Effects of frutalin and doxorubicin on growth, ultrastructure and gene expression in goat secondary follicles cultured in vitro.

This study evaluated the effects of frutalin (0.6, 6.0 or 60.0 µg/mL) and doxorubicin (0.3 µg/mL) on survival, growth and ultrastructure of in-vitro cultured goat secondary follicles. The effects of these substances on the levels of mRNA for Casp3, Casp6, Bax, and Bcl2 were also investigated. Results showed that, after 6 days of culture, frutalin or doxorubicin reduced the percentage of normal follicles (P < 0.05), but doxorubicin had higher toxicity than frutalin. Except for follicles cultured with 0.6 µg/mL frutalin, follicular growth rate was reduced after culture with doxorubicin or frutalin (P < 0.05). The presence doxorubicin or 60.0 µg/mL frutalin increased the levels of mRNA for Casp3, Casp6, Bax, and Bcl2 (P < 0.05). Higher mRNA levels for Casp3, Casp6 and Bax were found in follicles cultured with doxorubicin, but higher levels of Bcl2 mRNA were found in follicles cultured with frutalin (P < 0.05). In conclusion, frutalin has lower toxic effects than doxorubicin on secondary follicles cultured in vitro.

Expression of TNF-α system members in bovine ovarian follicles and the effects of TNF-α or dexamethasone on preantral follicle survival, development and ultrastructure in vitro.

This study was conducted to detect the protein expression of TNF-α system members (TNF-α/TNFR1/TNFR2) in bovine ovarian follicles and to evaluate the effects of TNF-α or dexamethasone on the survival and growth of primordial follicles in vitro, as well as on gene expression in cultured ovarian tissue. It was hypothesized that TNF-α induces follicular atresia in ovarian tissues cultured in vitro, and that dexamethasone suppresses the production of endogenous TNF-α, which can improve follicle viability in vitro. Ovarian fragments were cultured for 6days in α-MEM+ supplemented with TNF-α (0, 1, 10, 100 or 200ng/ml) or dexamethasone (0, 1, 10, 100 or 200ng/ml). After culture, the expression of mRNAs for BCL-2, BAX, P53, TNF-α, and CASP3 and CASP6 were evaluated. Immunohistochemical results showed that the TNF-α system members, were detected in bovine preantral and antral follicles. After 6days, the TNF-α (10ng/ml) treatment reduced the percentage of normal preantral follicles and increased the number of TUNEL-positive cells in cultured tissue. Dexamethasone (10ng/ml) during 6days of culture did maintain the percentage of normal follicles and the ultrastructure of follicles, while the presence of TNF-α or dexamethasone did not influence primordial follicle activation. However, TNF-α or dexamethasone had no effect on the levels of mRNA for P53, BCL-2, BAX and CASP6, in cultured tissues, but the presence of dexamethasone reduced the levels of CASP3 compared to ovarian slices cultured in control medium (α-MEM+). In conclusion, proteins of the TNF-α system are expressed at different bovine follicle stages. The addition of TNF-α in culture reduces follicle survival and increases the number of apoptotic cells in ovarian tissue, while the presence of dexamethasone maintains follicle ultrastructure in cultured tissue.

mRNA expression profile of the TNF-α system in LH-induced bovine preovulatory follicles and effects of TNF-α on gene expression, ultrastructure and expansion of cumulus-oocyte complexes cultured in vitro.

This study evaluated (1) the effects of in vivo GnRH treatment on mRNA expression of TNF-α system (TNF-α, TNFR1 and TNFR2) in granulosa cells of bovine preovulatory follicles, (2) the in vitro influence of gonadotropins on mRNA expression of TNF-α system in cultured cumulus cells, (3) the protein expression of the TNF-α system in late antral follicles and, (4) the influence of TNF-α on cumulus cells expansion, ultrastructure and on expression of HAS2, CASP3 and CASP6 in follicular cells cultured for 24 h. An increased expression of TNF-α and TNFR1 was observed after 3, 6 and 12 h of GnRH treatment when compared to 0 and 24h. Higher TNFR2 mRNA levels were observed 3, 6 and 12 h after GnRH, when compared to 0 and 24 h. Proteins of TNF-α system were also expressed in late antral follicles. In vitro, TNF-α did not affect cumulus cells expansion, but reduced the HAS2, CASP3 and CASP6 mRNA levels in cumulus cells after 12 h. After 24 h of culture, TNF-α increased the mRNA levels for CASP6 in mural granulosa cells, while the TNF-α, TNFR1 and TNFR2 mRNA levels were increased in cumulus-oocyte complexes (COCs) cultured for 12 h with gonadotropins, but not after 24 h. Ultrastructural analysis confirmed the integrity of COCs cultured in presence of TNF-α. In conclusion, TNF-α system members are present in bovine antral follicles and expression of TNF-α is influenced by gonadotropins in vivo and in vitro. In vitro, TNF-α maintained cumulus cells ultrastructure during COC culture.

Protein and messenger RNA expression of interleukin 1 system members in bovine ovarian follicles and effects of interleukin 1ß on primordial follicle activation and survival in vitro.

This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1ß on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1ß (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1ß, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1ß (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1ß promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.

Effects of BMP-4 and FSH on growth, morphology and mRNA expression of oocyte-secreted factors in cultured bovine secondary follicles

This study evaluated the effect of different concentrations of bone morphogenetic protein-4 (BMP-4), as well as the interaction of BMP-4 and follicle stimulating hormone (FSH) on growth, ultrastructural integrity, and expression of mRNA for growth differentiation factor-9 (GDF-9), BMP-15, maternal antigen that the embryo requires (Mater) and nucleoplasmin-2 (Npm-2) in bovine secondary follicles cultured in vitro for 18 days. Follicles cultured in the presence of 50 ng/ml BMP-4 had a progressive increase in their diameters with the increase of culture period from 0 to 6 and 12 days, but no significant differences were observed among treatments. The presence of both FSH and BMP-4 in a culture medium did not stimulate follicle growth when compared to the control medium. After 12 days, the percentage of normal follicles was maintained similar to that of day 0 in the medium supplemented with both FSH and BMP-4, but no significant differences among treatments were observed after 18 days of culture. BMP-4 maintained the ultrastructural integrity of follicles after 18 days of culture, while follicles cultured in medium supplemented with FSH or both BMP-4 and FSH had oocyte with irregular zona pellucida, vesicular bodies, and an abundance of vacuoles. Follicles cultured in the presence of BMP-4 had an increase in the levels of BMP-15 mRNA, when compared to those cultured in medium supplemented with FSH alone. In conclusion, the addition of BMP-4 in culture medium contributes to preserve follicular ultrastructure, but BMP-4 did not interact positively with FSH. Regarding secondary follicles cultured in the presence of FSH, BMP-4 increases the expression of mRNA for BMP-15.

Effects of BMP-4 and FSH on growth, morphology and mRNA expression of oocyte-secreted factors in cultured bovine secondary follicles

This study evaluated the effect of different concentrations of bone morphogenetic protein-4 (BMP-4), as well as the interaction of BMP-4 and follicle stimulating hormone (FSH) on growth, ultrastructural integrity, and expression of mRNA for growth differentiation factor-9 (GDF-9), BMP-15, maternal antigen that the embryo requires (Mater) and nucleoplasmin-2 (Npm-2) in bovine secondary follicles cultured in vitro for 18 days. Follicles cultured in the presence of 50 ng/ml BMP-4 had a progressive increase in their diameters with the increase of culture period from 0 to 6 and 12 days, but no significant differences were observed among treatments. The presence of both FSH and BMP-4 in a culture medium did not stimulate follicle growth when compared to the control medium. After 12 days, the percentage of normal follicles was maintained similar to that of day 0 in the medium supplemented with both FSH and BMP-4, but no significant differences among treatments were observed after 18 days of culture. BMP-4 maintained the ultrastructural integrity of follicles after 18 days of culture, while follicles cultured in medium supplemented with FSH or both BMP-4 and FSH had oocyte with irregular zona pellucida, vesicular bodies, and an abundance of vacuoles. Follicles cultured in the presence of BMP-4 had an increase in the levels of BMP-15 mRNA, when compared to those cultured in medium supplemented with FSH alone. In conclusion, the addition of BMP-4 in culture medium contributes to preserve follicular ultrastructure, but BMP-4 did not interact positively with FSH. Regarding secondary follicles cultured in the presence of FSH, BMP-4 increases the expression of mRNA for BMP-15.(AU)

Accelerated growth of bovine preantral follicles in vitro after stimulation with both FSH and BMP-15 is accompanied by ultrastructural changes and increased atresia.

The objective of the present study was to determine the effects of bone morphogenetic protein (BMP)-15 and FSH on the growth, viability, and expression of mRNA for FSH (FSH-R) and BMP-15 (BMPR-IB and BMPR-II) receptors in cultured bovine secondary follicles. Secondary follicles were microdissected and cultured for 12 days in minimum essential medium-α alone or supplemented with BMP-15, sequential FSH, both BMP-15 and FSH, or BMP-15 from days 0 to 6, and FSH from days 7 to 12. Thereafter, the effect of these treatments on the follicular volume, viability, and antrum formation and the levels of mRNA for BMPR-IB, BMPR-II, and FSH-R were assessed. Compared with day 0, the follicles cultured with FSH or BMP-15, or both, had a significant and progressive increase in volume (P < 0.05). However, the follicles cultured for 12 days with both BMP-15 and FSH had the greatest volume and a greater rate of antrum formation than those in control medium, but results similar to those cultured with FSH (days 0 to 12) or BMP-15 (days 0 to 6) and FSH (days 7 to 12). Together with their accelerating effect on in vitro follicle growth, the combination of FSH and BMP-15 induced ultrastructural changes in the cultured follicles and increased atresia. However, adding either BMP-15 or FSH to the culture medium, not only promoted follicular growth and follicular antrum formation, but also maintained follicular viability during culture. Except for follicles cultured in minimal essential medium-α, the levels of mRNA for BMPR-IB were reduced, and the levels of mRNA for FSH-R were significantly greater in follicles cultured in medium supplemented with BMP-15. In conclusion, all in vitro follicle treatments supported growth of bovine preantral follicles; however, adding both BMP-15 and FSH to the culture medium (minimal essential medium-α) for 12 days provided the greatest stimulation. Furthermore, the viability and ultrastructural integrity of cultured follicles were only maintained when only BMP-15 or FSH was added to the culture medium.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles.

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

Levels of mRNA for bone morphogenetic proteins, their receptors and SMADs in goat ovarian follicles grown in vivo and in vitro.

This study investigated the stability of housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase, ß-tubulin, ß-actin, phosphoglycerate kinase (PGK), 18S rRNA, ubiquitin and ribosomal protein 19) and the levels of mRNA for bone morphogenetic protein-2 (BMP-2), -4 (BMP-4), -6 (BMP-6), -7 (BMP-7) and -15 (BMP-15), their receptors (BMPR-IA, -IB and -II) and Similar to Mothers Against Decapentaplegic (SMADs) (-1, -5 and -8) in goat follicles of 0.2, 0.5 and 1.0mm, as well as in secondary follicles before and after culture for 18 days. ß-tubulin and PGK were the most stable housekeeping genes and the levels of mRNA for BMP-2 in follicles of 0.2mm were higher than in follicles of 0.5 and 1.0mm. For BMP-4, -6 and -7, the highest levels of mRNA were found in follicles of 1.0mm. The expression of BMPR-IB was higher in follicles of 0.2mm, whereas the levels of BMPR-II were higher in follicles of 0.5mm. The levels of mRNA for SMAD-5 were higher in follicles of 0.2mm, whereas SMAD-8 had higher levels in 0.5-mm follicles. After culture, follicles showed increased levels of mRNA for BMP-2 and reduced mRNA for BMP-4, BMP-7, BMPR-IA and SMAD-5. In conclusion, ß-tubulin and PGK are the most stable reference genes, and BMPs, their receptors and SMADs have variable levels of mRNA in the follicular size classes analysed.

FSH and LH enhance the development of goat preantral follicles cultured in vitro

This study evaluated th e effect of increased follicle stimulating hormone (FSH) concentrations on the expression of mRNA for LH receptors after in vitro culture of goat preantral follicles ( ≥ 150 μm) for 18 days. It also investigated whether the addition of luteinizing hormone (LH) to the culture medium, which contained increasing concentrations of FSH throughout the culture period, influenced the surv ival, growth and antrum formation of in vitro cultured goat preantral follicles. In experiment 1, preantral follicles were cultured in α -MEM + or α -MEM + supplemented with increasing concentrations of FSH throughout the culture period (sequential medium: FSH 100 ng/ml (days 0 to 6), FSH 500 ng/ml (days 6 to 12) and FSH 1000 ng/ml (days 12 to 18). The expression of luteinizing hormone receptor (LHR) was analyzed in noncultured and cultured follicles using real time RT-PCR. In experiment 2, isolated preantral follicles were cultured for 18 days in a sequential medium containing FSH (control) or a control medium supplemented with LH (50 or 100 ng/ml) from day 12 of culture onwards. Follicle development was evaluated on the basis of antr al cavity formation as well as follicular and oocyte growth after in vitro maturation. FSH stimulated a significant increase in the expression of mRNA for LH receptors after 18 days of culture. Furthermore, after 18 days, all tested media promoted follicular survival and antrum formation; however, a significant increase in the rate of follicular growth and resumption of meiosis was ob served when LH was used compared to the control. In conclusion, preantral follicles cultured in a medium supplemented with FSH increased LH receptor mR NA levels. Moreover, the addition of LH to the culture medium containing increasing concentrations of FSH (sequential medium) improved the in vitro development of goat preantral follicles.(AU)

Proteínas morfogenéticas ósseas (BMPs) e seu papel na regulação da oogênese e da foliculogênese ovariana em mamíferos / The bone morphogenetic proteins (BMPs) and its role in the regulation of ovarian folliculogenesis and oogenesis in mammalian

O processo de foliculogênese é controlado por uma variedade de gonadotrofinas e de fatores de crescimento locais, que agem em conjunto para regular a formação e o desenvolvimento dos folículos ovarianos. Dentre esses fatores, destacam-se as proteínas morfogenéticas ósseas (BMPs), que representam uma família de fatores de crescimento amplamente estudados e que se caracterizam por controlar as funções ovarianas em diferentes estágios do desenvolvimento folicular. Dessa forma, a presente revisão tem como foco principal descrever os locais de expressão das BMPs dos tipos 2, 4, 6, 7 e 15 e discutir o papel delas, bem como das gonadotrofinas FSH e LH durante a foliculogênese em mamíferos.(AU)

The process of folliculogenesis is controlled by a variety of gonadotropins and local growth factors that act together to regulate formation and development of ovarian follicles. Among these factors, the bone morphogenetic proteins (BMPs) represent a family of growth factors widely studied that have important functions at different stages of ovarian follicular development. Thus, this review aims to describe the local of expression and to discuss the role of BMPs 2, 4, 6, 7 and 15 as well as the gonadotropins FSH and LH during folliculogenesis in mammals. (AU)

Proteínas morfogenéticas ósseas (BMPs) e seu papel na regulação da oogênese e da foliculogênese ovariana em mamíferos / The bone morphogenetic proteins (BMPs) and its role in the regulation of ovarian folliculogenesis and oogenesis in mammalian

O processo de foliculogênese é controlado por uma variedade de gonadotrofinas e de fatores de crescimento locais, que agem em conjunto para regular a formação e o desenvolvimento dos folículos ovarianos. Dentre esses fatores, destacam-se as proteínas morfogenéticas ósseas (BMPs), que representam uma família de fatores de crescimento amplamente estudados e que se caracterizam por controlar as funções ovarianas em diferentes estágios do desenvolvimento folicular. Dessa forma, a presente revisão tem como foco principal descrever os locais de expressão das BMPs dos tipos 2, 4, 6, 7 e 15 e discutir o papel delas, bem como das gonadotrofinas FSH e LH durante a foliculogênese em mamíferos.

The process of folliculogenesis is controlled by a variety of gonadotropins and local growth factors that act together to regulate formation and development of ovarian follicles. Among these factors, the bone morphogenetic proteins (BMPs) represent a family of growth factors widely studied that have important functions at different stages of ovarian follicular development. Thus, this review aims to describe the local of expression and to discuss the role of BMPs 2, 4, 6, 7 and 15 as well as the gonadotropins FSH and LH during folliculogenesis in mammals.

FSH and LH enhance the development of goat preantral follicles cultured in vitro

This study evaluated th e effect of increased follicle stimulating hormone (FSH) concentrations on the expression of mRNA for LH receptors after in vitro culture of goat preantral follicles ( ≥ 150 μm) for 18 days. It also investigated whether the addition of luteinizing hormone (LH) to the culture medium, which contained increasing concentrations of FSH throughout the culture period, influenced the surv ival, growth and antrum formation of in vitro cultured goat preantral follicles. In experiment 1, preantral follicles were cultured in α -MEM + or α -MEM + supplemented with increasing concentrations of FSH throughout the culture period (sequential medium: FSH 100 ng/ml (days 0 to 6), FSH 500 ng/ml (days 6 to 12) and FSH 1000 ng/ml (days 12 to 18). The expression of luteinizing hormone receptor (LHR) was analyzed in noncultured and cultured follicles using real time RT-PCR. In experiment 2, isolated preantral follicles were cultured for 18 days in a sequential medium containing FSH (control) or a control medium supplemented with LH (50 or 100 ng/ml) from day 12 of culture onwards. Follicle development was evaluated on the basis of antr al cavity formation as well as follicular and oocyte growth after in vitro maturation. FSH stimulated a significant increase in the expression of mRNA for LH receptors after 18 days of culture. Furthermore, after 18 days, all tested media promoted follicular survival and antrum formation; however, a significant increase in the rate of follicular growth and resumption of meiosis was ob served when LH was used compared to the control. In conclusion, preantral follicles cultured in a medium supplemented with FSH increased LH receptor mR NA levels. Moreover, the addition of LH to the culture medium containing increasing concentrations of FSH (sequential medium) improved the in vitro development of goat preantral follicles.

Effects of BMP-7 and FSH on the development of goat preantral follicles and levels of mRNA for FSH-R, BMP-7 and BMP receptors after in-vitro culture

This study aimed to evaluate the effects of FSH and BMP - 7 on growth and on expression of FSH - R , BMP - 7 and BMP receptors in cultured secondary follicles. Goat secondary follicles (~200 μm) were isolated and cultured in vitro, with 5% CO 2 in air at 39°C, for 6 days in the presence of BMP - 7 (50 ng/m l ) supplemented or not with FSH (50 ng/m l ). Follicular diameter and the formation of the antrum were evaluated before and after culture. For each treatment, at the end of culture period, groups of 6 follicles were collected and, after extraction of total RNA and cDNA synthesis, the levels of mRNA for FSH - R, BMP - 7 and BMP receptors in cultured secondary follicles were quantified by real time PCR. The results showed that addition of BMP - 7 or F SH to culture medium stimulated growth of secondary follicles , while addition of both BMP - 7 and FSH was needed to significantly increase the percentage of follicles forming an antrum and the follicular levels of mRNA for both BMP - 7 and FSH - R. For BMP receptors, FSH reduced the levels of mRNA for BMPR - IA and BMP - RII in comparison with those follicles cultured in MEM alone and supplemented with BMP - 7, respectively. In conclusion , l ike FSH, BMP - 7 affect s in vitro growth of cultured secondary follicle s , but it stimulates antrum formation and expression of the mRNA s for BMP - 7 and FSH - R only in presence of both BMP - 7 and FSH . However, the levels of mRNA f or BMP - RIA and BMP - RII are reduced in follicles culture d in medium supplemented with FSH.(AU)

Effects of BMP-7 and FSH on the development of goat preantral follicles and levels of mRNA for FSH-R, BMP-7 and BMP receptors after in-vitro culture

This study aimed to evaluate the effects of FSH and BMP - 7 on growth and on expression of FSH - R , BMP - 7 and BMP receptors in cultured secondary follicles. Goat secondary follicles (~200 μm) were isolated and cultured in vitro, with 5% CO 2 in air at 39°C, for 6 days in the presence of BMP - 7 (50 ng/m l ) supplemented or not with FSH (50 ng/m l ). Follicular diameter and the formation of the antrum were evaluated before and after culture. For each treatment, at the end of culture period, groups of 6 follicles were collected and, after extraction of total RNA and cDNA synthesis, the levels of mRNA for FSH - R, BMP - 7 and BMP receptors in cultured secondary follicles were quantified by real time PCR. The results showed that addition of BMP - 7 or F SH to culture medium stimulated growth of secondary follicles , while addition of both BMP - 7 and FSH was needed to significantly increase the percentage of follicles forming an antrum and the follicular levels of mRNA for both BMP - 7 and FSH - R. For BMP receptors, FSH reduced the levels of mRNA for BMPR - IA and BMP - RII in comparison with those follicles cultured in MEM alone and supplemented with BMP - 7, respectively. In conclusion , l ike FSH, BMP - 7 affect s in vitro growth of cultured secondary follicle s , but it stimulates antrum formation and expression of the mRNA ’s for BMP - 7 and FSH - R only in presence of both BMP - 7 and FSH . However, the levels of mRNA f or BMP - RIA and BMP - RII are reduced in follicles culture d in medium supplemented with FSH.