This study evaluated the receptor- and/or antioxidant stress-mediated mechanisms by which melatonin prevents the ovarian toxicity of cisplatin treatment. The expression of the MT1 receptor in mouse ovaries was investigated by immunohistochemistry. Pretreatment with melatonin (5, 10, or 20 mg/kg body weight, i.p.) before cisplatin (5 mg/kg body weight, i.p.) was administered to mice once daily for 3 days (phase I). The pharmacological modulation via melatonin type 1 and/or 2 receptors was analyzed by administration of receptor antagonists (luzindole: nonselective MT1/MT2 antagonist; 5 mg/kg body weight or 4-phenyl-2-propionamidotetralin: selective MT2 antagonist; 4 mg/kg body weight) once daily for 3 days, 15 min before the treatment with melatonin and cisplatin (phase II). Thereafter, the ovaries were harvested and used for histological (morphology and activation), immunohistochemical (PCNA, activated caspase-3 and bcl-2 expression), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and fluorescence (reactive oxygen species [ROS], glutathione [GSH], and active mitochondria levels) analyses. The expression of the MT1 protein in mouse ovaries was documented. Pretreatment with 20 mg/kg melatonin before cisplatin administration preserved the normal follicular morphology and cell proliferation rate, reduced apoptosis, ROS production, mitochondrial damage and increased GSH expression, as compared to the cisplatin treatment alone. Additionally, administration of the nonselective MT1/MT2 receptor antagonist inhibited the melatonin ovarian protection from the cytotoxic effects of cisplatin. However, administration of a selective MT2 antagonist did not modify the protective effects observed at 20 mg/kg melatonin. In conclusion, pretreatment with 20 mg/kg melatonin effectively protected the ovaries against cisplatin-induced damage. Moreover, the MT1 receptor and melatonin antioxidant effects mediated this cytoprotective activity.
The aim of the present study was to evaluate the in vitro and in vivo effect of the addition of superoxide dismutase (SOD) and reduced glutathione (GSH) to ram semen freezing extender. Significant differences (p < 0.05) were detected between groups regarding total motility (TM), straightness (STR) and wobble (WOB), for which the GSH 7 mM group had lesser TM and better STR than the other groups and the GSH 5 and 7 mM groups had higher wobble values than the control, SOD 25 and 100 U/ml groups. The ultrastructural analysis revealed that the acrosome was better preserved after freezing in the SOD 100 U/ml and GSH 2 and 5 mM (p < 0.05) groups than the other groups, whereas mitochondria in both the control group and the 7 mM GSH group suffered the greatest damage. The plasma membrane remained preserved after freezing, regardless of the group. For in vivo fertilization, the SOD group achieved better results than the GSH group (p > 0.05). It can therefore be concluded that the addition of SOD 100 U/ml and GSH 2 and 5 mM preserves the acrosome integrity of frozen ram spermatozoa, while the addition of SOD 100 U/ml to Tris egg-yolk extender offers protection to the membranes of sperm cells after thawing.
Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF-2 protein expression in ovine ovaries and to verify the effect of FGF-2 on the morphology, apoptosis and growth of ovine pre-antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM(+) ) alone or supplemented with FGF-2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF-2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF-2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF-2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF-2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF-2 (p < 0.05). However, the percentage of apoptotic cells only decreased (p < 0.05) in ovarian tissues cultured in 1 or 10 ng/ml FGF-2 compared with the control medium and other FGF-2 treatments. In conclusion, this study demonstrated the presence of FGF-2 in ovine ovaries. Furthermore, 10 ng/ml FGF-2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.
Neoplastic transformation is the abnormal proliferation of cells. These transformations are often related to changes in cell surface glycoconjugates which can be detected by lectins. We evaluated the anti-tumor potential of BlL, a galactoside-binding lectin isolated from Bothrops leucurus venom as well as its cytotoxicity and hemolysis activity. The phosphatidylserine externalization and mitochondrial membrane potential were also determined. BlL exhibited cytotoxic activity against all tumor cell lines tested by induced phosphatidylserine externalization and mitochondrial depolarization, indicating cell death by apoptosis.
The objective of this study was to determine ruminal degradability of the dry matter (DM) and crude protein (CP) of 10 varieties of spineless cactus (Opuntia spp.) grown in north-eastern Brazil. Two ruminally fistulated steers were used in a randomized complete block design. Ash, CP, acid detergent fiber, and acid detergent lignin levels ranged from 10.4 to 13.3 %, 6.2-7.7 %, 19.8-24.8 % and 3.4-5.4 %, respectively. Relative to the other cactus varieties, Redonda had the highest (P < 0.05) in situ soluble DM fraction and effective DM degradability. No differences in effective DM degradability were observed between the other cactus varieties. In situ soluble CP fraction ranged from 1.7 % of CP for the 69 IPA ⁄ UFRPE variety to 11.1 % of CP for the Gigante variety. Slowly degradable CP fraction and its rate of degradation were similar among the cactus varieties (average 90 % of CP and 9 % h )1 , respectively). The cactus variety 69 IP ⁄ UFRPE had a lower (P < 0.05) effective ruminal CP degradability (ECPD) than the other varieties, which had a similar ECPD (average 66 % of CP). It was concluded that differences in ruminal degradability exist between cactus varieties, with more variations observed for ruminal DM than for CP degradability.
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