Unexpected effect of the vehicle (grain ethanol) of homeopathic FSH on the <i>in vitro</i> survival and development of isolated ovine preantral follicles

Lima, Laritza Ferreira de; Rocha, Rebeca Magalhães Pedrosa; Duarte, A.B.G.; Brito, I.R.; Silva, Gerlane M.; Rodrigues, Giovanna Quintino; Nunes-Pinheiro, Diana Célia Sousa; Sales, A.D.; Moura, Arlindo A.; Wheeler, Matthew B.; Rodrigues, Ana Paula Ribeiro; Campello, C.C.; Figueiredo, J.R.

doi:10.1002/jemt.22810

Cited by 8 publications

(7 citation statements)

References 48 publications

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“…insularis extract on the in vitro culture and maturation of ovine isolated follicles. While FSH has been widely used to maintain follicle survival [41], promote the antrum formation [42] and stimulate follicular growth and oocyte maturation [11, 36], similar effects were obtained with 0.3 mg/mL of J . insularis .…”

Section: Discussionmentioning

confidence: 94%

“…Immediately postmortem, pairs of ovaries were washed once in 70% alcohol and then twice in minimum essential medium (MEM) plus HEPES (MEM-HEPES). The ovaries were placed into tubes containing 15 mL of MEM-HEPES, supplemented with penicillin (100 μg/mL) and streptomycin (100 μg/mL) and then transported to the laboratory at 4°C within 1 h since they were collected [36]. A total of 342 secondary follicles obtained from those ovaries were randomly in vitro cultured for 18 days in five different treatments as described below.…”

Section: Methodsmentioning

confidence: 99%

“…Then, secondary follicles (approximately 150–250 μm in diameter) were visualized under a stereomicroscope (SMZ 645 Nikon, Tokyo, Japan) and manually dissected from the slices of ovarian cortex using 26-gauge needles. After isolation, only secondary follicles with a visible oocyte, surrounded by granulosa cells, an intact basement membrane and no antral cavity [36] were selected to in vitro culture in different media conditions, corresponding to five different treatments. Therefore, secondary follicles were cultured in α-MEM (M5650, pH 7.2–7.4), supplemented with 3 mg/mL of bovine serum albumin (BSA), ITS (10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium), 2 mM of glutamine, 2 mM of hypoxanthine, 50 ng/mL of Leukemia Inhibitory Factor (LIF) and 50 ng/mL of Kit Ligant (KL), referred as α-MEM + [37], considered the Control .…”

Section: Methodsmentioning

confidence: 99%

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Supplementation of in vitro culture medium with FSH to grow follicles and mature oocytes can be replaced by extracts of Justicia insularis

et al. 2018

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The present study evaluated the effect of supplementing in vitro culture medium with J. insularis compared to FSH on isolated secondary follicles and in vitro maturation of oocytes from those follicles. Secondary follicles were isolated from sheep ovaries and individually cultured for 18 days in α-MEM+ (Control), α-MEM+ supplemented with 100 ng/mL recombinant bovine follicle stimulating hormone (FSH) or with 0.3, 1.25, or 2.5 mg/mL of J. insularis extract (JI0.3, JI1.25, and JI2.5, respectively). Culture medium collected every 2 days was used to measure ROS levels. At the end of the culture period, cumulus oocytes complex (COCs) were collected and matured in vitro. Follicular walls were used for mRNA quantitation. JI0.3 led to a higher (P < 0.05) percentages of intact follicles than other groups after 18 days of culture. While follicular diameter remained unchanged from Day 6 onwards with JI0.3 and FSH, percentages of antral cavity formation were higher (P < 0.05) with JI0.3 at Day 6 than in all other treatments. No differences were observed between controls and treatment groups regarding ROS levels and mRNA expression of genes. Viability of resulting oocytes was higher (P < 0.05) in JI0.3 compared to FSH. Interestingly, in control experiment, supplementation of maturation medium with JI0.3 led to higher (P < 0.05) percentages of metaphase II compared to controls. Although more validations will be needed, it seems that this natural extract could be used as a cheap and easily available alternative to commercial FSH.

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Section: Discussionmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Supplementation of in vitro culture medium with FSH to grow follicles and mature oocytes can be replaced by extracts of Justicia insularis

et al. 2018

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“…Final products are colorless and odorless and contain 0.1% ethanol. Traces of ethanol present in highly diluted medicines may exert direct effects on cell behavior in vitro (44,45). For this reason, the last 3 M1 and M8 dilutions were performed in distilled water, and water was used as a control.…”

Section: Methodsmentioning

confidence: 99%

In vitro attenuation of classic metastatic melanoma‑related features by highly diluted natural complexes: Molecular and functional analyses

et al. 2019

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Metastasis is responsible for the majority of deaths among patients with malignant melanoma. Despite recent advances, the majority of current and modern therapies are ineffective and/or financially unfeasible. Thus, in this study, we investigated two low-cost highly-diluted natural complexes (HDNCs) that have been shown to be effective against malignant melanoma in a murine model in vivo. The aim of this study was to determine the mechanisms through which these HDNCs directly affect melanoma cells, either alone or in an artificial tumor microenvironment, suppressing the metastatic phenotype, thus explaining previous in vivo effects. For this purpose, HDNC in vitro treatments of B16-F10 melanoma cells, alone or in co-culture with Balb/3T3 fibroblasts, were carried out. Molecular biology techniques and standard functional assays were used to assess the changes in molecule expression and in cell behaviors related to the metastatic phenotype. Melanoma progression features were found to be regulated by HDNCs. Molecules related to cell adhesion (N-cadherin, β1-integrin and CD44), and migration, extracellular matrix remodeling and angiogenesis were modulated. The cell migratory, invasive and clonogenic capacities were reduced by the HDNCs. No loss of cell proliferation or viability were observed. On the whole, the findings of this study indicate that HDNCs directly reprogram, molecularly and functionally, melanoma cells in vitro, modulating their metastatic phenotype. Such findings are likely to be responsible for the attenuation of tumor growth and lung colonization previously observed in vivo.

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“…After isolation, follicles were transferred to 100 µl drops of medium under mineral oil to further evaluate follicular quality. Secondary follicles with a visible oocyte, surrounded by granulosa cells, an intact basement membrane were selected for in vitro culture according to Lima et al ().…”

Section: Methodsmentioning

confidence: 99%

Early ovine preantral follicles have a potential to grow until antral stage in two‐step culture system in the presence of aqueous extract of Justicia insularis

Mbemya

Sá

Guerreiro

et al. 2019

Reprod Domestic Animals

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The objective of this study was to determine whether preantral follicles cultured in vitro for 7 days within ovine ovarian cortical strips could be isolated at the secondary follicles (SF) and grown until antral stage during an additional 6 days period of in vitro culture in the presence of aqueous extract of Justicia insularis. Fresh ovarian fragments from 16 adult sheep were fixed for histological analysis (Control 1) or in vitro cultured individually in α‐MEM+ supplemented with 0.3 mg/ml J. insularis (Step 1) for 7 days. Part of the fragments then were fixed for histological analysis (in vitro culture group). Remaining fragments were exposed stepwise to increasing trehalose concentrations before immediate isolation of SF and viability assessment (Control 2) or after 6 days of culture in α‐MEM++ supplemented with 0.3 mg/ml J. insularis (Step 2). In Step 1, percentage of follicular activation was 80%. In Step 2, a significant increase (p < 0.05) in follicular diameter and antrum formation within 6 days in vitro culture of isolated follicles was achieved. The total antioxidant capacity from both steps significantly increase (p < 0.05) from day 2 to day 6. Confocal analysis of oocytes showed 57.14% oocytes with homogeneous distribution and 42.86% with peri‐cortical distribution. In conclusion, SF can be successfully isolated from sheep ovarian cortex after 7 days of culture and are capable of surviving and forming an antral cavity if cultured in vitro for an additional 6 days in the presence of 0.3 mg/ml J. insularis.

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Unexpected effect of the vehicle (grain ethanol) of homeopathic FSH on the in vitro survival and development of isolated ovine preantral follicles

Cited by 8 publications

References 48 publications

Supplementation of in vitro culture medium with FSH to grow follicles and mature oocytes can be replaced by extracts of Justicia insularis

Supplementation of in vitro culture medium with FSH to grow follicles and mature oocytes can be replaced by extracts of Justicia insularis

In vitro attenuation of classic metastatic melanoma‑related features by highly diluted natural complexes: Molecular and functional analyses

Early ovine preantral follicles have a potential to grow until antral stage in two‐step culture system in the presence of aqueous extract of Justicia insularis

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