Renin mRNA is upregulated in testes and testicular cells in response to treatment with aflatoxin B 1

Abstract

Aflatoxin B 1 (AFB 1) has been shown to affect fertility in many species; however, the exact molecular mechanisms associated with the disruption are not known. Our objectives were to determine changes in testicular gene expression due to exposure to AFB 1 and to investigate which cell types were affected by treatment with AFB 1. Male mice 4 wk of age were administered a daily placebo (control; N = 9) or 50 μg/kg AFB 1 (AFB 1 treated; N = 10) daily for 45 days. Males were then mated to four females each for 8 days. Male mice were characterized as being “Tolerant” (N = 3) or “Intolerant” (N = 3) to the effects of AFB 1 based on positive terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in the testes and the number of pups sired. Tolerant males produced a similar average number of fetuses (mean ± SEM) (12.5 ± 1.2) per male as selected control males (13.4 ± 1.2), but more fetuses (P = 0.01) than Intolerant males (7.6 ± 1.2). The number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells in Intolerant males tended to be (P = 0.10) greater (136.5 ± 27.2) than in Tolerant (55.0 ± 22.2) and selected control (54.3 ± 22.2) males. Affymetrix microarray (Sunnyvale, CA, USA) analysis revealed differential expression (P < 0.05) of 193 extra cellular space and signaling genes, 49 signal transduction genes, 45 immune regulation genes, and 230 cell differentiation genes in the testis. Renin was commonly represented amongst many clusters and was chosen for further analyses. Upregulation (P < 0.001) of Renin in Tolerant mice (N = 3) compared with Intolerant mice (N = 3) was confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR) (P = 0.05). This upregulation (P = 0.01) was also observed in representative AFB 1 treated males (N = 8) compared with control males (N = 8) selected for real-time reverse transcription polymerase chain reaction analysis. Spermatogonia cultured in vitro and treated with 0, 5, 10, or 20 μg/mL AFB 1 (N = 6 per treatment) resulted in a 10-fold upregulation (P = 0.01) of Renin message at the 20 μg/mL level, whereas Leydig tumor cells showed similar differences (P = 0.03) in message for Renin in treated (10 and 20 μg/mL) versus control cell cultures. Based on these results, we inferred a role for Renin at the molecular level in the response to the adverse effects of AFB 1 in male mice.