174 EFFECT OF HUMAN ENDOTHELIAL PROGENITOR CELLS ON IN VITRO MATURATION OF PORCINE OOCYTES AND PARTHENOGENETIC EMBRYO DEVELOPMENT COMPETENCE

Abstract

In oocyte maturation, hepatocyte growth factor and vascular endothelial growth factor (VEGF) contribute to promote granulosa cell proliferation and cumulus cell expansion. It is well known that human endothelial progenitor cells (hEPC), which are isolated from monocytes and macrophages, secrete a variety of growth factors, such as hepatocyte growth factor and VEGF, and improve the process of angiogenesis. Therefore, the aim of this study was to investigate the effects of hEPC on in vitro oocyte maturation and subsequent embryo development in pigs. To isolate and culture hEPC, human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated. The peripheral blood mononuclear cells were seeded into flask with defined Keratinocyte-SFM-based medium and incubated at 37°C, 5% CO₂. The hEPC were cultured and cryopreserved until use for co-culturing with porcine oocytes obtained from a local slaughterhouse ovaries. Cumulus-oocyte complexes were randomly cultured in 2 groups; 1) co-culturing with hEPC and 2) culturing without hEPC. Cumulus-oocyte complexes were cultured in the in vitro maturation (IVM) medium containing TCM-199 supplemented with 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL⁻¹ of insulin-transferrin-selenium solution 100X (Invitrogen, Seoul, South Korea), 10% porcine follicular fluid, 10 IU mL⁻¹ of eCG, and 10 IU mL⁻¹ of hCG. After IVM, the first polar body extrusion was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and cultured in porcine zygote medium-5 for 7 days. The cleavage and blastocyst formation rates were observed on Day 2 and 7, respectively. Also, blastocysts were stained with Hoechst 33342 and total blastocyst cell numbers were evaluated under a fluorescence microscope. As a result, the oocyte maturation rate or first polar body extrusion rate of the hEPC co-culture group (90.06 ± 0.75) was significantly higher than the control group (90.06 ± 0.75 v. 85.79 ± 0.59; P < 0.05). There was no significant difference between the hEPC co-cultured and the control groups in cleavage rate. However, a significant difference in blastocyst formation rate was observed between the hEPC co-cultured and the control groups (28.45 ± 4.92 v. 15.87 ± 2.27; P < 0.05), whereas total blastocyst cell numbers did not show significant difference between the 2 groups. The all data were analysed by unpaired t-test using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Values are means ± standard error of mean. In conclusion, the results in the present study demonstrated that co-culturing with hEPC improved the in vitro oocyte maturation and blastocyst formation rate. Also, we are underway in analysing the concentration of VEGF families in the hEPC co-culture medium after IVM. For further study, we will analyse the genes of the VEGF signaling pathway in the cumulus cells and matured oocytes derived from the 2 groups.

This research was supported by Nature Cell (#550-20150030), global PH.D Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), and Research Institute for Veterinary Science, the BK21 plus program.