Characterization of PRAMEY isoforms and their involvement in bovine sperm capacitation and acrosome reaction
Open Access
- Author:
- Kern, Chandlar
- Graduate Program:
- Animal Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- July 29, 2020
- Committee Members:
- Wansheng Liu, Thesis Advisor/Co-Advisor
Daniel M Kniffen, Committee Member
Francisco Javier Diaz, Committee Member
Terry D Etherton, Program Head/Chair
Peter Sutovsky, Committee Member - Keywords:
- PRAMEY
Capacitation
Acrosome Reaction
spermatogenesis - Abstract:
- Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal gametogenic tissues and a variety of tumors. The PRAME protein was discovered initially in a human melanoma cell line with early studies in cancer biology finding PRAME to be a dominant repressor involved in the retinoic acid receptor (RAR) signaling in melanoma cells. PRAME is a leucine-rich repeat (LRR) protein that folds into a horseshoe shape, providing a structural framework for the formation of protein–protein interactions. Like other CTAs, PRAME is a multi-copy gene family representing one of the most amplified gene families in eutherian mammals. In bovine, there are multiple copies of PRAME on chromosome 16, and a single copy on chromosome 17. PRAME on the bovine chromosome 17 underwent an autosome-to-Y transposition and amplification resulting in a PRAME Y-linked (PRAMEY) gene subfamily, which is expressed solely in testis. Copy number variation (CNV) of PRAMEY is associated with testis size and male fertility in cattle highlighting the importance of this protein in male reproduction. While PRAME has been widely studied in cancer biology, the role of PRAME during spermatogenesis or physiological processes that spermatozoa experience thereafter (i.e. capacitation and acrosome reaction) have not been extensively evaluated. The objective of this study is to characterize the isoforms of the Y-linked PRAME (PRAMEY) and to explore their potential functions during spermatozoal maturation, capacitation and the acrosome reaction (AR). Using a custom peptide-specific antibody, PRAMEY was characterized in tissue, reproductive fluid, and spermatozoa from bovine testes (n=5) and caput and cauda segments of the epididymis (n=5) to evaluate PRAMEY as spermatozoa mature. Western blot (WB) analysis revealed that the testis expressed solely the 58 and 30 kDa PRAMEY isoforms, suggesting their involvement in spermatogenesis. The 13 kDa isoform was characterized as likely being involved in sperm motility, as it was highly present in spermatozoa tails from the cauda epididymis. To evaluate PRAMEY during capacitation and the AR, freshly ejaculated spermatozoa were induced to capacitate and AR in vitro (n=5). We found that the changes in sperm protein concentration of 26 and 13 kDa isoforms in the 4 hr. control and capacitation groups suggest the involvement of the PRAMEY protein in sperm capacitation. Furthermore, the absence of the PRAMEY isoforms in AR sperm suggests that the PRAMEY protein is being released during the AR, suggesting PRAMEY’s function during fertilization. The results obtained from this project will help elucidate PRAMEY’s role in fertilization and reproduction as a whole.