Antioxidants enhance the recovery of three cycles of bleomycin, etoposide, and cisplatin–induced testicular dysfunction, pituitary-testicular axis, and fertility in rats  Narayana Kilarkaje, Ph.D., Alyaa M. Mousa, Ph.D., Maie M. Al-Bader, Ph.D., Khalid M. Khan, Ph.D.  Fertility and Sterility  Volume 100, Issue 4, Pages 1151-1159.e5 (October 2013) DOI: 10.1016/j.fertnstert.2013.06.019 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Body weight and fertility parameters in control, three cycles (TC) of bleomycin, etoposide, and cisplatin (BEP) and antioxidant cocktail (AC) with recovery period (TC + R) groups. Data are shown as mean ± SD for each group (n = 10–15; except hormone studies: n = 5). *P<.05, control vs. experimental groups; $P<.05, AC vs. experimental groups; #P<.05, BEP vs. BEP + AC groups; εP<.05, TC vs. TC + R. STD = seminiferous tubular diameter. Fertility and Sterility 2013 100, 1151-1159.e5DOI: (10.1016/j.fertnstert.2013.06.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of three cycles of BEP on testicular structure. In control (A and E) and AC (B and F) groups, the normal tubules (N) with normal cell associations are indicated. Boxes in A and B indicate areas shown in high magnification in E and F, respectively, showing late stage VII tubules with normal cell associations. (C) In the BEP group, all tubules are atrophic (At) with a very few germ cells, and most of the nuclei observed are of Sertoli cells. Boxed area in C is shown in G under high magnification. Note that the Sertoli cell nuclei appear round due to tubular atrophy. A germ cell nucleus (Gc) and rounded Sertoli cell (Sc) nucleus are indicated. Note that the eosinophilic material is formed by the cytoplasm of Sertoli cells. The basement membrane is thicker than that in E and F. (D) In the BEP + AC group, a considerable recovery is observed and the number of tubules with the germ cells is increased indicating the tubules with partial atrophy (Ap), although the atrophic tubules (At) are still present. Boxed area in D is indicated in H under high magnification. Repopulated germ cells (Gc), probably primary spermatocytes and spermatogonia (Sg), are indicated. The Sertoli cell nuclei show rounded or triangular profiles due to missing germ cells. Note that the basement membrane is still thicker than that seen in G. Periodic acid–Schiff plus hematoxylin. El = elongating spermatids; Ps = primary spermatocytes; Sd = round spermatids; other abbreviations as in Figure 1. A–D: ×200; E–H: ×1,000. Fertility and Sterility 2013 100, 1151-1159.e5DOI: (10.1016/j.fertnstert.2013.06.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Testicular structure at the end of the recovery period. In (A) control and (B) AC-treated rats, the testes show normal tubules (N) with normal structure and cell associations in seminiferous epithelium. (C–G) In the BEP group, although the epithelium shows recovery in some animals (C) with normal (N) and some tubules with disrupted epithelium (D), in some other animals (D and E), the tubules are atrophic (At). The atrophic tubules show only Sertoli cells and the epithelium is formed by their cytoplasm (E inset: arrows). The tubules with disrupted epithelium show abnormal cell associations, chromatin margination in round spermatids (Cm), pyknotic nuclei (Py) of germ cells (F) and epithelial degeneration (Ed) (G). (H–J) In the BEP + AC group, the tubules show epithelial degeneration and the number of atrophic tubules (At) is less than that in BEP group (D and E). However, a few tubules show atrophy, especially at the peripheral parts of the organ. (I) The atrophic tubules show only disfigured Sertoli cell nuclei and cytoplasm (S). (J) The epithelium in some tubules still show abnormal germ cells indicating germ cell death (Nc). Cp = capsule; asterisk = basement membrane; other abbreviations as in Figure 1. A and B: ×200; C–E and H: ×100; F, G, I, J, and E inset: ×400. Fertility and Sterility 2013 100, 1151-1159.e5DOI: (10.1016/j.fertnstert.2013.06.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effects of three cycles of BEP with or without AC and the recovery period on expression of proliferating cell nuclear antigen (PCNA) and transferrin in the testes. Western blotting for the proteins (A) at the end of treatment and (B) at the end of the recovery period. β-Actin is included as an endogenous control. PCNA (∼36 kDa) and transferrin (∼78–82 kDa) were probed by primary antibodies (see Materials and Methods). Densitometric analyses of (C) PCNA and (D) transferrin indicate a significant decrease in the protein expression in the BEP and BEP + AC groups at the end of treatment (*P<.05, control vs. experimental groups; $P<.05, AC vs. experimental groups). An increase in PCNA level is observed at the end of the recovery period in both BEP and BEP + AC groups, but the transferrin level is elevated only in the BEP group, compared with control groups (#P<.05, control vs. experimental groups; θP<.05, AC vs. experimental groups). Transferrin expression recovered to control level in the BEP + AC group compared with BEP group (P<.05). Compared with the effect at the end of treatment, the expression of the proteins shows significant increase at the end of the recovery period (σP<.05). Data are shown as mean ± SD for each group (n = 4–6). Abbreviations as in Figure 1. Fertility and Sterility 2013 100, 1151-1159.e5DOI: (10.1016/j.fertnstert.2013.06.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Effects of three cycles of bleomycin, etoposide, and cisplatin (BEP) with the recovery period on sperm morphology. At both sample points, the control (A and E) and the antioxidant cocktail (AC)–treated (B and F) rats show normal sperm morphology. The normal sperm show sickle-shaped heads and long tails, indicating normal structure. (C) The three cycles of BEP induced detachment of sperm heads from their tails (descending arrows), but a very few sperm show intact tails with cephalocaudal junction defect (ascending arrow). (D) At the end of treatment in the BEP + AC group, protection of sperm morphology is observed, although a few sperm show the detachment of heads (solid arrow) and some other intact sperm show cephalocaudal bending (open arrow). (G) At the end of the recovery period, the majority of sperm show normal morphology although a few sperm also show the detachment of sperm heads (arrow). (H) At the end of the recovery period in the BEP + AC group, the sperm morphology is completely recovered and similar to that in the control groups. Periodic acid–Schiff plus hematoxylin; ×1,000. Fertility and Sterility 2013 100, 1151-1159.e5DOI: (10.1016/j.fertnstert.2013.06.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Effects of three cycles of BEP with or without AC on expression of PCNA in the testis. In the (A) control and (B) AC-treated groups, intensely labeled cells are seen near the basal compartment of the seminiferous epithelium, indicating active cell division. Boxed areas in A and B are shown in E and F, respectively, under high magnification. This late stage VII or early stage VIII tubule indicates localization of the protein in nuclei of spermatogonia (Sg) and primary spermatocytes (Ps) and mild labeling in round spermatids (Sd). In the (C) BEP and (D) BEP + AC groups, the germ cell nuclei in both atrophic (At) and partly atrophic (Ap) tubules show proliferating cell nuclear antigen labeling. Boxed areas in C and D are shown in G and H, respectively, under high magnification. Note that the number of germ cells in the BEP and BEP + AC groups (C, D, G, and H) is less than in the control groups, but they are intensely labeled. The Sertoli cell (Sc) nuclei are either labeled lightly or not labeled at all. El = elongating spermatids; N = normal tubule; Gc = germ cell; other abbreviations as in Supplemental Figure 1. A–D: ×20; E–H: ×1,000. Counterstained with Mayer hematoxylin. Fertility and Sterility 2013 100, 1151-1159.e5DOI: (10.1016/j.fertnstert.2013.06.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Effects of three cycles of BEP with or without AC and the recovery period on proliferating cell nuclear antigen (PCNA) expression in the testis. In the (A) control and (B) AC-treated groups, the normal tubules (N) with PCNA-labeled germ cells at the basal epithelial layers indicate cell division. In the BEP groups (C–E), the protein expression is observed in almost all cells in the epithelium. In atrophic tubules (At), the surviving germ cells (Gc), and Sertoli cells (S) are stained intensely. The intensity of the labeling is more in the BEP group. In the BEP + AC group (F–H), the expression is similar to that seen in the BEP group. Insets in E and H show intensely labeled nuclei of spermatogonia (Sg), primary spermatocytes (Ps), and the round spermatid (Sd) nuclei. The latter show more peripheral labeling owing to chromatin margination. D = the tubules with epithelial disruption; El = elongating spermatids; V = vacuole; other abbreviations as in Supplemental Figure 1. Counterstained with hematoxylin. A, B, and F: ×200; C: ×100; D, E, G, H, and insets: ×400. Fertility and Sterility 2013 100, 1151-1159.e5DOI: (10.1016/j.fertnstert.2013.06.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 4 Effects of three cycles of BEP with or without AC on transferrin expression. In the control (A, E, and I) and AC-treated (B, F, and J) groups, cytoplasmic expression of transferrin is observed, mainly in Sertoli cells (Sc). The cytoplasm of spermatogonia (Sg), primary spermatocytes (Ps), and round spermatids (Sd) show light staining for the protein. Epithelial gaps observed are due to microwave treatment. In atrophic tubules (At) in the BEP group (C, G, and K), the cytoplasm of Sertoli cells shows transferrin expression, thus giving a brown appearance in images because there are few or no germ cells. In the BEP + AC group (D, H, and L), the similar pattern of staining is observed as seen in BEP group. Ap = partly atrophic tubule; El = elongating spermatids; N = normal tubule; Ps = primary spermatocytes; other abbreviations as in Supplemental Figure 1. A–D: ×20; E–H: ×40; I–L: ×1,000. Counterstained with Mayer hematoxylin. Fertility and Sterility 2013 100, 1151-1159.e5DOI: (10.1016/j.fertnstert.2013.06.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 5 Effects of three cycles of BEP with or without AC and with the recovery period on transferrin expression. In all groups, only normal tubules (N) are indicated. Tubules in the (A) control, (B) AC, (C) BEP, and (D) BEP + AC groups, showing cytoplasmic expression of the protein. In the BEP group, expression of the protein is up-regulated. Insets indicate Sertoli cells (Sc) and germ cells showing cytoplasmic expression of the protein. El = elongating spermatids; Ps = primary spermatocytes; Sd = round spermatid; Sg = spermatogonia; other abbreviations as in Supplemental Figure 1. ×400. Asterisk = artifact. Counterstained with Mayer hematoxylin. Fertility and Sterility 2013 100, 1151-1159.e5DOI: (10.1016/j.fertnstert.2013.06.019) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions