The major reproductive peculiarity of the bitch is that ovulation releases prophase I (germinal vesicle, GV, immature) oocytes. Resumption of meiotic maturation, as well as fertilisation and embryonic development to the morula stage occur in the oviduct. Because the dog is a biomedical model for human diseases and also a model for endangered canid species, the development of assisted reproduction techniques would be of great interest. To date, in vitro-produced canine embryos remain exceptional and no puppy has been born. The main limiting factors of in vitro embryo production are the low oocyte maturation rates, the poor oocyte quality and the high polyspermy. A better knowledge of the composition of oviductal fluid during the periovulatory period may help to mimic the in vivo conditions for in vitro oocyte culture and, thereafter, their fertilisation and embryonic development. The objective of this study was to analyse the oviductal fluid by a label-free quantitative proteomic workflow based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein separation, nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis and quantitative method using spectral counting. Ovarian cycles were followed by vaginal smears, ultrasonography and progesterone blood assays. Oviductal fluids were collected from 3 beagle bitches, after ovariectomies performed 3.5 days after ovulation. After dissection, the ampulla and isthmus were separated and flushed with 50 μL of PBS. Oviductal fluids were submitted to 1D SDS-PAGE and all bands were digested with trypsin. Peptide extracts were analysed on an Ettan multidimensional LC (MDLC) system coupled to a linear ion trap quadrupole (LTQ) mass spectrometer. After protein identification using Mascot server and with Swiss-Prot and National Center for Biotechnology Information (NCBI) databases, bioinformatic processing of data and statistic analysis (t-test with P < 0.05) were performed using the spectral counting quantitative module of the Scaffold software. Using this strategy, 427 proteins were qualitatively identified in canine oviductal fluid. Three proteins were specific of the ampulla, 10 specific of the isthmus and 414 were found in both oviductal parts. Among these common proteins, some were differentially expressed, from 1.25 to 9 times higher (HV303_Human, RLA2_Horse, SPRL1_Human, SODC_CANFA, PROF1_Human, ARF4_Bovin and TRXR1_Bovin). The gene ontology analysis displayed biological pathways specific to the biology of reproduction (6 proteins; RUVB1_Human, OVGP1_Pig, STAT3_Human, PLAK_Human, GPX3_Rat and DYL1_Human). These candidate proteins and especially oviduct-specific glycoprotein and glutathione peroxidase, will now be validated by immunodetection methods.
Improved LC-MS chromatographic alignment increases the accuracy of label-free quantitative proteomics: Comparison of spectral counting versus ion intensity-based proteomic quantification strategies
