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research-article |
Center for Animal Transgenesis and Germ Cell Research, Department of Clinical Sciences, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania 19348
ABSTRACT
During mammalian development, morphogenesis of the testis requires the coordinated interplay of somatic cells to form seminiferous cords in which the primitive germ cells reside. These cords are the precursor of the functional male gonad and as such form the basis of male fertility. Cell migration during mammalian organogenesis and formation of complex tissues, such as the testis, are difficult to study in situ. Herein, we report extensive rearrangement of cells to regenerate complete functional testis tissue after implantation of isolated neonatal porcine testis cells under the skin of immunodeficient mice. Somatic cells and germ cells reorganized into structures that have remarkable morphologic and physiologic similarity to normal testis tissue, forming the endocrine and spermatogenic compartment of the testis. This unique in vivo system provides an accessible model for the study of testicular morphogenesis that could be especially useful in nonrodent species.
assisted reproductive technology, germ cells, morphogenesis, pig, spermatogenesis, testis
The factors controlling development of the mammalian testis are not completely understood. In the presence of the Y-linked gene Sry, the fetal gonad develops into a testis [1, 2]. This transformation requires testicular cord formation involving the aggregation of Sertoli cells and germ cells, followed by differentiation and polarization of Sertoli cells [3–5]. Preperitubular myoid cells from the mesonephros then migrate into the male gonad and surround the aggregating Sertoli and germ cells to form the cords.
Postnatal development of the testis involves proliferation and maturation of Sertoli cells to transform testicular cords into seminiferous tubules, followed by sequential divisions and differentiation of germ cells to generate sperm. While it has been demonstrated that Sertoli and peritubular myoid cells isolated from neonatal porcine testes could form testicular cords when transplanted under the kidney capsule of immunocompromised mice [6, 7], it was not known if these cords contained a functional niche for germline stem cells and, if so, whether germ cells would be able to home to this niche and initiate spermatogenesis. It was previously demonstrated that small fragments of testis tissue from immature rodents, domestic animals, and primates that were grafted subcutaneously into immunodeficient mice undergo complete development, resulting in the production of functional sperm [8–11]. The objective of this study was to investigate if ectopic grafting of isolated neonatal porcine testis cells would support formation of functional seminiferous cords capable of maturation and support of spermatogenesis.
Sixteen testes from four groups of male piglets (1–2 wk of age) were subjected to a two-step enzymatic digestion [12, 13]. After enzymatic treatment, cells were filtered through 60-µm mesh to remove any remaining cell clumps and to assure that a single-cell suspension was obtained. Within 1 h after cell isolation, aliquots of 50 x 106 cells in Dulbecco modified Eagle medium (DMEM) were concentrated by centrifugation at 1000 x g for 5 min to obtain cell pellets and were immediately transplanted to recipient mice.
Immunodeficient recipient mice (9 SCID and 3 NCr nude mice, 2-4 recipients per donor cell preparation) were anesthetized, castrated, and given four linear incisions (0.5 cm in length) into the dorsal skin on each side of the midline, spaced between the shoulder and the rump. A small pocket of fascia was made by blunt dissection under the skin, and a cell pellet was gently implanted into each pocket. The skin incision was closed with wound clips (Michel Clips, 7.5 mm; Miltex, York, PA). The care and use of research animals were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
Sample Recovery and Processing
Mice were randomly assigned to analysis time points between 4 days and 53 wk after transplantation. To monitor morphogenesis of testicular cords, mice were killed, the newly formed tissue grafts were visually identified, excised from the skin, measured, fixed in Bouin solution, and stored in 70% ethanol until processing for histology. The weight of seminal vesicles was documented as an indicator of bioactive testosterone produced by the newly formed tissue. Photographic documentation of the histology was performed to monitor the development of seminiferous tubules over time. To assess the degree of reconstitution of functional spermatogenic tissue, all cross sections of tubular structures in all formed grafts recovered at 30 wk or more after grafting were examined for the most advanced germ cell type present in the tubule.
Ubiquitin carboxyl-terminal esterase L1 (UCHL1) immunohistochemistry was used to identify gonocytes and spermatogonia [14] in the tissue samples, with some modifications [15]. Slides were treated with 3% hydrogen peroxide in distilled H2O to block the endogenous peroxidase activity and were incubated at room temperature in 1% BSA in PBS for 1 h to block the nonspecific antibody binding. Samples were incubated with primary antibody (PGP 9.5; Biogenesis, Exeter, NH) diluted to 1:1000 overnight at 4°C, followed by peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) as the secondary antibody (1:500). Peroxidase activity was detected using VIP (Vector Laboratories, Burlingame, CA). Hematoxylin (Vector Laboratories) was used to counterstain the sections.
To determine the percentages of different cell types present in the initial cell suspension before grafting, cells (500 000 cells in 0.5 ml of DMEM per chamber) were allowed to attach to poly-L-lysine-coated Permanox slide chambers for 2 h and were fixed in 2% paraformaldehyde for 30 min. Samples were blocked with 5% normal horse sera, avidin, and biotin and were incubated in the primary antibodies overnight at 4°C. Primary antibodies were against UCHL1 (1:250) to identify germ cells, 
-smooth muscle actin (1:1000; Sigma, St. Louis, MO) to identify peritubular myoid cells, GATA-binding protein 4 (GATA4) (1:20; Santa Cruz Biotechnology, Santa Cruz, CA) to identify Sertoli cells, and steroidogenic acute regulator (STAR) (1:1000; a gift from Jerome F. Strauss III) to identify Leydig cells. The secondary antibody was a universal (goat, mouse, and rabbit) biotinylated pan-specific antibody made in horse (Vector Laboratories) used at 6 µg/ml for 30 min, followed by horseradish peroxidase-streptavidin (Vector Laboratories) at 3 µg/ml for 30 min at room temperature. Visualization of specific immunolocalization was developed using the DAB kit (Vector Laboratories). At least 500 cells per chamber were counted per antibody and replicate.
To document target cell specificity of the antibodies used, indirect immunostaining was performed on Bouin-fixed sections from piglet testes that were 1–2 wk old after antigen retrieval (Antigen Unmasking Solution; Vector Laboratories). Staining was performed as already described except that sections were incubated in CAS Block (Zymed, San Francisco, CA) to block nonspecific binding and were incubated for 1 h at room temperature in the aforementioned antibodies at the following dilutions: -smooth muscle actin (1:500), GATA4 (1:100), and STAR (1:500). Immunohistochemical identification of germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells in donor piglet testis is shown in Figure 1.
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Single-cell suspensions were obtained from neonatal pig testes by enzymatic digestion [12, 13]. At this age in the pig, the testis tissue consists of seminiferous cords containing early germ cells (gonocytes) and immature Sertoli cells, surrounded by peritubular myoid cells and by interstitial Leydig cells (Figs. 2a and 3a). Although the initial enzymatic treatment is designed to remove most of the interstitial cells to yield mostly Sertoli cells and germ cells, some peritubular cells and Leydig cells will remain in the cell suspension. Based on immunocytochemistry for UCHL1, GATA4, -smooth-muscle actin, and STAR, the cell suspension contained a mean ± SD of 4.5% ± 1.6% germ cells, 46.8% ± 2.2% Sertoli cells, 18.6% ± 0.7% peritubular myoid cells, and 9.8% ± 2.3% Leydig cells, respectively (n = 5). Cells were filtered through 60-µm mesh to assure that a single-cell suspension was obtained; at this stage, porcine germ cells can be readily identified in the cell suspension based on size and morphology (Figs. 2b and 3b). Cells were concentrated by centrifugation, and the cell pellets were gently implanted under the back skin of immunodeficient recipient mice.
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Of the 96 cell pellets transplanted, 79 (82%) had given rise to easily identifiable grafts. The mean ± SD size of recovered grafts ranged from 23.5 ± 8.8 mm3 (n = 7) at 4 days after transplantation to 139 ± 27.3 mm3 (n = 11) at 30 wk after transplantation (range, 1–256 mm3; n = 79). At the time of transplantation, cells in the pellets were randomly distributed, with no organized pattern (Fig. 2c). As early as 4 days after transplantation, some cells were aligned in discernible structures (Fig. 2d), and some of these structures contained germ cells (Fig. 3c). By Day 7, cords had formed (Fig. 2e), and by 2 wk after grafting, these cords (Fig. 2f) resembled those of the neonatal pig testis (Fig. 2a), although fewer germ cells per cord were observed (Fig. 3d). While some germ cells were initially (
1 wk after transplantation) observed outside of the forming tubules, germ cells were only present inside the tubules at later time points. Sertoli cells seemed polarized, with their nuclei along the basal membrane of the tubules, and gonocytes were present in cords at 4 wk after transplantation (Figs. 2g and 3e). The organization of seminiferous cords had progressed further by 10 wk after grafting (Fig. 2h), and by 25 wk, germ cells had started to proliferate along the basement membrane of the tubules (Figs. 2i and 3f). By 30 wk after grafting, complete organized spermatogenesis occurred in the seminiferous tubules that had arisen from the grafted isolated cells (Fig. 2j). Spermatogenesis in grafts originating from single-cell pellets was morphologically identical to spermatogenesis occurring in a graft of intact testis tissue at 30 wk after grafting (shown for comparison in Fig. 2k). Complete morphologically normal spermatogenesis was also found in grafts recovered at 41 wk after cell transplantation (Fig. 2l), while grafts recovered from two recipients after 52 and 53 wk showed massive cellular infiltrates, indicating that a cellular reaction at the graft site had occurred in these recipient mice. Overall, in grafts collected at 30 wk or more after grafting, a mean ± SD of 11.3% ± 9.7% of seminiferous tubules contained elongated spermatids, and an additional 4.7% ± 5.3% contained round spermatids as the most advanced germ cell type (n = 4 mice, with 11 grafts). There were no apparent differences in tubular morphogenesis and development of spermatogenesis between SCID and nude recipient mice. In all recipient mice except one with massive cellular infiltrates, the weight of the seminal vesicles (>200 mg) was comparable to that in intact male mice [11], indicative of the production of bioactive testosterone by the cell grafts.
The present article documents the fascinating morphogenic capacity of isolated testis cells from neonatal pigs not only to form structures resembling seminiferous cords but also to create a functional germ cell niche and the microenvironment to support complete spermatogenesis. It has been shown previously that small fragments of neonatal testis tissue from a variety of mammalian species grafted underneath the skin of recipient mice led to complete xenogeneic spermatogenesis [8, 9, 16]. However, in the present study, neonatal pig testes were enzymatically dissociated and filtered through a 60-µm mesh to ensure that only single cells were isolated (Fig. 2b). Therefore, the cells underwent considerable rearrangements to form a tissue that resembled and functioned analogous to normal testis tissue. In the testis, the accurate spatial positioning of the different cell types is crucial to its normal functioning. The basement membrane of the seminiferous cords and tubules is shaped by the peritubular myoid cells and Sertoli cells. Germ cells are supported by Sertoli cells, and the location of the putative stem cell niche at the basement membrane seems to be affected by the spatial proximity to peritubular myoid cells. Sertoli cell tight junctions provide the compartmentalization between the basal compartment housing premeiotic germ cells and the adluminal compartment where the haploid germ cell population resides. Tubular morphogenesis from isolated neonatal porcine Sertoli cells and myoid cells has been described [6, 7], and the timing of cord formation in the present study was similar to that reported previously [7]. Recently, de novo formation of seminiferous tubules after short-term culture and subsequent xenografting of isolated rat testis cells has been reported; although germ cells were present in some tubules, germ cell differentiation did not occur [17]. It is possible that in vitro culture of testis cells before grafting negatively affected the germ cell differentiation potential compared with the complete spermatogenic differentiation achieved after grafting of freshly isolated cells used in the present study. In addition, species differences in the efficiency of spermatogenesis in testis xenografts could account for the different outcomes [8]. The present article significantly extends the previous observations of tubular morphogenesis by demonstrating that somatic and germ cells present in the testis cell suspension can interact in a controlled fashion to recreate functional testicular tissue.
Although the two-step enzymatic digestion protocol used in our study is designed to harvest a cell population enriched in intratubular Sertoli cells and germ cells [13], there seemed to be sufficient numbers of peritubular myoid cells and Leydig cells present to allow for formation of a tubular basement membrane and for production of testosterone at levels sufficient to support spermatogenesis. Although a large number of tubular structures were formed by the isolated cells after transplantation, not all tubules observed contained germ cells. This may be because of a comparatively higher loss of germ cells during the early stages of engrafting or because of a lower proliferative potential compared with Sertoli cells. A similar phenomenon of relative germ cell loss was observed after xenografting of bovine testicular tissue into mouse hosts [15]. Relative enrichment of the cell suspension for germ cells before grafting [18] may overcome this limitation. From the present study, it cannot be determined if localization of germ cells to the developing tubules represents an active homing process or relies on random incorporation into tubular structures during their formation after grafting.
In the fetal gonad, seminiferous cords are composed of germ cells and somatic cells, including epithelialized Sertoli cells, which are surrounded by a layer of peritubular myoid cells. In XY mice, the expression of Sry occurs between 10.5 and 12.5 days post coitum, inducing the migration of somatic cells from the mesonephros to the gonad [19–22]. However, Sry and its downstream targets are no longer expressed in the postnatal testis. Therefore, the ability of testicular somatic cells to form tubular structures capable of supporting spermatogenesis must be cell autonomous.
The present study provides testimony for the cell-inherent morphogenic potential of isolated testicular somatic cells and germ cells to recreate functional gonadal tissue. Recapitulation of testicular morphogenesis and sperm production has not been accomplished in vitro, to our knowledge. The present experiment focused on testicular morphogenesis; therefore, isolation of sperm from the tissue formed de novo was not attempted.
The system of using ectopic grafting to a mouse host provides an accessible in vivo culture system that will allow us to explore conditions necessary to recreate the essential cell associations to support spermatogenesis in vitro. Production of fertilization-competent sperm from immature isolated germ cells in vitro would be an invaluable tool to study the molecular and cellular control of spermatogenesis and to preserve male fertility. The in vivo culture system used in the present study provides a crucial first step in obtaining this thus-far elusive goal. Manipulation of specific pathways in germ cells or somatic cells before reaggregation will provide a controlled accessible system to study processes governing cell-cell interactions during testicular morphogenesis and spermatogenesis. While the approach of transplantation of germ cells into a germ cell-depleted testis of a recipient animal has already contributed significantly to our understanding of the control of spermatogenesis mainly in rodents [23], xenografting of isolated testis cells will provide a vastly more versatile and technically easier system that can be expected to be applicable to diverse mammalian species, including humans [24].
In conclusion, we describe a novel system for regeneration of functional testis tissue from dissociated porcine cells in mice. This remarkable system shows the persistence of cell migration elements and organogenesis potential during the postnatal period. This model is expected to be widely applicable to other species and to prove valuable in the study of intrinsic factors and mechanisms involved in testicular development and formation of the stem cell niche. It may also provide a unique tool to study the effects of manipulation of isolated testis cells in vitro before implantation on the development and function of the testis in vivo.
ACKNOWLEDGMENTS
We thank Janet Turpin and Terry Jordan for animal care and James Hayden for preparation of illustrations.
FOOTNOTES
3Current address: Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada S7N 5B4. 
1Supported by National Research Initiative Competitive Grant 2003-35203-13486 from the United States Department of Agriculture Cooperative State Research, Education, and Extension Service and by grant 5 R01 RR17359-05 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or the NIH.
Correspondence: 2 FAX: 610 925 8121; e-mail: dobrinsk{at}vet.upenn.edu
Received: 23 June 2006.
First decision: 20 July 2006.
Accepted: 15 September 2006.
REFERENCES
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  L. Arregui, R. Rathi, S. O Megee, A. Honaramooz, M. Gomendio, E. R S Roldan, and I. Dobrinski Xenografting of sheep testis tissue and isolated cells as a model for preservation of genetic material from endangered ungulates Reproduction, July 1, 2008; 136(1): 85 - 93. [Abstract] [Full Text] [PDF]
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 Z. Zhang, J. Hill, M. Holland, Y. Kurihara, and K. L. Loveland Bovine Sertoli Cells Colonize and Form Tubules in Murine Hosts Following Transplantation and Grafting Procedures J Androl, July 1, 2008; 29(4): 418 - 430. [Abstract] [Full Text] [PDF]
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 V. Keros, K. Hultenby, B. Borgstrom, M. Fridstrom, K. Jahnukainen, and O. Hovatta Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment Hum. Reprod., May 1, 2007; 22(5): 1384 - 1395. [Abstract] [Full Text] [PDF]
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 K. Kita, T. Watanabe, K. Ohsaka, H. Hayashi, Y. Kubota, Y. Nagashima, I. Aoki, H. Taniguchi, T. Noce, K. Inoue, et al. Production of Functional Spermatids from Mouse Germline Stem Cells in Ectopically Reconstituted Seminiferous Tubules Biol Reprod, February 1, 2007; 76(2): 211 - 217. [Abstract] [Full Text] [PDF]
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