Effects of coculture with cumulus-derived somatic cells on in vitro maturation of porcine oocytes

초록

In the process of IVM, cumulus-oocyte complexes (COCs) separate from the follicular microenvironment, leading to the loss of endocrine interactions between follicular mural somatic cells and COCs. To restore the microenvironment, a coculture system was established using cumulus-derived somatic cells (CSCs) for IVM. The CSCs were cultured in Dulbecco's modified Eagle's medium for 48 hours with varying numbers of CSCs (0.0, 2.5 x 10(4), 5.0 x 10(4), and 10.0 x 10(4)) and then cultured in tissue culture medium 199 (TCM 199) for 4 hours before adding the oocytes. Cumulus-oocyte complexes from 3- to 6-mm follicles were matured in 500 mu L of TCM 199 with eCG and hCG for 22 hours and then cultured in TCM 199 without hormones for 22 hours. After IVM, the group with 2.5 x 10(4) CSCs showed a significant increase in intracellular glutathione levels compared with the control group. In the evaluation of sperm penetration, efficient fertilization was increased in the groups with 2.5 x 10(4) and 5.0 x 104 CSCs compared with controls (44.9 and 46.5 vs. 32.1, respectively). The mRNA expression pattern analysis in matured COCs showed a significant upregulation of PCNA, COX-2, Has2, Ptx3, and Nrf2 in the 2.5 x 10(4) CSC group compared with controls. During COC maturation at 0, 11, 22, 33, and 44 hours, the 2.5 x 10(4) and 5.0 x 10(4) CSC groups showed a significantly altered mRNA expression of BMP15 and GDF9. The developmental competence of the matured oocytes in all groups was evaluated after IVF and parthenogenetic activation (PA). After IVF, the 2.5 x 10(4) CSC group showed significantly higher cleavage, blastocyst formation rate, and total cell numbers compared with controls (60.0%, 35.7%, and 127.3 vs. 43.2%, 21.1%, and 89.3, respectively). After PA, the 2.5 x 10(4) CSC group had significantly higher blastocyst formation rate and total cell number than the control group (52.0% and 120.4 vs. 35.4% and 90.9, respectively). In conclusion, these results suggest that the presence of a population of 2.5 x 10(4) CSCs during IVM synergistically improved the developmental potential of 1VF- and PA-derived porcine embryos by increasing the intracellular glutathione level via changing of a specific gene expression pattern during oocyte maturation. (C) 2015 Elsevier Inc. All rights reserved.

키워드