Progress 07/01/19 to 06/06/24
Outputs
Target Audience:The primary target audience are research scientists with a focus on embryology, reproductive biology, and epigenetics. The results of the research are also relevant to dairy farmers, cattle ranchers, and embryo technology practitioners. Efforts will be made at the appropriate time to convey results and recommendations for changes in reproductive management and embryo production to these groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In the project period, research contributed to the research dissertations of four PhD students. How have the results been disseminated to communities of interest?Hao Ming, PhD student graduated from my laboratory won the finalist of graduate research competition at the annual conference of International Embryo Technology Society (IETS) and presented his research at New York, 2020. Eva Wang, PhD student graduated from my laboratory won the first place of graduate research competition at the annual conference of IETS and presented her research at Savannah, Georgia, 2022. Linkai Zhu, PhD student in my laboratory won the second place of graduate research competition at the annual conference of IETS and presented her research at Savannah, Georgia, 2022. Giovanna Scatolin, PhD student in my laboratory won the first place of graduate student poster competition and presented her research at the annual conference of IETS, Lima, Peru, 2023 What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A. Under specific Aim 1: using immunostaining, we have characterized the 6mA dynamics in bovine oocytes and pre-implantation embryos at 2-, 4-, 8-, 16-cell, morula, blastocyst stage as well as peri-implantation embryos at day 14. Immunofluorescence analysis showed that 6mA first appeared at 8-cell stage and was predominantly present at trophectoderm cell lineages in early embryos. We then sought to determine the 6mA genome-wide deposition in bovine embryos. However, all recently studies have confirmed the low abundance of 6mA in the mammalian genome, which making it extremely challenge to profile 6mA in the bovine pre-implantation development with limited embryo materials. Alternatively, we have been working to develop the protocols to establish bovine stem cell models to study 6mA regulated mechanisms. To this end, we have successfully developed bovine trophoblast stem cells (TSCs, Wang et al., Cell Reports, 2023) and bovine blastocyst-like structures via assembly of bovine embryonic stem cells (ESCs) and TSCs, termed, bovine blastoids (Pinzo-Arteaga and Wang et al., Cell Stem Cell, 2023). These bovine stem cell cultures will provide a powerful cell model to study the 6mA mediated epigenetic mechanisms. Additionally, in collaboration with Dr. Andrew Xiao (collaborator of this application), in a mouse model, we found 6mA functions at the boundaries between euchromatin and heterochromatin to restrict the spread of euchromatin; The repression of SATB1 mediated by 6mA is essential for gene regulation during trophoblast development in cell culture models and in vivo (Li et al., Nature, 2020). Overall, our findings demonstrate an unexpected molecular mechanism for 6mA function via SATB1, and reveal connections between DNA modification, DNA secondary structures and large chromatin domains in early embryonic development. B. Under specific Aim 2: using IVF embryo platform and CRISPR/CAS9 gene editing system, we have established bovine embryo mutants via genetic manipulation (overexpression or OE, and knockout or KO) of ALKBH1 (6mA demethylase) and METTL7A (6mA putative methyltransferase). We've also characterized the phenotypes from mutant embryos. We found that ALKBH1 and METTL7A KO/OE embryos develop normally to 8-cell stage. Interestingly, overexpression of METTL7A significantly improved the embryo developmental potential, an increase of the blastocyst rate. Additionally, overexpression of ALKBH1 impaired blastocyst formation while knockout of ALBKH1 moderately improved the embryo developmental potential. To further determine the developmental competence of ALKBH1 KO and METTL7A OE embryos, we performed embryo transfer to synchronized surrogates and found both edited groups developing into peri-implantation stage with normal morphology, lineage composition and allocation, and elicit maternal recognition of pregnancy. Finally, we demonstrated that overexpression of METTL7A is beneficial to bovine embryo competence through reducing embryonic cell oxidative stress and DNA damage, which providing a therapeutical target to improve the fertility of animals. A manuscript regarding the biological roles of ALKBH1 and METTL7A in the bovine pre-implantation development is being prepared for publication. C. Under Specific Aim 3: We've characterized the DNA 5mC methylome dynamics during bovine pre-implantation development by whole genome bisulfite sequencing (Duan and Jiang et al., Frontiers in Genetics, 2019) and revealed the negative correlation of 5mC and 6mA dynamics during bovine pre-implantation development. We have also determined the underlying mechanisms of 6mA modulation by ALKBH1 and METTL7A in the regulating transcriptomes and chromatin accessibilities of bovine embryos. We interrogated the transcriptional profiles and chromatin landscapes on embryos at 2-, 8-cell, and blastocyst (with inner cell mass and trophectoderm separated) with ALKBH1 and METTL7A gene perturbation by RNA-seq and ATAC-seq analysis, respectively. We found that the trophoblast genes were significantly regulated. We also demonstrated that over expression of METTL7A tends to attenuate mitochondrial activity and DNA damage level, which is consistent with the beneficial action of METTL7A in the promoting bovine embryo competence. We have also characterized the chromatin dynamics (Hao et al., Epigenetics, 2020) and translatome (Zhu et al., Development, 2022) during bovine pre-implantation embryo development. Comparative analysis of multiple levels of functional genome (transcriptome, translatome, DNA methylome, and chromatin accessibility) of bovine pre-implantation embryo development has been established, providing a reference database for understanding the molecular programs of bovine embryogenesis. C. Besides of the abovementioned significant accomplishments, we have produced a total of 27 publications and 2 patents (see products) with part of support of this USDA-AFRI-NIFA award
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Xu S, Wang N, Zuccaro M, Gerhardt J, Iyyappan R, Scatolin GN, Jiang Z, Baslan T, Koren A, Egli D. DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility. Nature Communications, 2024 (accepted)
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Lamacova L, Jansova D, Jiang Z, Dvoran M, Iyyappan R, Fan H, Jiao Y, Susor A. CPEB3 maintains developmental competence of the oocyte. Cells, 2024. PMID: 38786074.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Scatolin GN*, Ming H*, Wang Y, Iyyappan R, Gutierrez-Castillo E, Zhu L, Sagheer M, Song C, Bondioli, K, Jiang Z#. Single-cell transcriptional landscapes of bovine peri-implantation development. iScience. 2024 Mar 27. PMID: 38633001.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Ming H, Zhang M, Rajput S, Logsdon D, Zhu L, Schoolcraft WB, Krisher RL, Jiang Z#, Yuan Y#. In vitro culture alters cell lineage composition and cellular metabolism of bovine blastocyst. Biol Reprod. 2024 Feb 26. PMID: 38408205
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Fendereski M, Ming H, Jiang Z, Guo Y. Mouse trophoblast cells have attenuated responses to TNF? and IFN? and can avoid synergic cytotoxicity of the two cytokines. Journal of Immunology. 2024 Jan 15. PMID: 38054905.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Jiang Z#. Molecular and cellular programs underlying the development of bovine pre-implantation embryos. Reproduction, Fertility and Development. 2023 December. PMID: 38064195
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Iyyappan R, Aleshkina D, Ming H, Dvoran M, Kakavand K, Jansova D, Del Llano E, Gahurova L, Bruce AW, Masek T, Pospisek M, Horvat F, Kubelka M, Jiang Z, Susor A. The translational oscillation in oocyte and early embryo development. Nucleic Acids Research. 2023 Nov 10. PMID: 37950888.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Hoorn QA, Rabaglino MB, Maia TS, Sagheer M, Fuego D, Jiang Z, Hansen PJ. Transcriptomic profiling of the bovine endosalpinx and endometrium to identify putative embryokines. Phyiol Genomics. 2023 Nov 1. PMID: 37720990
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
11. Zhu L, Tillquist N, Scatolin G, Gately R, Kawaida M, Reiter A, Reed S, Zinn S, Govoni KE, Jiang Z#. Maternal restricted- and over-feeding during gestation perturbs offspring sperm epigenome in sheep. Reproduction. 2023 Aug 1. PMID: 37647207.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Stanton DL, Graf A, Maia TS, Blum H, Jiang Z, Hansen PJ. Absence of a molecular circadian clock in the preimplantation embryo is a conserved characteristic in the mammal. Reproduction. 2023 Jul 31;166(3):199-207. PMID: 37387479.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Pinzon-Arteaga C*, Wang Y*, Wei Y, Li L, Orsi A, Scatolin G, Liu L, Sakurai M, Ye J, Yu L, Li B, Jiang Z#, Wu J#. Bovine blastocyst like structures derived from stem cell cultures. Cell Stem Cell. 2023 May 4. PMID: 37146582.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Wang Y*, Yu L, Li J, Zhu L, Ming H, Pinzon-Arteaga C, Sun H, Wu J, Jiang Z#. Establishment of bovine trophoblast stem cells. Cell Reports. 2023 May 30. PMID: 37146606
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Iyyappan R, Aleshkina D, Zhu L, Jiang Z, Kinterova V, Susor A. Oocyte specific LncRNA variant Rose influences oocyte and embryo development. Noncoding RNA research 2021 Jun 26. PMID: 34278057.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Li C, Sun J, Liu Q, Ming H, Wang L, Li Y, Li R, Jiang Z, Francis J, Fu X. The landscape of accessible chromatin in quiescent and post-myocardial infarction cardiac fibroblasts. Epigenetics 2021 Sep 23. PMID: 34551670.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Zhu L, Marjani SL, Jiang Z#. The epigenetics of gametes and early embryos and potential long-range consequences in livestock species - filling in the picture with epigenomic analyses. Front Genet. 2021 Mar 2; 12:557934. PMID: 33747031.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Gutierrez E, Ming H, Foster B, Gatenby L, Mak CK, Pinto C, Bondioli K, Jiang Z#. Effect of vitrification on global gene expression dynamics of bovine elongating embryos. Reprod Fertil Dev. 2021 February 19. PMID: 33602389.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Li Z, Zhao S, Nelakanti RV, Lin K, Wu TP, Alderman III MH, Guo C, Wang P, Zhang M, Wang M, Jiang Z, Wang Y, Li H, Xiao AZ. N6-methyladenine in DNA antagonizes SATB1 in early development. Nature 2020 Jul: 583, 625630. PMID: 32669713
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Zhu L, Zhou T, Iyyappan R, Ming H, Wang Y, Dvoran M, Chen Q, Roberts RM, Susor A, Jiang Z#. High-resolution ribosome profiling reveals translational selectivity for transcripts in bovine preimplantation embryo development. Development, 2022. PMID: 36227586
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Dodlapati S, Jiang Z, Sun J. Completing single-cell DNA methylome profiles via transfer learning together with KL-divergence. Front Genet. 2022. PMID: 35938031.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Llano ED, Iyyappan R, Aleshkina D, Dvoran M, Jiang Z, Kubelka M, Susor A. SGK1 is essential for meiotic resumption in mammalian oocytes. European Journal of Cell Biology. 2022 Feb 25. PMID: 35240557.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Smith R, Susor A, Ming Hao, Tait J, Conti M, Jiang Z#, Lin CJ#. The H3.3 chaperone Hira complex orchestrates oocyte developmental competence. Development. 2022 Feb 03. PMID: 35112132.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Diaz FA, Gutierrez-Castillo EJ, Foster BA, Hardin PT, Bondioli, KR, Jiang Z#. Evaluation of seasonal heat stress on transcriptomic profiles and global DNA methylation of bovine oocytes. Front Genet. 2021 Oct 29. PMID: 34777457.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Sang L, Xiao Y, Jiang Z, Forde N, Tian X, Lonergan P, Hansen PJ. Atlas of receptor genes expressed by the bovine morula and corresponding ligand-related genes expressed by uterine endometrium. Molecular Reproduction and Development. 2021. DOI: 10.1002/mrd.23534.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Ming H, Sun J, Pasquariello R, Gatenby L, Herrick J, Yuan Y, Pinto C, Bondioli KR, Krisher RL, Jiang Z#. The landscape of accessible chromatin in bovine oocytes and early embryos. Epigenetics. 2020 Jul 14. PMID:32663104
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Li Y, Sun J, Ling Y, Hao M, Chen Z, Fang F, Liu Y, Cao H, Ding J, Cao Z, Zhang X, Bondioli K, Jiang Z#, Zhang Y#. Transcriptional profiles of oocytes during maturation and embryos during preimplantation development in vivo in the goat. Reprod Fertil Dev. 2020 Apr;32(7):714-725. PMID: 32317096.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
West R*, Ming H*, Logsdon DM*, Sun J, Rajput SK, Kile RA, Schoolcraft WB, Roberts RM, Krisher RL, Jiang Z#, Yuan Y#. Dynamics of trophoblast differentiation in peri-implantation stage human embryos. PNAS. 2019 Oct 21. PMID: 31636193.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Duan J*, Jiang Z*, Alqahtani F, Mandoiu I, Hong D, Zheng X, Marjani SL, Chen J, Tian XC. Methylome dynamics of bovine gametes and in vivo early embryos. Front Genet. 2019 May 28; 10:512. PMID: 31191619.
|
Progress 07/01/22 to 06/30/23
Outputs
Target Audience:The primary target audience are research scientists with a focus on embryology, reproductive biology, and epigenetics. The results of the research are also relevant to dairy farmers, cattle ranchers, and embryo technology practitioners. Efforts will be made at the appropriate time to convey results and recommendations for changes in reproductive management and embryo production to these groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In the project period, research contributed to the research programs of three PhD students. How have the results been disseminated to communities of interest?Giovanna Scatolin, PhD student in my laboratory presented her research at the annual conference of International Embryo Technology Society, Lima, Peru. She has won the first place in the graduate student poster competition. What do you plan to do during the next reporting period to accomplish the goals?A. Since extracellular vesicles (EV) have been utilized to deliver the genes into cells for gene therapy, we will employ and engineer EV as carriers to modulate METTL7A and ALKBH1 in bovine embryos through IVF embryo culture, which will have several advantages over conventional gene manipulation via microinjection. We plan to develop promising new therapeutical targets and embryo gene delivery approach to improve animal fertility. B. We will use the established bovine stem cell models to profile 6mA genome-wide distributions in the bovine genome.
Impacts
What was accomplished under these goals?
A. We utilized in vitro produced embryos and zygotic intervention through microinjecting CRISPR/Cas9 RNP complex targeting ALKBH1 (6mA demethylase)/METTL7A (6mA putative methyltransferase) or in vitro transcribed mRNA to establish ALKBH1/METTL7A knockout (KO) or overexpression (OE) bovine embryos. Immunofluorescence analysis showed that 6mA first appeared at 8-cell stage and was predominantly present at trophectoderm cell lineages in early embryos. We found that ALKBH1 and METTL7A KO/OE embryos develop normally to 8-cell stage. Interestingly, overexpression of ALKBH1 impaired blastocyst formation while knockout of ALBKH1 significantly improved the embryo developmental potential, an increase of the blastocyst rate. An elevated blastocyst rate was also observed in METTL7A OE group. B. We performed RNA-seq and ATAC-seq analysis of edited embryos at 2-, 8-cell, and blastocyst (with inner cell mass and trophectoderm separated) and accessed the impact of 6mA remodeling in the transcriptome and other epigenomes (chromatin accessibility) of pre-implantation embryos. We also revealed that mitochondrial function and stress, and energy metabolism were the most repressed in embryos overexpressing METTL7A at the 8-cell and blastocyst stage. C. To further determine the developmental competence of ALKBH1 KO and METTL7A OE embryos, we performed embryo transfer to synchronized surrogates and found both edited groups developing into peri-implantation stage with normal morphology, lineage composition and allocation, and elicit maternal recognition of pregnancy. D. Two manuscripts regarding the biological roles of ALKBH1 and METTL7A in the bovine pre-implantation development are preparing for publication. E. Since ALBKH1 KO and METTL7A OE embryos have increased developmental blastocyst rate. We sought to develop an efficient method to use these molecules to improve the embryo competence. In our ongoing experiments, we employed and engineered extracellular vesicles (EV) as carriers to modulate METTL7A and ALKBH1 in bovine embryos through IVF embryo culture, which will have several advantages over conventional gene manipulation via microinjection. F. Our findings from this study and published studies have shown that 6mA present in the mammalian genome with low abundance, we sought to develop stem cell-based embryo models to profile 6mA at genome-wide resolution. To this end, we have established bovine trophoblast stem cells and blastocyst like structures (blastoids). These bovine stem cell cultures will provide a powerful cell model to study the N6-mA mediated epigenetic mechanisms during bovine pre-implantation development.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Zhu L, Tillquist N, Scatolin G, Gately R, Kawaida M, Reiter A, Reed S, Zinn S, Govoni KE, Jiang Z. Maternal restricted- and over-feeding during gestation perturbs offspring sperm epigenome in sheep. Reproduction. 2023 Aug 1. doi: 10.1530/REP-23-0074. Online ahead of print. PMID: 37647207
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Zhu L, Zhou T, Iyyappan R, Ming H, Dvoran M, Wang Y, Chen Q, Roberts RM, Susor A, Jiang Z. High-resolution ribosome profiling reveals translational selectivity for transcripts in bovine preimplantation embryo development. Development. 2022 Nov 1;149(21):dev200819. doi: 10.1242/dev.200819. Epub 2022 Nov 3.PMID: 36227586
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Stanton DL, Graf A, Maia TS, Blum H, Jiang Z, Hansen PJ. Absence of a molecular circadian clock in the preimplantation embryo is a conserved characteristic in the mammal. Reproduction. 2023 Jul 31;166(3):199-207. doi: 10.1530/REP-23-0104. Print 2023 Sep 1. PMID: 37387479
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Wang Y, Ming H, Yu L, Li J, Zhu L, Sun HX, Pinzon-Arteaga CA, Wu J, Jiang Z. Establishment of bovine trophoblast stem cells. Cell Rep. 2023 May 30;42(5):112439. doi: 10.1016/j.celrep.2023.112439. Epub 2023 May 4.PMID: 37146606
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Pinz�n-Arteaga CA, Wang Y, Wei Y, Ribeiro Orsi AE, Li L, Scatolin G, Liu L, Sakurai M, Ye J, Hao Ming, Yu L, Li B, Jiang Z, Wu J. Bovine blastocyst-like structures derived from stem cell cultures. Cell Stem Cell. 2023 May 4;30(5):611-616.e7. doi: 10.1016/j.stem.2023.04.003.PMID: 37146582
- Type:
Other
Status:
Under Review
Year Published:
2023
Citation:
Scatolin GN, Ming H, Wang Y, Zhu L, Castillo EG, Bondioli K, Jiang Z. Single-cell transcriptional landscapes of bovine peri-implantation development. . bioRxiv. 2023 Jun 14:2023.06.13.544813. doi: 10.1101/2023.06.13.544813. Preprint. PMID: 37398069 Free PMC article.
- Type:
Other
Status:
Under Review
Year Published:
2023
Citation:
Ming H, Zhang M, Rajput S, Logsdon D, Zhu L, Schoolcraft WB, Krisher R, Jiang Z, Yuan Y. In Vitro Culture Alters Cell Lineage Composition and Cellular Metabolism of Bovine Blastocyst. bioRxiv. 2023 Jun 11:2023.06.09.544379. doi: 10.1101/2023.06.09.544379. Preprint. PMID: 37333292
|
Progress 07/01/21 to 06/30/22
Outputs
Target Audience:The primary target audience are research scientists with a focus on embryology, reproductive biology, and epigenetics. Theresults of the research are also relevant to dairy farmers, cattle ranchers, and embryo technology practitioners. Efforts will bemade at the appropriate time to convey results and recommendations for changes in reproductive management and embryoproduction to these groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In the project period, research contributed to the research programs of four graduatestudents (three Ph.D and one M.S students) How have the results been disseminated to communities of interest?A. Eva Wang, PhD student in my laboratory presented his research at the 48th annual conference of International Embryo Technology Society, Savannah, GA. She has won the first place in the graduate research competition. B. Linkai Zhu, PhD student in my laboratory presented his research at the 48th annual conference of International Embryo Technology Society, Savannah, GA. He has won the runner up in the graduate research competition. What do you plan to do during the next reporting period to accomplish the goals?A.We will perform single cell RNA sequencinganalysis of elongated embryos (Day 14, Day 16, and Day 18) derived from ALKBH1 and METTL7A edited embryos, and characterize the lineage composition and allocation compared to the controls (matched elongated embryos flushed in vivo and embryos flushed from IVF blastocyst transfers). B. We will utilize the bovine TSC and stem cell-based embryos as cell models tostudy theN6-mA mediated epigenetic mechanisms that control bovine embryo development. C. We will optimize the small molecules to target N6mA enzymes to improve the IVF embryo culture condtion. D. We plan to transfer the edited embryos [in parallel with conventional IVF embryos (control)] into a large group of recipient heifers and see if they are producing successful pregnancies and healthy offspring.
Impacts
What was accomplished under these goals?
A. We have established KO or OE ofALKBH1andMETTL7Abovine embryos by CRISPR/Cas9 mediated gene targeting by zygote injection. We found all manipulation groups produce normal cleavage and 8-cell embryos. OE ofALKBH1and KO ofMETTL7Aimpair bovine blastocyst formation, while KO ofALBKH1and OE ofMETTL7Asignificantly improves embryo developmental potential, as calculated by the blastocyst rate. For example, OverexpressionMETTL7Aresulted in 13% blastocyst rate increase, 47% compared to 34% in control group. B. We have shown thatMETTL7Aoverexpression significantly increased the trophectoderm cell number in the blastocysts. The transcriptome analysis of trophectoderm and inner cell mass of edited embryos also suggest trophoblast program was modulated. C. We have transferredALKBH1KO andMETTL7AOE blastocysts into recipient heifers, the gene edited embryos developed normally as evidenced by the flushing of Day 14 elongated embryos as compared to control IVF blastocyst transfer. D. We have optimized a culture condition and established bovine trophoblast stem cell (TSC). This culture condition could sustain both bovine TSC and embryonic stem cells (ESCs) culture,which sets a foundation to establish stem cell derived embryos. Because of the relative low abundance of N6mA in mammalian genome, the proposed profiling of N6mA in bovine early embryos are extremely challenge, in this regard,the bovine TSC and stem cell derived embryos will provide a powerful cell model to study theN6-mA mediated epigenetic mechanisms that control bovine trophoblast development and function. E. Besides of the abovementioned significant progresses towards our goals, we have published sixmanuscripts (see products) with part of support of this USDA-AFRI-NIFA award.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Iyyappan R, Aleshkina D, Zhu L, Jiang Z, Kinterova V, Susor A. Oocyte specific lncRNA variant Rose influences oocyte and embryo development. Noncoding RNA Res. 2021 Jun 26;6(2):107-113. doi: 10.1016/j.ncrna.2021.06.001. PMID: 34278057; PMCID: PMC8258604.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Li C, Sun J, Liu Q, Dodlapati S, Ming H, Wang L, Li Y, Li R, Jiang Z, Francis J, Fu X. The landscape of accessible chromatin in quiescent cardiac fibroblasts and cardiac fibroblasts activated after myocardial infarction. Epigenetics. 2021 Oct 25:1-20. doi: 10.1080/15592294.2021.1982158. Epub ahead of print. PMID: 34551670.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Sang L, Xiao Y, Jiang Z, Forde N, Tian XC, Lonergan P, Hansen PJ. Atlas of receptor genes expressed by the bovine morula and corresponding ligand-related genes expressed by uterine endometrium. Mol Reprod Dev. 2021 Oct;88(10):694-704. doi: 10.1002/mrd.23534. Epub 2021 Oct 1. PMID: 34596291; PMCID: PMC8558826.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Diaz FA, Gutierrez-Castillo EJ, Foster BA, Hardin PT, Bondioli KR, Jiang Z. Evaluation of Seasonal Heat Stress on Transcriptomic Profiles and Global DNA Methylation of Bovine Oocytes. Front Genet. 2021 Oct 29;12:699920. doi: 10.3389/fgene.2021.699920. PMID: 34777457; PMCID: PMC8585773.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Smith R, Susor A, Ming H, Tait J, Conti M, Jiang Z, Lin CJ. The H3.3 chaperone Hira complex orchestrates oocyte developmental competence. Development. 2022 Mar 1;149(5):dev200044. doi: 10.1242/dev.200044. Epub 2022 Feb 28. PMID: 35112132; PMCID: PMC8959146.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Del Llano E, Iyyappan R, Aleshkina D, Masek T, Dvoran M, Jiang Z, Pospisek M, Kubelka M, Susor A. SGK1 is essential for meiotic resumption in mammalian oocytes. Eur J Cell Biol. 2022 Apr;101(2):151210. doi: 10.1016/j.ejcb.2022.151210. Epub 2022 Feb 25. PMID: 35240557.
|
Progress 07/01/20 to 06/30/21
Outputs
Target Audience:The primary target audience are research scientists with a focus on embryology, reproductive biology, and epigenetics. The results of the research are also relevant to dairy farmers, cattle ranchers, and embryo technology practitioners. Efforts will be made at the appropriate time to convey results and recommendations for changes in reproductive management and embryo production to these groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In the project period, research contributed to the doctoral research programs of three students. How have the results been disseminated to communities of interest?Linkai Zhu, PhD student in my lab presented his research at 47thannual conference of International Embryo Technology Society and won the second place Poster Award. What do you plan to do during the next reporting period to accomplish the goals?A. We will add more replicates and the embryo injection numbers to our KO and OE experiments and validate our findings. B. We will perform embryo N6mA-DIP-seq to analysis the regions in the bovine genome that contain N6mA and the N6mA deposition alternations in response to the gene manipulation. These data will be integratedly analyzed with transcriptome, chromatin accessibility and DNA methylome data. C. We will profile other epigenetics in bovine early embryos, such as histone modification and small RNAs. Then integratedly analysis will be performed to assess the epigenetic regulation during bovine early embryo development. D. We will conduct more comprehensivecharacterizations on generatedbovine TSC. E. Bovine TSC would provide great resources to study the role of N6mA in bovine trophoblast development. We will utilize genetics, biochemistry, genomics, and developmental approaches to interrogate the function of N6mA in trophoblast development.
Impacts
What was accomplished under these goals?
A. We have established ALKBH1 knockout (KO) and overexpression (OE) bovine embryos though microinjecting CRISPR/Cas9 and in vitro transcript RNA and have shown N6-mA alternation via ALKBH1 modulation affect embryo development. B. We have identified a potential N6mA methyltransferase (pending further characterization) andleveraged KO and OE approaches to survey its function in the bovine early development. We have shown that OE of this N6mA methyltransferase results insignificantly increase ofblastocyst formation, mainly increasingtrophectoderm(TE; Cdx2 positive) cells in the embryos with no changein the inner cells (ICM; OCT4 positive). These phenotyping results indicate that N6mAis essential in TE lineage specification and proliferation. C. We have collected bovine oocytes and embryos at 2-, 4-, 8-, 16-cell, blastocyst, and day 14 elongating embryos, and performed immunostaining analysis tovalidatethe N6mA dynamicsin bovine early embryo development. Our analysis hasshownthat N6mA first appears at 8-cell stage coinciding with trophoblast stem cell (TSC) emergence in bovine, then increases significantly with development, reaching its maximum in blastocysts.We have also found N6mA ispredominantlypresent at TE of blastocystsand trophoblastsof day 14 embryos. These datafurther confirm the regulatory role of N6mA in the trophoblast development, which isconsistent with theKO and OE experiments. D.We haveperformed RNA-seq and ATAC-seq analysis of 2-cell, 8-cell, and blastocyst stage embryos fromKO, OE, and control groups. Our initial RNA-seq analysis identified a number of genes and pathways regulated bythe N6mA demethylase and methyltransferase, which may contribute to the blastocyst competence and trophectoderm cell allocation. We are currently further analyzing the data. E. We have collected 8-cell and blastocyst stage embryos fromKO, OE, and control groups (1000 embryos per stage per treatment). We are on the process ofperform N6mA-DIP-seq to identify the regions in the bovine genome that contain N6mA and the N6mA deposition alternations in response to the gene manipulation. F. Despite the disruption of the pandemic, we managed to set up new experimental systems to address the role of N6mA in the trophoblast development. We have optimized a culture condition and established bovine trophoblast stem cell (TSC) lines from blastocysts. Bovine TSC would provide great resources to study the role of N6mA in bovine trophoblast development. A few characterizations has shown they are bovine TSCs, 1) these cells are able to maintain over 40 passages now, 2) qPCR and immunofluorescence approaches have shown the expression of trophoblast marker genes (CDX2, GATA3, KRT8, GATA6, etc.), but no SOX2 or OCT4 expression, 3) they are able to differentiate to binuclear trophoblasts and secrete IFN during the differentiation, 4) RNA-seq analysis of TSC, differentiated cells, TE from blastocysts and day 14 elongating embryos have further confirmed their trophoblast stem identities. G. Besides of the abovementioned significant progresses towards our goals. We have published three manuscripts to 1) obtain the global gene expression dynamics of trophoblast lineages in bovine elongating embryos (Gutierrez-Castillo et al., Reproduction Fertility and Development 2021), 2) describe the function of N6mA in regulating trophoblast lineage emergence in a mouse model (Li et al., nature 2020), and 3) review the epigenetics (including N6mA) of early embryos and potential long-range consequences (Zhu et al., Frontiers in Genetics 2021).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Li Z, Zhao S, Nelakanti RV, Lin K, Wu TP, Alderman MH 3rd, Guo C, Wang P, Zhang M, Min W, Jiang Z, Wang Y, Li H, Xiao AZ. N6-methyladenine in DNA antagonizes SATB1 in early development. Nature. 2020 Jul;583(7817):625-630. doi: 10.1038/s41586-020-2500-9. Epub 2020 Jul 15. PMID: 32669713.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Zhu L, Marjani SL, Jiang Z. The Epigenetics of Gametes and Early Embryos and Potential Long-Range Consequences in Livestock Species-Filling in the Picture With Epigenomic Analyses. Front Genet. 2021 Mar 3;12:557934. doi: 10.3389/fgene.2021.557934. PMID: 33747031; PMCID: PMC7966815.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Gutierrez-Castillo E, Ming H, Foster B, Gatenby L, Mak CK, Pinto C, Bondioli K, Jiang Z. Effect of vitrification on global gene expression dynamics of bovine elongating embryos. Reprod Fertil Dev. 2021 Mar;33(5):338-348. doi: 10.1071/RD20285. PMID: 33602389; PMCID: PMC8049512.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Li C, Sun J, Liu Q, Ming H, Wang L, Li Y, Li R, Jiang Z, Francis J, Fu X. The landscape of accessible chromatin in quiescent and post-myocardial infarction cardiac fibroblasts. bioRxiv. 2021 March 4.
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Progress 07/01/19 to 06/30/20
Outputs
Target Audience:?The primary target audience are research scientists with a focus on embryology, reproductive biology, and epigenetics. The results of the research are also relevant to dairy farmers, cattle ranchers, and embryo technology practitioners. Efforts will be made at the appropriate time to convey results and recommendations for changes in reproductive management and embryo production to these groups. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In the project period, research contributed to the doctoral research programs of two students. How have the results been disseminated to communities of interest?The results from this research was selected as finalists for graduate student research competition and awarded travel scholarships. Ming Hao, PhD student in mylab presentedhis research titled "The landscape of accessible chromatin in bovine oocytes and early embryos"at the 2019 Annual Conference ofInternational Embryo Technologies Society (IETS) at New York. What do you plan to do during the next reporting period to accomplish the goals?1. A large number of oocytes and embryos arerequried to determine the level andgenomic distribution of 6mA through LC-MS/MS and6mA ChIP-seq. Therefore, production andcollection of sufficientoocytes and embryos will be continued in the next reporting period. 2. More comprehensive characterization of thecompositions ofthe ICM andTE in blastocystafter Alkbh1 KO and OE will performed. 3.To understand the role of 6mA mediated by Alkbh1 duringthe bovine early embryo development, we will KO or OE Albkh1 in embryo, and perform RNA sequencing on 8-cell embryos and blastocysts (ICM and TE) todetermine the effect of 6mA remodeling on embryonic genome activation and first differentiation at blastocysts.
Impacts
What was accomplished under these goals?
1. Weproduced the bovine oocytes and embryos at different deveopmental stages including 2-, 4-, 8-, 16-cell, morula and blastocyst by in vitro fertilizaton (IVF). 2. Chromatin reorganization governs the regulation of gene expression during preimplantation development tegother with other epigenetic mechanims such as6mA. Weconstructed a genome-wide map of accessible chromatin in bovine oocytes and early embryos using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), whichrevealed unique features of the accessible chromatin during bovine early embryo development. We found that chromatin accessibility is low in oocytes and 2-/4-cell embryos, followed by a significant increase in embryos during major embryonic genome activation (EGA), and peaked in elongating day 14 embryos. By integrated analysis of chromatin accessibility with transcriptomes and DNA methylomes in bovine early embryos, we showed that promoter accessibility was positively correlated with gene expression, especially during major EGA, and was strongly correlated to DNA methylation and CpG density. Future analysis will beintegrating ATAC-seqwith the 6mA ChIP-seq data at the same developmental stage of embryos. 3. We designed siRNAs to target Alkbh1 and N6amt1. We also produced the IVT-RNA forAlkbh1 and N6amt1via in vitro transcription. Knockdown(KO)oroverepression(OE) of Alkbh1 andN6amt1 throughmicroinjection ofsiRNAs orIVT-mRNAs in the zygote stage embryos were performed, followed by embryo culture and evaluation ofcleavage ragte and and blastocyst rate. Manipulation ofN6amt1 didnot affect cleavage rate and blastocyst rate. However, Either KO or OE ofAlkbh1 also did not affect cleavage rate, butsignificantly impared blastocyst formation. More comprehensive characterization of embryo developmental potential and 6mA level will be performed. 4. We havealso collaborated researchersto study the role of 6mA in early embryogenesis using mice models. The examination of the cohort of embryos from mutant mice lacking Albkh1demonstrated that significant reduction of trophoblast cell lineages. The results from mosue studies suggest that, if the role of 6mA is conserved across mammlian species, 6mA may play an important role in the trophobalst lineagedevelopment in bovine as well. Future studies we will focus on the characterization of compositions ofthe inner cell mass (ICM)andtrophectoderm (TE)cells in blastocystafter Alkbh1 KO and OE.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Li Z, Zhao S, Nelakanti RV, Lin K, Wu TP, Alderman III MH, Guo C, Wang P, Zhang M, Wang M, Jiang Z, Wang Y, Li H, Xiao AZ. N6-methyladenine in DNA antagonizes SATB1 in early development. Nature. 2020 Jul 15: 583, 625630. PMID: 32669713.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Ming H, Sun J, Pasquariello R, Gatenby L, Herrick J, Yuan Y, Pinto C, Bondioli KR, Krisher RL, Jiang Z. The landscape of accessible chromatin in bovine oocytes and early embryos. Epigenetics. 2020 July 14. PMID: 32663104.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Li Y, Sun J, Ling Y, Hao M, Chen Z, Fang F, Liu Y, Cao H, Ding J, Cao Z, Zhang X, Bondioli K, Jiang Z, Zhang Y. Transcriptional profiles of oocytes during maturation and embryos during preimplantation development in vivo in the goat. Reprod Fertil Dev. 2020 Apr;32(7):714-725. PMID: 32317096.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Esencan E, Jiang Z, Wang T, Zhang M, Soylemez-Imamoglu G, Seli E. Impaired mitochondrial stress response due to CLPP deletion is associated with altered mitochondrial dynamics and increased apoptosis in cumulus cells. Reproduction Sciences. 2020 Jan 14. PMID: 31939198.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
West R, Ming H, Logsdon D, Sun J, Rajput S, Kile R, Schoolcraft W, Roberts M, Krisher R, Jiang Z, Yuan Y. Dynamics of trophoblast differentiation in peri-implantation stage human embryos. Proc Natl Acad Sci U S A. 2019 Oct 21. PMID: 31636193.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Smith R, Jiang Z, Susor A, Ming Hao, Tait J, Conti M, Lin CJ. The H3.3 chaperone Hira complex orchestrates oocyte developmental competence. bioRxiv. 2020 May 26, doi: https://doi.org/10.1101/2020.05.25.114124.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Zhang M, Bener MB, Jiang Z, Wang T, Esencan E, Scott R, Horvath T, Seli E. Mitofusin 1 is required for female fertility and to maintain ovarian follicular reserve. Cell Death & Disease, 2019 Jul 22;10(8):560. PMID: 31332167.3.
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