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Regular Article |
a Departments of Theriogenology,
b Animal Science,
c Veterinary Pharmacology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
ABSTRACT
Angiotensin II (Ang II) and atrial natriuretic peptide (ANP) may be involved in local regulation of the oviductal contraction during the estrous cycle. Thus, the in vitro effects of Ang II and ANP on the secretion and contraction of bovine oviduct during the follicular, postovulatory, and luteal phases were investigated. An in vitro microdialysis system (MDS) was utilized to determine the intraluminal release of prostaglandins (PGs), Ang II, and endothelin-1 (ET-1) from the bovine oviducts as well as to observe the effect of Ang II and ANP on the local secretion of these substances. The basal release of PGs, ET-1, and Ang II was higher (P < 0.05) during the follicular and postovulatory phases than during the luteal phase. Stimulation by infusion of Ang II (10-6 M) or ANP (10-7 M) into the MDS was carried out for 4 h between 4 and 8 h of incubation. In the oviducts from the follicular and postovulatory phases, the infusion of ANP increased the release of Ang II, but not of ET-1. Infusion of Ang II stimulated the release of ET-1. Both Ang II and ANP increased PGE2 and PGF2
release. In the contraction study, direct administration of Ang II (10-7 M) or ANP (10-8 M) into the medium during the follicular and postovulatory phases increased the amplitude of oviductal contraction. In contrast, these substances did not show any effect in the contraction and secretion of oviducts from cows during the midluteal phase. These results indicate that during the periovulatory period, Ang II and ANP stimulate the contractile amplitude of the oviduct in vitro. In addition to their direct action on oviductal contraction, Ang II may activate oviductal secretion of ET-1 and PGs. Likewise, ANP stimulates oviductal secretion of PGs and Ang II. Hence, the overall results suggest the existence of a functional endothelin-angiotensin-ANP system in the bovine oviduct during the periovulatory period, which may regulate the oviductal contraction to ensure maximum efficiency of gamete/embryo transport through the oviduct.
hormone action, oviduct, ovum pick-up/transport
INTRODUCTION
The oviduct plays an essential role in the establishment of pregnancy by providing the proper environment for sperm transport and capacitation, oocyte transport and maturation, fertilization, and early embryonic development [1, 2]. The timing of the passage of the embryo into the endometrial environment is an essential step for establishment of implantation. Oviductal contraction and secretion are regulated by several factors, among which the key role of prostaglandins (PGs) and gonadal steroids are well established [3].
The spontaneous motility of the cow oviducts increases significantly following ovulation. The response to PGF2 is reduced during the follicular phase, but it increases markedly following both spontaneous and human chorionic gonadotropin-induced ovulation [4]. Our own recent study [5] suggested that the preovulatory LH surge stimulates the maximum oviductal production of PGs and endothelin-1 (ET-1), resulting in an active oviductal contraction for a rapid transport of gametes [5].
Vasoactive peptides such as angiotensin II (Ang II), atrial natriuretic peptide (ANP), and ET-1 have been involved in the autocrine/paracrine regulation of the oviducts [6–8]. Angiotensin-converting enzyme (ACE) has been found at high concentrations on the epithelial cells of the oviducts of nonpregnant sheep [9]. The ACE activity has also been found in the isthmus of the periovulatory sow oviduct [10]. Moreover, the ACE activity found in the ejaculate may modulate the local generation of Ang II and smooth muscle tonus of the uterus, and the oviduct could facilitate sperm transport [6]. On the other hand, the ANP system was suggested to exist in the rat oviduct, which is involved in regulation of the secretion and contraction of the oviduct [7]. In addition, we recently demonstrated that ET-1 stimulates the contraction and enhances the secretion of PGs in the bovine oviduct isolated at the follicular and postovulatory phases [5]. However, the role of Ang II and ANP in the local regulation of bovine oviductal secretion and contraction are still unclear.
Therefore, this study aimed to investigate in vitro 1) the effect of Ang II and ANP on the release of PGE2, PGF2, ET-1, and Ang II from epithelial cells of the intact oviduct using an in vitro microdialysis system (MDS) and 2) the direct effect of Ang II and ANP on the contraction of bovine oviductal segments isolated during the follicular, postovulatory, and luteal phases.
MATERIALS AND METHODS
Animals and Sample Collection
Reproductive tracts were collected from nonpregnant Holstein cows at a local slaughterhouse. The phase of the estrous cycle was determined according to the criteria described by Ireland et al. [11] by visual observation of the ovarian structures and the uterine mucus characteristics. The oviducts were classified as either follicular phase with a regressed corpus luteum (CL) and a Graafian follicle (Days 18–20), postovulatory phase with a corpus hemorrhagicum (Days 1–2), or luteal phase with an active CL (diameter, <2 cm, Days 11–17). They were then separated from the uterotubal junction and surrounding connective tissues and trimmed. Oviducts for MDS experiments were transported to the laboratory in Medium 199 (M199; 25 mM Hepes, Earle salts, 365 mg/L L-glutamine, 0.85 g/L of NaHCO3, 60 mg/L of penicillin, 100 mg/L of streptomycin, 56 mg/L of ascorbic acid, and 2 mg/L of amphotericin B, pH 7.4; Sigma Chemical Co., St. Louis, MO), which was maintained at 38°C. Oviducts for the contraction experiments were transported in ice-cold Locke Ringer solution (154 mM NaCl, 5.6 mM KCl2, 2.4 mM CaCl2, 6.0 mM HaHCO3, and 5.6 mM dextrose, pH 7.4). In a preliminary experiment, differences in response associated with the side of ovulation or region of the oviduct were not observed. Therefore, each oviduct was divided into two segments, and four oviductal segments from each cow were maintained in one organ-culture chamber. Each segment was assigned randomly to different treatments as described below. A small segment of the ampullary and isthmic regions of oviducts were used in the contraction experiment.
Estimation of the Doses of Substances Administered in MDS and Contraction Experiments
The concentrations of different substances used in this study were chosen based on measured concentrations in the bovine oviduct during the normal estrous cycle [12]. Diffusion through the MDS capillary membrane was estimated as 1% for PGs and 0.1% for peptides [13, 14]. Thus, the doses of Ang II and ANP used were 1000-fold greater than the concentrations estimated at the site of action.
In Vitro MDS of Bovine Oviduct
The in vitro MDS of the oviduct [5] was adapted from the method described for the bovine CL [13]. Briefly, the lumen of each 10-cm-long oviductal segment was implanted with a 7-cm-long, 0.2-mm-diameter dialysis capillary membrane (cutoff Mr = 40 kDa; Fresenius SPS 600 Hollow fibers; Fresenius AG, St. Wendel, Germany) with each end glued to 5-cm-long, 0.3-mm-diameter silastic tubing. Both ends of the oviduct were then fixed to the silastic tubing by Histoacryl Blau (B. Braun Melsungen AG, Melsungen, Germany) and incubated in M199 with 0.5% (w/v) BSA (Sigma Chemical Co., St. Louis, MO) in simple organ-culture chambers (modified 50-ml Falcon tubes; Becton Dickinson & Co., Franklin Lakes, NJ). Medium was continuously exchanged at a flow rate of 50 ml/h during the whole period of incubation at 38°C. Both ends of the silastic tubing were connected to Teflon tubing. Ringer solution was continuously perfused (1.3 ml/h) from one end using a multichannel peristaltic pump, whereas the other end was connected to a multichannel fraction collector. After a 2-h preincubation, the perfusate was collected in 4-h fractions for 16 h. Control (i.e., Ringer solution only) or Ang II (10-6 M; Peptide Institute, Inc., Osaka, Japan) or ANP (10-7 M; Peptide Institute) diluted in Ringer solution were infused for 4 h between 4 and 8 h of incubation. The 4-h fractions of perfusate were stored at -20°C until extraction for PGs and peptides. After the experiment, the MDS capillary membranes implanted in the oviduct lumens were confirmed to be in tight contact with the surface of the epithelial cells [5].
Hormone Extraction
The MDS fractions collected were warmed to room temperature, adjusted to pH 3.5 with 1 N HCl, and placed at room temperature for 1 h. The PG, ET-1, and Ang II extractions from MDS samples were performed as described earlier [15]. As a result of the extractions, PGs and peptides (ET-1 and Ang II) were concentrated 12- and 52-fold, respectively. Samples were dissolved in assay buffers for enzyme immunoassays (EIAs) for PGs (40 mM PBS, 0.1% [w/v] BSA, pH 7.2) and for peptides (42 mM Na2HPO4, 8 mM KH2PO4, 20 mM NaCl, 4.8 mM EDTA, 0.05% [w/v] BSA, pH 7.5), respectively. The recovery rates, which were estimated by adding three different concentrations of PGE2 (1, 0.5, and 0.1 ng/ml), PGF2 (1, 0.5, and 0.1 ng/ml), ET-1 (10, 5, and 1 pg/ml), and Ang II (100, 10, and 1 pg/ml) to the Ringer solution, were 78% for PGE2, 75% for PGF2, 63% for ET-1, and 90% for Ang II. The results presented are corrected for differences in the recoveries of PGs, Ang II, and ET-1.
PGs, ET-1, and Ang II Determinations
Concentrations of PGE2, PGF2, ET-1, and Ang II in Ringer extracts were determined in duplicate by second-antibody EIA after extraction using 96-well ELISA plates (Corning Glass Works, Corning, NY).
The EIA for PGE2 determination was performed as described previously [12]. The standard curve for PGE2 ranged from 30 to 14 200 pg/ml, and the median effective dose (ED50) of the assay was 350 pg/ml. The intra- and interassay coefficients of variation (CVs) were 7.3% and 11.4%, respectively. The method of EIA for PGF2 is described elsewhere [16]. 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 8.2% and 11.8%, respectively. The ET-1 assay was performed as described earlier [14], the standard curve ranged from 10 to 20 000 pg/ml, and the ED50 of the assay was 375 pg/ml. The intra- and interassay CVs were 9.6% and 13.2%, respectively. The EIA for Ang II was performed as described elsewhere [17]. The standard curve ranged from 2.5 to 2500 pg/ml, and the ED50 of the assay was 250 pg/ml. The intra- and interassay CVs were 5.6% and 9.2%, respectively.
Measurement of Oviductal Contraction
One end of each 10-mm-long oviductal segment was horizontally fixed through the lumen to a plastic holder, and the free end was attached to the arm of a force-displacement transducer (TB-625T; Nihon Koden, Tokyo, Japan) with a stainless-steel hook and a silk ligature. A basal loading tension of 1 gf was maintained throughout the experiment. The preparation was soaked in Locke Ringer solution in double-jacketed organ-culture chambers (Iwaki Glass Co., Chiba, Japan) and maintained at 36 ± 0.5°C (mean ± SEM). The temperature of the solution in the organ bath was held constant with an external water jacket and a thermoregulatory device (Thermominder Mini-80; Taiyo, Tokyo, Japan) and was monitored with a thermistor (Modal MAG-II; Shibaura Electric Co., Tokyo, Japan). The solution was bubbled with a mixture of 95% O2 and 5% CO2 and exchanged at a rate of 60 ml/h using a peristaltic pump. The signals were amplified by an isometric amplifier (RAT-1200M; Nihon Kohden), digitized with an analog-to-digital converter (MacLab/4S; AD Instruments Pty Ltd., Castle Hill, Australia), and stored on a hard disk of a personal computer (Macintosh PowerBook 190cs; Apple Computer, Inc., Cupertino, CA).
The preparation was equilibrated in a bathing solution for 2 h, and then the data were recorded for 7 h. After the start of the 7-h experimental period, Ang II (10-7 M) or ANP (10-8 M) were added to the Locke Ringer solution for 1 h between 1 and 2 h. Controls were maintained only in Locke Ringer solution throughout the complete period of the experiment.
Statistical Analysis
The mean concentrations of each substance in the first 4-h fraction (i.e., perfusion with Ringer 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 capillary membranes implanted in the various oviducts. All values were expressed as a percentage of the corresponding baseline. The CVs in the absolute concentration of each hormone in the MDS perfusates collected from different oviducts during the first 4 h (i.e., baseline) were as follows: PGE2, 80–254%; PGF2, 72–243%; ET-1, 35–62%; and Ang II, 23–54%. This transformation enables an evaluation of relative changes of hormonal values between the different oviducts. The effects of the infused substances (Ang II and ANP) on the release of PGE2, PGF2, ET-1, and Ang II were compared with control values obtained during the same time period using ANOVA followed by Duncan new multiple-range test. Differences were considered to be significant at a probability of less than 5% (P < 0.05).
Amplitude and frequency of oviductal contractions in the control and treatment groups were averaged over 10-min intervals and analyzed using the MacLab Chat data acquisition/analysis program (AD Instruments) [18]. The basal oviduct amplitude varied considerably among individual oviducts. Therefore, the baseline of oviduct amplitude (defined as 100%) was calculated by averaging the data for the first 1 h, and the amplitude of each 10-min interval was then expressed as a percentage of this individual baseline. The effects of the added substances on the contraction were compared with control values obtained during the same time point using ANOVA followed by Duncan new multiple-range test. Probabilities of less than 0.05 (P < 0.05) were considered to be significant.
RESULTS
The basal release of each substance into the MDS capillaries in the control group (perfused with Ringer solution only) as well as the spontaneous amplitude and frequency of the oviductal contraction in the control group (maintained in Locke Ringer solution only) were constant and stable during the experimental periods. The basal release of all substances measured and the spontaneous amplitude and frequency of the oviductal contractions were higher (P < 0.05) during the follicular and postovulatory phases than during the luteal phase (Table 1). Moreover, the contraction pattern of the oviducts was more regular during the follicular and postovulatory phases than during the luteal phase.
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Effect of Ang II and ANP on PG Secretion from Microdialyzed Oviducts
Both Ang II and ANP stimulated the release of PGE2 (Fig. 1) and PGF2 (Fig. 2) (P < 0.05–0.001) in the oviduct from cows in the follicular and postovulatory phases. Neither Ang II nor ANP stimulated all substances measured during the luteal phase (Figs. 1 and 2).
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Effect of Ang II and ANP on ET-1 Secretion from Microdialyzed Oviducts
Angiotensin II stimulated ET-1 release (P < 0.05) in the follicular and postovulatory phases. The ET-1 release was not affected by ANP infusion at any stage of the estrous cycle (Fig. 3).
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Effect of ANP on Ang II Secretion from Microdialyzed Oviducts
The infusion of ANP significantly increased Ang II release in the oviducts from the postovulatory (P < 0.05) and follicular (P < 0.001) phases (Fig. 4). Neither Ang II nor ANP stimulated the secretion of any of the above-measured substances during the luteal phase (Figs. 1–4).
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Effect of Ang II and ANP on Oviductal Contraction
Administration of Ang II and ANP into the medium increased significantly (P < 0.05) the amplitude of contraction in both follicular- and postovulatory-phase oviducts. The stimulation of the amplitude of oviductal contraction by Ang II persisted for as long as 30–50 min, whereas ANP stimulation persisted for as long as 80–90 min after treatment. None of these treatments altered the basal frequency of the oviductal contraction during any stage of the estrous cycle. Moreover, in the oviducts from the luteal phase, the above treatments did not affect the amplitude or frequency of contractions (Fig. 5).
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DISCUSSION
The results of this study provide, to our knowledge, the first evidence that Ang II and ANP enhance the in vitro secretion of PGE2 and PGF2 as well as the contractile amplitude of bovine oviducts from the follicular and postovulatory phases, but not those from the luteal phase. Furthermore, ANP increased the Ang II secretion, and Ang II stimulated the release of ET-1 in the MDS experiment. We recently reported that the bovine oviductal tissue contains the highest concentrations of PGs and ET-1 during the periovulatory period [12]. This was confirmed by the present results, in which the secreted levels of PGs and ET-1 were also higher during the follicular and postovulatory phases than in the luteal phase.
Several lines of evidence suggest that Ang II is involved in the contraction and that ANP is responsible for the relaxation of smooth muscles. Angiotensin II dose dependently increases contractile activity in the pregnant rat uterus [19] and human uterine artery [20]. On the other hand, it was reported that ANP induced the relaxation of cultured vascular smooth muscles [21] and guinea pig cecal smooth muscles [22]. Furthermore, ANP has a vasorelaxing effect on human uteroplacental vessel [23]. In the present contraction study, however, ANP clearly stimulated the contractile amplitude of the oviducts from the follicular and postovulatory phases. It has been reported that the concentration of ANP and its receptor in the rat myometrial smooth muscle layers are highest during the periovulatory period [24], when the peak uterine motility in rat is reported [25, 26]. Interestingly, ANP stimulated Ang II and PG release in the present study. Collectively, the above observations suggest that the high levels of ANP and its receptor during the periovulatory period may directly modify the tonus and increase the amplitude of the oviductal contraction, acting directly on the myosalpinx or indirectly by stimulating Ang II and PG production by the endosalpinx in an autocrine and/or paracrine manner.
Angiotensin 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 result principally from a stimulation of ET-1 release by a mechanism involving receptor-mediated mobilization of intracellular Ca2+ and activation of protein kinase C [27] as well as activation of preproendothelin-1 mRNA expression [28]. It was also reported that Ang II increased PG production in bovine mature follicles [29] and cultured astrocytes [30]. The observation in the present MDS study that Ang II stimulates the secretion of PGs and ET-1 is comparable with the above results. On the other hand, it has been reported that ANP stimulates renin release from the isolated rat kidney [31]. Similarly, stimulatory effects of ANP on the oviductal secretions of PGs and Ang II were observed in the present study. Furthermore, ANP has been shown to inhibit the synthesis and release of ET-1 in cultured endothelial cells [32, 33]. In the present study, ANP did not affect the ET-1 secretion in the oviduct, suggesting a possible tissue-specific interaction.
The steroid hormones play an important modulatory role in oviductal secretion and contraction. Our previous study using cow oviductal epithelial cell culture indicated that the administration of estradiol (E2) increased the production of oviductal PGs and ET-1, and that the simultaneous administration of progesterone (P4) blocked the effect of E2 [34]. The P4 modulates the action of E2 by a rapid reduction of the estrogen receptor concentration [35], and it makes the tissue less responsive to E2 [36]. Moreover, P4 blocks the effect of E2 on protein secretion and oviductal epithelial cell differentiation [37]. In addition, P4 reduces PG production by lowering phospholipase A2 activity in the oviductal epithelial cells [38]. The concentration of binding sites for Ang II was highest in the estrogen-dominant myometrium of women, and P4 blunted this estrogenic effect [39]. It was also reported that Ang II receptor in bovine theca cells is up-regulated by LH [40]. The fact that Ang II and ANP stimulated both the secretion and the contractile activity of oviducts only during the periovulatory period suggests that these effects reflect the high levels of functional binding sites for the vasoactive peptides during this period after the LH surge.
In vivo studies reported that the amplitude of oviductal contraction was low during the luteal phase, increased 3–5 days before estrus, reached a maximum during estrus, and diminished progressively 3–5 days after estrus in the cow [4, 41]. Bennet et al. [4] suggested that the higher level of motility in the oviduct ipsilateral to the active ovary may be due to a local effect mediated by the functional ovary or the ovum. However, in the present study, we did not detect any clear differences in the action of Ang II and ANP on oviductal contraction associated with the side of ovulation or region of the oviducts. Therefore, the phase of the estrous cycle appears to be one of the main factors determining the response of the oviductal smooth muscle to these vasoactive peptides in vitro.
We demonstrated that LH enhances ET-1, PGE2, and PGF2 secretion, and that these substances increase the amplitude of the contraction of oviducts from the follicular and postovulatory phases in the same in vitro models [5]. However, these substances did not affect the secretion and contraction of oviducts from the luteal phase. In the present study, ANP stimulated fallopian Ang II release, and Ang II increased ET-1 secretion. Based on these data, we propose the existence of a dynamic interaction among these peptides that may locally amplify the effect of the LH surge by enhancing ET-1 and PG secretion. Besides their direct effect on the smooth muscle, Ang II and ANP may collaborate with the ovarian steroids and LH to increase the secretion of PGs and ET-1 and to maximize the oviductal contraction observed during the periovulatory period [4, 41]. The PGs modulate oviductal contraction through the effects of E-series PGs on relaxation of smooth muscle and of F-series PGs on contraction [3]. Human oviductal musculature responds to PGF2 by increasing tonus, and PGF2 is recognized as a prerequisite for maintenance of normal tubular contractions [42].
These results indicate that during the periovulatory period, Ang II and ANP stimulate the contractile amplitude of the oviduct in vitro. In addition to the direct action on oviductal contraction, Ang II may activate oviductal secretion of ET-1 and PGs; likewise, ANP stimulates oviductal secretion of PGs and Ang II. Hence, the overall results suggest the existence of a functional endothelin-angiotensin-ANP system in the bovine oviduct during the periovulatory period, which may regulate the oviductal contraction to ensure maximum efficiency of gamete/embryo transport through the oviduct.
ACKNOWLEDGMENTS
The authors wish to thank Dr. D. Schams, Technical University of Munich, Germany, for ET-1 antiserum; Dr. S. Ito, Kansai University of Medicine, Japan, for PG antiserum; Dr. K. Wakabayashi, Gunma University, Japan, for Ang II antiserum; and Fresenius AG, St. Wendel, Germany for the MDS capillary membranes.
FOOTNOTES
First decision: 16 January 2001.
1 Correspondence. FAX: 81 155 49 5462; akiomiya{at}obihiro.ac.jp 
2 Current address: Department of Animal Science, University of Peradeniya,
Peradeniya, Sri Lanka.
Accepted: April 19, 2001.
Received: December 4, 2000.
REFERENCES
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