Effect of Passage Number of Conditioned Medium Collected from Equine Amniotic Fluid Mesenchymal Stem Cells: Porcine Oocyte Maturation and Embryo Development
Abstract
:1. Introduction
2. Results
2.1. Effects of eAFMSC-CM Passage Numbers on Cumulus Expansion during IVM (Experiment 1)
2.2. Effects of eAFMSC-CM Passages during IVM on Oocyte Cytoplasmic Maturation (Experiment 2)
2.3. Levels of Antioxidant Biomarkers in Fresh CM and IVM Medium with eAFMSC-CM (Experiment 3)
2.4. Effects of eAFMSC-CMp7 during IVM on Porcine Embryo Development after IVF (Experiment 4)
3. Discussion
4. Materials and Methods
4.1. Ethics
4.2. Reagents and Chemicals
4.3. Preparation of Equine Amniotic Fluid Stem Cell-Conditioned Medium (eAFMSC-CM)
4.4. Maturation In Vitro
4.5. Evaluation of Cumulus Cell Expansion
4.6. Detection of Intracellular Glutathione (GSH) and ROS Levels
4.7. Assessment of Mitochondrial Distribution
4.8. Assessment of Cortical Granule Distribution
4.9. Antioxidant Enzyme Activity Assays
4.10. In Vitro Fertilization
4.11. Embryo Development and Total Blastocyst Counts
4.12. Immunofluorescence Staining
4.13. Analysis of Gene Expression by Quantitative Real-Time PCR
4.14. Experimental Design
4.15. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Sequence (5′-3′) | Product Size (bp) | NCBI Accession No. |
---|---|---|---|
GAPDH | F: gtc ggt tgt gga tct gac ct | 207 | NM_001206359.1 |
R: ttg acg aag tgg tcg ttg ag | |||
BAX | F: ggt cgc gct ttt cta ctt tg | 111 | XM_003127290 |
R: cga tct cga agg aag tcc ag | |||
BCL2 | F: aaa caa tgc agc agc tga ga | 85 | NM_214285 |
R: aac cac ccc agc tag agt ca | |||
HAS2 | F: cag gga caa ttc agc cac tt | 100 | NM_214053.1 |
R: ggt gac atg ttg gga gct tt | |||
TNFAIP6 | F: gaa gca cgg tcg ggc aag | 141 | NM_001159607.1 |
R: cat cca ccc agc agc aca g | |||
PTGS2 | F: tcc tga aca cct ccg ctt tg | 147 | NM_214321.1 |
R: agc cgt tca tcg tcc cat tc | |||
Cx37 | F: cac cct gtc cct acc tcg ta | 101 | NM_001244224.1 |
R: gag cac cag gga gat gag tc | |||
Cx43 | F: gct ggt cgt atc ctt ggt gt | 83 | NM_001244212.1 |
R: tct ttc cct tca cac gat cc | |||
SOD2 | F: tgg agg cca cat caa tca ta | 136 | NM_214127.2 |
R: agc ggt caa ctt ctc ctt ga | |||
NRF-2 | F: gtg cct ata agt ccc ggt ca | 108 | MH101365.1 |
R: atg cag agc ttt tgc cct ta | |||
BECN1 | F: gat agt ggc gga aaa tct cg | 160 | NM_001044530.1 |
R: cat ctg ggc ata acg cat ct | |||
SOX2 | F: cgc aga cct aca tga acg | 103 | NM_001123197.1 |
R: tcg gac ttg acc act gag | |||
NANOG | F: ccc gaa gca tcc att tcc ag | 171 | NM_001129971.1 |
R: tgt gga aga atc agg gct gt |
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Park, A.; Oh, H.J.; Ji, K.; Choi, E.M.; Kim, D.; Kim, E.; Kim, M.K. Effect of Passage Number of Conditioned Medium Collected from Equine Amniotic Fluid Mesenchymal Stem Cells: Porcine Oocyte Maturation and Embryo Development. Int. J. Mol. Sci. 2022, 23, 6569. https://doi.org/10.3390/ijms23126569
Park A, Oh HJ, Ji K, Choi EM, Kim D, Kim E, Kim MK. Effect of Passage Number of Conditioned Medium Collected from Equine Amniotic Fluid Mesenchymal Stem Cells: Porcine Oocyte Maturation and Embryo Development. International Journal of Molecular Sciences. 2022; 23(12):6569. https://doi.org/10.3390/ijms23126569
Chicago/Turabian StylePark, Ahyoung, Hyun Ju Oh, Kukbin Ji, Eunha Miri Choi, Dongern Kim, Eunyoung Kim, and Min Kyu Kim. 2022. "Effect of Passage Number of Conditioned Medium Collected from Equine Amniotic Fluid Mesenchymal Stem Cells: Porcine Oocyte Maturation and Embryo Development" International Journal of Molecular Sciences 23, no. 12: 6569. https://doi.org/10.3390/ijms23126569