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Cancer Chemother Pharmacol ; 87(4): 567-578, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33471160


PURPOSE: 5-Fluorouracil (5-FU), an anti-cancer drug, has been used for hepatoblastoma (HB) chemotherapy in children, who may have impaired  ovarian follicle pool reserve with lasting effects to reproduction. Therefore, this study aimed to investigate 5-FU effects on survival, growth, and morphology of ovarian preantral follicles from C57BL6J young mice. METHODS: Experiments were carried-out both in vivo and in vitro. Mice were treated with 5-FU injection (450 mg/kg i.p) or saline and sacrificed 3 days after to obtain ovaries for histology and molecular biology. Ovaries for in vitro studies were obtained from unchallenged mice and cultured under basic culture medium (BCM) or BCM plus 5-FU (9.2, 46.1, 92.2 mM). Preantral follicles were classified according to developmental stages, and as normal or degenerated. To assess cell viability, caspase-3 immunostaining was performed. Transcriptional levels for apoptosis (Bax, Bcl2, p53, Bax/Bcl2) and Wnt pathway genes (Wnt2 and Wnt4) were also analyzed. Ultrastructural analyses were carried-out on non-cultured ovaries. In addition, ß-catenin immunofluorescence was assessed in mouse ovaries. RESULTS: The percentage of all-types normal follicles was significantly lower after 5-FU challenge. A total loss of secondary normal follicles was found in the 5-FU group. The highest 5-FU concentrations reduced the percentage of cultured normal primordial follicles. Large vacuoles were seen in granulosa cells and ooplasm of preantral follicles by electron microscopy. A significantly higher gene expression for Bax and Bax/Bcl2 ratio was seen after 5-FU treatment. A marked reduction in ß-catenin immunolabeling was seen in 5-FU-challenged preantral follicles. In the in vitro experiments, apoptotic and Wnt gene transcriptions were significantly altered. CONCLUSION: Altogether, our findings suggest that 5-FU can deleteriously affect the ovarian follicle reserve by reducing preantral follicles survival.

Mol Pharm ; 16(7): 2912-2921, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31083895


Silica-based nanoparticles have been developed as powerful platforms for drug delivery and might also prevent undesired side effects of drugs. Here, a fast method to synthesize positively charged mesoporous silica nanoparticles (ζ = 20 ± 0.5 mV, surface area = 678 m2 g-1, and 2.3 nm of porous size) was reported. This nanomaterial was employed to anchor sodium nitroprusside (SNP), a vasodilator drug with undesired cyanide release. A remarkable incorporation of 323.9 ± 7.55 µmol of SNP per gram of nanoparticle was achieved, and a series of studies of NO release were conducted, showing efficient release of NO along with major cyanide retention (ca. 64% bound to nanoparticle). Biological assays with mammalian cells showed only a slight drop in cell viability (13%) at the highest concentration (1000 µM), while SNP exhibited an LC50 of 228 µM. Moreover, pharmacological studies demonstrated similar efficacy for vasodilation and sGC-PKG-VASP pathway activation when compared to SNP alone. Altogether, this new SNP silica nanoparticle has great potential as an alternative for wider and safer use of SNP in medicine with lower cyanide toxicity.

Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Doadores de Óxido Nítrico/efeitos adversos , Doadores de Óxido Nítrico/química , Nitroprussiato/efeitos adversos , Nitroprussiato/química , Dióxido de Silício/química , Animais , Aorta Torácica/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Liberação Controlada de Fármacos , Cobaias , Masculino , Óxido Nítrico/metabolismo , Porosidade , Artéria Pulmonar/efeitos dos fármacos , Ratos , Ratos Wistar , Propriedades de Superfície , Células Vero
Reprod Biol Endocrinol ; 12: 78, 2014 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-25117631


Preantral follicles are the majority of the ovarian follicle population and their use as a source of homogeneous oocytes for bovine reproductive biotechnologies could result in a substantial advance in this field. However, while in other species embryos and offspring have been produced, in bovine species the results have been limited to the follicular activation of small (primordial) preantral follicles and formation of early antral follicles from large (secondary) preantral follicles after in vitro culture. Therefore, this review will highlight the basic aspects of bovine folliculogenesis by focusing on preantral follicles, the methods of harvesting preantral follicles, the main results from in vitro follicular culture during the last 20 years, and the potential candidate substances (basic supplements, growth factors, and hormones) for improving the efficiency of in vitro follicle growth.

Bovinos/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oogênese , Folículo Ovariano/citologia , Animais , Feminino , Oogônios/citologia , Técnicas de Reprodução Assistida/veterinária , Técnicas de Cultura de Tecidos/veterinária
Cell Tissue Res ; 344(3): 539-50, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21503599


Our aim was to verify the steady-state level of epidermal growth factor (EGF) mRNA in goat follicles at various developmental stages and to investigate the influence of EGF on the survival, antrum formation and growth of secondary follicles cultured for 6 days. Primordial, primary and secondary goat follicles and small and large antral follicles were obtained to quantify EGF mRNA by real-time reverse transcription with the polymerase chain reaction. The influence of EGF and the presence or absence of follicle-stimulating hormone (FSH) on the development of secondary follicles and on mRNA expression for EGF and FSH receptor (FSH-R) was determined after 6 days of culture. Survival, antrum formation and follicular diameter were evaluated every other day of culture. EGF mRNA levels in secondary follicles were significantly higher than those in primordial follicles, whereas in small and large antral follicles, EGF mRNA levels in cumulus-oocyte complexes (COCs) were significantly higher than in granulosa/theca cells. During culture, EGF in the presence or absence of FSH increased the follicular daily growth rate of secondary follicles when compared with that in enriched alpha minimal essential medium. FSH, EGF or both reduced EGF mRNA levels, whereas EGF reduced FSH-R mRNA levels after follicle culture for 6 days. Thus, EGF mRNA levels are higher in secondary follicles than in earlier stages, with both FSH and EGF promoting the growth of goat secondary follicles. EGF and/or FSH reduce EGF mRNA levels, whereas EGF decreases FSH-R mRNA levels, in cultured secondary follicles.

Fator de Crescimento Epidérmico/metabolismo , Cabras/metabolismo , Folículo Ovariano/citologia , RNA Mensageiro/metabolismo , Animais , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/farmacologia , Feminino , Hormônio Foliculoestimulante/metabolismo , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , RNA Mensageiro/genética
Pesqui. vet. bras ; 30(4): 305-310, abr. 2010. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-548881


This study was conducted in order to verify the effect of different concentrations of BMP-7 in the in vitro survival and development of caprine preantral follicles. Fragments of caprine ovarian cortical tissue were cultured for 1 or 7 days in Minimum Essential Medium (MEM+) supplemented with different concentrations of BMP-7 (1, 10, 50 or 100ng/ml). Non-cultured fragments or those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM). Parameters such as follicular survival, activation and growth were evaluated. The results showed that, after 1 or 7 days of culture, the percentage of morphologically normal follicles was significantly reduced in all treatments when compared with fresh control, except at 1ng/ml of BMP-7 for 1 day. In addition, the concentration of 10ng/ml of BMP-7 significantly increases follicular diameter from day 1 to 7 of culture. There was no influence of the other concentrations of BMP-7 regarding to the follicular and oocyte diameter. Ultrastructure studies confirmed follicular integrity after 7 days of culture in 1ng/ml BMP-7. In conclusion, small concentrations of BMP-7 can improve the survival and growth of caprine preantral follicles during in vitro culture.

O presente trabalho foi conduzido de modo a se verificar o efeito de diferentes concentrações da BMP-7 no desenvolvimento in vitro de folículos pré-antrais caprinos. Fragmentos de tecido cortical ovariano caprino foram cultivados por 1 ou 7 dias em Minimum Essential Medium (MEM+) suplementado com diferentes concentrações de BMP-7 (1, 10, 50 ou 100ng/ml). Os fragmentos não cultivados ou aqueles cultivados por 1 ou 7 dias foram processados para histologia clássica e microscopia eletrônica de transmissão (TEM), sendo avaliados parâmetros morfológicos indicativos de viabilidade, ativação e crescimento. Os resultados mostraram que o percentual de folículos morfologicamente normais diminuiu significativamente em todos os tratamentos quando comparados ao controle, exceto na concentração de 1ng/ml por 1 dia de cultivo. Já no D7 todos os tratamentos reduziram significativamente os percentuais de folículos morfologicamente normais. Utilizando 10ng/ml de BMP-7 foi observado um aumento significativo no diâmetro folicular quando comparados os diferentes períodos de cultivo. Não houve influência das demais concentrações de BMP-7 quando avaliados além do diâmetro folicular o diâmetro oocitário. A análise por TEM confirmou a integridade ultra-estrutural nos folículos após 7 dias de cultivo com 1ng/ml de BMP-7 . Em conclusão, o BMP-7 em baixas concentrações pode melhorar a sobrevivência e o crescimento durante o cultivo in vitro de folículos pré-antrais caprinos.

Animais , Ovário/anatomia & histologia , Proteínas Morfogenéticas Ósseas/administração & dosagem , Cabras