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.

Melatonin improves development, mitochondrial function and promotes the meiotic resumption of sheep oocytes from in vitro grown secondary follicles.

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.

Effects of melatonin on the in vitro growth of early antral follicles and maturation of ovine oocytes.

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.

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.

In vivo and in vitro strategies to support caprine preantral follicle development after ovarian tissue vitrification.

The aim of the present study was to compare fresh and vitrified goat ovarian tissue after autotransplantation and in vitro culture. Adult goats were completely ovariectomised and each ovarian pair was sliced and distributed among six different treatment groups: fresh control, fresh transplant, fresh culture, vitrified control, vitrified transplant and vitrified culture. Follicular morphology, development, growth, density, revascularisation and hormone production were evaluated in all groups. Three antral follicles (two in the fresh transplant and one in the vitrified transplant groups) were observed on the surface of the graft 90 days after transplantation. The percentage of morphologically normal follicles was similar in the fresh control, fresh transplant and vitrified transplant groups. The percentage of developing (transition, primary and secondary) follicles was higher after in vitro culture of fresh or vitrified tissue. Transplantation resulted in a lower follicle density. Serum oestradiol concentrations remained constant during the entire transplantation period. In contrast, progesterone production decreased significantly. Expression of CD31 mRNA was lower in fresh culture. In conclusion, restoration of goat ovarian function can be successfully achieved following transplantation of both fresh and vitrified goat ovarian tissue. However, transplantation induced higher follicle loss than in vitro culture.

Expectations and limitations of ovarian tissue transplantation.

Constant progress in the diagnosis and treatment of cancer disease has increased the number and prognosis of cancer survivors. However, the toxic effects of chemotherapy and radiotherapy on ovarian function have resulted in premature ovarian failure. Patients are, therefore, still expecting methods to be developed to preserve their fertility successfully. Several potential options are available to preserve fertility in patients who face premature ovarian failure, including immature or mature oocyte and embryo cryopreservation. However, for children or prepubertal women needing immediate chemotherapy, cryopreservation of ovarian tissue is the only alternative. The ultimate aim of this strategy is to implant ovarian tissue into the pelvic cavity (orthotopic site) or in a heterotopic site once oncological treatment is completed and the patient is disease free. Transplantation of ovarian tissue with sufficiently large numbers of follicles could potentially restore endocrine function and allow multiple cycles for conception. However, the success of ovarian tissue transplantation still has multiple challenges, such as the low number of follicles in the graft that may affect their longevity as well as the survival of the tissue during ex vivo processing and subsequent transplantation. Therefore, this review aims to summarize the achievements of ovary grafting and the potential techniques that have been developed to improve ovarian graft survival.

Effect of the aqueous extract of Senecio biafrae (Oliv. & Hiern) J. Moore on some fertility parameters in immature female rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Senecio biafrae is a plant from the huge family of Asteraceae used in the African pharmacopoeia for the treatment of many ailments among which is infertility. MATERIAL AND METHODS: The aqueous extract, which was primarily subjected to polyphenol analysis, has been administered to immature female rats for 20 days at 8, 32, 64 and 128 mg/kg of body weight. The day following the treatment, the animals were sacrificed; their serum, ovary and uterus were retained respectively for reproductive hormones, ovarian and uterine proteins, and ovarian cholesterol assays. RESULTS: Light body weight gain variation of treated animals was observed during the experimental period. A significant increase (p ˂ 0.05) in serum estradiol and proteins as well as in uterine weight (p ˂ 0.01) of all Senecio biafrae treated animals was noted. No significant variation was noticed in the ovarian weight and follicle numbers. CONCLUSION: The various biochemical and physiological parameters of fertility were significantly improved with the aqueous extract of Senecio biafrae, thus attesting some of its traditional usage.