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.
Highlights
In mammalian females, oocytes originate from embryonic precursors termed the primordial germ cells (PGCs) [1,2,3]
In order to detect the in vitro meiotic maturation ability of the oocytes cultured under different conditions described above, germinal vesicle breakdown (GVBD) and polar body emission were evaluated (Fig. 1B–D)
In the Denuded oocytes (DO)+preantral granulosa cell (PAGC)+Activin A (ActA) group, the percents of GVBD and metaphase II (MII) oocytes were 27.6061.20% and 24.5061.09% (n = 1120), respectively, significantly higher than those in the DO+ PAGCs group (20.561.01% and 20.560.95% (n = 1233), respectively) and in the groups without PAGCs (P,0.05 or P,0.01). In these latter groups the capability of oocytes to undergo GVBD and reach MII was not influenced by the presence of ActA (Fig. 1B–C)
Summary
Oocytes originate from embryonic precursors termed the primordial germ cells (PGCs) [1,2,3]. PGC precursors arise in the proximal epiblast at around 6.25 days post coitum (dpc), and move into the extraembryonic mesoderm at the base of the allantois where by 7.25 dpc they are specified as PGCs [4,5]. Most oocytes have entered meiosis by 14.5 dpc, and become arrested at the diplotene stage of the first meiosis around birth (19.5 dpc -1-2 days post partum, dpp) [7,10]. At this stage, oocytes are individually surrounded by pregranulosa cells to form primordial follicles [2,11,12]
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