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Growth of Mouse Oocytes to Maturity from Premeiotic Germ Cells In Vitro (Mature Oocytes from Premeiotic Germ Cells In Vitro)

In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12–14 day- old mice to reach meiotic ma... Full description

Journal Title: PLoS ONE 2012, Vol.7(7), p.e41771
Main Author: Zhang, Zhi-Peng
Other Authors: Liang, Gui-Jin , Zhang, Xi-Feng , Zhang, Guo-Liang , Chao, Hu-He , Li, Lan , Sun, Xiao-Feng , Min, Ling-Jiang , Pan, Qing-Jie , Shi, Qing-Hua , Sun, Qing-Yuan , De Felici, Massimo , Shen, Wei
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: E-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0041771
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recordid: plos10.1371/journal.pone.0041771
title: Growth of Mouse Oocytes to Maturity from Premeiotic Germ Cells In Vitro (Mature Oocytes from Premeiotic Germ Cells In Vitro)
format: Article
creator:
  • Zhang, Zhi-Peng
  • Liang, Gui-Jin
  • Zhang, Xi-Feng
  • Zhang, Guo-Liang
  • Chao, Hu-He
  • Li, Lan
  • Sun, Xiao-Feng
  • Min, Ling-Jiang
  • Pan, Qing-Jie
  • Shi, Qing-Hua
  • Sun, Qing-Yuan
  • De Felici, Massimo
  • Shen, Wei
subjects:
  • Research Article
  • Biology
  • Physiology
  • Cell Biology
  • Developmental Biology
ispartof: PLoS ONE, 2012, Vol.7(7), p.e41771
description: In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12–14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6–7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16–18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.
language: eng
source:
identifier: E-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0041771
fulltext: fulltext
issn:
  • 1932-6203
  • 19326203
url: Link


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titleGrowth of Mouse Oocytes to Maturity from Premeiotic Germ Cells In Vitro (Mature Oocytes from Premeiotic Germ Cells In Vitro)
creatorZhang, Zhi-Peng ; Liang, Gui-Jin ; Zhang, Xi-Feng ; Zhang, Guo-Liang ; Chao, Hu-He ; Li, Lan ; Sun, Xiao-Feng ; Min, Ling-Jiang ; Pan, Qing-Jie ; Shi, Qing-Hua ; Sun, Qing-Yuan ; De Felici, Massimo ; Shen, Wei
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ispartofPLoS ONE, 2012, Vol.7(7), p.e41771
identifierE-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0041771
subjectResearch Article ; Biology ; Physiology ; Cell Biology ; Developmental Biology
descriptionIn the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12–14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6–7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16–18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.
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titleGrowth of Mouse Oocytes to Maturity from Premeiotic Germ Cells In Vitro (Mature Oocytes from Premeiotic Germ Cells In Vitro)
descriptionIn the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12–14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6–7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16–18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.
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abstractIn the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12–14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6–7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16–18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage.
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