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.

Derivation of embryonic stem cells from wild-derived mouse strains by nuclear transfer using peripheral blood cells.

Wild-derived mouse strains have been extensively used in biomedical research because of the high level of inter-strain polymorphisms and phenotypic variations. However, they often show poor reproductive performance and are difficult to maintain by conventional in vitro fertilization and embryo transfer. In this study, we examined the technical feasibility of derivation of nuclear transfer embryonic stem cells (ntESCs) from wild-derived mouse strains for their safe genetic preservation. We used leukocytes collected from peripheral blood as nuclear donors without sacrificing them. We successfully established 24 ntESC lines from two wild-derived strains of CAST/Ei and CASP/1Nga (11 and 13 lines, respectively), both belonging to Mus musculus castaneus, a subspecies of laboratory mouse. Most (23/24) of these lines had normal karyotype, and all lines examined showed teratoma formation ability (4 lines) and pluripotent marker gene expression (8 lines). Two male lines examined (one from each strain) were proven to be competent to produce chimeric mice following injection into host embryos. By natural mating of these chimeric mice, the CAST/Ei male line was confirmed to have germline transmission ability. Our results demonstrate that inter-subspecific ntESCs derived from peripheral leukocytes could provide an alternative strategy for preserving invaluable genetic resources of wild-derived mouse strains.

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.

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.

Progress of genome editing technology and developmental biology useful for radiation research.

Following the development of genome editing technology, it has become more feasible to create genetically modified animals such as knockout (KO), knock-in, and point-mutated animals. The genome-edited animals are useful to investigate the roles of various functional genes in many fields of biological science including radiation research. Nevertheless, some researchers may experience difficulty in generating genome-edited animals, probably due to the requirement for equipment and techniques for embryo manipulation and handling. Furthermore, after obtaining F0 generation, genome-edited animals generally need to be expanded and maintained for analyzing the target gene function. To investigate genes essential for normal birth and growth, the generation of conditional KO (cKO) animals in which a tissue- or stage-specific gene mutation can be introduced is often required. Here, we describe the basic principle and application of genome editing technology including zinc-finger nuclease, transcription-activator-like effector nuclease, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas) systems. Recently advanced developmental biology methods have enabled application of the technology, especially CRISPR/Cas, to zygotes, leading to the prompt production of genome-edited animals. For pre-implantation embryos, genome editing via oviductal nucleic acid delivery has been developed as an embryo manipulation- or handling-free method. Examining the gene function at F0 generation is becoming possible by employing triple-target CRISPR technology. This technology, in combination with a blastocyst complementation method enables investigation of even birth- and growth-responsible genes without establishing cKO strains. We hope that this review is helpful for understanding and expanding genome editing-related technology and for progressing radiation research.

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.

Improving ovulation in gilts using anti-inhibin serum treatment combined with fixed-time artificial insemination.

For successful batch farrowing, porcine oestrus and ovulation must be synchronized using fixed-time artificial insemination (FTAI). However, exogenous gonadotropins, which are currently used in FTAI, negatively affect gilt ovulation. Here, we aimed to improve sexually mature gilt superovulation efficiency using passive immunization against inhibin during FTAI. Altrenogest-treated gilts were challenged with 10 ml anti-inhibin serum (AIS group, n = 6), 1,000 IU pregnant mare serum gonadotropin (PMSG group, n = 6), or 10 ml goat serum (control group, n = 6). Gilts in the AIS and PMSG groups were inseminated according to the FTAI protocol, and gilts in the control group were inseminated during natural oestrus. When PMSG was replaced by AIS during FTAI of gilts, ovulation rate and embryos recovered were significantly greater in the AIS group as compared to the other two groups (p < .05). Especially the average number of 6-8-cell embryos in the AIS group was significantly higher than that in the PMSG group (p < .01). Moreover, the blastocyst number in the AIS group was significantly higher than that in the PMSG group and the control group (p < .05). But there was no significant difference in the blastocyst number between the PMSG group and the control group (p > .05). Besides, plasma levels of estradiol-ß (E2) and progesterone (P4) were significantly greater in the AIS group as compared to the other two groups on Day 23 and D 27, respectively (p < .01). In summary, we devised an improved high-yield FTAI protocol for sexually mature gilts using AIS; this protocol had a greater superovulation efficiency than the FTAI using PMSG.

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.

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.

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.

Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice.

The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells.

Telomere shortening by transgenerational transmission of TNF-α-induced TERRA via ATF7.

Various stresses increase disease susceptibility and accelerate aging, and increasing evidence suggests that these effects can be transmitted over generation. Epidemiological studies suggest that stressors experienced by parents affect the longevity of their offspring, possibly by regulating telomere dynamics. Telomeres are elongated by telomerase and shortened by certain stresses as well as telomere repeat-containing RNA (TERRA), a telomere transcript. However, the mechanism underlying the transgenerational effects is poorly understood. Here, we show that TNF-α, which is induced by various psychological stresses, induces the p38-dependent phosphorylation of ATF7, a stress-responsive chromatin regulator, in mouse testicular germ cells. This caused a release of ATF7 from the TERRA gene promoter in the subtelomeric region, which disrupted heterochromatin and induced TERRA. TERRA was transgenerationally transmitted to zygotes via sperm and caused telomere shortening. These results suggest that ATF7 and TERRA play key roles in paternal stress-induced telomere shortening in the offspring.

Aberrant imprinting in mouse trophoblast stem cells established from somatic cell nuclear transfer-derived embryos.

Although phenotypic abnormalities frequently appear in the placenta following somatic cell nuclear transfer (SCNT), mouse trophoblast stem cells (TSCs) established from SCNT embryos reportedly show no distinct abnormalities compared with those derived from normal fertilization. In this study, we reexamined SCNT-TSCs to identify their imprinting statuses. Placenta-specific maternally imprinted genes (Gab1, Slc38a4, and Sfmbt2) consistently showed biallelic expression in SCNT-TSCs, suggesting their loss of imprinting (LOI). The LOI of Gab1 was associated with decreased DNA methylation, and that of Sfmbt2 was associated with decreased DNA methylation and histone H3K27 trimethylation. The maternal allele of the intergenic differentially methylated region (IG-DMR) was aberrantly hypermethylated following SCNT, even though this region was prone to demethylation in TSCs when established in a serum-free chemically defined medium. These findings indicate that the development of cloned embryos is associated with imprinting abnormalities specifically in the trophoblast lineage from its initial stage, which may affect subsequent placental development.

Loss of H3K27me3 Imprinting in Somatic Cell Nuclear Transfer Embryos Disrupts Post-Implantation Development.

Animal cloning can be achieved through somatic cell nuclear transfer (SCNT), although the live birth rate is relatively low. Recent studies have identified H3K9me3 in donor cells and abnormal Xist activation as epigenetic barriers that impede SCNT. Here we overcome these barriers using a combination of Xist knockout donor cells and overexpression of Kdm4 to achieve more than 20% efficiency of mouse SCNT. However, post-implantation defects and abnormal placentas were still observed, indicating that additional epigenetic barriers impede SCNT cloning. Comparative DNA methylome analysis of IVF and SCNT blastocysts identified abnormally methylated regions in SCNT embryos despite successful global reprogramming of the methylome. Strikingly, allelic transcriptomic and ChIP-seq analyses of pre-implantation SCNT embryos revealed complete loss of H3K27me3 imprinting, which may account for the postnatal developmental defects observed in SCNT embryos. Together, these results provide an efficient method for mouse cloning while paving the way for further improving SCNT efficiency.

Application of auxin-inducible degron technology to mouse oocyte activation with PLCζ.

In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/µl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ-AID-EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ-AID-EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ-AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.

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

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 KitlSl/KitlSl-d mouse testes with defective Sertoli cells received Kitl-expressing AAVs, spermatogenesis regenerated and offspring were produced. None of the offspring contained the AAV genome. Thus, AAV1/9 allows efficient germline and niche manipulation by penetrating the BTB and basement membrane, providing a promising strategy for the development of gene therapies for reproductive defects.

MAFB is dispensable for the fetal testis morphogenesis and the maintenance of spermatogenesis in adult mice.

The transcription factor MAFB is an important regulator of the development and differentiation of various organs and tissues. Previous studies have shown that MAFB is expressed in embryonic and adult mouse testes and is expected to act as the downstream target of retinoic acid (RA) to initiate spermatogenesis. However, its exact localization and function remain unclear. Here, we localized MAFB expression in embryonic and adult testes and analyzed its gene function using Mafb-deficient mice. We found that MAFB and c-MAF are the only large MAF transcription factors expressed in testes, while MAFA and NRL are not. MAFB was localized in Leydig and Sertoli cells at embryonic day (E) 18.5 but in Leydig cells, Sertoli cells, and pachytene spermatocytes in adults. Mafb-deficient testes at E18.5 showed fully formed seminiferous tubules with no abnormal structure or differences in testicular somatic cell numbers compared with those of control wild-type mice. Additionally, the expression levels of genes related to development and function of testicular cells were unchanged between genotypes. In adults, the expression of MAFB in Sertoli cells was shown to be stage specific and induced by RA. By generating Mafbfl/fl CAG-CreER™ (Mafb-cKO) mice, in which Cre recombinase was activated upon tamoxifen treatment, we found that the neonatal cKO mice died shortly upon Mafb deletion, but adult cKO mice were alive upon deletion. Adult cKO mice were fertile, and spermatogenesis maintenance was normal, as indicated by histological analysis, hormone levels, and germ cell stage-specific markers. Moreover, there were no differences in the proportion of seminiferous stages between cKO mice and controls. However, RNA-Seq analysis of cKO Sertoli cells revealed that the down-regulated genes were related to immune function and phagocytosis activity but not spermatogenesis. In conclusion, we found that MAFB is dispensable for fetal testis morphogenesis and spermatogenesis maintenance in adult mice, despite the significant gene expression in different cell types, but MAFB might be critical for phagocytosis activity of Sertoli cells.

Histone H3 Methylated at Arginine 17 Is Essential for Reprogramming the Paternal Genome in Zygotes.

At fertilization, the paternal genome undergoes extensive reprogramming through protamine-histone exchange and active DNA demethylation, but only a few maternal factors have been defined in these processes. We identified maternal Mettl23 as a protein arginine methyltransferase (PRMT), which most likely catalyzes the asymmetric dimethylation of histone H3R17 (H3R17me2a), as indicated by in vitro assays and treatment with TBBD, an H3R17 PRMT inhibitor. Maternal histone H3.3, which is essential for paternal nucleosomal assembly, is unable to be incorporated into the male pronucleus when it lacks R17me2a. Mettl23 interacts with Tet3, a 5mC-oxidizing enzyme responsible for active DNA demethylation, by binding to another maternal factor, GSE (gonad-specific expression). Depletion of Mettl23 from oocytes resulted in impaired accumulation of GSE, Tet3, and 5hmC in the male pronucleus, suggesting that Mettl23 may recruit GSE-Tet3 to chromatin. Our findings establish H3R17me2a and its catalyzing enzyme Mettl23 as key regulators of paternal genome reprogramming.