Copy number determination of sperm-borne small RNAs implied in the intergenerational inheritance of metabolic...

Mammalian spermatocytes harbor small RNAs that are mostly degradation products of abundant noncoding RNAs, including ribosomal RNA-derived small RNAs (rsRNAs) and tRNA-derived RNAs (tDRs). Notably, tDRs have been implicated in inheriting paternally acquired traits in rodents. Direct experimental proof for this notion comes from manipulating fertilized murine oocytes through microinjection of small RNA preparations, resulting in metabolic changes measurable in the offspring. How these paternally transmitted small RNAs could function mechanistically in the developing zygote remains to be understood. Since nothing is known about how many small RNAs are required for functional impact, we aimed to determine the copy numbers of specific small RNAs contained in a single spermatocyte. Using hybridization-based methods that avoid amplification-induced biases, we estimated average copy numbers for specific tDRs and rsRNAs per murine spermatocyte. While the measured numbers allow an approximation of how many rRNA- and tRNA-derived RNAs enter a murine oocyte during fertilization, the magnitude of these numbers underscores the need for remaining cautious when interpreting the effects of nonphysiological copy numbers of small RNAs that were used to manipulate a biological system.