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Endocrinology Vol. 140, No. 6 2679-2684
Copyright © 1999 by The Endocrine Society

ARTICLES

Characterization and Localization of ß2-Adrenergic Receptors in the Bovine Oviduct: Indication for Progesterone-Mediated Expression1

R. Einspanier, C. Gabler, A. Kettler and W. Kloas

Institute of Physiology, Technical University Munich-Weihenstephan (R.E., C.G., A.K.), Freising, D-85350 Germany; and the Department of Zoology II, University of Karlsruhe (W.K.), D-76128 Karlsruhe, Germany

Address all correspondence and requests for reprints to: Dr. R. Einspanier, Institute of Physiology, Technical University Munich, Weihenstephaner Berg 5, D-85350 Freising, Germany.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ß2-Adrenergic receptors were detected in bovine oviductal epithelium by use of receptor binding studies and expression analysis. Complementary DNA cloning gave use to the first full-length bovine ß2-adrenoceptor messenger RNA sequence (2030 bases). Receptor bioactivity in oviduct epithelial cells was characterized by specific ligand interaction and consequent cAMP generation. Expression studies demonstrated an estrous cycle-dependent regulation, with higher transcript levels and significantly increased binding capacity during the luteal phase. After progesterone supplementation, oviduct epithelial cells showed elevated receptor expression in culture, supporting the hypothesis that progesterone up-regulates the ß2-adrenergic receptor within these cells. It seems likely that catecholamines from the circulation or from innervation might be able to influence reproductive success by regulating oviductal secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FEMALE reproductive tract is controlled by a finely balanced interaction of different hormone systems. At the site of fertilization of the mammalian egg and subsequent development of the early embryo, the oviduct provides an optimum local environment, which is thought to be modulated by a variety of hormones. Especially the adrenergic hormone system is known to rapidly affect metabolic turnover as well as smooth muscle contractility in a tissue-specific manner. Therefore, small and specific effects of catecholamines might be of considerable physiological interest also in the regulation of reproductive organs. In this context, it has been suggested that ovum transport could be mediated through changing adrenoceptors in the oviduct muscle layer (1). Another important property of catecholamines to be recognized in reproductive tissues was their ability to stimulate steroid hormone secretion, for example progesterone in the human, rat, or bovine ovary (2, 3, 4). Furthermore, ß-adrenergic receptors were found on porcine ovarian luteal cells, with significant physiological variations during the development of the corpus luteum (5). Although changing catecholamine levels are found during the sexual cycle in human and porcine oviducts (6, 7), a biological function distinct from muscle constriction remains speculative. Up until now, the ß-adrenoceptor has only been described in the muscle cell layer of the oviduct or uterus, implying an inhibitory effect on contractility during the sexual cycle (8).

The aim of the present study was to investigate possible adrenergic reception in the bovine oviductal epithelium. Thereafter, we analyzed the complete molecular structure of the ß2-adrenoceptor and its localization and bioactivity. Finally, the effects of steroid hormones on ß2-receptor expression were investigated in the oviduct at the site of fertilization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue preparation and cell culture
Oviducts and liver pieces from German Fleckvieh cows were collected at a local slaughterhouse within 20 min of death. The stage of the cycle was defined from the appearance of the ovaries and uterus (9). The oviducts were accordingly classified into four groups: secretory phase (postovulation on days 1–5 and early to midluteal stage on days 6–12), proliferative phase (late luteal stage on days 13–18 and preovulatory stage on days 19–21). Where required, ampulla and isthmus were separated. For RNA analysis of the epithelial layer, the oviducts were flushed twice with 1 ml Ringer solution (Fresenius AG, Bad Homburg, Germany). After centrifugation at 570 x g for 3 min at 4 C, cell pellets were stored at -80 C. The identity of these flushed cells was verified as described previously (10), and as the majority (>80%) of the flushed cells have an epithelial phenotype, they are referred to here as oviduct epithelial cells. The viability of the cells used for cell culture was checked by microscopic observation of beating cilia as well as the exclusion of trypan blue. Epithelial cells (105/ml) were cultured in medium 199 (Sigma Chemical Co., Deisenhofen, Germany) containing 2% FCS and a mixture of antibiotics (100 µg/ml streptomycin and 10 U/ml penicillin G) at 39 C in 5% CO2 and saturated humidity with increasing progesterone (1–50 ng/ml) and estradiol (1–10 pg/ml) concentrations or with adrenaline (10-4-10-6 M) for the cAMP assay. For immunohistochemistry, fresh oviducts were dissected into ampulla and isthmus, mounted in Tissue-Tek (Miles, Inc., Elkhart, IN), and stored at -80 C until examined. Total protein was determined by the bicinchonic acid assay using BSA as standard (11).

Messenger RNA (mRNA) analysis and complementary DNA (cDNA) cloning
Total RNA was isolated from epithelial cells after flushing oviducts twice with 1 ml Ringer’s solution (>80% epithelial character) or liver following the single step method of Chomczynski and Sacchi (12) using Trizol reagent (Life Technologies, Gaithersburg, MD). Total RNA was spectroscopically quantified at 260 nm. The quantity and quality of oviductal RNA (8 µg) were verified after electrophoresis on a 1% (wt/vol) denaturing agarose gel followed by ethidium bromide staining. Northern blot analysis was thereafter performed using a ß2-receptor-specific PCR-probe labeled with digoxigenin to detect specific mRNAs, as described by the supplier (Boehringer Mannheim, Mannheim, Germany).

Four micrograms of oviductal or liver total RNA were used for RT-PCR to first generate single strand cDNA in a 60-µl reaction mixture as previously described (10). Commercially synthesized PCR primers (Pharmacia Biotech, Piscataway, NJ) were used to amplify bovine transcripts specific for {alpha}2- and ß2-adrenergic receptors, each newly designed on the basis of cross-species sequence homologies: ß2-adrenergic receptor: forward, 5'-AGACTTTAGGCATTATCATGGG-3'; reverse, 5'-TGGAAGGCAATCCTGAAATC-3'; and {alpha}2-adrenergic receptor: forward, 5'-GAAGAGAGGCAGGAGTGGC-3'; reverse, 5'-TTGGCGATCAGGTAGATGC-3'.

All cDNA samples were assayed for integrity and relative quantity by performing PCR for ubiquitin mRNA using the following primers: forward, 5'-ATG CAG ATC TTT GTG AAG AC-3'; and reverse, 5'-CTT CTG GAT GTT GTA TC-3'. The predicted sizes of the resulting RT-PCR products were 192 bp for the ß2-adrenergic receptor, 150 bp for the {alpha}2-adrenergic receptor, and 189 and 417 bp for ubiquitin, corresponding to identical multimeric gene cassettes.

PCR reactions were performed as described previously (10) in a final volume of 25 µl. Individually adjusted amplification programs were applied for ß2- or {alpha}2-receptors (28 or 35 cycles at 94 and 60 C, 1 min each) and ubiquitin (24 cycles at 94, 55, and 72 C, 45 sec each). Subsequently, 5 µl PCR product were subjected to 1.5% agarose gel electrophoresis followed by ethidium bromide staining. For each phase of the estrous cycle, five independent PCR reactions were carried out. Sequence determination of the complete ß2-receptor was performed after generating a complex bovine oviductal {lambda} cDNA library ({lambda} gt11 kit, Stratagene, San Diego, CA), successfully screened for a large ß2-receptor-specific clone by PCR as previously described (13). This clone as well as each RT-PCR product were sequenced commercially (TopLab, Munich, Germany). Subcloning of the ß2-adrenoceptor cDNA into a transcription vector (pCR-Script, Stratagene) enabled the generation of synthetic standard complementary RNA (cRNA) for relative RT-PCR quantitation.

In vitro autoradiography
Ten-micron oviduct sections were thaw-mounted onto gelatin-coated glass slides and dried under vacuum at 4 C. After blocking, sections were incubated with 150 pM [125I]iodocyanopindolol (125ICP; 2200 Ci/mmol; Amersham, Braunschweig, Germany) for 3 h at 37 C, washed, and air-dried according to the method of Booze (14). Nonspecific binding was detected in parallel on adjacent sections, but in the presence of the nonselective ß-adrenoceptor antagonist propranolol at 1 µM. Localization of ligand binding was achieved by autoradiography for 1 day (Hyperfilm, Amersham). The morphology of the sections was carried out in a similar way, but staining was performed using the Masson-Goldner method. Semiquantitative analyses of autoradiographs for total and nonspecific binding were performed using computerized image analysis (MCID, Imaging Research, Inc., St. Catherines, Canada).

RRA
RRAs of catecholamines were performed as previously described (15) using oviduct epithelial cell membranes prepared by differential centrifugation. Frozen epithelial cells, obtained after gentle flushing of bovine oviducts, were homogenized in 5 vol buffer (1 mM NaHCO3, 5 mM CaCl2, and 0.02% bacitracin, pH 7.75), filtered at last through 50-µm pore size gauze, and centrifuged for 15 min at 3,000 x g. These pellets were resuspended in 6.5 vol homogenization buffer, adjusted to a sucrose concentration of 35%, and centrifuged for 90 min at 90,000 x g through a discontinuous sucrose gradient (49%, 45%, and 41% sucrose). The resulting purified membranes were diluted and pelleted by centrifugation (15 min, 30,000 x g), and the final pellet was suspended in incubation buffer (1 mM NaHCO3, 100 mM HEPES, 120 mM NaCl, 1.2 mM MgSO4, 15 mM CH3COONa, 1 mM sodium EDTA, 2.5 mM KCl, 10 mM glucose, 0.02% bacitracin, and 0.1% ascorbic acid, pH 7.75) and, after total protein determination, stored at -80 C. Binding assays were performed with 50 µl purified oviductal plasma membranes (0.5 mg protein) and incubated with radiolabeled catecholamines. Total and nonspecific binding were determined in the absence or presence of 1 µM propranolol. Separation of bound and free ligand was performed by centrifugation through 3% albumin. Supernatants were discarded, pellets were washed, and the remaining radioactivity was measured using a {gamma}-counter. For saturation experiments, purified plasma membranes were incubated for total and nonspecific binding over 45 min using increasing concentrations of 125ICP (32–2,500 pM). The dissociation constant (Kd) and maximum binding capacity (Bmax) were analyzed by Scatchard plot (16). Competitive displacement experiments of membrane preparations were carried out over 45 min with 250 pM 125ICP in the absence or presence of unlabeled propranolol, adrenaline, noradrenaline, dopamine, and phentolamine at concentrations ranging from 10-11–10-3 M. IC50 values were determined by transforming the displacement curve in a logit-log plot and calculating the intersection of the linear regression and the x-axis.

cAMP measurement
Oviduct epithelial cells and culture supernatants were measured for cAMP using a commercial cAMP enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). Dispersed oviduct epithelial cells were cultured for 18 h, harvested, washed, and resuspended in PBS to a final concentration of about 1.5 x 106 cells/ml. Except for the control, 300 µl of this cell suspension were treated with adrenaline (10-5 M) or propranolol (10-5 M) together with adrenaline (10-5 M) and incubated for 30 min at 39 C, and cells or supernatants were separately examined for cAMP. Cells were extracted with ice-cold ethanol for 5 min, cell debris was pelleted by centrifugation, and supernatants were vacuum-dried and resuspended in PBS. All samples were acetylated to increase assay sensitivity.

Statistical analysis
IC50 values and binding assays were analyzed by the Mann-Whitney U test. The cAMP data were compared for statistical significance using Student’s t test. All data are expressed as the mean ± SD of the single value.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A comparison of autoradiographic images for total 125ICP binding with the histology of the corresponding stained sections revealed a distinct epithelial localization of ß-adrenoceptors (Fig. 1Go). The specificity of this binding was verified by competing 125ICP binding with 1 µM propranolol (nonspecific binding; Fig. 1cGo) where semiquantitative image analyses comparing the optical densities from total and nonspecific binding indicated 59% specific binding. Saturation experiments resulted in a strong and specific binding of the ß-adrenergic ligand 125ICP to isolated bovine oviduct membranes. Specific binding was saturable, and Scatchard plot analyses represented by linear regressions (Fig. 2Go) revealed the existence of a single class of ß-adrenergic receptors with a Kd value of 339 ± 74 pM and a Bmax of 2.24 ± 0.24 fmol/mg protein (mean ± SD; n = 4). The specificity of 125ICP binding to epithelial oviduct cell membranes, as shown by IC50 values from displacement experiments, resulted in the following affinity ranking for unlabeled ligands: propranolol > ad-renaline > noradrenaline > dopamine > phentolamine (Table 1Go).



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  		Figure 1. Representative histological (A) and autoradiographic images of bovine oviduct freeze sections showing specific ß-ligand (125ICP) binding (B) and the control (C). Magnification, x30.

 


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  		Figure 2. Saturation curve (mean ± SD; n = 4) and Scatchard plot (Kd, 339 ± 74 pM; Bmax, 2.224 ± 0.2415 fmol/mg protein; mean ± SD; n = 4) for ß-adrenoceptors in oviduct epithelial membrane preparations.

 

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  		Table 1. Specificity of [125I]CP binding to epithelial oviduct cell membranes

 
Furthermore, significant differences in specific 125ICP binding to epithelial membranes were measured at different sexual stages for both regions of isthmus and ampulla of the bovine oviduct. In the early secretory phase (days 1–5 after ovulation), ampulla membranes bound 0.60 ± 0.18 fmol/mg protein, whereas corresponding isthmus membranes bound 0.30 ± 0.03 fmol/mg protein (mean ± SD; n = 10). In the early proliferative phase (days 13–18 after ovulation), the values of specific 125ICP binding in the ampulla and isthmus regions were 1.45 ± 0.35 and 0.65 ± 0.30 fmol/mg protein (mean ± SD; n = 8), respectively. All groups, except ampulla days 1–5 and isthmus days 13–18, differed highly significantly from each other (P < 0.005). Saturation and displacement experiments demonstrated the existence of a single class of ß-adrenoceptors possessing the highest affinity for the natural ligand adrenaline.

After selecting cross-species homologous primers, bovine transcripts of the ß2-adrenoceptor were analyzed by RT-PCR, providing initial expression data for the ß2-adrenergic receptor mRNA in the bovine oviduct. Subsequently, Northern blot analysis revealed a single mRNA of about 2 kb in oviduct epithelial cells, representing a smaller size than that from the liver (Fig. 3AGo). Obvious sexual cycle-dependent fluctuations of ß2-adrenoceptor transcripts were evident in epithelium from individual oviducts: during the luteal phase (days 6–18; late secretory and early proliferative phase) receptor transcripts were clearly enriched, but lower levels of ß2-receptor mRNA were measured around ovulation on days 19–22 and 1–5 (Fig. 3BGo). Ampullary transcripts were increased at all stages compared with those in the isthmus. The low abundant {alpha}2-specific transcripts showed little variation, with no obvious cycle or region dependency. Equal mRNA concentrations in all samples were judged by the use of the housekeeping gene ubiquitin for the control PCR. Sequence determining of all resulting PCR products identified homologous {alpha}2- and ß2-adrenergic sequences to the known human ones. Sequencing of the resulting {alpha}2-adrenoceptor product (EMBL accession no. AJ006452) indicated an extension of the sequence information compared with that previously available (17).



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  		Figure 3. A, ß2-Adrenoceptor specific mRNA in bovine oviduct epithelial RNA (O) and liver (L) shown by Northern blot (1 ). In the lower part (2 ), the total RNA of both samples (O and L) is depicted after ethidium bromide staining. B, Expression of ß2-adrenoceptor RNA (ß2; 192 bp) detected by RT-PCR in epithelial cells from single oviducts during the sexual cycle (1 = days 1–5; 2 = days 6–12; 3 = days 13–18; 4 = days 19–21) and in liver (L). Transcript signal strength was separated into ampulla (A) and isthmus (I) regions compared with increasing amounts of standard ß2-adrenoceptor cRNA (1–1000 fg). In the lower part, corresponding ubiquitin signals were shown (Ub; 189 and 417 bp). One of five independent experiments is shown.

 
Screening of a complex bovine oviduct epithelial cDNA library in bacteriophage {lambda} enabled the isolation and sequencing of a full-length ß2-adrenoceptor clone, with a length of 2032 bp (EMBL accession no. Z86037). This first complete bovine cDNA indicated an open reading frame between 224-1480 bases, followed by a 3'-untranslated region with polyadenylation signal (2011–2016 bases; Fig. 4Go). The deduced 418-amino acid receptor protein provided highest homologies with the following known sequences: 90.2% with dog, 88.8% with pig, 88% with human, and 87% with rodents.



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  		Figure 4. Complete cDNA sequence and deduced amino acid composition of the bovine ß2-adrenergic receptor in oviduct epithelial cells. The putative polyadenylation signal is underlined.

 
To assess a possible correlation of the observed ß2-transcript increase during the luteal phase to high blood progesterone levels, oviduct epithelial cell cultures were studied; after overnight administration of progesterone and estradiol concentrations known to span the bovine physiological blood range (18), ß2-adrenergic receptor transcripts were increased (>50%) under the influence of progesterone, but not in a striking dose-dependent manner compared with untreated controls (Fig. 5Go). Estradiol had no immediate influence on receptor expression, but may antagonize the progesterone effect. A densitometric analysis of the staining intensity of each PCR product supported these visual data. In contrast to the ß2-receptor, the {alpha}2-specific receptor appeared to be expressed at very low levels and was unmodulated. When external standards (synthetic ß2- and {alpha}2-receptor cRNAs) were introduced during the RT-PCR procedures (Figs. 3Go and 5Go), relative transcript levels could be estimated in bovine oviductal epithelial cells as 0.1–1 pg for the {alpha}2-receptor mRNA and 2–40 pg for the ß2-receptor mRNA/µg total RNA. This indicates a more than 10 times higher mRNA level for ß2-receptors in the bovine oviduct epithelium compared with that for {alpha}2-receptor.



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  		Figure 5. RT-PCR specific for ß2-adrenocetpors (ß2), {alpha}2-adrenoceptors ({alpha}2), or ubiquitin (ub) in oviduct epithelial cells after culture without (C) or with 1 or 100 ng/ml progesterone (P1, P100), 1 or 100 pg/ml estradiol (E1, E100), or progesterone and estradiol in combination (PE1, PE100), as indicated. Increasing standard adrenoceptor cRNAs were introduced for ß2 (10, 100, and 1000 fg; 192 bp) and {alpha}2 (1, 10, and 100 fg; 150 bp) as well as a water control (W). Ubiquitin products (ub) served as internal quality control. One of five independent experiments is shown.

 
The functional effects of the ß2-adrenoceptor system in bovine oviducts were tested by generation of the second messenger cAMP after catecholamine stimulation. Adrenaline significantly elevated the intracellular cAMP concentration in cultured oviduct epithelium by 94.8%, whereas the ß-adrenoceptor blocker propranolol completely inhibited this effect (Table 2Go). A significantly elevated cAMP release into the supernatant (88.3%) was also measured by comparison to the control after adrenaline stimulation; propranolol was able to completely block this effect (Table 2Go).


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  		Table 2. cAMP generation in cultured oviductal cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The complete cDNA sequence of the ß2-adrenoceptor and its bioactivity were verified for the first time in bovine reproductive epithelial cells. This new structural information additionally confirmed the high protein sequence homology found for the ß-adrenoceptors throughout mammals, comprising seven transmembrane domains and highly conserved ligand binding as well as G protein coupling sites (19). Possibly due to variations in the untranslated regions, different transcript lengths are evident in different tissues, as shown here for the bovine liver and oviduct. Supported by ligand binding and RNA expression studies, the ß2-subtype of the adrenergic receptor was mainly found in the epithelial cells of the oviduct, with a significant cycle-specific regulation. The less prominent {alpha}2-subtype appeared not to vary. It is known that adrenoceptors are located in smooth muscles of the female reproductive tract (20), and recently, the ß-adrenergic dominance in oviduct muscle mediating its relaxation was reported (21). In the rat uterus, increased ß2-receptor levels were found in the myometrium during midpregnancy, possibly under progesterone control (22), supporting a steroid-regulated adrenergic receptivity that has also been described for human oviduct muscle cells during the menstrual cycle (23). However, in smooth muscles only the ß-type receptor appears to be progesterone dependent in the rabbit, rat, and human uterus or oviduct, never the {alpha}-adrenoceptors (23, 24, 25). Some researchers speculate that this observation is correlated with smooth muscle constriction promoting gamete transportation (1) or triggering the blood flow through vasomotor control of oviductal vessels (26). Here we have additionally found a progesterone-dependent increase in ß2-adrenoceptor transcripts in vitro, possibly correlating with an increased expression and density of the receptor during the luteal phase in vivo. A direct connection between gonadal steroids and the adrenergic system in the female appears obvious.

Only very limited data are available about adrenergic receptivity in the epithelial cell. It cannot be excluded that a main adrenergic nerval innervation of the isthmus region in the bovine oviduct (27) could mediate in vivo effects through locally liberated catecholamines. Previously, secretion of oxytocin and tissue-type plasminogen activator has been reported for bovine and rat ovarian cells under the influence of catecholamines (28, 29). Furthermore, increased fluid formation, effects on Cl ion transport, and electrical potential differences induced by catecholamines have been described for rabbit oviductal cells (30, 31). As shown here, catecholamine stimulation of oviduct epithelial cells in vitro induced a rapid intra- as well as extracellular cAMP surge, possibly followed by cell-specific metabolic responses. In the human endometrium, an elevated catecholamine-induced adenylate cyclase activity has been described during the secretory phase (32). Although uterine tissue is not directly comparable with that of the oviduct, in this study a similar increase in ß2-receptivity was observed during the late secretory phase in the oviduct epithelium. Possibly, catecholamines might mediate protein phosphorylation and glycosylation via the cAMP pathway (33). Extending these findings, it is possible that ß2-adrenoceptor-mediated functions could contribute to the substantial variations observed during the secretory phase in the bovine oviduct, which have been thought to be mainly mediated by steroids (34). Our findings support potent interactions of the adrenergic system within the bovine oviduct, with adrenaline as the most potent ligand, although noradrenaline is found in 2-fold higher concentrations in the blood circulation of the cow (35, 36). The local supply of catecholamines within the bovine oviduct is uncertain, and only limited information is available for human and rabbit oviductal fluids, showing a higher nor-/adrenaline concentration in the isthmus region compared with that in the ampulla as well as just before ovulation (6, 37). Thus, the main source of oviductal catecholamines could be local, possibly delivered by innervation, which was found to be enriched in the isthmus region (27). However, we found higher levels of ß2-adrenoceptors in the ampulla, suggesting that adrenergic receptivity follows a different gradient along the bovine oviduct. The results presented here imply important functions for the abundant ß2-adrenergic receptors, especially within the ampullary part of the bovine oviduct. The observed progesterone-dependent up-regulation of these receptors during the early luteal phase may stimulate oviductal turnover events but are less likely to support the embryo.

In conclusion, it appears probable that the local secretion of substances into the bovine oviduct lumen might be subject to a fine tuning by catecholamines supplied mainly from innervation, but possibly also from the circulation. Future work should address the physiological effects of catecholamines on reproductive success in mammals. Possibly more attention should be given to the adrenergic innervation of reproductive secretory tissue and thus lead to an improved understanding of female fertility.


   Acknowledgments
 
We thank Sandra Rode for motivated technical assistance, and Richard Ivell for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ei 296/4–3). Back

Received September 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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