The Pennsylvania State University The Graduate School Department of Animal Science CHARACTERIZATION OF THE PRAME/PRAMEY GENE FAMILY DURING SPERMATOGENESIS A Thesis in Animal Science by Yaqi Zhao 2013 Yaqi Zhao Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2013 The thesis of Yaqi Zhao was reviewed and approved* by the following: Wansheng Liu Associate Professor of Animal Genomics Thesis Advisor Francisco J. Diaz Assistant Professor of Reproductive Biology Joy L. Pate Professor of Reproductive Physiology Terry D. Etherton Distinguished Professor of Animal Nutrition Head of the Department of Animal Science *Signatures are on file in the Graduate School iii ABSTRACT Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis (CT) antigen that is predominantly expressed in normal gametogenic tissues and a variety of tumors. Like other CT antigens, PRAME is a multi-copy gene family representing one of the most amplified gene families in eutherian mammals. Members of the PRAME gene family encode leucine-rich repeat (LRR) proteins that fold into a horseshoe shape and provide a versatile structural framework for the formation of protein-protein interactions. PRAME has been extensively studied in cancer biology and is believed to play a regulatory role in cancer cells. The function of PRAME during testicular development and spermatogenesis remains unknown. The objective of this study is to characterize the expression of PRAME during spermatogenesis, to identify protein(s) that interact with PRAME and to explore the possible functions of PRAME. We chose the bovine PRAMEY (on chromosome Y) and mouse Pramel1 (on chromosome 4) as representatives of the PRAME gene family in this study. Using a custom peptide-specific antibody, PRAMEY was characterized as a testis- and spermatozoa-specific protein in bovine. PRAMEY was expressed in the acrosome of spermatids, as well as the acrosome and flagellum of spermatozoa. Immunogold electron microscopy revealed the subcellular localization of PRAMEY in spermatids and spermatozoa: PRAMEY was firstly localized in the acrosomal granule of step 4 spermatids, migrated with the content of acrosomal granule during spermiogenesis, and was finally present in the acrosome lumen of mature spermatozoa. To identify the PRAME interactive protein(s), we performed co- immunoprecipitation (co-IP) using the PP1γ2 and PRAMEY antibodies in bovine iv spermatozoa. The results showed that PRAMEY was co-immunoprecipitated with PP1γ2 in caput and caudal epididymal sperm. By blocking PRAMEY during in vitro fertilization (IVF), fewer embryos were formed while the incidence of polyspermy increased, suggesting a role of PRAMEY in fertilization and blocking of polyspermy. In mice, the expression level of the PRAMEL1 protein was very low in newborn testes, then was increased gradually from 1- to 3-week-old testes, and remained constant after three weeks of age. Immunofluorescent staining on testis cross sections revealed that PRAMEL1 was localized in the cytoplasm of spermatocytes in 2-week-old testis, and translocated to the acrosomal region of round spermatids in 3-week-old testis. Taken together, these results suggest that PRAME is involved in acrosome formation during spermatogenesis, functions in block to polyspermy during fertilization, may interact with PP1γ2 and play a role in the regulation of sperm motility. v TABLE OF CONTENTS List of Figures .............................................................................................................. vii List of Tables ............................................................................................................... viii Acknowledgements ...................................................................................................... ix Chapter 1 Review of the Literature .............................................................................. 1 1. Cancer/testis (CT) antigen ................................................................................ 1 1.1 CT antigens in cancer .............................................................................. 1 1.2 CT antigens in testis ................................................................................ 4 2. Preferentially expressed antigen in melanoma (PRAME) ................................ 5 2.1 The discovery of PRAME ....................................................................... 5 2.2 PRAME is a largely expanded gene family in eutheria ........................... 6 2.3 PRAME is a leucine rich repeat (LRR) protein ....................................... 7 2.4 Expression and function of PRAME in cancer ........................................ 10 2.5 Expression and Function of PRAME in reproduction ............................. 12 3. Spermatogenesis ............................................................................................... 14 3.1 Phases of spermatogenesis ...................................................................... 15 3.2 The first wave of spermatogenesis .......................................................... 19 3.3 Acrosome biogenesis ............................................................................... 21 3.4 Structure of the mature acrosome ............................................................ 22 3.5 Molecular mechanism of acrosome biogenesis ....................................... 24 3.6 Modulation of sperm motility by PP1γ2 complexes ............................... 25 4. Fertilization and block of polyspermy .............................................................. 27 5. Summary and the objectives of this study ........................................................ 28 Chapter 2 Characterization of the Bovine PRAMEY During Spermiogenesis, and its Potential Role in the Block to Polyspermy During Fertilization ..................... 30 1. Introduction ....................................................................................................... 30 2. Materials and methods ...................................................................................... 33 3. Results............................................................................................................... 42 4. Discussion ......................................................................................................... 75 Chapter 3 The Expression and Localization of the Mouse PRAMEL1 during the First Wave of Spermatogenesis ............................................................................ 81 1. Introduction ....................................................................................................... 81 2. Materials and Methods ..................................................................................... 83 3. Results............................................................................................................... 86 vi 4. Discussion ......................................................................................................... 92 Summary ...................................................................................................................... 94 References .................................................................................................................... 95 vii LIST OF FIGURES Figure 1-1. The 3D structure of PRAME protein showing its horseshoe shape (Chang et al., 2011). ............................................................................................. 9 Figure 1-2. Mouse stages in the cycle of the seminiferous epithelium (Hess and Renato de Franca, 2008). ...................................................................................... 17 Figure 1-3. Bovine stages in the cycle of the seminiferous epithelium (Berndston and Desjardins, 1974). .......................................................................................... 18 Figure 2-1. Identification of the bovine PRAMEY protein in caudal spermatozoa by western blot with the custom-made PRAMEY antibody. ............................... 45 Figure 2-2. Comparison of the bovine sperm PRAMEY proteins extracted either by different buffers or at different freeze-thaw times. .......................................... 46 Figure 2-3. Temporal expression of the bovine PRAMEY protein during testis development. ......................................................................................................... 48 Figure 2-4. Localization of the bovine PRAMEY during spermiogenesis. ................. 50 Figure 2-5. Localization of the bovine PRAMEY in testicular spermatozoa, caput and caudal epididymal spermatozoa ..................................................................... 52 Figure 2-6. The subcellular localization of the bovine PRAMEY in spermatids and spermatozoa revealed by immunogold electron microscopy. ........................ 57 Figure 2-7. Co-immunoprecipitation analysis of PP1γ2 with PRAMEY in bovine. ... 66 Figure 2-8. 2-D electrophoresis of the bovine caudal sperm proteins. ........................ 68 Figure 2-9. Enrichment of the bovine sperm PRAMEY protein by immunoprecipitation. ............................................................................................ 70 Figure 2-10. Sperm-oocyte binding and IVF using caudal sperm incubated with the PRAMEY antibody. ........................................................................................ 73 Figure 3-1. Temporal expression analysis of Pramel1 by RT-PCR during the mouse testis development. ...................................................................................