Use Of Spermatogonial Stem Cells Research Articles

Identification and Manipulation of Spermatogonial Stem Cells with the Aim of Inducing Spermatogenesis in Vitro.

Assisted reproduction techniques for infertile men with non-obstructive azoospermia require a sufficient number of functional germ cells produced in vitro. Understanding the mechanisms that allow the resumption of spermatogenesis outside the testicular environment is crucial for fertility preservation in these patients. A review of the literature was conducted using databases such as PubMed, Scopus and Web of Science, with keywords including "spermatogonial stem cell," "germ cells," "male factor infertility," and "enrichment and propagation of SSCs in vitro." Currently, two models-"in vivo" and "in vitro"-have been developed for producing haploid germ cells. The "in vivo" models include spermatogonial stem cell transplantation and testicular xenograft techniques. In contrast, the "in vitro" models consist of conventional culture systems, organ culture, and three-dimensional culture systems, all designed to induce spermatogenesis in vitro. These culture systems enable the simulation of the seminiferous epithelium in vitro, which facilitates better regulation of cell-signaling pathways that control the self-renewal and differentiation of SSCs. This review provides up-to-date information on the organization of SSCs, focusing on the identification, proliferation, and differentiation of spermatogonia in vitro.

Comparing the adult and pre-pubertal testis: Metabolic transitions and the change in the spermatogonial stem cell metabolic microenvironment.

Survivors of childhood cancer often suffer from infertility. While sperm cryopreservation is not feasible before puberty, the patient's own spermatogonial stem cells could serve as a germ cell reservoir, enabling these patients to father their own children in adulthood through the isolation, in vitro expansion, and subsequent transplantation of spermatogonial stem cells. However, this approach requires large numbers of stem cells, and methods for successfully propagating spermatogonial stem cells in the laboratory are yet to be established for higher mammals and humans. The improvement of spermatogonial stem cell culture requires deeper understanding of their metabolic requirements and the mechanisms that regulate metabolic homeostasis. This review gives a summary on our knowledge of spermatogonial stem cell metabolism during maintenance and differentiation and highlights the potential influence of Sertoli cell and stem cell niche maturation on spermatogonial stem cell metabolic requirements during development. Fetal human spermatogonial stem cell precursors, or gonocytes, migrate into the seminiferous cords and supposedly mature to adult stem cells within the first year of human development. However, the spermatogonial stem cell niche does not fully differentiate until puberty, when Sertoli cells dramatically rearrange the architecture and microenvironment within the seminiferous epithelium. Consequently, pre-pubertal and adult spermatogonial stem cells experience two distinct niche environments potentially affecting spermatogonial stem cell metabolism and maturation. Indeed, the metabolic requirements of mouse primordial germ cells and pig gonocytes are distinct from their adult counterparts, and novel single-cell RNA sequencing analysis of human and porcine spermatogonial stem cells during development confirms this metabolic transition. Knowledge of the metabolic requirements and their changes and regulation during spermatogonial stem cell maturation is necessary to implement laboratory-based techniques and enable clinical use of spermatogonial stem cells. Based on the advancement in our understanding of germline metabolism circuits and maturation events of niche cells within the testis, we propose a new definition of spermatogonial stem cell maturation and its amendment in the light of metabolic change.

Viral Transduction of Mammalian Spermatogonial Stem Cells.

Lentiviral vectors have been major tools for genetic manipulation of spermatogonial stem cells (SSCs) in vitro. Adeno-associated viral vectors are promising emerging tools for in vivo SSC transduction that are less invasive, compared to lentivirus, since AAV DNA is not integrated into the host genome and the host genome remains intact. In this chapter, we describe protocols using lentiviral and adeno-associated viral vectors to transduce SSCs in vitro and vivo, respectively.

The use of spermatogonial stem cells to correct a mutation causing meiotic arrest.

The use of spermatogonial stem cells to correct a mutation causing meiotic arrest.

Stem cell-based therapies for fertility preservation in males: Current status and future prospects.

With the decline in male fertility in recent years, strategies for male fertility preservation have received increasing attention. In this study, by reviewing current treatments and recent publications, we describe research progress in and the future directions of stem cell-based therapies for male fertility preservation, focusing on the use of spermatogonial stem cells (SSCs), SSC niches, SSC-based testicular organoids, other stem cell types such as mesenchymal stem cells, and stem cell-derived extracellular vesicles. In conclusion, a more comprehensive understanding of the germ cell microenvironment, stem cell-derived extracellular vesicles, and testicular organoids will play an important role in achieving male fertility preservation.

Stage-specific embryonic antigen 4 is a membrane marker for enrichment of porcine spermatogonial stem cells.

Spermatogonial stem cells (SSCs), as tissue-specific stem cells, are capable of both self-renewal and differentiation and supporting the continual and robust spermatogenesis for male fertility. As a rare sub-fraction of undifferentiated spermatogonia, SSCs share most molecular markers with the progenitor spermatogonia. Thus, the heterogeneity of the progenitor cells often obscures the characteristics of stem cells. Distinguishing SSCs from the progenitors is of paramount importance to understand the regulatory mechanisms governing their actions. The present study was designed to reveal that SSEA4 can be a marker for putative porcine SSCs that distinguished it from the progenitors and to build a sorting program for efficient enrichment of porcine SSCs. To explore expression of SSEA4 within the undifferentiated spermatogonial population, we performed co-immunofluorescent staining for SSEA4 and common molecular markers (VASA, DBA, PLZF, c-KIT, and SOX9) in the 7-, 90-, and 150-day-old porcine testicular tissues. SSEA4-positive cells were isolated from the 90-day-old porcine testes by flow cytometry. Immunofluorescent, RNA-sequencing, and transplantation analysis were used to reveal that SSEA4-positive fraction holds the stem cell capacity. We found that SSEA4 was expressed in a rare sub-fraction of porcine undifferentiated spermatogonia, and RNA-sequencing analysis revealed that the genes for stem cell maintenance and SSC-specific markers (ID4 and PAX7) were up-regulated in the SSEA4-sorted fraction, compared with undifferentiated spermatogonia. In addition, germ cell transplantation assay demonstrated that SSEA4-positive spermatogonia colonized in the recipient testicular tubules. Sorting of the undifferentiated spermatogonia with anti-SSEA4 antibody resulted in a 2.5-fold enrichment of SSCs compared with the germ cell gate group, and 21-fold enrichment of SSCs compared with the SSEA4-negative spermatogonia group. Our findings revealed that SSEA4 is a new surface marker for porcine undifferentiated spermatogonia. This finding helps to elucidate the characteristics of porcine SSCs and facilitates the culture and manipulation of SSCs.

The study and manipulation of spermatogonial stem cells using animal models.

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.

Oncofertility in adult and pediatric populations: options and barriers.

Cancer and its treatments can affect fertility in a variety of ways, and recent advances in cancer detection and treatment have led to an increasing number of cancer survivors for whom future fertility is a primary concern. Oncofertility is the study of interactions between cancer, anti-cancer therapy, fertility, and reproductive health. Fertility preservation aims to optimize fertility potential before initiation of gonadotoxic therapies. Sperm cryopreservation from an ejaculated sample is the gold standard for adults and post-pubertal adolescents, though added maneuvers such as medical therapy, penile vibratory stimulation, and electroejaculation can be employed when appropriate. When all these approaches fail, testicular sperm extraction can be used to obtain and cryopreserve testicular sperm from the azoospermic patient. Fertility preservation in the pre-pubertal pediatric patient is still experimental, but recent scientific breakthroughs with use of spermatogonial stem cells and testicular tissue transplantation offer great promise for the future. While there may be several practical, cultural, religious, and other barriers to fertility preservation, the establishment of a dedicated fertility preservation team can help to overcome these obstacles and optimize the utilization of fertility preservation in cancer patients of all ages.

Possible use of spermatogonial stem cells in the treatment of male infertility

Spermatogonial stem cells, which are already present at birth in the testicles, are the progenitors of male gametes. These cells cannot produce mature sperm before puberty due to their dependence on hormonal stimuli. This feature of the reproductive system limits preservation of fertility only to males who can produce an ejaculate. Therefore, the use of cancer treatment which can lead to fertility loss has made sperm cryopreservation a standard practice. Prepubertal cancer boys – who are prescribed chemotherapy that is toxic to their reproductive system – are deprived of this fertility management procedure. This review focuses on the problem of obtaining and preserving spermatogonial stem cells for future transplantation to restore spermatogenesis. Development of these methods is becoming increasingly urgent due to higher survival rates in childhood cancer over the past decades thanks to improvements in diagnosis and effective treatment. Restoring and preserving fertility using spermatogonial stem cells may be the only option for such patients.

Clearing, immunofluorescence, and confocal microscopy for the three-dimensional imaging of murine testes and study of testis biology

Optical clearing techniques provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three-dimensional structure of intact biological systems. There is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high-impact applications in fertility medicine and reproductive biotechnology. The objective of our study is to apply optical clearing, immunofluorescence, and confocal microscopy to testicular tissue in order to reconstruct its three-dimensional microstructure in intact samples. We used Triton-X/DMSO clearing in combination with refractive index matching to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C-terminal hydrolase was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. We were able to successfully clear testicular tissue and utilize immunofluorescent probes. Additionally, we successfully visualized the histological compartments of testicular tissue in three-dimensional reconstructions. Optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the macro-scale perspective of the intact system. Considering the importance of the murine model, our developed method represents a significant contribution to the field of male reproductive biology, enabling the study of testicular function.

In Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated Viruses

SummaryAdeno-associated virus (AAV) penetrates the blood-brain barrier, but it is unknown whether AAV penetrates other tight junctions. Genetic manipulation of testis has been hampered by the basement membrane of seminiferous tubules and the blood-testis barrier (BTB), which forms between Sertoli cells and divides the tubules into basal and adluminal compartments. Here, we demonstrate in vivo genetic manipulation of spermatogonial stem cells (SSCs) and their microenvironment via AAV1/9. AAV1/9 microinjected into the seminiferous tubules penetrated both the basement membrane and BTB, thereby transducing not only Sertoli cells and SSCs but also peritubular cells and Leydig cells. Moreover, when congenitally infertile KitlSl/KitlSl-d mouse testes with defective Sertoli cells received Kitl-expressing AAVs, spermatogenesis regenerated and offspring were produced. None of the offspring contained the AAV genome. Thus, AAV1/9 allows efficient germline and niche manipulation by penetrating the BTB and basement membrane, providing a promising strategy for the development of gene therapies for reproductive defects.

Transfection of early and late stage sheep spermatogonial stem cells in culture

The genetic manipulation of spermatogonial stem cells (SSCs) can be used as an alternative to somatic cell nuclear transfer method for the production of transgenic animals. SSCs are now in vitro cultured and transplanted in sheep, however, there are no known protocol for DNA transfection of sheep SSCs. The aim of present study was to define the optimal transfection conditions of spermatogonial stem cells (SSCs) in early and late ovine SSC colony formation stages in culture. SSCs were isolated from the slaughterhouse ram testis tissue using a two-step enzymatic digestion process. Results showed that, 2 µl of DNA with 0.5 µg of Lipofectamin or Turbofect were able to transfect SSC colonies at late stage. Since the colonies of SSCs were not in logarithmic growth phase, around 15% of colonies were transfected and no significant difference between Lipofectamin or Turbofect was observed. However, more cells were transfected on early stages of SSC colony formation (7th days), especially when Turbofect was used (around 40 and 45% for Lipofectamin and Turbofect, respectively). Although the early stages SSCs were more suitable for transfection, but the formation of colonies were impaired on transfected cells.

Optical Clearing and 3D‐Histology of Murine Testis

Optical clearing techniques (e.g., CLARITY, Scale, SeeDB, and 3DISCO) provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three‐dimensional structure of intact biological systems. Although these methods have been used primarily to investigate nervous system tissues, there is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high‐impact applications in fertility medicine and reproductive biotechnology. Therefore, the objective of our study is to apply optical clearing and immunofluorescence to testicular tissue in order to reconstruct its three‐dimensional microstructure in intact large samples. We used Triton‐X detergent‐based clearing in combination with Refractive Index Matching Solution to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin (SMA) was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C‐terminal hydrolase (UCHL1) was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. Our results demonstrate that we were able to successfully clear testicular tissue and utilize immunofluorescent probes. Even with a basic confocal microscope and conventional objective lenses, we were able to image through as much as 500 microns of cleared tissue (the mechanical limit of our microscope). Moreover, we successfully visualized the histological compartments of testicular tissue in three‐dimensional reconstructions. The anti‐SMA antibody labeled the peritubular myoid cells in striking detail and exhibited their characteristic polygonal morphology. The antibody against UCHL1 provided clear visualization of mouse Sertoli cells and spermatogonia. When complemented with DAPI nuclear staining, the progressive stages of spermatogenesis could be observed proceeding from the basal compartment to the adluminal compartment of the seminiferous epithelium. In summary, optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the large‐scale perspective of the intact system.Support or Funding InformationThis research was generously funded by the Arizona Alzheimer's Consortium and Midwestern University.

The Use of Spermatogonial Stem Cells In Cancer-Induced Infertility

The Use of Spermatogonial Stem Cells In Cancer-Induced Infertility

Beyond the mouse monopoly: studying the male germ line in domestic animal models.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and essential to maintain the continuous production of spermatozoa after the onset of puberty in the male. The study of the male germ line is important for understanding the process of spermatogenesis, unravelling mechanisms of stemness maintenance, cell differentiation, and cell-to-cell interactions. The transplantation of SSCs can contribute to the preservation of the genome of valuable individuals in assisted reproduction programs. In addition to the importance of SSCs for male fertility, their study has recently stimulated interest in the generation of genetically modified animals because manipulations of the male germ line at the SSC stage will be maintained in the long term and transmitted to the offspring. Studies performed mainly in the mouse model have laid the groundwork for facilitating advancements in the field of male germ line biology, but more progress is needed in nonrodent species in order to translate the technology to the agricultural and biomedical fields. The lack of reliable markers for isolating germ cells from testicular somatic cells and the lack of knowledge of the requirements for germ cell maintenance have precluded their long-term maintenance in domestic animals. Nevertheless, some progress has been made. In this review, we will focus on the state of the art in the isolation, characterization, culture, and manipulation of SSCs and the use of germ cell transplantation in domestic animals.

Generation of transgenic mice by exploiting spermatogonial stem cells in vivo.

The protocols in this chapter describe two techniques for the generation of transgenic mice by in vivo manipulation of spermatogonial stem cells (SSCs) with a high rate of success. SSCs in prepubescent animals can either be infected in vivo with recombinant lentiviruses expressing the transgene of interest or DNA can be injected into the testis followed by the application of an electric current resulting in integration of the linearized DNA containing a transgene downstream of the appropriate promoter into SSCs. All male pre-founder mice produced transgenic pups using both protocols with the transgene being heritable. Further, the pre-founder mice could be used in multiple mating experiments resulting in the generation of multiple progeny. These protocols could be extended to perform over-expression/knockdown screens in vivo using bar-coded lentiviruses/plasmid constructs, thus permitting the design of genetic screens in the mouse. Further, these protocols could be adapted to achieve transgenesis in other laboratory animals resulting in the generation of model systems that closely approximate human development and disease.

Spermatogonial stem cells from domestic animals: progress and prospects

Spermatogenesis, an elaborate and male-specific process in adult testes by which a number of spermatozoa are produced constantly for male fertility, relies on spermatogonial stem cells (SSCs). As a sub-population of undifferentiated spermatogonia, SSCs are capable of both self-renewal (to maintain sufficient quantities) and differentiation into mature spermatozoa. SSCs are able to convert to pluripotent stem cells during in vitro culture, thus they could function as substitutes for human embryonic stem cells without ethical issues. In addition, this process does not require exogenous transcription factors necessary to produce induced-pluripotent stem cells from somatic cells. Moreover, combining genetic engineering with germ cell transplantation would greatly facilitate the generation of transgenic animals. Since germ cell transplantation into infertile recipient testes was first established in 1994, in vivo and in vitro study and manipulation of SSCs in rodent testes have been progressing at a staggering rate. By contrast, their counterparts in domestic animals, despite the failure to reach a comparable level, still burgeoned and showed striking advances. This review outlines the recent progressions of characterization, isolation, in vitro propagation, and transplantation of spermatogonia/SSCs from domestic animals, thereby shedding light on future exploration of these cells with high value, as well as contributing to the development of reproductive technology for large animals.

201 TRANSPLANTATION OF SSEA-1+ AND SSEA-4+ SPERMATOGONIAL CELL SUBPOPULATIONS IN UNTREATED SEXUALLY IMMATURE DOMESTIC CATS

Captive breeding efforts in felids, including assisted reproduction techniques, have had varied success depending on species. Spermatogonial stem cells (SSC), comprising a small percentage of germ cells in the testis, are progenitor cells with the ability to both self-renew and differentiate into spermatozoa throughout the life of the male. Manipulation of SSC for transplantation (SSCT) may allow the propagation of genetically important males, as demonstrated by the production of ocelot sperm following transplantation of ocelot mixed germ cells to domestic cat testes (Silva et al. 2012 J. Androl. 33, 264–276). Using specific cell surface markers, SSC have been isolated from mixed germ cells in several other species for SSCT, culture, and studying germ cell biology; however, expression may differ with species. Using the domestic cat as a model for exotic felids, we recently began evaluating the expression of surface markers in feline SSC. Previously, we determined that pluripotent markers SSEA-1, SSEA-4, TRA-1–60, and TRA-1–81 were more specific to cat spermatogonia than SSC surface markers GFRα1 and GPR125 used in other species, with SSEA-1 and SSEA-4 expressed in the fewest cells (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222; Powell et al. 2012 Reprod. Fertil. Dev. 25, 290–291). Our current goal was to 1) confirm the presence of SSC within SSEA-1+ and SSEA-4+ cell populations by the ability to colonize following SSCT; 2) compare the effectiveness of transplanting SSC purified by flow cytometry versus mixed germ cells; and 3) show that depletion of endogenous germ cells before SSCT, usually performed by irradiation or chemotherapy in other studies, is not necessary when using sexually immature recipients. Mixed germ cells from 8 to 12 adult testes were pooled, stained for SSEA-1 or SSEA-4, and sorted by flow cytometry. SSEA-1+, SSEA-4+, or mixed germ cells were then labelled with the membrane dye PKH26 (Sigma MINI26) and injected into the testes of six 5-month-old and six 6-month-old cats at the site of the external rete testis after carefully microdissecting the head of the epididymis away from the testis. Injections contained an average of 230 000 sorted or 10 × 106 mixed germ cells suspended in 80 μL of DMEM/F12 + 3 μL of Trypan Blue (T8154, Sigma, St. Louis, MO, USA). Testes were harvested 10 to 12 weeks post-SSCT and bisected, half snap-frozen for later cryosectioning and the other half enzymatically digested to loosen seminiferous tubules for immediate evaluation. Fluorescence was detected in the testes of both 6-month-old males that received injections of mixed germ cells, one 6-month-old male injected with SSEA-4+ cells, and two 5-month-old males, one injected with SSEA-4+ cells and one with SSEA-1+ cells. Results indicate that SSC are found in both SSEA-1+ and SSEA-4+ cell populations, but that purification of SSC is not necessary for successful SSCT. Additionally, SSC colonization in cats is possible without depletion of endogenous cells in sexually immature recipients.

Effect of vitamin C on growth of caprine spermatogonial stem cells in vitro

The genetic manipulation of spermatogonial stem cells (SSCs) can be used for the production of transgenic animals in a wide range of species. However, this technology is limited by the absence of an ideal culture system in which SSCs can be maintained and proliferated, especially in domestic animals like the goat. The aim of this study therefore was to investigate whether the addition of vitamin C (Vc) in cell culture influences the growth of caprine SSCs. Various concentrations of Vc (0, 5, 10, 25, 40, and 50 μg/mL−1) were added to SSC culture media, and their effect on morphology and alkaline phosphatase activity was studied. The number of caprine SSC colonies and area covered by them were measured at 10 days of culture. The expression of various germ cell and somatic cell markers such as VASA, integrins, Oct-4, GATA-4, α-SMA, vimentin, and Thy-1 was studied to identify the proliferated cells using immunostaining analyses. Further, the intracellular reactive oxygen species (ROS) level was measured at the 3rd, 6th, and 9th day after culture, and expression of Bax, Bcl-2, and P53, factors involved in the regulation of apoptosis, were analyzed on the 7th day after culture using reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction. The results showed that the SSCs formed compact colonies and had unclear borders in the different Vc-supplemented groups at 10 days, and there were no major morphologic differences between the groups. The number and area of colonies were both the highest in the 40 μg/mL−1 Vc group. Differential expression of markers for germ cells, undifferentiated spermatogonia, and testis somatic cells was observed. Cultured germ cell clumps were found to have alkaline phosphatase activity regardless of the Vc dose. The number of Thy-1– and Oct-4–positive cells was the most in the 40 μg/mL−1 Vc group. Moreover, the level of ROS was dependent on the Vc dose and culture time. The Vc dose 40 μg/mL−1 was found to be optimum with regard to decreasing ROS generation, and increasing the expression of the antiapoptotic gene Bcl-2 and decreasing the expression of the proapoptotic genes Bax and P53. In conclusion, the addition of 40 μg/mL−1 Vc can maintain a certain physiological level of ROS, trigger the expression of the antiapoptosis gene Bcl-2, suppress the proapoptotic gene P53 and Bax pathway, and further promote the proliferation of caprine SSCs in vitro.

Possible Therapeutic Use of Spermatogonial Stem Cells in the Treatment of Male Infertility: A Brief Overview

Development of germ cells is a process starting in fetus and completed only in puberty. Spermatogonial stem cells maintain spermatogenesis throughout the reproductive life of mammals. They are undifferentiated cells defined by their ability to both self-renew and differentiate into mature spermatozoa. This self-renewal and differentiation in turn is tightly regulated by a combination of intrinsic gene expression as well as the extrinsic gene signals from the local tissue microenvironment. The human testis is prone to damage, either for therapeutic reasons or because of toxic agents from the environment. For preservation of fertility, patients who will undergo radiotherapy and/or chemotherapy have an attractive possibility to keep in store and afterwards make a transfer of spermatogonial stem cells. Germ cell transplantation is not yet ready for the human fertility clinic, but it may be reasonable for young cancer patients, with no other options to preserve their fertility. Whereas this technique has become an important research tool in rodents, a clinical application must still be regarded as experimental, and many aspects of the procedure need to be optimized prior to a clinical application in men. In future, a range of options for the preservation of male fertility will get a new significance.