Protective effect of gallic acid on doxorubicin-induced ovarian toxicity in mouse.

The aims of the present study were to evaluate the protective effects of gallic acid against doxorubicin-induced ovarian toxicity in mice, and to verify the possible involvement of PI3K and mTOR signaling pathway members (PTEN, Akt, FOXO3a and rpS6) in the gallic acid protective actions. Mice were pretreated with NaCl (0.15 M, p.o.) (control and doxorubicin groups) or gallic acid (50, 100 or 200 mg/kg body weight, p.o.) once daily, for 5 days, and on the third day of treatment, after 1 h of treatment administration, the mice received saline solution (i.p.) (control group) or doxorubicin (10 mg/kg of body weight, i.p.). Next, the ovaries were harvested for histological (follicular morphology and activation), fluorescence (GSH and mitochondrial activity), and immunohistochemical (PCNA, cleaved caspase-3, TNF-α, p-PTEN, Akt, p-Akt, p-rpS6 and p-FOXO3a) analyses. The results showed that cotreatment with 50 mg/kg gallic acid plus doxorubicin preserved the percentage of normal follicles and cell proliferation, reduced the percentage of cleaved caspase-3 follicles, prevented inflammation, and increased GSH concentrations and mitochondrial activity compared to doxorubicin treatment alone. Furthermore, cotreatment 50 mg/kg gallic acid plus doxorrubicin increased expression of Akt, p-Akt, p-rpS6 and p-FOXO3a compared to the doxorubicin alone. In conclusion, 50 mg/kg gallic acid protects the mouse ovary against doxorubicin-induced damage by improving GSH concentrations and mitochondrial activity and cellular proliferation, inhibiting inflammation and apoptosis, and regulating PI3K and mTOR signaling pathway.

Involvement of PTEN and FOXO3a Proteins in the Protective Activity of Protocatechuic Acid Against Cisplatin-Induced Ovarian Toxicity in Mice.

The present study evaluated the effects of protocatechuic acid (PCA) after cisplatin-induced ovarian toxicity in mice and if PTEN and FOXO3a proteins are involved in PCA action. The mice were divided into five experimental groups (five animals per group) and treated once a day for 3 days as follows: (1) the control group was pretreated with oral administration (o.p.) of saline solution, followed by an intraperitoneal (i.p.) injection of saline solution. The other groups were pretreated (o.p.) with (2) saline solution (cisplatin group), (3) N-acetylcysteine (150 mg/kg of body weight), or with (4) 20 or (5) 50 mg/kg body weight of PCA, followed by 5 mg/kg body weight (i.p.) of cisplatin. Next, the ovaries were destined to histological (morphology and activation), immunohistochemical (PCNA and cleaved caspase-3 expression), and fluorescence (reactive oxygen species [ROS], glutathione [GSH], and active mitochondria levels) analyses. Moreover, the immunoreactivity for p-PTEN and p-FOXO3a was evaluated to investigate a potential mechanism by which PCA could prevent the cisplatin-induced ovarian damage. Pretreatment with N-acetylcysteine or 20 mg/kg PCA before cisplatin preserved the percentage of normal follicles and cell proliferation as observed in the control, reduced apoptosis and ROS levels, and showed higher active mitochondria and GSH levels than the cisplatin treatment (P < 0.05). Moreover, pretreatment with 20 mg/kg PCA decreased cisplatin-induced p-PTEN and increased (P < 0.05) nuclear export of p-FOXO3a. In conclusion, PCA at 20 mg/kg reduced apoptosis, maintained cell proliferation and mitochondrial function, reduced ROS production, and increased GSH expression likely through the involvement of PTEN and FOXO3a proteins.

Follicular survival, activation of primordial follicles and DNA fragmentation after storage of goat ovaries at 35ºC in supplemented Minimal Essential Medium

This study evaluated the effect of caprine ovarian tissue transportation conditions (medium supplementation and transportation duration) on the morphology, DNA fragmentation and development of cultured and non-cultured preantral follicles. After the fragmentation of ovaries, one fragment was fixed (fresh control) while the remaining slices were placed individually in two different conservation media (Minimal Essential Medium - MEM without supplementation or supplemented MEM, i.e. MEM+) and stored at 35ºC for 6 or 12 h without (non-cultured) or with a subsequent 5-day in vitro culture in supplemented alfa-MEM. After transportation, followed or not by in vitro culture, the fragments were processed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) examination. For the preserved and non-cultured fragments, the percentages of normal follicles after the storage of ovarian tissue in MEM+ for 6 h and the DNA fragmentation rates after preservation in MEM for 6 h and MEM+ for 6 or 12 h were maintained similar to the fresh control. However, all cultured treatments reduced the proportion of normal follicles and increased the percentage of TUNEL-positive cells as compared to the fresh control and non-cultured treatments. On the contrary, all culture conditions (except after preservation in MEM for 6 h) promoted an increase in primordial follicle activation. In conclusion, the use of an enriched medium (MEM+) during ovary transportation is preferable to maintain satisfactory rates of normal follicles after the preservation of caprine ovarian tissue at 35ºC for up to 6 h, without affecting the ability of the primordial follicle to grow in vitro.

Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

The aims of this study were to verify the effects of Epidermal Growth Factor (EGF) on the morphology, primordial follicle activation, growth and proliferation of granulosa cells of ovine follicles cultured in situ, as well as the effect of a PI3K inhibitor on the follicular activation. Ten ovine ovaries were divided into fragments, being one fixed for histological analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+) alone or supplemented with EGF (1, 10, 50, 100 or 200 ng/mL). Follicles were classified as normal or atretic, as primordial or growing, and the oocyte and follicle diameters were measured. PCNA immunohistochemistry was performed in the fresh control and in treatment that showed the bestresults for follicular activation. Pharmacologic inhibition of PI3K activity was performed through pretreatment in media added with 50 μMLY294002 for 1 h. The percentage of normal follicles decreased (P 0.05). In conclusion, PI3K pathway mediates the in vitrospontaneous activation of sheep primordial follicles. Moreover, EGF may act indirectly on follicular activation by promoting granulosa cell proliferation at 1 ng/mL, and EGF inhibited follicle activation in concentrations similar or higher than 10 ng/mL.

Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

The aims of this study were to verify the effects of Epidermal Growth Factor (EGF) on the morphology, primordial follicle activation, growth and proliferation of granulosa cells of ovine follicles cultured in situ, as well as the effect of a PI3K inhibitor on the follicular activation. Ten ovine ovaries were divided into fragments, being one fixed for histological analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+) alone or supplemented with EGF (1, 10, 50, 100 or 200 ng/mL). Follicles were classified as normal or atretic, as primordial or growing, and the oocyte and follicle diameters were measured. PCNA immunohistochemistry was performed in the fresh control and in treatment that showed the bestresults for follicular activation. Pharmacologic inhibition of PI3K activity was performed through pretreatment in media added with 50 μMLY294002 for 1 h. The percentage of normal follicles decreased (P < 0.05) after 7 days of culture in all treatments compared to the fresh control. A significantreduction in the percentage of primordial follicles and an increase (P < 0.05) in the growing ones were observed in all treatments compared to fresh control. Furthermore, both the control medium and 1 ng/mL EGF promoted an increase (P < 0.05) in follicular activation compared to other EGF treatments. The PCNA-positive cells in the EGF treatment were higher (P < 0.05) than in fresh control and α-MEM+. Pretreatment of ovarian tissue with PI3K inhibitor significantly inhibited (P < 0.05) α-MEM+-stimulated primordial follicle activation, but had no effect on EGF-stimulated activation (P > 0.05). In conclusion, PI3K pathway mediates the in vitrospontaneous activation of sheep primordial follicles. Moreover, EGF may act indirectly on follicular activation by promoting granulosa cell proliferation at 1 ng/mL, and EGF inhibited follicle activation in concentrations similar or higher than 10 ng/mL.(AU)

Follicular survival, activation of primordial follicles and DNA fragmentation after storage of goat ovaries at 35ºC in supplemented Minimal Essential Medium

This study evaluated the effect of caprine ovarian tissue transportation conditions (medium supplementation and transportation duration) on the morphology, DNA fragmentation and development of cultured and non-cultured preantral follicles. After the fragmentation of ovaries, one fragment was fixed (fresh control) while the remaining slices were placed individually in two different conservation media (Minimal Essential Medium - MEM without supplementation or supplemented MEM, i.e. MEM+) and stored at 35ºC for 6 or 12 h without (non-cultured) or with a subsequent 5-day in vitro culture in supplemented alfa-MEM. After transportation, followed or not by in vitro culture, the fragments were processed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) examination. For the preserved and non-cultured fragments, the percentages of normal follicles after the storage of ovarian tissue in MEM+ for 6 h and the DNA fragmentation rates after preservation in MEM for 6 h and MEM+ for 6 or 12 h were maintained similar to the fresh control. However, all cultured treatments reduced the proportion of normal follicles and increased the percentage of TUNEL-positive cells as compared to the fresh control and non-cultured treatments. On the contrary, all culture conditions (except after preservation in MEM for 6 h) promoted an increase in primordial follicle activation. In conclusion, the use of an enriched medium (MEM+) during ovary transportation is preferable to maintain satisfactory rates of normal follicles after the preservation of caprine ovarian tissue at 35ºC for up to 6 h, without affecting the ability of the primordial follicle to grow in vitro.(AU)