The differentiation of round spermatids into spermatozoa, known as spermiogenesis, is a complex process involving the formation of the acrosome, the flagellum, and condensation of the nucleus. In preparation for condensation, the spermatid nucleus undergoes dramatic chromatin remodeling including genome-wide cessation of transcription, dismantling of the nucleosomal organization, and histone to protamine transition. This process is unique to the male germ line. Mouse models have shown that defects in nuclear condensation lead to male infertility; however, the mechanisms regulating this complex genome-wide process are not well understood. Our working hypothesis is that the genome organizer protein CTCF coordinates the chromatin remodeling events accompanying spermatid differentiation. The evolutionarily conserved eleven zinc finger protein CCCTC-binding factor (CTCF) is exclusively expressed within the round and early elongating spermatids in mice, coinciding with transcriptional shutdown and the onset of histone replacement. In this pilot, feasibility grant proposal we will test the hypotheses that: 1) CTCF functions as a transcription factor as well as an organizer of spermatid genome to facilitate chromatin remodeling and 2) CTCF is essential for the completion of spermatogenesis and male fertility. We will perform ChIP-seq to determine genome-wide occupancy of CTCF in round spermatids and generate CTCF conditional knockout mice using the Cre-loxP technology to test the requirement of CTCF for spermatogenesis and male fertility. CTCF is an ideal candidate for this role because it is a multifunctional DNA binding protein with diverse roles including that of a chromatin organizer. Floxed CTCF mice as well as the male germ cell-specific cre-deleter strain (Stra8-iCre) are readily available. This proposal will explore the role of CTCF in male fertility fr the first time. Infertility affects 1 in 6 couples in the reproductive age group, with the male facor accounting for 50% of those cases. If depletion of CTCF causes sperm maturation arrest, this study will provide a novel mouse model for male infertility. Given the role that CTCF plays in establishing and / or maintaining epigenetic marks, the proposed knockout mouse may be a useful model to understand the risks involved in Assisted Reproductive Technology using incompletely developed spermatids or sperm. Thus, the studies are highly significant from the point of view of male reproductive health.
Infertility affects 1 in 6 couples in the reproductive age group in the United States and the male factor accounts for roughly 50% of those cases. Due to lack of understanding of the causative factors, treatment options for male infertility are currently limite. Consequently, the use of assisted reproductive technology (ART) has increased, accounting for 1-3% of births in developed countries. Studies show that miscarriage, imprinting disorders, intrauterine growth restriction, and childhood cancer are associated with ART. This highlights the importance of understanding how the genetic and epigenetic information delivered by the sperm influences embryo development. Here we test the role of the genome organizer protein CTCF in spermatogenesis using the mouse as a model system. Insights into the function of CTCF in spermiogenesis and the CTCF male germ cell-specific knockout mouse model will be made available through this study. Our work has translational potential with implications for ART, and development of new methods for screening and treating male infertility.
