Tributyltin chloride alters the structural, genomic, and epigenomic integrity of postejaculatory mammalian sperm
A global top priority for ameliorating male factor infertility includes identification of environmental factors and mechanisms that impact sperm function. Detection of endocrine disruptors (EDs) in seminal plasma and within the female reproductive tract has created an urgent need to understand how environmental stressors alter postejaculatory sperm function. Tributyltin chloride (TBT) is a model organotin, ED and epigenetic modifier that causes reproductive disorders in testicular sperm; however, consequences of TBT exposure on postejaculatory sperm prior to fertilization remain unknown. Our previous data indicate that TBT-exposed sperm affects initial cleavage and lowers embryo development potential. However, identifying reasons for idiopathic male factor subfertility is challenged by lack of routine clinical assessment for subtle phenotypes that compromise sperm function and therefore remain undetected with current diagnostic tools. Results 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. Bovine sperm were exposed to TBT (0 ,1, 10, 100nM) for 24 h under non-capacitating conditions at 25°C followed by quantification of sperm kinematics at 37°C, DNA integrity, and methylation status. No differences in sperm kinematics or capacitation status were detected after 24 h. However, acrosome integrity was compromised at both 0 and 24 h (P = 0.05). Sperm DNA integrity was also negatively affected after 24 h. The methylation levels of select loci were quantified by PacBio single-molecule real-time (SMRT) long-read high-fidelity sequencing. Whole genome methyl-seq revealed 750 differentially methylated regions (DMRs) associated with exposure to TBT for 24 h. IPA and GO analyses identified embryo development, cell signaling, and transcriptional regulation as the most relevant bio-functions of TBT altered DMRs. Conclusions Postejaculatory mammalian sperm exposure to TBT negatively affects parameters important for sperm function while altering DNA integrity and the methylation profile of promotor regions. Consequences of sperm exposure to TBT include cellular and molecular mechanisms that are important for sperm function but remain undetected by routine clinical analyses. These findings provided new insight towards environmental impacts on postejaculatory sperm function. Overall design: Frozen-thawed sperm from two different commercial bulls were used for all experiments except where stated otherwise. Four exposure level of TBT (100nm, 10nm, 1nM and 0nM) were used in all experimnets unless stated otherwise.In each replicate,sperm were isolated from four frozen straws on a discontinuous Percoll gradient (90:45%).DNA was isolated from TBT exposed sperm following TBT exposure at 24 h followed by enzymatic Methyl seq Conversion. The enzymatic methyl seq converted DNA from sperm were PCR amplified for three hypermethylated loci (PTK2B, HDAC11, PAK1) and three imprinted loci(SNRPN, H19, KCNQ1) and sequenced for loci specific PacBio single molecule real time long-read high-fidelity sequencing (SMRT).In SMRT sequencing, only two treatments (VC and TBT 10nM) were used (n = 2 bulls, 4 pooled independent reps/ bull/trt). For whole genome methyl-seq analysis, three treatments were used (VC, TBT 100nM and TBT 10nM) n = 1 bull, 4 pooled independent reps/bull/trt).Data were analyzed by logistic regression using a generalized linear mixed effect model. Treatment and time were considered fixed effects while bull and replicate were assigned as random effects.