Spermatogenesis, epididymis morphology and plasma sex steroid secretion in the male lizard Podarcis sicula exposed to diuron

The present study investigates the effects of diuron, a substituted urea-based herbicide, in the male lizard Podarcis sicula utilizing quantitative and qualitative morphological features of the reproductive system and endocrinological analysis. Besides the control group, lizards were divided into three groups ([a–c]) (n=6/group) and placed for 3 weeks in terraria on polluted soil substrate sprayed with 3.75 L/ha of herbicide Toterbane 50F (50% diuron). Each terrarium was supplemented either with drinking water contaminated by herbicide (i.e. 1.08 μg/mL of diuron; group [a]), or with food contaminated by herbicide (i.e. 5.4 mg of diuron; group [b]), or with drinking water and food contaminated as described above (group [c]).None of the animals exposed to the contaminant showed any signs of general toxicity or death during the course of the experiments. Severe testicular effects are evidenced in all herbicide-treated groups, although, such effects are of a greater magnitude in lizards exposed to contaminated water (groups [a] and [c]). The main degenerative changes observed include: (1) a significant decrease in the mean gonadosomatic index of 55% in group [a] (P<0.001), 21% in group [b] (P<0.01) and 34% in group [c] (P<0.001) compared with control group; (2) a significant shrinking (P<0.001) of seminiferous tubule diameter (more than 60% of the control) in groups [a] and [c], and about 18% in group [b] (P<0.01); (3) a significant decrease in the crude numbers of spermatogonia of 92% in group [a] (P<0.001), 27% in group [b] (P<0.01) and 62% in group [c] (P<0.001) compared with control group. A complete loss of meiotic and mature germ cells in groups [a] and [c], and a reduction of primary spermatocytes, secondary spermatocytes and spermatids (more than 27% of the control) and a decrease of spermatozoa (more than 90% of the control) in group [b]; and (4) an hypertrophy of interstitial connective tissue which contains numerous lymphocytes, neutrophils and monocytes.The decrease and/or loss of germ cells seems to be related to an induction of inflammation (necrosis) rather than to apoptotic processes. Indeed, this hypothesis is supported by a TUNEL-assay, which failed to reveal any apoptotic cells either in the seminiferous epithelium or in the interstitial space in the testis of all exposed groups.Also the epididymis appears affected by diuron exposure. In particular, in experimental groups [a] and [c] it is regressed with abundant connective tissue and low epithelial cells without secretory granules, whereas in group [b] it appears partially regressed, with some secretory granules still present.At the same time, an impairment of the plasma sex-hormone levels is observed in treated lizards, as evidenced by RIA analysis. Testosterone values significantly decreased by 43% in group [a] (P<0.001), 34% in group [b] (P<0.01) and 52% in group [c] compared with control group. Instead, 17β-estradiol plasma content is undetectable in all diuron-exposed lizards.Taken together, the results presented here indicate that diuron exposure resulted in direct male reproductive toxicity and reveal that this lizard is suitable as a laboratory reptile species for toxicological investigations.