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)

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.

Steady-state levels of vasoactive intestinal peptide (VIP) mRNA in goat ovaries and the effect of VIP on the in vitro development of isolated preantral follicles

The aims of this study were to verify the steady-state level of vasoactive intestinal peptide (VIP) mRNA in goat follicles at various developmental stages and to investigate the influence of VIP on the survival, antrum formation and growth of secondary follicles cultured for 6 days. Primordial, primary and secondary goat follicles and small and large antral follicles were obtained to quantify VIP mRNA by real-time reverse transcription with the polymerase chain reaction. The influence of VIP and the presence or absence of follicle-stimulating hormone (FSH) on the development of secondary follicles and on mRNA expression for VIP and FSH receptor (FSHR) were determined after 6 days of culture. Survival, antrum formation and follicular diameter were evaluated every other day of culture. The levels of VIP mRNA in primary and secondary follicles were significantly higher than in primordial follicles. Cumulus–oocyte complexes (COCs) from both small and large antral follicles had significantly higher levels ofVIP mRNA than their respective granulosa/theca cells. During culture, the addition of VIP and/or FSH had o effect on follicular development. However, the presence of FSH and/or VIP in the culture medium significantly reduced VIP mRNA levels, but did not alter FSHR mRNA levels. In conclusion, VIP mRNA was detected in all goat follicular categories and cellular types, VIP and/or FSH did not affect the development of secondary follicles and reduce the expression of VIP mRNA levels.(AU)

Steady-state levels of vasoactive intestinal peptide (VIP) mRNA in goat ovaries and the effect of VIP on the in vitro development of isolated preantral follicles

The aims of this study were to verify the steady-state level of vasoactive intestinal peptide (VIP) mRNA in goat follicles at various developmental stages and to investigate the influence of VIP on the survival, antrum formation and growth of secondary follicles cultured for 6 days. Primordial, primary and secondary goat follicles and small and large antral follicles were obtained to quantify VIP mRNA by real-time reverse transcription with the polymerase chain reaction. The influence of VIP and the presence or absence of follicle-stimulating hormone (FSH) on the development of secondary follicles and on mRNA expression for VIP and FSH receptor (FSHR) were determined after 6 days of culture. Survival, antrum formation and follicular diameter were evaluated every other day of culture. The levels of VIP mRNA in primary and secondary follicles were significantly higher than in primordial follicles. Cumulus–oocyte complexes (COCs) from both small and large antral follicles had significantly higher levels ofVIP mRNA than their respective granulosa/theca cells. During culture, the addition of VIP and/or FSH had o effect on follicular development. However, the presence of FSH and/or VIP in the culture medium significantly reduced VIP mRNA levels, but did not alter FSHR mRNA levels. In conclusion, VIP mRNA was detected in all goat follicular categories and cellular types, VIP and/or FSH did not affect the development of secondary follicles and reduce the expression of VIP mRNA levels.

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.

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)

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.

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)

Expression of protein and mRNA encoding Insulin Growth Factor-I (IGF-I) in goat ovarian follicles and the influence of IGF-I on in vitro development and survival of caprine preantral follicles

The aim of this study was to investigate the protein and mRNA expression levels of IGF-I and the effects of IGF-I on preantral follicle survival and development, using an in vitro goat ovarian cortical culture system. The ovaries were used for immunohistochemical localization of IGF-I protein or used to demonstrate mRNA expression of IGF-I. For the latter goal, preantral and antral follicles, cumulus-oocyte complex, mural granulosa and theca cells were collected to study mRNA expression. For in vitro studies, ovarian cortex were cultured for 1 and 7 days with MEM supplemented IGF-I (0, 1, 10, 50, 100 or 200 ng/ml). Immunohistochemical results showed strong reactions for IGF-I in oocytes and granulosa cells of all follicular stages, except in granulosa cells of primordial and primary follicles. mRNA expression analysis demonstrated a discrete increase in the production of IGF-I during the transition from primordial to the primary and secondary follicle stages. After 7 days of culture, addition of 50 ng/ml of IGF-I to the medium showed the greatest percentage of normal follicles when compared with other concentrations. Furthermore, the highest percentage of primary follicles was observed after 7 days of culture in MEM+ plus 10 and 50 ng/ml of IGF-I. Culture of ovarian tissue for 7 days in MEM+ plus 50 ng/ml of IGF-I promoted the greatest increase (P < 0.05) in follicular diameter of all of the concentrations tested. In conclusion, IGF-I protein and mRNA were expressed in all follicular categories of goat. Furthermore, IGF-I maintained preantral follicle survival, promoted primordial follicle activation and stimulated the transition from intermediate to primary follicles.(AU)

Expression of protein and mRNA encoding Insulin Growth Factor-I (IGF-I) in goat ovarian follicles and the influence of IGF-I on in vitro development and survival of caprine preantral follicles

The aim of this study was to investigate the protein and mRNA expression levels of IGF-I and the effects of IGF-I on preantral follicle survival and development, using an in vitro goat ovarian cortical culture system. The ovaries were used for immunohistochemical localization of IGF-I protein or used to demonstrate mRNA expression of IGF-I. For the latter goal, preantral and antral follicles, cumulus-oocyte complex, mural granulosa and theca cells were collected to study mRNA expression. For in vitro studies, ovarian cortex were cultured for 1 and 7 days with MEM supplemented IGF-I (0, 1, 10, 50, 100 or 200 ng/ml). Immunohistochemical results showed strong reactions for IGF-I in oocytes and granulosa cells of all follicular stages, except in granulosa cells of primordial and primary follicles. mRNA expression analysis demonstrated a discrete increase in the production of IGF-I during the transition from primordial to the primary and secondary follicle stages. After 7 days of culture, addition of 50 ng/ml of IGF-I to the medium showed the greatest percentage of normal follicles when compared with other concentrations. Furthermore, the highest percentage of primary follicles was observed after 7 days of culture in MEM+ plus 10 and 50 ng/ml of IGF-I. Culture of ovarian tissue for 7 days in MEM+ plus 50 ng/ml of IGF-I promoted the greatest increase (P < 0.05) in follicular diameter of all of the concentrations tested. In conclusion, IGF-I protein and mRNA were expressed in all follicular categories of goat. Furthermore, IGF-I maintained preantral follicle survival, promoted primordial follicle activation and stimulated the transition from intermediate to primary follicles.

Regulation of ovarian folliculogenesis by Kit Ligand and the c-Kit system in mammals

The system comprised of Kit Ligand (KL) and its receptor c-Kit has proven to play a role in normal female reproduction and fertility in mammals. Gene expression studies have revealed that biological activities of ligands and receptors of the KL/c-Kit system are important in controlling apoptosis and cellular proliferation in reproductive tissues. Collectively, these studies have provided a better understanding of ovarian physiology and female fertility through the establishment of the concept that the KL/c-Kit system regulates the viability of primordial germ cells and follicles, initiation of primordial follicle growth, and further oocyte and follicular development through different signaling proteins. The purpose of this article is to review the importance of the KL/c-Kit system in ovarian follicular development, especially in the preantral phase of folliculogenesis.(AU)

Regulation of ovarian folliculogenesis by Kit Ligand and the c-Kit system in mammals

The system comprised of Kit Ligand (KL) and its receptor c-Kit has proven to play a role in normal female reproduction and fertility in mammals. Gene expression studies have revealed that biological activities of ligands and receptors of the KL/c-Kit system are important in controlling apoptosis and cellular proliferation in reproductive tissues. Collectively, these studies have provided a better understanding of ovarian physiology and female fertility through the establishment of the concept that the KL/c-Kit system regulates the viability of primordial germ cells and follicles, initiation of primordial follicle growth, and further oocyte and follicular development through different signaling proteins. The purpose of this article is to review the importance of the KL/c-Kit system in ovarian follicular development, especially in the preantral phase of folliculogenesis.

Mechanisms of atresia in ovarian follicles

There are thousands to millions of follicles in the mammalian ovary, and the majority (99.9%) are eliminated by a process known as atresia. This phenomenon occurs in any stage of follicular development, through an apoptotic manner or the degenerative process of necrosis. Thus, a better understanding of the mechanisms involved in atresia is necessary to avoid the great follicular loss that occurs in vivo and to maximize female reproductive potential. The present review focuses on aspects related to follicular population and atresia, mechanisms of atresia (apoptosis or the degenerative process of necrosis), techniques used to analyze atresia in ovarian follicles, and the occurrence of the atretic process during different follicular stages.(AU)

Angiogenic factors and ovarian follicle development

Ovarian follicles require an adequate blood supply for oxygen, nutrients and hormones, in addition to eliminating CO2 and other metabolites. Acquisition of an adequate vascular supply is probably a limiting step in the selection and maturation of the dominant follicle. In this way, there is a progressive interest in the study of the growth factors involved in the angiogenic process. In addition, a better understanding about the mechanisms that regulate the expression and action of these factors could be a key point to increase the reproductive performance in females. Therefore, this review aims to summarize current data on the importance of the pro- and anti-angiogenic growth factors which regulate angiogenesis in ovarian follicle development.(AU)

Angiogenic factors and ovarian follicle development

Ovarian follicles require an adequate blood supply for oxygen, nutrients and hormones, in addition to eliminating CO2 and other metabolites. Acquisition of an adequate vascular supply is probably a limiting step in the selection and maturation of the dominant follicle. In this way, there is a progressive interest in the study of the growth factors involved in the angiogenic process. In addition, a better understanding about the mechanisms that regulate the expression and action of these factors could be a key point to increase the reproductive performance in females. Therefore, this review aims to summarize current data on the importance of the pro- and anti-angiogenic growth factors which regulate angiogenesis in ovarian follicle development.

Mechanisms of atresia in ovarian follicles

There are thousands to millions of follicles in the mammalian ovary, and the majority (99.9%) are eliminated by a process known as atresia. This phenomenon occurs in any stage of follicular development, through an apoptotic manner or the degenerative process of necrosis. Thus, a better understanding of the mechanisms involved in atresia is necessary to avoid the great follicular loss that occurs in vivo and to maximize female reproductive potential. The present review focuses on aspects related to follicular population and atresia, mechanisms of atresia (apoptosis or the degenerative process of necrosis), techniques used to analyze atresia in ovarian follicles, and the occurrence of the atretic process during different follicular stages.