Human luteinized granulosa cells secrete apoB100-containing lipoproteins
2010; Elsevier BV; Volume: 51; Issue: 8 Linguagem: Inglês
10.1194/jlr.m005181
ISSN1539-7262
AutoresThomas Gautier, Steffi Becker, Véronique Drouineaud, Franck Ménétrier, Paul Sagot, Jerzy–Roch Nofer, Sören von Otte, Laurent Lagrost, David Masson, Uwe J.F. Tietge,
Tópico(s)Ovarian function and disorders
ResumoThus 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 the ovary, as well as in corresponding plasma samples (n = 12). HDL cholesterol within FF correlated well with plasma HDL cholesterol (r = 0.80, P < 0.01), whereas VLDL cholesterol did not, indicating that VLDL in FF might originate directly from the granulosa cells producing FF. Primary human granulosa cells expressed apoB, microsomal triglyceride transfer protein, and apoE, but not the apoB-editing enzyme apobec-1. Using 3H-leucine, we show that granulosa cells secrete apoB100-containing lipoproteins and that secretion can be stimulated by adding oleate to the medium (+83%). With electron microscopy, apoB-containing lipoproteins within the secretory pathway of human granulosa cells were directly visualized. Finally, we found a positive relationship between apoB levels in FF and improved fertility parameters in a population of 27 women undergoing in vitro fertilization. This study demonstrates that human granulosa cells assemble and secrete apoB100-containing lipoproteins, thereby identifying a novel cell type equipped with these properties. These results might have important implications for female infertility phenotypes as well as for the development of drugs targeting the VLDL production pathway. 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 the ovary, as well as in corresponding plasma samples (n = 12). HDL cholesterol within FF correlated well with plasma HDL cholesterol (r = 0.80, P < 0.01), whereas VLDL cholesterol did not, indicating that VLDL in FF might originate directly from the granulosa cells producing FF. Primary human granulosa cells expressed apoB, microsomal triglyceride transfer protein, and apoE, but not the apoB-editing enzyme apobec-1. Using 3H-leucine, we show that granulosa cells secrete apoB100-containing lipoproteins and that secretion can be stimulated by adding oleate to the medium (+83%). With electron microscopy, apoB-containing lipoproteins within the secretory pathway of human granulosa cells were directly visualized. Finally, we found a positive relationship between apoB levels in FF and improved fertility parameters in a population of 27 women undergoing in vitro fertilization. This study demonstrates that human granulosa cells assemble and secrete apoB100-containing lipoproteins, thereby identifying a novel cell type equipped with these properties. These results might have important implications for female infertility phenotypes as well as for the development of drugs targeting the VLDL production pathway. The ability to assemble and secrete apolipoprotein (apo) B-containing lipoproteins has long been known for enterocytes as well as hepatocytes (1.Shelness G.S. Ledford A.S. Evolution and mechanism of apolipoprotein B-containing lipoprotein assembly.Curr. Opin. Lipidol. 2005; 16: 325-332Crossref PubMed Scopus (87) Google Scholar, 2.Blasiole D.A. Davis R.A. Attie A.D. The physiological and molecular regulation of lipoprotein assembly and secretion.Mol. Biosyst. 2007; 3: 608-619Crossref PubMed Scopus (90) Google Scholar). These cells use chylomicrons to secrete resorbed cholesterol and triglycerides (TG) in the case of enterocytes (3.Hussain M.M. Fatma S. Pan X. Iqbal J. Intestinal lipoprotein assembly.Curr. Opin. Lipidol. 2005; 16: 281-285Crossref PubMed Scopus (98) Google Scholar), and VLDL to export endogenously synthesized or internalized cholesterol and TG in the case of hepatocytes (4.Olofsson S.O. Boren J. Apolipoprotein B: a clinically important apolipoprotein which assembles atherogenic lipoproteins and promotes the development of atherosclerosis.J. Intern. Med. 2005; 258: 395-410Crossref PubMed Scopus (225) Google Scholar, 5.Goldberg I.J. Ginsberg H.N. Ins and outs modulating hepatic triglyceride and development of nonalcoholic fatty liver disease.Gastroenterology. 2006; 130: 1343-1346Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). ApoB-containing lipoproteins are also secreted by the human placenta, supposedly to transfer lipids from the maternal circulation to the fetus (6.Madsen E.M. Lindegaard M.L. Andersen C.B. Damm P. Nielsen L.B. Human placenta secretes apolipoprotein B-100-containing lipoproteins.J. Biol. Chem. 2004; 279: 55271-55276Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Recently, another cell type in the body has been recognized to be capable of producing apoB-containing lipoproteins, namely cardiomyocytes (7.Nielsen L.B. Veniant M. Boren J. Raabe M. Wong J.S. Tam C. Flynn L. Vanni-Reyes T. Gunn M.D. Goldberg I.J. et al.Genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in the heart: evidence that the heart has the capacity to synthesize and secrete lipoproteins.Circulation. 1998; 98: 13-16Crossref PubMed Scopus (114) Google Scholar). In the heart, VLDL secretion is thought to represent a means of protecting the organ against toxicity associated with TG accumulation (8.Veniant M.M. Nielsen L.B. Boren J. Young S.G. Lipoproteins containing apolipoprotein B-100 are secreted by the heart.Trends Cardiovasc. Med. 1999; 9: 103-107Crossref PubMed Scopus (14) Google Scholar, 9.Nielsen L.B. Bartels E.D. Bollano E. Overexpression of apolipoprotein B in the heart impedes cardiac triglyceride accumulation and development of cardiac dysfunction in diabetic mice.J. Biol. Chem. 2002; 277: 27014-27020Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In contrast, other peripheral cells utilize apoB-containing lipoproteins to meet their supply with TG and cholesterol. Developing oocytes within ovarian follicles grow rapidly and require supply with energy and cholesterol (10.Stouffer R.L. Xu F. Duffy D.M. Molecular control of ovulation and luteinization in the primate follicle.Front. Biosci. 2007; 12: 297-307Crossref PubMed Scopus (88) Google Scholar). Oocytes are surrounded by follicular fluid (FF) that in contrast to human plasma mainly contains HDL cholesterol, the smallest lipoprotein subclass (11.Jaspard B. Fournier N. Vieitez G. Atger V. Barbaras R. Vieu C. Manent J. Chap H. Perret B. Collet X. Structural and functional comparison of HDL from homologous human plasma and follicular fluid. A model for extravascular fluid.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1605-1613Crossref PubMed Scopus (54) Google Scholar, 12.Simpson E.R. Rochelle D.B. Carr B.R. MacDonald P.C. Plasma lipoproteins in follicular fluid of human ovaries.J. Clin. Endocrinol. Metab. 1980; 51: 1469-1471Crossref PubMed Scopus (81) Google Scholar). Although expression of the LDL receptor as well as of LDL receptor-related protein 4 has been reported for mammalian oocytes (13.Sato N. Kawamura K. Fukuda J. Honda Y. Sato T. Tanikawa H. Kodama H. Tanaka T. Expression of LDL receptor and uptake of LDL in mouse preimplantation embryos.Mol. Cell. Endocrinol. 2003; 202: 191-194Crossref PubMed Scopus (19) Google Scholar, 14.Yamaguchi Y.L. Tanaka S.S. Kasa M. Yasuda K. Tam P.P. Matsui Y. Expression of low density lipoprotein receptor-related protein 4 (Lrp4) gene in the mouse germ cells.Gene Expr. Patterns. 2006; 6: 607-612Crossref PubMed Scopus (17) Google Scholar), most research at present has been focused on HDL within FF. Granulosa cells, the major estrogen-producing cell type of the follicle, line the follicle and are shielded against the blood compartment by a basal membrane (15.Azhar S. Tsai L. Medicherla S. Chandrasekher Y. Giudice L. Reaven E. Human granulosa cells use high density lipoprotein cholesterol for steroidogenesis.J. Clin. Endocrinol. Metab. 1998; 83: 983-991Crossref PubMed Scopus (92) Google Scholar, 16.Havelock J.C. Rainey W.E. Carr B.R. Ovarian granulosa cell lines.Mol. Cell. Endocrinol. 2004; 228: 67-78Crossref PubMed Scopus (151) Google Scholar, 17.Okamura H. Katabuchi H. Ohba T. What we have learned from isolated cells from human ovary?.Mol. Cell. Endocrinol. 2003; 202: 37-45Crossref PubMed Scopus (31) Google Scholar). Whereas HDL are thought to enter the FF by diffusion (11.Jaspard B. Fournier N. Vieitez G. Atger V. Barbaras R. Vieu C. Manent J. Chap H. Perret B. Collet X. Structural and functional comparison of HDL from homologous human plasma and follicular fluid. A model for extravascular fluid.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1605-1613Crossref PubMed Scopus (54) Google Scholar), apoB-containing lipoproteins, which are considerably larger in size, are not expected to do so. However, given the presence of receptors for apoB-containing lipoproteins on oocytes, we hypothesized that granulosa cells might display an intrinsic ability to assemble and secrete apoB-containing lipoproteins. Therefore, we tested this hypothesis in the present study. Our results provide evidence that human granulosa cells represent a novel cell type that is capable of assembly and secretion of apoB-containing lipoproteins. Human FF was obtained from women who were undergoing in vitro fertilization (IVF) (Center of Sterility, CHU Dijon or Klinik fuer Frauenheilkunde und Geburtshilfe, Luebeck) and were following a follicle-stimulation regimen, including the injection of 10,000 IU of human chorionic gonadotrophin 36 h before transvaginal follicle puncture under ultrasound guidance exactly as previously described (18.von Otte S. Paletta J.R. Becker S. Konig S. Fobker M. Greb R.R. Kiesel L. Assmann G. Diedrich K. Nofer J.R. Follicular fluid high density lipoprotein-associated sphingosine 1-phosphate is a novel mediator of ovarian angiogenesis.J. Biol. Chem. 2006; 281: 5398-5405Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 19.Drouineaud V. Sagot P. Garrido C. Logette E. Deckert V. Gambert P. Jimenez C. Staels B. Lagrost L. Masson D. Inhibition of progesterone production in human luteinized granulosa cells treated with LXR agonists.Mol. Hum. Reprod. 2007; 13: 373-379Crossref PubMed Scopus (39) Google Scholar). After oocyte isolation, FFs were obtained, centrifuged for 5 min at 1,500 rpm, and stored at −80°C until analysis. Granulosa cells were then isolated and cultured in DMEM medium supplemented with 10% FBS and antibiotics as previously published (19.Drouineaud V. Sagot P. Garrido C. Logette E. Deckert V. Gambert P. Jimenez C. Staels B. Lagrost L. Masson D. Inhibition of progesterone production in human luteinized granulosa cells treated with LXR agonists.Mol. Hum. Reprod. 2007; 13: 373-379Crossref PubMed Scopus (39) Google Scholar). At the same time, a fasting peripheral venous blood sample was obtained from the patients, and plasma was stored frozen at −80°C. All patients had given informed consent, and the protocol was approved by the respective responsible local ethics committees. To assess the cholesterol distribution over the different lipoprotein subclasses, plasma as well as FF samples were subjected to fast protein liquid chromatography (FPLC) gel filtration using a superose 6 column (GE Healthcare, Uppsala, Sweden) as described (20.Tietge U.J.F. Maugeais C. Cain W. Grass D. Glick J.M. de Beer F.C. Rader D.J. Overexpression of secretory phospholipase A(2) causes rapid catabolism and altered tissue uptake of high density lipoprotein cholesteryl ester and apolipoprotein A-I.J. Biol. Chem. 2000; 275: 10077-10084Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Total cholesterol levels were enzymatically measured within individual fractions using commercially available reagents (Wako Pure Chemical Industries, Neuss, Germany). To correlate plasma and FF levels of lipoproteins, the respective values obtained for all fractions of a given subclass (VLDL, LDL, HDL) were added. Total RNA from granulosa cells, HepG2 cells (obtained from LGC Standards, Middlesex, UK) and human livers (samples from healthy donor livers intended for liver transplantation but not used due to technical reasons, provided by Dr. Matthias Bahr, Dept. of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Germany) was isolated using Trizol (Invitrogen) and quantified with a NanoDrop ND-100 UV-Vis spectrophotometer. cDNA synthesis was performed from 1 μg of total RNA using reagents from Applied Biosystems (Darmstadt, Germany). Real-time quantitative PCR was carried out on an ABI-Prism 7700 (Applied Biosystems) sequence detector with the default settings (21.Nijstad N. Wiersma H. Gautier T. van der Giet M. Maugeais C. Tietge U.J.F. Scavenger receptor BI-mediated selective uptake is required for the remodeling of high density lipoprotein by endothelial lipase.J. Biol. Chem. 2009; 284: 6093-6100Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). PCR primers and fluorogenic probes were designed with the Primer Express Software (Applied Biosystems) and synthesized by Eurogentec (Seraing, Belgium). mRNA expression levels presented were calculated relative to the average of the housekeeping gene cyclophilin and further normalized to the relative expression level determined in granulosa cells. Human primary granulosa cells cultured for 3 days were preincubated for 20 h in serum-free DMEM containing 1.5% BSA alone or 1.5% BSA + 0.4 mM oleate. Following a wash with PBS, cells were pulsed for 25 min with 3H-Leu (100 µCi/ well) in leucine-free MEM, washed three times with PBS, and chased for 1 h in serum-free medium. Then media were collected and centrifuged for 3 h at 100,000 rpm in a Beckman TLX tabletop ultracentrifuge with the density adjusted to 1.006 g/ml with KBr solution. Counts within the supernatant representing apoB-containing lipoproteins produced were measured by liquid scintillation counting. To the cells 0.1M NaOH was added, protein content was determined by the bicinchoninic acid method (Pierce, Rockford, IL), and counts were corrected for cellular protein content. For Western blots, samples were mixed with a loading buffer containing SDS and a reducing agent (Invitrogen, Carlsbad, CA) and then incubated for 10 min at 70°C. Samples were subsequently applied onto 4–12% polyacrylamide gradient gels (NuPage, Invitrogen) in a X-Cell SureLock system (Invitrogen) and then blotted to nitrocellulose membranes (Protran, Schleicher and Schuell, Dassel, Germany). The resulting blots were blocked for 1 h in 5% low-fat dried milk in PBS (100 mM, pH 7.4) containing 0.1% Tween and then washed with PBS/Tween. Human apoB was detected by incubation with an anti-apoB antibody (Boehringer Mannheim, Germany) followed by the appropriate horseradish peroxidase-coupled secondary antibody (Sigma Life Sciences, St Louis, MO). Blots were finally developed using the ECL Plus Detection System (GE Healthcare) and were analyzed with a GelDoc 2000 system (BioRad, Hercules, CA) and the QuantityOne software. VLDL purified from fasting human plasma by ultracentrifugation (d < 1.006) were used as positive control for apoB100. The composition of granulosa- derived VLDL (d < 1.006) was determined using enzymatic methods for total cholesterol (Diasys, Holzheim, Germany) and free cholesterol (Wako), TGs (Diasys), phospholipids (Wako), and bicinchoninic acid for proteins. Transmission electron microscopy was performed on primary human granulosa cells after 3 days of culture. Briefly, cells were detached from culture plates after trypsin incubation and pelleted with a short spin. The cells were then fixed for 30 min with 4% paraformaldehyde and 1.5% glutaraldehyde in 0.1 M phosphate buffer solution (pH 7.4), postfixed in 2% osmium tetroxide for 1 h, dehydrated with graded ethanol series, and finally embedded in Epon. Sections were stained with uranyl acetate and lead citrate prior to examination with a H-7500 electron microscope (Hitachi, Bron, France). FFs were obtained from follicular aspirates of 27 women (age, 25–41 years; BMI, 17–29) undergoing IVF and embryo transfer (ET) at the Center of Sterility, CHU, Dijon. Infertility was due to polycystic ovary syndrome, ovulatory dysfunction, endometriosis, tubal abnormalities, male infertility, or was unexplained. All patients gave informed consent. The stimulation protocol was standard and included downregulation with a gonadotropin-releasing hormone agonist and hyperstimulation with recombinant follicle stimulating hormone. Administration of recombinant chorionic gonadotropin (Ovitrelle, Serono, Boulogne, France) was performed when at least three follicles exceeded 17 mm in diameter. Oocytes were retrieved approximately 36 h after hCG administration by transvaginal ultrasound-guided aspiration. After retrieval, intracytoplasmic sperm injection (ICSI; n = 15) or conventional IVF (n = 12) was performed. Fertilization was assessed 16–18 h after insemination or microinjection by the presence of two pronuclei and two polar bodies. The fertilized oocytes were maintained in culture medium (Global medium, LifeGlobal, USA) and transferred to the uterus transcervically under transabdominal ultrasound guidance 48 h after oocyte retrieval. All embryos were scored on day 2 according to the Giorgetti classification system (22.Giorgetti C. Terriou P. Auquier P. Hans E. Spach J.L. Salzmann J. Roulier R. Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers.Hum. Reprod. 1995; 10: 2427-2431Crossref PubMed Scopus (347) Google Scholar). Embryo scoring (1–4 points) was determined as follows: segmented embryo, 1 point; 4-cells embryo, 1 point; absence of irregular cells, 1 point; absence (0–20%) of cytoplasmic remainders, 1 point. For each oocyte retrieval, the number of top-quality embryos was determined. An embryo was considered a top-quality embryo if there were four or five blastomeres on day 2 with <20% of fragments and the total absence of multinucleated blastomeres (23.Van Royen E. Mangelschots K. De Neubourg D. Valkenburg M. Van de Meerssche M. Ryckaert G. Eestermans W. Gerris J. Characterization of a top quality embryo, a step towards single-embryo transfer.Hum. Reprod. 1999; 14: 2345-2349Crossref PubMed Scopus (439) Google Scholar). All ETs were performed 2 days after oocyte retrieval using Frydman catheters (CCD Laboratories, Paris, France). A clinical pregnancy was defined as the observation of a gestational sac on ultrasound scanning between 6 and 7 weeks after ET. In parallel with IFV-ET, FF were analyzed for apoB content. By the day of oocyte retrieval, FF aliquots were collected and examined by an embryologist to detect and remove cumulus-oocyte complexes, centrifuged at 3,000 g for 15 min to eliminate cellular elements, and supernatants were stored at – 80°C before analysis. ApoB concentration in FF was determined by an immunoturbidimetric method (24.Riepponen P. Marniemi J. Rautaoja T. Immunoturbidimetric determination of apolipoproteins A-1 and B in serum.Scand. J. Clin. Lab. Invest. 1987; 47: 739-744Crossref PubMed Google Scholar) using a commercial kit with a goat anti- human apoB antibody (Apolipoprotein B FS kit, DiaSys, Holzheim, Germany) on a Dimension Xpand automated device (Siemens) according to the manufacturer's instructions. Briefly, samples were diluted 1:100 in a 100 mM Tris solution containing polyethylenglycol and detergent, and incubated for 5 min before reading background absorbance at 340 nm. Diluted samples were then incubated for 5 min at 37°C in the presence of the anti-apoB antibody prior to endpoint absorbance reading at 340 nm. ApoB concentrations in samples were calculated from a calibration curve obtained with the TruCal calibrator set (DiaSys). Statistical analyses were performed using SPSS (SPSS Inc., Chicago, IL). Data are presented as means ± SEM unless otherwise indicated. Student's t-test was used to compare values of two different groups and the Pearson correlation coefficient to assess possible associations between different parameters. For the studies on human infertility, values were compared by using Mann-Whitney U-test or by the chi-square test, as appropriate. Statistical significance for all comparisons was assigned at P < 0.05. FPLC analysis of lipoprotein distribution over the different subclasses revealed that 83% of the cholesterol is contained within the HDL fraction, indicating that the major cholesterol carrier within FF is HDL (Fig. 1A). However, a small but consistently present VLDL peak was discernible in all FPLC profiles performed on FF (Fig. 1A). In contrast, in plasma, 61% of the cholesterol is found within apoB-containing lipoproteins and on average only 39% within HDL (Fig. 1A). Relating plasma lipoprotein cholesterol to FF cholesterol levels, plasma contains 5.6-fold more VLDL, 15.4-fold more LDL, and 1.8-fold more HDL cholesterol compared with FF. Interestingly, HDL cholesterol levels in plasma were correlated with FF HDL cholesterol (r = 0.80, P < 0.01, Fig. 1B) consistent with diffusion being the conceivable mechanism of entry for HDL lipoproteins into FF (11.Jaspard B. Fournier N. Vieitez G. Atger V. Barbaras R. Vieu C. Manent J. Chap H. Perret B. Collet X. Structural and functional comparison of HDL from homologous human plasma and follicular fluid. A model for extravascular fluid.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1605-1613Crossref PubMed Scopus (54) Google Scholar, 25.Le Goff D. Follicular fluid lipoproteins in the mare: evaluation of HDL transfer from plasma to follicular fluid.Biochim. Biophys. Acta. 1994; 1210: 226-232Crossref PubMed Scopus (44) Google Scholar). On the other hand, VLDL cholesterol levels in plasma and FF were not correlated (r = 0.05, n.s., Fig. 1C). These data demonstrate that VLDL is present in human FF and indicate that, in contrast to HDL, FF-VLDL might not be derived directly from plasma. To further test the hypothesis that FF-VLDL are assembled and secreted on site, we first investigated if genes relevant for the production of apoB-containing lipoproteins are expressed in granulosa cells, which are in direct contact with FF and have a major impact on the composition of FF (16.Havelock J.C. Rainey W.E. Carr B.R. Ovarian granulosa cell lines.Mol. Cell. Endocrinol. 2004; 228: 67-78Crossref PubMed Scopus (151) Google Scholar, 17.Okamura H. Katabuchi H. Ohba T. What we have learned from isolated cells from human ovary?.Mol. Cell. Endocrinol. 2003; 202: 37-45Crossref PubMed Scopus (31) Google Scholar). As shown in Fig. 2, granulosa cells express apoB and microsomal triglyceride transfer protein (MTP), the two genes absolutely required for VLDL production (26.Hussain M.M. Shi J. Dreizen P. Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly.J. Lipid Res. 2003; 44: 22-32Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 27.Olofsson S.O. Asp L. Boren J. The assembly and secretion of apolipoprotein B-containing lipoproteins.Curr. Opin. Lipidol. 1999; 10: 341-346Crossref PubMed Scopus (188) Google Scholar, 28.Veniant M.M. Kim E. McCormick S. Boren J. Nielsen L.B. Raabe M. Young S.G. Insights into apolipoprotein B biology from transgenic and gene-targeted mice.J. Nutr. 1999; 129: 451S-455SCrossref PubMed Google Scholar), as well as apoE, a major modifier of VLDL secretion (29.Kuipers F. Jong M.C. Lin Y. Eck M. Havinga R. Bloks V. Verkade H.J. Hofker M.H. Moshage H. van Berkel T.J. et al.Impaired secretion of very low density lipoprotein-triglycerides by apolipoprotein E- deficient mouse hepatocytes.J. Clin. Invest. 1997; 100: 2915-2922Crossref PubMed Scopus (150) Google Scholar, 30.Maugeais C. Tietge U.J.F. Tsukamoto K. Glick J.M. Rader D.J. Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice.J. Lipid Res. 2000; 41: 1673-1679Abstract Full Text Full Text PDF PubMed Google Scholar). However, the expression level of these respective genes was by far lower in granulosa cells compared with the human hepatoma cell line HepG2 (apoB: 303-fold, MTP: 72-fold, apoE: 2.1-fold; Fig. 2) as well as human liver (apoB: 395-fold, MTP: 21-fold, apoE: 15.2-fold; Fig. 2). Expression of apobec-1 (31.Chan L. Chang B.H. Nakamuta M. Li W.H. Smith L.C. Apobec-1 and apolipoprotein B mRNA editing.Biochim. Biophys. Acta. 1997; 1345: 11-26Crossref PubMed Scopus (61) Google Scholar), the editing enzyme responsible for the generation of apoB48, was absent in granulosa cells. In addition, granulosa cells expressed 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase in comparable amounts to human liver, but less than HepG2 cells, while mRNA expression of the LDL receptor and fatty acid synthase was greatly increased in granulosa cells (Fig. 2). These results indicate that primary human granulosa cells express the relevant genes required for assembly and secretion of apoB-containing lipoproteins. Next, we explored if the expression of the relevant genes for assembly and secretion of apoB-containing lipoproteins in granulosa cells also translates into actual VLDL production. Therefore, we initially performed Western blots on cell culture supernatants of granulosa cells grown in FBS-free medium. As shown in Fig. 3A, an apoB100 band could be detected; consistent with the absent expression of apobec1, no immunoreactive apoB48 was found. Interestingly, the intensity of the apoB band appeared to be increased following incubation of the cells in the presence of 0.4 mM oleate. VLDL present in the media were subsequently isolated by ultracentrifugation and analyzed for their lipid and protein content. The relative composition (as percent of total weight) of granulosa-derived VLDL was 5.83 ± 0.86% for total cholesterol, 3.69 ± 1.59% for unesterified cholesterol, 2.80 ± 0.26% for cholesteryl esters, 73.23 ± 2.27% for TGs, 5.70 ± 0.92% for phospholipids, and 10.62 ± 1.91% for proteins. This composition pattern closely resembled native plasma VLDL but with a slightly higher TG enrichment, mainly at the expense of cholesterol content (32.Yang C.Y. Gu Z.W. Valentinova N. Pownall H.J. Lee B. Yang M. Xie Y.H. Guyton J.R. Vlasik T.N. Fruchart J.C. et al.Human very low density lipoprotein structure: interaction of the C apolipoproteins with apolipoprotein B-100.J. Lipid Res. 1993; 34: 1311-1321Abstract Full Text PDF PubMed Google Scholar). Subsequently, a pulse-chase experiment was performed using a pulse of 3H-leucine for 25 min on primary human granulosa cells preincubated either in the absence or the presence of oleate. After a chase for 1 h, supernatants were collected, and counts within apoB-containing lipoproteins produced were assessed. Using this approach, we could demonstrate that significant amounts of tracer are incorporated into apoB secreted by granulosa cells and that the addition of oleate resulted in a 83% increase of apoB protein secretion from these cells (Fig. 3B). In addition to demonstrating VLDL production by granulosa cells, we also investigated these cells by electron microscopy. Interestingly, small lipid-staining particles with a diameter ranging from 50 to 150 nm, which in terms of appearance were consistent with the presence of apoB-containing lipoproteins in the secretory pathway (33.Chao F.F. Stiers D.L. Ontko J.A. Hepatocellular triglyceride synthesis and transfer to lipid droplets and nascent very low density lipoproteins.J. Lipid Res. 1986; 27: 1174-1181Abstract Full Text PDF PubMed Google Scholar), were detected within the Golgi apparatus of granulosa cells (Fig. 4A–D). These results demonstrate that apoB-containing lipoproteins can be directly visualized by electron microscopy within granulosa cells, providing further evidence that these cells assemble and secrete VLDL. FFs from 27 women undergoing IVF were collected during oocyte retrieval and analyzed for their apoB content. ApoB was readily detectable in all patients, although the concentration in FF (mean, 14.0 ± 3.1 mg/L) was 40–50 times lower than in normolipidemic plasma [640 ± 90 mg/L, (34.Pont F. Duvillard L. Florentin E. Gambert P. Verges B. Early kinetic abnormalities of apoB-containing lipoproteins in insulin-resistant women with abdominal obesity.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1726-1732Crossref PubMed Scopus (47) Google Scholar)]. Patients were subsequently divided into two groups according to the median apoB level in FF, with values ≤ 13.6 mg/L and > 13.6 mg/L in the low apoB group and in the high apoB group, respectively (Table 1). Age distribution, body mass index, and the IVF to ICSI ratio were not statistically different between groups. When apoB stratification was applied to the whole population (n = 27), the number of grade 4 embryos was significantly higher in high apoB patients compared with low apoB patients (2.8 ± 2.2 vs. 1.6 ± 2.7, respectively; P< 0.05), and this was accompanied by a higher pregnancy rate in the high apoB group compared with the low apoB group (69.1% vs. 23.1%, respectively; P < 0.05) (Table 1, left). Because the population studied includes patients with direct ovarian disorders that might introduce confounding factors to follicular function and oocyte quality, the same stratification was performed after exclusion of patients with polycystic ovary syndrome, ovarian endometriosis, ovulatory dysfunction, or those who underwent ovarian surgery. Again, in the remaining population (n = 18), high apoB levels in FF were associated with a higher number of grade 4 embryos (3.6 ± 1.5 vs. 0.6 ± 0.9 in the low apoB group; P < 0.05), a higher number of top-quality embryos (4.0 ± 1.6 vs. 1.2 ± 1.3 in the low apoB group; P < 0.05), and also a strikingly higher clinical pregnancy rate (60.0% vs. 0.0% in the low apoB group; P < 0.05, Table 1, right). These data suggest that the concentration of apoB present in FF might predict oocyte quality for the generation of viable embryos and further succe
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