Virgin Controls Research Articles

Pregnancy-induced adaptations in the intrinsic structure of rat pelvic floor muscles

Maternal birth trauma to the pelvic floor muscles (PFMs) is a major risk factor for pelvic floor disorders. Modeling and imaging studies suggest that demands placed on PFMs during childbirth exceed their physiologic limits; however many parous women do not sustain PFM injury. Here we determine whether pregnancy induces adaptations in PFM architecture, the strongest predictor of muscle function, and/or intramuscular extracellular matrix (ECM), responsible for load bearing. To establish if parallel changes occur in muscles outside of the PFM, we also examined a hind limb muscle. Coccygeus, iliocaudalis, pubocaudalis, and tibialis anterior of 3-month-old Sprague-Dawley virgin, mid-pregnant, and late-pregnant; 6-month-old virgin; and 4- and 12-week postpartum rats (N = 10/group) were fixed in situ and harvested. Major architectural parameters determining muscle's excursion and force-generating capacity were quantified, namely, normalized fiber length (Lfn), physiologic cross-sectional area, and sarcomere length. Hydroxyproline content was used as a surrogate for intramuscular ECM quantity. Analyses were performed by 2-way analysis of variance with Tukey post hoc testing at a significance level of .05. Pregnancy induced a significant increase in Lfn in all PFMs by the end of gestation relative to virgin controls. Fibers were elongated by 37% in coccygeus (P < .0001), and by 21% in iliocaudalis and pubocaudalis (P < .0001). Importantly, no Lfn change was observed in the tibialis anterior. Physiologic cross-sectional area and sarcomere length were not affected by pregnancy. By 12 weeks' postpartum, Lfn of all PFMs returned to the prepregnancy values. Relative to virgin controls, ECM increased by 140% in coccygeus, 52% in iliocaudalis, and 75% in pubocaudalis in late-pregnant group, but remained unchanged across time in the tibialis anterior. Postpartum, ECM collagen content returned to prepregnancy levels in iliocaudalis and pubocaudalis, but continued to be significantly elevated in coccygeus (P < .0001). This study demonstrates that pregnancy induces unique adaptations in the structure of the PFMs, which adjust their architectural design by adding sarcomeres in series to increase fiber length as well as mounting a substantial synthesis of collagen in intramuscular ECM.

Pharmacological Activation of Fxr Attenuates Pregnancy‐Induced Repression of Hepatic and Ileal Bile Acid Transporters in Mice

The Farnesoid X Receptor (Fxr) controls bile acid homeostasis by coordinately regulating the expression of bile acid synthesis enzymes (Cyp7a1) and transporters in the intestine (Asbt, Ostβ) and liver (Ntcp, Oatp1a4, Bsep, Mrp3, Ostβ), as well as endocrine (Fgf15) and transcription (Shp) factors. We have demonstrated that expression of bile acid synthesis enzymes and transporters is dysregulated in pregnant mice. To determine the role of Fxr in the adaptive gene responses of pregnancy, mRNA and protein expression were quantified in livers and intestines from wild‐type and Fxr‐null mice, as well as wild‐type mice administered the Fxr agonist GW4064 (GW). In response to pregnancy, ileal Fgf15, Asbt and Ostβ mRNAs decreased by 25 to 57% in wild‐type mice. Expression of these genes was also constitutively reduced 40 to 99% in Fxr‐null mice, although unchanged by pregnancy. In the liver, pregnancy‐mediated repression of Bsep mRNA was observed in wild‐type, but not Fxr‐null mice. Similar to wild‐types, pregnancy further reduced hepatic Shp, Ntcp, Oatp1a4 and Mrp3 mRNA levels of Fxr‐null mice. Treatment of pregnant mice with GW induced hepatic Shp, Ntcp, Bsep, Mrp3 and Ostβ, and ileal Fgf15 and Ostβ mRNA levels by 20 to 300%. GW also enhanced protein levels of Ntcp and Bsep in pregnant mice. Likewise, the induction of Cyp7a1 in pregnant mice was reduced by GW to levels lower than virgin controls. Collectively, these data implicate both intestinal and hepatic Fxr signaling in pregnancy‐related procholestatic gene changes, and suggest that Fxr activation may restore bile acid homeostasis in maternal cholestasis. Supported by AFPE, R01ES020522, GM104037 and T32ES007148.

Effects of stress early in gestation on hippocampal neurogenesis and glucocorticoid receptor density in pregnant rats

Pregnancy is a time of marked neural, physiological and behavioral plasticity in the female and is often a time when women are more vulnerable to stress and stress-related diseases, such as depression and anxiety. Unfortunately the impact of stress during gestation on neurobiological processes of the mother has yet to be fully determined, particularly with regard to changes in the hippocampus; a brain area that plays an important role in stress-related diseases. The present study aimed to determine how stress early in pregnancy may affect hippocampal plasticity in the pregnant female and whether these effects differ from those in virgin females. For this purpose, adult age-matched pregnant and virgin female Sprague–Dawley rats were divided into two conditions: (1) Control and (2) Stress. Females in the stress condition were restrained during days 5–11 of gestation and at matched time-points in virgin females. All pregnant females received an injection of bromodeoxyuridine (BrdU) on day 1 of gestation and were sacrificed 21days later. The same procedure was carried out at matched time points in virgin females. Results show that for number of Ki67-immunoreactive (ir) cells and doublecortin (DCX)-ir cells, there were significant interactions between reproductive state (pregnant/virgin) and stress exposure (p=.05, p=.04, respectively) with control virgin and stressed pregnant females having more Ki67-ir cells than control pregnant females and more DCX-ir cells than stressed virgin females. Results also show that pregnant females had significantly greater glucocorticoid receptor (GR) density in the CA1, CA3 and granule cell layer compared to virgin females. In addition, there was a main effect of stress on GR density in the CA3 region, with stressed females having significantly lower GR density compared to control females (p=.01). This work adds to our understanding of how stress and reproductive state affect plasticity in the female hippocampus.

Behavioural changes in mothers and maternally sensitised female mice

The maternal motivation and depression-like behaviour of primiparous mother and maternally sensitised virgin female mice were investigated. During a 1-h test period, dams and sensitised female mice spent significantly more time in pup-associated than in control cages when they could freely choose between them, while virgin control and ovariectomised females had no such preference. In the forced swim test, the time spent in active (swimming and struggling) and passive (floating) behaviours was measured for 6 min. Mother mice spent more time engaged in active behaviours than virgin and sensitised female mice, while the latter two groups did not differ from each other in the forced swim test. The results suggest that maternal motivation is increased in postpartum mothers and maternally sensitised female mice. We also provide the first demonstration that postpartum mother mice display anti-depression-like behaviours in the forced swim test, while maternally sensitised females do not show such emotional changes.

Influenza A(H1N1) vaccination during early pregnancy transiently promotes hippocampal neurogenesis and working memory. Involvement of Th1/Th2 balance

The 2009 influenza A(H1N1) pandemic led to a particularly high risk of morbidity and mortality among pregnant women. Therefore, inactivated influenza vaccines have been widely recommended for women in any period of gestation. Recent studies have shown that the peripheral adaptive immune system plays an important role in the function of the central nervous system (CNS). The present study was conducted to explore if influenza vaccination, aiming to induce protective immune activation, affects maternal neurogenesis and cognitive ability. The results showed that A(H1N1) pregnant mice (AIV+Pre) had superior spatial working memory performance compared with pregnant controls (Pre). At the cellular level, a transient increase in both cell proliferation and neuronal differentiation in the dentate gyrus (DG) was found in the AIV+Pre group compared with the Pre group when BrdU was injected on gestational day 14 (G14). However, there were no obvious differences between A(H1N1) virgin mice (AIV+Vir) and virgin controls (Vir) in both hippocampal neurogenesis and working memory. Our findings further indicated that prolactin (PRL) concentrations were not overtly different between the AIV+Pre group and the Pre group at any time. Interestingly, IL-4 and IFN-γ levels were obviously increased both in the serum and hippocampus of the AIV+Pre group (with a T helper-1 like response; Th1) compared with the Pre group (with a T helper-2 like response; Th2) at G14, whereas the expression of IL-6 and TNF-α, the proinflammatory factors, was significantly reduced. Altogether, the results suggest that A(H1N1) vaccination during early pregnancy may contribute to adult hippocampal neurogenesis and spatial working memory and that the improvements were, at least in part, associated with Th1/Th2 balance.

Abstract 126: Pregnancy Hormones Induce An Increase Of Capillary Density In The Mouse Heart

During pregnancy the maternal organism undergoes a variety of adaptive physiological changes. The cardiovascular system is strongly affected and its response is characterized by cardiac hypertrophy. The molecular mechanisms underlying these adaptations are still poorly understood. The recent postulation of resident organotypic stem cells in the cardiovascular system prompted us to explore the potential involvement of these cells and the related signaling cascades involved in this adaptation process in the heart. We have analyzed cell proliferation using immunostainings in mouse hearts at different stages during pregnancy. The average percentage of proliferating cells in cardiac sections increased from virgin controls to gestational day 3 (GD3), peaked at GD14, and immediately stopped after delivery. Co-staining revealed that the majority of proliferating cells were fibroblasts and endothelial cells (ECs). The lack of cardiomyocyte proliferation was corroborated using BrdU pulse chase experiments analyzed in single cells upon Langendorff perfusion. The proliferation of fibroblasts and ECs reflects angiogenesis, which was underscored by an increase of capillary density, and extracellular matrix remodeling known to occur during pregnancy induced hypertrophy. To mimic the action of pregnancy hormones we implanted pellets with constant release of hormone for 21 days subcutaneously in ovariectomized mice. Capillary density was significantly increased in mice receiving progesterone-pellets alone or in combination with estrogen-pellets without induction of hypertrophy. Thus, increase of capillary density is directly induced by pregnancy hormones. Assessment of differentially expressed genes after hormone treatment revealed induction of angiogenetic factors and their receptors. We are currently investigating the mechanisms underlying the pregnancy hormone-induced proliferation in cardiac ECs and fibroblasts.

Experimental Study of Hot Recycled Asphalt Mixtures with High Percentages of Reclaimed Asphalt Pavement and Different Recycling Agents

Hot recycled mixture has been widely used as binder course on highways in the Jiangsu Province, China. The objectives of this study were to evaluate recycled mixture's performance with different Reclaimed Asphalt Pavement (RAP) contents and recycling agents (RA). At first, optimum RA contents were determined according to asphalt blending charts based on penetration index. Secondly, the performance of recycled mixtures and control virgin asphalt mixture was evaluated based on retained stability, Tensile Strength Ratio (TSR), dynamic stability (DS), failure strain, and fatigue life. Thirdly, statistical significance on the basis of analysis of variance was evaluated as a function of RAP contents and RA type. The results indicated that adding RA could improve moisture stability, low temperature cracking resistance, while decreasing high temperature stability and strength performance of recycled mixture. Recycled asphalt mixture Sup-20 with RA has similar performance compared to virgin asphalt mixture Sup-20. The former has better moisture stability and high temperature stability than that of the latter, with a slight reduction in low temperature cracking and fatigue resistance. With a fixed RA-2, TSR, and failure strain of recycled mixes decreases, and DS first slightly decreases and then increases, while fatigue life first increases and then decreases with increasing RAP contents. The results indicated that the interface status and blending level between recycled asphalt and aggregate is variable due to the mixing process and material compositions, which results in the varied performance results of recycled mixes due to the different test temperatures, loading modes, and testing periods, etc. Furthermore, the results of analysis of variance are consistent with the experimental test results.

Medial prefrontal cortex: genes linked to bipolar disorder and schizophrenia have altered expression in the highly social maternal phenotype.

The transition to motherhood involves CNS changes that modify sociability and affective state. However, these changes also put females at risk for post-partum depression and psychosis, which impairs parenting abilities and adversely affects children. Thus, changes in expression and interactions in a core subset of genes may be critical for emergence of a healthy maternal phenotype, but inappropriate changes of the same genes could put women at risk for post-partum disorders. This study evaluated microarray gene expression changes in medial prefrontal cortex (mPFC), a region implicated in both maternal behavior and psychiatric disorders. Post-partum mice were compared to virgin controls housed with females and isolated for identical durations. Using the Modular Single-set Enrichment Test (MSET), we found that the genetic landscape of maternal mPFC bears statistical similarity to gene databases associated with schizophrenia (5 of 5 sets) and bipolar disorder (BPD, 3 of 3 sets). In contrast to previous studies of maternal lateral septum (LS) and medial preoptic area (MPOA), enrichment of autism and depression-linked genes was not significant (2 of 9 sets, 0 of 4 sets). Among genes linked to multiple disorders were fatty acid binding protein 7 (Fabp7), glutamate metabotropic receptor 3 (Grm3), platelet derived growth factor, beta polypeptide (Pdgfrb), and nuclear receptor subfamily 1, group D, member 1 (Nr1d1). RT-qPCR confirmed these gene changes as well as FMS-like tyrosine kinase 1 (Flt1) and proenkephalin (Penk). Systems-level methods revealed involvement of developmental gene networks in establishing the maternal phenotype and indirectly suggested a role for numerous microRNAs and transcription factors in mediating expression changes. Together, this study suggests that a subset of genes involved in shaping the healthy maternal brain may also be dysregulated in mental health disorders and put females at risk for post-partum psychosis with aspects of schizophrenia and BPD.

Asphalt Pavements with High Reclaimed Asphalt Pavement Content

Today's sustainability-driven systems require a product or process to be environmentally beneficial as well as cost-effective. This study used life-cycle cost analysis (LCCA) and life-cycle assessment (LCA) to consider the economic and environmental feasibility of using a high content of reclaimed asphalt pavement (RAP) in asphalt mixtures. The LCCA conducted in the study incorporated agency cost as well as the costs incurred by the user in the construction work zone. Initial construction as well as future maintenance and rehabilitation activities were considered in the analysis. The hybrid LCA took into account the material, construction, and maintenance and rehabilitation phases of the pavement life cycle. The results showed savings in terms of costs and energy use and reduction in greenhouse gas emissions with an increase in RAP content. On the basis of the LCCA and LCA performed under various performance scenarios, break-even performance levels were identified for mixtures with up to 50% RAP content. Break-even performance levels underscored the importance of achieving field performance for recycled mixtures equivalent to that for control virgin mixtures.

Pregnancy‐induced remodelling and enhanced endothelium‐derived hyperpolarization‐type vasodilator activity in rat uterine radial artery: transient receptor potential vanilloid type 4 channels, caveolae and myoendothelial gap junctions

In pregnancy, the vasculature of the uterus undergoes rapid remodelling to increase blood flow and maintain perfusion to the fetus. The present study determines the distribution and density of caveolae, transient receptor potential vanilloid type 4 channels (TRPV4) and myoendothelial gap junctions, and the relative contribution of related endothelium-dependent vasodilator components in uterine radial arteries of control virgin non-pregnant and 20-day late-pregnant rats. The hypothesis examined is that specific components of endothelium-dependent vasodilator mechanisms are altered in pregnancy-related uterine radial artery remodelling. Conventional and serial section electron microscopy were used to determine the morphological characteristics of uterine radial arteries from control and pregnant rats. TRPV4 distribution and expression was examined using conventional confocal immunohistochemistry, and the contribution of endothelial TRPV4, nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-type activity determined using pressure myography with pharmacological intervention. Data show outward hypertrophic remodelling occurs in uterine radial arteries in pregnancy. Further, caveolae density in radial artery endothelium and smooth muscle from pregnant rats was significantly increased by ~94% and ~31%, respectively, compared with control, whereas caveolae density did not differ in endothelium compared with smooth muscle from control. Caveolae density was significantly higher by ~59% on the abluminal compared with the luminal surface of the endothelium in uterine radial artery of pregnant rats but did not differ at those surfaces in control. TRPV4 was present in endothelium and smooth muscle, but not associated with internal elastic lamina hole sites in radial arteries. TRPV4 fluorescence intensity was significantly increased in the endothelium and smooth muscle of radial artery of pregnant compared with control rats by ~2.6- and 5.5-fold, respectively. The TRPV4 signal was significantly higher in the endothelium compared with the smooth muscle in radial artery of both control and pregnant rats, by ~5.7- and 2.7-fold, respectively. Myoendothelial gap junction density was significantly decreased by ~37% in radial artery from pregnant compared with control rats. Pressure myography with pharmacological intervention showed that NO contributes ~80% and ~30%, and the EDH-type component ~20% and ~70% of the total endothelium-dependent vasodilator response in radial arteries of control and pregnant rats, respectively. TRPV4 plays a functional role in radial arteries, with a greater contribution in those from pregnant rats. The correlative association of increased TRPV4 and caveolae density and role of EDH-type activity in uterine radial artery of pregnant rats is suggestive of their causal relationship. The decreased myoendothelial gap junction density and lack of TRPV4 density at such sites is consistent with their having an integral, albeit complex, interactive role in uterine vascular signalling and remodelling in pregnancy.

Interleukin 6 Deficiency Modulates the Hypothalamic Expression of Energy Balance Regulating Peptides during Pregnancy in Mice

Pregnancy is associated with hyperphagia, increased adiposity and multiple neuroendocrine adaptations. Maternal adipose tissue secretes rising amounts of interleukin 6 (IL6), which acts peripherally modulating metabolic function and centrally increasing energy expenditure and reducing body fat. To explore the role of IL6 in the central mechanisms governing dam's energy homeostasis, early, mid and late pregnant (gestational days 7, 13 and 18) wild-type (WT) and Il6 knockout mice (Il6-KO) were compared with virgin controls at diestrus. Food intake, body weight and composition as well as indirect calorimetry measurements were performed in vivo. Anabolic and orexigenic peptides: neuropeptide Y (Npy) and agouti-related peptide (Agrp); and catabolic and anorectic neuropeptides: proopiomelanocortin (Pomc), corticotrophin and thyrotropin-releasing hormone (Crh and Trh) mRNA levels were determined by in situ hybridization. Real time-PCR and western-blot were used for additional tissue gene expression and protein studies. Non-pregnant Il6-KO mice were leaner than WT mice due to a decrease in fat but not in lean body mass. Pregnant Il6-KO mice had higher fat accretion despite similar body weight gain than WT controls. A decreased fat utilization in absence of Il6 might explain this effect, as shown by increased respiratory exchange ratio (RER) in virgin Il6-KO mice. Il6 mRNA levels were markedly enhanced in adipose tissue but reduced in hypothalamus of mid and late pregnant WT mice. Trh expression was also stimulated at gestational day 13 and lack of Il6 blunted this effect. Conversely, in late pregnant mice lessened hypothalamic Il6 receptor alpha (Il6ra), Pomc and Crh mRNA were observed. Il6 deficiency during this stage up-regulated Npy and Agrp expression, while restoring Pomc mRNA levels to virgin values. Together these results demonstrate that IL6/IL6Ra system modulates Npy/Agrp, Pomc and Trh expression during mouse pregnancy, supporting a role of IL6 in the central regulation of body fat in this physiological state.

Amyloid Precursor Protein Mutation Disrupts Reproductive Experience-Enhanced Normal Cognitive Development in a Mouse Model of Alzheimer's Disease

Women experience dramatic changes in hormones, mood and cognition through different periods of their reproductive lives, particularly during pregnancy and giving birth. While limited human studies of early pregnancy and motherhood showed alteration of cognitive functions in later life, researches on rodents showed a persistent improvement of learning and memory performance in females with history of giving birth compared to virgin controls. Alzheimer's disease (AD), the most common dementia in elderly, is more prevalent in women than in men. One of the risk factors is related to the sharp reduction of estrogen in aged women. It is unknown whether the history of fertility activity plays any roles in altering risk of AD in females, such as altering cognitive function. Would reproductive experience alter the risk of AD in females? If so, what might be the mechanisms of the change? In this study, we examined the effects of reproductive experience on cognitive function in an AD transgenic mouse model (APP23) and age-matched wild-type non-transgenic control mice (WT). Our data showed an age-dependent effect of reproductive experience on learning and memory activity between breeders (had one or more litters) and non-breeders (virgins). More importantly, our data, for the first time, demonstrated a genotype-dependent effect of parity on cognitive function between APP23 and WT mice. At the age of 12months, WT breeders outperform non-breeders in spatial working and reference memory while APP23 breeders performed worse in spatial learning and memory than age-matched APP23 non-breeders. These genotype- and age-dependent effects of reproductive activity on cognitions are significantly associated with changes of neuropathology of AD in the APP23 mice, expression of proteins related to synaptic plasticity and cognitive functions in the brain.

Reversal of Peripheral Nerve Injury-induced Hypersensitivity in the Postpartum Period

Physical injury, including surgery, can result in chronic pain; yet chronic pain following childbirth, including cesarean delivery in women, is rare. The mechanisms involved in this protection by pregnancy or delivery have not been explored. We examined the effect of pregnancy and delivery on hypersensitivity to mechanical stimuli of the rat hindpaw induced by peripheral nerve injury (spinal nerve ligation) and after intrathecal oxytocin, atosiban, and naloxone. Additionally, oxytocin concentration in lumbar spinal cerebrospinal fluid was determined. Spinal nerve ligation performed at mid-pregnancy resulted in similar hypersensitivity to nonpregnant controls, but hypersensitivity partially resolved beginning after delivery. Removal of pups after delivery prevented this partial resolution. Cerebrospinal fluid concentrations of oxytocin were greater in normal postpartum rats prior to weaning. To examine the effect of injury at the time of delivery rather than during pregnancy, spinal nerve ligation was performed within 24 h of delivery. This resulted in acute hypersensitivity that partially resolved over the next 2-3 weeks. Weaning of pups resulted only in a temporary return of hypersensitivity. Intrathecal oxytocin effectively reversed the hypersensitivity following separation of the pups. Postpartum resolution of hypersensitivity was transiently abolished by intrathecal injection of the oxytocin receptor antagonist, atosiban. These results suggest that the postpartum period rather than pregnancy protects against chronic hypersensitivity from peripheral nerve injury and that this protection may reflect sustained oxytocin signaling in the central nervous system during this period.

Abstract PD09-10: DNA Damage and Repair in Mammary Gland Development

Abstract Numerous factors are linked to breast cancer risk including reproductive history, early exposure to radiation, age and genetic background. Events that occur during specific stages of mammary development are believed to alter the lifetime risk of breast cancer. Epidemiological studies over the past 3 decades have suggested an association between parity and decreased lifetime breast cancer risk. Studies have also shown that breast cancer risk is significantly higher in women who received radiotherapy to the chest as adolescents compared to those who received chest radiation as adults. These associations implicate development specific changes in mammary epithelium that affect the molecular mechanisms whereby cells respond to DNA damage and, importantly, are at subsequent cancer risk. Because many of the genes linked to hereditary breast cancer are associated with DNA double-strand break (DSB) repair, we hypothesize that there are differences in overall DSB repair, homologous recombination (HR), and cell fate following damage in mammary epithelium at different stages of mammary gland development—puberty, pregnancy, lactation and post-involution. To assess these differences we are using mice containing the DR-GFP reporter targeted to chromosome 17 to study HR at chromosome breaks induced by the transient expression of I-SceI in mammary epithelial cells. DR-GFP is a widely used repair reporter in which direct repeats of two defective GFP genes are induced to recombine by I-SceI endonuclease cleavage of one of the repeats. We have observed that the efficiency of HR in primary mammary epithelial cells derived from 8-week old virgin mice is similar to that observed in other primary cells of somatic origin (approximately 0.65% of cells heterozygous for the DR-GFP reporter). Interestingly, the amount of HR is increased in mammary epithelium derived from pregnant mice compared to age-matched virgin controls (0.98 versus 0.65%). This 1.5 fold increase in HR suggests that in pregnancy, mammary epithelium is more proficient in the accurate repair of DNA DSBs. We are also assessing DNA damage signaling in different periods of mammary gland development. Preliminary analysis of histone H2AX phosphorylation following whole-body irradiation suggests more rapid kinetics of DNA damage signaling response in mammary tissue derived from pregnant as compared to age-matched virgin mice. We have recently generated DR-GFP containing mice that express I-SceI from a randomly integrated I-SceI transgene under the control of a tetracycline-inducible promoter. We are currently using these mice to assess HR in an entirely in vivo system and are in the process of breeding this reporter line with mice carrying mutations in Brca1 or Brca2. This mouse model will be used to assess HR in vivo in the mammary tissue of these mice during different stages of mammary gland development. This project will provide valuable insight into how developmental changes unique to mammary gland epithelium affect cellular responses to DNA damage. We hope to consider therapeutic manipulation of identified stage-specific differences to decrease breast cancer susceptibility. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD09-10.

Regulation of Hepatic Phase II Metabolism in Pregnant Mice

Phase II enzymes, including Ugts, Sults, and Gsts, are critical for the disposition and detoxification of endo- and xenobiotics. In this study, the mRNA and protein expression of major phase II enzymes, as well as key regulatory transcription factors, were quantified in livers of time-matched pregnant and virgin control C57BL/6 mice on gestation days (GD) 7, 11, 14, 17, and postnatal days (PND) 1, 15, and 30. Compared with virgin controls, the mRNA expression of Ugt1a1, 1a6, 1a9, 2a3, 2b1, 2b34, and 2b35 decreased 40 to 80% in pregnant dams. Protein expression of Ugt1a6 also decreased and corresponded with reduced in vitro glucuronidation of bisphenol A in S9 fractions from livers of pregnant mice. Similar to Ugts levels, Gsta1 and a4 mRNAs were reduced in pregnant dams in mid to late gestation; however no change in protein expression was observed. Conversely, Sult1a1, 2a1/2, and 3a1 mRNAs increased 100 to 500% at various time points in pregnant and lactating mice and corresponded with enhanced in vitro sulfation of acetaminophen in liver S9 fractions. Coinciding with maximal decreases in Ugts as well as increases in Sults, the expression of transcription factors CAR, PPARα, and PXR and their target genes were downregulated, whereas ERα mRNA was upregulated. Collectively, these data demonstrate altered regulation of hepatic phase II metabolism in mice during pregnancy and suggest that CAR, PPARα, PXR, and ERα signaling pathways may be candidate signaling pathways responsible for these changes.

Learning about prospective mates in male fruit flies: effects of acceptance and rejection

Neurogenetic research in fruit flies (Drosophila melanogaster) has established numerous protocols that illustrate flies' rich behavioural repertoire as well as excellent learning and memory abilities. To place that information in an evolutionary context, we followed up on earlier studies by evaluating the effect of experience with a variety of relevant female categories on males' courtship behaviour. We found, for the first time, that the experience of mating with conspecific females caused males to subsequently show lower levels of courtship towards heterospecific females compared to control males. These courtship levels, however, were higher than those of males that had experienced rejection by heterospecific females. Courtship reduction was selective: males that mated conspecifically did not subsequently show low levels of courting other conspecific females, and males that were rejected by recently mated conspecific females did not show low levels of courting heterospecific females. Interestingly, males that mated with virgin conspecifics and males that were rejected by virgin heterospecifics subsequently showed similar levels of low courtship towards recently mated conspecific females, suggesting that males find mated females unattractive after encountering virgin females. Males, however, showed higher levels of courtship towards mated females after mating with simulated mated females than after mating with control virgin females. Overall, our results indicate that adaptive use of learning by male fruit flies enhances mating success and reduces time and effort pursuing relatively less desirable or attainable females.

The inhibitory effect of dietary fructose on lactation‐induced increases in rat epithelial calcium transport is mediated by fructose‐induced reductions in vitamin D levels

Pregnancy and lactation require marked increases in consumption and transport of dietary Ca2+ to maintain Ca2+ homeostasis and bone health. This increase in epithelial Ca2+ transport is regulated by increasing levels of calcitriol whose genomic effects are mediated by the vitamin D receptor (VDR). We recently showed that fructose (F) consumption reduced intestinal Ca2+ transport. We used lactating rats as animal model to test whether the deleterious effects of excessive F intake on Ca2+ transport is regulated by calcitriol and lead to bone defects. Pregnant and virgin (control) rats were fed isocaloric 63% F or, as controls, glucose and starch diets from d 2 of gestation to end of lactation. Compared to virgins, lactating dams fed glucose or starch had higher rates of intestinal Ca2+ transport, elevated intestinal and renal expression of Ca2+ transporters, as well as increased levels of calcitriol and 1‐alpha‐hydroxylase. F consumption prevented these lactation‐induced increases, and reduced VDR binding to promoter regions of TRPV6 and CaBP9k. Bone mineral density, content and mechanical strength each decreased with lactation, and this decrease was exacerbated by F diet. In lactating rats with enhanced Ca2+ needs, excessive F intake prevents, through a calcitriol‐dependent pathway, adaptive increases in epithelial Ca2+ transport, likely compromising bone quality (NSF IOS‐1121049, Benjamin Delessert)

Expression and regulation of chemerin during rat pregnancy

BackgroundChemerin is an adipocytokine that is expressed in different fat deposits and has been shown to play an important role in adaptive and innate immunity due to its activity as a chemoattractant. Chemerin acts as a ligand for the G protein-coupled receptor chemokine - like receptor 1 (CMKLR1). Chemerin has been shown to regulate the development and metabolic function of adipocytes, liver and muscle tissue. ObjectiveThere is evidence indicating that several adipocytokines play an important role in placenta. This study aimed to investigate the regulation of chemerin in rat and human placentas throughout gestation. Design and settingChemerin was examined in rat and human placentas using immunohistochemistry. The chemerin expression pattern in the placenta and adipose tissue of female Sprague Dawley rats on days 12, 16, 19 and 21 of gestation (each of these days represents a group of 12 rats) was determined using TaqMan probe-based quantitative real-time PCR. Rat chemerin serum levels were analyzed with ELISA on days 8, 12, 16, 19 and 21 and compared to virgin controls. ResultsChemerin expression was detected in the cytoplasm of rat placental trophoblastic cells and third trimester human placental cytotrophoblast and Hofbauer’s cells. The serum chemerin levels of rats decreased significantly as gestation progressed. Furthermore, placental chemerin mRNA levels rose significantly at day 16 of gestation and decreased significantly towards the end of the gestation period. ConclusionTaken together, this data suggests that chemerin may be an important regulator of maternal-fetal metabolism and metabolic homeostasis during pregnancy.

Effect of Recycled Concrete Aggregate Properties on Mixture Proportions of Structural Concrete

This study focuses on characterizing several recycled concrete aggregate (RCA) sources, developing concrete mixture proportions that incorporate RCA as coarse aggregate, and investigating the effect of coarse aggregate properties on the main mixture proportion parameters [i.e., cement content, water demand, and water–cement (w/c) ratio]. Four aggregate types were investigated: one control virgin aggregate source and three RCAs produced from the crushing of hardened concrete. Numerous aggregate tests, including density, absorption, abrasion resistance, adhered mortar content, and crushing value, were performed. Fourteen mixture proportions were developed with the use of three mixture proportion scenarios (control, direct replacement, and strength based) and two compressive strength levels (40 and 60 MPa). The effect of RCA on compressive strength and workability was evaluated by replacement of natural coarse aggregate with RCA. Contrary to numerous studies, one of the RCA concretes (RCA-1) had compressive strengths up to 12% higher than the equivalent control mixture. Mixture proportions (water, cement, and w/c ratio) were later adjusted to ensure that the RCA concretes had compressive strength and slump values similar to the control concretes. Variations in water demand, cement content, and w/c ratio could then be directly attributed to the properties of the RCA source. RCA-1 concrete required less cement (and a higher w/c ratio) to achieve strengths and slumps similar to the control concrete. The findings and recommendations of this research will assist concrete producers, engineers, and field technicians involved in the selection of RCA sources in developing mixture proportions for structural-grade RCA concrete.

Dietary fructose inhibits lactation‐induced adaptations in rat 1,25‐(OH) 2 D 3 synthesis and calcium transport

We recently showed that excessive fructose consumption, already associated with numerous metabolic abnormalities, reduces rates of intestinal Ca(2+) transport. Using a rat lactation model with increased Ca(2+) requirements, we tested the hypothesis that mechanisms underlying these inhibitory effects of fructose involve reductions in renal synthesis of 1,25-(OH)(2)D(3). Pregnant and virgin (control) rats were fed isocaloric fructose or, as controls, glucose, and starch diets from d 2 of gestation to the end of lactation. Compared to virgins, lactating dams fed glucose or starch had higher rates of intestinal transcellular Ca(2+) transport, elevated intestinal and renal expression of Ca(2+) channels, Ca(2+)-binding proteins, and CaATPases, as well as increased levels of 25-(OH)D(3) and 1,25-(OH)(2)D(3). Fructose consumption prevented almost all of these lactation-induced increases, and reduced vitamin D receptor binding to promoter regions of Ca(2+) channels and binding proteins. Changes in 1,25-(OH)(2)D(3) level were tightly correlated with alterations in expression of 1α-hydroxylase but not with levels of parathyroid hormone and of 24-hydroxylase. Bone mineral density, content, and mechanical strength each decreased with lactation, but then fructose exacerbated these effects. When Ca(2+) requirements increase during lactation or similar physiologically challenging conditions, excessive fructose consumption may perturb Ca(2+) homeostasis because of fructose-induced reductions in synthesis of 1,25-(OH)(2)D(3).