Shi Ying Jin, Ph. D. , Lei Lei, Ph. D. , Ariella Shikanov, Ph. D

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A novel two-step strategy for in vitro culture of early-stage ovarian follicles in the mouse Shi Ying Jin, Ph.D., Lei Lei, Ph.D., Ariella Shikanov, Ph.D., Lonnie D. Shea, Ph.D., Teresa K. Woodruff, Ph.D. Fertility and Sterility Volume 93, Issue 8, Pages 2633-2639 (May 2010) DOI: 10.1016/j.fertnstert.2009.10.027 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Representative photomicrographs of H&E-stained paraffin sections of whole ovaries before and after culture. (A) Control, uncultured 8-day-old mouse ovary. (B) Eight-day-old mouse ovary after 4 days of organ culture. (C) H&E staining of uncultured 8-day-old mouse ovary, which contains mainly primordial follicles with a few primary and secondary follicles. (D) H&E staining of 8-day-old mouse ovary after 4 days of organ culture. More secondary follicles were observed. (E) H&E staining of uncultured 12-day-old mouse ovary. (F) Follicle distribution in mouse ovaries before and after 4-day organ culture. 0, primordial follicle; 1, primary follicle; 2, secondary follicle; scale bars in A and B = 150 μM; other scale bars = 200 μM; letters indicate a statistically significant difference between groups (P < 0.05). Fertility and Sterility 2010 93, 2633-2639DOI: (10.1016/j.fertnstert.2009.10.027) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Development and differentiation of representative secondary follicles cultured in vitro. (A) Secondary follicles with centrally located immature oocytes isolated from cultured ovarian tissues. (B, C) Follicles maintained their 3D structure with proliferation of granulosa cells, antrum formation (white arrowhead), and development of theca cell layers (black arrowhead) after 12 days of culture in 0.25% alginate or FA. (D) Follicle diameter in both culture systems increased significantly during the culture period. (E) Oocyte size increased significantly over the culture period. Statistically significant differences were observed between groups as indicated with different letters (P < 0.05). Scale bar =100 μM. (F, G) Average values of E2 (F) and P (G) secretion were measured in conditioned culture media from secondary follicle cultures. Fertility and Sterility 2010 93, 2633-2639DOI: (10.1016/j.fertnstert.2009.10.027) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Meiotic and fertilization competence of oocytes from follicles cultured for 12 days in alginate (A–D) or FA (E–H) were assessed by in vitro maturation (IVM) and in vitro fertilization (IVF). CEOs isolated from antral follicles retrieved from alginate (A) or FA (E) culture systems were induced with hCG for 18 hours in vitro. (B, F) In both environments, cumulus cells around the oocytes expanded. (C, G) Oocytes resumed meiosis and extruded the first polar body (arrowhead). (D, H) Two-cell embryos were obtained by IVF of MII oocytes. Scale bar = 50 μM. Fertility and Sterility 2010 93, 2633-2639DOI: (10.1016/j.fertnstert.2009.10.027) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions