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Spermatogonial stem cells (SSCs) undergo self-renewal division to sustain spermatogenesis. Although it is possible to derive SSC cultures in most mouse strains, SSCs from a 129 background never proliferate under the same culture conditions, suggesting they have distinct self-renewal requirements. Here, we established long-term culture conditions for SSCs from mice of the 129 background (129 mice). An analysis of 129 testes showed significant reduction of GDNF and CXCL12, whereas FGF2, INHBA and INHBB were higher than in testes of C57BL/6 mice. An analysis of undifferentiated spermatogonia in 129 mice showed higher expression of Chrna4, which encodes an acetylcholine (Ach) receptor component. By supplementing medium with INHBA and Ach, SSC cultures were derived from 129 mice. Following lentivirus transduction for marking donor cells, transplanted cells re-initiated spermatogenesis in infertile mouse testes and produced transgenic offspring. These results suggest that the requirements of SSC self-renewal in mice are diverse, which has important implications for understanding self-renewal mechanisms in various animal species., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Spermatogonial stem cell (SSC) transplantation is a valuable tool for studying stem cell-niche interaction. However, the conventional approach requires the removal of endogenous SSCs, causing damage to the niche. Here we introduce WIN18,446, an ALDH1A2 inhibitor, to enhance SSC colonization in nonablated recipients. Pre-transplantation treatment with WIN18,446 induced abnormal claudin protein expression, which comprises the blood-testis barrier and impedes SSC colonization. Consequently, WIN18,446 increased colonization efficiency by 4.6-fold compared with untreated host. WIN18,446-treated testes remained small despite the cessation of WIN18,446, suggesting its irreversible effect. Offspring were born by microinsemination using donor-derived sperm. While WIN18,446 was lethal to busulfan-treated mice, cyclophosphamide- or radiation-treated animals survived after WIN18,446 treatment. Although WIN18,446 is not applicable to humans due to toxicity, similar ALDH1A2 inhibitors may be useful for SSC transplantation into nonablated testes, shedding light on the role of retinoid metabolism on SSC-niche interactions and advancing SSC research in animal models and humans., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) are generated from NADPH oxidases and mitochondria; they are generally harmful for stem cells. Spermatogonial stem cells (SSCs) are unique among tissue-stem cells because they undergo ROS-dependent self-renewal via NOX1 activation. However, the mechanism by which SSCs are protected from ROS remains unknown. Here, we demonstrate a crucial role for Gln in ROS protection using cultured SSCs derived from immature testes. Measurements of amino acids required for SSC cultures revealed the indispensable role of Gln in SSC survival. Gln induced Myc expression to drive SSC self-renewal in vitro, whereas Gln deprivation triggered Trp53-dependent apoptosis and impaired SSC activity. However, apoptosis was attenuated in cultured SSCs that lacked NOX1. In contrast, cultured SSCs lacking Top1mt mitochondria-specific topoisomerase exhibited poor mitochondrial ROS production and underwent apoptosis. Gln deprivation reduced glutathione production; supra-molar Asn supplementation allowed offspring production from SSCs cultured without Gln. Therefore, Gln ensures ROS-dependent SSC-self-renewal by providing protection against NOX1 and inducing Myc., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Spermatogonial transplantation has been used as a standard assay for spermatogonial stem cells (SSCs). After transplantation into the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) between Sertoli cells and settle in a niche. Unlike in the repair of other self-renewing systems, SSC transplantation is generally performed after complete destruction of endogenous spermatogenesis. Here, we examined the impacts of recipient conditioning on SSC homing. Germ cell ablation downregulated the expression of glial cell line-derived neurotrophic factor, which has been shown to attract SSCs to niches, implying that nonablated niches would attract SSCs more efficiently. As expected, SSCs colonized nonablated testes when transplanted into recipients with the same genetic background. Moreover, although spermatogenesis was arrested at the spermatocyte stage in Cldn11-deficient mice without a BTB, transplantation not only enhanced donor colonization but also restored normal spermatogenesis. The results show promise for the development of a new transplantation strategy to overcome male infertility., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The spermatogonial stem cell (SSC) population in testis is small, and the lack of SSC markers has severely handicapped research on these cells. During our attempt to identify genes involved in SSC aging, we found that CD2 is expressed in cultured SSCs. Flow cytometric analysis and spermatogonial transplantation experiments showed that CD2 is expressed in SSCs from mature adult mouse testes. Cultured SSCs transfected with short hairpin RNAs (shRNAs) against CD2 proliferated poorly and showed an increased frequency of apoptosis. Moreover, functional analysis of transfected cells revealed impairment of SSC activity. Fluorescence activated cell sorting and spermatogonial transplantation experiments showed that CD2 is expressed not only in mouse but also in rat SSCs. The results indicate that CD2 is a novel SSC surface marker conserved between mouse and rat SSCs.

The blood-testis barrier (BTB) is thought to be indispensable for spermatogenesis because it creates a special environment for meiosis and protects haploid cells from the immune system. The BTB divides the seminiferous tubules into the adluminal and basal compartments. Spermatogonial stem cells (SSCs) have a unique ability to transmigrate from the adluminal compartment to the basal compartment through the BTB upon transplantation into the seminiferous tubule. Here, we analyzed the role of Cldn11 , a major component of the BTB, in spermatogenesis using spermatogonial transplantation. Cldn11 -deficient mice are infertile due to the cessation of spermatogenesis at the spermatocyte stage. Cldn11 -deficient SSCs failed to colonize wild-type testes efficiently, and Cldn11 -deficient SSCs that underwent double depletion of Cldn3 and Cldn5 showed minimal colonization, suggesting that claudins on SSCs are necessary for transmigration. However, Cldn11 -deficient Sertoli cells increased SSC homing efficiency by >3-fold, suggesting that CLDN11 in Sertoli cells inhibits transmigration of SSCs through the BTB. In contrast to endogenous SSCs in intact Cldn11 -deficient testes, those from WT or Cldn11 -deficient testes regenerated sperm in Cldn11 -deficient testes. The success of this autologous transplantation appears to depend on removal of endogenous germ cells for recipient preparation, which reprogrammed claudin expression patterns in Sertoli cells. Consistent with this idea, in vivo depletion of Cldn3 / 5 regenerated endogenous spermatogenesis in Cldn11 -deficient mice. Thus, coordinated claudin expression in both SSCs and Sertoli cells expression is necessary for SSC homing and regeneration of spermatogenesis, and autologous stem cell transplantation can rescue congenital defects of a self-renewing tissue., Competing Interests: The authors declare no competing interest.

Spermatogonial stem cells (SSCs) serve as a resource for producing genetically modified animals. However, genetic manipulation of SSCs has met with limited success. Here, we show efficient gene transfer into SSCs via a lentivirus (FV-LV) using a fusion protein (F), a Sendai virus (SV) envelope protein involved in virion/cell membrane fusion. FV-LVs transduced cultured SSCs more efficiently than conventional LVs. Although SSCs infected with SV failed to produce offspring, those transduced with FV-LVs were fertile. In vivo microinjection showed that FV-LVs could penetrate not only the basement membrane of the seminiferous tubules but also the blood-testis barrier, which resulted in successful transduction of both spermatogenic cells and testicular somatic cells. Cultured SSCs transfected with FV-LVs that express drug-inducible CRISPR/Cas9 against Kit or Sycp3 showed impaired spermatogenesis upon transplantation and drug treatment in vivo. Thus, FV-LVs provide an efficient method for functional analysis of genes involved in SSCs and spermatogenesis., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) undergo continuous self-renewal division in response to self-renewal factors. The present study identified ephrin type-A receptor 2 (EPHA2) on mouse SSCs and showed that supplementation of glial cell-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), which are both SSC self-renewal factors, induced EPHA2 expression in cultured SSCs. Spermatogonial transplantation combined with magnetic-activated cell sorting or fluorescence-activated cell sorting also revealed that EPHA2 was expressed in SSCs. Additionally, ret proto-oncogene (RET) phosphorylation levels decreased following the knockdown (KD) of Epha2 expression via short hairpin ribonucleic acid (RNA). Although the present immunoprecipitation experiments did not reveal an association between RET with EPHA2, RET interacted with FGFR2. The Epha2 KD decreased the proliferation of cultured SSCs and inhibited the binding of cultured SSCs to laminin-coated plates. The Epha2 KD also significantly reduced the colonization of testis cells by spermatogonial transplantation. EPHA2 was also expressed in human GDNF family receptor alpha 1-positive spermatogonia. The present results indicate that SSCs express EPHA2 and suggest that it is a critical modifier of self-renewal signals in SSCs., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Stem cell homing is a complex phenomenon that involves multiple steps; thus far, attempts to increase homing efficiency have met with limited success. Spermatogonial stem cells (SSCs) migrate to the niche after microinjection into seminiferous tubules, but the homing efficiency is very low. Here we report that reversible disruption of the blood-testis barrier (BTB) between Sertoli cells enhances the homing efficiency of SSCs. We found that SSCs on a C57BL/6 background are triggered to proliferate in vitro when MHY1485, which stimulates MTORC, were added to culture medium. However, the cultured cells did not produce offspring by direct injection into the seminiferous tubules. When acyline, a gonadotropin-releasing hormone (GnRH) analogue, was administered into infertile recipients, SSC colonization increased by ~5-fold and the recipients sired offspring. In contrast, both untreated individuals and recipients that received leuprolide, another GnRH analogue, remained infertile. Acyline not only decreased CLDN5 expression but also impaired the BTB, suggesting that increased colonization was caused by efficient SSC migration through the BTB. Enhancement of stem cell homing by tight junction protein manipulation constitutes a new approach to improve homing efficiency, and similar strategy may be applicable to other self-renewing tissues.

Adeno-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 Kitl Sl /Kitl Sl-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., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The spermatogonial transplantation technique was developed by Dr. Ralph Brinster in 1994. Transplanted spermatogonial stem cells (SSCs) produce germ cell colonies after microinjection into the seminiferous tubules of infertile mice. This technique provided the first functional assay for SSCs. Although it became possible to produce transgenic animals using this transplantation technique in 2001, the lack of SSC culture systems prevented efficient genetic manipulation. To overcome this problem, a long-term SSC culture technique was developed in 2003. Cultured SSCs, designated as germline stem cells, allow drug selection of transfected SSCs, and knockout mice were produced in 2006. Using these techniques, it is now possible to address basic biological questions of SSC biology. They also open up new possibilities for male germline manipulation. In this review, we will briefly summarize our findings on SSCs and discuss unresolved issues that remain to be addressed., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

The introduction of megabase-sized large DNA fragments into the germline has been a difficult task. Although microcell-mediated chromosome transfer into mouse embryonic stem cells (ESCs) allows the production of transchromosomic mice, ESCs have unstable karyotypes and germline transmission is unreliable by chimera formation. As spermatogonial stem cells (SSCs) are the only stem cells in the germline, they represent an attractive target for germline modification. Here, we report successful transfer of a mouse artificial chromosome (MAC) into mouse germline stem cells (GSCs), cultured spermatogonia enriched for SSCs. MAC-transferred GSCs maintained the host karyotype and MAC more stably than ESCs, which have significant variation in chromosome number. Moreover, MAC-transferred GSCs produced transchromosomic mice following microinjection into the seminiferous tubules of infertile recipients. Successful transfer of MACs to GSCs overcomes the problems associated with ESC-mediated germline transmission and provides new possibilities in germline modification., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Vast amounts of sperm are produced from spermatogonial stem cells (SSCs), which continuously undergo self-renewal. We examined the possible effect of laterality in male germline transmission efficiency of SSCs using a spermatogonial transplantation technique. We transplanted the same number of wild-type and Egfp transgenic SSCs in the same or different testes of individual recipient mice and compared the fertility of each type of recipient by natural mating. Transgenic mice were born within 3 months after transplantation regardless of the transplantation pattern. However, transgenic offspring were born at a significantly increased frequency when wild-type and transgenic SSCs were transplanted separately. In addition, this type of recipient sired significantly more litters that consisted exclusively of transgenic mice, which suggested that left and right testes have different time windows for fertilization. Thus, laterality plays an important role in germline transmission patterns from SSCs., (© The Author(s) 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Myc plays critical roles in the self-renewal division of various stem cell types. In spermatogonial stem cells (SSCs), Myc controls SSC fate decisions because Myc overexpression induces enhanced self-renewal division, while depletion of Max, a Myc-binding partner, leads to meiotic induction. However, the mechanism by which Myc acts on SSC fate is unclear. Here we demonstrate a critical link between Myc/Mycn gene activity and glycolysis in SSC self-renewal. In SSCs, Myc/Mycn are regulated by Foxo1, whose deficiency impairs SSC self-renewal. Myc/Mycn-deficient SSCs not only undergo limited self-renewal division but also display diminished glycolytic activity. While inhibition of glycolysis decreased SSC activity, chemical stimulation of glycolysis or transfection of active Akt1 or Pdpk1 (phosphoinositide-dependent protein kinase 1 ) augmented self-renewal division, and long-term SSC cultures were derived from a nonpermissive strain that showed limited self-renewal division. These results suggested that Myc-mediated glycolysis is an important factor that increases the frequency of SSC self-renewal division., (© 2016 Kanatsu-Shinohara et al.; Published by Cold Spring Harbor Laboratory Press.)

Spermatogenesis originates from self-renewal of spermatogonial stem cells (SSCs). Previous studies have reported conflicting roles of gonadotropic pituitary hormones in SSC self-renewal. Here, we explored the role of hormonal regulation of SSCs using Fshb and Lhcgr knockout (KO) mice. Although follicle-stimulating hormone (FSH) is thought to promote self-renewal by glial cell line-derived neurotrophic factor (GDNF), no abnormalities were found in SSCs and their microenvironment. In contrast, SSCs were enriched in Lhcgr-deficient mice. Moreover, wild-type SSCs transplanted into Lhcgr-deficient mice showed enhanced self-renewal. Microarray analysis revealed that Lhcgr-deficient testes have enhanced WNT5A expression in Sertoli cells, which showed an immature phenotype. Since WNT5A was upregulated by anti-androgen treatment, testosterone produced by luteinizing hormone (LH) is required for Sertoli cell maturation. WNT5A promoted SSC activity both in vitro and in vivo. Therefore, FSH is not responsible for GDNF regulation, while LH negatively regulates SSC self-renewal by suppressing WNT5A via testosterone., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genes are thought to be transmitted to offspring by random fertilization of a small number of oocytes with numerous spermatozoa. Here we analyzed the dynamics of male germline transmission by genetic marking and transplantation of spermatogonial stem cells (SSCs). We found that offspring deriving from a small number of specific SSCs appear within a limited time. Interestingly, the same SSC clones reappear later with an average functional lifespan of ∼124.4 days. Cyclic offspring production from SSCs was not caused by changes in SSC self-renewal activity because lineage-tracing analyses suggested that all SSCs actively proliferated. Selection appears to occur during the differentiating spermatogonia stage, when extensive apoptosis was observed. The pattern of germline transmission could be predicted using a mathematical model in which SSCs repeat cycles of transient spermatogenic burst and refractory periods. Thus, spermatogenesis is a regulated process whereby specific SSC clones are repeatedly recruited for fertilization with long-term cycles., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) comprise a small population of germ cells with self-renewal potential. Previous studies have shown that SSCs share several common features with stem cells in other self-renewing tissues, including surface markers and proliferative machinery. However, studies of SSCs are severely handicapped by the small number of SSCs and the lack of SSC-specific markers. In the present study, we examined the utility of CDy1 and Rh123, both of which are used for the collection of stem cells in several self-renewing tissues. CDy1 stained germline stem (GS) cells, cultured spermatogonia enriched for SSC activity, after in vitro incubation without exerting toxic effects. Unlike previously reported stem cell-specific dyes, CDy1 was also useful for enrichment of SSCs in both GS cell culture and mature adult testes. Spermatogonial transplantation showed that ∼1 in 66.7 cells exhibited SSC activity after CDH1-based magnetic cell selection and CDy1 staining. In contrast, although Rh123 was previously used successfully to collect SSCs from cryptorchid testes, it was not possible to recover SSCs from both GS cell cultures and wild-type testes. Thus, CDy1 staining will provide a useful strategy for the enrichment of SSCs and may be used in conjunction with other reagents for the enrichment of SSCs., (© 2016 by the Society for the Study of Reproduction, Inc.)

Spermatogonial stem cells (SSCs) represent a unique population of germ cells with self-renewal potential. Although reactive oxygen species (ROS) are considered toxic to germ cells, we recently showed that moderate levels of ROS are required for SSC self-renewal and that Nox1 is involved in ROS generation. In this study, we showed that self-renewal factor treatment induces Nox3 to trigger SSC self-renewal. Nox3 was transiently expressed in cultured spermatogonia by FGF2 and GDNF stimulation, whereas Nox1 was expressed predominantly during the stable phase of proliferation. Nox3 inhibition by short hairpin RNA reduced cytokine-induced ROS generation and limited the proliferation of cultured spermatogonia. Although Nox3 overexpression revealed no apparent effect, depletion of Nox3 decreased the number of SSCs in both cultured spermatogonia and freshly isolated testis cells. Our results suggest that self-renewal of SSCs is regulated by sequential activation of different Nox genes, and underscore the complexity of ROS regulation in the self-renewal division of SSCs., (© 2015 by the Society for the Study of Reproduction, Inc.)

Spermatogonial stem cells (SSCs) are required for spermatogenesis. Earlier studies showed that glial cell line-derived neurotrophic factor (GDNF) was indispensable for SSC self-renewal by binding to the GFRA1/RET receptor. Mice with mutations in these molecules showed impaired spermatogenesis, which was attributed to SSC depletion. Here we show that SSCs undergo GDNF-independent self-renewal. A small number of spermatogonia formed colonies when testis fragments from a Ret mutant mouse strain were transplanted into heterologous recipients. Moreover, fibroblast growth factor 2 (FGF2) supplementation enabled in vitro SSC expansion without GDNF. Although GDNF-mediated self-renewal signaling required both AKT and MAP2K1/2, the latter was dispensable in FGF2-mediated self-renewal. FGF2-depleted testes exhibited increased levels of GDNF and were enriched for SSCs, suggesting that the balance between FGF2 and GDNF levels influences SSC self-renewal in vivo. Our results show that SSCs exhibit at least two modes of self-renewal and suggest complexity of SSC regulation in vivo., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) undergo self-renewal divisions to provide the foundation for spermatogenesis. Although Rb1 deficiency is reportedly essential for SSC self-renewal, its mechanism has remained unknown. Here we report that Rb1 is critical for cell cycle progression and protection of SSCs from DNA double-strand breaks (DSBs). Cultured SSCs depleted of Cdkn1b proliferated poorly and showed diminished expression of CDK4 and RB1, thereby leading to hypophosphorylation of RB1. Rb1 deficiency induced cell cycle arrest and apoptosis in cultured SSCs, which expressed markers for DNA DSBs. This DNA damage is caused by increased E2F1 activity, the depletion of which decreased DNA DSBs caused by Rb1 deficiency. Depletion of Cdkn1a and Bbc3, which were upregulated by Trp53, rescued Rb1-deficient cells from undergoing cell cycle arrest and apoptosis. These results suggest that the CDKN1B-RB1-E2F1 pathway is essential for SSC self-renewal and protects SSCs against genomic damage.

Diploid germ cells are thought to have pluripotency potential. We recently described a method to derive pluripotent stem cells (PSCs) from cultured spermatogonial stem cells (SSCs) by depleting Trp53 and Dmrt1, both of which are known suppressors of teratomas. In this study, we used this technique to analyze the effect of this protocol in deriving PSCs from the male germline at different developmental stages. We collected primordial germ cells (PGCs), gonocytes and spermatogonia, and the cells were transduced with lentiviruses expressing short hairpin RNA against Dmrt1 and/or Trp53. We found that PGCs are highly susceptible to reprogramming induction and that only Trp53 depletion was sufficient to induce pluripotency. In contrast, gonocytes and spermatogonia were resistant to reprogramming by double knockdown of Dmrt1 and Trp53. PSCs derived from PGCs contributed to chimeras produced by blastocyst injection, but some of the embryos showed placenta-only phenotypes suggestive of epigenetic abnormalities of PGC-derived PSCs. These results show that PGCs and gonocytes/spermatogonia have distinct reprogramming potential and also suggest that fresh and cultured SSCs do not necessarily have the same properties.

Germ cells are thought to exhibit a unique DNA damage response that differs from that of somatic stem cells, and previous studies suggested that Trp53 is not involved in the survival of spermatogonial stem cells (SSCs) after irradiation. Here, we report a critical role for the Trp53-Trp53inp1-Tnfrsf10b pathway during radiation-induced SSC apoptosis. Spermatogonial transplantation revealed that Trp53 deficiency increased the survival of SSCs after irradiation. Although Bbc3, a member of the intrinsic apoptotic pathway, was implicated in apoptosis of germ and somatic stem cells, Bbc3 depletion inhibited apoptosis in committed spermatogonia, but not in SSCs. In contrast, inhibition of Tnfrsf10b, an extrinsic apoptosis regulator, rescued SSCs. Tnfrsf10b, whose deficiency protected SSCs, was upregulated by Trp53inp1 upon irradiation. These results suggest that the Trp53-Trp53inp1-Tnfrsf10b pathway responds to genotoxic damage in SSCs and that stem and progenitor cells exhibit distinct DNA damage responses in self-renewing tissue., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) undergo self-renewal divisions to support spermatogenesis throughout life. Although several positive regulators of SSC self-renewal have been discovered, little is known about the negative regulators. Here, we report that F-box and WD-40 domain protein 7 (FBXW7), a component of the Skp1-Cullin-F-box-type ubiquitin ligase, is a negative regulator of SSC self-renewal. FBXW7 is expressed in undifferentiated spermatogonia in a cell cycle-dependent manner. Although peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1), essential for spermatogenesis, is thought to destroy FBXW7, Pin1 depletion decreased FBXW7 expression. Spermatogonial transplantation showed that Fbxw7 overexpression compromised SSC activity whereas Fbxw7 deficiency enhanced SSC colonization and caused accumulation of undifferentiated spermatogonia, suggesting that the level of FBXW7 is critical for self-renewal and differentiation. Screening of putative FBXW7 targets revealed that Fbxw7 deficiency up-regulated myelocytomatosis oncogene (MYC) and cyclin E1 (CCNE1). Although depletion of Myc/Mycn or Ccne1/Ccne2 compromised SSC activity, overexpression of Myc, but not Ccne1, increased colonization of SSCs. These results suggest that FBXW7 regulates SSC self-renewal in a negative manner by degradation of MYC.

Spermatogonial stem cells (SSCs) migrate to the niche upon introduction into the seminiferous tubules of the testis of infertile animals. However, only 5-10% of the transplanted cells colonize recipient testes. In this study, we analyzed the impact of cell cycle on spermatogonial transplantation. We used fluorescent ubiquitination-based cell cycle indicator transgenic mice to examine the influence of cell cycle on SSC activity of mouse germline stem (GS) cells, a population of cultured spermatogonia enriched for SSCs. GS cells in the G1 phase are more efficient than those in the S/G2-M phase in colonizing the seminiferous tubules of adult mice. Cells in the G1 phase not only showed higher expression levels of GFRA1, a component of the GDNF self-renewal factor receptor, but also adhered more efficiently to laminin-coated plates. Furthermore, this cell cycle-dependency was not observed when cells were transplanted into immature pup recipients, which do not have the blood-testis barrier (BTB) between Sertoli cells, suggesting that cells in the G1 phase may passage through the BTB more readily than cells in the S/G2-M phase. Thus cell cycle status is an important factor in regulating SSC migration to the niche.

Reactive oxygen species (ROS) generation is implicated in stem cell self-renewal in several tissues but is thought to be detrimental for spermatogenesis as well as spermatogonial stem cells (SSCs). Using cultured SSCs, we show that ROS are generated via the AKT and MEK signaling pathways under conditions where the growth factors glial cell line-derived neurotrophic factor and fibroblast growth factor 2 drive SSC self-renewal and, instead, stimulate self-renewal at physiological levels. SSCs depleted of ROS stopped proliferating, but they showed enhanced self-renewal when ROS levels were increased by the addition of hydrogen peroxide, which induced the phosphorylation of stress kinases p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Moreover, ROS depletion in vivo decreased SSC number in the testis, and NADPH oxidase 1 (Nox1)-deficient SSCs exhibited reduced self-renewal division upon serial transplantation. These results suggest that ROS generated by Nox1 play critical roles in SSC self-renewal via the activation of the p38 MAPK and JNK pathways., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) provide the foundation of spermatogenesis, but studies are hampered by their scarcity. Although the cryptorchid operation is often used to obtain an enriched SSC population, making cryptorchid testes is time-consuming and the technique is not applicable to many animal species. In the present study, we screened for a new surface antigen on SSCs using germline stem (GS) cells (i.e., cultured SSCs). Germ cell transplantation experiments showed that SSCs express melanoma cell adhesion molecule (MCAM), which belongs to the immunoglobulin superfamily and mediates cation-independent adhesion. Although MCAM overexpression in GS cells did not influence SSC colony formation frequency or subsequent spermatogenesis after transplantation, MCAM knockdown in GS cells by short-interfering RNA treatment reduced colony numbers, suggesting that MCAM plays a role in sustaining SSC potential. Multiparameter selection of wild-type adult testis cells with a CD9⁺EPCAM(low)MCAM⁺KIT⁻ phenotype resulted in a 561-fold enrichment of SSCs. Development of a new strategy for SSC enrichment from mature adult testes will facilitate analyses of SSCs in the normal testicular microenvironment.

Spermatogonial stem cells (SSCs) reside in specific niches within seminiferous tubules. These niches are thought to secrete chemotactic factors for SSCs, because SSCs migrate to them upon transplantation. However, the identity of these chemotactic molecules remains unknown. Here, we established a testis feeder cell culture system and used it to identify SSC chemotactic factors. When seeded on testis cells from infertile mice, SSCs migrated beneath the Sertoli cells and formed colonies with a cobblestone appearance that were very similar to those produced by hematopoietic stem cells. Cultured cells maintained SSC activity and fertility for at least 5 months. Cobblestone colony formation depended on GDNF and CXCL12, and dominant-negative GDNF receptor transfection or CXCL12 receptor deficiency reduced SSC colonization. Moreover, GDNF upregulated CXCL12 receptor expression, and CXCL12 transfection in Sertoli cells increased homing efficiency. Overall, our findings identify GDNF and CXCL12 as SSC chemotactic factors in vitro and in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Fibroblast growth factor 2 (FGF2) and glial cell line-derived neurotrophic factor (GDNF) are required to recapitulate spermatogonial stem cell (SSC) self-renewal in vitro. Although studies have revealed the role of the GDNF signaling pathway in SSCs, little is known about how FGF2 is involved. In the present study, we assessed the role of the FGF2 signaling pathway using a mouse germline stem (GS) cell culture system that allows in vitro expansion of SSCs. Adding GDNF or FGF2 induced phosphorylation of MAPK1/3, and adding the MAP2K1 inhibitor PD0325091 reduced GS cell proliferation and MAPK1/3 phosphorylation. Moreover, GS cells transfected with an activated form of Map2k1 not only upregulated Etv5 and Bcl6b gene expression, but also proliferated in an FGF2-independent manner, suggesting that they act downstream of MAP2K1 signaling to drive SSC self-renewal. Although GS cells transfected with Map2k1, Etv5 or Bcl6b showed normal spermatogonial markers, transplanting GS cells expressing Bcl6b into infertile mouse testes resulted in the formation of a germ cell tumor, suggesting that excessive self-renewal signals causes tumorigenic conversion. These results show that FGF2 depends on MAP2K1 signaling to drive SSC self-renewal via upregulation of the Etv5 and Bcl6b genes.

The homing ability of spermatogonial stem cells (SSCs) allows them to migrate into niches after being transplantated into infertile testes. Transplanted SSCs attach to Sertoli cells and transmigrate through the blood-testis barrier (BTB), formed by inter-Sertoli tight junctions, toward niches on the basement membrane. The most critical step is the passage through the BTB, which limits the homing efficiency to <10%. Here we demonstrated the involvement of Rac1 in SSC transmigration. Rac1-deficient SSCs did not colonize the adult testes, but they reinitiated spermatogenesis when transplanted into pup testes without a BTB. Moreover, a dominant-negative Rac1 construct not only reduced the expression of several claudin proteins, which comprise the BTB, but also increased SSC proliferation both in vitro and in vivo. Short hairpin RNA (shRNA) -mediated suppression of claudin3, which was downregulated by Rac inhibition, reduced the SSC homing efficiency. Thus, Rac1 is a critical regulator of SSC homing and proliferation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Spermatogonial stem cells (SSCs) are the only stem cells in the body with germline potential, which makes them an attractive target for germline modification. We previously showed the feasibility of homologous recombination in mouse SSCs and produced knockout (KO) mice by exploiting germline stem (GS) cells, i.e., cultured spermatogonia with SSC activity. In this study, we report the successful homologous recombination in rat GS cells, which can be readily established by their ability to form germ cell colonies on culture plates whose surfaces are hydrophilic and neutrally charged and thus limit somatic cell binding. We established a drug selection protocol for GS cells under hypoxic conditions. The frequency of the homologous recombination of the Ocln gene was 4.2% (2 out of 48 clones). However, these GS cell lines failed to produce offspring following xenogeneic transplantation into mouse testes and microinsemination, suggesting that long-term culture and drug selection have a negative effect on GS cells. Nevertheless, our results demonstrate the feasibility of gene targeting in rat GS cells and pave the way toward the generation of KO rats.

Stem cells of the side population (SP) phenotype are found in many self-renewing tissues and can be identified by their unique ability to effectively exclude the dye Hoechst 33342. We previously established a method for expanding spermatogonial stem cells (SSCs) in vitro, but the frequency of SSCs is only about 1 to 2%, limiting detailed SSC analyses. In this study, we sought to isolate SSCs from in vitro cultures by exploiting their ability to exclude Hoechst 33342. In contrast to the findings of previous in vivo studies, we found that SP cells developed in a stochastic manner in vitro. Moreover, SP cells in culture were not enriched in SSCs, but they were interconvertible with non-SP cells. Although SP cells were consistently found in testes after transplantation of cultured cells, they were not enriched in SSCs. These results show that SSCs have an unstable SP phenotype and provide evidence that SSCs change their phenotype characteristics in response to their microenvironment.

Spermatogonial stem cells (SSCs) undergo self-renewal divisions to support spermatogenesis. Although several in vitro SSC culture systems have been developed, these systems include serum or fibroblast feeders, which complicate SSC self-renewal analyses. Here, we developed a serum- and feeder-free culture system for long-term propagation of SSCs. In addition to the SSC self-renewal factors, including glial cell line-derived neurotrophic factor, supplementation with fetuin and lipid-associated molecules was required to drive SSC proliferation in vitro. Cultured cells proliferated for at least 6 mo at a rate comparable to that of serum-supplemented cultured cells. However, germline potential was reduced under serum- and feeder-free conditions, as indicated by a lower SSC frequency after germ cell transplantation. Nevertheless, the cultured cells completed spermatogenesis and produced offspring following spermatogonial transplantation into seminiferous tubules of infertile mice. This culture system provides a basic platform for understanding the regulation of SSC fate commitment in vitro and for improving SSC culture medium.

Background: Spermatogonial stem cells (SSCs) have the unique ability to undergo self-renewal division. However, these cells are morphologically indistinguishable from committed spermatogonia, which have limited mitotic activity. To establish a system for SSC purification, we analyzed the expression of SSC markers CD9 and epithelial cell adhesion molecule (EPCAM), both of which are also expressed on embryonic stem (ES) cells. We examined the correlation between their expression patterns and SSC activities., Methodology and Principal Findings: By magnetic cell sorting, we found that EPCAM-selected mouse germ cells have limited clonogenic potential in vitro. Moreover, these cells showed stronger expression of progenitor markers than CD9-selected cells, which are significantly more enriched in SSCs. Fluorescence-activated cell sorting of CD9-selected cells indicated a significantly higher frequency of SSCs among the CD9(+)EPCAM(low/-) population than among the CD9(+)EPCAM(+) population. Overexpression of the active form of EPCAM in germline stem (GS) cell cultures did not significantly influence SSC activity, whereas EPCAM suppression by short hairpin RNA compromised GS cell proliferation and increased the concentration of SSCs, as revealed by germ cell transplantation., Conclusions/significance: These results show that SSCs are the most concentrated in CD9(+)EPCAM(low/-) population and also suggest that EPCAM plays an important role in progenitor cell amplification in the mouse spermatogenic system. The establishment of a method to distinguish progenitor spermatogonia from SSCs will be useful for developing an improved purification strategy for SSCs from testis cells.

Spermatogonial stem cells (SSCs) undergo self-renewal division to support spermatogenesis. Although several positive regulators of SSC self-renewal have been identified, little is known about the mechanisms that negatively regulate SSCs. Here we developed a novel transplantation assay for SSCs and demonstrate that p21 and p27 cyclin-dependent kinase inhibitors play critical roles in SSC self-renewal and differentiation. Overexpression of p21 or p27 abrogated proliferation of cultured SSCs in vitro, and their expression levels were downregulated by exogenous self-renewal signals. In contrast, no apparent defects were found in p21 or p27-deficient SSCs by spermatogonial transplantation. However, competitive spermatogonial transplantation with WT SSCs revealed that the loss of either gene causes distortion of germline transmission: p21-deficiency facilitated mutant offspring production, whereas germline transmission was limited by p27-deficiency. Serial transplantation also showed that the loss of p27, but not p21, decreases secondary colony formation, suggesting that appropriate amounts of p27 are necessary for sustaining SSC self-renewal. Thus, p21 and p27 cyclin-dependent kinase inhibitors play critical roles in germline transmission by regulating the balance between SSC self-renewal and differentiation, and competitive spermatogonial transplantation technique will be useful for analyzing subtle defects in spermatogenesis that are not evident by traditional spermatogonial transplantation.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis, and are unique tissue-specific stem cells because of their ability to transmit genetic information to offspring. Generation of knockout mice using mouse SSCs became feasible after the successful establishment of protocols for the transplantation and long-term culture of these cells, called germline stem (GS) cells. Furthermore, SSCs can acquire pluripotentiality similar to that of embryonic stem (ES) cells, in addition to their highly differentiated spermatogenic potential. These ES-like cells, called multipotent GS (mGS) cells, are capable of generating knockout mice in a manner similar to that of ES cells. The use of GS and mGS cells for animal transgenesis has added a new dimension to gene-targeting technology using ES cells and somatic cell nuclear transfer, which has limited application. Furthermore, for regenerative medicine purposes, the use of mGS will settle problems such as ethics issues and immunological rejection associated with ES cells, as well as risks of insertional mutagenesis associated with integrated genes into induced pluripotent stem cells.

Spermatogonial stem cells (SSCs) are slowly dividing cells that undergo self-renewal division to support spermatogenesis. Although the effects of genetic background in stem cell self-renewal have been well studied in hematopoietic stem cells, little is known about its effect on stem cells in other self-renewing tissues, including SSCs. To examine whether genetic factors are involved in regulation of SSC self-renewal, we first studied spermatogenesis in different inbred mouse strains (C57BL/6, DBA/2, AKR, BALB/C and C3H) after chemical damage caused by busulfan. Spermatogenesis in the DBA/2 and AKR strains was relatively resistant to busulfan treatment, whereas spermatogenesis was diminished in C57BL/6 mice and nearly ablated in C3H and BALB/C mice. Serial germ cell transplantation experiments provided functional evidence that SSCs with the DBA/2 background expanded more rapidly than those with the B6 background. Finally, we also employed the Germline Stem (GS) cell culture technique to examine the self-renewal activity in vitro. Although genetic manipulation of GS cells has been limited to those from the DBA/2 background, we produced transgenic offspring of the C3H background by electroporation of GS cells with a plasmid vector. Our results underscore the importance of genetic factors in SSC self-renewal. Furthermore, application of genetic modification techniques to GS cells with non-DBA/2 backgrounds extends the potential of a SSC-based approach in male germline modification.

Spermatogonial stem cells (SSCs) in the testes are a new target for germline modification. With the development of an in vitro culture system and spermatogonial transplantation technique, SSCs can now be manipulated and used as an alternative to embryonic stem cells for knockout mice production. The genetic and epigenetic stability of SSCs provide new possibilities for the application of germline mutagenesis in a wide range of animals., (2010 Elsevier Inc. All rights reserved.)

Background: Spermatogonial stem cells (SSCs) continuously undergo self-renewal division to support spermatogenesis. SSCs are thought to have a fixed phenotype, and development of a germ cell transplantation technique facilitated their characterization and prospective isolation in a deterministic manner; however, our in vitro SSC culture experiments indicated heterogeneity of cultured cells and suggested that they might not follow deterministic fate commitment in vitro., Methodology and Principal Findings: In this study, we report phenotypic plasticity of SSCs. Although c-kit tyrosine kinase receptor (Kit) is not expressed in SSCs in vivo, it was upregulated when SSCs were cultured on laminin in vitro. Both Kit(-) and Kit(+) cells in culture showed comparable levels of SSC activity after germ cell transplantation. Unlike differentiating spermatogonia that depend on Kit for survival and proliferation, Kit expressed on SSCs did not play any role in SSC self-renewal. Moreover, Kit expression on SSCs changed dynamically once proliferation began after germ cell transplantation in vivo., Conclusions/significance: These results indicate that SSCs can change their phenotype according to their microenvironment and stochastically express Kit. Our results also suggest that activated and non-activated SSCs show distinct phenotypes.

Spermatogonial stem cells (SSCs) undergo self-renewal division and support spermatogenesis. Although several cytokines coordinate to drive SSC self-renewal, little is known about the mechanisms underlying this process. We investigated the molecular mechanism by reconstructing SSC self-renewal in vitro without exogenous cytokines. Activation of Ras or overexpression of cyclins D2 and E1, both of which were induced by Ras, enabled long-term self-renewal of cultured spermatogonia. SSCs with activated Ras responded properly to differentiation signals and underwent spermatogenesis, whereas differentiation was abrogated in cyclin transfectants after spermatogonial transplantation. Both Ras- and cyclin-transfected cells produced seminomatous tumors, suggesting that excessive self-renewing stimulus induces oncogenic transformation. In contrast, cells that overexpressed cyclin D1 or D3 failed to make germ cell colonies after transplantation, which indicated that cyclin expression pattern is an important determinant to long-term SSC recolonization. Thus, the Ras-cyclin D2 pathway regulates the balance between tissue maintenance and tumorigenesis in the SSC population.

Spermatogonial stem cells (SSCs) continue to proliferate in the testis to support spermatogenesis throughout life, which makes them ideal targets for germline modification. Although recent success in the production of transgenic and knockout animals using SSCs has opened up new experimental possibilities, several problems, including the low efficiency of germ cell transplantation and poor fertility rates, remain to be resolved. In the present study, we took advantage of the xenogeneic transplantation to resolve these problems. Rat SSCs were transduced in vitro with a lentiviral vector that expressed enhanced green fluorescent protein (EGFP), and then transplanted into the testes of immunodeficient mice. The transduced rat SSCs produced EGFP-expressing spermatogenic cells, and microinsemination using these cells was used to produce transgenic rats, which stably transmitted the transgene to the next generation. Thus, xenogeneic transplantation is a powerful strategy for transgenesis, and smaller xenogeneic surrogates can be used for male germline modification using SSCs.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. In a manner comparable to hematopoietic stem cell transplantation, SSCs colonize the niche of recipient testes and reinitiate spermatogenesis following microinjection into the seminiferous tubules. However, little is known about the homing mechanism of SSCs. Here we examined the role of adhesion molecules in SSC homing. SSCs isolated from mice carrying loxP-tagged beta1-integrin alleles were ablated for beta1-integrin expression by in vitro adenoviral cre transduction. The beta1-integrin mutant SSCs showed significantly reduced ability to recolonize recipient testes in vivo and to attach to laminin molecules in vitro. In contrast, genetic ablation of E-cadherin did not impair homing, and E-cadherin mutant SSCs completed normal spermatogenesis. In addition, the deletion of beta1-integrin on Sertoli cells reduced SSC homing. These results identify beta1-integrin as an essential adhesion receptor for SSC homing and its association with laminin is critical in multiple steps of SSC homing.

Although pluripotent stem cells were recently discovered in postnatal testis, attempts to analyze their developmental potential have led to conflicting claims that spermatogonial stem cells are pluripotent or that they lose spermatogenic potential after conversion into pluripotent stem cells. To examine this issue, we analyzed the developmental fate of a single spermatogonial stem cell that appeared during transfection experiments. After transfection of a neomycin-resistance gene into germline stem cells, we obtained an embryonic stem-like, multipotent germline stem cell line. Southern blot analysis revealed that the germline stem and multipotent germline stem clones have the same transgene integration pattern, demonstrating their identical origin. The two lines, however, have different DNA methylation patterns. The multipotent germline stem cells formed chimeras after blastocyst injection but did not produce sperm after germ cell transplantation, whereas the germline stem cells could produce only spermatozoa and did not differentiate into somatic cells. Interestingly, the germline stem cells expressed several transcription factors (Pou5f1, Sox2, Myc, and Klf4) required for reprogramming fibroblasts into a pluripotent state, suggesting that they are potentially pluripotent. Thus, our study provides evidence that a single spermatogonial stem cell can acquire pluripotentiality but that conversion into a pluripotent cell type is accompanied by loss of spermatogenic potential.

Spermatogonial stem cells provide the foundation for spermatogenesis in male animals. We recently succeeded in culturing and genetically engineering mouse spermatogonial stem cells, but little is known regarding the culture and growth requirements of spermatogonial stem cells in other animal species. In this study, we report the successful long-term culture of spermatogonial stem cells from hamster testes. Spermatogonial stem cells were purified using an anti-ITGA6 antibody and cultured in the presence of glial cell line-derived neurotrophic factor. The cells continued to proliferate for at least 1 year. During this period, they were genetically modified using a lentivirus and underwent spermatogenesis after transplantation into the testes of immunodeficient nude mice. However, germ cells generated in the surrogate xenogeneic recipients did not differentiate beyond the spermatid stage, and these round spermatids could not produce offspring through in vitro microinsemination. These results suggest that the germ cells may not have acquired characteristics necessary for fertility in the xenogeneic microenvironment. Nevertheless, the successful establishment of culture conditions conducive for hamster spermatogonial stem cell growth and maintenance indicates that this technique can be extended to other animal species in which current genetic modification techniques are impossible or inefficient.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. During the last decade, several techniques for the manipulation of this cell type have been developed; as a result, SSCs can now be subjected to long-term in vitro expansion and genetically manipulated for knockout mouse production. These techniques have allowed SSCs to serve as a new target for animal transgenesis, which may provide an alternative to embryonic stem (ES) cells. Furthermore, SSCs may be converted into ES-like cells, demonstrating that the postnatal testis is a source of pluripotent stem cells. These techniques were first established in mice, but they are currently being extended to other animal species. SSC-based technologies will be useful in agriculture and medicine and will also provide valuable opportunities to study SSC biology. The mechanisms of self-renewal division and differentiation and the regulation of pluripotency in SSCs are now being studied at the molecular level. However, some technical and conceptual pitfalls must be kept in mind when designing and analyzing experimental results. Nevertheless, these advances in SSC research will provide valuable insight into the study of mammalian stem cell systems.

Spermatogenesis depends on a population of cells called spermatogonial stem cells, which self-renew to support male reproduction throughout life. In 2003, the long-term culture of spermatogonial stem cells of mice proved to be successful. In the presence of glial cell-line-derived neurotrophic factor, germline stem (GS) cells were established from postnatal mouse testis. These cells proliferated over a 2-year period (>10(85)-fold) and restored fertility to congenitally infertile recipient mice following transplantation into the seminiferous tubules. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, the GS cells retained their euploid karyotype and androgenetic imprint during the 2-year experimental period, and they produced normal fertile offspring. Mutagenization of the GS cells was successful using gene trapping and gene targeting vectors to produce homozygous knockout offspring, thereby providing a new approach to germline modification. In the course of the gene targeting experiments, establishment of embryonic-stem (ES)-like cells was also successful [i.e., multipotent germline stem (mGS) cells] from postnatal mouse testis. These mGS cells were phenotypically similar to the ES/embryonic germ cells, except for their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under the conditions used to induce the differentiation of the ES cells, and the mGS cells formed germline chimeras when injected into blastocysts. These new spermatogonial stem-cell lines will be useful for studying the mechanism of spermatogenesis, and they have important implications for developing new transgenic or medical technologies.

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, we assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells.

Spermatogonial stem cells represent a self-renewing population of spermatogonia, and continuous division of these cells supports spermatogenesis throughout the life of adult male animals. Previous attempts to introduce adenovirus vectors into spermatogenic cells, including spermatogonial stem cells, have failed to yield evidence of infection, suggesting that male germ cells may be resistant to adenovirus infection. In this study we show the feasibility of transducing spermatogonial stem cells by adenovirus vectors. When testis cells from ROSA26 Cre reporter mice were incubated in vitro with a Cre-expressing adenovirus vector, Cre-mediated recombination occurred at an efficiency of 49-76%, and the infected spermatogonial stem cells could reinitiate spermatogenesis after transplantation into seminiferous tubules of infertile recipient testes. No evidence of germ-line integration of adenovirus vector could be found in offspring from infected stem cells that underwent Cre-mediated recombination, which suggests that the adenovirus vector infected the cells but did not stably integrate into the germ line. Nevertheless, these results suggest that adenovirus may inadvertently integrate into the patient's germ line and indicate that there is no barrier to adenovirus infection in spermatogonial stem cells.

Spermatogonial stem cells continuously divide in the testis to support spermatogenesis throughout the life of adult male animals. Although very few spermatogonial stem cells are present in vivo, we recently succeeded in expanding these cells in vitro. Germ cells from postnatal testes were able to proliferate in the presence of several types of cytokines, and they formed uniquely shaped colonies of spermatogonia (germline stem or GS cells). These cells reinitiated normal spermatogenesis when transplanted into seminiferous tubules. However, much remains unknown about the contributions of cytokines to successful stem cell culture. In the present study, we examined the role of leukemia inhibitory factor (LIF) in GS cell culture. We found that the addition of LIF to newborn testis cell culture enhances the formation of germ cell colonies. Ciliary neurotrophic factor, but not oncostatin M, had the same effect, although they both bind to the IL-6ST (gp130) receptor. On the other hand, GS cells could be established from pup or adult testes in the absence of LIF. No phenotypic or functional difference was found between GS cells established from different stages, and normal offspring were born from pup-derived GS cells that had been maintained in the absence of LIF, indicating that LIF per se is not involved in the self-renewal of GS cells. These results demonstrate that LIF is useful in the initiation of GS cell culture and suggest that LIF or a related cytokine is involved in the maturation of gonocytes into spermatogonia.

Spermatogonial transplantation has demonstrated a unique opportunity for studying spermatogenesis and provided an assay for spermatogonial stem cells. However, it has remained unknown whether germ cells that matured in a xenogeneic environment are functionally normal. In this investigation, we demonstrate the successful production of xenogeneic offspring by using spermatogonial transplantation. Rat spermatogonial stem cells were collected from immature testis and transplanted into the seminiferous tubules of busulfan-treated nude mouse testis. Using rat spermatids or spermatozoa that developed in xenogeneic surrogate mice, rat offspring were born from fresh and cryopreserved donor cells after microinsemination with rat oocytes. These offspring were fertile and had a normal imprinting pattern. The xenogeneic offspring production by interspecies germ cell transplantation and in vitro microinsemination will become a powerful tool in animal transgenesis and species conservation.

Spermatogenesis originates from a small number of spermatogonial stem cells that can reinitiate spermatogenesis and produce germ cell colonies following transplantation into infertile recipient testes. Although several previous studies have suggested a single-cell origin of germ cell colonies, only indirect evidence has been presented. In this investigation, we tested the clonal origin hypothesis using a retrovirus, which could specifically mark an individual spermatogonial stem cell. Spermatogonial stem cells were infected in vitro with an enhanced green fluorescence protein-expressing retrovirus and subsequently transplanted into infertile recipient mice. Live haploid germ cells were recovered from individual colonies and were microinjected into eggs to create offspring. In total, 45 offspring were produced from five colonies, and 23 (51%) of the offspring were transgenic. Southern blot analysis indicated that the transgenic offspring from the single colony carried a common integration site, and the integration site was different among the transgenic offspring from different colonies. These results provide evidence that germ cell colonies develop from single spermatogonial stem cells.