Non-pregnant Mice Research Articles

TRPM4 contribution in mouse uterine contractions.

Inappropriate uterine contractions are a matter of concern during pregnancy or menses. We identified the transient receptor potential melastatin 4 (TRPM4) ion channel as a new actor in mouse uterine contractions highlighting this protein as a potential pharmacological target for a better control of myometrial activity. Control of uterine contractions is of interest in the context of inappropriate myometrial activity during pregnancy and at time of delivery, but it is also a matter for menstrual pain. While several molecular determinants of myometrial contractions have been described, the complete distribution of roles to the various actors is far from understood. A key phenomenon is a variation in cytoplasmic Ca2+ which leads to the activation of calmodulin in smooth muscle and also in the phosphorylation of myosin allowing contraction. The Ca2+ - TRPM4 channel which is known to modulate Ca2+- fluxes in several cell types was shown to participate in vascular as well as detrusor muscle contraction. We thus designed a study to determine whether it also participates in myometrial contraction. Uterine rings were isolated from Trpm4+/+ and Trpm4-/- non-pregnant adult mice and contractions were recorded using an isometric force transducer. In basal conditions, spontaneous contractions were similar in both groups. Application of 9-phenanthrol, a pharmacological TRPM4 inhibitor, dose-dependently reduced contraction parameters in Trpm4+/+ rings with an IC50 around 2.10-6 mol/L. The effect of 9-phenanthrol was significantly reduced in Trpm4-/- rings. The effect of oxytocin was tested and was found to be stronger in Trpm4+/+ rings compared to Trpm4-/-. Under a constant stimulation by oxytocin, 9-phenanthrol still reduced contraction parameters in Trpm4+/+ rings with a smaller effect on Trpm4-/-. Altogether it indicates that TRPM4 participates in uterine contractions in mice and may thus be evaluated as a new target to control such contractions.

Does cardio-hepatic communication contribute to pregnancy-induced cardiac growth and its reversal?

Introduction: During pregnancy, the maternal heart undergoes physiological cardiac growth, which reverses following parturition; however, the underlying processes that contribute are largely unknown. Changes in both systemic and cardiac metabolism appear to be associated with cardiac growth, which could also contribute during pregnancy. We have shown that several cardiac metabolic transcripts and proteins and liver-derived metabolites are elevated during pregnancy and postpartum and are associated with cardiac growth. Therefore, the goal of this study was to examine the extent to which the cardio-hepatic signaling axis contributes to pregnancy-induced cardiac growth and reversal. Methods and Results: Timed pregnancy studies in 12-week-old FVB/NJ mice revealed that both heart weight (HW:TL; p < 0.0001) and liver weight (LIV:TL; p <0.0001) were highest in lactating 1-week post-birth (PB) mice versus mid-pregnant (8 d), late-pregnant (LP; 16 d), and non-pregnant (NP) mice. Interestingly, we also found that HW:TL ( p < 0.01) and LIV:TL ( p < 0.0001) were significantly reduced in non-lactating 1-week post-birth mice (PBNL) compared with PB mice. Hearts and livers both remained significantly larger, irrespective of lactation status, following parturition through 6 weeks post birth (PB) versus NP. Plasma proteomics revealed significant increases in prolactin at LP in addition to increases in liver-derived proteins including hepatocyte growth factors; however, these levels returned to NP levels by PB. Examination of the liver proteome from the same mice revealed significant increases in fatty acid and ketone body metabolism proteins. Cardiac and liver immunoblot analysis revealed significant reductions in Pdk4 between 1- and 3-week PB and PBNL groups that was reversed by 6-weeks postpartum and a significant increase in Pdk4 expression at 6 weeks postpartum, respectively. Interestingly, we see a sustained increase in cardiac Bdh1 expression that persisted at 6-weeks postpartum which could contribute to the sustained increases in HW:TL versus NP. Conclusion: Changes in both lactation status and metabolism are likely contributing to similar patterns of growth seen in the heart and liver during and following pregnancy. NIGMS COBRE Pilot project grant P30 GM127607; NIH R01 HL147844 (Hill/ Jones); Jewish Heritage Fund for Excellence Faculty Support; Jewish Heritage Fund for Excellence Research Enhancement Award; UofL School of Medicine Basic Science award; NIGMS IDeA state funded proteomics voucher. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelial cell vasodilator dysfunction mediates progressive pregnancy-induced hypertension in endothelial cell tetrahydrobiopterin deficient mice

Background and purposePregnancy-associated vascular remodelling is essential for both maternal and fetal health. We have previously shown that maternal endothelial cell tetrahydrobiopterin (BH4) deficiency causes poor pregnancy outcomes. Here, we investigated the role and mechanisms of endothelial cell-mediated vasorelaxation function in these outcomes. Experimental approachThe vascular reactivity of mouse aortas and uterine arteries from non-pregnant and pregnant endothelial cell-specific BH4 deficient mice (Gch1fl/flTie2cre mice) was assessed by wire myography. Systolic blood pressure was assessed by tail cuff plethysmography. Key resultsIn late pregnancy, systolic blood pressure was significantly higher (∼24 mmHg) in Gch1fl/flTie2cre mice compared with wild-type littermates. This was accompanied by enhanced vasoconstriction and reduced endothelial-dependent vasodilation in both aorta and uterine arteries from pregnant Gch1fl/flTie2cre mice. In uterine arteries loss of eNOS-derived vasodilators was partially compensated by upregulation of intermediate and large-conductance Ca2+-activated K+ channels. In rescue experiments, oral BH4 supplementation alone did not rescue vascular dysfunction and pregnancy-induced hypertension in Gch1fl/flTie2cre mice. However, combination with the fully reduced folate, 5-methyltetrahydrofolate (5-MTHF), restored endothelial cell vasodilator function and blood pressure. Conclusions and implicationsWe identify a critical requirement for maternal endothelial cell Gch1/BH4 biosynthesis in endothelial cell vasodilator function in pregnancy. Targeting vascular Gch1 and BH4 biosynthesis with reduced folates may provide a novel therapeutic target for the prevention and treatment of pregnancy-related hypertension.

Mechanisms of Lactation-induced Infertility in Female Mice.

Lactation in mammals is associated with a period of infertility, which serves to direct maternal metabolic resources toward caring for the newborn offspring rather than supporting another pregnancy. This lactational infertility is characterized by reduced pulsatile luteinizing hormone (LH) secretion and lack of ovulation. The mechanisms mediating suppression of LH secretion during lactation are unclear. There are potential roles for both hormonal cues such as prolactin and progesterone, and pup-derived cues such as suckling, on the inhibition of reproduction. To enable future studies using transgenic animals to investigate these mechanisms, in the present study our aim was to characterize lactational infertility in mice, and to investigate the effect of removing pup-derived cues on LH secretion, time to ovulation, and kisspeptin immunoreactivity. We first confirmed that C57BL/6J mice experience prolonged anestrus during lactation, which is dependent on establishment of lactation, as removal of pups the day of parturition led to immediate resumption of pulsatile LH secretion and normal estrous cycles. Once lactation is established, however, the lactational anestrus persisted for several days even after premature removal of pups. Pharmacological suppression of prolactin following premature weaning significantly reduced this period of lactational infertility. Progesterone does not appear to play a significant role in the suppression of fertility during lactation in mice, as levels measured during lactation were not different from nonpregnant mice. These data suggest that prolactin plays a key role in mediating anestrus during early lactation in mice, even in the absence of the suckling stimulus.

Tumor Necrosis Factor-α Alters Maternal and Embryonic Zinc Metabolism and Is Developmentally Toxic in Mice

We tested the hypothesis that tumor necrosis factor-α (TNF-α) would be teratogenic in mice due in part to its effects on zinc metabolism. In Experiment 1, nonpregnant mice were injected with a single dose of TNF-α (40,000 U) or PBS and then received a 65Zn-labeled meal. Mice killed 10 h after TNF-α treatment had high liver 65Zn and low plasma 65Zn, compared with controls. In Experiment 2, gestation day 8 (GD 8) mice were injected with PBS or TNF-α and then received a 65Zn-labeled meal. Dams killed 10 h after TNF-α treatment had higher liver and kidney 65Zn and lower plasma and embryonic 65Zn accumulation than controls. In Experiment 3, TNF-α dosing from GD 7–12 was associated with high maternal liver Zn and metallothionein concentrations on GD 13 and a high frequency of exencephaly on GD 18. In Experiment 4, dams fed diets containing 4.5, 12.5 or 50.0 µg Zn/g were given PBS or TNF-α on GD 7–12. Gross fetal defects were not observed in the PBS-treated litters evaluated on GD 18. In contrast, TNF-α-treated litters were characterized by multiple defects, with the incidence and severity being highest in the low Zn diet group. In Experiment 5, embryos cultured in serum from TNF-α-treated animals exhibited a high frequency of defects; the developmental toxicity of this serum was ameliorated when it was supplemented with Zn. Thus, the developmental toxicity of TNF-α is due in part to its influence on Zn metabolism.

Effects of High-Fat Feeding on Expression of Genes Regulating Fatty-1 Acid Metabolism in Hearts of Pregnant C57BL/6 Mice

Cardiovascular complications in pregnancy increase risk for subsequent heart disease, suggesting adverse events during pregnancy may permanently alter maternal heart health. The heart undergoes physiologic hypertrophy with pregnancy, which is distinct from pathological remodeling associated with obesity. We previously demonstrated that C57BL/6 mice fed a high-fat diet, a model of diet-induced obesity, had attenuated cardiac hypertrophy with pregnancy compared to low-fat controls, associated concentric remodeling. Dual effects of pregnancy and obesity on cardiac metabolism during hypertrophy have not been studied. We investigated whether expression of genes regulating fatty acid metabolism in the heart were altered in pregnant mice fed a high-fat diet. The Nanostring Metabolic Pathways Panel and nCounter analysis system was used to quantify individual mRNA transcripts of genes regulating fatty acid metabolism from the left ventricles of pregnant and non-pregnant female C57BL/6 mice fed a high-fat or control low-fat diet. Pregnancy increased expression of genes regulating fatty acid transport (Cd36, Slc27a1, Cpt1b) and β-oxidation (Acaa2, Acadl, Acox1, Ehhadh, Mlycd), but the effect was observed in low-fat mice only. Increases in gene expression with high-fat feeding were pronounced in non-pregnant mice, but effects not additive with pregnancy. Further, three genes with functions related to energy metabolism (Glul, Mat2a, Ogdhl,) were increased in low fat–fed pregnant mice only. Obesity during pregnancy may “max out” cardiac fatty acid utilization through upregulation of transporters and oxidation of long-chain fatty acids, and also downregulate metabolic pathways essential to cardiac adaptation. These 48 results suggest pre-existing obesity could disrupt cardiac physiologic remodeling during pregnancy.

The Effects of Pregnancy on the Pulmonary Immune Response in a Mouse Model of LPS-Induced Acute Lung Injury.

This study evaluated the effect of pregnancy on the pulmonary innate immune response in a mouse model of acute lung injury (ALI) using nebulized lipopolysaccharide (LPS). Pregnant (day 14) C57BL/6NCRL mice and nonpregnant controls received nebulized LPS for 15 minutes. Twenty-four hours later, mice were euthanized for tissue harvest. Analysis included blood and bronchoalveolar lavage fluid (BALF) differential cell counts, whole-lung inflammatory cytokine transcription levels by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and whole-lung vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and BALF albumin by western blot. Mature bone marrow neutrophils from uninjured pregnant and nonpregnant mice were examined for chemotactic response using a Boyden chamber and for cytokine response to LPS by RT-qPCR. In LPS-induced ALI, pregnant mice had higher BALF total cell (p < 0.001) and neutrophil counts (p < 0.001) as well as higher peripheral blood neutrophils (p < 0.01) than nonpregnant mice, but a similar increase (as compared with unexposed mice) in airspace albumin levels. Whole-lung expression of interleukin 6, tumor necrosis factor-α (TNF-α), and keratinocyte chemoattractant (CXCL1) was also similar. In vitro, marrow-derived neutrophils from pregnant and nonpregnant mice had similar chemotaxis to CXCL1 and N-formylmethionine-leucyl-phenylalanine, but neutrophils from pregnant mice expressed lower levels of TNF (p < 0.001) and CXCL1 (p < 0.01) after LPS stimulation. In uninjured mice, VCAM-1 was higher in lungs from pregnant versus nonpregnant mice (p < 0.05). In this model, pregnancy is associated with an augmented lung neutrophil response to ALI without increased capillary leak or whole-lung cytokine levels relative to the nonpregnant state. This may stem from increased peripheral blood neutrophil response and intrinsically increased expression of pulmonary vascular endothelial adhesion molecules. Differences in lung innate cell homeostasis may affect the response to inflammatory stimuli and explain severe lung disease in respiratory infection during pregnancy. · Inhalation of LPS in midgestation versus virgin mice is associated with increased neutrophilia.. · This occurs without a comparative increase in cytokine expression.. · This may be explained by pregnancy-enhanced pre-exposure expression of VCAM-1 and ICAM-1..

The role of thymus- and extrathymus-derived regulatory T cells in maternal-fetal tolerance.

Regulatory T (Treg) cells could be divided into thymus-derived Treg (tTreg) cells and peripherally derived Treg (pTreg) cells, and in vitro induced Treg (iTreg) cells. To date, the functions of tTreg versus pTreg and their relative contributions to maternal-fetal immune tolerance remain insufficiently defined due to a lack of a specific marker to distinguish tTreg cells from pTreg cells. In this study, we investigated the role of thymus- and extrathymus-derived Treg cells in pregnancy tolerance using transgenic ACT-mOVA, Foxp3DTR and Foxp3GFP mice, and Treg cell adoptive transfer, etc. We found that the frequencies of Treg cells in the thymus, spleen and lymph nodes (LNs) in either syngeneically- or allogeneically-mated pregnant mice were not different from non-pregnant mice. However, percentages of blood Treg cells in pregnant mice increased at mid-gestation, and percentages of decidua Treg cells in pregnant mice increased as the pregnancy progressed compared with non-pregnant mice, and were significantly higher in allogeneic mice than those in syngeneic group. Compared with syngeneic mice, levels of CCR2 and CCR6 on blood and decidua Treg cells and CCL12 in the decidua significantly increased in allogeneic mice. A surrogate fetal antigen mOVA that was recognized by naïve T cells from OT-IIFoxp3GFP mice induced the generation of pTreg cells in vivo. Transfusion of thymus and spleen Treg cells significantly decreased diphtheria toxin (DT)-increased embryo resorption rates (ERRs) and IFN-γ levels in the blood and decidua. iTreg cells also decreased ERRs and IFN-γ levels in the blood and decidua to an extent lower than thymus and spleen Treg cells. In conclusion, increased blood and decidua Treg cells in pregnancy and increased ERRs in DT-treated Foxp3DTR mice suggest an important immunosuppressive role of Treg cells in pregnancy. Elevated decidua Treg cells in pregnancy could be derived from the recruitment of tTreg cells to the decidua, or from the transformation of naïve T cells in the decidua to pTreg cells. While the immune-suppression effects of thymus and spleen Treg cells are comparable, iTreg cells might play a weaker role in maternal-fetal tolerance.

Immunization with a Multivalent Listeria monocytogenes Vaccine Leads to a Strong Reduction in Vertical Transmission and Cerebral Parasite Burden in Pregnant and Non-Pregnant Mice Infected with Neospora caninum.

Neospora caninum is an apicomplexan parasite that causes abortion and stillbirth in cattle. We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified version of the attenuated Listeria monocytogenes vaccine vector Lm3Dx_NcSAG1, which expresses the major N. caninum surface antigen SAG1. Multivalent vaccines were generated by the insertion of gra7 and/or rop2 genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7 and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Six experimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1 × 107 CFU of the respective vaccine strains. Seven days post-mating, mice were challenged by the subcutaneous injection of 1 × 105N. caninum NcSpain-7 tachyzoites. Non-pregnant mice, dams and their offspring were observed daily until day 25 post-partum. Immunization with Lm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival, whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almost all pups had died at the end of the experiment in the infection control. The triple mutant was the most promising vaccine candidate, providing the highest rate of protection against vertical transmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1 resulted in lower vertical transmission and enhanced protection against cerebral infection in dams and in non-pregnant mice.

Dynamics of neuropilin1 (Nrp1)-positive thymus-derived and Nrp1-negative peripherally induced paternal antigen specific regulatory T cells in the uterus and spleen during pregnancy in mice

Paternal antigen-specific regulatory T (PA-Treg) cells suppress the immune response against the fetus. Naturally occurring Treg (nTreg) cells derived from the thymus and peripherally induced Treg (iTreg) cells are functional for sustaining pregnancy. This study aimed to compare the variation in PA-Treg cells between the feto-maternal interface and the spleen and to elucidate the dynamics of nTreg and iTreg cells during the gestational period. PA-Treg cells, defined as Treg cells with paternally derived Mls-1a antigen-specific T cell receptors Vβ6, from allogeneic pregnant mice on days 3.5, 5.5, 11.5, and 18.5 post-coitum (pc) were evaluated by flow cytometry. The percentage of Vβ6+ Ki67+ PA-Treg cells activated by the paternal antigen increased on day 11.5 pc in the decidua (p < 0.05) compared to non-pregnant mice. On day 18.5 pc, this percentage in the decidua parietalis decreased to the level of the non-pregnant state but was significantly higher (p < 0.05) in the decidua basalis. No changes were observed in the spleens. We used two nTreg cell markers, neuropilin1 (Nrp1) and Helios, to distinguish between nTreg cells and iTreg cells. Nrp1+ PA-Treg cell levels decreased in late pregnancy compared to those observed in early pregnancy (day 3.5 pc: 57.14 ± 6.16% vs. day 18.5 pc: 30.43 ± 3.09%; p < 0.05), whereas Helios+ cell levels did not change. In conclusion, PA immune tolerance is induced by Nrp1+ nTreg cells in early pregnancy and Nrp1-negative Treg cells in late pregnancy.

RANKL up-regulated by progesterone aggravates lipopolysaccharide-induced acute lung injury during pregnancy

Acute lung injury (ALI) is a common acute respiratory disease with high morbidity and mortality rate in pregnant women. Receptor activator of NF-κB ligand (TNFSF11, also known as RANKL) exerts either pro-inflammatory or anti-inflammatory effects on the immune response. LPS administration reduced the survival time (n = 10, p < 0.01), increased wet/dry ratio (n = 10, p < 0.001) and lung injury score (n = 10, p < 0.001), the elevated proportions of plasmacytoid dendritic cells (pDCs) (n = 10, p < 0.0001), tissue-resident DCs (resDCs) (n = 10, p < 0.0001), macrophages (n = 10, p < 0.0001), and neutrophils (n = 10, p < 0.0001), and the expressions of costimulatory molecules and inflammation cytokines (n = 10, p < 0.05) in lungs of pregnant mice, compared with non-pregnant mice. In vitro, progesterone up-regulated the expression of RANKL (n > 6, p < 0.05) on pulmonary fibroblasts. The results of cytokine arrays showed that the cytokines associated with inflammatory response and leukocyte differentiation were decreased in pulmonary fibroblasts after treatment with anti-RANKL neutralizing antibody, compared with control pulmonary fibroblasts. More notably, we found that Tnfsf11-/- pregnant mice had longer survival durations (n = 10, p < 0.01), lower lung injury scores (n = 10, p < 0.05), and lower immune cell infiltration (n = 10, p < 0.05). These data imply that the RANKL/RANK axis plays an essential role in LPS-induced ALI during pregnancy possibly through a variety of pathways.

Gut and lung microbiome profiles in pregnant mice.

In recent years, microbiome research has expanded from the gastrointestinal tract to other host sites previously thought to be abacterial such as the lungs. Yet, the effects of pregnancy in the lung and gut microbiome remains unclear. Here we examined the changes in the gut and lung microbiome in mice at 14 days of gestation. Lung tissue and stool samples were collected from pregnant and non-pregnant female BALB/c mice, DNA was isolated, amplified, and bacterial specific V4 16S rRNA gene was sequenced. Using an in-house bioinformatic pipeline we assessed the microbial composition of each organ using stool and lung tissue samples. The stool data showed that Lachnospiraceae and Lactobacillaceae were more abundant in the pregnant mice. Likewise, Lactobacillaceae were dominant in the lungs of pregnant mice. However, Streptococcaceae were dominant in the lungs of non-pregnant mice with a low microbial abundance in the pregnant mice. A permutation test showed that pregnancy significantly contributes to the variance in both the lung and stool microbiome. At the same time, we estimate that 49% of the total detected operational taxonomic units were shared between the stool and lung data. After removing common stool-associated bacteria from the lung dataset, no microbial differential abundance was detected between the pregnant and non-pregnant lung microbial community. Thus, pregnancy contributes to variance to the lung and stool microbiome but not in the unique lung microbiota.

Hyperuricemia during Pregnancy Leads to a Preeclampsia-Like Phenotype in Mice.

Hyperuricemia is a common feature in pregnancies compromised by pre-eclampsia, a pregnancy disease characterized by hypertension and proteinuria. The role of uric acid in the pathogenesis of pre-eclampsia remains largely unclear. The aim of this study was to investigate the effect of elevated uric acid serum levels during pregnancy on maternal blood pressure and neonatal outcome using two different murine knockout models. Non-pregnant liver-specific GLUT9 knockout (LG9KO) mice showed elevated uric acid serum concentrations but no hypertensive blood pressure levels. During pregnancy, however, blood pressure levels of these animals increased in the second and third trimester, and circadian blood pressure dipping was severely altered when compared to non-pregnant LG9KO mice. The impact of hyperuricemia on fetal development was investigated using a systemic GLUT9 knockout (G9KO) mouse model. Fetal hyperuricemia caused distinctive renal tissue injuries and, subsequently an impaired neonatal growth pattern. These findings provide strong evidence that hyperuricemia plays a major role in the pathogenesis of hypertensive pregnancy disorders such as pre-eclampsia. These novel insights may enable the development of preventive and therapeutic strategies for hyperuricemia-related diseases.

Abstract 11489: Pregnancy Increases Cardiac Vulnerability to Post-Partum Pathological Stimuli

Introduction: The hemodynamic demands on the heart during normal pregnancy induce physiological cardiac hypertrophy. Together with exercise induced cardiac hypertrophy and cardiac enlargement during post-natal growth, these physiological cardiac adaptations are distinct from pathological hypertrophy caused by heart diseases including volume/pressure overload. Exercise-induced cardiac hypertrophy can induce a protective phenotype that leads to resistance from ischemic injury or pressure overload induced pathological remodeling. Whether pregnancy induced cardiac hypertrophy can also protect the heart from post-partum (PP) pathological stimuli remains unknown and is the focus of this study. Methods: We challenged both post-partum and age matched non-pregnant female C57BL/6 mice with 7-day osmotic minipump infusion of Angiotensin II/phenylephrine (AngII/PE) and determined the cardiac alterations. Results: PP mice had more severe cardiac pathological remodeling after AngII/PE infusion compared with non-pregnant counterparts, with greater increases in heart weight, cardiomyocyte hypertrophy and fibrosis. Echocardiography showed both left and right ventricular hypertrophy without overt systolic dysfunction. PP mice had greater elevations in expression levels of fetal genes and fibrotic genes compared with non-pregnant mice after AngII/PE infusion. RNA-sequencing analysis suggested a more robust change in gene expression level in PP mice after AngII/PE treatment compared to non-pregnant counterparts, with a substantial role for extracellular matrix organization genes. We also found a significant alteration of gene expression levels for enriched extracellular structural organization at 1-day PP, which underlies ongoing structural re-organization in the myocardium shortly after deliver. These PP changes may predispose the heart to adverse remodeling when simultaneously challenged with pathological stimuli. Conclusions: Pregnancy induced cardiac remodeling does not protect the heart against PP pathological stimuli. In fact, the rapid remodeling in the PP heart may predispose it to exacerbation of pathological remodeling.

Gut microbiota involved in desulfation of sulfated progesterone metabolites: A potential regulation pathway of maternal bile acid homeostasis during pregnancy.

Abnormally raised circulating bile acids (BA) during pregnancy threat fetal and offspring health. Our previous study has identified sulfated progesterone metabolites (PMSs) in part account for dysregulation of maternal BA homeostasis during pregnancy, however, limited intervention strategies to remedy increased serum BA through PMSs during pregnancy are available. The purpose of this study is to test the feasibility of manipulating BA homeostasis and progesterone metabolism through steering gut microbiota. A total of 19 pregnant sows were randomly treated with standard diet or vancomycin-supplemented diet, to investigate the intercorrelation of PMSs, intestinal microbiota, and maternal BA metabolism from day 60 of gestation (G60) until farrowing (L0). Pregnant mice orally gavaged with epiallopregnanolone sulfate (PM5S) or vehicle and nonpregnant mice were sampled and further analyzed to verify the effect of PM5S on maternal BA metabolism. The present study revealed that oral vancomycin reduced maternal fasting serum total BA (TBA) levels and postprandial serum TBA levels at day 90 of gestation (G90). BA profile analysis showed the decreased TBA after vancomycin treatment was attributed to the decrease of primary BA and secondary BA, especially hyodeoxycholic acid (HDCA). By using newly developed UPLC-MS/MS methods, we found vancomycin increased fecal excretion of allopregnanolone sulfate (PM4S) and PM5S during late gestation and thus maintaining the relative stability of serum PM4S and PM5S, which play an important role in BA metabolism. Further study in mice showed that pregnant mice have higher serum and liver TBA levels compared with nonpregnant mice, and PM5S administration induced higher gallbladder TBA levels and TBA pool in pregnant mice. In addition, after oral vancomycin, the continuously decreased Parabacteroides genus, potentially enriched with genes encoding steroids sulfatase, may explain the increased fecal PMSs excretion in pregnant sows. Taken together, our study provides the evidence that pregnancy-induced elevation of BA levels in sow is likely regulated by manipulation of gut microbiota, which offer new insights into the prevention and treatment of disrupted BA homeostasis during pregnancy by targeting specific microbiota.

Establishment of a useful in vitro decidual induction model using eCG-primed nonpregnant mouse endometrial stromal cells†.

Uterine endometrial differentiation is essential for developmental continuity and female health. A convenient in vitro model mimicking the physiological status is needed to effectively evaluate implantation and uterine response mechanisms. Thus, we developed a promising in vitro model, the FSS (FSH mimic-stimulated synchronized) model, by using primary mouse uterine stromal cells (mUSCs) obtained from equine chorionic gonadotropin (eCG)-primed mice. These mUSCs could be differentiated into decidualized cells with 17 beta-estradiol (E2) and progesterone (P4). The pregnancy day 4 (PD4) model, in which mUSCs are obtained at day 4 of pregnancy, was used as a control. The cell shape index and polyploidy rates were similar between the two models. The staining intensities of lipids and glycogen were significantly higher in the induced groups in both models but stronger in the FSS model than in the PD4 model. The expression levels of AP-TNAP, cathepsin L, Prl8a2, Gja1, Cebpb, and Igfbp1 were increased at 24h after decidual induction. PR-alpha and PR-beta levels were also increased at 24h after decidual induction in both models. These results indicate that the FSS model provides a convenient method for obtaining USCs that are usable for various experimental approaches due to their physiological competence and flexibility for triggering induction. This may serve as a model system for the study of pathogeneses originating from the endometrium or communication with other tissues and lead to a better understanding of embryo implantation mechanisms. Furthermore, the results of this study will be integral for further refinements of 3D uterine culture manipulation techniques.

Abstract P141: Phosphodiesterases Mediate The Facilitatory Effect Of Inhibitory G Proteins On Myogenic Constriction And Are Negatively Regulated By The Dual Action Of Rgs2 And 5

G protein regulation by RGS (regulators of G protein signaling) proteins of the R4/B family plays a key role in vascular tone maintenance; However, the regulatory mechanisms are unclear. Previous studies showed that the loss of regulation of G i/o and G q/11 class G proteins by RGS2 is involved in augmented myogenic tone and decreased uterine blood flow in non-pregnant mice. Additionally, RGS5, another R4/B member, was shown to regulate vascular tone by regulating G q/11 signaling. RGS2 and 5 are structurally and functionally closely related and are co-expressed in the resistance vasculature, including the uterine vascular bed. However, whether and how RGS2 and 5 coordinate their regulatory activities to fine-tune G protein signaling and vascular tone are unknown. Here, we tested the hypothesis that the integrated activity of RGS2 and 5 decreases myogenic tone by inhibiting G i/o signaling, promoting cAMP-dependent smooth muscle relaxation in the uterine vascular bed. Using pressure myography, we examined myogenic constriction of uterine artery segments isolated from non-pregnant wild type, Rgs5 KO, Rgs2 KO, and Rgs2/5 dbKO mice in the absence and presence of exogenous cAMP or various pharmacological inhibitors of G i/o signaling, while nitric oxide synthesis was continuously blocked with L-NAME. We found that myogenic constriction was reduced in Rgs5 KO relative to WT or Rgs2 KO arteries. Activating G i/o with dopamine increased, whereas cAMP decreased myogenic tone in Rgs5 KO arteries to WT level. Dual deletion of Rgs2 and 5 abolished the reduced myogenic tone in the absence of only Rgs5 and enhanced dopamine induced G i/o effects in Rgs2/5 dbKO arteries. Conversely, G i/o inhibition with pertussis toxin decreased myogenic constriction in Rgs2/5 dbKO to similar levels in Rgs5 KO arteries. The application of the pan-phosphodiesterase (PDE) inhibitor, IBMX, normalized myogenic tone, and blocked dopamine-mediated myogenic tone augmentation in Rgs2 KO, Rgs5 KO, and Rgs2/5 dbKO arteries. We conclude that activated G i/o augments myogenic tone by promoting PDE-mediated inhibition of cAMP-dependent vasodilatation. While RGS2 and 5 inhibit this novel G i/o -PDE-cAMP signaling pathway, RGS5 dampens the inhibitory efficacy of RGS2 towards G i/o in uterine arteries.

Abstract P095: Arginine Vasopressin Infusion Is Sufficient To Cause Pregnancy-specific Cardiac Hypertrophy

Preeclampsia is a prevalent gestational disorder characterized by hypertension, proteinuria, and renal damage. Mothers with a history of preeclampsia (PE) are at increased risk of future cardiovascular disease (CVD). Yet, the mechanism by which preeclampsia predisposes patients to future CVD is unknown. Previously published data demonstrated that arginine vasopressin (AVP) infusion in mice recapitulates clinical features of PE including pregnancy-specific hypertension. We hypothesized that chronic AVP infusion in pregnant mice would cause cardiac hypertrophy. C57Bl6/J mice were implanted with an osmotic minipump to deliver 24ng/hr of AVP throughout gestation until necropsy on gestational day 18. Blood pressures were monitored by tail cuff plethysmography and showed no increase in mean arterial pressure (MAP) in nonpregnant mice. However in pregnant mice, an increase of 18.8 ± 7.4 mmHg (82.1 vs 100.8, p=0.05) in MAP was observed in AVP-infused mice compared to saline-infused pregnant mice. Similarly, there was pregnancy-specific 13.4 ± 0.9 mg (93.6 vs 106.9, p=0.0001) increase in the heart weight of AVP-infused mice. Plasma levels of NT-ProBNP were measured by ELISA and showed a significant 163.6 ± 61.00 pg/ml (426.0 vs 589.6, p=0.01) increase in pregnant AVP-infused mice. Human 3rd trimester maternal plasma samples were obtained from the Iowa Maternal Fetal Tissue Bank (IRB# 200910784) for a case control study. In plasma samples from preeclamptic patients there was a significant 264.6 ± 79.72 pg/ml (185.1 vs 449.8, p=0.001) and a 27.9 ± 11.5 pg/ml (64.6, n=32 vs 92.5, n=66, p=0.02) increase in NT-ProBNP and MR-proANP respectively compared to normal pregnancy plasma. H9C2 rat cardiomyocytes treated with pooled plasma from preeclamptic patients showed a 6.6 and 1.8 fold increase in rat ANP and BNP expression respectively. These data suggest that AVP infusion in mice specifically during pregnancy is sufficient to cause cardiac hypertrophy beyond typical hypertrophy seen in a normal pregnancy and that unknown circulating factors in preeclamptics may contribute to the development of hypertrophy. Importantly, these data support using AVP-infusion as a novel mouse model of pregnancy-specific cardiac hypertrophy to study postpartum cardiomyopathies.

Abstract P097: Effect Of Maternal Fecal Microbiome And Adverse Pregnancy Outcomes In The Obese BPH/5 Female Mice

Obesity is a worldwide pandemic and of women childbearing age in the US, two-thirds are considered overweight/obese. Maternal microbial dysbiosis associated with obesity may play a role in adverse pregnancy outcomes. To better understand the relationship of microbiome and adverse pregnancy outcomes, we utilized the obese hypertensive BPH/5 mouse model of preeclampsia (PE). We hypothesized that the maternal fecal microbiome during pregnancy will differ in the BPH/5 mice compared to lean normotensive C57 controls as well as non-pregnant (NP) BPH/5 females. We have shown that adult BPH/5 female mice have an adverse cardiometabolic phenotype: hypertension and obesity that is exaggerated by pregnancy and phenocopies PE. To test our hypothesis, fecal samples were collected from NP and pregnant (P) BPH/5 and C57 control females in late gestation and Illumina sequencing of 16S v4 rRNA amplicon libraries were used to compare microbiomes. Microbial community composition of BPH/5 compared to C57 P mice were significantly different (variance of 33.59% in PCo1 and Pco2 11.48%, n=9-17, p=0.001, PERMANOVA with Bray-Curtis dissimilarity of 16S Amplicon Sequence Variant’s relative abundance). Alpha diversity was increased in BPH/5 compared to C57 P mice (n=9-17, p=0.005). There was a difference in beta diversity within the communities in BPH5 P mice compared to BPH5 NP mice, suggesting that the fecal microbiome was altered with pregnancy (n=9-17, p=0.006), but no difference was seen in the alpha diversity meaning that the richness and evenness were similar (n=9-17, p=0.115). The most abundant phyla are represented by 49.39% Firmicutes, 38.54% Bacteroidetes, and 7.48% Epsilonbacteraeota. The most abundant genera including 22.59% Alistipes, 19.57% Lachnospiraceae genus unknown, and 9.85% Helicobacter within the BPH5 samples. Muribaculum spp (p=1.58e-06) and Lachnospiraceae_NK4A136_unclassified (p=2.46e-03) showing significant decreases with pregnancy. In conclusion, obese BPH5 dams have altered fecal microbial communities associated with obesity and adverse pregnancy outcomes. Further investigations are necessary to further understand the importance of these microbial community changes and how they play a role in maternal hypertension and offspring.

Abstract P2019: Do Changes In Metabolism Or Lactation Mediate The Reversal Of Pregnancy-induced Cardiac Growth?

During pregnancy, the maternal heart undergoes physiologic growth, which reverses following parturition and is associated with the restoration of hemodynamic parameters. However, the molecular processes underlying the reversal of pregnancy-induced cardiac growth and the time frame in which these changes occur remain unclear. Because changes in cardiac metabolism have been shown to influence the remodeling in the maternal heart, changes in metabolism may also contribute to the reversal of pregnancy-induced remodeling. Therefore, the goal of this study was to examine the underlying processes that contribute to the structural and functional changes occurring in the maternal heart following parturition. Timed pregnancy studies in 12-week-old female FVB/NJ mice showed that heart weight to tibia length (HW:TL) and cardiomyocyte size were highest in lactating 1-week post-birth (PB) mice versus mid-pregnant (8 d), late-pregnant (LP; 16 d), and non-pregnant (NP) mice. Interestingly, we also found that HW:TL was significantly reduced in non-lactating 1-week post-birth mice (PBNL) compared with LP mice. To determine the time-course of the reversal of pregnancy-induced remodeling and to examine the impact of lactation, we examined HW:TL in PB and PBNL mice 1, 3, and 6 weeks following parturition. We find that HW:TL is significantly higher in lactating PB mice versus non-lactating PBNL mice at 1 (8.71 ± 0.05 v 7.09 ± 0.26 mg/mm) and 3 weeks following parturition (8.90 ± 0.40 v 6.92 ± 0.56 mg/mm); however, 6 weeks after parturition, HW:TL was comparable between PB (7.35 ± 0.29 mg/mm) and PBNL groups (7.05 ± 0.18 mg/mm). Nevertheless, HW:TL remained elevated for 6 weeks after parturition in PB and PBNL mice compared with NP mice. RNA-Seq showed reductions in metabolic transcripts in PB hearts, including Pdk4 . Immunoblot analysis of key metabolic proteins showed that Pdk4 was reduced in hearts from 1 and 3 PB versus 1 and 3 weeks PBNL and NP. Together, these findings suggest that lactation influences the reversal of pregnancy-induced cardiac growth, which may be due in part to changes in cardiac metabolism.