Down-regulation of mt 1 melatonin receptors in rat ovary following estrogen exposure

Abstract

In this study, we have demonstrated that 2-[ 125I]-iodomelatonin binds specifically to rat ovarian granulosa cell (GC) membranes with high affinity (K D=83 pM; B max=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt 1 melatonin receptor antibody, we have also detected mt 1 melatonin receptors in rat ovary. Because melatonin has been reported to alter the steroidogenic responses of ovarian tissues to gonadotropins, a physiological role for intra-ovarian melatonin may exist. Thus, in order to investigate a possible intra-ovarian role for melatonin, we have used both an in vivo and in vitro model of follicular development. Treatment of immature (day 21) female rats with estradiol (E; 0.2 mg/d × 3 d; subcutaneous) was used to induce follicular growth. Membranes from both untreated (U) and E-treated animals' ovaries contained high-affinity 2-[ 125I]-iodomelatonin (I-MEL) binding sites (Kd=83 and 23 pM, respectively). Estradiol treatment in vivo caused a significant decrease (P<0.05) in binding of 2-[ 125I]-iodomelatonin to ovarian membranes with untreated animals' ovaries having a B max=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a B max=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt 1 melatonin receptors in rat ovary were down-regulated (∼95%) using immunoblot analysis. Granulosa cells isolated from E-treated rats were further matured in vitro with testosterone (T) and the pituitary gonadotropin follicle-stimulating hormone (FSH). Granulosa cells were cultured with either T (10 ng/ml) or FSH (5.71 ng ovine FSH-20/ml) alone, or both FSH and T for 48 h. There was no statistically significant specific binding of 2-[ 125I]-iodomelatonin to GC membranes cultured with T or FSH alone. However, following a 48-h exposure to FSH and T in vitro specific 2-[ 125I]-iodomelatonin binding occurred with total 2-[ 125I]-iodomelatonin binding =5.5 fmol/mg protein. Therefore, the existence of hormonally-regulated expression of high-affinity melatonin binding sites suggests that melatonin may have an important intra-ovarian physiological role.