Restoration of fertility in nonablated recipient mice after spermatogonial stem cell transplantation.

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.

Intracytoplasmic sperm injection induces transgenerational abnormalities in mice.

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.

Incomplete activation of Alyref and Gabpb1 leads to preimplantation arrest in cloned mouse embryos.

Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog, whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.

UHRF1 is essential for proper cytoplasmic architecture and function of mouse oocytes and derived embryos.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is a protein essential for the maintenance of DNA methylation in somatic cells. However, UHRF1 is predominantly localized in the cytoplasm of mouse oocytes and preimplantation embryos, where it may play a role unrelated to the nuclear function. We herein report that oocyte-specific Uhrf1 KO results in impaired chromosome segregation, abnormal cleavage division, and preimplantation lethality of derived embryos. Our nuclear transfer experiment showed that the phenotype is attributable to cytoplasmic rather than nuclear defects of the zygotes. A proteomic analysis of KO oocytes revealed the down-regulation of proteins associated with microtubules including tubulins, which occurred independently of transcriptomic changes. Intriguingly, cytoplasmic lattices were disorganized, and mitochondria, endoplasmic reticulum, and components of the subcortical maternal complex were mislocalized. Thus, maternal UHRF1 regulates the proper cytoplasmic architecture and function of oocytes and preimplantation embryos, likely through a mechanism unrelated to DNA methylation.

Glutamine protects mouse spermatogonial stem cells against NOX1-derived ROS for sustaining self-renewal division in vitro.

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.

Birth of mice from meiotically arrested spermatocytes following biparental meiosis in halved oocytes.

Microinjection of spermatozoa or spermatids into oocytes is a major choice for infertility treatment. However, the use of premeiotic spermatocytes has never been considered because of its technical problems. Here, we show that the efficiency of spermatocyte injection in mice can be improved greatly by reducing the size of the recipient oocytes. Live imaging showed that the underlying mechanism involves reduced premature separation of the spermatocyte's meiotic chromosomes, which produced much greater (19% vs. 1%) birth rates in smaller oocytes. Application of this technique to spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans, resulted in the production of live offspring. Thus, the microinjection of primary spermatocytes into oocytes may be a potential treatment for overcoming a form of nonobstructive azoospermia caused by meiotic failure.

Regeneration of spermatogenesis by mouse germ cell transplantation into allogeneic and xenogeneic testis primordia or organoids.

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 × 104 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.

Highly rigid H3.1/H3.2-H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells.

The placenta is a highly evolved, specialized organ in mammals. It differs from other organs in that it functions only for fetal maintenance during gestation. Therefore, there must be intrinsic mechanisms that guarantee its unique functions. To address this question, we comprehensively analyzed epigenomic features of mouse trophoblast stem cells (TSCs). Our genome-wide, high-throughput analyses revealed that the TSC genome contains large-scale (>1-Mb) rigid heterochromatin architectures with a high degree of histone H3.1/3.2-H3K9me3 accumulation, which we termed TSC-defined highly heterochromatinized domains (THDs). Importantly, depletion of THDs by knockdown of CAF1, an H3.1/3.2 chaperone, resulted in down-regulation of TSC markers, such as Cdx2 and Elf5, and up-regulation of the pluripotent marker Oct3/4, indicating that THDs maintain the trophoblastic nature of TSCs. Furthermore, our nuclear transfer technique revealed that THDs are highly resistant to genomic reprogramming. However, when H3K9me3 was removed, the TSC genome was fully reprogrammed, giving rise to the first TSC cloned offspring. Interestingly, THD-like domains are also present in mouse and human placental cells in vivo, but not in other cell types. Thus, THDs are genomic architectures uniquely developed in placental lineage cells, which serve to protect them from fate reprogramming to stably maintain placental function.

Spermatogonial stem cell transplantation into nonablated mouse recipient testes.

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.

Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†.

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.

Tsga8 is required for spermatid morphogenesis and male fertility in mice.

During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We have previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X-chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility.

An interplay of NOX1-derived ROS and oxygen determines the spermatogonial stem cell self-renewal efficiency under hypoxia.

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.

OGG1 protects mouse spermatogonial stem cells from reactive oxygen species in culture†.

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.

Germ cell depletion in recipient testis has adverse effects on spermatogenesis in orthotopically transplanted testis pieces via retinoic acid insufficiency.

Germ cell depletion in recipient testes is indispensable for successful transplantation of spermatogonial stem cells. However, we found that such treatment had an adverse effect on spermatogenesis of orthotopically transplanted donor testis tissues. In the donor tissue, the frequency of stimulated by retinoic acid (RA) 8 (STRA8) expression was reduced in germ cells, suggesting that RA signalling indispensable for spermatogenesis was attenuated in germ cell-depleted recipient testes. In this context, germ cell depletion diminished expression of testicular Aldh1a2, which is responsible for testicular RA synthesis, while Cyp26b1, which is responsible for testicular RA metabolism, was still expressed even after germ cell depletion, suggesting an alteration of the RA synthesis/metabolism ratio. These observations suggested that RA insufficiency was one of the causes of the defective donor spermatogenesis. Indeed, repetitive RA administrations significantly improved donor spermatogenesis to produce fertile offspring without any side effects. These findings may contribute to improving fertility preservation techniques for males, especially to prevent iatrogenic infertility induced by chemotherapy in prepubertal cancer patients.

A non-mosaic transchromosomic mouse model of down syndrome carrying the long arm of human chromosome 21.

Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we "clone" the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.

Loss of H3K27me3 imprinting in the Sfmbt2 miRNA cluster causes enlargement of cloned mouse placentas.

Somatic cell nuclear transfer (SCNT) in mammals is an inefficient process that is frequently associated with abnormal phenotypes, especially in placentas. Recent studies demonstrated that mouse SCNT placentas completely lack histone methylation (H3K27me3)-dependent imprinting, but how it affects placental development remains unclear. Here, we provide evidence that the loss of H3K27me3 imprinting is responsible for abnormal placental enlargement and low birth rates following SCNT, through upregulation of imprinted miRNAs. When we restore the normal paternal expression of H3K27me3-dependent imprinted genes (Sfmbt2, Gab1, and Slc38a4) in SCNT placentas by maternal knockout, the placentas remain enlarged. Intriguingly, correcting the expression of clustered miRNAs within the Sfmbt2 gene ameliorates the placental phenotype. Importantly, their target genes, which are confirmed to cause SCNT-like placental histology, recover their expression level. The birth rates increase about twofold. Thus, we identify loss of H3K27me3 imprinting as an epigenetic error that compromises embryo development following SCNT.

Autologous transplantation of spermatogonial stem cells restores fertility in congenitally infertile mice.

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.

Early production of offspring by in vitro fertilization using first-wave spermatozoa from prepubertal male mice.

Mature male mice (aged 10-12 weeks or older) are conventionally used for in vitro fertilization (IVF) in order to achieve high fertilization rates (e.g., > 70%). Here, we sought to determine the earliest age at which male mice (C57BL/6J strain) can be used efficiently for producing offspring via IVF. Because we noted that the addition of reduced glutathione (GSH) to the IVF medium significantly increased the fertilizing ability of spermatozoa from prepubertal males, we used this IVF protocol for all experiments. Spermatozoa first reached the caudal region of the epididymides at day 35; however, they were unable to fertilize oocytes. Caudal epididymal spermatozoa first became competent for oocyte fertilization at day 37, albeit at a low rate (2.9%). A high fertilization rate (72.0%) was obtained at day 40, and 52.4% of the embryos thus obtained developed into offspring after embryo transfer. Moreover, we found that corpus epididymal spermatozoa in prepubertal mice could fertilize oocytes; however, the fertilization rates were always < 50%, regardless of the age of the males. Caput epididymal spermatozoa failed to fertilize oocytes irrespective of the age of the males. Therefore, we propose that caudal epididymal spermatozoa from male mice aged 40 days can be efficiently used for IVF, to obtain offspring in the shortest attainable time. This protocol will reduce the turnover time required for the generation of mice by ~1 month compared with that of the conventional IVF protocol.

Birth of a marmoset following injection of elongated spermatid from a prepubertal male.

The common marmoset is a small nonhuman primate in which the application of transgenesis and genetic knockout techniques allows the generation of gene-modified models of human diseases. However, its longer generation time than that of rodents is a major obstacle to the widespread use of gene-modified marmosets for biomedical research. In this study, we examined the feasibility of shortening the generation time by using prepubertal marmoset males as gamete donors. We collected late round stage spermatids (Steps 5-7), elongated spermatids, and testicular spermatozoa from the testis of a prepubertal 11-month-old male marmoset and injected them into in vitro-matured oocytes. After 7 days in culture, two embryos from elongated spermatid injection and two embryos from sperm injection were transferred into two separate recipient females. The recipient female that received elongated spermatid injection-derived embryos became pregnant and gave birth to one female infant. This is the first demonstration that a spermatid from a prepubertal male primate can support full-term development. Using this method, we can expect to obtain offspring of gene-modified males 6 months to a year earlier than with natural mating.

ROS amplification drives mouse spermatogonial stem cell self-renewal.

Reactive oxygen species (ROS) play critical roles in self-renewal division for various stem cell types. However, it remains unclear how ROS signals are integrated with self-renewal machinery. Here, we report that the MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification. The activation of MAPK14 induced MAPK7 phosphorylation in cultured SSCs, and targeted deletion of Mapk14 or Mapk7 resulted in significant SSC deficiency after spermatogonial transplantation. The activation of this signaling pathway not only induced Nox1 but also increased ROS levels. Chemical screening of MAPK7 targets revealed many ROS-dependent spermatogonial transcription factors, of which BCL6B was found to initiate ROS production by increasing Nox1 expression via ETV5-induced nuclear translocation. Because hydrogen peroxide or Nox1 transfection also induced BCL6B nuclear translocation, our results suggest that BCL6B initiates and amplifies ROS signals to activate ROS-dependent spermatogonial transcription factors by forming a positive feedback loop.