Melatonin Mediates StAR Expression In Human Granulosa Cells And The Molecular Mechanism

Sex hormones including estrogen,testosterone and progesterone,belong to steroid hormones.The endocrine profile of normal female changes periodically,under the stimulation of gonadal axis.Steroidogenesis is a complex process that involves multiple enzymatic reactions.Once free cholesterol has been transported to the mitochondria,it will be moved from the outer to the inner mitochondrial membrane by steroidogenic acute regulatory protein(StAR),which has been well-recognized as the key regulatory protein involved in the rate-limiting step of steroidogenesis.On the inner mitochondrial membrane,cholesterol is catalyzed to pregnenolone by the cholesterol side-chain cleavage enzyme complex.Pregnenolone is transferred to the cytoplasm and is then catalyzed to progesterone by 3?-hydroxysteroid dehydrogenase(3?-HSD).Progesterone(P4)is the precursor of other steroid hormones.Melatonin(N-acetyl-5-methoxytryptamine)is a compound extracted from pineal glands and peripheral nerve.As a neurohormone,melatonin has numerous important physiological functions and regulates varieties of central and peripheral actions related to circadian rhythms and reproduction.Melatonin's activity is mostly performed through membrane-bound receptors MT1 and MT2,which are members of the superfamily of G protein-coupled receptors.Earlier studies have demonstrated that MT1 and MT2 are widely distributed in the female reproductive system,such as the uterus,ovaries,the epithelial cells of the mammary gland,and the granulosa cells.The broad-spectrum free radical scavenging capacity and antioxidant capacity of melatonin play an important role in the reproductive system.Melatonin may directly affect ovarian function.It also participates in the regulation of hypothalamicpituitary-gonadal axis function and reproductive endocrine activity.The cyclic adenosine monophosphate/protein kinase A(cAMP/PKA)signaling pathway is a well-defined mechanism for regulating StAR expression in the reproductive system.In addition,many other hormones,growth factors and cytokines can participate in the regulation of StAR expression by activating different intracellular signaling pathways.In granulosa cells,LH/hCG also induces StAR expression.Epidermal growth factor receptor(EGFR)and its ligands are expressed in female reproductive tissues and have been shown to regulate various important reproductive functions.In granulosa cells and follicle fluids,it has been indicated that the mRNA levels and concentrations of AREG,BTC and EREG increase,could induce cumulus-oocyte-complex(COC)expansion,oocyte maturation and COX-2 expression in vivo and in vitro,which is called “LH-like” effect.As the most abundantly expressed EGFR ligand in human follicular,AREG had been indicated can upregulate the StAR expression.Melatonin also rise rapidly after the LH/hCG peak,and can also promote oocyte maturation and ovulation.Furthermore,melatonin can also activate the cAMP/PKA signaling pathway.However,whether Melatonin mediates the StAR expression and whether it has the synergistic effect with AREG on the upregulation of the StAR expression in human granulosa cells remains unknown.Thus,we aim at exploring whether Melatonin regulate the expression of StAR and defining the underlying mechanism.Objective:To investigate the role of melatonin in the regulation of StAR expression,whether it has the synergistic effect with AREG on the upregulation of the StAR expression and the underlying molecular mechanisms in human granulosa cells.Methods:Human primary granulose cells were used as the cell model to explore whether melatonin played a role in the regulation of StAR expression and progesterone production.Level of mRNA and protein was measured by RT-qPCR and Western-blotting respectively.The protein levels of Melatonin in follicular fluid were measured by enzyme-linked immune sorbent assay(ELISA).The levels of progesterone in serum were measured by electrochemiluminescence immunoassay(ECLIA).Results:In human primary granulosa cells,treatment with 0.5mmol/L Melatonin for 24 h could promote the expression of StAR expression.Inhibition of MT by Luzindole could abolish Melatonin-induced StAR expression.Moreover,activation of PKA/CREB signaling pathway was participated for Melatonin-induced up-regulation of StAR expression.Inhibition of PKA by H89 could abolish Melatonin-induced StAR expression.Melatonin strengthens the effect of AREG on the upregulation of the StAR expression in human granulosa cells by activating the ERK1/2 signaling pathway.Inhibition of EGFR by AG1478 could abolish its synergistic effect.Follicular fluid Melatonin levels were positively correlated with progesterone levels in serum on hCG day and oocyte-pick up(OPU)day.Conclusion:Melatonin mediates StAR expression in primary human granulosa cells through MT and activating PKA/CREB signaling pathway.Melatonin also can upregulate AREG-induced StAR expression by activating the ERK1/2 signaling pathway through binding EGFR.