Derivation of developmentally competent oocytes by the culture of preantral follicles retrieved from adult ovaries: maturation, blastocyst formation, and embryonic stem cell transformation  In Wook Kim, M.S., Seung Pyo Gong, B.Sc., Cho Rong Yoo, B.Sc., Jun Hee Choi, B.Sc., Dae Yong Kim, Ph.D., D.V.M., Jeong Mook Lim, Ph.D., D.V.M.  Fertility and Sterility  Volume 92, Issue 5, Pages 1716-1724 (November 2009) DOI: 10.1016/j.fertnstert.2008.08.084 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Comparison of the number of preantral follicles retrieved from 2-week-old prepubertal and 8-week-old adult B6CBAF1 mice. Preantral follicles at the primary, early secondary, and late secondary stages were mechanically collected from the donor ovaries. Overall, the retrieval number (A) was larger in the prepubertal than in the adult group. In the case of the primary (B) and early secondary (C) stages, the follicle number was larger in the prepubertal than in the adult group, whereas more late secondary follicles (D) were retrieved from the adults than from the prepubertal mice. The model effects of the treatments (indicated by the P value) were .0567, .0211, .0623, and .0927 for the total number of preantral follicles retrieved and the numbers of primary, early secondary, and late secondary follicles retrieved, respectively. Fertility and Sterility 2009 92, 1716-1724DOI: (10.1016/j.fertnstert.2008.08.084) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The establishment of ESCs derived from blastocysts of various origins (A–E) or parthenogenetic activation (F). Evenly cleaved blastomeres with clearly defined cytoplasms were visible in the embryos. Two-cell (B) and four-cell embryos (B), morula (C), and expanded (D) and hatching blastocysts (E) derived from IVF. (F) A blastocyst derived from parthenogenetic activation. Bars = 50 μm. Fertility and Sterility 2009 92, 1716-1724DOI: (10.1016/j.fertnstert.2008.08.084) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Characterization of colony-forming cells derived from the subculture of blastocyst ICM cells. The blastocysts were generated by the in vitro insemination of intrafollicular oocytes matured in in vitro–cultured, preantral follicles retrieved from adult (8-week-old) B6CBAF1 mice. (A) Morphology of colony-forming cells subpassaged 10 times (A1) and staining of the colony-forming cells with alkaline phosphatase (A2) and anti-SSEA-1 (A3), anti-SSEA-3 (A4), anti-SSEA-4 (A5), anti-integrin α6 (A6), and anti-integrin β1 (A7) antibodies. The colony-forming cells were positive for SSEA-1, integrin α6, integrin β1, and alkaline phosphatase, whereas they were negative for SSEA-3 and SSEA-4. Bars = 50 μm. (B) Pluripotent cell-specific gene expression in the established colony-forming cells was monitored by reverse transcriptase polymerase chain reaction; the colony-forming cells expressed ESC-specific genes. (C) Sex-specific Xist (X-chromosome specific) and Zfy1 (Y-chromosome specific) expression was detected in the established cells. Fertility and Sterility 2009 92, 1716-1724DOI: (10.1016/j.fertnstert.2008.08.084) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Spontaneous differentiation of colony-forming cells in vitro and in vivo. Colony-forming cells were derived from the subculture of ICM cells from blastocysts generated by the in vitro insemination of oocytes matured in in vitro–cultured, adult preantral follicles. (A) In vitro differentiation of colony-forming cells into EBs by culturing them in LIF-free culture medium. (A1) Embryoid body observed on day 4 of culture. Immunocytochemistry was used to detect the differentiation of the three germ layers using the markers S-100 (A2; ectodermal), smooth muscle actin (A3; mesodermal), nestin (A4; ectodermal), desmin (A5; mesodermal), α-fetoprotein (A6; endodermal), and troma-1 (A7; endodermal). Bars = 100 μm. (B) In vivo differentiation of colony-forming cells by subcutaneous transplantation into non-obese diabetic (NOD) severe combined immunodeficient mice. Staining of the teratoma with hematoxylin and eosin revealed the presence of (B1) squamous epithelia with keratin production, (B2) adipose and muscular tissues, (B3) osseum, (B4) cartilage, (B5) glandular epithelia, and (B6) ciliated epithelia. Bars = 50 μm. Fertility and Sterility 2009 92, 1716-1724DOI: (10.1016/j.fertnstert.2008.08.084) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions