Two Hits of EDCs Three Generations Apart: Effects on Social Behaviors in Rats, and Analysis by Machine Learning

All individuals are directly exposed to extant environmental endocrine-disrupting chemicals (EDCs), and indirectly exposed through transgenerational inheritance from our ancestors. Although direct and ancestral exposures can each lead to deficits in behaviors, their interactions are not known. Here we focused on social behaviors based on evidence of their vulnerability to direct or ancestral exposures, together with their importance in reproduction and survival of a species. Using a novel “two hits, three generations apart” experimental rat model, we investigated interactions of two classes of EDCs across six generations. PCBs (a weakly estrogenic mixture Aroclor 1221, 1 mg/kg), Vinclozolin (antiandrogenic, 1 mg/kg) or vehicle (6% DMSO in sesame oil) were administered to pregnant rat dams (F0) to directly expose the F1 generation, with subsequent breeding through paternal or maternal lines. A second EDC hit was given to F3 dams, thereby exposing the F4 generation, with breeding through the F6 generation. Approximately 1200 male and female rats from F1, F3, F4 and F6 generations were run through tests of sociability and social novelty as indices of social preference. We leveraged machine learning using DeepLabCut to analyze nuanced social behaviors such as nose touching with accuracy similar to a human scorer. Surprisingly, social behaviors were affected in ancestrally exposed but not directly exposed individuals, particularly females from a paternally exposed breeding lineage. Effects varied by EDC: Vinclozolin affected aspects of behavior in the F3 generation while PCBs affected both the F3 and F6 generations. Taken together, our data suggest that specific aspects of behavior are particularly vulnerable to heritable ancestral exposure of EDC contamination, that there are sex differences, and that lineage is a key factor in transgenerational outcomes.

The Role of Fatty Acid Signaling in Islet Beta-Cell Adaptation to Normal Pregnancy

BackgroundMaintenance of a normal fetal nutrient supply requires major adaptations in maternal metabolic physiology, including of the islet beta-cell. The role of lipid signaling processes in the mechanisms of islet beta-cell adaptation to pregnancy has been minimally investigated.ObjectiveTo determine the effects of pregnancy on islet fatty acid (FA) metabolic partitioning and FA augmentation of glucose-stimulated insulin secretion (GSIS).MethodsAge matched virgin, early pregnant (gestational day-11, G11) and late pregnant (G19) Sprague-Dawley rats were studied. Fasted and fed state biochemistry, oral glucose tolerance tests (OGTT), and fasted and post-OGTT liver glycogen, were determined to assess in vivo metabolic characteristics. In isolated islets, FA (BSA-bound palmitate 0.25 mmol/l) augmentation of GSIS, FA partitioning into esterification and oxidation processes using metabolic tracer techniques, lipolysis by glycerol release, triacylglycerols (TG) content, and the expression of key beta-cell genes were determined.ResultsPlasma glucose in pregnancy was lower, including during the OGTT (glucose area under the curve 0-120 min (AUC0-120); 655±24 versus 849±13 mmol.l-1.min; G19 vs virgin; P<0.0001), with plasma insulin concentrations equivalent to those of virgin rats (insulin AUC0-120; 97±7 versus 83±7 ng.ml-1.min; G19 vs virgin; not significant). Liver glycogen was depleted in fasted G19 rats with full recovery after oral glucose. Serum TG increased during pregnancy (4.4±0.4, 6.7±0.5; 17.1±1.5 mmol/l; virgin, G11, G19, P<0.0001), and islet TG content decreased (147±42, 172±27, 73±13 ng/µg protein; virgin, G11, G19; P<0.01). GSIS in isolated islets was increased in G19 compared to virgin rats, and this effect was augmented in the presence of FA. FA esterification into phospholipids, monoacylglycerols and TG were increased, whereas FA oxidation was reduced, in islets of pregnant compared to virgin rats, with variable effects on lipolysis dependent on gestational age. Expression of Ppargc1a, a key regulator of mitochondrial metabolism, was reduced by 51% in G11 and 64% in G19 pregnant rat islets compared to virgin rat islets (P<0.001).ConclusionA lowered set-point for islet and hepatic glucose homeostasis in the pregnant rat has been confirmed. Islet adaptation to pregnancy includes increased FA esterification, reduced FA oxidation, and enhanced FA augmentation of glucose-stimulated insulin secretion.

Preclinical Trial of Traditional Plant Remedies for the Treatment of Complications of Gestational Malaria

Background: Most pregnant women living in high malaria endemic regions of Nigeria use herbal remedies for the management of malaria-in-pregnancy, rather than the commonly prescribed drugs. Remedies common to this area involve a suspension of A. indica (AI) leaves and in some cases, a suspension containing a mixture of AI and D.edulis (PS). Aim: This study examined the therapeutic efficacies of AI, PS, or a combination of AI and PS in a pregnant rat model for exoerythrocytic stages of Plasmodium falciparum parasite. Method: A predetermined sample size of 30 dams was used (for a power level and confidence interval of 95%), and divided equally into six groups made up of non-malarous dams, untreated malarous dams, and malarous dams either treated exclusively with 1 mL of 3000 mg/kg b.w AI, 1000 mg/kg b.w PS, AI + PS (50% v/v), or 25 mg/kg b.w CQ. Result: No maternal mortality was recorded. AI significantly improved maternal weight gain from 32.4 to 82.2 g and placental weight from 0.44 to 0.53 g. In the curative test, AI and AI + PS significantly reduced the average percentage parasitemia (APP) in the pregnant rats from >80% to <20%. No significant difference in the APP was found between the pregnant rats treated with any of CQ or AI during the suppressive test. Results for the prophylactic test of the study groups showed that the APP was significantly reduced from 24.69% to 3.90% when treated with AI and 3.67% when combined with PS. AI + PS reduced diastolic blood pressure from 89.0 to 81.0 mm/Hg and compared with that of the non malarous dams. AI or AI + PS significantly increased the platelet counts (103 µL) from 214.1 to 364.5 and 351.2, respectively. AI and AI + PS improved birth weight from 2.5 to 3.9 g and crown rump length from 2.6 to 4.1 cm. For biomarkers of preeclampsia, combining AI and PS led to the reversal of the altered levels of creatine kinase, lactate dehydrogenase, cardiac troponin, soluble Fms-Like Tyrosine Kinase-1, and placental growth factor. Conclusions: This study validates the use of A. indica for the treatment of gestational malaria due to its antiplasmodial and related therapeutic effects and in combination with pear seeds for the management of malaria-in-pregnancy-induced preeclampsia.

Adaptive Changes in single-nephron GFR, Tubular Morphology, and Transport in a Pregnant Rat Nephron: Modeling and Analysis

Normal pregnancy is characterized by massive increases in plasma volume and electrolyte retention. Given that the kidneys regulate homeostasis of electrolytes and volume, the organ undergoes major adaptations in morphology, hemodynamics, and transport to achieve the volume and electrolyte retention required in pregnancy. These adaptations are complex, sometimes counterintuitive, and not fully understood. In addition, the demands of the developing fetus and placenta change throughout the pregnancy. For example, during late pregnancy, K+ retention and thus enhanced renal K+ reabsorption is required despite many kaliuretic factors. The goal of this study is to unravel how known adaptive changes along the nephrons contribute to the ability of the kidney to meet volume and electrolyte requirements in mid- and late pregnancy. We developed computational models of solute and water transport in the superficial nephron of the kidney of a rat in mid- and late pregnancy. The mid-pregnant and late-pregnant rat superficial nephron models predict that morphological adaptations and increased activity of the sodium hydrogen exchanger 3 (NHE3) and epithelial sodium channel (ENaC) are essential for enhanced Na+ reabsorption observed during pregnancy. Model simulations showed that for sufficient K+ reabsorption, increased H +-K +-ATPase activity and decreased K+ secretion along the distal segments is required in both mid- and late-pregnancy. Furthermore, certain known sex differences in renal transporter pattern (e.g., the higher NHE3 protein abundance but lower activity in the proximal tubules of virgin female rats compared to male) may serve to better prepare the female for the increased transport demand in pregnancy.

Possible Roles of Calreticulin in Uterine Decidualization and Receptivity in Rats and Humans

Previous in vitro studies have suggested that calreticulin (CALR), which is responsible for the folding and quality control of glycoproteins, may be associated with decidualization. However, its precise role in regulating decidualization has not been explored in vivo. Here, we used pregnant rat models to examine endometrial CALR expression during the peri-implantation period. We also examined whether polypectomy, a procedure that could ameliorate infertility, alters the endometrial expression levels of CALR and several implantation factors in women diagnosed as infertile. In rats, uterine CALR was expressed at a high level at the implantation site, and a marked increase in CALR expression was observed in decidual cells of normal pregnancy. In addition, endometrial CALR expression was enhanced by either administration of estradiol-17β in the delayed implantation rat model or the artificial induction of decidualization in the pseudopregnant rat. In cultured stromal cells, siRNA-mediated silencing of CALR inhibited the decidual stimulus-induced expression of prolactin, decidual/trophoblast prolactin-related protein, and connexin 43. In humans, the endometrial expression levels of the mRNAs encoding CALR and the implantation-related factor insulin-like growth factor binding protein (IGFBP)-7 tended to increase after polypectomy. The strongest positive correlation between expression levels before polypectomy was observed for IGFBP-7 and CALR, and the strength of this correlation increased after the surgery. Thus, endometrial CALR may play a role in the formation of decidua, and the polypectomy of infertile patients may result in the co-operative expression of endometrial factors, including CALR, that could enhance endometrial receptivity.

Endothelium-Derived Hyperpolarizing Factor (EDHF) Mediates Acetylsalicylic Acid (Aspirin) Vasodilation of Pregnant Rat Mesenteric Arteries

Acetylsalicylic acid (aspirin) exhibits a broad range of activities, including analgesic, antipyretic, and antiplatelet properties. Recent clinical studies also recommend aspirin prophylaxis in women with a high risk of pre-eclampsia, a major complication of pregnancy characterized by hypertension. We investigated the effect of aspirin on mesenteric resistance arteries and found outdiscovered the molecular mechanism underlying this action. Aspirin (10−12–10−6 M) was tested on pregnant rat mesenteric resistance arteries by a pressurized arteriography. Aspirin was investigated in the presence of several inhibitors of: (a) nitric oxide synthase (L-NAME 2 × 10−4 M); (b) cyclooxygenase (Indomethacin, 10−5 M); (c) Ca2+-activated K+ channels (Kca): small conductance (SKca, Apamin, 10−7 M), intermediate conductance (IKca, TRAM34, 10−5 M), and big conductance (BKca, paxilline, 10−5 M); and (d) endothelial-derived hyperpolarizing factor (high KCl, 80 mM). Aspirin caused a concentration-dependent vasodilation. Aspirin-vasodilation was abolished by removal of endothelium or by high KCl. Furthermore, preincubation with either apamin plus TRAM-34 or paxillin significantly attenuated aspirin vasodilation (p < 0.05). For the first time, we showed that aspirin induced endothelium-dependent vasodilation in mesenteric resistance arteries through the endothelial-derived hyperpolarizing factor (EDHF) and calcium-activated potassium channels. By activating this molecular mechanism, aspirin may lower peripheral vascular resistance and be beneficial in pregnancies complicated by hypertension.

NEP1-40-overexpressing neural stem cells enhance axon regeneration by inhibiting Nogo-A/NgR1 signaling pathway

Background: Nogo-66 antagonistic peptide (NEP1-40) offers the potential to improve spinal cord injury (SCI). Objective: To explore the effect of NEP1-40 overexpression on neural stem cells (NSCs) regulating the axon regeneration of injured neurons. Methods: We isolated NSCs from brain tissues of pregnant rat fetuses and used Nestin immunofluorescence to identify them. The NEP1-40 overexpressing NSCs were constructed by transfection with the NEP1-40-overexpressing vector. The expression of NSCs differentiation associated markers including Tuj-1, GFAP, Oligo2 and MBP, were detected by RT-PCR, western blotting and immunofluorescence. NeuN immunofluorescence staining was used to measure the number of neurons. And western blotting was used to detect the phosphorylation levels of LIMK1/2, cofilin and MLC-2 and the protein levels of GAP-43, MAP-2 and APP. Results: The NEP1-40 overexpression promoted the expression level of Tuj-1, Oligo2 and MBP, and increased the number of Tuj-1, Oligo2 and MBP positive cells. NEP1-40-overexpressing NSCs (NEP-NSCs) improved NeuN positive cells of co-culture with injured neurons. And NEP-NSCs also increased the protein levels of axon regeneration indicators (GAP-43, MAP-2) and decreased APP protein level. In addition, the phosphorylation level of LIMK1/2, cofilin and MLC-2 were markedly decreased in NEP-NSCs. Conclusion: NEP1-40 overexpression enhanced the ability of NSCs differentiation into neurons and promoted axon regeneration by inhibiting the Nogo-A/NgR1 signaling pathway. This study provides an alternative gene modified transplantation NSCs for the SCI treatment.

Neuritogenic Activities of Various Kappaphycus alvarezii Extracts in Hippocampal Neurons

The carrageenophyte Kappaphycus alvarezii seaweed is known to have neuritogenic activities. Post-harvest treatment of seaweed affects its biological activities. Five drying treatments of K. alvarezii, including oven-drying, sun-drying, freeze-drying, shade-drying, and salting followed by shade-drying, were evaluated for their effects on neuritogenic activities. We also evaluated the neuritogenic activity of different parts of K. alvarezii thalli and its carrageenan waste. Neuron cells of the 19th day pregnant rat fetuses were collected from the hippocampus by brain dissection. Neuron cells were isolated by dissociation of the hippocampal tissue. Cells were plated onto poly-DL-lysine-coated glass coverslips in 24-well plates and treated with extracts. All tested extracts were obtained from maceration using 95% ethanol. Freeze and shade-drying extracts exhibited significantly higher neuritogenic activities (p 0.05) compared to that of the vehicle control. Carrageenan waste also significantly promoted the neuritogenic activities (p 0.05) with an optimal dose at 1 µg mL-1. Old and young thalli showed insignificant differences in neuritogenic activities. The carrageenan waste extract retained neuritogenic activities. Thus, the utilization of carrageenan waste for neuritogenic material provides added value to the waste in the carrageenan industry. Freeze and shade dried K. alvarezii can be used as a neuritogenic agent to provide optimum biological activity.