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Unit of Reproductive Biology, Research Institute for the Biology of Farm Animals, Dummerstorf, Germany
1 Faculty of Medicine, Unit of Medical Biology, University of Rostock, Germany
(Requests for offprints should be addressed to T Viergutz; Email: viergutz{at}fbn-dummerstorf.de)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Another pathway includes oocyte–somatic cell interactions (Elvin et al. 2000). Oocytes secrete factors, such as the growth differentiation factor-9, that stimulates progesterone synthesis in both granulosa cells, which line the follicle wall, and granulosa-cumulus cells, which are closest to the oocyte. Granulosa-cumulus cells do not express LH receptors. Their preovulatory expansion and increase in progesterone production is mediated through a prostaglandin (PG) E2–PGE2 receptor pathway. This pathway requires LH surge-induced transcription of cyclooxygenase-2 (COX-2) (Wu & Wiltbank 2002) that is closely connected with COX-2 protein expression (Elvin et al. 2000, Sirois 1994, Sirois et al. 2004). The COX-2 enzyme activity is rate limiting for prostaglandin synthesis from arachidonic acid released from phospholipids by A2-phospholipases (Smith et al. 2000). The COX-2 activity has an absolute requirement for hydroperoxides including alkyl hydroperoxides (Smith et al. 1996). They are generated by strong oxidants that arise from very reactive superoxide anions with other radicals. The LH surge appears also to induce the ovulatory process via interconnected cyclooxygenase and lipoxygenase pathways (Mikuni et al. 1998). These activities can generate oxylipids, such as oxy and epoxy eicosatrienoic acids and oxidized forms of phospholipids, including platelet-activating factor (PAF)-like compounds, all present in the follicular fluid (Lopez Bernal et al. 1992, Narahara et al. 1996, Newman et al. 2004).
Together with the progesterone–progesterone receptor pathway, these activities have been implicated in an increase in follicular blood flow, vascularization, vascular permeability, degradation of the follicle wall matrix by induction of proteolytic enzymes, contraction of follicular thecal smooth muscle, oocyte maturation by superoxide anions, cumulus expansion, granulosa cell differentiation into the luteal stage, follicle rupture and capture of the cumulus–oocyte complex by the fimbria of the oviduct (Robker & Richards 1998, Tsafriri & Reich 1999, Behrman et al. 2001, Jo et al. 2002).
However, lipids undergo oxidation not only within the follicle. An uptake of circulatory lipids is likely to involve lipoproteins oxidized by tissues with high aerobic metabolism, including liver and vascular endothelium with phospholipids of low density lipoprotein (LDL) as a major target. Then, LDL and its phospholipids change their bioactivity (Heery et al. 1995, Marathe et al. 1999, Löhrke et al. 2005a). Thecal vascularization and vascular permeability increase during preovulatory development, and preovulatory follicles have been reported to contain LDL and high density lipoprotein (HDL) (Simpson et al. 1980, Volpe et al. 1991, Jaspard et al. 1997). These lipoproteins have been shown to support synthesis of progesterone by supplying cholesterol from LDL (Bao et al. 1995) but also by cholesterol-dependent and -independent actions of HDL (Azhar et al. 1998, Ragoobir et al. 2002). Follicle cells express receptors of the LDL superfamily, including very low density lipoprotein (VLDL) receptors, LDL receptors and lipoprotein receptor-related protein (LRP) receptors (Argov et al. 2004). The uptake of LDL is mediated via an endocytotic pathway (Golos et al. 1986) and the 
2-macroglobulin receptor (Chu & Pizzo 1994, Ireland et al. 2004). The uptake of lipoprotein particle remnants and HDL is mediated via the scavenger receptor, class B, type I and LRP receptors (Acton et al. 1996, Reaven et al. 1998, Argov et al. 2004). While intact LDL is known to stimulate luteal steroidogenesis (Pate & Condon 1989, Brannian et al. 1995), oxidatively modified LDL (oxLDL) prepared in a laboratory setting has been reported to inhibit luteal progesterone production (Carroll et al. 1992). Anti-steroidogenic and anti-gonadotrophic activities have been observed also for synthetic alkyl hydroperoxides (Kodaman et al. 1994). In turn, the level of circulating oxLDL increases in dairy cows with increasing yield whereas no correlation was observed with the oxidized state of VLDL (Löhrke et al. 2005a). However, the role of endogenously oxidized circulating lipids in the ovulatory process of single-ovulating species with follicular waves, such as human and cattle, is unknown. Thecal vascularization during the preovulatory stage may favour exposure to circulatory oxLDL. Moreover, there is a relative paucity of information on the local regulatory factors affecting theca function (Glister et al. 2005). Therefore, experiments were designed to investigate the thecal response of maturing bovine follicles to phospholipids from endogenous oxLDL while testing the hypothesis that such lipids interfere with essential components of the ovulatory process.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cloprostenol and depherelin were purchased from Veyx Pharma (Schwarzenborn, Germany), antibodies from Miltenyi (Bergisch-Gladbach, Germany), SYBR gold from MoBiTec (Göttingen, Germany), the lipohydroperoxide assay kit from Alexis Biochemicals (Grünberg, Germany), and the RNA extraction kit from Invitek (Berlin, Germany). Oligonucleotides were synthesised by TIB Mol (Berlin, Germany), and the iScript cDNA synthesis kit and iQ-SYBR green supermix were obtained from BIO-RAD (München, Germany). Organic solvents were from Roth (Karlsruhe, Germany), enzymes from Serva (Heidelberg, Germany), and culture plates from Greiner (Frinkenhausen, Germany). Molecular mass standards were from Roth and Fermantas (StLeon-Rot, Germany). All other chemicals and biochemicals were from Sigma (Taufkirchen, Germany).
Procedures to isolate mature follicles
Five cyclic Holstein heifers (2–2.5 years old, 528 ± 31 kg in weight) were treated on day 9 ± 1 of the oestrous cycle with a luteolytic dose of the PGF2 analogue cloprostenol (0.5 mg/2 ml), 58 h after the administration of an analogue of the gonadotrophin releasing hormone (GnRH) depherelin – Gonavet (100 µg/2 ml) to induce the LH surge. Evidence for a comparable response among Holstein cows (with a similar age and weight) was obtained by a parallel experiment (Schneider et al. 2002), indicating a circulatory LH concentration of 60 ± 15 ng/ml (n=6) which peaked 2 h post GnRH with a decline to about 45% 2 h after the peak and then to baseline values of 0.2–9.9 ng/ml. All animals developed oestrus (=day 0 of the oestrous cycle) and were slaughtered in the abattoir of the institute 18–19 h subsequent to the depherelin treatment. The ovaries were removed about 15 min after the death of the animal and transported within 10 min to the laboratory. Treatments were approved by the governmental Committee on Animal Use and Care (Mecklenburg-Vorpommern). Because of limited material, a second experiment was designed to complete determinations of the dose–response to LH and the effect of the phospholipid, platelet activation factor (PAF), on LH responses. Four follicles were used 20 x 25 (n=2) and 25 x 25 (n=2) mm in size from four ovaries obtained from cows slaughtered at an abattoir. Presence of regressing corpus luteum in the ipsilateral or contra-lateral ovary and vascularization were recorded to assess the clinical state of all ovaries.
Preparation of cells, thecal tissue and culture conditions
Follicles with at least one diameter of at least 15 mm per pair of ovaries were aspirated, the follicle fluid volume was measured, the granulosa cells spun off (60 x g, 4 °C, 10 min) after addition of EDTA (to 2 mM, 200 mM stock, pH 7.2) to minimize aggregation, and aliquots were stored (–80 °C) for analyses. The cellular sediment was resuspended in double-distilled ice-cold H2O for 10 s to lyse the erythrocytes. Lysis was terminated by the addition of NaCl (to 150 mM, 1.5 M stock autoclaved and filtered through a 0.2 µm filter). An aliquot was used to determine contamination with monocytes and neutrophils by labelling surface antigens as recommended by the manufacturer. Fluorescence of single cells was measured by flow cytometry (Coulter Elite XL) as described previously (Löhrke et al. 1998). The proportion of immunoreactive cells was <1% in all granulosal preparations. The remaining cells were collected by centrifugation (60 x g, 4 °C, 10 min) and resuspended in serum–free Ham’s F12 medium diluted (1 vol/1 vol) with HBS ( 20 mM Hepes, pH 7.4, 150 mM NaCl, 0.2 mM EDTA) containing BSA (0.1%), insulin (100 ng/ml) and L-glutamine (3.0 mM).
The cells were counted (Coulter counter), cells >12 µm in diameter were recorded, the concentration was adjusted to 0.5 x 106/ml and aliquots (100 µl) were plated on 96-well culture plates. Viability was tested by trypan blue exclusion. Cells were then treated with additives in a final volume (110 ± 1 µl) to concentrations as indicated. Cell number and integrity were also determined at the end of the culture period (2.5 h). Next, the follicle wall was excised and the thecal layers were dissected under microscopic inspection using sterile surgical tools. Separation of the thecal layers took advantage of the loose theca interna during the vascularized periovulatory stage. After peeling away the theca interna, the theca externa fragments (smooth muscle with attached thecal steroidogenic cells) were prepared without vessels as assessed by microscopy on the analogy of a described technique (Sirois 1994). Three pieces (about 1.0 x 5.0 mm in size and 20 ± 2 mg in wet weight) were plated per well of a 96-well plate using the medium described above. Each treatment for cells and thecal fragments from each follicle was accomplished in triplicate or quadruplicate when not otherwise stated. Incubations were for 2.5 h at 37 °C. Next, lucigenin was added and chemiluminescence was counted as described below. Then, tissue fragments and aliquots of the supernatant were stored frozen (–80 °C) for further analyses. Lucigenin did not interfere with steroidogenesis and transcript levels as examined by preliminary experiments comparing data with and without lucigenin additions.
Hormone assays
Progesterone was measured by a direct, single-antibody RIA using purified rabbit antibodies and [1,2,6,7-3H]progesterone as tracer. The sensitivity of the assay was 0.4 ng/ml. Intra- and interassay coefficients of variation were 10% and 15% respectively. The concentration of 17ß-oestradiol in follicle fluids was determined by a 3H-RIA after extraction of 17ß-oestradiol by ethyl-ether using purified rabbit antibody (Schneider et al. 2002). The sensitivity was 5 pg/ml, intra- and interassay coefficients of variation were 7% and 12% respectively. LH was determined in a parallel study (Schneider et al. 2002) to assess the response of Holstein heifers to the GnRH analogue used to evoke the LH surge for inducing the development of the large follicles studied here. LH determination was performed by an electrochemiluminescence immunoassay (ECLIA). The assay was based on a sandwich technique using ruthenium (II)–labelled monoclonal antibody and purified rabbit polyclonal antibody against the ß-subunit of LH (Schneider et al. 2002). Chemiluminescence triggered by an electric potential was measured by an ORIGEN 1.5 analyser (IGEN). The sensitivity of the assay was 0.03 ng LH/ml. The intra-and interassay coefficients of variation were 6.4% and 8.9% respectively.
RNA extraction
Thecal tissue was ground into powder under liquid nitrogen using a diethyl pyrocarbonate–treated, autoclaved mortar and pestle. About 10 mg powder were homogenized (Ultra Turrax homogenizer) and total RNA was extracted using an Invitek kit according to the manufacturer’s protocol. The concentration of RNA was determined by absorbance at 260 nm. The purity and integrity was assessed by the 260/280 nm ratio and electrophoresis using 2% agarose in TBE buffer (89 mM Tris, 89 mM boric acid, 1 mM EDTA, pH 8.0) and the SYBR gold stain in a final concentration as recommended by the manufacturer.
Quantification of transcripts
Fragments of mRNA encoding COX-2, P450 cholesterol side chain cleavage enzyme (P450 scc), 3ß-hydroxy-
5-steroid dehydrogenase (3ß-HSD), LDL-receptor (LDL-R), LRP-receptor (LRP-R), also called 2-macroglobulin proteinase complex receptor, and lipoprotein lipase (LPL) were obtained from reverse transcription (RT) performed by an iScript cDNA synthesis kit following the protocol of the manufacturer using 100 ng total RNA. The cDNA amount was amplified by real time PCR (iCycler, BIO-RAD) using an iQ-SYBR green supermix. One microlitre of each RT reaction (1/20 of total) was in each 10 µl PCR primed with gene-specific oligonucleotides.
The PCR was accomplished by a hot start (3 min, 94 °C; 30 s, 60 °C; 45 s, 70 °C) followed by 45 cycles (10 s, 94 °C; 30 s, 60 °C; 45 s, 70 °C with an additional 5 s each cycle) and terminated by 10 s, 94 °C; 30 s, 60 °C; 7 min, 70 °C for denaturation, annealing and elongation respectively. The primers were designed to span a corresponding intron and to anneal between 60 °C and 70 °C based on the published cDNA and gene sequences. The primer sets (sense/antisense) are shown in Table 1
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Detection of lipohydroperoxide (LHP)
We used an assay kit (Cayman, Grünberg, Germany) following the instructions of the manufacturer with some modifications based on a previous study (Mihaljevic et al. 1996) in order to meet the requirements of limited sample material. Lipids were extracted from LDL or follicle fluid (FF) by vortexing (2 min) equal volumes (routinely 200 µl) of LDL or FF and deoxygenated acidic CH3OH that contained the component R (5 mg/ml) of the kit using polypropylene tubes. Deoxygenation of CH3OH and CHCl3 was performed by the addition of bubbling nitrogen for 50 min on ice. Next, lipids were transferred into CHCl3 by vortexing with one volume deoxygenated CHCl3 (2 min). After centrifugation (3000 x g, 5 min, 0 °C), the aqueous top layer was removed and the bottom CHCl3 layer collected without the middle solid protein layer by inserting a polypropylene tip down the side of the tube. The lipid extract (CHCl3 layer) was stored on ice. Next, standards were prepared from an ethanolic solution of 50 µM 13-hydroperoxyoctadienoic acid (HPODA). A deoxy-genated mixture of 2 vol CHCl3/1 vol CH3OH was mixed with HPODA yielding final amounts of 0 (background), 0.2, 0.3, 0.4 and 0.6 nmol (total volume of 120 µl) on ice. Then, to an extract aliquot (55 µl) CHCl3/CH3OH (60 µl) was added, the chromogen prepared (1 vol 4.5 mM ferrous sulphate in 0.2 M HCl/1 vol 3% methanolic ammonium thiocyanate) and added (5 µl). Parallel reactions were prepared using triphenylphosphin (to 0.9 mM that abolished colour development using 0.6 nmol HPODA) to reduce LHP of extracts. The resulting colour was used as background absorbance. The mixtures were immediately transferred into a 96-well glass plate at room temperature and the colour recorded after 20 min at 490 nm. Absorbances of the background (determinations run in triplicate) were averaged and subtracted from all values. Standard values were plotted vs nmol HPODA. An analysis of linear regression provided the equation (routinely r2=0.99) used to calculate LHP of samples.
Superoxide anion assay
Superoxide anions (O2•–) were directly measured by an assay based on the chemilumininescence of lucigenin (bis-N-methylacridinium nitrate) as described previously (Ohara et al. 1993). Briefly, the degree of univalent reduction of oxygen (producing O2•–) in our culture conditions was determined spectrophotometrically with xanthine oxidase (0.004 U) and the reduction of ferricytochrome c in the presence of different xanthine concentrations (0.1–1 µM). An analysis of linear regression of the transient chemiluminescence signals produced in a manner dependent on the concentration of xanthine resulted in the yield of O2•– (37% of the xanthine concentration present in the reaction). This value was used to calibrate the lucigenin signal obtained by reactions of xanthine and xanthine oxidase. An analysis of linear regression provided the equation (r2=0.99) to calculate O2•– production from the lucigenin signal elicited by thecal fragments. Lucigenin (6 mM stock in HBS) was added (to 0.20 mM) into the wells with and without tissue, and transient chemiluminescence signals were immediately measured using a total period of 40 min and a Fluostar Optima microtitre plate counter (BMG Labtechnologies), a gain of 4095, an emission filter lens for lucigenin and 40 s records at 37 °C. Reactions without tissue (background control) that were intended to be substracted did not generate signals. This allowed a direct use of the data for calculating O2•– production.
Polar phospholipids from endogenous oxLDL
Specimens were from Holstein cows on postpartum days 50–55 with high (>40 kg/day) and medium (<40 kg/day) lactation associated with different lipohydroperoxide concentrations (Löhrke et al. 2004, 2005b). Blood was collected via coccygeal arterio-venipuncture, placed on ice and centrifuged after clotting (3000 x g, 15 min, 4 °C). Serum was treated with butylated hydroxytoluene (to 20 µM) and 4-(2-aminoethyl) benze-nesulphonylfluoride (to 150 µM) to inhibit artificial oxydations and deacylation of phospholipids. Serum (4 ml) was overlaid with 150 mM NaCl (2 ml) to transfer chylomicrons and very low density lipoproteins into the supernatant by ultracentrifugation (150 000 x g, 12–14 h, 10 °C). The supernatant was removed and the infranatant was diluted with 1 vol 150 mM NaCl. LDL was precipitated by addition of an aqueous dextrane sulphate-400 (to 0.015%)-MgCl2 (to 105 mM) mixture. After 3 min on ice, LDL was spun off (5000 x g, 5 min, 4 °C) and dissolved in HBS. LDL particles were protected by sucrose (to 0.43 M) and stored at –80 °C. Preparation of LDL from follicular fluid followed this protocol; however, LDL was precipitated by 0.03% dextrane sulphate-400 (MgCl2, 210 mM). HDL of the supernatant was precipitated through an increase in dextrane sulphate-400 (to 0.045%). After thawing, lipids were extracted as described under LHP detection, the organic phase evaporated by a stream of nitrogen and the residue resuspended in HBS. Undissolved material was spun off (10 000 x g, 3 min, 2 °C). The supernatant was chromatographed (after adjusting the pH to 5.2–5.3 with 1 M HCl) using an amberlite XAD-2 column (Salari 1986). The column was prepared as recommended by the manufacturer using 0.5 g of the dry resin. After loading, the column was washed with H2O (25 ml, flow rate of 1 ml/min) and eluted with CH3CN (0.5 ml/min) collecting fractions of 1 ml. Polar phospholipids in the first fraction enriched with 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (Salari 1986) were determined spectrometrically and by phosphorus analysis using a sensitive microassay (Ames & Dubin 1960). CH3CN was evaporated under a stream of nitrogen and the polar phospholipids were dissolved in HBS (to 10 pmol phosphorus/µl). The content of short-chain fatty acid residues bound at the sn-2 position of polar glycerophospholipids was measured after hydrolysis with phospholipase A2 (Tojo et al. 1988) by gas chromatography using isocaproic acid as an internal standard (to 12 mM) and PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) as a positive control. Phospholipase A2 released acetic acid from PAF (mol%, 0.04 ± 0.008) and short-chain fatty acids from polar phospholipids isolated from oxLDL with 0.5 and 1.5 nmol/ml lipohydroperoxides as follows (mol%): acetic acid (0.0042 ± 0.0005 and 0), n-butyric acid (0 and 0.003 ± 0.001), n-valeric acid (0 and 0.002 ± 0.001), iso-valeric acid (0.001 ± 0.0005 and 0.001 ± 0.0004).
Statistical analysis
Results are presented as means ± S.E.M. of data from all follicles or from individual follicles (S.E.M. then represents the variability of determinations run at least in triplicate) as indicated.
Pair-wise comparisons and comparisons vs control group (before converting data to relative values) were accomplished by one-way ANOVA and post hoc Newman-Keuls procedure providing ANOVA yielded a significant F ratio. P values <0.05 were considered statistically significant. Tests, logarithmic (lg) transformations of non-linear data sets to linearize the data and analyses of linear regression were performed using SAS statistical package and Sigma Stat of the Jandel Scientific Software. Graphic presentations were accomplished by Sigma Plot of the Jandel Scientific program package (Erkrath, Germany); SAS Institute Inc., Cary, NC, USA.
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Results |
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In response to treatment with GnRH, ovaries developed large follicles differing in size, follicle fluid volume, and lipohydroperoxides in the follicle fluid on oestrous cycle day 0 (Table 2). Data from GnRH-induced large follicles were comparable to those from naturally cyclic ovaries (data not shown). The concentration of lipohydroperoxides (LHP) in total lipids extracted from the follicle fluid varied between 0.28 and1.22 nmol/ml (Table 2). LDL and HDL isolated from the fluid of follicles with a fluid volume >1 ml contained 0.70 ± 0.06 and 0.86 ± 0.02 nmol/ml LHP. The LHP values of LDL corresponded to the LHP level of circulatory LDL (0.50 ± 0.022 nmol/ml). The concentration of LHP in circulatory HDL was 1.55 ± 0.45 nmol/ml. To ascertain the developmental stage of the follicles at the time of their isolation and whether they had responded to the GnRH analogue, follicular fluid concentrations of progesterone and 17ß-oestradiol were determined. Concentrations of progesterone and progesterone/17ß-oestradiol ratios (Table 2) varied among follicles isolated at an almost identical time (20 ± 1 and 22 ± 1 h postGnRH and after the LH surge respectively). The corpus luteum of all ovaries exhibited a strong structural regression indicated by small size, cavitation and absent vascularization. Follicular size or fluid parameters did not correspond with an ipsilateral or contralateral localization of the corpus luteum. No relationship was found between follicle size and follicle fluid characteristics (Table 2).
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The number of viable cells after preparation was 98 ± 2%. A small decrease in the cell number was observed at a low level of PAF (270 pmol/ml) at the end of the culture period. A decrease occurred at the end of the culture period at levels of PAF higher than 500 pmol/ml (540 and 750 pmol/ml, 90 ± 4 and 88 ± 5%, P>0.05 and P<0.05 vs control) as opposed to the progesterone production of granulosa cells (data not shown). Cells tended to aggregate into small clumps at the end of a culture period particularly in the presence of PAF, rendering it more difficult to determine cell number. LH used as a positive control (1 ng/ml) did not affect cell number and stimulated progesterone secretion moderately (120 ± 10% vs control) while polar phospholipids (PL) did not significantly influence steroidogenesis of granulosa cells.
Response of thecal progesterone production in vitro to oxLDL and oxLDL-derived PL
When thecal fragments were exposed to endogenously oxidized LDL containing 0.5 or 1.5 nmol/ml lipohydroperoxides, the progesterone level of the culture medium decreased significantly (Fig. 1A). A marked increase in response to forskolin which triggers the PKA pathway was observed (Fig. 1A). LDL with 0.5 nmol/ml LHP tended to inhibit the forskolin effect (Fig. 1A) but the decrease did not reach statistical significance (P=0.052). Phospholipids from mildly oxLDL (containing 0.5 nmol/ml lipohydroperoxides) tended to exert greater effects than those from moderately oxLDL (1.5 nmol/ml lipohydroperoxides; Fig. 1A).
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). A block by WEB 2086 of receptors mediating activity of PL with a structure similar to PAF, tended to mitigate the PL effect on the progesterone level. However, progesterone level differed significantly from the control (Fig. 1B). Thecal progesterone production in response to PAF
Hydrolysis of the polar phospholipids with phospholipase A2 indicated the presence of short-chain fatty acid derivatives similar to other studies (Heery et al. 1995, Lehr et al. 1997, Marathe et al. 1999). Such phospholipids may possess PAF-like features. Therefore, thecal fragments were exposed to varying amounts of PAF (Fig. 2A) to test whether oxLDL-derived polar phospholipids mimic PAF effects. These treatments decreased thecal progesterone synthesis (Fig. 2A). The inhibitory impact was lost when WEB 2086 was administered (Fig. 2A). A possible action between PAF and LH was tested at putative physiologic levels (circulatory LH concentrations fluctuated between 0.2 and 9.0 ng/ml plasma apart from the values of the LH surge with a peak roughly 10-times the basal level). Interference of PAF with the LH-induced response was detected (Fig. 2B) acting in a dose-dependent manner (Fig. 2B).
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The effects of polar phospholipids from endogenously oxidized LDL on thecal progesterone synthesis may involve responses at the level of mRNA encoding steroidogenic enzymes. To test this hypothesis, thecal tissue was incubated with PAF (Fig. 3A,B), oxLDL-derived phospholipids and LH (Fig. 3B). The transcripts encoding the P450 scc and 3ß-HSD that direct the P450 scc product, pregnenolone, towards progesterone synthesis were detected in the thecal fragments of all follicles (Fig. 3). The P450 scc mRNA concentration was found to be 1.6-times the 3ß-HSD mRNA level. A dose-dependent response of thecal mRNA accumulation to PAF was observed with negative maximal effects of PAF at a level of 270 pmol/ml (Fig. 3A). A PAF receptor block reversed the negative regulation induced by PAF (Fig. 3A). The thecal response to LH was small (Fig. 3B) while dose-dependent effects of PAF in the presence of LH on mRNA levels were detectable (Fig. 3B). A significant decrease in P450 scc and 3ß-HSD mRNA levels was induced by 270 pmol/ml PAF in the presence of LH (Fig. 3B). In contrast, PAF at higher levels induced an increase in thecal 3ß-HSD and P450 scc mRNA concentrations (Fig. 3B). Forskolin elevated the thecal mRNA accumulation of 3ß-HSD and P450 scc about 6- and 3-times the basal values but PL or PAF did not significantly change the forskolin effect (data not shown). Phospholipids isolated from oxLDL containing 0.5 nmol/ml lipohydroperoxides decreased mRNA abundance (Fig. 3B). Thecal expression of mRNA encoding some common types of receptors mediating uptake of lipoproteins including oxLDL was tested as a prerequisite for specific lipid effects on intracellular activities. The levels of these transcripts (Table 3) exceeded about 10-fold the basal level of mRNA encoding steroidogenic enzymes but were 3- to 6-times lower than those of the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 3). Higher concentrations were found in the preovulatory theca interna than in the theca externa (Table 3), in contrast to the GAPDH values (Table 3).
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COX-2 transcripts were assayed as an indicator of the preovulatory stage which is associated with elevated production of oxylipids involved in remodelling the follicular wall. The thecal mRNA level of individual follicles varied between 0.01 and 11 pg/µg total RNA (the detection limit of the quantitative RT-PCR was about 5 fg mRNA). These variations were not related to the follicle size or follicle fluid characters shown in Table 2. When thecal tissue was exposed to endogenously oxLDL with 0.5 nmol/ml lipohydroperoxides (0.5 LHPLDL, Fig. 4A) or PAF, the COX-2 mRNA decreased significantly (Fig. 4A). The decrease was expressed relative to the control treated with the solvent of the phospholipids because of the strong individual basal fluctuations observed also for P450 scc and 3ß-HSD mRNA (data not shown). Endogenously oxLDL with 1.5 nmol/ml lipohydroperoxides (1.5 LHPLDL, Fig. 4A) only tended to exert an effect on relative thecal COX-2 transcript levels (Fig. 4A). To test whether the decrease in thecal COX-2 mRNA levels in vitro has relevance to follicle character in vivo, forskolin-mediated COX-2 mRNA responses (modelling the LH surge in vivo) were plotted compared with the corresponding progesterone/17ß-oestradiol ratios found in the follicular fluid (Fig. 4B). An almost linear function was obtained after log transforming the data, with a significantly negative slope and significant coefficient of determination (Fig. 4B), indicating that COX-2 mRNA levels responded to forskolin after incubation for 2.5 h in a manner dependent on the ratio between both steroids in the follicle fluid (Fig. 4B). The effect of phospholipids on this response was negligible (data not shown). The function occurred because forskolin increased the COX-2 mRNA levels 212.5-times the basal level at a low progesterone/17ß-oestradiol ratio of the follicle fluid, but the drug almost failed to induce COX-2 transcripts at higher progesterone to 17ß-oestradiol concentrations (Fig. 4B). In contrast to this relationship, basal COX-2 mRNA levels of thecal tissue (cultured without LH or forskolin) correlated with neither thecal steroidogenesis in vitro nor with concentrations of progesterone, 17ß-oestradiol and LHP in the follicle fluid (data not shown).
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As PAF and oxLDL (0.5 LHPLDL, Fig. 4A) induce a significant change in thecal COX-2 mRNA levels, processes related to COX-2 enzyme activity would be expected to change. The activity of the cyclooxygenase enzyme expressed by COX-2 mRNA has an absolute requirement for hydroperoxides such as alkyl peroxides. Such peroxides are precursors for production of reduced oxygen required to form cyclic endoperoxides, converting arachidonic acid into PGG2/PGH2. Reduced oxygen may also contribute to intrafollicular cell–cell communication during preovulatory remodelling of the follicular wall. We measured thecal generation of superoxide as an important reduced oxygen species. The thecal production of superoxide anions was found to correlate linearly with the COX-2 mRNA (Fig. 5A) using logarithmically transformed COX-2 mRNA concentrations (Fig. 5A). PAF and 0.5 LHPLDL (Fig. 4A) inhibited COX-2 mRNA abundance and PAF and oxLDL-derived phospholipid (PL, Fig. 5B) thecal superoxide production, and in this way probably generated the correlation shown (Fig. 5A). WEB 2086 did not completely reverse the effects of PAF (Fig. 5B) but the results no longer differed from the basal value (carrier-treated control, Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Various pathways regulate progesterone synthesis, including the supply of substrate mediated by several types of lipoprotein receptors and intracellular transporters (Ragoobir et al. 2002, Argov et al. 2004), and cAMP/PKA- and PGE2-signalling (Elvin et al. 2000). We observed a marked preovulatory thecal expression of transcripts encoding lipoprotein receptors. In agreement with previous studies on other ovarian cells or follicle stages (Brannian et al. 1995, Ragoobir et al. 2002, Argov et al. 2004), these observations allow us to conclude that circulating lipoproteins appear to be an important source for preovulatory steroidogenesis. In our study, the cAMP/PKA pathway was experimentally induced by LH or the PKA-activator, forskolin, to obtain a positive control. A significant increase in mRNA encoding 3ß-HSD and P450 scc together with the progesterone level was already evident after 2.5 h incubation with forskolin, indicating cAMP signalling was primed in vivo by the LH surge. Phospholipids failed to impair the forskolin-triggered increase in progesterone synthesis as opposed to LH-stimulated progesterone production, suggesting that postreceptor cAMP signalling is little affected by these lipids. Long-term regulation of mammalian steroid hormone synthesis occurs principally by transcriptional regulation of the gene for the rate-limiting cholesterol side-chain cleavage enzyme P450 scc (Moore et al. 1990). Its expression is positively regulated via the cAMP–PKA pathway; negative regulation includes elevation of the intracellular free Ca2+ level (Moore et al. 1990). Our culture conditions avoided increases in Ca2+ levels by the presence of a low final concentration of EDTA (0.1 mM), thereby allowing, perhaps, thecal cells to maintain higher concentration of P450 scc mRNA relative to the 3ß-HSD mRNA level.
Ovarian regulation of bovine 3ß-HSD expression is not clear (Bao et al. 1997). Differentiation of bovine dominant follicles into the preovulatory stage has been reported to be accompanied by an increase in thecal 3ß-HSD mRNA (Tian et al. 1995) but also by a decrease immediately after the LH surge (Voss & Fortune 1993). Progesterone has been reported to decrease and LH, follicle-stimulating hormone and 17ß-oestradiol to increase human 3ß-HSD activity (Sasano et al. 1990). We found that a significant decrease in progesterone in response to PAF corresponded to a significant decline in thecal 3ß-HSD and P450 scc mRNA. However, negative effects were highest when the preovulatory theca fragments were exposed to 270 pmol/ml PAF. Higher PAF concentrations (>540 nmol/ml) did not significantly decrease P450 scc mRNA. A PAF receptor block fully abolished the negative impact, indicative of specific responses. Dose-dependent effects were also detected when the preovulatory theca was exposed to PAF in the presence of LH, indicating that PAF interferes with thecal LH signalling depending on the concentration of PAF. A comparison with progesterone responses reveals that these responses of mRNA encoding steroidogenic enzymes do not always resemble those of progesterone. The dose-dependent responses of mRNA-encoding steroidogenic enzymes to PAF or oxLDL-derived PL in the absence and presence of LH may be explained by the effects of these phospholipids on a pathway additional to cAMP/PKA signalling. Recently, COX-2 mRNA expression has been reported to be regulated by a switch from cAMP/PKA to PKC dependence during luteinization of preovulatory bovine granulosa cells cultured with forskolin (Wu & Wiltbank 2002). Further experiments are required to elucidate whether thecal transcripts encoding COX-2 and steroidogenic enzymes are regulated in this manner.
Basal thecal progesterone synthesis differed among individual follicles. For that reason the effect of phospholipids on progesterone production of cultured thecal tissue was expressed relative to the basal rate. A significant inhibitory impact was found for polar phospholipids isolated from oxLDL containing 0.5 nmol/ml and 1.5 nmol/ml lipohydroperoxides, but the latter tended to exert a weaker effect. Cell lysis cannot be fully excluded as a source of progesterone released into the culture medium. Such an effect may contribute to a loss of significant difference compared with basal progesterone levels with increasing PL concentration. In turn, phospholipids have also been reported to assist the complex intracellular mechanism splitting off the side chain of cholesterol by P450 scc (Solano et al. 1984), providing, in this manner, more precursors for progesterone synthesis (but also byproducts, such as 4-methyl-pental and NADP+ through oxidation of NADPH, consequently inducing possible changes in the intracellular redox state and in the activity of redox-sensitive transcription factors such as nuclear factor kappa B (NF
B)). Stronger responses to mildly compared with moderately oxidized LDL have recently been reported using cell death analysis of macrophages and of smooth muscle cells. The differences were explained by divergent content of degradation products of oxidized lipids (Carpenter 2003). The amberlite XAD-2 column used to isolate polar phospholipids does not retain such products after thorough washing. However, the first fraction of the eluation with acetonitril contains polar phospholipids, mainly acyl and alkyl phosphatidylcholines (Salari 1986) generated by oxidation of phosphatidylcholines that induces a plethora of chemically related phosphatidylcholines (Marathe et al. 1999). In agreement with previous reports (Lehr et al. 1997, Marathe et al. 1999), this was indicated by the release of short-chain fatty acids by phospholipase A2 hydrolysis of polar phospholipids isolated from endogenously oxidized LDL.
In contrast to phosphatidylethanolamines, fragmented alkyl phosphatidylcholines have PAF-like bioactivities (Heery et al. 1995, Marathe et al. 1999). Therefore, PAF was tested to see whether it exerts effects similar to isolated polar phospholipids. Our results indicate that the thecal responses to polar phospholipids from oxLDL with 0.5 nmol/ml lipohydroperoxides were mimicked by PAF, suggesting PAF-like phospholipids may be responsible for the inhibitory effects. Additionally, these data suggest that dilution of isolated polar phospholipids with related, but less active, diacyl homologues is unlikely to induce the effects observed. Inhibitory activity may occur at the intracellular level, since synthetic oxylipids have been reported to interrupt transmitochondrial cholesterol transport (Kodaman et al. 1994). In addition, a block of PAF receptors by an antagonist, WEB 2086 (Lehr et al. 1997), did not always fully reverse the inhibitory actions.
A preovulatory LH surge not only increases progesterone production that triggers expression of thecal proteolytic enzymes (Robker et al. 2000) capable of degrading the perifollicular matrix but also induces the production of local mediators stimulating follicular blood flow, vascular permeability and follicular volume (Tsafriri & Reich 1999). These mediators include eicosanoids and oxygen radicals that are also required for the resumption of meiosis (Behrman et al. 2001). Both lipoxygenase and cyclooxygenase pathways seem to play a role in the ovulatory process and are expressed in response to the preovulatory LH surge (Mikuni et al. 1998). Little information exists about bovine thecal expression of COX-2 mRNA; therefore we focused on COX-2 mRNA expression reported to be a marker of the preovulatory follicle (Sirois et al. 2004). Owing to suicidal COX-2 inactivation (Kulmacz et al. 1994), mRNA levels are closely related to COX-2 enzyme activity. This activity requires hydroperoxides and generates oxygen radicals (Smith et al. 1996) that are necessary for the conversion of arachidonic acid and O2 to PGH2, the committed step in prostanoid biosynthesis. We observed an interference of PAF and oxLDL with thecal COX-2 mRNA levels, superoxide generation and LH-induced progesterone production. Some evidence for the relevance of this in vivo was obtained by the significant relationship between these findings in vitro and the data from analysis of the follicle fluid representing the follicle state in vivo.
Such a significant correlation between the progesterone/17ß-oestradiol ratio of the follicle fluid and the relative thecal COX-2 mRNA levels was detected only after a thecal response to forskolin that might model a preovulatory LH surge. The drug strongly stimulated the COX-2 abundance up to >200 times the basal value in a low progesterone/17ß-oestradiol ratio of the follicle fluid, indicating an early, transient period of the preovulatory stage. The result is consistent with bovine data indicating acute regulation of COX-2 mRNA by cAMP signalling about 18 h after the LH surge (Sirois 1994). The thecal basal COX-2 mRNA levels varied between individual follicles but corresponded to reported bovine values (Tsai et al. 1996). Notably, basal concentrations were related neither to the follicle size nor to the progesterone/17ß-oestradiol ratio of the follicle fluid. These observations suggest individual differences in follicle development because COX-2 mRNA elevation occurs only during a narrow periovulatory window (Ando et al. 1998, Wu & Wiltbank 2002, Sirois et al. 2004). Thus, the analysis of follicles with a divergent developmental stage allowed us to elicit a forskolin-induced correlation between COX-2 mRNA expression and the progesterone/17ß-oestradiol ratio of the follicle fluid, i.e. the microenvironment within the follicle in vivo, a result consistent with previous data (Sirois 1994, Tsai et al. 1996).
Isolated phospholipids and PAF decreased basal thecal COX-2 abundance whereas forskolin-evoked responses were not changed, suggesting that phospholipids did not exert significant effects on forskolin-induced signalling towards COX-2 mRNA transcription. These data resemble those of the progesterone response. Since prostanoid metabolism has been reported as a source of superoxide anions in other tissues (Kontos et al. 1985, Ohara et al. 1993), the basal COX-2 mRNA level was plotted against thecal superoxide production. This plot resulted in a significant slope, however, with an apparent intercept (because of log transformation of the data) of roughly 2 pmol O2•–/min. This indicates that the portion of COX-2 activity that may account for about 30% of the total thecal superoxide production arises from numerous sources such as the mitochondrial electron transport chain (Loschen et al. 1974) and cytochrome P450 enzymes (Sligar et al. 1974). The relationship may be part of the interaction, since superoxide stimulates the transcription factor NFB involved in the stimulation of the COX-2 mRNA transcription (Allen & Tresini 2000). Polar phospholipids decreased thecal superoxide production to a level corresponding to the apparent intercept of the linearized regression between basal COX-2 mRNA concentration and thecal superoxide production. The result suggests that phospholipids may inhibit COX-2 sources of superoxide. However, more specific experiments are required to clarify the mechanism, since forskolin-induced COX-2 mRNA expression did not parallel superoxide production linearly.
The follicle fluid of all large oestrous cycle day 0 follicles contained between 0.28 and 1.22 nmol/ml lipohydroperoxides. The LDL-associated LHP concentration was found to correspond to these values, suggesting that the concentration used to elicit these effects was physiologic. Apparently, no information about lipohydroperoxide concentration in bovine follicle fluid is available. Previous investigations have reported that phospholipids and their oxidized forms are present in the human follicle fluid and may arise from diffusion/transudation via the follicular wall vascularized and permeabilized during preovulatory maturation (Lopez Bernal et al. 1992, Narahara et al. 1996).
In the porcine fluid, cytochrome P450 (lipoxygenase and cyclooxygenase)-derived metabolites of polyunsaturated fatty acids, including arachidonate and linoleate epoxides, were detected by liquid chromatography (HPLC) tandem mass spectrometry (Newman et al. 2004). The concentrations were up to 0.15 nmol/ml with a higher epoxide/diol ratio (0.45) in samples from the pre-oestrous group compared with other stages (0.06–0.07). The colourimetric method used in the present study has been reported to assay lipohydroperoxides (Mihaljevic et al. 1996). Non-hydroperoxide-generated colour was determined by addition of triphenylphosphine into parallel reactions to reduce hydroperoxides. Specific colour was about 25% of the total colour. In turn, the HPLC tandem mass spectrometry recoveries of epoxyeicosanoids, which may give rise to very reactive lipid alkoxyl radicals and may produce colour in the assay used here, was also only approximately 25% (Newman et al. 2002). Therefore, alternative techniques to measure peroxidized lipids more exactly seem to be lacking. Consistent with the porcine data (Newman et al. 2004), the concentration of oxylipids in the bovine follicle fluid did not correlate with other follicle parameters. However, uptake of or exposition to circulatory reactive phospholipids, such as PAF and PAF-like phospholipids, may be a source of interference with oxylipids produced by thecal cyclooxygenase and lipoxygenase pathways (Spector et al. 2004) essential for ovulatory regulation (Mikuni et al. 1998, Acosta et al. 2003).
In summary, the present study indicates an impairment of thecal progesterone production, COX-2 mRNA concentration and superoxide anion generation by polar phospholipids isolated from circulatory, endogenous oxLDL. These responses were mimicked by PAF but were not found to be unequivocally mediated via the PAF receptor; therefore, the role of this receptor should be fully explored in future studies. Unfavourable effects of lipids have been implicated in reproductive abnormalities, such as amenorrhaea and anovulation frequently observed in disorders of energy metabolism (Mu et al. 2001). These effects on female reproduction seem to occur in single-ovulating species at a preovulatory stage, since the development of dominant follicles was not a limiting factor in the bovine reproductive recovery postpartum at different energy balance and dietary fat levels (Beam & Butler 1997). Such responses mimic COX-2 gene knock-out and COX-2 inhibition in vivo and in vitro (Lim et al. 1997, Mikuni et al. 1998, Davis et al. 1999). Thus, the present observations may contribute to a better understanding of the effects of lipid on female reproductive dysfunction.
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Acknowledgements |
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ppel, Mrs S. Radewald and Mrs R. Hantel for excellent assistance. |
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Received 31 August 2005
Accepted 26 September 2005
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