Effect of DETA-NO on apoptotic mitochondrial pathway activation.

<p>Testicular fragments obtained from normal rats were incubated with medium containing or not DETA-NO for 18h. Representative Western blots and semiquantitative results obtained by densitometric analysis of Western blots. A.U.: arbitrary units. Data from medium were arbitrarily set at 1. A) Cytochrome c content in the cytosolic fractionand B) Ratio of Bax/Bcl-2 content in the mitochondrial fraction (n = 10 rats) C) Caspase 9 processing and D) activity (colorimetric assay) in the cytosolic fraction (n = 10 rats). Each bar represents mean±SEM. *p<0.05 and **p<0.01 vs. medium. One sample t Test.</p

Effect of DETA-NO on testosterone secretion.

<p>A) Determination of nitrites generated from different concentrations of DETA-NO in DMEM/F12 medium without phenol red. Each circle represents mean of three wells (B) Testicular fragments obtained from normal rats were incubated with media containing or not DETA-NO in the presence or absence of NAC for 18h. Testosterone content in the media was evaluated by RIA. Each bar represents mean±SEM (n = 5–8 rats) from two independent experiments. ***p<0.001 and ^Δp<0.05 vs. medium. Bonferroni Multiple Comparison Test.</p

Involvement of oxidative stress in the apoptotic effect of DETA-NO on germ cell (GC) apoptosis.

<p>A) Testicular fragments (TF) obtained from a normal rat were incubated with medium containing or not DETA-NO in the presence or absence of NAC for 18h. Apoptosis was evaluated by TUNEL technique on sections of TF. The % of seminiferous tubules (ST) with apoptotic cells in the TF immediately removed from the testis was 2.482±0.967; data represent mean±SEM of n = 6–9 non consecutive sections of TF from one representative experiment of three. 300 seminiferous tubules were counted in each condition. ***p<0.001 vs. medium and ^ΔΔp<0.01 vs. DETA. Bonferroni Multiple Comparison Test.</p

Effect of DETA-NO on caspase 3 activation.

<p>Testicular fragments obtained from normal rats were incubated with medium containing or not DETA-NO for 18h. A) Representative Western blot of caspase 3 content in cytosolic lysates B) Semiquantitative results of caspase 3 fragment content obtained by densitometric analysis of Western blots. A.U.: arbitrary units. Data from medium were arbitrarily set at 1. Each bar represents mean±SEM of 10 rats. *p<0.05 vs. medium. One sample t Test.</p

Serum testosterone (T), FSH and LH levels and testicular T content in untreated normal rats or immunized with TH and adjuvants (E) and injected with saline or L-NAME.

<p>Hormone levels were measured by RIA in sera and testes from untreated normal rats (n = 9) or rats immunized with TH and adjuvants and injected with saline (E+saline, n = 11) or L-NAME (E+L-NAME, n = 12). Data are expressed as mean±SEM.</p><p>*p<0.01 versus respective normal and</p><p>^ΔΔ p<0.05 vs. E+saline.</p><p>Dunn Multiple Comparison Test.</p><p>Serum testosterone (T), FSH and LH levels and testicular T content in untreated normal rats or immunized with TH and adjuvants (E) and injected with saline or L-NAME.</p

Effect of NO released by testicular macrophages on germ cell (GC) apoptosis (TUNEL technique) and testosterone secretion (RIA).

<p>A) Representative microphotographs of sections of testicular fragments (TF) from normal rats incubated with medium alone or testicular macrophages (TM) isolated from EAO rats in the presence of D-NAME (2mM) or L-NAME (2mM) for 18h; apoptotic GC are blue stained. Scale bar indicates 20μm B) % of seminiferous tubules (ST) with apoptotic GC, the % of ST with apoptotic cells in the TF immediately removed from the testis was 3.140±0.890. Data represent mean±SEM of (n = 30–40) non consecutive sections of TF obtained from four animals in four independent experiments. 300 ST were counted in each condition/rat. ***p<0.001 vs medium D-NAME; ^ΔΔΔp<0.001 vs D-NAME TM. Two-way ANOVA followed by Bonferroni Multiple Comparison Test. C) Testosterone content in the media. Data represent the mean±SEM (n = 5–7 wells/group from two experiments); ***p<0.001 vs medium D-NAME, ^ΔΔ p<0.01 vs D-NAME TM. Two-way ANOVA followed by Bonferroni Multiple Comparison Test. D) Nitrite production measured in the medium (fluorometric kit). Data represent mean±SEM (n = 5–7 wells/group from two experiments). *p<0.05 vs D-NAME TM. Student t Test.</p

Effect of DETA-NO on caspase 8 activation.

<p>Testicular fragments obtained from normal rats were incubated with medium containing or not DETA-NO for 18h. A) Representative Western blot of caspase 8 content in cytosolic lysates B) Semiquantitative results of caspase 8 fragment obtained by densitometric analysis of Western blots. A.U.: arbitrary units. Data from medium was arbitrarily set at 1. Each bar represents mean±SEM of 10 rats. *** p<0.01 vs. medium. One sample t Test.</p

Effect of NO released by testicular macrophages on apoptotic mitochondrial pathway activation.

<p>Testicular fragments from normal rats incubated with medium alone or testicular macrophages (TM) isolated from EAO rats in the presence of D-NAME (2mM) or L-NAME (2mM) for 18h. Representative Western blots and semiquantitative results obtained by densitometric analysis of Western blots. A.U.: arbitrary units. Data from medium were arbitrarily set at 1. A) Cytochrome c content in the cytosolic fraction. B) Ratio of Bax/Bcl-2 content in the mitochondrial fraction. C) Caspase 9 processing in the cytosolic fraction. Each bar represents mean±SEM of three rats in three independent experiments.</p