RESUMO
Ovarian fragments were exposed to 0.5 M sucrose and 1 M ethylene glycol (freezing solution; FS) with or without selenium or Trolox. Histological and ultrastructural analyses showed that the percentages of normal follicles in control tissue and in tissue after exposure to FS + 50 µM Trolox were similar. Trolox prevented endoplasmic reticulum (ER)-related vacuolization, which is commonly observed in oocytes and stromal tissue after exposure to FS. From the evaluated stress markers, superoxide dismutase 1 (SOD1) was up-regulated in ovarian tissue exposed to FS + 10 ng/ml selenium. Ovarian fragments were subsequently frozen-thawed in the presence of FS with or without 50 µM Trolox, followed by in vitro culture (IVC). Antioxidant capacity in ovarian fragments decreased after freeze-thawing in Trolox-free FS compared with FS + 50 µM Trolox. Although freezing itself minimized the percentage of viable follicles in each solution, Trolox supplementation resulted in higher rates of viable follicles (67 %), even after IVC (61 %). Furthermore, stress markers SOD1 and ERp29 were up-regulated in ovarian tissue frozen-thawed in Trolox-free medium. Relative mRNA expression of growth factors markers was evaluated after freeze-thawing followed by IVC. BMP4, BMP5, CTGF, GDF9 and KL were down-regulated independently of the presence of Trolox in FS but down-regulation was less pronounced in the presence of Trolox. Thus, medium supplementation with 50 µM Trolox prevents ER stress and, consequently, protects ovarian tissue from ER-derived cytoplasmic vacuolization. ERp29 but not ERp60, appears to be a key marker linking stress caused by freezing-thawing and cell vacuolization.
Assuntos
Cebus/metabolismo , Cromanos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Congelamento , Ovário/efeitos dos fármacos , Ovário/patologia , Vitamina E/análogos & derivados , Animais , Calreticulina/metabolismo , Crioprotetores/farmacologia , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Ovário/metabolismo , Ovário/ultraestrutura , Superóxido Dismutase/metabolismoRESUMO
The experiment described the morphological and morphometrical characteristics as well as estimate the population of primordial, primary and secondary ovarian follicles from common squirrel monkey (Saimiri sciureus). Ovaries (n=10) from five senile squirrel monkeys were collected after natural death and processed for classical histology. The mean ovarian population was estimated as 915.04 ± 78.83, 230.46 ± 20.82 and 115.88 ± 15.72 primordial, primary and secondary follicles per ovary, respectively. 73.30% were classified as primordial, 18.62% as primary, and 8.09% as secondary follicles. From all these developmental stages, the mean diameters of follicles, oocytes, oocytes nuclei and the mean number of granulosa cells were described. The number of granulosa cells surrounding normal primordial follicles (5.65 ± 0.001) was lower (P<0.05) than the number of granulosa cells (13.17 ± 0.02) surrounding the primary follicles. Secondary follicles presented the highest (P<0.001) number of granulosa cells surrounding each oocyte (273.73 ± 20.80). We have estimated the follicular population, as well as described the morphometric and morphological characteristics of preantral follicles from senile squirrel monkeys, which may be a valuable animal model for female ovarian aging studies.
Assuntos
Envelhecimento/fisiologia , Folículo Ovariano/anatomia & histologia , Folículo Ovariano/citologia , Saimiri , Animais , Contagem de Células , Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Tamanho Celular , Feminino , Células da Granulosa/citologia , Células da Granulosa/ultraestrutura , Oócitos/citologia , Oócitos/ultraestrutura , Tamanho do Órgão , Folículo Ovariano/ultraestrutura , Saimiri/anatomia & histologia , Saimiri/fisiologia , Estatística como AssuntoRESUMO
In this study, we analysed the effect on morphology and viability of ovine primordial follicles, when ascorbic acid (AA) was added to vitrification and in vitro culture (IVC) media. For morphological analysis, ovarian tissue was vitrified using DMSO or ethylene glycol (EG), to which AA was added or omitted. After warming, the tissue was fixed for histology or 1-day cultured in the presence or absence of AA. Isolated primordial follicles from ovine ovarian tissue vitrified with DMSO or EG, both supplemented with AA were stained with trypan blue for viability analysis, or 5-day cultured with or without AA followed by a viability analysis. In this study, we report on the successful vitrification protocol developed for ovine ovarian tissue using EG. Vitrification using DMSO reduced the percentage of morphological normal primordial follicles, whereas addition of AA to the vitrification and culture media did enhance these results (p < 0.05). However, vitrification in a DMSO + AA medium followed by 5-day IVC resulted in a significant decrease in the follicular viability, independently of the presence of AA in the IVC medium.