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Original Studies |
) Biosynthesis and Release by Human Luteal Cells: Evidence of a New Paracrine/Autocrine Regulation of Luteal Function
Departments of Obstetrics and Gynecology (Fi.M., Fr.M., M.G.P., S.M., R.A.) and Pharmacology (P.N.), Università Cattolica del Sacro Cuore, 00168 Rome, Italy; and OASI Institute for Research (C.P., A.L.), 94018 Troina, Italy
Address all correspondence and requests for reprints to: Rosanna Apa, M.D., Department of Obstetrics and Gynecology, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, Italy. E-mail: krimisa{at}libero.it
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Abstract |
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and
PGE2 were assayed in the culture medium to investigate
whether ETs also influence cyclooxygenase activity in these cells. We
found that both ETs are able to significantly stimulate
PGF2 and PGE2 release in a dose- and
time-dependent manner. ET-1 was always more effective than ET-3.
Experiments with two endothelin receptor antagonists (the BQ485 and
BQ788 compounds, which block the ET-A and ET-B receptors, respectively)
showed that the two endothelins induce PG production through different
receptors and signaling pathways. In conclusion, here we demonstrate
the ability of ETs to influence PG synthesis and release from human
luteal cells. As PGs are deeply involved in corpus luteum activity, and
ETs were also able to influence progesterone production, the present
new data suggest an interesting interplay among progesterone, PGs, and
ETs in the control of corpus luteum physiology. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two ET receptor subtypes, ET-A and ET-B, have been identified in a variety of tissues. ET-1 has a higher selectivity for ET-A; its binding affinity is almost 100 times higher than that of ET-3 (10), whereas ET-B is nonselective and is bound by all ETs with similar affinities (11, 12). ET-1 binds the ET-A and ET-B receptors with equal affinities, although the majority of its effects are mediated by ET-A receptors. The latter are coupled, via G protein, to the activation of phospholipase C in all tissues investigated (7, 13, 14). Conversely, in cultured porcine granulosa cells ETs attenuate gonadotropin-stimulated P secretion by inhibiting adenylyl cyclase, and this effect is mediated by the ET-B subtype, indicating that the latter is coupled to activation of the adenylyl cyclase-cAMP pathway (15).
It is well established that the regression of CL is essential for the
normal menstrual cycle because it allows the development of a new
ovulatory follicle, whereas prevention of luteolysis is necessary for
the maintenance of pregnancy. In CL physiology, a key role is played by
both PGF2 and PGE2. The
latter seems to exert a luteotropic action; in animals it stimulates
luteal P production (16), and in humans a positive
correlation between P and PGE2 has been
demonstrated throughout the menstrual cycle (17).
PGE2 was also shown to stimulate P synthesis
(18, 19) and activate the cAMP pathway (18, 20) in human CL in vitro. Conversely,
PGF2 seems to be involved in CL regression; a
negative correlation has been demonstrated between the concentrations
of P and PGF2 throughout the human menstrual
cycle (21), and the intraluteal injection of
PGF2 caused both an immediate fall in serum P
and a shortening of the luteal phase (22).
The present study was designed to continue our investigation about the role played by ETs in CL physiology; in particular, we sought to determine whether these peptides are able to influence PGs synthesis in human luteal cells in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic ET-1 and ET-3 were obtained from Roche (Mannheim, Germany); BQ485 and BQ788 were purchased from NovaBiochem (La Jolla, CA). hCG and phorbol 12-myristate 13-acetate (PMA) were purchased from Serono (Rome, Italy). The following chemicals were purchased from Sigma (St. Louis, MO): collagenase type IV, antibiotics, glutamine, HEPES and cAMP. Ham’s F-12 medium was obtained from Flow Laboratories (Milan, Italy), and FCS was purchased from Biological Industries (Kibbutz Beit HaEmek, Israel).
Luteal cell culture preparation and experimental procedure
CL were obtained at the time of hysterectomy performed for nonendocrine gynecological diseases (leiomyomatosis) in the midluteal phase of the menstrual cycle (days 5–6 from ovulation). Patients were between 30–43 yr of age. All of them had a history of regular menstrual cycles. A total of 13 experiments were performed. Informed consent was obtained from each patient, and the study was approved by the internal review board. The age of the CL was determined as follows. All patients were monitored until ovulation by daily measurement of basal body temperature (BBT) and ultrasound examination of follicular growth. When the maximal follicular diameter had reached 18 mm, daily determination of plasma P values was made. The time of ovulation (day 0) was detected by the biphasic pattern of BBT, by the typical ultrasound disappearance of the dominant follicle or the ultrasound detection of CL, and by the rise in plasma P concentrations. At the time of surgery, plasma samples were collected immediately before anesthesia to determine plasma P concentrations. The luteal tissue removed was immediately freed from blood vessels and ovarian stroma under a dissecting microscope, dissected, and minced. Human CL cultures were performed as previously described (23) with some modification. The luteal tissue was placed in 10 mL prewarmed Ham’s F-12 HEPES medium containing type IV collagenase (200 U/mL), then incubated at 37 C in a shaking water bath for 45 min. The medium containing the cell suspension was filtered through a 40-µm nylon mesh, and the cells obtained were centrifuged and resuspended twice in fresh medium. This procedure was repeated once with the remaining undigested tissue to obtain highly purified luteal cells. Cells were counted in a hemocytometer, and viability was determined by the trypan blue exclusion test. The cells were diluted to a final concentration of 250,000 live cells/mL medium supplemented with 2 mmol/L L-glutamine, 100 IU penicillin, 100 mg/mL streptomycin, and 10% FCS and cultured in 48-multiwell plates for 24 h in 5% CO2 and 95% air at 37 C. After this time the cells were attached to the wells; the medium was then removed and replaced with fresh serum-free medium alone (controls) or containing ET-1 (10-9–10-6 mol/L), ET-3 (10-9–10-6 mol/L), BQ 485 (10-9–10-6 mol/L) alone or combined with ET-1 (10-7 mol/L), and BQ 788 (10-9–10-6 mol/L) alone or combined with ET-3 (10-7 mol/L). In another group of experiments, we stimulated the cells with cAMP (1 mmol/L) and PMA (10-7 mol/L) either alone or combined with ET-1 or ET-3 (10-7 mol/L). At the end of experiments the cells were stained for lipids with oil red O (14) and counted. More than 90% of the luteal cells stained positively for lipids. The remainder did not stain for lipids, and there were occasional vascular cells, including erythrocytes and leukocytes. The medium was harvested after 12, 24, and 48 h of culture and stored at -20 C until assayed for PG immunoreactivity.
PG assays
RIAs for PGE2 and
PGF2 adopted in this study were first
characterized for measurement of the prostanoids in human urine
(24) and later were used successfully to measure PGs
produced and released by several cell types in vitro,
including cells from human ovaries (20). Briefly, for each
assay, incubation mixtures of 1.5 mL were prepared in disposable
plastic tubes in which 50 µL incubation medium were diluted to 250
µL with 0.025 mol/L phosphate buffer (pH 7.5). Tritiated
PGE2 or PGF2
500–3(2,500–3,500 cpm) and appropriately diluted antisera were added
together to a final volume of 1.5 mL. The antisera (provided by Prof.
G. Ciabattoni) were employed at a final dilution of 1:120,000 or
1:150,000 (for PGE2 or
PGF2, respectively). A duplicate standard
curve ranging from 2–100 pg/tube was run for each assay. All tubes
were incubated for 24 h at 4 C. Separation of antibody-bound PGs
was obtained with 2.5 mg charcoal (Norit-A), which absorbs 95–98% of
free PGs; a charcoal suspension (2.5 mg/50 µL) in 0.025 mol/L
phosphate buffer, pH 7.5, was added to each tube after the addition of
100 µL 5% BSA. The tubes were briefly shaken and then centrifuged
for 10 min at 4 C. Supernatants were decanted into 10 mL scintillation
liquid. Radioactivity was measured by liquid scintillation counting.
The detection limit of the assay was 2 pg/tube in all cases. The inter-
and intraassay variability coefficients were 2.7% and 2.9% for
PGE2, and 3.2% and 2.8% for
PGF2, respectively.
[3H]PGE2 and
[3H]PGF2 were obtained
from NEN Life Science Products (Milan, Italy).
Data analysis
Data were first analyzed by Kolmorogov-Smirnov test to assess differences in the general shapes of distribution. Normally distributed data were then analyzed by one-way ANOVA with Bonferroni correction to perform pairwise comparisons between group means.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and PGE2
are produced and released in sizable amounts from human luteal cells.
After release, PGs do not appear to be further taken up or metabolized
by cultured cells, as they tend to accumulate within the incubation
medium in a time-related fashion (Fig. 2
). As PGs are not stored in
these cells, the fraction released in the medium can be taken as a
reliable index of total PG biosynthesis.
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and
PGE2 synthesis by human luteal cells
Luteal cells were cultured for 24 h with graded doses of ET-1
and ET-3
(10-9–10-6
mol/L). As shown in Fig. 1A, both ETs
were able to increase PGF2 release in a
dose-dependent manner. The effect was statistically significant
(P < 0.05) for ET-1 from
10-8 mol/L onward, whereas
for ET-3 it was significant from
10-7 mol/L onward. In a
similar way, both ETs positively affected PGE2
production (Fig. 1B) with the same potencies as those observed for
PGF2. In fact, in this case also ET-1
significantly stimulated PGE2 release from
10-8 mol/L
(P < 0.05) onward, whereas ET-3 was effective from
10-7 mol/L
(P < 0.001). A plateau was reached by both ETs at a
concentration of 10-7
mol/L.
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(left panel) shows how the highest
PGF2 levels were induced by ET-1 after 24
h of culture (P < 0.001 vs. controls) even
though a significant effect was already present after 12 h
(P < 0.05). After 48 h of incubation,
PGF2 concentrations fell to values similar to
those observed in the control group. The effect of ET-3 was
statistically significant only after 24 h of culture
(P < 0.05), whereas no effect was seen at 12 and
48 h. ET-1 was overall more effective than ET-3 in stimulating
PGF2 release, as it was already effective
after 12 h of culture, and at 24 h its effect was stronger
compared with that of ET-3 (ET-1 vs. controls,
P < 0.001; ET-3 vs. controls,
P < 0.05). Figure 2 (right panel) shows the
time-dependent effects of both ETs on PGE2
release. ET-1 exerted a time-dependent effect that was already
significant after 12 h (P < 0.05) and further
increased after 24 (P < 0.001) and 48 h
(P < 0.01 vs. controls). ET-3 also
significantly stimulated PGE2 at 12
(P < 0.05 vs. controls) and 24 h
(P < 0.001), whereas at 48 h PG levels were
similar to those observed in the control group. Only at 48 h was
ET-1 more effective than ET-3. Receptor subtypes mediating the effects of ETs on PGs release
To determine the subtype(s) of receptor mediating the effects of
ETs on PG release, luteal cells were preincubated for 30 min with BQ
485
(10-9–10-6
mol/L) or BQ 788
(10-9–10-6
mol/L). The former is a potent and selective ET-A receptor antagonist,
whereas BQ 788 selectively blocks ET-B receptors. Cells were then
treated for 24 h with ET-1 (the groups receiving BQ 485) and ET-3
(the groups receiving BQ 788), both at a concentration of
10-7 mol/L. As shown in
Fig. 3, in the presence of BQ 485 the
stimulatory effect of ET-1 on PGF2 (left
panel) and PGE2 release (right
panel) was dose dependently reduced; at its highest concentration,
BQ 485 completely abolished stimulation by ET-1. A remarkably similar
effect was observed when the cells were incubated with ET-3 and the
ET-B receptor antagonist BQ788 (Fig. 4).
Also in this case, we observed a graded decrease in ET-3-induced PG
release that paralleled the increase in BQ788 concentrations. At the
highest concentration of BQ 788, the effect of ET-3 was completely
abolished. Neither receptor antagonist had an intrinsic effect on PGs
release when given alone.
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In an attempt to clarify the pathway(s) through which ETs exert
their effects on PGs, luteal cells were incubated for 24 h with
PMA (100 ng/mL) or cAMP (1 mmol/L) alone or combined with ET-1 and ET-3
(10-7 mol/L). It is
interesting to notice that the results obtained for
PGF2 (Fig. 5A)
and PGE2 (Fig. 5B) were superimposable. We found
that both PMA and cAMP, when given alone, produced a much higher
increase in PGs release compared with control and ET-treated groups
(cAMP, P < 0.001 vs. control and ET groups;
PMA, P < 0.001 vs. control and ET groups).
When they were used in association with the ETs, we found that only
ET-1 was able to further increase the positive effect of cAMP on both
PGs (P < 0.05 vs. cAMP alone). In all other
cases, the ETs were unable to further enhance cAMP- or PMA-stimulated
PGs release.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGs are closely related to the luteal function. Human CL produces
both PGE2 and PGF2
(29, 30). In various species, including humans,
PGF2 seems to be of importance for luteolysis
(20, 31, 32, 33, 34), whereas there is increasing evidence of
luteotropic activity exerted by PGE2. Within this
frame, the present study was designed to continue our ongoing research
about the effects of ETs on CL in an attempt to discover the novel
action mechanism(s) by which ETs influence luteal physiology. We found
that both ET-1 and ET-3 are able to significantly stimulate
PGF2 and PGE2 synthesis
and release from human luteal cells in a dose- and time-dependent
manner. For both PGs, the effects of ETs were observed at
concentrations suggesting a physiological, rather than a
pharmacological, action of the peptides. In addition, it is unlikely
that these effects are secondary to a possible mitogenic action of ETs,
as luteal cells were highly differentiated, and no mitosis was observed
during the culture period (35).
The observed stimulatory effect of ETs on PGE2
release was unexpected. In fact, based on the inhibition exerted by
ET-1 on P production (9) and the fact that ET-1 mRNA
expression is increased during the late luteal phase, when
PGE2 levels are lowest (26, 36), we
were postulating that the negative effect of ET-1 on P production might
be mediated by an increase in PGF2 and a
decrease in luteotropic PGE2 production,
respectively. The fact that our results did not confirm our hypothesis
might be explained in two ways. 1) PGs might influence the
steroidogenic activity of CL in part through well established,
classical ways and in part through alternative, as yet poorly
understood mechanisms; this assumption seems to be confirmed by the
fact that in the present study ET-3 has been shown to stimulate the
synthesis of both PGs, whereas it had no effect on P production in the
same experimental paradigm (9). 2) An alternative, or
additional, hypothesis involves the vascular component of CL.
Endothelial cells are particularly abundant in CL (>50% of total CL
cells) (37, 38), where they are in contact with
steroidogenic cells (39). Furthermore, endothelial cells
produce ETs. It has been demonstrated that PGE2
inhibits ET-1 production and secretion from cultured bovine aortic
endothelial cells (40), whereas
PGF2 increases ET-1 content and inhibits P
production in bovine CL slices (22). If these mechanisms
are also present in human CL, we can hypothesize the presence of a
feedback loop within the CL, where ETs and PGs mutually modulate each
other’s production and action. Interestingly, both ETs and PGs can
play a dual role: on steroidogenic cells and on arterial tone. Indeed,
PGE2 has a luteotropic action (19)
and is a potent vasodilator, whereas ET-1 inhibits P synthesis and is
the most potent vasoconstrictor known (1).
Conflicting are data regarding the location and type of ET receptors in the ovary. Several studies have demonstrated the presence of ET-A receptors in bovine luteal cells (22). In contrast, porcine and rat granulosa cells contain ET-B receptors, characterized by binding studies and mRNA expression (27, 41, 42). As far as the human ovary is concerned, Macina and colleagues (43) reported that ET-A and ET-B receptors are mostly expressed in the blood vessel of the ovary, rather than in the tissue. On the contrary, another group showed the presence of both receptor subtypes in luteinized granulosa cells, with the expression of the ET-A receptor mRNA being much greater than that of ET-B (44). In the present study we did not perform binding or molecular biology studies; however, the high degree of purity of our luteal cultures and the established selectivity of pharmacological tools indicate, albeit indirectly, that both ET receptor subtypes are expressed in cultured human luteal cells. In addition, findings with the antagonists of both ET receptors suggest that the action of ET-1 on PG release is mostly mediated by the ET-A receptor, whereas the effect of ET-3 is mediated by the ET-B subtype. The latter statement is based on the assumption that if the effects of ET-1 and ET-3 were in part mediated by both receptor subtypes, in both cases we could not have achieved complete inhibition of ET’s effects by blocking a single subtype.
Our findings in experiments with cAMP and phorbol esters provide some additional evidence to the concept that in the ovary the ET-A receptor is coupled to the activation of phospholipase C via G protein (11, 12, 13, 44), whereas ET-B receptors signal via the adenylyl cyclase pathway (15). When used alone, cAMP and PMA stimulated PG release more strongly compared with ETs alone, and PMA was more effective than cAMP, probably because the protein kinase C pathway, activated by PMA, stimulates phospholipase A2, which, in turn, triggers the enzymatic cascade ending in PG biosynthesis. When ETs were added to the cultures containing PMA or cAMP, we found that ET-1 was able to further increase cAMP-induced PG release, whereas ET-3 had no such effect. These results might be explained assuming that ET-1, but not ET-3, activates the phospholipase C-protein kinase C pathway, which synergizes with the adenylyl cyclase pathway to produce maximal PG release. Conversely, the lack of potentiation by ET-3 suggests that this peptide signals through the adenylyl cyclase pathway, which is already maximally stimulated by cAMP and therefore is not capable of further increase. Although specifically exploring second messenger mechanisms, these experiments lend further support to the concept that in human luteal cells, ET-1 acts mainly via the activation of ET-A receptors, whereas the ET-B subtype mediates the effect of ET-3.
In conclusion, we report that ET-1 and ET-3 are able to stimulate PG synthesis and release by human luteal cells. Taken collectively, the present data and our previous findings showing the presence of ET-1 mRNA in these cells as well as the ability of ET-1 to influence luteal steroidogenesis strongly support the idea that ETs play a primary role in CL physiology.
Received April 27, 2000.
Revised August 2, 2000.
Revised September 30, 2000.
Accepted October 13, 2000.
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References |
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in the human corpus
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, P
< 0.05; 
