Follicular survival, activation of primordial follicles and DNA fragmentation after storage of goat ovaries at 35ºC in supplemented Minimal Essential Medium

This study evaluated the effect of caprine ovarian tissue transportation conditions (medium supplementation and transportation duration) on the morphology, DNA fragmentation and development of cultured and non-cultured preantral follicles. After the fragmentation of ovaries, one fragment was fixed (fresh control) while the remaining slices were placed individually in two different conservation media (Minimal Essential Medium - MEM without supplementation or supplemented MEM, i.e. MEM+) and stored at 35ºC for 6 or 12 h without (non-cultured) or with a subsequent 5-day in vitro culture in supplemented alfa-MEM. After transportation, followed or not by in vitro culture, the fragments were processed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) examination. For the preserved and non-cultured fragments, the percentages of normal follicles after the storage of ovarian tissue in MEM+ for 6 h and the DNA fragmentation rates after preservation in MEM for 6 h and MEM+ for 6 or 12 h were maintained similar to the fresh control. However, all cultured treatments reduced the proportion of normal follicles and increased the percentage of TUNEL-positive cells as compared to the fresh control and non-cultured treatments. On the contrary, all culture conditions (except after preservation in MEM for 6 h) promoted an increase in primordial follicle activation. In conclusion, the use of an enriched medium (MEM+) during ovary transportation is preferable to maintain satisfactory rates of normal follicles after the preservation of caprine ovarian tissue at 35ºC for up to 6 h, without affecting the ability of the primordial follicle to grow in vitro.

Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

The aims of this study were to verify the effects of Epidermal Growth Factor (EGF) on the morphology, primordial follicle activation, growth and proliferation of granulosa cells of ovine follicles cultured in situ, as well as the effect of a PI3K inhibitor on the follicular activation. Ten ovine ovaries were divided into fragments, being one fixed for histological analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+) alone or supplemented with EGF (1, 10, 50, 100 or 200 ng/mL). Follicles were classified as normal or atretic, as primordial or growing, and the oocyte and follicle diameters were measured. PCNA immunohistochemistry was performed in the fresh control and in treatment that showed the bestresults for follicular activation. Pharmacologic inhibition of PI3K activity was performed through pretreatment in media added with 50 μMLY294002 for 1 h. The percentage of normal follicles decreased (P 0.05). In conclusion, PI3K pathway mediates the in vitrospontaneous activation of sheep primordial follicles. Moreover, EGF may act indirectly on follicular activation by promoting granulosa cell proliferation at 1 ng/mL, and EGF inhibited follicle activation in concentrations similar or higher than 10 ng/mL.

Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

The aims of this study were to verify the effects of Epidermal Growth Factor (EGF) on the morphology, primordial follicle activation, growth and proliferation of granulosa cells of ovine follicles cultured in situ, as well as the effect of a PI3K inhibitor on the follicular activation. Ten ovine ovaries were divided into fragments, being one fixed for histological analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+) alone or supplemented with EGF (1, 10, 50, 100 or 200 ng/mL). Follicles were classified as normal or atretic, as primordial or growing, and the oocyte and follicle diameters were measured. PCNA immunohistochemistry was performed in the fresh control and in treatment that showed the bestresults for follicular activation. Pharmacologic inhibition of PI3K activity was performed through pretreatment in media added with 50 μMLY294002 for 1 h. The percentage of normal follicles decreased (P < 0.05) after 7 days of culture in all treatments compared to the fresh control. A significantreduction in the percentage of primordial follicles and an increase (P < 0.05) in the growing ones were observed in all treatments compared to fresh control. Furthermore, both the control medium and 1 ng/mL EGF promoted an increase (P < 0.05) in follicular activation compared to other EGF treatments. The PCNA-positive cells in the EGF treatment were higher (P < 0.05) than in fresh control and α-MEM+. Pretreatment of ovarian tissue with PI3K inhibitor significantly inhibited (P < 0.05) α-MEM+-stimulated primordial follicle activation, but had no effect on EGF-stimulated activation (P > 0.05). In conclusion, PI3K pathway mediates the in vitrospontaneous activation of sheep primordial follicles. Moreover, EGF may act indirectly on follicular activation by promoting granulosa cell proliferation at 1 ng/mL, and EGF inhibited follicle activation in concentrations similar or higher than 10 ng/mL.(AU)

Follicular survival, activation of primordial follicles and DNA fragmentation after storage of goat ovaries at 35ºC in supplemented Minimal Essential Medium

This study evaluated the effect of caprine ovarian tissue transportation conditions (medium supplementation and transportation duration) on the morphology, DNA fragmentation and development of cultured and non-cultured preantral follicles. After the fragmentation of ovaries, one fragment was fixed (fresh control) while the remaining slices were placed individually in two different conservation media (Minimal Essential Medium - MEM without supplementation or supplemented MEM, i.e. MEM+) and stored at 35ºC for 6 or 12 h without (non-cultured) or with a subsequent 5-day in vitro culture in supplemented alfa-MEM. After transportation, followed or not by in vitro culture, the fragments were processed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) examination. For the preserved and non-cultured fragments, the percentages of normal follicles after the storage of ovarian tissue in MEM+ for 6 h and the DNA fragmentation rates after preservation in MEM for 6 h and MEM+ for 6 or 12 h were maintained similar to the fresh control. However, all cultured treatments reduced the proportion of normal follicles and increased the percentage of TUNEL-positive cells as compared to the fresh control and non-cultured treatments. On the contrary, all culture conditions (except after preservation in MEM for 6 h) promoted an increase in primordial follicle activation. In conclusion, the use of an enriched medium (MEM+) during ovary transportation is preferable to maintain satisfactory rates of normal follicles after the preservation of caprine ovarian tissue at 35ºC for up to 6 h, without affecting the ability of the primordial follicle to grow in vitro.(AU)

Efeito de diferentes meios e períodos para conservação de ovários caprinos e ovinos sobre a morfofisiologia, a apoptose e o crescimento in vitro de folículos pré-antrais

O objetivo deste estudo foi avaliar o efeito de diferentes períodos e meios para conservação de tecido ovariano ovino e caprino no transportador de oócitos sobre a sobrevivência, apoptose e desenvolvimento in vitro de folículos pré-antrais. Para isto dois experimentos foram conduzidos nas diferentes espécies utilizando a mesma metodologia. Após a coleta, ovários (n=10), foram divididos em 13 fragmentos, sendo um dos fragmentos diretamente fixado e destinado à análise histologia (controle). Os fragmentos restantes foram conservados individualmente por 6 ou 12 h no TO, à 35ºC, em criotubos contendo 2 mL de diferentes meios: Meio Essencial Mínimo acrescido de HEPES, 100 mg/mL de penicilina e 100 mg/mL de estreptomicina (MEM) ou o meio MEM suplementado com BSA (3 mg/mL), glutamina (2 mM), hipoxantina (2 mM), ITS (6,25 ?g/mL de insulina, 6,25 ?g/mL de transferrina e 6,25 ng/mL de selênio), ácido ascórbico (50 ?g/ml), FSH recombinante (50 ng/ml), penicilina (100 mg/mL) e estreptomicina (100 mg/mL), correspondendo ao meio MEM+. Após os períodos de conservação, uma parte dos fragmentos foram fixados para HC e os demais fragmentos foram destinados ao cultivo in vitro por 1 ou 5 dias em incubadora a 39ºC com 5% de CO2. Além disso, para avaliação das taxas de sobrevivência, ativação e crescimento folicular, folículos foram classificados em normais ou atrésicos, em primordiais ou em desenvolvimento (transição, primários e secundários), bem como, os diâmetros folicular e oócitário foram mensurados. Para análise de apoptose, foi realizada através da técnica de TUNEL. As taxa de apoptose foram submetidas ao teste Qui-quadrado e os dados de sobrevivência e ativação e crescimento folicular submetidos à ANOVA e teste Tukey. Os valores foram considerados significativos quando P<0,05. Os resultados mostraram que após os períodos de conservação, não houve diferença (P>0,05) na percentagem de folículos pré-antrais ovinos normais em ambos os meios e períodos comparados ao controle. Por outro lado, em caprinos, apenas folículos conservados em MEM+ em ambos os períodos mantiveram as percentagens de folículos normais similares ao controle fresco (P>0,05). Além disso, para ambos os experimentos, observou-se uma redução (P<0,05) de folículos normais em todos os tratamentos quando o tecido ovariano foi cultivado por 5 dias e ativação folicular após 1 dia de cultivo (exceto em caprinos quando folículos foram conservados por 6 h em MEM). Houve aumento (P<0,05) do diâmetro folicular e oócitário após 5 dias de cultivo em todos os tratamentos para ambas as espécies exceto, em ovinos quando folículos foram conservados por 6 h em MEM. Células TUNEL positivas aumentaram (P<0,05) após 5 dias de cultivo em tecido ovariano conservado em MEM em ambos os períodos e aqueles conservados por 12 h em MEM+ para as diferentes espécies. Em conclusão, o tecido ovariano ovino pode ser conservado com sucesso em MEM e MEM+ a 35 ºC por até 12 h utilizando um transportador de oócitos, sem afetar a sua sobrevivência e posterior capacidade de se desenvolver in vitro. Por outro lado, o tecido ovariano caprino só obtém o mesmo sucesso quando conservado em MEM+ pelo mesmo período. Palavras-chave: Cabras, ovelhas, preservação, folículo ovariano, cultivo in vitro, sobrevivência