Introduction: Versican is a large extracellular matrix (ECM) proteoglycan that regulates adhesion, survival, proliferation and migration of the cells, as well as ECM assembly. In rodent follicles, versican V0/V1 expression increases about 4 h after hCG induction, while in bovine granulosa cells no increase was observed post hCG injection, suggesting that versican expression may varies significantly between species. Interestingly, the versican V1 cleaved product G1-DPEAA accumulates in the mouse cumulus matrixin vivo few hours prior ovulation, likely contributing to its expansion. We investigated the spatiotemporal expression of G1-DPEAA cleaved product in porcine follicles during the periovulatory period.
Methods: Porcine oocyte cumulus complexes (OCCs) stimulated in vivo and OCCs and mural granulosa cells (MGCs) stimulated in vitro with FSH/LH were undigested or digested with either chondroitinase ABC or Streptomyces hyaluronidase. Total, matrix and cell pellet extracts were then analyzed by Western blot by using a versican antibody recognizing the neoepitope DPEAAE.
Results: Both in vivo expanded and in vitro FSH/LH stimulated porcine OCCs accumulated V1 versican cleaved form (~70 kDa) in the ECM. Our in vitro analysis clearly indicated that the versican ~70 kDa cleaved product accumulated in the matrix with time, since it was barely visible at 26 h and became quite evident at 44 h of culture. Interestingly, the OCCs samples treated with hyaluronidase showed an additional band of about 75 kDa MW. This ~75 kDa form was quite evident at 26 h after stimulation, increased slightly at 44 h and was absent in MGCs. Conclusion: V1 versican cleaved form (~70 kDa) was detected both in in vivo and in vitro matrix extracts of gonadotropin stimulated OCCs as well as in matrix extracts of MGCs after stimulation with FSH/LH. The identity of ~75 kDa band is under investigation. Supported by Grant Agency of the Czech Republic (grant: 15-22765S).
11 Jul 2016 - 11 Jul 2016