Publications by authors named "Yasuo Kiso"

47 Publications

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

J Reprod Dev 2021 Aug 9;67(4):265-272. Epub 2021 Jul 9.

Graduate School of Agricultural Science, Utsunomiya University, Tochigi 321-8505, Japan.

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.
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http://dx.doi.org/10.1262/jrd.2021-018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8423609PMC
August 2021

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

J Vet Med Sci 2021 Aug 14;83(8):1244-1247. Epub 2021 Jun 14.

Laboratory of Developmental Biology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.

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.
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http://dx.doi.org/10.1292/jvms.21-0231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8437718PMC
August 2021

Morphology of placentome in Korean water deer Hydropotes inermis argropus.

J Vet Med Sci 2021 Jul 10;83(7):1081-1085. Epub 2021 May 10.

Laboratory of Veterinary Anatomy, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.

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.
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http://dx.doi.org/10.1292/jvms.21-0158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8349818PMC
July 2021

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

J Vet Med Sci 2021 Apr 19;83(3):512-521. Epub 2021 Feb 19.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753-8515, Japan.

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.
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http://dx.doi.org/10.1292/jvms.20-0570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025434PMC
April 2021

Hyper-polyploid embryos survive after implantation in mice.

Zygote 2020 Jun 10;28(3):247-249. Epub 2020 Mar 10.

Laboratory of Veterinary Developmental Biology, United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan.

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.
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http://dx.doi.org/10.1017/S0967199420000064DOI Listing
June 2020

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

Biochem Biophys Res Commun 2020 01 18;521(1):24-30. Epub 2019 Oct 18.

Laboratory of Veterinary Developmental Biology, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2019.10.026DOI Listing
January 2020

Aggregation recovers developmental plasticity in mouse polyploid embryos.

Reprod Fertil Dev 2019 Jan;31(2):404-411

Laboratory of Veterinary Anatomy, Joint-Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi, 7538515, Japan.

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.
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http://dx.doi.org/10.1071/RD18093DOI Listing
January 2019

Paraffin-embedded vertical sections of mouse embryonic stem cells.

J Vet Med Sci 2018 Oct 9;80(10):1479-1481. Epub 2018 Aug 9.

Laboratory of Veterinary Anatomy and Developmental Biology, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753-0841, Japan.

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.
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http://dx.doi.org/10.1292/jvms.18-0352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207513PMC
October 2018

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

J Vet Med Sci 2018 Mar 26;80(3):413-420. Epub 2018 Jan 26.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753-8515, Japan.

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.
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http://dx.doi.org/10.1292/jvms.17-0418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880819PMC
March 2018

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

J Reprod Dev 2016 Dec 29;62(6):571-576. Epub 2016 Aug 29.

Laboratory of Veterinary Anatomy and Embryology, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753-8515, Japan.

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.
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http://dx.doi.org/10.1262/jrd.2016-037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5177974PMC
December 2016

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

Reprod Med Biol 2016 07 16;15(3):183-186. Epub 2015 Dec 16.

United Graduate School of Agricultural Science Tokyo University of Agriculture and Technology 183-8509 Fuchu-shi Japan.

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.
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http://dx.doi.org/10.1007/s12522-015-0229-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715853PMC
July 2016

Histocytological specificities of adrenal cortex in the New World Monkeys, Aotus lemurinus and Saimiri boliviensis.

J Vet Med Sci 2016 Jan 28;78(1):161-5. Epub 2015 Aug 28.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.

The New World monkey Aotus spp. (night monkeys) are expected for use of valuable experimental animal with the close species of Saimiri spp. (squirrel monkeys). Saimiri is known to show spontaneous hypercortisolemia, although few reports in Aotus. We compared basic states of blood steroid hormones and histological structure of the adrenal glands in two monkeys. Serum cortisol and ACTH levels were statistically lower in Aotus than Saimiri. Conversely, Aotus adrenocortical area showed significant enlargement, especially at the zona fasciculata. Electron microscopic observation at Aotus fasciculata cells revealed notable accumulation of large lipid droplets and irregular shapes of the mitochondrial cristae. These results suggest potential differences in cellular activities for steroidogenesis between Aotus and Saimiri and experimental usefulness in adrenocortical physiology and pathological models.
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http://dx.doi.org/10.1292/jvms.15-0290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751139PMC
January 2016

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers.

PLoS One 2015 19;10(6):e0130585. Epub 2015 Jun 19.

Laboratory of Veterinary Anatomy, Joint-Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130585PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4474668PMC
May 2016

Nutrient starvation affects expression of LC3 family at the feto-maternal interface during murine placentation.

J Vet Med Sci 2015 Mar 25;77(3):305-11. Epub 2014 Nov 25.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753-8515, Japan.

LC3 - the mammalian homolog of Atg8 - was found as autophagosome membrane binding protein in mammals and widely used as an autophagosomal marker. LC3A, B and C show different expression patterns in each tissue. The aim of this study was to reveal the differences of expression patterns among LC3 families in mouse placenta under normal condition and nutrient starving condition. LC3A and B were highly expressed in decidual cells. LC3A and B were increased in D14 compared with D12 and D16 in mouse placenta, while LC3C was decreased. Starvation induced increase in LC3B expression specifically. Immunohistochemistry showed different expression patterns among LC3A, B and C. LC3A expression in syncytiotrophoblast was vanished by starvation. The results of real time RT-PCR suggested differences between D12 and D16 in autophagic cascade induced by starvation. Taken together, this study suggests that autophagy could play a role in placental invasion system and that nutrient starvation affects LC3B expression.
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http://dx.doi.org/10.1292/jvms.14-0490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383776PMC
March 2015

Dynamics and reproductive effects of complement factors in the spontaneous abortion model of CBA/J×DBA/2 mice.

Immunobiology 2014 May 10;219(5):385-91. Epub 2014 Jan 10.

Department of Integrated Structural Biosciences, Division of Veterinary Science, Graduate School of Life and Environmental Science, Osaka Prefecture University, Osaka 598-8531, Japan.

The complement system is one component of innate immunity that could participate in fetal loss. We have already reported that adipsin, a complement activator in the alternative pathway, is stably expressed in the placenta and that an increase in this expression is related to spontaneous abortion. However, complement inhibitor Crry was concurrently expressed in the placenta, and the role of complement factors during pregnancy was not clear. In the present study, we examined the endogenous regulation of complement factors in placenta and serum by using another model mouse for spontaneous abortion and studied the effect of exogenous complement disruption on pregnancy. Compared to control mice, the CBA/J×DBA/2 model mice had higher expression levels of adipsin in the placenta and serum. Adipsin and complement C3 were localized in the metrial gland and labyrinth regions, and both positive reactive ranges were limited in the maternal blood current in normal implantation sites. These results suggest that extrauterine adipsin hematogenously reaches the placenta, activates complement C3, and promotes destruction of the feto-maternal barrier in aborted implantation sites. Crry was consistently expressed in the placenta and serum and reduced in the resorption sites of CBA/J×DBA/2 mice as compared to normal sites. Injection of recombinant adipsin increased the resorption rate and changed the expression of Th-type cytokines toward a Th1 bias. The present study indicates that adipsin could induce the fetal loss that accompanies the Th1 bias and may be a crucial cause of spontaneous abortion. In addition, the local expression of Crry prevents complement activation in placenta in response to a systemic increase of adipsin.
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http://dx.doi.org/10.1016/j.imbio.2014.01.001DOI Listing
May 2014

Influence of atopic dermatitis on reproduction and uterine natural killer cells.

J Vet Med Sci 2014 Jun 27;76(6):913-6. Epub 2014 Feb 27.

Laboratory of Basic Veterinary Science, the United Graduate School of Veterinary Science, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan.

The causal relationship between severe allergic conditions and successful pregnancy remains unclear. We aimed to evaluate reproductive performance in an experimental mouse model of atopic disease (AD), and the appearance of uterine natural killer (uNK) cells that have crucial roles in placental formation was examined. In the NC/Nga pregnant mice with moderate skin allergic lesions and an 8.6-fold elevation of plasma IgE, significant differences were not detected in the reproductive indices of the number of normal fetuses, abortion rate and placental size. There were few uNK cells in the placenta of AD mice, and they showed a significant decrease regarding the immature subtype as compared with controls. These findings revealed that AD disturbs uNK cell differentiation and provides disadvantageous effects on placental formation, although it does not arrest the pregnancy process. It may be possible that specific immunological conditions behind AD operate favorably to recover the reproductive performance.
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http://dx.doi.org/10.1292/jvms.13-0547DOI Listing
June 2014

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

Transgenic Res 2014 Feb 14;23(1):165-75. Epub 2013 Sep 14.

Laboratory of Applied Genetics, Graduate School of Agricultural and Life Science, University of Tokyo, Tokyo, 113-8657, Japan.

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.
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http://dx.doi.org/10.1007/s11248-013-9751-2DOI Listing
February 2014

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

Med Mol Morphol 2014 Jun 23;47(2):76-82. Epub 2013 Apr 23.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, 1677-1 Yoshida, Yamaguchi, 753-8515, Japan.

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.
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http://dx.doi.org/10.1007/s00795-013-0044-xDOI Listing
June 2014

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

J Reprod Dev 2012 22;58(2):231-6. Epub 2011 Dec 22.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753-8515, Japan.

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.
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http://dx.doi.org/10.1262/jrd.11-128tDOI Listing
October 2012

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

J Reprod Dev 2011 Dec 12;57(6):700-7. Epub 2011 Aug 12.

Laboratory of Animal Morphology and Function, Division of Biofunctional Development, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

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.
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http://dx.doi.org/10.1262/jrd.11-048hDOI Listing
December 2011

Differentiation of uterine natural killer cells in pregnant SCID (scid/scid) mice.

J Vet Med Sci 2011 Oct 30;73(10):1337-40. Epub 2011 May 30.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, Yoshida, Yamaguchi, Japan.

To determine whether functional T- and B-cells can affect differentiation and/or proliferation of uterine natural killer (uNK) cells, their numbers in SCID mice (genotype, C.B.-17/Icr-scid/scid) were compared with those of control mice (genotype, C.B.-17/Icr-+/+) on days 8, 12 and 16 of pregnancy. Using biotinylated-Dolichos biflorus agglutinin (DBA) lectin staining, uNK cells can be readily classified into 4 subtypes, I to IV, from immature to mature types. The number of uNK cells was significantly lower in the decidua basalis of SCID mice than in that of control mice on day 8 of pregnancy. Particularly, the number of uNK cells of immature subtype II was significantly lower in SCID mice than in the control mice. By day 12, however, the uNK cell number in the SCID mice reached the same level as that of the control mice. It is likely that uNK cell differentiation in SCID mice was delayed during the early placentation period due to a lack of functional T and B cells.
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http://dx.doi.org/10.1292/jvms.11-0189DOI Listing
October 2011

Quantitative expression and immunohistochemical detection of glucose transporters, GLUT1 and GLUT3 in the rabbit placenta during successful pregnancy.

J Vet Med Sci 2011 Sep 25;73(9):1177-83. Epub 2011 May 25.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, 1677–1 Yoshida, Yamaguchi 753–8515, Japan.

Glucose is essential for the development of the fetus. We address here the quantitative expression and immunohistochemical localization of glucose transporter (GLUT1 and GLUT3) in the rabbit placenta during successful pregnancy. Blood glucose level showed a significant decrease at the gestation period in comparison with non-pregnancy. Maternal serum glucose was gradually increased according to fetal development. Quantitative RT-PCR results showed that expression of GLUT1 was significantly increased from day 13 to day 18, while GLUT3 mRNA level was significantly decreased during the same periods. Western blot analysis demonstrated that GLUT1 protein did not change significantly in the placenta during pregnancy when compared to non-pregnant uteri. Immunohistochemistry indicated that distribution of GLUT1 was observed mainly to the surface of the outer trophoblasts, whereas GLUT3 mainly localized to the basal site of the inner trophoblasts and fetal blood vessels. These results suggest that glucose is transported through GLUT1 from the maternal blood stream for use as a placental fuel and for further transport through GLUT3 to the fetal circulation, thus signifying the distinct anatomical localization of GLUT1 and GLUT3 in the rabbit placenta during successful pregnancy.
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http://dx.doi.org/10.1292/jvms.11-0144DOI Listing
September 2011

Bisphenol-A (BPA) affects reproductive formation across generations in mice.

J Vet Med Sci 2011 Sep 2;73(9):1211-5. Epub 2011 May 2.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, 1677–1 Yoshida, Yamaguchi 753–8515, Japan.

To understand effects of Bisphenol-A (BPA) exposure on the reproductive organ across generations, we analyzed morphology of the uterus and ovary, and the methylation pattern of HOXA10 gene of the 2(nd) generation. Pregnant mice (F0) were treated with sc injection of BPA in sesame oil at various doses of 0-1,000 mg/kg Bwt on days 12-16 of gestation. Their offspring (F1) were bred by foster mice, and the offspring (F2) from F1 mice were prepared. That is, F1 mice experienced in utero BPA exposure during the developmental period of reproductive organs, while F2 mice did not at all. Using these F2 mice, the present study was carried out. Comparing to the control, the body weights in BPA exposure groups were significantly increased. Correlating with the increase of body weight, the relative weights of the ovary and uterus in each group were decreased. The histological analysis revealed expansion or emphraxis of the uterine lumen and partial loss of the uterine epithelium. Unmethylation of HOXA10 gene in the uterus was observed in the intron region. The present study suggested that BPA exposure to F0 mice could affect reproductive organ of F2 mice who were not exposed to BPA.
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http://dx.doi.org/10.1292/jvms.11-0135DOI Listing
September 2011

Spacing of the embryo in the uterus is disrupted by the supine position of the body during the peri-implantation period in mice.

J Reprod Dev 2010 Apr 25;56(2):191-4. Epub 2009 Dec 25.

Laboratory of Basic Veterinary Science, United Graduate School of Veterinary Science, Yamaguchi University, Japan.

This study aimed to clarify the mechanism of the spacing of murine embryos along the metrial and anti-metrial (MA) axis of the uterus using our newly developed experimental model. The model mice were produced by keeping mice in the supine position from the pre-implantation to implantation period. The starting points and periods of restraint of the mice in the supine position were set variously during the peri-implantation. Then, the position of the embryo was evaluated morphologically. In only one group (set in the instrument from the second day of pregnancy, Day 2, to Day 5), strong disruption of embryo spacing along the MA axis was observed. On the other hand, there was little abnormality in embryo positioning in the groups that were treated from Day 3 to Day 5 or from Day 3 to Day 6. These results suggested that determination of the position of the embryo in the MA axis is not related to duration of the experiment (2 days or 3 days), but is related to the starting time-point of the experiment, at Day 2 or Day 3. In conclusion, the period between Days 2 and 3 is critical for determination of the position of the embryo along the MA axis.
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http://dx.doi.org/10.1262/jrd.09-064kDOI Listing
April 2010

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

J Vet Med Sci 2009 Apr;71(4):519-23

The United Graduate School of Veterinary Science, Yamaguchi University, Japan.

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.
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http://dx.doi.org/10.1292/jvms.71.519DOI Listing
April 2009

Agrin pathway is controlled by leukemia inhibitory factor (LIF) in murine implantation.

J Reprod Dev 2009 Jun 26;55(3):293-8. Epub 2009 Mar 26.

Department of Veterinary Anatomy, Faculty of Agriculture, Yamaguchi University.

Agrin is the heparan sulfate proteoglycan (HSPG) that is well known as the molecule that aggregates acetylcholine receptor (AChR) through muscle specific kinase (MuSK) and rapsyn at neuromuscular junctions. HSPGs are spatiotemporally expressed in embryonic and maternal tissues during implantation. The present study examined the role of agrin in the mouse embryo using leukemia inhibitory factor (LIF)-deficient mice, which show complete sterility. Agrin was detected widely in the cytoplasm of uterine luminal epithelial cells at the third day of pregnancy (Day 3) and Day 4. At Day 5, agrin moved to the apical surface of the luminal epithelium. This migration was not found in LIF-deficient mice. AChR was also found in the apical surface of the uterine epithelium at Day 4 and Day 5 in normal mice. LIF-deficient mice did not show this pattern of expression. Only nAChR b1 subunit mRNA was increased at Day 5 in normal mice. Furthermore, acetylcholinesterase was active in the uterine stroma of normal mice throughout the implantation period and was exclusively active in the uterine epithelium at Day 4. Taken together, agrin signaling was activated in the uterus during embryo implantation in the mice. Here, we suggest that the agrin pathway is involved in closure of the uterine epithelium toward placentation.
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http://dx.doi.org/10.1262/jrd.20162DOI Listing
June 2009

Spontaneous endometrial hyperplasia in the uteri of IL-2 receptor beta-chain transgenic mice.

J Reprod Dev 2009 Jun 16;55(3):273-7. Epub 2009 Mar 16.

Department of Laboratory Animal Science, Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University.

We found frequent and spontaneous proliferation of glandular epithelium and dilated cysts in the uteri of interleukin-2 receptor (IL2R) beta-chain transgenic (Tg2Rbeta) mice. The aim of this study was to examine the involvement of IL2R beta-chain in the pathogenesis of endometrial hyperplasia (EH). Mouse uteri and serum were collected from Tg2Rbeta and normal littermates (NL), which were classified into three groups according to age. The incidence of EH increased in an age-dependent manner in both types of mice. However, in old age, Tg2Rbeta mice showed more serious phenotypes of EH than NL. Immunohistochemical analysis revealed specific localization of IL2R beta-chain in the glandular epithelial cells, with a correlation to the degree of EH, not only in the Tg2Rbeta uteri but also in the NL uteri. Immunoreactions of CD3 and CD25 were detected in the uteri of Tg2Rbeta but were weak in the uteri of NL, and CD25-positive cells were distributed in the endometrial stroma and myometrium in the Tg2Rbeta mice. These findings suggest that the IL2R beta-chain induces growth potential for glandular epithelial cells and an immune-privileged condition mediated by CD25+regulatory-T cells.
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http://dx.doi.org/10.1262/jrd.20171DOI Listing
June 2009

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

J Vet Med Sci 2009 Feb;71(2):251-3

The United Graduate School of Veterinary, Yamaguchi University, Yoshida, Yamaguchi, Japan.

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.
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http://dx.doi.org/10.1292/jvms.71.251DOI Listing
February 2009

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

Immunobiology 2008 22;213(2):143-50. Epub 2007 Oct 22.

Department of Veterinary Anatomy, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8511, Japan.

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.
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http://dx.doi.org/10.1016/j.imbio.2007.08.003DOI Listing
May 2008
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