ABSTRACT. 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. Bisphenol-A (BPA) is a nonsteroidal estrogen that is ubiquitous in the environment. There are a lot of reports about effects of BPA on formation and function of female reproductive organs. Among these reports, well known are (i) that BPA exposure advanced puberty [6], (ii) that BPA exposure changed patterns of estrous cycle [11], (iii) that BPA exposure brought about the loss of uterine decidualization [13], and (iv) that BPA exposure decreased the endometrial weight and increased expressions of estrogen receptor  and progesterone receptor [7]. We previously reported that BPA exposure during implantation and placentation periods decreased the number of fetus and pups, and the survival rate before weaning [14]. These suggest that in utero BPA exposure altered reproductive performance and formation of reproductive organs. DNA methylation is supposed to be one of the ways that BPA induces the endocrine disruption. DNA methylation regulates gene expression that is involved with growth and development [3,8]. There are many reports that unmethylation by BPA occurred in the promoter region of phosphodiesterase type 4 [5], that BPA induced unmethylation of agouti gene, using the viable yellow agouti (Avy) mouse [4], and that in utero BPA exposure brought about unmethylation of HOXA10 gene [2,12]. These suggest that BPA exposure can also affect molecular biologically next generation. The present study is designed to understand effects of BPA exposure on the reproductive organ across generations. We analyzed the morphology of uterus and ovary, and the methylation pattern of HOXA10 gene using the next generation of the offspring that was born from mother exposed to BPA. ICR mice obtained from Kyudo Company (Saga, Japan) were used in this study. Mice were housed in standard polypropylene cages in a temperature-controlled room (22°C) with a 12 hr li...

Abstract. 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. Key words: eNOS, iNOS, Rabbit placenta, Vascular formation (J. Reprod. Dev. 58: [231][232][233][234][235][236] 2012) I n pregnant mammals, the placenta acts as exchange interface for nutrients and waste products between the fetal and maternal circulation. The placenta is a very fast growing tissue with corresponding high metabolic demand from the embryo or fetus that requires an active blood supply and rapid vascular development [1][2][3]. Failure of placental growth during early and mid pregnancy is directly associated with inadequate uterine and umbilical blood flow, which adversely affects transportation of fetal nutrients [4]. Extensive increase of the transplacental exchange during the last half of gestation is closely dependent upon the dramatic growth of the vascular architecture and the resultant large volume of uterine and umbilical blood flow [1].Nitric oxide (NO), a multifunctional biomolecule, is produced from the essential amino acid L-arginine via nitric oxide synthase (NOS), which is classified into the calcium-independent or constitutive calcium/calmodulin-sensitive isoforms. The former is represented by inducible NOS (iNOS), and the latter is represented by the endothelial and neuronal NOS (eNOS and nNOS) [5]. NO plays crucial roles in the mediation of a wide variety of physiological processes including vasodilation, angiogenesis, platelet aggression, immune functions, connective tissue remodeling and smooth muscle activity [6]. iNOS and eNOS are known to dynamically regulate normal physiological events during successful pregnancy such as ovulation, implantation, trophoblast invasion, placental formation, fetal development and delivery [7][8][9].In the developing placenta, specific vascular formation occurs through the processes of destruction of preexisting vessels, de novo angiogenesis and convergence of blood path in association with the invitation of plenty ...

ABSTRACT. 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.KEY WORDS: GLUT1, GLUT3, immunohistochemistry, mRNA, rabbit placenta.

Authors' Contribution DK and HK designed the study. NA, MTT did statistical analysis. MT, MSK and RUK wrote the article.

ABSTRACT. 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.KEY WORDS: differentiation, SCID mouse, uterine NK cell.J. Vet. Med. Sci. 73(10): 1337-1340, 2011 Significant numbers of uterine natural killer (uNK) cells are found in the murine uterus only during pregnancy [7,10,13,15,19]. The uNK cells are observed in the metrial gland (MG) and decidua basalis (DB) of each implantation site, proliferate in the MG by day 12 of pregnancy, differentiate in the DB by day15 and degenerate during late pregnancy [1,9]. The uNK cells can produce several cytokines and growth factors, having crucial roles in pregnancy maintenance, particularly decidual health and modification of spiral arteries [12,16]. Peripheral NK cells and splenic NK cells are known to be affected in their differentiation and/or proliferation by cytokines and growth factors derived from functional T-and B-cells [14]. Since uNK cells are members of the NK cell lineage, their differentiation and/or proliferation may also be affected by T-and B-cells. We previously reported that the morphology of uNK cells in severe combined immunodeficient (SCID) mice deficient in functional T and B cells was similar to control mice on day 12 of pregnancy [18] and that the appearance of uNK cell precursors in SCID mice after birth was delayed compared with that of normal mice [5]. However, differentiation and/ or proliferation of uNK cells in SCID mice during successful pregnancy remains to be fully understood. We wished to determine whether differentiation and/or proliferation of uNK cells were altered by absence of functional T and B cells using SCID mice. SCID mice (genotype, C.B-17/Icr-scid/scid) and control mice (C.B-17/Icr-+/+) obtained from CLEA Japan (Osaka, Japan) were used in this study. Studies were performed according to protocols for animal use approved by the Yamaguchi University Animal Experimental Guidelines. Both mice were housed within the barrier containment facility at our University. Female mice were selected for estrus and paired with control males, and the morning of vaginal plug detection was called day 1 of pregnancy. Mated females were sa...

Abstract. This study aimed to clarify the mechanism of the spacing of murine embryos along the metrial and antimetrial (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. Key words: Embryo implantation, Gravity, Mice, Spacing (J. Reprod. Dev. 56: [191][192][193][194] 2010) mbryo implantation is one of the most important events in mammalian reproduction [1]. This implantation consists of two major phenomena; the first is positioning of the embryo in the uterus before apposition of the blastocyst to the uterine luminal epithelium (LE), and the second is initiation of direct feto-maternal communication after completion of positioning (above "apposition"). In polytocous species, such as the mouse, precise positioning of individual embryos in the right place is required before the implantation window opens. The spacing of the mouse embryo is defined as follows: (1) the embryo aligns along the long axis of the uterine horn (LA axis) in equally spaced manner and (2) the embryo is localized exclusively at the anti-metrial side of the uterine horn the metrial and anti-metrial axis (MA axis).In mice, fertilization occurs at the ampulla of the oviduct. Fertilized eggs migrate to the uterine horn during the 2-to 16-cell stage at Day 2 of pregnancy (the day of the vaginal plug is defined as the first day of pregnancy, Day 1). The morula moves to the uterus around noon on Day 3. Then, the embryo develops to the blastocyst stage, and it completes spacing in the morning on Day 4. Finally, the blastocyst begins to attach to the LE [1][2][3].Leukemia inhibitory factor (LIF) is an essential factor for initiation of pregnancy after positioning of the embryo in mice [4]. LIF is involved in the process of implantation; it is involved in apoptosis of the LE and successive decidualization. The molecular pathway of LIF leading to successful implantation has gradually been revealed. In addition to LIF, some molecules have been suggested to be indispensable factors for the progress of implantation [4][5][6]. On the other hand, the mechanisms of embryo spacing ...

Authors' Contribution HK conceived the idea of the research and wrote the manuscript. AW carried out the experimental work and YM, Summaya K and Salman K helped hiim. AK and IK helped in the execution of the experiments. SA and NK analysed of the data statically.

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.