Identification of an Ovarian Voltage-Activated Na+-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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
An endocrine type of voltage-activated sodium channel (eNaCh) was identified in the human ovary and human luteinized granulosa cells (GC). Whole-cell 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 Na+ 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 Na+ 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 cellular 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). After sequencing of the PCR products, we found that they corresponded to the {alpha}-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. 2Go), 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).



View larger version (22K):
[in this window]
[in a new window]
 
Figure 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).

 


View larger version (69K):
[in this window]
[in a new window]
 
Figure 2. Detection of eNaCh mRNA in Untreated and hCG-Treated 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 ß-actin (1.3 kb), which was used for normalization of the results.

 
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. 3AGo) as described previously (24 ). With the solution containing CsCl/ tetraethylammoniumchloride (TEA)-Cl, outward currents were blocked (Fig. 3BGo), indicating the presence of voltage-activated K+ channels. The remaining transient inward currents were sensitive to tetrodotoxin (TTX, Fig. 4AGo), 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. 4BGo). 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.



View larger version (28K):
[in this window]
[in a new window]
 
Figure 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.

 


View larger version (17K):
[in this window]
[in a new window]
 
Figure 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).

 
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. 5Go) 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%.



View larger version (22K):
[in this window]
[in a new window]
 
Figure 5. Example of Na+ Current Activation (•) and Steady-State Inactivation ({circ}) 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.

 
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 24–48 h, eNaCh mRNA levels were suppressed (Fig. 2Go). 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. 6Go). Moreover, after this period the specific Na+ currents were significantly reduced (Fig. 6Go; 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. 7Go).



View larger version (17K):
[in this window]
[in a new window]
 
Figure 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.

 


View larger version (22K):
[in this window]
[in a new window]
 
Figure 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.

 
Consequences of eNaCh Activation and Blockage in GC: Progesterone Production and Lysosomes
Activation of eNaCh by 50 µM veratridine for 24–48 h decreased media progesterone levels by half (P < 0.05), whereas TTX showed no statistically significant effect (Fig. 8Go). Blockage of eNaCh by TTX for 24–48 h preserved a highly differentiated cellular phenotype (Fig. 9Go). 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. 10Go). 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. 9FGo). None of the described changes were seen when human carcinoma cells (A431) lacking NaCh were incubated with veratridine or TTX (not shown).



View larger version (33K):
[in this window]
[in a new window]
 
Figure 8. Consequences of eNaCh Activation and Blockage on Progesterone Production

Incubation of GC with veratridine (50 µM) 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 µM) showed no statistically significant effect. Bars represent means ± SEM.

 


View larger version (110K):
[in this window]
[in a new window]
 
Figure 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 µm. B, Ultrastructure of a GC cultured for 24 h in basal medium and then treated with TTX (5 µM) for 24 h: note the abundantly present mitochondria (double arrow), few lysosomes (arrows) and lipid droplets (asterisks). Bar, 2 µm. C, Veratridine (VERA, 50 µM) 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 µm. D and E, Examples of the morphology of the secondary lysosomes found in veratridine-treated GC. Bars, 0.8 µm. F, TTX treatment blocked the effects of veratridine. Note presence of abundant mitochondria and absence of lysosomes. Bar, 1.5 µm.

 


View larger version (30K):
[in this window]
[in a new window]
 
Figure 10. Increased Lysosomes in GC after Veratridine Treatment (VERA, 50 µM) 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 µm.

 
Expression of eNaCh in the Primate CL
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. 11Go). Human and monkey eNaCh sequences did not differ. The monkey sequence was submitted to Genbank (accession no. AF164965).



View larger version (38K):
[in this window]
[in a new window]
 
Figure 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).

 
Electron Microscopy of Luteal Cells
Electron microscopic examination of the CL from nonhuman primates revealed that on days 7 and 11, i.e. when the CL is functional, luteal cells contain abundant primary lysosomes (Fig. 12Go), 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.



View larger version (102K):
[in this window]
[in a new window]
 
Figure 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 µm. B, A luteal cell from day 11 of the luteal phase showed primary and secondary lysosomes (arrows). Bar, 1 µm. C, Secondary lysosomes were present in a regressing luteal cell CL (day 18 of the luteal phase). Bar, 1.3 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study we report that the gene for a member of the voltage-activated, TTX-sensitive family 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 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 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. 8Go). 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 ).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 µM), TTX (5 and 50 µM), or human CG (hCG; 10 IU/µl). 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 ).

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{Omega}. Seal resistances typically ranged from 2 to 5 G{Omega} 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) {alpha}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. 8Go; paired t test) and change of V50 with time (F test against slope value of zero).


    ACKNOWLEDGMENTS
 
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.


    FOOTNOTES
 
Address requests for reprints to: Dr. Artur Mayerhofer, Anatomisches Institut, Technische Universität München, Biedersteiner Strasse 29, D-80802 München, Germany.

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 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. Lippincott-Raven, 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[Abstract/Free Full Text]
  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[Abstract/Free Full Text]
  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[Abstract/Free Full Text]
  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[Abstract/Free Full Text]
  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[Abstract]
  9. Wyllie AH, Kerr JF, Currie AR 1980 Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306[Medline]
  10. Quirk SM, Cowan RG, Joshi SG, Henrikson KP 1995 Fas antigen-mediated apoptosis in human granulosa/luteal cells. Biol Reprod 52:279–287[Abstract]
  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[Abstract]
  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[Abstract]
  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[Abstract/Free Full Text]
  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[Abstract/Free Full Text]
  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[Abstract]
  17. Rodger FE, Fraser HM, Duncan WC, Illingworth PJ 1995 Immunolocalization of bcl-2 in the human corpus luteum. Hum Reprod 10:1566–1570[Abstract]
  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[Abstract]
  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[Abstract]
  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[Abstract/Free Full Text]
  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[Abstract]
  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[Medline]
  23. Takenaka A 1981 An ultrastructural and cytochemical study of human corpora lutea. Acta Obstet Gynaecol Jpn 33:907–915[Medline]
  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[Free Full Text]
  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[Abstract]
  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 voltage-dependent persistent sodium current in mammalian hippocampal neurons. J Gen Physiol 95:1139–1157[Abstract]
  28. Crill WE 1996 Persistent sodium current in mammalian central neurons. Annu Rev Physiol 58:349–362[CrossRef][Medline]
  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[Abstract/Free Full Text]
  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[Abstract/Free Full Text]
  31. Dees WL, Hiney JK, Schultea TD, Mayerhofer A, Danilchik M, Dissen GA, Ojeda SR 1995 The primate ovary contains a population of catecholaminergic neuron-like cells expressing nerve growth factor receptors. Endocrinology 136:5760–5768[Abstract]
  32. 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[Abstract/Free Full Text]
  33. 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[Abstract]
  34. Sontheimer H, Black JA, Waxman SG 1996 Voltage-gated Na+ channels in glia: properties and possible functions. Trends Neurosci 19:325–331[CrossRef][Medline]
  35. 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[Abstract]
  36. 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[CrossRef][Medline]
  37. Catterall WA 1992 Cellular and molecular biology of voltage-gated sodium channels. Physiol Rev 72:S15–S48
  38. Catterall WA 1995 Structure and function of voltage-gated ion channels. Annu Rev Biochem 64:493–531[CrossRef][Medline]
  39. 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[CrossRef][Medline]
  40. Taylor CP, Meldrum BS 1995 Na+ channels as targets for neuroprotective drugs. Trends Pharmacol Sci 16:309–316[CrossRef][Medline]
  41. 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[Abstract/Free Full Text]
  42. 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[Abstract]
  43. Murialdo G, Galimberti CA, Magri F, Sampaolo P, Copello F, Gianelli MV, Gazzerro E, Rollero A, Deagatone 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[Medline]
  44. 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[Abstract/Free Full Text]
  45. 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[Medline]
  46. 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[Abstract/Free Full Text]
  47. 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[Abstract]
  48. 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[Abstract]
  49. 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[Abstract/Free Full Text]
  50. 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[Abstract]
  51. 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[CrossRef][Medline]
  52. 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[Medline]