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Department of Animal Science (R.M., N.L., R.M.), Faculty of Agriculture, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel; Department of Obstetrics and Gynecology (B.R., J.S.D.), Women’s Research Institute, University of Kansas School of Medicine, Wichita, Kansas 67214; and VA Medical Center (B.R., J.S.D.), Wichita, Kansas 67214
Address all correspondence and requests for reprints to: Dr. Rina Meidan, Department of Animal Sciences, Faculty of Agriculture, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot 76100, Israel. E-mail: rina{at}agri.huji.ac.il
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Abstract |
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receptor mRNA. The ETA receptor
gene was expressed by all three major cell populations of the bovine
CL; i.e. small and large luteal cells, as well as in
luteal endothelial cells. Among these various cell populations, the
highest ETA receptor mRNA levels were found in endothelial cells. cAMP
elevating agents, forskolin and LH, suppressed ETA receptor mRNA
expression in luteinized theca cells (LTC). This inhibition was
dose dependent and was evident already after 24 h of incubation.
In luteinized granulosa cells (LGC), 10 and 100 ng/ml of insulin-like
growth factor I and insulin (only at a concentration of 2000 ng/ml)
markedly decreased ETA receptor mRNA levels. In both LGC and LTC there
was an inverse relationship between ETA receptor gene expression and
progesterone production; insulin (in LGC) and forskolin (in LTC)
enhanced progesterone production while inhibiting ETA receptor mRNA
levels. Our findings may therefore suggest that, during early stages of
luteinization when peak levels of both LH and insulin-like growth
factor I exist, the expression of ETA receptors in the gland are
suppressed. This study demonstrates physiologically relevant regulatory
mechanisms controlling ETA receptor gene expression and further
supports the inhibitory role of ET-1 in CL function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endothelins bind two major receptor subtypes, both of which belong to the seven-transmembrane G protein-coupled receptor superfamily (4, 5). The two receptor subtypes have been termed ETA (for aorta) and ETB (for bronchus). The ETA receptor has a high specificity for ET-1 (ET-1 affinity is about 100 times that of ET-3, whereas the other type, ETB, binds all three endothelins (ET-1, ET-2, and ET-3) with the equipotent affinity (4, 5). The binding of endothelins to its receptors activates several transmembrane signalling pathways that most likely contribute to the diversity of the biological responses induced by this peptide (1, 3, 6, 7).
ET-1, isolated only a decade ago, has been described as the most potent
vasoconstrictive agent yet identified (8); however, it quickly gained
immense attention due to its role in numerous organs (1, 3, 6). Within
the ovary, high concentrations of ET-1 were found in follicular fluids
and in CL (9, 10, 11, 12). In addition, ET-1 inhibited cAMP and progesterone
production in gonadotropin-stimulated granulosa cells of various
species (10). Recently, we and others have shown that ET-1 is an
essential paracrine regulator of corpus luteum (CL) function in which
it mediates the antisteroidogenic effects of prostaglandin F2
(PGF2: 13–14). The identification of the receptor subtypes that
subserve the inhibitory effects of endothelins in these ovarian cells
has been controversial as both ETA and ETB receptors were identified
(15, 16, 17).
In mesengial, smooth muscle and endothelial cells, ET-1 binding capacity is regulated by growth factors, hormones and proinflammatory agents (18, 19); however, the hormonal regulation of ET-1 binding sites in ovarian cells has not yet been studied. In our studies, ET-1 expression in the bovine CL varied throughout the estrous cycle, with maximal levels being expressed during luteal regression (9). It is still unknown whether these changes are accompanied by alterations in ET-1 receptor gene expression in the CL.
The present study was undertaken to characterize ETA receptor gene expression in bovine ovarian cells: 1) throughout the estrous cycle; 2) in different luteal cell populations; and 3) in theca and granulosa cells luteinized in vitro under different hormonal milieu.
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Materials and Methods |
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CL collection and luteal cell enrichment
The bovine luteal tissue collected at specific days of the
luteal phase were collected for use in earlier studies and have been
previously reported (20). Briefly, Lutalyse was administered (25
mg, im 2x) to Western range cattle to synchronize their estrous
cycles. The cows were observed twice daily for estrus (estrus =
day 0). On the day of collection the CL were either surgically removed
or collected immediately following slaughter. The CL were snap frozen
in liquid nitrogen and stored at -70 C. All animal studies described
in this study were reviewed and approved by the appropriate
institutional animal care and use committee. Total RNA was extracted
from tissues by the guanidinium thiocyanate method (21). For studies
with dispersed luteal cells, CL were collected from a local slaughter
house. The highly enriched populations of bovine large and small luteal
cells were collected as has been previously described (22). Briefly,
collagenase-dissociated cells were injected into a Sanderson
elutriation chamber, and the elutes were collected with continuous flow
as follows. The first fraction contained predominantly erythrocytes and
endothelial cells (< 10 µm) and variable degree of small luteal
cells (SLC), the second fraction contained predominantly SLC (10–20
µm; SLC 81.6 ± 2.7%, 0.3 ± 0.2% large luteal cells
(LLC) and 15.7 ± 2.1 endothelial cells). The fourth fraction, was
highly enriched in LLC (> 30 µm) and contained less than 5%
enucleated cells (59.6 ± 6.6% LLC, 1.6 ± 5.0% SLC, and
18.1 ± 1.8% endothelial cells). Mixed luteal cells, counted
before elutriation, were composed of SLC (60.1 ± 2.2%), LLC
(4.3 ± 0.2%), and endothelial cells (35.2 ± 2.7%).
Cell cultures
Granulosa and theca cells were isolated from healthy bovine
preovulatory follicles, as previously described (22, 23).
Short-term cultures
These studies were performed with cells cultured in the presence
of insulin (2 µg/ml) and forskolin (10 µM); conditions
previously shown to induce the differentiation/luteinization of
granulosa and theca cells into large and small luteal-like cells (22).
Media were replaced every 2 days. On day 8, cells were washed several
times with fresh media and incubated for an additional 24 h in
media containing the various treatments as specified in the text. At
the end of the 24 h incubation period, total RNA was extracted by
the guanidinium thiocyanate method (21).
Long-term cultures
Freshly isolated granulosa cells were cultured in basal media
(containing 1% FCS) only or basal media containing insulin (2 µg/ml)
for 8 days. Freshly isolated theca cells were cultured in basal media
only or in the presence of forskolin (0.1, 1, or 10 µM).
On day 8, media were collected for progesterone determination from all
triplicate culture wells. DNA was extracted (24) from one of the
triplicates and total RNA from the two remaining wells.
Semiquantitative RT-PCR
Semiquantitative RT-PCR was carried out as described previously
(22, 25) with the housekeeping gene, glyceraldehyde 3-phosphate
dehydrogenase (G3PDH), as an internal standard. G3PDH is constitutively
expressed in both granulosa-derived luteal cells and theca-derived
luteal cells and has been used effectively in studies on the regulation
of gene expression in ovarian cells (26, 27). The RT-PCR amplification
of luteinized theca and granulosa cells [luteinized theca cells
(LTC) and luteinized granulosa cells (LGC), respectively] was
calibrated using G3PDH and ETA receptor primer pairs. G3PDH
oligonucleotide primer pair (sense 5'- TGTTCCAGTATGATTCCACCC-3';
antisense 5'-TCCACCACCCTGTTGCTGTA-3') and ETA receptor oligonucleotide
primer pair (sense 5'- ATGGCCCCAACGCGCTGATAGC-3'; antisense
5'-GACGTGGCATTGAGCATACAGG-3') were synthesized according to bovine ETA
receptor complementary DNA (cDNA) sequence described by Arai et
al. (4). To prevent coamplification of ETB cDNA, primers were
designed to span a region with low homology between the two ET receptor
subtypes. The expected PCR product lengths were: 850 bp for G3PDH and
420 bp for ETA receptor. Number of cycles used for the PCR reactions of
LGC (G3PDH, 23 cycles, ETA -28 cycles) and LTC (G3PDH, 23 cycles, ETA,
25 cycles).
Computer searches and sequence alignments were performed by means of software from Genetics Computer Group Inc. (Madison, WI). The number of cycles was varied to determine the optimal number that would allow detection of the appropriate messenger RNA (mRNA) transcripts, while still keeping amplification for these genes in the log phase [primer dropping method (28)].
To verify the specificity of the PCR product for the ETA receptor, it
was cleaved at a unique site using BamHI. Figure 1
demonstrates the amplified cDNA and the
expected 107- and 313-bp fragments obtained after cleavage.
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Statistical analysis
All experiments were repeated at least three times; each
experiment represents an individual corpus luteum or follicle (in
either case one per cow). ETA receptor and PGF2 receptor mRNA were
expressed in relation to G3PDH mRNA. Data are presented as means
± SEM. One-way ANOVA was used to determine the statistical
significance of individual treatments, 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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) and in CL
collected during various stages of the cycle (Fig. 2B). The expression
of ETA receptor was compared with that of PGF2 receptor, which was
previously characterized (29, 30). As expected, PGF2 receptor mRNA
levels were almost undetectable in follicular cells and greatly
enhanced in CL. On the contrary, granulosa and theca cells of
preovulatory follicles contained high levels of ETA mRNA and those
levels were reduced in CL of early or mid-luteal phases (Fig. 2). A
marked up-regulation of ETA mRNA occurred in luteal tissue collected at
later stages of the estrous cycle- during the time of luteal regression
(on days 18–21 of the bovine cycle). At this stage elevated ETA
receptor mRNA coincided with low levels of PGF2 receptor mRNA levels
(Fig. 2).
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. However,
the resident endothelial cells of the CL had significantly higher
levels of ETA receptor mRNA (5.84 ± 0.22; P <
0.01) than either large or small luteal cells. These two steroidogenic
cell types contained similar levels of ETA receptor mRNA (1.25 ±
0.27 and 1.85 ± 0.36 in small and large luteal cells,
respectively, P > 0.05). In dispersed mixed luteal
cell preparations, which reflects the cell composition within the
bovine CL, ETA receptor mRNA level (2.85 ± 1.45) appear to be the
average value for endothelial and steroidogenic cells.
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. In LTC, incubation with forskolin
reduced ETA receptor mRNA levels by approximately 2-fold compared with
cells incubated in serum alone (Fig. 4, P < 0.05). The
effect of forskolin was similar in LTC cultured with or without
insulin, suggesting that the presence of insulin, per se,
did not affect ETA receptor gene expression in LTC.
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).
To further examine the role of elevated cAMP levels in regulating ETA
receptor mRNA levels in LTC, cells were incubated for 24 h with
basal media only or in the presence of various concentrations of
forskolin (0.1, 1, 10 µM) or LH (1, 10, 100 ng/ml). As
shown in Fig. 5, both LH and forskolin,
down-regulate ETA receptor gene expression in LTC in a
concentration-dependent manner.
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). Insulin significantly decreased ETA
receptor mRNA only at pharmacological concentrations, whereas IGF-1
down-regulated ETA receptor gene expression at both 10 and 100 ng/ml.
Together, these data suggest that the inhibitory effects of insulin are
exerted via type I IGF-1 receptors.
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High levels of ETA receptor mRNA were present in both granulosa and
theca cell cultured in basal media (containing only 1% FCS; Figs. 7 and 8).
Similar, to its short-term effect on luteal cells (Figs. 4 and 6),
8-day cultures of LGC in the presence of insulin reduced the ETA
receptor mRNA levels to one-third of the levels observed in cells
cultured in basal media (Fig. 6). No such effect was observed in LTC
cultured in the presence of insulin (data not shown). However, in this
cell type there was a significant effect of forskolin
(P < 0.01), which inhibited ETA receptor mRNA gene
expression in a dose-dependent manner (Fig. 8).
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and 8). Insulin (in LGC) and forskolin (in LTC) enhanced progesterone
production on the one hand and inhibited ETA receptor mRNA levels on
the other hand. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies examining the function of endothelins in ovarian cells suggested that endothelins inhibited gonadotrophic hormone action in porcine and rat granulosa cells (10, 17). Similarly, we had reported that basal and bLH -or cAMP-stimulated progesterone secretion from bovine luteal cells was inhibited by ET-1 in a dose-dependent manner (13). Direct binding competition assays using a selective ETA receptor antagonist (BQ123) and bioactivity studies demonstrated that the inhibitory actions of ET-1 in luteal cells were exerted via the ETA receptor binding sites (13). The findings reported in the present study confirm and extend our previous observations and demonstrated that the ETA receptor gene is expressed in the two luteal cell types enriched from bovine CL or obtained after luteinization in vitro. Flores et al. (15), detected ETA receptor binding exclusively in granulosa cells of healthy large preovulatory follicles of pigs; their study also claimed that ET binding to porcine CL was confined to blood vessels, whereas the parenchyma of the CL were devoid of ET binding. These data are at odds with the findings reported in the present study and could be explained either by differences between species or, perhaps, by the use of different methodologies. The present study used isolated bovine luteal cells and RT-PCR, whereas the previous study examined ET binding to porcine CL slices using autoradiography (15). In light of the elaborate capillary network in the CL (32), and the higher expression of ETA receptor in endothelial cells, the presence of ET-1 binding to steroidogenic cells, may have been overlooked. Nevertheless, these two studies clearly demonstrate that endothelial cells of the CL, similar to endothelial cells of many other tissues, contain ETA receptor. The role of endothelial ETA receptors in CL physiology remains to be determined.
The two steroidogenic luteal cell types, i.e. the large and small luteal cells, are both engaged in progesterone production; nevertheless, they exhibit differing morphological and functional properties [reviewed by Hansel and Blair (33)]. Data reported here demonstrate that these cells also differ in their hormonal regulation of ETA receptor; in theca-derived luteal cells, elevation of cAMP levels down-regulated ETA receptor, whereas in the granulosa-derived luteal cells IGF-I (or insulin) inhibited the ET-1 receptor mRNA levels. These findings are important for the understanding of ETA receptor regulation in general, but in addition, they have significant physiological implications for CL function. The LH surge triggers the formation of the CL and in the course of this process, follicular cells are transformed into luteal cells and acquire high steroidogenic capacity. Not only LH (acting via cAMP-stimulated pathway), but also IGF-I (acting via tyrosine kinase-stimulated pathway) was shown to have profound luteotrophic effects (31, 34). IGF-I produced within the ovary (35) or that produced by the liver may both affect ovarian cells, however only ovarian IGF-I varies throughout the estrous cycle (36, 37).
Our findings may therefore suggest that during early stages of luteinization, when peak levels of both LH and IGF-I exist, the expression of ETA receptor in the gland are suppressed. The findings on the in vitro regulation of ETA mRNA levels are corroborated by the in vivo data showing a decrease from the levels in preovulatory follicles to those present during early to mid luteal phase CL. In light of the inhibitory role of ET-1 on luteal steroid production (13), this mechanism may function to ensure an undisturbed progesterone production. Recently we have obtained data (25) showing that StAR mRNA levels in LTC were induced mainly by elevated cAMP levels whereas in LGC, IGF-I, induced and maintained high StAR mRNA levels. It is noteworthy, therefore, that LH and IGF-I inhibited ETA receptor expression in LTC and LGC, respectively. Data on the regulation of StAR mRNA (25) and on progesterone production (the present report) may therefore indicate that for each of the two luteal cell types steroidogenic capacity was inversely correlated with ETA receptor expression.
High ETA receptor mRNA levels were observed in this study during luteal
regression. At this stage of the cycle, PGF2 receptors mRNA dropped
to their lowest values, as also shown by Juengel et al.
(30). These data, when combined with previous studies, suggest the
following sequence of intraluteal events during luteolysis: PGF2
triggers the expression of ET-1 (9, 14, 38), PGF2 receptor content
declines, ETA receptor expression then increases allowing ET-1 to
inhibit progesterone secretion (12, 13). This sequence of events is in
accord with the notion that ET-1 is a mediator of the antisteroidogenic
actions of PGF2.
Collectively, these findings demonstrate that physiologically significant mechanisms regulate ETA receptor expression in the bovine CL and further support the essential role for ET-1 in CL function.
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Footnotes |
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Received October 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promotes the
inhibitory effects of endothelin-1 on the bovine luteal function
in vitro. J Endocrinol 152:R7–R11
induced anti-steroidogenic action. Endocrinology 137:1306–1312[Abstract]
and LH receptors
mRNA expression in different bovine luteal cell types. Biol Reprod 58:849–856
receptor in bovine preovulatory follicles. Endocrinology 137:3348–3355[Abstract]
receptor (FP) during
luteolysis and early pregnancy in the ovine corpus luteum. Endocrine 3:781–787
receptor in ovine corpus luteum. Biol
Reprod 54:1096–1102[Abstract]
-induced luteolysis in the cow. Biol Reprod 58:103–108This article has been cited by other articles:
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