Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling

Abstract

Different semen storage and sperm purification methods may affect the integrity of isolated spermatozoal RNA. RNA-Seq was applied to determine whether semen storage methods (pelleted vs. liquefied) and somatic cell lysis buffer (SCLB) vs. PureSperm (PS) purification methods affect the quantity and quality of sperm RNA. The results indicate that the method of semen storage does not markedly impact RNA profiling whereas the choice of purification can yield significant differences. RNA-Seq showed that the majority of mitochondrial and mid-piece associated transcripts were lost after SCLB purification, which indicated that the mid-piece of spermatozoa may have been compromised. In addition, the number of stable transcript pairs from SCLB-samples was less than that from the PS samples. This study supports the view that PS purification better maintains the integrity of spermatozoal RNAs.

Keywords::

• mitochondrial RNA
• preferentially isolated transcripts
• stable transcript pairs

Acknowledgments

We are grateful to Meritxell Jodar and Selvaraju Sellappan for their review of the manuscript.

Declaration of interest: The authors declare no conflicts of interest. This work was supported in part by the Charlotte B. Failing Professorship to SAK, a GENI pilot grant to SAK and RH from Harvard School of Public Health; National Institute of Environmental Health Sciences (Grant Number ES017285) to RH and SAK and in part by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Contract 25PM6 in collaboration with the LIFE Study Working Group, Division of Epidemiology, Statistics, and Prevention Research who provided semen samples for analysis.

Disclaimers: Mention of company names and/or products does not constitute endorsement by the National Institute for Occupational Safety and Health. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.

Author contributions: Analyzed the data and wrote the manuscript: SM; Prepared libraries for RNA-Seq and reviewed the manuscript: RJG; Reviewed and edited the manuscript: RH; Collected samples and designed the experiment: SS; Reviewed the manuscript and accomplished the statistical analysis of the data and: ZC; Oversaw the project and edited the manuscript: SAK.