233 Conditioned medium derived from amniotic stem cells support telomere-related genes during in vitro maturation of porcine oocytes until embryo development
E. M. Choi A , H. J. Oh A , E. Kim B , T.-Y. Kil C and M. Kim A BA Division of Animal and Dairy Science, College of Agriculture and Life Science, Chungnam National University, Daejeon, South Korea
B MK Biotech Inc., Daejeon, South Korea
C Department of Social Welfare, Joongbu University, Keumsan-gun, Chungnam, South Korea
Reproduction, Fertility and Development 35(2) 245-246 https://doi.org/10.1071/RDv35n2Ab233
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Telomere is important in germ cells undergoing fertilisation; therefore, dysfunction causes unstable cellular maintenance, leading to abnormal cleavage during embryo development. The aim of this study is to investigate the supplementary effect of conditioned medium (CM) from equine amniotic fluid stem cells (eAFSCs) in passages 3, 5, and 7 during porcine in vitro maturation (IVM) on oocyte maturation, embryo development, and expression of telomere-related genes. eAFSCs were cultured in DMEM and changed to serum-free medium 48 h before CM collection in passages, 3, 5, and 7. The experiment groups were: (1) Control: 100% TCM199; (2) CM-P3: TCM199 with 50%; CM-P3; (3) CM-P5: TCM199 with 50% CM-P5; (4) CM-P7: TCM199 with 50% CM-P7. For the first experiment, eAFSCs for passages 3, 5, and 7 were characterised using FACS, and RT-qPCR was used to analyse telomere-related genes: telomerase reverse transcriptase (TERT), sirtuin1 (SIRT1), and sirtuin6 (SIRT6). The second experiment investigated the supplementary effect of CM-P3, CM-5, and CM-7 during IVM of oocytes. Evaluation of oxidative stress was done by reactive oxygen species (ROS), and glutathione (GSH) detection in mature oocytes. RT-qPCR of telomere-related genes was held with cumulus cells (CC). After IVM, mature oocytes with first polar body went through parthenogenesis and in vitro culture in PZM-5 for six days. Third, in vitro development of embryos and telomere-related gene expressions and ROS were observed in parthenogenetic blastocysts (BLs). ELISA assay of antioxidant enzymes; superoxide dismutase (SOD) and catalase (CAT) were examined with IVM medium from three statuses: Medium before culture (pre-medium), first Medium (0–22 hr IVM), second Medium (22 hr–44 hr IVM). Surface markers of eAFSCs at passages 3, 5, and 7 were all positive for CD14, CD34, CD45, and negative for CD29 and CD44. In passage 7, eAFSCs, TERT, SIRT1, and SIRT6 were remarkably more increased than in passages 3 and 5. Despite no difference in oocyte nuclear maturation, the ROS value of the CM groups was significantly lower than that of the control group by 0.3 folds, and the GSH level in CM-P7 was 1.82 folds higher than control (P < 0.001). TERT, SIRT1, and SIRT6 in CC were significantly upregulated in the CM groups than control (P < 0.05), and there was no significant difference between TERT and SIRT1 between CM-P3 and CM-P7. BL formation was highest in CM-P7 (43.86%) than other groups (P < 0.001), and there was no difference among control (30.44%), CM-P3 (35.58%), and CM-P5 (36.04%). TERT, SIRT1 in CM-P7 BLs were visibly higher than control and CM-P3 (P < 0.05). ROS was markedly lower in BLs of CM-P7 compared to those of control and CM-P3 (P < 0.05). SOD and CAT showed higher activity in pre-medium of CM groups than control, however, in second-medium-only, CM-P7 maintained high activity (P < 0.05). In conclusion, this study demonstrated that CM-P7 of eAFSCs supports the expression of antioxidant and telomere-related genes improving oocyte quality, consequently enhancing embryonic development.
