Gene Therapy (2003) 10, 43–50 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt RESEARCH ARTICLE Seminiferous tubule cannulation (STC): a new, sensitive technique for detecting gene transfer in developing sperm JW Gordon Department of Obstetrics and Gynecology, Mt Sinai School of Medicine, New York, NY 10029, USA As gene therapy vectors, strategies, and disease targets delivery of high quantities of gene therapy vector directly to continue to expand and diversify, the likelihood that devel- spermatogenic cells without significantly disturbing the oping germ cells will be exposed to gene transfer vectors cytoarchitecture of the seminiferous tubule. To demonstrate increases. Insertion of exogenous genetic material into the the effectiveness of this technique, three promoters driving germ line might have devastating effects on normal devel- lacZ gene expression in adenovirus vectors were tested for opment which could be heritable. Accordingly, it is important their ability to transduce cells within the seminiferous tubule. that vectors be tested for their potential to insert genes into Results indicate that the cytomegalovirus promoter, but not developing gametes. Such tests are most difficult in males, the Rous sarcoma virus or elongation factor 1a promoters, is where differentiating sperm are sequestered behind the active within the seminiferous tubule. Further development of blood–testis barrier. In this communication we report the this technique promises to lead to a standardized test for development of a new technique, which we call seminiferous male germ cell transduction by gene therapy vectors. tubule cannulation (STC). We demonstrate that STC allows Gene Therapy (2003) 10, 43–50. doi:10.1038/sj.gt.3301850 Keywords: germ cell transduction; seminiferous tubule; adenovirus Introduction therapeutic strategies diversify and become promul- gated. Assessment of germ line integration is more As with any other therapy, gene therapy can have problematic for spermatogenic cells than for developing adverse effects. However, unlike other treatments, a eggs. Development of oocytes within preovulatory potential risk of gene therapy is genetic modification of follicles mainly involves growth; however, spermatogen- the germ line by addition of new genetic material. While esis is characterized by a complex differentiative process some conventional remedies can alter the germ line whereby the nuclear material condenses, chromosomal through mutagenesis, gene therapy has the potential to proteins are replaced, and a flagellum forms. A cell at add new coding functions to the genome. Expression of any of the many stages of spermatogenesis might have new genetic material in the embryo, fetus, or adult could unique susceptibility to insertion of gene therapy have serious harmful effects which might be heritable. vectors. Further complicating the problem is that For this reason it is important that experimental systems spermatogenesis takes place behind the blood:testis be developed which sensitively test for the potential of barrier, which prevents large molecules from accessing gene therapy vectors to access the germ line. developing sperm from the bloodstream.11 A variety of tests have thus far been developed to Some laboratories, including our own,12,13 have per- address this problem. Most of these assays have been formed direct testicular injection of adenovirus vectors indirect, relying upon tests of progeny after systemic and examined seminiferous tubules for expression of the 1–4 administration of vectors, tests for reporter gene reporter gene lacZ, which was encoded in the vectors. expression or genetic material in gonads at various times Expression within tubules is seen in some13 but not all12 2,4,5–9 after vector administration, or statistical analyses experiments, and these tests are crude for several which extrapolate probabilities of germ line integration reasons: even the smallest injection needles are far larger 10 based upon negative DNA assays of gonadal tissues. than the lumen of the mouse seminiferous tubule. Thus, Unfortunately, none of these testing paradigms ad- it is necessary to rupture the wall of the tubule with the dresses the question of whether direct exposure of sharp edge of the needle in order to access the lumen, gametogenic cells, gametes, or early embryos will lead and the lumen can be accessed only at sites immediately to gene transfer. Such direct exposure is likely to occur as adjacent to the rupture point. Because spermatogenesis gene therapy vectors, routes of administration, and proceeds in waves longitudinally along the seminiferous tubule, such cross-sectional approaches are unlikely to expose cells at all stages of spermatogenesis to the vector. Correspondence: JW Gordon, Department of Obstetrics and Gynecology, Mt Sinai School of Medicine, 1 Gustave L Levy Place, New York, NY Moreover, these procedures are traumatic, and can 10029, USA disrupt the normal relationship between gametogenic Received 26 August 2001; accepted 15 June 2002 cells and Sertoli cells of the tubule. Experimental models A new test for gene transfer in developing sperm JW Gordon 44 wherein vectors are inserted into the male sex ducts as establish it as a standardized test for male germ cell models for retrograde transport of vectors to the testis in transduction. the course of gene therapy for prostate or bladder disease have been utilized.4,7,8 However, these methods require successful cannulation of ductular tubules, with retro- Results grade flow of substantial material back to the testis. As such they are technically challenging and do not Experience with the STC procedure guarantee that high quantities of vector will reach the Twelve STC procedures were performed with the lumen of the seminiferous tubule. cytomegalovirus (CMV) vector, 13 with the Rous In an effort to develop direct tests for gene insertion sarcoma virus (RSV) vector, and 14 with the EF-1 vector. into gametes, our laboratory has exposed sperm directly The following observations were made from cannula- to adenovirus vector, performed in vitro fertilization, and tions of seminiferous tubules with microneedles and 12 examined early embryos for reporter gene expression. instillation of concentrated suspensions of adenovirus We have also exposed oocytes to vectors and examined gene therapy vectors carrying the reporter gene lacZ. subsequent embryos and fetuses for gene expression or These observations were facilitated by addition of a 14 integration. These experiments are a step forward small amount of trypan blue to the vector solution (see because the embryos studied after in vitro fertilization Methods): are known to be derived from gametes that were directly (1) Even the smallest conventional injection needles are exposed to the vector. However, because they involve far too large to access the lumen of the seminiferous only mature sperm these tests are also imperfect: they do tubule. Figure 1 shows seminiferous tubules exposed not allow exposure of spermatogenic cells at various through an incision in the testis capsule. Included in this stages of meiosis and spermiogenesis to vectors, as these photograph is a 30g injection needle. These needles have differentiation events occur within the seminiferous been used in previous attempts to expose spermatogenic tubule. cells to gene therapy vectors,12 and they are the smallest In this communication we describe a new technique commercially available needles. As can be readily we have developed, which we call seminiferous tubule appreciated from this photograph, a 30g needle is far cannulation (STC). This procedure allows perfusion of too large to access the lumen of the seminiferous tubule, substantial lengths of the seminiferous tubule with and can only expose spermatogenic cells to gene therapy highly concentrated gene therapy vector preparations vectors by cutting through the tubule wall. without disturbing the structural integrity of the tubule. (2) STC allows perfusion of substantial lengths of the These attributes indicate promise for development of this seminiferous tubule with gene therapy vector prepara- methodology into a definitive test for insertion of gene tions. therapy vectors into spermatogenic cells. We describe Figure 1 shows a testis subjected to a single STC this technique and use it to test three different promoters injection. In order to better demonstrate the effectiveness for their ability to drive reporter gene expression in the of this procedure, blue ink was instilled into the tubules seminiferous tubule. We also discuss improvements in for improved visualization. As demonstrated here, the STC method which must be made in order to several loops of the seminiferous tubule have been Figure 1 View through a dissecting microscope of seminiferous tubules exposed through an incision in the testis capsule and subjected to STC. The tubules were perfused with a single injection of blue ink (arrows) to indicate that length of tubule that can be perfused in one injection. Note that several loops of tubule are perfused. Also included in this figure is a 30g injection needle. This needle is far too large to access the lumen of the seminiferous tubule. Gene Therapy A new test for gene transfer in developing sperm JW Gordon 45 perfused (arrows). This length of tubule is equivalent to resolution. Figure 2 shows the results of this experiment. several thousand cell diameters, and thus STC can The distribution of expression was similar to that seen expose germ cells