(334) Mature Mammalian Sperm are Susceptible to Novel Epigenetic Modifications and Structural Damage from Environmental Toxins

Muhammad S. Siddique¹; Santosh Anand⁵, Marie Gauthier³, Christopher J. Martyniuk⁴, Michael Kladde³, Ramji K. Bhandhari⁵, Bradford W. Daigneault^1,2

¹ Department of Animal Sciences, University of Florida, Gainesville, Florida, USA

² Department of Large Animal and Clinical Sciences, Gainesville, Florida, USA

³ Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida, USA

⁴Department of Physiological Sciences, University of Florida, Gainesville, Florida, USA

⁵ Department of Biological Sciences, University of Missouri, Columbia, Missouri, USA

Abstract Text:

A key global priority in addressing male factor infertility is identifying environmental factors and mechanisms that influence sperm function. Identifying causes of male factor infertility is often masked by sperm that appear clinically acceptable yet harbor structural and cellular abnormalities that remain undetected with routine clinical analyses. Impacts of environmental stressors to sperm at the testicular level have been described, but consequences of short-term exposure to postejaculatory sperm are severely lacking. Detection of endocrine disruptors (EDs) in seminal plasma and within the female reproductive tract has created an urgent need to understand how environmental stressors and EDs alter postejaculatory sperm function. Tributyltin chloride (TBT) is a model organotin, ED and epigenetic modifier that causes reproductive disorders, but consequences of TBT exposure on postejaculatory sperm prior to fertilization remain unknown. Previous reports from our group demonstrate a reduction in embryo development from bovine oocytes fertilized with TBT-exposed sperm of normal motility. Therefore, the present study was aimed at identifying structural, genomic and epigenomic consequences of TBT exposure to postejaculatory sperm in a temporal manner reflective of sperm transport prior to fertilization within the female reproductive tract. Cryopreserved bovine sperm from two bulls were independently exposed to TBT (0 ,1, 10, 100nM; n = 4 individual replicates/bull) for 24 h under non-capacitating conditions at 25°C followed by quantification of sperm kinematics at 37°C. TBT and vehicle control (0.1%DMSO) exposed sperm were collected in replicate for all analyses following immediate addition of TBT (0 h) and again at 24 h. DNA damage, acrosome integrity, and changes to DNA methylation were quantified. Data were analyzed by logistic regression with a generalized linear mixed effect model. No differences were detected via computer-assisted sperm assessment for total motility, progressive motility and average-path velocity in TBT-exposed sperm compared to vehicle-control after 24 h. Likewise, anti-tyrosine phosphorylation analysis revealed no changes to the capacitation status of TBT-exposed sperm. However, acrosome integrity was compromised in an acute and dose dependent manner as detected by FITC-PSA (P ≤ 0.05).  Similarly, DNA integrity, assessed using the TUNEL assay, showed a dose-dependent decline after 24 h of TBT exposure (P ≤ 0.05). Alterations to the methylation landscape of mature sperm were determined following 24 h of TBT exposure using two independent approaches. Loci-specific methylation changes to the promoter regions of select hypermethylated (PTK2B, HDAC11, PAK1) and imprinted (SNRPN, H19, KCNQ1) loci were initially measured using PacBio single-molecule real-time (SMRT) long-read high-fidelity sequencing to identify differential amounts of CpG methylation among individual males. Whole-genome Enzymatic Methyl Sequencing (EM-Seq) identified approximately 2,200 differentially methylated regions (DMRs) (≥ 25%) between TBT-exposed sperm and vehicle-control samples, spanning promoter regions across the entire genome. In conclusion, postejaculatory mammalian sperm exposure to TBT affects acrosome integrity, induces DNA damage, and alters the methylation status at regions important for early embryonic development. Collectively, these data suggest a novel phenotype and paradigm shift to understanding of environmentally induced epigenetic modifications to transcriptionally quiescent and translationally repressed mature sperm. These findings implicate new considerations of sperm exposure to endocrine disruptors and epigenetic modifiers that appear to impact cellular and molecular mechanisms important for sperm function, but with consequences that remain undetected by routine clinical analyses.