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.

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.

Discoidin domain receptor 2 (DDR2) regulates body size and fat metabolism in mice.

Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that is activated by fibrillar collagens, which act as its endogenous ligand. DDR2 regulates cell proliferation, cell adhesion, migration, extracellular matrix remodeling and reproductive functions. Both DDR2 null allele mice and mice with a recessive, loss-of-function allele for Ddr2 exhibit dwarfing and a reduction in body weight. However, the detailed mechanisms by which DDR2 exerts its positive systemic regulation of whole body size, local skeletal size and fat tissue volume remain to be clarified. To investigate the systemic role of DDR2 in body size regulation, we produced transgenic mice in which the DDR2 protein is overexpressed, then screened the transgenic mice for abnormalities using systematic mouse abnormality screening. The modified-SHIPRA screen revealed that only the parameter of body size was significantly different among the genotypes. We also discovered that the body length was significantly increased, while the body weight was significantly decreased in transgenic mice compared to their littermate controls. We also found that the epididymal fat pads were significantly decreased in transgenic mice compared to normal littermate mice, which may have been the cause of the leptin decrement in the transgenic mice. The new insight that DDR2 might promote metabolism in adipocyte cells is very interesting, but more experiments will be needed to elucidate the direct relation between DDR2 and adipose-derived hormones. Taken together, our data demonstrated that DDR2 might play a systemic role in the regulation of body size thorough skeletal formation and fat metabolism.

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.

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.

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.

Embryo implantation is blocked by intraperitoneal injection with anti-LIF antibody in mice.

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (i.p.) injections with anti-LIF antibody (7.5 µg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after i.p. injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.

Biological potentials for a family of disintegrin and metalloproteinase (ADAMDEC)-1 in mouse normal pregnancy.

Our previous research has indicated local expression of ADAMDEC-1, a family of disintegrin and metalloproteinase, was confirmed in the mouse placentas and enhancement was found in the sites for spontaneous abortion. Present study was aimed to identify biological effects of ADAMDEC-1 in pregnancy process. Syngeneic pairs of C57BL/6J mice and heterogenic mating pairs of CBA/J and DBA/2 mice were used. Pregnant mice were treated with recombinant ADAMDEC-1 protein. Vasculogenesis effects was evaluated using the Matrigel plugs including vascular endothelial growth factor singularity or combination with ADAMDEC-1. ADAMDEC-1 single effects were evaluated by tubal formation and proliferation assays using HuEht-1 endothelial cells. Expression of ADAMDEC-1 was not exactly corresponded with the time periods for miscarriage initiation. ADAMDEC-1 was distributed in normal placentas and fetuses, especially at extraembryonic ectoderm, decidua cells, uterine natural killer (uNK) cells in decidua, trophoblasts in labyrinthine zone, and hematopoietic cells in umbilical blood and fetal liver. ADAMDEC-1 treatment did not affect reproductive performances, while it elevated uNK cell recruitment in placenta and enlarged lumen sizes of the intraplacental vessels. In vitro analysis also indicated ADAMDEC-1 promoting effect on tubal formation and cell length of HuEht-1. qPCR analysis showed that ADAMDEC-1 modified placental gene expression especially for linkage of actin filament rearrangement. Our findings suggested that ADAMDEC-1 is correlated on cell shape, stability, and movement via modification of actin cytoskeleton. ADMADEC-1 suspected to regulate cellular activity of endothelial cells, trophoblasts, and uNK cells and may support normal developing of mouse placentas.

Characterization and expression of DNA sequences encoding the growth hormone gene in African Pygmy Mouse (Mus minutoides).

We determined the nucleotide sequence of the growth hormone (Gh) gene in Mus minutoides, one of the smallest mammals, where was predicted to be distinct in the functional regions between M. minutoides and Mus musculus. To investigate the evolutionary characteristics of Gh in M. minutoides, we constructed a phylogenetic tree based on the putative amino acid sequences of Gh, suggesting that the Gh of M. minutoides diverged earlier than M. musculus. Furthermore, the Gh gene expressed higher in M. minutoides than in M. musculus. Our results suggest that the specific feature of the Gh in M. minutoides is in rather the regulatory mechanism than the sequence.

Morphology of placentome in Korean water deer Hydropotes inermis argropus.

The placenta of the Korean water deer was anatomically examined to accumulate basic information regarding its reproductive system. The convex placentomes with five to nine well-developed pedicles were observed in the whole uterine horns, and therefore, the placenta was classified as oligocotyledonary. The evidence indicating the migration of binucleate cells (BNCs) from trophectoderm to the uterine epithelium led to the histological classification of the placenta as synepitheliochorial. The number of fetuses was markedly higher than that in other ruminant species. However, the number of placentomes was found to be similar to the other Cervidae species. Therefore, these results suggest that the Korean water deer may possess special mechanisms or structures at the fetus attachment site to maintain this unusally high number of fetuses.

Reproductive performance in diabetes mice with a special reference to uterine natural killer cells and placental growth factor.

To determine the effect of diabetes on reproductive performance, two kinds of diabetes mice, i.e., KK/TaJcl mice with Type-II diabetes and Streptozotocin-induced diabetes mice with Type-I diabetes, were used in this study. Particular attention was paid to uterine natural killer (uNK) cells and placental growth factor (PlGF). The number of fetuses, the fetal and placental weights in both diabetes mice were significantly decreased when compared to controls. Surprisingly, uNK cells in both diabetes mice persisted in the metrial gland even at the term of pregnancy. Although PlGF expression in both diabetes mice was significantly decreased, PlGF protein did not change. These results show that diabetes condition affects reproductive performance, particularly uNK cell behavior, but not PlGF production.

Uterine natural killer cells in perforin and beta(2)-microglobulin deficient mice.

Uterine natural killer (uNK) cells have roles for immune responses at the feto-maternal interface in mice. We studied the effects of beta(2)-microglobulin (beta(2)m) and perforin on proliferation and differentiation of uNK cells in pregnancy, using beta(2)-microglobulin-deficient (beta(2)m(-/-)) mice and perforin-deficient (P(-/-)) mice. The cell population of uNK cells in the metrial gland of P(-/-) mice was tended to be higher than the control B6 mice. The cell population of uNK cells in the metrial gland of beta(2)m(-/-) mice was significantly increased at Day 12 of pregnancy comparing to B6 and P(-/-) mice. On the other hand, the cell population of uNK cells in the decidua basalis of beta(2)m(-/-) mice was tended to be lower than B6 and P(-/-) mice. These results indicate that beta(2)m may be involved in proliferation of uNK cells in the metrial gland, and that beta(2)m may affect the maturation of uNK cells in the decidua basalis.

Effects of leukemia inhibitory factor on lectin-binding patterns in the uterine stromal vessels of mice.

Lectin histochemistry was performed on mouse uteri to determine what effects leukemia inhibitory factor (LIF) has on carbohydrate epitope expressions at the time of implantation. Twenty-two biotinylated lectins were used in this study. Following injection of LIF, specific binding to the apical surface of the uterine glandular epithelium (GE) was recognized by six lectins. Particularly, binding of the lectin from Griffonia (Bandeiraea) simplicifolia was specific to the glandular epithelium close to the luminal epithelium. Succinylated wheat germ agglutinin (WGA), which has specificity for oligosaccharides recognized by WGA without sialic acid residues, showed weaker binding to the uterine luminal epithelium (LE) and the stroma than WGA, suggesting that terminal residues of glyco-conjugates on these tissues may be modified by sialic acids. Lectin binding to the glandular and luminal epithelium was not influenced by LIF. However, three lectins including a lectin from Dolichos biflorus showed specificity for stromal vessels 6h after LIF injection. Since the lectin from D. biflorus binds to neo-vascular vessels, LIF may play a role in regulating maternal angiogenesis directly and/or indirectly during implantation.

Paraffin-embedded vertical sections of mouse embryonic stem cells.

Cultured cells are generally observed through the bottom of dishes or flasks using an inverted microscope. Two-dimensional and horizontal observation is insufficient for histological analysis of several cell lines, such as embryonic stem cells or cancer cells, because they form three-dimensional colonies. In the present study, we aimed to establish a more informative method for analysis of such stereoscopic cultured cells. We cultured mouse embryonic stem cells using a temperature-sensitive culture dish, embedded these cells in paraffin, and successfully observed vertical sections of embryonic stem cells. This vertical analysis of the stereoscopic colony emphasized structural features such as the dome shape of naïve pluripotent stem cells. This method could have the potential for analysis of three-dimensional structures and histological preservation in cultured cells.

Morphological analyses of the retinal photoreceptor cells in the nocturnally adapted owl monkeys.

Owl monkeys are the only one species possessing the nocturnal lifestyles among the simian monkeys. Their eyes and retinas have been interested associating with the nocturnal adaptation. We examined the cellular specificity and electroretinogram (ERG) reactivity in the retina of the owl monkeys by comparison with the squirrel monkeys, taxonomically close-species and expressing diurnal behavior. Owl monkeys did not have clear structure of the foveal pit by the funduscope, whereas the retinal wholemount specimens indicated a small-condensed spot of the ganglion cells. There were abundant numbers of the rod photoreceptor cells in owl monkeys than those of the squirrel monkeys. However, the owl monkeys' retina did not possess superiority for rod cell-reactivity in the scotopic ERG responses. Scanning electron microscopic observation revealed that the rod cells in owl monkeys' retina had very small-sized inner and outer segments as compared with squirrel monkeys. Owl monkeys showed typical nocturnal traits such as rod-cell dominance. However, the individual photoreceptor cells seemed to be functionally weak for visual capacity, caused from the morphological immaturity at the inner and outer segments.