Optimization of sperm RNA processing for developmental research

Abstract

Recent studies have demonstrated the significance of sperm RNA function as a transporter of important information directing the course of life. To determine the message contained in sperm RNA, it is necessary to optimize transcriptomic research tools. The current study was performed to optimize the processing of sperm RNA from sample storage to quantitative real-time PCR and assess the corresponding method with to evaluate male fertility and its representative markers, equatorin (EQTN) and peroxiredoxin (PRDX). Following successive steps of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, several options were compared using boar spermatozoa. To evaluate the optimized procedures, the relationship between mRNA expression of EQTN and PRDX in spermatozoa and the fertility (litter size) of 20 individual boars was assessed. Unexpectedly, DNase treatment during RNA isolation had the deleterious effect by decreasing the RNA concentration by 56% and eliminating the correlation between EQTN and PRDX4 mRNA expression and male fertility. Moreover, when sperm RNA was processed using the corresponding method, the results showed the highest exon sequence expression, male fertility prediction power, and consistency. This optimized protocol for predicting male fertility can be used to study the transport of messages directing the life course from spermatozoon to offspring.

Urinary extracellular vesicles: Assessment of pre‐analytical variables and development of a quality control with focus on transcriptomic biomarker research

“…However, the amount of DNA is generally low in cell‐free samples, including urine (Oreskovic et al., 2019 ; Streleckiene et al., 2018 ). As the RNA content of EV samples is also low in comparison to cell or tissue RNA and the DNAse treatment reduces RNA yields (Norhazlin et al., 2015 ; Pang et al., 2020 ), the risk of losing the precious EV‐RNA due to the treatment is high. We thus regarded relevant to investigate the necessity of the DNAse treatment by analysing RNA yield and mRNA‐seq results from uEV.…”

Establishment of a male fertility prediction model with sperm RNA markers in pigs as a translational animal model

“…Sperm RNA was isolated according to a previous study [ 38 ]. Briefly, sperm numbers were adjusted to 40–50 × 10 6 cells/mL.…”

Sperm Transcriptome Analysis Accurately Reveals Male Fertility Potential in Livestock

“…Many factors determine the results of the transcriptomic analysis. Pang et al [ 27 ] found that the average RNA concentration of sperm with DNase was 56% lower in the 15 min of incubation treatment with DNase (44.28 ± 8.12 ng/µL) than without DNase (101.7 ± 13.5 ng/µL) but produced a RNA quality that was not different between treatment with DNase (1.79 ± 0.03) and without DNase (1.80 ± 0.01). Using an RNA stabilizer solution is also considered significant [ 42 ].…”

“…Sperm RNA has been identified in many mammalian species, such as humans, cattle, pigs, horses, and rodents. Some of the functions of sperm RNA include contributing during fertilization by delivering new paternal RNA to the oocyte and signaling in mitochondrial translation [ 27 ]. This sperm transcriptome also has an important role in the developmental stages of cell division in oocytes after fertilization and placenta development [ 26 ], zygote development [ 28 ], early embryonic development [ 29 ], and even epigenetic inheritance between generations [ 30 ].…”