Estela Bevilacqua

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.

Participation of the Mouse Implanting Trophoblast in Nitric Oxide Production During Pregnancy

Abstract While considerable progress has been made in elucidating nitric oxide (NO) regulatory mechanisms in the later stages of gestation, much less is known about its synthesis and role during embryo implantation. Thus, to evaluate the participation of the trophoblast in the production of NO during this phase, this study focused on NADPH-diaphorase activity and the distribution of NO synthase isoforms (NOS) using immunohistochemistry in pre- and postimplantation mouse embryos in situ and in vitro, as well as on NO production itself, measured as total nitrite, in trophoblast culture supernatants (Griess reaction). No NADPH-diaphorase activity was found in preimplanting embryos except after culturing for at least 48 h, when a few trophoblastic giant cells were positive. Conversely, postimplantation trophoblast cells either lodged into the implantation chamber (in situ) or after culturing (in vitro) showed intense NADPH-diaphorase activity. Also in the postimplantation trophoblast, the endothelial and inducible NOS (eNOS and iNOS) isoforms were immunodetected, under both in situ and in vitro conditions, although in different patterns. Extracts of ectoplacental cone also revealed bands of 135 and 130 kDa on SDS-PAGE that reacted with anti-eNOS and anti-iNOS, respectively, on Western blot. Analysis of the culture supernatant demonstrated that the nitrite concentration was 1) proportional to the number of cultured trophoblast cells, 2) almost completely abolished in the presence of Nω-nitro-l-arginine methyl ester, and 3) increased 2-fold in cultures stimulated with γ-interferon. These results strongly suggest the production of NO from constitutive and inducible isoforms of NOS by the implanting mouse trophoblast. They also emphasize the possibility of the participation of these cells in vasodilatation and angiogenesis, and in cytotoxic mechanisms involved in the intense phagocytosis of injured maternal cells, which occur during the implantation process.

The role of polymorphonuclear leukocytes in the resistance to cutaneous Leishmaniasis

The massive infiltration by polymorphonuclear leukocytes (PMN) soon after skin infection with Leishmania major suggests that PMN could participate in reducing parasite load and controlling the spreading of leishmanial infection. Yet, direct evidence for the participation of PMN in host defense against L. major was lacking. We investigated L. major infection in susceptible and resistant mice treated with the monoclonal (mAb) antibody RB6-8C5 that depletes the population of mature neutrophils and eosinophils. Both BALB/c and C57BL/6 mice depleted of PMN show accelerated parasite spreading and more severe footpad swelling than similarly infected untreated mice. In addition, significant higher parasite numbers were found in the lesion draining lymph nodes from PMN-depleted C57BL/6 mice. Histopathological analysis of the paw confirmed neutrophils containing ingested parasites as the dominant cell type in the infiltrate of the first days after infection and the nearly absolute neutrophil depletion in mAb-treated mice. Our data show the importance of PMN in early control of parasite load and parasitism spreading in cutaneous leishmaniasis.

Nitric oxide modulates eosinophil infiltration in antigen-induced airway inflammation in rats.

The influence of nitric oxide (NO) on eosinophil infiltration into the airways was investigated in rats actively sensitized with ovalbumin. The animals were treated chronically with the NO synthase inhibitor, N omega-Nitro-L-arginine methyl ester (L-NAME; 75 mumol rat-1 day-1), for 4 weeks. Bronchoalveolar lavage was performed at 6, 24, 48 and 72 h after intratracheal injection of ovalbumin. Intratracheal challenge of the sensitized rats with ovalbumin caused a significant increase in total leucocyte infiltration in bronchoalveolar lavage fluid both 24 and 48 h post-ovalbumin injection. Neutrophils and eosinophils peaked, respectively, at 24 h (29%) and 48 h (30%) in bronchoalveolar lavage fluid whereas the mononuclear cell did not differ significantly from the counts in non-sensitized rats at any time. At both 6 and 24 h post-ovalbumin injection, the chronic treatment of the animals with L-NAME affected neither the total nor the differential leucocyte content. However, at 48 h post-ovalbumin challenge, the total cell count was reduced by approximately 48% in the L-NAME-treated animals and this was associated with a marked inhibition (81%) of the eosinophil influx. Histological examination of the lungs from these animals (48 h post-ovalbumin challenge) also showed a prominent reduction (69.5%; P < 0.05) of the eosinophil infiltration in the respiratory segments. Our results demonstrate that NO plays a pivotal role in the eosinophil infiltration in airways of actively sensitized rats.

Collagen remodeling during decidualization in the mouse

SummaryAn ultrastructural study of the features and distribution of collagen fibrils was performed in the endometrium of virgin and pregnant (2nd to 11th day) mice. Collagen-containing structures were observed in the cytoplasm of fibroblasts on the 2nd day of pregnancy. Treatment of tissues with lanthanum nitrate established that these structures were intracytoplasmic. Their association with lysosome-like bodies suggested the occurrence of intracellular digestion of collagen, probably connected with remodeling of the endometrial stroma prior to decidualization. On the 4th day of pregnancy, very few collagen fibrils were present in the intercellular space. From the 6th day of pregnancy onwards, “thick” collagen fibrils were observed between decidual cells. The diameter of these fibrils measured up to 300 nm whereas the fibrils present in the endometrium of virgin mice measured 40–68 nm.

Effect of Maytenus ilicifolia Mart. on pregnant mice

Maytenus ilicifolia Mart. is used in Brazilian herbal medicine particularly for stomach disorders, but it is also used, as in other parts of South America, for fertility control. To verify its potential as an abortifacient, the lyophilized hydroalcoholic extract of its leaves was administered orally at a dose of 1000 mg/kg/day to mice between the first and third day of pregnancy (DOP), between the forth and sixth DOP, or between the seventh and ninth DOP. The extract caused a pre-implantation embryonic loss, but it did not have an effect on implantation or organogenesis. Morphological alterations of the reproductive system, not an embryotoxic effect, were not found. Estrogenic activity of the extract, exhibited by an uterotrophic effect, suggests that it may be interfering with the uterine receptivity to the embryo.

LPS exposure increases maternal corticosterone levels, causes placental injury and increases IL-1Β levels in adult rat offspring: relevance to autism.

Maternal immune activation can induce neuropsychiatric disorders, such as autism and schizophrenia. Previous investigations by our group have shown that prenatal treatment of rats on gestation day 9.5 with lipopolysaccharide (LPS; 100 μg/kg, intraperitoneally), which mimics infections by gram-negative bacteria, induced autism-like behavior in male rats, including impaired communication and socialization and induced repetitive/restricted behavior. However, the behavior of female rats was unchanged. Little is known about how LPS-induced changes in the pregnant dam subsequently affect the developing fetus and the fetal immune system. The present study evaluated the hypothalamic-pituitary-adrenal (HPA) axis activity, the placental tissue and the reproductive performance of pregnant Wistar rats exposed to LPS. In the adult offspring, we evaluated the HPA axis and pro-inflammatory cytokine levels with or without a LPS challenge. LPS exposure increased maternal serum corticosterone levels, injured placental tissue and led to higher post-implantation loss, resulting in fewer live fetuses. The HPA axis was not affected in adult offspring. However, prenatal LPS exposure increased IL-1β serum levels, revealing that prenatal LPS exposure modified the immune response to a LPS challenge in adulthood. Increased IL-1β levels have been reported in several autistic patients. Together with our previous studies, our model induced autistic-like behavioral and immune disturbances in childhood and adulthood, indicating that it is a robust rat model of autism.

Vascular endothelial growth factor (VEGF) and VEGF-receptor expression in placenta of hyperglycemic pregnant women

Hyperglycemia occurs in a variety of conditions such as overt diabetes, gestational diabetes and mild hyperglycemia, all of which are generally defined based on the oral glucose tolerance test and glucose profiles. Whereas diabetes has received considerable attention in recent decades, few studies have examined the mechanisms of mild hyperglycemia and its associated disturbances. Mild gestational hyperglycemia is associated with macrosomia and a high risk of perinatal mortality. Morphologically, the placenta of these women is characterized by an increase in the number of terminal villi and capillaries, presumably as part of a compensatory mechanism to maintain homeostasis at the maternal-fetal interface. In this study, we analised the expression of VEGF and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) in placentas from mildly hyperglycemic women. This expression was compared with that of normoglycemic women and women with gestational and overt diabetes. Immunohistochemistry revealed strong staining for VEGF and VEGFR-2 in vascular and trophoblastic cells of mildly hyperglycemic women, whereas the staining for VEGFR-1 was discrete and limited to the trophoblast. The pattern of VEGF and VEGF-receptor reactivity in placentas from women with overt diabetes was similar to that of normoglycemic women. In women with gestational diabetes, strong staining for VEGFR-1 was observed in vascular and trophoblastic cells whereas VEGF and VEGFR-2 were detected only in the trophoblast. The expression of these proteins was confirmed by western blotting, which revealed the presence of an additional band of 75 kDa. In the decidual compartment, only extravillous trophoblast reacted with all antibodies. Morphological analysis revealed collagen deposition around large arteries in all groups with altered glycemia. These findings indicate a placental response to altered glycemia that could have important consequences for the fetus. The change in the placental VEGF/VEGFR expression ratio in mild hyperglycemia may favor angiogenesis in placental tissue and could explain the hypercapillarization of villi seen in this gestational disturbance.

Differentiation-dependent expression of gelatinase B/matrix metalloproteinase-9 in trophoblast cells

Abstract The purpose of this study was to evaluate the Rcho-1 trophoblast culture system as a model for studying trophoblast invasion and to examine stage-specific expression of enzyme(s) potentially participating in rat trophoblast giant cell invasive behavior. The invasive behavior of the differentiating Rcho-1 trophoblast cells was demonstrated using Matrigel invasion chambers. Gelatin zymography and Western blot analysis of conditioned medium from differentiating Rcho-1 trophoblast cell cultures and rat ectoplacental cone outgrowths revealed a differentiation-dependent increase in gelatinase B/matrix metalloproteinase (MMP-9). Nothern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of Rcho-1 trophoblast or ectoplacental cone cells also showed increasing expression of MMP-9 accompanying cell differentiation. Rcho-1 trophoblast cells stably transfected with MMP-9 promoter/luciferase reporter constructs exhibited a differentiation-dependent increase in MMP-9 promoter activation. In conclusion, trophoblast giant cell differentiation is characterized by transcriptional activation of the MMP-9 gene and appearance of the invasive phenotype.

Macrophage migration inhibitory factor is up-regulated in human first-trimester placenta stimulated by soluble antigen of Toxoplasma gondii, resulting in increased monocyte adhesion on villous explants.

Considering the potential role of macrophage migration inhibitory factor (MIF) in the inflammation process in placenta when infected by pathogens, we investigated the production of this cytokine in chorionic villous explants obtained from human first-trimester placentas stimulated with soluble antigen from Toxoplasma gondii (STAg). Parallel cultures were performed with villous explants stimulated with STAg, interferon-gamma (IFN-gamma), or STAg plus IFN-gamma. To assess the role of placental MIF on monocyte adhesiveness to human trophoblast, explants were co-cultured with human myelomonocytic THP-1 cells in the presence or absence of supernatant from cultures treated with STAg (SPN), SPN plus anti-MIF antibodies, or recombinant MIF. A significantly higher concentration of MIF was produced and secreted by villous explants treated with STAg or STAg plus IFN-gamma after 24-hour culture. Addition of SPN or recombinant MIF was able to increase THP-1 adhesion, which was inhibited after treatment with anti-MIF antibodies. This phenomenon was associated with intercellular adhesion molecule expression by villous explants. Considering that the processes leading to vertical dissemination of T. gondii remain widely unknown, our results demonstrate that MIF production by human first-trimester placenta is up-regulated by parasite antigen and may play an essential role as an autocrine/paracrine mediator in placental infection by T. gondii.