Mouse embryonic stem cells maintain differentiation potency into somatic lineage despite alternation of ploidy.

Vertebrates, including mammals, are considered to have evolved by whole genome duplications. Although some fish have been reported to be polyploids that have undergone additional genome duplication, there have been no reports of polyploid mammals due to abnormal development after implantation. Furthermore, as the number of physiologically existing tetraploid somatic cells is small, details of the functions of these ploidy-altered cells are not fully understood. In this present study, we aimed to clarify the details of the differentiation potency of tetraploids using tetraploid embryonic stem cells. To clarify the differentiation potency, we used mouse tetraploid embryonic stem cells derived from tetraploid embryos. We presented tetraploid embryonic stem cells differentiated into neural and osteocyte lineage in vitro and tetraploid cells that contributed to various tissues of chimeric embryos ubiquitously in vivo. These results revealed that mouse embryonic stem cells maintain differentiation potency after altering the ploidy. Our results provide an important basis for the differentiation dynamics of germ layers in mammalian polyploid embryogenesis.

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis).

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells.

BACKGROUND: Cell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells. METHODS: Fused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells. RESULTS: Colonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells. CONCLUSIONS: Our results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.

Hyper-polyploid embryos survive after implantation in mice.

Polyploids generated by natural whole genome duplication have served as a dynamic force in vertebrate evolution. As evidence for evolution, polyploid organisms exist generally, however there have been no reports of polyploid organisms in mammals. In mice, polyploid embryos under normal culture conditions normally develop to the blastocyst stage. Nevertheless, most tetraploid embryos degenerate after implantation, indicating that whole genome duplication produces harmful effects on normal development in mice. Most previous research on polyploidy has mainly focused on tetraploid embryos. Analysis of various ploidy outcomes is important to comprehend the effects of polyploidization on embryo development. The purpose of this present study was to discover the extent of the polyploidization effect on implantation and development in post-implantation embryos. This paper describes for the first time an octaploid embryo implanted in mice despite hyper-polyploidization, and indicates that these mammalian embryos have the ability to implant, and even develop, despite the harmfulness of extreme whole genome duplication.

Aggregation recovers developmental plasticity in mouse polyploid embryos.

Tetraploid embryos normally develop into blastocysts and embryonic stem cells can be established from tetraploid blastocysts in mice. Thus, polyploidisation does not seem to be so harmful during preimplantation development. However, the mechanisms by which early mammalian development accepts polyploidisation are poorly understood. In this study, we aimed to elucidate the effect of polyploidisation on early mammalian development and to further comprehend its tolerance using hyperpolyploid embryos produced by repetitive whole genome duplication. We successfully established several types of polyploid embryos (tetraploid, octaploid and hexadecaploid) and studied their developmental potential invitro. We demonstrated that all types of these polyploid embryos maintained the ability to develop to the blastocyst stage, which implies that mammalian cells might have basic cellular functions in implanted embryos, despite polyploidisation. However, the inner cell mass was absent in hexadecaploid blastocysts. To complement the total number of cells in blastocysts, a fused hexadecaploid embryo was produced by aggregating several hexadecaploid embryos. The results indicated that the fused hexadecaploid embryo finally recovered pluripotent cells in the blastocyst. Thus, our findings suggest that early mammalian embryos may have the tolerance and higher plasticity to adapt to hyperpolyploidisation for blastocyst formation, despite intense alteration of the genome volume.

A novel spontaneous mutation of BCAR3 results in extrusion cataracts in CF#1 mouse strain.

A substrain of mice originating from the CF#1 strain (an outbred colony) reared at Osaka Prefecture University (CF#1/lr mice) develops cataracts beginning at 4 weeks of age. Affected mice were fully viable and fertile and developed cataracts by 14 weeks of age. Histologically, CF#1/lr mice showed vacuolation of the lens cortex, swollen lens fibers, lens rupture and nuclear extrusion. To elucidate the mode of inheritance, we analyzed heterozygous mutant hybrids generated from CF#1/lr mice and wild-type BALB/c mice. None of the heterozygous mutants were affected, and the ratio of affected to unaffected mice was 1:3 among the offspring of the heterozygous mutants. For the initial genome-wide screening and further mapping, we used affected progeny of CF#1/lr × (CF#1/lr × BALB/c) mice. We concluded that the cataracts in CF#1/lr mice are inherited through an autosomal recessive mutation and that the mutant gene is located on mouse chromosome 3 between D3Mit79 and D3Mit216. In this region, we identified 8 genes associated with ocular disease. All 8 genes were sequenced and a novel point mutation (1 bp insertion of cytosine) in exon 7 of the Bcar3 gene was identified. This mutation produced a premature stop codon and a truncated protein. In conclusion, we have identified the first spontaneous mutation in the Bcar3 gene associated with lens extrusion cataracts. This novel cataract model may provide further knowledge of the molecular biology of cataractogenesis and the function of the BCAR3 protein.

Effects of whole genome duplication on cell size and gene expression in mouse embryonic stem cells.

Alterations in ploidy tend to influence cell physiology, which in the long-term, contribute to species adaptation and evolution. Polyploid cells are observed under physiological conditions in the nerve and liver tissues, and in tumorigenic processes. Although tetraploid cells have been studied in mammalian cells, the basic characteristics and alterations caused by whole genome duplication are still poorly understood. The purpose of this study was to acquire basic knowledge about the effect of whole genome duplication on the cell cycle, cell size, and gene expression. Using flow cytometry, we demonstrate that cell cycle subpopulations in mouse tetraploid embryonic stem cells (TESCs) were similar to those in embryonic stem cells (ESCs). We performed smear preparations and flow cytometric analysis to identify cell size alterations. These indicated that the relative cell volume of TESCs was approximately 2.2-2.5 fold that of ESCs. We also investigated the effect of whole genome duplication on the expression of housekeeping and pluripotency marker genes using quantitative real-time PCR with external RNA. We found that the target transcripts were 2.2 times more abundant in TESCs than those in ESCs. This indicated that gene expression and cell volume increased in parallel. Our findings suggest the existence of a homeostatic mechanism controlling the cytoplasmic transcript levels in accordance with genome volume changes caused by whole genome duplication.

Intracytoplasmic sperm injection into oocytes matured in vitro and early embryonic development in the owl monkey (Aotus lemurinus).

Purpose: We explored the possibility of employing intracytoplasmic sperm injection (ICSI), involving oocytes and sperm of owl monkeys, to increase the availability of this species for investigations relating to malaria, etc., by increasing the number of animals in our laboratory. Methods: Two owl monkeys (a female and a male), raised at the Amami Laboratory of the University of Tokyo, were used. Follicular oocytes surrounded with cumulus cells were cultured in vitro for approximately 25 h and cumulus cells were removed with 0.1 % hyaluronidase. Because of the poor motility of caudal epididymal sperm, sperm were injected without adding polyvinylpyrrolidone to immobilize them. The ICSI procedure was performed by an individual with considerable experience of human ICSI. Results: We were able to produce two owl monkey embryos using ICSI of oocytes that matured to MII stage. Both embryos reached the 10-cell stage at 98 h after ICSI and showed signs of compaction, but failed to cleave further. Conclusions: Although we successfully produced owl monkey embryos after ICSI, the embryos did not develop to the blastocyst stage. Many parameters need to be studied further, including superovulation, selection of culture media, and selection of good quality sperm in order to achieve successful ICSI in the owl monkey.

Identification and characterization of the interactive proteins with cytotoxic T-lymphocyte antigen-2α.

Cytotoxic T-lymphocyte antigen-2α (CTLA-2α) is a potent inhibitor of cathepsin L-like cysteine proteases. Recombinant CTLA-2α is known to be a potent, competitive inhibitor of cathepsin L-like cysteine proteases. In this study, cathepsin L, cathepsin C, and tubulointerstitial nephritis antigen-related protein 1 (TINAGL1) were identified as novel interactive proteins of CTLA-2α by the yeast two-hybrid screening system. The direct interactions and co-localization of these proteins with CTLA-2α were confirmed using co-immunoprecipitation and immunofluorescence staining, respectively. The disulfide-bonded CTLA-2α/cathepsin L complex was isolated from mouse tissue. CTLA-2α was found to be specific and consistently expressed on the maternal side of the mouse placenta. Double immunofluorescence analysis showed that CTLA-2α was co-localized with cathepsin L, cathepsin C, and TINAGL1 in placenta. A simple cell-based fluorescence assay revealed that CTLA-2α exhibited inhibitory activity toward cathepsin C in live cells, which indicated that CTLA-2α is a novel endogenous inhibitor of cathepsin C.

Characteristic patterns of maternal and fetal arterial construction in the rabbit placenta.

We studied vascular structure of the rabbit placenta, especially on three-dimensional morphological patterns and developmental process. Basic structure of maternal arterial system was re-constructed during day 13-18 of pregnancy, forming main routes for blood supply through the arterial sinuses and radial arteries. Intra-villous spaces were drastically developed showing as branches from the terminal radial arteries. Fetal arterial system was generated accompanied with maternal vascular development, showing characteristic features such as the perforating linear artery, hairpin flexion, and circular anastomoses in the capillaries. From the correlation of maternal and fetal blood currents, gas-exchange style in the rabbit placenta was considered as counter-current and pool mixed patterns. These data demonstrated an original feature for the placental arterial systems in rabbits, which differed from other animals having a property for discoid placenta.

Appearance of small extracellular vesicles in the mouse pregnant serum and the localization in placentas.

Exosomes or small extracellular vesicles (sEVs) are present in the blood of pregnant mice and considered to be involved in pregnancy physiology. Although sEVs in pregnant periods are proposed to be derived from placentas, sEVs-producing cells are not well known in mouse placentas. We studied the dynamics and localization of sEVs in pregnant serum and placentas, and examined gestational variation of microRNA (miRNA). Serums and placentas were collected from non-pregnant (NP) and pregnant mice throughout the entire gestational day (Gd). EVs were purified from serums and total RNA was isolated from EVs. Nanoparticle-tracking assay (NTA) revealed that the rates of sEVs in EVs are 53% at NP, and increased to 80.1% at Gd 14.5 and 97.5% at Gd 18.5. Western blotting on EVs showed positive reactivity to the tetraspanin markers and clarified that the results using anti-CD63 antibody were most consistent with the sEVs appearance detected by NTA. Serum EVs also showed a positive reaction to the syncytiotrophoblast marker, syncytin-1. Immunohistostaining using anti-CD63 antibody showed positive reactions in mouse placentas at the syncytiotrophoblasts and endothelial cells of the fetal capillaries. Quantitative PCR revealed that significantly higher amounts of miRNAs were included in the sEVs of Gd 18.5. Our results suggested that sEVs are produced in the mouse placenta and transferred to maternal or fetal bloodstreams. sEVs are expected to have a miRNA-mediated physiological effect and become useful biomarkers reflecting the pregnancy status.

Verification of the efficacy of deciduae for determining the genetic type of mouse embryos.

Mouse embryos in the early-implantation stage require manipulation under a microscope. While the extraction of DNA, RNA and proteins from a single sample allows for both determination of genetic type and analysis of gene expression, whole mount analysis is not possible. In this study, we explored the applicability of PCR using extraembryonic tissues, especially the decidual side tissue after isolating the embryos from implantation sites to establish a method for determining the genetic type of embryos. The implantation site was resected at each day from the date of vaginal plug confirmation, separated into embryos and deciduae. Genomic DNA were isolated separately from the embryos and the deciduae. PCR was performed using these genomic DNA, and the band patterns were compared after electrophoresis. As a result, we demonstrated that detecting embryo-derived cells in the decidua allows determination of the sex and presence of transgenes without harming the mouse embryos themselves, from 8.5 days of age. This method enables the determination of the genetic type of mouse embryos without damaging. This technique would expand the adaptations for analysis of mouse implanted embryos.

Specific expression and blood kinetics for relaxin 2, lipocalin 2, and tissue factor pathway inhibitor 2 at the canine placenta and pregnant bloods.

In general, humoral factors released from the placenta influence pregnancy progression, but the involvement of the canine placenta is often unidentified. We investigated specific genes in canine placentas and analyzed the blood dynamics of the translated proteins. Furthermore, RNAs are known to be released from placentas embedding in exosomes, a type of extracellular vesicles. Here, the presence of cell-free RNAs in pregnant serums was also confirmed. RNA specimens were purified from the normal healthy dog placentas and applied to RNA-Seq analysis. Expressions of frequent genes were confirmed by RT-PCR using placentas from other individuals and breeds. Relaxin (RLN) 2, lipocalin (LCN) 2, and tissue factor pathway inhibitor (TFPI) 2 were selected as high-expressed and placenta-specific genes. By western blot, the three factors were clearly detected in the pregnant serums. Quantitative analysis revealed that the amount of RLN2 increased significantly from non-pregnancy to day 41 of pregnancy. Regarding LCN2 and TFPI2, the protein serum levels elevated during pregnancy, but the statistical differences were not detected. Exosomes were found in all pregnant serums; however, the percentage was less than 6% in total extracellular vesicles. The cell-free RNA related to RLN2 was detected, but no elevation was confirmed during pregnancy. We found specific genes in the canine placenta and the transition of their translated protein into the blood. These factors may become useful tools for research on canine pregnancy and monitoring of reproductive management. Exosomes and cell-free RNA could not be found to be valid in canine reproduction.

Establishment of African pygmy mouse induced pluripotent stem cells using defined doxycycline inducible transcription factors.

Mus minutoides is one of the smallest mammals worldwide; however, the regulatory mechanisms underlying its dwarfism have not been examined. Therefore, we aimed to establish M. minutoides induced pluripotent stem cells (iPSCs) using the PiggyBac transposon system for applications in developmental engineering. The established M. minutoides iPSCs were found to express pluripotency markers and could differentiate into neurons. Based on in vitro differentiation analysis, M. minutoides iPSCs formed embryoid bodies expressing marker genes in all three germ layers. Moreover, according to the in vivo analysis, these cells contributed to the formation of teratoma and development of chimeric mice with Mus musculus. Overall, the M. minutoides iPSCs generated in this study possess properties that are comparable to or closely resemble those of naïve pluripotent stem cells (PSCs). These findings suggest these iPSCs have potential utility in various analytical applications, including methods for blastocyst completion.

Characteristics of pectens in diurnal and nocturnal birds and a new functional proposal relating to non-visual opsins.

The pecten is a fold-structured projection at the ocular fundus in bird eyes, showing morphological diversity between the diurnal and nocturnal species. However, its biological functions remain unclear. This study investigated the morphological and histological characteristics of pectens in wild birds. Additionally, the expression of non-visual opsin genes was studied in chicken pectens. These genes, identified in the chicken retina and brain, perceive light periodicity regardless of visual communication. Similar pleat numbers have been detected among bird taxa; however, pecten size ratios in the ocular fundus showed noticeable differences between diurnal and nocturnal birds. The pectens in nocturnal brown hawk owl show extremely poor vessel distribution and diameters compared with that of diurnal species. RT-PCR analysis confirmed the expression of Opn5L3, Opn4x, Rrh and Rgr genes. In situ hybridization analysis revealed the distribution of Rgr-positive reactions in non-melanotic cells around the pecten vessels. This study suggests a novel hypothesis that pectens develop dominantly in diurnal birds as light acceptors and contribute to continuous visual function or the onset of periodic behaviour.

Effects of transient forebrain ischemia on the hippocampus of the Mongolian gerbil (Meriones unguiculatus): an immunohistochemical study.

In the Mongolian gerbil, bilateral common carotid artery occlusion (BCCAO) for several minutes induces ischemia and delayed neuronal cell death in the CA1 region of the hippocampus due to their incomplete Circle of Willis. In the present study, the expression of fibroblast growth factor 2 (FGF2), its receptors (FGFR1 and FGFR2), glial fibrillary acidic protein (GFAP), and isolectin B4 (ISLB4) was investigated by immunohistochemical and lectin-binding methods after BCCAO was performed for 5 min in gerbils. One day after BCCAO, the pyramidal cells of the CA1 region of the hippocampus showed degenerative changes and lowered expression of FGF2, FGFR1, and FGFR2. Three days after BCCAO, there was an increase in GFAP-positive astrocytes and ISLB4-positive microglial cells. From five to 10 days after BCCAO, intense neuronal cell death in the stria pyramidale of the hippocampal CA1 region was observed, as well as an increase in GFAP-positive astrocytes and decrease in ISLB4-positive microglial cells. These results indicate that transient forebrain ischemia induces neuronal cell death with lowered expression of FGF2 and its receptors, and that the activation of glial cells may not directly lead to neuronal cell death.

Effect of platelet lysate on Schwann-like cell differentiation of equine bone marrow-derived mesenchymal stem cells.

Currently, treatment for peripheral nerve injuries in horses primarily relies upon physical therapy and anti-inflammatory drugs. In humans, various treatments using mesenchymal stem cells (MSCs) are being attempted. Therefore, in this study, Schwann-like cell differentiation cultures of equine MSCs were prepared using fetal bovine serum (FBS) and equine platelet lysate (ePL). ePL increased the platelet count to 1 × 106/µl, the optimal concentration for culture. In both groups, an elongated morphology at both ends, characteristic of Schwann cells, was observed under the microscope. Real-time PCR analysis of the expression levels of neuronal markers showed that the ePL group tended to express higher levels of Nestin, Musashi1, and Pax3 than the FBS group. p75 was expressed at low levels in both groups. Immunostaining results showed localization of Nestin in both groups of differentiated cells, but the positive cell rate was significantly higher in the ePL group than in the FBS group. Overall, the ePL gro showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease. This knowledge could be applied translationaly in the treatment of amyotrophic lateral sclerosis in humans.Overall, the ePL group showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease and in the treatment of amyotrophic lateral sclerosis in humans.

Characteristic amino acid residues in the growth hormone receptor gene on Mus minutoides underlying dwarfism.

The African pygmy mouse ( Mus minutoides ) displays a dwarfism phenotype distinctive from closely related species. This study aimed to investigate the growth hormone receptor (Ghr) gene sequence in M. minutoides . We identified several amino acid variations, including the P469L mutation. Our findings suggest that this mutation affects Ghr protein functionality, decreasing Igf1 expression and contributing to the dwarfism observed in M. minutoides . Further studies utilizing genome editing technology are necessary to elucidate the mechanisms involved in mammalian body size determination.

Expression dynamics of Crry at the implantation sites in normal pregnancy and response against miscarriage induction.

In mammals, immune tolerance against fetal tissue has been established for normal pregnancy progression. It is known that Crry regulates complemental activity to prevent injury of the mouse embryo and extra-embryonic tissue. This study aimed to examine the expression appearance and normal localization sites of Crry in the mouse placenta. Also, the emergency responses of Crry were verified at the time of experimental miscarriage induction. Moreover, we investigated an existing similar protein of Crry in animal placentas other than mice. Crry expression level showed a peak at day 8.5 of pregnancy. Trophoblast giant cells and decidual cells indicated a positive reaction to anti-Crry antibody. After treatments of interferon-γ, Crry expression was increased significantly in the survived implantation sites as compared with the controls. However, there was no significant difference in the miscarriage-initiated sites. It disclosed that Crry distributes from the early to middle periods of the placentas and involves complement regulation at the extraembryonic tissue and decidua basalis. Crry also showed an ability to respond to risk against external initiation for urgent miscarriage. Finally, we found anti-mouse Crry antibody-bound proteins in the placenta of many animals. It suggests a potency of Crry to make an environment of immune tolerance in many types of mammal placentas.

Expression and localization of NO synthase isoenzymes (iNOS and eNOS) in development of the rabbit placenta.

Nitric oxide synthase (NOS) is a key regulator of angiogenesis and embryogenesis in the mammalian reproductive process. Here, we attempted to clarify the expression and localization of inducible and endothelial NOS (iNOS and eNOS) in the developing rabbit placenta. Real-time RT-PCR analysis indicated that iNOS mRNA was significantly upregulated till the complete development of the placenta (d18), and then significantly decreased at the end of fetal growth stage (d28) during successful pregnancy. The eNOS mRNA was also enhanced in the pregnant uteri and gradually decreased near the term of pregnancy. Western blot analysis also showed elevation of the iNOS and eNOS protein levels during the course of successful pregnancy till the functional maturation of the placenta (d18). Immunohistochemical study revealed distinct localizations of iNOS along the radial arteries and eNOS at the spiral arteries and arterial sinuses in the developing placenta. This may reflect that iNOS and eNOS participate in pregnancy success through placentation-specific vascular formation and by supporting adequate blood circulation in the rabbit placenta.