Characterization of the PRAME/PRAMEY Gene Family During Spermatogenesis
Open Access
- Author:
- Zhao, Yaqi
- Graduate Program:
- Animal Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- December 06, 2013
- Committee Members:
- Wansheng Liu, Thesis Advisor/Co-Advisor
Francisco Javier Diaz, Thesis Advisor/Co-Advisor
Joy Lee Pate, Thesis Advisor/Co-Advisor - Keywords:
- PRAME
spermatogenesis
fertilization
polyspermy
acrosomal granule
cancer/testis antigen - 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 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.