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b Departments of Theriogenology
c and Animal Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF), and interleukin-1ß (IL-1ß), a microdialysis system was implanted into the theca layer of preovulatory bovine follicles that were maintained in organ culture chambers. The effects of LH, ET-1, TNF, and IL-1ß on the local release of steroids, prostaglandin E2 (PGE2), and ET-1 from the cells surrounding the implanted capillary membrane were investigated.
Each preovulatory follicle (selected based on the concentrations of steroids and PGE2) was dissected from surrounding stromal tissue and implanted with 4 capillary dialysis membranes (control, LH, cytokines or ET-1, and LH+cytokine or LH+ET-1) into the theca layer. They were then incubated in organ culture chambers and perfused with Ringer's solution for 14 h after pre-perfusion for 2 h. The stimulation with LH (5 µg/ml) between 4 and 6 h increased the release of progesterone (P4), androstenedione (A), estradiol-17ß (E2), PGE2 (p < 0.001), and ET-1 (p < 0.05). The infusion of ET-1 (250 ng/ml) between 8 and 10 h inhibited P4 and A release but stimulated E2 release (p < 0.05). The infusion of TNF (100 ng/ml) between 8 and 10 h after LH exposure suppressed the release of A and E2 (p < 0.05). IL-1ß (10 ng/ml) between 8 and 10 h stimulated E2 release but inhibited A release (p < 0.05). Moreover, ET-1 and cytokines clearly stimulated PGE2 release (p < 0.05). ET-1 and TNF induced further release of PGE2 stimulated by LH (p < 0.05). Also, TNF and IL-1ß induced further release of ET-1 stimulated by LH (p < 0.05).
These results show that ET-1 is released from the theca layer of mature bovine follicles in vitro and that it affects follicular steroids and PGE2 secretion. The overall results suggest that interactions among ET-1, PGE2, and cytokines may have key roles in a local intermediatory/amplifying system of the LH-triggered ovulatory cascade in the bovine preovulatory follicle.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ovarian follicle has been reported to be a site of synthesis, reception, and action of endothelin-1 (ET-1) [8, 11–13], tumor necrosis factor (TNF) [10, 14, 15], and interleukin-1ß (IL-1ß) [16]. These peptides directly affect the secretory function of theca and/or granulosa cells with a stimulatory or inhibitory effect on prostaglandin biosynthesis and steroidogenesis [10, 12, 15]. Furthermore, ET-1 inhibits cAMP-mediated progesterone production in LH-stimulated granulosa cells in the rat [8, 11] and the pig [17]. However, the specific roles of ET-1 and its interrelationships with LH, cytokines, and PGE2 during the preovulatory period in the bovine follicle still remain unclear.
Therefore, we attempted to observe the local secretion of ET-1 and its effect on the release of steroid hormones and PGE2 in the theca layer of bovine mature follicles in vitro. The study was further extended to examine the possible interaction of ET-1 with LH, TNF, and IL-1ß. For this purpose, a microdialysis system (MDS) was implanted into the theca layer of isolated bovine mature follicles, where the cells maintain the integrity of follicular structures, thus enabling a real-time observation of local release of substances that may play a role in cell-to-cell communication, as was originally done in freely moving pigs [18].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries from Holstein cows containing a mature, presumably preovulatory follicle were collected within 20 min after slaughter from a local slaughterhouse and were transported to the laboratory in sterile saline solution (0.9% NaCl) containing 100 000 IU penicillin and 100 mg/L streptomycin at 38°C. Ovaries were visually inspected. The preovulatory stage was defined by the presence of a graafian follicle (between 1.5 and 2.0 cm in diameter) and a regressing corpus luteum in the ipsilateral or contralateral ovary [19]. The uterine characteristics (size, color, tonus, consistency, and mucus) were also considered. Furthermore, at the end of the experiment, follicular fluid from each follicle was collected and the follicular wall was prepared for routine histological observation. Steroid (progesterone, P4; androstenedione, A; and estradiol-17ß, E2), PGE2, and ET-1 concentrations in the follicular fluid were determined after extraction as described below. The concentrations are shown in Table 1. Only dominant follicles that had not yet been exposed to the endogenous LH surge were used in this experiment. Such follicles were identified by the presence of a PGE2 concentration in the follicular fluid of less than 2000 pg/ml. The position of implantation of the MDS capillary membrane in the theca layer was also confirmed with a light microscope. Furthermore, to confirm that the MDS was functional in the theca layer, pregnenolone (10-7 M) was infused for 4 h into the MDS, and A and E2 releases were determined. A significant increase in A release (179 ± 19%, mean ± SEM, of baseline; p < 0.01) but not E2 release (108 ± 14% of baseline) was observed, which is a typical response of theca cells.
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MDS In Vitro
The MDS for bovine mature follicles was based on the method developed for porcine follicles in vivo [18], with some modifications for an in vitro organ culture system [20]. Basically each follicle was dissected from surrounding stromal tissue, and four capillary dialysis membranes (Fresenius SPS 900 Hollow Fibers, cutoff molecular size 1000 kDa, 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. The four capillaries (for control, LH, cytokines or ET-1, and LH+cytokine or LH+ET-1) were affixed to the surface of the follicular tissue by Histoacryl blue (B. Braun-Dexon GmbH, Spangenberg, Germany). Both ends of the capillary were glued to silicone elastomer tubing (i.d. 0.3 mm). For perfusion, one end of the tube was connected to a peristaltic pump and the other was routed to a 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, follicles were perfused with Ringer's solution at a flow rate of 1.8 ml/h. After 2 h of pre-perfusion, fractions of the perfusate were collected every 2 h up to 14 h. These conditions were selected because the follicular tissues were found to produce a constant release of steroids, PGE2, and ET-1. Collected samples were stored at -20°C until hormone determination.
Bovine LH (USDA-bLH-B-6), human ET-1 (Peptide Institute Inc., Osaka, Japan), recombinant human TNF, and recombinant human IL-1ß (both donated by Dainippon Pharm. Co., Osaka, Japan) were diluted in Ringer's solution to obtain the required final concentrations of 5 µg/ml, 250 ng/ml, 100 ng/ml, and 10 ng/ml, respectively. The doses were determined based on the result of a preliminary experiment and on the transfer capacity of the membrane, which was previously estimated to be 1% for steroids and prostaglandins and 0.1% for peptides and LH [20, 21]. The solutions were then infused into the MDS for 2 h (LH between 4 and 6 h; ET-1, TNF, or IL-ß between 8 and 10 h).
Steroids and PGE2 Extraction
The perfusates (3 ml) and follicular fluids (300 µl) were adjusted to pH 3.5 using 1 N HCl and 5 N HCl, respectively, and allowed to stand at room temperature for 1 h. Then 3 volumes of diethyl ether was added and the mixture was shaken for 30 min at a frequency of 250 cycles/min (using Shaker SA 31; Yamato Co., Tokyo, Japan). The samples were then allowed to stand for 15 min and placed in a -80°C freezer for 1 h. The upper diethyl ether fraction was decanted and evaporated. The residue was dissolved in 300 µl assay buffer for steroid and prostaglandin enzyme immunoassays (EIAs) (40 mM PBS, 0.1% BSA, pH 7.2). To estimate the recovery rate, A, P4, E2, and PGE2 were added to the Ringer's solution (30, 100, 20, and 100 pg/ml, respectively), and the obtained values were 85%, 88%, 75%, and 72%, respectively.
ET-1 Extraction
After the diethyl ether extraction, the remaining Ringer's solution was used for ET-1 extraction. BSA 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 acetic acid. The samples were then applied to a Sep-Pak C18 Cartridge (Waters, Millford, MA) as described previously [21]. 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 EIA. Thus, the samples were concentrated about 15-fold as a result of the process. The recovery rate of ET-1 (10 pg/ml) that had been added to the Ringer's solution was 63%.
Hormone Determinations
The concentrations of the various hormones were determined in duplicate by double-antibody EIAs using 96-well ELISA plates (Corning Glass Works, Corning, NY).
The EIA for P4 was done as previously described [22]. The standard curve ranged from 0.05 to 25 ng/ml, and the ED50 of the assay was 2.8 ng/ml. The average intra- and interassay coefficients of variation (CVs) were 5.8% and 8.2%, respectively.
The EIA for E2 was carried out as described previously [23]. The standard curve ranged from 2 to 2000 pg/ml, and the ED50 of the assay was 120 pg/ml. The intra- and interassay CVs were 6.1% and 8.9%, respectively.
The A EIA was identical to the P4 EIA. Basically, standards or samples were incubated with 100 µl polyclonal antibody (raised in a rabbit against A-3-CMO-BSA; Cosmo Bio Co., Tokyo, Japan) solution (1:500 000) and 100 µl A-3-CMO-horseradish peroxidase (1:70 000; Cosmo Bio Co.) for 16 h at 4°C. The standard curve ranged from 2 to 1000 pg/ml, and the ED50 of the assay was 130 pg/ml. The intra- and interassay CVs were 6.8% and 8.1%, respectively. The cross-reactivities of the antibody were 100% for A, 35% for 5-androstenedione, 4.5% for dehydroepiandrosterone (DHA), 1.5% for A, 1.2% for P4, 6.0% for testosterone, 0.3% for cortisol, 0.2% for corticosterone, and 0.01% for estradiol.
The EIA for PGE2 is described elsewhere [23]. The standard curve 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 cross-reactivities of the antibody were 100% for PGE2, 60% for PGB2, 15% for PGE1, 4% for PGA2, 3% for PGF2, 0.1% for PGD2, and 0.04% for 15-keto-PGE2.
The EIA for ET-1 was performed as described previously [21]. The standard curve ranged from 10 to 20 000 pg/ml. The intra- and interassay CVs were 8.5% and 13.5%, respectively.
Statistical Analysis
The mean hormone (P4, A, E2, PGE2, and ET-1) concentrations in the first two perfusates (first 4-h perfusion with Ringer's solution only) were used to calculate the individual baselines because of a large variation in the absolute amount of hormones released into each of the MDS lines implanted in the various follicles. All hormone concentrations in the perfusate fractions were expressed as a percentage of the baseline levels. To simplify the view of figures, the values of the first two perfusates were pooled, and they are shown in a single column as a baseline (100%). The effects of the infused substances (LH, ET-1, TNF, and IL-1ß) on the releases of steroids, PGE2, and ET-1 were tested by comparison with the individual baselines using an analysis of variance 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Infusion of LH clearly increased the release of P4, A, E2, and PGE2 (201 ± 15% for P4, 163 ± 9% for A, 186 ± 11% for E2, and 212 ± 12% of baseline for PGE2; mean ± SEM; p < 0.001). The increase in A release was observed only during the LH infusion, while the release of P4 remained at higher levels until 2 h after LH infusion. LH infusion induced increases in the release of both E2 (p < 0.05; Figs. 1–3) and PGE2 (p < 0.05; Fig. 4), and these stimulatory effects were maintained until the end of the experiment. The release of ET-1 was also increased after LH exposure (165 ± 23% of baseline; mean ± SEM; p < 0.05) and remained at high levels until the end of the experiment (p < 0.05; Fig. 5).
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Effect of ET-1, TNF, and IL-1ß on the Release of Steroid Hormones
ET-1 inhibited the release of A and P4, but it stimulated the release of E2 (p < 0.05; Fig. 1). The decrease in the release of A was observed only during the ET-1 infusion. However, the effect on P4 and E2 remained for a prolonged period. TNF alone did not affect basal production of P4, A, or E2, but it suppressed the LH-stimulated release of A and E2 (p < 0.05; Fig. 2). IL-1ß inhibited the release of A but stimulated the release of E2 (p < 0.05) without having any distinct effect on the release of P4 (Fig. 3).
Effects of ET-1, TNF, and IL-1ß on the Release of PGE2
ET-1, TNF, and IL-1ß stimulated PGE2 release for a prolonged period (p < 0.05; Fig. 4). The infusion of ET-1 or TNF after LH exposure induced a further increase of PGE2 release as compared with LH infusion alone (p < 0.05; Fig. 4).
Effects of LH and Cytokines on the Release of ET-1
LH clearly stimulated ET-1 release, and the effect continued during the period of the experiment (p < 0.05; Fig. 5). The infusion of TNF- or IL-1ß after LH exposure further increased the ET-1 release as compared with LH infusion alone (p < 0.05). However, TNF or IL-1ß alone did not have any noticeable effect on ET-1 release.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microscopic observations, as well as the selective increase of the release of A rather than the release of E2 in response to pregnenolone infusion, confirmed that the MDS capillary membranes were placed in the theca layer, where the predominant secretory product is A [24, 25]. This is further supported by the fact that the basal concentration of A in the perfusate was more than 2-fold higher than that of E2. LH and the peptides infused into the capillary membrane are considered to diffuse along a concentration gradient into the extracellular fluid and modify the cellular secretory function in a paracrine manner [26]. Based on the observation from the preliminary experiment that substances infused in one line did not affect the hormonal release in other lines separated by at least a 5-mm distance, a maximum of four lines per follicle were implanted. The individual response of each line to the infused peptides suggests that the microenvironment of each line was independent from that of other lines.
LH stimulated the release of steroids (P4, A, and E2) and PGE2 from the theca layer. The results are comparable to those of previous studies on isolated preovulatory follicles from rabbits [1], on the capacity for prostaglandin synthesis in preovulatory follicles from sheep [2], and on bovine theca cell cultures, in which LH increased the release of E2, A, P4, and PGE2 in vitro [3, 4]. In the present model, it is likely that an increase of local P4 concentration by LH stimulation accelerates the cascade of steroid biosynthesis and that this influence could overcome the well-known direct inhibitory effect of LH on the E2 secretion, thus resulting in the increase in both A and E2 secretion. Additionally, the present results provide evidence that LH stimulates the release of ET-1 from the theca layer of bovine mature follicles in vitro. Similar findings were shown in a study by Girsh et al. [27] in which LH stimulated the ET-1 content in a bovine corpus luteum slice incubation. The effect may be a direct stimulation of LH on the endothelial cells in the theca layer, since we could not find measurable concentrations of ET-1 in the medium used to culture bovine granulosa and theca cells (unpublished results). To support this hypothesis, mRNA for LH receptor must be detected in endothelial cells in the theca layer, which requires further investigation.
As expected, a significant concentration of ET-1 was found in the perfusate from the theca layer. Based on the ET-1 concentration in the perfusate (2–7 pg/ml) and the transfer capacity of the microdialysis membrane for ET-1 (0.1%), the possible concentration of ET-1 in the extracellular fluid in the theca layer can be calculated to be in the range 2–7 ng/ml. This range is almost the same as that of the intraluteal ET-1 concentration as previously reported [21] but about 100-fold higher than plasma ET-1 concentrations in the cyclic cow [28]. These findings strongly support the concept that the bovine mature follicle is also a site of ET-1 production. A recent study by our group revealed that plasma ET-1 concentration increases during PGF2-induced luteolysis as well as during the periovulatory period [28]. These findings suggest that ET-1 may play an important role in ovarian functions in relation to acute changes in blood capillary functions [29] during those periods.
ET-1 affected steroidogenesis in the present MDS study. A previous study using a rat granulosa cell culture showed a direct action of ET-1 on steroidogenesis. This action involves selective modulation of key steroidogenic steps concerned with formation and degradation of steroids: ET-1 attenuates enzymatic action common to P4 and A production, such as that of cholesterol side-chain cleavage and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but increases the activity of those enzymes that participate in their degradation (5-reductase and 3ß-HSD) [11]. Indeed, ET-1 also inhibited the release of P4 and A from the bovine ovary in the present MDS study. In contrast, E2 release was stimulated by ET-1 for a prolonged period, suggesting that ET-1 may stimulate aromatase activity. Although the effect of ET-1 on the release of E2 in the MDS might be through an indirect pathway, a direct stimulatory effect of ET-1 on the E2 secretion is also highly possible. Specifically, some of the ET-1 infused into the MDS may pass through the theca interna into the granulosa cell layer, so that it may directly stimulate the E2 secretion from granulosa cells. This would be a better explanation for the opposite effects of ET-1 on P4 and A secretion (inhibition) and E2 secretion (stimulation) that were observed in the present study. Moreover, ET-1 stimulated PGE2 release in this study. ET-1 has been shown to activate multiple biochemical pathways within the target cells including phospholipase C, phospholipase D, and the arachidonate cascade [30]. Theca cells as well as endothelial cells and granulosa cells could be responsive to such activations, since all these cell types are capable of PGE2 production (our unpublished results).
TNF and IL-1ß were shown to stimulate P4 secretion in the isolated rat ovary perfused in vitro [9] and in a rat follicle culture [15], whereas they did not exert any clear effect on basal or LH-stimulated P4 production in the present MDS study. Our data are partly comparable with the results of a study of porcine theca cells [31] in which TNF- did not affect basal secretion of P4 from the cells but caused a marked dose-dependent inhibition of LH-stimulated P4 production. As the present data are the first on the effects of cytokines in an MDS study in the bovine follicle, a detailed evaluation of these effects must await an accumulation of data from different systems of bovine follicles, rather than from other species.
Theca cells in the preovulatory follicle have been demonstrated to release prostaglandin upon cytokine stimulation in vitro [9]. The present MDS study demonstrated that LH increased local ET-1 release from the theca layer and that ET-1 stimulation after LH exposure induced a further PGE2 release. These results strongly suggest that ET-1 locally regulates the LH-triggered ovulatory process. ET-1 and PGE2 could possibly act synergistically to control blood flow, modify vascular permeability, and activate proteolytic enzymes involved in follicular rupture. LH-induced PGE2 release was amplified also by TNF. This local increase in PGE2 may modulate collagenolytic activity and hemodynamic changes associated with the ovulatory process [32, 33].
In conclusion, ET-1 is released from the theca layer of mature bovine follicles in vitro and affects follicular steroids and PGE2 secretion. The overall results suggest that interactions among ET-1, PGE2, and cytokines may play key roles in a local intermediatory/amplifying system of the LH-triggered ovulatory cascade in the bovine preovulatory follicle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted: April 3, 1998.
Received: December 29, 1997.
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H. Hirayama, K. Sawai, S. Moriyasu, M. Hirayama, Y. Goto, E. Kaneko, A. Miyamoto, K. Ushizawa, T. Takahashi, and A. Minamihashi Excess estrogen sulfoconjugation as the possible cause for a poor sign of parturition in pregnant cows carrying somatic cell clone fetuses Reproduction, November 1, 2008; 136(5): 639 - 647. [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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