Microarray assessment of the influence of the conceptus on gene expression in the mouse uterus during decidualization.

During pregnancy in several species including humans and rodents, the endometrium undergoes decidualization. This process of differentiation from endometrial to decidual tissue occurs only after the onset of implantation in mice. It can also be artificially induced causing the formation of deciduomal tissue. The purpose of this study was to compare the gene expression profile of the developing decidua in pregnant mice with the deciduoma formed after artificial induction in an effort to identify conceptus-influenced changes in uterine gene expression during decidualization. We induced decidualization artificially by transferring blastocyst-sized ConA-coated agarose beads into the uterus on day 2.5 of pseudopregnancy. Recently published work has found this model to be more 'physiological' than other methods. Total RNA was isolated from blastocyst and bead-induced 'implantation' sites of the uteri of day 7.5 pregnant (decidua) and pseudopregnant (deciduoma) mice respectively. This RNA was then used for microarray analysis using Mouse Illumina BeadArray chips. This analysis revealed potential differential mRNA levels of only 45 genes between the decidua and bead-induced deciduoma tissues. We confirmed the differential mRNA levels of 31 of these genes using quantitative RT-PCR. Finally, the level and localization of some of the mRNAs for select genes (Aldh3a1, Bcmo1, Guca2b, and Inhbb) identified by our microarray analysis were examined in more detail. This study provides the identity of a small set of genes whose expression in the uterus during decidualization may be influenced by molecular signals from the conceptus.

Maternal vitamin D intake during pregnancy increases gene expression of ILT3 and ILT4 in cord blood.

BACKGROUND: Recent studies indicate that prenatal vitamin D intake may protect against the development of atopic diseases in young children. Vitamin D has been shown to induce tolerogenic antigen-presenting cells such as dendritic cells. Whether the allergy-protective potential of prenatal vitamin D is mediated through such mechanisms is, however, unknown. OBJECTIVE: To evaluate the association between prenatal vitamin D supplementation and tolerogenic antigen-presenting cells in cord blood (CB) as determined by mRNA measurement of immunoglobulin-like transcripts (ILT)3 and ILT4. METHODS: A prospective multi-centre birth cohort was established in rural areas of five European countries. Information on maternal exposures including vitamin D intake was collected by questionnaires during pregnancy. The gene expression of ILT3 and ILT4 was analysed by real-time PCR in the CB of 927 children. Maternal vitamin D supplementation was assessed in Finland and France (n=349). RESULTS: Maternal vitamin D supplementation during pregnancy was associated with an increase in the gene expression of ILT3 (P=0.012) and ILT4 (P<0.001). This association remained significant for ILT4 (P=0.020) and showed a positive trend for the gene expression of ILT3 (P=0.059) after multivariate analysis controlling for various confounders. CONCLUSIONS: Vitamin D supplementation during pregnancy may increase the mRNA levels of ILT3 and ILT4 in CB. This finding may point towards an early induction of tolerogenic immune responses by maternal vitamin D intake.

Gene expression measurements in the context of epidemiological studies.

BACKGROUND: Gene expression measurements became an attractive tool to assess biological responses in epidemiological studies. However, collection of blood samples poses various technical problems. We used gene expression data from two epidemiological studies to evaluate differences between sampling methods, comparability of two methods for measuring RNA levels and stability of RNA samples over time. METHODS: For the PARSIFAL study, PBLC of 1155 children were collected using EDTA tubes in two countries. In the PASTURE study, tubes containing RNA-stabilizing solutions (PAXgene) Blood RNA Tubes; PreAnalytiX) were used to collect cord blood leucocytes of 982 children in five countries. Real-time PCR (conventional single tube assay and high-throughput low density arrays) was used to quantify expression of various innate immunity genes. In 77 PARSIFAL samples, gene expression was measured repeatedly during prolonged storage. RESULTS: In PARSIFAL (EDTA tubes) the median RNA yield after extraction significantly differed between the two centres (70 and 34 ng/microl). Collecting blood into an RNA-stabilizing solution markedly reduced differences in RNA yield in PASTURE (range of medians 91-107 ng/microl). The agreement [Spearman rank correlation (r)] between repeated measurements of gene expression decreased with increasing storage time [e.g., for CD14: r (first/second measurement) = 0.35; r (first/third measurement) = 0.03]. RNA levels measured with either the conventional method or low-density arrays were comparable (r > 0.9). CONCLUSION: Collecting blood samples into tubes containing an RNA-stabilizing solution increases RNA yield and reduces its variability. Long-term storage of samples may lead to RNA degradation, requiring special attention in longitudinal studies.

Soy protein influences the development of the metabolic syndrome in male obese ZDFxSHHF rats.

Previous investigations have demonstrated a marked effect of soy protein on the metabolic syndrome (MS). The purpose of this preliminary study was to identify the effects of soy-based diets on male obese ZDFxSHHF (fa/ fa-cp/?) rats. Animals were randomly assigned to one of four diets: control, casein (C); low-isoflavone (LIS) soy protein; high-isoflavone (HIS) soy protein; or casein + rosiglitazone (CR). Physiological, biochemical, and molecular parameters were determined at sacrifice. Body weight (p < 0.01) and food intake (p < 0.05) were lower in LIS-fed rodents. Rosiglitazone-treated animals had higher body weight and adiposity (p < 0.05). LIS and CR groups exhibited better glycemic control (p < 0.05), but with a limited effect in rosiglitazone-treated animals. HIS fed rats had higher glucose and triacylglyceride levels (p < 0.01), and lower plasma insulin (p < 0.01). Renal function parameters with the exception of an increase in systolic blood pressure (p < 0.05) were all suppressed in the LIS group (p < 0.01). The CR group had twofold PPARalpha and PPARgamma mRNA abundance (p < 0.01). LIS-fed animals also exhibited greater abundance of PPARgamma mRNA (p < 0.001), and nearly threefold FAS and CPT-1 mRNA levels (p < 0.05). HIS-fed rats also had higher abundance of CPT-1 mRNA, as well as a lower abundance of ACC mRNA (p < 0.05). Soy-based diets, influenced by isoflavone content and distinct from rosiglitazone, improved several metabolic parameters in obese ZDFxSHHF rats.

Soy protein influences insulin sensitivity and cardiovascular risk in male lean SHHF rats.

Previous investigations have demonstrated a marked effect of soy protein on multiple physiological parameters associated with the metabolic syndrome (MS). This preliminary study investigated the physiological effects of soy-based diets on cardiovascular risk in a unique rodent model that reflects early stages of MS. Briefly, lean male SHHF (+/cp) rats were randomly assigned to the following treatment groups: casein (control, C); low-isoflavone (LIS) soy protein isolate; high-isoflavone (HIS) soy protein isolate; or C+ 0.01 % rosiglitazone (CR). Rats were fed for thirty-six weeks. Liver weight, heart weight, total plasma cholesterol, fasting blood glucose were lower in soy-fed animals compared to control (p < 0.01). Body weight, kidney weight, alanine aminotransferase (ALT), fasting plasma insulin, and homeostasis model assessment (HOMA) score were also lower in LIS-fed rodents (p < 0.05) compared to casein treatment. All diet groups exhibited lower urine protein (p < 0.01) and small arteriole content (p < 0.05) compared to controls. LIS feed had a slightly more profound influence on body weight, liver metabolism, and insulin sensitivity. However, both soy diets exhibited marked improvements over a casein-based diet.

Sensory but not parasympathetic nerves are required for ocular vascular remodeling following chronic sympathectomy in rat.

Choroidal vascularity increases following chronic sympathetic denervation in rats. The mechanisms of this remodeling are unclear. Since both nitric oxide and substance P/CGRP have been suggested as angiogenic factors in other targets, we hypothesized that sensory or parasympathetic nerves may also participate in ocular vascular remodeling. To test this hypothesis, sympathetic denervation was accomplished by superior cervical ganglionectomy. Sensory denervation was induced by subcutaneous injections of capsaicin on postnatal days 2 and 9, and ocular parasympathetic innervation was ablated by pterygopalatine ganglion excision on postnatal day 60. Eyes were processed and sectioned for light microscopic histomorphometry. Sympathetic denervation for 6 weeks resulted in increased choroidal thickness, vascular luminal area, numbers of large venules and large arterioles, and capillaries in the outer nuclear layer. Capsaicin pretreatment prevented sympathectomy-induced increases in choroidal thickness, vascular luminal area and large venules and large arterioles, whereas pterygopalatine ganglionectomy was without effect. Both sensory and parasympathetic denervation attenuated increases in outer nuclear layer capillaries. These studies indicate that increased choroidal vascularity noted after chronic sympathectomy requires intact sensory innervation.

Presynaptic muscarinic facilitation of parasympathetic neurotransmission after sympathectomy in the rat choroid.

The effect of sympathectomy on parasympathetic regulation of ocular perfusion was investigated. Uveal blood flow through the vortex veins was measured by laser Doppler flowmetry during electrical stimulation of the superior salivatory nucleus, which activates ocular parasympathetic nerves, in adult rats with intact innervation and 2 days or 6 weeks after excision of the ipsilateral superior cervical ganglion. In all groups, parasympathetic stimulation produced comparable increases in flux, which were abolished by the selective neuronal nitric-oxide synthetase inhibitor, 1-(2-trifluoromethylphenyl) imidazole. Atropine had no effect in control and acutely sympathectomized rats but abolished the flux increase in four of six chronically sympathectomized animals, and 1-(2-trifluoromethylphenyl) imidazole eliminated the residual response. The muscarinic receptor agonist bethanechol did not affect basal flow in control or sympathectomized rats. However, bethanechol enhanced parasympathetically mediated vasodilation, but only in rats studied at 6 weeks after sympathectomy, a finding consistent with the appearance of muscarinic prejunctional facilitation of nitrergic transmission. In chronically sympathectomized rats, the M(2) and M(4) receptor antagonists methoctramine and tropicamide did not affect choroidal flow during parasympathetic activation. However, pirenzepine increased flux, implying the presence of M(1) inhibitory autoreceptors on these nerves. Parasympathetically mediated increased flux was partially blocked by the M(3) antagonist 4-diphenylacetoxy-N-methylpiperdine, and the remaining vasodilation was blocked by atropine. We conclude that parasympathetic prejunctional facilitatory M(3) and probably M(5) receptors adopt a crucial role after chronic sympathectomy in maintaining nitrergic vasodilatory ocular neurotransmission in the face of down-regulated nitric oxide transmitter mechanisms.

Regional regulation of choroidal blood flow by autonomic innervation in the rat.

Regional influences of parasympathetic and sympathetic innervation on choroidal blood flow were investigated in anesthetized rats. Parasympathetic pterygopalatine neurons were activated by electrically stimulating the superior salivatory nucleus, whereas sympathetic neurons were activated by cervical sympathetic trunk stimulation and uveal blood flow was measured by laser Doppler flowmetry. Parasympathetic stimulation increased flux in the anterior choroid and nasal vortex veins but not in the posterior choroid. Vasodilation was blocked completely by the neuronal nitric oxide synthase inhibitor 1-(2-trifluoromethylphenyl)imidazole but was unaffected by atropine. Sympathetic stimulation decreased flux in all regions, and this was blocked by prazosin. Parasympathetic stimulation did not affect vasoconstrictor responses to sympathetic stimulation in the posterior choroid but attenuated the decrease in blood flow through the anterior choroid and vortex veins via a nitrergic mechanism. We conclude that sympathetic alpha-noradrenergic vasoconstriction occurs throughout the choroid, whereas parasympathetic nitrergic vasodilation plays a selective role in modulating blood flow in anterior tissues of the eye.