Endocrinology Vol. 138, No. 8 3452-3458
Copyright © 1997 by The Endocrine Society
Cyclic Adenosine Monophosphate Signaling in the Primate Corpus Luteum: Maintenance of Protein Kinase A Activity throughout the Luteal Phase of the Menstrual Cycle1
Deborah Fairchild Benyo and
Anthony J. Zeleznik
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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Recent studies from our laboratory demonstrating that the expression of
cAMP-dependent nuclear transcription factor CREB (cAMP response element
binding protein) is lost following ovulation in macaques has revealed a
novel mechanism by which the cytoplasmic and nuclear actions the
cAMP-protein kinase A (PKA) intracellular signaling system may be
regulated independently. Implicit in this hypothesis is the assumption
that PKA activity is maintained throughout the luteal phase of the
menstrual cycle, yet to date there have been no published reports
regarding PKA activity in the primate corpus luteum. PKA activity was
assessed by the incorporation of 32P from radiolabeled ATP
into a PKA-specific peptide substrate (kemptide) in the presence or
absence of cAMP. Luteal cytosolic fractions were obtained from corpora
lutea collected during the spontaneous luteal phase (days 3–5, 7–8,
10–11, 13–15, and postmenses) or obtained from animals on days 11 or
16 of the luteal phase after the animals received seven days of
exogenous human CG (hCG) treatment. Examination of PKA activity in
luteal slices from various aged CL maintained in short-term organ
culture in the presence or absence of recombinant cynomolgus monkey LH
was also performed. There were no significant differences in basal or
cAMP-stimulated PKA activities in corpora lutea collected throughout
the spontaneous luteal phase. Further, Western immunoblot analyses of
the catalytic subunit of PKA (PKA C
) 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.
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Introduction
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ALTHOUGH LH is absolutely required for
progesterone production by the primate corpus luteum (1), it does not
appear that luteal regression at the termination of nonfertile
menstrual cycles is due directly to alterations in LH secretion. Thus,
our previous studies demonstrated that neither a reduction in LH pulse
frequency like that which occurs during the mid to late luteal phase of
the menstrual cycle nor a 50% reduction in the absolute plasma LH
concentration during the luteal phase results in premature luteal
regression (2, 3). On the basis of these observations, we concluded
that luteal regression at the termination of nonfertile menstrual
cycles is the result of a reduced capacity of the corpus luteum to
respond to the ambient concentration of LH rather than to a reduction
in LH secretion per se, in complete support of Hisaw’s
conclusion made over 50 yr ago that "menstruation is not due
necessarily to a lack or absence of pituitary gonadotropin but, rather,
to a failure of the corpus luteum" (4).
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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Animals
Female cynomolgus monkeys (Macaca fascicularis) with
normal menstrual cycle histories were used in this study and were
housed under standard husbandry conditions at the University of
Pittsburgh Primate Research Laboratory. All experimental procedures
were approved by the University of Pittsburgh Institutional Animal Care
and Use Committee. Daily blood samples were collected by femoral
venipuncture under ketamine HCl (10 mg/kg) sedation and serum samples
were assayed for estradiol and progesterone levels as previously
described (8). Day 1 of the luteal phase was defined as the first day
after the midcycle estradiol peak with progesterone levels >0.2 ng/ml.
Corpora lutea were collected at defined stages of the spontaneous cycle
or in response to hCG treatment given in increasing doses twice daily
as described (8, 15) for 7 days during the early luteal phase (days
5–11) or the mid-to-late luteal phase (days 10–16). Luteectomies were
performed by midventral laparotomy under ketamine anesthesia, and
corpora lutea were snap frozen and stored in liquid nitrogen until the
PKA assays were performed. The corpora lutea from hCG-treated animals
were collected from animals used in a previous study (8). Ovaries from
pseudopregnant rats were used as a standardized source of luteal PKA
activity for determining the inter and intra assay variabilities of the
PKA assay. Twenty-three-day-old female rats were treated with 50 IU PMS
gonadotropin followed 28 h later by 50 IU hCG and killed 7 days
after the hCG injection.
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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Characterization of PKA in macaque corpora lutea
Incorporation of 32P into kemptide peptide substrate
by soluble fractions of luteal homogenates was linear with respect to
protein concentration from 20–100 µg protein per tube (data not
shown) and was stimulated upon cAMP addition to the assay (Table 1
). 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.
PKA activity in macaque CL: effects of luteal age
PKA activity was assessed in luteal homogenates obtained from
macaque corpora lutea collected at various stages of the spontaneous
luteal phase. PKA activity in the presence of cAMP is shown in Fig. 1A, 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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Figure 1. PKA activity in luteal homogenates (25 µg
protein) obtained at various times throughout the spontaneous luteal
phase (PM = postmenses CL). Luteal homogenates were assayed for
PKA activity in the presence (A) and absence (B) of cAMP to determine
stimulated and basal activities, respectively. Values represent the
mean ± SEM (n = 4 animals/group).
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Detection of the catalytic subunit of PKA in corpora lutea by
immunoblotting
Figure 2 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).
PKA activity in macaque CL: effects of in vitro LH treatment
Data presented in Fig. 3 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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Figure 3. Effect of recombinant cynomolgus monkey LH on PKA
activity ratios of corpora lutea collected during the early, mid, and
late luteal phases. Luteal tissues were collected on days 3–5, 9–10,
and 13–15 of the luteal phase and cultured for 30 min in the absence
or presence of recombinant cynomolgus LH (50 and 500 ng/ml) as
described in Material and Methods. Values represent the
mean ± SEM (n = 3 CL/group) of the ratio of
basal and cAMP-stimulated PKA activities. *, Statistical difference
between luteal age for a given LH treatment. Although not depicted in
graph, treatment with the high concentration of LH (500 ng/ml)
significantly elevated PKA activities above untreated controls in CL
from all days of the luteal phase.
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Effect of hCG treatment in vivo on PKA and CREB in macaque CL
Figure 4 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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Figure 4. PKA activity in luteal homogenates (25 µg
protein) obtained on days 11 or 16 of the control luteal phase or from
animals after receiving hCG injections twice daily for 7 days. Luteal
homogenates were assayed for PKA activity in the presence (A) and
absence (B) of cAMP to determine stimulated and basal activities,
respectively. Values represent the mean ± SEM (n
= 4 animals/group). *, Statistically different outcome
(P < 0.05) as compared with other groups.
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Figure 5A 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).
Figure 5B 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.
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Discussion
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Results from this study indicate that neither basal nor
cAMP-stimulated PKA activity in luteal cell homogenates differ between
corpora lutea collected during the early, mid, or late luteal phase of
the menstrual cycle. Moreover, Western immunoblot analyses of extracts
of corpora lutea collected throughout the luteal phase using an
antiserum directed against the catalytic subunit of PKA revealed no
pronounced differences in catalytic subunit immunoreactivity.
Collectively, these results demonstrate that PKA activity is maintained
at equivalent levels in corpora lutea throughout the luteal phase. A
caveat to this interpretation, however, is that measurement of
cAMP-dependent PKA activity in whole luteal homogenates reflects enzyme
activity in both luteal parenchymal (steroidogenic) cells as well as
other nonsteroidogenic cells within the corpus luteum such as
fibroblasts and vascular endothelial cells. To exclude the contribution
of these non-LH-responsive cells, we also examined PKA activation of
luteal fragments in vitro by recombinant macaque LH. Results
of these studies demonstrated that corpora lutea collected during the
early, mid, and late luteal phases exhibited both LH and
Bt2cAMP-stimulated PKA activities. Although LH-dependent
PKA activity was higher in corpora lutea collected during the early
luteal phase of the menstrual cycle, there were no significant
differences in PKA activities between tissue collected during the mid
and late luteal phases when the maximum decline in steroidogenesis
occurs. Whether the differences between LH-stimulated PKA in early
luteal phase tissues when compared with mid and late luteal phase
samples reflects an absolute difference in PKA activity in luteal
steroidogenic cells or whether lower activity in mid to late luteal
phase tissues reflects the fact that these tissues would contain a
greater percentage of nonsteroidogenic cells (25) cannot be determined.
However, despite the lower degree of LH stimulation during the late
luteal phase, the relative stimulation between basal and LH dependent
PKA activity was greatest in the late luteal phase tissues (2.9-fold
vs. 2.3- and 1.9-fold for early and mid luteal phase
samples, respectively). The greater relative stimulation of PKA by LH
in late luteal phase corpora lutea is in agreement with the studies of
Fisch et al. (6), which demonstrated that the relative
stimulation of progesterone production by hCG was greater in luteal
cells collected during the late luteal phase as compared cells
collected from early and mid luteal phase tissues as well as in
vivo observations that revealed a close temporal relationship
between the occurrence of pulses of LH and episodes of progesterone
secretion during the mid and late luteal phase of the menstrual cycle
(11).
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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We wish to thank Michael Cicco and Bob Beidler of the University
of Pittsburgh Primate Research Laboratory for providing daily animal
care. Sincere gratitude is also expressed to Dr. Mary Hunzicker-Dunn
for her guidance in developing this assay and helpful discussions and
to Dr. K.M.J. Menon for his suggestions.
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Footnotes
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1 This work was supported by NRSA HD-07691 (DFB) and NIH Grants
HD-08610, HD-16842 (to A.J.Z.). 
Received February 17, 1997.
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Top
Abstract
Introduction
