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.

Epigallocatechin-3-gallate (EGCG) reduces apoptosis of preantral follicles through the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway after in vitro culture of sheep ovarian tissue.

The aims of this study were to analyze the effects of different concentrations of epigallocatechin-3-gallate (EGCG) on the primordial follicle survival and development after in vitro culture of ovarian tissue, and to verify the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway in the EGCG actions in the sheep ovary. Ovarian fragments were fixed for histological analysis (fresh control) or cultured in α-minimum essential medium alone (α-MEM+: control medium) or with different concentrations of EGCG (0.01; 0.1; 1; 10 or 100 µg/mL) for 7 days. Inhibition of PI3K activity was performed in fragments cultured with 1 µg/mL EGCG plus LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3 and AKT phosphorylation (p-AKT). The results showed that 1 µg/mL EGCG maintained the follicular survival similar (P > 0.05) to that of the fresh control and higher (P < 0.05) than that of the α-MEM+ and other EGCG treatments. No difference (P > 0.05) in the follicular activation was observed. However, both follicle and oocyte diameters increased after in vitro culture with 1 µg/mL EGCG compared to other treatments (P < 0.05), except for 10 µg/mL EGCG (P > 0.05). After PI3K inhibition, there was an increase (P < 0.05) of the follicular apoptosis and a reduction of p-AKT immunolocalization. In conclusion, EGCG at 1 µg/mL reduces apoptosis of preantral follicles through the PI3K/AKT pathway after in vitro culture of sheep ovarian tissue.

Reply to Comment "Assessing Porous Media Permeability in Non-Darcy Flow: A Re-Evaluation Based on the Forchheimer Equation".

In the name of the authors of the paper "Permeability versus Design in TPMS Scaffolds", published in Materials in 2019 and already counting six citations in Google Scholar to date, I would like to formally respond to the comment manuscript now entitled "Assessing Porous Media Permeability in Non-Darcy Flow: A Re-Evaluation Based on the Forchheimer Equation" [...].

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.

Permeability versus Design in TPMS Scaffolds.

Scaffolds for bone tissue engineering are porous structures that serve as support for cellular growth and, therefore, new tissue formation. The present work assessed the influence of the porous architecture of triply periodic minimal surface (TPMS) scaffolds on their macroscopic permeability behavior, combining numerical and experimental methods. The TPMS scaffolds considered were Schwartz D, Schwartz P, and Gyroid, which have been previously studied for bone tissue engineering, with 70% porosity. On the experimental side, these scaffolds were produced by MultiJet 3D printing and tested for fluid passage to calculate their permeability through Darcy's Law. On the numerical side, finite element (FE) models of the scaffolds were simulated on ABAQUS® for fluid passage under compression to assess potential fluid concentration spots. The outcomes revealed that the design of the unit cell had a noticeable effect on both calculated permeability and FE computed fluid flow velocity, regardless of the identical porosity, with the Gyroid scaffold having higher permeability and the Schwartz P a lower probability of fluid trapping. Schwartz D had the worst outcomes in both testing modalities, so these scaffolds would most likely be the last choice for promoting cell differentiation onto bone cells. Gyroid and Schwartz P would be up for selection depending on the application and targeted bone tissue.

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.

In vitro survival, growth, and maturation of sheep oocytes from secondary follicles cultured in serum-free conditions: impact of a constant or a sequential medium containing recombinant human FSH.

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.

Kaempferol promotes primordial follicle activation through the phosphatidylinositol 3-kinase/protein kinase B signaling pathway and reduces DNA fragmentation of sheep preantral follicles cultured in vitro.

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.

Quercetin influences in vitro maturation, apoptosis and metabolically active mitochondria of goat oocytes.

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.

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.

Use of protocatechuic acid as the sole antioxidant in the base medium for in vitro culture of ovine isolated secondary follicles.

This study evaluated the effect of the protocatechuic acid (PCA) as the sole antioxidant in the base medium for in vitro culture of ovine secondary follicles. Secondary follicles (200-230 µm) were isolated and cultured in α-minimal essential medium supplemented with BSA, insulin, glutamine and hypoxanthine (α-MEM: antioxidant-free medium) or α-MEM also added by transferrin, selenium and ascorbic acid (α-MEM+: with antioxidant) or α-MEM added by PCA (56.25; 112.5; 225; 450; or 900 µg/ml). Moreover, after culture, oocytes were matured and the chromatin configuration and DNA fragmentation were evaluated. After 12 days, the treatment containing 56.25 µg/ml PCA showed higher percentage of normal follicles than control medium or the other treatments (p < .05), except for 900 µg/ml PCA (p > .05). The antrum formation was significantly higher in treatments containing 56.25, 112.5 or 900 µg/ml PCA, compared to the α-MEM and similar (p > .05) to the other treatments. The rates of fully grown oocytes (≥110 µm) were similar (p > .05) among all treatments containing PCA and α-MEM+, and those were superior (p < .05) than α-MEM, except for 450 µg/ml PCA (p > .05). GSH levels and mitochondrial activity were higher (p < .05) in α-MEM+ than in α-MEM and similar (p > .05) to all PCA treatments. The rates of meiotic resumption and DNA fragmentation were similar (p > .05) among α-MEM+ and 56.25 µg/ml PCA. In conclusion, PCA at 56.25 µg/ml as the sole antioxidant added to the medium for ovine isolated secondary follicle culture maintains follicular survival, GSH and active mitochondria levels, meiotic developmental competence and DNA integrity of cultured oocytes.

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.

Kit ligand promotes the transition from primordial to primary follicles after in vitro culture of ovine ovarian tissue.

This study evaluated the effects of kit ligand (KL) on the morphology and development of ovine preantral follicles (fresh control) and after 7 days of in vitro culture in α-Minimal Essential Medium (α-MEM; control medium) or the presence of KL (1, 10, 50, 100 or 200 ng/ml). There was an increase in the percentage of primary follicles at the concentration of 100 ng/ml KL, compared with the fresh control, control medium (α-MEM) and the other KL concentrations. Follicle diameter was significantly higher than the control medium only at concentrations of 50 and 100 ng/ml KL. In conclusion, 100 ng/ml KL promoted the transition from primordial to primary follicles (follicular activation) after in vitro culture of ovine ovarian tissue.

Lower apoptosis rate in ovine preantral follicles from ovaries stored in supplemented preservation media.

The aim of this study was to investigate the effect of ovarian tissue transportation conditions (medium and period of time) on the morphology, apoptosis and development of ovine preantral follicles cultured in vitro. Each ovarian pair was cut into nine slices, with one fragment being fixed immediately (fresh control). The remaining fragments were placed individually in cryotubes containing conservation medium (minimal essential medium (MEM) without supplementation or MEM+ - with supplementation) and stored at 35ºC for 6 or 12 h without (non-cultured) or with subsequent culture for 5 days. Then, the fragments were processed for histological and terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labelling (TUNEL) examination. Preservation of ovarian slices in MEM or MEM+ (non-cultured) resulted in similar percentages of normal follicles when compared with the fresh control. Nevertheless, compared with the fresh control, a decrease in the percentage of normal follicles was observed in tissues cultured for 5 days. Only for tissues preserved in supplemented medium (MEM+) for 6 h, the percentage of TUNEL positive cells was similar between non-cultured tissues and tissues cultured for 5 days. Follicular activation and growth (follicular and oocyte diameter) were higher in cultured tissues than in fresh control or non-cultured tissues, except those from fragments preserved for 6 h in MEM and then cultured for 5 days in which no growth was observed. In conclusion, ovine ovarian tissue was successfully preserved in supplemented medium (MEM+) at a temperature close to physiological values (35°C) for up to 6 h without affecting apoptosis in the ovarian follicles and their ability to develop in vitro.

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.