In vitro characterization of endonuclease activity in bovine sperm cells

• Grant number: 13/16087-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2013
• End date: September 30, 2014
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Mayra Elena Ortiz D' Avila Assumpcao
• Grantee: Catarina Augusta de Queiroz Nunes
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Sperm-mediated gene transfer (SMGT) is a method considered easy and inexpensive for producing transgenic animals. This technique uses spermatozoa as a vector for introducing foreign DNA into an oocyte during the fertilization process. It was first applied in murine species, in which it was observed dose-dependent activation of sperm endonucleases in response to interaction with foreign DNA. Endonucleases are enzymes present in the sperm nucleus, which promote chromosomal and foreign DNA digestion when activated. This phenomenon may be responsible for reducing the technique efficiency and for the discrepancy in the results. However, in bovine spermatozoa submitted to the same technique, there was no evidence of chromosomal DNA digestion by endonucleases. The knowledge of the activity of these proteins in bovine is still limited, which may limit the application and efficiency of SMGT and other reproductive biotechnologies in this species. This study aims to evaluate the activity of bovine endonucleases by in vitro plasmid digestion, checking the start time of endonuclease activity and the amount of foreign DNA required in activating them. The results of this study will offer subsidies on bovine sperm cells basic biology, allowing protocols improvement and new biotechnologies increased efficiency, as SMGT.