Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts

Krishna Chaitanya Pavani; Tim Meese; Osvaldo Bogado Pascottini; XueFeng Guan; Xiaoyuan Lin; Luc Peelman; Joachim Hamacher; Filip Van Nieuwerburgh; Dieter Deforce; Annekatrien Boel; Björn Heindryckx; Kelly Tilleman; Ann Van Soom; Bart M Gadella; An Hendrix; Katrien Smits

doi:10.1073/pnas.2122708119

Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts

Krishna Chaitanya Pavani, Tim Meese, Osvaldo Bogado Pascottini, XueFeng Guan, Xiaoyuan Lin, Luc Peelman, Joachim Hamacher, Filip Van Nieuwerburgh, Dieter Deforce, Annekatrien Boel, Björn Heindryckx, Kelly Tilleman, Ann Van Soom, Bart M Gadella, An Hendrix, Katrien Smits

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Extracellular vesicles (EVs) and their cargo microRNAs (miRNAs) are important regulators of embryo development to the blastocyst stage and beyond. Before implantation can take place, hatching of blastocysts from their zona pellucida is required. However, underlying mechanisms by which blastocyst formation and hatching are initiated remain largely unknown. Here, we provide evidence that embryonic EVs containing bta-miR-378a-3p play a crucial role in blastocyst hatching, using a bovine model. A customized procedure was used to isolate EV-miRNAs from culture droplets conditioned by individual bovine embryos that either developed to the blastocyst stage or did not (nonblastocyst). RNA sequencing identified 69 differentially expressed miRNAs between EVs derived from blastocyst conditioned medium (CM) and nonblastocyst CM. Among the miRNAs up-regulated in blastocyst CM, we selected bta-miR-378a-3p for further validation by functionality testing on developing in vitro embryos by means of mimics and inhibitors. Supplementing the embryo culture medium with miR-378a-3p mimic significantly improved blastocyst quality, with higher cell numbers and reduced apoptosis, and improved hatching, while the opposite was found after supplementation with miR-378a-3p inhibitor (P < 0.01). Transcriptomic analysis of embryos treated with miR-378 mimic/inhibitor showed differential expression (P < 0.01) of genes associated with embryo development and implantation, including RAP1GAP, ARFGEF2, SLC7A6, CENPA, SP1, LDLR, PYCR1, MYD88, TPP1, and NCOA3. In conclusion, miR-378a-3p is up-regulated in EVs secreted by embryos that develop to the blastocyst stage, and this EV-derived miR-378a-3p increases blastocyst quality and regulates embryo hatching, which is essential for embryo implantation.

Original language	English
Article number	e2122708119
Pages (from-to)	1-12
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	119
Issue number	12
DOIs	https://doi.org/10.1073/pnas.2122708119
Publication status	Published - 22 Mar 2022

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS. We thank Petra Van Damme and Sofie De Geyter for their excellent technical assistance. We thank Roćıo Melissa Rivera from the University of Missouri for her careful revision of the manuscript. This work was supported by Ghent University (Grant: Bijzonder Onderozoeksfonds Geconcerteerde Onderzoeksactie 2018000504 [GOA030-18 BOF]) and by the European Union (H2020 Marie Sklodowska-Curie Innovative Training Network: project Biology and Technology of Reproductive Health or REP-BIOTECH 675526). K.C.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 1228821N; O.B.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 12Y5220N. The confocal microscope was supported by grants to B.H. (FWO.HMZ.2016.0002.01, Hercules project and UGent BOF.BAS.2018.0018.01).

Funding Information:
We thank Petra Van Damme and Sofie De Geyter for their excellent technical assistance. We thank Rocıo Melissa Rivera from the University of Missouri for her careful revision of the manuscript. This work was supported by Ghent University (Grant: Bijzonder Onderozoeksfonds Geconcerteerde Onderzoeksactie 2018000504 [GOA030-18 BOF]) and by the European Union (H2020 Marie Sklodowska-Curie Innovative Training Network: project Biology and Technology of Reproductive Health or REP-BIOTECH 675526). K.C.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 1228821N; O.B.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 12Y5220N. The confocal microscope was supported by grants to B.H. (FWO.HMZ.2016.0002.01, Hercules project and UGent BOF.BAS.2018.0018.01).

Publisher Copyright:
Copyright © 2022 the Author(s).

Keywords

Blastocyst
Extracellular vesicles
Hatching
Mirnas

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Cite this

Pavani, K. C., Meese, T., Pascottini, O. B., Guan, X., Lin, X., Peelman, L., Hamacher, J., Van Nieuwerburgh, F., Deforce, D., Boel, A., Heindryckx, B., Tilleman, K., Van Soom, A., Gadella, B. M., Hendrix, A., & Smits, K. (2022). Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts. Proceedings of the National Academy of Sciences of the United States of America, 119(12), 1-12. Article e2122708119. https://doi.org/10.1073/pnas.2122708119

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title = "Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts",

abstract = "Extracellular vesicles (EVs) and their cargo microRNAs (miRNAs) are important regulators of embryo development to the blastocyst stage and beyond. Before implantation can take place, hatching of blastocysts from their zona pellucida is required. However, underlying mechanisms by which blastocyst formation and hatching are initiated remain largely unknown. Here, we provide evidence that embryonic EVs containing bta-miR-378a-3p play a crucial role in blastocyst hatching, using a bovine model. A customized procedure was used to isolate EV-miRNAs from culture droplets conditioned by individual bovine embryos that either developed to the blastocyst stage or did not (nonblastocyst). RNA sequencing identified 69 differentially expressed miRNAs between EVs derived from blastocyst conditioned medium (CM) and nonblastocyst CM. Among the miRNAs up-regulated in blastocyst CM, we selected bta-miR-378a-3p for further validation by functionality testing on developing in vitro embryos by means of mimics and inhibitors. Supplementing the embryo culture medium with miR-378a-3p mimic significantly improved blastocyst quality, with higher cell numbers and reduced apoptosis, and improved hatching, while the opposite was found after supplementation with miR-378a-3p inhibitor (P < 0.01). Transcriptomic analysis of embryos treated with miR-378 mimic/inhibitor showed differential expression (P < 0.01) of genes associated with embryo development and implantation, including RAP1GAP, ARFGEF2, SLC7A6, CENPA, SP1, LDLR, PYCR1, MYD88, TPP1, and NCOA3. In conclusion, miR-378a-3p is up-regulated in EVs secreted by embryos that develop to the blastocyst stage, and this EV-derived miR-378a-3p increases blastocyst quality and regulates embryo hatching, which is essential for embryo implantation.",

keywords = "Blastocyst, Extracellular vesicles, Hatching, Mirnas",

author = "Pavani, {Krishna Chaitanya} and Tim Meese and Pascottini, {Osvaldo Bogado} and XueFeng Guan and Xiaoyuan Lin and Luc Peelman and Joachim Hamacher and {Van Nieuwerburgh}, Filip and Dieter Deforce and Annekatrien Boel and Bj{\"o}rn Heindryckx and Kelly Tilleman and {Van Soom}, Ann and Gadella, {Bart M} and An Hendrix and Katrien Smits",

note = "Funding Information: ACKNOWLEDGMENTS. We thank Petra Van Damme and Sofie De Geyter for their excellent technical assistance. We thank Ro{\'c}ıo Melissa Rivera from the University of Missouri for her careful revision of the manuscript. This work was supported by Ghent University (Grant: Bijzonder Onderozoeksfonds Geconcerteerde Onderzoeksactie 2018000504 [GOA030-18 BOF]) and by the European Union (H2020 Marie Sklodowska-Curie Innovative Training Network: project Biology and Technology of Reproductive Health or REP-BIOTECH 675526). K.C.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 1228821N; O.B.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 12Y5220N. The confocal microscope was supported by grants to B.H. (FWO.HMZ.2016.0002.01, Hercules project and UGent BOF.BAS.2018.0018.01). Funding Information: We thank Petra Van Damme and Sofie De Geyter for their excellent technical assistance. We thank Rocıo Melissa Rivera from the University of Missouri for her careful revision of the manuscript. This work was supported by Ghent University (Grant: Bijzonder Onderozoeksfonds Geconcerteerde Onderzoeksactie 2018000504 [GOA030-18 BOF]) and by the European Union (H2020 Marie Sklodowska-Curie Innovative Training Network: project Biology and Technology of Reproductive Health or REP-BIOTECH 675526). K.C.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 1228821N; O.B.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 12Y5220N. The confocal microscope was supported by grants to B.H. (FWO.HMZ.2016.0002.01, Hercules project and UGent BOF.BAS.2018.0018.01). Publisher Copyright: Copyright {\textcopyright} 2022 the Author(s).",

year = "2022",

month = mar,

day = "22",

doi = "10.1073/pnas.2122708119",

language = "English",

volume = "119",

pages = "1--12",

journal = "Proceedings of the National Academy of Sciences of the United States of America",

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Pavani, KC, Meese, T, Pascottini, OB, Guan, X, Lin, X, Peelman, L, Hamacher, J, Van Nieuwerburgh, F, Deforce, D, Boel, A, Heindryckx, B, Tilleman, K, Van Soom, A, Gadella, BM, Hendrix, A & Smits, K 2022, 'Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts', Proceedings of the National Academy of Sciences of the United States of America, vol. 119, no. 12, e2122708119, pp. 1-12. https://doi.org/10.1073/pnas.2122708119

Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts. / Pavani, Krishna Chaitanya; Meese, Tim; Pascottini, Osvaldo Bogado et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 119, No. 12, e2122708119, 22.03.2022, p. 1-12.

Research output: Contribution to journal › Article › Academic › peer-review

TY - JOUR

T1 - Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts

AU - Pavani, Krishna Chaitanya

AU - Meese, Tim

AU - Pascottini, Osvaldo Bogado

AU - Guan, XueFeng

AU - Lin, Xiaoyuan

AU - Peelman, Luc

AU - Hamacher, Joachim

AU - Van Nieuwerburgh, Filip

AU - Deforce, Dieter

AU - Boel, Annekatrien

AU - Heindryckx, Björn

AU - Tilleman, Kelly

AU - Van Soom, Ann

AU - Gadella, Bart M

AU - Hendrix, An

AU - Smits, Katrien

N1 - Funding Information: ACKNOWLEDGMENTS. We thank Petra Van Damme and Sofie De Geyter for their excellent technical assistance. We thank Roćıo Melissa Rivera from the University of Missouri for her careful revision of the manuscript. This work was supported by Ghent University (Grant: Bijzonder Onderozoeksfonds Geconcerteerde Onderzoeksactie 2018000504 [GOA030-18 BOF]) and by the European Union (H2020 Marie Sklodowska-Curie Innovative Training Network: project Biology and Technology of Reproductive Health or REP-BIOTECH 675526). K.C.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 1228821N; O.B.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 12Y5220N. The confocal microscope was supported by grants to B.H. (FWO.HMZ.2016.0002.01, Hercules project and UGent BOF.BAS.2018.0018.01). Funding Information: We thank Petra Van Damme and Sofie De Geyter for their excellent technical assistance. We thank Rocıo Melissa Rivera from the University of Missouri for her careful revision of the manuscript. This work was supported by Ghent University (Grant: Bijzonder Onderozoeksfonds Geconcerteerde Onderzoeksactie 2018000504 [GOA030-18 BOF]) and by the European Union (H2020 Marie Sklodowska-Curie Innovative Training Network: project Biology and Technology of Reproductive Health or REP-BIOTECH 675526). K.C.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 1228821N; O.B.P. is supported by Fonds Wetenschappelijk Onderzoek: Grant 12Y5220N. The confocal microscope was supported by grants to B.H. (FWO.HMZ.2016.0002.01, Hercules project and UGent BOF.BAS.2018.0018.01). Publisher Copyright: Copyright © 2022 the Author(s).

PY - 2022/3/22

Y1 - 2022/3/22

N2 - Extracellular vesicles (EVs) and their cargo microRNAs (miRNAs) are important regulators of embryo development to the blastocyst stage and beyond. Before implantation can take place, hatching of blastocysts from their zona pellucida is required. However, underlying mechanisms by which blastocyst formation and hatching are initiated remain largely unknown. Here, we provide evidence that embryonic EVs containing bta-miR-378a-3p play a crucial role in blastocyst hatching, using a bovine model. A customized procedure was used to isolate EV-miRNAs from culture droplets conditioned by individual bovine embryos that either developed to the blastocyst stage or did not (nonblastocyst). RNA sequencing identified 69 differentially expressed miRNAs between EVs derived from blastocyst conditioned medium (CM) and nonblastocyst CM. Among the miRNAs up-regulated in blastocyst CM, we selected bta-miR-378a-3p for further validation by functionality testing on developing in vitro embryos by means of mimics and inhibitors. Supplementing the embryo culture medium with miR-378a-3p mimic significantly improved blastocyst quality, with higher cell numbers and reduced apoptosis, and improved hatching, while the opposite was found after supplementation with miR-378a-3p inhibitor (P < 0.01). Transcriptomic analysis of embryos treated with miR-378 mimic/inhibitor showed differential expression (P < 0.01) of genes associated with embryo development and implantation, including RAP1GAP, ARFGEF2, SLC7A6, CENPA, SP1, LDLR, PYCR1, MYD88, TPP1, and NCOA3. In conclusion, miR-378a-3p is up-regulated in EVs secreted by embryos that develop to the blastocyst stage, and this EV-derived miR-378a-3p increases blastocyst quality and regulates embryo hatching, which is essential for embryo implantation.

AB - Extracellular vesicles (EVs) and their cargo microRNAs (miRNAs) are important regulators of embryo development to the blastocyst stage and beyond. Before implantation can take place, hatching of blastocysts from their zona pellucida is required. However, underlying mechanisms by which blastocyst formation and hatching are initiated remain largely unknown. Here, we provide evidence that embryonic EVs containing bta-miR-378a-3p play a crucial role in blastocyst hatching, using a bovine model. A customized procedure was used to isolate EV-miRNAs from culture droplets conditioned by individual bovine embryos that either developed to the blastocyst stage or did not (nonblastocyst). RNA sequencing identified 69 differentially expressed miRNAs between EVs derived from blastocyst conditioned medium (CM) and nonblastocyst CM. Among the miRNAs up-regulated in blastocyst CM, we selected bta-miR-378a-3p for further validation by functionality testing on developing in vitro embryos by means of mimics and inhibitors. Supplementing the embryo culture medium with miR-378a-3p mimic significantly improved blastocyst quality, with higher cell numbers and reduced apoptosis, and improved hatching, while the opposite was found after supplementation with miR-378a-3p inhibitor (P < 0.01). Transcriptomic analysis of embryos treated with miR-378 mimic/inhibitor showed differential expression (P < 0.01) of genes associated with embryo development and implantation, including RAP1GAP, ARFGEF2, SLC7A6, CENPA, SP1, LDLR, PYCR1, MYD88, TPP1, and NCOA3. In conclusion, miR-378a-3p is up-regulated in EVs secreted by embryos that develop to the blastocyst stage, and this EV-derived miR-378a-3p increases blastocyst quality and regulates embryo hatching, which is essential for embryo implantation.

KW - Blastocyst

KW - Extracellular vesicles

KW - Hatching

KW - Mirnas

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U2 - 10.1073/pnas.2122708119

DO - 10.1073/pnas.2122708119

M3 - Article

C2 - 35298333

SN - 0027-8424

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JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 12

M1 - e2122708119

ER -

Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts

Abstract

Bibliographical note

Keywords

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