYCP3 in a subset of germ cells, illustrating a transition between pre-meiotic and early meiotic stages (Figure 1A).
Conclusions: This study identified the surface marker TSP50 for FACS isolation of STRA8-expressing germ cells in pre-meiotic to early meiotic stages of spermatogenesis. Isolation of these cells provides an avenue for in depth characterization of STRA8 function in spermatogenesis.
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