197 EXPRESSION OF MELATONIN-RELATED GENES IN RAT CUMULUS–OOCYTE COMPLEXES

Abstract

Melatonin is a hormone usually associated with the modulation of circadian rhythms and the regulation of seasonal reproductive function. There is evidence that melatonin acts directly on the regulation of ovary function. The mRNA expression of melatonin membrane receptors genes was detected in mammalian and non-mammalian ovaries. In spite of melatonin receptors and Asmt (limiting enzyme in melatonin biosynthesis) genes being present in cattle cumulus–oocyte complexes (COC), no information regarding rat COC has been reported. The aim of this study was to investigate the expression of melatonin receptor (Mt2) and melatonin synthesis enzyme (Asmt) genes at different meiotic cell cycle stages in COC from 27-day-old female rats. All the procedures involving animals were approved by the Animal Care Committee of the Institute of Biomedical Sciences. To obtain germinal vesicle (GV) immature COC from ovarian follicles, rats were treated with 20 IU equine chorionic gonadotropin (eCG) for induction of follicular development and killed by euthanasia 48 h later. To obtain COC at MII oocyte stage from oviducts, rats were injected with 20 IU of eCG and 48 h later with 20 IU of human chorionic gonadotropin (hCG), and then killed after 14 to 16 h. The oocytes in COC were separated from their cumulus cells by repeated pipetting through a narrow-bore pipette in culture medium. Oocytes and cumulus cells total RNA were isolated using a Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). Pools of 80 COC per cDNA sample were used. Quantitative real-time PCR (qRT-PCR) was performed (7500 Real-Time PCR System; Applied Biosystems Inc., Foster City, CA, USA) with 25-µL reactions containing 2 µL of cDNA (10 ng µL–1), SYBR green (Power SYBR Green; Applied Biosystems Inc.), and 400 nM specific intron-spanning primers. A set of 10-fold serial dilutions of each internal standard (102 to 106 copies/2 µL) was used to generate a standard curve. Transcript numbers were determined by the system software and normalized using the geometric mean calculated from the reference genes: Actb and Rpl37a. All the results were plotted as the mean ± SEM, and 4 replicates were performed. Unpaired t-test was used to evaluate the cell type (oocyte v. cumulus cells) differences. The qRT-PCR analyses revealed the presence of transcripts of the Mt2 gene only in oocytes from immature (GV) and mature (MII) COC. At both maturational stages, the copy numbers for Asmt in oocytes were significantly higher than in cumulus cells. The results confirm the presence of the Asmt gene in rat COC and suggest the possible involvement of these cells on melatonin biosynthesis. The presence of Mt2 transcript in immature and mature oocytes also suggests the potentially important role of melatonin in regulating the rat meiotic cellular cycle. Research supported by FAPESP.