Opposite effects of methanandamide on lipopolysaccharide-induced prostaglandin E2 and F2α synthesis in uterine explants from pregnant mice

Prostaglandins (PG) are effective abortifacients and are important mediators of lipopolisaccharide (LPS)-induced embryonic resorption (ER). Besides, anandamide (AEA) has been described as one of the major endocannabinoids present in the uterus suggesting that it might play a role in reproduction. It has been reported that high levels of AEA are associated with pregnancy failure and that LPS increases AEA production. Also, it has been observed that AEA modulates PG production in different tissues. In this sense, we studied whether LPS-induced PG production is modulated by AEA and we also assessed the effect of this endocannabinoid on PG metabolism in an in vitro model. Uterine explants from BALB/c implantation sites were cultured in the presence of LPS plus cannabinoid receptor (CB) specific antagonists and PG production was assessed. Then, we studied the effect of exogenous AEA on different steps of PG metabolic pathway. We showed that AEA is involved in LPS-induced PG biosynthesis. Also, we observed that AEA exerts opposite effects on PGE2 and PGF2α biosynthesis, by inhibiting PGE2 production and increasing PGF2α levels. We suggest that AEA could be involved in the mechanisms implicated in LPS-induced ER. A better understanding of how AEA could be affecting ER could help developing specific interventions to prevent this pathology.Fil: Vercelli, Claudia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Aisemberg, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Cella, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Salazar, Ana Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Wolfson, Manuel Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Franchi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

Maternal exposure to an enrichment environment promotes uterine vascular remodeling and prevents embryo loss in mice.

Implantation-related events are crucial for pregnancy success. In particular, defects in vascular remodeling at the maternal-fetal interface are associated with spontaneous miscarriage and recurrent pregnancy loss. Physical activity and therapies oriented to reduce stress improve pregnancy outcomes. In animal models, environmental stimulation and enrichment are associated with enhanced well-being, cognitive function and stress resilience. Here, we studied whether the exposure of BALB/c mice to an enriched environment (EE) regulates crucial events during early gestation at the maternal-fetal interface. Pregnant BALB/c mice were exposed to the EE that combines non-invasive stimuli from the sensory pathway with voluntary physical activity. The pregnancy rate was evaluated. Implantation sites were investigated microscopically and macroscopically. Vascular adaptation parameters at the maternal-fetal interface were analyzed. We found that exposure to the EE prevented pregnancy loss between gestational days 7 and 15. Also, it increased the diameter of the uterine artery and decreased the wall:lumen ratio of the mesometrial decidual vessels, suggesting that EE exposure promotes vascular remodeling. Moreover, it increased nitric oxide synthase activity and inducible nitric oxide synthase expression, as well as prostaglandin F2a production and endoglin expression in the implantation sites. Exposure of pregnant females to the EE regulates uterine physiology, promoting vascular remodeling during early gestation. These adaptations might contribute to preventing embryo loss. Our results highlight the importance of the maternal environment for pregnancy success. The design of an 'EE-like' protocol for humans could be considered as a new non-pharmacologic strategy to prevent implantation failure and recurrent miscarriage.Fil: de la Cruz Borthiry, Fernanda Luz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Schander, Julieta Aylen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Cella, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Beltrame, Jimena Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Franchi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Ribeiro, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

Maternal obesity reverses the resistance to LPS-induced adverse pregnancy outcome and increases female offspring metabolic alterations in cannabinoid receptor 1 knockout mice

Maternal overnutrition negatively impacts the offspring's health leading to an increased risk of developing chronic diseases or metabolic syndrome in adulthood. What we eat affects the endocannabinoid system (eCS) activity, which in turn modulates lipogenesis and fatty acids utilization in hepatic, muscle, and adipose tissues. This study aimed to evaluate the transgenerational effect of maternal obesity on cannabinoid receptor 1 knock-out (CB1 KO) animals in combination with a postnatal obesogenic diet on the development of metabolic disturbances on their offspring. CB1 KO mice were fed a control diet (CD) or a high-fat diet (HFD; 33% more energy from fat) for 3 months. Offspring born to control and obese mothers were also fed with CD or HFD. We observed that pups born to an HFD-fed mother presented higher postnatal weight, lower hepatic fatty acid amide hydrolase activity, and increased blood cholesterol levels when compared to the offspring born to CD-fed mothers. When female mice born to HFD-fed CB1 KO mothers were exposed to an HFD, they gained more weight, presented elevated blood cholesterol levels, and more abdominal adipose tissue accumulation than control-fed adult offspring. The eCS is involved in several reproductive physiological processes. Interestingly, we showed that CB1 KO mice in gestational day 15 presented resistance to LPS-induced deleterious effects on pregnancy outcome, which was overcome when these mice were obese. Our results suggest that an HFD in CB1 receptor-deficient mice contributes to a “nutritional programming” of the offspring resulting in increased susceptibility to metabolic challenges both perinatally and during adulthood.Fil: Bariani, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Correa, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Domínguez Rubio, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Wolfson, Manuel Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Schander, Julieta Aylen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Cella, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Aisemberg, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Franchi, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

Role of Nitric Oxide in Shiga Toxin-2-Induced Premature Delivery of Dead Fetuses in Rats

Shiga toxin-producing Escherichia coli (STEC) infections could be one of the causes of fetal morbimortality in pregnant women. The main virulence factors of STEC are Shiga toxin type 1 and/or 2 (Stx1, Stx2). We previously reported that intraperitoneal (i.p.) injection of rats in the late stage of pregnancy with culture supernatant from recombinant E. coli expressing Stx2 and containing lipopolysaccharide (LPS) induces premature delivery of dead fetuses. It has been reported that LPS may combine with Stx2 to facilitate vascular injury, which may in turn lead to an overproduction of nitric oxide (NO). The aim of this study was to evaluate whether NO is involved in the effects of Stx2 on pregnancy. Pregnant rats were i.p. injected with culture supernatant from recombinant E. coli containing Stx2 and LPS (sStx2) on day 15 of gestation. In addition, some rats were injected with aminoguanidine (AG), an inducible isoform inhibitor of NO synthase (iNOS), 24 h before and 4 h after sStx2 injection. NO production was measured by NOS activity and iNOS expression by Western blot analysis. A significant increase in NO production and a high iNOS expression was observed in placental tissues from rats injected with sStx2 containing 0.7 ng and 2 ng Stx2/g body weight and killed 12 h after injection. AG caused a significant reduction of sStx2 effects on the feto-maternal unit, but did not prevent premature delivery. Placental tissues from rats treated with AG and sStx2 presented normal histology that was indistinguishable from the controls. Our results reveal that Stx2-induced placental damage and fetus mortality is mediated by an increase in NO production and that AG is able to completely reverse the Stx2 damages in placental tissues, but not to prevent premature delivery, thus suggesting other mechanisms not yet determined could be involved

The Effect of Anandamide on Uterine Nitric Oxide Synthase Activity Depends on the Presence of the Blastocyst

Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot−1 h−1) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies

Anandamide Induces Sperm Release from Oviductal Epithelia through Nitric Oxide Pathway in Bovines

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 µM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 µM L-NAME, a NO synthase inhibitor, or 30 µg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM