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

Area of Science:

• Reproductive Biology
• Molecular Genetics
• Avian Science

Background:

• Sperm-borne RNAs play vital roles in embryonic development and epigenetic inheritance.
• Existing protocols often include somatic cell removal, which is not established for avian species.
• A need exists for a reliable method to isolate diverse sperm-borne RNA populations in birds.

Purpose of the Study:

• To evaluate somatic cell contamination in avian semen.
• To assess RNA isolation methods for avian sperm.
• To characterize the miRNA and mRNA content of purified avian sperm-borne RNA.

Main Methods:

• Semen samples from chickens and quails were examined for somatic cells using fluorescent staining.
• Somatic cell lysis buffer (SCLB) efficacy and impact on sperm integrity were tested.
• Three RNA isolation approaches (two mammalian, one commercial) were compared for efficiency, quality, and purity; RT-(q)PCR was used to detect specific RNA molecules.

Main Results:

• No somatic cells were detected in chicken or quail semen.
• SCLB lysed liver cells but damaged sperm; thus, it was not used.
• A commercial kit yielded the most consistent and pure RNA, free of DNA contamination. Detected RNAs included avian equivalents of mammalian sperm miRNAs and mRNAs (e.g., gga-miR-100-5p, GAPDH), but not leukocyte or Sertoli cell markers.

Conclusions:

• Pre-treatment to remove somatic cells is unnecessary for avian sperm RNA isolation.
• A validated protocol using a commercial kit enables isolation of diverse sperm-borne RNAs (miRNA, mRNA) from avian species.
• This method is applicable to valuable avian models like chickens and quails, advancing research in reproductive biology and epigenetics.