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Growth of mouse oocytes to maturity from premeiotic germ cells in vitro.

Zhang ZP, Liang GJ, Zhang XF, Zhang GL, Chao HH, Li L, Sun XF, Min LJ, Pan QJ, Shi QH, Sun QY, De Felici M, Shen W - PLoS ONE (2012)

Bottom Line: Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established.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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, Qingdao Agricultural University, Qingdao, China.

ABSTRACT
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.

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Characterization of the maturation status of the oocytes generated in vitro from 12.5 dpc embryonic ovaries.(A) An example of GVDB and MII oocytes observed under Nikon optics (left) and after DNA staining with Hoechst (right). (B, C) Percents of GVDB and MII oocytes generated within the ovarian explants after 28 days of culture in the presence of ActA and cocultured onto PAGCs for 7 days in the presence or absence of ActA. (D) MI and MII spindle morphology in oocytes. Oocytes generated in vitro from premeiotic germ cells (In vitro+PAGCs+ActA) show normal MI spindle, MII spindle and chromosome assembly as compared to those of control oocytes isolated from ovaries of 21–22-day-old mice (In vivo control). In contrast, oocyte generated in vitro as described above but cocultured onto PAGCs in the absence of ActA (In vitro+PAGCs-ActA) show anomalous MI spindle and chromosome misalignment. (E) Phosphorylation (activation) of ERK1/2 in oocytes as detected by immunoblotting. Expression of ERK2 protein was analyzed in oocytes obtained from ovarian explants at 14, 21 and 28 days of culture + or – ActA and from 7, 14 and 21 dpp ovaries; only 28 day oocytes in vitro and 21 dpp oocytes in vivo showed detectable ERK2 expression. Phosphorylation of ERK1/2 (pERK1/ERK2) was analyzed in GV, GVDB and MII oocytes generated in vitro (28 days within ovary explants+ActA and 7 days onto PAGCs+ActA) or isolated from ovaries of adult females (21 d); only in vitro and in vivo MII oocytes showed pERK1/ERK2. *P<0.05; **P<0.01.
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pone-0041771-g005: Characterization of the maturation status of the oocytes generated in vitro from 12.5 dpc embryonic ovaries.(A) An example of GVDB and MII oocytes observed under Nikon optics (left) and after DNA staining with Hoechst (right). (B, C) Percents of GVDB and MII oocytes generated within the ovarian explants after 28 days of culture in the presence of ActA and cocultured onto PAGCs for 7 days in the presence or absence of ActA. (D) MI and MII spindle morphology in oocytes. Oocytes generated in vitro from premeiotic germ cells (In vitro+PAGCs+ActA) show normal MI spindle, MII spindle and chromosome assembly as compared to those of control oocytes isolated from ovaries of 21–22-day-old mice (In vivo control). In contrast, oocyte generated in vitro as described above but cocultured onto PAGCs in the absence of ActA (In vitro+PAGCs-ActA) show anomalous MI spindle and chromosome misalignment. (E) Phosphorylation (activation) of ERK1/2 in oocytes as detected by immunoblotting. Expression of ERK2 protein was analyzed in oocytes obtained from ovarian explants at 14, 21 and 28 days of culture + or – ActA and from 7, 14 and 21 dpp ovaries; only 28 day oocytes in vitro and 21 dpp oocytes in vivo showed detectable ERK2 expression. Phosphorylation of ERK1/2 (pERK1/ERK2) was analyzed in GV, GVDB and MII oocytes generated in vitro (28 days within ovary explants+ActA and 7 days onto PAGCs+ActA) or isolated from ovaries of adult females (21 d); only in vitro and in vivo MII oocytes showed pERK1/ERK2. *P<0.05; **P<0.01.

Mentions: In order to promote the meiotic maturation, oocytes with a diameter >50 µm after culture in the presence of ActA for 28 days were isolated and further cocultured with PAGCs for 6–7 days in the presence or absence of ActA (Fig. S5). During this period, no significant increase in the oocyte diameters was observed (data no shown). At the end of this period, the capability of the oocytes to undergo GVBD and reach MII in IVM was evaluated. The results showed that 95/581 (16.35%) of the oocytes cocultured onto PAGCs with ActA underwent GVBD, amont which 17/95 (17.89%) reached MII stages. In contrast, only 15/183 (8.20%) of the oocytes cocultured onto PAGCs without ActA underwent GVBD and none of them (0/15) were able to reach MII (P<0.01) (Fig. 5A–C). Oocytes cultured with PAGCs in the presence of ActA showed normal MI and MII spindles. In contrast, although part of the oocytes cocultured with PAGCs in the absence of ActA were able to undergo GVBD, they failed to form typical spindle structure and the chromosomes were misaligned (Fig. 5D).


Growth of mouse oocytes to maturity from premeiotic germ cells in vitro.

Zhang ZP, Liang GJ, Zhang XF, Zhang GL, Chao HH, Li L, Sun XF, Min LJ, Pan QJ, Shi QH, Sun QY, De Felici M, Shen W - PLoS ONE (2012)

Characterization of the maturation status of the oocytes generated in vitro from 12.5 dpc embryonic ovaries.(A) An example of GVDB and MII oocytes observed under Nikon optics (left) and after DNA staining with Hoechst (right). (B, C) Percents of GVDB and MII oocytes generated within the ovarian explants after 28 days of culture in the presence of ActA and cocultured onto PAGCs for 7 days in the presence or absence of ActA. (D) MI and MII spindle morphology in oocytes. Oocytes generated in vitro from premeiotic germ cells (In vitro+PAGCs+ActA) show normal MI spindle, MII spindle and chromosome assembly as compared to those of control oocytes isolated from ovaries of 21–22-day-old mice (In vivo control). In contrast, oocyte generated in vitro as described above but cocultured onto PAGCs in the absence of ActA (In vitro+PAGCs-ActA) show anomalous MI spindle and chromosome misalignment. (E) Phosphorylation (activation) of ERK1/2 in oocytes as detected by immunoblotting. Expression of ERK2 protein was analyzed in oocytes obtained from ovarian explants at 14, 21 and 28 days of culture + or – ActA and from 7, 14 and 21 dpp ovaries; only 28 day oocytes in vitro and 21 dpp oocytes in vivo showed detectable ERK2 expression. Phosphorylation of ERK1/2 (pERK1/ERK2) was analyzed in GV, GVDB and MII oocytes generated in vitro (28 days within ovary explants+ActA and 7 days onto PAGCs+ActA) or isolated from ovaries of adult females (21 d); only in vitro and in vivo MII oocytes showed pERK1/ERK2. *P<0.05; **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3404094&req=5

pone-0041771-g005: Characterization of the maturation status of the oocytes generated in vitro from 12.5 dpc embryonic ovaries.(A) An example of GVDB and MII oocytes observed under Nikon optics (left) and after DNA staining with Hoechst (right). (B, C) Percents of GVDB and MII oocytes generated within the ovarian explants after 28 days of culture in the presence of ActA and cocultured onto PAGCs for 7 days in the presence or absence of ActA. (D) MI and MII spindle morphology in oocytes. Oocytes generated in vitro from premeiotic germ cells (In vitro+PAGCs+ActA) show normal MI spindle, MII spindle and chromosome assembly as compared to those of control oocytes isolated from ovaries of 21–22-day-old mice (In vivo control). In contrast, oocyte generated in vitro as described above but cocultured onto PAGCs in the absence of ActA (In vitro+PAGCs-ActA) show anomalous MI spindle and chromosome misalignment. (E) Phosphorylation (activation) of ERK1/2 in oocytes as detected by immunoblotting. Expression of ERK2 protein was analyzed in oocytes obtained from ovarian explants at 14, 21 and 28 days of culture + or – ActA and from 7, 14 and 21 dpp ovaries; only 28 day oocytes in vitro and 21 dpp oocytes in vivo showed detectable ERK2 expression. Phosphorylation of ERK1/2 (pERK1/ERK2) was analyzed in GV, GVDB and MII oocytes generated in vitro (28 days within ovary explants+ActA and 7 days onto PAGCs+ActA) or isolated from ovaries of adult females (21 d); only in vitro and in vivo MII oocytes showed pERK1/ERK2. *P<0.05; **P<0.01.
Mentions: In order to promote the meiotic maturation, oocytes with a diameter >50 µm after culture in the presence of ActA for 28 days were isolated and further cocultured with PAGCs for 6–7 days in the presence or absence of ActA (Fig. S5). During this period, no significant increase in the oocyte diameters was observed (data no shown). At the end of this period, the capability of the oocytes to undergo GVBD and reach MII in IVM was evaluated. The results showed that 95/581 (16.35%) of the oocytes cocultured onto PAGCs with ActA underwent GVBD, amont which 17/95 (17.89%) reached MII stages. In contrast, only 15/183 (8.20%) of the oocytes cocultured onto PAGCs without ActA underwent GVBD and none of them (0/15) were able to reach MII (P<0.01) (Fig. 5A–C). Oocytes cultured with PAGCs in the presence of ActA showed normal MI and MII spindles. In contrast, although part of the oocytes cocultured with PAGCs in the absence of ActA were able to undergo GVBD, they failed to form typical spindle structure and the chromosomes were misaligned (Fig. 5D).

Bottom Line: Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established.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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, Qingdao Agricultural University, Qingdao, China.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus