Adenovirus-Mediated Gene Transfer to rat Testis in Vivo

Abstract

To study transgene expression in the adult rat testis in vivo, an adenovirus vector carrying a lacZ transgene with a nuclear localization signal was used as a marker. The adenovirus vector was first tested on rat Sertoli cell-germ cell cocultures in vitro; it efficiently mediated transgene expression in Sertoli cells but not germ cells. This vector was then delivered to the interstitial compartment of adult rat testes by intratesticular injection, resulting in Leydig cells expressing the transgene. Alternatively, delivering the vector to the intratubular compartment by rete testis injection resulted in expression of the transgene by Sertoli cells of the seminiferous epithelium and principal cells of the epididymis. In vivo, each cell type expressed the transgene by 2 days postinfection, and expression persisted for at least 10 days; however, later time points were associated with a loss of transgene expression and focal interstitial inflammation. This study documents the ability of adenovirus to mediate gene transfer to specific testicular cells, providing a powerful tool to study the short-term effects of specific genes on spermatogenesis in vivo.

Adenovirus-mediated gene delivery and in vitro microinsemination produce offspring from infertile male mice

“…We extended the findings reported in other studies (18) by demonstrating that adenoviruses could be used to infect Sertoli cells in infertile mouse testes. Furthermore, we found that the introduction of full-length Sl factor cDNA into the Sertoli cells partially restored spermatogenesis up to round spermatid stage in the Sl͞Sl d testes, and offspring were produced after microinsemination.…”

“…Earlier attempts at using adenoviruses to infect Sertoli cells in wild-type testes had limited success; transgene expression lasted for a few weeks and was accompanied by a severe inflammatory reaction (18). In contrast, our results show that infected Sertoli cells in germ cell-depleted testes continued to express the LacZ gene for up to 3 mo.…”

“…The discrepancies between the various studies, in terms of successful adenovirus infection of Sertoli cells, may have arisen from differences in the promoters and vectors used. In several tested cell lines, the activity of CAG promoter was more than 10-fold when compared to Rous sarcoma virus promoter used in the previous study (18,22). However, we also believe that Sertoli cells in the infertile testis offered at least two additional advantages for adenovirus infection.…”

“…Furthermore, genetic modification of Sertoli cells could be used to correct defective Sertoli cells, thus providing a method to treat those cases of male infertility that might be the results of defects in Sertoli cell function. However, although germ cells can be transfected by using several methods (15)(16)(17), there is currently no technique that allows long-term, widespread transgene expression in Sertoli cells (16,18). Adenoviruses have potential as gene therapy vectors in human patients because of their relatively high cloning capacity and amenability to production in high titers (19).…”

“…Earlier attempts to infect Sertoli cells in wild-type testes met with limited success, as transgene expression was transient and inflammation was apparent (18). Nonetheless, we hoped that adenovirus infection of infertile testes might provide a better outcome.…”