Effect of ovarian tissue storage in Morus nigra extract on the morphology and DNA fragmentation of ovine preantral follicles

Abstract

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 analyses (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 analyses. 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. ResumoEste 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 às análises histológica e 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 (6 h) também foram destinados às análises 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 h em todos os tratamentos em comparação com o controle fresco. Além diss...

Supplemented Morus nigra extract‐based medium associated with FSH enables the survival and growth of isolated ovine secondary ovarian follicles

“…A previous study has shown that the ethanolic extract of M. nigra , which was the same used in the current work, contains compounds with antioxidant properties: isoquercetin (22.3 ± 3.02 μg/ml), rutin (5.05 ± 1.3 μg/ml) and kaempferitrin (Cavalcante et al., ). We should take into account that environments in which the plant develops (temperature, relative humidity, solar radiation, soil) exert a direct influence on the chemical composition of the extracts (Floss, ; Suarez, Gil, & Lorenzo, ).…”

“…The leaves were dried in an oven at 40°C and pulverized and extracted at room temperature with 95% ethanol (Vetec, Duque de Caxias‐RJ, Brazil) for 72 hr. The extract was dried at 45°C for some hours under vacuum using rotavapor and the yield was approximately of 10% obtaining the crude ethanolic extract (CEE) of the leaves of M. nigra (Cavalcante et al., ), which was dissolved in 0.9% saline solution, corresponding to concentrations of 0.025, 0.05 or 0.1 mg/ml.…”

“…To verify the influence of the plant, the follicles (approximately 60 follicles per treatment) were randomly distributed into α‐MEM (control medium) or into medium composed of different concentrations of M. nigra extract, obtained from the dilution in 0.9% saline solution (MN 0.025, 0.05 or 0.1 mg/ml; pH 7.2–7.4). The concentrations of the M. nigra extract were chosen based on our previous study, in which this extract has been used to preserve ovine ovarian tissue (Cavalcante et al., ). In all treatments, 60 μl of the culture media was replaced with fresh media in each droplet every 2 days.…”

“…The mulberry has been widely used in folk medicine as hepatoprotective, hypotensive, antipyretic, analgesic, diuretic, expectorant and antidiabetic (Chen, Nakashima, Kimura, & Kimura, ; Nomura, ). The ethanolic extract of M. nigra has been used to preserve at 4°C for 6 hr (Cavalcante et al., ) or culture for 7 days (B. B. Gouveia, R. S. Barberino, V. G. Menezes, T. J. S. Macedo, A. Y. P. Cavalcante, R. J. S. Gonçalves, J. R. G. S. Almeida, M. H. T. Matos, unpublished data) ovine ovarian tissue, maintaining 54% of normal follicles and lower DNA damage levels than the control medium (MEM or α‐MEM) or promoting primordial follicle activation, respectively.…”

“…B. Gouveia, R. S. Barberino, V. G. Menezes, T. J. S. Macedo, A. Y. P. Cavalcante, R. J. S. Gonçalves, J. R. G. S. Almeida, M. H. T. Matos, unpublished data) ovine ovarian tissue, maintaining 54% of normal follicles and lower DNA damage levels than the control medium (MEM or α‐MEM) or promoting primordial follicle activation, respectively. Both studies used the same M. nigra extract in which the following compounds with antioxidant properties (Mata‐Campuzano et al., ; Mazimba, Majinda, & Motlhanka, ; Volpato et al., ) were identified: rutin, isoquercetin and kaempferitrin (Cavalcante et al., ).…”