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Spermatogonial stem cells (SSCs) are a rare group of cells in the testis that undergo self-renewal and complex sequences of differentiation to initiate and sustain spermatogenesis, to ensure the continuity of sperm production throughout adulthood. The difficulty of unequivocal identification of SSCs and complexity of replicating their differentiation properties in vitro have prompted the introduction of novel in vivo models such as germ cell transplantation (GCT), testis tissue xenografting (TTX), and testis cell aggregate implantation (TCAI). Owing to these unique animal models, our ability to study and manipulate SSCs has dramatically increased, which complements the availability of other advanced assisted reproductive technologies and various genome editing tools. These animal models can advance our knowledge of SSCs, testis tissue morphogenesis and development, germ-somatic cell interactions, and mechanisms that control spermatogenesis. Equally important, these animal models can have a wide range of experimental and potential clinical applications in fertility preservation of prepubertal cancer patients, and genetic conservation of endangered species. Moreover, these models allow experimentations that are otherwise difficult or impossible to be performed directly in the target species. Examples include proof-of-principle manipulation of germ cells for correction of genetic disorders or investigation of potential toxicants or new drugs on human testis formation or function. The primary focus of this review is to highlight the importance, methodology, current and potential future applications, as well as limitations of using these novel animal models in the study and manipulation of male germline stem cells.

Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genes onto the next generation. SSCs also have the potential to provide important knowledge about stem cells in general and to offer critical in vitro and in vivo applications in assisted reproductive technologies. After century-long research, proof-of-principle culture systems have been introduced to support the in vitro differentiation of SSCs from rodent models into haploid male germ cells. Despite recent progress in organotypic testicular tissue culture and two-dimensional or three-dimensional cell culture systems, to achieve complete in vitro spermatogenesis (IVS) using non-rodent species remains challenging. Successful in vitro production of human haploid male germ cells will foster hopes of preserving the fertility potential of prepubertal cancer patients who frequently face infertility due to the gonadotoxic side-effects of cancer treatment. Moreover, the development of optimal systems for IVS would allow designing experiments that are otherwise difficult or impossible to be performed directly in vivo, such as genetic manipulation of germ cells or correction of genetic disorders. This review outlines the recent progress in the use of SSCs for IVS and potential in vivo applications for the restoration of fertility.

The purpose of this study was to evaluate the long-term outcome of testis tissue xenografting from immature deer. Testis tissue was collected post-mortem from a 2-mo-old white-tailed deer fawn (Odocoileus virginianus) and small fragments of the tissue were grafted under the back skin of immunodeficient recipient mice (n = 7 mice; 8 fragments/mouse). Single xenograft samples were removed from representative recipient mice every 2 mo from grafting for up to 14 mo post-grafting. The retrieved xenografts were evaluated for seminiferous tubular density (per mm(2)) and tubular diameter, as well as for seminiferous tubular morphology and identification of the most advanced germ cell type present in each tubule cross section. Overall, 63% of the grafted testis fragments were recovered as xenografts. Testis tissue xenografts showed a gradual testicular development starting with tubular expansion by 2 mo, presence of spermatocytes by 6 mo post-grafting, round and elongated spermatids by 8 mo, followed by fully-formed sperm by 12 mo post-grafting. The timing of complete spermatogenesis generally corresponded to the reported timing of sexual maturation in white-tailed deer. This study demonstrated, for the first time, that testis tissue xenografting from immature deer donors into recipient mice can successfully result in testicular maturation and development of spermatogenesis in the grafts up to the stage of sperm production. These results may therefore provide a model for salvaging genetic material from immature male white-tailed deer that die before reaching sexual maturity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Spermatogenesis from many mammalian species occurs in fragments of normal testis tissue xenografted to mice. Here we apply xenografting to the study of testicular pathology. Using the horse model, we investigated whether exposure to a permissive extratesticular environment in the mouse host would rescue spermatogenesis in cryptorchid testicular tissue or in tissue affected by idiopathic testicular degeneration (ITD). In cryptorchid tissue, where the extratesticular environment is abnormal, xenografting induced spermatogenesis up to meiosis in a subpopulation of seminiferous tubules. Thus, spermatogonia survive and partially retain their potential to differentiate in cryptorchid horse testes. In contrast, the primary defect in equine ITD is hypothesised to be tissue autologous. In support of this, xenografting did not restore spermatogenesis to tissue affected by ITD, thus confirming that the testis itself is primarily diseased. This outcome was not affected by supplementation of exogenous gonadotropins to the mouse host or by reconstitution of a normal reproductive regulatory axis supplied by functional porcine testicular xenografts. These studies demonstrate the usefulness of xenografting for the study of testicular pathology.

Spermatogenesis can occur in testis tissue from immature bulls ectopically grafted into mouse hosts; however, efficiency of sperm production is lower than in other donor species. To elucidate a possible mechanism for the impaired spermatogenesis in bovine testis xenografts, germ cell fate and xenograft development were investigated at different time points and compared with testis tissue from age-matched calves as controls. Histologically, an initial decrease in germ cell number was noticed in xenografts recovered up to 2 months post-grafting without an increase in germ cell apoptosis. From 2 months onward, the number of germ cells increased. In contrast, a continuous increase in germ cell number was seen in control tissue. Pachytene spermatocytes were observed in some grafts before 4 months, whereas in the control tissue they were not present until 5 months of age. Beyond 4 months post-grafting spermatogenesis appeared to be arrested at the pachytene spermatocyte stage in most grafts. Elongated spermatids were observed between 6 and 8 months post-grafting, similar to the controls, albeit in much lower numbers. Lumen formation started earlier in grafts compared with controls and by 6 months post-grafting tubules with extensively dilated lumen were observed. A donor effect on efficiency of spermatogenesis was also observed. These results indicate that the low efficiency of sperm production in bovine xenografts is due to an initial deficit of germ cells and impaired meiotic and post-meiotic differentiation. The characterization of spermatogenic efficiency will provide the basis to understand the control of spermatogenesis in testis grafts.

The efficiency of germ cell transplantation, the procedure of transferring germ cells from a donor male into the testes of recipient males, can be greatly increased by reduction of endogenous germ cells in recipient animals. To develop effective methods for suppression of endogenous spermatogenesis in potential pig and goat recipients, we either administered busulfan to pregnant sows or irradiated the testes of immature goats. Piglets from sows treated twice with busulfan (7.5 mg/kg) at days 98 and 108 of gestation showed reduced gonocyte numbers at 2, 4, and 8 weeks of age and reduced initiation of spermatogenesis at 16 weeks of age. For goats, groups of 3 kids at 1, 5, or 9.5 weeks of age received fractionated irradiation of the testes with 3 doses of 2 Gy on 3 consecutive days. At 2 months after irradiation, 5%-10% of seminiferous tubule cross sections contained pachytene spermatocytes, compared with 50%-100% in controls. At 3 months after irradiation, spermatozoa appeared in 20% of tubule cross sections in all treated goats and in 100% of tubules in control goats. By 6 months after irradiation, spermatogenesis had recovered in 60% of tubules in goats treated at 5 or 9.5 weeks of age but in only 29% of tubules after treatment at 1 week of age. Therefore, late gestation in utero treatment of pigs with low doses of busulfan and testicular irradiation of goats at 1 week of age will result in a reduction in the endogenous germ cell population that could facilitate donor cell colonization after germ cell transplantation.

Loss of genetic diversity because of infertility or the premature death of valuable individuals is a significant problem in the conservation of rare and endangered felid species, as well as in the maintenance of lines of cats used to study inherited feline and human disease. Attempts to overcome loss of genetic diversity have focused on freezing sperm; however, sperm cannot be collected from immature males. Previously, we reported completion of spermatogenesis in testis tissue from newborn pigs and goats grafted ectopically into host mice. The objective of this study was to extend the technique of testis tissue xenografting to the domestic cat as a model for felid species. Testes from 1- to 5-week-old domestic shorthaired kittens (n = 9) were cut into small fragments (about 0.5-1 mm3 each), and up to 8 fragments were grafted under the back skin of each castrated immunodeficient host mouse (n = 16). Histologic examination of the testis xenografts was performed between 5 and 54 weeks posttransplantation. At the time of grafting, the seminiferous cords of the donor testis tissue contained only immature Sertoli cells and gonocytes. At 14 weeks after grafting, tubular expansion was evidently caused by the proliferation of Sertoli cells and tubular lumen formation. By 18 weeks after transplantation, the seminiferous epithelium contained spermatocytes, and by 20 weeks, round spermatids were the most advanced types of germ cells. By 36 weeks after transplantation, xenografts of cat testis tissue had completed spermatogenesis. These results demonstrate the potential of xenografting to achieve full spermatogenesis in testis tissue from kittens. Therefore, sperm production in a mouse host can provide an alternative for germ line preservation from immature felids where sperm cryopreservation is not an option.

Spermatogenesis is a productive and highly organized process that generates virtually unlimited numbers of sperm during adulthood. Continuous proliferation and differentiation of germ cells occur in a delicate balance with other testicular compartments, especially the supporting Sertoli cells. Many complex aspects of testis function in humans and large animals have remained elusive because of a lack of suitable in vitro or in vivo models. Germ cell transplantation has produced complete donor-derived spermatogenesis in rodents but not in other mammalian species. Production of sperm in grafted tissue from immature mammalian testes and across species has not yet been accomplished. Here we report the establishment of complete spermatogenesis by grafting testis tissue from newborn mice, pigs or goats into mouse hosts. This approach maintains structural integrity and provides the accessibility that is essential for studying and manipulating the function of testes and for preserving the male germ line. Our results indicate that this approach is applicable to diverse mammalian species.

Simple Summary: One out of every 650 children is affected by cancer, and about half of survivors become permanently infertile due to the side effects of cancer treatments. Pre-treatment testicular biopsies can be collected and cryopreserved for future use to uphold biological fatherhood. Currently, the only safe approach to produce haploid germ cells is through in vitro spermatogenesis (IVS), which has only been fully achieved in mice. Pigs are a more suitable animal model for the study of testicular development and IVS, not only because of their anatomical and physiological similarities to humans but also their long prepubertal period. This study explored optimal conditions for the long-term culture of neonatal porcine testicular tissues as a first step in achieving IVS from pigs, as a model. In this study, we investigated the effects of testicular tissue size, tissue preparation method, and serum source in media on long-term culture of neonatal porcine testicular tissues. We showed that small intact testicular tissue fragments (~2 mg) cultured in knockout serum replacement could be effectively maintained in vitro for up to 4 weeks of culture. Moreover, the presence of viable germ cells after 4 weeks of culture indicates that this approach may be applicable for IVS in pigs. Long-term culture of testicular tissue has important applications, including the preservation of fertility potential of prepubertal boys undergoing gonadotoxic cancer treatment. This study was designed to define optimal conditions for the long-term culture of neonatal porcine testicular tissue as an animal model for preadolescent individuals. Testes from 1 wk old donor piglets were used to examine the effects of tissue fragment size (~2, 4, 6, or 8 mg), preparation method (intact, semi-digested, or physically dispersed fragments), and serum source in the media (fetal bovine serum—FBS—or knockout serum replacement—KSR). Testicular fragments were examined weekly for 4 weeks for tissue integrity, seminiferous cord density and morphology, and gonocyte counts. Testicular tissue integrity was dependent on fragment size and preparation method, where the smallest size (2 mg, p < 0.05) and intact preparation method were advantageous (p < 0.05). Seminiferous cord density decreased over the culture period (p < 0.05). Although the relative number of gonocytes decreased over time for all sizes and methods (p < 0.01), smaller intact fragments (2 and 4 mg) had greater numbers of gonocytes (p < 0.05). Our findings suggest that intact or physically dispersed testicular fragments of the smallest size (2 mg) cultured in KSR-supplemented media could be effectively maintained in vitro for the duration of 4 weeks. [ABSTRACT FROM AUTHOR]

Ectopic implantation of donor testis cell aggregates in recipient mice results in de novo formation or regeneration of testis tissue and, as such, provides a unique in vivo model for the study of testis development. However, currently the results are inconsistent and the efficiency of the model remains low. This study was designed to: (1) examine several factors that can potentially improve the consistency and efficiency of this model and (2) explore the use of ultrasound biomicroscopy (UBM) for the non-invasive in vivo evaluation of implants. Testis cell aggregates, containing ~40% gonocytes, from 1-week-old donor piglets were implanted under the back skin of immunodeficient mice through skin incisions using gel matrices or through subcutaneous injection without using gel matrices. The addition of gel matrices led to inconsistent tissue development; gelatin had the greatest development, followed by collagen, whereas agarose resulted in poor development. The results also depended on the implanted cell numbers since implants with 100 × 106 cells were larger than those with 50 × 106 cells. The injection approach for cell implantation was less invasive and resulted in more consistent and efficient testis tissue development. UBM provided promising results as a means of non-invasive monitoring of implants. We have previously shown that implantation of dissociated neonatal pig testis cells under the back skin of immunodeficient mice can lead to regeneration of functional testis tissue, although the results were inconsistent and inefficient. Here, by examining several factors, we dramatically improved the consistency, efficiency and predictability of the model. The testis cell implantation model can be used as an accessible system to study and manipulate testis tissue development and regeneration, and as a unique model for fertility preservation of prepubertal boys undergoing gonadotoxic cancer treatments. [ABSTRACT FROM AUTHOR]

Background: Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and in vitro manipulation for potential application in reproductive technologies. Using six experiments in a step-wise design, we examined the effects of several culture conditions on the maintenance, proliferation, and colony formation of porcine gonocytes. Testis cells from neonatal piglets were cultured for 7 d in DMEM supplemented with 10% fetal bovine serum. The examined culture conditions included using different cell seeding densities, gonocyte proportions, incubation temperatures, sampling strategies, and medium changing regimens. Results: Confluency of cells was optimal (>90% by ~6 d) when 3.0 x 104 testis cells/cm² containing ~40% gonocytes were used. Incubating the cells at 35 °C or 37 °C resulted in similar cell number and viability at confluency, but incubation at 35 °C resulted in a delayed confluency. In the first 2 d of culture, gonocytes remained mostly floating in the medium and gradually settled over the next 5 d. Consequently, not changing the medium for 7 d (as opposed to changing it every 2 d) led to a significant increase in the number of gonocyte colonies by reducing the loss of "floating gonocytes". Conclusion: We found that gonocytes require the presence of a critical minimum number of somatic cells for settlement, and can proliferate and form growing colonies even in a basic medium. Large numbers of viable gonocytes remain floating in the medium for several days. The optimized culture conditions in the present study included seeding with 3.0 x 104 testis cells/cm² containing ~40% gonocytes, incubating at 37 °C, and without changing the medium in the first week, which can result in improved colony formation of porcine gonocytes. [ABSTRACT FROM AUTHOR]

Abstract: The objective was to evaluate the long-term outcome of testis tissue xenografting from neonatal bison calves as a model for closely related rare or endangered ungulates. Testis tissue was collected postmortem from two newborn bison calves (Bison bison bison) and small fragments of the tissue were grafted under the back skin of immunodeficient recipient mice (n = 15 mice; eight fragments/mouse). Single xenograft samples were removed from representative recipient mice every 2 mo after grafting (for up to 16 mo). The retrieved xenografts were evaluated for seminiferous tubular density, tubular diameter, seminiferous tubular morphology, and identification of the most advanced germ cell type. Overall, 69% of the grafted testis fragments were recovered as xenografts. Xenografts weight increased (P < 0.02) approximately four-fold by 2 mo and 10-fold by 16 mo post-grafting. In testis xenografts, gradual maturational changes were evident, manifested as the first detection of the following at the times specified: seminiferous tubule expansion, 2 mo; spermatocytes, 6 mo; round spermatids, 12 mo; and elongated spermatids, 16 mo. Furthermore, there were differences between the two donor calves regarding the efficiency of spermatogenesis in xenografts. The timing of complete spermatogenesis approximately corresponded to the reported timing of sexual maturation in bison. This study demonstrated, apparently for the first time, that testis tissue xenografting from neonatal bison donors into recipient mice resulted in testicular maturation and complete development of spermatogenesis in the grafts. [Copyright &y& Elsevier]

Testis tissue xenografting and testis cell aggregate implantation from various donor species into recipient mice are novel models for the study and manipulation of testis formation and function in target species. Thus far, the analysis of such studies has been limited to surgical or post-mortem retrieval of samples. Here we used ultrasound biomicroscopy (UBM) to monitor the development of neonatal porcine testis grafts and implants in host mice for 24 wk, and to correlate UBM and (immuno)histologic changes. This led to long-term visualization of gradual changes in volume, dimension and structure of grafts and implants; detection of a 4 wk developmental gap between grafts and implants; and revelation of differences in implant development depending on the craniocaudal site of implantation on the back of host mice. Our data support the reliability and precision of UBM for longitudinal study of transplants, which eliminates the need for frequent surgical sampling. [ABSTRACT FROM AUTHOR]