Germ cell apoptosis was quantified by TUNEL and expressed as apoptotic index (AI, number of apoptotic germ cells per 100 Sertoli cells)(A). The Western blots were used to assess the BAX expression in mitochondrial (B) and cytosolic (C) fractions in rat testis. A: GnRH-A treatment increased male germ cell apoptosis which is reduced by concomitant treatment with synthetic HN. Apoptosis is expressed as a number of apoptotic cells/Sertoli cells stained with TUNEL and counted manually. B: GnRH-A treatment increased BAX in the mitochondrial fractions. HN reversed the increased BAX observed with GnRH-A treatment. COX IV was used as loading control (normalization factor for density analysis in mitochondrial fractions). C: GnRH-A±HN treatment had no effect on BAX level in cytosolic fractions. GAPDH was used as loading control (normalization factor for density analysis). In each experiment, groups of 3 or 4 animals received treatment with vehicle (control, n=3), GnRH-A (n=3), HN (n=4), or GnRH-A+HN (n=4) as described in the methods. Values are means ± SD. Means with unlike superscripts are significantly (P<0.05) different.