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REPRODUCTION-DEVELOPMENT |
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
Address all correspondence and requests for reprints to: Rina Meidan, Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel. E-mail: rina.meidan{at}huji.ac.il
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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, elevation 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 endothelial 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 parenchymal and
endothelial cells may cooperate in the biosynthesis of mature bioactive
ET-1. In the mature CL, ECE-1 mRNA increase occurred both in
steroidogenic and endothelial 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 PGF2-induced luteolysis
(7, 8, 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
PGF2. These observations were made in ovine,
bovine, and human luteal cells (6, 10, 11, 12, 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
PGF2 under both in vitro
(10) and in vivo (5, 6)
conditions. The administration of either ET-1 or a subluteolytic dose
of PGF2 to ewes and cows significantly reduced
progesterone concentrations of jugular venous blood without shortening
of luteal life span; however, the combination of these treatments 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 different 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). Tripiciano 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 temporal 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 2–4) and midluteal phase (d 9–14). For studies
involving luteal RNA extractions, CL were frozen in liquid
N2 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 containing collagenase was added. This procedure was
repeated 7–8 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 at 4 C on a rocking
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
described (24). To induce luteinization, granulosa and
theca cells were cultured in the presence of 1% FCS, insulin (2
µg/ml), and forskolin (10 µM). 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
guanidinium thiocyanate method (25). Semiquantitative
RT-PCR was performed 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. Alternative 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 (1277–1906).
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
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Determination of ET-1 and big ET-1 peptide concentrations
ET-1 concentrations in CL extracts and in culture media were
determined 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 C18 cartridges preequilibrated 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 ED50 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 ED50 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 µM) for
24 h. ET-1 concentrations in supernatants were determined by
EIA.
Western analyses
CL were homogenized in lysis buffer [20 mM Tris
HCl, pH8.6; 1% SDS; phenylmethylsulfonyl fluoride (1
mM); and protease inhibitor cocktail] and were immediately
boiled for 10 min. After chilling, homogenates were centrifuged for 15
min at 2000 x g, and protein concentrations of the
supernatant were determined using DC reagents (Bio-Rad Laboratories, Inc., Hercules, CA). Samples containing 10 µg
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], membranes
were incubated with anti-ECE-1 antiserum (produced at M. Yanagisawa’s
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-conjugated goat antirabbit IgG for 1 h at room
temperature, and binding was detected with an enhanced
chemiluminescence detection system (Amersham 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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).
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). The elevated CD31 expression in
total dispersed cells at midcycle indicates a higher proportion of
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 enriched 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 markers, CD31 and StAR; BS-1 positive cells expressed
only 3–5% of the steroidogenic cell marker, and BS-1 negative cells
expressed 1–3% of the endothelial cells marker.
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), 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 midluteal 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 corresponding
endothelial cell fraction at early cycle. The difference between total
dispersed CL cells and enriched endothelial 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 steroidogenic 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 agreement with data obtained using enriched luteal cell
preparations (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 observed both at
early and midluteal phases (Fig. 5, A and C). 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, respectively; Fig. 5, A and C). No
specific hybridization was observed when hybridizing with the sense
probe (Fig. 5E).
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, 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 medium; Both cells types efficiently converted
big ET-1 into mature ET-1 (Fig. 6B). Addition of the metalloprotease
inhibitor, phosphoramidon, which inhibits ECE activity, significantly
inhibited the production of ET-1 peptide (Fig. 6B).
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, 5, and 6 indicate that
luteal steroidogenic cells express functional ECE-1 capable of
producing mature ET-1 peptide. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ET-1 plays an important role in the female reproductive cycle: its
quick ascent during luteal regression (7, 8, 9), ability to
inhibit steroidogenesis in vitro and in vivo
(6, 10, 11, 13), combined with the observation that the
luteolytic effects of PGF2 were decreased by
pretreatment with a ETA antagonists (6, 10), suggest that
this peptide functions as an important element of the luteolytic
cascade. In addition, ET-1 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 establishment
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 Sertoli cells (32, 33, 34). 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 endothelial cells within the CL may cooperate in the biosynthesis 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 physiologically 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 developmental 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; however, 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, steroidogenic 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 demonstrated such regulatory roles for ECE-1 under physiological 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 mature ET-1 to a defined zone during appropriate developmental 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, during 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 Meidan, 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 PGF2 exerts its
luteolytic effects was a subject of vigorous research in recent years;
and several mediators [NO (41), luteal
PGF2 (42), voltage-activated
sodium 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 PGF2, 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 PGF2 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
PGF2 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
PGF2 injection failed to increase its levels
(9). Low ECE-1 levels could also account for this
phenomenon; 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 PGF2.
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Acknowledgments |
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Footnotes |
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Abbreviations: aa, Amino acid; BME, ß-mercaptoethanol; BS-1, Bandeiraea 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.
Received February 13, 2001.
Accepted for publication August 22, 2001.
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References |
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- induced luteal regression in the
ewe. Biol Reprod 64:1619–1623.
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promotes the
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regulates distinct physiological changes in early and
mid-cycle bovine corpora lutea. Biol Reprod 58:346–352This article has been cited by other articles:
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