R. S. Barberino

Melatonin protects against cisplatin-induced ovarian damage in mice via the MT1 receptor and antioxidant activity

Abstract 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; 4mg/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. Summary Sentence Melatonin attenuated cisplatin-induced ovarian damage in mice, and the MT1 receptor could be used as a promising therapeutic target to the development of novel agents for preserving ovarian function during chemotherapy.

Use of protocatechuic acid as the sole antioxidant in the base medium for in vitro culture of ovine isolated secondary follicles

This study evaluated the effect of the protocatechuic acid (PCA) as the sole antioxidant in the base medium for in vitro culture of ovine secondary follicles. Secondary follicles (200-230xa0μm) were isolated and cultured in α-minimal essential medium supplemented with BSA, insulin, glutamine and hypoxanthine (α-MEM: antioxidant-free medium) or α-MEM also added by transferrin, selenium and ascorbic acid (α-MEM+: with antioxidant) or α-MEM added by PCA (56.25; 112.5; 225; 450; or 900xa0μg/ml). Moreover, after culture, oocytes were matured and the chromatin configuration and DNA fragmentation were evaluated. After 12xa0days, the treatment containing 56.25xa0μg/ml PCA showed higher percentage of normal follicles than control medium or the other treatments (pxa0<xa0.05), except for 900xa0μg/ml PCA (pxa0>xa0.05). The antrum formation was significantly higher in treatments containing 56.25, 112.5 or 900xa0μg/ml PCA, compared to the α-MEM and similar (pxa0>xa0.05) to the other treatments. The rates of fully grown oocytes (≥110xa0μm) were similar (pxa0>xa0.05) among all treatments containing PCA and α-MEM+, and those were superior (pxa0<xa0.05) than α-MEM, except for 450xa0μg/ml PCA (pxa0>xa0.05). GSH levels and mitochondrial activity were higher (pxa0<xa0.05) in α-MEM+ than in α-MEM and similar (pxa0>xa0.05) to all PCA treatments. The rates of meiotic resumption and DNA fragmentation were similar (pxa0>xa0.05) among α-MEM+ and 56.25xa0μg/ml PCA. In conclusion, PCA at 56.25xa0μg/ml as the sole antioxidant added to the medium for ovine isolated secondary follicle culture maintains follicular survival, GSH and active mitochondria levels, meiotic developmental competence and DNA integrity of cultured oocytes.

Influence of the ovarian fragmentation before storage at 4°C on the apoptosis rates and in vitro development of ovine preantral follicles

The aim of this study was to evaluate the effect of ovarian tissue fragmentation before preservation at 4°C in MEM on the morphology, apoptosis, and growth of ovine preantral follicles. After collection, the ovaries were divided into two halves, being one divided into two fragments (1/4 of the ovary). One fragment was subdivided into two fragments (1/8), being one fixed for histology (fresh control). The remaining whole ovary, 1/2, 1/4 and 1/8 of the ovary were preserved in MEM at 4°C for 6, 12 or 24 h. The tissue was further destined to histology. In vitro culture and TUNEL technique were performed in treatments that showed the best results of follicular survival after preservation. Storage of 1/8 of the ovary increased the normal follicles compared with half or whole ovary. After preservation in 1/8 of the ovary and culture for 7 days, the percentage of apoptotic cells was similar to the fresh control and non-cultured fragments. The percentage of growing follicles increased after preservation of 1/8 of the ovary compared with 1/4. In conclusion, ovine preantral follicles can be preserved in fragments of 1/8 of the ovary in MEM at 4°C for 24 h, without affecting apoptosis and their ability to grow in vitro .

Extract of Amburana cearensis maintains the survival of ovine preantral follicles during long-term ovarian tissue transport and promotes primordial follicle activation after in vitro culture

This study evaluated the effect of Amburana cearensis extract as a preservation or culture medium for ovine ovarian tissue. Ovarian fragments were fixed in 4% buffered formaldehyde for 18 h (fresh control), stored in Minimal Essential Medium (MEM) or in A. cearensis extract (0.1; 0.2 or 0.4 mg/mL) at a temperature of 4oC for 6, 12 or 24 h (preservation - experiment 1) or cultured for 7 days in ?-MEM+ or in A. cearensis extract without (0.1; 0.2 or 0.4 mg/mL) or with supplements (0.1+ ; 0.2+ or 0.4+ mg/ mL; experiment 2). The percentages of morphologically normal follicles and follicular activation were submitted to analysis of variance (ANOVA) and Tukey´s test. The values of TUNEL-positive cells were submitted to Chi-square test (P 0.05). Moreover, after in vitro culture, A. cearensis at a concentration of 0.1 mg/mL maintained the percentage of TUNEL positive cells (30.0%) in a way that is similar to that observed in the fresh control (22%) (P > 0.05). In conclusion, ovine preantral follicles can be preserved at 4°C in MEM for 6 h. For longer periods of preservation (24 h), MEM and 0.2 mg/mL A. cearensis are recommended. Moreover, after in vitro culture, A. cearensis extract (0.1 mg/mL) showed higher activation and lower DNA fragmentation in ovine preantral follicles.

Resveratrol promotes in vitro activation of ovine primordial follicles by reducing DNA damage and enhancing granulosa cell proliferation via phosphatidylinositol 3-kinase pathway: XXXX

We aimed to study the effects of resveratrol on the morphology, DNA fragmentation, follicular activation and cell proliferation after in vitro culture of ovine ovarian tissue, and to verify if PI3K pathway is involved in resveratrol action in the sheep ovary. Ovaries were collected and divided into fragments. One fragment was fixed for histology (fresh control). The remaining fragments were cultured for 7xa0days in control medium (α-MEM+ ) alone or with resveratrol (2, 10 or 30xa0µM). After culture, ovarian tissue was destined to morphological analysis. TUNEL and proliferating cell nuclear antigen (PCNA) analyses were performed in the fresh control, α-MEM+ and 2xa0µM resveratrol. Inhibition of PI3K activity was performed through pre-treatment with LY294002. The percentage of normal follicles was similar between α-MEM+ and 2xa0µM resveratrol, and higher than those in other resveratrol treatments. An increase in follicular activation was observed in all treatments compared to fresh control. DNA fragmentation decreased in tissues cultured in 2xa0µM resveratrol compared to α-MEM+ . Moreover, PCNA-positive cells were higher in 2xa0µM resveratrol than in α-MEM+ . LY294002 inhibited follicular activation stimulated by α-MEM+ and 2xa0µM resveratrol. In conclusion, 2xa0µM resveratrol promotes primordial follicle activation compared to the fresh control by reducing DNA fragmentation and stimulating granulosa cell proliferation through activation of the PI3K pathway.

Sperm quality, and morphology and apoptosis of germinal epithelium cells of ram lambs receiving water of different salinities

The aim of the present study was to evaluate the influence of water salinity on semen quality, and on the morphology and apoptosis of germinal epithelial cells in prepubertal Morada Nova male lambs. Thirty-two lambs were allocated into four treatments with different amounts of sodium chloride (NaCl) added to the drinking water to simulate different water salinities; consequently, the concentrations of total dissolved solids (TDS) were as follows: 640 (control), 3188; 5740 and 8326 mg/L TDS. After 78 days, sperm was collected for analysis. The animals were slaughtered and histological and morphometric analyses and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay were performed on the testis tissue. The thickness of the germinal epithelium and diameter of the seminiferous tubules were measured. A quadratic effect (P 0.05) from the other salinity treatments. Moreover, treatments with 3188 mg/L or 5740 mg/L TDS showed a higher (P < 0.05) spermatic vigour than did the other treatments. There was an increase (P < 0.05) in the number of TUNEL-positive cells in the treatment with the highest salinity (8326 mg/L TDS) compared with the control and other treatments. In conclusion, water used for drinking should contain between 3188 and 5740 mg/mL TDS so as to improve the concentration, vigour, motility and volume of semen, and to decrease sperm abnormalities in germinal cells of seminiferous tubule of Morada Nova ram lambs.