Grafting Of Testicular Tissue Research Articles

Restoring male fertility using immature testicular tissue: what role can testicular tissue grafting and spermatogonial stem cell transplantation play?

Restoring male fertility using immature testicular tissue: what role can testicular tissue grafting and spermatogonial stem cell transplantation play?

Spermatogonial Stem Cell-Based Therapies: Taking Preclinical Research to the Next Level.

Fertility preservation via biobanking of testicular tissue retrieved from testicular biopsies is now generally recommended for boys who need to undergo gonadotoxic treatment prior to the onset of puberty, as a source of spermatogonial stem cells (SSCs). SSCs have the potential of forming spermatids and may be used for therapeutic fertility approaches later in life. Although in the past 30 years many milestones have been reached to work towards SSC-based fertility restoration therapies, including transplantation of SSCs, grafting of testicular tissue and various in vitro and ex vivo spermatogenesis approaches, unfortunately, all these fertility therapies are still in a preclinical phase and not yet available for patients who have become infertile because of their treatment during childhood. Therefore, it is now time to take the preclinical research towards SSC-based therapy to the next level to resolve major issues that impede clinical implementation. This review gives an outline of the state of the art of the effectiveness and safety of fertility preservation and SSC-based therapies and addresses the hurdles that need to be taken for optimal progression towards actual clinical implementation of safe and effective SSC-based fertility treatments in the near future.

Study on cryopreservation of mouse single seminiferous tubule

Cryopreservation of single seminiferous tubule is significant for fertility preservation, especially for male patients with cryptorchidism, Y-chromosome deletion and orchitis. However, there are few studies on cryopreservation of single seminiferous tubule. This study proposes several improved strategies for cryopreservation of single seminiferous tubule in mice. First, single seminiferous tubule was cryopreserved with modified slow freezing and vitrification methods. The results showed that the apoptosis negative rates of spermatogenic cells in single seminiferous tubule with modified slow freezing method were significantly higher than vitrification with plastic slide. Then, plastic slide and two metal vitrification carriers with high thermal conductivity, copper mesh and aluminum foil, were used to vitrify single seminiferous tubule. The metal carriers could improve the outcome of vitrification than plastic slide. The apoptosis negative rates of spermatogenic cells in the aluminum foil group was significantly higher than that in the copper mesh group. Finally, single seminiferous tubule was perfused with CPAs by micro-injection technique and vitrified. The results of cryo-microscopy experiments showed that the ice crystals formed inside the injected seminiferous tubule was reduced during the cooling process, the apoptosis negative rate of spermatocytes, spermatids and Sertoli cells were significantly higher than that of the non-injection group, indicating that the injection technique can effectively improve the effect of vitrification. This study has potential clinical value for in-vitro culture of spermatogonial stem cells and autologous testicular tissue grafting in patients with azoospermia.

P–056 To graft or not to graft? Intratesticular grafting of testicular tissue from Klinefelter boys to the mouse testis as possible novel in vivo model

Abstract Study question Can intratesticular transplanted testis tissue from Klinefelter boys to the mouse testis be used to study the mechanisms behind testicular fibrosis? Summary answer Grafting of testicular tissue from Klinefelter boys to the mouse testis is not a valuable new in vivo model to study Klinefelter-related testicular fibrosis. What is known already Klinefelter syndrome (KS; 47, XXY) affects 1–2 in 1000 males. Most KS men suffer from azoospermia due to a loss of spermatogonial stem cells. Additionally, testicular fibrosis is detected from puberty onwards. However, mechanisms responsible for fibrosis and germ cell loss remain unknown. An optimal in vivo model to study the KS testicular fibrotic process is not available. This study aimed to evaluate a possible in vivo model to study KS-related testicular fibrosis. In addition, the effect of the mast cell blocker ketotifen, which showed positive effects on fertility in infertile non-KS patients, was evaluated in this graft model. Study design, size, duration First, the survival time of the KS graft was established, since it was the first time KS tissue was transplanted to the mouse testis. Testes were collected after two, four, six and eight weeks after which histological and immunohistochemical evaluations were performed. Next, the effect of daily ketotifen injections on the fibrotic appearance of intratesticular grafted testicular tissue from KS and controls was evaluated. Participants/materials, setting, methods Testicular biopsy samples from pre- and peripubertal KS (n = 22) and age-matched control samples (n = 22) were transplanted to the testes of six weeks old Swiss Nu/Nu mice (n = 22). Prior to grafting, testicular tissue pieces were cultured in vascular endothelial growth factor (VEGF) for five days. Next, tissues were transplanted to the mouse testes. Testicular transplants were analysed by immunohistochemistry. In the second experiment, mice were given daily subcutaneous injections of ketotifen or saline. Main results and the role of chance Four weeks after transplantation, all KS grafts could still be retrieved. At a later timepoint, degeneration of the tissue could be detected. In the grafts, recovered four weeks after transplantation, about 30% of the tubules in peripubertal grafts showed a good integrity, while in the prepubertal tissue, 83% of the tubules were intact. A fibrotic score was assigned to each graft. No significant changes in fibrotic score was observed between testicular biopsies before or after transplantation. However, an increased (p < 0.01) fibrotic score was observed after in-vitro treatment with VEGF both in control and KS tissue. Based on recovery and tubule integrity grafts were recovered after four weeks in the second experiment. Treatment with ketotifen did not result in significant histological differences compared to non-treated grafts (KS and control tissue). The survival potential of grafts from KS testicular biopsies of pre- and peripubertal boys was patient- and age-dependent. After four weeks, most KS tissue starts to degenerate. In prepubertal tissue, seminiferous tubules were mostly intact, while tissue from adolescent boys was impaired. Interestingly, no loss of germ cells was observed after transplantation of the testicular tissue. Limitations, reasons for caution The availability of tissue from young KS patients is very scarce, leading to a low number of included patients (n = 8). Testicular tissue pieces from the same patient were included to evaluate the differences before and after transplantation. However, histological variability between testicular tissue biopsy pieces is well-known in KS patients. Wider implications of the findings Since testicular tissue from KS boys, transplanted to the mouse testes, already starts to degenerate after four weeks and the integrity is not optimal, we conclude that this is not a valuable model for future studies. In vitro models to study the KS-testicular fibrosis should be investigated. Trial registration number NA

Oxidative Stress Disrupted Prepubertal Rat Testicular Development after Xenotransplantation.

In the past two decades, testicular tissue grafting and xenografting have been well established, with the production of fertilization-competent sperm in some studies. However, few studies have been carried out to observe the development of grafted prepubertal testicular tissue of rats and compare the biological differences between in situ testis and grafted testis. In this study, we established the prepubertal testicular tissue xenografting model using a 22-day-old rat and evaluated certain parameters, including testicular histology, testosterone production, and ultrastructure of the grafted testes. We also assessed gene expression of cell proliferation markers, testicular cell markers, and antioxidative defense system. Our results showed that 47 days after transplantation, intratesticular testosterone concentration was not significantly altered; however, cell proliferation, spermatogenesis, and Sertoli cell markers in the transplanted testes were significantly disrupted compared with the control group, accompanied by aggravated apoptosis and oxidative damage. Moreover, the transplanted testes showed smaller tubular diameter and disrupted spermatogenic epithelium with apparent vacuoles, distorted and degenerated germ cells with obscure nuclear margin, and no spermatids in the center of the tubules. Although testis xenografting has been extensively tested and attained great achievement in other species, the prepubertal rat testicular tissue xenografting to immunodeficient mice exhibited obvious spermatogenesis arrest and oxidative damage. The protocol still needs further optimization, and there are still some unknown factors in prepubertal rat testes transplantation.

Autologous grafting of cryopreserved prepubertal rhesus testis produces sperm and offspring.

Testicular tissue cryopreservation is an experimental method to preserve the fertility of prepubertal patients before they initiate gonadotoxic therapies for cancer or other conditions. Here we provide the proof of principle that cryopreserved prepubertal testicular tissues can be autologously grafted under the back skin or scrotal skin of castrated pubertal rhesus macaques and matured to produce functional sperm. During the 8- to 12-month observation period, grafts grew and produced testosterone. Complete spermatogenesis was confirmed in all grafts at the time of recovery. Graft-derived sperm were competent to fertilize rhesus oocytes, leading to preimplantation embryo development, pregnancy, and the birth of a healthy female baby. Pending the demonstration that similar results are obtained in noncastrated recipients, testicular tissue grafting may be applied in the clinic.

Testicular organoids: a new model to study the testicular microenvironment in vitro?

In recent decades, a broad range of strategies have been applied to model the testicular microenvironment in vitro. These models have been utilized to study testicular physiology and development. However, a system that allows investigations into testicular organogenesis and its impact in the spermatogonial stem-cell (SSC) niche in vitro has not been developed yet. Recently, the creation of tissue-specific organ-like structures called organoids has resurged, helping researchers to answer scientific questions that previous in vitro models could not help to elucidate. So far, a small number of publications have concerned the generation of testicular organoids and their application in the field of reproductive medicine and biology. Here, we aim to elucidate whether testicular organoids might be useful in answering current scientific questions about the regulation and function of the SSC niche as well as germ cell proliferation and differentiation, and whether or not the existing in vitro models are already sufficient to address them. Moreover, we would like to discuss how an organoid system can be a better solution to address these prominent scientific problems in our field, by the creation of a rationale parallel to those in other areas where organoid systems have been successfully utilized. We comprehensively reviewed publications regarding testicular organoids and the methods that most closely led to the formation of these organ-like structures in vitro by searching for the following terms in both PubMed and the Web of Science database: testicular organoid, seminiferous tubule 3D culture, Sertoli cell 3D culture, testicular cord formation in vitro, testicular morphogenesis in vitro, germ cell 3D culture, in vitro spermatogenesis, testicular de novo morphogenesis, seminiferous tubule de novo morphogenesis, seminiferous tubule-like structures, testicular in vitro model and male germ cell niche in vitro, with no restrictions to any publishing year. The inclusion criteria were based on the relation with the main topic (i.e. testicular organoids, testicular- and seminiferous-like structures as in vitro models), methodology applied (i.e. in vitro culture, culture dimensions (2D, 3D), testicular cell suspension or fragments) and outcome of interest (i.e. organization in vitro). Publications about grafting of testicular tissue, germ-cell transplantation and female germ-cell culture were excluded. The application of organoid systems is making its first steps in the field of reproductive medicine and biology. A restricted number of publications have reported and characterized testicular organoids and even fewer have denominated such structures by this method. However, we detected that a clear improvement in testicular cell reorganization is recognized when 3D culture conditions are utilized instead of 2D conditions. Depending on the scientific question, testicular organoids might offer a more appropriate in vitro model to investigate testicular development and physiology because of the easy manipulation of cell suspensions (inclusion or exclusion of a specific cell population), the fast reorganization of these structures and the controlled in vitro conditions, to the same extent as with other organoid strategies reported in other fields. By way of appropriate research questions, we might use testicular organoids to deepen our basic understanding of testicular development and the SSC niche, leading to new methodologies for male infertility treatment.

Establishment of a strain of haemophilia-A pigs by xenografting of foetal testicular tissue from neonatally moribund cloned pigs

Grafting of testicular tissue into immunodeficient mice makes it possible to obtain functional sperm from immature donor animals that cannot be used for reproduction. We have developed a porcine model of human haemophilia A (haemophilia-A pigs) by nuclear transfer cloning from foetal fibroblasts after disruption of the X-linked coagulation factor VIII (F8) gene. Despite having a recessive condition, female F8+/− cloned pigs died of severe bleeding at an early age, as was the case for male F8−/Y cloned pigs, thus making it impossible to obtain progeny. In this study, therefore, we produced sperm from F8−/Y cloned pigs by grafting their foetal testicular tissue into nude mice. Two F8+/− female pigs were generated from oocytes injected with xenogeneic sperm. Unlike the F8+/− cloned pigs, they remained asymptomatic, and delivered five F8−/Y and four F8+/− pigs after being crossed with wild-type boars. The descendant F8−/Y pigs conserved the haemophilia phenotype. Thus, the present F8+/− pigs show resolution of the phenotypic abnormality, and will facilitate production of F8−/Y pigs as founders of a strain of haemophilia-A pigs for the development of new therapeutics for haemophilia A. This strategy will be applicable to other genetically modified pigs.

Optimal methods for recovery of transplantable stem cells from frozen/thawed human testicular tissue

Spermatogonial stem cell (SSC)-based technologies may have application for preserving and/or restoring male fertility. We evaluated the recovery of stem and progenitor spermatogonia from frozen/thawed testicular cell suspensions versus testicular tissue fragments frozen at a controlled rate (CR) or by vitrification. Laboratory study using human tissue. Human testicular tissue was obtained through the Center for Organ Recovery and Education with approval from the University of Pittsburgh Institutional review board (#0506140). Testicular tissue was digested with enzymes to produce a cell suspension and frozen at CR or minced into large (6-10 mm3) or small (3-5 mm3) testicular tissue fragments and frozen at CR or by vitrification. The efficiency of each technique was analyzed by immunocytochemistry (ICC) for the spermatogonia marker UTF1 and human to nude mouse xenotransplantation. Cryopreservation method had a significant effect on the recovery of UTF1 positive spermatogonia per gram of tissue (p<0.001). Large testicular tissue fragments frozen at CR had the greatest number of UTF1 cells/g tissue (14.9±2.2), followed by CR small (10.4±1.3), vitrified small (5.4±0.9), vitrified large (4.4±0.5), and frozen/thawed cell suspension (1.5±0.2). The difference in UTF1 positive cell recovery from CR large tissue fragments versus CR cell suspension was statistically significant (14.9 vs. 1.5, p=0.027). Human to nude mouse xenotransplants confirmed that recovery of transplantable SSCs (colonies/g tissue) from CR small (1045.9±240.6) and CR large (570.0±147.1) testicular tissue fragments was greater than the CR cell suspension (62.6±12.3, p<0.05). Recovery of transplantable stem cells from vitrified small (562.4±186.0) and vitrified large (75.4±33.2) testicular tissue fragments was not significantly greater than the CR cell suspension (p>0.05). CR freezing of testicular tissue fragments (large or small) leads to the greatest recovery of UTF1 positive spermatogonia and transplantable SSCs. Recovery of SSCs from vitrified small testicular tissue fragments was also very good. Freezing and thawing of intact testicular tissue is compatible with several downstream applications, including testicular tissue grafting, testicular tissue organ culture or digestion with enzymes to produce a cell suspension for SSC transplantation.

Préservation de la fertilité chez le garçon prépubère : transplantation de cellules souches spermatogoniales et greffe testiculaire

Spermatogonial stem cells (SSC) are the founder cells of spermatogenesis and are responsible for the lifelong production of spermatozoa. The cryopreservation and transplantation of these cells has been proposed as a fertility preservation strategy for young boys at risk for stem cell loss, i.e. patients undergoing chemotherapy for cancer or as a conditioning treatment for bone marrow transplantation. To prevent lifelong sterility in boys, two fertility restoration strategies are being developed: the injection of SSC and the grafting of testicular tissue containing SSC. Depending on the disease of the patient one of these two approaches will be applicable. Grafting has the advantage that SSC can reside within their natural niche, preserving the interactions between germ cells and their supporting cells and may therefore be regarded as the first choice strategy. However, in cases where the risk for malignant contamination of the testicular tissue is real, e.g. leukemia, transplantation of SSC by injection is preferable over grafting.

Mouse germ cells go through typical epigenetic modifications after intratesticular tissue grafting

Since spermatogonial stem cell transplantation (SSCT) and testicular tissue grafting (TTG) may have important clinical applications, the safety of these promising techniques has to be proved. This study was designed to characterize epigenetic modifications in prepubertal and adult mouse germ cells and to study these epigenetic mechanisms after SSCT and TTG. Testicular cell suspensions were transplanted to the testes of genetically sterile W/W(v) recipients. Intratesticular tissue grafting was performed between green fluorescent protein (GFP(+)) donors and GFP(-) acceptor mice. DNMT1 and DNMT3A expression, the general methylation status and the histone modifications H3K4me3, H3K9ac, H4K5ac, H4K8ac, H4K12ac and H4K16ac were studied in a stage-dependent manner by immunohistochemistry and compared with data from adult control mice. The expression levels of DNMT1 and DNMT3A, the DNA methylation status and most of the stage-specific histone modifications after SSCT or TTG were not different from fertile adult controls. Although, in elongated spermatids, the acetylation pattern was as expected, the stage-dependent expression of H4K5ac and H4K8ac was altered after SSCT. Intratesticular tissue grafting might be the better choice for fertility restoration. Disrupting the stem cell niche might influence epigenetic patterns. Since the function of H4K5ac and H4K8ac in spermatogonia and spermatocytes still has to be explored, in-depth epigenetical analyses are warranted.

Xenografting of testicular tissue from an infant human donor results in accelerated testicular maturation

Grafting of testicular tissue into immunodeficient mice has been used to differentiate the neonatal testes from different animal species up to the level of complete spermatogenesis; however, this approach has not been successful for human testicular tissue. The aim of this study was to evaluate the capacity for differentiation of infant human testicular tissue grafts. Testicular tissue from a 3-month-old patient with testicular cancer was grafted into immunodeficient nude mice. At the time of grafting, A spermatogonia were the only germ cells present in the testicular tissue. B spermatogonia and first spermatocytes were observed at 7 months and 1 year after grafting, respectively. Positive immunostaining with antibodies against BOULE and CDC25A suggested that spermatocytes in the graft were not arrested but in meiosis. Furthermore, ultrastructural and immunohistochemical analyses showed that the onset of both Sertoli cell maturation and partial differentiation of Leydig cells preceded the appearance of spermatocytes. Differentiation of testicular cells was accelerated compared with in vivo development. Spermatogenesis in the xenograft of infant human testicular tissues proceeded successfully from the stage of spermatogonial stem cells until pachytene spermatocyte formation. The differentiation of Sertoli cells and Leydig cells was reproduced in a manner similar to that in normal testicular development. Grafting of infant human testicular tissue may be a powerful tool to examine the early period of human spermatogenesis and may pave the way for fertility preservation among infant patients.

Limited survival of adult human testicular tissue as ectopic xenograft

Grafting of testicular tissue into immunodeficient mice has become an interesting and promising scientific tool for the generation of gametes and the study of testicular function. This technique might potentially be used to generate sperm from patients whose testes need to be removed or are destroyed due to therapeutic intervention or as a consequence of disease. Here we explore whether adult human testicular tissue from patients with different testicular pathologies survives as xenograft. Testis tissue from adult patients with varying degrees of spermatogenesis was grafted into two strains of immunodeficient mice (severe combined immunodeficiency, Nu/Nu). Tissue with active spermatogenesis prior to grafting largely regressed. However, testicular tissue survival was better in cases where spermatogenesis was suppressed prior to grafting and occasionally spermatogonial stem cells survived. Cases with spermatogenic disruption were not corrected by the xenografting. Superior survival of the germinal epithelium and spermatogonia when spermatogenesis was suppressed prior to grafting could provide a novel strategy for germline preservation in pre-pubertal cancer patients. This approach could also be valuable to study the early stages of human spermatogenesis.

Congélation du tissu germinal chez le garçon

An increased incidence of cancer is observed in the population of adolescents and young adults since thirty years. Major progress in cancer diagnosis and therapy is unfortunately associated to high degree of toxicity on gonad function. Cryopreservation of ovarian tissue is performed in girls and women before cancer treatment with high risk of infertility. Procedures for ejaculated or testicular extracted spermatozoa are well defined. However, for prepubertal boys or after ejaculated sperm collection failure, mature or immature testicular tissue banking should be proposed. Still, an optimal cryopreservation protocol is a prerequisite for clinical application and does not exist for the moment. Furthermore, the future applications of immature testicular tissue banking should be developed, not solely germ cell in vitro maturation but also autologous testicular tissue grafting.

Generation of Normal Progeny by Intracytoplasmic Sperm Injection Following Grafting of Testicular Tissue from Cloned Mice That Died Postnatally1

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.

Cryopreservation and Transplantation of Spermatogonia and Testicular Tissue for Preservation of Male Fertility

The existence of spermatogonial stem cells in the testis offers clinically relevant options for preservation and restoration of male fertility. New approaches based on male germ cell transplantation and testicular tissue grafting can be applied to generate a limited number of sperm cells and could therefore be considered important new avenues for restoration of fertility in oncological patients. We have developed approaches to infuse germ cells into rodent and primate testes and shown that germ cell transplantation is a procedure for restoration of spermatogenesis in the testis that might be adaptable to primates. As a promising alternative, grafting of testicular tissue has been used to produce fertile sperm. The rapid progress in the development of novel experimental strategies to generate sperm by transplantation of spermatogonial stem cells or by grafting of testicular tissue should stimulate oncologists to consider the cryopreservation of testicular tissue. This review introduces the reader to the physiology of spermatogonial stem cells and summarizes the current and potential future options for fertility preservation in male oncological patients.

Notch-1, c-kit and GFRα-1 are developmentally regulated markers for premeiotic germ cells

Culture, transfection and immortalization of mouse germ line stem cells, germ cell transplantation and grafting of testicular tissue are milestones of recent biotechnological breakthroughs. Alone and in combination they offer new pathways to explore the cellular mechanisms responsible for pluripotency and the requirements of cells to enter the germ line. Efficient markers, isolation and culture systems as well as transfection approaches are developed to elucidate the molecular and cellular mechanisms leading to the development of male germ line cells. Here, we describe the localization pattern of c-kit, Notch-1 and GFRα-1 in postnatal, immature and adult testes. All three proteins are potentially useful markers for spermatogonial characterization and enrichment. First attempts and various future perspectives to use spermatogonial stem cells as pathway for the introduction of transgenes are discussed.

022.Spermatogonial stem cells: from basic research to clinical applications

The testis contains undifferentiated spermatogonia and is therefore the only adult organ populated with proliferating germline cells. Whereas the biology of these cells is quite well understood in rodents, their modes of mitotic expansion and differentiation are poorly understood in primates. The existence of these cells offers clinically relevant options for preservation and restoration of male fertility. New approaches based on male germ cell transplantation and testicular tissue grafting can be applied to generate a limited number of sperm and could therefore be considered as important new avenues applicable to a variety of disciplines like animal conservation, genetic germline modification or restoration of fertility in oncological patients. In principle, germ cell transplantation presents a removal of the stem cell from the donor's niche and a transfer into the niche of a recipient. Grafting can be considered as a transplantation of the stem cell in conjunction with its niche. Germ cell transplantation of human spermatogonia into mouse testes revealed that the stem cells survive and expand but are not able to differentiate and complete spermatogenesis. We have developed an approach to infuse germ cells into monkey and human testes and showed that germ cell transplantation is feasible as an autologous approach in primates. Furthermore, we applied germ cell transplantation in the monkey model mimicking a gonadal protection strategy for oncological patients. Ectopic xenografting of testicular tissue was applied to generate fertile sperm from a variety of species. Newborn testicular tissue was grafted into the back skin of immunodeficient mice and developed up to qualitatively complete spermatogenesis. The rapid progress in the development of novel experimental strategies to generate sperm from cryopreserved spermatogonial stem cells or immature testicular tissue will lead to many new options for germline manipulation and fertility preservation.

Progeny from sperm obtained after ectopic grafting of neonatal mouse testes.

Ectopic grafting of testicular tissue is a promising new approach that can be used to preserve testicular function. This technique has been used recently to differentiate the neonatal testes of different species, up to the level of complete spermatogenesis. This approach can be applied successfully to generate live progeny using sperm extracted from grafts originating from testes of newborn donors. The sperm are capable of supporting normal development and producing fertile male and female offspring after intracytoplasmic injection into mouse oocytes and embryo transfer into surrogate mothers. The grafted tissue was also capable of significantly normalizing reproductive hormone levels in the castrated recipients. This technique presents new avenues for experimentation. The recipient mouse can be regarded as a living incubator and a culture system of testicular tissue, allowing the experimental manipulation of several aspects of testis development and spermatogenesis. The successful generation of pups indicates that this technique can be used to study the testicular phenotype and to breed mutant or transgenic mouse strains with lethal postnatal phenotypes. The ability to generate sperm from the germ line ex vivo also paves the way for the development of new strategies for preserving fertility in boys undergoing cancer therapy.

Spermatogenesis and steroidogenesis in mouse, hamster and monkey testicular tissue after cryopreservation and heterotopic grafting to castrated hosts.

Retrieval, extracorporal storage and autotransplantation of testicular tissue could become an important strategy for preserving male gonadal function. The present study used syngeneic and immunodeficient nude mice as hosts, and immature and adult mice, neonatal and adult photoregressed Djungarian hamsters and neonatal marmosets to identify the potential of testicular tissue grafting to maintain the morphological and functional integrity of the testis. Testicular tissue was grafted s.c. either as fresh tissue or after cryopreservation into adult, orchidectomized hosts. The mice that received rodent testis tissue were autopsied 50 days later, and blood samples were collected. Sixty-five per cent of mouse isografts contained morphologically normal testicular tissue and seminiferous tubules with some degree of spermatogenic recovery. Mature spermatozoa were recovered after enzymatic disaggregation. Although the recovery of spermatogenesis was limited in adult mouse and hamster tissue, complete spermatogenesis was observed in grafts from immature rodents. Testicular tissue from neonatal marmosets developed up to the stage of spermatocytes at day 135 after xenografting. Androgen concentrations were comparable in intact control mice and in mice receiving fresh mouse and hamster grafts, slightly lower in mice receiving cryopreserved grafts and adult photoregressed hamster tissue, and low in castrated control mice and in mice receiving marmoset tissue. These results show that isografts and xenografts of immature and adult testicular tissue become functionally active as a s.c. graft in the mouse and that this approach might be useful in combination with cryopreservation as a tool for storage and activation of the male germ line and androgen replacement therapy in patients.