0013-7227/99/$03.00/0
Endocrinology
Copyright © 1999 by The Endocrine Society
Vol. 140, No. 6
Printed in U.S.A.
Characterization and Localization of b2-Adrenergic
Receptors in the Bovine Oviduct: Indication for
Progesterone-Mediated Expression*
R. EINSPANIER, C. GABLER, A. KETTLER,
AND
W. KLOAS
Institute of Physiology, Technical University Munich-Weihenstephan (R.E., C.G., A.K.), Freising, D85350 Germany; and the Department of Zoology II, University of Karlsruhe (W.K.), D-76128 Karlsruhe,
Germany
ABSTRACT
b2-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 b2adrenoceptor 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
T
HE 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– 4). Furthermore, b-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 b-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.
Received September 1, 1998.
Address all correspondence and requests for reprints to: Dr. R. Einspanier, Institute of Physiology, Technical University Munich, Weihenstephaner Berg 5, D-85350 Freising, Germany. E-mail: einspani@
weihenstephan.de.
* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ei 296/4 –3).
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 b2-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. (Endocrinology 140: 2679 –2684, 1999)
Thereafter, we analyzed the complete molecular structure of
the b2-adrenoceptor and its localization and bioactivity.
Finally, the effects of steroid hormones on b2-receptor
expression were investigated in the oviduct at the site of
fertilization.
Materials and Methods
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 3 g for 3 min at 4 C, cell pellets
were stored at 280 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 mg/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 (1024-1026 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 280 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
2679
2680
b2-ADRENERGIC RECEPTORS IN BOVINE OVIDUCT
Trizol reagent (Life Technologies, Gaithersburg, MD). Total RNA was
spectroscopically quantified at 260 nm. The quantity and quality of
oviductal RNA (8 mg) 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 b2-receptorspecific 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-ml reaction mixture
as previously described (10). Commercially synthesized PCR primers
(Pharmacia Biotech, Piscataway, NJ) were used to amplify bovine transcripts specific for a2- and b2-adrenergic receptors, each newly designed
on the basis of cross-species sequence homologies: b2-adrenergic receptor: forward, 59-AGACTTTAGGCATTATCATGGG-39; reverse, 59-TGGAAGGCAATCCTGAAATC-39; and a2-adrenergic receptor: forward,
59-GAAGAGAGGCAGGAGTGGC-39; reverse, 59-TTGGCGATCAGGTAGATGC-39.
All cDNA samples were assayed for integrity and relative quantity
by performing PCR for ubiquitin mRNA using the following primers:
forward, 59-ATG CAG ATC TTT GTG AAG AC-39; and reverse, 59-CTT
CTG GAT GTT GTA TC-39. The predicted sizes of the resulting RT-PCR
products were 192 bp for the b2-adrenergic receptor, 150 bp for the
a2-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 ml. Individually adjusted amplification programs were
applied for b2- or a2-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 ml 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 b2-receptor was performed after generating
a complex bovine oviductal l cDNA library (l gt11 kit, Stratagene, San
Diego, CA), successfully screened for a large b2-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 b2-adrenoceptor cDNA into a transcription vector
(pCR-Script, Stratagene) enabled the generation of synthetic standard
complementary RNA (cRNA) for relative RT-PCR quantitation.
Endo • 1999
Vol 140 • No 6
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 b-adrenoceptor antagonist propranolol at 1 mm. 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 MassonGoldner 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-mm
pore size gauze, and centrifuged for 15 min at 3,000 3 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 3 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 3 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 280 C. Binding assays were performed with 50 ml 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 mm
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 g-counter. For saturation experiments, purified plasma membranes
were incubated for total and nonspecific binding over 45 min using
FIG. 1. Representative histological (A) and autoradiographic images of bovine oviduct freeze sections showing specific b-ligand (125ICP) binding
(B) and the control (C). Magnification, 330.
b2-ADRENERGIC RECEPTORS IN BOVINE OVIDUCT
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
10211–1023 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 3 106 cells/ml. Except for the control, 300 ml
of this cell suspension were treated with adrenaline (1025 m) or propranolol (1025 m) together with adrenaline (1025 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
2681
Student’s t test. All data are expressed as the mean 6 sd of the single
value.
Results
A comparison of autoradiographic images for total 125ICP
binding with the histology of the corresponding stained sections revealed a distinct epithelial localization of b-adrenoceptors (Fig. 1). The specificity of this binding was verified
by competing 125ICP binding with 1 mm propranolol (nonspecific binding; Fig. 1c) 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
b-adrenergic ligand 125ICP to isolated bovine oviduct membranes. Specific binding was saturable, and Scatchard plot
analyses represented by linear regressions (Fig. 2) revealed
the existence of a single class of b-adrenergic receptors with
a Kd value of 339 6 74 pm and a Bmax of 2.24 6 0.24 fmol/mg
protein (mean 6 sd; n 5 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 . adrenaline . noradrenaline . dopamine . phentolamine
(Table 1).
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 6 0.18 fmol/mg
protein, whereas corresponding isthmus membranes bound
TABLE 1. Specificity of [125I]CP binding to epithelial oviduct cell
membranes
FIG. 2. Saturation curve (mean 6 SD; n 5 4) and Scatchard plot (Kd,
339 6 74 pM; Bmax, 2.224 6 0.2415 fmol/mg protein; mean 6 SD; n 5
4) for b-adrenoceptors in oviduct epithelial membrane preparations.
FIG. 3. A, b2-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 b2-adrenoceptor RNA (b2;
192 bp) detected by RT-PCR in epithelial cells from single oviducts during the
sexual cycle (1 5 days 1–5; 2 5 days
6 –12; 3 5 days 13–18; 4 5 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
b2-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.
Ligand
M
Propranolol
Adrenaline
Noradrenaline
Dopamine
Phentolamine
6.7 6 3.4 3 10210
1.1 6 0.3 3 1025
1.1 6 0.4 3 1024
8.5 6 0.8 3 1024
1.8 6 1.9 3 1023
Values are the mean 6
SD
(n 5 4).
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b2-ADRENERGIC RECEPTORS IN BOVINE OVIDUCT
Endo • 1999
Vol 140 • No 6
FIG. 4. Complete cDNA sequence and deduced amino acid composition of the bovine b2-adrenergic receptor in oviduct epithelial cells. The
putative polyadenylation signal is underlined.
0.30 6 0.03 fmol/mg protein (mean 6 sd; n 5 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 6 0.35 and 0.65 6 0.30 fmol/mg protein (mean 6
sd; n 5 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 b-adrenoceptors possessing the highest affinity for the natural
ligand adrenaline.
After selecting cross-species homologous primers, bovine
transcripts of the b2-adrenoceptor were analyzed by RT-PCR,
providing initial expression data for the b2-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. 3A). Obvious sexual cycle-dependent fluctuations
of b2-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 b2-receptor
mRNA were measured around ovulation on days 19 –22 and
1–5 (Fig. 3B). Ampullary transcripts were increased at all
stages compared with those in the isthmus. The low abundant a2-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 a2- and b2-adrenergic sequences to the known
human ones. Sequencing of the resulting a2-adrenoceptor
product (EMBL accession no. AJ006452) indicated an extension of the sequence information compared with that previously available (17).
Screening of a complex bovine oviduct epithelial cDNA
library in bacteriophage l enabled the isolation and sequencing of a full-length b2-adrenoceptor clone, with a length of
2032 bp (EMBL accession no. Z86037). This first complete
bovine cDNA indicated an open reading frame between 2241480 bases, followed by a 39-untranslated region with polyadenylation signal (2011–2016 bases; Fig. 4). The deduced
418-amino acid receptor protein provided highest homolo-
b2-ADRENERGIC RECEPTORS IN BOVINE OVIDUCT
2683
TABLE 2. cAMP generation in cultured oviductal cells
Treatment
pmol cAMP/5 3 105 cells
(intracellular)
fmol/mg protein
(extracellular)
Control
Adrenaline
Adrenaline 1 propranolol
59.8 6 17.5
116.5 6 57.9a
51.3 6 8.5
138.7 6 10.7
261.2 6 83.8a
70.3 6 61.3
Values are the mean 6 SD (n 5 5).
a
Significant difference vs. control, P , 0.05.
FIG. 5. RT-PCR specific for b2-adrenocetpors (b2), a2-adrenoceptors
(a2), 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 b2 (10, 100, and 1000 fg; 192 bp) and a2 (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.
gies with the following known sequences: 90.2% with dog,
88.8% with pig, 88% with human, and 87% with rodents.
To assess a possible correlation of the observed b2-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), b2-adrenergic receptor transcripts
were increased (.50%) under the influence of progesterone,
but not in a striking dose-dependent manner compared with
untreated controls (Fig. 5). 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 b2-receptor, the a2-specific receptor appeared to be expressed at very low levels and was unmodulated. When external standards (synthetic b2- and a2-receptor
cRNAs) were introduced during the RT-PCR procedures
(Figs. 3 and 5), relative transcript levels could be estimated
in bovine oviductal epithelial cells as 0.1–1 pg for the a2receptor mRNA and 2– 40 pg for the b2-receptor mRNA/mg
total RNA. This indicates a more than 10 times higher mRNA
level for b2-receptors in the bovine oviduct epithelium compared with that for a2-receptor.
The functional effects of the b2-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
b-adrenoceptor blocker propranolol completely inhibited
this effect (Table 2). 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 2).
Discussion
The complete cDNA sequence of the b2-adrenoceptor and
its bioactivity were verified for the first time in bovine re-
productive epithelial cells. This new structural information
additionally confirmed the high protein sequence homology
found for the b-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 b2-subtype
of the adrenergic receptor was mainly found in the epithelial
cells of the oviduct, with a significant cycle-specific regulation. The less prominent a2-subtype appeared not to vary. It
is known that adrenoceptors are located in smooth muscles
of the female reproductive tract (20), and recently, the b-adrenergic dominance in oviduct muscle mediating its relaxation was reported (21). In the rat uterus, increased b2-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 b-type receptor appears to be progesterone dependent in the rabbit, rat, and human uterus or oviduct, never
the a-adrenoceptors (23–25). 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 b2-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 b2-receptivity was observed during the late secretory
2684
b2-ADRENERGIC RECEPTORS IN BOVINE OVIDUCT
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 b2-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 b2-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 b2-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.
10.
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27.
28.
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