Avian sperm-borne RNAs: optimisation of a new isolation protocol

1. Sperm-borne RNAs are involved in sperm and embryonic protein translation, the regulation of early development and the epigenetic inheritance of the paternal phenotype. Sperm-borne RNA purification protocols generally include a cell purification stage to discard contamination by somatic cells. In avian species, no protocol is currently available to isolate all the populations composing sperm-borne RNAs.2. This study evaluated the presence of somatic cells in semen samples of chickens and quails using visual examination after fluorescent nuclei staining. The efficiency of somatic cell lysis buffer (SCLB) on chicken liver cells and its impacts on chicken sperm cell integrity was explored. Three different approaches were tested to isolate RNA: two developed for mammalian sperm cells and a commercial kit for somatic cells. The efficiency and reliability of each approach was determined based on RNA quality and purity. Eventually, the presence of miRNA and mRNA in purified avian sperm-borne RNAs was investigated by RT-(q)PCR.3. No somatic cells were found in chicken and quail semen. The SCLB totally lysed chicken liver cells but also induced sperm cell necrosis. Consequently, this treatment wasn't performed on samples prior to RNA isolation. Among the tested RNA purification protocols, the commercial one was the least variable and isolated RNA with the highest purity levels. No DNA contamination was observed. Furthermore, the samples contained miRNA and mRNA already known as present in mammalian sperm cells (gga-miR-100-5p, gga-miR-191-5p, GAPDH and PLCZ1), but mRNAs associated with leucocytes (CD4) and Sertoli cells (SOX4, CLDN11) were not detected. This protocol was successfully applied to quail sperm cells.4. Altogether, the study reveals that it is unnecessary to pre-treat samples to remove somatic cell contamination before RNA purification and successfully describes an isolation protocol for sperm-borne RNAs, including small non-coding and long coding RNAs, in two distinct avian species highly valuable as biological models.