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Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health Sciences Center, and National Science Foundation Center for Biology Timing, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Dr. Jorge A. Flores, Biology Department, West Virginia University, Brooks Hall, P.O. Box 6057, Morgantown, West Virginia 26506-6057.
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Abstract |
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In summary, the present single cell investigations in mature granulosa cells demonstrate that LH drives initial intracellular Ca2+ mobilization followed by transmembrane divalent cation influx. The PLC inhibitor U-73122 antagonizes this action of LH. By analyzing [Ca2]i responses in individual living granulosa cells, we further show that, despite within-follicle diversity, the LH dose biphasic [Ca2+]i response arises via the recruitment of a larger number of responding gonadal cells rather than by increased [Ca2+]i signal amplitude. Finally, the percentage of individual LH (but not endothelin-1)-responding granulosa cells increases with follicular maturation. Collectively, these data highlight the potential importance of the LH-stimulatable, PLC-transduced [Ca2+]i signaling mechanism in the later stages of granulosa cell differentiation.
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Introduction |
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Here we implement monitoring of cytoplasmic concentration of free calcium ions ([Ca2+]i) with high temporal resolution by semiquantitative fluorescence video microscopy in single (swine) granulosa cells to examine the mechanisms of LH’s stimulation of [Ca2+]i responses within individual homologous target (granulosa) cells expressing native LH receptors. We show that LH’s activation of the [Ca2+]i signaling pathway is marked by significant cell to cell heterogeneity. Mechanistic investigations using the manganese quench technique further reveal that the LH-stimulated biphasic [Ca2+]i signal arises by way of both intracellular Ca2+ mobilization and transmembrane cation uptake. Moreover, we observe that the mechanism subserving the LH dose response of [Ca2+]i in single ovarian cells embodies progressive recruitment of responding granulosa cells at higher LH concentrations. The percentage of responding cells is also higher for granulosa cells harvested from more mature follicles. Lastly, we show that a specific PLC inhibitor antagonizes LH-driven [Ca2+]i signal generation in individual granulosa cells, thus supporting the relevance of activation of PLC by LH in [Ca2+]i signaling in mature granulosa cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effects of LH on
[Ca2+]i
Calcium measurements. Granulosa cells were collected as
described previously (12) from immature (<70 kg) swine ovaries into
HEPES-buffered MEM from individually dissected follicles measuring 1.0
or 5.0 mm in diameter. Cells were counted in a hemocytometer, and cell
density was adjusted to 1 x 105 cells/ml by adding
bicarbonate-buffered MEM supplemented with 0.1% FCS. This initial
concentration of FCS in the MEM allowed granulosa cell attachment to
the microscope slides. A 60-µl aliquot of the granulosa cell
suspension was applied to a poly-L-lysine-coated microscope
slide with a Cunningham chamber (13, 14). The Cunningham chambers were
maintained overnight in a humidified incubator (37 C, 95% air-5%
CO2).
The following day the cells were used for calcium measurements. The medium in these experiments contained 127 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 2 mM MgCl2, 5 mM KHPO4, 5 mM NaHCO3, 10 mM HEPES, 10 mM glucose, and 0.1% BSA, pH 7.4. Manganese-containing medium was prepared by replacing CaCl2 with MnCl2 (1.8 mM). Granulosa cells were loaded with 2 µM fura-2/AM in experimental medium (without hormones) for 20 min at 37 C. The cells were then washed with experimental medium and incubated for an additional 20 min at 37 C to allow cytoplasmic deesterification of the fura-2/AM dye.
After dye loading, the Cunningham chamber was placed on the stage of a Zeiss Axioplan microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with epifluorescence illumination. All experiments were performed at room temperature (22–23 C). The excitation light was supplied by a high pressure xenon arc UV lamp, and the excitation wavelengths were selected by 360- and 380-nm filters (2 nm half-bandwidth; Corion, Hollinston, MA) mounted in a rotating filter wheel (MAC 2000, Ludl Electronic Products, Hawthorne, NY) between the UV lamp and the microscope. Fluorescence emission was collected via the objective (UV-F, Nikon, Melville, NY; x20) and passed through a barrier filter (490–600 nm transmission) to the face of a silicon-intensified target camera (series 68, DAGE-MTI, Michigan City, IN). The resultant video signal was stored on broadcast quality tape at 33 frames/sec (U-matic Vo-5600, Sony Corp., Tokyo, Japan).
LH-stimulated [Ca2+]i responses. The ability of different LH concentrations (1.0, 10, 100, 1,000, and 10,000 pM) to stimulate a rise in [Ca2+]i in individual granulosa cells was tested as previously described (12). The granulosa cells were collected from individual, well vascularized follicles at least 5.0 mm in diameter, except where indicated otherwise. Each LH concentration was evaluated in three slides per experimental day. In other experiments, the same cell was exposed to progressively increasing concentrations of LH or repetitively to the same concentration of LH. At least six independent experiments were performed to address each question using granulosa cells from a different follicle collected on a different day.
To test whether LH’s ability to stimulate a transient rise in [Ca2+]i was a function of follicular development, the responsiveness to a 10-nM LH challenge was investigated in individual granulosa cells collected from different sized follicles (1 vs. 5 mm diameter). Each cell was also exposed to 10 nM endothelin-1 (ET-1). ET-1 served as a positive control, which has been previously shown to stimulate a transient rise in [Ca2+]i in swine granulosa cells (12). The ET-1 challenge was performed before or after the LH challenge. Results were independent of order. This protocol was performed in six independent experiments.
As activation of PLC-linked receptors can mobilize both intracellular and external sources of Ca2+ (15, 16, 17, 18, 19, 20, 21), we investigated the Ca2+ source(s) for the LH-stimulated biphasic rise in [Ca2+]i by removal of extracellular Ca2+ before or immediately after a 10-nM LH challenge. However, the relative contribution and timing of intracellular Ca2+ discharge and transmembrane Ca2+ entry cannot be distinguished with these protocols (20). Thus, we used the Mn2+ quench technique of Merritt et al. to identify transmembrane cation uptake (22). In various nonexcitable cells, extracellular Mn2+ can serve as a surrogate cation for Ca2+ entry (22). When Mn2+ enters the cell, it binds to fura-2 and quenches the fluorescence signal (20, 22). Agonist-stimulated fluorescence signal quenching of fura-2-loaded cells in the presence of extracellular Mn2+ is thus evidence of agonist-stimulated influx of extracellular divalent cations. Fura-2 fluorescence intensity was measured at two excitation wavelengths, 360 nm (Ca2+ independent) and 380 nm (Ca2+ dependent), to allow sequential monitoring of changes in [Ca2+]i (380 nm) and Mn2+ influx (360 nm). Approximately 15–25 sec after initiation of a recording, experimental Ca2+-containing medium was replaced with Mn2+-containing medium to record the basal rate of Mn2+ influx. Subsequently, LH-stimulated Ca2+ mobilization and Mn2+ influx were studied by delivering 10 nM LH prepared in Mn2+ medium. The specificity of LH-induced Mn2+ influx was assessed by testing the ability of a second delivery of Mn2+-containing but LH-deficient medium to affect the basal rate of Mn2+ influx. At least 20 cells in three independent experiments were studied with this protocol.
To directly test whether the ability of LH to stimulate a transient rise in [Ca2+]i was mediated by PLC, the responsiveness of [Ca2+]i to a 200-nM LH challenge was investigated in individual granulosa cells pretreated with U-73122, a PLC inhibitor, or its inactive congener, U-73343. In this protocol, we also tested the responsiveness to a 10-µM ET-1 challenge (12). Cells were pretreated with 10 µM of the PLC inhibitor U-73122, its inactive analog U-73343 (23), or vehicle [0.44% (vol/vol) DMSO] for 2 min. Subsequently, the cells were exposed to 200 nM LH or 10 µM ET-1 in the continuing presence of the respective pretreatment compound for an additional 2 min. At the end of each treatment the calcium ionophore, ionomycin (10 µM), was delivered to verify cellular [Ca2+]i responsiveness. Two or three slides of granulosa cells, allowed to anchored to slides as described above and in the presence of estradiol (0.5 µg/ml) for 48 h, were used per treatment in each of 4 independent experiments with separate batches of ovaries. A total of 210 and 221 cells were studied using LH or ET-1, respectively. Cells were classified as responding with an increase in [Ca2+]i if the Fo (initial fluorescence emission intensity at each wavelength/Fi (fluorescence at time i) ratio was greater than baseline + 3SD. The percentage of cells responding and the times required to achieve the half-maximal [Ca2+]i value (t1/2) were calculated for each treatment. These data are presented as the mean ± SEM For statistical analysis, data were transformed to natural logarithm values and subjected to ANOVA followed by the Tukey highest significant difference multiple comparison test.
Analysis. The video-recorded fluorescence intensity signal was captured and digitized using software (RADTIME) run on a QX-7 image analysis system (Quantex Corp., Sunnyvale, CA), which creates an ASCII file of the mean radiance values observed every 30 msec within the cell of interest. The ASCII file is exported to a spreadsheet program (Lotus 1–2-3, version 2.0, Lotus Development Corp.), where the 380 and 360 nm fluorescence values are converted to ratio values using the equation R = Fo/Fi. The data in graphic form represent the plot of the converted fluorescence ratio (y-axis) over time (x-axis).
Effect of LH on inositol phosphate accumulation
Swine ovaries were obtained from a local abattoir, and granulosa
cells were harvested and pooled from 3- to 5-mm (diameter) Graafian
follicles by fine needle aspiration. To allow cell anchorage, monolayer
cultures were initially established in medium 199 (which contains 0.05
mg/liter inositol) supplemented with 3% FCS, ovine FSH (100 ng/ml),
and porcine insulin (3 µg/ml) at a plating density of 1 x
107 viable cells/35-mm tissue culture dish. These culture
conditions promote LH responsiveness as defined by LH stimulation of
progesterone biosynthesis and cAMP accumulation. After 48 h, the
incubation medium was replaced by serum-free medium 199
supplemented with 0.1% BSA. After 24 h of serum-free culture,
granulosa cells were equilibrium labeled with
[3H]myo-inositol L-myo-[1,2-3H(N)]inositol
by exposing them to medium 199 containing 0.1% BSA and 2.5 µCi/ml
[3H]myo-inositol (60 Ci/mM) for 18 h.
Thereafter, monolayers were washed twice in medium 199 with 0.1% BSA
and incubated for 1 h in the presence of 10 mM
unlabeled inositol. During the last 15 min of this "cold chase"
period, 30 µl of a 0.5-M stock LiCl solution was added to
each dish (final concentration, 10 mM). At designated
intervals, medium alone (control) or LH (final concentration, 10
nM) was added in 80-µl aliquots. The reaction was stopped
by addition of ice-cold chloroform-methanol (1:2, vol/vol). Cells were
immediately scraped from the culture dishes maintained on ice, and
centrifuged in biovials. [3H]Inositol phosphates were
separated by anion exchange chromatography, as described previously
(23A ). [3H]Inositol mono-, bis-, and triphosphate
([3H]IP1, [3H]IP2,
and [3H]IP3) were sequentially eluted into
scintillation vials with 6 ml 0.2, 0.4, and 0.8 M ammonium
formate containing 0.1 M formic acid, respectively. Ten
milliliters of scintillation solution were added to each vial, and the
radioactivity was determined in a liquid scintillation counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). There was a
consistent enhancement in the number (percentage) of responding cells
in the presence of increasing LH concentrations. For example, when 10
granulosa cells in a given microscopic field were challenged with 1
pM LH, no responders were observed. When the same cells
were exposed to higher LH concentrations (10, 100, and 1000
pM, we observed 2, 3, and up to 5 cells that responded with
a spike and plateau rise in [Ca2+]i. Similar
observations were made when the order of LH doses was reassigned in 6
different experiments using granulosa cells collected from different
follicles (44 cells total). Augmentation of the number of LH-responsive
cells at higher LH concentrations was not observed if the cells were
challenged serially with the same (1.0 nM) LH concentration
(40 cells total). Figure 1 also shows the
[Ca2+]i responses elicited in single
granulosa cells exposed to LH concentrations greater than 100
pM. This stimulus evoked the rapid (within 10–30 sec)
onset of a single spike-like rise in [Ca2+]i,
which was followed by a more sustained plateau-like elevation of
[Ca2+]i.
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). In contrast, only 4 ± 1.5%
cells from 1.0-mm follicles responded similarly (6 experiments, total
of 175 cells). The percentages of granulosa cells that responded to
ET-1 in 5.0- and 1.0-mm follicles were not different (33 ± 8.5%
and 32 ± 11%, respectively; 6 experiments, 95 cells). In
addition, LH and ET-1 delivered sequentially 10 min apart in either
order increased [Ca2+]i in the same
individual granulosa cell that was ET-1 responsive (8 experiments, 30
cells).
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, upper panel). However, the apparent plateau increase in
[Ca2+]i after the
[Ca2+]i spike was inhibited or abolished by
removal of extracellular Ca2+ (Fig. 3). In the presence of
extracellular Ca2+, the peak of the
[Ca2+]i rise averaged a 2.9 ± 0.6-fold
increase over basal (assuming basal = 1.0), and then maintained a
value that was 1.6 ± 0.1-fold basal for 2.5 min (n = 40
cells in 6 independent experiments). In the absence of extracellular
Ca2+, only the fold increase in the plateau-like
[Ca2+]i increase sustained after the spike
declined significantly (1.2 ± 0.1; P < 0.05, by
Student’s two-tailed unpaired t test; n = 26 cells in
4 independent experiments). Similar reductions in the plateau
[Ca2+]i level were observed when
extracellular Ca2+ was removed during the postspike
interval (n = 24 cells in 4 independent experiments).
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). In all 20 cells, LH-stimulated
Mn2+-induced quenching of fura-2 fluorescence was always
preceded by the spike-like increase in intracellular (mobilized)
Ca2+. There was a subpopulation of granulosa cells (20
cells) in which no increased rate of Mn2+ quench was
detected within 2.5 min after the spike of mobilized calcium induced by
the 10-nM LH challenge (not shown).
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).
Similarly, we found that 91% of granulosa cells responded with a rapid
biphasic increase in [Ca2+]i to a
10-µM ET-1 challenge. Pretreatment of granulosa cells
with 10 µM U-73343 (inactive congener) or vehicle did not
significantly affect the percentage of cells responding to either LH or
ET-1. On the other hand, the number of responding cells was
significantly reduced to 25% in the presence of 10 µM
U-73122 (P < 0.001) in LH-stimulated cells and to 36%
(P < 0.001) in ET-1 stimulated cells. Furthermore,
granulosa cells responding to LH or ET-1 in the U-73122 pretreatment
group took a significantly longer time to achieve half-maximal
[Ca2+]i value (t1/2). LH- plus
U-73343-treated cells exhibited a t1/2 of 21.7 ± 1.5
sec (n = 27), and LH plus U-73122-treated cell a t1/2
of 39.1 ± 7.2 sec (n = 12; P = 0.002; see
Fig. 5). Similarly, in ET-1-stimulated cells, the t1/2 of
13.9 ± 2.6 sec (n = 34) for U-73343 cotreatment
vs. the t1/2 of 43.6 ± 14 sec (n = 7;
P < 0.002) for U-73122 cotreatment indicated a greater
latency to half-maximal [Ca2+]i increases
during PLC inhibitor treatment.
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). Half-maximal stimulation of inositol
phosphate production was observed at a LH concentration of 1.0
nM. A maximally stimulating concentration of LH (10
nM) induced a rapid increase in the accumulation of
inositol phosphates recognized within 30 sec. In contrast, inositol
phosphate accumulation in the control (medium-treated) cells remained
unchanged throughout the experiment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By single cell fluorescence video microscopy, we could demonstrate granulosa cell to cell heterogeneity in the actions of a given dose of LH in inducing a rapid onset biphasic rise in [Ca2+]i. Of physiological interest, the fraction of individual granulosa cells responding with a spike and plateau [Ca2+]i signal was significantly higher when granulosa cells were collected from more developmentally mature follicles. This finding is in agreement with the reported developmental expression of the LH receptor in populations of more highly differentiated granulosa cells (24), with recent studies using staged avian Graafian follicles (25), and with greater LH receptor messenger RNA expression in large preovulatory follicles (26). Moreover, transfection experiments have demonstrated that LH receptor signaling via PLC is dependent on receptor density (27). Of interest, we did not observe heightened ligand-induced [Ca2+]i signaling with greater follicular maturation for the alternative PLC agonist, ET-1. As ET-1-mediated [Ca2+]i signaling was preserved in the same individual granulosa cell that was unresponsive to LH, we infer that single cell unresponsiveness to LH is not due to cellular phosphatidylinositol substrate or PLC enzymic deficiency, endoplasmic IP3 receptor insensitivity, or depletion of intracellular Ca2+ stores. Accordingly, other factors, such as variations in the cellular expression or activation of functional G protein-coupled LH receptors, may offer a basis for granulosa cell to cell heterogeneity in [Ca2+]i signaling.
Using a single cell experimental strategy, we could investigate the relationship between the LH concentration and the [Ca2+]i signaling response in the individual target (ovarian) cell bearing native LH receptors. We discovered that lower concentrations of LH elicit [Ca2+]i signals of typically lower amplitude and delayed onset in granulosa cells, which may reflect an apparently incremental (quantal) nature of IP3-mediated intracellular Ca2+ mobilization via activation of IP3-sensitive Ca2+ channels. For example, submaximal IP3 concentrations generated by low LH concentrations may only activate release of Ca2+ from localized intracellular stores; on the other hand, stimulation of granulosa cells with higher LH concentrations may initiate more generalized propagation of Ca2+ release, as has been inferred for intact and permeabilized cells (28). Indeed, Ca2+ release by hormonal stimulation or IP3 delivery to permeabilized cells or membrane-reconstituted IP3 receptors appears to be a quantal rather than a continuous process (28, 29).
In addition to the qualitative differences in the time course of [Ca2+]i responses to varying concentrations of LH, our single granulosa cell studies reveal that the most significant mechanism by which higher concentrations of LH elicit a greater overall population response is by cell recruitment. In particular, granulosa cells within any given follicle were heterogeneous with respect to the threshold concentration of LH required to elicit a spike- and plateau-like [Ca2+]i signal. Thus, higher LH concentrations progressively exceed the threshold requirements of a larger fraction of the ovarian (granulosa) cell population. Accordingly, the number of granulosa cells responding, rather than the magnitude of the individual cell’s response, primarily governs the population [Ca2+]i response to any given LH concentration. A similar cellular dose-response mechanism has been recognized recently for the stimulation by FSH and ET-1 of [Ca2+]i rises in (rat) Sertoli cells (30, 31), but not, to our knowledge, previously for LH actions.
The present study demonstrates that the LH-stimulated biphasic
[Ca2+]i signal in granulosa cells involves
both intracellular Ca2+ mobilization and transmembrane
cation influx. This inference is consistent with the idea that the
native LH receptor of differentiated granulosa cells is coupled to PLC,
as are other receptors that typically evoke these
[Ca2+]i dynamics (32, 33, 34). We used
extracellular Ca2+ depletion and Mn2+ quench
experiments to show that LH-stimulated Ca2+ influx across
the plasma membrane occurs only after the LH-stimulated mobilization of
intracellular Ca2+ stores. Initial agonist-mediated
[Ca2+]i mobilization followed by
Mn2+ entry has been observed in other cells with
PLC-mediated second messenger signaling, e.g. human
umbilical vein endothelial cells (33), and is compatible with the
so-called capacitance model of Ca2+ influx. This model
asserts that the IP3-driven initial discharge of an
internal Ca2+ store establishes the basis for secondary
(delayed) Ca2+ entry (34). Interestingly, some granulosa
cells (viz. 
50%) exhibited an apparently typical rapid
initial spike pattern of intracellular Ca2+ mobilization
without evident Mn2+ influx. This suggests that at the
single cell level, Ca2+ release from IP3 stores
is not always coupled to full activation of capacitative
Ca2+ influx and/or that effective
[Ca2+]i depletion is not achieved uniformly
by a single IP3-stimulated
[Ca2+]i spike-like response. Additional
biochemical requirements may exist in granulosa and other cells before
the IP3 signal and/or other mediators can bring about
delayed Ca2+ influx. This disparity has also been reported
in rat basophilic leukemia cells, where sustained Ca2+
release evoked by IP3 failed to activate capacitative
Ca2+ entry (35). Also, in some nonexcitable cells,
depletion of the IP3-sensitive Ca2+ stores does
not result in capacitative Ca2+ influx (36, 37).
In summary, the present investigations of the mechanisms of actions of LH in motivating [Ca2+]i signaling in single untransformed granulosa cells support the idea that native LH receptors are coupled to PLC. Based on single cell analysis, we infer further that LH stimulates a biphasic rise in [Ca2+]i by way of immediate intracellular Ca2+ mobilization and delayed transmembrane [Ca2+]i influx. Stimulation of individual granulosa cells reveals significant cell to cell heterogeneity in [Ca2+]i responses to fixed or increasing concentrations of LH. Moreover, higher concentrations of LH elicit a greater population [Ca2+]i response in granulosa cells by recruiting a larger number of responding target cells without markedly altering the intensity of the second messenger Ca2+ signal in any given granulosa cell.
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Acknowledgments |
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Footnotes |
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2 Present address: Organon, Inc., West Orange, New Jersey 07052.
Received January 28, 1998.
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References |
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initiates polyphosphatidylinositol hydrolysis and membrane
translocation of protein kinase C in swine ovarian cells. Biochem
Biophys Res Commun 149:112–117[CrossRef][Medline]
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