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Department of Human Physiology (J.L.T., S.M.V., D.W.W.), School of Medicine, University of California, Davis, California 95616; and Division of Life Sciences (G.S.), Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720
Address all correspondence and requests for reprints to: Dr. Judith L. Turgeon, Department of Human Physiology, School of Medicine, University of California, Davis, California 95616. E-mail: jlturgeon{at}ucdavis.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogen consistently has been observed to increase the number of PRs in female pituitary tissue from rats and primates (5, 7, 8, 9, 10, 11, 12) and in cultured female rat pituitary cells (13), based on analysis by steroid binding assays; in addition, estrogen has been reported to increase pituitary PR, as determined by immunoblot analysis (14). There is much less known about pituitary PR messenger RNA (mRNA) and its regulation by estrogen; but importantly, Bethea et al. (15) report that estrogen treatment increases PR mRNA in monkey anterior pituitary, as assessed by in situ hybridization. However, at present, there is little known regarding the regulation of rat pituitary PR at the level of mRNA. Multiple PR transcripts have been detected in nonpituitary tissue for several species, including the rat (16, 17, 18, 19, 20, 21, 22), but information is lacking on the multiple PR transcripts and their modulation by estrogen in the pituitary of any species.
The action of progesterone on PR regulation has been found to be tissue- and even cell type-specific within the female reproductive tract, mammary gland, and brain (reviewed in Ref. 23). We have been examining the role of progesterone and the PR in the regulation of LH secretion using an in vitro model of the rat preovulatory gonadotrope (24, 25, 26, 27). During the preovulatory surge, the action of progesterone on gonadotropin secretion is biphasic, moving from a severalfold augmentation of the release action of GnRH to a suppression of the efficacy of GnRH, which occurs in rats over a period of about 12 h, based on in vivo and in vitro studies (1, 24, 28, 29, 30, 31). The status of the PR during this period, particularly with regard to dynamic changes in expression, is largely unknown. The aims of the present study were to use a cultured female pituitary cell model to define the colocalization of PR with gonadotropes and to determine the effect of 17ß-estradiol (E2) and progesterone on the PR mRNA levels and PR protein within the temporal context of the rat preovulatory period. Part of this study has been presented in preliminary form (32).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pituitary cell culture
Rats were ovariectomized and maintained for 2 weeks, after which
pituitaries were removed after CO2 narcosis and
decapitation. Anterior pituitary cells, obtained by trypsin dispersion
(day 0), were cultured in Eagle’s MEM containing d-valine,
0.2 mM kanamycin sulfate, 10% charcoal-treated FBS
(FBS-CT), plus or minus 0.2 nM E2 in a
humidified atmosphere of 5% CO2 in air, as described (24).
Residual steroid concentrations in the FBS-CT were 10 pM
for progesterone and less than 1 pM for E2, as
determined by RIA. On day 2, cells were changed to fresh media.
Northern blot experiments
For PR mRNA studies, pituitary cells were plated at 3–4 x
106 in 60-mm dishes. On day 3, the cells were changed to
serum-free media containing 1 mg/ml BSA and plus or minus
E2, as appropriate. For some of the cells, the medium
included 200 nM progesterone for different periods (from
3–12 h). Termination was accomplished by rinsing the cells in cold PBS
and extracting total RNA using guanidine thiocyanate, by the method of
Chomczynski and Sacchi (33). For comparison, anterior pituitary and
uterus were removed from an adult rat after CO2 narcosis
and decapitation; the tissues were trimmed and immediately frozen in
liquid N2 and stored at -70 C until processed for total
RNA as above. For all groups, poly(A+) RNA was isolated
from total RNA using oligo(dt)-cellulose (New England Biolabs, Inc., Beverly, MA) minicolumns with a bed vol of 0.23 ml.
Northern blotting was performed as described by Sambrook et
al. (34). Briefly, RNA was separated on formaldehyde agarose gels
and transferred overnight onto Duralon-UV membrane and cross-linked
with a UV Stratalinker (Stratagene, La Jolla, CA). The
single-stranded [
-32P]deoxycytidine
triphosphate-labeled probe was generated by assymetric (single-primer)
PCR from an approximately 0.8-kb complementary DNA (cDNA) fragment from
the hormone binding domain of the mouse PR (20). Prehybridization and
hybridization were carried out in QuickHyb (Stratagene) at
68 C followed by washes in 2 x saline-sodium citrate (SSC)
(20 x SSC stock = 3 M NaCl, 0.3 M
sodium citrate, pH 7.0) with 0.1% SDS at room temperature and a wash
at 60 C in 0.2 x SSC/0.1% SDS. Message size was determined by
comparison with migration of an RNA ladder (0.24–9.5 kb; Gibco BRL, Grand Island, NY), run in an adjacent lane, transferred,
and stained with methylene blue. For normalization to RNA loading, the
membranes were stripped and rehybridized using a mouse actin probe
generated by random priming using a kit from Amersham Pharmacia Biotech (Piscataway, NJ). Blots were analyzed by phosphorimaging
(Molecular Imager Scanner, Bio-Rad Laboratories, Inc.,
Richmond, CA) and then subjected to autoradiography (X-OMAT,
Eastman Kodak Co., Rochester, NY).
Immunofluorescence experiments
For LH and PR dual immunocytochemistry experiments, cells were
plated at 6 x 104 on
poly-L-lysine-coated glass coverslips inserted into
22-mm wells. On day 3, the cells were changed to serum-free media
containing 1 mg/ml BSA and plus or minus E2, as
appropriate. For some of the cells, the medium included 20
nM progesterone for varying periods (from 3–12 h).
Termination was accomplished by rinsing the cells in PBS at room
temperature, followed by fixation with methanol:acetone (1:1) for 7 min
at -20 C; fixed cells were stored in PBS at 4 C until stained.
Immunofluorescence staining was carried out at room temperature by
successive incubation of the coverslips in the following solutions: 1)
5% normal goat serum (NGS; Vector Laboratories, Inc.,
Burlingame, CA), 1 h; 2) anti-LH (1:500) and anti-PR (1:500),
2 h; 3) PBS-0.1% BSA (PBSA) washes x 3; 4) 5% NGS, 30 min;
5) tetramethyl rhodamine isothiocyanate (TRITC) conjugated goat
antimouse IgG (1:100, Sigma Chemical Co.), 1 h; 6)
PBSA washes x 3; 7) fluorescein isothiocyanate (FITC)-labeled
goat antirabbit IgG (1:160; Sigma Chemical Co.), 1 h;
8) PBSA washes x 3; and 9) distilled water wash and mount using
Vectashield mounting medium containing the nuclear dye,
4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc.). As controls, either or both of the primary antibodies
were omitted for some coverslips. All antibodies were diluted in 5%
NGS. Mouse monoclonal antibody 518B7 to bovine LHß (35) was provided
by Jan Roser (University California, Davis). The polyclonal antibody
used for analysis of PR is against a synthetic peptide corresponding to
amino acids 376–394, selected from the amino-terminal half of the
mouse PR sequence (20), and reacts with both the A and B forms of
murine PR (36).
Immunofluorescence analysis
Immunofluorescence was viewed using either a 25x or 40x
objective on a Leitz Orthoplan microscope equipped with a
multiband pass filter photometer and a Leitz Vario
Orthomat2 camera (Leico Microsystems, Inc., Deerfield,
IL). Photographs of DAPI-, FITC-, and TRITC-labeled cells were taken
using Kodak Ektachrome ASA400 film (Eastman Kodak Co.,
Rochester, NY); the photographic slides were scanned at high resolution
to generate computer images [Photoshop (Adobe Systems, Inc., San Jose,
CA)]. The percent of LH- and PR-positive cells was determined by
counting DAPI-stained cells, FITC-labeled cells, and TRITC-labeled
cells in parallel projected photographic slides and in parallel
Photoshop images. From 1–3 fields were examined for each coverslip,
and each field was analyzed independently by two individuals.
Intensity analysis. The quantitation of PR staining intensity in the nucleus, as distinct from the cytoplasm, was determined on TIFF images exported from Photoshop; cells were analyzed using Scion Image (Scion Corp., Frederick, MD). The cohort of coverslips used for this analysis had been stained and photographed all within the same 3-day period. Photographs were obtained at the same magnification and light exposure for all fields; the exposure time was allowed to vary to adjust for time-dependent loss of fluorescence and photobleaching. Because one objective was a comparison of PR staining intensity in the two compartments across treatment groups, it was important to minimize, as much as possible, the interference caused by subtle variations in staining reagents, fluorescence stability, and photographic exposures that might occur over time. Because the nuclear-to-cytoplasmic ratio is calculated from data obtained from the same cell, it is independent of these potential variations.
Data analysis
Data are presented as mean ± SEM. For the
Northern blot experiments, n refers to the number of times an
experiment was repeated, and each experiment represents a separate pool
of dispersed pituitary cells. For the immunofluorescence experiments, n
refers to the number of single cells examined. Each treatment group
reflects coverslips from three or more separate pools of dispersed
pituitary cells; however, for the selected data used for intensity
analysis, the data were derived from 2–5 fields/treatment group. In
some cases, the t distribution was used to test the
hypothesis that the response was significantly different than 100%.
All other statistical analyses were done using SigmaStat (SPSS, Inc., Chicago, IL). For multiple comparisons, differences
between groups were determined by ANOVA and the Student-Neuman-Keuls
method; where differences are indicated as being significant,
P < 0.05. For the ratio data presented (see Table 2
),
the treatment groups had unequal variances; therefore, ANOVA on ranks
and Dunn’s test for multiple comparisons were used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). For comparison,
poly(A+) RNA samples prepared from anterior pituitary and
uterine tissue samples are included in Fig. 1. There are no apparent
differences in relative size of pituitary PR mRNA species from in
vitro, compared with in vivo, samples. Among the
multiple RNA species displayed in the uterine samples, the most
abundant transcripts were approximately 10.1 and 5.8 kb (Fig. 1). The
finding of multiple transcripts is in general agreement with reports
for tissues (including those from human, rat, mouse, rabbit, and
chicken) and cell types analyzed by Northern blot using cDNA-derived
probes, although the functional significance of multiple PR mRNA
transcripts has not been precisely determined (16, 17, 18, 19, 20, 21, 22). Whether
variations in the size and the number of reported transcripts is
related to differences in functional expression or to methodological
differences, e.g. in cDNA probes, has not been established.
Fig. 1, however, which shows the results using the same probe for rat
pituitary and uterus, suggests that the expression of certain PR mRNA
transcripts could be tissue specific.
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, 3 days of E2 treatment in culture leads to a marked
increase in steady-state levels of PR mRNA in pituitary cells. The
E2-induced increase was 5- to 6-fold: in the absence of
estrogen, PR mRNA levels are 19 ± 7% for the 5.1-kb transcript
and 15 ± 5% for the 10.1-kb transcript, relative to that found
for the E2-treated group (n = 4). We next questioned
whether progesterone could modulate PR mRNA levels in
E2-treated pituitary cells. Analysis of steady-state levels
of the predominant transcripts shows that, by as little as 3 h of
progesterone exposure, there is a significant decrease in the
approximately 10.1-kb fragment and that the depression remains over the
next 6 h. By 12 h of progesterone treatment, there is an
apparent recovery, with the PR mRNA levels not significantly different
than in the estrogen-only group, although there was considerable
variability within the 12-h group (Fig. 2B). For the approximately
5.1-kb transcript, the pattern was similar, but there was less of a
decline associated with progesterone treatment, and the first
significant decrease was not observed until 6 h; by 12 h, the
recovery was complete (Fig. 2C). Whether this difference in temporal
pattern between two PR mRNA transcripts has functional consequences,
e.g. leading to alterations in the translation and ratio of
the protein isoforms of PR, is not known. Overall, however, these data
show that progesterone treatment of cultured rat pituitary cells can
lead to a reversible decrease in steady-state levels of PR mRNA.
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and Table 1, nuclear PR essentially is undetectable
by immunofluorescence in anterior pituitary cells cultured in the
absence of estrogen (panel b), but strong PR immunoreactivity is found
after 3 days of E2 treatment (panel e). To determine the
extent of colocalization of PR with gonadotropes, we used LH
immunoreactivity as a marker. Under our conditions, LH-positive cells
represent 7–8% of the total cells, which is in general agreement with
that reported for pituitaries from intact females but lower than
reported for pituitaries from longer-term ovariectomized rats (37). We
found no significant differences in the percent of LH-positive cells
related to the presence or absence of E2 in the culture
medium (Table 1). When cells are exposed to E2, essentially
all of the LH-positive cells are also positive for PR (Fig. 3, panels d
and e; also see Fig. 4). Of the nearly
300 cells identified as PR-immunopositive, only four did not colocalize
with LH immunoreactivity (Table 1); the possibility that these cells
were gonadotropes expressing only FSH was not investigated.
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, the addition of progesterone to
the medium for varying duration did not significantly affect either the
percent of LH cells identified as positive for PR, or the percent of
pituitary cells negative for LH but positive for PR immunoreactivity.
The scoring for this analysis was based on a simple yes/no
determination of PR immunoreactivity without regard for potential
gradations in staining intensity. When the pattern and intracellular
distribution of PR immunoreactivity is examined, a more complex result
is obtained. As shown in Fig. 4, gonadotropes exhibit the
characteristic predominant nuclear staining for PR in the presence of
E2. When the cells are exposed to progesterone, there seems
to be a gradual increase in cytoplasmic staining, particularly after 9
and 12 h of progesterone treatment (panels h and j). All
gonadotropes, however, did not follow the same temporal pattern of
apparent changes in PR staining intensity; for example, in panel h, two
of the three cells exhibit distinct cytoplasmic PR staining, whereas a
third gonadotrope in the field shows the characteristic predominant
nuclear PR staining similar to that found in the absence of
progesterone (compare with panel b).
To quantitatively assess the distribution of PR, we analyzed the
staining intensity in the cytoplasm and the nucleus (Table 2). In the absence of progesterone, the
high ratio of nuclear to cytoplasmic staining intensity is consistent
with the previously reported predominant nuclear localization of the
PR. When cells are incubated in the presence of progesterone, there is
a gradual change, over time, in the PR staining intensity in the two
compartments, such that by 9 h and 12 h, the
nucleus-to-cytoplasm ratio is significantly decreased to near unity.
Examination of the individual compartmental changes shown in Table 2
suggests that the decrease in the ratio is caused, at least in part, by
a gradual increase in PR staining intensity in the cytoplasm, which is
significant by 12 h of progesterone exposure. The nuclear
compartment shows a more complex pattern; in comparing the groups that
were incubated with progesterone for varying durations, we found that
nuclear PR staining intensity significantly decreased by 9 and 12
h, compared with that found for cells exposed to progesterone for
3 h.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Concomitant with the onset of the preovulatory gonadotropin surge is an acute rise in progesterone that extends past the termination of the LH surge and that has a duration of about 12 h in rats (28, 39). The pituitary is prepared for this almost 10-fold increase in progesterone; early reports suggested that pituitary PR protein content is highest in tissue taken from proestrous rats (11, 40) and that the pituitary PR protein in vivo is induced by estrogen (7, 9, 11). We now show that E2 induction of pituitary PR occurs in vitro for mRNA and for protein and that PR protein essentially localizes exclusively to the gonadotrope. The finding of colocalization of PR with LH cells is consistent with that reported for intact tissue (6).
At the other end of the preovulatory rise in progesterone, is the decrease in pituitary PR abundance on estrus a consequence of progesterone? Whether, in general, progesterone can down-regulate its own receptor has been a difficult experimental question because of a variety of tissue and even cell specificities and because of the complexity associated with the ability of progesterone to interfere with estrogen’s action in some tissues. For the rat pituitary gland, results from early studies, using exchange assays, are conflicting but suggest that progesterone treatment in vivo is related to a transient increase in nuclear PR within 1–2 h (11, 12) and to a decrease in cytosolic PR at 24 h, possibly attributable to a suppression of estrogen’s action (9, 12). For the monkey pituitary, extended progesterone treatment in vivo (14 days) suppressed PR mRNA (15), but the effect on PR protein was variable (5, 14). Our results for rat pituitary cells in vitro establish that progesterone exposure can lead to a reduction of about 50% in steady-state PR mRNA levels by 6 h. This is of interest, in light of reports that the ability of progesterone to augment GnRH-stimulated LH secretion only occurs within a narrow window of about 1–6 h of exposure to progesterone (24, 29) and is consistent with the temporal relationship between progesterone and LH secretion during the preovulatory period. Whether the restricted time course for the progesterone stimulatory action is caused simply by a diminution in the number of available PRs or, more likely, by a combination of possible mechanisms, including a progesterone-induced decrease in GnRH receptors (30) and an alteration in the ratio of the various isoforms (A, B, and possibly C) of the PR and a decrease in PRs remains to be determined.
The restoration of PR message levels by 12 h of progesterone exposure in cultured pituitary cells is less easy to reconcile with in vivo events, given that the number of PRs is reported to be reduced on estrus, compared with proestrus (11, 40). One difference between the in vitro model used for this study and the case in vivo is that the pituitary cells in vitro were in the continual presence of E2 during exposure to progesterone. On proestrus, once the gonadotropin surge is initiated and while progesterone levels are rising, E2 levels drop precipitously and are at their nadir by the end of proestrus and during estrus, thus allowing progesterone to exert its effect in the relative absence of E2 (39, 41). There is little direct information available on progesterone modulation of estrogen receptor message or interference with estrogen’s action in adult pituitary cells, although there is a report that progesterone treatment had no consistent effect on pituitary estrogen receptor mRNA isoforms (42). Taken together, one interpretation of our results is that, under these temporal conditions, progesterone does not substantially interfere with estrogen’s action and that the restoration of PR message levels at 12 h reflects an E2-induced wave of activity after the earlier progesterone-induced down-regulation of PR mRNA. The cultured pituitary cell model could be useful to directly test whether progesterone does indeed affect estrogen’s regulation of PR in the gonadotrope.
Consistent with the observed change in steady-state PR mRNA levels at 12 h, we found an increase in cytosolic PR protein that could be interpreted as reflecting an increase in PR synthesis. This speculation is based on our use of the immunofluorescence approach for assessing PR, which allows for localization of the protein in situ without cell fractionation and also for the manipulation of progesterone levels without compromising the assay-detection system. PR mRNA stability and protein half-life certainly contribute to the observed changes in PR levels, but no information on these variables is available for gonadotropes. In T47D human breast cancer cells, the half-life of PR mRNA was estimated at 2–2.5 h and to be unaffected by the presence of progesterone (43); a 21-h half-life was estimated for PR protein, with a reduction to 6 h in the presence of a progesterone analog (44) and a requisite 8 h or more before cytoplasmic replenishment is observed after progesterone-induced nuclear processing (45). These studies generally support our speculation regarding synthesis of PR in pituitary cells, but comparisons between dividing cells and terminally differentiated cells are difficult, because of the influence of cell cycle on kinetic estimates for the tumor cells.
In summary, we show that E2 increases steady-state levels of PR mRNA and PR protein in cultured rat gonadotropes and that exposure to progesterone leads to a rapid, but transient, down-regulation of PR message and a change in PR protein distribution in these cells. The time course of the observed progesterone effect is significant for its concordance with the temporal context associated with rat preovulatory gonadotropin secretion.
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Acknowledgments |
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Footnotes |
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2 Current address: Genzyme Transgenics Corp., One
Mountain Road, Framingham, Massachusetts 01701.
Received October 16, 1998.
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References |
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-hydroxyprogesterone binding, and
nuclear receptors. J Steroid Biochem 9:419–427[CrossRef][Medline]
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