ABSTRACT
Blastocyst formation is a primordial event of pre-implantation development since it is required for pregnancy establishment and progression. The blastocyst plays a pivotal role because it is the stage at which the embryo starts coordinated cross-talk with the mother. It is also the terminal step before transfer in bovine; it reflects all stresses the embryo may have faced during the process of in vitro treatment. Achieving the formation of a morphologically healthy blastocyst following normal kinetics is good, but remains a poor indicator of embryo quality. Considering the limitation of the invasive methods for competence assessment, the analysis of blastocysts' gene expression is a promising way to improve our understanding of blastocyst formation and to study the effects of different treatments on gene expression. To that end, early, expanded, and hatched blastocysts, derived from in vitro fertilization and culture, were collected for RNA extraction, amplification, and cDNA microarray hybridization. Gene candidates (IFNt, PLAC8, SSLP1, AKR1B1, HNRNPA2B1, ARGFX, NANOS, CCNB1) were selected and confirmed using real-time RT-PCR to validate the microarray data. Our analysis showed that hatched blastocysts are enriched in transcripts implicated in attachment, cell adhesion, and extracellular matrix digestion. Early blastocysts expressed genes mainly involved in cell cycle control, transcription, and translation. Real-time RT-PCR validated most microarray results (87.5%). Overall, our study provides new insights into the molecular regulation of blastocyst formation. In addition, it could help assess blastocyst staging and select better embryos based on the expression of quality markers.
Subject(s)
Blastocyst/metabolism , Embryo Implantation/genetics , Embryo Transfer/methods , Embryonic Development/genetics , Gene Expression Regulation, Developmental , Animals , Cattle , Embryo Transfer/veterinary , Embryo, Mammalian/metabolism , Female , Fertilization in Vitro/methods , Gene Expression Profiling/methods , Oligonucleotide Array Sequence Analysis/methods , Pregnancy , RNA-Binding Proteins/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Crab digestive phospholipase (CDPL) was purified from the hepatopancreas of Carcinus mediterraneus crabs. Homogeneous enzyme was obtained after two chromatography steps: anion exchange and size exclusion HPLC column. Homogeneous CDPL has a molecular mass of 14 kDa as determined by SDS/PAGE analysis. Unlike known digestive phospholipases like porcine PLA(2) (PPPL), CDPL displayed its maximal activity at 50 degrees C and not at 37 degrees C. A specific activity of 40 U/mg for the purified CDPL was measured using PC as substrate under optimal conditions (pH 8 and 50 degrees C) in the presence of 8 mM sodium deoxycholate (NaDC) and 10 mM CaCl(2). In contrast to PPPL, purified CDPL was completely inactivated at 60 degrees C. The N-terminal sequence was determined by automatic Edman degradation. No similarity between 12 N-terminal amino acid residues of CDPL was found with those of known digestive phospholipases. CDPL appears to be a new member of invertebrate phospholipases, and it is potentially useful for treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds which can be used in the food industry.
