A.P. Almeida

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.

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.

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.

The Effects of Insulin and Follicle-Simulating Hormone (FSH) During In Vitro Development of Ovarian Goat Preantral Follicles and the Relative mRNA Expression for Insulin and FSH Receptors and Cytochrome P450 Aromatase in Cultured Follicles

ABSTRACT The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 μg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles. Low concentration of insulin with follicle-stimulating hormone is essential for promoting oocyte meiosis resumption in goat preantral follicles.

Eight-Cell Parthenotes Originated From In Vitro Grown Sheep Preantral Follicles

We investigated the effect of the leukemia inhibitory factor (LIF) alone or in association with follicle-stimulating hormone (FSH) on the in vitro growth and antrum formation of sheep preantral follicles. To evaluate oocyte quality, parthenogenetic activation of the oocytes recovered from in vitro grown preantral follicles was performed. Preantral follicles >110 μm in diameter were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/mL) in the absence or presence of FSH. Every 6 days the follicular survival, growth, and antrum formation were evaluated. When compared to control (P < .05), antrum formation was increased in follicles cultured in the presence of LIF10 and FSH. At the end of the culture, the oocytes underwent in vitro maturation (IVM); their viability and chromatin configuration were assessed. Although IVM was not affect by the treatments (P > .05), the numerically highest maturation rates (29.63%) were obtained when follicles were cultured in 50 ng/mL LIF (LIF50). Therefore, their oocytes were submitted to parthenogenetic activation; from which 58.3% of the mature oocytes resulted in 8-cell stage parthenotes. In conclusion, although LIF10 + FSH increases antrum formation when compared to a nonsupplemented medium (minimum essential medium), oocytes from sheep preantral follicles are capable of growing and maturing in vitro independent of LIF addition to the medium, which resulted in the formation of 8-cell parthenotes.

Canine preantral follicles cultured with various concentrations of follicle-stimulating hormone (FSH).

The objective was to evaluate the effects of various concentrations of exogenous FSH during in vitro culture of isolated canine preantral follicles. Preantral secondary follicles (>200 microm) were isolated by microdissection and cultured for 18 d in supplemented alpha-Minimum Essential Medium (alpha-MEM). There were three treatment groups: 1) absence of FSH (control medium); 2) FSH100 (fixed concentration of 100 ng/mL throughout the entire culture period); and 3) sequential FSH (FSHSeq - 100, 500, and 1,000 ng/mL were added sequentially). Following culture, all follicles from all treatments were still viable (marked green by calcein-AM). The initial (D0) average follicle diameter for the control, FSH100, and FSHSeq was (mean +/- SEM) 298.96 +/- 7.02, 286.00 +/- 5.87, and 275.39 +/- 174 6.55 microm, respectively (P > 0.05). Mean diameter of follicles treated with FSHSeq on Day 18 (D18-439.80 +/- 14.08 microm) was greater than those of the other treatments (P < 0.05). Daily follicular growth rate (microm/d) of follicles in the FSHSeq treatment (6.47 +/- 0.55) was significantly faster than for both the control (3.67 +/- 0.32) and FSH100 (4.47 +/- 0.38) treatments. Furthermore, FSH100 and FSHSeq treatments had a significantly higher rate of antrum formation than the control group on D12 of culture, whereas after D12, FSH100 had a significantly higher rate of extrusion compared to the control (P < 0.05). In conclusion, the sequential addition of FSH to the culture medium maintained the survival of isolated canine preantral follicles and promoted an increased rate of follicular growth and antrum formation.

Steady-state level of insulin-like growth factor-I (IGF-I) receptor mRNA and the effect of IGF-I on the in vitro culture of caprine preantral follicles

The objectives were to quantify insulin-like growth factor receptor-1 (IGFR-1) mRNA in preantral follicles on Days 0 and 18 of in vitro culture in the presence or absence of FSH, and to evaluate the effects of IGF-I on the rate of normal follicles, antral cavity formation, and in vitro growth and maturation of caprine oocytes on Days 0, 6, 12, and 18 of culture. The expression of IGFR-1 was analyzed using real-time RT-PCR before and after follicle culture. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the presence or absence of bovine IGF-I (50 or 100 ng/mL). At the end of the culture period, the oocytes were submitted to IVM. The expression of IGFR-1 mRNA in preantral follicles cultured in vitro only approached being significantly higher in follicles supplemented with FSH when compared to follicles immediately after recovery (P<0.06) and cultured without FSH (P<0.1). There was a higher (P<0.05) percentage of normal follicles on Days 6, 12, and 18 of culture in IGF-I 50 (97, 92, 67%, respectively) and IGF-I 100 (100, 90, 80%) groups versus the control (90, 64, 36%). In addition, the rate of antrum formation at 6 and 12 d of culture was higher (P<0.05) in IGF-I groups (IGF-I 50: 72 and 90% and IGF-I 100: 69 and 85%) than the control group (41 and 59%). After 18 d of culture, the percentages of grown oocytes acceptable for IVM were higher (P<0.05) in follicles cultured in the presence of IGF-I (82 vs 49%). Furthermore, follicles cultured in the presence of IGF-I 50 and IGF-I 100 had higher (P<0.05) meiotic resumption rates (63 and 66%, respectively) than the control group (11%). In conclusion, treatment with FSH tended to increase IGFR-1 mRNA expression during the in vitro culture of preantral follicles and the addition of IGF-I to the culture medium clearly improved the in vitro development of caprine preantral follicles.

Gene Expression and Immunolocalization of Fibroblast Growth Factor 2 in the Ovary and Its Effect on the In Vitro Culture of Caprine Preantral Ovarian Follicles

This study quantified Fibroblast growth factor 2 (FGF-2) mRNA and localized FGF-2 protein in different categories of follicles isolated from goat ovaries. In addition, we verified the effects of this factor on the in vitro culture of preantral follicles isolated from goats. For mRNA quantification, we performed real-time PCR using primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa and theca cells of small and large antral follicles. For FGF-2 protein localization, the ovaries were subjected to conventional immunohistochemical procedures. Preantral follicles were isolated and cultured in vitro for 12 days in either control (basic) or supplemented with FGF-2 medium. The expression of FGF-2 mRNA was detected in all categories of follicles and there was no difference in preantral follicles and COCs or granulosa/theca cells from small and large antral follicles. However, in large antral follicles, COCs showed expression levels significantly lower than in granulosa/theca cells (p < 0.05). We observed moderate expression of FGF-2 protein in preantral follicles but not in granulosa cells of primordial follicles and theca cells of secondary follicles. In both small and large antral follicles, strong, moderate and weak staining was observed in oocytes, granulosa and theca cells, respectively. The addition of FGF-2 caused a significant increase in the daily follicular growth rate compared to the control group. We conclude that FGF-2 mRNA is expressed throughout follicular development and that its protein can be found in different patterns in preantral and antral follicles. Furthermore, FGF-2 increases the follicular growth rate in vitro.

Insulin-like growth factor II (IGF-II) and follicle stimulating hormone (FSH) combinations can improve the in vitro development of grown oocytes enclosed in caprine preantral follicles

OBJECTIVE Evaluate the possible role of IGF-II alone or in association with FSH on in vitro development of isolated caprine preantral follicles. METHODS Preantral follicles (≥150 μm) were isolated from goat ovaries and cultured for 18 days in basic αMEM medium (control) or supplemented with IGF-II alone at 20 or 50 ng/ml, named IGF20 and IGF50, respectively, or in combination with recombinant FSH (FSH, IGF20F or IGF50F). During in vitro culture, the follicles were analyzed by using morphology criteria, antrum formation and growth rate as parameters. After 18 days of follicular culture, oocytes equal to or larger than 110 μm were used for in vitro maturation (IVM). Oocyte viability and meiosis resumption were assessed by fluorescence microscopy after labeling with calcein-AM, ethidium homodimer and Hoechst 33342. RESULTS The IGF20 treatment was the only treatment capable of maintaining the percentage of morphologically normal follicles from D0 until D6 and from D12 to D18 (p>0.05), while in all other treatments the percentage of morphologically normal follicles decreased progressively during 18 days of in vitro culture (p<0.05). At D18, all treatments with IGF-II or FSH resulted in a significantly higher percentage of normal follicles when compared to αMEM alone. The IGF50F treatment provided a significantly higher early antrum formation rate when compared to αMEM and FSH alone. The addition of IGF-II alone (20 or 50 ng/ml) or in combination with FSH prevented oocyte degeneration after IVM. Moreover, the FSH treatment demonstrated a lower percentage of oocyte degeneration when compared to control (4.35% vs. 26.3%, respectively; p<0.05). Regarding meiosis resumption, the IGF20F treatment was the only treatment that significantly differed from αMEM alone. All treatments except the control (αMEM alone) presented oocytes at metaphase II. CONCLUSION IGF-II associated with FSH stimulated in vitro follicular development, oocyte viability and meiotic resumption of caprine oocytes after IVM.

The Effect of LIF in the Absence or Presence of FSH on the In Vitro Development of Isolated Caprine Preantral Follicles

We investigated the effect of the leukaemia inhibitory factor (LIF) alone or in association with FSH on the in vitro culture (IVC) of caprine preantral follicles. Preantral follicles >200 μm in size were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/ml) in the absence or presence of FSH. Every 6 days, follicular survival, growth and antrum formation were evaluated. At the end of the culture period, the oocytes underwent in vitro maturation (IVM), and their viability and chromatin configuration were assessed. Follicles of the control group and those cultured in 10 ng/ml LIF maintained the structural integrity (particularly the preservation of the basement membrane) when compared to the oocytes cultured in 50 ng/ml LIF, regardless the presence of FSH. In the absence of FSH, the percentage of antrum formation after 18 days of culture in the 50 ng/ml LIF group was significantly lower than in either the control group or the 10 ng/ml LIF group. However, this effect was not observed in the presence of FSH. The rate of resumption of meiosis was significantly higher in the 50 ng/ml LIF group in the absence of FSH in comparison with the control and 10 ng/ml LIF groups. Metaphase II was observed only when follicles were cultured in a combination of FSH and 50 ng/ml LIF. In conclusion, LIF alone does not interfere with antral formation and oocyte growth, but at concentration of 50 ng/ml and combined with FSH, it promotes oocyte maturation.