Preliminary Investigation on the Ameliorative Role Exerted by D-Aspartic Acid in Counteracting Ethane Dimethane Sulfonate (EDS) Toxicity in the Rat Testis

Abstract

Herein is reported the first evidence of the protective role of D-aspartic acid (D-Asp) in preventing the toxic effect exerted by the alkylating agent ethane dimethane sulfonate (EDS) in the rat testis. We confirmed that EDS treatment specifically destroyed Leydig cells (LC), resulting in the drastic decrease of the serum testosterone level and producing morphological changes in the germinal tubules, i.e., altered organization of the epithelium, loss of cell contacts and the consequent presence of empty spaces between them, and a reduce number of spermatozoa. Moreover, an increase of TUNEL-positive germ cells, other than alteration in the protein level and localization of two LC "markers", StAR and PREP, were observed. Interestingly, results obtained from rats pre-treated with D-Asp for 15 days before EDS-injection showed that all the considered parameters were quite normal. To explore the probable mechanism(s) involved in the protection exerted by D-Asp, we considered the increased oxidative stress induced by EDS and the D-Asp antioxidant effects. Thiobarbiturc acid-reactive species (TBARS) levels increased following EDS-injection, while no change was observed in the D-Asp + EDS treated rats. Our results showed that D-Asp may be used as a strategy to mitigate the toxic effects exerted by environmental pollutants, as endocrine disrupters, in order to preserve the reproductive function.

Keywords: D-aspartic acid; EDS; PREP; StAR; endocrine disrupters; testis; testosterone.

Figure 1

Serum testosterone (ng/mL) level from control, ethane dimethane sulfonate (EDS), and D-Aspartic acid (D-Asp) + EDS treated rats. In the EDS-treated rats, testosterone concentration strongly decreased, while in D-Asp + EDS treated animals, the decrease is less pronounced. Values are expressed as mean ± SEM from 5 animals in each group.

Figure 2

Hematoxylin-eosin and TUNEL staining of rat testicular paraffin-embedded sections. (A–C): Hematoxylin-eosin staining of control (A), EDS (B), and D-Asp + EDS (C) treated rat testes. The absence of Leydig cells in the EDS-treated group is evident; (D,E): Determination of apoptotic cells through the detection of TUNEL-positive cells (green) in control (D), EDS (E), and D-Asp + EDS (F) treated rats. Slides were counterstained with DAPI-fluorescent nuclear staining (blue). The images were captured at ×20 magnification. Scale bars represent 20 μm. Asterisks: Leydig cells. Triangles: luminal Spermatozoa. Arrowheads: Spermatogonia; Striped Arrows: Spermatocytes.

Figure 3

Testicular steroidogenic acute regulatory (StAR) and prolyl endopeptidase (PREP) protein levels in EDS and D-Asp + EDS treated rats. (A) Western blot analysis of StAR (32 kDa) and PREP (80 kDa) protein levels in the testis from control, EDS, and D-Asp + EDS treated rats. (B) The amount of StAR was quantified using ImageJ program and normalized with respect to α-tubulin (50 kDa). (C) The amount of PREP was quantified using ImageJ program and normalized with respect to α-tubulin (50 kDa). Values are expressed as mean ± SEM from 5 animals in each group.

Figure 4

(A) StAR-PCNA and PREP-TUB co-localization in the testis from controls, EDS, and D-Asp + EDS treated rats. a-c: StAR (green) and PCNA (red) immunolocalization in the testis of control (a), EDS (b) and D-Asp + EDS (c) treated rats. d-e: PREP (green) and tubulin (red) immunolocalization in the testis of control (d), EDS (e), and D-Asp + EDS (f) treated rats. The yellow-orange intermediate tint indicates the two proteins co-localization. Slides were counterstained with DAPI-fluorescent nuclear staining (blue). The images were captured at ×20 magnification. Scale bars represent 20 μm. Asterisks: Leydig cells. Arrowheads: Spermatogonia; Arrows: Sertoli cells cytoplasm. (B) Histogram showing the quantification of PCNA fluorescence signal intensity using ImageJ. Values are expressed as means ± SEM from 5 animals in each group.

Figure 5

Lipid peroxidation evaluated by thiobarbituric acid-reactive species (TBARS) assay. Oxidative stress status evaluated via TBARS assay in testicular samples from control, EDS, and D-Asp + EDS treated rats. Values are expressed as means ± SEM from 5 animals in each group.