R. Rossetto

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.

Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles.

This study evaluates the effects of ascorbic acid and its interaction with follicle-stimulating hormone (FSH) on the morphology, activation, and in vitro growth of caprine preantral follicles. Ovarian fragments were cultured for 1, 7, or 14 d in minimum essential medium (MEM) containing ascorbic acid (50 or 100microg/mL), FSH (50ng/mL), or both of these substances. Ovarian tissue that was either fresh (control) or cultured for 1, 7, or 14 d was processed for histological and ultrastructural evaluation. The results showed that after 14 d of culture, medium supplemented with 50microg/mL of ascorbic acid alone or combined with FSH showed higher rates of follicular survival compared with MEM. After 7 d of culture, FSH, ascorbic acid at 50microg/mL with or without FSH, and ascorbic acid at 100microg/mL increased the percentage of follicular activation compared to fresh control. In addition, FSH alone significantly increased the percentage of growing follicles after 14 d. The combination of 50microg/mL of ascorbic acid and FSH promoted a significant increase in oocyte and follicular diameter after 7 d of culture. Ultrastructural and fluorescent analysis confirmed the integrity of follicles cultured with 50microg/mL of ascorbic acid and FSH after 14 d. In conclusion, the combination of 50microg/mL of ascorbic acid and FSH maintained follicular integrity and promoted follicular activation and growth after long-term in vitro culture of caprine preantral follicles.

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3%), and the highest percent of primary follicles was achieved with IGF-I (57.7%). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively.

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 medium composition on the in vitro culture of bovine pre-antral follicles: morphology and viability do not guarantee functionality

Summary This study investigated the effect of three different culture media (α minimum essential medium (α-MEM), McCoy or TCM199 during the in vitro culture (IVC) of bovine isolated pre-antral follicles. Pre-antral follicles greater than 150 μm in size were isolated and cultured for 0 (control), 8 or 16 days in one of the abovementioned culture media. Follicles were evaluated for survival, growth and antrum formation at days 8 and 16. The results showed that TCM199 was the most suitable medium to preserve follicular viability and ultrastructure, resulting in the highest rates of antrum formation. In conclusion, TCM199 promotes the in vitro development of isolated pre-antral follicles without hampering follicular functionality by sustaining in vitro growth and antrum formation.

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.

Dynamic medium containing kit ligand and follicle-stimulating hormone promotes follicular survival, activation, and growth during long-term in vitro culture of caprine preantral follicles.

The aim of this study was to evaluate the effects of a dynamic medium containing kit ligand (KL) and follicle-stimulating hormone (FSH) on the in vitro culture of caprine preantral follicles for 16 days. Ovarian fragments were cultured in α-MEM+ containing or not containing KL (50 ng/ml) and/or FSH (50 ng/ml) added during the first (days 0–8) and/or second half (days 8–16) of the culture period. Noncultured (control) and cultured fragments were processed for histological and ultrastructural evaluation. After 1 day of culture, only the treatments performed with KL or FSH maintained a percentage of normal follicles similar to that of the control. After 16 days, all treatments using KL until day 8 (KL/KL, KL/FSH, and KL/FSH+KL) and only FSH during the entire culture period (FSH/FSH) showed higher rates of follicular survival compared to α-MEM+ alone. After 1 and 8 days, the treatments initially cultured with KL increased the percentage of follicular activation in comparison to α-MEM+ alone and other treatments. The highest follicular diameter after 16 days was observed in follicles cultured with KL until day 8 followed by FSH (KL/FSH). Furthermore, this treatment promoted, as early as after 1 day of culture, an increase in oocyte growth compared to α-MEM+ alone. Ultrastructural analysis confirmed the integrity of follicles cultured in KL/FSH after 16 days. In conclusion, a dynamic medium containing KL and FSH maintained follicular integrity and promoted follicular activation and growth during the long-term in vitro culture of caprine preantral follicles.

Expression of keratinocyte growth factor in goat ovaries and its effects on preantral follicles within cultured ovarian cortex.

The aims of this study were to evaluate the expression of keratinocyte growth factor (KGF) in goat ovaries and to study its effects on preantral follicle survival and development. The ovaries were used for immunohistochemistry or for in vitro culture for 1 or 7 days with KGF (0, 1, 10, 50, 100, 150, or 200 ng/mL). Noncultured (fresh control) and cultured ovarian slices were processed for histological analysis and transmission electron microscopy (TEM). The results showed that after 7 days of in vitro culture, all treatments had a significant reduction in the percentage of normal follicles compared with the fresh control. After 7 days of culture, the highest KGF concentrations (150 and 200 ng/mL) induced a significant reduction in the percentage of normal follicles compared with the tissues cultured in the absence (α-MEM+ alone) or presence of 1, 10, and 50 ng/mL KGF. Transmission electron microscopy confirmed follicular integrity after 7 days of culture in 1 ng/mL KGF. In addition, compared with the fresh control, the percentage of growing follicles was significantly increased in all treatments after 1 or 7 days of culture. Immunohistochemical analyses showed the expression of KGF in oocytes and granulosa cells in all follicle developmental stages as well as in thecal and stromal cells. In conclusion, this study demonstrated that, at the lowest concentration (1 ng/mL), KGF maintained the ultrastructure of goat preantral follicles cultured in vitro for up to 7 days. Furthermore, the KGF protein was widely distributed in goat ovaries, especially in ovarian follicles.

Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro.

The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control - FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control - CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.