Targeted anti-apoptosis activity for ovarian protection against chemotherapy-induced ovarian gonadotoxicity Shun-Jen Tan, Li-Jen Lee, Chii-Ruey Tzeng,

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Targeted anti-apoptosis activity for ovarian protection against chemotherapy-induced ovarian gonadotoxicity Shun-Jen Tan, Li-Jen Lee, Chii-Ruey Tzeng, Chia-Woei Wang, Ming-I Hsu, Chi-Huang Chen Reproductive BioMedicine Online Volume 29, Issue 5, Pages 612-620 (November 2014) DOI: 10.1016/j.rbmo.2014.07.014 Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Experimental design. The ovaries of mice in the S1P + B groups were injected with different doses of sphingosine-1-phosphate (0.5 mM or 2.0 mM), followed by the administration of busulfan. Mice in the B group were treated with busulfan alone, and those in the control group were treated with vehicle only. PET, 5% polyethylene glycol, 2.5% ethanol, and 0.8% Tween-80; S1P, sphingosine-1-phosphate. Reproductive BioMedicine Online 2014 29, 612-620DOI: (10.1016/j.rbmo.2014.07.014) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Estimated primordial, primary, secondary, and antral follicle counts in mice in the busulfan (B), low-dose sphingosine-1-phosphate (S1P) (0.5 mM) plus busulfan (S1PL + B), high-dose S1P (2.0 mM) plus busulfan (S1PH + B), and control (vehicle) groups. A significant difference was found in the number of follicles at all stages between the B and control groups, and greater numbers of primordial follicles were found in the S1PL + B and S1PH + B groups compared with the B group. A significant decline was found in the numbers of primary, secondary and antral follicles in the B and S1P + B groups. **P < 0.01; *P < 0.05. Values expressed as mean ± SEM. Reproductive BioMedicine Online 2014 29, 612-620DOI: (10.1016/j.rbmo.2014.07.014) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Histological examination with immunohistochemical localization of active caspase-3 in the mouse ovary. (A) Follicles with normal morphology were observed in the control group. Most follicles had no immunoreactivity for active caspase-3; (B) numerous immunopositive follicles were found in the busulfan group. Active caspase-3 was detected mainly in granulosa cells and only occasionally in oocytes (arrows); (C) active caspase-3 was detected in granulosa cells of primary, secondary, and antral follicles, with weaker staining in the S1P + B group (scale bars, 50 µm). Reproductive BioMedicine Online 2014 29, 612-620DOI: (10.1016/j.rbmo.2014.07.014) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Expression of the anti-Müllerian hormone (AMH) and growth differentiation factor 9 (GDF-9) mRNAs in the busulfan (B), low-dose sphingosine-1-phosphate (S1P) (0.5 mM) plus busulfan (S1PL + B), high-dose S1P (2.0 mM) plus busulfan (S1PH + B), and control groups. (A) The level of AMH in the B group was significantly lower than that observed in the three remaining groups; (B) No significant differences were found in GDF-9 levels among the four groups. **P < 0.01; *P < 0.05. Values expressed as mean ± SEM. Reproductive BioMedicine Online 2014 29, 612-620DOI: (10.1016/j.rbmo.2014.07.014) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions