Novel dynamic culture system to support initiation of primordial follicle growth in prepubertal mouse ovaries  Katharina Winkler-Crepaz, M.D., Verena Nederegger, M.D., Sarrah Ayuandari, M.D., Doris Rosenfellner, B.Sc., Ioannis Zervomanolakis, M.D., Susanne Hofer, Ph.D., M.Sc., Ludwig Wildt, M.D., Ph.D., Stephanie C. Ziehr, M.D.  Fertility and Sterility  Volume 102, Issue 3, Pages 864-870.e2 (September 2014) DOI: 10.1016/j.fertnstert.2014.05.038 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Experimental design. (A) Ovaries were isolated from 8-day-old mice and subsequently cultured in a dynamic or static culture system. Uncultured ovaries from 8- and 12-day-old animals served as baseline or in vivo control, respectively. (B) To generate dynamic in vitro culture conditions a peristaltic pump continuously flushes fresh medium through the culture chambers, leading to a constant medium exchange over 24 hours. The total in vitro culture period was 4 days. Fertility and Sterility 2014 102, 864-870.e2DOI: (10.1016/j.fertnstert.2014.05.038) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Percentage of secondary follicles present in the ovarian tissue before culture (Baseline, day 8), after dynamic culture or static culture for 4 days, and on day 12 in vivo (In vivo control, day 12). The percentage of secondary follicles was significantly higher after dynamic and static culture compared with baseline controls (∗P<.001). After dynamic culture the percentage of secondary follicles was equivalent compared with in vivo controls (P=ns), whereas after static culture the percentage of secondary follicles was significantly higher than in vivo (∗∗P=.038). Asterisks indicate significantly different results between treatments according to nonparametric Mann-Whitney U test (P<.05). Box represents the median and the upper and lower quartiles. Whiskers illustrate the expected data range. Small circles display normal outliers. Fertility and Sterility 2014 102, 864-870.e2DOI: (10.1016/j.fertnstert.2014.05.038) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Histologic sections of prepubertal ovaries isolated from the baseline control (A), after 4 days in dynamic culture (B), from the in vivo control (C), and after 4 days in static culture (D). Asterisks display groups of primordial follicles, and arrows display early secondary follicles. After dynamic as well as after static culture the viability of the central area of the tissue appears to be impaired, showing degenerated secondary follicles (arrowheads), whereas the outer part remains intact. This might be due to the serum-free culture conditions. Hematoxylin and eosin staining. Fertility and Sterility 2014 102, 864-870.e2DOI: (10.1016/j.fertnstert.2014.05.038) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Immunofluorescent viability staining. Isolated primary and secondary follicles are stained with calcein acetoxymethyl ester (Calcein AM, green) and ethidium-homodimer (EthD-1, red). Completely viable follicles (A) are predominantly stained with Calcein AM (green) with only few degenerated cells (EthD-1, red) on their surface. Intermediate viable follicles are subdivided into follicles consisting of >50% viable cells (B) or <50% viable cells (C). Completely nonviable follicles are only stained with EthD-1 without showing any green fluorescence (D). Fertility and Sterility 2014 102, 864-870.e2DOI: (10.1016/j.fertnstert.2014.05.038) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions