Thymol increases primordial follicle activation, protects stromal cells, collagen fibers and down-regulates expression of mRNA for superoxide dismutase 1, catalase and periredoxin 6 in cultured bovine ovarian tissues.

This study aims to investigate the influence of thymol on primordial follicle growth and survival, as well as on collagen fibers and stromal cells density in bovine ovarian tissues cultured in vitro. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the thiol levels and the expression of mRNAs for SOD1, CAT, periredoxin 6 (PRDX6) and GPX1 were also investigated. Ovarian cortical tissues were cultured in α-MEM+ alone or with thymol (400, 800, 1600 or 3200 µg/mL) for six days. Before and after culture, the tissues were processed for histological analysis to evaluate follicular activation, growth, morphology, ovarian stromal cell density and collagen fibers. The levels of mRNA for SOD1, CAT, GPX1 and PRDX6 were evaluated by real-time PCR. The results show that tissues cultured with thymol (400 and 800 µg/mL) had increased percentages of normal follicles, when compared to tissues cultured in other treatments. At concentrations of 400 and 800 µg/mL, thymol maintained the rate of normal follicles similar to the uncultured control. In addition, 400 µg/mL thymol increased follicle activation, collagen fibers and stromal cell density of when compared to tissues cultured in control medium. The presence of 800 µg/mL thymol in culture medium increased CAT activity, while 400 or 800 µg/mL thymol reduced mRNA levels for SOD1, CAT and PRDX6, but did not alter GPX1 expression. In conclusion, 400 µg/mL thymol increases primordial follicle activation, preserves stromal cells, collagen fibers, and down-regulates expression of mRNA for SOD1, CAT and PRDX6 in cultured bovine ovarian tissues.

Effects of N-acetylcysteine on Growth, Viability, and Ultrastructure of In Vitro Cultured Bovine Secondary Follicles.

This study aimed to investigate the effects of different concentrations of N-acetylcysteine (NAC) on the growth, antrum formation, viability, and ultrastructure of bovine secondary follicles cultured in vitro for 18 days. To this end, the follicles were cultured in TCM-199+ medium alone or supplemented with 1.0, 5.0, or 25.0 mM NAC. Follicular growth, antrum formation, viability (calcein-AM and ethidium homodimer-1) and ultrastructure were evaluated at the end of culture period. The results showed that 1.0 mM NAC increased the percentage of growing follicles and the fluorescence intensity for calcein-AM when compared to other treatments (p < 0.05). On the other hand, follicles cultured with 25.0 mM NAC had higher fluorescence intensity for ethidium homodimer-1, which is a sign of degeneration. Ultrastructural analysis showed that oocytes from follicles cultured in control medium alone or with 1 mM NAC had intact zonae pellucidae in close association with oolemmae, but the ooplasm showed mitochondria with a reduced number of cristae. On the other hand, oocytes from follicles cultured with 5 or 25 mM NAC had extremely vacuolated cytoplasm and no recognizable organelles. In conclusion, 1 mM NAC increases cytoplasmic calcein staining and the growth rate in bovine secondary follicles cultured in vitro, but the presence of 5 or 25 mM NAC causes damage in cellular membranes and organelles, as well as reducing the percentages of growing follicles.

Effects of Aloe vera extract on growth, viability, ultrastructure and expression of mRNA for antioxidant enzymes in bovine secondary follicles cultured in vitro.

This study aimed to investigate the effects of Aloe vera extract on follicular growth, viability, ultrastructure, and mRNA levels for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 1 (GPX1) and peroxiredoxin 6 (PRDX6) in bovine secondary follicles cultured in vitro. To this end, secondary follicles were mechanically isolated from the ovarian cortex and cultured at 38.5 °C, with 5% CO2 in air, for 18 days in TCM-199+ alone or supplemented with 2.5%, 5.0%, 10.0% and 20.0% Aloe vera extract. Follicular growth, morphology and antrum formation were evaluated every 6 days, while ultrastructure was evaluated at the end of culture. Analysis of viability was performed by calcein-AM and ethidium homodimer-1, while mRNA levels for SOD, CAT, GPX1 and PRDX6 were evaluated by real-time PCR at the end of culture. The results show that follicles cultured with 2.5% Aloe vera had increased the rate of antrum formation, while 2.5% and 5.0% Aloe vera improved follicular viability rate. Follicles cultured with 2.5% and 10.0% Aloe vera increased the levels of mRNA for SOD and GPX1 respectively, but the levels of CAT were reduced in follicles cultured with 2.5%, 5.0%, 10.0% and 20.0%. Additionally, follicles cultured with 2.5% of Aloe vera had their ultrastructure well preserved, while those cultured with 5.0%, 10.0% and 20.0% exhibited increased oocyte vacuolization and damaged organelles. In conclusion, 2.5% Aloe vera increases antrum formation, viability and expression of mRNA for SOD in cultured secondary follicles, but higher concentrations of Aloe vera have negative effects on follicular ultrastructure.

Protective Effect of Cimicifuga racemosa (L.) Nutt Extract on Oocyte and Follicle Toxicity Induced by Doxorubicin during In Vitro Culture of Mice Ovaries.

This study evaluated the potential of Cimicifuga racemosa (L.) Nutt extract (CIMI) to reduce the deleterious effects of doxorubicin (DOXO) in oocytes, follicles and stromal cells in mice ovaries cultured in vitro. In experiment 1, mice ovaries were cultured in DMEM+ alone or supplemented with 5, 50 or 500 ng/mL CIMI, while in experiment 2, mice ovaries were cultured in DMEM+ alone or supplemented with 5 ng/mL CIMI (better concentration), 0.3 µg/mL DOXO or both. Thereafter, the ovaries were processed for histological (morphology, growth, activation, extracellular matrix configuration and stromal cell density), immunohistochemical (caspase-3) analyses. Follicle viability was evaluated by fluorescence microscopy (ethidium homodimer-1 and calcein) while real-time PCR was performed to analyses the levels of (mRNA for SOD, CAT and nuclear factor erythroid 2-related factor 2 (NRF2) analyses. The results showed that DOXO reduces the percentage of normal follicles and the density of stromal cells in cultured ovaries, but these harmful effects were blocked by CIMI. The DOXO reduced the percentage of primordial follicles, while the presence of CIMI alone did not influence percentage of primordial follicles. A higher staining for caspase-3 was seen in ovaries cultured in control medium alone or with DOXO when compared with those cultured with CIMI alone or both CIMI and DOXO. In addition, follicles from ovaries cultured with both CIMI and DOXO were stained by calcein, while those follicles cultured with only DOXO were stained with ethidium homodimer-1. Furthermore, ovaries cultured with CIMI or both CIMI and DOXO had higher levels of mRNA for SOD and CAT, respectively, than those cultured with only DOXO. In conclusion, the extract of CIMI protects the ovaries against deleterious effects of DOXO on follicular survival and ovarian stromal cells.