A mature cumulus-oocyte complex (COC) is formed by the egg, the zona pellucida (ZP), a glycoprotein matrix surrounding the egg, and a hyaluronic acid jelly containing expanded cumulus cells. When the sperm reaches the oocyte interacts with the cumulus cells and later with the ZP to finally cross it, fuse with the oolemma and enter within the ooplasm. The aim of this study was to develop a novel 3D model mimicking the COC’ size and shape as a new tool to study gametes interaction in depth in porcine species. The 3D model consists of magnetic Sepharose® beads coated with ZP proteins and with cumulus cells attached to the surface. In a first step, the recombinant porcine ZP proteins (ZP2, ZP3 and ZP4) were designed and expressed in mammalian cells (Chinese Hamster Ovary, CHO, cells). The culture medium containing the secreted proteins was collected and characterized by SDS-PAGE and Western Blot by the addition of peptide tags within the sequence and the molecular weights obtained were 100 kDa, 55 kDa and 65 kDa for ZP2, ZP3 and ZP4 respectively. To obtain the 3D models, in a second step, the recombinant proteins were conjugated to Sepharose® beads thus obtaining three different models: beads coated with ZP2 (BZP2), beads coated with ZP3 (BZP3) and beads coated with ZP4 (BZP4). A control model (BCtrl) consisted of beads incubated with growth CHO-cell medium. Protein adhesion to beads surface was confirmed by SDS-PAGE and Western Blot. In a third step, cumulus cells were obtained by denuding in vitro matured porcine cumulus-oocyte complexes. The beads (BZP2, BZP3, BZP4 and BCtrl) were co-incubated with cumulus cells in TALP medium for 24 h at a ratio of 3,000 cumulus cells per bead, 5% CO2 and 38.5ºC under constant movement by an oscillating shaker. After 24 h, beads were mechanically washed, fixed (0.5% glutaraldehyde) and stained (1% Hoescht 33342) to assess the number of cumulus cells attached to the bead surface. Three replicates were performed and results assessed by one-way ANOVA. More than 95% of BZP had at least one cumulus cell attached. BCtrol showed a lower number of cumulus cells per bead (5.99 ± 0.45, n=201) than BZP2 (8.79 ± 0.54, n=195), BZP3 (8.92 ± 0.52, n=212) and BZP4 (10.01 ± 0.49, n=199) (P<0.001). No differences were found among the three BZP models. Cumulus-oocyte complexes-like 3D models by means of magnetic Sepharose® beads coated with recombinant porcine ZP proteins and cumulus cells were generated in this preliminary study. In a next future, these models might be an alternative to native COCs to study the molecular basis of gametes interaction.
Supported by MINECO and FEDER (AGL2015-70159-P) and Fundación Seneca-Agencia de Ciencia y Tecnología de la Región de Murcia (18931/JLI/13).