Materials and methods: supplementary details

Morphometric and histological analyses

When the tubular sections were oblique, only the minor distance was measured (Miraglia and Hayashi 1993; Stumpp et al. 2004). Fifty random seminiferous tubule cross sections per testicular section were examined at x380 final magnification, totaling 100 seminiferous tubule sections per animal.

Apoptotic germ cell numerical density

With the aim of investigating whether carnitine protects germ cells against apoptosis caused by etoposide, the numerical density of apoptotic cells detected in the seminiferous epithelium was obtained. The numerical density (Nv) is expressed by units of volume and corresponds to the ratio between the total number of scored germ cells and total volume of tubular tissue examined (V). To detect apoptosis, the TUNEL method and the analysis of the nuclear morphology were performed (Stumpp et al. 2004; Lirdi et al. 2008) as follows.

TUNEL staining

The apoptotic cells were detected by the TUNEL method by using the ApopTag Plus Peroxidase kit (Intergen Company, NY), as previously described (Sasso-Cerri and Miraglia 2002). Two 7-µm-thick testicular sections from each animal were hydrated and treated with 20µg/ml proteinase K for 12 min at room temperature (25ºC). The sections were incubated in 3% hydrogen peroxide for 10 min for the inactivation of endogenous peroxidases, washed in phosphate-buffered saline (pH7.3, 0.05M) and treated with equilibration buffer for 20 min. The sections were then incubated with terminal deoxynucleotidyl transferase (TdT) enzyme at 37ºC in a humidified chamber for 1h. The tissue was washed in Stop Wash buffer for 30 min and incubated with antidigoxigenin antibody conjugated to peroxidase, at room temperature, for 30min. The reaction was revealed by 3,3’-diaminobenzidine (DAB), and the sections were counterstained with Harris’ hematoxylin. Negative controls were carried out following the same protocol, except for the incubation with TdT. Mammary gland sections supplied by the ApopTag kit were used as positive controls.

Cells with morphological characteristics of apoptosis

The TUNEL method detects DNA fragmentation associated with late apoptosis (Barroso et al. 2000; Huerta et al. 2007). However, this method does not label cells in the more advanced and final stages of apoptosis (Stumpp et al. 2004). Thus, the TUNEL-negative cells with abnormal nuclear morphology suggestive of apoptosis were also counted (Lirdi et al. 2008). According to Hasegawa et al. (1997), cells in the advanced and final stages of apoptosis show condensed and peripheral chromatin with sharp delineation; this standard description was adopted to recognize the TUNEL-negative cells. Cells with abnormal nuclear morphology (ANM) were scored in the same sections submitted to TUNEL reaction (Stumpp et al. 2004; Lirdi et al. 2008), as these sections were counterstained with Harris’ hematoxylin.

Numerical density

The numerical densities of TUNEL-positive (Nvt1) and TUNEL-negative cells with abnormal nuclear morphology (Nvt2) were calculated. Then, the total numerical density of apoptotic cells per cm3, including the TUNEL-positive cells and TUNEL-negative cells with ANM (Nvt3), was obtained according to Stumpp et al. (2004) and Lirdi et al. (2008). Fifty tubular sections were randomly analyzed per testicular section, totaling 100 seminiferous tubule sections per animal, by means of a computer-assisted image analysis system (Leica-Q550IW; Cambridge-England) coupled to a binocular microscope. To avoid overestimation, we focused on only one chosen plane of a tubular section to quantify the apoptotic germ cells (Cruz-Orive and Weibel 1990). TUNEL-positive cells with weak labeling were not scored.

Concentration and morphological analyses of spermatozoa in the epididymal fluid

Samples of epididymal fluid were obtained from the caudal region of the epididymis of 127-day-old rats for the analyses of the concentration and morphology of spermatozoa. Samples of 3 µl epididymal fluid obtained from the left epididymis caudal region were homogenized in 2 ml twice-distilled water. One drop of the solution was smeared onto a glass slide and air-dried. The smears were stained by the Shorr/hematoxylin method. For morphological evaluation, 200 spermatozoa were randomly analyzed, and the percentage of abnormal spermatozoa was obtained (Filler 1993). The abnormal characteristics considered were: (1) the shape and size of the spermatozoa head, including large or small heads, with lighter or accentuated curvature; (2) intermediate piece defects resulting in united heads; (3) defects of tails, including short, multiple, folded, or broken tails. Subsequently, another 2ml twice-distilled water were added to the solution, and 9 µl were collected for spermatozoa counts in a hematocytometer chamber (Neubauer Bright Line Improved, 0.100 mm) according to Strasinger (1989).

References

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Cruz-Orive LM, Weibel ER (1990) Recent stereological methods for cell biology: a brief survey. Am J Physiol 258:L148–L156

Filler R (1993) Methods for evaluation of rat epididymal sperm morphology. In: Chapin RE, Heindel JJ (eds) Methods in toxicology, part A: male reproductive toxicology. Academic Press, California, pp 335-343

Hasegawa M, Wilson G, Russel LD, et al (1997) Radiation-induced cell death in the mouse testis: relationship to apoptosis. Radiat Res 147:457-467

Huerta S, Goulet EJ, Huerta-Yepez S, Livingston EH (2007) Screening and detection of apoptosis. J Surg Res 139:143–156

Lirdi LC, Stumpp T, Sasso-Cerri E, et al (2008) Amifostine protective effect on cisplatin-treated rat testis. Anat Rec 291:797-808

Miraglia SM, Hayashi H (1993) Histomorphometry of immature rat testis after heating. J Morphol 217:65-74

Sasso-Cerri E, Miraglia SM (2002) In situ demonstration of both TUNEL-labeled germ cell and Sertoli cell in the cimetidine-treated rats. Histol Histopathol 17:411-417

Strasinger SK (1989) Uroanálise e fluidos biológicos: texto auto-instrumental. Panamericana, São Paulo

Stumpp T, Sasso-Cerri E, Freymüller E, et al (2004) Apoptosis and testicular alterations in albino rats treated with etoposide during the prepubertal phase. Anat Rec [A] 279:611-622