Kit ligand decreases the incidence of apoptosis in cultured vitrified whole mouse ovaries  Shabnam Abdi, Mojdeh Salehnia, Saman Hosseinkhani  Reproductive BioMedicine Online  Volume 30, Issue 5, Pages 493-503 (May 2015) DOI: 10.1016/j.rbmo.2015.01.009 Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Photomicrographs of mouse ovary sections stained with haematoxylin and eosin: non-vitrified- non-cultured whole ovaries (low magnification) (A); high magnification of one preantral follicle and several primordial follicles from the same group (B); vitrified-non-cultured whole ovaries (low magnification) (C) and high magnification of the same group (D); non-vitrified -cultured ovary in the absence of Kit ligand (E,F); non-vitrified cultured ovary in the presence of Kit ligand (G,H); vitrified cultured ovary in the absence of Kit ligand (I,J) and vitrified cultured ovary in the presence of Kit ligand (K,L). KL, Kit ligand. Reproductive BioMedicine Online 2015 30, 493-503DOI: (10.1016/j.rbmo.2015.01.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Photomicrographs of mouse ovaries viewed under an inverted microscope: non-vitrified-non-cultured (A); non-vitrified cultured in the absence of Kit ligand (B); non-vitrified cultured in the presence of Kit ligand (C); vitrified-non-cultured (D); vitrified cultured in the absence of Kit ligand (E); vitrified cultured in the presence of Kit ligand (F). In the same magnification on day 7, the area of non-vitrified cultured ovary in the presence of Kit ligand (C) is larger than group not treated with Kit ligand (B). The follicles with centrally located oocyte are prominent at the cortical part of the tissue (arrow). KL, kit ligand. A comparison of the mean surface area of mouse ovary during the culture period (G). a: Significantly different compared with the respective group not treated with Kit ligand (P < 0.02). b: Significantly different compared with the respective non-vitrified group (P < 0.001). Reproductive BioMedicine Online 2015 30, 493-503DOI: (10.1016/j.rbmo.2015.01.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 The level of 17-beta oestradiol (A), progesterone (B) and dehydroepiandrosterone (DHEA) (C) in collected media of mouse ovaries during culture in all groups. a: Significantly different compared with the respective group not treated with Kit ligand (P < 0.001). b: Significantly different compared with the respective non-vitrified group (P < 0.001). *In all groups of study, the level of hormones (oestradiol, progesterone and DHEA) is significantly different on day 7 compared with day 3 of culture (P < 0.001). Reproductive BioMedicine Online 2015 30, 493-503DOI: (10.1016/j.rbmo.2015.01.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Fluorescence microscopy images of mouse ovaries subjected to TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end-labelling (TUNEL) staining, the second and fourth columns of rows one to three show their dark field images. In non-vitrified, non-cultured (A, B) and vitrified, non-cultured group (C,D) no TUNEL-positive labelling cells were seen in the oocytes and follicles of both groups before culture. Some TUNEL-positive cells were observed within the stroma and follicular cells of non-vitrified cultured tissue (E, F) and these were more prominent in vitrified cultured ovaries (G, H). The representative figures of cultured ovarian tissues treated with Kit ligand in non-vitrified (I,K) and in the vitrified group (L,M). High magnification of image non-vitrified cultured Kit ligand− tissue group (N) and vitrified cultured kit ligand− ovaries group (O). Oocytes did not show TUNEL positive signals in both the vitrified and non-vitrified groups (yellow arrow), whereas TUNEL signals were observed mainly in follicular and stromal cells (white arrow). Induced thymic tissue used as a positive control showed TUNEL positive reaction as green staining (P) and the negative control did not show any positive signals (Q). Reproductive BioMedicine Online 2015 30, 493-503DOI: (10.1016/j.rbmo.2015.01.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Analysis of total cellular DNA in mouse ovaries using gel electrophoresis. No DNA laddering pattern was seen in both vitrified-non-cultured (V) and non-vitrified-non-cultured (NV) ovaries (A). The DNA laddering pattern, indicative of apoptosis, is clearly present in the control induced thymic tissue (T) and in the 21-day-old mouse ovary (OV). As shown in panel B, no DNA laddering pattern is seen in non-vitrified cultured ovaries in the absence of Kit ligand (NVK−) or in non-vitrified cultured ovaries in the presence of KL (NVK+); however, as shown in panel C, DNA laddering was observed in vitrified ovaries cultured in the presence of KL (VK+) and in vitrified ovaries cultured in the absence of KL (VK−) groups; Ladder (L). Reproductive BioMedicine Online 2015 30, 493-503DOI: (10.1016/j.rbmo.2015.01.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions