Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Tissue-specific inhibition of urokinase-type plasminogen activator expression in the testes of mice by inducible lentiviral RNA interference causes male infertility

Kai Zhao A * , Yan Liu B * , Zhe Xiong C , Lian Hu A B D and Cheng-liang Xiong A B
+ Author Affiliations
- Author Affiliations

A Family Planning Research Institute and Center of Reproductive Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.

B Wuhan Tongji Reproductive Medicine Hospital, Wuhan, 430014, China.

C Department of Physiology, Medical School of Jianghan University, Wuhan, 430056, China.

D Corresponding author. Email: hulian02@163.com

Reproduction, Fertility and Development 29(11) 2149-2156 https://doi.org/10.1071/RD16477
Submitted: 7 September 2016  Accepted: 24 January 2017   Published: 16 March 2017

Abstract

Urokinase-type plasminogen activator (uPA) is involved in many physiological processes, including male infertility. To explore the effects of uPA in male reproduction, we constructed an inducible uPA short hairpin RNA (shRNA) system expressed by lentiviral vectors. After proving inhibition of uPA expression in the mouse Sertoli cell line TM4 by 1 μg mL−1 doxycycline (Dox), two lentivirus (pLenti4-shRNA and pLenti6/TR) were co-microinjected into mouse testes to produce TetR&shuPA mice model. Though oral gavage by 0.75 mg mL−1 Dox each day for 1 week, the Plau mRNA expression, uPA protein level and uPA enzyme activity in mice testis decreased significantly in TetR&shuPA mice model. After Dox induction of 1 week, the TetR&shuPA mice mated with female mice. Our results show that the pregnancy rate was reduced by approximately 40% and the sperm motility also decreased significantly. These data indicated that downregulation of uPA could decrease the fertility of male mice, which may be caused by a reduction in sperm motility. To investigate the reversible effect and safety of the inducible uPA shRNA system, we withdraw Dox and found the mating rate and sperm motility gradually recovered after 2 weeks. The histopathology structure of the testis, epididymis, and main organs was not altered significantly. The results of the present study indicating that uPA may be regarded as a novel target for the regulation of male fertility.

Additional keywords: doxycycline, lentivirus.


References

Bahi, A., Boyer, F., Kafri, T., and Dreyer, J.-L. (2006). Silencing urokinase in the ventral tegmental area in vivo induces changes in cocaine-induced hyperlocomotion. J. Neurochem. 98, 1619–1631.
Silencing urokinase in the ventral tegmental area in vivo induces changes in cocaine-induced hyperlocomotion.CrossRef | 1:CAS:528:DC%2BD28XhtVymtLbN&md5=ca0d087feda6472fecea1efe8ced176bCAS |

Barde, I., Zanta-Boussif, M. A., Paisant, S., Leboeuf, M., Rameau, P., Delenda, C., and Danos, O. (2006). Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector. Mol. Ther. 13, 382–390.
Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector.CrossRef | 1:CAS:528:DC%2BD28XksVChtA%3D%3D&md5=0e1d13455e6200808f0769f74817f6d2CAS |

Blasi, F., and Sidenius, N. (2010). The urokinase receptor: focused cell surface proteolysis, cell adhesion and signaling. FEBS Lett. 584, 1923–1930.
The urokinase receptor: focused cell surface proteolysis, cell adhesion and signaling.CrossRef | 1:CAS:528:DC%2BC3cXkvFOjsrk%3D&md5=5197a27e7fc89ccdb216f4a3a2b167ceCAS |

Castellino, F. J., and Ploplis, V. A. (2005). Structure and function of the plasminogen/plasmin system. Thromb. Haemost. 93, 647–654.
| 1:CAS:528:DC%2BD2MXjs1ersb0%3D&md5=ff939ab89f5a74146add0f25a6014e3dCAS |

Cibula, D., Gompel, A., Mueck, A. O., La Vecchia, C., Hannaford, P. C., Skouby, S. O., Zikan, M., and Dusek, L. (2010). Hormonal contraception and risk of cancer. Hum. Reprod. Update 16, 631–650.
Hormonal contraception and risk of cancer.CrossRef | 1:CAS:528:DC%2BC3cXhtlShu7%2FL&md5=3586eb9de5d48ea58c209bbb42b75822CAS |

Coy, P., Jiménez-Movilla, M., García-Vázquez, F. A., Mondéjar, I., Grullón, L., and Romar, R. (2012). Oocytes use the plasminogen–plasmin system to remove supernumerary spermatozoa. Hum. Reprod. 27, 1985–1993.
Oocytes use the plasminogen–plasmin system to remove supernumerary spermatozoa.CrossRef | 1:CAS:528:DC%2BC38XptVGisbs%3D&md5=63e9b92f330966c03381a385fe824a4fCAS |

Das, A. T., Zhou, X., Metz, S. W., Vink, M. A., and Berkhout, B. (2016). Selecting the optimal Tet-on system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells. Biotechnol. J. 11, 71–79.
Selecting the optimal Tet-on system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells.CrossRef | 1:CAS:528:DC%2BC2MXhsFCit7vF&md5=e6074e87f8bb03f2a7e0c735e64764dbCAS |

Ebisch, I. M., Steegers-Theunissen, R. P., Sweep, F. C., Zielhuis, G. A., Geurts-Moespot, A., and Thomas, C. M. (2007). Possible role of the plasminogen activation system in human subfertility. Fertil. Steril. 87, 619–626.
Possible role of the plasminogen activation system in human subfertility.CrossRef | 1:CAS:528:DC%2BD2sXksVyltb4%3D&md5=38400182883b95c1eda14ba312bf4786CAS |

Gossen, M., and Bujard, H. (1992). Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl Acad. Sci. USA 89, 5547–5551.
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.CrossRef | 1:CAS:528:DyaK38Xks1equr4%3D&md5=a288a15afac4f2abd644c0eb306c1b9eCAS |

Grullón, L. A., Gadea, J., Mondéjar, I., Matás, C., Romar, R., and Coy, P. (2013). How is plasminogen/plasmin system contributing to regulate sperm entry into the oocyte? Reprod. Sci. 20, 1075–1082.
How is plasminogen/plasmin system contributing to regulate sperm entry into the oocyte?CrossRef |

Huang, X. B., Xia, W., Xiong, C. L., Xiao, D. Z., Shen, J. Y., and Zhou, J. L. (1997). Studies on the relationship between urokinase plasminogen activator (uPA) and human sperm motility. J. Tongji Med. Univ. 17, 213–217.
Studies on the relationship between urokinase plasminogen activator (uPA) and human sperm motility.CrossRef |

Huang, D. H., Zhao, H., Tian, Y. H., Li, H. G., Ding, X. F., and Xiong, C. L. (2007). Gene expression changes of urokinase plasminogen activator and urokinase receptor in rat testes at postnatal stages. Asian J. Androl. 9, 679–683.
Gene expression changes of urokinase plasminogen activator and urokinase receptor in rat testes at postnatal stages.CrossRef | 1:CAS:528:DC%2BD2sXhtFaksb7L&md5=14d15aa1b5143cd630e51af17302ba29CAS |

Law, R. H., Abu-Ssaydeh, D., and Whisstock, J. C. (2013). New insights into the structure and function of the plasminogen/plasmin system. Curr. Opin. Struct. Biol. 23, 836–841.
New insights into the structure and function of the plasminogen/plasmin system.CrossRef | 1:CAS:528:DC%2BC3sXhsl2gs7zO&md5=1c17d9c59a10648514cac572d267bf7cCAS |

Le Magueresse-Battistoni, B. (2007). Serine proteases and serine protease inhibitors in testicular physiology: the plasminogen activation system. Reproduction 134, 721–729.
Serine proteases and serine protease inhibitors in testicular physiology: the plasminogen activation system.CrossRef | 1:CAS:528:DC%2BD1cXmt1eiuw%3D%3D&md5=2528b0ed1e4ed2fc28fdef4e39a46c31CAS |

Liu, Y. X. (2007). Involvement of plasminogen activator and plasminogen activator inhibitor type 1 in spermatogenesis, sperm capacitation, and fertilization. Semin. Thromb. Hemost. 33, 29–40.
Involvement of plasminogen activator and plasminogen activator inhibitor type 1 in spermatogenesis, sperm capacitation, and fertilization.CrossRef |

Liu, Y., Pang, X. B., Liao, J. J., Hu, L., and Xiong, C. L. (2008). Correlation of the content and expression of urokinase plasminogen activator with asthenospermia in rat models. Zhonghua Nan Ke Xue 14, 786–791.
| 1:CAS:528:DC%2BD1MXhtVGmt73M&md5=bb12432d76983f4ab8eb5264aaf6e132CAS |

McLaughlin, E. A., Ford, W. C., and Hull, M. G. (1992). Motility characteristics and membrane integrity of cryopreserved human spermatozoa. J. Reprod. Fertil. 95, 527–534.
Motility characteristics and membrane integrity of cryopreserved human spermatozoa.CrossRef | 1:STN:280:DyaK38zovFaltw%3D%3D&md5=78d349bf0ed535213c210758c7ae61a7CAS |

Merino, P., Diaz, A., Jeanneret, V., Wu, F., Torre, E., Cheng, L., and Yepes, M. (2016). Urokinase-type plasminogen activator (uPA) binding to the uPA receptor (uPAR) promotes axonal regeneration in the central nervous system. J. Biol. Chem. 292, 2741–2753.
Urokinase-type plasminogen activator (uPA) binding to the uPA receptor (uPAR) promotes axonal regeneration in the central nervous system.CrossRef |

Montuori, N., and Ragno, P. (2014). Role of uPA/uPAR in the modulation of angiogenesis. Chem. Immunol. Allergy 99, 105–122.
Role of uPA/uPAR in the modulation of angiogenesis.CrossRef | 1:CAS:528:DC%2BC2cXotlOgtbY%3D&md5=a529b6c07d0795d9ad5a7d35b8aa35f8CAS |

Morimoto, H., Lee, J., Tanaka, T., Ishii, K., Toyokuni, S., Kanatsu-Shinohara, M., and Shinohara, T. (2012). In vitro transformation of mouse testis cells by oncogene transfection. Biol. Reprod. 86, 148.
In vitro transformation of mouse testis cells by oncogene transfection.CrossRef |

National Research Council (2011). ‘Guide for the care and use of laboratory animals, eighth edition.’ (National Academies Press: Washington, D.C.)

Noh, H., Hong, S., and Huang, S. (2013). Role of urokinase receptor in tumor progression and development. Theranostics 3, 487–495.
Role of urokinase receptor in tumor progression and development.CrossRef |

Popa, N. L., Wergedal, J. E., Lau, K.-H. W., Mohan, S., and Rundle, C. H. (2014). Urokinase plasminogen activator gene deficiency inhibits fracture cartilage remodeling. J. Bone Miner. Metab. 32, 124–135.
Urokinase plasminogen activator gene deficiency inhibits fracture cartilage remodeling.CrossRef | 1:CAS:528:DC%2BC2cXktlSqsr0%3D&md5=a8f194b847f8f8fbbd8b4fbd52fc3761CAS |

Tian, Y. H., Xiong, C. L., Wan, H., Huang, D. H., Guan, H. T., Ding, X. F., and Shang, X. J. (2007). Inhibition of the urokinase-type plasminogen activator by triplex-forming oligonucleotides in rat Sertoli cells: a new contraceptive alternative? Oligonucleotides 17, 174–188.
Inhibition of the urokinase-type plasminogen activator by triplex-forming oligonucleotides in rat Sertoli cells: a new contraceptive alternative?CrossRef | 1:CAS:528:DC%2BD2sXnsl2gu7Y%3D&md5=1cd49d822bcdbc75fe0436ea406f6511CAS |

Vieyra, D. S., and Goodell, M. A. (2007). Pluripotentiality and conditional transgene regulation in human embryonic stem cells expressing insulated tetracycline-ON transactivator. Stem Cells 25, 2559–2566.
Pluripotentiality and conditional transgene regulation in human embryonic stem cells expressing insulated tetracycline-ON transactivator.CrossRef | 1:CAS:528:DC%2BD2sXht1CltLzN&md5=8711f26780bdd6a5deeaa8bc5e67e97bCAS |

Vu, Q., Micks, E., McCoy, E., and Prager, S. (2016). Efficacy and safety of long-acting reversible contraception in women with cardiovascular conditions. Am. J. Cardiol. 117, 302–304.
Efficacy and safety of long-acting reversible contraception in women with cardiovascular conditions.CrossRef |

Wang, L., Guan, H. T., Tian, Y. H., and Xiong, C. L. (2006). Urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor in the seminal plasma and sperm of fertile and oligoasthenozoospermia males. Zhonghua Nan Ke Xue 12, 791–793.
| 1:CAS:528:DC%2BD1cXhvFantg%3D%3D&md5=af66cfe754444cec8086aa89cc48e1b7CAS |

Wu, F., Catano, M., Echeverry, R., Torre, E., Haile, W. B., An, J., Chen, C., Cheng, L., Nicholson, A., Tong, F. C., Park, J., and Yepes, M. (2014). Urokinase-type plasminogen activator promotes dendritic spine recovery and improves neurological outcome following ischemic stroke. J. Neurosci. 34, 14219–14232.
Urokinase-type plasminogen activator promotes dendritic spine recovery and improves neurological outcome following ischemic stroke.CrossRef | 1:CAS:528:DC%2BC2cXitVSlsrfL&md5=2fe079b7912933c812fe3813e1ce2c67CAS |

Zapata, L. B., Steenland, M. W., Brahmi, D., Marchbanks, P. A., and Curtis, K. M. (2013). Effect of missed combined hormonal contraceptives on contraceptive effectiveness: a systematic review. Contraception 87, 685–700.
Effect of missed combined hormonal contraceptives on contraceptive effectiveness: a systematic review.CrossRef | 1:CAS:528:DC%2BC38XhsFKksrbK&md5=c8fbf55dd3c410fda703837a39f3d789CAS |

Zhang, J., Wang, C., Ke, N., Bliesath, J., Chionis, J., He, Q. S., Li, Q., Chatterton, J. E., Wong-staal, F., and Zhou, D. (2007). A more efficient RNAi inducible system for tight regulation of gene expression in mammalian cells and xenograft animals. RNA 13, 1375–1383.
A more efficient RNAi inducible system for tight regulation of gene expression in mammalian cells and xenograft animals.CrossRef |

Zhao, K., Chen, Y., Yang, R., Bai, Y., Li, C., Li, H., and Xiong, C. (2015). miR-424/322 is downregulated in the semen of patients with severe DNA damage and may regulate sperm DNA damage. Reprod. Fertil. Dev. , .
miR-424/322 is downregulated in the semen of patients with severe DNA damage and may regulate sperm DNA damage.CrossRef |



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