Yasuo Kiso

Distribution Patterns of Uterine Glands and Embryo Spacing in the Mouse

To clarify the mechanism of implantation, relationship between positioning of the mouse embryo in the uterus and distribution of uterine glands along the long axis of the uterine horn was examined by three‐dimensional remodelling of the uterine endometrium. There were two unique regions in the endometrium. Uterine glands were distributed widely from mesometrial to anti‐mesometrial side in one region. It was localized from lateral to anti‐mesometrial side in another. These different regions were alternately aligned throughout the uterine horn. The number and position of embryos was consistent with that of the latter region. This study suggests that the type of distribution of uterine glands is closely related to the positioning of the embryo in mice.

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

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.

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

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.

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

AbstractDiscoidin 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.n

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.

Morphological characterization of spermatozoa of the night monkey

Abstract n The morphology, sizes and abnormality rates of spermatozoa in the night monkey were revealed in the present paper. Motile spermatozoa of three males, 7, 8 and 12–13 years old, were squeezed from the ducts of the cauda epididymis after cutting the ducts in cryopreservation media. The morphology of the spermatozoa and abnormalities in them were observed, and the sizes of the spermatozoa were measured in smear samples. The spermatozoa of the night monkey had heads with rounded and thick shapes. Measurement of the spermatozoa revealed that the average widths and lengths of the heads, average lengths of the middle pieces, and average total lengths from the head to tail tip were 4.7 ± 0.8, µm and 2.8 ± 0.4 µm, 6.6 ± 2.2, µm and 55.1 ± 6.2 µm, respectively (average ± SD). The total rates of abnormal spermatozoa were different among the 7-, 8- and 12–13- year-old night monkeys, 41.8%, 24.0% and 36.5%, respectively. Freezing of semen was also attempted using the procedure contained in a commercial kit for the mouse. Although the motility of the spermatozoa from the night monkeys was poor in fresh samples, the motility of their spermatozoa frozen-thawed with the commercial kit was similar to that before freezing.

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

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.

Morphological Characterization and in vitro Maturation of Follicular Oocytes from the Owl Monkey (Aotus lemurinus)

Abstract: The morphological characterization of ovaries and ovarian follicular oocytes obtained from the owl monkey is detailed in the present paper. In vitro maturation of oocytes, using a method which has proved successful for other mammalian oocytes, was also evaluated and the maturation rate was compared with that obtained with squirrel monkey oocytes. The ovaries of the owl monkey are oval in shape (long axis of about 10 mm, short axis about 7 mm) and their oocytes are spherical. The mean diameter of owl monkey oocytes is significantly larger than that of squirrel monkey oocytes (145.7 ± 16.3 µm vs 112.0 ± 12.4 µm). The owl monkey oocytes were incubated in HTF medium containing 10% FBS at 37 °C (5% CO2 in air) for 22–23 hours. The in vitro maturation rate of the owl monkey oocytes was higher than that of the squirrel monkey oocytes (83.3% vs 40.0%); therefore, our maturation conditions were as suitable for them. This study is the first to detail differences in the follicular oocytes of the owl monkey and squirrel monkey. Further studies using owl monkey gametes might yield results leading to a greater understanding of primate reproduction.

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

PurposeWe 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.MethodsTwo 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 25xa0h and cumulus cells were removed with 0.1xa0% 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.ResultsWe 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 98xa0h after ICSI and showed signs of compaction, but failed to cleave further.ConclusionsAlthough 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.

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

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.