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research-article |
Departments of Urology,3 Molecular Pathology,4 and Regenerative Medicine,5 Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan
Mitsubishi Kagaku Institute of Life Science,6 Tokyo 194-8511, Japan
RIKEN,7 Bioresource Center, Ibaraki 305-0074, Japan
Department of Science for Laboratory Animal Experimentation,8 Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
ABSTRACT
Testicular germ cell transplantation into the seminiferous tubules is at present the only way to induce spermatogenesis from a given source of spermatogonial stem cells. Here we show an alternative method that harnesses the self-organizing ability of testicular somatic cells. The testicular cells of embryonic or neonatal mice or rats and of newborn pigs were dissociated into single cells. Each of them reorganized into a tubular structure following implantation into the subcutis of immunodeficient mice. When mouse germline stem (GS) cells derived from spermatogonial stem cells and expanded in culture were intermingled with testicular cells of rodents, they were integrated in the reconstituted tubules and differentiated beyond meiosis into spermatids. Normal offspring were produced by the microinjection of those spermatids into oocytes. This method could be applicable to various mammalian species and useful for producing functional gametes from GS cells in a xenoectopic environment.
assisted reproductive technology, gametogenesis, meiosis, spermatogenesis, spermatogonial stem cell, testis
Spermatogenesis is a complex cell differentiation process in which spermatogonial stem cells develop into spermatozoa, which takes 35 days in mice and 74 days in humans [1]. The spermatogonial stem cells maintain their number by self-renewing division, which is the fundamental mechanism of the lifelong process of spermatogenesis. Both the differentiation and the self-renewal of stem cells take place in the seminiferous tubules, which consist of Sertoli cells, peritubular myoid cells, and germ cells, as well as extracellular matrix components in the basement membrane. Therefore, the seminiferous tubule is the structure that provides the microenvironment necessary for mammalian spermatogenesis. In order to elucidate the mechanism of and the conditions for spermatogenesis, researchers have long attempted to recapitulate the process in vitro [2, 3]. Several reports claimed the completion of meiosis from spermatocytes to spermatids [4, 5]. Nonetheless, it is not possible yet to promote meiosis from spermatogonial stem cells to produce haploid gametes in vitro.
Since its technical development in 1994, transplantation into the lumen of seminiferous tubules has been the only method of inducing complete spermatogenesis from spermatogonial stem cells [6, 7]. The transplantation technique had been applied to different animal species, including livestock and monkeys [8]. However, the procedure has to be modified for individual animal species depending on the histoanatomical structure of their testicles, and the efficiency was quite low compared with the mouse experiment. Xenogeneic transplantation of testicular cells from different donor species to recipient immunodeficient mice was explored but proved to be successful only from rats and hamsters [9, 10]. In addition, to achieve satisfactory colonization of donor cells, the recipient animals have to be pretreated with the cytoablasive drug busulfan or irradiation, which could be detrimental to the physical condition of the recipients [11].
In our search for alternative ways to induce spermatogenesis from spermatogonial stem cells, we used the reorganizing ability of immature testicular cells to produce a supportive microenvironment for the spermatogenesis. In fact, reconstruction of testicular structures both in vivo and in vitro was reported in several mammalian species, including mice, rats, and pigs [12–16]. Starting the present study, we postulated that those reconstituted seminiferous tubules could serve as a microenvironment for spermatogenesis even at an ectopic site.
C57BL/6 and ICR mice, nude mice (BALB/cAJcl-nu/nu), Scid mice (C.B-17/lcr-scid/scidJcl), and Sprague Dawley rats were purchased from Clea (Tokyo, Japan). Neonatal pig testes were a gift from a breeding farm. Two lines of transgenic mice (C57BL/6 genetic background), one carrying the pCXN-eGFP transgene [17] (pCXN-GFP transgenic mouse) and the other carrying the haspin-promoter-eGFP transgene (haspin-GFP transgenic mouse, line name BiPRO-2) [18], were used to produce germline stem (GS) cell lines. All of the experiments with animals conformed to the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation (Research Institute for Yokohama City University, Yokohama, Japan).
To establish green fluorescent protein (GFP)-expressing GS cell lines, male pCXN-GFP transgenic mice and male haspin-GFP transgenic mice were mated with wild-type ICR females to produce F1 pups. The testes of F1 pups were dissected out and used as a source of GS cells. As the pCXN-GFP transgenic mice express GFP in every cell type, including germ cells, the established GS cells (GFP-GS cells) also expressed GFP [17]. The GS cells (haspin-GS cells) derived from F1 pups of haspin-GFP transgenic males and ICR females expressed GFP specifically in haploid germ cells [18]. As both GS cell lines were derived from F1 pup testes, they were hemizygous for each GFP transgene. The derivation of GS cells was described elsewhere [19, 20]. The cells were cultured for more than 3 mo and passaged at least eight times before being used for this experiment.
Transplantation of Testicular Cell Suspension
Donor cells for transplantation were prepared from testes of mice and rats in the embryonic (15.5~19.5 days postcoitum [dpc]) and neonatal (1–2 days postpartum [dpp]) periods, and from testes of pigs within a day after delivery. The testes were decapsulated, and seminiferous cords or tubules were exposed. They were digested by a two-step enzymatic treatment [14]. Briefly, testicular tissue was treated with digestion solution I containing collagenase type II (2 mg/ml) and DNAse (10 µg/ml) in PBS at 37°C for 20 min. The specimens were centrifuged and then treated with digestion solution II, which contained collagenase type II (2 mg/ml), DNAse (10 µg/ml), and hyaluronidase (2 mg/ml) in Dulbecco modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA) at 37°C for 5 min. The cell suspension was washed twice with PBS and filtered through mesh with a pore size of 40 µm. Average yield of the cell harvests were 0.3~0.6 x 106 cells from a neonatal mouse and 0.5~1.0 x 106 cells from a neonatal rat. One or two litters of mice or rats were used in a single experiment. The cell suspension was mixed with the same volume of growth factor-reduced Matrigel Matrix (MGM; Becton Dickinson Labware, Bedford, MA) on ice, with a final concentration of 10~30 x 106 cells/ml. Male BALB/c nude or Scid mice aged 4~8 wk were anesthetized with an i.p. injection of a ketamine-xylazine cocktail. The MGM cell suspension (0.05 ml) was injected subcutaneously using a 26-G needle into the dorsal region of the mice. Each animal was injected at two to four sites. The mice were then castrated through a lower midline abdominal incision. GS cells were harvested by the vigorous pipetting of culture dishes and were mixed with the digested testicular cells. The cell number ratio of GS cells to testicular cells was 1:3~5. The mixed cell suspension then was added to the same amount of MGM and injected into the back of immunodeficient mice in the manner described above.
Gross and Histological Examination
Each reorganized tissue was analyzed at 4–10 wk after the transplantation. Subcutaneous specimens were dissected out, and their diameter was measured. They were observed under a stereomicroscope with transillumination from underneath to identify tubular or cystic structures of the cell mass. For the observation of GS cells, specimens were also observed under a stereomicroscope equipped with an excitation light for GFP (Olympus SZX12; Olympus, Tokyo, Japan) to identify GFP-positive GS cells and their daughter cells. The specimens were then fixed in Bouin solution at room temperature overnight. After being rinsed in 70% ethanol for 1 to 2 days, the tissues were embedded in paraffin. Histological 3-µm-thick serial cross sections were cut at 50-µm intervals and stained with hematoxylin and eosin. For the identification of GFP-expressing cells in histological sections, immunohistochemical staining was performed. The sectioned samples were incubated with anti-GFP polyclonal antibody (1:200; Molecular Probes, Eugene, OR) overnight at 4°C. For immunodetection, DAKO ENVISION kit (DAKO Japan, Kyoto, Japan) was used according to the manufacturer's instructions. The color was developed using a diaminobenthidine reaction.
Reorganized tissues in the subcutis of nude mice were cut out, and foci of GFP-positive cell assemblies were dissected out under a stereomicroscope. GFP-positive round spermatids were collected and injected into the ooplasm of wild-type mature oocytes of B6D2F1 using a Piezo-driven micromanipulator [21, 22]. Fertilized oocytes were cultured for 24 hours, and two-cell embryos were transferred into the oviducts of pseudopregnant ICR females. Live fetuses retrieved on Day 19.5 were raised by lactating foster ICR dams.
The genomic DNA was extracted from the placenta of each newborn mouse with a DNeasy Tissue kit (QIAGEN, Hilden, Germany). The DNA samples (100 ng/5 µl) were added to 45 µl reaction mixture containing 0.5 µM of each enhanced green fluorescent protein (EGFP)-specific primer, 160 µM deoxyribonucleotide triphosphate (dNTP) mixture, 1x GeneAmp PCR buffer, and 2.5 U AmpliTaq Gold (Applied Biosystems, Foster City, CA). EGFP-specific primers: 5'-TACGGCAAGCTGACCCTGAA-3' and 5'-TGTGATCGCGCTTCTCGTTG-3'. The reaction profile was: 35 cycles of denaturation at 96°C for 60 sec, annealing at 60°C for 60 sec, and extension at 72°C for 60 sec.
Ectopic Reconstitution of Seminiferous Tubules
The testicular tissues were harvested from mice and rats in the embryonic (15.5~19.5 dpc) and neonatal (1~2 dpp) periods. A single-cell suspension made from the testicular tissue and containing 0.5~1.5 x 106 cells was mixed with MGM and then injected subcutaneously into the backs of nude mice. The site of the injection instantly bulged with the cell suspension but flatted out the next day. Within 2 wk, the injected sites bulged again. Their size gradually increased up to around 10 mm in diameter and 3 mm in height during the subsequent observation period. At 4 to 6 wk, the mice were killed, and the subcutaneous cellular masses were dissected. These masses often showed evident neovascularization (Fig. 1a). Stereomicroscopic observation with transillumination from underneath revealed tubular or cystic structures in the dissected specimens (Fig. 1b). Tubular structures were observed in 17 of 24 rat-testis-cell transplants and 17 of 31 mouse-testis-cell transplants. Histological examination confirmed the presence of quasiseminiferous tubular structures in transplants of both rats and mice, some of which could not be distinguished from the original seminiferous tubules (Fig. 1, c and d). Sertoli cells were located regularly on the basement membrane, where they formed tubules, whereas Leydig cells were located between the tubules (Fig. 1c). Although most of those tubules were composed of Sertoli cells only (Fig. 1d), we occasionally observed tubules with germ cells, mostly spermatogonia. A few tubules contained meiotic germ cells (Fig. 1c). To confirm the reorganizational ability of testicular somatic cells in other species, we tested pig testicular cells (Fig. 2a). The cells from testes of newborn pigs were enzymatically dissociated and filtered. They were treated in the same manner as the rodent testicular cells and were injected into the backs of nude and Scid mice. At 3–7 wk after the injection, the cell transplants enlarged in size up to 5 mm in diameter (Fig. 2b). Of nine transplants in five recipient mice, we observed the reorganization of tubules histologically in three transplants (Fig. 2c). Their architecture was quite similar to that of the seminiferous tubules of testicular tissue fragments grafted under the skin of the same mice as a control (Fig. 2d). This result reinforced the notion that immature testicular somatic cells of many, if not all, mammalian species have a general ability to reconstitute seminiferous tubules under xenoectopic conditions.
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Integration of GS Cells into Reconstituted Tubules and Spermatogenesis
We then tested whether spermatogenic stem cells of a different source mixed with testicular somatic cells would be integrated into the reconstituted seminiferous tubules. It was reported recently that spermatogonial stem cells harvested from mouse testes can be propagated exponentially in vitro [19, 20, 23]. These cultured spermatogonial stem cells were termed germline stem (GS) cells [19]. For the subsequent experiments, we used GFP-expressing GS cells (GFP-GS cells), which were established from the testes of pCXT-GFP transgenic mice [17]. The GFP-GS cells were mixed with dissociated testicular cells from embryonic or neonatal mice or rats in MGM and then injected into the backs of nude mice. In this case again, the injected cell mixtures produced bulges during the observation period. In several prominent cases, they grew into what appeared to be a single organ with its own blood supply (Fig. 3a). After 4~9 wk, the transplanted cells formed a mass 5~7 mm in diameter (Table 1) comprising tubular or cystic structures. With GFP excitation light, GS cells were observed to lie along the rim of the tubules at 4 wk, indicating that they had settled in their designated niche, namely, in the basal compartment of the seminiferous tubules (Fig. 3b). The tubular structures, the diameters of which were 100~150 µm, showed a convoluted arrangement with occasional branching. Histological observation confirmed that some reconstituted tubules had integrated germ cells (Fig. 3c). These germ cells either were intrinsic to the testicular tissue or were derived from GS cells. Among the transplant specimens that contained germ cells—10 in mouse and 24 in rat (Table 1)—tubules harboring germ cells were observed in 23.2% (6.0%~51.7%) and 12.9% (2.0%~44.5%) of all tubules counted in histological sections in mice and rats, respectively. The immunohistochemical analysis using four sections of reconstituted samples with anti-GFP antibody showed that of 57 reconstituted tubules with germ cells, 33 tubules (58%) harbored GFP-positive germ cells (derived from GS cells; Fig. 3d). Spermatogenesis up to or beyond the meiotic phase was observed occasionally (Table 1). Some of the meiotic and postmeiotic cells appeared to be derived from the original germ cells of the mouse and rat testes cells (Fig. 3, e and f). Also, there were foci at which spermatogenesis had progressed to generate round spermatids, which originated from GS cells as confirmed by immunohistochemical analysis (Fig. 3, g and h).
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Production of Fertilization-Competent Gametes
To further verify the spermatogenic differentiation of GS cells in the reconstituted tubules, we used another line of GS cells (haspin-GS cells) derived from haspin-GFP transgenic mice (BiPRO-2; unpublished results). This GFP is expressed specifically in haploid germ cells due to the haspin-promoter sequence [18]. The haspin-GS cells were mixed with embryonic or neonatal testicular cells of mice and rats to inject them into the subcutis of nude mice. After 7–10 wk, groups of cells expressing haspin-GFP were observed sporadically in the reconstituted tubules (Fig. 4, a and b). The haspin-GFP-positive cells, namely, spermatids derived from mouse GS cells, were recognized in 3 of 14 rat-testis-cell transplants and in 4 of 13 mouse-testis-cell transplants. In order to finally test their fertilizing ability, GFP-positive round spermatids released from tubules of mouse-testis-cell transplants were injected into mature oocytes (Fig. 4, c and d). Of 69 embryos thus constructed, 59 (86%) progressed to the two-cell stage by 24 h in culture. They were transferred to four pseudopregnant females. A total of 10 pups (17% per transfer, four males and six females) were produced from three of four recipients (two, four, and four pups each; Fig. 4e). The donor origin (haspin-GS cell) was confirmed by detecting the GFP gene in 4 of 10 pups from their placental DNA by PCR analysis (Fig. 4f). As haspin-GS cells were hemizygous for GFP gene, six pups that were not positive for GFP also could have originated from GS cell-derived spermatids. These results demonstrated that the spermatids produced in the ectopically reconstituted tubules were competent for fertilization to create the progeny.
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There have been several reports that embryonic male gonads of rodents and pigs can reconstitute seminiferous tubules in vitro and in vivo after being dissociated into single cells [12–16]. However, there have been few reports documenting the behavior of endogenous germ cells of the original gonads. A recent study has shown that when germ cells derived from embryonic stem cells in vitro were aggregated with embryonic testicular cells (13.5 dpc) and transplanted into the interstitium of the adult mouse testis, the organization of seminiferous tubules took place, and spermatogenesis was completed [24]. In the present study, we used testicular tissues at more developed stages than embryonic 13.5 dpc, GS cells instead of embryonic stem cell-derived germ cells, and subcutaneous instead of intratesticular space. With such arrangements, we showed that spermatogenesis of mice and rats can take place in seminiferous tubules reconstituted subcutaneously from dissociated embryonic or neonatal testicular cells. These reconstituted seminiferous tubules integrated GS cells and supported their spermatogenesis to produce haploid gametes by which normal progeny were delivered.
This method of reconstitution provides unique opportunities for the study of spermatogenesis. First, the reconstitution of seminiferous tubules from neonatal testicular tissue of a variety of mammalian species would be possible. As spermatogenesis in testicular tissue fragments of different mammalian species proceeded in the subcutis of castrated nude mice [25], spermatogenesis of such species also could be possible in ectopically reconstituted seminiferous tubules. In fact, a recent study showed both reconstitution of seminiferous tubules from dissociated immature porcine testicular cells in the subcutis of immunodeficient mice and complete spermatogenesis of germ cells in the tubules [26]. These data support the concept that the subcutaneous reconstitution methods can be applicable to various mammalian species for producing ectopic spermatogenesis from spermatogonial stem cells, or GS cells when available. Second, reconstitution may be possible with a combination of two sources of testicular cells, such as different species or different genetic backgrounds, with one having particular mutations, for instance. Third, by combining cell sorting technologies, certain types of testicular somatic cells, such as Leydig cells, could be omitted from and/or added to the cell mixture to examine their effects on the reconstitution and spermatogenesis within. Use of the subcutaneous space, instead of renal subcapsular or intratesticular spaces, for reconstitution has additional advantages. As the implanted sites are easily identified over the skin, supplemental treatments such as hormone injections would be possible at the sites at any time during the experiment. These experimental manipulations applicable with the reconstitution method would elucidate the cellular and molecular mechanism of spermatogenesis, not only in rodents but in other species as well.
In vitro expansion of murine spermatogonial stem cells [19, 20, 23] and transgenesis through them [27] is a new strategy for making transgenic mice and possibly other mammalian species. In fact, it was reported recently that spermatogonial stem cells of rats also could proliferate in culture, and gene manipulation is possible [28, 29]. In order to establish GS cells in other species, it is necessary to develop an assay system for them to verify their functional characteristics. The reconstitution method would be a convenient and reliable assay system for possible GS cells from a variety of mammalian species. It also could serve for the production of gametes from gene-manipulated GS cells, which would then be useful for the production of gene-manipulated animals. In addition, the reconstitution methods also could be used in future clinical settings to preserve the fertility of males or to treat infertile male patients. With the prevalence of assisted reproduction technologies, which are represented by microinsemination techniques, one needs very few sperm or spermatids to produce offspring. When no sperm can be found in the testicles of azoospermic patients, harvesting spermatogonia from the testicle to nurture them into sperm ex vivo would be the next challenge for reproductive medicine. Our result is a substantial step toward such goals.
ACKNOWLEDGMENTS
We acknowledge the technical assistance of K. Katagiri and M. Taniguchi.
FOOTNOTES
1Supported in part by the 2005 Strategic Research Project grant K17002 from Yokohama City University, Japan. 
Correspondence: 2Takehiko Ogawa, Department of Urology, Yokohama City University Graduate School of Medicine, 3–9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. FAX: 81 45 786 5775; e-mail: ogawa{at}med.yokohama-cu.ac.jp
Received: 30 August 2006.
First decision: 14 September 2006.
Accepted: 5 October 2006.
REFERENCES
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