Search

Current infertility treatment strategies focus on mature gametes, leaving a significant proportion of cases with gamete progenitors that stopped complete differentiation. On the other hand, recent advancements in next-generation sequencing have identified many candidate genes that may promote maturation of germ cells. Although gene therapy has shown success in mice, concerns about the integration of DNA vectors into oocytes hinder clinical applications. Here, we present the restoration of fertility in female mice through Sendai virus (SeV)-mediated RNA delivery. Ovaries lacking Kitl expression exhibit only primordial follicles due to impaired signaling to oocytes expressing the KIT tyrosine kinase. Despite SeVs being immunogenic and larger than the blood-follicle barrier, the administration of Kitl -expressing SeVs reinitiated oogenesis in genetically infertile mice that have only primordial follicles, resulting in the birth of normal offspring through natural mating. This virus also effectively addressed iatrogenic infertility induced by busulfan, a widely used cancer chemotherapy agent. Offspring born through SeV administration and natural mating displayed normal genomic imprinting patterns and fertility. Since SeVs pose no genotoxicity risk, the successful restoration of fertility by SeVs represents a promising approach for treating congenital infertility with somatic cell defects and protecting fertility of cancer patients who may become infertile due to loss of oocytes during cancer therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

Intracytoplasmic sperm injection (ICSI) is clinically used to treat obstructive/nonobstructive azoospermia. This study compared the efficacy of ICSI with cauda epididymal and testicular sperm in Wistar (WI) and Brown-Norway (BN) rats. The transfer of ICSI oocytes with cryopreserved epididymal and testicular WI sperm resulted in offspring production of 26.2% and 3.7%-4.7%, respectively (P < 0.05). Treatments for artificial oocyte activation (AOA) and acrosome removal improved pronuclear formation in BN-ICSI oocytes; however, only AOA treatment was effective in producing offspring (3.7%-6.5%). In the case of ICSI with testicular sperm (TESE-ICSI), one offspring (0.6%) was derived from the BN-TESE-ICSI oocytes. The application of AOA or a hypo-osmotic sperm suspension did not improve the production of TESE-ICSI offspring. Thus, outbred WI rat offspring can be produced by using ICSI and less efficiently by using TESE-ICSI. Challenges in producing offspring by using ICSI/TESE-ICSI in inbred BN strain require further investigation.

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.)

J Reprod Dev, Vol. 70, No. 4, p. 255 Table 2 have been corrected. For the bottom of Survived (%) row, the Survived value, which read "69", should be replaced with "86".

Spermatogonial stem cells (SSCs) possess a unique ability to recolonize the seminiferous tubules. Upon microinjection into the adluminal compartment of the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) to the basal compartment of the tubule and reinitiate spermatogenesis. It was recently discovered that inhibiting retinoic acid signaling with WIN18,446 enhances SSC colonization by transiently suppressing spermatogonia differentiation, thereby promoting fertility restoration. In this study, we report that WIN18,446 increases SSC colonization by disrupting the BTB. WIN18,446 altered the expression patterns of tight junction proteins (TJPs) and disrupted the BTB in busulfan-treated mice. WIN18,446 upregulated the expression of FGF2, one of the self-renewal factors for SSCs. While WIN18,446 enhanced SSC colonization in busulfan-treated wild-type mice, it did not increase colonization levels in busulfan-treated Cldn11-deficient mice, which lack the BTB, indicating that the enhancement of SSC colonization in wild-type testes depended on the loss of the BTB. Serial transplantation analysis revealed impaired self-renewal caused by WIN18,446, indicating that WIN18,446-mediated inhibition of retinoic acid signaling impaired SSC self-renewal. Strikingly, WIN18,446 administration resulted in the death of 45% of busulfan-treated recipient mice. These findings suggest that TJP modulation is the primary mechanism behind enhanced SSC homing by WIN18,446 and raise concerns regarding the use of WIN18,446 for human SSC transplantation.

In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are 2 major assisted reproductive techniques (ARTs) used widely to treat infertility. Recently, spermatogonial transplantation emerged as a new ART to restore fertility to young patients with cancer after cancer therapy. To examine the influence of germ cell manipulation on behavior of offspring, we produced F1 offspring by a combination of two ARTs, spermatogonial transplantation and ICSI. When these animals were compared with F1 offspring produced by ICSI using fresh wild-type sperm, not only spermatogonial transplantation-ICSI mice but also ICSI-only control mice exhibited behavioral abnormalities, which persisted in the F2 generation. Furthermore, although these F1 offspring appeared normal, F2 offspring produced by IVF using F1 sperm and wild-type oocytes showed various types of congenital abnormalities, including anophthalmia, hydrocephalus, and missing limbs. Therefore, ARTs can induce morphological and functional defects in mice, some of which become evident only after germline transmission.

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.)

Characterization of spermatogonial stem cells (SSCs) has been hampered by their low frequency and lack of features that distinguish them from committed spermatogonia. Few conserved SSC markers have been discovered. To identify a new SSC marker, we evaluated SIRPA expression in mouse and rat SSCs. SIRPA was expressed in a small population of undifferentiated spermatogonia. SIRPA, and its ligand CD47 were expressed in cultured SSCs. Expression of both SIRPA and CD47 was upregulated by supplementation of GDNF and FGF2, which promoted SSC self-renewal. Sirpa depletion by short hairpin RNA impaired the proliferation of cultured SSCs, and these cells showed decreased MAP2K1 activation and PTPN11 phosphorylation. Immunoprecipitation experiments showed that SIRPA associates with PTPN11. Ptpn11 depletion impaired SSC activity in a manner similar to Sirpa depletion. SIRPA was expressed in undifferentiated spermatogonia in rat and monkey testes. Xenogenic transplantation experiments demonstrated that SIRPA is expressed in rat SSCs. These results suggest that SIRPA is a conserved SSC marker that promotes SSC self-renewal division by activating the MAP2K1 pathway via PTPN11., (© The Author(s) 2023. 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.)

The testis is an immune-privileged organ. It is considered that the testis somatic microenvironment is responsible for immune suppression. However, immunological properties of spermatogonial stem cells (SSCs) have remained unknown. Here, we report the birth of allogeneic offspring by enhanced expression of immunosuppressive PD-L1 in SSCs. In vitro supplementation of GDNF and FGF2 increased expression of PD-L1 in SSCs. Cultured SSCs maintained allogeneic spermatogenesis that persisted for >1 year. However, depletion or gene editing of Pd-l1 family genes in SSCs prevented allogeneic spermatogenesis, which suggested that germ cells are responsible for suppression of the allogeneic response. PD-L1 was induced by activation of the MAPK14-BCL6B pathway, which drives self-renewal by reactive oxygen species (ROS) generation. By contrast, reduced ROS or Mapk14 deficiency downregulated PD-L1. Allogeneic offspring were born after SSC transplantation into congenitally infertile and chemically castrated mice. Thus, SSCs have unique immunological properties, which make allogeneic recipients into "surrogate fathers.", Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Oogenesis depends on close interactions between oocytes and granulosa cells. Abnormal signaling between these cell types can result in infertility. However, attempts to manipulate oocyte-granulosa cell interactions have had limited success, likely due to the blood-follicle barrier (BFB), which prevents the penetration of exogenous materials into ovarian follicles. Here, we used adenoviruses (AVs) to manipulate the oocyte-granulosa cell interactions. AVs penetrated the BFB and transduced granulosa cells through ovarian microinjection. Although AVs caused transient inflammation, they did not impair fertility in wild-type mice. Introduction of Kitl-expressing AVs into congenitally infertile Kitl Sl-t /Kitl Sl-t mutant mouse ovaries, which contained only primordial follicles because of a lack of Kitl expression, restored fertility through natural mating. The offspring showed no evidence of AV integration and exhibited normal genomic imprinting patterns for imprinted genes. These results demonstrate the usefulness of AVs for manipulating oogenesis and suggest the possibility of gene therapies for human female infertility.

A small number of offspring are born from the numerous sperm generated from spermatogonial stem cells (SSCs). However, little is known regarding the rules and molecular mechanisms that govern germline transmission patterns. Here we report that the Trp53 tumor suppressor gene limits germline genetic diversity via Cdkn1a. Trp53-deficient SSCs outcompeted wild-type (WT) SSCs and produced significantly more progeny after co-transplantation into infertile mice. Lentivirus-mediated transgenerational lineage analysis showed that offspring bearing the same virus integration were repeatedly born in a non-random pattern from WT SSCs. However, SSCs lacking Trp53 or Cdkn1a sired transgenic offspring in random patterns with increased genetic diversity. Apoptosis of KIT + differentiating germ cells was reduced in Trp53- or Cdkn1a-deficient mice. Reduced CDKN1A expression in Trp53-deficient spermatogonia suggested that Cdkn1a limits genetic diversity by supporting apoptosis of syncytial spermatogonial clones. Therefore, the TRP53-CDKN1A pathway regulates tumorigenesis and the germline transmission pattern., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Oocytes and granulosa cells closely interact with each other during follicular development, and a lack of appropriate signaling between them results in infertility. Attempts to manipulate oocyte microenvironment have been impeded by the impermeability of the blood-follicle barrier (BFB). To establish a strategy for manipulating oogenesis, we use adeno-associated viruses (AAVs), which have a unique ability of transcytosis. Microinjecting of AAVs into the ovarian stroma penetrates the BFB and achieves long-term gene expression. Introduction of an AAV carrying the mouse Kitl gene restores oogenesis in congenitally infertile Kitl Sl-t /Kitl Sl-t mutant mouse ovaries, which lack Kitl expression but contain only primordial follicles. Healthy offspring without AAV integration are born by natural mating. Therefore, AAV-mediated gene delivery not only provides a means for studying oocyte-granulosa interactions through the manipulation of the oocyte microenvironment but could also be a powerful method to treat female infertility resulting from somatic cell defects., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Gametogenesis requires close interactions between germ cells and somatic cells. Derivation of sperm from spermatogonial stem cells (SSCs) is hampered by the inefficiency of spermatogonial transplantation technique in many animal species because it requires a large number of SSCs and depletion of endogenous spermatogenesis. Here we used mouse testis primordia and organoids to induce spermatogenesis from SSCs. We microinjected mouse SSCs into embryonic gonads or reaggregated neonatal testis organoids, which were transplanted under the tunica albuginea of mature testes. As few as 1 × 10 4 donor cells colonized both types of transplants and produced sperm. Moreover, rat embryonic gonads supported xenogeneic spermatogenesis from mouse SSCs when transplanted in testes of immunodeficient mice. Offspring with normal genomic imprinting patterns were born after microinsemination. These results demonstrate remarkable flexibility of the germ cell-somatic cell interaction and raise new strategies of SSC manipulation for animal transgenesis and analysis of male infertility., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Germline mutations underlie genetic diversity and species evolution. Previous studies have assessed the theoretical mutation rates and spectra in germ cells mostly by analyzing genetic markers and reporter genes in populations and pedigrees. This study reported the direct measurement of germline mutations by whole-genome sequencing of cultured spermatogonial stem cells in mice, namely germline stem (GS) cells, together with multipotent GS (mGS) cells that spontaneously dedifferentiated from GS cells. GS cells produce functional sperm that can generate offspring by transplantation into seminiferous tubules, whereas mGS cells contribute to germline chimeras by microinjection into blastocysts in a manner similar to embryonic stem cells. The estimated mutation rate of GS and mGS cells was approximately 0.22 × 10 -9 and 1.0 × 10 -9 per base per cell population doubling, respectively, indicating that GS cells have a lower mutation rate compared to mGS cells. GS and mGS cells also showed distinct mutation patterns, with C-to-T transition as the most frequent in GS cells and C-to-A transversion as the most predominant in mGS cells. By karyotype analysis, GS cells showed recurrent trisomy of chromosomes 15 and 16, whereas mGS cells frequently exhibited chromosomes 1, 6, 8, and 11 amplifications, suggesting that distinct chromosomal abnormalities confer a selective growth advantage for each cell type in vitro. These data provide the basis for studying germline mutations and a foundation for the future utilization of GS cells for reproductive technology and clinical applications., (© 2021. The Author(s).)

Spermatogonial stem cells (SSCs) are maintained in a special microenvironment called a niche. However, much is unknown about components that constitute the niche. Here, we report that Cdc42 is essential for germline niche development. Sertoli cell-specific Cdc42-deficient mice showed normal premeiotic spermatogenesis. However, germ cells gradually disappeared during haploid cell formation and few germ cells remained in the mature testes. Spermatogonial transplantation experiments revealed a significant loss of SSCs in Cdc42-deficient testes. Moreover, Cdc42 deficiency in Sertoli cells downregulated GDNF, a critical factor for SSC maintenance. Cdc42-deficient Sertoli cells also exhibited lower nuclear MAPK1/3 staining. Inhibition of MAP2K1 or depletion of Pea15a scaffold protein downregulated GDNF expression. A screen of transcription factors revealed that Cdc42-deficient Sertoli cells downregulate DMRT1 and SOX9, both of which are critical for Sertoli cell development. These results indicate that Cdc42 is essential for niche function via MAPK1/3-dependent GDNF secretion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.)

Although reactive oxygen species (ROS) are required for spermatogonial stem cell (SSC) self-renewal, they induce DNA damage and are harmful to SSCs. However, little is known about how SSCs protect their genome during self-renewal. Here, we report that Ogg1 is essential for SSC protection against ROS. While cultured SSCs exhibited homologous recombination-based DNA double-strand break repair at levels comparable with those in pluripotent stem cells, they were significantly more resistant to hydrogen peroxide than pluripotent stem cells or mouse embryonic fibroblasts, suggesting that they exhibit high levels of base excision repair (BER) activity. Consistent with this observation, cultured SSCs showed significantly lower levels of point mutations than somatic cells, and showed strong expression of BER-related genes. Functional screening revealed that Ogg1 depletion significantly impairs survival of cultured SSCs upon hydrogen peroxide exposure. Thus, our results suggest increased expression of BER-related genes, including Ogg1, protects SSCs from ROS-induced damage., (© The Author(s) 2020. 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.)

Reactive oxygen species (ROS) produced by NADPH1 oxidase 1 (NOX1) are thought to drive spermatogonial stem cell (SSC) self-renewal through feed-forward production of ROS by the ROS-BCL6B-NOX1 pathway. Here we report the critical role of oxygen on ROS-induced self-renewal. Cultured SSCs proliferated poorly and lacked BCL6B expression under hypoxia despite increase in mitochondria-derived ROS. Due to lack of ROS amplification under hypoxia, NOX1-derived ROS were significantly reduced, and Nox1 -deficient SSCs proliferated poorly under hypoxia but normally under normoxia. NOX1-derived ROS also influenced hypoxic response in vivo because Nox1 -deficient undifferentiated spermatogonia showed significantly reduced expression of HIF1A, a master transcription factor for hypoxic response. Hypoxia-induced poor proliferation occurred despite activation of MYC and suppression of CDKN1A by HIF1A, whose deficiency exacerbated self-renewal efficiency. Impaired proliferation of Nox1 - or Hif1a -deficient SSCs under hypoxia was rescued by Cdkn1a depletion. Consistent with these observations, Cdkn1a -deficient SSCs proliferated actively only under hypoxia but not under normoxia. On the other hand, chemical suppression of mitochondria-derived ROS or Top1mt mitochondria-specific topoisomerase deficiency did not influence SSC fate, suggesting that NOX1-derived ROS play a more important role in SSCs than mitochondria-derived ROS. These results underscore the importance of ROS origin and oxygen tension on SSC self-renewal., (© 2021 Morimoto et al.; Published by Cold Spring Harbor Laboratory Press.)

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.)

Because spermatogonial stem cells (SSCs) are immortal by serial transplantation, SSC aging in intact testes is considered to be caused by a deteriorated microenvironment. Here, we report a cell-intrinsic mode of SSC aging by glycolysis activation. Using cultured SSCs, we found that aged SSCs proliferated more actively than young SSCs and showed enhanced glycolytic activity. Moreover, they remained euploid and exhibited stable androgenetic imprinting patterns with robust SSC activity despite having shortened telomeres. Aged SSCs showed increased Wnt7b expression, which was associated with decreased Polycomb complex 2 activity. Our results suggest that aberrant Wnt7b expression activated c- jun N-terminal kinase (JNK), which down-regulated mitochondria numbers by suppressing Ppargc1a Down-regulation of Ppargc1a probably decreased reactive oxygen species and enhanced glycolysis. Analyses of the Klotho -deficient aging mouse model and 2-y-old aged rats confirmed JNK hyperactivation and increased glycolysis. Therefore, not only microenvironment but also intrinsic activation of JNK-mediated glycolysis contributes to SSC aging., Competing Interests: The authors declare no conflict of interest.

Spermatogonial stem cells (SSCs) provide the foundation of spermatogenesis. However, because of their small number and slow self-renewal, transfection of SSCs has met with limited success. Although several viral vectors can infect SSCs, genome integration and an inability to maintain long-term gene expression have hampered studies on SSCs. Here we report successful SSC infection by Sendai virus (SV), an RNA virus in the Paramyxoviridae. The SV efficiently transduced germline stem (GS) cells, cultured spermatogonia with enriched SSC activity, and maintained gene expression for at least 5 months. It also infected freshly isolated SSCs from adult testes. The transfected GS cells reinitiated spermatogenesis following spermatogonial transplantation into seminiferous tubules of infertile mice, suggesting that SV transfection does not interfere with spermatogenesis progression. On the other hand, microinjection of SV into the seminiferous tubules of immature mice transduced SSCs and Sertoli cells, but did not transduce Leydig or peritubular cells by interstitial virus injection. SV-infected hamster GS cells, and freshly isolated rabbit or monkey SSC-like cells were identified following xenogeneic spermatogonial transplantation, suggesting that SV transduces SSCs from several mammalian species. Thus, SV is a useful vector that can transduce both SSCs and Sertoli cells and overcome problems associated with other viral vectors., (© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

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 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.)

Spermatogenesis is a complicated process that originates from spermatogonial stem cells (SSCs), which have self-renewal activity. Because SSCs are the only stem cells in the body that transmit genetic information to the next generation, they are an attractive target for germline modification. Although several virus vectors have been successfully used to transduce SSCs, cell toxicity or insertional mutagenesis of the transgene has limited their usage. Adeno-associated virus (AAV) is unique among virus vectors because of its target specificity and low toxicity in somatic cells, and clinical trials have shown that it has promise for gene therapy. However, there are conflicting reports on the possibility of germline integration of AAV into the genome of male germ cells, including SSCs. Here, we examined the usefulness of AAV vectors for exploring germline gene modification in SSCs. AAV1 infected cultured SSCs without apparent toxicity. Moreover, SSCs that were infected in fresh testis cells generated normal appearing spermatogenic colonies after spermatogonial transplantation. A microinsemination experiment produced offspring that underwent excision of the floxed target gene by AAV1-mediated Cre expression. Analysis of the offspring DNA showed no evidence of AAV integration, suggesting a low risk of germline integration by AAV infection. Although more extensive experiments are required to assess the risk of germline integration, our results show that AAV1 is useful for genetic manipulation of SSCs, and gene transduction by AAV will provide a useful approach to overcome potential problems associated with previous virus vector-mediated gene transduction., (© The Authors 2016. 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.)

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) provide the foundation for spermatogenesis. Earlier studies have shown that the transplantation of SSCs restores fertility to infertile recipients. However, most of the previously described experiments have depended on transplantation using sexually immature animals, and the effectiveness of spermatogonial transplantation in mature animals has not been examined in detail. In this study, we evaluated the efficiency of offspring production by adult recipients of spermatogonial transplantation using germline stem (GS) cells, cultured spermatogonia with enriched SSC activity. GS cells were transplanted into mature WBB6F1-W/W(v) (W) or busulfan-treated mice, which were then mated with female mice to obtain offspring from donor cells. We found that GS cells produced offspring most efficiently by transplantation into busulfan (44 mg/kg)-treated mice and all recipients produced progeny within 4 mo (76-111 days) after transplantation. When the dose dependence of offspring production was examined in W mice, approximately 40-80 SSCs were estimated to be required for fertility restoration. Efficient offspring production using GS cells and spermatogonial transplantation will be useful for analyzing factors involved in male fertility., (© 2016 by the Society for the Study of Reproduction, Inc.)

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 division, which can be recapitulated in vitro. Attempts to establish serum-free culture conditions for SSCs have met with limited success. Although we previously reported that SSCs can be cultured without serum on laminin-coated plates, the growth rate and SSC concentration were relatively low, which made it inefficient for culturing large numbers of SSCs. In this study, we report on a novel culture medium that showed improved SSC maintenance. We used Iscove modified Dulbecco medium, supplemented with lipid mixture, fetuin, and knockout serum replacement. In the presence of glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), SSCs cultured on laminin-coated plates could proliferate for more than 5 mo and maintained normal karyotype and androgenetic DNA methylation patterns in imprinted genes. Germ cell transplantation showed that SSCs in the serum-free medium proliferated more actively than those in the serum-supplemented medium and that the frequency of SSCs was comparable between the two culture media. Cultured cells underwent germline transmission. Development of a new serum- and feeder-free culture method for SSCs will facilitate studies into the effects of microenvironments on self-renewal and will stimulate further improvements to derive SSC cultures from different animal species., (© 2014 by the Society for the Study of Reproduction, Inc.)

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.