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...

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...

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3 percent), and the highest percent of primary follicles was achieved with IGF-I (57.7 percent). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively.

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.

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.