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Anatomisches Institut der Technischen Universität
München (A.B., M.G., A.M.) D-80802 München,
Germany
Frauenklinik der Ludwig Maximilians-Universität
(F.D.B., U.B.) D-80333 München, Germany
Division of Reproductive Sciences (D.M.D., R.L.S.) and
Division of Neuroscience (S.R.O.) Oregon Regional Primate Research
Center Oregon Health Sciences University Beaverton, Oregon
97006
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tight control of the luteolytic process during the menstrual cycle and after pregnancy is of pivotal importance for ovarian and reproductive physiology. However, the mechanisms responsible for the initiation of luteolysis at the cellular level are not well understood. In many species, apoptosis of luteal cells has been implicated in this process. In the rat, for example, apoptosis-associated genes become expressed in luteal cells (5 6 ), and the typical programmed cell death signs including DNA fragmentation are well documented (7 8 9 ). In nonhuman primates and humans, the situation is less clear. Although cultured human granulosa cells express both the Fas antigen (10 ) and the apoptosis-inducing protooncogene product BAX, no evidence for apoptosis was found in luteal cells in human CL examined for DNA fragmentation using the terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end labeling (TUNEL) method (11 12 ). In another report, some scattered TUNEL-positive cells were reported in the degenerating human CL (13 ). Likewise, apoptotic cells were found in the regressing CL (14 ), which, however, were mainly vascular cells. It is possible that these cells may account for DNA fragmentation reported by other investigators (13 15 16 ) in degenerating human CL. Endocrine cells of the human CL may be protected by the product of the protooncogene bcl-2, an inhibitor of apoptosis, which was demonstrated in luteal cells throughout the luteal phase (17 ). It is thus unclear whether apoptosis is a major contributor to either the functional or the structural luteolysis in humans. In the nonhuman primate ovary of the marmoset (18 19 20 ), apoptosis of luteal cells was detected, but another morphologically distinct form of cell regression became apparent. These luteal cells were characterized by the formation of cytoplasmic vacuoles due to cellular atrophy and phagocytosis of cytoplasmic debris. This process in primates, beyond its phenomenological description at the light microscopic and ultrastructural level (19 20 ), was not further examined. Equally unknown is the relationship that may exist between this form of cellular regression and the drastic increase in the number of lysosomes and lysosomal activity described, in particular, in the regressing CL of several species (21 22 ) including the human (20 23 ).
In a preliminary study (24 ), we reported the presence of functional voltage-activated potassium and sodium channels (NaCh) in human luteinized granulosa cells (GC). The type present in freshly isolated and cultured GC was found to be homologous to the one originally described in neuroendocrine cells of the adrenal and thyroid C cells (25 ). We named it endocrine (e) NaCh, rather than neuroendocrine, because of its presence in steroidogenic cells. In an attempt to characterize ovarian eNaCh and to elucidate its function, we report here evidence that the eNaCh type is also expressed in the human ovary and in the CL of nonhuman primates. Our results indicate hormonal down-regulation of these ion channels and show the consequences of eNaCh activation on the secretory function and lysosomal activation of progesterone-producing cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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).
After sequencing of the PCR products, we found that they corresponded
to the 
-subunit of an endocrine (e) voltage-activated NaCh. The form
in GC (two clones from culture day 2; two clones from culture day 3;
one clone from day 9; one clone from ovary) was 100% identical to a
previously identified neuroendocrine NaCh (25 ). When Northern blot
analyses were performed with RNA extracted from human GC (Fig. 2), an eNaCh transcript of approximately
7.5 kb was detected in untreated cells cultured for short or longer
periods of time (days in vitro, DIV, 1–10).
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) as
described previously (24 ). With the solution containing CsCl/
tetraethylammoniumchloride (TEA)-Cl, outward currents were blocked
(Fig. 3B), indicating the presence of voltage-activated
K+ channels. The remaining transient inward
currents were sensitive to tetrodotoxin (TTX, Fig. 4A), indicating the presence of voltage-
activated Na+ channels. Increasing
concentrations of TTX allowed determination of the half-maximal
blocking concentration (IC50) of 6.8
nM (Fig. 4B). A Hill slope of 0.67 was determined,
differing from the expected value of 1. Maximum inward currents were
obtained at voltage steps from -120 mV to a test potential of -15 mV,
and the resulting Na+ current densities ranged
from -2 to -30 picoamperes (pA)/picofarads (pF). Assuming a general
value for NaCh single channel conductance of 15 picoSiemens (pS) (26 ),
the maximum density was estimated to be 0.3 channels per
µm2 cell membrane.
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) of steady-state
inactivation revealed a mean V50 value of
-70.4 ± 8.8 mV (n = 13; 3–11 DIV) that was independent of
current density or culture time. In contrast, V50
values of activation showed a significant shift to more negative
potentials (P < 0.01, F Test, n = 30) from
approximately -10 to -20 mV over a culture period of 10 days (not
shown). For individual cells, the proportion of open eNaCh allowing
persisting Na+ fluxes, based on the model of
Hodgkin and Huxley (27 28 ) (window current), is between 0.3% and
4.0%.
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Results in three independent experiments showed reduction beyond
detection level of eNaCh in one experiment (24 h treatment with hCG),
reduction to 36% of control levels in another experiment (24 h), and
reduction to 68% when cells were treated for 48 h.
Incubation of human GC with hCG for 48 h reduced the resting
membrane potential (P < 0.05; n = 30 control
cells, 14 treated cells; 1–3 DIV) from -26.8 mV to -19.3 mV (Fig. 6). Moreover, after this period the
specific Na+ currents were significantly reduced
(Fig. 6; control group 30 cells: -9.50 pA/pF; hCG group: -3.46 pA/pF;
n = 21 cells; P < 0.01). In addition, hCG was
found to have an acute and profound effect on the peak amplitude of
voltage-activated Na+ current. In 10 of 10
experiments (using 5 cells) we observed that within seconds after hCG
application, the peak amplitude of the Na+
current was reversibly lowered (Fig. 7).
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). Blockage of eNaCh by TTX for 24–48 h
preserved a highly differentiated cellular phenotype (Fig. 9). Electron microscopy showed GC with
abundant intact mitochondria, smooth and rough endoplasmic reticulum,
and lipid droplets. In contrast, activation of eNaCh by veratridine
induced abundant secondary lysosomes, characterized by their
heterogeneous content. Most of them appeared to be autophagosomes
containing remnants of cytoplasmic organelles. The increase in
lysosomes resulting from veratridine treatment of GC was also confirmed
by the visualization of accumulation of a lysosomal-specific dye in GC
(Fig. 10). There was no evidence for
apoptosis; in particular, nuclei of veratridine-treated cells were not
altered. Concomitant incubation with TTX was able to prevent the
veratridine effects (Fig. 9F). None of the described changes were seen
when human carcinoma cells (A431) lacking NaCh were incubated with
veratridine or TTX (not shown).
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). Human and monkey eNaCh sequences
did not differ. The monkey sequence was submitted to Genbank (accession
no. AF164965).
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), characterized by their homogenous
content, and also a few secondary lysosomes. Numerous secondary
lysosomes prevailed later in the regressing CL (days 15, 16, and 18),
indicating that they can be induced not only in GC in vitro,
but are also present in normal cells of the regressing CL near the end
of the menstrual phase. Thus the observations in GC are mirrored by
results obtained in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The mRNA encoding the eNaCh was present throughout the life span of the monkey CL, during development (day 3), function (day 10), and regression (day 14) in the luteal phase of the menstrual cycle. A preliminary semiquantitative RT-PCR study of small CL samples (our unpublished observation) suggested that overall mRNA levels of the eNaCh increase toward the end of the life span of the CL. The CL is a heterogeneous organ, composed of different cell types (endocrine, vascular, immune, and connective type of cells), and their composition changes dramatically during the life of the CL (2 33 ). Examination of only a part of the CL is therefore not truly reflecting the complex events inside this endocrine organ and thus, we did not follow up on this preliminary result. Rather we attempted to study eNaCh function and regulation in a pure cell culture system of luteinized GC. In this system, we found, unexpectedly, that the luteotrophic hormone hCG markedly decreased eNaCh mRNA levels, probably indicating a negative hormonal regulation of eNaCh transcription. This was accompanied by altered resting membrane potential and Na+ currents. Moreover, hCG directly reduced Na+ peak currents within seconds. These effects of a gonadotropin are important in view of CL function in vivo. In the primate, progesterone production of the aging CL drops and LH secretion pattern changes (2 ). Alterations of LH frequency and amplitude, however, appear not to account for the regression of the function of this organ. Luteolysis can, as recently shown in an elegant study, be overcome simply by additional LH/hCG infusions (4 ). Thus the decline in CL function can be prevented by a stronger gonadotropin support. This result proves that it is the responsiveness of this temporary endocrine organ to LH/hCG that becomes dramatically reduced at the end of its life span (4 ). The precise reasons for this reduced responsiveness are not fully clear. One likely reason is a drop in vascular support of this tissue. Consequently, an individual luteal cell in the CL experiences a reduction in available or bioactive LH/hCG, i.e. a situation simulated in human GC cultured without hCG.
In general, the factors regulating the expression of the different NaCh genes are largely unknown (29 ) and, to our knowledge, a repressive function on NaCh gene expression has not been reported in any other cells. In contrast, that a closely related NaCh, the one described in astrocytes, can be positively regulated by factors originating from neurons, is well documented (34 ). But the nature of these, as yet undefined, factors present in conditioned medium from cultured neurons is unknown. In GC, it remains to be clarified whether hCG itself or hCG-induced gene products are the true inhibitors of NaCh gene transcription. For example, in addition to secretion of progesterone, hCG stimulates release of relaxin and oxytocin (35 ). That hCG has an acute and direct effect on the function of existing eNaCh, however, was clearly demonstrated by its ability to reduce the peak amplitude of Na+ currents in repeated pulse experiments within seconds. It is important to mention that this immediate effect of hCG is also of inhibitory nature. It is conceivable that hCG may act via cAMP and subsequent phosphorylation of the channel protein, because such a mechanism has been shown to exist in neurons that express another type of a NaCh (36 ). Thus, our data indicate that eNaChs are negatively regulated directly and/or indirectly by the luteotrophic hormone LH/hCG, which is crucial for the functional competence of the CL. Our results support the hypothesis that LH/hCG suppresses both eNaCh expression and function and thus is able to prevent deleterious downstream events that may result from the activation of this ion channel (see below).
Voltage-activated sodium channels constitute a family of related ion
channels, which are expressed mainly, but not exclusively, in excitable
cells, such as neurons, smooth muscle, skeletal and heart muscle, and
aminergic and peptidergic endocrine cells (25 37 ). In these typical
excitable cells, NaCh is responsible for the generation of electrical
signals, i.e. the generation of action potentials (38 ).
However, this may not be their sole function, since nonexcitable
cells, namely glial cells (34 ), have also been found to express NaCh.
The glial form expressed by Schwann cells is closely related to eNaCh
(
93% homology). The density of NaCh in glial cells and GC appear
also to be comparable (34 ). Moreover, GC and luteal cells in the ovary
are tightly coupled via gap junctions (see references in Ref. 39 ). Gap
junctions provide a low resistance shunt to adjacent cells, and
coupling will therefore prevent large membrane depolarizations. These
facts and our experimental results (e.g. most GC tested had
a membrane potential where the main portion of
Na+ channels would be in the inactivated state)
make it rather unlikely that GC or luteal cells are excitable.
Provided that generation of action potentials is not a role of eNaCh, what is the function of eNaCh in GC and in the CL? To address this question we took advantage of the fact that NaChs can be activated by veratridine or blocked by TTX (25 37 38 40 ). Addition of TTX to human GC, while causing no significant change in progesterone production of these cells, induced an ultrastructurally highly differentiated cellular phenotype with abundant mitochondria, which was distinct from untreated control cells. Thus, functional TTX-sensitive eNaChs appear to be present, although progesterone production alone did not reflect this supposition. In contrast, pharmacological activation of eNaCh by veratridine produced both a decrease in progesterone release and conspicuous signs of cellular regression. These included increases of secondary lysosomes, indicative of autophagocytosis. Reduced progesterone could most likely be a direct consequence of autophagy of cellular compartments involved in steroidogenesis, such as mitochondria and smooth endoplasmic reticulum. That these processes were indeed initiated and/or sustained by activation of eNaCh on GC became clear from two types of experiments: 1) concomitant treatment of GC with TTX prevented the striking effect of veratridine; 2) veratridine or TTX treatment of human epithelial A431 carcinoma cells, which do not posses NaCh, induced no ultrastructural change (no increase in lysosomes).
These results, in particular the morphological changes observed in
TTX-treated GC, allow the conclusion that eNaChs are present and
functional in GC. From our current point of view, this functionality
pertains to the possibility of persistent Na+
influx mediated by eNaCh. The membrane potential of most cells tested
was in a range where a possible window current would be maximal (-20
to -30 mV), with a probability of open channels of up to 4%.
Untreated cells tended to more negative V50
values of eNaCh activation with cultivation time, possibly resulting in
a slow increase of Na+ influx. Interestingly, hCG
treatment led to a shift of the resting membrane potential to more
positive values after 48 h and therefore would reduce persistent
Na+ current influx. We do not expect high levels
of Na+ influx to occur within the first days of
GC cultivation in the absence of hCG, because TTX, apart from its
morphological effects, provoked only a small and not statistically
significant increase in progesterone production (Fig. 8). However, if
cells change their properties as described above, an increase in
Na+ influx can be expected at later time
points.
Clearly, we currently do not have information about the probability of persistent Na+ fluxes to occur in luteal cells in vivo, which express the eNaCh gene. Given this possibility, however, how are these linked to regression of GC and luteal cells? Support for such a link comes from our observations and also from experiments performed in other species and organ systems. Our ultrastructural studies performed on sections from the Rhesus monkey CL show that similar events associated with eNaCh activation in vitro (in GC) occur also in vivo. These include lysosomal activity leading to autophagy of luteal cells. Both primary and secondary lysosomes were present in monkey luteal cells on day 7 of the luteal phase, i.e. in the functional CL. Primary and secondary lysosomes were more readily detected around day 11 of the luteal phase, and the number and size of secondary lysosomes were increased in the regressing CL. In several species, including guinea pig, pig, monkey, and human, lysosomes and/or increased lysosomal activity or autophagy were previously described in the regressing CL (20 21 22 23 ). In humans and nonhuman primates, luteolysis occurring at the level of the luteal cell appears to differ from the processes reported for the rat (see Introduction). The available reports addressing this issue describe vacuolated cells and autophagy involving lysosomes, e.g. in the marmoset CL (19 20 ). Thus, the observed changes in GC and luteal cell morphology are corroborated by other authors and mirror normal physiological processes.
That the family of NaCh, by altering intracellular ion concentrations, are involved in the regulation of cell viability and forms of cell death in a variety of cell types is clearly shown by several reports (34 41 ). For instance, in glial cells it has been proposed (34 ) that their NaCh may serve as a return pathway for Na+ ions, thus fueling the Na+/K+ ATPase. Experimental blockage of astrocyte NaCh by TTX produced a dose- dependent reduction in glial cell viability (42 ). Such a possibility can almost be ruled out for GC, in which TTX treatment resulted in the opposite and preserved a structurally well differentiated cellular phenotype. In neuronal cells, intracellular Na+ overload, initiated by NaCh activity, has been linked to necrosis occurring in cerebral ischemia or trauma (40 ). Unfortunately, a detailed picture of the events leading to these changes in neurons can presently not be given and certainly neuronal cell necrosis cannot be compared with the regressive changes observed in GC and luteal cell, namely the accumulation of secondary lysosomes and the lack of typical signs of necrotic cell death or of apoptosis.
In summary, we propose the following hypothesis: The gonadotropins LH/hCG negatively regulate both eNaCh gene expression and eNaCh activity, as well as membrane potential of GC and presumably luteal cells. LH/hCG thus act to prevent the observed deleterious effect after expression and function of eNaCh on GC and luteal cells. This implies that reduced LH values/or reduced accessibility/bioactivity of this hormone to luteal cells causes the expression of active eNaChs in luteal cells during the menstrual cycle. From the current point of view, eNaChs allow persistent Na+ influx to occur, which then initiates a process of luteal cell regression involving autophagocytosis. The regulated expression and function of these channels may represent an, as yet unknown, way by which functional luteolysis occurs in the primate CL at the level of individual cells. Ovarian NaCh may therefore be a key molecular element in luteolysis.
Our findings could have implications for human physiology and human diseases: NaChs are targets for various drugs, including local anesthetics and systemically applied antiarrhythmics, as well as antiepileptic drugs such as phenytoin (40 ). Our results raise the question of whether alterations of CL function(s) may be a possible consequence of systemic treatment with such substances in women (43 ).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tissues
The care and housing of rhesus macaques (Macaca
mulatta) at the Oregon Regional Primate Research Center (ORPRC)
were previously described (47 ). Animal protocols and experiments were
approved by the Oregon Regional Primate Research Center Animal Care and
Use Committee, and studies were conducted in accordance with the NIH
Guide for the Care and Use of Laboratory Animals. Ovaries were
collected from rhesus macaques undergoing ovariectomy (see below) for
other purposes or were obtained at necropsy from the tissue
distribution program at the ORPRC (n = 3). In total, ovaries from
14 adult monkeys were examined. Upon collection, all tissues were
rapidly frozen on dry ice (for extraction of RNA) or immersed in
fixative for electron microscopy (see below). CL samples were obtained
as described previously (47 ). Adult female monkeys with regular
menstrual cycles were bled daily by saphenous venipuncture beginning on
day 8 after the onset of menses. Serum concentrations of estradiol
(E2) and progesterone were measured by RIA in the
Endocrine Services Laboratory at the ORPRC (48 ). The day of the
precipitous fall in circulating E2 levels after
the midcycle peak was designated day 1 of the luteal phase (48 ).
Corpora lutea were surgically removed from anesthetized monkeys on
different days of the luteal phase for RNA extraction (days 3, 10 and
14; total of 4 samples) or for fixation for electron microscopy (days
7, 11, 15, 16, 18; total of 7 samples), as previously described
(47 ).
Electrophysiology
Patch pipettes were manufactured from borosilicate glass
capillaries and fire polished on a DMZ-Universal puller (Engel,
Augsburg, Germany), with resistances between 3 and 7 M
. Seal
resistances typically ranged from 2 to 5 G and were established
within 30 sec. Before rupture of the cell membranes, the potential was
clamped to -70 mV. All recordings were performed in the whole-cell
configuration at room temperature using an EPC-9 amplifier controlled
by PULSE (Heka, Lambrecht, Germany). Patch pipettes were filled with a
solution containing (in millimolar concentration): 140 KCl, 10 HEPES, 5
EGTA, 1 CaCl2, 1 MgCl2, (pH
7.4/KOH, free Ca2+ 100 nM). For
investigation of Na+ currents alone, a solution
was used containing (in millimolar concentration): 120 CsCl, 20 TEA-Cl,
5 EGTA, 0.5 CaCl2, 1 MgCl2,
10 HEPES (pH 7.4 with CsOH, free Ca2+ 10
nM). The bathing solution consisted of (in millimolar
concentration): 140 NaCl, 3 KCl, 1 CaCl2, 10
HEPES, 10 glucose (pH 7.4 with NaOH). For local and fast application of
TTX or hCG (Sigma, Deisenhofen, Germany) a seven-channel
superfusion system was used. Activation of Na+
currents was investigated with a cyclic pulse protocol (protocol 1).
Within each sequence the cell was hyperpolarized to -120 mV for 100
msec. Subsequently, a variable test potential from -90 to +60 mV was
applied. For investigation of steady-state inactivation, the cell was
clamped to the hyperpolarized potential, then to a variable
prepotential, and finally to the test potential of -20 mV (protocol
2). In both protocols, the time interval between sequences was 500
msec. For determination of TTX inhibition curves, constant test pulses
were applied in intervals of 1 sec. Cells tended to form branches and
were coupled by gap junctions. This was supported by the observation
that within a group of neighboring cells, the patched cell had an
apparently very low input resistance. To avoid this problem, isolated
cells were used for the experiments described.
RT-PCR
Preparation of total GC mRNA was done as described (44 ) using
the RNeasy kit from QIAGEN (Hilden, Germany), the acid
phenol-extraction method, as described previously (32 ), or by a cesium
chloride ultracentrifugation method (3 ). In addition, a commercial
human cDNA (2 µl), reverse transcribed from pooled adult ovarian
mRNA, was used for PCR (Invitrogen, DeSchelp, The
Netherlands). For reverse transcription, 200 ng of RNA together with
18-mer polydeoxythymidine primer and Moloney’s murine leukemia virus
(Promega Corp., Mannheim, Germany) were incubated for
2 h at 37 C. For amplification of the sodium channel (NaCh)
subunit, primers were constructed to match common sequences of
different channel types from human (thyroid, brain) and rat (peripheral
nerve, brain; (5'-ATC GGA ATC TGA AGA CAG C-3', sense and 5'-CTG TGC
TCA TCA TCG GCA A-3', antisense). In some cases cyclophilin was
coamplified with the sodium channel using primers and conditions
described previously (49 ). PCR amplification was performed in a PTC-200
thermocycler (MJ Research, Inc., Watertown, MA) using
Taq polymerase (Promega Corp.) starting with a
94 C step for initial denaturation (5 min) followed by 35 cycles of 1
min annealing at 54 C, 2 min extension at 72 C, and 15 sec denaturation
at 94 C. PCR products were resolved on a 2% agarose gel and visualized
with ethidium bromide. For sequence analysis, they were either
sequenced directly using one of the primers or they were first
subcloned into the pGEMT vector (Promega Corp.).
Sequencing was performed as described previously (44 ) using a
fluorescence-based dideoxy sequencing reaction on an ABI model 373A DNA
sequencer (Perkin-Elmer Corp., Überlingen,
Germany).
Northern Blotting
Northern blotting was performed as described (50 ) using 10 µg
of total RNA. Riboprobes (eNaCh and actin) were prepared by in
vitro transcription using 32P-UTP and T7- or
SP6-RNA-polymerase (Promega Corp.). Transcripts were
purified with Nick-columns (Pharmacia Biotech, Freiburg,
Germany) and hybridized to the membrane containing the test RNA at 60 C
overnight. Subsequently, blots were washed five times at 65 C in
0.1 x SSC, 0.1% SDS and dried. Autoradiograms were developed
after 1–5 days. For densitometric measurements, blots were digitized
using an image documentation system (MWG-Biotech, Ebersberg, Germany).
Integrated optical densities were determined using a noncommercial
program. Densities obtained from the actin signal were used to
normalize the values obtained from eNaCh signals.
Fluorescence Microscopy: Lysosomal Dye
The density and size of lysosomes were analyzed in GC treated
with veratridine or TTX for 24 h using the LysoTracker L-7528
lysosomal dye (Molecular Probes, Inc., Eugene, OR). Cells
on glass cover slides were incubated with the dye (10 nM)
for 2 min at room temperature and subsequently observed and
photographed using an inverse fluorescence microscope (Axiovert
135TV;Carl Zeiss, Jena, Germany). The experiments were
repeated with three different preparations of cells.
Electron Microscopy
For ultrastructural studies, human A 431 carcinoma cells and GC
were incubated for 24 and 48 h with or without veratridine, TTX,
and hCG and were then fixed with 4% paraformaldehyde/0.5%
glutaraldehyde and postfixed with 4%
OsO4/potassium hexacyanoferrate (II). Another
control group, in which we tested whether veratridine effects can be
antagonized by TTX, consisted of GC pretreated with TTX (8 h) and
subsequent incubation with veratridine for 24 h. After embedding
in Epon, thin sections were cut, contrasted with uranylacetate
(2%)/lead citrate (2.7%) as described previously (51 52 ), and
examined with an EM10 electron microscope (Carl Zeiss).
Three different preparations of GC were examined. Small fragments of
nonhuman CL tissues obtained at different times of the luteal phase
(days 7, 11, 15, and 16, and day 18; total of 7) were also examined. In
this case, samples were immersed in 5% glutaraldehyde in 0.1
M cacodylate acid [pH 7.4, (52 )] and subsequently
processed as described above.
Progesterone Assay
Culture media from various stimulation experiments were
collected and frozen at -20 C until determination of progesterone
concentrations, using the Serozyme-M kit from BioChem (Freiburg,
Germany), as described (44 ).
The amount of protein was determined as described (44 ), and progesterone values were expressed as nanograms/mg protein.
Statistics
For statistical analyses, unpaired and two-tailed t
tests were performed with the exception of progesterone values (Fig. 8;
paired t test) and change of V50 with
time (F test against slope value of zero).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by DFG grants Ma1080/12–1 and Graduiertenkolleg 333, Volkswagen-Stiftung (A.M.), as well as by NIH Grants HD-20869, HD-24870, and RR00163 (R.L.S., S.R.O.).
Received for publication October 5, 1999. Revision received February 21, 2000. Accepted for publication March 10, 2000.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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S. Fritz, L. Kunz, N. Dimitrijevic, R. Grunert, C. Heiss, and A. Mayerhofer Muscarinic Receptors in Human Luteinized Granulosa Cells: Activation Blocks Gap Junctions and Induces the Transcription Factor Early Growth Response Factor-1 J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1362 - 1367. [Abstract] [Full Text] [PDF]
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N. Levy, M. Gordin, R. Mamluk, M. Yanagisawa, M. F. Smith, J. H. Hampton, and R. Meidan Distinct Cellular Localization and Regulation of Endothelin-1 and Endothelin-Converting Enzyme-1 Expression in the Bovine Corpus Luteum: Implications for Luteolysis Endocrinology, December 1, 2001; 142(12): 5254 - 5260. [Abstract] [Full Text] [PDF]
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 J. S. Richards New Signaling Pathways for Hormones and Cyclic Adenosine 3',5'-Monophosphate Action in Endocrine Cells Mol. Endocrinol., February 1, 2001; 15(2): 209 - 218. [Abstract] [Full Text]
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A. Mayerhofer, S. Fritz, R. Grünert, S. L. Sanders, D. M. Duffy, S. R. Ojeda, and R. L. Stouffer D1-Receptor, DARPP-32, and PP-1 in the Primate Corpus Luteum and Luteinized Granulosa Cells: Evidence for Phosphorylation of DARPP-32 by Dopamine and Human Chorionic Gonadotropin J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4750 - 4757. [Abstract] [Full Text]
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