Melatonin receptor depletion suppressed hCG-induced testosterone expression in mouse Leydig cells

Gao et al. Cellular & Molecular Biology Letters https://doi.org/10.1186/s11658-019-0147-z (2019) 24:21 Cellular & Molecular Biology Letters RESEARCH Open Access Melatonin receptor depletion suppressed hCG-induced testosterone expression in mouse Leydig cells Yuan Gao1*† , Xiaochun Wu2†, Shuqin Zhao1, Yujun Zhang1, Hailong Ma1, Zhen Yang1, Wanghao Yang1, Chen Zhao1, Li Wang1 and Quanwei Zhang1,2 * Correspondence: yyshpy@gmail. com † Yuan Gao and Xiaochun Wu contributed equally to this work. Yuan Gao and Xiaochun Wu are considered co-first authors. 1 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, Gansu, China Full list of author information is available at the end of the article Abstract Melatonin receptors MT1 and MT2 (genes officially named MTNR1A and MTNR1B, respectively) play crucial roles in melatonin-mediated regulation of circadian rhythms, the immune system, and control of reproduction in seasonally breeding animals. In this study, immunolocalization assay showed that MT1 and MT2 are highly expressed in Leydig cell membrane. To understand the biological function of melatonin receptors in hCG-induced testosterone synthesis, we generated melatonin receptor knockdown cells using specific siRNA and performed testosterone detection after hCG treatment. We found that knockdown of melatonin receptors, especially MTNR1A, led to an obvious decrease (> 60%) of testosterone level. Our further study revealed that knockdown of melatonin receptors repressed expression, at both the mRNA level and the protein level, of key steroidogenic genes, such as p450scc, p450c17 and StAR, which are essential for testosterone synthesis. hCG triggered endoplasmic reticulum (ER) stress to regulate steroidogenic genes’ expression and apoptosis. To further investigate the potential roles of melatonin receptors in hCG-induced regulation of ER stress and apoptosis, we examined expression of some crucial ER stress markers, including Grp78, Chop, ATF4, Xbp1, and IRE1. We found that inhibition of melatonin receptors increased hCG-induced expression of Grp78, Chop and ATF4, but not Xbp1 and IRE1, suggesting that hCG may modulate IRE1 signaling pathways in a melatonin receptor-dependent manner. In addition, our further data showed that knockdown of MTNR1A and MTNR1B promoted hCG-induced expression of apoptosis markers, including p53, caspase-3 and Bcl-2. These results suggested that the melatonin receptors MTNR1A and MTNR1B are essential to repress hCG-induced ER stress and cell apoptosis. Our studies demonstrated that the mammalian melatonin receptors MT1 and MT2 are involved in testosterone synthesis via mediating multiple cell pathways. Keywords: Melatonin receptor, Testosterone, ER stress, Apoptosis Introduction Melatonin (N-acetyl-5-methoxytryptamine), a neuro-hormone that is mainly secreted from the pineal gland in all mammals, influences various physiological activities such as neuroendocrine function, regulation of seasonal reproduction, sexual maturation, immunoregulation, thermoregulation, some aspects of aging and strong antioxidant activity [1–5]. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gao et al. Cellular & Molecular Biology Letters (2019) 24:21 Melatonin’s physiological actions are mainly mediated by two types of melatonin receptors, MT1/Mel1a and MT2/ Mel1b (genes officially named MTNR1A and MTNR1B, respectively). Both the MT1 and MT2 receptors are classified as class A rhodopsin type G-protein coupled receptors (GPCRs) with typically seven transmembrane domains, connected to each other by three extracellular regions and three intracellular loops [6, 7]. The two receptors have 60% homology and have been reported in rats, mice, and humans [1, 8, 9]. Nevertheless, a third subtype, MT3/Mel1c, has also been identified but only found in non-mammalian species, such as birds, amphibians, and fish [10, 11]. Additionally, in mammals, a third subtype, initially identified as melatonin receptor MT3, has been further characterized as a cytosolic, non-G coupled-binding site for melatonin. It belongs to the quinone reductase family and is named quinone reductase 2 (NQO2) [12, 13]. Melatonin acts as a non-substrate inhibitor to bind to and inhibit this enzyme [14]. As members of GPCRs, activation of melatonin receptors MT1 and MT2 alters the levels of second messengers to modulate intracellular signal transduction [15]. Both MT1 and MT2 receptors inactivated adenylate cyclase (AC) and decreased intracellular cAMP production, and resulted in a decrease in protein kinase A (PKA) activity [6, 16]. Melatonin receptors also can dimerize as homo- or heterodimers to regulate cell physiological activity [17, 18]. Intriguingly, MT1 and MT2 receptors are also capable of activating very different signaling cascades in different tissues, organs or species. The MT1 receptor can increase phosphorylation of mitogen-activated protein kinase 1/2 (MAPK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) to active the MAPK cascade. The MT2 receptor inhibits both forskolin (forsk)-induced cAMP and cGMP formation, leading to activation of protein kinase C (PKC) in the suprachiasmatic nucleus (SCN) and decrease of calcium-dependent dopamine release in the retina [19]. A growing body of evidence shows that melatonin receptors are involved in reproductive regulation [20, 21]. Leydig cells, which are located between the seminiferous tubules of the testis, are the primary cells to synthesize and secrete testosterone, an important hormone to promote the development of male reproductive tissues such as testes and prostate, as well as maintaining spermatogenesis and secondary sexual characteristics [22, 23]. Testosterone synthesis is induced by luteinizing hormone (LH) or chorionic gonadotropin (CG). Human CG (hCG) is widely used to induce testosterone synthesis [24, 25]. Testis Leydig cells, a type of endocrine secretory cells with strong testosterone synthesis and secretion in response to LH/CG stimulation, express key steroidogenic enzymes for the regulation of testosterone synthesis [24]. Treatment with LH/hCG increased intracellular levels of cAMP, and promoted the transfer of cholesterol to the inner mitochondrial membrane through steroidogenic acute regulatory protein (StAR). Then, cholesterol is converted into pregnenolone via cytochrome p450 cholesterol side chain cleavage enzymes (p450scc/CYP11A1). Afte (...truncated)