(226) ARRDC5 Regulation of Histone-to-Protamine Exchange During Spermiogenesis

Abstract Authors: LeeLa D. Robinson¹, Melissa J. Oatley¹, and Jon M. Oatley¹
1. School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, USA

Abstract Text:

Spermiogenesis is the final phase of spermatogenesis when haploid round spermatids undergo a transformation to yield specialized elongated cells that are released from the seminiferous epithelium as spermatozoa. During this process, the paternal genome is condensed into toroid structures via an exchange of histones with protamines. Disruption in the histone-to-protamine exchange can lead to sperm head malformations and elevated DNA damage, both of which are prominent abnormalities associated with the most common clinical description of male infertility, oligoasthenoteratospermia (OAT). In previous studies, we discovered that the molecule arrestin domain containing 5 (Arrdc5) is an essential regulator of spermiogenesis. Knockout of Arrdc5 in mice leads to genesis of sperm with severe head malformations and a significant increase in DNA damage compared to sperm from wild-type mice. Here, we explored whether the histone-to-protamine exchange is abnormal in the absence of ARRDC5. Sperm from Arrdc5 knockout mice were found to stain more intensely with the preferential histone binding dye aniline blue compared to sperm from wild-type mice, indicating possible abnormal histone content. In addition, outcomes of immunoblot analyses revealed significantly greater amounts of histone variants in the DNA of sperm from Arrdc5 knockout mice compared to sperm from wild-type counterparts. Furthermore, protamine content was found to be reduced and the ratio of protamine 1 and 2 variants altered in sperm of Arrdc5 knockout mice compared to sperm from wild-type mice. Following from these findings, direct interaction between ARRDC5 and various histones was investigated. Testis-specific histones variants were assessed initially for potential interaction with ARRDC5 using alpha-fold which indicated likely direct binding with TH2A. Next, a split-luciferase expression construct was engineered to test physical interaction of ARRDC5 and TH2A in a mammalian cell system via transfection of human embryonic kidney cells (HEK). Outcomes confirmed that ARRDC5 and TH2A are binding partners. Ongoing studies are utilizing state-of-the-art mass spectrometry to determine whether post-translational modifications of TH2A and other histones are abnormal in spermatids generated by Arrdc5 knockout mice which will aid in explaining the disrupted histone-to-protamine exchange that occurs in the absence of ARRDC5.