Dose-dependent adverse effects of salinomycin on male reproductive organs and fertility in mice

Abstract

Salinomycin is used as an antibiotic in animal husbandry. Its implication in cancer therapy has recently been proposed. Present study evaluated the toxic effects of Salinomycin on male reproductive system of mice. Doses of 1, 3 or 5 mg/kg of Salinomycin were administered daily for 28 days. Half of the mice were sacrificed after 24 h of the last treatment and other half were sacrificed 28 days after withdrawal of treatment. Effects of SAL on body and reproductive organ weights were studied. Histoarchitecture of testis and epididymis was evaluated along with ultrastructural changes in Leydig cells. Serum and testicular testosterone and luteinizing hormones were estimated. Superoxide dismutase, reduced glutathione, lipid peroxidation, catalase and lactate dehydrogenase activities were measured. Spermatozoa count, morphology, motility and fertility were evaluated. Expression patterns of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage proteins (CYP11A1) were assessed by Western blotting. Salinomycin treatment was lethal to few mice and retarded body growth in others with decreased weight of testes and seminal vesicles in a dose dependent manner. Seminiferous tubules in testes were disrupted and the epithelium of epididymis showed frequent occurrence of vacuolization and necrosis. Leydig cells showed hypertrophied cytoplasm with shrunken nuclei, condensed mitochondria, proliferated endoplasmic reticulum and increased number of lipid droplets. Salinomycin decreased motility and spermatozoa count with increased number of abnormal spermatozoa leading to infertility. The testosterone and luteinizing hormone levels were decreased in testis but increased in serum at higher doses. Depletion of superoxide dismutase and reduced glutathione with increased lipid peroxidation in both testis and epididymis indicated generation of oxidative stress. Suppressed expression of StAR and CYP11A1 proteins indicates inhibition of steroidogenesis. Spermatogenesis was however observed in testis 28 days after Salinomycin withdrawal. The results indicate reversible dose-dependent adverse effects of Salinomycin on male reproductive system of mice.

Figure 1. Mice treated with SAL a) 1mg/kg; b) 3mg/kg and c) 5mg/kg.

Note the hind limb paralysis.

Figure 2. Transverse sections of testes of mice treated with SAL for 28 days (a) control (b) 1mg/kg (c) 3mg/kg and (d) 5mg/kg.

Note the shrunken seminiferous tubules and vacuolation in the germinal epithelium (b, c & d) in SAL treated mice. Note the signs of recovery 28 days after withdrawal in treated mice; e (3mg/kg) and f (5mg/kg).

Figure 3. Transverse sections of caput epididymidis of mice following SAL treatment (a) control; (b) 1mg/kg; (c) 1mg/kg; (d) 3mg/kg; (e) 3mg/kg (f) 5mg/kg & (g) 5mg/kg.

Note: Figures c, e & g are from the recovery groups.

Figure 4. Transverse sections of cauda epididymidis of mice following SAL treatment (h) control; (i) 1mg/kg; (j) 1mg/kg; (k) 3mg/kg; (l) 3mg/kg; (m) 5mg/kg & (n) 5mg/kg.

Note: Figures j, l & n are from the recovery groups.

Figure 5. Electron micrographs of testes sections focusing on Leydig cells in controls (a & b) and SAL treated mice (c & d: I mg/kg; e & f: 3mg/kg and g & h: 5mg/kg).

Note: Mitochondria-M; Nucleus -N; Lipid droplet -L.

Figure 6. Effect of SAL on sperm count.

Figure 7. Effect of SAL on sperm motility.

Figure 8. Effect of SAL on sperm morphology.

Figure 9. Expression levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage (CYP11A1) in the testis of mice (a) Western blot (b) densitometric quantitation of the Western blot.