RESUMO
This study aims to investigate the effects of melatonin on follicular growth, viability and ultrastructure, as well as on the levels of mRNA for antioxidant enzymes, reactive oxygen species (ROS) and meiotic progression in oocytes from in vitro cultured bovine early antral follicles. To this end, isolated early antral follicles (500-600 µm) were cultured in TCM-199+ alone or supplemented with 10-6 , 10-7 or 10-8 M melatonin at 38.5°C with 5% CO2 for 8 days. Follicle diameters were evaluated at days 0, 4 and 8 of culture. At the end of culture, ultrastructure, chromatin configuration, viability (calcein-AM and ethidium homodimer-1 staining), and the levels of ROS and mRNA for catalase (CAT), superoxide dismutase (SOD) and peroxiredoxin 6 (PRDX6) and glutathione peroxidase (GPx) were investigated in oocyte-granulosa cell complexes (OGCs). The results showed that early antral follicles cultured with 10-6 and 10-8 M melatonin had a progressive and significant increase in their diameters throughout the culture period (p < .05). Additionally, oocytes from follicles cultured with 10-7 or 10-8 M melatonin had increased fluorescence for calcein-AM, while those cultured with 10-6 or 10-7 M had reduced fluorescence for ethidium homodimer-1. Different from follicles cultured in other treatments, those cultured with 10-8 M melatonin had well-preserved ultrastructure of oocyte and granulosa cells. Melatonin, however, did not influence the levels of ROS, the mitochondrial activity, oocyte meiotic resumption and expression mRNA for SOD, CAT, GPX1 and PRDX6. In conclusion, the presence of 10-8 M melatonin in culture medium improves viability and preserves the ultrastructure of oocyte and granulosa cells of early antral follicles cultured in vitro.
Assuntos
Fluoresceínas , Melatonina , Feminino , Animais , Bovinos , Melatonina/farmacologia , Melatonina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oócitos , Superóxido Dismutase , RNA Mensageiro/metabolismoRESUMO
The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy's (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy's solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted.
Assuntos
Folículo Ovariano , Ovário , Animais , Cavalos , Feminino , Fixadores/farmacologia , Folículo Ovariano/anatomia & histologia , OócitosRESUMO
This study investigated the effects of cyclic adenosine monophosphate modulating during cumulus-oocyte complexes (COCs) pre-maturation and the role of melatonin on in vitro maturation (IVM) of bovine COCs. In experiment one, COCs were pre-matured for 8 h in control medium or with 3-isobutyl-1-methylxanthine (IBMX) and forskolin, IBMX and C-type natriuretic peptide, c-type natriuretic peptide and forskolin or IBMX, forskolin and c-type natriuretic peptide. Then, meiotic progression was evaluated. In experiment two, COCs were pre-matured, followed by IVM in control medium alone or with 10-6, 10-7 or 10-8 M melatonin. After IVM, chromatin configuration, transzonal projections (TZPs), reactive oxygen species, mitochondrial distribution, ultrastructure and mRNA expression for antioxidant enzymes were evaluated. In experiment 1, COCs pre-matured with both C-type natriuretic peptide and forskolin or C-type natriuretic peptide, forskolin and IBMX had lower meiotic resumption rate when compared to control. Considering that IBMX had not an additional effect to potentiate inhibition of meiotic resumption, a combination of C-type natriuretic peptide and forskolin was chosen. In experiment 2, COCs matured with 10-8 M melatonin had greater rates of meiotic resumption when compared to the other treatments (P < 0.05). The COCs matured with 10-7 or 10-8 M melatonin had greater mitochondrial activity (P < 0.05), while those matured with 10-6 or 10-8 M of melatonin had greater levels of TZPs. Ultrastructure of oocyte and cumulus cells after IVM with melatonin was relatively well preserved. COCs matured with 10-8 M melatonin increased mRNA expression for superoxide dismutase (SOD) and catalase (CAT) (P < 0.05), when compared to non-cultured and pre-matured COCs, respectively. In conclusion, bovine COC pre-maturation with C-type natriuretic peptide and forskolin, followed by IVM with 10-8 M melatonin improves meiotic resumption rates, TZPs, mitochondrial distribution and mRNA expression for SOD and CAT.