150 PROTEOME OF BOVINE CUMULUS CELLS AS RELATED TO OOCYTE MORPHOLOGY AND IN VITRO EMBRYO PRODUCTION

Abstract

The present study was conducted to study the effect of cumulus-oocyte complex (COC) morphology on subsequent in vitro embryo development and to assess the proteome of their corresponding cumulus cells (CC). Cow ovaries were obtained at an abattoir and COC aspirated from 3–8 mm follicles. The COC were defined as type I (TI): homogeneous ooplasma and ≥4 layers of compact CC; type II (TII): granular ooplasma and ≥4 layers of slight expanded CC. Fifty COC had ~500,000 CC. Cumulus cells were frozen in ammonium bicarbonate and immediately lyophilized for proteome analysis. Other selected COC were matured in vitro in TCM-199-supplemented media for 24 h. After maturation, CC were collected (T24) and processed as described above. The remaining COC were fertilized with sperm from a fertile bull and zygotes, cultured in vitro until Day 7. Ten blastocysts per group were stained (Hoechst 33342) and blastomeres, counted for assessment of embryo quality. The CC proteins were obtained from the following groups: immature type I (TIT0) and type II (TIIT0), and in vitro matured type I (TIT24) and type II (TIIT24). For protein extraction, we used sonication (30 min, 4°C), freezing and unfreezing in liquid nitrogen, and maceration. The CC proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by ESI-MS/MS. Differences in cleavage, embryo rates, and blastomere numbers were analysed by t-test. Protein expression difference was set at 2.5-fold (P < 0.05). In silico protein interactions were investigated using STRING v. 10.0. There were no differences in cleavage (88 ± 4 v. 89 ± 8%) and embryo rates (36 ± 7 v. 33 ± 8%) between COC of TI (n = 220) and TII (n = 161), respectively. Blastomeres were also similar in TI (101) and TII (104) groups. Major proteins expressed in all CC were α-enolase, β-actin, oestradiol 17-β-dehydrogenase 1, glutathione S-transferase, glyceraldehyde 3-phosphate dehydrogenase, heat shock protein β-1, histone H2B type 1-N, histone H4, mitochondrial malate dehydrogenase 2, protein disulfide isomarase A6, triosephosphate isomerase, tubulin α-1C chain, and vimentin. Glyceraldehyde 3-phosphate dehydrogenase appeared to be more expressed in TIT0, whereas tubulin α-1C chain and vimentin had greater expression in TIIT24. As evidenced by in silico analysis, most CC proteins interact among themselves, participating in complex networks involving intracellular signalling and other events. In conclusion, there are no difference in embryo development when using compact and early-expanded COC, indicating that both types can be selected for IVP. Protein profile of cumulus cell may serve as a marker for in vitro embryo competence.