DAZL Knockout Pigs as Recipients for Spermatogonial Stem Cell Transplantation.

Spermatogonial stem cell (SSC) transplantation into the testis of a germ cell (GC)-depleted surrogate allows transmission of donor genotype via donor-derived sperm produced by the recipient. Transplantation of gene-edited SSCs provides an approach to propagate gene-edited large animal models. DAZL is a conserved RNA-binding protein important for GC development, and DAZL knockout (KO) causes defects in GC commitment and differentiation. We characterized DAZL-KO pigs as SSC transplantation recipients. While there were GCs in 1-week-old (wko) KO, complete GC depletion was observed by 10 wko. Donor GCs were transplanted into 18 DAZL-KO recipients at 10-13 wko. At sexual maturity, semen and testes were evaluated for transplantation efficiency and spermatogenesis. Approximately 22% of recipient seminiferous tubules contained GCs, including elongated spermatids and proliferating spermatogonia. The ejaculate of 89% of recipients contained sperm, exclusively from donor origin. However, sperm concentration was lower than the wild-type range. Testicular protein expression and serum hormonal levels were comparable between DAZL-KO and wild-type. Intratesticular testosterone and Leydig cell volume were increased, and Leydig cell number decreased in transplanted DAZL-KO testis compared to wild-type. In summary, DAZL-KO pigs support donor-derived spermatogenesis following SSC transplantation, but low spermatogenic efficiency currently limits their use for the production of offspring.

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

BACKGROUND: 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. AIM: 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. RESULTS AND CONCLUSIONS: 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.

A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture.

Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Alternatively, testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, and in vitro culture systems are required to increase numbers of SSCs for treatment, demanding culture systems for SSC propagation. Using the pig as a non-rodent model, we developed culture systems to expand spermatogonia from immature testis tissue, comparing different feeders (Sertoli cells, peritubular myoid cells (PMCs) and pig fetal fibroblasts (PFFs)). Spermatogonia co-cultured with Sertoli cells, PMCs and PFFs had comparable rates of proliferation and apoptosis. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes are incorporated by spermatogonia, and inhibition of exosomal release reduces spermatogonial proliferation. Together, these results show that PMCs, PFFs and Sertoli cells promote spermatogonial proliferation in co-culture, with exosomal exchange representing one possible mechanism. Further characterization of exosomal cargo may ultimately allow the development of feeder-free culture systems for clinical use.

The Proliferation of Pre-Pubertal Porcine Spermatogonia in Stirred Suspension Bioreactors Is Partially Mediated by the Wnt/ß-Catenin Pathway.

Male survivors of childhood cancer are at risk of suffering from infertility in adulthood because of gonadotoxic chemotherapies. For adult men, sperm collection and preservation are routine procedures prior to treatment; however, this is not an option for pre-pubertal children. From young boys, a small biopsy may be taken before chemotherapy, and spermatogonia may be propagated in vitro for future transplantation to restore fertility. A robust system that allows for scalable expansion of spermatogonia within a controlled environment is therefore required. Stirred suspension culture has been applied to different types of stem cells but has so far not been explored for spermatogonia. Here, we report that pre-pubertal porcine spermatogonia proliferate more in bioreactor suspension culture, compared with static culture. Interestingly, oxygen tension provides an avenue to modulate spermatogonia status, with culture under 10% oxygen retaining a more undifferentiated state and reducing proliferation in comparison with the conventional approach of culturing under ambient oxygen levels. Spermatogonia grown in bioreactors upregulate the Wnt/ ß-catenin pathway, which, along with enhanced gas and nutrient exchange observed in bioreactor culture, may synergistically account for higher spermatogonia proliferation. Therefore, stirred suspension bioreactors provide novel platforms to culture spermatogonia in a scalable manner and with minimal handling.

Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells.

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1-/- mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1-/- mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1-/- compared to wild-type mice. Furthermore, cultured Uch-l1-/- SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

Unique metabolic phenotype and its transition during maturation of juvenile male germ cells.

Human male reproductive development has a prolonged prepubertal period characterized by juvenile quiescence of germ cells with immature spermatogonial stem cell (SSC) precursors (gonocytes) present in the testis for an extended period of time. The metabolism of gonocytes is not defined. We demonstrate with mitochondrial ultrastructure studies via TEM and IHC and metabolic flux studies with UHPLC-MS that a distinct metabolic transition occurs during the maturation to SSCs. The mitochondrial ultrastructure of prepubertal human spermatogonia is shared with prepubertal pig spermatogonia. The metabolism of early prepubertal porcine spermatogonia (gonocytes) is characterized by the reliance on OXPHOS fuelled by oxidative decarboxylation of pyruvate. Interestingly, at the same time, a high amount of the consumed pyruvate is also reduced and excreted as lactate. With maturation, prepubertal spermatogonia show a metabolic shift with decreased OXHPOS and upregulation of the anaerobic metabolism-associated uncoupling protein 2 (UCP2). This shift is accompanied with stem cell specific promyelocytic leukemia zinc finger protein (PLZF) protein expression and glial cell-derived neurotropic factor (GDNF) pathway activation. Our results demonstrate that gonocytes differently from mature spermatogonia exhibit unique metabolic demands that must be attained to enable their maintenance and growth in vitro.

Development and function of smooth muscle cells is modulated by Hic1 in mouse testis.

In mammalian testis, contractile peritubular myoid cells (PMCs) regulate the transport of sperm and luminal fluid, while secreting growth factors and extracellular matrix proteins to support the spermatogonial stem cell niche. However, little is known about the role of testicular smooth muscle cells during postnatal testicular development. Here we report age-dependent expression of hypermethylated in cancer 1 (Hic1; also known as ZBTB29) in testicular smooth muscle cells, including PMCs and vascular smooth muscle cells, in the mouse. Postnatal deletion of Hic1 in smooth muscle cells led to their increased proliferation and resulted in dilatation of seminiferous tubules, with increased numbers of PMCs. These seminiferous tubules contained fewer Sertoli cells and more spermatogonia, and fibronectin was not detected in their basement membrane. The expression levels of genes encoding smooth muscle contractile proteins, Acta2 and Cnn1, were downregulated in the smooth muscle cells lacking Hic1, and the seminiferous tubules appeared to have reduced contractility. These data imply a role for Hic1 in determining the size of seminiferous tubules by regulating postnatal smooth muscle cell proliferation, subsequently affecting spermatogenesis in adulthood.

Mammalian germ cells are determined after PGC colonization of the nascent gonad.

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL's absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis-after organogenesis has begun-in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer.

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.

Brinster Spermatogonial Stem Cell Transplantation 25th Anniversary Symposium.

The Symposium, co-sponsored by the Institute of Regenerative Medicine, the University Research Foundation, the Center for Research on Reproduction and Women's Health, the Penn Center for the Study of Epigenetics in Reproduction, and Penn Vet at the University of Pennsylvania, commemorated the 25th anniversary of the first publications describing spermatogonial stem cell (SSC) transplantation in mice. This transformative approach has propelled advances in our understanding of germ cell biology, has been translated to a variety of vertebrate species, and holds translational potential for fertility restoration in patients. The symposium opened with a lecture by Dr. Brinster reflecting on the origin of the work, as well as advances over the 25 years up to present ongoing studies. Following Dr. Brinster's remarks, 10 lectures were presented by distinguished scientists, including several of Dr. Brinster's former trainees and colleagues. The symposium closed with a keynote lecture by Dr. David Page. Topics ranged from aspects of basic SSC biology to applications in large animal models and potential translation to treating human male infertility. Many of the studies presented directly resulted from SSC transplantation technology highlighting its tremendous impact in advancing the field. The Symposium program and the lectures can be found at https://spark.adobe.com/page/jS0cDLzLHvOiJ.

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.

De novo morphogenesis of testis tissue: an improved bioassay to investigate the role of VEGF165 during testis formation.

De novo formation of testis tissue from single-cell suspensions allows manipulation of different testicular compartments before grafting to study testicular development and the spermatogonial stem cell niche. However, the low percentages of newly formed seminiferous tubules supporting complete spermatogenesis and lack of a defined protocol have limited the use of this bioassay. Low spermatogenic efficiency in de novo formed tissue could result from the scarcity of germ cells in the donor cell suspension, cell damage caused by handling or from hypoxia during tissue formation in the host environment. In this study, we compared different proportions of spermatogonia in the donor cell suspension and the use of Matrigel as a scaffold to support de novo tissue formation and spermatogenesis. Then, we used the system to investigate the role of vascular endothelial growth factor 165 (VEGF165) during testicular morphogenesis on blood vessel and seminiferous tubule formation, and on presence of germ cells in the de novo developed tubules. Our results show that donor cell pellets with 10×10(6) porcine neonatal testicular cells in Matrigel efficiently formed testis tissue de novo. Contrary to what was expected, the enrichment of the cell suspension with germ cells did not result in higher numbers of tubules supporting spermatogenesis. The addition of VEGF165 did not improve blood vessel or tubule formation, but it enhanced the number of tubules containing spermatogonia. These results indicate that spermatogenic efficiency was improved by the addition of Matrigel, and that VEGF165 may have a protective role supporting germ cell establishment in their niche.

Goat embryonic stem-like cell derivation and characterization.

Embryonic stem (ES) cells are derived from the inner cell masses of preimplantation embryos. ES cells are pluripotent cells with the capacity for long-term propagation and broad differentiation plasticity. These cells have an exceptional functional feature in that they can differentiate into all tissues and organs, including germ cells. Established ES cell lines have been generated in mouse, human, and nonhuman primate but derivation of ES cells in farm animals has been problematic. Several ES-like cell lines from farm animals have been reported to exhibit properties of pluripotency in vitro. However, only a few of them morphologically resemble ES cells, or express markers that are associated with established ES cell lines from mouse and humans. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. In this chapter, we describe methods for isolation of goat ES (gES) cell lines from in vivo-derived blastocysts and characterization of markers indicative of pluripotency. In addition, we outline differentiation of gES cells into all three germ layers in vivo by forming teratomas as a hallmark of pluripotency.

MiRNA-20 and mirna-106a regulate spermatogonial stem cell renewal at the post-transcriptional level via targeting STAT3 and Ccnd1.

Studies on spermatogonial stem cells (SSCs) are of unusual significance because they are the unique stem cells that transmit genetic information to subsequent generations and they can acquire pluripotency to become embryonic stem-like cells that have therapeutic applications in human diseases. MicroRNAs (miRNAs) have recently emerged as critical endogenous regulators in mammalian cells. However, the function and mechanisms of individual miRNAs in regulating SSC fate remain unknown. Here, we report for the first time that miRNA-20 and miRNA-106a are preferentially expressed in mouse SSCs. Functional assays in vitro and in vivo using miRNA mimics and inhibitors reveal that miRNA-20 and miRNA-106a are essential for renewal of SSCs. We further demonstrate that these two miRNAs promote renewal at the post-transcriptional level via targeting STAT3 and Ccnd1 and that knockdown of STAT3, Fos, and Ccnd1 results in renewal of SSCs. This study thus provides novel insights into molecular mechanisms regulating renewal and differentiation of SSCs and may have important implications for regulating male reproduction.

Viral transduction of male germline stem cells results in transgene transmission after germ cell transplantation in pigs.

Genetic modification of germline stem cells (GSCs) is an alternative approach to generate large transgenic animals where transgenic GSCs are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objective of the present study was to explore the application of viral vectors in delivering an enhanced green fluorescent protein (EGFP) transgene into GSCs for production of transgenic gametes through germ cell transplantation. Both adeno-associated virus (AAV)- and lentivirus (LV)-based vectors were effective in transducing pig GSCs, resulting in the production of transgenic sperm in recipient boars. Twenty-one boars treated with busulfan to deplete endogenous GSCs and nine nontreated boars received germ cell transplantation at 12 wk of age. Semen was collected from recipient boars from 5 to 7 mo posttransplantation when boars became sexually mature, and semen collection continued for as long as 5 yr for some boars. The percentage of ejaculates that were positive for the EGFP transgene ranged from 0% to 54.8% for recipients of AAV vector-transduced germ cells (n = 17) and from 0% to 25% for recipients of LV vector-transduced germ cells (n = 5). When semen from two AAV recipients was used for in vitro fertilization (IVF), 9.09% and 64.3% of embryos were transgenic. Semen collected from two LV-vector recipients produced 7.7% and 26.3% transgenic IVF embryos. Here, we not only demonstrated AAV-mediated GSC transduction in another large animal model (pigs) but also showed, to our knowledge for the first time, that LV-mediated GSC transduction resulted in transgene transmission in pigs.

Testicular tissue grafting and male germ cell transplantation.

Testicular tissue grafting and male germ cell transplantation are techniques that offer unprecedented opportunities to study testicular function and development. While testicular tissue grafting allows recapitulation of testis development and spermatogenesis from immature males of different mammalian species in recipient mice, germ cell transplantation results in donor-derived spermatogenesis in recipient testes.Testicular tissue grafting results in spermatogenesis from a wide variety of large animal donor species and is therefore an attractive way to study testis development and spermatogenesis and preserve fertility of immature males. Germ cell transplantation represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs) in a given donor cell population and serves as a valuable tool to study stem cell biology and spermatogenesis. In this chapter we provide detailed methodology to successfully perform both techniques.

Endocrine modulation of the recipient environment affects development of bovine testis tissue ectopically grafted in mice.

Testis tissue xenografting is a powerful approach for the study of testis development and spermatogenesis, and for fertility preservation in immature individuals. In bovine testis xenografts, maturation and spermatogenesis are inefficient when compared to other species. To evaluate if exogenous modulation of the endocrine milieu in recipient mice will affect spermatogenic efficiency in xenografts from newborn calves, recipient mice were treated with the GnRH antagonist acyline (5 mg/kg s.c. every 2 weeks) to reduce testosterone production in xenografts, or with 6-N-propyl-2-thiouracil (PTU, 0.1% in drinking water for 4 weeks), to induce transient hypothyroidism in recipient mice respectively. Both treatments altered developmental parameters of testis xenografts and reduced germ cell differentiation. While the effects of acyline treatment can be attributed to inhibition of GnRH and gonadotropin action, lower Sertoli cell numbers and decreased seminiferous tubule length observed after PTU treatment were opposite to effects reported previously in rats. Regardless of treatment, Sertoli cells underwent only partial maturation in xenografts as Müllerian inhibiting substance and androgen receptor expression were lower than in donor and adult tissue controls respectively. In conclusion, although treatments did not result in improvement of maturation of bovine testis xenografts, the current study demonstrates that exogenous modulation of the endocrine milieu to affect xenograft development in recipient mice provides an accessible model to study endocrine control of spermatogenesis in large donor species.

Suppression of spermatogenesis before grafting increases survival and supports resurgence of spermatogenesis in adult mouse testis.

OBJECTIVE: To test whether absence of complete spermatogenesis in mature testicular tissue before grafting will increase graft survival. DESIGN: Prospective experimental study. SETTING: Laboratory. ANIMAL(S): Donor testes were obtained from adult untreated mice, adult mice rendered cryptorchid, and adult mice treated with a GnRH antagonist (acyline). INTERVENTION(S): Donor testes were ectopically grafted to nude mice and recovered at three time points. MAIN OUTCOME MEASURE(S): Most advanced germ cell type and presence of spermatogonia were assessed. Donor testes and grafts were analyzed by histology and by immunocytochemistry for ubiquitin C-terminal hydrolase-L1 to mark germ cells. RESULT(S): Suppression of spermatogenesis by inducing cryptorchidism or acyline treatment resulted in improved survival of grafted tissue compared with controls and recovery of complete spermatogenesis, whereas control testis grafts mostly degenerated and did not restore complete spermatogenesis. CONCLUSION(S): These results indicate that complete spermatogenesis at the time of grafting has a negative effect on graft survival. Grafting of adult testis tissue from donors with suppressed spermatogenesis leads to spermatogenic recovery and may provide a tool to study and preserve fertility and for conservation of genetic resources in individuals that lack complete germ cell differentiation.

Germ cell transplantation and testis tissue xenografting in mice.

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 1994(1,2). These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal(2). The use of donor males carrying the bacterial ß-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals(1). Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located(3). It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation(4,5), to study Sertoli cell-germ cell interaction(6,7), SSC homing and colonization(3,8), as well as SSC self-renewal and differentiation(9,10). Germ cell transplantation is also feasible in large species(11). In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species(12). An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm(13). Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice(14).

Isolation, characterization, and culture of human spermatogonia.

This study was designed to isolate, characterize, and culture human spermatogonia. Using immunohistochemistry on tubule sections, we localized GPR125 to the plasma membrane of a subset of the spermatogonia. Immunohistochemistry also showed that MAGEA4 was expressed in all spermatogonia (A(dark), A(pale), and type B) and possibly preleptotene spermatocytes. Notably, KIT was expressed in late spermatocytes and round spermatids, but apparently not in human spermatogonia. UCHL1 was found in the cytoplasm of spermatogonia, whereas POU5F1 was not detected in any of the human germ cells. GFRA1 and ITGA6 were localized to the plasma membrane of the spermatogonia. Next, we isolated GPR125-positive spermatogonia from adult human testes using a two-step enzymatic digestion followed by magnetic-activated cell sorting. The isolated GPR125-positive cells coexpressed GPR125, ITGA6, THY1, and GFRA1, and they could be cultured for short periods of time and exhibited a marked increase in cell numbers as shown by a proliferation assay. Immunocytochemistry of putative stem cell genes after 2 wk in culture revealed that the cells were maintained in an undifferentiated state. MAPK1/3 phosphorylation was increased after 2 wk of culture of the GPR125-positive spermatogonia compared to the freshly isolated cells. Taken together, these results indicate that human spermatogonia share some but not all phenotypes with spermatogonial stem cells (SSCs) and progenitors from other species. GPR125-positive spermatogonia are phenotypically putative human SSCs and retain an undifferentiated status in vitro. This study provides novel insights into the molecular characteristics, isolation, and culture of human SSCs and/or progenitors and suggests that the MAPK1/3 pathway is involved in their proliferation.