Human luteinized granulosa cells secrete apoB100-containing lipoproteins

• DOI: 10.1194/jlr.m005181
• Publication Date: 2010-04-21T02:48:46Z
• Authors: Thomas Gautier; Steffi Becker; Véronique Drouineaud; Franck Ménétrier; Paul Sagot; Jerzy-Roch Nofer; Sören von Otte; Laurent Lagrost; David Masson; Uwe J.F. Tietge
• Keywords: Follicular fluid; Granulosa cell; Phospholipid transfer protein

Thus far, liver, intestine, heart, and placenta have been shown to secrete apolipoprotein (apo)B-containing lipoproteins. In the present study, we first investigated lipoproteins in human follicular fluid (FF), surrounding developing oocytes within ovary, as well corresponding plasma samples (n = 12). HDL cholesterol FF correlated with (r 0.80, P < 0.01), whereas VLDL did not, indicating that might originate directly from granulosa cells producing FF. Primary expressed apoB, microsomal triglyceride transfer protein, apoE, but not apoB-editing enzyme apobec-1. Using 3H-leucine, show apoB100-containing secretion can be stimulated by adding oleate medium (+83%). With electron microscopy, apoB-containing secretory pathway of were visualized. Finally, found a positive relationship between apoB levels improved fertility parameters population 27 women undergoing vitro fertilization. This study demonstrates assemble lipoproteins, thereby identifying novel cell type equipped these properties. 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Overexpression impedes cardiac dysfunction diabetic mice.J. 2002; 277: 27014-27020Abstract (116) contrast, other peripheral utilize meet their supply cholesterol. Developing ovarian follicles grow rapidly require energy (10.Stouffer R.L. Xu F. Duffy D.M. Molecular control ovulation luteinization primate follicle.Front. Biosci. 12: 297-307Crossref (88) Oocytes surrounded (FF) contrast mainly contains cholesterol, smallest subclass (11.Jaspard B. Fournier N. Vieitez G. Atger V. Barbaras R. Vieu Manent Chap H. Perret Collet Structural functional comparison homologous fluid. A model extravascular fluid.Arterioscler. Thromb. Vasc. 1997; 17: 1605-1613Crossref (54) 12.Simpson E.R. Rochelle D.B. Carr B.R. MacDonald P.C. Plasma ovaries.J. Clin. Endocrinol. Metab. 1980; 51: 1469-1471Crossref (81) Although expression LDL receptor receptor-related 4 reported mammalian (13.Sato Kawamura K. Fukuda Honda Y. Sato Tanikawa Kodama Tanaka Expression uptake mouse preimplantation embryos.Mol. Cell. 2003; 202: 191-194Crossref (19) 14.Yamaguchi Y.L. S.S. Kasa Yasuda P.P. Matsui low density (Lrp4) gene germ cells.Gene Expr. Patterns. 6: 607-612Crossref (17) most research at focused on Granulosa cells, major estrogen-producing follicle, line follicle shielded blood compartment basal membrane (15.Azhar Tsai Medicherla Chandrasekher Giudice Reaven high steroidogenesis.J. 83: 983-991Crossref (92) 16.Havelock J.C. Rainey W.E. Ovarian lines.Mol. 228: 67-78Crossref (151) 17.Okamura Katabuchi Ohba What learned isolated ovary?.Mol. 37-45Crossref (31) Whereas enter diffusion considerably larger size, expected do so. However, given presence receptors oocytes, hypothesized display an intrinsic Therefore, tested this hypothesis study. Our provide was obtained who fertilization (IVF) (Center Sterility, CHU Dijon Klinik fuer Frauenheilkunde und Geburtshilfe, Luebeck) following follicle-stimulation regimen, including injection 10,000 IU chorionic gonadotrophin 36 h before transvaginal puncture under ultrasound guidance exactly previously described (18.von Otte Paletta J.R. Becker Konig Fobker Greb R.R. Kiesel Assmann Diedrich Nofer Follicular lipoprotein-associated sphingosine 1-phosphate mediator angiogenesis.J. 281: 5398-5405Abstract (61) 19.Drouineaud Sagot Garrido Logette Deckert Gambert Jimenez Staels Lagrost Masson D. Inhibition progesterone luteinized treated LXR agonists.Mol. Hum. Reprod. 13: 373-379Crossref (39) After oocyte isolation, FFs obtained, centrifuged 5 min 1,500 rpm, stored −80°C until analysis. then cultured DMEM supplemented 10% FBS antibiotics published (19.Drouineaud At same time, fasting venous sample patients, frozen −80°C. All patients had informed consent, protocol approved respective responsible local ethics committees. To assess distribution over different subclasses, subjected fast liquid chromatography (FPLC) gel filtration using superose 6 column (GE Healthcare, Uppsala, Sweden) (20.Tietge U.J.F. Maugeais Cain W. Grass Glick J.M. de Beer F.C. Rader D.J. phospholipase A(2) causes rapid catabolism altered tissue cholesteryl ester A-I.J. 2000; 275: 10077-10084Abstract (148) Total enzymatically measured individual fractions commercially available reagents (Wako Pure Chemical Industries, Neuss, Germany). correlate values all (VLDL, LDL, HDL) added. RNA HepG2 (obtained LGC Standards, Middlesex, UK) livers (samples healthy donor intended transplantation used due technical reasons, provided Dr. Matthias Bahr, Dept. Gastroenterology, Hepatology Endocrinology, Hannover Medical School, Germany) Trizol (Invitrogen) quantified NanoDrop ND-100 UV-Vis spectrophotometer. cDNA synthesis performed 1 μg total Applied Biosystems (Darmstadt, Real-time quantitative PCR carried out ABI-Prism 7700 (Applied Biosystems) sequence detector default settings (21.Nijstad Wiersma Gautier van der Giet Tietge Scavenger BI-mediated selective required remodeling endothelial lipase.J. 2009; 284: 6093-6100Abstract (55) primers fluorogenic probes designed Primer Express Software Eurogentec (Seraing, Belgium). mRNA presented calculated relative average housekeeping cyclophilin further normalized level determined cells. primary 3 days preincubated 20 serum-free 1.5% BSA alone + 0.4 mM oleate. Following wash PBS, pulsed 25 3H-Leu (100 µCi/ well) leucine-free MEM, washed three times chased medium. Then media collected 100,000 rpm Beckman TLX tabletop ultracentrifuge adjusted 1.006 g/ml KBr solution. Counts supernatant representing produced scintillation counting. 0.1M NaOH added, content bicinchoninic acid method (Pierce, Rockford, IL), counts corrected cellular content. For Western blots, mixed loading buffer SDS reducing agent (Invitrogen, Carlsbad, CA) incubated 10 70°C. Samples subsequently applied onto 4–12% polyacrylamide gradient gels (NuPage, Invitrogen) X-Cell SureLock system blotted nitrocellulose membranes (Protran, Schleicher Schuell, Dassel, resulting blots blocked 5% low-fat dried milk PBS mM, pH 7.4) 0.1% Tween PBS/Tween. detected incubation anti-apoB antibody (Boehringer Mannheim, followed appropriate horseradish peroxidase-coupled secondary (Sigma Life Sciences, St Louis, MO). Blots finally developed ECL Plus Detection System Healthcare) analyzed GelDoc 2000 (BioRad, Hercules, QuantityOne software. purified ultracentrifugation (d 1.006) apoB100. composition granulosa- derived enzymatic methods (Diasys, Holzheim, free (Wako), TGs (Diasys), phospholipids proteins. Transmission microscopy after culture. Briefly, detached culture plates trypsin pelleted short spin. fixed 30 4% paraformaldehyde glutaraldehyde 0.1 M phosphate solution (pH 7.4), postfixed 2% osmium tetroxide h, dehydrated graded ethanol series, embedded Epon. Sections stained uranyl acetate lead citrate prior examination H-7500 microscope (Hitachi, Bron, France). aspirates (age, 25–41 years; BMI, 17–29) IVF embryo (ET) Center CHU, Dijon. Infertility polycystic ovary syndrome, ovulatory dysfunction, endometriosis, tubal abnormalities, male infertility, unexplained. gave consent. stimulation standard included downregulation gonadotropin-releasing hormone agonist hyperstimulation recombinant stimulating hormone. Administration gonadotropin (Ovitrelle, Serono, Boulogne, France) when least exceeded 17 mm diameter. retrieved approximately hCG administration ultrasound-guided aspiration. retrieval, intracytoplasmic sperm (ICSI; n 15) conventional 12) performed. Fertilization assessed 16–18 insemination microinjection two pronuclei polar bodies. fertilized maintained (Global medium, LifeGlobal, USA) transferred uterus transcervically transabdominal 48 retrieval. embryos scored day 2 according Giorgetti classification (22.Giorgetti Terriou Auquier Hans Spach J.L. Salzmann Roulier Embryo score predict implantation in-vitro fertilization: based 957 single transfers.Hum. 1995; 10: 2427-2431Crossref (347) scoring (1–4 points) follows: segmented embryo, point; 4-cells absence irregular (0–20%) cytoplasmic remainders, point. each number top-quality determined. An considered if there four five blastomeres <20% fragments multinucleated (23.Van Royen Mangelschots De Neubourg Valkenburg Van Meerssche Ryckaert Eestermans Gerris Characterization top quality step towards single-embryo transfer.Hum. 14: 2345-2349Crossref (439) ETs retrieval Frydman catheters (CCD Laboratories, Paris, clinical pregnancy defined observation gestational sac scanning 7 weeks ET. parallel IFV-ET, By aliquots examined embryologist detect remove cumulus-oocyte complexes, 3,000 g 15 eliminate elements, supernatants – 80°C ApoB concentration immunoturbidimetric (24.Riepponen Marniemi Rautaoja Immunoturbidimetric determination apolipoproteins A-1 serum.Scand. Lab. Invest. 1987; 47: 739-744Crossref Scholar) commercial kit goat anti- (Apolipoprotein FS kit, DiaSys, Dimension Xpand automated device (Siemens) manufacturer's instructions. diluted 1:100 100 Tris polyethylenglycol detergent, reading background absorbance 340 nm. Diluted 37°C endpoint concentrations calibration curve TruCal calibrator set (DiaSys). Statistical analyses SPSS (SPSS Inc., Chicago, IL). Data ± SEM unless otherwise indicated. Student's t-test compare groups Pearson correlation coefficient possible associations parameters. studies compared Mann-Whitney U-test chi-square test, appropriate. significance comparisons assigned 0.05. FPLC analysis subclasses revealed 83% contained fraction, carrier (Fig. 1A). small consistently peak discernible profiles plasma, 61% only 39% Relating levels, 5.6-fold more VLDL, 15.4-fold 1.8-fold Interestingly, 0.01, Fig. 1B) consistent being conceivable entry into 25.Le Goff mare: evaluation fluid.Biochim. Biophys. Acta. 1994; 1210: 226-232Crossref (44) On hand, 0.05, n.s., 1C). data demonstrate indicate that, HDL, FF-VLDL plasma. test assembled site, genes relevant direct contact impact (16.Havelock As 2, express (MTP), absolutely (26.Hussain Shi Dreizen Microsomal its role apoB-lipoprotein assembly.J. Lipid Res. 44: 22-32Abstract (447) 27.Olofsson Asp lipoproteins.Curr. 341-346Crossref (188) 28.Veniant Kim McCormick Insights biology transgenic gene-targeted Nutr. 129: 451S-455SCrossref modifier (29.Kuipers Jong M.C. Lin Eck Havinga Bloks Verkade H.J. Hofker M.H. Moshage Berkel T.J. al.Impaired very lipoprotein-triglycerides E- deficient hepatocytes.J. 100: 2915-2922Crossref (150) 30.Maugeais Tsukamoto Hepatic E lipoprotein-apolipoprotein vivo 41: 1673-1679Abstract far lower hepatoma (apoB: 303-fold, MTP: 72-fold, apoE: 2.1-fold; 2) 395-fold, 21-fold, 15.2-fold; 2). apobec-1 (31.Chan Chang B.H. Nakamuta Li W.H. Smith L.C. Apobec-1 editing.Biochim. 1345: 11-26Crossref editing generation apoB48, absent addition, 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase comparable amounts less than while synthase greatly increased Next, explored translates actual production. initially grown FBS-free 3A, apoB100 band could detected; apobec1, no immunoreactive apoB48 found. intensity appeared lipid (as percent weight) granulosa-derived 5.83 0.86% 3.69 1.59% unesterified 2.80 0.26% esters, 73.23 2.27% TGs, 5.70 0.92% phospholipids, 10.62 1.91% pattern closely resembled native slightly higher enrichment, expense (32.Yang C.Y. Gu Z.W. Valentinova Pownall Lee Yang Xie Y.H. Guyton Vlasik T.N. Fruchart al.Human structure: interaction C B-100.J. 1993; 34: 1311-1321Abstract Subsequently, pulse-chase experiment pulse 3H-leucine either chase collected, assessed. approach, significant tracer incorporated addition resulted increase 3B). demonstrating microscopy. lipid-staining particles diameter ranging 50 150 nm, terms appearance (33.Chao F.F. Stiers D.L. Ontko J.A. Hepatocellular droplets nascent 1986; 27: 1174-1181Abstract Golgi apparatus 4A–D). visualized providing VLDL. during readily detectable although (mean, 14.0 3.1 mg/L) 40–50 normolipidemic [640 90 mg/L, (34.Pont Duvillard Florentin Verges Early kinetic abnormalities insulin-resistant abdominal obesity.Arterioscler. 22: 1726-1732Crossref (47) Scholar)]. Patients divided median FF, ≤ 13.6 mg/L > group group, respectively (Table 1). Age distribution, mass index, ICSI ratio statistically groups. When stratification whole 27), grade significantly (2.8 2.2 vs. 1.6 2.7, respectively; P< 0.05), accompanied rate (69.1% 23.1%, 0.05) 1, left). Because studied includes disorders introduce confounding factors function quality, exclusion those underwent surgery. Again, remaining 18), (3.6 1.5 0.6 0.9 group; (4.0 1.2 1.3 strikingly (60.0% 0.0% Table right). suggest viable succe