Epididymal post-transcriptional regulation mechanisms in varicocele: relation with male infertility.

Abstract

As varicocele alters the proteomic profile in seminal plasma, it is fundamental to understand what are the intrinsic mechanisms of protein regulation presents in testicular and epididymal environment. One of the major post- transcriptional mechanisms to control the protein expression is given by action of non-coding RNAs, denominated microRNAs (miRNAs). Thus, our hypotheses are: (i) there are differences in epididymosomes miRNAs composition in men with and without varicocele and; (ii) varicocele surgical repair (varicocelectomy) may restore the miRNA profile in epididymosomes, leading to the improvement of some epididymal function related to sperm. Therefore, the aim of the present study is to identify the miRNAs in epididymosomes of control men (without varicocele), men with varicocele and men post varicocelectomy, collected for the PhD study funded by FAPESP (process 2016/05487-3). Samples were already collected and are stored at -20 °C in the Urology Research Center (UNIFESP, Sao Paulo, Brazil; samples from controls (n=30), varicocele (n=30), and pre and post varicocelectomy groups (n=20). Epididymosomes will be separated from human seminal plasma after ultracentrifugation and after total protein quantification, between 240 and 410 ¼g of protein containing epididymosomes will be subjected to miRNA extraction and purification. Epididymosomal miRNAs will be extracted and purified using the mirVana TM miRNA Isolation Kit (Life Technologies). All samples that will be used in the experimental groups (controls, with varicocele and pre and post varicocelectomy), will be labeled and hybridized separately on 12 miRNA microarrays. For statistical analysis, the software PAWS (SPSS) 18.0 will be utilized. For controls,varicocele,pre and post varicocelectomy groups'comparison will be used unpaired Student's t test in normal distributed data or Mann Whitney test for non-normal data. Real-time PCR quantification of miRNAs expressed epididymosomes will be performed to validate results streaming from the microarray analysis. Pulsed RT with stem-loop primers followed by quantitative PCR using SYBR Green (Roche Diagnostics) will be carried out. Quantitative PCR will be performed on reverse transcribed templates with LightCycler FastStart SYBR Green I (Roche Diagnostics) according to the manufacturer's instructions. Fluorescence signals will be continuously collected at a wavelength of 530 nm from 65°C to 95°C at 0.2°C per second. The RT and quantitative PCR will be performed in duplicate on each sample. Four points will be used to plot the standard curve (nondiluted sample and template dilutions of 1:2, 1:4, and 1:8).