Causative alleles for chondrodysplastic dwarfism, factor XI deficiency, and factor XIII deficiency in the Kumamoto sub-breed of Japanese Brown cattle.

Japanese Brown cattle are the second most popular Wagyu breed, and the Kumamoto sub-breed shows better daily gain and carcass weight. One of the breeding objectives for this sub-breed is to reduce genetic defects. Chondrodysplastic dwarfism and factor VIII deficiency have been identified as genetic diseases in the Kumamoto sub-breed. Previously, we detected individuals in the Kumamoto sub-breed with causative alleles of genetic diseases identified in Japanese Black cattle. In the current study, 11 mutations responsible for genetic diseases in the Wagyu breeds were analyzed to evaluate the risk of genetic diseases in the Kumamoto sub-breed. Genotyping revealed the causative mutations of chondrodysplastic dwarfism, factor XI deficiency, and factor XIII deficiency and suggested the appearance of affected animals in this sub-breed. DNA testing for these diseases is needed to prevent economic loses in beef production using the Kumamoto sub-breed.

Differences in permissive cytomegalovirus infection between primary cultured human fetal membrane chorion and amnion cells.

Human cytomegalovirus (CMV) is the most common cause of viral intrauterine infection in developed countries. It has been shown that CMV DNA was frequently detected in the fetal membranes when the placenta was infected in utero. However, it is still not clear whether CMV replicates in constituent cells of the fetal membranes. We investigated CMV infection of primary cultured chorion and amnion cells prepared from human fetal membrane tissues. In both types of cell cultures, rounded cells were observed at day 8 and 12 after CMV inoculation, and virus yields in culture supernatants were increased after the inoculation. In both types of cells, viral immediately early 1 (IE1) protein-positive nuclei were scattered at day 4 after the inoculation, and IE1 mRNA was expressed throughout day 1 to 12 after CMV inoculation. In chorion cell cultures, the number of IE1 protein-positive nuclei increased significantly at day 8 and 12 after CMV inoculation as compared to day 4, by which foci were formed. Furthermore, an evident increase in levels of lactate dehydrogenase leakage from chorion cells was observed after CMV inoculation. Contrary, these phenomena were not observed in amnion cell cultures. These results demonstrated that both chorion and amnion cells were permissive to CMV infection, while the velocity of cell-to-cell spread of CMV infection in amnion cells was much lower than that in chorion cells. Therefore, the present study suggests that CMV may replicate rapidly in the chorion cell layer and slowly in the amnion cell layer during intrauterine infection.

Possible roles of proinflammatory and chemoattractive cytokines produced by human fetal membrane cells in the pathology of adverse pregnancy outcomes associated with influenza virus infection.

Pregnant women are at an increased risk of influenza-associated adverse outcomes, such as premature delivery, based on data from the latest pandemic with a novel influenza A (H1N1) virus in 2009-2010. It has been suggested that the transplacental transmission of influenza viruses is rarely detected in humans. A series of our study has demonstrated that influenza virus infection induced apoptosis in primary cultured human fetal membrane chorion cells, from which a factor with monocyte differentiation-inducing (MDI) activity was secreted. Proinflammatory cytokines, such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-ß, were identified as a member of the MDI factor. Influenza virus infection induced the mRNA expression of not only the proinflammatory cytokines but also chemoattractive cytokines, such as monocyte chemoattractant protein (MCP)-1, regulated on activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1ß, IL-8, growth-regulated oncogene (GRO)-α, GRO-ß, epithelial cell-derived neutrophil-activating protein (ENA)-78, and interferon inducible protein (IP)-10 in cultured chorion cells. These cytokines are postulated to associate with human parturition. This paper, therefore, reviews (1) lessons from pandemic H1N1 2009 in pregnancy, (2) production of proinflammatory and chemoattractive cytokines by human fetal membranes and their functions in gestational tissues, and (3) possible roles of cytokines produced by human fetal membranes in the pathology of adverse pregnancy outcomes associated with influenza virus infection.

Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes.

Arsenic trioxide (arsenite, As(III)) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As(III) on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As(III) on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As(III)-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As(III) were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As(III) than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As(III) in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As(III)-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As(III) cytotoxicity between these cells.

Direct contribution of inducible nitric oxide synthase expression to apoptosis induction in primary smooth chorion trophoblast cells of human fetal membrane tissues.

We have previously demonstrated that apoptosis induction is observed only in smooth chorion laeve trophoblast cells, and not in amnion epithelial cells of human fetal membrane tissues prepared at the term. Apoptosis induction was suppressed by the presence of an inhibitor specific for inducible nitric oxide synthase (iNOS), suggesting that intracellular oxidative stress plays a critical role in this process. In this study, we transfected the iNOS gene into primary cultured chorion and amnion cells to examine the direct contribution of iNOS gene expression to the apoptosis induction in these cells. We identified a significant increase in the levels of iNOS protein expression and nitrite accumulation in both chorion and amnion cells after the iNOS gene transfection. However, the induction of apoptosis was observed in an approximately 70% of chorion cells transfected with iNOS gene. Transfection of the iNOS gene into chorion cells resulted in the activation of p38 mitogen-activated protein kinase (MAPK) and downregulation of hemeoxygenase-1 protein expression, whereas no such events were observed in the transfected amnion cells. These results suggest that apoptosis induced in the chorion trophoblast cells by the iNOS gene expression is closely linked to a physiological consequence, such as the rupture of fetal membranes.

Expression of stathmin, a microtubule regulatory protein, is associated with the migration and differentiation of cultured early trophoblasts.

BACKGROUND: The microtubule-destabilizing protein stathmin is expressed by the villous cytotrophoblasts and invasive extravillous trophoblasts (EVTs) in the first-trimester human placenta. Here, we evaluated the significance of stathmin expression in terms of the functions of trophoblasts. METHODS: We employed two choriocarcinoma cell lines (BeWo and JEG-3), an EVT cell line (HTR-8/SVneo) and isolated first-trimester trophoblast cells. The effects of small-interfering (si) RNA-mediated stathmin knockdown on trophoblast proliferation and migration were measured by WST-1 and Transwell assays, respectively. Trophoblast differentiation was induced by dibutyryl (db)-cAMP treatment and evaluated by measuring human chorionic gonadotrophin beta (hCGbeta) and syncytin expression and cell fusion. We examined the effect of knockdown and induced stathmin expression on db-cAMP-induced differentiation. RESULTS: siRNA-induced silencing of stathmin expression had a marked inhibitory effect on BeWo, JEG-3 and HTR-8/SVneo cell migration and also suppressed their proliferation, albeit to a lesser extent. db-cAMP-enhanced hCGbeta and syncytin expression and cell fusion in BeWo cells was inhibited by stathmin knockdown. However, induced expression of stathmin reversed the hCGbeta and syncytin expression and cell fusion in the Tet-On BeWo cells. Suppression of stathmin expression also inhibited the migration of and hCGbeta production by first-trimester trophoblasts. CONCLUSIONS: Stathmin expression may be closely associated with early trophoblast migration and differentiation into syncytiotrophoblasts during placentation.