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Regular Article |
a Department of Animal Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
b Institute of Physiology, Technical University of Munich, D-80350 Freising-Weihenstephan, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corpus luteum function, ovulation, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The developing CL is characterized by highly active vascularization and mitosis of steroidogenic cells in parallel, and the majority of the steroidogenic cells of the mature CL are in contact with one or more capillaries [10]. Vascular endothelial cells (ETC) account for up to 50% of the bovine CL [11, 12], and the vasoactive peptides are released from ETC to regulate CL function as well as vascular tone. In particular, Ang II, which is converted from Ang I by angiotensin-converting enzyme (ACE), induces neovascularization [13, 14] and stimulates steroidogenesis [8]. Recently, we showed the existence of ACE in bovine ETC derived from developing CL [8]. Furthermore, in a study using an in vitro microdialysis system (MDS), the infusion of Ang II directly stimulated progesterone (P) and prostaglandin (PG) F2
release from bovine early CL [8].
On the basis of the aforementioned findings, we hypothesized that a similar local system of vasoactive peptides in the ovulatory follicle might exist in developing bovine early CL in which active angiogenesis is occurring. Because of a lack of in vivo information on the local secretion and relationship among vasoactive peptides within bovine early CL, the objective of the present study was to determine 1) the real-time changes in vasoactive peptide and P concentrations within the developing CL, along with the changes in concentration in both ovarian venous plasma (OVP) ipsilateral to the CL and jugular venous plasma (JVP) in the cow, 2) the effects of CL exposure to vasoactive peptides on Ang II and P secretion in the developing CL, and 3) the expression of mRNA for ANP type C receptor in the bovine CL and ETC derived from bovine CL assessed by reverse transcription-polymerase chain reaction (RT-PCR).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The in vivo MDS experiment was carried out at the Institute of Physiology, TU Munich-Weihenstephan, Germany. Six multiparous, nonlactating Brown Swiss cows were used for this study. The cows were induced to have multiple CL by administration of a normal dose (18 mg) of FSH (Ovagen; Immuno-chemical products LTD, Auckland, New Zealand) in total. A schematic time schedule of the superovulatory treatment and the MDS is shown in Figure 1. Two i.m. injections were given daily at 0700 h and 1800 h for 4 days, starting between Days 8 and 11 of the estrous cycle. At 0700 h on the third day of the FSH treatment, a luteolytic dose of 500 µg of cloprostenol (estrumate; Mallinckrodt GmbH, Burgwedel, Germany), a PGF2 analogue, was injected i.m., and then 2 days after PGF2 injection, 100 µg of GnRH was injected to induce an LH surge and ovulation. Three days after the GnRH injection, a laparotomy was performed as described previously [15] to surgically implant the MDS membranes into the newly formed CL and to cannulate the ovarian vein ipsilateral to the implanted MDS. A jugular venous catheter was also implanted. Before surgery, the ovaries were monitored by transrectal ultrasonography to verify ovulation. After surgery, the cows were moved to individual stanchions, where they were fed daily with corn silage and hay with free access to water. At the end of the experiment (Day 6 after the GnRH injection), the cows were ovariectomized, and the ovaries were visually inspected for the state of CL and the location of the MDS within the CL.
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Implantation of the MDS Capillaries Into the Early CL
The MDS for examining bovine CL in vivo was applied as described previously [15]. Basically, one or two dialysis membranes (Fresenius SPS 900 Hollow Fibers, cutoff Mr = 1000 kDa, 0.2 mm in diameter, 5 mm long; Fresenius AG, St. Wendel, Germany) were implanted into early CL with a 25-gauge hypodermic needle. Both ends of the membrane were glued to a 25-cm-long piece of silicone elastomer tubing (DETAKTA; Isolier-und Messtechnik GmbH & Co., Norderstedt, Germany) (i.d., 0.3 mm) and connected to the MDS. The tubing was fixed on the surface of the early CL by Histoacryl blau (B. Braun-Dexon GmbH, Spangenberg, Germany), and the dialysis pieces with silicone tubing were connected to Teflon tubing (Naflon tubing, Tombo 9003; Nichias Co., Tokyo, Japan) that lead to the outside of the abdomen. The exteriorized bundle of afferent and efferent Teflon tubing was fixed on the back of the cow. One end of the MDS was connected to a multiline peristaltic pump, and the other was connected to a multiline fraction collector. The MDS was continuously perfused with Ringer solution (Fresenius AG, St. Wendel, Germany) with or without stimulant at a flow rate of 2.5 ml/h throughout the experiments, and the fractions of the perfusates were collected at intervals of 4 h starting on Day 3 for the next 4 days. To determine the effect of vasoactive peptides on P and Ang II release, Ang II, ANP, and ET-1 (all from Peninsula Laboratories, Inc., Belmont, CA) were infused. All were diluted in Ringer solution to obtain the required final concentrations (10-7 M). These substances were infused at 24–28 h and 48–52 h (Fig. 1). The transfer capacity of the microdialysis membrane was about 0.1% of the concentration infused as determined for vasoactive peptides and about 1% for P. These values of transfer capacity were determined according to the method of Jarry et al. [16]. The concentrations of the infused substances were chosen based on both of the previously noted transfer capacities and the basal release of each substance as measured in this study.
At the time of surgery, an 18-gauge catheter (Medicut Catheter Kit; Argyle Co., Japan Sherwood, Tokyo, Japan) was inserted into the ovarian vein ipsilateral to the implanted MDS and was sutured in place. Samples of JVP and OVP for determination of peptide and P concentrations were collected at 4-h intervals into sterile 10-ml tubes containing 200 µl of a stabilizer solution (0.3 M EDTA, 1% acid acetyl salicylic, pH 7.4). All tubes were immediately chilled in ice water for 10 min and centrifuged at 2000 x g for 10 min at 4°C, and the obtained plasma was frozen at -30°C until further analysis.
Extraction of Peptides
The plasma samples (6 ml) were diluted with 5 ml of distilled water, and the pH was adjusted to 2.5. For the MDS perfusates (8 ml), BSA (Fraction V; Sigma Chemical Co., St. Louis, MO) was added to the MDS samples to a final concentration of 1 mg/ml, and the pH was adjusted to 2.5 with acetic acid. All samples were then applied to a Sep-Pak C18 Cartridge (Waters, Milford, MA) as described previously [17]. The residue was evaporated and then dissolved in 250 µl of assay buffer (42 mM Na2HPO4, 8 mM KH2PO4, 20 mM NaCl, 4.8 mM EDTA, 0.05% BSA, pH 7.5) for peptide enzyme immunoassays (EIAs). Thus, the samples were concentrated 24-fold for plasma and 32-fold for the MDS perfusate as the result of this process. This process put the concentrations within the range of the standard curve for their determination by EIA (described subsequently). The recovery rates of Ang II (100 pg/ml), ANP (100 pg/ml), and ET-1 (10 pg/ml) that had been added to the plasma were 89%, 76%, and 57%, respectively. The recovery rates of Ang II (10 pg/ml), ET-1 (5 pg/ml), and ANP (20 pg/ml) that had been added to the Ringer solution were 92%, 63%, and 67%, respectively.
Hormone Determination
Concentrations of P and Ang II were determined in the perfusate fractions from the MDS with second-antibody EIAs that were based on a competitive assay using horseradish peroxidase-labeled P or biotin-labeled peptides as tracers. However, ANP and ET-1 concentrations in the MDS perfusate were under the limit of the sensitivity of their respective standard curves. Concentrations of P were assayed as described in detail previously [18]. The standard curve of P ranged from 0.05 to 50 ng/ml, and the ED50 of the assay was 1.8 ng/ml. The intraassay and interassay coefficients of variation (CVs) were on average 6.2% and 9.3%, respectively. The EIA for Ang II was performed with rabbit antiserum raised against human Ang II as described in detail previously [7]. The standard curve of Ang II ranged from 2.4 to 10 000 pg/ml, and the ED50 of the assay was 110 pg/ml. The intraassay and interassay CVs were on average 6.4% and 8.7%, respectively. The EIA for ANP was performed as described elsewhere [9]. The standard curve ranged from 30 to 30 000 pg/ml, and the ED50 of the assay was 850 pg/ml. The intraassay and interassay CVs were 6.5% and 10.4%, respectively. The EIA for ET-1 was performed as described previously [17]. The standard curve of ET-1 ranged from 9.7 to 5000 pg/ml, and the ED50 of the assay was 450 pg/ml. The intraassay and interassay CVs of the ET-1 assay were on average 8.7% and 12.6%, respectively.
CL Collection for RNA Extraction
The CL from Brown Swiss cows were collected at a local slaughterhouse within 10–20 min after slaughter. The stage of the estrous cycle was defined by macroscopic observation of the uterus and ovaries (follicles and CL). The characteristics of the ovaries that were studied included size, color, consistency, connective tissue, thickness of the endometrium, mucus, and absence of elongated early embryos. The early luteal stage (stage I: Days 1–4 of the estrous cycle) was identified as described by Ireland et al. [19]. To estimate the quantity of mRNA for ANP type C receptor, CL were separated from the ovaries immediately after determination of the stages, frozen rapidly in liquid nitrogen, and then stored at -80°C until processed for studies of gene expression.
Culture of Microvascular ETC
Cytokeratin-negative ETC, type 3, derived from the microvascular bed of the developing bovine CL were used and were cultured as described previously [20]. These cytokeratin-negative cells are known to exhibit a cobblestone appearance. Only cells in passage 11 were used in the present study.
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Total RNA from bovine CL and ETC was isolated by the single-step method of Chomczynski and Sacchi [21] with Trizol reagent (Gibco BRL, Gaithersburg, MD) and spectroscopically quantified at 260 nm. Aliquots were subjected to 1% denaturing agarose gel electrophoresis and ethidium bromide staining to verify the quantity and quality of RNA. The RNA yield was then either immediately subjected to reverse transcription-polymerase chain reaction (RT-PCR) or stored at -80°C until analysis.
Two micrograms of total RNA was used to generate single-strand cDNA in a 60-µl reaction mixture as described previously [22]. The resulting cDNA templates were subjected to PCR amplification. The optimal amount of total RNA for RT was evaluated by testing different RNA concentrations. All samples were run on a single gel, allowing only a relative quantification. Conditions for enzymatic amplification were optimized for each PCR as follows: the ANP type C receptor PCR contained 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.6 µM of each primer, and 0.5 U of the thermostable polymerase PrimeZyme (Biometra, Göttingen, Germany) to 5 µl cDNA (final volume, 25 µl). The ubiquitin PCR was performed under the same condition as that for ANP type C receptor, but a higher concentration of primer (1.5 µM) was used. On the basis of optimization of temperature, amplification of ANP type C receptor analysis consisted of one denaturing step at 94°C for 2 min, followed by 30 cycles at 94°C for 30 sec and 60°C for 45 sec. One additional elongation step was carried out at 72°C for 2 min. Amplification of the housekeeping gene consisted of one denaturing step at 94°C for 2 min; 22 cycles at 94°C for 45 sec, 55°C for 45 sec, and 72°C for 45 sec; and one additional elongation at 72°C for 2 min.
To determine the optimal quantity of reverse transcript needed for PCR and to verify that the cDNA product was dependent on the amount of transcript used, varying quantities of transcript template were used in the PCR reaction process. The RT product from 3 µl was in the linear range for these amounts and produced a visible band. To exclude the possibility of amplification of genomic DNA, all experiments included reactions in which the RT enzyme or cDNA template was omitted. As a negative control, water was used instead of RNA for the RT-PCR to exclude any contamination from buffers and tubes. The primers were designed to encode the bovine sequences by using the European Molecular Biology Laboratory (EMBL) database or were commercially synthesized (Amersham-Pharmacia, Freiburg, Germany). The primers were chosen using the HUSAR online program package in Heidelberg (http://genome.dkfz-heidelberg.de). The primers were as follows: ANP type C receptor forward 5'-ACGTGAACAGTTTGTTGAAGG-3' and reverse 5'-CGCTGATTCTTCTAGGCCA-3'; ubiquitin forward 5'-ATGCAGATCTTTGTGAAGAC-3' and reverse 5'-CTTCTGGATGTTGTAGTC-3'. The predicted sizes of the resulting RT-PCR products were 392 base pairs (bp) for ANP type C receptor and 189 bp for ubiquitin, respectively.
Aliquots of the PCR reaction products (5 µl) were added to 1 µl bromphenol blue glycerin and fractionated by electrophoresis through a 1.5% agarose gel containing ethidium bromide in a constant 60-V field. To determine the length of the RT-PCR products, a mass ladder and 100-bp marker were used. The resultant band intensities were scanned by a video documentation system (Amersham-Pharmacia) and analyzed with the Image Master ID program (Amersham-Pharmacia). Confirmation of the PCR product identity was obtained by cDNA subcloning into a transcription vector (pCR-Script; Stratagene, La Jolla, CA) and by subjecting the products to commercial DNA sequencing (TopLab, Munich, Germany).
Statistical Analysis
The mean hormone (P and Ang II) concentrations in the MDS perfusate in the first 24-h fraction were used to calculate the individual baseline because of a large variation in the basal concentrations of each hormone released into the MDS lines implanted in the different CL (P: 1.8–11.4 ng/ml; Ang II: 1.4–10.3 pg/ml). All hormone concentrations were expressed as a proportion of this individual baseline. This treatment enables an evaluation of relative changes of hormonal values between the different CL. Means were analyzed by ANOVA followed by the Tukey-Kramer test as a multiple comparison test. For the figures showing MDS data, all hormone concentrations in the fractions were then expressed as a percentage of this individual baseline. The absolute concentrations of each hormone during the first 24 h (baseline) of an experiment are given in the figure legends. The mean concentrations of P, Ang II, ANP, and ET-1 in OVP and JVP samples collected at different time periods were compared on the basis of each 24-h period. To compare the values of concentrations between OVP and JVP, the mean values of each 24-h period were analyzed by ANOVA followed by the Student t-test. The mean absolute concentrations of Ang II and P were analyzed on the basis of the aforementioned 24-h period throughout the experiment by ANOVA followed by the Tukey-Kramer test as a multiple comparison test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in P concentrations in the MDS fractions and in OVP and JVP during CL development are shown in Figure 2. Intraluteal and plasma P concentrations gradually increased from Day 3 to Day 6 (P < 0.05; Fig. 2 and Table 1). The P levels in OVP were about 100 times higher than those in JVP (P < 0.001; Table 1).
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Intraluteal and Plasma Changes in Ang II Concentration During CL Development
Changes in Ang II concentrations in the MDS fractions and in OVP and JVP during CL development are shown in Figure 3. The Ang II concentration in OVP was higher than that in JVP throughout the experimental period (P < 0.05 and P < 0.001; Table 1), and the concentration at 48–72 h was significantly higher than that at 24–48 h (P < 0.05; Table 1). The intraluteal Ang II release and peripheral plasma concentrations showed no clear change during the experimental period.
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Plasma Changes in ANP and ET-1 Concentrations During CL Development
Changes in plasma ANP and ET-1 concentrations are shown in Figure 4. Concentrations of ANP in OVP were stable and higher than those in JVP until 48 h (P < 0.05 and P < 0.01; Table 1). The plasma ET-1 concentrations were stable at approximately 5–8 pg/ml in both OVP and JVP. The ANP and ET-1 concentrations in the MDS samples after extraction and concentration were not detectable in our EIAs (ANP: <1.5 pg/ml; ET-1: <0.5 pg/ml).
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Effects of Vasoactive Peptides on the Release of P and Ang II
Vasoactive peptides (10-7 M) were infused at 24–28 h and 48–52 h during the experimental period (Fig. 1). The first infusion (24–28 h) of three vasoactive peptides had no effect on P and Ang II release. However, the second infusion of Ang II (10-7 M) at 48–52 h significantly increased P secretion (P < 0.05), and the second infusion of ANP (10-7 M) stimulated Ang II secretion (P < 0.05; Table 2). No effect of ET-1 infusion (10-7 M) on secretory function was observed throughout the experimental period (Table 2).
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Expression of mRNA for ANP Type C Receptor
PCR products having the expected sizes of ANP type C receptor were found in bovine early CL and ETC derived from CL. The obtained partial PCR sequences for ANP type C receptor (392 bp) were 100% homologous to the known bovine genes after sequencing. A representative example for the RT-PCR products of ANP type C receptor in CL tissue and ETC is shown in Figure 5. The strong signal in ETC suggests that ANP type C receptor is located mainly in this cell type.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Ang II concentration in OVP was higher than that in JVP throughout the experiment. The intraluteal release of Ang II was stable during the experimental period. The follicular release of Ang II rose at the onset of the LH surge and then remained stable after 72 h [23]. This high level of Ang II release was continued in the period of observation in the present study, when the CL was growing rapidly. Based on the Ang II concentration in the MDS perfusate (1.4–10.3 pg/ml) and the transfer capacity of the dialysis membrane for Ang II (0.1%), the possible concentration of Ang II in the extracellular fluid in bovine early CL could be calculated to be in the range of 1–10 ng/ml. This range is more than 100-fold higher than the Ang II concentration in the peripheral plasma in this study. Thus, this finding strongly supports the concept that the bovine CL is a site of Ang II production [7, 8]. In our previous study of the bovine follicle in vivo, a high concentration of Ang II in the OVP was first observed after the LH surge, and these high Ang II levels in OVP persisted up to 72 h after the LH surge [23]. The Ang II concentration in the OVP remained at a high level during the following 72 h in this study. This finding implies that ovarian Ang II production is highly active throughout the time from the periovulatory period to early CL development. Angiotensin II, which is converted from its precursor Ang I by ACE on the surface of endothelial cells [8], binds to its type 1 and 2 receptors to stimulate angiogenesis [13, 14] as well as P and PGF2 release from bovine early CL in vitro [8].
Intraluteal infusion of Ang II at 24–28 h (Day 4 after GnRH administration) did not affect P release, whereas infusion of Ang II at 48–52 h stimulated P release (Fig. 1, Table 2). This result suggests that Ang II first affects the secretory mechanism at the later stage of luteal angiogenesis and not at the first stage just after ovulation. Therefore, this finding raises the possibility that the role of Ang II shifts from the stimulation of angiogenesis in the newly formed CL (Days 2 and 3 after ovulation) to the promotion of P production around 4 days after ovulation, although we cannot exclude the possibility that the first infusion affected the CL sensitivity to Ang II thereafter.
Atrial natriuretic peptide is also a vasoactive peptide that basically modulates vascular activity in the systemic circulation. In the present study, the ANP concentrations in OVP were significantly higher than those in JVP during Days 3–5 after GnRH administration. Intraluteal release of ANP was not detectable in our EIA system because the ANP concentration in the MDS perfusate at this stage was below limit of detection of our EIA (<1.5 pg/ml). However, immunoreactive ANP has been identified in bovine CL [24] and human aortic ETC [25]. Therefore, ETC in bovine CL may also be the site of ANP production. The present study illustrated the expression of mRNA for ANP type C receptor in ETC derived from bovine developing CL. Moreover, infusion of ANP into the MDS stimulated Ang II release during Days 5 and 6 after GnRH treatment but did not affect P secretion. Since Ang II infusion directly stimulated P release as described previously, the increased Ang II secretion stimulated by ANP could affect P release. Although the reason why ANP had no effect on P secretion is not clear, the absence of an effect may be explained by the technique that we used. Namely, in the MDS technique, the cells contacting the microdialysis membrane are continuously perfused by Ringer solution. Therefore, the locally secreted hormone (Ang II in this case), as well as the infused stimulant, is washed out continuously. Thus, the increased Ang II secretion stimulated by ANP infusion seems to also be washed away from the cell surface, so that P secretion was not affected by ANP infusion. Atrial natriuretic peptide has been shown to stimulate cGMP formation but to have no effect on cAMP accumulation and P production by bovine luteal cells in vitro [26]. Thus, the findings suggest that the circulating and/or locally produced ANP binds to its type C receptor on ETC within CL to stimulate intracellular cGMP production and directly increases Ang II production of ETC via stimulation of ACE activity. Thus, we speculate that ANP may indirectly support luteal function in the developing CL by stimulating Ang II secretion. However, there is little information to evaluate the physiologic impact of ANP on luteal function at this stage.
The plasma ET-1 concentration was similar in OVP and JVP, and it did not change throughout the experimental period. Furthermore, the ET-1 concentration in the MDS perfusate was not detectable in our EIA (<0.5 pg/ml) at this stage, in spite of the presence of concentrations in the detectable range at the midluteal phase [7, 17]. In fact, the ET-1 content and production in bovine CL are at their lowest levels during development [27]. In addition, the infusion of ET-1 did not affect P and Ang II release in this in vivo study. These findings emphasize that levels of ET-1 may not directly correlate with luteal function in the developing CL, but that ET-1 has an essential role in CL regression [15, 17, 28].
We previously proposed that an ET-Ang-ANP system exists in the bovine mature follicle and that this system stimulates PGF2 production in the days leading up to ovulation [23]. Several studies have shown a high production of PGF2 in bovine early CL [29, 30]. Angiotensin II and other angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor directly stimulate PGF2 release from bovine early CL in vitro [8, 31]. Thus, the active PGF2 production in the early luteal phase appears to be the result of active angiogenesis involving the action of the aforementioned angiogenic factors. The present and previous results [8] show that Ang II directly stimulates P release both in vivo and in vitro in the bovine early CL. Furthermore, the interaction of Ang II and PGF2 ensures the mechanism of P production in bovine early CL [8], suggesting that luteal Ang II and PGF2 fundamentally support P production in the early CL. On the other hand, ANP has no effect on P release but enhances Ang II release. Additionally, ANP is capable of stimulating PGF2 secretion of bovine early CL in vitro [32]. Therefore, ANP appears to be associated with the increase in Ang II and PGF2 levels in the developing CL, which is a prerequisite for active angiogenesis. Consequently, the ET-Ang-ANP system in the preovulatory follicle may switch to an Ang-ANP system to enhance both the angiogenesis and steroidogenesis that actively occur in developing CL.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by the German Research Foundation (Scha 257/14-1), Grant-in-Aid for Scientific Research (11660276 and 12556046) and the Japan-Germany joint research project of the Japan Society for the Promotion of Science (JSPS); the Novartis Foundation (Japan) for the Promotion of Science; and Morinaga Hoshikai Foundation (Japan). S.K. and K.H. were supported by H. Wilhelm Schaumann Stiftung, T.J.A. and K.H. were supported by JSPS Fellowships for Young Scientist, and A.M. was supported by Alexander von Humboldt Stiftung. 
2 Correspondence. FAX: 81 155 49 5462; akiomiya{at}obihiro.ac.jp
Accepted: August 28, 2001.
Received: June 19, 2001.
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  C. Boiti, M. Maranesi, C. Dall'Aglio, L. Pascucci, G. Brecchia, A. Gobbetti, and M. Zerani Vasoactive Peptides in the Luteolytic Process Activated by PGF2alpha in Pseudopregnant Rabbits at Different Luteal Stages Biol Reprod, July 1, 2007; 77(1): 156 - 164. [Abstract] [Full Text] [PDF]
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 J. R. Pepperell, G. Nemeth, Y. Yamada, F. Naftolin, and M. Merino Localized accumulation of angiotensin II and production of angiotensin-(1-7) in rat luteal cells and effects on steroidogenesis Am J Physiol Endocrinol Metab, August 1, 2006; 291(2): E221 - E233. [Abstract] [Full Text] [PDF]
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 K. Shirasuna, H. Asaoka, T. J Acosta, M. P B Wijayagunawardane, M. Ohtani, K.-G. Hayashi, M. Matsui, and A. Miyamoto Real-time dynamics of prostaglandin F2{alpha} release from uterus and corpus luteum during spontaneous luteolysis in the cow Reproduction, August 1, 2004; 128(2): 189 - 195. [Abstract] [Full Text] [PDF]
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