Extract of Cimicifuga racemosa (L.) Nutt protects ovarian follicle reserve of mice against in vitro deleterious effects of dexamethasone

The present study aims to investigate if Cimicifuga racemosa (L.) Nutt extract (CIMI) reduces deleterious effects of dexamethasone (DEXA) in ovaries cultured in vitro. Mouse ovaries were collected and cultured in DMEM+ only or supplemented with 5 ng/mL of CIMI, or 4 ng/mL DEXA, or both CIMI and DEXA. The ovaries were cultured at 37.5°C in 5% CO2 for 6 days. Ovarian morphology, follicular ultrastructure, and the levels of mRNA for Bax, Bcl-2, and Caspase-3 were evaluated. The results showed that DEXA reduced the percentage of morphologically normal follicles, while CIMI prevented the deleterious effects caused by DEXA. In addition, DEXA negatively affected the stromal cellular density, while CIMI prevented these adverse effects. Ovaries cultured with DEXA and CIMI showed similar levels of mRNA for Bax, Bcl-2, and Caspase-3 compared to those cultured in control medium, while ovaries cultured with DEXA had increased expression of the above genes. Additionally, the ultrastructure of the ovaries cultured with CIMI was well preserved. Thus, the extract of CIMI was able to prevent the deleterious effects caused by DEXA on cultured mouse ovaries.

Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.

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.

Conservação de folículos pré-antrais bovinos em solução salina 0,9 por cento ou TCM 199 / Preservation of bovine preantral follicles in 0.9 percent saline solution or TCM 199

Investigou-se a eficiência da solução salina 0,9 por cento (SS) e TCM 199 na conservação de folículos pré-antrais (FOPA) bovinos in situ em diferentes temperaturas e tempos de incubação. Cada par ovariano foi dividido em 25 fragmentos. Um fragmento foi escolhido aleatoriamente e fixado imediatamente após a coleta (controle). Os demais foram distribuídos em tubos contendo SS ou TCM 199 a 4, 20 ou 39ºC por 2, 4, 12 ou 24h. A análise histológica mostrou que a conservação a 4ºC em ambas as soluções manteve a porcentagem de FOPA normais similar ao controle. A conservação em SS a 20ºC por 12 ou 24h, TCM 199 a 20ºC por 24h e em ambas as soluções a 39ºC a partir de 2h aumentou (P<0,05) a porcentagem de FOPA degenerados comparada à porcentagem de folículos-controle. Em ambas as soluções, independente do tempo de incubação, a porcentagem de folículos normais, após conservação a 39ºC, foi (P<0,05) menor que a obtida com 4 e 20ºC. FOPA bovinos podem ser conservados eficientemente a 4ºC por até 24h em ambas as soluções, e a 20ºC por 4 e 12h em SS e TCM 199, respectivamente.

The efficiency of 0.9 percent saline solution (SS) and TCM 199 on the preservation of bovine preantral follicles (PF) in situ at different temperatures and incubation times was investigated. Each ovarian pair was divided into 25 fragments. One fragment was taken randomly and immediately fixed (control). The other fragments were distributed in tubes containing SS or TCM 199 at 4, 20 or 39ºC for 2, 4, 12 or 24h. The histological analysis showed that the storage at 4ºC in both solutions kept the percentage of normal follicles similar to control values. Preservation in SS at 20ºC for 12 or 24h, TCM 199 at 20ºC for 24h and in both solutions at 39ºC from 2 h onward (P<0.05) increased the percentage of degenerated follicles when compared with control. In both solutions, independent of incubation time, the percentage of normal follicles observed at 39ºC was (P<0.05) lower them those observed at 4 and 20ºC. Bovine PF can be preserved efficiently at 4ºC for up to 24h in both solutions, at 20ºC for 4 and 12h in SS and TCM 199, respectively.