Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, beta-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro.
Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development.
Bettegowda
Anilkumar
A
Laboratory of Mammalian Reproductive Biology and Genomics, Michigan State University, East Lansing, Michigan 48824, USA.
Patel
Osman V
OV
Ireland
James J
JJ
Smith
George W
GW
D000199:Actins / Q000235:genetics; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D021984:Cyclophilin A / Q000235:genetics; D047108:Embryonic Development / Q000235:genetics*; D005307:Fertilization in Vitro; D020869:Gene Expression Profiling; D005966:Glucuronidase / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006657:Histones / Q000235:genetics; D066298:In Vitro Techniques; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012269:Ribosomal Proteins / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D000199:Actins; D006657:Histones; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C043649:ribosomal protein L15; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017853:Phosphotransferases (Alcohol Group Acceptor); C023916:phosphoglycerol kinase; D005966:Glucuronidase; D021984:Cyclophilin A