Evaluation of miRNAs regulation of BDNF and IGF1 genes in T2DM insulin resistance in experimental models: bioinformatics based approach.

microRNAs (miRNAs) are recognized as diabetes mellitus type 2 (T2DM) biomarkers useful for disease metabolism comprehension and have great potential as therapeutics targets. BDNF and IGF1 increased expression are highly involved in the benefits of insulin and glucose paths, however, they are down-regulated in insulin resistance conditions, while their expression increase is correlated to the improvement of glucose and insulin metabolism. Studies suggest the microRNA regulation of these genes in several different contexts, providing a novel investigation approach for comprehending T2DM metabolism and revealing potential therapeutic targets. In the present study, we investigate in different animal models (human, rat, and mouse) miRNAs that target BDNF and IGF1 in skeletal muscle tissue with T2DM physiological conditions. Bioinformatics tools and databases were used to miRNA prediction, molecular homology, experimental validation of interactions, expression in the studied physiological condition, and network interaction. The findings showed three miRNAs candidates for IGF1(miR-29a, miR-29b, and miR-29c) and one for BDNF (miR-206). The experimental evaluations and the search for the expression in skeletal muscle from T2DM subjects confirmed the predicted interaction between miRNA-mRNA for miR-29b and miR-206 through human, rat, and mouse models. This interaction was reaffirmed in multiple network analyses. In conclusion, our results show the regulation relationship between miR-29b and miR-206 with the investigated genes, in several tissues, suggesting an inhibition pattern. Nevertheless, these data show a large number of possible interaction physiological processes, for future biotechnological prospects.

In vitro cytotoxic effects of 5-Fluorouracil on isolated murine ovarian preantral follicles.

5 fluorouracil (5FU), an antineoplastic drug, is often utilized in the therapeutic regimen for several types of cancer, including the hepatoblastoma in children. The effects of 5FU on the population of ovarian preantral follicles, which is the largest oocyte reservoir, is still poorly understood. The integrity of the ovarian preantral follicle pool is important for lifelong fertility. The better understanding of such effects may favor intervention strategies to protect fertility in 5FU-treated children and women coping with cancer. To analyze the effects of 5FU on isolated murine secondary follicles in vitro, ovaries were collected from young mice (28-30 days old), and secondary follicles were isolated and cultured for 12 days in basic culture medium, with or without 5FU at concentrations of 0.3 mM, 1 mM, 3 mM, 10 mM, and 30 mM. In the in vitro study, we analyzed the percentage of morphologically normal follicles, antrum formation, follicular diameter, and hormone production. On day 12, oocytes were recovered for in vitro maturation. 5FU treatment did not alter the percentage of morphologically normal follicles. On day 12, only 1, 10, and 30 mM 5FU significantly reduced the percentage of antrum. From day 4 onwards, 5FU treatments significantly reduced follicle diameter. The meiosis resumption rate was significantly lower in all 5FU treatments. 5FU concentrations ≥3 mM reduced estradiol levels. In conclusion, 5FU does not affect follicular morphology. However, 5FU deleteriously affects follicular growth, estradiol production, and oocyte maturation in isolated ovarian follicles.

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.

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.

Effect of bone morphogenetic proteins 2 and 4 on survival and development of bovine secondary follicles cultured in vitro.

This study evaluated the effect of bone morphogenetic proteins 2 (BMP2) and 4 (BMP2) on follicle development and mRNA expression for GDF9, Cyclin B1, BMPR1A, BMPR1B, BMPRII, FSHR and SMAD1 in bovine secondary follicles cultured in vitro. Isolated secondary follicles were cultured for 18 days in TCM199+ medium alone or supplemented with BMP2 (10 ng/mL), BMP4 (100 ng/mL) or combination of both BMP2 and 4. Real-time PCR was used to analyze mRNA levels in fresh and cultured follicles. After 18 days of culture, follicles cultured with BMP2 alone or with BMP4 alone had larger diameters when compared to control (P < .05). In addition, all treatments promoted antrum formation and maintained a high viability rate through the growing period. The presence of BMP2, BMP4 or both together did not influence mRNA expression for the tested genes. However, the in vitro culture induces down-regulation for mRNA expression of BMPR1A. In conclusion, the addition of BMP2 or BMP4 alone in cultured medium promotes follicular growth and antrum formation in bovine follicles after 18 days of 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.

In vitro growth and maturation of isolated caprine preantral follicles: Influence of insulin and FSH concentration, culture dish, coculture, and oocyte size on meiotic resumption.

The aims of this study were: (1) to evaluate the effect of different insulin concentrations, alone or in combination with either a fixed FSH concentration or increasing FSH concentrations on the in vitro culture of isolated caprine preantral follicles and (2) to analyze the efficiency of two IVM media and maturation culture systems (with or without coculture with in vivo grown oocytes) on the meiosis resumption. Secondary follicles were cultured for 18 days in a basic medium supplemented with low- or high-insulin concentration alone or with a fixed FSH concentration or with increasing FSH concentrations. Oocytes grown in vivo or in vitro were matured alone or cocultured. The high-insulin concentration associated with fixed FSH treatment had higher meiotic resumption rate (P < 0.05) and was the only treatment capable of producing oocytes in metaphase II. The rates of germinal vesicle, germinal vesicle breakdown, metaphase I, metaphase II (MII), meiotic resumption, and oocyte diameter were similar between the maturation media. In conclusion, a basic medium supplemented with 10-µg/mL insulin and 100-µg/mL FSH throughout the culture period improved meiotic resumption rate and produced MII oocytes from caprine preantral follicles cultured in vitro. The MII rate was similar between in vivo and in vitro grown oocytes ≥110 µm.

Anethole improves the in vitro development of isolated caprine secondary follicles.

The aim of this study was to investigate the effect of three concentrations of anethole (30, 300, and 2000 µg/mL) on survival, antrum formation, follicular diameter, and oocyte maturation in the caprine species. The study also evaluated the effects of anethole on transcripts of ICAM-1, CAV-1, TIMP-2, and PAI-1 genes and levels of reactive oxygen species (ROS) in isolated goat preantral ovarian follicles before and after in vitro culture for 18 days. Preantral follicles were isolated from goat ovaries and individually cultured in alpha minimum essential medium modified (α-MEM+), defined as the control treatment, α-MEM+ supplemented with ascorbic acid at a concentration of 100 µg/mL (AA), or α-MEM+ supplemented with three different concentrations of anethole (30, 300, 2000 µg/mL) for a period of 18 days. Treatments were named as α-MEM+, AA, AN30, AN300, and AN2000, respectively. After culture, the follicles were opened, the cumulus oocytes complex (COCs) were removed and matured in vitro. The walls of the follicles were used for the quantitation of mRNA by quantitative real-time polymerase chain reaction. Finally, the medium collected at the end of culture was used for the measurements of ROS. After 18 days of culture, the AA treatment showed the percentage of intact follicles and follicular diameter significantly higher compared with the other treatments. However, daily growth rate, antrum formation, and also oocyte diameter were similar among the treatments. In addition, compared with AA, the rate of oocytes for in vitro maturation (diameter ≥ 110 µm) and the meiosis resumption rate were significantly higher in the treatments AN30 and AN2000, respectively. When assessing gene related to remodeling of the basement membrane, significant differences in mRNA levels for ICAM-1, CAV-1, TIMP-2, and PAI-1 were observed in comparison with Day 0, i.e., in the noncultured control. In addition, the ROS from Day 12, all treatments with the addition of anethole have significantly lower values of ROS than α-MEM+ and AA. In conclusion, the addition of anethole to the in vitro culture medium was able to improve the development of goat preantral follicles by reducing concentrations of ROS and increasing the percentage of oocytes able to resume meiosis.

Effects of Growth Hormone on In Situ Culture of Bovine Preantral Follicles are Dose Dependent.

The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross-breeds defined were cultured in situ for 1 and 7 days in minimal essential medium (α-MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non-cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey's and Dunnett's tests) and chi-square test (χ(2) ). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ.

Effects of IGF-1 on In Vitro Culture of Bovine Preantral Follicles are Dose-Dependent.

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF-1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α-MEM(+) , supplemented with different concentrations of human recombinant IGF-1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO2 in 24-well plates with total replacement of the medium every 2 days. Non-cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF-1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF-1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF-1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF-1 tested. Ultrastructurally, the non-cultured control and IGF-1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF-1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.

Balance of insulin and FSH concentrations improves the in vitro development of isolated goat preantral follicles in medium containing GH.

The aim of this study was to evaluate the effect of different combinations of insulin and FSH concentrations in culture media containing GH on the in vitro follicle morphology, antrum formation, growth rates, estradiol (E2) production, oocyte viability and maturation as well as gene expression for FSHR, GHR, INSR, CYP19A1, CYP17, 3ßHSD. Secondary follicles were individually cultured for 18 days in a basic medium containing 50ng/mL GH supplemented with low insulin concentration (INS-LW: 10ng/mL) or high insulin concentration (INS-HG: 10µg/mL) alone or with a fixed FSH concentration (FSH100: 100ng/mL) or with increasing FSH concentrations (FSH-SEQ: 100ng/mL, days 0-6; 500ng/mL, days 6-12; 1000ng/mL days 12-18). In the INS-LW treatment was observed a higher (P<0.05) incidence of normal follicles at day 18 of culture. However, overall higher (P<0.05) follicular growth, oocyte diameter and meiotic resumption rates were obtained using INS-HG+FSH 100. The INS-HG and INS-HG+FSH100 treatments showed higher E2 production and mRNA levels for CYP19A1, CYP17, 3ßHSD when compared to INS-LW and INS-LW+FSH100. However, the addition of increasing FSH concentration, regardless of insulin concentration, did not improve the follicular growth, meotic resumption, E2 production or gene expression of steroidogenic enzymes when compared with INS-HG+FSH100. In conclusion, in presence of GH, a basic medium supplemented with 10µg/mL insulin and 100µg/mL FSH throughout the culture period, improves follicular and oocyte growth, oocyte meiotic resumption and E2 production from isolated preantral caprine follicles cultured in vitro.

Amburana cearensis leaf extract maintains survival and promotes in vitro development of ovine secondary follicles.

The antioxidant properties of Amburana cearensis extract may be a useful substitute for standard cell culture medium. Thus, the aim of this study was to evaluate the effect of this extract, with or without supplementation, on in vitro survival and development of sheep isolated secondary follicles. After collection of the ovaries, secondary follicles were isolated and cultured for 18 days in α-MEM+ supplemented with bovine serum albumin, insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (control medium) or into medium composed of different concentrations of A. cearensis extract without supplements (Amb 0.1; 0.2 or 0.4 mg/ml) or A. cearensis extract supplemented with the same substances described above for α-MEM+ supplementation. The A. cearensis supplemented medium was named Amb 0.1+; 0.2+ or 0.4+ mg/ml. There were more morphologically normal follicles in Amb 0.1 or Amb 0.4 mg/ml than in the control medium (α-MEM+) after 18 days of culture. Moreover, the percentage of antrum formation was significantly higher in Amb 0.1 or Amb 0.2 mg/ml than in α-MEM+ and Amb 0.1+ mg/ml, and similar to the other treatments. All A. cearensis extract media induced a progressive and significant increase in follicular diameter throughout the culture period. In conclusion, this study showed that 0.1 mg/ml of this extract, without supplementation, maintains follicular survival and promotes the development of ovine isolated secondary follicles in vitro. This extract can be an alternative culture medium for preantral follicle development.

Bone Morphogenetic Protein-6 (BMP-6) Stimulates the Antrum Formation by the Regulation of its Signalling Pathway in Caprine Pre-antral Follicles Cultured In Vitro.

BMP-6 has been found to be important to ovarian cells and oocyte, as well as to uterus. Thus, this study investigated the effect of bone morphogenetic protein (BMP-6) and recombinant follicle-stimulating hormone (rFSH) alone or in combination on the in vitro culture (IVC) of isolated caprine secondary follicles (Experiment 1) and the mRNA levels for BMP receptors/Smad signalling pathway (BMPR1A, BMPR2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7 and SMAD8) in vivo and in vitro using BMP-6 (Experiment 2). Secondary follicles were cultured in αMEM(+) alone (control medium) or supplemented with BMP-6 at 1 or 10 ng/ml and rFSH alone or the combination of both BMP-6 concentrations and rFSH. The results from Experiment 1 showed that the antrum formation rate was higher in the BMP-6 at 1 ng/ml (p < 0.05) than in MEM. In Experiment 2, the mRNA expression for BMPR2, SMAD1, SMAD5 and SMAD6 was detected in non-cultured control and after in vitro culture (MEM and 1 ng/ml BMP-6); while the expression of SMAD7 and SMAD8 mRNA was only detected after IVC, SMAD4 was only detected in the BMP-6 at 1 ng/ml treatment. In conclusion, the low BMP-6 concentration positively influenced antrum formation and ensured normal mRNA expression for BMP receptor and Smads after IVC of caprine secondary follicles.

Long-term in vitro culture of bovine preantral follicles: Effect of base medium and medium replacement methods.

Two culture media and replacement methods were compared during long-term in vitro culture of secondary follicles of cattle using α-MEM(+) or TCM-199(+) as base media. The medium replacement methods were: Conventional - removal and subsequent addition of the same amount (60µl) in a 100µl aliquot (MEM-C and TCM-C), and Small Supplementation - addition of 5µl of fresh medium to an initial small aliquot (50µl), resulting in a final volume of 125µl on the last day of culture (MEM-S and TCM-S). A total of 207 secondary follicles were cultured individually for 32 days at 38.5°C in 5% CO2 and medium replacement was performed every other day. The MEM-S treatment resulted in a larger (P<0.01) follicular diameter, greater (P<0.02) growth rate, greater (P<0.02) antrum formation, as well as greater (P<0.0001) estradiol concentrations when compared with the MEM-C treatment. The medium change methods did not affect (P>0.05) the follicular and estradiol end points for TCM-199(+). The expression of the FSHR gene was greater (P<0.03) with the TCM-C than TCM-S treatment, while the relative amounts of mRNA for IGF1 was greater (P<0.02) with MEM-S than TCM-S treatments and for VEGF was greater (P<0.02) with MEM-C than TCM-C treatment. In conclusion, the type of base medium and the effect of periodic addition of medium differentially affected follicle development, estradiol production, and gene expression. Furthermore, α-MEM(+) can be used to replace TCM-199(+) for culture of preantral follicles of cattle if progressive addition of medium is used for medium change.

In vitro development of secondary follicles from pre-pubertal and adult goats cultured in two-dimensional or three-dimensional systems.

The aim of this study was to evaluate the influence of two-dimensional (2D) and three-dimensional (3D) alginate culture systems on in vitro development of pre-antral caprine follicles. In addition, the influence of the reproductive age of the ovary donor on the in vitro culture success was investigated. Pre-antral follicles from pre-pubertal or adult goats were isolated and cultured directly on a plastic surface (2D) or encapsulated in an alginate-based matrix (3D). After 18 days, the oocytes underwent in vitro maturation (IVM) and in vitro fertilization (IVF) to produce embryos. The 3D system showed higher rates of follicle survival, lower rates of oocyte extrusion, and a greater number of recovered oocytes for IVM and IVF (P < 0.05). Only pre-antral follicles from adult animals produced MII oocytes and embryos. The estradiol concentrations increased from day 2 to day 12 of culture in all groups tested (P < 0.05). Conversely, progesterone concentrations were lower in 3D-cultured follicles than in 2D-cultured follicles, with differences on days 2 and 6 of culture (P < 0.05). We provide compelling evidence that a 2D or 3D alginate in vitro culture system offers a promising approach to achieving full in vitro development of caprine pre-antral follicles to produce mature oocytes that are capable of fertilization and viable embryos.

Effect of sequential medium with fibroblast growth factor-10 and follicle stimulating hormone on in vitro development of goat preantral follicles.

A sequential medium with fibroblast growth factor-10 (FGF-10) and follicle stimulating hormone (FSH) was evaluated on the survival, ultrastructure, activation and growth rate of caprine preantral follicles submitted to long-term culture, aiming to establish an ideal in vitro culture system. Ovarian fragments were cultured for 16 days in α-MEM(+) alone or supplemented with FGF-10 and/or FSH added sequentially on different days of culture. Ovarian fragments were cultured during the first (days 0-8) and second (days 8-16) halves of the culture period, generating 10 treatments: α-MEM(+)/α-MEM(+) (cultured control), FSH/FSH, FSH/FGF-10, FSH/FSH+FGF-10, FGF-10/FGF-10, FGF-10/FSH, FGF-10/FSH+FGF-10, FSH+FGF-10/FSH+FGF-10, FSH+FGF-10/FSH and FSH+FGF-10/FGF-10. Follicle morphology, viability and ultrastructure were analyzed. The FSH/FGF-10 treatment showed a higher (P<0.05) percentage of normal follicles compared to all other treatments. In addition, follicles from the FSH/FGF-10 treatment maintained ultrastructural integrity after the culture period. After 16 days of culture, the FSH/FGF-10 and FSH/FSH treatments showed a higher percentage of activation compared to the cultured control (α-MEM(+)/α-MEM(+)). Moreover, the FSH/FGF-10 treatment promoted greater follicular and oocyte diameters compared to the fresh control. In conclusion, this study showed that a sequential medium with FSH followed by FGF-10 (FSH/FGF-10 and FSH/FSH) maintains follicular viability and ultrastructure and promotes transition from the primordial to primary stage (activation) and growth in goat preantral follicles cultured in vitro.

In vitro development of bovine secondary follicles in two- and three-dimensional culture systems using vascular endothelial growth factor, insulin-like growth factor-1, and growth hormone.

The aim of this study was to evaluate the development and estradiol production of isolated bovine secondary follicles in two-dimensional (2D, experiment 1) and three-dimensional (3D using alginate, experiment 2) long-term culture systems in the absence (control group; only α-MEM(+)) or presence of vascular endothelial growth factor (VEGF), insulin-like growth factor-1, or GH alone, or a combination of all. A total of 363 isolated secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO2 in a humidified incubator with addition of medium (5 µL) every other day. In 2D culture system, follicular growth and antrum formation rates were higher (P < 0.05) in VEGF treatment compared with the other treatments. In 3D culture system, only estradiol concentration was greater (P < 0.05) in the GH than in the control group, whereas the other end points were similar (P > 0.05). In summary, this study demonstrated that the benefits of using a certain type of medium supplement depended on the culture system (2D vs. 3D). Vascular endothelial growth factor was an effective supplement for the in vitro culture of bovine secondary follicles when the 2D culture system was used, whereas GH only affected estradiol production using the 3D culture system. This study sheds light on advancements in methodology to facilitate subsequent studies on bovine preantral follicle development.

Protein localization of epidermal growth factor in sheep ovaries and improvement of follicle survival and antrum formation in vitro.

The aims of this study were to characterize EGF protein expression in ovine ovaries and to verify the effect of EGF on the in vitro development of isolated pre-antral follicles. After collection, ovarian tissue was fixed for immunohistochemical analysis. Additional pairs of ovaries were collected, and secondary follicles were cultured for 18 days in α-MEM(+) (control) alone or supplemented with EGF (1, 10 or 50 ng/ml). The immunostaining for EGF was observed in oocytes from pre-antral and antral follicles, in granulosa cells of primary and secondary follicles, as well as in cumulus and mural cells of antral follicles. After 18 days, the results showed that treatment with 50 ng/ml EGF significantly increased the percentage of morphologically normal follicles compared with the control group (α-MEM(+) ) and significantly reduced the precocious extrusion of oocytes and increased the percentage of antral follicles compared with the control and 1 ng/ml EGF. All the treatments induced a progressive and significant increase of the follicular diameter throughout the period of culture. However, there were no significant differences in follicular diameter or in the daily growth rate among treatments. In conclusion, this study demonstrated the presence of EGF in ovine ovaries. Moreover, 50 ng/ml EGF increased the percentage of normal follicles and improved antrum formation in isolated ovine follicles after 18 days of in vitro culture.

Immunolocalization of melatonin and follicle-stimulating hormone receptors in caprine ovaries and their effects during in vitro development of isolated pre-antral follicles.

The expression of melatonin type 1 (MT1) and FSH (FSHR) receptors in caprine ovaries and the effects of these hormones on the in vitro development of isolated pre-antral follicles were evaluated. Follicles (≤200 µm) were cultured for 12 days in α-MEM (control) or melatonin (100 or 1000 pg/ml) or sequential melatonin medium (100 pg/ml: from day 0 to day 6; 1000 pg/ml: from day 6 to day 12; experiment 1) and in control or sequential FSH (100 ng/ml from day 0 to day 6; 500 ng/ml from day 6 to day 12) or sequential melatonin or this latter plus sequential FSH (experiment 2). MT1 and FSHR expressions were observed in granulosa cells from secondary and antral follicles. The oocytes from primordial and primary follicles also express FSHR. Sequential melatonin increased the percentage of normal follicles and oocyte recovery compared with the control or melatonin (1000 pg/ml) at day 12. In experiment 2, all the treatments increased the normal follicles and growth compared with the control. In conclusion, this study demonstrated the presence of MT1 and FSHR in caprine ovaries. The addition of increased concentrations of melatonin (sequential medium) or FSH can be used to promote the in vitro development of caprine pre-antral follicles.