Transforming growth factor-β (TGF-β) maintains follicular ultrastructure and stimulates preantral follicle growth in caprine ovarian tissue cultured in vitro / Fator de crescimento transformante-β (TGF-β) mantém a ultraestrutura folicular e estimula o crescimento de folículos pré-antrais caprinos inclusos em tecido ovariano cultivado in vitro

The objectives of this study were to investigate whether TGF-β affect the survival, activation and further growth of goat primordial follicles enclosed in ovarian cortex after in vitro culture. Goat ovaries were collected from an abattoir and pieces of ovarian tissues were cultured for one or seven days in a supplemented alpha Minimum Essential Medium, alone or containing TGF-β (1, 5, 10 or 50ng/mL). Ovarian tissues from the fresh control as well as those cultured were processed for histological and ultrastructural studies. The results showed that when compared with fresh control, there was decrease in the percentages of histologically normal follicles in all treatments only after seven days culture. TGF-β did not affect the activation of preantral follicles regardless of its concentration, however, larger follicles diameter (P<0.05) was observed using 10ng/mL TGF-β than in the fresh control and other treatments. Moreover, this concentration maintained the normal ultrastructure after seven days of culture. In conclusion, TGF-β showed additional effect on the follicle growth and the maintenance of ultrastructural integrity of goat preantral follicles enclosed in ovarian tissue when used at 10ng/mL during seven days of culture.(AU)

O objetivo desse estudo foi investigar se o TGF-β afeta a sobrevivência, ativação e crescimento de folículos primordiais caprinos inclusos no córtex ovariano após o cultivo in vitro. Ovários de cabras foram coletados em abatedouro e fragmentos de tecido ovariano foram cultivados por um e sete dias em meio essencial mínimo alfa (α-MEM+) sozinho ou suplementado com TGF-β (1, 5, 10 ou 50ng/mL). Fragmentos ovarianos não cultivados e cultivados foram processados para análise histológica e ultraestrutural. Os resultados mostraram que, comparado ao controle fresco, houve diminuição no percentual de folículos morfologicamente normais em todos os tratamentos somente após sete dias de cultivo. O TGF-β não afetou a ativação folicular independente da concentração testada, contudo, o diâmetro folicular foi superior (P<0.05) no tratamento com 10ng/mL de TGF-β quando comparado ao controle fresco e aos demais tratamentos. Além disso, essa mesma concentração manteve a ultraestrutura normal dos folículos após sete dias de cultivo. Em conclusão, o TGF-β apresentou efeito adicional no crescimento folicular e na manutenção da integridade ultraestrutural de folículos pré-antrais caprinos inclusos no tecido ovariano quando utilizado na concentração de 10ng/mL durante sete dias de cultivo.(AU)

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)

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.