Effects of adenovirus mediated gene transfer to mouse testis in vivo on spermatogenesis and next generation.
OBJECTIVE
We directly injected DNA into mouse testes in vivo using an adenovirus vector to transfect testicular cells. We then analyzed the transfection efficiency, immunological problems and effects of gene transfer on spermatogenesis and the next generation. In this study we discuss the potential of gene therapy for male infertility.


METHODS
A replication incompetent human adenovirus serotype 5 contained 2 deletions (E1 and E3 deletions) and was constructed such that the transgene was driven by the chicken beta-actin promoter to promote over expression of the downstream target gene (Lac Z). This adenovirus vector or control solution was injected into the interstitial space (intratesticular injection) or seminiferous tubules (intratubular injection) of the mouse testis. We investigated beta-galactosidase gene expression by X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) staining, the effects of gene transfer on spermatogenesis by evaluating the frequency of apoptotic cells by the TUNEL method, the inflammatory response on testes by detecting CD4 and CD8 positive cells immunohistochemically, and interleukin (IL)-6 and IL-8 by immunoblot analysis, epididymides sperm motility and the reproductive response of each mouse 3, 7, 14 and 28 days after injection.


RESULTS
Intratesticular injection of adenovirus vector resulted in strong transgene expression in Leydig cells. In contrast, intratubular injection resulted in strong expression in Sertoli cells. Transgene expression was not detected in germ cells by either method. The peak of beta-galactosidase activity was on day 7, ie 0.674 +/- 0.20 (intratesticular) and 0.534 +/- 0.22 U (intratubular), and it decreased with time thereafter. The apoptosis index on day 7 was significantly higher in adenovirus injected groups than in noninjected groups, ie 0.46 +/- 0.20 vs 0.10 +/- 0.11 (intratesticular) and 0.78 +/- 0.31 vs 0.24 +/- 0.10 (intratubular). Transfected animals showed a slight mononuclear inflammatory response in the testes composed of CD4 and CD8 positive cells. Adenovirus vector stimulation resulted in the induction of IL-6 and IL-8 secretion in the testis. These immune responses subsided after day 7. There were no significant differences in the percent of motile sperm or the rate of abnormal sperm between the groups on any day after injection. Reproductive ability remained almost normal even after adenovirus mediated gene transfer with no effect observed in offspring.


CONCLUSIONS
Our results suggest that although slight spermatogenic damage and inflammatory response caused by these methods may present problems, adenovirus mediated gene transfer may be effective for transfecting testicular somatic cells and applicable for in vivo gene therapy for male infertility in the future.
Kojima
Yoshiyuki
Y
Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. yoshi.kojima@nifty.ne.jp
Sasaki
Shoichi
S
Umemoto
Yukihiro
Y
Hashimoto
Yoshihiro
Y
Hayashi
Yutaro
Y
Kohri
Kenjiro
K
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000235:genetics; D015496:CD4-Positive T-Lymphocytes / Q000473:pathology; D018414:CD8-Positive T-Lymphocytes / Q000473:pathology; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007248:Infertility, Male / Q000628:therapy*; D007267:Injections; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007985:Leydig Cells / Q000473:pathology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D011247:Pregnancy; D012708:Sertoli Cells / Q000473:pathology; D013081:Sperm Motility / Q000235:genetics; D013091:Spermatogenesis / Q000235:genetics*; D013737:Testis / Q000473:pathology*
D015850:Interleukin-6; D016209:Interleukin-8