1 Horizontal Medical Research Organization and 4 Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, 2 Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 and 3 The Institute of Physical and Chemical Research (RIKEN), Bioresource Center, Ibaraki 305-0074, Japan
5 To whom correspondence should be addressed. Email: tshinoha{at}virus.kyoto-u.ac.jp
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Abstract |
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Key words: infertility/niche/Sertoli cells/spermatogenesis/transplantation
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Introduction |
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Recently, we have shown the feasibility of transplanting Sertoli cells (Shinohara et al., 2003). Sertoli cells isolated from the donor testis could colonize the seminiferous tubules of the infertile recipient testis following microinjection. The colonizing activity was enhanced significantly when the recipient Sertoli cells were eliminated by cadmium treatment or when the donor Sertoli cells were prepared from immature testes in which the Sertoli cells were mitotically active. The dissociated donor cells reaggregated after transplantation to form a seminiferous tubule-like structure (minitubule), and spermatogenesis was detected in the recipient testis. Although these studies demonstrated spermatogenesis in the tubule-like aggregates, it has not been possible to induce the recovery of spermatogenesis from endogenous stem cells. This was an important goal, since it is necessary to use endogenous germ cells in the host to rescue infertility in clinical situations. Our present results provide evidence that a similar process of reorganization occurred in vivo with heterologous testis cells in the seminiferous tubules of the recipient mice.
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Materials and methods |
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For transplantation of the prepared testis cells, 3 µl of the donor cell suspensions were injected into the seminiferous tubules of the testes of each 46 week old Sl recipient mouse through the efferent duct (Ogawa et al., 1997
). The cells were injected at a concentration of 8.3x107 cells/ml. In some experiments, 4 weeks after the transplantation of cells from W mice, the recipient Sl mice were given an additional transplantation of 3x105 testis cells from another Sl mouse. The injections filled 7585% of the tubules in all recipient testes. The estimated concentration of Sertoli cells was
3.7x107 cells/ml. In both cases,
2.5x105 testis cells from W mice (approximately equivalent to one-half W testis) were microinjected into the testes of each Sl recipient.
A total of four experiments was performed. Eighteen Sl testes received only W testis cells, and six Sl testes received both W and additional Sl testis cells by injection into the seminiferous tubules. All of the animal experimentation protocols were approved by the Institutional Animal Care and Use Committee of Kyoto University.
Analysis
Two to 3 months after the transplantation, the mice were killed and the testes were fixed with 10% neutral-buffered formalin and processed for paraffin sectioning. Two histological sections were made from each recipient testis with an interval of 12 µm between sections. All sections were stained with haematoxylin and eosin. Each slide was viewed at a magnification of x400 to determine the extent of spermatogenesis. The number of tubule cross-sections with or without spermatogenesis, defined as the presence of multiple layers of spermatogenic cells in the entire circumference of the seminiferous tubule, was recorded for one histological section from each testis.
Microinsemination
Two recipients that had been transplanted with testis cells from W mice were killed at 6 months after transplantation. The testes were mechanically dissociated, and live spermatogenic cells were recovered by repeated pipetting of the seminiferous tubules. Round spermatids, as identified by a small round nucleus with a uniquely shaped chromatin mass, were microinjected into oocytes derived from C57BL/6xDBA/2 F1 mice. About 80% of oocytes survived the injection procedure irrespective of the day of experiment. Microinsemination was performed as previously described using round spermatids (Kimura and Yanagimachi, 1995). Embryos that reached the 2-cell stage after 24 h in culture were transferred to the oviducts of day 0.5 (the day following sterile mating) pseudopregnant (ICR) females. Live fetuses retrieved on day 19.5 were raised by lactating foster ICR mothers.
DNA analysis
The genomic DNA was isolated from tissue samples taken from the tail of each offspring using phenol/chloroform extraction, followed by ethanol precipitation. Ten micrograms of DNA were digested with EcoRI, and separated on 1.0% agarose gels. The DNA transfer and hybridization were performed as described previously (Kanatsu-Shinohara et al., 2002). An NciI-BglI fragment of the cDNA of the Sl gene (
430 bp, provided by Dr Y.Matsui) was used as a hybridization probe.
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Results |
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Generation of offspring by microinsemination
To confirm that the germ cells that developed in the host testes were fertile, we attempted to produce offspring from the Sl recipient mice using in vitro microinsemination, a technique commonly used to produce offspring from infertile animals and humans (Palermo et al., 1992; Kimura and Yanagimachi, 1995
). In two separate experiments, a total of 136 oocytes was constructed, using round spermatids (Figure 2I) and the 114 (83.8%) oocytes that developed to the 2-cell stage were transferred into the oviducts of Imperial Cancer Research (ICR) pseudopregnant recipients on the next day after microinsemination. The recipient females produced a total of four offspring, one male and three females (Figure 3A). These offspring had black coats with a white patch on the belly, which suggested that they were heterozygous Sl/+ or Sld/+ mice (Figure 3B). Southern blot analysis revealed that three of the live offspring were Sld/+ and one was Sl/+, indicating that the Sl and Sld haplotypes of the Sl/Sld germ cells had segregated during meiosis (Figure 3C). Breeding of the Sl/+ male and Sld/+ females produced completely white Sl/Sld offspring. These results indicated that the spermatogenic cells that developed in the testes of Sl mice after transplantation of W testis cells originated from endogenous Sl germ cells and that the offspring produced from these cells were fertile.
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Discussion |
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Several methods have been used to manipulate the microenvironment of spermatogonial stem cells. We and others have shown that virus particles microinjected into the seminiferous tubules can transduce Sertoli cells in situ (Ikawa et al., 2002; Kanatsu-Shinohara et al., 2002
). The transduction of Sl Sertoli cells in Sl mice with the wild-type Sl gene induced spermatogenesis from Sl stem cells, and normal offspring were produced. Sertoli cells can also be transduced by microinjection of DNA and subsequent electroporation of entire testis (Yomogida et al., 2002
). Although these approaches are useful in that they allow very efficient gene expression in Sertoli cells, there are at least two major drawbacks. First, the genetic rescue of defective Sertoli cell functions requires the identification of the responsible genes, which are currently not well characterized in humans. Second, the transduction of Sertoli cells is accompanied by the potential transduction of the germline cells. Recent studies have shown that virus vectors can be transmitted to offspring after the infection of stem cells (Nagano et al., 2001
; Kent Hamra et al., 2002
; Kanatsu-Shinohara et al., 2004
). These animal studies strongly suggest the potential risk of germline transduction, which precludes the use of virus vectors in clinical situations.
Sertoli cell transplantation provides an alternative method to overcome these problems. The success of the Sertoli transplantation technique probably depends on the unique ability of testicular cells to conserve their histogenic capacities. It was originally shown >25 years ago that dissociated newborn testis cells could reorganize in vitro into histotypic structures under conditions of slow rotation (Ohno et al., 1978; Zenzes et al., 1978
). The reconstituted tubule-like structure contained germ cells, Sertoli cells and myoid cells, among others. The morphogenic activity is suppressed at the onset of puberty, but it can be induced if germ cells are removed (Zenzes and Engel, 1981
). In our previous study, we extended this observation by demonstrating that dissociated testis cells reorganize into tubule-like structures in vivo after transplantation into seminiferous tubules (Shinohara et al., 2003
). The critical finding of this in vivo study, in contrast to the previous in vitro studies, was the induction of spermatogenesis in the tubule-like aggregates; whereas spermatogenesis could not be induced in the reaggregated tubules in vitro, spermatogenesis occurred efficiently in vivo. The normal Sertoli cells from the W mice mingled with the defective Sl Sertoli cells of the recipients to reform tubule structures and provide membrane-bound wild-type Sl factor to the germ cells of the Sl mice to permit spermatogenesis, indicating the considerable flexibility of the seminiferous tubule.
The most striking result of the present study was the production of offspring from the infertile Sl mice. The efficiency of offspring production was very low after microinsemination, which suggests that not all the germ cells may have undergone normal development. Another study also reported a lower development rate of embryos in microinsemination experiments using germ cells that developed after germ cell transplantation (Goossens et al., 2003). Nevertheless, our result clearly demonstrates that at least some of the germ cells produced after Sertoli cell transplantation were functionally normal. Thus, the transplantation technique not only provides a method for basic studies on spermatogenesis but may also provide a new method for the treatment of male infertility. Currently, very little is known about the genes responsible for human male infertility, and no effective treatments are available for infertile men with potential Sertoli cell defects. The testes of Sl mice have histological features similar to those observed in the clinical condition known as Sertoli cell-only syndrome, which is found in 515% of infertile men (Del Castillo et al., 1947
). Although germ cell transplantation may be useful in some cases, the technique is inevitably accompanied by ethical problems and is only useful for infertile men with germ cell defects. Nevertheless, given our results, it may be anticipated that healthy Sertoli cells from a donor testis could be transplanted into a heterologous recipient who has defective Sertoli cells to induce spermatogenesis from the normal stem cells of the patient. The present technique may also be used to achieve cross-species germ cell transplantation. When germ cells from phylogenetically distant donors, such as from primate or human, are transplanted into immunodeficient mouse recipients, their differentiation arrests at the stage of spermatogonial proliferation, possibly owing to the incompatibilities in the microenvironments (Nagano et al., 2001
, 2002
). The transplantation of Sertoli cells may enable differentiation of these donors in mouse surrogates, thereby providing a biological assay system to characterize human spermatogenesis.
While our results show the remarkable flexibility of the spermatogenic system, improving the efficiency of spermatogenesis is the next important step required to facilitate a wide range of studies using Sertoli cell transplantation. Although premeiotic germ cells continued to proliferate for several months and spermatogenesis occurred up to the round spermatid stage, it was less efficient after the elongated spermatid stage. One possible explanation is that the transplanted Sertoli cells failed to reconstruct the spatially coordinated, cyclic gene expression pattern of normal seminiferous tubules. Alternatively, the limited area of colonization established by the donor Sertoli cells could also influence germ cell differentiation. A previous transplantation study showed that extensive proliferation of the spermatogonia population occurred in small colonies in the recipient tubules, but that meiotic differentiation occurred exclusively in colonies longer than 1 mm (Nagano et al., 1999). However, in this study, Sertoli cell colonization occurs in a limited area without appropriate host conditioning. Endogenous Sertoli cells can be eliminated by treating the seminiferous tubules with cadmium; donor Sertoli cells can colonize extensive areas after such treatment (Shinohara et al., 2003
). Cadmium treatment, however, also removes the endogenous germ cells and is thus not appropriate in the present case (Shinohara et al., 2003
). It will be important to develop other methods to enhance Sertoli cell colonization without affecting the endogenous germ cell population. The resolution of these problems will not only improve the efficiency of Sertoli cell transplantation techniques but also increase our understanding of spermatogenesis.
Several new therapeutic approaches can be envisaged for human spermatogenic failure. A stem cell transplantation technique is available that allows offspring production from fresh or cryopreserved spermatogonial stem cells (Avarbock et al., 1996; Ogawa et al., 2000
; Kanatsu-Shinohara et al., 2003a
). This technique will be particularly useful for restoring fertility to those who become infertile after malignancy therapy by chemicals or radiation. Although no method for fertility protection is currently available for prepubertal boys who do not have sperm, stem cell transplantation will provide a method to recover their fertility, since spermatogenesis occurs by transplantation of spermatogonial stem cells even from immature donors (Shinohara et al., 2001
). Recent development of spermatogonial stem cell culture techniques enables in vitro expansion of stem cells from a small biopsy sample for autologous transplantation (Kanatsu-Shinohara et al., 2003b
, 2004
; Kubota et al., 2004
; Ogawa et al., 2004
). Future developments might even allow correction of defective genes at the spermatogonial stem cell level, and methods for genetic manipulations are being developed (Kanatsu-Shinohara et al., 2005
). In contrast, the Sertoli cell transplantation technique will be used to correct spermatogenic failure due to defects in Sertoli cells. Unlike stem cell transplantation, defective Sertoli cells can be replaced with healthy Sertoli cells from heterologous donors with less ethical restriction. As the method to culture Sertoli cells develops, it will be possible to correct the defect in Sertoli cells to be used for autologous transplantation in vitro. Our successful production of offspring from infertile Sl mice demonstrates the usefulness of the Sertoli cell transplantation technique, and indicates a promising opportunity to develop a new strategy for the treatment of human male infertility.
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Acknowledgements |
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Submitted on March 23, 2005; resubmitted on April 25, 2005; accepted on April 27, 2005.
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