Proteomic study to identify factors in follicular fluid and/or serum involved in in vitro cumulus expansion of porcine oocytes.

Follicular fluid, as a transudate of serum, constitutes the micro environment of the maturing oocyte. In a previous study, we have shown that follicular fluid (pFF) is superior to autologous serum in promoting cumulus expansion during in vitro maturation (IVM) of porcine oocytes (Bijttebier et al. 2008). After ultrafiltration of both fluids, the fraction containing molecules > 30kDa includes the factor(s) responsible for the observed differences in cumulus expansion Bifftebier, unpublished observations). This suggests the factor is likely to be a protein. The present study aimed to identify those proteins responsible for the observed differences in cumulus expansion after IVM in 10% serum ( > 30 kDa) versus 100/opFF (>30 kDa) obtained from sows in the preovulatory stage of the estrous cycle. Shotgun proteomics analysis of the pFF and serum fractions > 30kDa from 3 sows was performed by application of the 'isobaric Tag for Relative and Absolute Quantitation'(iTRAQ) technology (Applied Biosystems) followed by 2D-LC ESI-Q-TOF MSMS. Of each sample, 100pg protein material was loaded and runs were performed in duplicate. The processed data, obtained from Mascot Daemon, was searched against the pig ESTdatabase for protein identification (http:// pigest.ku.dk). Protein ratios resulting from duplicate runs were averaged, log-transformed and analyzed by Student's t-test. In addition, 600 prepubertal gilt oocytes were matured in vitro for 26h in NCSU23 supplemented with 100/0pFF ( > 30kDa) or 10% serum (> 30kDa) of each of the 3 sows. After IVM, the expanded cumulus matrices were collected and subjected to proteomic analysis. Proteins in the matrix extracts were separated using 2D-PAGE. Two spots that were absent in matrices matured in pFF were excised and submitted to mass spectrometric analysis using ESI-Q-TOF MSMS. The processed data, obtained from Mascot Daemon, was searched against the pig ESTdatabase for protein identification. First of all, serum and pFF were not depleted for high abundant proteins like albumin, because the depleted sample did not show the same biological effect on the IVM of porcine oocytes. Therefore an exclusion list was used based on the first run to exclude abundant peptides derived from albumin. Proteomic analysis of serum and pFF revealed 63 unique proteins present in both fluids of which 13 showed significantly (P< 0.05) different expression levels (10 proteins levels on P<0.01). Seven of these proteins were more abundant in serum whereas 6 of them were more abundant in the pFF fractions. Alpha2-macroglobulin (A2M) and ch4 and secrete domains of swine IgM, which were both down regulated in pFF, were also identified as the protein spots that were absent in the cumulus matrices after IVM in 10% pFF ( > 30 kDa) compared to IVM in 10% serum ( > 30 kDa). In conclusion, 2 proteins that are upregulated in autologous serum were also solely retrieved in the cumulus matrices of oocytes matured in 10% serum ( > 30 kDa). One of them, A2M,