RESUMO
BACKGROUND: Crocin is an active ingredient of saffron (Crocus sativus L.) and its antioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione (GSH) synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant. MATERIALS AND METHODS: In the present in vitro experimental study, we collected cumulus oocyte complexes (COCs) from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute (NMRI) mice. Oocytes were subjected to in vitro maturation (IVM) in the presence of either crocin (5 or 10 µg/ml), 5 mM buthionine-[S-R]- sulfoximine (BSO), or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes (n=631) were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II (MII) oocytes after IVM (n=240) were also assessed by the 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay. RESULTS: Supplementation of IVM media with 10 µg/ml crocin significantly (P<0.05) increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of 5 µg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 µg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group. CONCLUSION: Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential of mouse oocytes. This may occur by its beneficial effect in increasing GSH concentrations in MII oocytes.
RESUMO
OBJECTIVE: Reactive oxygen species have effects on gamete quality and gamete interaction; they influence spermatozoa, oocytes, embryos, and their environment. In this study, we evaluated the antioxidant effect of different concentrations of saffron (Crocus sativus L.) aqueous extract (SAE) and its ingredient, crocin, on the improvement of in vitro maturation (IVM) and subsequent in vitro fertilization (IVF) and embryo development of mouse oocytes. MATERIALS AND METHODS: Cumulus oocyte complexes were collected from ovaries, and germinal vesicle oocytes were cultured in the presence of SAE and crocin. SAE was added at dosages of 5 µg/mL, 10 µg/mL, and 40 µg/mL; dosages of crocin were 50 µg/mL, 100 µg/mL, and 400 µg/mL. All dosages were added to maturation medium and a group without SAE or crocin was considered as the control group. Following IVM, metaphase II oocytes were fertilized and cultured in vitro in order to observe embryo development. RESULTS: Both SAE and crocin improved the rate of IVM, IVF, and in vitro culture. Addition of 40 µg/mL SAE to maturation medium significantly increased the rate of IVM, IVF, and in vitro culture (p < 0.05). Furthermore 100 µg/mL crocin significantly increased the IVM rate compared to the control group (p < 0.05). CONCLUSION: Use of SAE during IVM can affect on IVM, IVF, and early embryo development in a dose-dependent manner. SAE appears to have a stronger effect than pure crocin.