Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-β-Cyclodextrin Treatment

Vitrification of oocytes is crucial for embryo biotechnologies, germplasm cryopreservation of endangered and excellent female animals, and the fertility of humans. However, vitrification significantly impairs the fertilization ability of oocytes, which significantly limits its widely used applicatio...

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Main Authors: Xi Xu, Tong Hao, Emma Komba, Baigao Yang, Haisheng Hao, Weihua Du, Huabin Zhu, Hang Zhang, Xueming Zhao
Format: Article
Language:English
Published: MDPI AG 2022-12-01
Series:International Journal of Molecular Sciences
Subjects:
Online Access:https://www.mdpi.com/1422-0067/24/1/590
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author Xi Xu
Tong Hao
Emma Komba
Baigao Yang
Haisheng Hao
Weihua Du
Huabin Zhu
Hang Zhang
Xueming Zhao
author_facet Xi Xu
Tong Hao
Emma Komba
Baigao Yang
Haisheng Hao
Weihua Du
Huabin Zhu
Hang Zhang
Xueming Zhao
author_sort Xi Xu
collection DOAJ
description Vitrification of oocytes is crucial for embryo biotechnologies, germplasm cryopreservation of endangered and excellent female animals, and the fertility of humans. However, vitrification significantly impairs the fertilization ability of oocytes, which significantly limits its widely used application. JUNO protein, a receptor for Izumo1, is involved in sperm-oocyte fusion and is an indispensable protein for mammalian fertilization, and its abundance is susceptible to vitrification. However, it is still unclear how vitrification reduces the fertilization capacity of bovine oocytes by affecting JUNO protein. This study was designed to investigate the effect of vitrification on the abundance and post-translational modifications of JUNO protein in bovine oocytes. Our results showed that vitrification did not alter the amino acid sequence of JUNO protein in bovine oocytes. Furthermore, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis results showed that vitrification significantly reduced the number and changed the location of disulfide bonds, and increased the number of both phosphorylation and glycosylation sites of JUNO protein in bovine oocytes. Finally, the fertilization capacity and development ability of vitrified oocytes treated with 200 pg JUNO mRNA microinjection and cholesterol-loaded methyl-β-cyclodextrin (CLC/MβCD) were similar to those of fresh oocytes. In conclusion, our results showed that vitrification of bovine oocytes did not alter the protein sequence of JUNO, but induced post-translational modifications and changed protein abundance. Moreover, the fertilization and development ability of vitrified bovine oocytes were improved by the combination treatment of JUNO mRNA microinjection and CLC/MβCD.
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spelling doaj.art-ec05e4321db5420d9031069fba5c09b02023-11-16T15:35:35ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-12-0124159010.3390/ijms24010590Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-β-Cyclodextrin TreatmentXi Xu0Tong Hao1Emma Komba2Baigao Yang3Haisheng Hao4Weihua Du5Huabin Zhu6Hang Zhang7Xueming Zhao8Institute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, ChinaInstitute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, ChinaInstitute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, ChinaInstitute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, ChinaInstitute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, ChinaInstitute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, ChinaInstitute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, ChinaInstitute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, ChinaInstitute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, ChinaVitrification of oocytes is crucial for embryo biotechnologies, germplasm cryopreservation of endangered and excellent female animals, and the fertility of humans. However, vitrification significantly impairs the fertilization ability of oocytes, which significantly limits its widely used application. JUNO protein, a receptor for Izumo1, is involved in sperm-oocyte fusion and is an indispensable protein for mammalian fertilization, and its abundance is susceptible to vitrification. However, it is still unclear how vitrification reduces the fertilization capacity of bovine oocytes by affecting JUNO protein. This study was designed to investigate the effect of vitrification on the abundance and post-translational modifications of JUNO protein in bovine oocytes. Our results showed that vitrification did not alter the amino acid sequence of JUNO protein in bovine oocytes. Furthermore, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis results showed that vitrification significantly reduced the number and changed the location of disulfide bonds, and increased the number of both phosphorylation and glycosylation sites of JUNO protein in bovine oocytes. Finally, the fertilization capacity and development ability of vitrified oocytes treated with 200 pg JUNO mRNA microinjection and cholesterol-loaded methyl-β-cyclodextrin (CLC/MβCD) were similar to those of fresh oocytes. In conclusion, our results showed that vitrification of bovine oocytes did not alter the protein sequence of JUNO, but induced post-translational modifications and changed protein abundance. Moreover, the fertilization and development ability of vitrified bovine oocytes were improved by the combination treatment of JUNO mRNA microinjection and CLC/MβCD.https://www.mdpi.com/1422-0067/24/1/590JUNOoocytesvitrificationbovinemicroinjection
spellingShingle Xi Xu
Tong Hao
Emma Komba
Baigao Yang
Haisheng Hao
Weihua Du
Huabin Zhu
Hang Zhang
Xueming Zhao
Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-β-Cyclodextrin Treatment
International Journal of Molecular Sciences
JUNO
oocytes
vitrification
bovine
microinjection
title Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-β-Cyclodextrin Treatment
title_full Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-β-Cyclodextrin Treatment
title_fullStr Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-β-Cyclodextrin Treatment
title_full_unstemmed Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-β-Cyclodextrin Treatment
title_short Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-β-Cyclodextrin Treatment
title_sort improvement of fertilization capacity and developmental ability of vitrified bovine oocytes by juno mrna microinjection and cholesterol loaded methyl β cyclodextrin treatment
topic JUNO
oocytes
vitrification
bovine
microinjection
url https://www.mdpi.com/1422-0067/24/1/590
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