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The flavonoid kaempferol has attracted research attention as a potential adjuvant during chemotherapy. This study aimed to evaluate the protective effects of kaempferol against ovarian damage in cisplatin-treated mice. Two groups of mice received saline solution (intraperitoneal injection [i.p.]; control) or a single dose of cisplatin (5 mg/kg body weight, i.p.). Moreover, two other mice groups were pretreated with kaempferol (1 or 10 mg/kg body weight, i.p.) 30 min before of the cisplatin administration. Thereafter, their ovaries were harvested and subjected to histological (follicular morphology and activation) and fluorescence (reactive oxygen species [ROS] production, glutathione [GSH] concentration, and mitochondrial activity) analyses. Compared with cisplatin treatment alone, pretreatment with 1 mg/kg kaempferol maintained normal follicular morphology, reduced ROS production and mitochondrial damage, and enhanced GSH concentration. However, pretreatment with 10 mg/kg kaempferol did not prevent cisplatin-induced damage. The rate of primordial follicle activation was greater in mice pretreated with 1 mg/kg kaempferol than in the other treatment groups. In conclusion, pretreatment with 1 mg/kg kaempferol prevents cisplatin-induced ovarian damage and stimulates primordial follicle activation in mice.

O flavonoide kaempferol tem atraído a atenção como um potencial adjuvante durante a quimioterapia. O presente estudo objetivou avaliar os efeitos do kaempferol contra os danos ovarianos em camundongos tratados com cisplatina. Fêmeas de camundongos receberam solução salina (injeção intraperitoneal [ip]; controle) ou uma dose única de cisplatina (5 mg/kg, ip) ou foram pré-tratadas com kaempferol (1 ou 10 mg/kg, ip) 30 min antes da administração de cisplatina. Os ovários foram recuperados e destinados para as análises histológicas (morfologia e ativação folicular) e de fluorescência (produção de espécies reativas de oxigênio [ERO], concentração de glutationa [GSH] e atividade mitocondrial). Em comparação ao tratamento apenas com cisplatina, o pré-tratamento com 1 mg/kg de kaempferol manteve a morfologia folicular normal, reduziu a produção de ERO, bem como os danos mitocondriais, e aumentou a concentração de GSH. Entretanto, o pré-tratamento com 10 mg/kg de kaempferol não preveniu os danos induzidos pela cisplatina. A taxa de ativação do folículo primordial foi maior em camundongos pré-tratados com 1 mg/kg de kaempferol do que nos outros grupos experimentais. Em conclusão, o pré-tratamento com 1 mg/kg de kaempferol previne o dano ovariano induzido pela cisplatina e estimula a ativação do folículo primordial em camundongos.

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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.

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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.

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The aim of this study was to evaluate follicular survival and development of ovine isolated secondary follicles cultured in medium containing fixed or sequential concentrations of melatonin and further oocyte maturation. Isolated secondary follicles were cultured for 18 days in α-MEM+ alone (control) or with different concentrations of melatonin (100, 500 or 1000 pg/mL) or sequential concentrations of melatonin (Mel Seq: Day 6 = 100; Day 12 = 500; Day 18 = 1000 pg/mL). The percentages of morphologically normal follicles and antral cavity formation increased significantly in 1000 pg/mL melatonin compared to the other treatments. After 18 days, 1000 pg/mL melatonin (Mel 100) showed a greater (P < 0.05) follicular diameter than α-MEM+, 100 and 500 pg/mL melatonin. In addition, the concentration of 500 pg/mL melatonin showed a higher (P < 0.05) percentage of fully grown oocytes than α-MEM+, Mel 100 and Mel Seq treatments. After oocyte maturation, the levels of ROS were lower (P < 0.05) in 1000 pg/mL melatonin (Mel 1000) than in other treatments. Both Mel 1000 and Mel Seq treatments showed significantly higher levels of mitochondrial activity than other treatments. There were no significant differences between 500 and 1000 pg/mL melatonin regarding meiotic stages. In conclusion, the concentration of 1000 pg/mL melatonin maintains survival, promotes follicular development and increases the levels of active mitochondria after in vitro culture of sheep secondary follicles. Moreover, this concentration promotes the meiotic competence of oocytes and decreases the production of ROS during oocyte maturation.

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This study aimed to evaluate the effect of melatonin on the in vitro culture and maturation of isolated sheep early antral follicles. Isolated early antral follicles were cultured for 12 d in α-minimum essential medium (MEM+) alone (control) or α-MEM+ added with fixed different concentrations (100, 500, or 1,000 pg/mL) or a sequential concentration of melatonin (MelSeq; day 6 = 100; day 12 = 500 pg/mL). The percentage of morphologically normal follicles was higher (P < 0.05) in 500 pg/mL melatonin than the other treatments at 6 d. Mel 500 also showed a higher rate of fully grown oocytes (P < 0.05) than other treatments. After in vitro culture, reactive oxygen species (ROS) levels in oocytes were similar between Mel 500 and MelSeq, with both being lower (P < 0.05) than other treatments. Oocytes cultured in both Mel 500 and Mel 1000 showed glutathione peroxidase levels similar (P > 0.05) to the control group and higher (P < 0.05) than other treatments. Mitochondrial activity was similar (P > 0.05) among control, Mel 500, and Mel 1000 treatments. Mel 500 treatment presented a higher percentage of germinal vesicle breakdown oocytes than the control group and similar percentages to the other treatments. Follicles cultured in melatonin followed by oocyte maturation with the addition of 500 pg/mL melatonin in maturation medium showed increased (P < 0.05) levels of mitochondrial activity compared to α-MEM+ alone. In conclusion, the concentration of 500 pg/mL of melatonin promotes development and decreases ROS levels of ovine oocytes from in vitro grown early antral follicles. Moreover, melatonin increases mitochondrial activity and promotes the acquisition of meiotic competence of these oocytes.

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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.

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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.

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This study evaluated the in vitro development and maturation of ovine oocytes from secondary follicles cultured in serum-free medium containing fixed or sequential concentrations of recombinant human FSH (rhFSH). Follicles were cultured in α-MEM+ alone or with constant (500, 750, or 1,000 ng/mL) or sequential concentrations of rhFSH (seq. 1: day 6 = 500; day 12 = 750; day 18 = 1,000 ng/mL and seq. 2: day 6 = 100; day 12 = 500; day 18 = 1,000 ng/mL). At the end of the experiment, follicular survival was higher (P < 0.05) in 750 ng/mL rhFSH than the control and 1,000 ng/mL rhFSH. As early as day 6 of culture, antral cavity formation was observed in all treatments. Follicular diameter increased progressively and significantly in all treatments throughout 18 d of culture. Furthermore, addition of rhFSH to the medium promoted a significant increase in the percentage of fully grown oocytes in all treatments compared to α-MEM+. Mitochondrial activity was higher in rhFSH treatments than in the control, except in rhFSH seq. 2 (P < 0.05). Maturation rates increased in oocytes from intact follicles cultured in 750 ng/mL rhFSH compared to the control (P < 0.05). In conclusion, rhFSH at 750 ng/mL maintained the survival of secondary follicles cultured in serum-free medium, improved oocyte growth, mitochondrial activity, and oocyte maturation.

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The aim of this study was to evaluate the effects of kaempferol on the morphology, follicular activation, growth, and DNA fragmentation of ovine preantral follicles cultured in situ, and the effects of a phosphatidylinositol 3 kinase (PI3K) inhibitor and the expression of phosphorylated protein kinase B (pAKT) after culture. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analyses (fresh control) or cultured in α-MEM+ alone (control) or with different concentrations of kaempferol (0.1, 1, 10, or 100 µM) for 7 days. Follicles were classified as normal or atretic, primordial or growing, and the oocyte and follicle diameters were measured. Proliferating cells were analyzed and DNA fragmentation was evaluated by the TUNEL assay. Inhibition of PI3K activity was performed through pretreatment in media added with 50 µM LY294002 for 1 hr and pAKT immunohistochemistry was performed after culture in the absence or presence of LY294002. After culture, the percentage of normal follicles was similar among the treatments (p > 0.05), except for 100 µM kaempferol, which had less normal follicles (p < 0.05). Moreover, kaempferol at 10 µM showed a higher percentage of follicular activation and cell proliferation than the other treatments (p < 0.05) and a percentage of TUNEL-positive cells similar to that in the fresh control and lower than other treatments (p < 0.05). LY294002 significantly inhibited primordial follicle activation stimulated by α-MEM+ and 10 µM kaempferol and reduced pAKT expression in those follicles. In conclusion, 10 µM kaempferol promotes primordial follicle activation and cell proliferation through the PI3K/AKT pathway and reduces DNA fragmentation of ovine preantral follicles cultured in vitro.

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SummaryThe present study aimed to investigate the effect of quercetin as an alternative antioxidant to cysteamine on in vitro maturation. Oocytes were collected from goat ovaries, destined for in vitro maturation and distributed into three groups: CIS group, oocytes were immersed in MIV base medium; in Groups Q4 and Q8, oocytes were immersed in the medium of the CIS group, adding 4 µM or 8 µM of quercetin, respectively, and cultured for 24 h at 38.5°C with 5% CO2. The CIS and Q4 groups presented the same percentage of expanded cumulus cells, but the per cent in the Q8 group was significantly lower than that of the other groups (P<0.05). The oocyte retraction rate in the Q8 group was higher (P<0.05) than in the CIS and Q4 groups. Treatment with 8 µM of quercetin presented a lower proportion of expanded oocytes than the CIS group and 4 µM of quercetin (P<0.05). The percentage of MII oocytes was higher in the Q4 group than in the CIS group (P<0.05), but the percentages in the CIS and Q8 groups were similar. The rate of apoptosis was higher in the CIS group than in the other groups (P<0.05). In addition, oocytes matured with 4 µM quercetin showed higher mitochondrial activity than matured oocytes in the CIS and Q8 groups (P<0.05). In conclusion, 4 µM of quercetin can be used as an alternative to cysteamine in the in vitro maturation of goat oocytes.

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