Leptin decreases apoptosis and promotes the activation of primordial follicles through the phosphatidylinositol-3-kinase/protein kinase B pathway in cultured ovine ovarian tissue.

This study evaluated the effects of leptin on primordial follicle survival and activation after in vitro culture of ovine ovarian tissue and if leptin acts through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway. Ovarian fragments were fixed for histology (fresh control) or cultured for 7 days in control medium (α-MEM+) alone or supplemented with leptin (1, 5, 10, 25 or 50 ng/ml). Follicle morphology, activation and apoptosis were analyzed. Next, the fragments were cultured in the medium that showed the best results in the absence or the presence of the PI3K inhibitor (LY294002), and immunohistostaining of p-Akt protein was assessed. After culture, the percentage of normal follicles decreased (P < 0.05) in all treatments compared with the fresh control. Moreover, control medium and 1 ng/ml leptin had similar (P > 0.05) percentages of normal follicles, which were significantly higher than those in other treatments. However, culture with 1 ng/ml leptin maintained apoptosis similarly (P > 0.05) to that of the fresh control and lower (P < 0.05) than that in α-MEM+. Leptin did not influence follicle activation (P > 0.05) compared with the control medium (α-MEM+). Culture in 1 ng/ml leptin with LY294002 decreased the normal follicles and increased apoptosis, inhibited follicle activation (P < 0.05), and reduced p-Akt immunostaining, compared with the medium containing 1 ng/ml leptin without PI3K inhibitor. In conclusion, leptin at 1 ng/ml reduces apoptosis and promotes the activation of primordial follicles compared with the fresh control after in vitro culture of ovine ovarian tissue possibly through the PI3K/Akt pathway.

Involvement of Phosphorylated Akt and FOXO3a in the Effects of Growth and Differentiation Factor-9 (GDF-9) on Inhibition of Follicular Apoptosis and Induction of Granulosa Cell Proliferation After In Vitro Culture of Sheep Ovarian Tissue.

This study aimed to evaluate the effects of growth and differentiation factor-9 (GDF-9) on the morphology, activation, apoptosis, and granulosa cell proliferation of ovine preantral follicles cultured within ovarian tissue slices and to verify whether GDF-9 could influence follicular activation through the phosphatidylinositol 3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway. Ovine ovarian fragments were cultured in α-MEM+ or α-MEM+ with GDF-9 (1, 50, 100, 200, or 400 ng/ml) for 7 days. Apoptosis and cell proliferation were analyzed. Next, the activation of the PI3K was inhibited with LY294002, and immunostaining for p-Akt and p-FOXO3a proteins was assessed. The concentration of 50 ng/ml GDF-9 had (P < 0.05) more morphologically normal follicles compared to all treatments, except 1 ng/ml GDF-9. Moreover, 50 ng/ml GDF-9 increased primordial follicle activation compared to all treatments, except α-MEM+ and 1 ng/ml GDF-9. However, the concentration of 50 ng/ml GDF-9 showed higher cell proliferation and lower apoptosis than α-MEM+ and 1 ng/ml GDF-9 treatments. Culture of the ovarian tissue with LY294002 inhibited the activation of primordial follicles and reduced p-Akt immunostaining in both α-MEM+ and 50 ng/ml GDF-9 treatments. In addition, after culture with LY294002, the percentage of oocytes with nuclear p-FOXO3 was higher in 50 ng/ml GDF-9 than in the control medium (α-MEM+). In conclusion, after culture of ovine ovarian cortical slices, the addition of 50 ng/ml GDF-9 reduces follicular apoptosis and promotes granulosa cell proliferation likely through the involvement of phosphorylated Akt and FOXO3a.

Immunohistochemical localization of insulin-like growth factor-1 (IGF-1) in the sheep ovary and the synergistic effect of IGF-1 and FSH on follicular development in vitro and LH receptor immunostaining.

This study analyzed IGF-1 protein immunostaining in sheep ovaries, the effect of IGF-1 alone or associated with FSH on the culture of secondary follicles, and the immunostaining of LHR protein in antral follicles before and after culture. Ovaries were collected for IGF-1 protein analysis. In experiment 1, secondary follicles were cultured in α-MEM+ (control) or α-MEM+ supplemented with IGF-1 (10, 50 or 100 ng/mL). In experiment 2, follicles were cultured in the same media of experiment 1 plus 750 ng/mL FSH. Moreover, LHR immunostaining was analyzed in fresh antral follicles and after culture in 50 ng/mL IGF-1 + FSH. The IGF-1 protein was immunolocalized in oocytes from all stages of follicle development and in the granulosa cells from secondary and antral follicles. IGF-1 did not influence (P > 0.05) follicular viability and growth (experiment 1). However, in experiment 2, 50 ng/mL IGF-1 + FSH stimulated oocyte growth (P < 0.05) and LHR immunostaining in antral follicles. Control medium, 10 or 50 ng/mL IGF-1 + FSH showed similar levels of reactive oxygen species, glutathione and active mitochondria (P > 0.05). In conclusion, the IGF-1 protein is present in all ovarian follicle stages in sheep. Moreover, the association between 50 ng/mL IGF-1 and FSH has a synergistic effect in vitro, increasing the percentage of fully grown oocytes and the intensity of immunostaining of LHR protein in oocytes and granulosa cells of cultured antral follicles.

Conditioned medium of ovine Wharton's jelly-derived mesenchymal stem cells improves growth and reduces ROS generation of isolated secondary follicles after short-term in vitro culture.

The aim of this study was to evaluate the effect of the conditioned medium of ovine Wharton's jelly-derived mesenchymal stem cells (oWJ-MSCs) on the morphology, growth, reactive oxygen species (ROS) and glutathione (GSH) intracellular levels, active mitochondria, and meiotic resumption of isolated ovine secondary follicles in vitro. The oWJ-MSCs were isolated and the medium where they were cultured was recovered (conditioned medium). Isolated ovine secondary follicles were cultured for 6 days in 1) supplemented α-MEM+ (control); 2) 50% α-MEM+ + 50% conditioned medium (α-MEM + CM group) or 3) conditioned medium only (CM group). The parameters analyzed were morphology, antrum formation, follicle and oocyte growth, ROS and GSH levels, mitochondrial activity and meiotic resumption. The percentage of normal follicles, antrum formation, and fully grown oocytes did not differ (P > 0.05) among treatments. Follicles cultured in α-MEM + CM group had greater (P < 0.05) diameter than other treatments after culture. Moreover, the diameter of the follicles cultured in CM alone was higher (P < 0.05) than in the α-MEM+. In addition, α-MEM + CM and CM treatments increased the growth rate compared to the α-MEM+. Treatments containing conditioned medium (α-MEM + CM or CM) significantly reduced ROS levels compared to the control medium. Moreover, mitochondrial activity was higher in α-MEM+ and α-MEM + CM than in CM alone. All treatments showed oocytes in GV, GVBD and MI. In conclusion, oWJ-MSCs conditioned medium, especially when associated with α-MEM, improves the growth of secondary follicles and reduces ROS generation after short-term culture.

Resveratrol has dose-dependent effects on DNA fragmentation and mitochondrial activity of ovine secondary follicles cultured in vitro.

The worldwide consumption of red wine, nuts and grapes has resulted in increased human exposure to resveratrol, which could affect reproductive function. However, the effect of resveratrol on in vitro culture of early-stage ovarian follicles has never been investigated. The aims of the present study were to evaluate the effect of resveratrol on sheep secondary follicle morphology, growth, DNA fragmentation, intracellular levels of glutathione (GSH) and active mitochondria. Secondary follicles were isolated from the ovaries and cultured for 18 days in supplemented α-MEM+ (control medium) or in control medium containing resveratrol (2, 10 or 30 µM). The parameters analyzed were morphology, antrum formation, follicle diameter, DNA fragmentation, GSH levels and mitochondrial activity. After 18 days, all resveratrol groups significantly decreased the percentages of morphologically normal follicles compared with the control group (α-MEM+). Antrum formation was higher in both α-MEM+ and 2 µM resveratrol groups than in the 10 µM resveratrol group. In addition, 30 µM resveratrol increased the percentage of oocytes with DNA damage compared with the control. Oocytes from follicles treated with 10 or 30 µM resveratrol significantly decreased intracellular GSH levels compared with the 2 µM resveratrol group. Moreover, follicles in α-MEM+ (control) showed more active mitochondria than those in 10 or 30 µM resveratrol. In conclusion, ovine isolated secondary follicles are able to grow to the antral stage after in vitro culture in medium containing 2 µM resveratrol, maintaining the same rates of DNA damage, GSH levels and mitochondrial function as the control medium. However, the addition of 30 µM resveratrol increased DNA fragmentation and oxidative stress through decreasing mitochondrial activity.

Rutin can replace the use of three other antioxidants in the culture medium, maintaining the viability of sheep isolated secondary follicles.

The present study evaluated the effect of addition of rutin alone or combined with other antioxidants (transferrin, selenium and ascorbic acid) present in the culture medium on the in vitro development of ovine isolated secondary follicles. After collection of the sheep ovaries, secondary follicles (200-230 µm) were isolated and cultured for 12 days in α-Minimal Essential Medium (α-MEM) supplemented with BSA, insulin, glutamine and hypoxanthine (α-MEM: antioxidant free-medium) or in this medium also added by transferrin, selenium and ascorbic acid (AO: base medium with antioxidants). Moreover, different concentrations of rutin (0.1; 1 or 10 µg/mL) were added to the different base media (α-MEM or AO). The parameters analyzed were morphology, antrum formation, extrusion rate, follicular diameter, growth and fully-grown oocytes (oocytes ≥ 110 µm) rates. In treatments that had the best results of morphology, follicular viability, apoptosis, glutathione (GSH), reactive oxygen species (ROS) levels and mitochondrial activity were also analyzed. After 12 days, the percentage of normal follicles was higher (P < 0.05) in α-MEM + 0.1 µg/mL rutin than the other treatments, except compared to AO medium (P > 0.05). There is no difference (P > 0.05) in the diameter and growth rate among treatments. Moreover, AO medium and α-MEM + 0.1 µg/mL rutin showed similar (P > 0.05) percentages of follicular viability, antrum formation, extruded follicles, fully-grown oocytes, levels of ROS and active mitochondria. However, α-MEM + 0.1 µg/mL rutin treatment showed higher (P > 0.05) GSH levels than AO medium. In conclusion, 0.1 µg/mL rutin can be used as the single antioxidant present in the base medium, replacing the addition of transferrin, selenium and ascorbic acid during in vitro culture of ovine secondary follicles, maintaining follicular viability and increasing GSH levels.