World J Mens Health.  2019 May;37(2):186-198. 10.5534/wjmh.180041.

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

• Yang L
• Lei L
• Zhao Q
• Gong Y
• Guan G
• Huang S

• KMID: 2443234
• DOI: http://doi.org/10.5534/wjmh.180041

Abstract

PURPOSE
This study investigated the role of natriuretic peptide receptor 2 (NPR2) on cell proliferation and testosterone secretion 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 monophosphate production, testosterone secretion, cell proliferation, cell cycle and cell apoptosis in mouse Leydig cells were analyzed 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 secretion in mouse Leydig cells.
CONCLUSIONS
Our study provides more evidences to better understand the function of CNP/NPR2 pathway in male reproduction, which may help us to treat male infertility.

Keyword

Germ cells; Leydig cells; Testicular diseases; Testosterone

MeSH Terms

Animals
Apoptosis
Blotting, Western
Cell Count
Cell Cycle
Cell Cycle Checkpoints
Cell Proliferation*
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Germ Cells
Guanosine Monophosphate
Humans
Infertility, Male
Lentivirus
Leydig Cells*
Male
Mice*
Natriuretic Peptide, C-Type
Polymerase Chain Reaction
Receptors, Peptide*
Reproduction
Reverse Transcription
RNA Interference
S Phase
Testicular Diseases
Testis
Testosterone*
Guanosine Monophosphate
Natriuretic Peptide, C-Type
Receptors, Peptide
Testosterone

Figure

• 

  Fig. 1 The expression of C-type natriuretic peptide (CNP)/natriuretic peptide receptor 2 (NPR2) at different postnatal stages of testes. (A) The expression of CNP/NPR2 mRNA in testes and Leydig cells. (B, C) The location by immunohistochemistry (×200) and expression by Western blot of NPR2 in testes and Leydig cells. (D) The expression of CNP at mouse different postnatal stages of testes. (E) The expression of NPR2 at mouse different postnatal stages of testes. The β-actin was used as internal control. The data are presented as the mean±standard error of mean of three independent experiments. Bars with different letters are significantly different (p<0.05).

• 

  Fig. 2 Effects of C-type natriuretic peptide (CNP) on cyclic guanosine monophosphate (cGMP) production, natriuretic peptide receptor 2 (NPR2) expression, cell proliferation, testosterone secretion in mouse Leydig cells. (A) The cGMP production in mouse Leydig cells. (B) The NPR2 expression in mouse Leydig cells. (C) The cell activity in mouse Leydig cells. (D) The testosterone secretion in mouse Leydig cells. The β-actin was used as internal control. The data are presented as the mean±standard error of mean of three independent experiments. Bars with different letters are significantly different (p<0.05).

• 

  Fig. 3 Effects of natriuretic peptide receptor 2 (NPR2) knockdown on cyclic guanosine monophosphate (cGMP) production and cell proliferation in mouse Leydig cells. (A and B) The NPR2 expression in mRNA and Western blot levels in mouse Leydig transducted with shRNA-NPR2 lentivirus. (C) The cGMP production in mouse Leydig cells transducted with shRNA-NPR2 lentivirus. (D) The cell activity in mouse Leydig cells transducted with shRNA-NPR2 lentivirus. (E) The mRNA expression of cyclin A1, cyclin B1, and cyclin D2 in mouse Leydig cells transducted with shRNA-NPR2 lentivirus. The β-actin was used as internal control. The protein of NPR2 were normalized to that of β-actin. The results are presented as the mean±standard error of mean and are representative of three independent experiments. *p<0.05 and **p<0.01 compared with each corresponding group in the control.

• 

  Fig. 4 Effects of natriuretic peptide receptor 2 (NPR2) knockdown on cells apoptosis in mouse Leydig cells. (A) Relative mRNA expression of cell apoptosis related genes (Bcl-2, Bax, p53, and Caspase-3) in mouse Leydig cells transducted with shRNA-NPR2 lentivirus. (B) The phosphorylation of AKT in mouse Leydig cells transducted with shRNA-NPR2 lentivirus. (C) Caspase-3 activity in mouse Leydig cells transducted with shRNA-NPR2 lentivirus. (D) Relative protein expression of Bcl-2 and Bax in mouse Leydig cells transducted with shRNA-NPR2 lentivirus. The β-actin was used as internal control. The phosphorylation of AKT were normalized to that of total AKT. The results are presented as the mean±standard error of mean and are representative of three independent experiments. *p<0.05 and **p<0.01 compared with each corresponding group in the control.

• 

  Fig. 5 Effect of natriuretic peptide receptor 2 (NPR2) knockdown on testosterone secretion in mouse Leydig cells. (A) The effects C-type natriuretic peptide (CNP) and human chorionic gonadotropin (hCG) on CNP/NPR2 expression. (B) Concentration of testosterone secretion in the culture medium transducted with shRNA-NPR2 lentivirus. (C) Relative mRNA expression of genes (Star, Cyp11a1, 3β-hydroxysteroid dehydrogenase [3β-HSD], insulin-like factor 3 [Insl3], and luteinizing hormone receptor [LHR]) in mouse Leydig cells transducted with shRNA-NPR2 lentivirus. (D) Relative protein expression of Star and Cyp11a1 in mouse Leydig cells transducted with shRNA-NPR2 lentivirus. The β-actin was used as internal control. The results are presented as the mean±standard error of mean and are representative of three independent experiments. Bars with different letters are significantly different (p<0.05).

• 

  Fig. 6 The mechanism of C-type natriuretic peptide (CNP), testosterone, and estradiol in promoting natriuretic peptide receptor 2 (NPR2) expression in mouse Leydig cells. (A) The expression of NPR2 after stimulation with CNP, testosterone and estradiol with or without flutamide and ICI182780 in mouse Leydig cells. (B) The expression of Cyp19a1 after stimulation with CNP, testosterone and estradiol with or without flutamide and ICI182780 in mouse Leydig cells. The Σ-actin was used as internal control. The data are presented as the mean±standard error of mean of three independent experiments. Bars with different letters are significantly different (p<0.05).

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