Various concentrations of the pooled FF in Ham’s F10 media (Sigma-Aldrich) (v/v), including 0, 25%, 50%, 75%, and 100%, and different concentrations of PAF in Ham’s F10, including 0, 10, 100, and 1000 nM, were incubated with the sperms for 0,

1, 2, and 4 h. The sperms were harvested and their motility was examined by light microscopy. Sample Groups and Motility Index Analysis The washed semen was divided into three groups: the first group was cultured in Ham’s F10 media for 2 h as the control group and the second and third groups were treated with 75% FF and 100 nM of PAF (Sigma-Aldrich) for Inhibitors,research,lifescience,medical 2 h, respectively, as the experimental groups. The sperm motility index was assessed and classified as progressive (rapid, slow, and total) and non-progressive. Immotile sperm was also

considered for the analysis. Sperm motility was manually assessed Inhibitors,research,lifescience,medical by a single skilled individual in duplicate before and after FF and PAF treatment. Quantitative real-Time Polymerase Chain Reaction (q RT-PCR) The total RNA of the control and the two experimental groups with FF or PAF were extracted using the Biozol® RNA isolation reagent (BioFlux, Tokyo, Japan), according to the manufacturer’s protocol. The purity of the RNA samples was determined by UV spectrophotometry at 260 / 280 nm. Total RNA was reverse-transcripted by RevertAidTM First Strand cDNA Synthesis (Fermentas, Finland), as is described by the manufacturer. Inhibitors,research,lifescience,medical Specific primers and TaqMan probes were designed13 Inhibitors,research,lifescience,medical for 18s rRNA (as housekeeping gene), LDH-C, and c-kit (table 1). The expressions of the genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR) choromo-4 detector system (BioRad,

USA). Gene expression was calculated based on the 2–ΔCt method in each condition (following formula).14 Table 1 Sequences of specific primers and TaqMan probes The result of the gene expression study revealed that c-kit was not expressed in our samples, showing that washing Inhibitors,research,lifescience,medical sperms with AllGrade excluded other cells such as germ cells in the specimens. Gene expression in each condition=2–ΔCt=2– (Ct LDH-C–Ct 18s rRNA) Western Blotting Sperm proteins were extracted in two steps. First, the sperms else were denatured in TCA buffer (50 mg of trichloroacetic acid, 0.5 ml 2-mercaptoethanol, and 50 ml acetone), incubated overnight at 4ºC, and centrifuged. Then, the sperm Hedgehog inhibitor protein pellets were dissolved in lysis buffer (0.2% CHAPS, 0.1%DDT, and 5M Urea). The sperm protein concentrations were determined by Bradford assay. Before LDH-C expression analysis, 10µg of each sample was pooled based on its primary progressive motility index. The sperm proteins were separated on 12% SDS-PAGE and blotted overnight into PVDF membrane. The membranes were blocked by 5% skimmed milk and stained overnight at 4ºC by HRP-conjugated anti LDH-C (ab7639, Abcam, USA) and HRP-conjugated anti β-actin (ab20272, Abcam, USA) as positive controls.