Identification of an Ovarian
Voltage-Activated Naⴙ-Channel
Type: Hints to Involvement in
Luteolysis
Andreas Bulling, Frank D. Berg, Ulrike Berg, Diane M. Duffy,
Richard L. Stouffer, Sergio R. Ojeda, Manfred Gratzl, and
Artur Mayerhofer
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
An endocrine type of voltage-activated sodium
channel (eNaCh) was identified in the human ovary
and human luteinized granulosa cells (GC). Wholecell patch-clamp studies showed that the eNaCh in
GC is functional and tetrodotoxin (TTX) sensitive.
The luteotrophic hormone human CG (hCG) was
found to decrease the peak amplitude of the sodium current within seconds. Treatment with hCG
for 24–48 h suppressed not only eNaCh mRNA levels, but also mean Naⴙ peak currents and resting
membrane potentials. An unexpected role for
eNaChs in regulating cell morphology and function
was indicated after pharmacological modulation of
presumed eNaCh steady-state activity in GC cultures for 24–48 h using TTX (NaCh blocker) and
veratridine (NaCh activator). TTX preserved a
highly differentiated cellular phenotype. Veratridine not only increased the number of secondary
lysosomes but also led to a significantly reduced
progesterone production. Importantly, endocrine
cells of the nonhuman primate corpus luteum (CL),
which represent in vivo counterparts of luteinized
GC, also contain eNaCh mRNA. Although the
mechanism of channel activity under physiological
conditions is not clear, it may include persistent
Naⴙ currents. As observed in GC in culture, abundant secondary lysosomes were particularly evident in the regressing CL, suggesting a functional
link between eNaCh activity and this form of cel0888-8809/00/$3.00/0
Molecular Endocrinology 14(7): 1064–1074
Copyright © 2000 by The Endocrine Society
Printed in U.S.A.
lular regression in vivo. Our results identify eNaCh
in ovarian endocrine cells and demonstrate that
their expression is under the inhibitory control of
hCG. Activation of eNaChs in luteal cells, due to
loss of gonadotropin support, may initiate a cascade of events leading to decreased CL function, a
process that involves lysosomal activation and autophagy. These results imply that ovarian eNaChs
are involved in the physiological demise of the temporary endocrine organ CL in the primate ovary
during the menstrual cycle. Because commonly
used drugs, including phenytoin, target NaChs,
these results may be of clinical relevance. (Molecular Endocrinology 14: 1064–1074, 2000)
INTRODUCTION
The corpus luteum (CL) in primates forms after ovulation and represents a temporary endocrine organ
within the ovary (1, 2). The main function of the CL
during its functional life span is the production of the
steroid progesterone, which is fundamental for preparation and maintenance of pregnancy. Both the CL
structure and progesterone are hormonally controlled
by the pituitary hormone LH or, during pregnancy, by
a closely related placental hormone (CG). The presence or absence of these luteotrophic hormonal signals determines growth and survival of the highly differentiated CL or its demise. In nonpregnant women
this process of formation, growth, and regression of
the CL occurs during each ovarian cycle. It is not clear
what initiates the regression of the CL at the end of
1064
Ovarian Na⫹-Channels
each menstrual cycle, in particular if the altered, lower
frequency of pituitary LH pulses is of importance (2, 3).
However, in a nonhuman primate species, cynomolgus monkeys (Macaca fascicularis), luteal regression
and reduced progesterone production are caused by a
reduction in the responsiveness of the aging CL to LH
or hCG (4). The observed shutting down of progesterone production is often referred to as functional luteolysis. The functional demise of the CL is followed by
a longer lasting process, structural luteolysis, a term
describing the remodeling of the CL into a scar, during
which the luteal cells are replaced by connective tissue.
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 (7–9). 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 (18–20),
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
1065
(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.
RESULTS
RT-PCR Identification of Ovarian eNaCh
We amplified cDNAs of 222 bp from human ovary and
human GC (cultured for 2, 3, and 9 days) (Fig. 1). 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).
Electrophysiological Characterization of
GC eNaCh
The mean resting membrane potential of human GC
measured directly after establishing the whole-cell
configuration was ⫺32.7 ⫾ 9.1 mV (n ⫽ 6 cells; 4, 7,
and 8 DIV). Using the standard internal solution, the
pulse protocol for activation gave rise to transient
inward and outward currents (Fig. 3A) 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
Fig. 1. Ethidium Bromide-Stained Agarose Gel Showing
cDNAs Obtained by RT-PCR
They correspond to eNaCh as confirmed by sequence
analysis and were identified in the human ovary (A) and
cultured human GC (B, 2 DIV).
MOL ENDO · 2000
1066
Fig. 2. Detection of eNaCh mRNA in Untreated and hCGTreated Human GC, Using Northern Blotting
An eNaCh transcript at approximately 7.5 kb is found in
human GC cultured for 24 h in the absence (Co) or presence
of 10 IU/ml hCG. The same blots were subsequently probed
for ␤-actin (1.3 kb), which was used for normalization of the
results.
(TTX, Fig. 4A), indicating the presence of voltageactivated 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
␮m2 cell membrane.
Although the measured current traces resembled
those underlying excitation processes, no action potentials could be triggered when depolarizing currents
were injected in current-clamp mode. Results from
Boltzmann fits (Fig. 5) 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%.
Regulation of eNaCh by hCG
hCG was able to regulate both eNaCh mRNA level and
eNaCh function. When the luteotrophic hormone hCG
(10 IU/ml) was added to cultures of human GC for
Vol 14 No. 7
Fig. 3. Results from Patch-Clamp Recordings of Human GC
A, Transient inward and outward currents are elicited by
stepwise depolarization (⫹15 mV per step) from a holding
potential of ⫺120 mV to various test potentials between ⫺90
and ⫹45 mV. Inward currents (arrow) are not resolvable at the
time scale shown. B, Patch pipettes filled with CsCl/TEA-Cl
blocked outward currents, leaving only TTX-sensitive inward
currents with maximum amplitudes of up to 1.5 nA. Increase
of test potentials ⫹5 mV per step.
24–48 h, eNaCh mRNA levels were suppressed (Fig.
2). 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).
Consequences of eNaCh Activation and Blockage
in GC: Progesterone Production and Lysosomes
Activation of eNaCh by 50 ␮M veratridine for 24–48 h
decreased media progesterone levels by half (P ⬍
0.05), whereas TTX showed no statistically significant
effect (Fig. 8). 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 endo-
Ovarian Na⫹-Channels
Fig. 4. TTX Sensitivity of eNaCh in GC
A, Effect of increasing concentrations of TTX on transient
inward currents of one cell. From bottom to top trace: 0, 0.1,
1, 10, 100, and 1000 nM TTX. B, Results from eight different
cells reveal a value for IC50 of 6.8 nM and a Hill slope of 0.67
for inhibition by TTX (circles are single values or means ⫾ SD).
1067
Fig. 6. Regulation of Na⫹ Current and Membrane Potential
by Chronic hCG Treatment
Incubation of human GC with hCG (10 IU/ml, 48 h, from
DIV 1–3), reduced specific Na⫹ currents, and membrane
resting potentials when compared with untreated control
cells. In panel A, the mean currents (marked by horizontal
lines) were ⫺9.50 pA/pF and ⫺3.46 pA/pF; P ⬍ 0.01 (t test);
n ⫽ 30 control cells/21 treated cells. In panel B, the mean
potentials were ⫺26.8 mV and ⫺19.3 mV; P ⬍ 0.05 (t test);
n ⫽ 30 control cells/14 treated cells.
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).
Expression of eNaCh in the Primate CL
Fig. 5. Example of Na⫹ Current Activation (F) and SteadyState Inactivation (E) in a GC
In this specific cell (3 DIV), superposition of both curves
would result in a window current (dashed line) of up to 2% of
maximal Na⫹ current at a potential of ⫺25 mV.
plasmic 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
Using RT-PCR followed by sequence analysis, we
found that the form of the eNaCh was also present in
the rhesus monkey ovary and CL on days 3 (two
clones), 10, and 14 (one clone each) corresponding to
the early, mid, and late life phase of this organ (Fig. 11).
Human and monkey eNaCh sequences did not differ.
The monkey sequence was submitted to Genbank
(accession no. AF164965).
Electron Microscopy of Luteal Cells
Electron microscopic examination of the CL from nonhuman primates revealed that on days 7 and 11, i.e.
MOL ENDO · 2000
1068
Vol 14 No. 7
Fig. 7. Acute Effect of hCG on eNaCh Activity
Application of hCG (10 IU/ml) to GC (3 DIV) reversibly
reduced the peak amplitude of voltage-activated Na⫹ currents. Similar results were seen in 10 of 10 experiments with
5 different cells. The inset shows current traces obtained 10
sec before, at application of, and 10, 12, 14, 16, and 18 sec
after application of hCG.
Fig. 8. Consequences of eNaCh Activation and Blockage on
Progesterone Production
Incubation of GC with veratridine (50 ␮M) for 24 h or 48 h
in GC (culture days 1 2 or 1–3, respectively) decreased progesterone production by half compared with untreated control cells (P ⬍ 0.05, marked by asterisks), whereas TTX (5 ␮M)
showed no statistically significant effect. Bars represent
means ⫾ SEM.
when the CL is functional, luteal cells contain abundant primary lysosomes (Fig. 12), 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.
DISCUSSION
In the present study we report that the gene for a
member of the voltage-activated, TTX-sensitive family
Fig. 9. Consequences of eNaCh Activation and Blockage on
Cell Morphology
A, Ultrastructural features of a GC cultured in basal medium. Note mitochondria (double arrow) and lipid droplets
(asterisks), as well as some lysosomes (arrows). Bar, 2.5 ␮m.
B, Ultrastructure of a GC cultured for 24 h in basal medium
and then treated with TTX (5 ␮M) for 24 h: note the abundantly
present mitochondria (double arrow), few lysosomes (arrows)
and lipid droplets (asterisks). Bar, 2 ␮m. C, Veratridine (VERA,
50 ␮M) treatment (24 h) led to the appearance of abundant
large secondary lysosomes (arrows). Most of them appeared
to be autophagosomes. Note that the nucleus is not altered.
Few mitochondria (double arrow) and lipid droplets (asterisks)
are seen. Bar, 2.5 ␮m. D and E, Examples of the morphology
of the secondary lysosomes found in veratridine-treated GC.
Bars, 0.8 ␮m. F, TTX treatment blocked the effects of veratridine. Note presence of abundant mitochondria and absence of lysosomes. Bar, 1.5 ␮m.
of Na⫹ channels is expressed in steroid-producing
endocrine cells of the primate ovary. Northern blotting
and RT-PCR experiments showed the presence of
eNaCh in vitro in human luteinized GC and in the
Ovarian Na⫹-Channels
1069
Fig. 10. Increased Lysosomes in GC after Veratridine Treatment (VERA, 50 ␮M) in a Lysosomal Dye Experiment
An increase in lysosomes (red) over the numbers seen in untreated cells (Co; A) was observed in veratridine-treated GC for 24 h
(B) as visualized by a lysosomal dye. Bars, 50 ␮m.
Fig. 11. Presence of eNaCh mRNA in the Monkey CL and
Ovary
Ethidium bromide visualization of RT-PCR-derived cDNAs
corresponding to eNaCh and cyclophillin (Cycloph.) are
shown. Identity of cDNAs was confirmed by sequencing. The
eNaCh mRNA is present in the early (E), mid (M), and late (L)
phase of the life cycle of the CL, as well as in a monkey ovary
(MK-Ov).
Fig. 12. Electron Microscopic Features of Monkey CL
A, Electron microscopical examination of a monkey luteal
cell of day 7 of the luteal phase. Note primary and secondary
lysosomes (arrows). Bar, 1.3 ␮m. B, A luteal cell from day 11
of the luteal phase showed primary and secondary lysosomes (arrows). Bar, 1 ␮m. C, Secondary lysosomes were
present in a regressing luteal cell CL (day 18 of the luteal
phase). Bar, 1.3 ␮m.
corresponding tissue in vivo, in the rhesus monkey CL.
This channel type was previously described in humans
to be expressed in neuroendocrine cells (C cell carcinoma), as well as in bovine adrenal and thyroid, but
not in pituitary or brain (25). Rat homologs were subsequently described in peripheral neurons of the dorsal root ganglion (29, 30). This widespread distribution
indicates that this form of NaCh is not as specific for
neuroendocrine cell types, as previously suggested.
Although the monkey ovary contains neuron-like cells
(31, 32), which may also express NaCh (our unpublished observation), these cells are found exclusively in
ovarian stroma and not within the CL. This fact and the
current observation, that the counterparts of luteal
cells, isolated human GC, express the eNaCh gene,
clearly identify steroidogenic cells as the sites of expression of eNaCh and exclude contamination by
other cells.
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 re-
MOL ENDO · 2000
1070
duced 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)
Vol 14 No. 7
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 TTXsensitive 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 sec-
Ovarian Na⫹-Channels
tions 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 (20–23). 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 dosedependent 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
1071
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).
MATERIALS AND METHODS
Cell Culture Chemicals and Treatments
Follicular fluid containing granulosa cells was derived from in
vitro fertilization patients (44). The experimental procedure
and the use of the cells were approved by the local ethics
committee, and the women gave their written consent. Isolation and culture of granulosa cells were performed as described previously (44). Cells were seeded in DMEM/Ham’s
F12 (1:1) medium with 10% FCS onto Falcon culture dishes
(Nunc, Wiesbaden, Germany), Labtek cell culture chambers
(Nunc), or on glass cover slides (for patch-clamp experiments). All surfaces were coated with laminin (Sigma, Deisenhofen, Germany), and cells were kept in culture for up to 11
days (44, 45). Culture medium was changed every 2 days.
The human carcinoma cell line (A431) was obtained from Dr.
M. Haasemann (Munich, Germany) and used for morphological studies (46). All chemicals used in stimulation experiments were purchased from Sigma. For lysosomal dye studies, ultrastructural examination, and progesterone release,
cells were incubated for 24 h or 48 h with veratridine (5 and
50 ␮M), TTX (5 and 50 ␮M), or human CG (hCG; 10 IU/␮l).
Ethanol was used to dilute veratridine and was therefore
added in a final concentration of 0.5% to cells not treated
with veratridine as an additional control.
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).
MOL ENDO · 2000
1072
Vol 14 No. 7
Electrophysiology
Northern Blotting
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.
Northern blotting was performed as described (50) using 10
␮g 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 ⫻ 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.
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 ␮l), 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).
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).
Ovarian Na⫹-Channels
1073
Acknowledgments
14. Gaytan F, Morales C, Garcia-Pardo L, Reymundo C,
Bellido C, Sanchez-Criado JE 1998 Macrophages, cell
proliferation, and cell death in the human menstrual corpus luteum. Biol Reprod 59:417–425
15. Yuan W, Giudice LC 1997 Programmed cell death in
human ovary is a function of follicle and corpus luteum
status [published erratum appears in J Clin Endocrinol
Metab 1998 Jan;83(1):240]. J Clin Endocrinol Metab 82:
3148–3155
16. Shikone T, Yamoto M, Kokawa K, Yamashita K, Nishimori K, Nakano R 1996 Apoptosis of human corpora
lutea during cyclic luteal regression and early pregnancy.
J Clin Endocrinol Metab 81:2376–2380
17. Rodger FE, Fraser HM, Duncan WC, Illingworth PJ 1995
Immunolocalization of bcl-2 in the human corpus luteum.
Hum Reprod 10:1566–1570
18. Fraser HM, Lunn SF, Cowen GM, Illingworth PJ 1995
Induced luteal regression in the primate: evidence for
apoptosis and changes in c-myc protein. J Endocrinol
147:131–137
19. Young FM, Illingworth PJ, Lunn SF, Harrison DJ, Fraser
HM 1997 Cell death during luteal regression in the marmoset monkey (Callithrix jacchus). J Reprod Fertil 111:
109–119
20. Fraser HM, Lunn SF, Harrison DJ, Kerr JB 1999 Luteal
regression in the primate: different forms of cell death
during natural and gonadotropin-releasing hormone antagonist or prostaglandin analogue-induced luteolysis.
Biol Reprod 61:1468–1479
21. Paavola LG 1978 The corpus luteum of the guinea pig. III.
Cytochemical studies on the Golgi complex and GERL
during normal postpartum regression of luteal cells, emphasizing the origin of lysosomes and autophagic vacuoles. J Cell Biol 79:59–73
22. Gregoraszczuk EL, Sadowska J 1997 Lysosomal acid
phosphatase activity and progesterone secretion by porcine corpora lutea at various periods of the luteal phase.
Folia Histochem Cytobiol 35:35–39
23. Takenaka A 1981 An ultrastructural and cytochemical
study of human corpora lutea. Acta Obstet Gynaecol Jpn
33:907–915
24. Bulling A, Brucker C, Berg U, Gratzl M, Mayerhofer A
1999 Identification of voltage-activated Na⫹ and K⫹
channels in human steroid-secreting ovarian cells. Ann
NY Acad Sci 868:77–79
25. Klugbauer N, Lacinova L, Flockerzi V, Hofmann F 1995
Structure and functional expression of a new member of
the tetrodotoxin-sensitive voltage-activated sodium
channel family from human neuroendocrine cells. EMBO
J 14:1084–1090
26. Hille B 1992 Counting channels. In: Hille B (ed) Ionic
Channels of Excitable Membranes. ed 2. Sinauer Associates, Sunderland, MA, p 329
27. French CR, Sah P, Buckett KJ, Gage PW 1990 A voltagedependent persistent sodium current in mammalian hippocampal neurons. J Gen Physiol 95:1139–1157
28. Crill WE 1996 Persistent sodium current in mammalian
central neurons. Annu Rev Physiol 58:349–362
29. Toledo-Aral JJ, Moss BL, He ZJ, Koszowski AG,
Whisenand T, Levinson SR, Wolf JJ, Silos-Santiago I,
Halegoua S, Mandel G 1997 Identification of PN1, a
predominant voltage-dependent sodium channel expressed principally in peripheral neurons. Proc Natl Acad
Sci USA 94:1527–1532
30. Sangameswaran L, Fish LM, Koch BD, Rabert DK, Delgado SG, Ilnicka M, Jakeman LB, Novakovic S, Wong K,
Sze P, Tzoumaka E, Stewart GR, Herman RC, Chan H,
Eglen RM, Hunter JC 1997 A novel tetrodotoxin-sensitive, voltage-gated sodium channel expressed in rat
and human dorsal root ganglia. J Biol Chem 272:
14805–14809
31. Dees WL, Hiney JK, Schultea TD, Mayerhofer A, Danilchik M, Dissen GA, Ojeda SR 1995 The primate ovary
We thank Mrs. G. Terfloth, Mrs. U. Fröhlich, Mrs. S. Boddien,
Mr. G. Prechtner, Mr. A. Mauermayer, and Mr. R. Grünert for
technical assistance and Dr. J. Grosse for the help with
Northern blotting.
Received October 5, 1999. Revision received February 21,
2000. Accepted March 10, 2000.
Address requests for reprints to: Dr. Artur Mayerhofer,
Anatomisches Institut, Technische Universität München,
Biedersteiner Strasse 29, D-80802 München, Germany. Email: [email protected].
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.).
REFERENCES
1. Zeleznik AJ, Benyo DF 1994 Control of follicular development, corpus luteum function, the recognition of pregnancy in higher primates. In: Knobil E, Neill JD (eds) The
Physiology of Reproduction. Raven Press, New York, pp
751–782
2. Stouffer RL 1996 Corpus luteum formation and demise.
In: Adashi EY, Rock JA, Rosenwaks Z (eds) Reproductive
Endocrinology, Surgery, and Technology. LippincottRaven, New York, pp 263–263
3. Duffy DM, Stewart DR, Stouffer RL 1999 Titrating luteinizing hormone replacement to sustain the structure and
function of the corpus luteum after gonadotropin-releasing hormone antagonist treatment in rhesus monkeys.
J Clin Endocrinol Metab 84:342–349
4. Zeleznik AJ 1998 In vivo responses of the primate corpus
luteum to luteinizing hormone and chorionic gonadotropin. Proc Natl Acad Sci USA 95:11002–11007
5. Guo K, Wolf V, Dharmarajan AM, Feng Z, Bielke W,
Saurer S, Friis R 1998 Apoptosis-associated gene expression in the corpus luteum of the rat. Biol Reprod.
58:739–746
6. Roughton SA, Lareu RR, Bittles AH, Dharmarajan AM
1999 Fas and Fas ligand messenger ribonucleic acid and
protein expression in the rat corpus luteum during apoptosis-mediated luteolysis. Biol Reprod 60:797–804
7. Gaytan F, Bellido C, Morales C, Sanchez-Criado JE 1998
Both prolactin and progesterone in proestrus are necessary for the induction of apoptosis in the regressing corpus luteum of the rat. Biol Reprod 59:1200–1206
8. Billig H, Furuta I, Hsueh AJ 1994 Gonadotropin-releasing
hormone directly induces apoptotic cell death in the rat
ovary: biochemical and in situ detection of deoxyribonucleic acid fragmentation in granulosa cells. Endocrinology 134:245–252
9. Wyllie AH, Kerr JF, Currie AR 1980 Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306
10. Quirk SM, Cowan RG, Joshi SG, Henrikson KP 1995 Fas
antigen-mediated apoptosis in human granulosa/luteal
cells. Biol Reprod 52:279–287
11. Rodger FE, Fraser HM, Krajewski S, Illingworth PJ 1998
Production of the proto-oncogene BAX does not vary
with changing in luteal function in women. Mol Hum
Reprod 4:27–32
12. Fukaya T, Funayama Y, Muakami T, Sugawara J, Yajima
A 1997 Does apoptosis contribute follicular atresia and
luteal regression in human ovary? Horm Res 48[Suppl
3]:20–26
13. Funayama Y, Sasano H, Suzuki T, Tamura M, Fukaya T,
Yajima A 1996 Cell turnover in normal cycling human
ovary. J Clin Endocrinol Metab 81:828–834
MOL ENDO · 2000
1074
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
contains a population of catecholaminergic neuron-like
cells expressing nerve growth factor receptors. Endocrinology 136:5760–5768
Mayerhofer A, Smith GD, Danilchik M, Levine JE, Wolf
DP, Dissen GA, Ojeda SR 1998 Oocytes are a source of
catecholamines in the primate ovary: evidence for a
cell-cell regulatory loop. Proc Natl Acad Sci USA 95:
10990–10995
Bukovsky A, Caudle MR, Keenan JA, Wimalasena J,
Upadhyaya NB, Van Meter SE 1995 Is corpus luteum
regression an immune-mediated event? Localization of
immune system components and luteinizing hormone
receptor in human corpora lutea. Biol Reprod 53:
1373–1384
Sontheimer H, Black JA, Waxman SG 1996 Voltagegated Na⫹ channels in glia: properties and possible functions. Trends Neurosci 19:325–331
Mayerhofer A, Sterzik K, Link H, Wiemann M, Gratzl M
1993 Effect of oxytocin on free intracellular Ca2⫹ levels
and progesterone release by human granulosa-lutein
cells. J Clin Endocrinol Metab 77:1209–1214
Schiffmann SN, Desdouits F, Menu R, Greengard P, Vincent JD, Vanderhaeghen JJ, Girault JA 1998 Modulation
of the voltage-gated sodium current in rat striatal neurons by DARPP-32, an inhibitor of protein phosphatase.
Eur J Neurosci 10:1312–1320
Catterall WA 1992 Cellular and molecular biology of voltage-gated sodium channels. Physiol Rev 72:S15–S48
Catterall WA 1995 Structure and function of voltagegated ion channels. Annu Rev Biochem 64:493–531
Mayerhofer A, Garfield RE 1995 Immunocytochemical
analysis of the expression of gap junction protein connexin 43 in the rat ovary. Mol Reprod Dev 41:331–338
Taylor CP, Meldrum BS 1995 Na⫹ channels as targets
for neuroprotective drugs. Trends Pharmacol Sci 16:
309–316
Bortner CD, Hughes FMJ, Cidlowski JA 1997 A primary
role for K⫹ and Na⫹ efflux in the activation of apoptosis.
J Biol Chem 272:32436–32442
Sontheimer H, Fernandez-Marques E, Ullrich N, Pappas
CA, Waxman SG 1994 Astrocyte Na⫹ channels are required for maintenance of Na⫹/K⫹-ATPase activity.
J Neurosci 14:2464–2475
Murialdo G, Galimberti CA, Magri F, Sampaolo P,
Copello F, Gianelli MV, Gazzerro E, Rollero A, Deagatone
Vol 14 No. 7
44.
45.
46.
47.
48.
49.
50.
51.
52.
C, Manni R, Ferrari E, Polleri A, Tartara A 1997 Menstrual
cycle and ovary alterations in women with epilepsy on
antiepileptic therapy. J Endocrinol Invest 20:519–526
Mayerhofer A, Hemmings HCJ, Snyder GL, Greengard P,
Boddien S, Berg U, Brucker C 1999 Functional dopamine-1 receptors and DARPP-32 are expressed in human ovary and granulosa luteal cells in vitro. J Clin Endocrinol Metab 84:257–264
Mayerhofer A, Engling R, Stecher B, Ecker A, Sterzik K,
Gratzl M 1995 Relaxin triggers calcium transients in human granulosa-lutein cells. Eur J Endocrinol 132:
507–513
Haasemann M, Cartaud J, Muller-Esterl W, Dunia I 1998
Agonist-induced redistribution of bradykinin B2 receptor
in caveolae. J Cell Sci 111:917–928
Vandevoort CA, Molskness TA, Stouffer RL 1988 Adenylate cyclase in the primate corpus luteum during
chorionic gonadotropin treatment simulating early
pregnancy: homologous versus heterologous desensitization. Endocrinology 122:734–740
Stouffer RL, Dahl KD, Hess DL, Woodruff TK, Mather JP,
Molskness TA 1994 Systemic and intraluteal infusion of
inhibin A or activin A in rhesus monkeys during the luteal
phase of the menstrual cycle. Biol Reprod 50:888–895
Mayerhofer A, Dissen GA, Costa ME, Ojeda SR 1997 A
role for neurotransmitters in early follicular development:
induction of functional follicle-stimulating hormone receptors in newly formed follicles of the rat ovary. Endocrinology 138:3320–3329
Mayerhofer A, Dissen GA, Parrott JA, Hill DF, Mayerhofer
D, Garfield RE, Costa ME, Skinner MK, Ojeda SR 1996
Involvement of nerve growth factor in the ovulatory
cascade: trkA receptor activation inhibits gap junctional
communication between thecal cells. Endocrinology
137:5662–5670
Höhne-Zell B, Gratzl M 1996 Adrenal chromaffin cells
contain functionally different SNAP-25 monomers
and SNAP-25/syntaxin heterodimers. FEBS Lett 394:
109–116
Mayerhofer A, Weis J, Bartke A, Yun JS, Wagner TE 1990
Effects of transgenes for human and bovine growth hormones on age-related changes in ovarian morphology in
mice. Anat Rec 227:175–186