Gene Transfer to Mouse Testes by Electroporation and Its Influence on Spermatogenesis

Abstract

We transferred the adventitious gene pCAGGS-lacZ to mouse testes with the use of a square-wave electroporator and investigated the efficiency of gene transfer (GT) and the influence of the procedure on testicular damage and spermatogenesis. Mice were divided into 5 groups: (1-2) injection of gene/phosphate-buffered saline (PBS) into the interstitial space followed by electroporation (EP), (3) EP alone, (4-5) injection of gene/PBS without EP. The presence of the lacZ gene was determined by X-gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) staining and the polymerase chain reaction (PCR). The influence of transfer on spermatogenesis was assessed by evaluating the seminiferous tubules according to the Johnsen score (JS). TdT-mediated dUTP-biotin nick end-labeling (TUNEL) staining was performed for the detection of apoptosis in the testes to evaluate the testicular damage caused by GT, and fertilization ability was assessed by mating male mice from each group with normal female mice at 1, 2, 4, 6, and 8 weeks after the procedure. LacZ expression was detected by X-gal staining and PCR for 4 weeks after GT in group 1. But in group 4, LacZ expression was not detected for all times. In groups 1 through 3, the JSs decreased gradually until 4 weeks and recovered at 6 and 8 weeks after GT. The JSs were significantly decreased at 4 weeks for groups 1 through 3 compared with groups 4 and 5. In groups 1 through 3, apoptotic cells were significantly more numerous at 1, 2, and 4 weeks after the procedure, and there were significant differences in their numbers between groups 1 through 3 and groups 4 and 5 until 4 weeks after the procedure. The number of offspring did not differ significantly between all groups. These results suggest that although spermatogenic damage caused by EP could present problems, GT by EP might be effective for transfecting germ cells or somatic cells and could be applicable for in vivo gene therapy for male infertility in the future.

<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.…”

Therapeutic Potential of Gene Transfer to Testis; Myth or Reality?

“…Another problem of electroporation is that the electric pulses themselves could result in testicular damage. In our previous reports, the Johnsen scores were significantly decreased and apoptotic cells were significantly increased in the testis several weeks after in vivo electroporation (Umemoto, 2002. These results suggest that spermatogenic damage caused by electroporation itself could be important problems for clinical application.…”

“…As a possible alternative and more effective method for generating transgenic mice, several researchers have attempted testis-mediated gene transfer using an in vivo electroporation method. After injection of exogenous DNA into the testis, the testes are held between a tweezer-type electrode, and square electric pulses are applied several times at 20-50 volts (Muramatsu, 1996(Muramatsu, , 1997Yamazaki, 1998Yamazaki, , 2000Umemoto, 2002. Expression of the exogenous gene was clearly observed in germ cells of seminiferous tubules 48 hours after transfecting the mouse testis by in vivo electroporation (Muramatsu, 1997).…”

“…We also directly injected DNA, which was constructed as a cytomegalovirus enhancer/chicken β-actin promoter connected with the β-galactosidase gene (Umemoto, 2002, into mouse testes using a square-wave electroporator, and investigated the efficiency of gene transfer. The β-galactosidase activity was detected in germ cells and Sertoli cells in seminiferous tubules and in Leydig cells for 4 weeks (Umemoto, 2002; however, although electroporation is a simple and convenient technique for gene transfer to germ cells in the testis, the gene expression was deemed transient and uncontrollable . Another problem of electroporation is that the electric pulses themselves could result in testicular damage.…”

Regulation of fertilization in male rats by CatSper2 knockdown

“…In vivo gene transfer into the testis by EP has been put forward as a non-hormonal approach to study the testis and sperm function, and as a novel way of creating transgenic animals. [41][42][43] Selective inhibitors of ion channels could, in principle, inhibit sperm function and prevent fertilization. By applying the patch-clamp technique to mature human spermatozoa, Lishko et al 44 and Strunker et al 45 independently found that the Catsper ion channel of human spermatozoa is synergistically activated by elevation The results were represented as mean6s.e.…”