Effect of ovarian tissue storage in Morus nigra extract on the morphology and DNA fragmentation of ovine preantral follicles / Efeito da conservação de tecido ovariano em extrato de Morus nigra sobre a morfologia e fragmentação de DNA de folículos pré-antrais ovinos

This study demonstrated the effect of Morus nigra leaf extract during ovine ovarian tissue transportation on the survival and apoptosis of preantral follicles in vitro. High Performance Liquid Chromatography (HPLC) was used to determine the fingerprint chromatogram of the crude ethanolic extract. Four pairs of ovaries from four sheep were collected. The ovarian cortex was fragmented and one fragment was fixed in 10% buffered formaldehyde and processed for histological and TUNEL analysis (fresh control). The other fragments were placed in Minimal Essential Medium (MEM control medium) or M. nigra extract (0.025; 0.05 or 0.1 mg/mL) and stored (simulating transport) at 4ºC for 6, 12 or 24 h. Preserved fragments (6 h) were also destined to histological and TUNEL analysis. HPLC analysis confirmed the presence of antioxidant compounds (rutin, isoquercetin e kaempferitrin) in the extract. There was a decrease (P < 0.05) in the percentage of morphologically normal preantral follicles after preservation in all treatments compared to the fresh control. The percentage of normal preantral follicles after preservation in M. nigra at 0.05 mg/mL for 6 h was higher (P < 0.05) than in MEM or 0.025 mg/mL M. nigra and similar (P > 0.05) to 0.1 mg/mL of the extract. Apoptosis increased (P < 0.05) after conservation for 6 h in all treatments compared to the fresh control. Moreover, TUNEL positive cells decreased (P < 0.05) after preservation in 0.05 or 0.1 mg/mL M. nigra compared to MEM or 0.025 mg/mL M. nigra. In conclusion, 0.05 mg/mL M. nigra extract can be used as a preservation medium for ovine ovarian tissue at 4°C for up to 6 h.(AU)

Este estudo demonstrou o efeito do extrato das folhas de Morus nigra durante o transporte na sobrevivência e apoptose de folículos pré-antrais in vitro. A CLAE (Cromatografia Líquida de Alta Eficiência) foi usada para determinar a impressão digital cromatográfica do extrato etanólico. Foram coletados 4 pares de ovários de 4 ovelhas. O córtex ovariano foi fragmentado e um dos fragmentos foi fixado em formaldeído tamponado a 10% e destinado à histologia clássica e análise de TUNEL (controle fresco). Os demais fragmentos foram colocados em Meio Essencial Mínimo (MEM - meio controle) ou extrato de M. nigra (0,025, 0,05 ou 0,1 mg/mL) e conservados (simulando transporte) à 4 ºC por 6, 12 ou 24 h. Os fragmentos conservados (6h) também foram destinados à análise histológica e de TUNEL. A análise de CLAE confirmou a presença de compostos antioxidantes (rutina, isoquercetina e canferitrina) no extrato. Houve uma redução (P < 0,05) na percentagem de folículos pré-antrais morfologicamente normais após conservação em todos os tratamentos em comparação com o controle fresco. A percentagem de folículos pré-antrais normais após conservação em M. nigra a 0,05 mg/mL por 6 h foi maior (P < 0,05) do que em MEM ou M. nigra a 0,025 mg/mL e similar (P > 0,05) ao extrato a 0,1 mg/mL. A apoptose aumentou (P < 0,05) após conservação durante 6 horas em todos os tratamentos em comparação com o controle fresco. Além disso, as células TUNEL positivas diminuíram (P < 0,05),após conservação em 0,05 ou 0,1mg/mL de M. nigra em comparação com MEM ou 0,025 mg/mL de M. nigra. Em conclusão, 0,05 mg/mL de M. nigra pode ser usado como meio de conservação de tecido ovariano ovino a 4 °C durante até 6 h.(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)

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) 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)

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.

Effect of ovarian tissue storage in Morus nigra extract on the morphology and DNA fragmentation of ovine preantral follicles / Efeito da conservação de tecido ovariano em extrato de Morus nigra sobre a morfologia e fragmentação de DNA de folículos pré-antrais ovinos

This study demonstrated the effect of Morus nigra leaf extract during ovine ovarian tissue transportation on the survival and apoptosis of preantral follicles in vitro. High Performance Liquid Chromatography (HPLC) was used to determine the fingerprint chromatogram of the crude ethanolic extract. Four pairs of ovaries from four sheep were collected. The ovarian cortex was fragmented and one fragment was fixed in 10% buffered formaldehyde and processed for histological and TUNEL analysis (fresh control). The other fragments were placed in Minimal Essential Medium (MEM control medium) or M. nigra extract (0.025; 0.05 or 0.1 mg/mL) and stored (simulating transport) at 4ºC for 6, 12 or 24 h. Preserved fragments (6 h) were also destined to histological and TUNEL analysis. HPLC analysis confirmed the presence of antioxidant compounds (rutin, isoquercetin e kaempferitrin) in the extract. There was a decrease (P 0.05) in the percentage of morphologically normal preantral follicles after preservation in all treatments compared to the fresh control. The percentage of normal preantral follicles after preservation in M. nigra at 0.05 mg/mL for 6 h was higher (P 0.05) than in MEM or 0.025 mg/mL M. nigra and similar (P > 0.05) to 0.1 mg/mL of the extract. Apoptosis increased (P 0.05) after conservation for 6 h in all treatments compared to the fresh control. Moreover, TUN

Este estudo demonstrou o efeito do extrato das folhas de Morus nigra durante o transporte na sobrevivência e apoptose de folículos pré-antrais in vitro. A CLAE (Cromatografia Líquida de Alta Eficiência) foi usada para determinar a impressão digital cromatográfica do extrato etanólico. Foram coletados 4 pares de ovários de 4 ovelhas. O córtex ovariano foi fragmentado e um dos fragmentos foi fixado em formaldeído tamponado a 10% e destinado à histologia clássica e análise de TUNEL (controle fresco). Os demais fragmentos foram colocados em Meio Essencial Mínimo (MEM - meio controle) ou extrato de M. nigra (0,025, 0,05 ou 0,1 mg/mL) e conservados (simulando transporte) à 4 ºC por 6, 12 ou 24 h. Os fragmentos conservados (6h) também foram destinados à análise histológica e de TUNEL. A análise de CLAE confirmou a presença de compostos antioxidantes (rutina, isoquercetina e canferitrina) no extrato. Houve uma redução (P 0,05) na percentagem de folículos pré-antrais morfologicamente normais após conservação em todos os tratamentos em comparação com o controle fresco. A percentagem de folículos pré-antrais normais após conservação em M. nigra a 0,05 mg/mL por 6 h foi maior (P 0,05) do que em MEM ou M. nigra a 0,025 mg/mL e similar (P > 0,05) ao extrato a 0,1 mg/mL. A apoptose aumentou (P 0,05) após conservação durante 6 horas em todos os tratamentos em comparação com o

Effect of ovarian tissue storage in Morus nigra extract on the morphology and DNA fragmentation of ovine preantral follicles / Efeito da conservação de tecido ovariano em extrato de Morus nigra sobre a morfologia e fragmentação de DNA de folículos pré-antrais ovinos

This study demonstrated the effect of Morus nigra leaf extract during ovine ovarian tissue transportation on the survival and apoptosis of preantral follicles in vitro. High Performance Liquid Chromatography (HPLC) was used to determine the fingerprint chromatogram of the crude ethanolic extract. Four pairs of ovaries from four sheep were collected. The ovarian cortex was fragmented and one fragment was fixed in 10% buffered formaldehyde and processed for histological and TUNEL analysis (fresh control). The other fragments were placed in Minimal Essential Medium (MEM control medium) or M. nigra extract (0.025; 0.05 or 0.1 mg/mL) and stored (simulating transport) at 4ºC for 6, 12 or 24 h. Preserved fragments (6 h) were also destined to histological and TUNEL analysis. HPLC analysis confirmed the presence of antioxidant compounds (rutin, isoquercetin e kaempferitrin) in the extract. There was a decrease (P 0.05) to 0.1 mg/mL of the extract. Apoptosis increased (P < 0.05) after conservation for 6 h in all treatments compared to the fresh control. Moreover, TUNEL positive cells decreased (P < 0.05) after preservation in 0.05 or 0.1 mg/mL M. nigra compared to MEM or 0.025 mg/mL M. nigra. In conclusion, 0.05 mg/mL M. nigra extract can be used as a preservation medium for ovine ovarian tissue at 4°C for up to 6 h.

Este estudo demonstrou o efeito do extrato das folhas de Morus nigra durante o transporte na sobrevivência e apoptose de folículos pré-antrais in vitro. A CLAE (Cromatografia Líquida de Alta Eficiência) foi usada para determinar a impressão digital cromatográfica do extrato etanólico. Foram coletados 4 pares de ovários de 4 ovelhas. O córtex ovariano foi fragmentado e um dos fragmentos foi fixado em formaldeído tamponado a 10% e destinado à histologia clássica e análise de TUNEL (controle fresco). Os demais fragmentos foram colocados em Meio Essencial Mínimo (MEM - meio controle) ou extrato de M. nigra (0,025, 0,05 ou 0,1 mg/mL) e conservados (simulando transporte) à 4 ºC por 6, 12 ou 24 h. Os fragmentos conservados (6h) também foram destinados à análise histológica e de TUNEL. A análise de CLAE confirmou a presença de compostos antioxidantes (rutina, isoquercetina e canferitrina) no extrato. Houve uma redução (P 0,05) ao extrato a 0,1 mg/mL. A apoptose aumentou (P < 0,05) após conservação durante 6 horas em todos os tratamentos em comparação com o controle fresco. Além disso, as células TUNEL positivas diminuíram (P < 0,05),após conservação em 0,05 ou 0,1mg/mL de M. nigra em comparação com MEM ou 0,025 mg/mL de M. nigra. Em conclusão, 0,05 mg/mL de M. nigra pode ser usado como meio de conservação de tecido ovariano ovino a 4 °C durante até 6 h.