Cetuximab intensifies cisplatin-induced testicular toxicity Mattan Levi, Aron Popovtzer, Moran Tzabari, Aviram Mizrachi, Naphtali Savion, Salomon M. Stemmer, Ruth Shalgi, Irit Ben-Aharon  Reproductive BioMedicine Online  Volume 33, Issue 1, Pages 102-110 (July 2016) DOI: 10.1016/j.rbmo.2016.04.004 Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Cetuximab detrimental effect on testicular function. Body weight (A), testis weight (B), epididymis weight (C), epididymal-spermatozoa total motile count (D), sperm motility (E) and sperm progressive motility (F) in mature male mice one week or one month after injection of saline (control), cetuximab (Cet; 10 mg/kg), cisplatin (Cis; 8 mg/kg) or the combination of cetuximab and cisplatin (Cis + Cet). Bars are mean ± SEM. (*) = significantly different from control value (P < 0.05). (**) = significantly different from control and Cis values (P < 0.05). Reproductive BioMedicine Online 2016 33, 102-110DOI: (10.1016/j.rbmo.2016.04.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Concentration of anti-Müllerian hormone (AMH) in the serum of male mice is increased after a combined treatment of cetuximab and cisplatin. Serum AMH (ng/ml) one week or one month after injection of saline, cetuximab, cisplatin or the combination of cetuximab and cisplatin. Bars are mean ± SEM. (*) = significantly different from the corresponding control value (P < 0.05). Reproductive BioMedicine Online 2016 33, 102-110DOI: (10.1016/j.rbmo.2016.04.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Testicular characterization one month after cetuximab and/or cisplatin administration. Images of spermatogenic milieu of mice testes excised one month after injection of saline (a − a'''), cetuximab (b − b'''), cisplatin (c − c''') or the combination of cetuximab and cisplatin (d − d'''). Representative bright field images of testes stained with hematoxylin (blue) and eosin (pink; a − d) or hematoxylin (blue) and Ki-67 (brown; a'−d'). Representative confocal laser-scanning microscopy images of testes stained with Hoechst 33280 (blue) and for DAZL (red; a''−d'') or testes stained with Hoechst 33280 (blue) and for GATA-4 (green) and PLZF (red; a'''−d'''). Bar = 100 µm. Reproductive BioMedicine Online 2016 33, 102-110DOI: (10.1016/j.rbmo.2016.04.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Rate of germ cell proliferation, and number of spermatocytes, Sertoli cells and spermatogonia in the testes of mice one month after cetuximab and/or cisplatin administration. The average number of Ki-67 (germ cells), DAZL (spermatocytes), GATA-4 (Sertoli cells), PLZF (spermatogonia) or epidermal growth factor receptor (EGFR) positive cells and elongated spermatids per tubule in testes of mice one month after injection of saline, cetuximab, cisplatin or the combination of cetuximab and cisplatin. Each bar is mean ± SEM. (*) = significantly different from control value (P < 0.05). (**) = significantly different from control and Cis values (P < 0.05). Reproductive BioMedicine Online 2016 33, 102-110DOI: (10.1016/j.rbmo.2016.04.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Vascularization, apoptosis and EGFR localization in the testes of mice one month after cetuximab and/or cisplatin administration. Images of vascularization, apoptosis and epidermal growth factor receptor (EGFR) localization in mice testes excised one month after injection of saline (a − a''), cetuximab (b − b''), cisplatin (c − c'') or the combination of cetuximab and cisplatin (d − d''). Representative confocal laser-scanning microscopy images of testes stained with Hoechst 33280 (blue) and with anti-CD34 (red; a − d), or by TUNEL assay (green; a'−d'), or anti-EGFR (green; a''−d''). Bar = 100 µm. Reproductive BioMedicine Online 2016 33, 102-110DOI: (10.1016/j.rbmo.2016.04.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Rate of vascularization and apoptosis in the testes of mice one month after cetuximab and/or cisplatin administration. The average number of TUNEL-positive cells per tubule in the field of view and the average number of CD34-positive vessels per section of testes excised from mice one month after injection of saline, cetuximab, cisplatin or the combination of cetuximab and cisplatin. Each bar is mean ± SEM. (*) = significantly different from control value (P < 0.05). (**) = significantly different from control and Cis values (P < 0.05). Reproductive BioMedicine Online 2016 33, 102-110DOI: (10.1016/j.rbmo.2016.04.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions