Effect of red propolis extract isolated or encapsulated in nanoparticles on the in vitro culture of sheep preantral follicle: Impacts on antrum formation, mitochondrial activity and glutathione levels.

This study aimed to characterize the hydroethanolic extract of red propolis (HERP) and nanoparticles containing HERP for using as an additive in the culture medium of isolated ovine preantral follicles. HERP was characterized by high-performance liquid chromatography (HPLC) and determination of flavonoid content, and the nanoparticles by the mean particle diameter, polydispersity index (PI) and encapsulation efficiency (EE). The effect of HERP (10 and 20 Î·g/ml-HERP10 and HERP20 groups) and nanoparticles (NP10 and NP20 groups) on isolated secondary follicles cultured in vitro for 12 days was observed by morphological evaluation, oxidative stress markers (reactive oxygen species-ROS and glutathione-GSH) and active mitochondria. HPLC showed formononetin as the major compound in the HERP (63.92 ± 0.21 µg/mL). The content of flavonoids ranged from 2.14% to 11.00%. The nanoparticles showed mean diameter of 287.5 ± 3.9 and 479 ± 18.1 Î·m; PI of 0.117 ± 0.018 and 0.316 ± 0.039; and EE of 67.15% and 41%, respectively, for the NP10 and NP20 groups. After 12 days of culture, HERP20 and NP20 increased (p < 0.05) the percentage of normal follicles compared to NP10. HERP20 showed significantly higher percentages of antrum formation than control medium (MEM) and NP10 (p < 0.05). HERP20 also showed an increase (p < 0.05) in mitochondrial activity compared to the other treatments, except NP20 (p > 0.05), and increased GSH levels (p < 0.05) compared to MEM and HERP10. In conclusion, use of HERP (20 Î·g/ml) on in vitro culture of isolated ovine preantral follicles can increase antrum formation, mitochondrial activity and GSH levels.

Insulin-like growth factor-1 (IGF-1) promotes primordial follicle growth and reduces DNA fragmentation through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signalling pathway.

We investigated the effects of insulin-like growth factor 1 (IGF-1) on the morphology and follicular activation of ovine preantral follicles cultured in situ and whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway is involved in IGF-1 action in the sheep ovary. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) analyses (fresh control) or cultured in supplemented alpha-minimum essential medium (α-MEM+; control) or α-MEM+ with IGF-1 (1, 10, 50, 100 or 200ngmL-1) for 7 days. Follicles were classified as normal or atretic, primordial or growing and the oocyte and follicle diameters were measured. DNA fragmentation was evaluated by TUNEL assay. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed on the fresh control, α-MEM+ and 100ngmL-1 IGF-1 samples. Inhibition of PI3K activity was performed through pretreatment with the PI3K inhibitor LY294002 and phosphorylated AKT (pAKT) expression was analysed after culture in the absence or presence of LY294002. IGF-1 at 100ngmL-1 increased (P<0.05) follicular activation compared with α-MEM+ and decreased TUNEL-positive cells (P<0.05) compared with other treatments. PCNA-positive cells also increased (P<0.05) in 100ngmL-1 IGF-1. LY294002 significantly inhibited follicular activation stimulated by α-MEM+ and 100ngmL-1 IGF-1 and reduced pAKT expression in follicles. Overall, IGF-1 at 100ngmL-1 promoted primordial follicle activation, cell proliferation and reduced DNA fragmentation after in situ culture through the PI3K/AKT pathway.

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

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 7 days in control medium (α-MEM+ ) alone or with resveratrol (2, 10 or 30 µM). 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 2 µM resveratrol. Inhibition of PI3K activity was performed through pre-treatment with LY294002. The percentage of normal follicles was similar between α-MEM+ and 2 µM 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 2 µM resveratrol compared to α-MEM+ . Moreover, PCNA-positive cells were higher in 2 µM resveratrol than in α-MEM+ . LY294002 inhibited follicular activation stimulated by α-MEM+ and 2 µM resveratrol. In conclusion, 2 µM 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.

Gallic acid promotes the in vitro development of sheep secondary isolated follicles involving the phosphatidylinositol 3-kinase pathway.

This study was conducted to evaluate the effect of addition of gallic acid as the single antioxidant to the base medium for in vitro culture of sheep secondary follicles and if the phosphatidylinositol 3-kinase (PI3K) pathway is involved in the action of gallic acid. Secondary follicles were isolated and cultured for 12 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium, and ascorbic acid (control medium: α-MEM+) or in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine and different concentrations of gallic acid (25, 50 or 100 µM), thus replacing transferrin, selenium and ascorbic acid in the medium. Follicle morphology, glutathione (GSH), and mitochondrial activity, and meiotic resumption were evaluated. Furthermore, inhibition of PI3K pathway was performed by pretreatment with LY294002. After 12 days of culture, the follicle survival in a medium containing 100 µM gallic acid was similar (P > 0.05) to α-MEM+ and greater (P < 0.05) compared with other gallic acid concentrations. Antrum formation, follicle diameter, GSH, and mitochondrial activity, and meiotic resumption, however, were greater (P < 0.05) when 100 µM gallic acid was included in the α-MEM+ culture medium compared with the control medium. Furthermore, LY294002 inhibited (P < 0.05) follicle survival, development, and meiotic resumption stimulated by 100 µM gallic acid. In conclusion, concentration of 100 µM of gallic acid can be a substitute for transferrin, selenium, and ascorbic acid in the base medium during in vitro culture of sheep secondary follicles, inducing follicle development likely through the PI3K pathway.