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a Departments of Theriogenology and
b Animal Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
c Institute of Physiology, Technical University of Munich, 80350 Freising-Weihenstephan, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Each preovulatory follicle obtained before the LH surge (based on the concentrations of steroids and PGs) received implants of 4 capillary dialysis membranes into the theca layer. The follicles were then incubated in organ culture chambers and perfused with Ringer's solution for 12 h. Stimulation by infusion of the different substances into the microdialysis system was carried out between 4 and 8 h. The infusion of ET-1 (10-7 M) stimulated the release of ANP and estradiol but inhibited the release of androstenedione and progesterone. The infusion of ANP (10-7 M) stimulated the release of Ang II, progesterone, and androstenedione. Moreover, the infusion of Ang II (10-5 M) inhibited the release of ANP but stimulated the release of ET-1, progesterone, and estradiol. All three peptides examined increased PGE2 and PGF2 release. In the reverse transcription-polymerase chain reaction analysis, expression of the mRNAs for ET type A and type B, and Ang II type 1 receptors did not change with the follicular size and the intrafollicular estradiol concentrations. Expression of the mRNA for the Ang II type 2 receptor dropped in follicles when the estradiol concentration ranged from 20 to 180 ng/ml and increased again when the estradiol concentration was > 180 ng/ml. The levels of expression of ANP type C receptor mRNA were slightly greater in follicles with estradiol concentrations > 20 ng/ml than in follicles with estradiol concentrations < 20 ng/ml.
These results demonstrate a complex interaction among Ang II, ET-1, and ANP that may contribute to increasing the follicular production of PGs and modulate steroidogenesis in the bovine mature follicle, thus providing evidence for a local functional endothelin-angiotensin-ANP system.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent findings have shown that Ang II and ET-1 locally modify the synthesis and secretion of hormones produced in the ovarian follicular cells in an autocrine and/or paracrine manner [5, 10, 14–17]. On the other hand, ANP counteracts the actions of Ang II on intracellular cyclic GMP and Ca2+ concentrations [18, 19] and affects steroidogenesis in vivo [20] and in vitro [21, 22]. On the basis of these findings, an active intraovarian endothelin-renin-angiotensin-ANP system has been postulated [22]. Furthermore, several lines of evidence indicate a regulatory role for ET-1 in ANP secretion in vascular endothelial cell cultures [23]. However, the local interrelationships among ET-1, Ang II, and ANP and their physiological roles in the local modulation of PG and/or steroid secretion in the bovine mature follicle remain unclear.
Therefore, we attempted to investigate 1) the local interrelationships among Ang II, ET-1, and ANP; 2) the effect of each vasoactive peptide on the secretion of steroid hormones and PGs in isolated bovine mature follicles; and 3) the expression of the mRNAs for Ang II, ET-1, and ANP receptors in the theca layer of follicles at different developmental stages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For the microdialysis system (MDS) study in vitro, ovaries from Holstein cows containing a mature, presumably preovulatory follicle were collected from a local slaughterhouse in Japan and transported to the laboratory at 37°C in sterile saline solution (0.9% NaCl) containing 100 000 IU penicillin and 100 mg streptomycin/L. Ovaries were visually inspected. The preovulatory stage was defined by the presence of a graafian follicle (1.5–2.0 cm in diameter) and a regressing corpus luteum (CL) in the ipsilateral or contralateral ovary [24]. The uterine characteristics (size, color, tonus, consistency, and mucus) were also considered. Concentrations of steroids (progesterone, androstenedione, and estradiol), PGE2, PGF2
, and peptides (Ang II, ET-1, ANP, and oxytocin) in the follicular fluid were determined at the end of the perfusion experiment as described below. These concentrations are shown in Table 1. Only the MDS perfusates (fractions) obtained from estradiol-dominant follicles that had not yet been exposed to the endogenous LH surge (based on the steroid, PG, and peptide concentrations in follicular fluid) were used for data analysis. Such follicles were identified by having a PGE2 concentration in the follicular fluid of less than 600 pg/ml.
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For the study of expression of receptor mRNAs by reverse transcription (RT)-polymerase chain reaction (PCR), ovaries from Simental cows were collected at a local slaughterhouse in Germany within 10–20 min after slaughter and were transported to the laboratory on ice. The follicles were classified into 5 groups (n = 5 in each group) according to their follicular fluid estradiol concentrations as follows (average follicular diameters ± SEM are in parentheses): < 0.5 ng/ml (6.6 ± 0.4 mm), 0.5–5 ng/ml (8.0 ± 0.5 mm), 5–20 ng/ml (12.2 ± 0.2 mm), 20–180 ng/ml (12.6 ± 0.4 mm), and > 180 ng/ml (14 ± 4.4 mm), respectively. The follicles of the last 3 groups were obtained from cows having a regressing CL in the ipsilateral or contralateral ovary. Only follicles that appeared healthy (i.e., well vascularized and having transparent follicular wall and fluid) and whose diameter was > 5 mm were used. Follicles with progesterone concentrations of more than 100 ng/ml were excluded. After aspiration of follicular fluid, follicles were bisected, and the inside wall was gently scraped and flushed with Ringer's solution to remove the granulosa cells. For RNA extraction, the surrounding tissue (theca externa) was removed with forceps under a stereo microscope. The theca tissues were snap-frozen in liquid nitrogen and stored at -80°C until RNA isolation.
MDS In Vitro
The MDS for bovine mature follicles has been previously described in detail [5]. Basically, each follicle was dissected from surrounding stromal tissue, and four capillary dialysis membranes (Fresenius SPS 900 Hollow Fibers, cut-off Mr = 1000, 0.2 mm diameter, 5 mm long; Fresenius AG, St. Wendel, Germany) were implanted into the theca layer with at least a 5-mm distance between capillaries. Both ends of the capillary were glued to silicone elastomer tubing (i.d. 0.3 mm) that was affixed to the surface of the follicular tissue by Hystoacryl blue (B. Braun-Dexon GmbH, Spangenburg, Germany). For the perfusion, one end of the tube was connected to a multiple-line peristaltic pump and the other was routed to a multiple-line fraction collector. The prepared follicles were then placed in organ culture chambers (modified 2070 tube; Falcon, Franklin Lakes, NJ) filled with 50 ml Medium 199 (Sigma Chemical Co., St. Louis, MO) containing Earle's salts, 10 mM NaHCO3, 365 mg/L L-glutamine, 25 mM Hepes, 5 g/L BSA, 60 mg/L penicillin, 100 mg/L streptomycin, 56 mg/L ascorbic acid, and 2 mg/L amphotericin B at pH 7.4. The chambers were maintained in a water bath at 38°C throughout the complete period of perfusion. The medium was continuously exchanged at a flow rate of 15 ml/h. During incubation, the MDS implanted in the follicular wall was perfused with Ringer's solution at a flow rate of 2 ml/h. In the LH infusion experiment, two lines were used as controls and two lines were used for LH infusion. For infusion of the vasoactive peptides, the four capillaries were used for control, ET-1, Ang II, and ANP infusion, respectively. After 2 h preperfusion, fractions of the MDS perfusate were collected up to 12 h (4 h/fraction). Collected samples were stored at -30°C until hormone determination.
Bovine LH (USDA-bLH-B-6), human Ang II, human ET-1, and human ANP (Peptide Institute Inc. Osaka, Japan) were diluted in Ringer's solution to obtain the required final concentrations of 5 µg/ml, 10-5 M, 10-7 M, and 10-7 M, respectively. The doses were determined on the basis of the concentrations of the different peptides in the perfusates and the transfer capacity of the membrane, which was previously estimated to be 1% for steroids and PGs, and 0.1% for peptides and LH [25, 26]. The solutions were then infused into the MDS for 4 h between 4 and 8 h.
Steroid and PG Extraction
The perfusates (4 ml) and follicular fluids (300 µl) were adjusted to pH 3.5 using 1N HCl and 5N HCl, respectively, and extracted using diethyl ether as described previously [3]. The residue was dissolved in 300 µl assay buffer for steroid and PG enzyme immunoassays (EIAs) (40 mM PBS, 0.1% BSA, pH 7.2). To estimate the recovery rate, androstenedione, progesterone, estradiol-17ß, PGE2, and PGF2 were added to the Ringer's solution (30, 100, 20, 100, and 100 pg/ml, respectively), and the obtained values were 85%, 88%, 75%, 72%, and 68%, respectively.
Extraction of Peptides
After the diethyl ether extraction, the remaining Ringer's solution was used for peptide extractions. BSA (Sigma Chemical Co.; Fraction V) was added to the samples to a final concentration of 1 mg/ml. Follicular fluids (2 ml) were diluted with the same volume of distilled water, and the pH was adjusted to 2.5 with 5N HCl. The samples were then applied to a Sep-Pak C18 Cartridge (Waters, Millford, MA) as described previously [26]. The residue was evaporated and then dissolved in 200 µl assay buffer (42 mM Na2HPO4, 8 mM KH2PO4, 20 mM NaCl, 4.8 mM EDTA, 0.05% BSA, pH 7.5) for peptide EIAs. The recovery rates of the Ang II (50 pg/ml), ET-1 (10 pg/ml), and ANP (20 pg/ml) that had been added to the Ringer's solution were 92%, 63%, and 67% respectively.
Hormone Determinations
The concentrations of the different hormones were determined in duplicate by second-antibody EIAs using 96-well ELISA plates (Corning Glass Works, Corning, NY). The EIA for progesterone was done as previously described [27]. The standard curve ranged from 0.05 to 25 ng/ml, and the ED50 of the assay was 2.6 ng/ml. The intra- and interassay coefficients of variation (CVs) were 4.8% and 7.5%, respectively. The EIA for estradiol was carried out as described previously [28]. The standard curve ranged from 2 to 2000 pg/ml, and the ED50 of the assay was 110 pg/ml. The intra- and interassay CVs were 6.3% and 8.5%, respectively. The androstenedione EIA was described earlier [5]. The standard curve ranged from 2 to 1000 pg/ml, and the ED50 of the assay was 115 pg/ml. The intra-and interassay CVs were 6.3% and 8.2%, respectively.
The EIAs for PGE2 and PGF2 are described elsewhere [28]. The standard curve for PGE2 ranged from 30 to 14 200 pg/ml, and the ED50 of the assay was 350 pg/ml. The intra- and interassay CVs were 9.5% and 12.5%, respectively. The standard curve for PGF2 ranged from 7 to 7000 pg/ml, and the ED50 of the assay was 250 pg/ml. The intra- and interassay CVs were 8.7% and 13.1%, respectively.
The EIAs for oxytocin and ET-1 were performed as described previously [26]. The standard curve for oxytocin ranged from 1.6 to 200 pg/ml, and the ED50 of the assay was 25 pg/ml. The intra- and interassay CVs were 5.9% and 8.4%, respectively. The ET-1 antibody was purchased from RD Laboratory (Diessen, Germany). This antibody shows specificity for the C terminus and cross-reacts 100% with ET-1, ET-2, big endothelin, and ET-3, and < 0.01% with atrial natriuretic factor 99–126 (ANF 99–126; human), ANF 99–126 (rat), vasoactive intestinal peptide (human), brain natriuretic peptide (porcine), neuropeptide Y, dynorphin A, Ang I, Ang II, Ang III, calcitocin gene-related peptide, bradykinin, and arginine vasopressin. The standard curve ranged from 10 to 500 pg/ml, and the ED50 of the assay was 60 pg/ml. The intra- and interassay CVs were 7.5% and 12.5%, respectively. The EIA for Ang II was performed as described previously [29]. The standard curve ranged from 2.5 to 2500 pg/ml, and the ED50 of the assay was 125 pg/ml. The intra- and interassay CVs were 5.5% and 8.3%, respectively.
The EIA for ANP was performed exactly the same as the ET-1 and Ang II EIAs. Basically, 20 µl of standard and samples was incubated with anti-a-human ANP developed in New Zealand white rabbits using human ANF 1–28 conjugated to bovine thyroglobulin as immunogen (RD Laboratory). The antiserum is monospecific for a-ANP, detecting the middle and the C terminus of the molecule. The following cross-reactivities were confirmed for other peptides: ANP (1–28, rat) 78%, atriopeptin II (rat) 30%, pro ANP (2–126, rat) 42%, and atriopeptin I and II (rat) 0.03%. The standard curve ranged from 30 to 30 000 pg/ml, and the ED50 of the assay was 850 pg/ml. The intra- and interassay CVs were 6.5% and 10.4%, respectively.
RT-PCR
Total RNA was isolated from thecal tissue by the single-step method of Chomczynski and Sacchi [30] using Trizol reagent (Gibco BRL, Gaithersburg, MD) and was spectroscopically quantified at 260 nm. In addition, samples were checked for evidence of RNA degradation by formaldehyde gel electrophoresis, seeking two distinct bands corresponding to 28S and 18S ribosomal RNAs. Four micrograms of total RNA was used to generate single-strand cDNA in a 60-µl reaction mixture as described previously [31]. 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: ET receptor type A (ETR-A), ET receptor type B (ETR-B), Ang II receptor type 1 (AT1), Ang II receptor type 2 (AT2), and ANP receptor type C (ANP-CR) 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 thermostable polymerase PrimeZyme (Biometra, Goettingen, Germany) to 3 µl cDNA (final volume 25 µl). Ubiquitin PCR was performed under the same conditions, but a higher concentration of primer (1.5 µM) was used. The samples for ETR-A, ETR-B, AT1, AT2, and ANP-CR were amplified for 30 cycles (one single denaturation step at 94°C for 2 min, each cycle at 94°C for 30 sec, 60°C for 45 sec (AT2 at 64°C for 45 sec), and afterwards one additional elongation step at 72°C for 2 min). Samples for the housekeeping gene ubiquitin were amplified by 22 cycles (one single denaturation step at 94°C for 2 min, each cycle at 94°C for 45 sec, at 55°C for 45 sec, at 72°C for 45 sec, and afterwards one additional elongation step at 72°C for 2 min). To confirm the integrity of the mRNA templates and RT-PCR protocol, the housekeeping gene ubiquitin was examined in all samples. To determine the optimal quantity of reverse transcript needed for PCR and to verify that the cDNA product was dependent on the input of transcript, varying quantities of transcript were used in the PCR reaction. 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 the buffers and tubes.
The primers (Table 2) were designed on the basis of the bovine sequences obtained from the EMBL database or were used as described elsewhere and commercially synthesized (Amersham-Pharmacia, Freiburg, Germany).
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Aliquots of the PCR reaction products (5 µl) were added to 1 µl bromophenol 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 products, a DNA mass ladder (Gibco BRL) was used. The ethidium bromide-stained gels were evaluated by a video documentation system (Amersham-Pharmacia). Band intensities were analyzed by computerized densitometry using the Image Master Program (Amersham-Pharmacia). To verify each PCR product, double-strand sequencing was performed directly or after subcloning (TopLab, Munich, Germany).
Statistical Analysis
The mean hormone concentrations in the first fraction (first 4-h perfusion with Ringer's solution only) were used to calculate the individual baseline for each hormone, because of a large variation in the absolute amount of hormones released into each of the MDS lines implanted in the various follicles. All values were expressed as a percentage of the corresponding baseline. The coefficient of variation (CV) in the absolute concentration of each hormone in the MDS perfusates collected from different follicles during the first 4 h (baseline) was as follows: progesterone = 19–45%, androstenedione = 10–32%, estradiol = 23–85%, PGE2 = 25–110%, PGF2 = 30–92%, Ang II = 27–45%, ET-1 = 23–38%, and ANP = 15–55%. This transformation enables an evaluation of relative changes of hormonal values between the different follicles. The effects of the infused substances (LH, Ang II, ET-1, and ANP) on the release of steroids, PGs, and peptides were compared with the control values obtained during the same time period using ANOVA followed by Student's t-test. Differences were considered significant at a probability less than 5% (P < 0.05). The absolute concentrations of the hormones in the MDS fractions (mean ± SEM) are given in the figure legends. The mRNA data for each receptor were expressed as optical densities, and means were analyzed by ANOVA followed by Fisher's Protected Least Significant Difference (PLSD) test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Infusion of LH increased the release of ET-1 (during infusion: 165 ± 18% of baseline, after infusion: 173 ± 15% of baseline; P < 0.05) without having any effects on the release of ANP (104 ± 8% of the baseline) or Ang II (98 ± 2% of the baseline). The basal release (100%) of each hormone was 45.5 ± 10.5 pg/ml for Ang II, 5.4 ± 1.8 pg/ml for ET-1, and 15 ± 5.3 pg/ml for ANP (n = 11; mean ± SEM). Infusion of LH also increased the release of PGF2 (180–450% of baseline) and PGE2 (162–490% of baseline) during and after exposure.
Interaction Among Vasoactive Peptides
Ang II release was stimulated by ANP infusion (P < 0.05). ET-1 release was stimulated by Ang II infusion (P < 0.01). Furthermore, ANP release was inhibited by Ang II (P < 0.05) but stimulated by ET-1 infusion (P < 0.05; Fig. 1).
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Effect of Vasoactive Peptides on the Release of Steroid Hormones
Ang II stimulated the release of progesterone and estradiol after infusion (P < 0.05). ET-1 inhibited the release of androstenedione and progesterone but stimulated the release of estradiol (P < 0.05). ANP increased the release of progesterone and androstenedione during the poststimulation period (P < 0.05; Fig. 2).
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Effects of Vasoactive Peptides on the Release of PGs
All ET-1, ANP, and Ang II strongly stimulated the release of PGE2 and PGF2 (P < 0.001–P < 0.05; Fig. 3).
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Expression of mRNAs for Receptors of Vasoactive Peptides
PCR products having the expected sizes of the receptors of each of the peptides were found in bovine theca tissue. The obtained partial PCR sequences for ETR-A (329 base pairs [bp]), ETR-B (296 bp), AT1 (324 bp), and ANP-CR (392 bp) were 100% homologous to the known bovine sequences. The sequence of the AT2 PCR product (335 bp; EMBL-S81979) was 97% homologous to the ovine sequences. A representative example for the RT-PCR products of all above receptors is given in Figure 4.
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The relative signal intensities for PCR products specific for each receptor were assessed after correction based on the ubiquitin signal intensities. The results of the densitometric analysis of mRNA for all receptors examined in thecal tissue are shown in Figure 5. The expression of mRNA for each type of receptor of vasoactive peptides was observed in all groups of follicles with different estradiol concentrations (Fig. 5). The expression of mRNA for ETR-A and ETR-B did not change in follicles with different estradiol concentrations (Fig. 5a). The levels of expression of the mRNA for AT2 transiently dropped when the intrafollicular estradiol concentration was in the range of 20–180 ng/ml, and they increased again in the follicles when the estradiol concentration was > 180 ng/ml (P < 0.05), whereas the levels of expression of AT1 mRNA were similar in all groups (Fig. 5b). The levels of expression of ANP-CR mRNA slightly increased (P < 0.05) in follicles with estradiol concentration at > 20 ng/ml (Fig. 5c).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous reports indicated that the LH surge stimulates the ovarian renin-angiotensin-system (RAS), including an increase in the renin and prorenin concentration in bovine follicular fluid [4] as well as an increase in Ang II concentration in rabbit ovarian perfusates and follicular fluid [36]. In addition, LH infusion increased ET-1 release from the theca layer of bovine mature follicles in vitro [5]. Thus, in this study we evaluated the possible direct effect of LH on the local release of Ang II and ANP using an MDS. Unexpectedly, LH did not affect Ang II and ANP release in the present study. It has been reported that the concentration of renin increases in atretic bovine follicular fluid [37], and the same authors found that granulosa cells secrete one or more factors that, even in the presence of LH, keep prorenin secretion low and inhibit LH-stimulated prorenin secretion in a dose-dependent manner from bovine theca cells in vitro [38]. These findings suggest that the model used to study RAS in the follicle may critically affect the experimental outcome. Thus, to investigate the interrelationship of local RAS with other local factors such as peptides, PGs, and steroids, the experimental model may need to preserve the integrity of follicular structures including the granulosa-theca complex, endothelial cells, and the follicular fluid, which was maintained in the present MDS study.
Ang II stimulated ET-1 release in the present study. Similar results have been reported in bovine endothelial cells, in which the increase of ET-1 secretion by Ang II may be principally due to a stimulation of ET-1 release by a mechanism involving receptor-mediated mobilization of intracellular Ca2+ and activation of protein kinase C [39], as well as activation of preproendothelin-1 mRNA expression [40]. In cow ovaries, theca cells have been shown to possess significant numbers of binding sites for 125I-labeled Ang II. Such binding sites are classified as the AT2 receptor and are up-regulated by LH [9]. It is interesting to note that in the present study, the level of AT2 receptor mRNA transiently dropped in follicles with estradiol concentrations ranging from 20 to 180 ng/ml (mean follicle diameter = 12.6 mm) but increased again when the estradiol concentration was more than 180 ng/ml (mean follicle diameter = 14.4 mm). These findings suggest that in the bovine mature follicle, Ang II might act via the AT2 receptor in the ovulatory process.
In the present study, ET-1 stimulated follicular ANP release. ET-1 has been found to regulate ANP gene expression in the brain and to increase ANP secretion from isolated perfused rat hearts [41, 42]. The facts that ET-1 stimulated follicular ANP release and that ANP stimulated follicular Ang II release indicate that these peptides may act in a local cascade-like reaction that might mediate LH action on follicular Ang II secretion to accelerate PG production during the preovulatory period. Indeed, an ongoing in vivo study in our laboratory revealed a peak of Ang II coincident with an increase in PG concentration in the ovarian venous plasma from 20 to 40 h after the endogenous LH surge, when ovulation was expected to occur (unpublished data), indicating that ovarian PG and Ang II secretions increase during the periovulatory period in the cow. Additionally, the present results showed that Ang II directly inhibits the release of ANP from the theca layer of the bovine mature follicle in vitro. This implies that Ang II in the theca extracellular fluid precisely modulates local ANP and ET production in bovine follicles (Fig. 6).
On the basis of the concentrations of Ang II and ANP in the perfusates and the transfer capacity of the MDS membrane, the extracellular concentrations of Ang II and ANP in the theca layer may be around 45 ng/ml and 15 ng/ml, respectively. Such concentrations are higher than those in the plasma (Ang II, 20 pg/ml, and ANP, 60 pg/ml; our unpublished data). These findings support the concept that mature follicles are sites of Ang II and ANP production, in addition to ET-1 production [5].
Regional changes in the blood flow have been observed in the preovulatory follicle [1]. The increase in ovarian PG production caused by the LH surge may also be mediated (at least in part) by the AT2 receptor-mediated effects of Ang II at the ovarian level [43]. Similarly, ET-1 and ANP also showed stimulatory effects on PGE2 and PGF2 secretion in the present study. ET-1 has been shown to activate multiple biochemical pathways within the target cells, including the phospholipase C pathway, the phospholipase D pathway, and the arachidonate cascade [44], which are directly involved in PG production. The specific importance of ANP in the ovulatory process is not currently understood, although it is possible that ANP augments the follicular blood flow and local PG secretion. This idea might be supported by the present data showing that the expression of the mRNA for the ANP type C receptor slightly increased in follicles when the intrafollicular concentration of estradiol was higher than 20 ng/ml. At the level of the granulosa cell, ANP has been shown to activate cyclic GMP, but not the cyclic AMP transduction sequence, resulting in increased biosynthesis of progesterone, but not estradiol [21]. These observations may be relevant to the role of ANP in the initiation of the luteinization of the preovulatory follicle that is associated with an increase in progesterone secretion.
In conclusion, the present results demonstrate a complex interaction among Ang II, ET-1, and ANP that may contribute to increasing the follicular production of PGs and modulate steroidogenesis in the bovine mature follicle, thus providing evidence for a local functional endothelin-angiotensin-ANP system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 81 155 49 5462; akiomiya{at}obihiro.ac.jp
Accepted: July 8, 1999.
Received: April 19, 1999.
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  V. M. Portela, P. B. D. Goncalves, A. M. Veiga, E. Nicola, J. Buratini Jr., and C. A. Price Regulation of Angiotensin Type 2 Receptor in Bovine Granulosa Cells Endocrinology, October 1, 2008; 149(10): 5004 - 5011. [Abstract] [Full Text] [PDF]
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B. Berisha, M. Steffl, W. Amselgruber, and D. Schams Changes in fibroblast growth factor 2 and its receptors in bovine follicles before and after GnRH application and after ovulation Reproduction, February 1, 2006; 131(2): 319 - 329. [Abstract] [Full Text] [PDF]
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K. Shirasuna, H. Asaoka, T. J. Acosta, M. P.B. Wijayagunawardane, M. Ohtani, M. Hayashi, M. Matsui, and A. Miyamoto Real-Time Relationships in Intraluteal Release among Prostaglandin F2{alpha}, Endothelin-1, and Angiotensin II During Spontaneous Luteolysis in the Cow Biol Reprod, November 1, 2004; 71(5): 1706 - 1711. [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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S.-i. Kobayashi, T. J. Acosta, T. Ozawa, K. Hayashi, B. Berisha, M. Ohtani, D. Schams, and A. Miyamoto Intraluteal Release of Angiotensin II and Progesterone In Vivo During Corpora Lutea Development in the Cow: Effect of Vasoactive Peptides Biol Reprod, January 1, 2002; 66(1): 174 - 179. [Abstract] [Full Text]
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K.H. Schauser, A.H. Nielsen, H. Winther, V. Dantzer, and K. Poulsen Localization of the Renin-Angiotensin System in the Bovine Ovary: Cyclic Variation of the Angiotensin II Receptor Expression Biol Reprod, December 1, 2001; 65(6): 1672 - 1680. [Abstract] [Full Text] [PDF]
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M. P.B. Wijayagunawardane, A. Miyamoto, Y. Taquahashi, T. J. Acosta, M. Nishimura, and K. Sato Angiotensin II and Atrial Natriuretic Peptide in the Cow Oviductal Contraction In Vitro: Direct Effect and Local Secretion of Prostaglandins, Endothelin-1, and Angiotensin II Biol Reprod, September 1, 2001; 65(3): 799 - 804. [Abstract] [Full Text] [PDF]
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J. A. Flores Gene Expression of Endothelin-1 in the Porcine Ovary: Follicular Development Biol Reprod, November 1, 2000; 63(5): 1377 - 1382. [Abstract] [Full Text]
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