Mara S. Hoshida

Obesity induced by high-fat diet promotes insulin resistance in the ovary.

Besides the effects on peripheral energy homeostasis, insulin also has an important role in ovarian function. Obesity has a negative effect on fertility, and may play a role in the development of the polycystic ovary syndrome in susceptible women. Since insulin resistance in the ovary could contribute to the impairment of reproductive function in obese women, we evaluated insulin signaling in the ovary of high-fat diet-induced obese rats. Female Wistar rats were submitted to a high-fat diet for 120 or 180 days, and the insulin signaling pathway in the ovary was evaluated by immunoprecipitation and immunoblotting. At the end of the diet period, we observed insulin resistance, hyperinsulinemia, an increase in progesterone serum levels, an extended estrus cycle, and altered ovarian morphology in obese female rats. Moreover, in female obese rats treated for 120 days with the high-fat diet, the increase in progesterone levels occurred together with enhancement of LH levels. The ovary from high-fat-fed female rats showed a reduction in the insulin receptor substrate/phosphatidylinositol 3-kinase/AKT intracellular pathway, associated with an increase in FOXO3a, IL1B, and TNFalpha protein expression. These changes in the insulin signaling pathway may have a role in the infertile state associated with obesity.

Modulation of the Induction of Lung and Airway Allergy in the Offspring of IFN-γ-Treated Mother Mice

Recent studies have highlighted the influence of fetal/maternal interactions on the development of asthma. Because IFN-γ reduces Th2-mediated allergic responses, we assessed its capacity to modulate asthma in the offspring when injected into mothers during pregnancy. IFN-γ was injected in CD1 female mice on day 6.5 of gestation. Immediately after birth, male newborns were housed in cages with interchanged mothers: the offspring from IFN-γ-treated mothers were breastfed by normal mothers (IFN/nor), and those from normal mothers were breastfed by IFN-γ-treated (Nor/IFN) or normal mothers (Nor/nor). Immediately after weaning, the spleen cells from IFN/nor and Nor/IFN mice produced less IL-4 and more IFN-γ than Nor/nor mice when stimulated with Con A. At the age of 6–7 wk, mice were immunized with OVA on days 0 and 7. From day 14 to 16, they were exposed to aerosolized OVA. The bronchoalveolar lavage fluid from Nor/nor mice showed eosinophilia, a large number of these cells being present in perivascular and peribronchial regions of lung tissues. IFN/nor or Nor/IFN mice showed greatly reduced eosinophil numbers in bronchoalveolar lavage fluid. In addition, lung sections from IFN/nor, but not Nor/IFN mice showed almost normal histology. In OVA-sensitized IFN/nor and Nor/IFN mice, the production of IFN-γ, IL-4, and IL-5 by spleen cells was significantly reduced as compared with cells from the OVA-sensitized Nor/nor group. IgE and anaphylactic IgG1 were also reduced in plasma of IFN/nor mice. In conclusion, the presence of IFN-γ during pregnancy confers to the fetus a protection against allergenic provocations in the adult life.

Spatiotemporal patterns of macrophage migration inhibitory factor (Mif) expression in the mouse placenta

BackgroundMacrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy.MethodsMif was immunolocalized at implantation sites on gestation days (gd) 7.5, 10.5, 13.5 and 17.5. Ectoplacental cones and fetal placentas dissected from the maternal tissues were used for Western blotting and qRT-PCR assays on the same gestation days.ResultsDuring the post-implantation period (gd7.5), trophoblast giant cells showed strong Mif reactivity. In later placentation phases (gds 10.5-17.5), Mif appeared to be concentrated in the junctional zone and trophoblast giant cells. Mif protein expression increased significantly from gd7.5 to 10.5 (p = 0.005) and from gd7.5 to 13.5 (p = 0.03), remaining at high concentration as gestation proceeded. Higher mRNA expression was found on gd10.5 and was significantly different from gd13.5 (p = 0.048) and 17.5 (p = 0.009).ConclusionsThe up-regulation of Mif on gd10.5 coincides with the stage in which the placenta assumes its three-layered organization (giant cells, spongiotrophoblast and labyrinth zones), fetal blood circulation begins and population of uNK cells reaches high proportions at the maternal counter part of the placenta, suggesting that Mif may play a role in either the placentation or in the adaptation of the differentiated placenta to the uterus or still in gestational immunomodulatory responses. Moreover, it reinforces the possibility of specific activities for Mif at the maternal fetal interface.

Interferon-gamma alters the phagocytic activity of the mouse trophoblast

Interferon-gamma (IFN-gamma) mediates diverse functions in bone marrow-derived phagocytes, including phagocytosis and microbe destruction. This cytokine has also been detected at implantation sites under both physiological and pathological conditions in many different species. At these particular sites, the outermost embryonic cell layer in close contact with the maternal tissues, the trophoblast exhibits intense phagocytic activity. To determine whether IFN-gamma affects phagocytosis of mouse-trophoblast cells, ectoplacental cone-derived trophoblast was cultured and evaluated for erythrophagocytosis. Phagocytic activity was monitored ultrastructurally and expressed as percentage of phagocytic trophoblast in total trophoblast cells. Conditioned medium from concanavalin-A-stimulated spleen cells significantly enhanced trophoblast phagocytosis. This effect was blocked by pre-incubation with an anti-IFN-gamma neutralizing antibody. Introduction of mouse recombinant IFN-gamma (mrIFN-gamma) to cultures did not increase cell death, but augmented the percentage of phagocytic cells in a dose-dependent manner. Ectoplacental cones from mice deficient for IFN-gamma receptor alpha-chain showed a significant decrease of the phagocytosis, even under mrIFN-gamma stimulation, suggesting that IFN-gamma-induced phagocytosis are receptor-mediated. Reverse transcriptase-PCR analyses confirmed the presence of mRNA for IFN-gamma receptor alpha and beta-chains in trophoblast cells and detected a significant increase in the mRNA levels of IFN-gamma receptor beta-chain, mainly, when cultured cells were exposed to IFN-gamma. Immunohistochemistry and Western blot analyses also revealed protein expression of the IFN-gamma receptor alpha-chain. These results suggest that IFN-gamma may participate in the phagocytic activation of the mouse trophoblast, albeit the exact mechanism was not hereby elucidated. Protective and/or nutritional fetal benefit may result from this physiological response. In addition, our data also shed some light on the understanding of trophoblast tolerance to inflammatory/immune cytokines during normal gestation.

NADPH oxidase as an important source of reactive oxygen species at the mouse maternal-fetal interface: putative biological roles

Oxygen derivatives that comprise the large family of reactive oxygen species (ROS) are actively involved in placental biology. They are generated at the maternal-fetal interface at the level of decidual, trophoblast and mesenchymal components. In normal conditions, ROS produced in low concentrations participate in different functions as signalling molecules, regulating activation of redox-sensitive transcription factors and protein kinases involved in cell survival, proliferation and apoptosis, hence much of cell functioning. Physiological ROS generation is also associated with such defence mechanisms as phagocytosis and microbiocidal activities. In mice, particularly but not exclusively, trophoblast cells phagocytose intensively during implantation and post-implantation periods and express enzymic machinery to address a ROS-producing response to changes in the environment. The cells directly associated with ROS production are trophoblast giant cells, which mediate each and every relationship with the maternal organism. In this review, the production of ROS by the implanting mouse trophoblast is discussed, focusing on NADPH oxidase expression, regulatory mechanisms and similarities with NOX2 from phagocytes. Some of the current controversies are assessed by attempting to integrate data from studies in human trophoblast and mouse models.

Localization of Cathepsins D and B at the Maternal-Fetal Interface and the Invasiveness of the Trophoblast during the Postimplantation Period in the Mouse

A survey of existing data suggests that trophoblast cells produce factors involved in extracellular matrix degradation. In this study, we correlated the expression of cathepsins D and B in the murine ectoplacental cone with the ultrastructural progress of decidual invasion by trophoblast cells. Both proteases were immunolocalized at implantation sites in lysosome-endosome-like compartments of trophoblast giant cells. Cathepsin D, but not cathepsin B, was also detected ultrastructurally in extracellular compartments surrounded by processes of the invading trophoblast containing extracellular matrix components and endometrial cell debris. The expression of cathepsins D and B by trophoblast cells was confirmed by RT-PCR in ectoplacental cones isolated from implantation chambers at gestation day 7.5. Our data addressed a positive relationship between the expression and presence of cathepsin D at the extracellular compartment of the maternal-fetal interface and the invasiveness of the trophoblast during the postimplantation period, suggesting a participation of invading trophoblast cells in the cathepsin D release. Such findings indicate that mouse trophoblast cells might exhibit a proteolytic ability to partake in the decidual invasion process at the maternal-fetal interface.

Preeclampsia and superimposed preeclampsia: The same disease? The role of angiogenic biomarkers

ABSTRACT Objective: We aimed to compare sFlt-1 and placental growth factor (PlGF) levels and the sFlt-1/PlGF ratio between women with preeclampsia and superimposed preeclampsia to, respectively, normotensive and chronic hypertensive ones. Study design: We performed a prospective two-armed cohort in a tertiary teaching hospital in Sao Paulo, Brazil, including 37 normotensive and 60 chronic hypertensive pregnant women. We assessed the serum levels of sFlt-1 and PlGF at 20, 26, 32, and 36 gestational weeks by enzyme-linked immunosorbent assay. Main outcome measures: Having preeclampsia and superimposed preeclampsia. Results: Among normotensive and chronic hypertensive pregnancies, 4 (10.8%) and 14 (23.3%) women developed preeclampsia and superimposed preeclampsia, respectively. Compared with those who remained normotensive, the preeclampsia women presented higher sFlt-1 levels at 32 gestational weeks (4323.45 pg/mL vs. 2242.04 pg/mL, p = 0.019), lower PlGF levels at 20 (183.54 pg/mL vs. 337.38 pg/mL, p = 0.034), 32 (169.69 pg/mL vs. 792.53 pg/mL, p = 0.001), and 36 gestational weeks (252.99 pg/mL vs. 561.81 pg/mL, p = 0.029), and higher sFlt-1/PlGF ratios at 26 (9.02 vs. 1.84, p = 0.004), 32 (23.61 vs. 2.55, p = 0.001), and 36 gestational weeks (49.02 vs. 7.34, p = 0.029). On the other hand, compared with those who remained chronic hypertensive, the superimposed preeclampsia women only presented a higher sFlt-1/PlGF ratio at 32 gestational weeks (9.98 vs. 2.51, p = 0.039). Conclusion: Although angiogenic imbalance is clearly related to preeclampsia, it seems to play a more modest role in superimposed preeclampsia, in which other mechanisms should also be investigated.

NADPH‐diaphorase activity and nitric oxide synthase isoforms in the trophoblast of Calomys callosus

The pattern of expression of a variety of placental nitric oxide synthase isoforms has contributed to elucidating the regulatory mechanisms of nitric oxide (NO) synthesis during gestation. The maintenance of vascular tone, attenuation of vasoconstriction, prevention of platelet and leukocyte adhesion to the trophoblast surface, and possible participation in uterine blood flow seem to be the main functions of NO generated at the fetal‐maternal interface in humans and mice. Extending this knowledge to other rodent species commonly used as laboratory animals, in this study we focus on NADPH‐diaphorase activity and the distribution of nitric oxide synthase isoforms (NOS) in the trophoblast cells of Calomys callosus during different phases of pregnancy. NADPH‐diaphorase activity was evaluated cytochemically and the presence of NOS isoforms detected by immunohistochemistry. These techniques were performed on pre‐ and postimplantation embryos in situ and in vitro, as well as in placentae on d 14 and 18 of pregnancy. Neither NADPH‐diaphorase activity nor inducible or endothelial NOS isoforms were found in pre‐implanting embryos except after culturing for at least 48 h, when some of the embryonic cells were positive for the diaphorase reaction. On d 6·5 of pregnancy, trophoblast cells showed intense diaphorase activity both in situ and under in vitro conditions. A positive reaction was also found in the different placental trophoblast cells on d 14 and 18 of pregnancy. The inducible NOS (iNOS) isoform, but not the endothelial isoform, was immunodetected in trophoblast cells from the placenta and from postimplantation embryos in situ and under in vitro conditions. These results strongly suggest the production of NO by the iNOS isoform in the trophoblast of Calomys callosus after embryo implantation. The data also emphasise a possible role for the trophoblast in producing and releasing cytotoxic molecules at the fetal‐maternal interface.

Signaling Molecules Involved in IFN-γ-Inducible Nitric Oxide Synthase Expression in the Mouse Trophoblast

Problem  We have previously shown that trophoblast can generate nitric oxide (NO) and express inducible isoform of nitric oxide synthase (iNOS). Moreover, this production was changed by the presence of interferon‐γ (IFN‐γ) establishing a relationship between trophoblast inductive response and this proinflammatory cytokine.

Expression of NADPH Oxidase by Trophoblast Cells: Potential Implications for the Postimplanting Mouse Embryo

ABSTRACT Cytochemical localization of hydrogen peroxide-generating sites suggests NADPH (nicotinamide adenine dinucleotide 3-phosphate [reduced form]) oxidase expression at the maternal-fetal interface. To explore this possibility, we have characterized the expression and activity of the NADPH oxidase complex in trophoblast cells during the postimplantation period. Implantation sites and ectoplacental cones (EPCs) from 7.5-gestational day embryos from CD1 mice were used as a source for expression analyses of NADPH oxidase catalytic and regulatory subunits. EPCs grown in primary culture were used to investigate the production of superoxide anion through dihydroxyethidium oxidation in confocal microscopy and immunohistochemical assays. NADPH subunits Cybb (gp91phox), Cyba (p22phox), Ncf4 (p40phox), Ncf1 (p47phox), Ncf2 (p67phox), and Rac1 were expressed by trophoblast cells. The fundamental subunits of membrane CYBB and cytosolic NCF2 were markedly upregulated after phorbol-12-myristate-13-acetate (PMA) treatment, as detected by quantitative real-time PCR, Western blotting, and immunohistochemistry. Fluorescence microscopy imaging showed colocalization of cytosolic and plasma membrane NADPH oxidase subunits mainly after PMA treatment, suggesting assembly of the complex after enzyme activation. Cultured EPCs produced superoxide in a NADPH-dependent manner, associating the NADPH oxidase-mediated superoxide production with postimplantation trophoblast physiology. NADPH-oxidase cDNA subunit sequencing showed a high degree of homology between the trophoblast and neutrophil isoforms of the oxidase, emphasizing a putative role for reactive oxygen species production in phagocytic activity and innate immune responses.