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Regular Article |
a Center for Animal Transgenesis and Germ Cell Research, Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania 19348
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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reproductive technology, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Spermatogonial transplantation is based on the introduction of stem spermatogonia into a recipient testis to establish donor-derived spermatogenesis. Transplantation of testicular germ cells from fertile donor mice into the testes of infertile recipient mice resulted in donor-derived spermatogenesis by the recipient animal [5], and the donor haplotype was passed on to the offspring [6–8]. The technique of spermatogonial transplantation has also been performed between rats [9, 10] and in cynomolgus monkeys [11]. This technique provides an invaluable tool for studying fundamental aspects of spermatogenesis and germ cell function [8, 12–14].
Genetic manipulation of farm animals has been directed toward improving productivity traits or development of transgenic animals for biomedical purposes. The development of genetically modified pigs is of interest because of its potential to provide tissues and organs for xenotransplantation to humans. To date, the majority of transgenic pigs have been produced by microinjection of individual zygotes [15], but the advent of somatic cell nuclear transfer to produce cloned pigs [16, 17] has recently opened another potential avenue for the production of transgenic animals. However, the efficiency of both these techniques is currently quite low, making them expensive approaches for producing transgenic animals. In addition, developmental abnormalities in cloned animals often result in embryonic and fetal losses as well as serious health problems in offspring born alive after nuclear transfer. As an alternate approach, transplantation of spermatogonial stem cells that have been transduced directly with the desired gene could be used to generate transgenic livestock using in vivo or in vitro fertilization. In mice, spermatogenesis was reestablished following spermatogonial transplantation into an infertile male, and up to 40% of the progeny that resulted from mating of the recipient male carried the donor-derived transgene [6].
Due to anatomical differences, transplantation techniques developed in mice cannot be easily extrapolated to large domestic animals. The large volume-to-surface ratio, stronger resistance of the lamina propria, and highly convoluted tubular mass of the bovine testis make microinjections into the seminiferous tubules of isolated immature bull testes very difficult and inefficient [11]. Cannulation of an efferent duct has been reported in testes of rams, bucks, and boars for the collection of testicular fluid but is also very difficult [18–20]. However, ultrasound-guided injection of dye into the rete testis of isolated bull testis has allowed access to the seminiferous tubules [11, 21].
Germ cells for transplantation into pig testes can be autologous (i.e., from a pig into its contralateral testis), homologous (i.e., from a pig into an unrelated pig testis), or heterologous (e.g., from mouse into pig testes or vice versa [22]). The purpose of the present study was to establish a technique for germ cell transplantation into pig testes. This will provide a first step for generating transgenic farm animals through manipulation of the male germ line.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolated porcine testes (n = 89) were used to develop the technique for germ cell transplantation. After refinement of the injection technique, prepubertal boars underwent intra-rete testis transplantation of germ cells (n = 18 testes). Donor testis cells were collected fresh from the contralateral testis of 10-wk-old recipient boars (autologous), from the testes of unrelated 1- or 10-wk-old boars (homologous), or from mouse testes (heterologous). Porcine donor cells were labeled with a fluorescent marker, and the mouse cells carried a transgene. Recipient testes were either removed immediately following transplantation or after 1–4 wk and examined for the presence and localization of the fluorescent-labeled, transplanted porcine cells by fluorescence microscopy. Staining with 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal) was employed to stain the transgenic mouse cells [23]. Animals were handled and treated according to the guidelines of the Animal Care and Use Committee at the University of Pennsylvania.
Preparation of the Cell Suspension
For experiments using isolated testes, a mouse embryonic fibroblast cell line (STO) served as a source of cells to explore the feasibility of cell injection. Before injection into testes, cells were labeled by incubation with the Red Fluorescent Cell Linker (PKH26; Sigma, St. Louis, MO) to allow identification of the injected cells in the recipient testis at analysis. PKH26 staining was performed according to the manufacturer's instructions. Briefly, cells were washed in Dulbecco modified Eagle medium (DMEM; Gibco BRL, Grand Island, NY), centrifuged at 400 x g for 5 min into a loose pellet, and resuspended in Diluent C (supplied with the kit). Immediately before staining, 24–32 x 10-6 molar of PKH26 dye was prepared using Diluent C, gently mixed with the cells, and incubated at 25°C for 2–6 min. Staining was stopped by the addition of fetal bovine serum (HyClone, Logan, UT), and cells were subsequently washed three times in DMEM. After the last wash, cells were resuspended to a final concentration of 5–50 x 106 cells/ml. Addition of trypan blue to the cell suspensions served to monitor the immediate outcome of cell infusion.
For in vivo experiments, donor testis cells were harvested from testes obtained at castration of 1- or 10-wk-old boars using a sequential enzymatic digestion procedure as previously described [22, 24] with minor modifications. Briefly, the tunica albuginea and visible connective tissue were removed. The exposed seminiferous tubules were then dissociated with collagenase (2 mg/ml, Type IV; Sigma) in DMEM at 37°C for 20–40 min with occasional agitation, followed by the addition of hyaluronidase (1 mg/ml; Sigma) for 15–20 min. The tissue was then rinsed twice in Dulbecco phosphate-buffered saline without Ca2+ and further digested with 0.25% (w/v) trypsin and 1 mM EDTA at 37°C for 5–10 min. DNase I (7 mg/ml; Sigma) in DMEM was added as needed. Fetal bovine serum was added to stop enzymatic digestion. The resulting cell suspension was filtered through a nylon mesh (pore size, 60 µm; Tetko, Kansas City, MO). The cells were then collected by centrifugation at 500 x g for 5 min at 16°C, and the pellet was resuspended in DMEM. Before transplantation, donor cells were labeled with the fluorescent marker (as described above). Cells were resuspended in DMEM to a final concentration of 75 x 106 cells/ml. Viability of cells was greater than 90% as determined by trypan blue exclusion. The cell suspension was kept on ice until transplantation.
To explore the possibility of heterologous transfer of germ cells from mice into pigs, donor mouse testis cells were also prepared for transplantation. To facilitate the detection of mouse cells in the pig testes, these cells were obtained from the transgenic mouse line B6,129-TgR(ROSA26)26SOR (Jackson Laboratory, Bar Harbor, ME) designated ROSA26. These mice carry the Escherichia coli ß-galactosidase structural gene, and their cells can be stained blue with X-gal [23]. In adult ROSA26 testes, all stages of germ cell differentiation stain blue, making them readily identifiable in the recipient testes [7]. Cells were obtained from adult mice using a two-step digestion procedure to produce a suspension of testis cells for transplantation [6] at a final concentration of 100 x 106 cells/ml.
Surgical Procedures
Prepubertal, 9-wk-old, Yorkshire-cross boars were obtained from a local farm and housed for at least 1 wk before undergoing transplantations. Transplantation of the testis cells into the rete testes of recipients was performed under general anesthesia and aseptic surgical conditions. Before surgery, feed and water were withheld overnight. Anesthesia was induced by i.m. administration of xylazine (2 mg/kg), ketamine (5 mg/kg), and atropine (0.04 mg/kg). Anesthesia was maintained by inhalation of isoflurane. An auricular vein and artery were catheterized to administer i.v. fluids and to monitor blood pressure throughout the procedure. The animal was placed in lateral recumbency, and the scrotal, inguinal, and perianal regions were prepared for surgery. A linear incision was made lateral and parallel to the median raphe, and the testis enclosed in the parietal vaginal tunic was exposed. After cell transplantation, testes were returned to the scrotum, the scrotal skin closed, and the animals allowed to recover.
Analysis of the Recipient Testes
Recipient pigs were castrated at different times following transplantation, and testes were dissected and examined to assess the extent of intratubular infusion. To examine the presence and distribution of the fluorescent-stained, transplanted porcine cells, samples of seminiferous tubules were dispersed using fine forceps and examined under transmitted light and fluorescence microscopy (rhodamine filters) at 100–400x magnification. For detection of ROSA26 mouse cells, dispersed seminiferous tubules were fixed for 2 h in 4% (w/v) paraformaldehyde at 4°C, washed in buffer, incubated with X-gal for 10–16 h at 37°C, and examined under light microscopy [6, 25]. Representative samples of the testes were obtained and either snap-frozen in liquid nitrogen for cryosectioning or fixed in Bouin solution overnight and stored in 70% (v/v) ethanol until processing for routine histology. Images of seminiferous tubules and histological sections were documented by a computer-imaging system.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The initial experiments were designed to establish an effective procedure for introducing donor cells into recipient pig seminiferous tubules. The transplantation techniques routinely used in laboratory rodents are not directly applicable to pig testes, because the size and anatomy of the testes and seminiferous tubules in these species differ dramatically (Fig. 1). Seminiferous tubules in immature pigs not only have a much greater total tubular length than those of the mouse testis (
600 m/testis in the 3-mo-old pig vs. 2 m/testis in the mouse) but also have a smaller tubular diameter (60 µm in the 3-mo-old pig vs. 150 µm in the busulfan-treated mouse) (Fig. 1, B and C). Seminiferous tubules in the pig testis are highly convoluted as well (see Fig. 3D), and not all of them are accessible through the surface of the testis. Therefore, microinjections directly into individual seminiferous tubules of large mammalian testes, similar to a technique described for mouse testis, would be technically difficult.
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Access to the efferent ducts of pig testes requires their exposure by careful surgical dissection, because the efferent ducts in the large mammalian testes, unlike in the mouse, are concealed within the head of the epididymis. Cannulation of these efferent ducts would be difficult, because they are fragile, translucent, and hard to recognize in the surrounding fat and connective tissue. The rete testis of the immature boar, however, provides more practical access to the seminiferous tubules. The rete testis is comprised of a network of irregular, interconnecting channels formed in the mediastinum testis. In pigs, the mediastinum testis is placed axially, has a very distinct structure (see Fig. 3A), and can be distinguished from the surrounding parenchyma of the developing testis by ultrasonography. Therefore, we used ultrasound scanning to guide insertion of an injection needle onto the network of channels that comprise the rete. Scanning was performed with a 7.5-MHz linear transducer attached to a B-mode scanner (Ausonics Impact VFI; Universal Medical Systems, Bedford Hills, NY). Following initial trials, we found that the most effective procedure was to insert an i.v. catheter (20-gauge x 1-1/4''; Surflo; Terumo Medical Co., Elkton, MD) through the cauda epididymis and testis into the rete testis (Fig. 2). The position of the catheter was monitored from longitudinal and cross-sectional planes, and its direction was adjusted to ensure positioning in the center of the rete testis. Once the catheter was inserted, a small drop of a tissue adhesive solution (Close Liquid Suture; Braun Veterinary Care, Bethlehem, PA) was applied to anchor the catheter to the testis. After removing the steel needle, an infusion set containing the dye solution or cell suspension was connected to the catheter (Fig. 2).
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Following experiments to establish an effective injection approach, 89 isolated testes from prepubertal Yorkshire-cross boars castrated at 8–12 wk of age were used to determine the efficiency of infusing a cell suspension into the seminiferous tubules of testes in vitro. Presence of the infused dye or labeled cells in the seminiferous tubules was confirmed in 70 of 89 injected pig testes (79%). Figure 3A illustrates the ultrasonographic appearance of the rete testis and the insertion of the infusion catheter under ultrasound guidance in a pig testis. In most cases, gravity or a low hydrostatic pressure was sufficient to start and to maintain flow of the cell suspension into the rete testis. The rate of infusion was approximately 1 ml/min, and a volume of 3–8 ml could be injected into each testis. An example of the pattern and extent of filling of the seminiferous tubules injected with trypan blue is shown in Figure 3, B–D. We determined that a successful injection procedure could fill the rete and up to 50% of seminiferous tubules. In the pig testis, the seminiferous tubules are arranged into lobules that are connected to the rete testis. Therefore, on infusion of dye or a cell suspension, individual lobules will completely fill with the infused fluid, whereas adjacent tubules may not fill. Figure 3, E and F, illustrates the appearance of fluorescent-labeled cells in seminiferous tubules isolated after injection ex vivo. Cells were found in the lumen of seminiferous tubules as single cells or small clumps of cells. A higher degree of tubular filling can be obtained by forceful injection of larger volumes, but increased intratesticular pressure is likely to compromise testicular blood flow in vivo.
Injection of Cell Suspensions into Testes In Vivo
In the next series of experiments, the techniques perfected in vitro were applied to the injection of testes in anesthetized animals. Ultrasound scanning of the testis and the injection procedure described for the isolated testes were readily applicable in vivo. The average volume of the cell suspension injected into each testis in vivo was approximately 5 ml, with a flow rate of approximately 0.5–1 ml/min. Because of the intricate and complex rete testis cannulation procedure and the time required to complete the infusion, general anesthesia of the recipient animal was necessary. The surgical procedure to exteriorize the testis, followed by cannulation of the rete testis and transplantation of germ cells, required approximately 10–20 min per testis to complete. As expected, the intratesticular resistance to flow of the cell suspension was slightly higher in vivo than in vitro, causing a slower rate of infusion. To prevent damage to the tissue and leakage of cells into the interstitial compartment of the testis, repeated reentry of the catheter into the rete and gross readjustments of the needle position were avoided. Using the technique described, we were able to efficiently transfer dye or labeled cells into up to 50% of the seminiferous tubules of pig testes in vivo.
Following transplantation of porcine testis cells, fluorescent-labeled donor cells were identified in multiple seminiferous tubules in 10 of 11 recipient testes (Table 1 and Fig. 4). No differences were apparent in the extent of donor cell presence between autologous and homologous transplantations, indicating that donor cells from unrelated pigs are not rejected by the recipient testis. In contrast, transgenic ROSA26 mouse cells could be identified in substantial numbers in one pig testis examined immediately after transplantation (Table 1 and Fig. 4A), but only a very small number of these heterologous cells remained in one of two testes analyzed 1 wk after transplantation (Fig. 4B). No mouse cells were found in the second testis. Porcine donor cells transplanted into pig recipients remained for long periods in the tubules (up to 4 wk). Individual seminiferous tubules were filled with fluorescent-labeled cells, whereas adjacent stretches of tubules did not contain labeled cells (Fig. 4, C and D). The numbers of tubules filled with donor cells or the extent of tubular filling was similar for autologous or homologous transplantations and for testes analyzed 1, 2, or 4 wk after transplantation (Fig. 4, C–H).
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Recipient testes were evaluated histologically for evidence of tissue damage and immune reaction. Surprisingly, injection of recipient testes was associated with very little damage. The histological appearance of recipient testes at 4 wk after autologous, homologous, or heterologous transplantation was very similar to that of the donor testes and did not reveal tissue damage or cellular immune reaction (Fig. 5, A–D). The only exception was the presence of limited inflammatory changes at the site of needle insertion. In one case, local inflammation of the testis injected with mouse cells was seen, along with minor systemic signs of infection that responded to antibiotics. No local or systemic signs of immunologic reactions to the transplantations were detectable.
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The prepubertal porcine seminiferous tubules used to supply donor cells contain many Sertoli cells and large, primitive germ cells (Fig. 5A), and all of these cells were labeled by the marker dye. In the recipient tubule, the location of the fluorescent cells can indicate the success of injection and the degree of association with endogenous Sertoli cells and tubule basement membranes. The microscopic appearance of fluorescent-labeled donor cells in the seminiferous tubule of recipient pigs 1 and 4 wk after transplantation is illustrated in Figure 5, E–H. The location and appearance of donor cells suggested that some of these cells had integrated into the organizational structure of the tubule. Thus, round cells, believed to represent germ cells, that fluoresced with marker dye were identified as being associated with Sertoli cells and, sometimes, located at the basement membrane.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, the seminiferous tubules of prepubertal recipient boars contained endogenous germ cells. Although colonization of the recipient testes by donor cells can likely be improved if the germ cell niche has been depleted of endogenous germ cells, engraftment studies involving the hematopoietic system have shown that donor stem cells can effectively compete with host stem cells in wild-type recipient animals [26]. Efficiency of engraftment will depend on the ratio of donor to host stem cells. In our study, donor cells represented a mixed cell suspension, harvested from donor testes, containing germ cells, Sertoli cells, and contaminating peritubular cells. It would be advantageous to increase the concentration of germ cells, and particularly of stem cells, in the donor cell preparation. Toward this aim, future experiments will attempt enrichment of germ cells by selection for cell-surface markers predominantly found in spermatogonial stem cells, as previously described for the mouse [27].
Germ cell transplantation experiments in mice utilize syngeneic strains of mice as donor and recipient animals [5, 28]. In one report of germ cell transplantation in a monkey, cells from the contralateral testis served as the source of donor cells for autologous transplantation [11]. Immunologically compromised recipients have been used for germ cell transplantation between different species to avoid rejection of donor germ cells in the recipient testis [22, 29–31]. However, we demonstrated earlier that germ cell transplantation from mice to rats was successful in immunologically competent Sprague-Dawley rats treated with CTLA4Ig to induce immunological tolerance [10]. In the present study, we addressed the possibility of immune reactions to germ cell transplantation by harvesting donor germ cells from the contralateral testis of some recipients (i.e., autologous transfer) as well as from unrelated recipient animals (i.e., homologous transfer). Surprisingly, no difference was observed in retention of autologous or homologous donor cells throughout the observation period, and no evidence of an immune reaction was seen at histological examination of recipient testes. The pigs used in the present study were obtained from one commercial herd. Donors and recipients did not originate from the same litters, however, and the herd uses a sire-rotation system, resulting in a high degree of genetic diversity between individual litters. It appears that, similar to our observation in rats, donor and recipient animals do not need to be genetically identical or immunosuppressed. This finding is very important to the future practical applicability of germ cell transplantation in domestic animals. In contrast, mouse germ cells transplanted into porcine seminiferous tubules were apparently eliminated in this experiment within the first week after transplantation, without overt signs of a cellular immune reaction. Germ cells from a phylogenetically distant donor species likely are not recognized or are rejected by pig Sertoli cells and, therefore, are quickly removed from the tubules, whereas porcine cells are actively retained.
Choice and preparation of recipient animals is very important for the success of germ cell transfer [10]. Recently, it was reported that the immature mouse testis provides a more favorable microenvironment than the adult testis for transplanted stem cells [32]. In most previous reports, endogenous recipient spermatogenesis has been suppressed to allow for colonization of recipient tubules by donor germ cells and subsequent donor spermatogenesis. Mouse strains inherently devoid of spermatogenesis, due to a genetic defect, represent ideal recipient animals [8, 33]. Another widely used strategy is the destruction of endogenous spermatogenesis by pretreatment of recipient animals with busulfan after they have reached sexual maturity [5, 30]. In another study, suppression of endogenous spermatogenesis was achieved by treating rats with busulfan immediately after birth or while still in utero [10]. Preliminary results demonstrating a reduction in germ cell numbers after treatment of immature pigs with busulfan have been reported [21]. Alternatively, spermatogenesis can be obliterated by irradiation of the testes [34, 35]. However, it is of great advantage to the testicular health of the recipient animal if cytotoxic treatments or irradiation can be avoided [10]. Preparation of recipient animals by experimentally induced cryptorchidism or hyperthermic treatment have also been suggested [36].
In the present study, we used prepubertal pigs as recipient animals without any treatment to reduce endogenous germ cell number. In these animals, seminiferous tubules contain only spermatogonia and Sertoli cells in a single cell layer; therefore, donor cells do not need to negotiate multiple layers of spermatogenic cells to reach the basement membrane as a first step for successful colonization [33]. Infusion of donor cells into juvenile testes also meets less resistance compared to that in testes of adult animals [11]. Previous work in the mouse has shown that donor cells can successfully colonize recipient testes that have maintained some endogenous spermatogenesis [6]. We have shown previously that further improvement of donor-derived spermatogenesis in recipient testes can be achieved by treatment of recipient mice and rats with a GnRH-agonist, most likely by suppression of high intratesticular testosterone levels [10, 37, 38]. Future experiments could be directed toward improving the prepubertal boar as a recipient model by depletion of endogenous germ cells and hormonal modulation of the testicular microenvironment. Based on the results reported here, the prepubertal pig testis represents a very promising recipient environment for germ cell transplantation.
Our primary goal was to demonstrate that germ cell transplantation in boars is possible, and that donor cells would remain in recipient seminiferous tubules. Therefore, recipient pig testes were analyzed at 7-day intervals for up to 1 mo after transplantation. We did not expect to see donor-derived spermatogenesis, because the pigs were prepubertal. Instead, we were primarily interested in the position and maintenance of donor cells. From the present results, it appears that porcine donor testis cells survive in, and possibly colonize, the recipient pig testis. Labeled donor cells were present in many seminiferous tubules and were found toward the periphery of the tubules in proximity to the basement membrane. In future experiments, we will maintain recipient boars through puberty to investigate whether donor cells will initiate spermatogenesis. Assessment of the presence and proportion of donor-derived sperm production will require a permanent or long-term, traceable labeling system for donor cells. Fluorescent markers provide a rather short-term labeling tool. In rats, intracerebrally transplanted PKH26-labeled cells were detectable up to 4 mo after transplantation in the host brain [39]. We have also found that most of the cultured pig germ cells retain the red fluorescent label for 3–4 mo (unpublished results). The use of donor cells from transgenic or phenotypically distinguishable breeds of pigs will be investigated as well as the introduction of a genetic marker into donor cells before transplantation.
Current methods of producing transgenic animals are very inefficient, and introduction of new techniques is critical. Murine spermatogonia can be frozen, cultured, and transfected and still maintain the ability to colonize the mouse seminiferous tubules [40–42]. Testis cells of several mammalian species, including the pig, can also be successfully cryopreserved [22, 30, 31]. Porcine spermatogonia have been shown to survive in culture for several days [43], but transplantation of cultured porcine germ cells into mouse testes has not yet resulted in colonization [22]. Based on reports in the mouse, enrichment of donor cell preparations for stem cells can certainly be developed. The combination of germ cell transplantation in the pig with these methodologies will potentially provide a novel means of manipulating the boar germ line.
In conclusion, this study demonstrates that germ cell transplantation is feasible in prepubertal pigs and can be achieved by ultrasound-guided injection into the rete testis of more than 90% of recipient testes with 50% tubular filling. Autologous or homologous porcine donor-derived cells are present in the recipient testis for at least 1 mo with no major adverse effects. To our knowledge, this is the first report of successful germ cell transplantation between individual animals in a livestock species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the National Institute of Health (NICHD HD39641-01) and U.S. Department of Agriculture/NRI Competitive Grants Program (99-35205-8620). 
2 Correspondence: I. Dobrinski, Center for Animal Transgenesis and Germ Cell Research, Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348. FAX: 610 925 8121; dobrinsk{at}vet.upenn.edu
Accepted: August 9, 2001.
Received: July 2, 2001.
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