Melatonin receptor depletion suppressed hCG-induced testosterone expression in mouse Leydig cells
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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.
KeywordsMelatonin receptor Testosterone ER stress Apoptosis
3β-hydroxysteroid dehydrogenase O5-O4-isomerase
Activating transcription factor 4
CCAAT/enhancer-binding homologous protein
Enzyme-linked immunosorbent assay
78 kDa glucose-regulated protein
human chorionic gonadotropin
Inositol-requiring enzyme 1
Mitogen-activated protein kinase
murine Leydig tumor cell line
cytochrome p450 cholesterol side chain cleavage enzymes
Protein kinase C
quantitative real-time PCR
small interfering RNA
Steroidogenic acute regulatory protein
Unfolded protein response
X-box binding protein 1
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, 2, 3, 4, 5].
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 .
As members of GPCRs, activation of melatonin receptors MT1 and MT2 alters the levels of second messengers to modulate intracellular signal transduction . 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 . 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 .
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). After movement from the mitochondria to the endoplasmic reticulum (ER), pregnenolone is converted into progesterone by 3β-hydroxysteroid dehydrogenase O5-O4-isomerase (3β-HSD) and subsequently metabolized to testosterone by 17a-hydroxylase/C17–20 lyase (CYP17) and 17β-hydroxysteroid dehydrogenase (17β-HSD) [24, 26].
In this study, we examined whether melatonin receptors are involved in regulation of hCG-induced testosterone synthesis, as well as whether melatonin receptors function via modulation of steroidogenic enzyme expression in mouse Leydig cells (mLTC-1). We further investigated the role of melatonin receptors in some cell processes including ER stress and apoptosis, attempting to find the potential signaling involved in melatonin receptor-mediated reproductive regulation.
Materials and methods
Male Kunming White outbred strain mice were purchased from the Laboratory Animal Center of the Fourth Military Medical University (Shaanxi, China). The mice were maintained in controlled conditions of temperature (23 ± 2 °C) and light (12 h light and 12 h dark cycle). The experimental procedures were performed in accordance with the Animal Ethical and Welfare Committee of Gansu Agricultural University.
The murine Leydig tumor cell line (mLTC-1, ATCC, Manassas, VA, USA) was cultured in RPMI 1640 Medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone) and 100 mg/L penicillin/streptomycin (Hyclone) at 37 °C under 5% CO2.
For testosterone assay, mLTC-1 cells were cultivated in 24-well plates (5 × 104 cells/well) for 24 h before stimulation with hCG (Yofoto. Ningbo, China). Then cells were washed with phosphate-buffered saline (PBS) and maintained in alternative FBS-free/phenol red medium containing hCG with a controlled concentration gradient. After 6 h incubation, the cells and culture supernatant were collected. Cells were used for RNA and protein assay, and culture supernatant for testosterone assay.
For production of tissue sections, the testis was dissected from mice (90 days old), fixed in 4% paraformaldehyde for 24 h, dehydrated through a graded ethanol series, and embedded in paraffin. Tissue sections 7 mm thick were cut and mounted on glass slides pre-coated with poly-L-lysine solutions. Then the dehydrated sections were placed in citrate buffer (0 .1M citrate, 0.1 M sodium citrate; pH 6.0). Antigen retrieval was performed by heating in a microwave oven (750 W for 10 min twice) and cooling slowly to room temperature. Endogenous catalase deactivation was performed by immersion of slides in 0.3% (v/v) hydrogen peroxide in methanol for 1 h at 37 °C.
For IHC staining, the sections were washed and then incubated with 10% goat serum for 30 min at 37 °C. The sections were washed with PBS and incubated with rabbit anti-MTNR1A/1B antibody (1:200, bs-0027R/bs-0963R, Bioss, Beijing, China) overnight at 4 °C. After washing in PBS, the sections were incubated with biotinylated anti-Rabbit IgG (Sigma-Aldrich, St. Louis, USA) for 10 min at 37 °C, and then immersed in horseradish peroxidase labeled streptavidin for 10 min at 37 °C. Appropriate negative control slides were run in parallel without a primary antibody. The slides were imaged using a digital microscope (Motic, Wetzlar, Germany).
Quantitative real-time PCR (qPCR)
Primer sequences used for qPCR analyses of gene mRNAs
Primer sequences (5′-3′)
mLTC-1 cells were cultured in a 35 mm dish with cover slips to a monolayer. Then cover slips were fixed in 4% formaldehyde for 30 min and permeabilized in 0.1% Triton X-100 for 15 min at room temperature. After being blocked with 5% BSA in PBS, cover slips were incubated with a rabbit anti-MTNR1A/1B primary antibody (1:200, Bioss) at 4 °C overnight, followed by incubation with an Alexa-Fluor 488 labeled goat anti-Rabbit IgG secondary antibody (1:300; Invitrogen) for 1 h at 37 °C. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Beyotime, Beijing, China) for 5 min. Images were captured with a digital camera under a Zeiss LSM800 confocal microscope (Carl Zeiss, Germany).
Western blotting (WB) assay
mLTC-1 cells after treatment were harvest for protein extraction using the Total Protein Extraction Kit (KeyGen, Nanjing, China). The protein concentration was determined using a BCA Protein Assay Kit (KeyGen). Equal total proteins were separated via 12% SDS-PAGE gel and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 10% skimmed milk in TBST for 2 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-β-actin antibody (Sanjian Biotech, Tianjing, China), anti-MTNR1A/1B antibody (Bioss), anti-StAR antibody (CusaBio, Wuhan, China), anti-p450c17 antibody (CusaBio), anti-Grp78 antibody (CusaBio), anti-ATF4 antibody (CusaBio), and anti-phospho-IRE1 antibody (CusaBio). Then, following three washes with TBST, the membranes were incubated with the corresponding secondary antibody conjugated to horseradish peroxidase (Sigma-Aldrich) for 1 h at room temperature. Finally, bands were visualized using the Gel Imaging System (Tannon Science & Technology, Shanghai, China) and then digitized by use of ImageJ software.
Small interfering RNA (siRNA) mediated knockdown assay
Sequences of small interfering RNA
Sense sequences (5′-3′)
antisense sequences (5′-3′)
Testosterone in culture supernatants were measured by the Testosterone ELISA kit (Beifang Biotech, Beijing, China) according to the manufacturer’s instructions. The minimum detectable concentration of testosterone was 0.05 ng/mL. The intra- and inter-assay coefficients of variation were < 10 and < 15%, respectively.
Data were analyzed with one-way ANOVA, followed by Fisher’s least significant difference test (Fisher’s LSD) and the independent-samples Student’s t test with SPSS software (Version 20.0; SPSS, Chicago, IL. USA). P < 0.05 was considered significant. All data are represented as the mean ± SEM of repeated experiments (n = 3).
Immunolocalization of MTNR1A and MTNR1B protein in mouse testes
To explore the function of melatonin receptors in vitro, we chose a mouse Leydig cell line, mLTC-1, for our research. To further confirm the expression and distribution of melatonin receptors MTNR1A and MTNR1B in mLTC-1, we performed immunofluorescence staining in mLTC-1 using specific anti-MTNR1A and MTNR1B antibody and Alexa-Fluor 488 labeled secondary antibody. We found that the melatonin receptors MTNR1A and MTNR1B are mainly located on the cell membrane (Fig. 1d-f). The result is consistent with previous reports [11, 28].
Knockdown of melatonin receptors inhibits hCG-induced testosterone synthesis
Knockdown of melatonin receptors inhibited hCG-induced steroidogenic gene expression
Inhibition of MTNR1A and MTNR1B altered hCG-induced endoplasmic reticulum (ER) stress
Knockdown of MTNR1A and MTNR1B promoted hCG-induced apoptosis genes
Melatonin is an important neuro-hormone mainly synthesized by the pineal gland in mammals [2, 4]. Melatonin influences various physiological activities such as neuroendocrine function, regulation of seasonal reproduction, sexual maturation, immunoregulation, thermoregulation and some aspects of aging [1, 2]. In addition, melatonin possesses strong antioxidant activity by which it protects cells, tissues and organs from the oxidative damage caused by reactive oxygen species (ROS) [3, 4, 5].
Melatonin receptors MT1 and MT2 play crucial roles in melatonin-mediated regulation of circadian rhythms , the immune system , and control of reproduction in seasonally breeding animals . Recent studies reported that melatonin facilitated spermatogenesis via MT1 and MT2 . Previous evidence also revealed that hCG-induced ER stress triggered apoptosis in Leydig cells of the testis and are involved in regulation of steroidogenic genes , which have an important function in testosterone synthesize [26, 38]. To further explore the biological mechanisms of melatonin receptors in hCG-induced testosterone synthesis and potential cellular regulation, siRNA-mediated melatonin receptor knockdown cells, mLTC-1, were established in this study to monitor the expression of related factors.
Melatonin receptors, including MT1 and MT2, are highly expressed in mouse Leydig cells both in tissue and the cell line (Fig. 1), and expectably, siRNA-mediated knockdown of melatonin receptors significantly inhibited hCG-induced testosterone synthesis (Fig. 3) and suppressed expression of steroidogenic genes, such as p450scc, p450c17 and StAR (Fig. 4). These results clearly demonstrated that melatonin receptors, including MT1 and MT2, play essential roles in hCG-induced steroid hormones synthesis.
The ER plays a crucial role in the synthesis and folding of secretory and membrane proteins in eukaryotic cells. Overload of ER functions, including excessive protein synthesis, Ca2+ homeostasis, and accumulation of unfolded and/or misfolded proteins in the ER lumen, lead to ER stress through activation of the UPR through three ER transmembrane proteins-mediated signaling: Perk, ATF6 and IRE1 . Previous evidence has confirmed that hCG-induced ER stress plays an important role in steroidogenic enzyme regulation through the activation of UPR pathways . In our studies, we found that knockdown of melatonin receptors obviously promoted hCG-induced major ER stress marker Grp78 protein expression (Fig. 5) and increased Chop and ATF4 expression, indicating that melatonin receptors play crucial roles in inhibiting hCG-induced ER stress. Interestingly, the expression of phospho-IRE1 protein, another ER stress indicator, dramatically decreased after knockdown of melatonin receptors. Together with the result that the expression of IRE1 and Xbp1 (a downstream functional transcriptional activator of IRE1) mRNA was also repressed significantly, these results demonstrated that hCG may modulate IRE1 signaling pathways in a melatonin receptor-dependent manner.
Although the UPR mediates physiological regulation or homeostasis of the ER, it can also mediate apoptotic signaling pathways under excessive ER stress [31, 32, 39, 40] through the activation of caspase-12 [33, 41]. In our study, we tested expression of some apoptosis markers after knockdown of melatonin receptors under hCG treatment. Expectably, inhibition of MTNR1A and MTNR1B promoted expression of key apoptosis markers, including p53 and caspase-3 (Fig. 6), which may contribute to the activation of UPR pathways after hCG stimulation. Curiously, Bcl-2, which is localized to the outer membrane of mitochondria and plays an important role in promoting cellular survival and inhibiting the actions of pro-apoptotic proteins , is also stimulated after knockdown of melatonin receptors under hCG treatment. These findings demonstrated that knockdown of melatonin receptors under hCG treatment can cause complex changes in cellular signaling pathways. The underlying mechanisms need to be further elucidated.
In conclusion, our study demonstrated that mammalian melatonin receptors MT1 and MT2 are involved in testosterone synthesis. Knockdown of melatonin receptors inhibits hCG-induced testosterone synthesis via inhibiting steroidogenic gene expression. In addition, melatonin receptors play variable roles in regulation of three ER stress pathways. In general, the melatonin receptors MTNR1A and MTNR1B are essential to repress hCG-induced ER stress and cell apoptosis, which indicated that melatonin receptors play a crucial role in maintaining homeostasis in Leydig cells. Notably, hCG may modulate IRE1 signaling pathways in a melatonin receptor-dependent manner. We will attempt to identify the underlying mechanisms of melatonin receptor-mediated regulation of ER stress and apoptosis pathways in a further study.
This work was funded by a Discipline construction fund project of Gansu Agricultural University (GAU-XKJS-2018-167/166) and Scientific research start-up funds for openly recruited doctors (2017RCZX-24), and a National Natural Science Foundation of China grant (31602083).
Availability of data and materials
XW, YZ, HM, WY, CZ and LW performed the experiments; YG, XW, QZ analyzed data; XW, SZ and ZY supervised the study; YG and XW designed the experiments and wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
The animal use protocol listed in this study was reviewed and approved by the Animal Ethical and Welfare Committee of Gansu Agricultural University in September 2018. Approval No. AEWC-GAU-2018153.
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