Transfer of maternal cholesterol to embryo and fetus in pregnant mice

Cholesterol is essential for antenatal development. However, the transport of maternal cholesterol to the embryo has not been sufficiently studied, and that to the fetus is still controversial. To this end, a 1 mg dose of [3,4-(13)C(2)]cholesterol was injected daily into pregnant mice and the labeled cholesterol was measured by gas chromatography-mass spectrometry. After venous injections from days 10 to 17 of gestation, [(13)C]cholesterol levels in total ((12)C and (13)C) cholesterol were increased to 5.1% and 2.8% in maternal and fetal plasma, respectively. Labeled cholesterol was identified in the liver, kidneys, and intestines, but not in the brain, of the fetus. After injections from days 1 to 8, [(13)C]cholesterol levels were increased to 12.4% and 8.0% of total cholesterol in maternal plasma and the embryo, respectively. The level of 11.5% in the yolk sac was higher than that in the embryo. Intrauterine transfer of maternal cholesterol to the embryo as well as the fetus was evident in mice, and both the placenta and the yolk sac appear to be sites of intermediate passage in murine pregnancy.     

Expression of the endothelial lipase gene in murine embryos and reproductive organs

Endothelial lipase (EL) is a recently discovered member of the triglyceride-lipase family that is involved in plasma HDL metabolism. In this study, we investigated the putative role of EL in mouse reproduction by studying EL gene expression in mouse embryos and adult reproductive organs. PCR analysis revealed that EL mRNA is expressed in mouse embryos on embryonic day 8.5 (E8.5) to E11.5, but not later in development. In situ hybridization studies on E10.5 whole embryos and embryonic sections showed expression of EL mRNA in multiple tissues, although of varying intensity. High expression was found in the neuroepithelium of the brain and the neural tube, the mesenchymal cells between organs, the optic lens and cup, and the otocyst. In adult mice, EL mRNA expression was high in ovaries from pregnant mice but low in ovaries from nonpregnant mice. EL mRNA was also highly expressed in placenta and testes. In situ hybridization studies demonstrated intense EL mRNA staining of lutein cells in corpora lutei in ovaries, of spermatocytes in the late pachytene and diplotene stages in testes, and of principal cells in epididymis. These results suggest that EL, in addition to its effects on plasma lipoprotein metabolism, plays a role in murine reproduction.     

Matrix metalloproteinase multiplex screening identifies increased MMP-2 urine concentrations in women predicted to develop preeclampsia

Background: Preeclampsia, a pregnancy disorder characterized by hypertension and proteinuria, represents the leading cause of fetal and maternal morbidity and mortality in developing countries. The identification of novel and accurate biomarkers that are predictive of preeclampsia is necessary to improve the prognosis of patients with preeclampsia.

Objective: The objective of this study is to evaluate the usefulness of nine urinary metalloproteinases to predict the risk of preeclampsia development.

Methods: MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12 and MMP-13 were analyzed in urine (early-pregnancy) from 17 women predicted to develop preeclampsia and 48 controls using the Bio-Plex Pro-Human MMP panel (Bio-Rad, Hercules, CA).

Results: Urinary MMP-2 showed differences between groups which allowed us to calculate an increased risk for PE development of up to 20 times among the study population.

Conclusion: Increased urinary concentration of MMP-2 at 12 and 16 weeks of gestation predicted an increased risk of developing preeclampsia in the study population.     

Effects of L-carnitine on reproductive performance,milk composition,placental development and IGF concentrations in blood plasma and placental chorions in sows

Recent studies have shown that L-carnitine supplementation of sows during pregnancy and lactation enhances their reproductive performance, but the underlying mechanisms are still needed to be further confirmed. This study was conducted to investigate the function of L-carnitine on placental development, milk nutrient content and release of hormones in sows. In this experiment, 40 multiparous crossbred sows (Yorkshire × Landrace) were allotted to two groups fed diets with or without a supplemental 50 mg/kg L-carnitine. The experimental diets were fed from d 1 post-coitus until d 21 post-partum. L-carnitine-treated sow had fewer weak piglets (p < 0.05) and a greater percentage of oestrus by 5 after 5-d post-partum (p < 0.05) than control sows. The percentage fat from colostrum was greater in L-carnitine-treated sow than control sows (p < 0.05). L-carnitine-treated sows had greater plasma concentrations of triglyceride and insulin-like growth factor (IGF)-1 and lesser plasma concentrations of glucose and IGF-binding protein (IGFBP-3) on day 60 of pregnancy (p < 0.05). A clearer structure of chorions, better-developed capillaries and absence of necrosis were observed in L-carnitine-treated sows compared with control sows. The protein abundance of IGF-1 and IGF-2 in placental chorions was greater in L-carnitine-treated sows compared with control sows (p < 0.05). This study suggests that sows fed an L-carnitine supplemented diet during pregnancy improved reproductive performance through enhancement of placental development and by increasing IGF concentrations in blood plasma and placental chorions.     

Long term effects of ART: What do animals tell us?

Early stages of mammalian embryonic development are now known to be very sensitive to their microenvironment, with long term effects on fetal, postnatal, and adult health, thus extending to these early stages the concept of Developmental Origin of Health and Disease (DoHaD). In this scientific context, and with 3% of births in developed countries, safety of Assisted Reproductive Techniques procedures becomes a matter of concern. Besides, embryo technologies in domestic mammals, using huge number of embryos, do not seem to evidence heavy impacts on adult phenotypes. This paper first discusses what can or cannot be concluded from farm animal data, then develops long term effects of ART procedures (ovarian stimulation, in vitro fertilization and embryo culture) evidenced in model species (mainly mouse model). Recent literature demonstrates both individual and cumulative effects of each ART procedure on fetal and postnatal phenotypes. In a second part, because they are sources for further perturbations, immediate effects of ART on early embryo phenotypes at the cellular and molecular levels are described in both farm animals and model species. Mechanistic hypotheses supporting these ART induced phenotypic alterations are subsequently considered. Finally, taking into account interspecies differences in the mechanisms likely to be involved, the relevance of results obtained in animal models for human ART are discussed.     

牛脑皮层G_i的纯化及其与AC的偶联

用1％胆酸钠和20％饱和度的硫酸铵抽提牛脑皮层细胞膜得到含G蛋白和腺苷酸环化酶（AC）的制剂,通过Sepharose6B柱将两者分开,再将含G蛋白的级分用庚胺-Sepharose4B疏水柱、羟基磷灰石柱将其它亚型的G蛋白（主要是Gs和Go）从抑制型G蛋白（Gi）中除去,获得纯化的高活力的Gi,其GTP结合活力为17．6nmol／mg,比细胞膜Gi活力提高50倍;并具有较高的产率,从1g膜蛋白中可获得0．66mg的Gi,同时可获得无G蛋白污染的AC和少量的Gs蛋白．SDS-PAGE显示分子量为41000和36000的两条蛋白带,证实是Gi的α基和β亚基．进一步用重建脂酶体的方法检测Gi对AC的抑制作用,结果显示Gi对AC活力的抑制达40％左右,表明CAMP信息跨膜转导通路中Gi与AC之间具有较好偶联功能．     

Application of multiple-quantum line narrowing with simultaneous 1H and 13C constant-time scalar-coupling evolution in PFG-HACANH and PFG-HACA(CO)NH triple-resonance experiments

Many triple-resonance experiments make use of one-bond heteronuclear scalar couplings toestablish connectivities among backbone and/or side-chain nuclei. In medium-sized(15–30 kDa) proteins, short transverse relaxation times of C single-quantum stateslimit signal-to-noise (S/N) ratios. These relaxation properties can be improved usingheteronuclear multiple-quantum coherences (HMQCs) instead of heteronuclear single-quantumcoherences (HSQCs) in the pulse sequence design. In slowly tumbling macromolecules, theseHMQCs can exhibit significantly better transverse relaxation properties than HSQCs.However, HMQC-type experiments also exhibit resonance splittings due to multiple two- andthree-bond homo- and heteronuclear scalar couplings. We describe here a family of pulsed-field gradient (PFG) HMQC-type triple-resonance experiments using simultaneous 1H and13C constant-time (CT) periods to eliminate the t1 dependence of these scalar couplingeffects. These simultaneous CT PFG-(HA)CANH and PFG-(HA)CA(CO)NH HMQC-typeexperiments exhibit sharper resonance line widths and often have better S/N ratios than thecorresponding HSQC-type experiments. Results on proteins ranging in size from 6 to 30 kDashow average methine CH HMQC:HSQC enhancement factors of 1.10 ± 0.15, withabout 40% of the cross peaks exhibiting better S/N ratios in the simultaneous CT-HMQCversions compared with the HSQC versions.     

High-level production of uniformly 15N-and 13C-enriched fusion proteins in Escherichia coli

Summary An approach to produce ¹³C-and ¹⁵N-enriched proteins is described. The concept is based on intracellular production of the recombinant proteins in Escherichia coli as fusions to an IgG-binding domain, Z, derived from staphylococcal protein A. The production method provides yields of 40–200 mg/l of isotope-enriched fusion proteins in defined minimal media. In addition, the Z fusion partner facilitates the first purification step by IgG affinity chromatography. The production system is applied to isotope enrichment of human insulin-like growth factor II (IGF-II), bovine pancreatic trypsin inhibitor (BPTI), and Z itself. High levels of protein production are achieved in shaker flasks using totally defined minimal medium supplemented with ¹³C₆-glucose and (¹⁵NH₄)₂SO₄ as the only carbon and nitrogen sources. Growth conditions were optimized to obtain high protein production levels and high levels of isotope incorporation, while minimizing ¹³C₆-glucose usage. Incorporation levels of ¹³C and/or ¹⁵N isotopes in purified IGF-II, BPTI, and Z were confirmed using mass spectrometry and NMR spectroscopy. More than 99% of total isotope enrichment was obtained using a defined isotope-enriched minimal medium. The optimized systems provide reliable, high-level production of isotope-enriched fusion proteins. They can be used to produce 20–40 mg/l of properly folded Z and BPTI proteins. The production system of recombinant BPTI is state-of-the-art and provides the highest known yield of native refolded BPTI.Abbreviations BPTI bovine pancreatic trypsin inhibitor - DTT dithiothreitol - Gdn-HCl guanidinium hydrochloride - IAA -indole acrylic acid - IGF-II insulin-like growth factor II - PBS phosphate-buffered saline - PDMS plasma desorption mass spectrometry - PFPA pentafluoro propionic acid - RP-HPLC reversed-phase high performance liquid chromatography - Z IgG-binding protein domain derived from staphylococcal protein A.