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.

Effects of leptin on the follicular development and mitochondrial activity of ovine isolated early antral follicles cultured in vitro.

This study evaluated the effect of leptin on the in vitro culture of isolated sheep early antral follicles. Early antral follicles (300-450 µm) were isolated and cultured for 12 days in tissue culture medium 199 (TCM 199) supplemented with glutamine, hypoxanthine, transferrin, insulin, selenium, ascorbic acid, bovine serum albumin (BSA) and recombinant follicle stimulating hormone (rFSH) (TCM 199+: control medium) or TCM 199+ supplemented with 2 or 10 ng/mL leptin. After culture, oocytes were subjected to in vitro maturation (IVM). The parameters analyzed were morphology, extrusion rate, follicular diameter, growth and fully-grown oocytes (oocytes ≥110 µm) rates. After IVM, reactive oxygen species (ROS) levels, mitochondrial activity, meiotic stages and meiotic resumption rates were also analyzed. After 12 days of culture, the concentration of 2 ng/mL of leptin showed a higher percentage of morphologically normal follicles, fully-grown oocytes (≥110 µm), active mitochondria and meiotic resumption compared to the control medium (TCM 199+; P < 0.05) but did not differ when compared to leptin concentration of 10 ng/mL (P > 0.05). After culturing, no significant differences existed among treatments in terms of the follicle diameter and ROS levels. In conclusion, the addition of 2 ng/mL leptin to the base culture medium is capable of improving follicular survival, oocyte growth, mitochondrial activity and meiotic resumption after the in vitro culture of isolated sheep early antral follicles.

Kaempferol can be used as the single antioxidant in the in vitro culture medium, stimulating sheep secondary follicle development through the phosphatidylinositol 3-kinase signaling pathway.

This study evaluated the effect of addition of kaempferol alone or combined with other antioxidants (transferrin, selenium and ascorbic acid) on in vitro culture of sheep isolated secondary follicles and if PI3K pathway is involved in kaempferol action. Secondary follicles 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 kaempferol (0.1; 1 or 10 µM) were added to the different base media (α-MEM or AO). After culture, glutathione (GSH) levels, mitochondrial activity and meiotic resumption were evaluated. In addition, inhibition of PI3K activity was performed through pretreatment in medium supplemented with LY294002. After 12 days, the percentage of normal follicles was higher (P < 0.05) in AO base medium than the other treatments and similar (P > 0.05) to α-MEM supplemented with 1 or 10 µM kaempferol Moreover, α-MEM plus 1 or 10 µM kaempferol and AO medium showed similar (P > 0.05) follicular diameter, fully-grown oocytes, and GSH levels. However, at the end of the culture, antrum formation was higher (P < 0.05) in α-MEM + 1 µM kaempferol than in AO, and similar (P > 0.05) to α-MEM + 10 µM kaempferol. In addition, oocytes cultured in α-MEM supplemented with 1 µM kaempferol showed greater (P < 0.05) levels of active mitochondria than α-MEM + 10 µM kaempferol and AO medium. The rates of meiotic resumption were similar (P > 0.05) among α-MEM + 1 µM kaempferol and AO medium. LY294002 significantly inhibited antrum formation, follicular diameter and the percentage of fully grown oocytes stimulated by 1 µM kaempferol. In conclusion, 1 µM kaempferol 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 survival, increasing active mitochondria levels, and promoting the oocyte meiotic resumption. Moreover, the development of the ovine secondary follicle stimulated by kaempferol is mediated by PI3K pathway.

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.

Supplemented base medium containing Amburana cearensis associated with FSH improves in vitro development of isolated goat preantral follicles.

The effects of Amburana cearensis ethanolic extract, with or without addition of a mix of supplements associated or not with FSH, on in vitro morphology and development of caprine secondary follicles were evaluated. In experiment 1, isolated follicles (250 µm in diameter) were cultured for 12 days in alpha-modified minimal essential medium (α-MEM) alone (control) or in medium composed of different concentrations of A. cearensis extract (Amb 0.1; 0.2, or 0.4 mg/mL). In experiment 2, culture media were α-MEM or Amb 0.2 mg/mL (both without supplements), or these same media supplemented with BSA, insulin, transferrin, selenium, glutamine, hypoxanthine, and ascorbic acid (referred as α-MEM(+) and Amb 0.2(+), respectively), or these last groups also supplemented with sequential FSH (100 ng/mL from Day 0 to Day 6; 500 ng/mL from Day 6 to Day 12), constituting groups α-MEM(+) + FSH and Amb 0.2(+) + FSH. At the end of culture in experiment 1, control medium (α-MEM) and Amb 0.2 mg/mL had higher percentages (P < 0.05) of morphologically normal follicles and percentage of fully grown oocytes, i.e., oocyte greater than 110 µm, compared to the other A. cearensis extract concentrations. In experiment 2, all supplemented media had higher percentages (P < 0.05) of normal follicles and antrum formation than nonsupplemented media. In addition, follicles cultured in Amb 0.2(+) + FSH showed an average increase in diameter higher (P < 0.05) than the other treatments. Oocytes cultured in both treatments supplemented with FSH showed greater glutathione and active mitochondria levels than nonsupplemented media but similar to the other treatments. In conclusion, A. cearensis extract (0.2 mg/mL) added by supplements and FSH improves follicular growth. Therefore, it can be an alternative culture medium for goat preantral follicle development.

Immunohistochemical localization of fibroblast growth factor-2 in the sheep ovary and its effects on pre-antral follicle apoptosis and development in vitro.

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.