Expression of a fluorescent recombinant form of sperm protein phospholipase C zeta in mouse epididymal sperm by in vivo gene transfer into the testis.
OBJECTIVE
To use in vivo gene transfer into the testis by electroporation to express a fluorescent recombinant form of a testis-specific gene in the mature epididymal sperm of mice and thus study the pattern of gene localization.


METHODS
Controlled animal study.


METHODS
Research laboratory at the University of Oxford.


METHODS
Four- to 6-week-old male mice.


METHODS
Phospholipase C zeta (PLCzeta), the putative mammalian egg activation factor, was fused to enhanced yellow fluorescent protein (EYFP), and in vivo gene transfer by electroporation was used to introduce this transgene (PLCzeta-EYFP) into mouse testis. Transgene expression in testis and sperm were analyzed at 20 and 40 days after electroporation.


METHODS
Transgene expression in testis and epididymal sperm was analyzed by fluorescence microscopy and an excitation light source suitable for EYFP.


RESULTS
Phospholipase C zeta-EYFP was successfully expressed in epididymal sperm when analyzed 40 days after gene transfer and was localized to the head and midpiece regions.


CONCLUSIONS
Our results provide the first demonstration that in vivo gene transfer can be used to study the localization of proteins in mature sperm and that this represents a powerful new technique for studying male infertility and gene function in sperm.
Coward
Kevin
K
Department of Pharmacology, University of Oxford, Oxford, United Kingdom. kevin.coward@pharm.ox.ac.uk
Kubota
Hiroki
H
Hibbitt
Olivia
O
McIlhinney
Jeff
J
Kohri
Kenjiro
K
Parrington
John
J
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods*; D030501:Epididymal Secretory Proteins / Q000096:biosynthesis* / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology; D018014:Gene Transfer Techniques; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D054801:Phosphoinositide Phospholipase C; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*; D014018:Tissue Distribution; D010738:Type C Phospholipases / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D030501:Epididymal Secretory Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C499234:Plcz1 protein, mouse