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J. Blanco-Rodríguez and C. Martínez-García

It is widely assumed that oestrogen administration in the male mimics hypophysectomy by suppressing gonadotrophin secretion. Nevertheless, oestradiol treatment can increase germ-cell apoptosis mainly at stages IV–X of the spermatogenic cycle, rather than at stage VII when apoptotic germ-cell death is mainly triggered by gonadotrophin withdrawal caused by hypophysectomy. Since the roles of testicular oestrogens in spermatogenic regulation, if any, are unknown, we re-evaluated the germ-cell types that undergo apoptosis after oestradiol treatment. Adult male rats were injected daily with 50 μg oestradiol, oestradiol plus testosterone propionate (25 mg every 3 days) or oestradiol plus human menopausal gonadotrophin (equivalent to 25 iu FSH plus 25 iu LH) for 15 days. Apoptosis was assessed by in situ 3′-end labelling of internucleosomal DNA fragments in plastic semithin sections; the germ-cell types involved were identified by high-resolution light microscopy. The quantitative analysis of our results shows that the apoptosis pattern elicited by oestradiol treatment of the seminiferous epithelium differs from that reported to be caused by gonadotrophin or testosterone withdrawal, suggesting a possible role for oestradiol in the modulation of germ-cell death in the adult testis of the rat.

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J Blanco-Rodriguez, C Martinez-Garcia and A Porras

In the seminiferous epithelium, both DNA synthesis and apoptosis occur at equivalent stages in various species, with apoptosis taking place mainly at the same stages as DNA replication in the second, third and fourth spermatogonial generations. As preservation of the cellular associations found at these stages may have some functional significance, it is important to determine whether there is a correlation between these cellular events. In this study, pairs of immunoperoxidase-stained adjacent testis sections from rats, mice, rabbits and cats in which either bromodeoxyuridine incorporated into the newly synthesized DNA strand (BrdU labelling) or DNA 3' end labelling of the apoptotic DNA fragments (TUNEL assay) were detected were compared. In addition, both events were analysed in double-labelled sections. These two methods revealed a clear correlation between the occurrence of DNA replication in the second to fourth generations of spermatogonia and most physiological apoptosis taking place in both spermatogonia and spermatocytes in the three different mammalian orders (Rodentia, Lagomorpha and Carnivora). This correlation may result from the synchronization of mitotic spermatogonial and meiotic spermatocyte cell cycle checkpoints operating at these stages.

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C Ortega Ferrusola, L González Fernández, J M Morrell, C Salazar Sandoval, B Macías García, H Rodríguez-Martinez, J A Tapia and F J Peña

Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (r=−0.789, P<0.001), and also between LPO and spermatozoa with high mitochondrial membrane potential (Δψm) post-thaw (r=−0.689, P<0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO was r=0.772, P<0.001. This LPO is unlikely to represent, per se, a sign of cryopreservation-induced injury, but it is apparently capable of triggering ‘apoptotic-like changes’ that could result in the sub-lethal cryodamage often seen among surviving spermatozoa.

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Felipe Martínez-Pastor, Eduardo Aisen, María Rocío Fernández-Santos, Milagros C Esteso, Alejandro Maroto-Morales, Olga García-Álvarez and J Julián Garde

Fe2 +/ascorbate, hydrogen peroxide (H2O2), and hypoxanthine/xanthine oxidase (XOD) are commonly used for inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed spermatozoa from red deer. First, we tested a high, medium, and low concentration of each agent: 100, 10, and 1 μM Fe2 + (hydroxyl radical generator); 1 mM, 100, and 10 μM H2O2; and 100, 10, and 1 mU/ml XOD (superoxide and H2O2 generator), incubated at 37 °C for 180 min. Intracellular reactive oxygen species (ROS; H2DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (Δψm) were considerably decreased by H2O2 (1 mM and 100 μM) and XOD (100 and 10 mU/ml). Only 1 mM H2O2 reduced viability. The antioxidant Trolox (10 μM) reduced intracellular ROS, but could not prevent the H2O2 or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H2O2 increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented Δψm loss, confirming that H2O2 was the causative agent, not superoxide. In a third experiment, caspase activation was tested using the (FAM-VAD-FMK) probe. Viable spermatozoa with activated caspases could be detected in untreated samples, and only H2O2 increased their proportion after 60 min. There were important differences between ROS generators, H2O2 being the most cytotoxic. Although H2O2 and XOD caused Δψm dissipation, this was not reflected in increasing apoptotic markers.