[Show abstract][Hide abstract] ABSTRACT: The efficiency of in vitro culture systems for premeiotic female germ cell is still low, mostly because our incomplete understanding of the mechanisms controlling oogenesis and the obvious difficulties in reproducing the complex in vivo environment of such process under in vitro conditions. Here we explored the possibility of recovering the developmental potential of mouse oocytes generated in vitro from premeiotic germ cells by transplantation under kidney capsule of adult animals. To this aim, mouse embryonic ovaries of 12.5 days post-coitum cultured in vitro in a serum-free medium for 7 or 14 days, were transplanted beneath the kidney capsule of immunodeficient mice and analyzed after 21 (7+21 group) or 14 days (14+14 group). Cultured ovaries before transplantation showed delayed oocyte meiotic progression and follicle development. Interestingly, grafted ovaries of both groups, especially those of the 7+21 group, seemed able to restore the reproductive cycle of recipients. While almost complete absence of primordial follicles was observed in grafted ovaries, oocytes from these ovaries showed transcript levels of genes associated to oocyte maturation similar to control. Moreover, the developmental stage of follicles and oocytes of the 7+21 group ovaries were comparable to that of 21 days post partum in vivo ovaries, whereas significant developmental delay were found in the 14+14 group ovaries. Nevertheless, oocytes retrieved from transplanted ovaries of both groups matured (around 80%) and were fertilized in vitro (around 20% - 45%). 2-cell embryos from the fertilized oocytes developed to hatching blastocysts (about 50%) or gave rise to healthy live offspring (from 6% to 10%) when transplanted in a host mother. In conclusion, our results indicate that premeiotic female germ cells cultured in vitro up to primordial/primary follicle stages preserve their capability to complete oogenesis and can be fertilized and generate live pups after transplantation into a suitable in vivo environment.
Stem cells and development 09/2012; 22(4). DOI:10.1089/scd.2012.0436 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulation of germ-cell-specific transcription is essential for the differentiation and other physiological processes of germ cells. Germ-cell-specific transcription factors, Sohlh2 and Figla, play key roles in gametogenesis. To elucidate whether an epigenetic mechanism is involved in the controlled expression of Sohlh2 and Figlα, we examined the dynamics of DNA methylation at two gene loci. Results showed changes in methylation patterns in the promoter regions of both genes during the period of germ cell differentiation, while the methylation patterns in first exons and first introns (near the transcription initiation sites) remained constant. The methylation reprogramming at the cytosine-phosphate-guanine (CpG) locus in the Figlα promoters (P1: -812 to -568 bp and P2: -692 to -438 bp) and in the Sohlh2 promoter (sohlh2-P: -202 to 173 bp) was found to correlate with the expression of Figlα and Sohlh2 transcripts, respectively. Data therefore suggested that a dynamic DNA CpG methylation in the Sohlh2 and Figlα promoters, but not in the intron and exon sequences, is linked to the regulation of gene expressions, even though CpG islands are also present in their introns or exons.
Molecular Human Reproduction 03/2011; 17(9):550-61. DOI:10.1093/molehr/gar017 · 3.75 Impact Factor