Analysis of melatonin in women with infertility under program in vitro fertilization: functional study in células da granulosa

Abstract

Melatonin is a hormone related to the light-dark cycle and it plays a role in the reproductive system. It acts in the ovary, mainly in theca-interstitial cells, which are androgen producing. In fact, synthesis of this steroid by the theca-interstitial (T-I) cells is primarily regulated by the luteinizing hormone (LH). Upon binding to its receptor, LH promotes increased steroid production through activation of a cAMP-dependent signal transduction cascade, which may be downregulated through a melatonin regulator. However, the intracellular mechanisms involved in the process are not clear. The aim of this study is to understand effects of melatonin on steroidogenesis in the ovarian theca-interstitial cells of rats and relevance to hormone therapy in polycystic ovary syndrome (PCOS). Fifty 25-day-old female Sprague-Dawley rats will be euthanized by CO2 asphyxiation. Their ovaries will be removed under sterile conditions and processed immediately for isolating the T-I cells. These will be isolated, dispersed, and cultured following a laboratory protocol published by Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan Medical School.The T-I cells will be divided into two experimental groups: Experimental Group I: GI, controls receiving vehicle (n=10); GII, controls receiving vehicle with (human chorionic gonadotropin [hCG]) (50 ng / ml) (n=10); GIII, experimental animals receiving hCG and melatonin (concentration of 10 uM), (n=10); GIV, experimental animals, receiving hCG and melatonin (concentration of 1.0 uM, (n=10); GV, experimental animals receiving hCG and melatonin (concentration of 0.1 uM), (n=10). All T-I cells will be treated for five consecutive days with hCG and melatonin, and at the end of the experiment the cells will be trypsinized for immunofluorescence analysis of CYP17A1, gene expression analysis by quantitative real-time polymerase chain reaction qRT-PCR for evaluating the androgen enzymatic pathway and Protein analysis through Western blotting technique. Experimental Group II: GI, controls receiving vehicle (n=10); GII, controls receiving vehicle with (human chorionic gonadotropin [hCG]) (50 ng/ml) (n=10); GIII, experimental animals receiving hCG and Melatonin (n=10); GIV, experimental animals, receiving hCG with Melatonin and 4-phenyl-2-propionamidotetraline [4P-PDOT] (0.1microM) (n=10); GV, experimental animals receiving hCG with Melatonin and Luzindole (0.1 microM) (n=10); GVI, experimental animals receiving hCG with Melatonin and Rapamycin (n=10). All T-I cells will be treated for five consecutive days with hCG, Melatonin, 4P-PDOT, Luzindole, Rapamycin, and at the end of the experiment the cells will be trypsinized for immunofluorescence analysis of CYP17A1, gene expression analysis by quantitative real-time polymerase chain reaction qRT-PCR for evaluating the androgen enzymatic pathway and Protein analysis through Western blotting technique. (AU)