Germ cell transplantation from large domestic animals into mouse testes

Donor-derived spermatogenesis after spermatogonial transplantation to recipient animals could serve as a novel approach to manipulate the male germ line in species where current methods of genetic modification are still inefficient. The objective of the present study was to investigate germ cell transplantation from boars, bulls, and stallions, which are economically important domestic animals, to mouse recipients. Donor testis cells (fresh, cryopreserved, or cultured for 1 month) were transplanted into testes of immunodeficient recipient mice in which endogenous spermatogenesis had been destroyed. Recipient testes were analyzed from 1 to > 12 months after transplantation for the presence of donor germ cells by donor-specific immunohistochemistry. Donor cells were present in most recipient testes with species-dependent differences in pattern and extent of colonization. Porcine donor germ cells formed chains and networks of round cells connected by intercellular bridges but later stages of donor-derived spermatogenesis were not observed. Transplanted bovine testis cells initially appeared similar but then developed predominantly into fibrous tissue within recipient seminiferous tubules. Few equine germ cells proliferated in mouse testes with no obvious difference between cells recovered from a scrotal or a cryptorchid donor testis. The pattern of colonization after transplantation of cultured cells did not resemble spermatogonial proliferation. These results indicate that fresh or cryopreserved germ cells from large animals can colonize the mouse testis but do not differentiate beyond the stage of spermatogonial expansion. Species-specific differences in the compatibility of large animal donors and mouse recipients were detected which cannot be predicted solely on the basis of phylogenetic distance between donor and recipient species.     

Intraperitoneal Germ Cell Transplantation in the Nile Tilapia Oreochromis niloticus

Germ cell transplantation offers promising applications in finfish aquaculture and the preservation of endangered species. Here, we describe an intraperitoneal spermatogonia transplantation procedure in the Nile tilapia Oreochromis niloticus. Through histological analysis of early gonad development, we first determined the best suitable stage at which exogenous germ cells should be transplanted into the recipients. For the transplantation procedure, donor testes from a transgenic Nile tilapia strain carrying the medaka β-actin/enhanced green fluorescent protein (EGFP) gene were subjected to enzymatic dissociation. These testicular cells were then stained with PKH26 and microinjected into the peritoneal cavity of the recipient fish. To confirm colonization of the donor-derived germ cells, the recipient gonads were examined by fluorescent and confocal microscopy. PKH26-labeled cells exhibiting typical spermatogonial morphology were incorporated into the recipient gonads and were not rejected within 22 days posttransplantation. Long-term survival of transgenic donor-derived germ cells was then verified in the gonads of 5-month-old recipients and in the milt and vitelogenic oocytes of 1-year-old recipients, by means of PCR using EGFP-specific primers. EGFP-positive milt from adult male recipients was used to fertilize non-transgenic oocytes and produced transgenic offspring expressing the donor-derived phenotype. These results imply that long-term survival, proliferation, and differentiation of the donor-derived spermatogonia into vitelogenic oocytes and functional spermatozoa are all possible. Upon further improvements in the transplantation efficiency, this intraperitoneal transplantation system could become a valuable tool in the conservation of genetic resources for cichlid species.     

Transplantation of germ cells from rabbits and dogs into mouse testes.

Spermatogonial stem cells of a fertile mouse transplanted into the seminiferous tubules of an infertile mouse can develop spermatogenesis and transmit the donor haplotype to progeny of the recipient mouse. When testis cells from rats or hamsters were transplanted to the testes of immunodeficient mice, complete rat or hamster spermatogenesis occurred in the recipient mouse testes, albeit with lower efficiency for the hamster. The objective of the present study was to investigate the effect of increasing phylogenetic distance between donor and recipient animals on the outcome of spermatogonial transplantation. Testis cells were collected from donor rabbits and dogs and transplanted into testes of immunodeficient recipient mice in which endogenous spermatogenesis had been destroyed. In separate experiments, rabbit or dog testis cells were frozen and stored in liquid nitrogen or cultured for 1 mo before transplantation to mice. Recipient testes were analyzed, using donor-specific polyclonal antibodies, from 1 to >12 mo after transplantation for the presence of donor germ cells. In addition, the presence of canine cells in recipient testes was demonstrated by polymerase chain reaction using primers specific for canine alpha-satellite DNA. Donor germ cells were present in the testes of all but one recipient. Donor germ cells predominantly formed chains and networks of round cells connected by intercellular bridges, but later stages of donor-derived spermatogenesis were not observed. The pattern of colonization after transplantation of cultured cells did not resemble spermatogonial proliferation. These results indicate that fresh and cryopreserved germ cells can colonize the mouse testis but do not differentiate beyond the stage of spermatogonial expansion.     

Recipient preparation is critical for spermatogonial transplantation in the rat

Testis cell transplantation from mice or rats into recipient mouse seminiferous tubules results in donor cell-derived spermatogenesis in nearly all host testes. Normal spermatozoa are produced and, in the most successful mouse transplantations, the donor haplotype is transmitted to progeny of the recipient. However, few studies have been performed in other species. In this report, we demonstrate that rat and mouse testis cells will generate donor cell-derived spermatogenesis in recipient rat seminiferous tubules. Depletion of endogenous spermatogenesis before donor cell transplantation was more difficult in rat than reported for mouse recipients. A protocol employing treatment of neonatal rats with busulfan was most effective in preparing recipients and allowed more than 90% of testes to be colonized by donor cells. Transplantation of mouse testis cells into rat seminiferous tubules was most successful in recipients made cryptorchid and treated with busulfan. In the best experiments, about 55% of rat testes were colonized by mouse cells. Both rat and mouse donor cell-derived spermatogenesis were improved by treatment of rat recipients with leuprolide, a gonadotropin-releasing hormone agonist. The studies indicated that recipient preparation for spermatogonial stem cell transplantation was critical in the rat and differs from the mouse. However, modification of currently used techniques should allow male germ line stem cell transplantation in many species.     

Expansion of murine spermatogonial stem cells through serial transplantation

Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.     

Primate spermatogonial stem cells colonize mouse testes

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.     

美洲鲥雄性生殖细胞冷冻保存及移植

采用分离细胞冻存和组织块直接冻存2种方法, 进行美洲鲥(American shad, Alosa sapidissima)精巢细胞的长时间冷冻保存(>250d), 并比较分析2种不同冻存方法对美洲鲥雄性生殖细胞的冻存效果。解冻复苏后用Hochest33342和PI共染细胞核, 分析统计各期雄性生殖细胞的存活率, 结果显示组织块冻存方法所得精原干细胞和精母细胞的存活率明显高于分离细胞冻存的; 而精细胞及其他细胞存活率在2种方法间无显著差异; 特别是, 镜检发现组织块冻存方法所得精子存活率高达93.83%, 说明此冻存方法能同时高效地冻存美洲鲥各期生殖细胞, 包括成熟的精子。同时, 将组织块冻存的美洲鲥生殖细胞用PKH26染色标记后移植到出苗第1天的斑马鱼仔鱼中, 在细胞植入后5d仍能在受体中检测到供体细胞, 且有部分供体细胞能与内源生殖细胞共定位, 表明经过长时间冷冻保存的美洲鲥生殖细胞仍具有生殖细胞特性, 且能整合到斑马鱼受体性腺原基。研究结果为进一步开展美洲鲥, 或其他洄游性鱼类的生殖细胞发育、培养及种质资源保存等研究工作奠定了技术理论基础。     

Germ cell transplantation and testis tissue xenografting in domestic animals

Transplantation of germ cells from fertile donor mice to the testes of infertile recipient mice results in donor-derived spermatogenesis and transmission of the donor's genetic material to the offspring of recipient animals. Germ cell transplantation provides a bioassay to study the biology of male germ line stem cells, develop systems to isolate and culture spermatogonial stem cells, examine defects in spermatogenesis and treat male infertility. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals. In domestic animals including pigs, goats and cattle, as well as in primates, germ cells can be transplanted to a recipient testis by ultrasonographic-guided cannulation of the rete testis. Germ cell transplantation was successful between unrelated, immuno-competent pigs and goats, whereas transplantation in rodents requires syngeneic or immuno-compromised recipients. Genetic manipulation of isolated germ line stem cells and subsequent transplantation will result in the production of transgenic sperm. Transgenesis through the male germ line has tremendous potential in domestic animal species where embryonic stem cell technology is not available and current options to generate transgenic animals are inefficient. As an alternative to transplantation of isolated germ cells to a recipient testis, ectopic grafting of testis tissue from diverse mammalian donor species, including horses and primates, into a mouse host represents a novel possibility to study spermatogenesis, to investigate the effects of drugs with the potential to enhance or suppress male fertility, and to produce fertile sperm from immature donors. Therefore, transplantation of germ cells or xenografting of testis tissue are uniquely valuable approaches for the study, preservation and manipulation of male fertility in domestic animals.     

Restoration of fertility by germ cell transplantation requires effective recipient preparation

Spermatogonial transplantation provides access to the mammalian germline and has been used in experimental animal models to study stem cell/niche biology and germline development, to restore fertility, and to produce transgenic models. The potential to manipulate and/or transplant the germline has numerous practical applications that transcend species boundaries. To make the transplantation technology more broadly accessible, it is necessary to develop practical recipient preparation protocols. In the current study, mouse recipients for spermatogonial transplantation were prepared by treating pregnant females with the chemotherapeutic agent busulfan at different times during gestation. Donor germ cells were introduced into the testes of male progeny between 5 and 12 days postpartum. Analysis of recipient animals revealed that busulfan treatment of pregnant females on 12.5 days postcoitum was the most effective; male progeny transplanted with donor germ cells became fertile and passed the donor genotype to 25% of progeny. This approach was effective because 1) the cytoablative treatment reduced (but did not abolish) endogenous spermatogenesis, creating space for colonization by donor stem cells, 2) residual endogenous germ cells contributed to a healthy testicular environment that supported robust donor and recipient spermatogenesis, and 3) fetal busulfan-treated males could be transplanted as pups, which have been established as better recipients than adults. Laboratory mice provide a valuable experimental model for developing the technology that now can be applied and evaluated in other species.     

Germ cell transplantation in goats

Transplantation of spermatogonial stem cells provides a unique approach for the study of spermatogenesis and manipulation of the male germ line. This technique may also offer an alternative to the currently inefficient methods of producing transgenic domestic animals. We have recently established the technique of spermatogonial transplantation, originally developed in laboratory rodents, in pigs, and this study was aimed to extend the technique to the goat. Isolated donor testis cells were infused into the seminiferous tubules of anesthetized recipient goats through an ultrasonographically-guided catheter inserted into the rete testis. Donor cells were obtained by enzymatic digestion of freshly collected testes from immature goats (either from the recipients' contralateral testis or from unrelated donors). Prior to transplantation, testis cells were labeled with a fluorescent marker to allow identification after transplantation. Recipient testes were examined for the presence and localization of labeled donor cells at 3-week intervals up to 12 weeks after transplantation. Labeled donor cells were found in the seminiferous tubules of all testes, comprising 10-35% of the examined tubules. Histological examination of the recipient testes did not reveal evident tissue damage, except for limited fibrotic changes at the site of needle insertion. Likewise there were no detectable local or systemic signs of immunologic reactions to the transplantations. These results indicate that germ cell transplantation is technically feasible in immature male goats and that donor-derived cells are retained in the recipient testis for at least three months and through puberty. This study represents the first report of germ cell transplantation in goats.     

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka,Oryzias latipes

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.     

Successful intra- and interspecific male germ cell transplantation in the rat

The lumen of the seminiferous tubules has hitherto been regarded as an immunologically privileged site. We report here the birth of young following transplantation of stem spermatogonia from Long-Evans rats to the seminiferous tubules of Sprague-Dawley rats after treatment with the immunosuppressive agent cyclosporin. Follicle-stimulating hormone was also given to stimulate Sertoli cell proliferation, and testosterone to stimulate the recovery of spermatogenesis. Donor germ cells underwent normal spermatogenesis, and progeny were repeatedly produced from the donor germ cells as demonstrated by microsatellite paternity analysis. In addition, donor germ cells from the cryptorchid testes of LacZ mice were also able to colonize the seminiferous tubules of Sprague-Dawley rats using this protocol. Morphologically normal rat and mouse spermatozoa were present in the epididymis and vas deferens of the recipient rats. This highlights the potential for transplantation of male germ cells between different species.     

CD9 is expressed on human male germ cells that have a long-term repopulation potential after transplantation into mouse testes

Human spermatogonial stem cells (SSCs) play critical roles in lifelong maintenance of male fertility and regeneration of spermatogenesis. These cells are expected to provide an important resource for male fertility preservation and restoration. A basic strategy has been proposed that would involve harvesting testis biopsy specimens from a cancer patient prior to cancer therapies, and transplanting them back to the patient at a later time; then, SSCs included in the specimens would regenerate spermatogenesis. To clinically apply this strategy, isolating live human SSCs is important. In this study, we investigated whether CD9, a known rodent SSC marker, is expressed on human male germ cells that can repopulate recipient mouse testes upon transplantation. Testicular tissues were obtained from men with obstructive azoospermia. Using immunohistochemistry, we found that CD9 was expressed in human male germ cells in the basal compartment of the seminiferous epithelium. Following immunomagnetic cell sorting, CD9-positive cells were enriched for germ cells expressing MAGEA4, which is expressed by spermatogonia and some early spermatocytes, compared with unsorted cells. We then transplanted CD9-positive cells into nude mouse testes and detected an approximately 3- to 4-fold enrichment of human germ cells that repopulated mouse testes for at least 4 mo after transplantation, compared with unsorted cells. We also observed that some cell turnover occurred in human germ cell colonies in recipient testes. These results demonstrate that CD9 identifies human male germ cells with capability of long-term survival and cell turnover in the xenogeneic testis environment.     

Germ cell transplantation in pigs.

Spermatogonial stem cells form the foundation of spermatogenesis, and their transplantation provides a unique opportunity to study spermatogenesis and may offer an alternative approach for animal transgenesis. This study was designed to extend the technique of spermatogonial transplantation to an economically important, large-animal model. Isolated immature pig testes were used to develop the intratesticular injection technique. Best results of intratubular germ cell transfer were obtained when a catheter was inserted into the rete testis under ultrasound guidance. The presence of infused dye or labeled cells was confirmed in the seminiferous tubules from 70 of 89 injected isolated testes. Infusion of 3-6 ml of dye solution or cell suspension could fill the rete and up to 50% of seminiferous tubules. The technique was subsequently applied in vivo. Donor cells included testis cells from 1- or 10-wk-old boars (from the recipients' contralateral testis or unrelated donors) and those from mice carrying a marker gene. Porcine testis cells were labeled with a fluorescent marker before transplantation. Testes were examined for the presence and localization of labeled donor cells immediately after transplantation or every week for 4 wk. Labeled porcine donor cells were found in numerous seminiferous tubules from 10 of 11 testes receiving pig cells. These results indicate that germ cell transplantation is feasible in immature pigs, and that porcine transplanted cells are retained in the recipient testis for at least 1 mo. This study represents a first step toward successful spermatogonial transplantation in a farm animal species.     

Germ cell transplantation in an azoospermic Klinefelter bull

Germ cell transplantation is a technique that transfers donor testicular cells into recipient testes. A population of germ cells can colonize the recipient testis, initiate spermatogenesis, and produce sperm capable of fertilization. In the present study, a nonmosaic Klinefelter bull was used as a germ cell recipient. The donor cell suspension was introduced into the rete testis using ultrasound-guided puncture. A pulsatile administration of GnRH was performed to stimulate spermatogenesis. The molecular approach to detect donor cells was done by a quantitative polymerase chain reaction with allele discrimination based on a genetic mutation between donor and recipient. Therefore, a known genetic mutation, associated with coat-color phenotype, was used to calculate the ratio of donor to recipient cells in the biopsy specimens and ejaculates for 10 mo. After slaughtering, meiotic preparations were performed. The injected germ cells did not undergo spermatogenesis. Six months after germ cell transplantation, the donor cells were rejected, which indicates that the donor cells could not incorporate in the testis. The hormone stimulation showed that the testosterone-producing Leydig cells were functionally intact. Despite subfertility therapy, neither the recipient nor the donor cells underwent spermatogenesis. Therefore, nonmosaic Klinefelter bulls are not suitable as germ cell recipients. Future germ cell recipients in cattle could be mosaic Klinefelters, interspecies hybrids, bulls with Sertoli cell-only syndrome, or bulls with disrupted germ cell migration caused by RNA interference.     

Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl

Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. We have previously adapted this approach for the domestic chicken and we present now an improvement of the germ cell transplantation technique by using an enriched subpopulation of c-Kit-positive spermatogonia as donor cells. Dispersed c-Kit positive testicular cells from 16 to 17 week-old pubertal donors were transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient's testes with c-Kit positive donor testicular cells, which resulted in the production of functional heterologous spermatozoa.Using manual semen collection, the first sperm production in the recipient males was observed about nine weeks after the transplantation. The full reproduction cycle was accomplished by artificial insemination of hens and hatching of chickens.     

Generation of normal progeny by intracytoplasmic sperm injection following grafting of testicular tissue from cloned mice that died postnatally

Animal cloning by nuclear transfer has been successful in several species and was expected to become an alternative reproductive technique. Among the problems associated with this cloning technique, however, are its low success rate and high mortality of cloned animals even if they develop to term. Nuclear transfer has thus come to be considered too difficult to apply as a reproductive technique. The transplantation of male germ cells or pieces of testicular tissue has enabled the induction of spermatogenesis from fetal or postnatal male mice. In the present study, we examined whether functional male gametes could be obtained by the transplantation of pieces of testicular tissue from cloned mice that died immediately after birth with typical aberrant phenotypes, such as large offspring syndrome. Donor testicular tissues were retrieved from cloned mice that died postnatally and were transplanted into the testes of recipient nude mice. Two to three months after transplantation, the grafted donor testicular tissue had grown in the host testis, and histological analysis showed that spermatogenesis occurred within the graft. Intracytoplasmic sperm injection demonstrated that the testicular sperm generated in the grafted donor tissue were able to support full-term development of progeny. These results clearly showed that functional spermatogenesis could be induced by transplanting testicular tissue from cloned mice that died postnatally into recipient mice. The strategy presented here will be applicable to cloned animals of other species, because the xenografting of testicular tissue into mice has been demonstrated previously to be possible.     

Production of donor-derived offspring by allogeneic transplantation of Spermatogonia in the yellowtail (Seriola quinqueradiata)

Although the yellowtail (Seriola quinqueradiata) is the fish most commonly farmed in Japan, breeding of this species has not yet started. This is primarily due to the lack of sufficiently sophisticated methods for manipulating gametogenesis, which makes it difficult to collect gametes from specific dams and sires. If it were possible to produce large numbers of surrogate fish by transplanting germ cells isolated from donor individuals harboring desirable genetic traits, then the probability of acquiring gametes carrying the donor-derived haplotype would increase, and breeding programs involving this species might increase as a result. As a first step, we established a method for the allogeneic transplantation of yellowtail spermatogonia and the production of donor-derived offspring. Donor cells were collected from immature (10-month-old) yellowtail males with testes containing abundant type A spermatogonia, labeled with PKH26 fluorescent dye, and transferred into the peritoneal cavities of 8-day-old larvae. Fluorescence observation at 28 days post-transplantation revealed that PKH26-labeled cells were incorporated into recipients' gonads. To assess whether donor-derived spermatogonia could differentiate into functional gametes in the allogeneic recipient gonads, gametes collected from nine male and four female adult recipients were fertilized with wild-type eggs and milt. Analysis of microsatellite DNA markers confirmed that some of the first filial (F(1)) offspring were derived from donor fish, with the average contribution of donor-derived F(1) offspring being 66% and the maximum reaching 99%. These findings confirmed that our method was effective for transplanting yellowtail spermatogonia into allogeneic larvae to produce donor-derived offspring.     

Restoration of spermatogenesis and male fertility by transplantation of dispersed testicular cells in the chicken

Transplantation of male germ cells into sterilized recipients has been widely used in mammals for conventional breeding and transgenesis purposes. This study presents a workable approach for germ cell transplantation between male chickens. Testicular cells from adult and prepubertal donors were dispersed and transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient seminiferous epithelium up to the production of heterologous sperm in about 50% of transplanted males. In comparison to males transplanted with testicular cell preparations from adult donors, in which the first ejaculates with sperm were recovered about 5 wk after transfer, a substantial interval (about 10 wk) was necessary to obtain ejaculates after the transfer of testicular cells from prepubertal donors. However, in both cases, recipient males produced ejaculates capable of fertilizing ova and producing progeny expressing donor genes.