Immunolocalization of melatonin receptors in bovine ovarian follicles and in vitro effects of melatonin on growth, viability and gene expression in secondary follicles

This study aims to investigate the (1) expression of melatonin receptors types 1A/B (MTNR1A/B) in bovine ovaries and (2) the in vitro effects of melatonin on secondary folli-cle development, antrum formation, viability, and expression of messenger ribonucleic acid (mRNA) for superoxide dismutase (SOD) , catalase (CAT) , glutathione peroxidase-1 (GPX1) and peroxiredoxin 6 (PRDX6). The expression of MTNR1A/B in bovine ovarian follicles was demonstrated by immunohistochemistry. To choose the most effective concentration of melatonin on follicular growth and viability, isolated secondary follicles were cultured in-dividually at 38.5 degrees C, with 5% CO2 in air, for 18 d in TCM-199(+) alone or supplemented with 10 (-11), 10 (-9), 10 (-7) or 10 (-5) M melatonin. Then, melatonin receptor antagonist, luzindole, was tested to further evaluate the mechanisms of actions of melatonin, that is, the follicles were cultured in control medium alone or supplemented with 10 (-7) M melatonin, 10 mu M luzin-dole and both 10 (-7) M melatonin and 10 mu M luzindole. Follicular growth, morphology and antrum formation were evaluated at days 6, 12 and 18. At the end of culture, viability of secondary follicles was analyzed by calcein-AM and ethidium homodimer-1, and the rela-tive levels of mRNA for SOD, CAT, GPX1 and PRDX6 were evaluated by real time polymerase chain reaction. Immunohistochemistry results showed expression of MTNR1A/B in oocyte and granulosa cells of primordial, primary, secondary and antral follicles. Secondary follicles cultured in medium supplemented with melatonin at different concentrations had well pre-served follicles after 18 d of culture. Furthermore, follicles cultured in presence of 10 (-7) M melatonin presented significantly higher diameters than those cultured in other treatments. The presence of melatonin receptor antagonist, luzindole, blocked the effects of melatonin on follicular growth and viability. In addition, follicles cultured in medium containing only melatonin had significantly higher rates of antrum formation. Follicles cultured in medium containing only melatonin had higher relative levels of mRNA for CAT, SOD and PRDX-6 than those cultured with both melatonin and luzindole. Follicles cultured with luzindole only or both melatonin and luzindole had lower relative levels of mRNA for PRDX6 and GPX1 than those cultured control medium. In conclusion, melatonin promotes growth of bovine secondary follicles through its membrane-coupled receptors, while luzindole blocks the effects of melatonin on follicle growth and reduces the expression of antioxidant enzymes in cul-tured follicles. (c) 2022 Elsevier Inc. All rights reserved.

Automated summary

This study investigates the expression of melatonin receptor types 1A/B in bovine ovaries and the effects of melatonin on follicle development and antioxidant enzyme expression. The results show that melatonin promotes follicle growth through its membrane-coupled receptors, while the melatonin receptor antagonist luzindole blocks its effects.