J.B. Bruno

Dynamic medium produces caprine embryo from preantral follicles grown in vitro.

The aim of this study was to develop a dynamic culture medium containing FSH, LH and EGF to promote the in vitro development of oocytes obtained from goat preantral follicles to complete maturation and to improve the capacity of these oocytes for in vitro fertilization (IVF) and embryo production. For experiment I, preantral follicles were cultured for 18 days in medium supplemented with increasing concentrations of FSH (T1 - control) or in control medium added LH alone or in association with EGF: T2 (LH 50 ng/ml), T3 (LH 50 ng/ml + EGF 50 ng/ml), T4 (LH 50 ng/ml + EGF 100 ng/ml), T5 (LH 100 ng/ml), T6 (LH 100 ng/ml + EGF 50 ng/ml) and T7 (LH 100 ng/ml + EGF 100 ng/ml). For experiment II, preantral follicles were cultured only in the culture medium used in T7, and after 18 days, their oocytes underwent in vitro maturation (IVM) followed by IVF. At the end of the culture period, T3, T4 and T7 had a positive influence on the daily follicular growth rate. Oocytes grown in T4 and T7 had a meiosis resumption percentage significantly superior to the other treatments. Two embryos were obtained, in which preantral follicles in medium supplemented with 100 ng/ml LH and 100 ng/ml EGF (T7). In conclusion, our sequential culture system was able to promote the in vitro growth of preantral follicles, promoting their oocyte maturation and caprine embryo production from preantral follicles.

Growth and differentiation factor-9 stimulates activation of goat primordial follicles in vitro and their progression to secondary follicles.

The aim of the present study was to investigate the effects of growth and differentiation factor-9 (GDF-9) on the survival and activation of preantral follicles, as well as their subsequent progression to secondary follicles, using goat ovarian cortical culture in vitro. Pieces of ovarian cortex were cultured for 1 and 7 days in minimum essential medium (MEM) with or without different concentrations of GDF-9 (1-200 ng mL(-1)). On Day 0 and after 1 and 7 days of culture, cortical pieces were fixed for histological and transmission electron microscopy evaluation. Preantral follicles were classified according to their development stage (primordial, intermediate, primary and secondary) and on the basis of morphological features (normal or degenerated). In addition, follicular and oocyte diameters were determined before and after culture. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P < 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL(-1) GDF-9. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P < 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 7 days of culture in MEM plus 200 ng mL(-1) GDF-9. At all concentrations of GDF-9 tested, follicular diameter increased significantly after 7 days of culture compared with non-cultured cortical tissue. In conclusion, the results of the present study indicate that 200 ng mL(-1) GDF-9 maintains the survival of preantral follicles and promotes activation of primordial follicles. Furthermore, GDF-9 stimulates the transition from primary to secondary follicles, maintaining ultrastructural integrity of the follicles.

Expression of follicle-stimulating hormone receptor (FSHR) in goat ovarian follicles and the impact of sequential culture medium on in vitro development of caprine preantral follicles

This study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 μm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus-oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.

Follicle stimulating hormone and fibroblast growth factor-2 interact and promote goat primordial follicle development in vitro

The aims of the present study were to investigate the effects of the interaction between follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on survival, follicular growth initiation and further growth of caprine preantral follicles. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM) supplemented with FSH, FGF-2 or FSH + FGF-2. Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM) to verify follicular morphology and growth. The results showed that, after 7 days culture, the highest percentages of normal follicles were observed in medium supplemented with FSH. After 7 days culture, the interaction between FSH and FGF-2 was most effective to promote the initiation of primordial follicles growth and oocyte growth. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in all treatments, except in those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that the interaction between FSH and FGF-2 stimulates the initiation of primordial follicles growth and the subsequent growth of developing follicles. Furthermore, these data showed that FSH is important to maintain follicular integrity after 7 days culture.

Steady-state level of kit ligand mRNA in goat ovaries and the role of kit ligand in preantral follicle survival and growth in vitro.

The aims of this study were to investigate steady‐state level of Kit Ligand (KL) mRNA and its effects on in vitro survival and growth of caprine preantral follicles. RT‐PCR was used to analyze caprine steady‐state level of KL mRNA in primordial, primary, and secondary follicles, and in small (1–3 mm) and large (3–6 mm) antral follicles. Furthermore, ovarian fragments were cultured for 1 or 7 days in Minimal Essential Medium (MEM+) supplemented with KL (0, 1, 10, 50, 100, or 200 ng/ml). Noncultured (control) and cultured fragments were processed for histology and transmission electron microscopy (TEM). RT‐PCR demonstrated an increase in steady‐state level of KL mRNA during the transition from primary to secondary follicles. Small antral follicles had higher steady‐state levels of KL mRNA in granulosa and theca cells than large follicles. After 7 days, only 50 ng/ml of KL had maintained the percentage of normal follicles similar to control. After 1 day, all KL concentrations reduced the percentage of primordial follicles and increased the percentage of growing follicles. KL at 10, 50, 100, or 200 ng/ml increased primary follicles, compared to MEM+ after 7 days. An increase in oocyte and follicular diameter was observed at 50 ng/ml of KL. TEM confirmed ultrastructural integrity of follicles after 7 days at 50 ng/ml of KL. In conclusion, the KL mRNAs were detected in all follicular categories. Furthermore, 50 ng/ml of KL maintained the integrity of caprine preantral follicle cultured for 7 days and stimulated primordial follicle activation and follicle growth. Mol. Reprod. Dev. 77: 231–240, 2010.

Expression of vascular endothelial growth factor (VEGF) receptor in goat ovaries and improvement of in vitro caprine preantral follicle survival and growth with VEGF.

The aim of the present study was to evaluate the effect of vascular endothelial growth factor (VEGF) on the survival and growth of goat preantral follicles after in vitro culture and to verify the expression of VEGF receptor (VEGFR)-2 in goat ovaries. Ovarian fragments were cultured for 1 or 7 days in minimal essential medium (MEM) with different concentrations of VEGF (1, 10, 50, 100 or 200 ng mL(-1)). Non-cultured (fresh control) and cultured tissues were processed for histological and ultrastructural studies. The results showed that 200 ng mL(-1) VEGF resulted in a similar percentage of normal preantral follicles after 1 and 7 days of culture compared with control. Compared with basic culture medium alone, an increase in follicular and oocyte diameters was observed in the presence of 10 ng mL(-1) VEGF after 7 days culture. Ultrastructural analysis confirmed follicular integrity after 7 days culture in the presence of 200 ng mL(-1) VEGF. Immunohistochemical studies demonstrated the expression of VEGFR-2 in oocytes and granulosa cells of all follicular stages, except in granulosa cells of primordial follicles. In conclusion, the present study has shown that VEGF maintains follicular ultrastructural integrity and promotes follicular growth. In addition, VEGFR-2 is expressed in oocytes of caprine ovarian follicles at all developmental stages and in granulosa cells of developing follicles.

Recombinant Epidermal Growth Factor Maintains Follicular Ultrastructure and Promotes the Transition to Primary Follicles in Caprine Ovarian Tissue Cultured In Vitro

We investigated the effects of epidermal growth factor on the survival and growth of caprine preantral follicles. Ovarian fragments were cultured for 1 and 7 days in enriched minimal essential medium with epidermal growth factor (0, 1, 10, 50, 100, or 200 ng/mL). Non-cultured and cultured tissues were processed for histological and ultrastructural studies. Results showed that after 7 days, the epidermal growth factor (1 and 10 ng/mL) maintained the percentage of normal follicles similar to control. An increase in the percentage of primary follicles was observed with 1, 10, and 50 ng/mL of epidermal growth factor compared to enriched minimal essential medium. Ultrastructural studies confirmed follicular integrity after 7 days in epidermal growth factor (1 and 10 ng/mL). In conclusion, the low concentrations of epidermal growth factor maintain caprine follicular viability and promote the transition from primordial to primary follicles.

Importance of vascular endothelial growth factor (VEGF) in ovarian physiology of mammals.

Ovarian folliculogenesis in mammals is a complex process. Several compounds have been tested during in vitro culture of follicular cells for a better understanding of the mechanisms and factors related to ovarian folliculogenesis in mammals. From these compounds, vascular endothelial growth factor (VEGF) can be highlighted, as it is strongly associated with angiogenesis and, in recent years, its presence in ovarian cells has been investigated extensively. Previous studies have shown that the presence of VEGF protein, as well as mRNA expression of its receptor 2 (VEGFR-2) increases during follicular development. Therefore, it is likely that the interaction between VEGF and VEGFR-2 is crucial to promote follicular development. However, few studies on the influence of this factor on follicular development have been reported. This review addresses aspects related to the structural characterization and mechanism of action of VEGF and its receptors, and their biological importance in the ovary of mammals.

Effect of the Medium Replacement Interval on the Viability, Growth and In Vitro Maturation of Isolated Caprine and Ovine Pre‐Antral Follicles

The aim of the present study was to evaluate the effects of different medium replacement intervals on the viability, antral cavity formation, growth and in vitro maturation (IVM) of oocytes from caprine and ovine pre-antral follicles. Pre-antral ovarian follicles (≥ 150 μm) were isolated from the ovarian cortex of goats and sheep and were individually cultured for 24 days using two different medium replacement intervals [2 days (T(1) ) or 6 days (T(2) )]. Follicle development was evaluated on the basis of antral cavity formation, increases in follicular diameter and the presence of healthy cumulus oocyte complexes and fully grown oocytes. For caprine species, results showed a higher percentage (p<0.05) of viable follicles in T(1) than T(2) from day 6 until the end of the culture. In addition, when comparing both treatments after the same culture duration, the rate of antrum formation was significantly higher in T(1) than in T(2) from day 12 onwards. Yet, in ovines, when both treatments were compared on day 24 of the culture, there were more viable follicles in T(2) than in T(1) (p<0.05). In the caprine species, percentages of fully grown oocytes (≥ 110 μm) acceptable for IVM after 24 days of culture were significantly higher in normal follicles cultured in T(1) (30.0%) than in T(2) (6.7%; p<0.05). On the other hand, in ovines, at the end of the culture, the percentage of oocytes destined for IVM was higher in T(2) than in T(1) (23.5% vs 2.9%; p<0.05). In conclusion, under the same conditions, the frequency of medium replacement significantly affected the in vitro development of caprine and ovine pre-antral follicles. To improve the efficiency of the culture system, the medium must be replaced every 2 and 6 days for goat and sheep pre-antral follicles, respectively.

Steady-state level of bone morphogenetic protein-15 in goat ovaries and its influence on in vitro development and survival of preantral follicles

This study investigates steady-state level of bone morphogenetic protein-15 (BMP-15) mRNA in caprine follicles, and the effects of BMP-15 on in vitro development of preantral follicles. Ovarian fragments were cultured for one or seven days in Minimal Essential Medium (MEM(+)) with BMP-15 (0, 1, 10, 50, 100 or 200 ng/mL), and further analyzed by histology, transmission electron and fluorescent microscopy. BMP-15 mRNA in secondary follicles was higher than in primordial and primary follicles. After seven days, 10, 50 or 100 ng/mL of BMP-15 maintained the percentage of normal follicles similar to the control (non-cultured), and increased the oocyte and follicle diameters when compared to the control and MEM(+). BMP-15 at 100 ng/mL increased the secondary follicles and maintained their ultrastructural integrity. In conclusion, the BMP-15 mRNAs were detected in all follicular categories. BMP-15 (100 ng/mL) maintained the integrity and promoted the growth of caprine preantral follicles cultured for seven days.