Down-regulation of mt1 melatonin receptors in rat ovary following estrogen exposure.
In this study, we have demonstrated that 2-[125I]-iodomelatonin binds specifically to rat ovarian granulosa cell (GC) membranes with high affinity (KD=83 pM; Bmax=3.28 fmol/mg protein). Using immunoblot analysis and an anti-mt1 melatonin receptor antibody, we have also detected mt1 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 x 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 Bmax=3.28 fmol/mg protein vs. estradiol-treated animals' ovaries having a Bmax=0.92 fmol/mg protein. In addition, following Estradiol treatment, mt1 melatonin receptors in rat ovary were down-regulated (approximately 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 =3.15 [corrected] 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.
Clemens
J W
JW
Department of Pharmacology and Toxicology, Duquesne University, Pittsburgh, PA 15282, USA.
Jarzynka
M J
MJ
Witt-Enderby
P A
PA
D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D015536:Down-Regulation / Q000187:drug effects; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects / Q000502:physiology; D007457:Iodine Radioisotopes; D007700:Kinetics; D008550:Melatonin / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000502:physiology; D006080:Ovarian Follicle / Q000187:drug effects / Q000502:physiology; D010053:Ovary / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000378:metabolism*; D044094:Receptors, Melatonin; D013739:Testosterone / Q000494:pharmacology
D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D013739:Testosterone; D004958:Estradiol; D005640:Follicle Stimulating Hormone; C044556:2-iodomelatonin; D008550:Melatonin