Abstract
Seminal neutral endopeptidase (NEP) has been suggested to regulate sperm motility based on the observation that addition of NEP inhibitors such as opiorphin (OP) to semen increase sperm motility (1). We conducted a peptidomic screen of semen to identify potential endogenous NEP inhibitors and identified a novel peptide, RSIY-11, which based on sequence analysis, may act as an NEP inhibitor. In these studies we evaluated its role as an NEP inhibitor and effects on sperm motility. Prospective laboratory in vitro studies. Peptidomics and mass spectrometry was performed on a semen sample with normal semen parameters. A highly expressed 11 amino acid (AA) peptide was identified, termed RSIY-11 (based on the last 4 AA at the carboxyl terminus) which, based on its AA sequence, could act as an NEP inhibitor. RSIY-11 was synthesized and a colorimetric assay performed with purified NEP to evaluate the inhibitory constant (Ki) of RSIY-11 and compared to that of OP. The effect of preincubation of NEP with RSIY-11 at 8.2μM was examined, comparing 0, 3 and 24 hour (hr) preincubation times. Dixon and Lineweaver Burk (LB) plots determined the inhibitory nature of RSIY-11 on NEP. Microscopy was used to evaluate sperm from 49 patients undergoing a semen analysis for an infertility workup for total motility (TM) and progressive motility (PM) at baseline and after addition of phosphate buffer solution (PBS) (negative control), OP at 200μM (positive control) and RSIY-11 at 0.75, 7.5, and 75μM at 0, 30, and 60 minutes following addition of peptides. TM and PM were done in duplicates and averages were calculated. STATA version 14.0 was used to analyze continuous data related to sperm motility using repeated measures ANOVA. P < 0.05 was considered significant. Data are presented as mean ± standard deviation. RSIY-11 acts as a competitive inhibitor of NEP with Ki of 18.4 ± 1.6 μM; considerably more potent than OP (Ki 264.5 ± 61.5 μM). Pre-incubation of RSIY-11 with NEP for 24 or 3 hr reduced its ability to inhibit NEP activity (26% and 29% inhibition, respectively), compared to no preincubation (47% inhibition), suggesting RSIY-11 is a substrate for NEP. Compared to baseline, OP and RSIY-11 at all concentrations significantly increased TM and PM at 0, 30, and 60 minutes (Table). No significant differences were found in TM and PM between OP and RSIY-11 at all concentrations. RSIY-11 is an endogenous, competitive, NEP inhibitor that when added to semen increases sperm motility. RSIY-11 is a potential biomarker for male factor infertility and a potential therapeutic strategy for treatment of asthenospermia.Tabled 1Motility Following Addition of PeptidesTotal Motility 0 minutes (%)Progressive Motility 0 minutes (%)Total Motility 30 minutes (%)Progressive Motility 30 minutes (%)Total Motility 60 minutes (%)Progressive Motility 60 minutes (%)Baseline49.7 ± 16.143.6 ± 16.449.6 ± 15.645.5 ± 16.148.0 ± 15.542.8 ± 16.0PBS50.8 ± 14.545.3 ± 15.649.8 ± 15.845.9 ± 15.749.5 ± 14.544.5 ± 15.1Opiorphin 200 μM59.1 ± 17.153.6 ± 18.457.6 ± 15.752.6 ± 16.857.1 ± 15.252.3 ± 16.6RSIY-11 0.75 μM60.2 ± 17.354.9 ± 17.657.6 ± 16.352.7 ± 17.054.4 ± 16.350.7 ± 15.7RSIY-11 7.5 μM58.7 ± 17.353.5 ± 17.758.4 ± 15.753.9 ± 16.054.9 ± 17.050.9 ± 16.7RSIY-11 75 μM58.7 ± 16.852.9 ± 17.957.2 ± 16.152.0 ± 17.657.9 ± 14.452.8 ± 14.9 Open table in a new tab
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