Interleukin 17D Enhances the Developmental Competence of Cloned Pig Embryos by Inhibiting Apoptosis and Promoting Embryonic Genome Activation
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Medium and Reagents
2.3. In Vitro Oocyte Maturation
2.4. In Vitro Fertilization
2.5. Somatic Cell Nuclear Transfer
2.6. IL17D Treatment
2.7. Transcriptome Sequencing
2.8. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
2.9. Terminal Deoxynucleotidyl Transferase (TdT)-Mediated 2′-Deoxyuridine-5′-Triphosphate-Digoxigenin Nick-End Labeling (TUNEL) Assay
2.10. Plasmid Construction
2.11. Microinjection
2.12. Statistical Analysis
3. Results
3.1. IL17D Expression Is Abnormal in Cloned Pig Embryos Compared to That in In Vivo-Fertilization-Derived Pig Embryos
3.2. IL17D Treatment Improved the Developmental Competence of Porcine SCNT and IVF Embryos
3.3. IL17D Treatment Inhibited the Apoptosis of Porcine SCNT and IVF Embryos at Blastocyst Stage
3.4. IL17D Treatment Increased the Expression of Global Genes in Porcine SCNT Embryos at 4-Cell Stage
3.5. IL17D Treatment Improved Porcine SCNT Embryo Development by Regulating Apoptosis-Related Pathways
3.6. The Effects of IL17D on Improving Porcine SCNT Embryo Development Might Be Related to the Up-Regulation of GADD45B Expression
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Types of Treated Embryos | Time Point of IL17D Addition | Groups | Evaluation Indexes | |
---|---|---|---|---|
Experiment 1 | SCNT | 1-cell stage | NC, 5, 25, 50 and 100 ng/mL | Cleavage rate, blastocyst rate |
Experiment 2 | IVF | 1-cell stage | NC, 50 ng/mL | Cleavage rate, blastocyst rate |
Experiment 3 | SCNT | 4-cell stage | NC, 50 ng/mL | Cleavage rate, blastocyst rate |
Experiment 4 | SCNT | 1-cell stage | NC, 50 ng/mL | Total cell number, number of apoptotic cells, percentage of apoptosis cells, apoptosis-related gene expression levels at blastocyst stage |
Experiment 4 | IVF | 1-cell stage | NC, 50 ng/mL | Total cell number at blastocyst stage |
Experiment 5 | SCNT | 1-cell stage | NC, 50 ng/mL | Transcriptome sequencing and qPCR validation at 4-cell stage |
Gene | Primer Sequences (5′-3′) | Product Size (bp) | Gene Accession Number |
---|---|---|---|
BCL2 | F: TCGCCCTGTGGATGACTGAGTAC | 131 | XM_021099593 |
R: AGACAGCCAGGAGAAATCAAATAGAGG | |||
BCL2L1 | F: TGAGCAGGTATTGAACGAACTCTTCC | 143 | NM_214285 |
R: CCATCCAAGTTGCGATCCGACTC | |||
TP53 | F: GCCCATCCTCACCATCATCACAC | 81 | NM_213824 |
R: GCACAAACACGCACCTCAAAGC | |||
CYCS | F: CGAGTGGTGGCTTGTCTGTTGAG | 101 | NM_001129970 |
R: GGCACTGGGCACACTTCTGAAC | |||
BAX | F: ATCGGCTGCTGGGCTGGATC | 124 | XM_003127290 |
R: ATGGTGAGCGAGGCGGTGAG | |||
BCL2L11 | F: TGCCAAGCCTTCAACCATTATCTCAG | 117 | NM_001160074 |
R: GTCTCCAATACGCCGTAACTCCTG | |||
BAD | F: CCGAGGAGGATGAAGGGACTGAG | 138 | XM_021082883 |
R: AGGAACCCTGGAACTCGTCACTC | |||
FOS | F: GGAGTCGTGAAGACCATGCC | 189 | NM_001123113 |
R: TAGCTGGTCTGTCTCCGCTTG | |||
JUN | F: AGGCGGAGAGGAAGCGTATGAG | 145 | NM_213880 |
R: CTGAGCATGTTGGCGGTGGAC | |||
MAP3K8 | F: CCAGTGAAGAGCCAGCAGTGTAC | 150 | XM_021064738 |
R: GCAAGTCCTCCACGGTTCCATATC | |||
MYC | F: TCAACGTCAGCTTCACCAACAGG | 85 | NM_001005154 |
R: AAGTTCTCCTCCTCGTCGCAGTAG | |||
GADD45B | F: TGAATTTGCTCTGTACCCAGGAACTC | 108 | XM_005654701 |
R: GTAAGCCTCCCATCTCTCTTTCAGTG | |||
GAPDH | F: TGACCCCTTCATTGACCTCC | 88 | XM_021091114 |
R: CTCCGCCTTGACTGTGCC |
Groups | No. of Repetition | No. of Cultured SCNT Embryos | No. of Cleaved Embryos (%) | No. of Blastocysts (%) |
---|---|---|---|---|
NC | 4 | 160 | 127 (79.12 ± 5.21 a) | 25 (15.48 ± 4.9 a) |
5 ng/mL IL17D | 3 | 105 | 78 (74.29 ± 2.86 a) | 18 (17.14 ± 5.71 ab) |
25 ng/mL IL17D | 3 | 107 | 76 (72.38 ± 7.19 a) | 21 (19.6 ± 2.54 ab) |
50 ng/mL IL17D | 4 | 166 | 131 (81.62 ± 6.99 a) | 46 (27.59 ± 3.09 c) |
100 ng/mL IL17D | 4 | 162 | 129 (79.47 ± 5.51 a) | 36 (22.06 ± 2.39 bc) |
Groups | No. of Repetition | No. of Cultured SCNT Embryos | No. of Cleaved Embryos (%) | No. of Blastocysts (%) |
---|---|---|---|---|
NC | 6 | 298 | 211 (69.64 ± 8.76 a) | 45 (14.97 ± 4.14 a) |
50 ng/mL IL17D | 6 | 289 | 210 (71.66 ± 8.32 a) | 67 (23.54 ± 3.34 b) |
Groups | No. of Repetition | No. of Cultured SCNT Embryos | No. of Cleaved Embryos (%) | No. of Blastocysts (%) |
---|---|---|---|---|
NC | 3 | 122 | 91 (74.57 ± 1.8 a) | 15 (12.28 ± 2.32 a) |
50 ng/mL IL17D | 3 | 117 | 88 (75.21 ± 2.96 a) | 14 (11.97 ± 1.48 a) |
Groups | No. of Repetition | No. of Cultured SCNT Embryos | No. of Cleaved Embryos (%) | No. of Blastocysts (%) |
---|---|---|---|---|
NC | 3 | 113 | 88 (77.9 ± 5.94 a) | 5 (4.41 ± 1.48 a) |
10 ng/µL | 3 | 113 | 77 (68.14 ± 0.49 a) | 8 (7.09 ± 1.58 a) |
50 ng/µL | 3 | 111 | 78 (69.61 ± 4.45 a) | 10 (8.89 ± 5.49 a) |
100 ng/µL | 3 | 112 | 82 (73.87 ± 7.8 a) | 12 (10.81 ± 2.7 b) |
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Wu, X.; Zhao, H.; Lai, J.; Zhang, N.; Shi, J.; Zhou, R.; Su, Q.; Zheng, E.; Xu, Z.; Huang, S.; et al. Interleukin 17D Enhances the Developmental Competence of Cloned Pig Embryos by Inhibiting Apoptosis and Promoting Embryonic Genome Activation. Animals 2021, 11, 3062. https://doi.org/10.3390/ani11113062
Wu X, Zhao H, Lai J, Zhang N, Shi J, Zhou R, Su Q, Zheng E, Xu Z, Huang S, et al. Interleukin 17D Enhances the Developmental Competence of Cloned Pig Embryos by Inhibiting Apoptosis and Promoting Embryonic Genome Activation. Animals. 2021; 11(11):3062. https://doi.org/10.3390/ani11113062
Chicago/Turabian StyleWu, Xiao, Huaxing Zhao, Junkun Lai, Ning Zhang, Junsong Shi, Rong Zhou, Qiaoyun Su, Enqin Zheng, Zheng Xu, Sixiu Huang, and et al. 2021. "Interleukin 17D Enhances the Developmental Competence of Cloned Pig Embryos by Inhibiting Apoptosis and Promoting Embryonic Genome Activation" Animals 11, no. 11: 3062. https://doi.org/10.3390/ani11113062