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The Effects of Retinoic Acid on Spermatogonial Stem Cell Differentiation in Vitro

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The Effects of Retinoic Acid on Spermatogonial Stem Cell Differentiation in Vitro The Effects of Retinoic Acid on Spermatogonial Stem Cell Differentiation In Vitro Sabrina Sicilia Department of Medicine Division of Experimental Medicine McGill University Montreal, Quebec, Canada November 2013 A thesis submitted to McGill University in partial fulfillment of the requirement of the degree of Masters of Science Sabrina Sicilia © 2013 i TABLE OF CONTENTS Abstract ...................................................................................................... page VI Résumé ..................................................................................................... page VIII Acknowledgements ..................................................................................... page X Contributions by author ........................................................................... page XI Abbreviations ........................................................................................... page XII Chapter 1: Introduction .............................................................................. page 1 The embryonic origin of male germ cells .......................................... page 1 Stages in the production of mature spermatozoa ............................... page 4 Spermatogonial stem cells ............................................................... page 10 Factors involved in the maintenance of SSCs ..................... page 11 Factors involved in the differentiation of SSCs .................. page 16 The role of retinoic acid in the differentiation of SSCs ................... page 19 Isolating SSCs – elucidating their specific markers ........................ page 23 Donor derived spermatogenesis in a recipient testis ....................... page 26 In vitro SSC culturing ...................................................................... page 27 Research hypothesis ........................................................................ page 30 Research objectives ......................................................................... page 31 Chapter 2: Materials and Methods .......................................................... page 33 Establishment of a Germ-Cell Cell Line ......................................... page 33 Immunocytochemistry against RARα – no treatment ..................... page 34 Cell Chain Counting ........................................................................ page 36 Cluster forming activity assay ......................................................... page 38 Spermatogonial Transplantation ...................................................... page 40 In vitro Apoptosis Assay ................................................................. page 43 Flow Cytometric Analysis of Apoptosis ......................................... page 44 ii Microarray ....................................................................................... page 45 Quantitative real-time RT-PCR ....................................................... page 46 ICC against LRPAP1 and Stra8 – with treatment ........................... page 48 Chapter 3: Results ..................................................................................... page 49 Expression pattern of RARα ............................................................ page 49 Overview of cluster cells’ morphology post-treatment ................... page 51 Overview of cluster cells’ morphology post-treatment ....... page 51 Cell chain kinetics post-treatment ....................................... page 52 Quantification of SSCs after RA treatment ..................................... page 56 Cluster forming activity assay ............................................. page 56 Transplantation Assay ......................................................... page 58 Transplantation – cultured with feeders .................. page 58 Feeder-Free Transplantation .................................... page 60 Cell death after RA treatment .......................................................... page 62 Gene expression pattern after RA treatment ................................... page 67 Protein expression pattern after RA treatment ................................ page 72 STRA8 expression after treatment .......................... page 72 LRPAP1 expression after treatment ........................ page 73 Chapter 4: Discussion and Future Experiments ..................................... page 77 Annex .......................................................................................................... page 84 Annex 1: Microarray data – Genes upregulated .............................. page 84 Annex 2: Microarray data – Genes downregulated ......................... page 91 Annex 3: Response to Thesis Evaluator’s Comments ................... page 105 References ................................................................................................ page 108 iii LIST OF FIGURES Chapter 1: Introduction Figure 1 Spermatogenesis .................................................................... page 7 Figure 2 Cellular bridges ..................................................................... page 8 Figure 3 Organization of the seminiferous tubule epithelium ........... page 12 Figure 4 Retinoic acid in the post natal testis .................................... page 20 Figure 5 Spermatogonia Markers ....................................................... page 26 Figure 6 In vitro germ cell cluster culture ......................................... page 28 Chapter 2: Materials and Methods Figure 7 Cell chain counting ............................................................ page 37 Figure 8 Morphology of cell chains ................................................. page 38 Figure 9 Cluster forming activity assay ........................................... page 40 Figure 10 In vivo donor derived GFP colonies .................................. page 43 Chapter 3: Results Figure 11 RARα expression pattern in cluster cells ............................ page 50 Figure 12 Cluster cells’ morphology after RA treatment .................... page 51 Figure 13 Kinetics of various cell chain formations ............................ page 54 Figure 14 Total number of cell chains formed post-treatment ............ page 56 Figure 15 Cluster forming activity assay post-treatment ..................... page 58 Figure 16 Transplantation assay with feeder culture conditions ......... page 60 iv Figure 17 Transplantation assay with feeder-free culture .................... page 62 Figure 18 In vitro apoptosis 24 hours post-treatment .......................... page 65 Figure 19 In vitro apoptosis 48 hours post-treatment .......................... page 66 Figure 20 Quantification of cellular apoptosis post-treatment ............ page 67 Figure 21 STRA8 expression pattern post-treatment ........................... page 73 Figure 22 LRPAP1 expression pattern post-treatment ........................ page 75 Figure 23 LRPAP1 expression pattern of cell chain post-treatment .... page 76 Chapter 4: Discussion and Future Experiments Figure 24 Study summary figure ......................................................... page 79 LIST OF TABLES Chapter 2: Materials and Methods Table 1 LRPAP1, RARα, and STRA8 concise ICC protocol .......... page 36 Table 2 qPCR forward and reverse primer sequence ....................... page 47 Chapter 3: Results Table 3 Microarray and qPCR data .................................................. page 71 v ABSTRACT The fundamental biological functions of spermatogonial stem cells (SSCs) is to support sperm generation and as such the propagation of genetic material to future generations. Like other stem cells, SSCs are defined by their ability to self-renew and also generate daughter cells which are committed to differentiation (J. M. Oatley & Brinster, 2008). It has been shown that retinoic acid (RA) is quintessential in spermatogenesis; its role is most clearly seen in its absence which results in infertility (Snyder, Small, & Griswold, 2010). In vivo RA acts on undifferentiated spermatogonia transitioning them into differentiating spermatogonia, consequently RA can serve as a key inducer of spermatogonial differentiation in vitro (Zhou et al., 2008). The objectives of my study were to assess the effects of RA on aggregates of undifferentiated spermatogonia in vitro (clusters), elucidate genes implicated in RA induced differentiation of SSCs and determine markers that coincide with the transition for SSC commitment to differentiation. I hypothesize that RA induces the differentiation of SSCs in vitro and further that SSCs can respond rapidly to RA. Results obtained support the hypothesis that RA rapidly induces SSC differentiation in vitro. These results included: observation of distinctive cell chain morphology in vitro post-treatment that is reflective of spermatogonia differentiation, SSC quantification confirms a decrease SSCs post-treatment and vi gene expression analysis reveals an increase in spermatogonial differentiation markers and a decrease of SSC specific markers following treatment. In addition to providing insight into RA directed SSC differentiation, gene expression analysis also revealed genes which were upregulated rapidly following RA treatment. These genes included LRPAP1 and Stxbp5; two genes have not been previously described in the context of SSC differentiation and may be novel markers for early SSC differentiation. These results indicate that the SSC-based, in vitro experimental paradigm that I used in this study provides an effective platform to further dissect
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