INVESTIGATING ARRDC5 SPATIO-TEMPORAL EXPRESSION PROFILE AND ITS ROLE WITHIN THE HISTONE-TO-PROTAMINE EXCHANGE

LeeLa  Robinson

Spermatogenesis is a highly conserved process across species, when genetic content of diploid cells is halved and haploid gametes are morphologically altered to achieve the sole goal of fertilization. The final phase, spermiogenesis, is where multiple highly dynamic processes take place that prepare the germ cell for fertilization. This includes processes such as cytoskeletal reorganization, acrosome biogenesis, and DNA condensation. A disruption in these can result in a loss of sperm, decrease motility, and abnormal morphology causing infertility. These severe phenotypes are often seen in wildlife species that have been bottlenecked due to a variety of reasons. Big cats, such as the Florida Panther and cheetah faced with small genetic pools, resulting in monoallelic genes that are often detrimental to the wellbeing of the population. Sperm from these animals often have low concentrations and abnormal morphology, which most attribute to poor DNA compaction. This lack of DNA compaction is often the result of a failure in the histone-to-protamine exchange (HPE). Using the Arrdc5 mouse model as previous studies have described its functional role as essential to proper spermiogenesis. The low sperm count, inhibited motility, and malformed sperm make it a perfect model to study a variety of failures during spermiogenesis. Within this study we will characterize the spatio-temporal expression profile of ARRDC5 in humans and mice through chromatin availability, transcript abundance, and using an eGfp-Arrdc5 mouse model to measure relative protein expression. Results showed that ARRDC5 in both humans and mice is solely expressed within the round and elongating spermatid population. Following this study, it was likely that Arrdc5 played a role in the HPE as the sperm from the knockout mouse had significant DNA damage and macrospermia. An aniline blue stain was used to measure significant histone retention in sperm from the knockout mouse. Immunoblotting was used to validate this with all four canonical histones, H2A, H2B, H3, and H4, H2A/H2B were significantly retained. Extracted protamines were ran on a poly-acrylamide gel to show there was significantly less protamine deposition in sperm from the knockout model. To measure protein binding affinity without an antibody, a luciferase construct was made with histones TH2A, TH2B, and H3.3 to determine if ARRDC5 directly interacted with histones involved in the HPE, TH2A and H3.3 were measured to bind to ARRDC5. Lastly, as the HPE is controlled by PTMs, they were measured using bottom-up LC/MS and two were found to be significantly lower in abundance. Together, these studies describe a clear picture of where ARRDC5 is expressed and its role within the HPE, further characterizing this highly dynamic process allowing a better understanding of this mechanism and hopefully solutions for wildlife infertility.

INVESTIGATING ARRDC5 SPATIO-TEMPORAL EXPRESSION PROFILE AND ITS ROLE WITHIN THE HISTONE-TO-PROTAMINE EXCHANGE

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