There is evidence that melatonin acts directly on the regulation of ovary function. This action is probably attributed in part to melatonin receptors, which are known to be present in granulosa and cumulus cells (Kang J-T et al. 2009 J. Pineal Res. 46, 2228). Melatonin is also known to be associated with the modulation of circadian rhythms and the regulation of seasonal reproductive function (Arendt J 1998 Rev. Reprod. 3, 1322). Circadian rhythms and clock genes appear to be involved in reproductive processes (Dolatshad H et al. 2009 Repro. Fertil. Dev. 21, 19). However, the presence of melatonin receptor genes and the clock genes has not been so widely studied or has never been reported to exist in mammalian oocytes.The aim of this study was to investigate the presence of nuclear (Rorα) and membrane (Mt1 and Mt2) melatonin receptors genes and the clock genes (Clock, Bmal1, Cry1, Cry21, Per1, Per2) in rat oocytes by reverse RT-PCR. Twenty-seven-day-old Wistar female rats were treated with 20 UI of pregnant mares serum gonadotropin for induction of follicular development and slaughtered 48 h later. All the procedures involving animals were approved by the Animal Care Committee of the Institute of Biomedical Sciences. The ovaries were removed and placed in TCM-199 supplemented with 100 UI mL-1 penicillin, 100 μg mL-1 streptomycin, and 0.1% polyvinyl alcohol (H-199 medium). Germinal vesicle-intact oocytes, isolated from the ovarian follicles, were denuded from cumulus cells by vortexing for 5 to 8 min. The denuded oocytes were incubated for 5 min in H-199 medium with 0.1% pronase for removal of the zona pellucida. Pools of 80 oocytes per cDNA sample were used.As an internal control for the sample integrity, additional primers for RPL37a were included in each PCR reaction. All the 3 control PCR replicates showed a repeatable amplification. Polymerase chain reaction amplifications of cDNA yielded Rorα, Clock, Bmal1, and Cry1 products in 2 of 3 replicates. No expression of the MT1 and MT2 mRNA was observed. The preliminary results suggest the presence of a nuclear melatonin receptor gene and some clock genes in rat oocytes. However, additional studies are necessary to confirm this hypothesis.
This research was supported by FAPESP.
Synthetic Melatoninergic Ligands: Achievements and Prospects
