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Distinct Cellular Localization and Regulation of Endothelin1 and EndothelinConverting Enzyme1 Expression in the Bovine Corpus Luteum: Implications for Luteolysis

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Endothelin-1 (ET)-1 within the corpus luteum (CL) is rapidly up-regulated during natural or PGF2-induced luteolysis; however, such an increase was not observed at early luteal stage, when the CL is refractory to PGF2. The mature and active form of ET-1 is derived from the inactive intermediate peptide, big ET-1, by ET-converting enzyme (ECE)-1. This study therefore examined the developmental and cell-specific expression of ECE-1 in bovine CL. A significant, 4-fold, eleva- tion in ECE-1 expression (mRNA and protein levels) occurred during the transition of the CL from early to midluteal phase. Analysis using in-situ hybridization and enriched luteal cell subpopulations showed that both steroidogenic and endothe- lial cells of the CL expressed high levels of ECE-1 mRNA; prepro ET-1 mRNA, on the other hand, was only expressed by resident endothelial cells. These data suggest that luteal pa- renchymal and endothelial cells may cooperate in the biosyn- thesis of mature bioactive ET-1. In the mature CL, ECE-1 mRNA increase occurred both in steroidogenic and endothe- lial cells and was accompanied by a significant rise in ET-1 peptide. However, in contrast to ECE-1, prepro ET-1 mRNA levels were similar in early and midluteal-phase CL. Low ECE-1 levels during the early luteal phase, restricting the production of active ET-1, may explain why the immature CL is able to withstand PGF2-induced luteolysis. (Endocrinology 142: 5254 -5260, 2001)
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Distinct Cellular Localization and Regulation of
Endothelin-1 and Endothelin-Converting Enzyme-1
Expression in the Bovine Corpus Luteum: Implications
for Luteolysis
NITZAN LEVY, MIRI GORDIN, RONI MAMLUK, MASASHI YANAGISAWA, MICHAEL F. SMITH,
JIM H. HAMPTON, AND RINA MEIDAN
Department of Animal Sciences (N.L., M.G., R.Ma., R.Me.), Faculty of Agricultural, Food and Environmental Quality
Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel; Howard Hughes Medical Institute and Department of
Molecular Genetics (M.Y.), University of Texas, Southwestern Medical Center, Dallas, Texas 75235; and Department of
Animal Sciences (M.F.S., J.H.H.), University of Missouri, Columbia, Missouri 65211
Endothelin-1 (ET)-1 within the corpus luteum (CL) is rapidly
up-regulated during natural or PGF
2
-induced luteolysis;
however, such an increase was not observed at early luteal
stage, when the CL is refractory to PGF
2
. The mature and
active form of ET-1 is derived from the inactive intermediate
peptide, big ET-1, by ET-converting enzyme (ECE)-1. This
study therefore examined the developmental and cell-specific
expression of ECE-1 in bovine CL. A significant, 4-fold, eleva-
tion in ECE-1 expression (mRNA and protein levels) occurred
during the transition of the CL from early to midluteal phase.
Analysis using in-situ hybridization and enriched luteal cell
subpopulations showed that both steroidogenic and endothe-
lial cells of the CL expressed high levels of ECE-1 mRNA;
prepro ET-1 mRNA, on the other hand, was only expressed by
resident endothelial cells. These data suggest that luteal pa-
renchymal and endothelial cells may cooperate in the biosyn-
thesis of mature bioactive ET-1. In the mature CL, ECE-1
mRNA increase occurred both in steroidogenic and endothe-
lial cells and was accompanied by a significant rise in ET-1
peptide. However, in contrast to ECE-1, prepro ET-1 mRNA
levels were similar in early and midluteal-phase CL. Low
ECE-1 levels during the early luteal phase, restricting the
production of active ET-1, may explain why the immature CL
is able to withstand PGF
2
-induced luteolysis. (Endocrinology
142: 5254 –5260, 2001)
E
NDOTHELIN (ET)-1, ORIGINALLY isolated from por-
cine aortic endothelial cells (1), belongs to a structurally
homologous peptide family, which includes ET-2, ET-3, and
sarafotoxins (2). These peptides bind two distinct subtypes of
G protein-coupled receptors, termed ETA and ETB (3, 4).
ET-1, initially identified as a potent vasoconstrictor (1), has
diverse biological functions. Studies carried out in recent
years have demonstrated that ET-1 is involved in the process
of luteal regression (5, 6). These studies showed that prepro
ET-1 (ppET-1) mRNA and peptide levels are rapidly up-
regulated during natural or PGF
2
-induced luteolysis (7–9).
Elevated ET-1, acting via the selective ETA binding sites,
caused a dose-dependent inhibition of basal and LH/human
CG-stimulated biosynthesis of progesterone and an increase
in PGF
2
. These observations were made in ovine, bovine,
and human luteal cells (6, 10–13). In accordance with the
inhibitory role of ET-1 in corpus luteum (CL) function, we
have recently reported that the expression of ETA receptors
was inversely correlated with steroidogenesis in luteal cells,
namely factors that stimulated steroidogenesis inhibited
ETA receptor levels (14). Blocking ET-1 action by means of
ETA antagonists decreased the antisteroidogenic effects of
PGF
2
under both in vitro (10) and in vivo (5, 6) conditions.
The administration of either ET-1 or a subluteolytic dose of
PGF
2
to ewes and cows significantly reduced progesterone
concentrations of jugular venous blood without shortening
of luteal life span; however, the combination of these treat-
ments acted synergistically to produce complete luteolysis
(6, 15). Collectively, these data strongly support the concept
that ET-1 plays a significant, physiological role during
luteolysis.
ET-1 is synthesized from a precursor of approximately 200
amino acids (aa), which is proteolytically cleaved into big
ET-1 (38 aa) and further processed to the active form of ET-1
(21 aa) by ET-converting enzyme (ECE) (16, 17). Unlike the
mature form of ET-1, the big ET-1 precursor has negligible
biological activity (16). There are 2 separate ECE genes,
termed ECE-1 and ECE-2 (17, 18). Both enzymes are zinc-
binding metalloendopeptidases, but they function at differ-
ent optimal pH levels and are different in tissue distribution.
ECE-2 is more abundant in neural tissues, whereas ECE-1 is
present in a wide variety of tissues (17, 19). ECE-1 mRNA
levels in steroidogenic tissues (adrenals, ovaries, and testis)
are significantly higher than those in the traditional ET-1-
producing tissues, such as kidney, lung, and heart (17). Tri-
piciano and colleagues (20) recently reported that the cyclic
contraction of the seminiferous tubules, which is regulated
by ET-1, is mediated by a cyclic expression of ECE-1 in Sertoli
cells. These data suggest that the spatial (cellular) and tem-
Abbreviations: aa, Amino acid; BME,
-mercaptoethanol; BS-1, Ban-
deiraea simplicifolia lectin-1; CL, corpus luteum; ECE, ET-converting
enzyme; EIA, antibody enzyme immunoassay; ET, endothelin; G3PDH,
glyceraldehyde 3-phosphate dehydrogenase; ppET-1, prepro ET-1; SSC,
saline sodium citrate; StAR, steroidogenic acute regulatory protein.
0013-7227/01/$03.00/0 Endocrinology 142(12):5254 –5260
Printed in U.S.A. Copyright © 2001 by The Endocrine Society
5254
poral patterns of ECE-1 expression can have profound effects
on ET-1 bioavailability.
This study was therefore undertaken to investigate the
developmental and cell-specific expression of ECE-1 and
ppET-1 in bovine CL during the early and midluteal phases
of the estrous cycle.
Materials and Methods
Materials
SuperScriptII RNase H
Reverse Transcriptase and Ultra pure elec
-
trophoresis agarose gel were obtained from Life Technologies, Inc.
(Gaithersburg, MD). Deoxynucleotide triphosphates, random hexamer
oligodeoxynucleotides, and Taq DNA polymerase were from Farmentas
(Vilnius, Lithuania). Oigonucleotide primers were synthesized by Bio-
Technology General Corp. (Rehovot, Israel); Dulbeccos modified MEM:
Hams F12 1:1 (vol/vol), M-199 nutrient mixture, bovine Big ET-1, phos-
phoramidon, protease inhibitor cocktail for mammalian cell extracts,
and horseradish peroxidase-conjugated goat antirabbit IgG were from
Sigma (St. Louis, MO). C18 cartridges were from Waters Corp. (Milford,
MA); Bandeiraea Simplicifolia Lectin-1 (BS-1) was from Vector Labo-
ratories, Inc. (Burlingame, CA); collagenase type IV was from Worth-
ington Biochemical Corp. (Freehold, NJ); and uncoated magnetic beads
(Dynabeads M-450) were from Dynal (Oslo, Norway).
CL Collection
CL were collected at a local slaughter house, and luteal stage was
determined by macroscopic examination, as described by M. J. Fields
and P. A. Fields (21). CL were divided into two groups: early luteal phase
(d 24) and midluteal phase (d 914). For studies involving luteal RNA
extractions, CL were frozen in liquid N
2
immediately after slaughter.
Luteal cell dispersion and purification
CL were dispersed using collagenase as previously described (22).
Briefly, CL were washed, sliced with a tissue slicer, and preincubated in
a shaking bath at 37 C for 10 min in M-199 media. Slices were than
incubated in M-199 containing 0.5% BSA and collagenase (420 U/ml).
Every 10 min, dissociated cells were removed, and fresh media con-
taining collagenase was added. This procedure was repeated 78 times.
Magnetic tosylactivated beads were coated with BS-1 lectin [0.15 mg/ml,
expressed in bovine endothelial cells (23)] for 24 h at room temperature.
The beads were than washed and stored at 4 C until use. Dispersed luteal
cells were suspended in 1% BSA in PBS, mixed with beads at a bead:
endothelial cell ratio of 1:3, and incubated for 25 min at4Conarocking
platform. The adherent cells were washed with 1% PBS and concentrated
using a magnet until the supernatant was free of cells. Both BS-1-positive
cells and nonadherent cells (BS-1-negative) were collected for RNA
extraction.
Follicular cell isolation and cultures
Ovaries were obtained at slaughter; granulosa and theca cells were
isolated from healthy bovine preovulatory follicles, as previously de-
scribed (24). To induce luteinization, granulosa and theca cells were
cultured in the presence of 1% FCS, insulin (2
g/ml), and forskolin (10
m). Total RNA was extracted on the ninth day of culture.
RNA extraction and semiquantitative RT-PCR
Total RNA was extracted from cells and tissues using the guani-
dinium thiocyanate method (25). Semiquantitative RT-PCR was per-
formed as previously described (22), with glyceraldehyde 3-phosphate
dehydrogenase (G3PDH) as an internal standard. Sequence analysis or
restriction mapping ascertained the identity of the PCR products. Al-
ternative splicing of the ECE-1 gene at the 5 end produces four isoforms
(26). Therefore, primers for ECE-1 were designed to span the 3 region
of the gene (12771906). Sequence analysis showed that these primers do
not amplify ECE-2. Computer searches and sequence alignments were
performed by using software from Genetics Computer Group (Madison,
WI). A list of primers is presented in Table 1.
In situ hybridization
Procedures for in situ hybridization were as previously described (27).
A 550-bp cDNA-encoding bovine ECE-1 was generated using RT-PCR.
The cDNA was ligated into pGEM-T-Easy vector (Promega Corp., Mad-
ison, WI) and sequenced. Both antisense and sense [
35
S]uridine triphos
-
phate-labeled cRNA probes (10
7
cpm/ml) were transcribed from lin
-
earized cDNA templates using a transcription kit (Promega Corp.)
according to the manufacturers recommendations. Hybridization was
performed using 100
l diluted probe in a humidified oven at 55 C for
20 h. After hybridization, slides were washed twice by shaking in dou-
ble-strength saline sodium citrate (SSC) (4552 C) for 15 min at room
temperature and were treated with ribonuclease (RNase-A; 50 mg/ml
in double-strength SSC) for1hat37C.Slides were then washed at 55
C in double-strength SSC containing 0.1%
-mercaptoethanol (BME) for
15 min, in single-strength SSC/0.1% BME for 15 min, in single-strength
SSC/50% formamide/0.1% BME for 30 min, and twice in 0.1-strength
SSC/BME for 15 min each. The slides were dehydrated, air-dried,
dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), and
exposed for 21 d at 4 C. Slides were developed, lightly counterstained
with hematoxylin and eosin, and mounted for microscopic examination.
For each CL, two sections were hybridized with the antisense probe, and
one section was hybridized to the sense probe. Sections from animals
ovariectomized at different times were balanced in each hybridization
run to minimize bias caused by variation among runs. Hybridization
intensity was measured using the Bioquant image analyses system
(R&M Biometrics, Nashville, TN); the image analysis system determined
the number of graphic pixels occupied by the silver grains.
Determination of ET-1 and big ET-1 peptide concentrations
ET-1 concentrations in CL extracts and in culture media were deter-
mined with double-antibody enzyme immunoassays (EIAs; 9, 11). ET-1
extraction was carried out as previously described (7). Tissues were
homogenized in 10 vol 1-m acetic acid and boiled. Homogenates were
centrifuged, and the supernatants were loaded on C
18
cartridges pre
-
equilibrated with 1 m acetic acid. The adsorbed materials were eluted
with 3 ml 60% acetonitrile in 0.1% trifluoroacetic acid. The eluates were
evaporated in vacuum to dryness and dissolved in EIA buffer. The
values of ET-1 measured in tissue extracts were corrected for recovery
losses (the recovery 56 3.0% was determined using synthetic ET-1
added to CL extracts). The ED
50
of the assay was 450 pg/ml. Cross-
reactivity of ET-1 antiserum with synthetic ET-1, ET-2, ET-3, and big
ET-1 were 100%, 50%, 22%, and 3%, respectively.
Big ET-1 concentrations in CL extracts (obtained as described above)
were determined using an enzyme-linked immunoassay kit (Biomedica,
Vienna, Austria). The ED
50
of big ET-1 assay was 20.9 pg/ml. The
cross-reactivity of ET-1 was lower than 1%.
Evaluation of ECE activity
Granulosa and theca-derived luteal cells were incubated in M-199
medium containing 0.5% BSA alone, or with the addition of big ET-1 (100
nm) in the presence or absence of phosphoramidon, a metalloprotease
inhibitor (17) (10
m) for 24 h. ET-1 concentrations in supernatants were
determined by EIA.
TABLE 1. Primer list
Gene Primers
G3PDH Sense 5-tgttccagtatgattccaccc-3
Antisense 5-tccaccaccctgttgctgta-3
ECE-1 Sense 5-tctccaccctcatcaacagcac-3
Antisense 5-cattgggtgaagagcgggtgta-3
StAR Sense 5-catggtgctccgccccttggct-3
Antisense 5-cattgcccacagacctcttga-3
CD-31 Sense 5-tgaggagcaagaccgtaccata-3
Antisense 5-acggagcagtaaaacacaatgg-3
ET-1 Sense 5-tgtcttcatcagcagctcg-3
Antisense 5-gtttctccctgaaatgtgcc-3
Levy et al. ET-1 and ECE in Bovine CL Endocrinology, December 2001, 142(12):52545260 5255
Western analyses
CL were homogenized in lysis buffer [20 mm Tris HCl, pH8.6; 1%
SDS; phenylmethylsulfonyl fluoride (1 mm); and protease inhibitor cock-
tail] and were immediately boiled for 10 min. After chilling, homoge-
nates were centrifuged for 15 min at 2000 g, and protein concentrations
of the supernatant were determined using DC reagents (Bio-Rad Lab-
oratories, Inc., Hercules, CA). Samples containing 10
g protein were
separated by 7.5% SDS/PAGE, under reducing conditions, and were
electrically transferred to nitrocellulose membrane (Schleicher &
Schuell, Inc., Keene, NH). After 2 h blocking [in TBST (20 mm Tris HCl
buffered saline containing 0 15% Tween-20) containing 5% BSA], mem-
branes were incubated with anti-ECE-1 antiserum (produced at M.
Yanagisawas lab, Ref. 18), directed against ECE1 C-terminal peptide for
2 h. The membranes reacted with primary antibodies (diluted 1:4000),
were washed, and than incubated with horseradish peroxidase-conju-
gated goat antirabbit IgG for1hatroom temperature, and binding was
detected with an enhanced chemiluminescence detection system (Am-
ersham Pharmacia Biotech, Piscataway, NJ). Western blots were exposed
to x-ray films and subsequently scanned and quantitated using NIH
Image (version 1.61).
Statistical analyses
Data are presented as means sem. The expression of the various
genes was quantified using the densitometric analysis, relative to an
internal standard (G3PDH). Statistical analysis was carried out using the
JMP package [version 3.2; SAS Institute, Inc., Cary, NC (28)]. One-way
ANOVA was used to determine the statistical significance of individual
groups, as indicated in the text. A value of P 0.05 was considered
significant.
Results
Expression of ppET-1 and ECE-1 during early and
midluteal phases
We first determined the expression of ET-1 during early
and midluteal stages. ET-1 peptide concentrations in early
CL tissue (d 24) were 7-fold lower than in CL at the mid-
luteal phase (d 9 14; P 0.02, Fig. 1). Surprisingly, however,
levels of ppET-1 mRNA were similar during the early and
midluteal phases. This large discrepancy between ppET-1
mRNA expression and the levels of the mature ET-1 peptide
form could indicate that ECE-1 expression varies with CL
age. Indeed, as demonstrated in Fig. 2, ECE-1 expression,
measured at both the mRNA (Fig. 2B) and the protein level
(Fig. 2A), were 3- to 4-fold higher in CL collected at midcycle
vs. CL in the early luteal phase. In agreement with these
findings, concentrations of big ET-1 at early luteal phase were
higher than those measured at midcycle CL (5.9 1.22 vs.
3.4 0.02 pg/mg protein, P 0.05, respectively); and the
ratio of ET-1/big ET-1, reflecting ECE-1 activity, increased
from 0.28 (early luteal phase) to 3.54 (mature CL).
Cellular localization of ECE-1 and ppET-1 mRNA during
early and midluteal phases
The CL is a heterogeneous tissue, containing mainly en-
dothelial and steroidogenic cell types. Therefore, it was of
interest to determine the cellular localization of ppET-1 and
ECE-1 in this gland. Early and mature CL tissues were en-
zymatically dispersed, and endothelial cells were enriched
by the use of magnetic beads coated with the endothelial
cell-specific lectin, BS-1, as detailed in Materials and Methods.
The identification of BS-1 positive cells as endothelial cells
was confirmed by presence of the endothelial cell marker,
CD31 (29) (Fig. 3A). The elevated CD31 expression in total
dispersed cells at midcycle indicates a higher proportion of
FIG. 1. Luteal ET-1 expression during the early and midbovine lu-
teal phases. ppET-1 mRNA levels were determined by semiquanti-
tative RT-PCR using G3PDH as an internal standard. ET-1 peptide
concentrations in CL extracts were determined by EIA. *, Significant
statistical difference (P 0.02) between early and midluteal phases.
FIG. 2. ECE-1 expression during early and midluteal phases. A,
ECE-1 mRNA levels (determined by semiquantitative RT-PCR using
G3PDH as an internal standard); left panel, inverse picture of
ethidium bromide stained agarose gel shows a typical RT-PCR reac-
tion with two replicates; right panel, bars, means SEM of the den-
sitometric analysis (n 6 for each luteal stage). B, ECE-1 protein
levels; left panel, Western blot using specific anti-ECE-1 antibody,
showing a typical reaction with two replicates; right panel, densito-
metric analysis of the ECL stained 130-kD bands; bars, means SEM
of four samples for each stage of the cycle; *, significant statistical
difference (P 0.01).
5256 Endocrinology, December 2001, 142(12):52545260 Levy et al. ET-1 and ECE in Bovine CL
endothelial cells at this stage and is in agreement with other
studies (30). As expected, the levels of CD31 in enriched
endothelial cells of early and midcycle CL were similar (Fig.
3A). The remaining BS-1 negative cells comprised an en-
riched steroidogenic cell population, as judged by the high
mRNA levels of steroidogenic acute regulatory protein
(StAR; Fig. 3B). Purity of endothelial and steroidogenic cell
fractions was estimated using these two cell-specific mark-
ers, CD31 and StAR; BS-1 positive cells expressed only 35%
of the steroidogenic cell marker, and BS-1 negative cells
expressed 13% of the endothelial cells marker.
We then determined levels of ppET-1 and ECE-1 in these
two cell fractions at various stages of CL development;
ppET-1 mRNA was mainly localized to the endothelial cell
fraction (Fig. 4A), both in early and mature CL. In accord
with data presented in Fig. 1, ppET-1 mRNA levels were
similar in dispersed cells of CL collected at early and mid-
luteal phases. In contrast, the endothelial cell fraction derived
from the mature CL expressed lower levels of ppET-1 (which
were not statistically significant; P 0.06) than the corre-
sponding endothelial cell fraction at early cycle. The differ-
ence between total dispersed CL cells and enriched endo-
thelial cells is perhaps a result of two opposing events during
CL maturation: an increase in endothelial cell number, and
a decrease in ppET-1 mRNA per endothelial cell. Unlike
ppET-1, the ECE-1 mRNA levels were expressed to a similar
level by both steroidogenic and endothelial cell fractions (Fig.
4B). ECE-1 mRNA levels increased significantly as the CL
matured, and this increment was observed both in the ste-
roidogenic and endothelial cells (Fig. 4B). The finding that
the mature CL contains higher levels of ECE-1 mRNA is
consistent with the data obtained from nondispersed CL
tissues (Fig. 2). To visualize the cellular localization of ECE-1
in intact luteal tissue, in situ hybridization with an antisense
probe to ECE-1 was performed (Fig. 5). In excellent agree-
ment with data obtained using enriched luteal cell prepara-
tions (Fig. 4), in situ hybridization studies showed that ECE-1
mRNA was localized to both the endothelial cell layer of
blood vessels and to parenchymal cells of CL; this was ob-
served both at early and midluteal phases (Fig. 5, A and C).
FIG. 3. mRNA levels of CD31 (A) and StAR (B) in luteal cell populations enriched from early luteal (early, e) and midluteal (mature, m) phase
CL. Dispersed cells (enzymatically dispersed CL cells), BS-1 positive cells (cells adherent to BS-1-coated magnetic beads), BS-1 negative cells
(nonadherent cells). mRNA levels were determined by semiquantitative RT-PCR using G3PDH as an internal standard. Data are the means
SEM (n 4 for each luteal stage) of the densitometric analysis. *, Significant (P 0.05) statistical difference between young and mature CL.
FIG. 4. mRNA levels of ppET-1 (A) and ECE-1 (B) in luteal cell populations enriched from early luteal and midluteal phase CL. For details,
refer to legend to Fig. 4. *, Significant (P 0.05) statistical difference between young and mature CL.
Levy et al. ET-1 and ECE in Bovine CL Endocrinology, December 2001, 142(12):52545260 5257
Also in accordance with data presented in Figs. 2 and 4, lower
hybridization of the ECE-1 probe was observed in early vs.
mature CL (320 61 and 634 35 arbitrary units, respec-
tively; Fig. 5, A and C). No specific hybridization was ob-
served when hybridizing with the sense probe (Fig. 5E).
mRNA expression and bioactivity of ECE-1 in steroidogenic
luteal cells
Because the methods used above could not determine
whether both steroidogenic luteal cells express ECE-1 gene,
we have determined its presence in granulosa and theca-
derived luteal cells obtained after in vitro luteinization. Data
presented in Fig. 6A, show that ECE-1 mRNA was readily
detectable in both of these luteal cell types. To examine
whether these cells indeed express the enzymatic activity,
exogenous big bovine ET-1 was added to the culture me-
dium; Both cells types efficiently converted big ET-1 into
mature ET-1 (Fig. 6B). Addition of the metalloprotease in-
hibitor, phosphoramidon, which inhibits ECE activity, sig-
nificantly inhibited the production of ET-1 peptide (Fig. 6B).
Together, the findings presented in Figs. 4B, 5, and 6 in-
dicate that luteal steroidogenic cells express functional ECE-1
capable of producing mature ET-1 peptide.
Discussion
The present study shows that ECE-1 mRNA was expressed
by both steroidogenic and endothelial cells of the bovine CL,
whereas ppET-1 mRNA was only expressed by the resident
endothelial cells. Additionally, ECE-1, but not ppET-1, in-
creased during the transition of the CL from early to mid-
luteal phase. The increment in ECE-1 levels (mRNA and
protein) was accompanied by increased ET-1 peptide
concentrations.
ET-1 plays an important role in the female reproductive
cycle: its quick ascent during luteal regression (79), ability
to inhibit steroidogenesis in vitro and in vivo (6, 10, 11, 13),
combined with the observation that the luteolytic effects of
PGF
2
were decreased by pretreatment with a ETA antago
-
nists (6, 10), suggest that this peptide functions as an im-
portant element of the luteolytic cascade. In addition, ET-1
FIG. 5. Detection of ECE-1 mRNA in sections of bovine CL by in situ
hybridization of a [
35
S]cRNA probe; Darkfield (A, C) and brightfield
(B, D) views of early and mature CL. Both luteal (L) and endothelial
cells (EC) expressed ECE-1; surrounding smooth muscle cells were
negative. The intensity of ECE-1 mRNA hybridization was higher in
midcycle CL then in early CL. E, Darkfield view of a midcycle CL
hybridized with the sense probe, showing no specific hybridization.
FIG. 6. mRNA expression and biological activity of ECE-1 in lutein-
ized granulosa cells (LGC) and luteinized theca cells (LTC). A, De-
tection of ECE-1 mRNA by RT-PCR, two replicates; B, conversion of
big ET-1. Luteinized granulosa and theca cells were incubated in
medium containing big ET-1 (0.1
M) in the presence or absence of
phosphoramidon (10
M) for 24 h at 37 C. ET-1 concentrations were
determined by EIA, as detailed in Materials and Methods. *, Signif-
icant (P 0.01) statistical difference of the respective cell types
treated with big ET-1 alone.
5258 Endocrinology, December 2001, 142(12):52545260 Levy et al. ET-1 and ECE in Bovine CL
may act to prevent premature luteinization of follicular cells
(31). Hence, changes in spatial and/or temporal expression
of ppET-1 and ECE-1, which modulate ET-1 peptide levels,
could affect processes such as ovulation and the establish-
ment of pregnancy. Moreover, given the short half-life of
ET-1, the cellular localization of ECE-1 may determine its
bioavailability in various ovarian compartments.
ET-1 is synthesized by the endothelium of virtually all
blood vessels and in nonvascular cell types such as cardiac
myocytes, adrenal zona glumerulosa cells, and testicular Ser-
toli cells (3234). These cells express both ppET-1 and ECE-1
genes, so that a single cell type is presumably capable of
producing bioactive ET-1 peptide. Steroidogenic cells of the
CL are unique in that they express high levels of ECE-1
mRNA, whereas ppET-1 is undetectable. This observation
has two important implications: that parenchymal and en-
dothelial cells within the CL may cooperate in the biosyn-
thesis of mature bioactive ET-1; also, that conversion of big
ET-1 into active ET-1, adjacent to its site of action (the ETA
receptor) on the plasma membrane of a steroidogenic cell
(14), may be necessary to ensure that short-lived, mature
ET-1 is active. The findings demonstrating that ECE-1 /
mice had a phenotype similar to that of ET-1 or ETA deficient
mice, despite the presence of substantial amounts of mature
ET-1 peptide (35), support the notion that to achieve its
biological effect, ET-1 must be produced within a physio-
logically relevant microenvironment.
ECE-1 knockout mice and also the double, ECE-1 and -2
knockouts, contained immunoreactive ET-1, suggesting that
other peptidases are also capable of converting big ET1.
Indeed, it was shown that matrix metalloproteinase-2 (36)
and chymase (37) can cleave big ET-1; nevertheless, it should
not be ignored that these ECE knockout mice had develop-
mental defects, such as mice lacking ET-1, indicating the
importance of ECE-1 in the generating bioavailable ET-1.
The existence of several different isoforms of ECE-1 that
are transcribed from a single gene by alternative promoters
was reported (26). These forms have comparable enzymatic
activity, but they differ in their intracellular localization.
Whereas some are highly expressed on the cell surface, others
are almost exclusively intracellular (38). The identity of
ECE-1 isoforms in steroidogenic cells is still unknown; how-
ever, because steroidogenic luteal cells can cleave exogenous
big ET-1 and do not express ppET-1, they may express the
isoform confined to the plasma membrane. However, further
investigation is warranted to characterize ECE-1 isoforms in
steroidogenic cells.
Using various detection methods (Figs. 2, 4, and 5), we
were able to demonstrate that, as the CL matures, an increase
in ECE-1 expression and activity occurs. Increased ECE-1
expression in endothelium may be attributed to the presence
of vascular endothelial growth factor in the mature CL (30),
as demonstrated previously for this cell type (39). What may
cause such an increase in steroidogenic cells is still unknown.
The transcription of ECE-1 in different luteal cell types, ste-
roidogenic and endothelial, would allow for cell type-
specific regulation of the enzyme, so that the gland could
accommodate the varying needs for ET-1 under different
physiological conditions.
Genetic manipulation studies have demonstrated that
changes in ECE-1 expression can affect ET-1 biosynthesis (35,
40). Nevertheless, only a small number of studies have dem-
onstrated such regulatory roles for ECE-1 under physiolog-
ical conditions. Tripiciano et al. (20) reported that rhythmic
tubular contractility of testicular tissue was controlled by
cyclic expression of ECE-1, restricting the production of ma-
ture ET-1 to a defined zone during appropriate develop-
mental stages of the tissue. An analogous event is reported
here for CL development. The increase in ET-1 peptide in
midcycle CL was accompanied by increased expression of
ECE-1. Because ppET-1 mRNA did not rise concurrently,
these results also suggest that ECE-1 expression may restrict
ET-1 biosynthesis at this stage of the cycle. Conversely, dur-
ing luteal regression, when ET-1 peptide concentrations
again increase (7, 8), ppET-1 mRNA rises in parallel (7, 9),
whereas ECE-1 mRNA remains unchanged (Levy and Mei-
dan, data not shown). Therefore, our results may indicate
that the significance of ppET-1 and ECE-1 expression to ET-1
biosynthesis could vary throughout CL life span: ECE-1
plays an important role at early luteal phase; and ppET-1,
possibly at later stages of the cycle.
The mechanism by which PGF
2
exerts its luteolytic effects
was a subject of vigorous research in recent years; and several
mediators [NO (41), luteal PGF
2
(42), voltage-activated so
-
dium channels (43), reactive oxygen species (44), and ET-1 (6,
9, 15)] were all implicated in this phenomenon. However, the
other facet of this process, namely the refractory nature of the
immature CL to the luteolytic actions of PGF
2
, has remained
unresolved. We have recently shown that, in contrast to the
responsive CL during midcycle, neither ET-1 nor ETA-R
mRNA were elevated when a luteolytic dose of PGF
2
was
administered early in the cycle. The current study also points
out the importance of uninterrupted ET-1 biosynthesis to the
process of luteal regression. Low ECE-1 levels during the
early luteal phase restrict the production of active ET-1. If
ET-1 functions as an essential mediator of PGF
2
actions, then
its absence may result in CL being unable to respond to this
PG. However, not only was ET-1 peptide very low in the
untreated early CL, but PGF
2
injection failed to increase its
levels (9). Low ECE-1 levels could also account for this phe-
nomenon; during luteolysis, there is a sustained increase in
ET-1 peptide concentrations (8). This increase is driven by an
autoregulatory positive feedback mechanism, in which ET-1
in itself (45), and ET-1-induced hypoxia (46), synergize in
stimulating ppET-1 transcription. These would drastically
augment ET-1 production, provided that sufficient amounts
of ECE-1 are present. In the early CL, low ECE-1 levels may
limit the propagation of the positive feedback loop, causing
the early CL to be refractory to the luteolytic actions of PGF
2
.
Acknowledgments
We are grateful to Dr. Anna Grazul-Bilska for helpful advice con-
cerning luteal endothelial cell enrichment procedure, to Dr. E. Girsh for
his help in the ET-1 determinations, and to Dr. D. Wolfenson and Mr.
Z. Roth for their help in ovary collection.
Received February 13, 2001. Accepted August 22, 2001.
Address all correspondence and requests for reprints to: Rina Mei-
dan, Department of Animal Sciences, Faculty of Agricultural, Food and
Levy et al. ET-1 and ECE in Bovine CL Endocrinology, December 2001, 142(12):52545260 5259
Environmental Quality Sciences, The Hebrew University of Jerusalem,
Rehovot 76100, Israel. E-mail: rina.meidan@huji.ac.il.
This work was supported by a grant from the United States-Israel
Binational Agricultural Research & Development Foundation.
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5260 Endocrinology, December 2001, 142(12):52545260 Levy et al. ET-1 and ECE in Bovine CL
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Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease.
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Prostaglandin F2alpha (PGF2alpha) has been recognized as the physiological luteolysin in ruminants and other species for more than three decades; however, the mechanisms involved in its action are poorly understood. We previously have shown that endothelin-1 (ET-1) mediates, at least in part, the action of PGF2alpha, and the current study examines the effect of PGF2alpha on the expression of ET-1 in bovine corpus luteum (CL). Endothelins (ETs) were extracted from CL, collected at various times of the estrous cycle, and highest levels were found during luteolysis. The expression of prepro-ET-1 was also highest in regressing CL, suggesting that PGF2alpha may have elevated ET-1 expression. This was confirmed by demonstrating that administration of PGF2alpha to heifers at midcycle elevated luteal ET-1 expression. Levels were induced as soon as 2 h after PGF2alpha treatment and 24 h later were 7-fold higher than preinjection levels. Endothelial cells isolated from bovine CL produced ET-1, and addition of PGF2alpha, oxytocin (OT), and vasopressin-augmented ET biosynthesis. Induction of ET-1 expression by PGF2alpha in these cells was evident after a short incubation time (15-90 min). Taken together, these data suggest that stimulation of luteal ET-1 expression by PGF2alpha may be achieved by several nonmutually exclusive mechanisms: 1) by acting directly on luteal endothelial cells; 2) indirectly, via OT release from large luteal cells; and 3) by causing hypoxia in the CL (as a result of ET-1-induced vasoconstriction). The latter mechanism may serve to augment ET-1 secretion in a positive-feedback process.
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LH and prostaglandin F2 alpha (PGF2 alpha) control the life span and function of the corpus luteum (CL). Nevertheless, identification of the various cell types (steroidogenic and nonsteroidogenic) expressing the receptors for these hormones remains controversial. In this study we characterized LH and PGF2 alpha receptor (r) expression in the various luteal cell types using quantitative reverse transcription-polymerase chain reaction. We found, in agreement with previously described functions of PGF2 alpha, that the two steroidogenic cell types, as well as luteal endothelial cells, expressed PGFr. In contrast, LHr was mainly expressed by small luteal cells. A similar pattern of PGFr and LHr expression was observed in steroidogenic cells luteinized in vitro and in cells derived from the mature CL. The expression of these two receptors was inversely affected by increased levels of cAMP (achieved by incubating cells with varying doses of forskolin); LHr expression was down-regulated by 50% in the presence of 10 microM forskolin (p < 0.05), while an increase was observed in PGFr expression. In granulosa-derived luteal cells, maximal expression of PGFr was higher (approximately by 3-fold, p < 0.05) than in the theca-derived luteal cells. PGF2 alpha, mimicking its in vivo effect, markedly down-regulated LHr expression in thecaderived luteal cells, abolishing expression at a concentration of 100 ng/ml. In summary, these studies depict cAMP and PGF2 alpha as major regulators of PGFr and LHr expression in the two steroidogenic cell types. All three major cell types of the CL (steroidogenic and endothelial) express PGFr. LHr mRNA, on the other hand, was detected mainly in small luteal cells. Such broad cellular distribution of PGFr may highlight the significant role played by this prostaglandin in the bovine CL.
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The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific “stages” of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.
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A diversified series of experiments was conducted to determine the potential role of endothelin-1 (ET-1) in ovine luteal function. Endothelin-1 inhibited basal and LH-stimulated progesterone production by dispersed ovine luteal cells during a 2-h incubation. This inhibition was removed when cells were preincubated with cyclo-d-Asp-Pro-d-Val-Leu-d-Trp (BQ123), a highly specific endothelin ETA receptor antagonist. Administration of a luteolytic dose of prostaglandin F2α (PGF2α) rapidly stimulated gene expression for ET-1 in ovine corpora lutea (CL) collected at midcycle. Intraluteal administration of a single dose of BQ123 to ewes on Day 8 or 9 of the estrous cycle mitigated the luteolytic effect of PGF2α. Intramuscular administration of 100 μg ET-1 to ewes at midcycle reduced plasma progesterone concentrations for the remainder of the estrous cycle. Following pretreatment with a subluteolytic dose of PGF2α, i.m. administration of 100 μg ET-1 caused a rapid decline in plasma progesterone and shortened the length of the estrous cycle. These data complement and extend previously published reports in the bovine CL and are the strongest evidence presented to date in support of a role for ET-1 in PGF2α-mediated luteal function in domestic ruminants.
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Luteal regression is initiated by prostaglandin F 2 (PGF 2). In domestic species and primates, demise of the corpus luteum (CL) enables development of a new preovulatory follicle. However , during early stages of the cycle, which are characterized by massive neovascularization, the CL is refractory to PGF 2. Our previous studies showed that endothelin-1 (ET-1), which is produced by the endothelial cells lining these blood vessels, plays a crucial role during PGF 2-induced luteolysis. Therefore, in this study, we compared the effects of PGF 2 administered at the early and mid luteal phases on ET-1 and its type A receptors (ETA-R) along with plasma ET-1 and progesterone concentrations , and the mRNA levels of PGF 2 receptors (PGF 2-R) and steroidogenic genes. As expected, ET-1 and ETA-R mRNA levels were markedly induced in midcycle CL exposed to luteolytic dose of PGF 2 analogue (Cloprostenol). In contrast, neither ET-1 mRNA nor its receptors were elevated when the same dose of PGF 2 analogue was administered on Day 4 of the cycle. In accordance with ET-1 expression within the CL, plasma ET-1 concentrations were significantly elevated 24 h after PGF 2 injection only on Day 10 of the cycle. The steroidogenic capacity of the CL (plasma progesterone as well as the mRNA levels of steroido-genic acute regulatory protein and cytochrome P450 scc) was only affected when PGF 2 was administered during midcycle. Nevertheless , PGF 2 elicited certain responses in the early CL: pro-gesterone and oxytocin secretion were elevated, and PGF 2-R was transiently affected. Such effects probably result from PGF 2 acting on luteal steroidogenic cells. These findings may suggest, however, that the cell type mediating the luteolytic actions of PGF 2 , possibly the endothelium, could yet be nonresponsive during the early luteal phase.
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Thesis--University of Tsukuba, D.M.S.(A), no. 592, 1988. 9. 30 Offprint. Originally published in: Nature, v. 332, no. 6163, pp. 411-415, 1988 Joint authors: Hiroki Kurihara ... et al Includes supplementary treatises
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The hypothesis that hydrogen peroxide generation occurs in the corpora lutea of superovulated rats during luteolysis was tested using a peroxide-dependent inhibitor of catalase, 3-amino-1,2,4-triazole (AT). Luteal regression was induced during midpseudopregnancy by injection of 500 micrograms prostaglandin F2 alpha (PGF2 alpha) 1 h before administration of AT (0.1 g/kg, ip) and was confirmed by progesterone analysis of peripheral blood serum. Within groups of both PGF2 alpha-treated and untreated control rats, other rats also received ethanol (0.2 g/kg, ip), which prevents hydrogen peroxide-mediated inhibition of catalase by AT. Diluted homogenates of ovaries removed 1 h after AT administration were assayed for catalase activity by measuring the decrease in absorbance at 240 nm for 30 sec after the addition of hydrogen peroxide (10 mM). Ethanol-sensitive catalase inhibition by AT was significantly higher (47.9 +/- 3.38%) in samples from PGF2 alpha-treated groups than in controls (23.1 +/- 4.82%; P less than 0.01; n = 9). Similar increases in catalase inhibition by AT were found in luteal tissue of rats treated with PGF2 alpha 24 h earlier and in rats in which luteolysis was allowed to occur spontaneously in late pseudopregnancy. Hemoglobin an AT assays revealed that the changes in catalase activity were not the result of altered blood contamination or AT concentration in the luteal homogenates. Since catalase inhibition by AT is only seen in the presence of hydrogen peroxide, these results support the conclusion that an early and sustained component of corpus luteum regression is the generation of hydrogen peroxide in luteal tissue.