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Forschungsinstitut für Molekulare Pharmakologie, D-10315 Berlin, Germany (N.H., J.F., M.B., H.B.) and Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, D-52425 Jülich, Germany (M.R.M., A.S., W.B.)
Address all correspondence and requests for reprints to: Dr. Hartmut Berger, Research Institute of Molecular Pharmacology, Alfred-Kowalke-Strasse 4, D-10315 Berlin, Germany. E-mail: berger{at}fmp-berlin.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-helical
CRF9–41, and accompanied by intracellular cAMP elevation. The rank
order of potency of the natural CRF agonists, hCRF, ovine CRF,
sauvagine, and urotensin, corresponded to that of their activities on
CRF-R1 in rat pituitary cells and also to that reported for this
receptor, but not for CRF-R2, when transfected into various cell lines.
Furthermore, the difference in response of mouse Leydig cells to
[11-D-Thr,12-D-Phe]- and
[13-D-His,14-D-Leu]-ovine CRF corresponded to
that measured when COS cells expressing CRF-R1 were activated, but was
considerably smaller than that observed for activation of COS cells
expressing CRF-R2 or -R2ß. The messenger RNA encoding the mouse
CRF-R1 was detected by RT-PCR in mouse Leydig cell preparations. In
contrast to mouse Leydig cells, CRF agonists had no influence on the
basal testosterone and cAMP production by rat Leydig cells, nor did the
agonists or antagonist change the hCG-stimulated testosterone and cAMP
production by these cells. It is concluded that mouse Leydig cells
express CRF-R1, mediating elevation of testosterone production by CRF
agonists through cAMP. Because potencies of CRF agonists in activating
mouse Leydig cells were more than 10-fold lower compared with their
potencies in stimulating rat pituitary cells, it is suggested that the
coupling of the CRF-R1 to intracellular signaling in Leydig cells is
different from that in corticotropic pituitary cells, at least in
quantitative terms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to pituitary and central nervous system effects, some effects of CRF in vitro and in vivo have been found at various peripheral sites, where specific binding sites for CRF or messenger RNA (mRNA) for CRF receptors have been localized as well. Such sites include the immune system (3, 4, 5, 6), cardiovascular system (7, 8, 9, 10), retina (11), uterine sites including placenta (12, 13), and the gonads, ovary (14), and testis (15, 16, 17, 18), thereby implicating CRF not only as central mediator of stress responses, but also in a variety of stress-related or -unrelated peripheral functions.
In 1993, one type of receptor for CRF (CRF-R), now designated CRF-R1,
was cloned from several species, including human (19, 20), rat (21, 22), and mouse (20), and shown to be a member of the
seven-transmembrane helix receptor family. Recently, the cloning and
characterization of two alternatively spliced forms of a novel subtype
of CRF-R (CRF-R2) from rat brain were reported (23). They result in two
putative receptor proteins of 411 and 431 amino acids, now designated
as CRF-R2 and CRF-R2ß, respectively. CRF-R2 and CRF-R2ß have
also been cloned from human (24) and mouse (7, 8, 9), respectively.
In rat central nervous system, CRF-R1 and CRF-R2 mRNA showed different
anatomical distribution (25), as did CRF-R2 and CRF-R2ß (26). From
the two CRF-R2 forms, R2 mRNA was found to be primarily expressed in
central nervous system while R2ß mRNA was additionally observed at
peripheral sites (26). On the other hand, CRF-R1, but neither form of
CRF-R2, is thought to be present in the rat (23, 25, 26) or mouse
pituitary (8). The heterogeneous distribution patterns of CRF-R1,
-R2, and -R2ß mRNA suggest distinct functional roles for each
receptor type.
One of the peripheral sites for which a putative function for CRF has been suggested is the Leydig cell of the testis. By one group, the hCG-stimulated testosterone production by the rat Leydig cell was found to be inhibited by CRF through specific receptors for CRF (15, 17). On the other hand, in mouse Leydig cells CRF directly stimulated the steroid production but did not influence the maximum hCG-stimulated production (18).
From these results it might be suggested that different types of CRF-R or different coupling to the intracellular signaling occur in the Leydig cells from rat and mouse. Rat cells were found to express, at very low levels, mRNAs of the pituitary form and of two splice variants of the CRF-R1, the pituitary receptor sequence being the predominant (27). However, in agreement with others (18), we could not find any biological effect of different CRF agonists on the rat cells but found a stimulatory effect on testosterone production by mouse Leydig cells. The aim of this study was to examine, first, the responses of the mouse Leydig cells to CRF peptides and to characterize their pharmacological profiles. This was thought to lead to some conclusion about the type of receptor, because it has been shown that CRF agonists differ in their rank order of potency in binding to (28), or in stimulation of adenylate cyclase activity (7, 23, 24, 29) in, CRF-R1- and CRF-R2-transfected cells. With this information we then sought to determine the expression of the respective mRNAs in mouse Leydig cell preparations. As a result, we found Leydig cells of mice to respond to CRF peptides corresponding to the specificity of CRF-R1 and to express mRNA for this receptor type.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helical CRF9–41
were synthesized as previously described (30). Furthermore, a series of
21 analogs of oCRF, each of them with two neighboring amino acids
(position 1,2; 3,4 ... 39,40; 40,41) systematically substituted by
their D-isomers (DD-replacement set), were synthesized.
Buserelin was a gift from Hoechst AG (Frankfurt am Main, Germany). hCG
with purity >95% was obtained from Calbiochem (Bad Soden, Germany).
Percoll was purchased from Pharmacia (Uppsala, Sweden), MEM was
purchased from GIBCO BRL (Eggenstein, Germany). Collagenase type 1A and
3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma
(Deisenhofen, Germany). Earle’s salt solution without
CaCl2 was prepared from substances of analytical grade. BSA
(fraction V) was from SERVA (Heidelberg, Germany). For the isolation
and incubation of the Leydig cells, MEM containing 25 mM
HEPES buffer (pH 7.4) and 0.5 g/liter BSA was used. Continuous density
gradients of 0–90% Percoll were prepared with Earle’s salt solution
containing 1 g/liter BSA and 25 mM HEPES buffer (pH
7.4).
Isolation and purification of Leydig cells
Leydig cells were prepared from NMRI-mice (25–30 g) (31), adult
Wistar, Sprague-Dawley, and Fischer rats (
250 g), all of which were
supplied by Charles River, Sulzfeld, Germany, and from immature Wistar
rats (21 days). The animals were maintained on a 12-h light, 12-h dark
cycle, with food and water available ad libitum. The testes
of mice were decapsulated, mechanically dispersed in medium, and freed
from tubuli and tissue fragments by filtration through gauze. The cells
were collected by centrifugation at 800 x g for 15 min
at 20 C, resuspended in medium, and layered onto the Percoll gradient.
After centrifugation at 1,800 x g for 25 min at 20 C,
four bands were obtained; from the third band, highly purified Leydig
cells were recovered and washed twice by centrifugation. The cells were
identified by their yellow halo under phase-contrast microscope and
counted in the Neubauer chamber under microscope. Cells (100,000 or
165,000) in 2 ml incubation medium containing 15 mM
NaHCO3 were plated in 12-well culture plates and allowed to
attach to the surface at 37 C under 5% CO2/air for
3 h. The testes of rats were dispersed using 0.25 mg/ml
collagenase in medium for about 20 min in a shaking water bath (100
rpm) at 37 C, after which the Leydig cells were purified as
described for mice; however, with immature cells the density
gradient centrifugation was omitted. Cells of adult rats
(500,000–1,000,000 cells) and of immature rats (1,000,000 cells) were
allowed to attach to the well plates for 2 and 1 h,
respectively.
Incubations of Leydig cells with hCG and peptides and determination
of testosterone and cAMP
The medium was removed from the attached cells and replaced with
1 ml of fresh incubation medium containing the phosphodiesterase
inhibitor IBMX (2.5 mM) and hCG, CRF agonists, the CRF
antagonist, the peptides of the DD-replacement set of oCRF, or
buserelin at the indicated concentrations. When the CRF antagonist was
tested, it was added to the cells 15 min before addition of the CRF
agonists. The incubations were performed in a shaking water bath (35
rpm) at 37 C for the indicated times. One hundred microliters of the
medium were frozen for the determination of testosterone by RIA (DPC
Biermann GmbH, Bad Nauheim, Germany) and 200 µl were dried in a Speed
Vac and stored frozen for the determination of extracellular cAMP by
RIA (Immuno Biological Laboratories, Hamburg, Germany) after
acetylation of samples and standards. Intracellular cAMP was determined
after the extraction of the cells with 80% (vol/vol) ethanol and
drying in a Speed Vac.
ACTH-releasing activity of the peptides of the oCRF DD-replacement
set
Pituitary cells were obtained from the anterior pituitary of
male Wistar rats and used for determining the ACTH-releasing activities
of the CRF peptides as described earlier (10). The activities of the
peptides were determined at the concentration of 0.3 nM,
corresponding to the EC50 value of oCRF, and compared with
their activities on mouse Leydig cells at 10 nM peptide on
a normalized scale. Additionally, their ACTH-releasing activities were
measured at 1 µM peptide to ensure that the effect of the
peptides of the set further increased and reached the same maximum
value as oCRF (data not shown).
cAMP-accumulating activities of CRF analogs on COS cells expressing
rCRF-R1, rCRF-R2, or rCRF-R2ß
COS-1 cells were transiently transfected by a modified calcium
phosphate coprecipitation method (32). The cells were incubated at 37 C
and 5% CO2/air for 22 h in 9-cm dishes containing 9
ml DMEM and 30 µg plasmid DNA [complementary DNAs (cDNAs) of
rCRF-R1, rCRF-R2, and rCRF-R2ß subcloned into pcDNAI,
Invitrogen]. Afterward, the cells were trypsinized and seeded into
24-well plates at a density of 100,000 cells per well. Two days after
transfection, the cells were washed for 10 min with PBS/0.2
mM IBMX and incubated with different concentrations of
[11-D-Thr,12-D-Phe]-,
[13-D-His,14-D-Leu]-, or
[16-L-Lys]-oCRF in PBS/0.2 mM IBMX for 30 min
at 37 C. The supernatant was aspirated and the cells were extracted
with chilled ethanol (-20 C). The cell lysates were dried and the cAMP
was quantified from duplicate wells using the cAMP binding protein kit
(Amersham).
Data analysis
EC50 values for the activities of the peptides in
stimulating and inhibiting the production of testosterone in Leydig
cells were calculated from the concentration-response curves by a
four-parameter logistic curve-fitting program. Differences between
EC50 values of the native CRF agonists were assessed by the
unpaired Student’s t test.
Nested PCR cloning and sequence analysis of a mouse (m)CRF-R1-cDNA
segment
Poly(A)+-RNA was isolated from the mouse crude
Leydig cell preparation (without purification by gradient
centrifugation) by using the Fast track 2.0-Kit (Invitrogen, NV Leek,
The Netherlands). First-strand complementary DNA (cDNA) was synthesized
using random hexamer primers and Moloney murine leukemia virus-RT (Life
Technologies, Eggenstein, Germany). Synthetic oligonucleotides were
designed based on segments of the mouse CRF-R1-cDNA (20). First round
of PCR amplification (40 cycles at 94 C, 50 C, and 72 C; each for 50
sec) was performed by using primer [5'-TGGACCTCATTGGCACC-3']
(N-terminal domain, bases 174–190) and primer
[5'-TGTGTGCAGGTAGCAGC-3'] (intracellular loop 2/transmembrane domain
3, bases 676–660). After a second round of PCR amplification (40
cycles at 94 C, 56 C, and 72 C; each for 50 sec) with primer
[5'-CTACGGTGTCCGCTACAAC-3'] (N-terminal domain, bases 247–265) and
primer [5'-GCTCACGGTGAGCTGGAC-3'] (extracellular loop 1/transmembrane
domain 2, bases 562–545), one single band was obtained. The PCR
fragment was subcloned into pBluescript SK(-) (Stratagene, Heidelberg,
Germany) and sequenced by the dideoxynucleotide chain-termination
method (33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) stimulated the testosterone production
of the mouse Leydig cells with EC50 of 2.84 nM.
Within 30 and 60 min, the testosterone production was maximally
stimulated by about 13- and 22-fold, respectively, over its basal
activity. The CRF antagonist -helical CRF9–41 did not influence the
basal testosterone production (Fig. 1) but dose-dependently inhibited
the testosterone production stimulated by hCRF with an IC50
of 65.0 nM (Fig. 2). The
superactive GnRH agonist buserelin showed no effect on the basal
testosterone production (Fig. 1).
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). The maximum stimulation of
testosterone production by CRF within 30–60 min incubation was about
35% of that elicited by hCG. The antagonist -helical CRF9–41 had
no influence on hCG-induced testosterone (data not shown).
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, where their EC50 values
are given, together with those for their ACTH-releasing activities on
rat pituitary cells as obtained earlier (10). Urocortin was the most
active agonist, whereas hCRF, oCRF, sauvagine, and urotensin had lower
but nearly the same activity on both cell types, but the
EC50 values of their activities on mouse Leydig cells were,
on average, 16.5-fold higher than on rat pituitary cells (Table 1).
Although the scrotal temperature is some degrees below 37 C, the
incubations of Leydig cells were generally performed at 37 C to be
compared with data on pituitary cells. When, in one experiment, the
testosterone production stimulated by oCRF was compared at 37 C and 25
C, no significant difference in the EC50 values was
found.
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demonstrates that intracellular
cAMP was increased by hCRF at concentrations that also stimulated
testosterone production. However, the EC50 for the
CRF-stimulated increase of intracellular cAMP (50 nM)
was about 20 times higher than that for the CRF-stimulated testosterone
production.
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and Fig. 5 the neighboring positions of the two
amino acid residues in the sequence of oCRF that were replaced with
their D-isomers are indicated for all 21 analogs synthesized. Their
potencies in stimulating the testosterone production in mouse Leydig
cells differed widely. Full concentration-response curves in the
concentration range up to 1 µM were only obtained for one
third of the peptides for which the EC50 values are given
in Table 2. With all other peptides, at least some response was
obtained at concentrations up to 1 µM. When the
activities of the DD-analogs in stimulating the testosterone production
of mouse Leydig cells were compared with their activities in the ACTH
release from rat pituitary cells, a good correspondence between the two
activities was obtained (Fig. 5). Figure 6D compares the testosterone production
by mouse Leydig cells evoked by two selected peptides from the
replacement set ([11-D-Thr,12-D-Phe]- and
[13-D-His,14-D-Leu]-oCRF) with that evoked by
[16-L-Lys]-oCRF. The ratio between the potencies of the
three peptides corresponded well with that obtained for their effect on
the cAMP production in cells transfected with rCRF-R1 (see below).
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-helical CRF9–41, no influence of the CRF
peptides on the basal and hCG-stimulated testosterone production was
observed at any time. This was true for cells from Wistar, Fischer, and
Sprague-Dawley rats as well as for cells from 21-day-old rats.
Furthermore, the peptides did not change the extracellular cAMP. To
determine whether only a subpopulation of rat Leydig cells was able to
respond to CRF, cells from adult rats were separated into a low-
(<1.07 g/ml) and high-density (>1.07 g/ml) fraction. The cells in the
two fractions strongly differed in their testosterone production, which
was stimulated by hCG in the low-density cells only by about 20% of
that observed with the high-density cells. However, CRF peptides did
not influence the testosterone production stimulated by hCG in either
cells. Also in contrast to mouse Leydig cells, the GnRH agonist
buserelin was found to acutely stimulate the basal testosterone
production of rat cells with an EC50 value of 0.29
nM.
cAMP-accumulating activities of CRF analogs on COS cells expressing
rCRF-R1, rCRF-R2, or rCRF-R2ß
From the DD-replacement set,
[11-D-Thr,12-D-Phe]- and
[13-D-His,14-D-Leu]-oCRF had comparable and,
compared with their neighbors in the set, relatively high activities on
the pituitary as well as the Leydig cells (Fig. 5
). The ratio between
their potencies in accumulating cAMP in COS cells expressing the
rCRF-R1 (EC50 0.94 and 0.45 nM, respectively;
Fig. 6A) was found to be very close to that observed in their action on
pituitary (Fig. 5) and Leydig cells (Fig. 6D).
However, in COS cells transfected with rCRF-R2 or rCRF-R2ß, the
analog [11-D-Thr,12-D-Phe]-oCRF stimulated
cAMP concentration with much lower potency (EC50 5.73 and
3.12 nM, respectively) than
[13-D-His,14-D-Leu]-oCRF (EC50
0.73 and 0.57 nM, respectively; Fig. 6, B and C). This
shift in potency for only
[11-D-Thr,12-D-Phe]-oCRF of the two analogs
is further underlined by the fact that its potency on both CRF-R2
variants became comparable with that of [16-L-Lys]-oCRF,
in contrast to [13-D-His,14-D-Leu]-oCRF and
to the results on CRF-R1 (Fig. 6A). [16-L-Lys]-oCRF was
earlier shown to possess very low activity in the ACTH release from rat
pituitaries (30) and in the present study was found to be nearly
equally active on CRF-R1, CRF-R2, and CRF-R2ß expressed in COS
cells (EC50 4.51, 5.11, and 2.71 nM,
respectively). Furthermore, nearly the same ratios in the potencies of
[11-D-Thr,12-D-Phe]-,
[13-D-His,14-D-Leu]-, and
[16-L-Lys]-oCRF in stimulating the testosterone
production by mouse Leydig cells (EC50 ratio 1:0.48:4.8,
Fig. 6D) were obtained when compared with their cAMP-accumulating
activities on COS cells that expressed CRF-R1 (EC50 ratio
1:0.43:6.3, Fig. 6A) but not CRF-R2 or CRF-R2ß (Fig. 6, B and
C).
Isolation of CRF-R1-cDNA fragment from mouse crude Leydig cell
preparation
The second round of PCR amplification of CRF-R1-cDNA obtained from
poly(A)+-RNA of crude mouse Leydig cells resulted in a
single band according to the expected size of 316 bp. After subcloning,
the sequence of the PCR product was found to correspond to the bases
247–562 of the mouse CRF-R1-cDNA (20).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In agreement with an earlier report (18), our results showed that hCRF,
which is identical in its amino acid sequence to rat and mouse CRF
(34), directly stimulated the testosterone production in Leydig cells
from mice in a time- and dose-dependent manner (Fig. 1). The effect was
saturable (Fig. 1), characterized by a low EC50 of 2.84
nM hCRF (Table 1), antagonized by the specific CRF
antagonist -helical CRF9–41 (Fig. 2), and obviously mediated by
cAMP (Fig. 4). These findings strongly argue for a CRF
receptor-mediated process.
It has been shown that the overall production of steroids in Leydig
cells and other steroidogenic cells is controlled by events that
facilitate the transport of cholesterol from lipid droplets and other
cellular stores to the inner mitochondrial membrane, where the
synthesis of the cell-specific steroid is initiated (for a review see
Ref.35). Several proteins have been implicated in this cholesterol
transfer (35), regulated by LH/hCG or other trophic hormone receptors.
Since CRF did not increase the testosterone production in the mouse
Leydig cell additively to hCG (Fig. 3), the CRF and hCG signaling
pathways probably converge, at the latest, at the step of the
cholesterol transfer, but CRF is able to exploit only about one third
of the hCG-stimulated capacity (Fig. 3).
When the specificity of the effect of CRF was studied by testing
natural CRF agonists (Table 1), rat urocortin, together with human
urocortin, the latest member of the CRF family (28), was found to
possess the highest potency. This agreed with the fact that urocortin,
of all CRF agonists, also exhibited the highest activity in the cAMP
accumulation by, and highest affinity to, all known types of CRF
receptors, i.e. CRF-R1 (28, 36), CRF-R2 (36), and
CRF-R2ß (28, 36), transfected into various cell lines. Furthermore,
rat urocortin was most active in the release of ACTH from rat pituitary
cells (Refs. 28 and 36 and Table 1), which contain almost exclusively
CRF-R1 (23).
The other agonists, hCRF, oCRF, sauvagine, and urotensin, differed only
slightly in their potencies in stimulating testosterone production in
mouse Leydig cells, as seen from their EC50 values (Table 1). This is in line with the specificity of CRF-R1 toward these
peptides, as shown with rat pituitaries (Table 1 and Refs. 10, 28, 36,
and 37) and cells expressing CRF-R1 (7, 21, 23, 24, 28, 29). However,
cells expressing one of the splice variants of CRF-R2, CRF-R2 (23, 24, 29) or CRF-R2ß (7), accumulated cAMP in response to sauvagine
with a potency, i.e. 1/EC50, 9- to 40-fold
higher as compared with hCRF or oCRF. Similarly, the affinity of
sauvagine to both receptors was more than 9-fold (8, 28, 29, 36) higher
than that of hCRF and oCRF. Therefore, the specificity of the
testosterone-stimulating effect of the natural CRF agonists on mouse
Leydig cells (Table 1) was the same as obtained for CRF-R1 and
different from that for both variants of CRF-R2.
Two further pharmacological indications for the mouse Leydig cell
receptor to be of type 1 were obtained. First, the widely differing
activities on the Leydig cell testosterone production of the 21 analogs
of oCRF (Table 2), obtained by systematically replacing two neighboring
amino acids with their D-isomers (DD-replacement set),
corresponded well with their activities on rat pituitary cells (Fig. 5). Second, two peptides from the DD-replacement set,
[11-D-Thr,12-D-Phe]- and
[13-D-His,14-D-Leu]-oCRF, differed
considerably in their cAMP-accumulating activities on CRF-R2 and
CRF-R2ß, expressed in COS cells, but only slightly on CRF-R1 (Fig. 6, A–C) as they did in their activities on the Leydig (Fig. 6D) and
pituitary cell (Fig. 5). This is further underlined by the nearly
constant ratios between the potencies of both peptides and the potency
of [16-L-Lys]-oCRF in their activities on Leydig cell
(Fig. 6D) and on COS cells expressing CRF-R1 (Fig. 6A), in contrast to
cells expressing CRF-R2 (Fig. 6B) or CRF-R2ß (Fig. 6C).
The identity of the Leydig cell receptor was further confirmed by RT-PCR analysis of the mRNA obtained from a crude Leydig cell preparation. Using primer pairs targeted to the mouse CRF-R1 sequence (20), the nucleotide sequence of the PCR fragment of 316 bp obtained was found to be identical to the corresponding segment of the mouse CRF-R1 cDNA. Further studies are under way to verify the possible existence of splice variants of this type as has been demonstrated for rat Leydig cells where two minor variants of CRF-R1 cDNA were derived from the cell mRNA (27).
Taken together, from the pharmacological and molecular data it is
concluded that mouse Leydig cells possess CRF receptors of type 1.
Remarkably, the CRF antagonist -helical CRF9–41 showed no partial
agonist activity (Figs. 1 and 2), in contrast to its action on
pituitary cells (10, 38). Furthermore, the potencies of the CRF
agonists in their effects on the CRF-R1 in mouse Leydig cells were 10-
to 20-fold lower as compared with their effects on the same receptor
type in pituitary cells of rat (Table 1) and, as reported for CRF (39),
of mouse. This was observed with the natural agonists (Table 1) as well
as with the peptides of the DD-replacement set, as shown by their
approximately equal activities on the pituitary and Leydig cells at 0.3
and 10 nM peptide concentrations, respectively (Fig. 5).
Different mechanisms could be operative to explain this difference. The most straightforward possibility is that the affinity of the CRF receptor for the ligands in mouse Leydig and rat pituitary cells might differ. Because the rat and mouse cDNAs encode CRF-R1 proteins that are 98% identical over their full length of 415 amino acids (40), it does not seem very likely that different receptor affinities account for the different potencies. In fact, a survey of the numerous reported studies on CRF binding to the CRF receptors in various tissues and cells of rat and mouse, as well as human, revealed almost identical, species-independent affinities of CRF to its receptors in all tissues with Kd values of about 1–4 nM for hCRF and oCRF (4, 10, 41).
Another possibility would be that more than 90% of the peptides were metabolized during incubations with Leydig cells, which appears to be unlikely to have constantly occurred with all the peptides tested including the CRF analogs of the DD-replacement set. Furthermore, increasing cell numbers and incubation times did not influence their EC50 values, but increased proportionally the testosterone production. After incubation of hCRF with Leydig cells at cell numbers exceeding by far those normally used, no loss of biological active peptide was observed as assessed by RRA (10) (data not shown). These observations excluded any significant influence of peptide metabolism on the results.
Therefore, it is suggested that the coupling of the CRF-R1 to
intracellular signaling in Leydig cells is different from that in
corticotropic pituitary cells at least in quantitative terms. The
EC50 values of hCRF and oCRF found here for the stimulation
of testosterone production in mouse Leydig cells (Table 1) corresponded
closely to the Kd values found in all binding studies on
CRF receptors in different tissues (see above). Furthermore, their
EC50 values and those of sauvagine and urotensin were
almost identical to their Kd values in the binding to
CRF-R1 in rat brain membranes (10). In conclusion, it is suggested that
the stimulation of the testosterone production in mouse Leydig cells by
CRF is to a great extent determined stoichiometrically by the
fractional occupancy of the receptor. Due to the more than 10-fold
higher potency of CRF in activating the same CRF receptor type 1 in
pituitary cells from rat and mouse, the coupling of the pituitary
receptor to the signaling events in these cells may be more efficient
when compared with the mouse Leydig cell. Therefore, the pituitary
cell, but not the Leydig cell, might be described as exhibiting spare
receptors.
There apparently is also a great disparity in the coupling of the
CRF-R1 to cAMP between the mouse Leydig cell and COS cells expressing
CRF-R1. The EC50 found here for CRF in the cAMP stimulation
in Leydig cells is much higher than the Kd reported for
binding of CRF to CRF-R1 (EC50/Kd about 20).
Similarly, rat pituitary cells show relations
EC50/Kd much higher than 1 (4, 41). The
opposite is the case with COS-7 cells (EC50/Kd
0.04; 20, 42). This was confirmed in our studies, seeing the great
difference between the EC50 values (around 1
nM) of [11-D-Thr,12-D-Phe]- and
[13-D-His,14-D-Leu]-oCRF in stimulating cAMP
content through CRF-R1 in COS cells (Fig. 6A) and their
EC50 for the stimulation of testosterone production in
Leydig cells (20 nM, Table 2). On the basis of these
facts it is concluded that among pituitary, Leydig, and COS cells the
coupling of CRF-R1 to the signaling cascade is at least in quantitative
terms quite different.
It remains unclear why we and others (18), in contrast to another report (15, 16), did not find any response of rat Leydig cells to CRF, although various rat strains, adult and 21-day-old rats (this study), and subpopulations of the cells (Ref. 18 and present study) were included in the studies. It may be argued that even slight differences in the isolation procedure for the rat cells could be responsible for different grades of damage to the CRF receptor, while the hCG receptor remains almost intact. Therefore, we looked for possible impairment of another receptor for a short peptide, the GnRH receptor, which was shown to be present and activated in Leydig cells of rats but not of mice (43, 44). In agreement with the literature, we did not find buserelin, a superactive GnRH agonist, to influence the testosterone production of mouse Leydig cells. However, the high potency response of our preparation of rat Leydig cells to buserelin (EC50 0.29 nM) confirmed that these cells were of high functional integrity.
In summary, our pharmacological and molecular data provide evidence that mouse Leydig cells express CRF receptors of type 1, the same type occurring in the pituitary. When activated by CRF agonists, the basal testosterone production is stimulated, similar to the pituitary cAMP-mediated, but at seemingly lower cellular coupling efficiency.
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Acknowledgments |
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Received July 17, 1997.
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),
and buserelin (
) on the testosterone production (mean ±
SD) in mouse Leydig cells.
),
[13-D-His,14-D-Leu]- (