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.

Involvement of vasoactive intestinal peptide (VIP) on ovarian physiology

In addition to the central nervous system, vasoactive intestinal peptide (VIP) containing nerves have been described throughout the female genital tract. VIP is reported to be produced by nerves fibers innervating follicles at all stages of development in rodents. There is growing evidence that VIP and their receptors play important roles in the local regulation of ovarian physiology mostly through cAMP pathway. It has been reported that VIP regulates the ovarian follicle survival and growth, oocyte maturation, ovulation and steroidogenesis. Studies also demonstrated that VIP inhibits apoptosis of rat follicles and is an important factor for the growth of preantral follicles enclosed in caprine ovarian tissue. Even though the addition of VIP to the culture medium did not improve in vitro maturation and fertilization of oocytes, it has been shown to stimulate ovulation in perfused rat ovaries. VIP is also involved in the regulation of steroidogenic activity. Therefore, this review aims to summarize current data on the importance of VIP on ovarian physiology.

Involvement of vasoactive intestinal peptide (VIP) on ovarian physiology

In addition to the central nervous system, vasoactive intestinal peptide (VIP) containing nerves have been described throughout the female genital tract. VIP is reported to be produced by nerves fibers innervating follicles at all stages of development in rodents. There is growing evidence that VIP and their receptors play important roles in the local regulation of ovarian physiology mostly through cAMP pathway. It has been reported that VIP regulates the ovarian follicle survival and growth, oocyte maturation, ovulation and steroidogenesis. Studies also demonstrated that VIP inhibits apoptosis of rat follicles and is an important factor for the growth of preantral follicles enclosed in caprine ovarian tissue. Even though the addition of VIP to the culture medium did not improve in vitro maturation and fertilization of oocytes, it has been shown to stimulate ovulation in perfused rat ovaries. VIP is also involved in the regulation of steroidogenic activity. Therefore, this review aims to summarize current data on the importance of VIP on ovarian physiology.(AU)

Bovine serum albumin improves in vitro development of caprine preantral follicles

This work investigated the effects of different protein supplements (fetal calf serum (FCS) and bovine serum albumin (BSA)) on the in vitro development of caprine preantral follicles. Preantral follicles (> 150 um) were isolated from ovarian cortex fragments and individually cultivated in a-MFM medium in an incubator at 37ºC for 24 days with 5% atmospheric Co2, and supplemented with either BSA at 1,25 or 3,0 mg/ml or FCS at 5 10%. An evaluation of follicular development was conducted based on survival rate, antrum formation, increase in follicular diameter, oocyte viability and obtainment of fully-grown oocytes. It was observed that from the 12th cultivation day, the oercentage of surviving follicles under treatment with BSA at 3.0 mg/ml was greater than that of the other treatments (p< 0,05) when compared to those treated with BSAA on the last day of cultivation the mean diameter and antrum formation of follicles treated with BSA at 3,0 mg/ml were greater than those of follicles under other treatments (P < 0,05). With oocyte growth, the percentage of oocytes cultivated with BSA (3,0 mg/ml) thatwere destined for in vitro maturation (IVM: >110 diameter( was higher than that of other treatments. Moreover, under thistreatment, 86% of oocytes presented a germinal vesicle and 14% restarted meiosis, out of wich 3% were mature (metaphase II). In conclusion supplementing cultuvation medium with BSA at 3,0 mg/ml not only improves follicular development but also provides meiotically-competent oocytes after in vitro cultivation of caprine preantral isolated follicles.(AU)

Bovine serum albumin improves in vitro development of caprine preantral follicles

This work investigated the effects of different protein supplements (fetal calf serum (FCS) and bovine serum albumin (BSA)) on the in vitro development of caprine preantral follicles. Preantral follicles (> 150 um) were isolated from ovarian cortex fragments and individually cultivated in a-MFM medium in an incubator at 37ºC for 24 days with 5% atmospheric Co2, and supplemented with either BSA at 1,25 or 3,0 mg/ml or FCS at 5 10%. An evaluation of follicular development was conducted based on survival rate, antrum formation, increase in follicular diameter, oocyte viability and obtainment of fully-grown oocytes. It was observed that from the 12th cultivation day, the oercentage of surviving follicles under treatment with BSA at 3.0 mg/ml was greater than that of the other treatments (p110 diameter( was higher than that of other treatments. Moreover, under thistreatment, 86% of oocytes presented a germinal vesicle and 14% restarted meiosis, out of wich 3% were mature (metaphase II). In conclusion supplementing cultuvation medium with BSA at 3,0 mg/ml not only improves follicular development but also provides meiotically-competent oocytes after in vitro cultivation of caprine preantral isolated follicles.

Androstenedione and follicle stimulating hormone involvement in the viability and development of goat preantral follicles in vitro

This study investigated the effects of androstenedione and follicle-stimulating hormone (FSH) on the viability and growth of caprine preantral follicles. Ovarian tissues were cultured for 1 or 7 days in Minimum. Essential Medium (MEM) containing androstenedione (0, 1, 10, 50 or 100 ng/ml). FSH (50 ng/ml), or a combination of these two hormones. Cultured and non-cultured control tissues were processed for histological and fluorescence analysis. In comparison with non-cultured control, a sinfnificant reduction was noted in the percentage of normal follicles in all treatments after 1 and 7 days of culture (except in all treatment with 1 ng/ml of androstenedione for 1 day). As the culture period progressed from 1 to 7 days, treatments with 10 ng/ml of androstenedione + FSH or 50 ng/ml of androstenedione alone maintained the percentage of normal follicles. After 1 day, treatments with 10, 50, or 100 ng/ml of androstenedione + FSH, or with 50 ng/ml of androstenedione alone had more developing follicles than fresh control tissue. When comparing the culture periods, treatments with 1, 10 or 100 ng/ml of androstenedione alone, or FSH alone, or FSH with 1 ng/ml of androstenedione, showed an increase in the percentage of developing follicles. After 1 and 7 days, there were no differences in oocyte and follicular diameter among the treated samples and non-cultured control or MEM+ cultured tissue. Fluorescence analysis demonstrated that only fragments cultured in 50 or 100 ng/ml of androstenedione + FSH had viable preantral follicles similar to those observed in MEM+ alone. In conclusion, androstenedione at 50 or 100 ng/ml, either associated with FSH or at 50 ng/ml alone, plays an important role in the maintenance of caprine preantral follicle viability and activation after only a short in vitro culture period. In addition, after 7 days MEM+ alone was efficient in the maintenance of viability and in follicular activation, showing the importance of basic medium composition.(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)

Androstenedione and follicle stimulating hormone involvement in the viability and development of goat preantral follicles in vitro

This study investigated the effects of androstenedione and follicle-stimulating hormone (FSH) on the viability and growth of caprine preantral follicles. Ovarian tissues were cultured for 1 or 7 days in Minimum. Essential Medium (MEM) containing androstenedione (0, 1, 10, 50 or 100 ng/ml). FSH (50 ng/ml), or a combination of these two hormones. Cultured and non-cultured control tissues were processed for histological and fluorescence analysis. In comparison with non-cultured control, a sinfnificant reduction was noted in the percentage of normal follicles in all treatments after 1 and 7 days of culture (except in all treatment with 1 ng/ml of androstenedione for 1 day). As the culture period progressed from 1 to 7 days, treatments with 10 ng/ml of androstenedione + FSH or 50 ng/ml of androstenedione alone maintained the percentage of normal follicles. After 1 day, treatments with 10, 50, or 100 ng/ml of androstenedione + FSH, or with 50 ng/ml of androstenedione alone had more developing follicles than fresh control tissue. When comparing the culture periods, treatments with 1, 10 or 100 ng/ml of androstenedione alone, or FSH alone, or FSH with 1 ng/ml of androstenedione, showed an increase in the percentage of developing follicles. After 1 and 7 days, there were no differences in oocyte and follicular diameter among the treated samples and non-cultured control or MEM+ cultured tissue. Fluorescence analysis demonstrated that only fragments cultured in 50 or 100 ng/ml of androstenedione + FSH had viable preantral follicles similar to those observed in MEM+ alone. In conclusion, androstenedione at 50 or 100 ng/ml, either associated with FSH or at 50 ng/ml alone, plays an important role in the maintenance of caprine preantral follicle viability and activation after only a short in vitro culture period. In addition, after 7 days MEM+ alone was efficient in the maintenance of viability and in follicular activation, showing the importance of basic medium composition.

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.

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.

Effects of alpha-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured / Efeitos dos antioxidantes alpha-tocoferol e ternatina na morfologia e na ativação de folículos pré-antrais caprinos cultivados in vitro

The effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15µM, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2 percent and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture.(AU)

Os efeitos do α-tocoferol e da ternatina sobre morfologia, ativação e crescimento de folículos pré-antrais caprinos cultivados in vitro, por um ou cinco dias, foram avaliados. Os fragmentos ovarianos foram imediatamente fixados (controle não-cultivado) ou cultivados in vitro, por um ou cinco dias, em Meio Essencial Mínimo (MEM) com ou sem suplementação com α-tocoferol ou ternatina nas concentrações de 5, 10 ou 15µM, formando os tratamentos MEM, TOC5, TOC10, TOC 15, TER5, TER10, TER15. O percentual de folículos pré-antrais normais no controle não-cultivado foi de 73,2 por cento, depois de cinco dias de cultivo, houve redução desse percentual em todos os tratamentos, quando comparados com o controle não-cultivado (P<0,05). O cultivo por cinco dias aumentou a ativação folicular em todos os tratamentos (P<0,05). A análise ultra-estrutural não mostrou folículos pré-antrais íntegros após cinco dias de cultivo em meio contendo antioxidantes. Concluiu-se que o α-tocoferol e a ternatina podem promover a ativação folicular, no entanto a adição desses antioxidantes nas concentrações testadas reduziu a viabilidade folicular após o cultivo in vitro.(AU)

Característica histológica, ultra-estrutural e produção de nitrito de folículos pré-antrais caprinos cultivados in vitro na ausência ou presença de soro / Histological and ultrastructural feature and nitrite production of caprine preantral follicles in vitro cultured in the presence or absence of serum

Avaliou-se o efeito da adição de diferentes tipos e concentrações de soro sobre o desenvolvimento e a sobrevivência de folículos ovarianos pré-antrais (FOPA) caprinos in vitro. Além disso, verificou-se a relação entre as concentrações de nitrito presentes no meio de cultivo e a viabilidade folicular. Cada par ovariano foi dividido em 29 fragmentos, sendo um destinado ao controle. Os fragmentos foram cultivados por um ou sete dias em meio essencial mínimo suplementado (MEM+) ou MEM+ com diferentes concentrações (10 ou 20 por cento) de soro fetal bovino (SFB), soro de cabra em estro (SCE) ou soro de cabra em diestro (SCD). Na análise morfológica após sete dias, apenas o tratamento com 10 por cento de SFB apresentou percentual de FOPA normais similar ao MEM+ (P>0,05). A análise ultra-estrutural dos folículos cultivados por sete dias com MEM+ ou MEM+ com 10 por cento de SFB mostrou danos oocitários, porém células da granulosa normais. A análise do meio de cultivo revelou correlação positiva entre a viabilidade folicular e a produção de nitrito. A suplementação com soro não melhorou a viabilidade de FOPA e a concentração de nitrito no meio de cultivo funcionou como um indicador da viabilidade das células da granulosa de FOPA caprinos cultivados in vitro.(AU)

The effect of the addition of different types and concentrations of sera on the viability and development of caprine preantal follicles (PAF) in vitro cultured was analyzed. In addition, it was evaluated the correlation between nitrite concentrations in culture medium and folicular viability. Each ovarian pair was divided in 29 fragments and one was used as control. The fragments were cultured for one or seven days in minimal essential medium (MEM+) or MEM+ with different concentrations of (10 or 20 percent) bovine fetal serum (BFS), estrous goat serum (EGS), or diestrous goat serum (DGS). After seven days, the morphological analysis showed that only the treatment with 10 percent BFS maintained the percentage of normal PAF similar to MEM+ (P>0.05). The ultrastructural analysis of follicles cultured for seven days in MEM+ or MEM+ with 10 percent BFS showed some oocyte damage, although the granulosa cells were normal. Analysis of culture medium revealed a positive correlation between follicular viability and nitrite production. Supplementation with serum did not improve the viability of PAF and nitrite levels in culture medium served as an indicator of viability of granulose cells from caprine PAF in vitro cultured.(AU)