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C-Type Natriuretic Peptide/Natriuretic Peptide Receptor 2 Is Involved in Cell Proliferation and Testosterone Production in Mouse Leydig Cells

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C-Type Natriuretic Peptide/Natriuretic Peptide Receptor 2 Is Involved in Cell Proliferation and Testosterone Production in Mouse Leydig Cells Original Article pISSN: 2287-4208 / eISSN: 2287-4690 World J Mens Health Published online Oct 23, 2018 https://doi.org/10.5534/wjmh.180041 C-Type Natriuretic Peptide/Natriuretic Peptide Receptor 2 Is Involved in Cell Proliferation and Testosterone Production in Mouse Leydig Cells Lei Yang1,3 , Lanjie Lei2 , Qihan Zhao1 , Ying Gong1,3 , Gaopeng Guan2,3 , Shaoxin Huang1 1College of Basic Medical Science, 2Clinical Skills Center, Affiliated Hospital of Jiujiang University, 3Key Laboratory of System Bio-medicine of Jiangxi Province, Jiujiang University, Jiujiang, China Purpose: This study investigated the role of natriuretic peptide receptor 2 (NPR2) on cell proliferation and testosterone secre- tion in mouse Leydig cells. Materials and Methods: Mouse testis of different postnatal stages was isolated to detect the expression C-type natriuretic peptide (CNP) and its receptor NPR2 by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Leydig cells isolated from mouse testis were cultured and treated with shNPR2 lentiviruses or CNP. And then the cyclic guanosine mono- phosphate production, testosterone secretion, cell proliferation, cell cycle and cell apoptosis in mouse Leydig cells were ana- lyzed by ELISA, RT-qPCR, Cell Counting Kit-8, and flow cytometry. Moreover, the expression of NPR2, cell cycle, apoptosis proliferation and cell cycle related gene were detected by RT-qPCR and Western blot. Results: Knockdown of NPR2 by RNAi resulted in S phase cell cycle arrest, cell apoptosis, and decreased testosterone secre- tion in mouse Leydig cells. Conclusions: Our study provides more evidences to better understand the function of CNP/NPR2 pathway in male reproduc- tion, which may help us to treat male infertility. Keywords: Germ cells; Leydig cells; Testicular diseases; Testosterone This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. INTRODUCTION Each of them is configured with a different location and has specific functions: Germ cells produce sperm Testes, as a critical component of the male reproduc- and are located in the seminiferous tubules; Leydig tive and endocrine tract, the primary functions is to cells produce sex hormones and are distributed in the produce sperm and androgens. Mammalian testes con- connective tissue of the convoluted seminiferous tu- sists of several well-defined cell types, including germ bules; Sertoli cells form the basement membrane of the cells of different stages, Sertoli cells and Leydig cells. seminiferous tubules and offer the environment neces- Received: May 21, 2018 Revised: Sep 10, 2018 Accepted: Sep 10, 2018 Published online Oct 23, 2018 Correspondence to: Lei Yang https://orcid.org/0000-0001-8859-9692 College of Basic Medical Science, Jiujiang University, 17 Lufeng Rd, Xunyang Qu, Jiujiang Shi, Jiangxi Sheng 332000, China. Tel: +86-0792-8570043, Fax: +86-0792-8577050, E-mail: [email protected] Correspondence to: Shaoxin Huang https://orcid.org/0000-0001-9801-4133 College of Basic Medical Science, Jiujiang University, 17 Lufeng Rd, Xunyang Qu, Jiujiang Shi, Jiangxi Sheng 332000, China. Tel: +86-0792-8570043, Fax: +86-0792-8577050, E-mail: [email protected] Copyright © 2018 Korean Society for Sexual Medicine and Andrology https://doi.org/10.5534/wjmh.180041 sary for the differentiation and maturation of germ 3β-hydroxysteroid dehydrogenase (3β-HSD) staining cells [1]. The importance of Leydig cells in male repro- using the modified Wiebe method. Simply, Leydig cells duction is exemplified by the fact that loss of func- were fixed in 1% paraformaldehyde for 20 minutes tion of gene such as the luteinizing hormone receptor and then washed with phosphate buffer saline (PBS) (LHR) [2], insulin-like factor 3 (Insl3) [3], and enzymes for three times. After washed the cells were incubated involved in testosterone biosynthesis [4]. with 1 mL PBS containing 1 mg bovine serum albumin, C-type natriuretic peptide (CNP) and its receptor 1.5 mg nicotinamide adenine dinucleotide, 0.2 mg nitro- (natriuretic peptide receptor 2, NPR2) play a role as blue tetrazolium and 0.25 mg dehydroepiandrosterone an oocyte maturation inhibitor in mammalian [5]. Re- for 2 hours at 37°C in the dark. Then the cells were cent studies suggested that CNP can improve oocyte gently rinsed with PBS and observed microscopically. maturation and developmental competence in vitro [6]. During all the culture, the cells were cultured with Compared with numerous studies about CNP/NPR2 on Dulbecco’s modified Eagle’s medium/F-12 containing female reproduction, there is little research about CNP/ 10% fetal bovine serum with or without CNP (CNP NPR2 in male reproduction. Previous studies reported were used to active CNP/NPR2 signaling pathway), that CNP regulates blood-testis barrier dynamics and is unless otherwise stated. related with spermatogenesis [7]. Sogawa et al [8] inves- tigated phenotype of NPR2-defcient short-limbed-dwarfs 3. Transfection of cells with shRNA- mice, and found that the developmental onset and ac- natriuretic peptide receptor 2 lentiviral quisition of spermatogenic function is delayed in NPR2 shRNA-NPR2 and shRNA-negative lentiviral vec- mutant mice. Subsequently, two studies reported that tor were purchased from Genechem (Shanghai, China). CNP/NPR2 is related with sperm motility, acrosome The mouse Leydig cells were seeded into 6-well plates, reaction and induces sperm attraction for fertilization, which were cultured to 30% to 40% confluence and in- thus regulating the reproductive function of males [9,10]. fected by addition of 1×108 TU/mL lentivirus, 5 µg/mL However, the function of CNP/NPR2 in male repro- polybrene and complete medium. After 12 hours, the duction remains largely unknown. In this study, we lentivirus solution was replaced by complete culture aimed to study the role of CNP/NPR2 on cell prolif- medium and cultured for 36 hours. And the cells were eration, testosterone secretion and related regulatory observed under a fluorescence microscope to evaluate mechanisms in mouse Leydig cells. the transfection efficiency. The efficiency and specific- ity of siRNA mediated knockdown were examined by MATERIALS AND METHODS Western blot and quantitative reverse transcription polymerase chain reaction (RT-qPCR). After determine 1. Chemicals and mice the NPR2 knockdown efficiency, the cells were cul- All chemicals were purchased from Sigma-Aldrich tured for subsequent experiments. (St. Louis, MO, USA) unless otherwise stated. Male Kunming mice were purchased from the Laboratory 4. Cell proliferation assay Animal Central of Jiujiang University. All mice were After culturing with CNP for 24 hours, Cell Count- fed a typical diet of lab chow and housed in a single ing Kit-8 (CCK8) was added to the cells (10 µL/well). room under conditions of constant temperature (25°C– Then the cells were incubated for 1 hour at 37°C and 28°C), humidity (55%±5%) and lighting (12 hours light, measured at 450 nm by a Microplate Reader (Bio-Rad 12 hours dark cycle) [11]. All procedures were approved 680, Hercules, CA, USA). The experiments were per- by the Committee for the Ethics on Animal Care and formed in triplicate. Experiments of Jiujiang University (approval No. SYXK(GAN)2017-0001). 5. Cyclic guanosine monophosphate measurement 2. Isolation and cultures of Leydig cells After 30 minutes of treatment, the cells were col- Leydig cells were isolated from the testes of 42- to lected to measure the cyclic guanosine monophosphate 49-day-old Kunming mice and cultured as previous (cGMP). The cGMP measurement was according to the report [12]. The purity of Leydig cells was assessed by procedure described as our previous report [6]. The lev- 2 www.wjmh.org Lei Yang, et al: CNP/NPR2 Promotes Testosterone Production els of cGMP were determined using cGMP-EIA kits ob- 10. Immunohistochemistry tained from Cayman Chemicals (Ann Arbor, MI, USA). Testes of 42- to 49-day-old Kunming mice were fixed Each sample was measured in triplicate. in 4% paraformaldehyde in PBS for one week, dehy- drated through a graded ethanol series, and embed- 6. Testosterone measurement ded in paraffin. Sections 7 mm thick were mounted After 24 hours of treatment, mouse Leydig cells onto glass slides precoated with Poly-L-Lysine Solution were counted. The concentration of testosterone in the and incubated overnight at 37°C. After dehydrating, culture supernatants (100,000 cell/mL culture superna- samples were placed in citrate buffer (pH=6.0). Antigen tant) was measured with ELISA kits (Ji Yin Mei; Co. retrieval was performed by treating samples in a mi- Ltd., Wuhan, China) according to the manufacturer’s crowave oven at 92°C for 15 minutes; slides were cooled instructions. Each sample was measured in triplicate. and then washed in PBS. The sections were pretreated with 3% (vol/vol) H2O2 in methanol to quench endog- 7. Cell cycle analysis enous peroxidase activity. After being washed with Mouse Leydig cells of the respective experimental PBS, sections were incubated with 10% goat serum for groups were collected and fixed in ice-cold 70% etha- 30 minutes at 37°C. After blocking, sections were incu- nol overnight at 4°C after the cells were treatment bated overnight at 4°C with rabbit polyclonal antibody and count. Then, the cells were washed with PBS and against NPR2 (Sigma-Aldrich; 1:50 dilutions) in a hu- stained with propidium iodide (PI) solution for 20 midified chamber. After washing, followed by incuba- minutes at 25°C in the dark. Finally, the cells were tion with biotinylated anti-rabbit immunoglobulin G analyzed by flow cytometry using a BD FACS Calibur antibody (Beijing 4A Biotech Co., Ltd, Beijing, China) at system and Mod Fit LT for MacV3.0 software. For each 37°C for 1 hour, and then sections were incubated with analysis, a minimum of 10,000 cells were analyzed.
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