Using the histone H2a transcript as an endogenous standard to study relative transcript abundance during bovine early...

Area of Science:

• Reproductive Biology
• Developmental Biology
• Molecular Biology

Background:

• Accurate RNA abundance assessment relies on standardized housekeeping genes.
• Previous studies identified histone H2a as a stable target in early bovine embryonic development.
• Discrepancies in primer sets targeting histone H2a variants have been noted.

Purpose of the Study:

• To re-evaluate histone H2a variants (H2a.1, H2a.z, H2a.o) as potential housekeeping genes in bovine oocyte-to-blastocyst development.
• To investigate the impact of polyadenylation status and transcriptional origin (maternal vs. de novo) on RNA abundance.
• To assess the influence of in vitro production systems on transcript stability.

Main Methods:

• RNA abundance profiling of histone H2a.1, H2a.z, and H2a.o across developmental stages from immature oocyte to blastocyst.
• Analysis of polyadenylation status and transcriptional origin (maternal or de novo) for each variant.
• Evaluation of serum's effect on H2a.1 RNA levels at the blastocyst stage.

Main Results:

• Histone H2a.z is primarily regulated by de novo transcription post-embryonic genome activation.
• Histone H2a.1 and H2a.o are predominantly of maternal origin, showing greater stability across development.
• H2a.z is polyadenylated, while H2a.1 and H2a.o are more abundant in a non-adenylated form; serum impacts H2a.1 at blastocyst stage.

Conclusions:

• Histone H2a.1 and H2a.o are more suitable housekeeping targets than H2a.z for early bovine embryonic development studies.
• Understanding transcript origin and polyadenylation is crucial for selecting reliable RNA abundance markers.
• The choice of primers and culture conditions can significantly affect RNA abundance measurements.