In Vivo Gene Transfer by Electroporation Allows Expression of a Fluorescent Transgene in Hamster Testis and Epididymal Sperm and Has No Adverse Effects upon Testicular Integrity or Sperm Quality1

Abstract

The study of gene function in testis and sperm has been greatly assisted by transgenic mouse models. Recently, an alternative way of expressing transgenes in mouse testis has been developed that uses electroporation to introduce transgenes into the male germ cells. This approach has been successfully used to transiently express reporter genes driven by constitutive and testis-specific promoters. It has been proposed as an alternative method for studying gene function in testis and sperm, and as a novel way to create transgenic animals. However, the low levels and transient nature of transgene expression that can be achieved using this technique have raised concerns about its practical usefulness. It has also not been demonstrated in mammals other than mice. In this study, we show for the first time that in vivo gene transfer using electroporation can be used to express a fluorescent transgene in the testis of a mammal other than mice, the Syrian golden hamster. Significantly, for the first time we demonstrate expression of a transgene in epididymal sperm using this approach. We show that expression of the transgene can be detected in sperm for as long as 60 days following gene transfer. Finally, we provide the first systematic demonstration that this technique does not lead to any significant long-term adverse effects on testicular integrity and sperm quality. This technique therefore offers a novel way to study gene function during fertilization in hamsters and may also have potential as a way of creating transgenic versions of this important model species.

Expression of a fluorescent recombinant form of sperm protein phospholipase C zeta in mouse epididymal sperm by in vivo gene transfer into the testis

“…Although transgene expression was detected in hamster epididymal sperm for as long as 60 days after in vivo gene transfer, we were unable to detect any adverse effects upon testicular integrity or sperm quality (17).…”

“…More recently, studies in our own laboratory have resulted in the expression of a transgene in the epididymal sperm of the hamster (17). Although transgene expression was detected in hamster epididymal sperm for as long as 60 days after in vivo gene transfer, we were unable to detect any adverse effects upon testicular integrity or sperm quality (17).…”

<em>In Vivo</em> Microinjection and Electroporation of Mouse Testis

“…This increase in transfection efficiency 12,13,15 by elevated voltage is accompanied by the adverse effect of testicular shrinking [12][13][14] . The most favorable voltage range seems to be between 30-50 V, guaranteeing small effects upon testicular integrity, a normal sperm quality 16 , a normal mating behavior 17 , the maintenance of offspring production ability [16][17][18][19][20] as well as a sufficient transfection efficacy.…”

Future Perspectives in the Diagnosis and Management of Unexplained Male Infertility

“…In vivo electroporation is another technique that involves injecting a DNA construct into the testes with application of electric pulses that permeates cell membranes and allow DNA to enter the cells. Using in vivo electroporation, groups have demonstrated the ability to express transgenes in mature epididymal sperm in mice [49] and hamsters [50]. They also demonstrated that the use of electroporation in vivo does not lead to adverse effects on testicular integrity and sperm quality [49].…”

“…However, the expression of these transgenes is typically transient and may last as long as 60 days. Additionally, only 5-10 % of sperm were found to carry the transgene indicating a low transfer rate [50].…”