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Departments of Cell Biology and Physiology and Obstetrics, Gynecology and Reproductive Sciences (A.J.Z.) and Magee Women’s Research Institute (D.F.B., A.J.Z.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
Address all correspondence and requests for reprints to: A. J. Zeleznik, Ph.D., Magee-Women’s Research Institute, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) in corpora lutea collected
throughout the luteal phase revealed immunoreactive protein bands with
similar intensities. In vitro addition of recombinant
cynomolgus LH and dibutyryl cAMP stimulated PKA activity in corpora
lutea collected during the early, mid, and late luteal phases. In
corpora lutea obtained from animals treated with hCG during the
midluteal phase, basal PKA activity was decreased 65% as compared with
untreated day 11 controls and in late luteal phase, hCG-exposed CL
basal PKA activity was decreased 30% as compared with untreated day 16
controls. However, there were no measurable differences in
cAMP-stimulated PKA activity in CL exposed to prior hCG treatment
in vivo and Western immunoblot analyses for PKA C in
these tissues revealed immunoreactive protein bands that were
comparable with corpora lutea collected from untreated animals.
Further, immunoblot analyses for CREB in corpora lutea collected from
hCG-treated animals revealed that CREB immunoreactivity remained
undetectable following a treatment regimen with hCG that mimics early
pregnancy. These results demonstrate that, although CREB expression
ceases following ovulation, PKA activity is maintained throughout the
luteal phase, which provides a mechanism by which the acute
steroidogenic actions of LH may be separated from longer term trophic
actions that may rely the transcriptional activity of CREB. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The notion that the functional capacity of the corpus luteum declines
with age is supported by both in vitro and in
vivo observations. Luteal cells isolated from both the monkey and
the human corpus luteum exhibit a progressive age-related decline in
their ability to produce progesterone (5, 6). In vivo, the
expression of messenger RNAs (mRNAs) for 3ß-hydroxysteroid
dehydrogenase, 
5–4 isomerase (3ß-HSD) and cytochrome
P450 cholesterol side chain cleavage enzyme (P450scc) is maximal
shortly after ovulation and declines progressively thereafter,
independently of the overall pattern of progesterone production (7).
Moreover, whereas treatment of monkeys with hCG during the early luteal
phase elevates mRNAs for 3ß-HSD and P450scc, an identical hCG
treatment regimen initiated during the midluteal phase does not
increase the luteal content of these mRNAs (8). The diminished function
of the corpus luteum that occurs with age does not appear to be due to
reductions in LH/CG receptor content or LH-dependent adenylyl cyclase
activity because, as shown previously by others, decreases in the
activity of this transmembrane signaling system appear to occur after
the initial fall in progesterone during the late luteal phase (9, 10).
Moreover, the observation that isolated luteal cells exhibit an
age-dependent decline in their responsiveness to cAMP (5) suggests that
changes distal to the formation of cAMP could be responsible for the
decline in function that accompanies luteal aging.
Despite the overall decline in luteal steroidogenesis that occurs during aging, progesterone production by the regressing corpus luteum remains highly responsive to LH/hCG as assessed by comparing the ratio of basal to gonadotropin-stimulated progesterone secretion (6, 11). These observations support our earlier conclusions from hypothalamic lesioned, GnRH-replaced monkeys that the acute actions of LH on the stimulation of progesterone secretion may be functionally separated from its longer term trophic actions (12). Our recent demonstration that the expression of the cAMP/protein kinase A (PKA)-dependent nuclear transcription factor CREB (cAMP response element binding protein) is extinguished following ovulation and luteinization in cynomolgus monkeys (13) revealed a novel mechanism whereby some nuclear actions of the cAMP-PKA signaling system may suppressed without compromising the ability of the corpus luteum to produce progesterone acutely in response to LH, the latter presumably regulated in the cytoplasm by the cAMP and PKA-dependent mobilization of cholesterol to the inner mitochondrial membrane (14). Implicit in this hypothetical model is that PKA activity is maintained throughout the luteal phase. However, to date there have been no studies published regarding PKA activity in the primate corpus luteum. Accordingly, the goal of the current study was to assess PKA activity in the primate corpus luteum throughout the luteal phase of the menstrual cycle and in response to in vivo hCG treatment. In addition, we also sought to determine whether an in vivo hCG treatment regimen that mimics early pregnancy results in the resurrection of CREB expression in association with the "rescue" of the corpus luteum.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Organ culture
Corpora lutea collected on days 3–5, 9–11, or 13 of the luteal
phase were sliced into 1-mm sections using a McIlwain tissue chopper
(Brinkman, Westbury, NY). An equal number of luteal slices were then
placed on stainless steel mesh screens which were floated at the
air-medium interface above the center well of an organ culture dish
(Falcon no. 3027, Becton Dickinson, Franklin Lakes, NJ). Cells were
preincubated for 2 h at 37 C in Medium 199 containing HEPES,
L-glutamine (2 mM), 5% calf serum, and
penicillin-streptomycin (GIBCO-BRL, Grand Island, NY) and 0.5
mM 3-isobutyl-1-methyl xanthine (Sigma Chemical Co., St.
Louis, MO). After preincubation, the screens and tissues were
transferred to fresh medium with or without the addition of recombinant
cynomolgus LH (AFP6936A, provided by NICHD, NIH, Bethesda, MD) at
concentrations of 50 or 500 ng/ml or 0.2 mM dibutyryl cAMP
(Bt2cAMP; Sigma). After a 30-min incubation at 37 C,
tissues were snap frozen until PKA assays were performed. In two
experiments, progesterone (P4) production was monitored in
the spent medium via RIA (16) and verified that P4
production was stimulated by LH treatment (mean P4 values
n = 2: control, 18.8 ng/ml/30 min; 50 ng/ml LH, 35.3 ng/ml/30 min;
500 ng/ml LH, 41.8 ng/ml/30 min).
PKA assay
All reagents used in the PKA assay were from Sigma unless
otherwise indicated and were prepared fresh on the day of use. Luteal
tissue (5–8 mg) was kept frozen on dry ice, where it was pulverized to
a fine powder and placed in 100 µl of homogenization buffer
consisting of 20 mM Tris (pH 7.5), 0.5 mM
1-methyl-3-isobutylxanthine, 25 mM benzamidine, 50 µg/ml
leupeptin (Boehringer-Mannheim, Indianapolis, IN), 2 mM
EDTA, and 5 mM EGTA. Tissue was then homogenized at 4 C
with 10 pulses of a Kontes tissue homogenization unit (with pestle
fitted for 1.5 ml tubes) and centrifuged at 16,000 x g
for 1 min. The supernatant was used immediately to determine protein
concentration (Bradford method, Bio-Rad, Richmond, CA). Samples were
diluted in 20 mM Tris, pH 7.5, to 25 µg protein per 40
µl to yield a provide a protein content within the previously
determined linear range of the PKA assay (20–100 µg protein). The
PKA assay was initiated with the timed addition of a 40 µl aliquot of
the supernatant. Each tissue sample was assayed in triplicate. The PKA
assay was performed within 10 min of homogenization to minimize any
shifts in enzyme dissociation kinetics after homogenization (17).
The PKA assay was a modification of that reported using rat ovarian
tissues (18). The activity of PKA was determined by measuring the
incorporation of 32P from 
-32P-ATP (40
µM, specific activity 10 Ci/mmol purchased from New
England Nuclear, Boston, MA) using 71 µM kemptide, a
PKA-specific peptide substrate (19) in 200 µl buffer (pH 7.0)
consisting of 26.7 mM -glycerol phosphate, 7.8
mM sodium fluoride, 8 mM magnesium acetate,
1.25 mM theophylline, and 0.6 mM dithiothreitol
in the presence or absence of 2 µM cAMP-Tris salt (10
µl). Incubations were done at 30 C for 5 min and were terminated with
the addition of 1 ml 75 mM phosphoric acid. Tubes were
placed on ice until vacuum filtration onto phosphocellulose discs
(Whatman P-81 papers, Whatman Lab Sales, Hillsboro, OR) as discussed
(20). After initial filtration, the assay tubes were rinsed with 75
mM phosphoric acid, filtered, and then the P81 discs were
subsequently washed five times with 1 ml 75 mM phosphoric
acid. The discs were then placed into liquid scintillation vials with 5
ml Ecolite Counting Cocktail (ICN Biomedicals, Irvine, CA) and counted
for 32P in a Wallec liquid scintillation counter
(Gaithersburg, MD). Background activity was considered to be that
detected in the absence of added kemptide substrate and was subtracted
for each sample.
Characterization of luteal PKA enzyme
PKA activity in cytosolic fractions was assessed by assay in the
absence or presence of a specific inhibitor of PKA activity (type II
protein kinase inhibitor, Sigma no. P-8140), which binds to the
catalytic subunit of PKA and inhibits its phosphotransferase activity
(21, 22). Preliminary studies were conducted to determine optimal
homogenization conditions for the maintenance of dissociation kinetics
of the PKA enzyme in macaque luteal tissue as it has been reported that
tissues possessing type I regulatory subunits of PKA dissociate from
catalytic subunits in the presence of NaCl, leading to an artificial
elevation in the PKA determination (17). Thus, macaque luteal tissue
was homogenized in the absence or presence of NaCl (0.15 or 0.5
M), and PKA activity was assayed. In addition, a batch
separation method on DEAE-cellulose similar to that described for the
purification of rat ovarian protein kinase C (23) was used to further
characterize the macaque luteal PKA enzyme. The DEAE ion exchange
medium (DE-52, Whatman) was equilibrated in Buffer A (20 mM
Tris, pH 7.5, 2 mM EDTA, 5 mM EGTA, and 10
mM ß-mercaptoethanol) in a small column, the fines were
decanted, and the DEAE was resuspended in a 50:50 slurry with Buffer A.
The slurry (750 µl) was placed in a test tube, and an equivalent
volume of luteal cytosol (diluted in H2O) was added and
incubated on ice for 1 h with frequent mixing. After incubation,
the mixture was centrifuged (1500 x g, 5 min) and the
supernatant was discarded. The pellet was washed twice (15 min
incubation on ice with mixing) with Buffer A, centrifuged, and then
again incubated with Buffer A for 15 min with each subsequent
incubation containing an increased concentration of NaCl (0.05, 0.1,
0.15, 0.2, 0.4 M NaCl). The supernatants collected after
centrifuging each incubation were assayed (40 µl) for PKA activity as
describe above.
Western immunoblotting
Frozen tissues were pulverized on dry ice and then homogenized
(1:5 wt/vol) in TE buffer (50 mM Tris-HCl, pH 7.4, 1.0
mM EDTA) supplemented with 20 µg/ml PMSF, and 1 µg/ml
leupeptin. Whole cell lysates (50 µg/lane) were separated on 12% SDS
discontinuous polyacrylamide gels and the resolved proteins were
electrophoretically transferred to nitrocellulose membranes. PKA
catalytic subunit detection was performed by immunoblotting using a
mouse monoclonal antibody directed against a protein fragment
corresponding to amino acids 18–347 of the human PKA C subunit
(Transduction Laboratories, Lexington, KY; no. P28320) at a
concentration of 1 µg/ml. Detection of CREB was accomplished using a
rabbit antirat CREB antiserum (Upstate Biotechnology, Lake Placid, NY)
at a 1:5000 dilution as previously described (13). Chemiluminescent
detection was accomplished using the BM Chemiluminescence Western
Blotting Kit according to the manufacturer’s directions (Boehringer
Mannheim, Indianapolis, IN).
Data analysis
Absolute PKA activity (pmol 32P
incorporated/min·mg protein) was determined in both the absence and
presence of cAMP to indicate the relative content of the enzyme. In
addition, the ratio of these measurements (-cAMP/+cAMP) was calculated
to determine the extent of activation in vivo
(i.e. a ratio of 1 would indicate maximum endogenous
stimulation of PKA) as discussed by Corbin (17).
Variation between and within assays was calculated from the addition of cytosolic fractions of rat luteal ovarian homogenates that were prepared from a pool of frozen tissue powder. The PKA activity of these pools was determined in triplicate samples at the beginning and end of each assay. The intra and interassay coefficients of variation were 11.4 and 21.1%, respectively. Corpora lutea from three or four macaques per age or treatment group were used in this experiment. The significance of results was assessed by ANOVA and comparison of treatment means was accomplished by least significant difference analysis (24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In addition, PKA activity was inhibited in a
dose-dependent manner upon the addition of a heat-stable PKA inhibitor
(Sigma no. P-8140; Table 1). The addition of NaCl to luteal homogenates
was evaluated for its effect on the PKA activity ratio (Table 2). The addition of NaCl resulted in an increase in the
PKA activity ratio and thus may represent the spontaneous dissociation
of the holoenzyme complex during homogenization. In addition,
preliminary characterization of the PKA regulatory subunits using
DEAE-cellulose ion exchange in a batch method demonstrated that
cAMP-dependent protein kinase activity was detectable primarily in salt
fractions 
0.15 M (data not shown). For these reasons, no
NaCl was included in the homogenization buffer in subsequent
assays.
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, whereas basal activity in the absence of cAMP is
shown in Fig. 1B. There were no differences in basal or stimulated PKA
activity levels throughout the menstrual cycle. PKA activity was
stimulated by cAMP approximately 8-fold in all groups
(P < 0.05).
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illustrates Western immunoblot analyses of
whole tissue lysates of corpora lutea collected during the early, mid,
and late luteal phases of the menstrual cycle using an antibody
directed against the catalytic subunit of human PKA C. An signal of
approximately 42 kDa was observed at each luteal age. The
chemiluminescent signal for the late luteal phase sample appeared more
intense than those of the early and mid luteal phases. A similar result
was observed with a second late luteal phase sample, whereas the
intensity of a third late luteal phase sample was slightly less than
those collected during the early and mid luteal phase (data not
shown).
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depict the PKA activity
ratio of luteal tissue slices obtained throughout the luteal phase and
maintained in short-term organ culture in the absence or presence of a
low (50 ng/ml) or high concentration (500 ng/ml) of recombinant
cynomolgus LH. Treatment of CL slices with the higher LH concentration
resulted in an elevation in the PKA activity ratio above untreated
controls, regardless of the day of the luteal phase from which the CL
were obtained (P < 0.05 on days 3 and 13,
P < 0.07 on day 9). The extent of PKA activation by LH
was greater in day 3 CL compared with CL isolated during the late
luteal phase. Although the PKA activity ratios obtained from CL during
the midluteal phase (days 9–11), either in the presence or absence of
LH, were lower than those observed with slices obtained from CL during
the early luteal phase, these differences did not reach statistical
significance. Relative stimulation of PKA by the 500 ng/ml dose of LH
was 2.3-fold in early luteal phase samples, 1.9-fold in mid luteal
phase samples, and 2.9-fold for late luteal phase tissues. Similar to
that seen with the addition of LH, PKA activity ratios measured
following the addition of 0.2 mM Bt2cAMP
revealed a greater response in CL collected during the early luteal
phase (0.755 ± 0.15) when compared with CL collected during
either the mid (0.49 ± 0.16) or late luteal phase (0.54 ±
0.12). Absolute basal and cAMP-stimulated PKA activities in the tissues
stimulated in vitro by LH and Bt2cAMPwere
similar to those observed in tissues collected from animals throughout
the luteal phase as presented in Fig. 1
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depicts PKA activity levels in luteal
homogenates on day 11 or 16 of the control luteal phase or on days 11
and 16 after animals were treated with hCG in vivo for 7
days. Stimulated and basal PKA activities are shown in Fig. 4, A and B,
respectively. There were no significant differences in cAMP-stimulated
PKA activity in hCG-treated vs. control corpora lutea. Basal
activity in day 11 corpora lutea following hCG treatment was reduced
65% as compared with corpora lutea from the corresponding day of the
control cycle (P < 0.05) and was significantly
attenuated as compared with all other groups (P <
0.05). Although not statistically different, basal PKA in corpora lutea
from animals treated with hCG on days 10–16 of the luteal phase was
reduced 31% as compared with day 16 control luteal PKA levels.
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illustrates Western analyses of PKA C
immunostaining of whole tissue lysates from corpora lutea collected
during the mid and late luteal phases of spontaneous menstrual cycles
and of lysates from corpora lutea collected from animals which had
received hCG in vivo as described above. Extracts of corpora
lutea collected during the spontaneous luteal phase as well as from
corpora lutea following hCG treatment in vivo expressed
immunoreactive PKA C. Similar results were observed with two
additional sets of tissues from hCG-treated animals (data not
shown).
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illustrates immunoblot analyses of CREB in whole cell lysates
of corpora lutea collected during the spontaneous luteal phase and from
animals that had received hCG in vivo. As reported
previously (13), immunoreactive CREB expression was absent in corpora
lutea collected during the spontaneous luteal phase and treatment of
animals with hCG in vivo did not resurrect the expression of
CREB. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While there was an acute increase in PKA activity in response to LH in vitro, we also demonstrated that an in vivo treatment regimen with hCG that has been shown to increase both progesterone production as well as 3ß-HSD and P450scc mRNA expression (8) resulted in a significant decline in basal PKA activity with no statistical difference in cAMP-stimulated PKA activity. Previous studies by VandeVoort et al. (26) demonstrated that a similar hCG treatment regimen of monkeys in vivo resulted in a significant desensitization of hCG responsive cAMP production in isolated luteal cells. Our observation that basal PKA activity in corpora lutea declines following hCG treatment could therefore reflect the consequences of desensitization of the LH receptor/adenylyl cyclase effector system. However, despite this decline in basal PKA activity, progesterone production was stimulated (8), indicating that the absolute activity of PKA in the corpus luteum throughout the luteal phase is not limiting with respect to the acute regulation of progesterone production.
Our finding that PKA activity in the primate corpus luteum is maintained throughout the luteal phase, together with our recent observations that the expression of CREB is extinguished following ovulation and luteinization (13), defines a novel regulation of the LH/cAMP/PKA signaling system in the monkey ovary that permits the acute regulation of progesterone secretion by LH while concurrently eliminating the transcriptional effects of cAMP/PKA signaling pathway that rely on CREB. The physiological significance of these observations is that the patent cAMP/PKA signaling system in the cytoplasm would ensure that the corpus luteum maintains the ability to produce progesterone that is essential for the maintenance of early pregnancy while at the same time the loss of the nuclear component of the cAMP/PKA/CREB signaling pathway could result in the cessation of trophic actions of LH that may rely on CREB. Results of the present study also demonstrate that in vivo treatment of monkeys with a regimen of hCG that mimics early pregnancy did not reactivate the expression of CREB in the corpus luteum. This outcome was not unexpected in view of recent studies that have addressed the cellular aspects of the rescue of the primate corpus luteum during pregnancy. Thus, we previously demonstrated that treatment of monkeys with hCG during the mid through late luteal phase, while stimulating progesterone production, did not increase mRNA levels for P450scc or 3ß-HSD (8), an observation that has recently been confirmed during spontaneous pregnancy in baboons (27). In addition, hCG treatment of monkeys during the luteal phase did not result in the resumption of luteal cell proliferation as assessed by immunostaining for Ki-67 (28). These findings suggest that the rescue of the corpus luteum during pregnancy does not represent its rejuvenation but rather the maintenance of the steroidogenic capacity of the declining corpus luteum by the heroic concentrations of CG achieved during early pregnancy.
The unresolved question relates to what specific genes are regulated by CREB in the ovary, as it now appears that neither the transcription of mRNAs for P450scc nor 3ß-HSD are CREB dependent (29). A major feature that distinguishes luteal cells from their progenitor granulosa cells is that while granulosa cells proliferate in response to FSH, luteal cells fail to proliferate in response to LH (30). Evidence obtained by employing dominant-negative mutants have implicated CREB in the control of proliferation in a number of cell types including somatotrophs, thyroid cells, and thymic lymphocytes (31, 32, 33), and recent studies have shown that, as fibroblasts become senescent they, like the corpus luteum, cease to express CREB but retain cAMP-dependent PKA activity (34). In addition, CREB has also been implicated in the control of bcl-2 expression and the rescue of human B cells from apoptosis (35). If CREB plays similar roles in the ovary, the loss of CREB following luteinization could be responsible not only for the cessation of proliferation of luteal cells but also for their ultimate demise that accompanies luteolysis.
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Acknowledgments |
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Footnotes |
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Received February 17, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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