Poster abstracts
Poster number 62 submitted by Manuel Torres
Promoter Mutation in Ccna2 Reveals Novel Functions of the Protein in Spermatogenesis
Manuel Torres (Department of Molecular Genetics, The Ohio State University), Lindsey Kent (Medical University of South Carolina, Hollings Cancer Center)
Abstract:
Spermatogenesis is the process by which germ cells called Spermatogonia (SSCs) undergo subsequent differentiation events until spermatozoa are produced (1). As they cycle, SSCs express Cyclin A2 (CCNA2/Ccna2), which regulates the phosphorylation of proteins that drive the S phase and G2/M transition phase of the cell cycle (2). CCNA2 mediates cellular processes like double-stranded break (DSB) repair and DNA replication termination and is expressed exclusively in proliferating spermatogonia and preleptotene spermatocytes (3). These stem cells regulate the expression of cell cycle genes (such as Ccna2) via the E2F family of transcription factors, which can bind to a specific DNA sequence and alter gene expression (4). The relevance of E2F-driven Ccna2 expression has not been thoroughly studied in vivo, so to evaluate the importance of E2F-mediated regulation of Ccna2, we generated mice with a null E2F binding site in its promoter. Although mice are viable and appear healthy, we observed infertility and early testicular atrophy in males homozygous for the promoter mutation. Mouse testes were collected at 1, 2, 3, 4 6, and 12 weeks of age and were fixed in either bouins or formalin for further experiments. Histological analysis revealed a cellular arrest at the preleptotene stage, an expanded SSC population, and lack of mature sperm in the mutants. Using RT-qPCR, Ccna2 levels were found to be upregulated at two weeks and through adulthood, while anti-yH2AX stains revealed elevated DNA damage in the mutants, suggesting unresolved DSBs. Although the molecular mechanism is still not clear, the data strongly suggests that E2F regulation of Ccna2 is vital for fertility and a successful first wave of spermatogenesis.
References:
1.Hermo, L., Pelletier, R., Cyr, D.. G., & Smith, C. E. (2009). Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells (Part 1). Microscopy Research and Technique Microsc. Res. Tech., 73(4), 241-278
2.Wolgemuth, D. J., Manterola, M., & Vasileva, A. (2013). Role of cyclins in controlling progression of mammalian spermatogenesis. Int. J. Dev. Biol. The International Journal of Developmental Biology, 57(2-3-4), 159-168. doi:10.1387/ijdb.130047av
3.Ravnik, S. E., & Wolgemuth, D. J. (1996). The Developmentally Restricted Pattern of Expression in the Male Germ Line of Murine Cyclin A2 Suggests Roles in Both Mitotic and Meiotic Cell Cycles. Developmental Biology, 173(1), 69-78.
4.Thurlings, I., & Bruin, A. D. (2016). E2F Transcription Factors Control the Roller Coaster Ride of Cell Cycle Gene Expression. Methods in Molecular Biology Cell Cycle Oscillators, 71-88.
Keywords: Spermatogenesis, Cell Cycle, Transcriptional Regulation