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Department of Human Physiology (J.L.T., 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: Judith L. Turgeon, Ph.D., Department of Human Physiology, University of California, Davis, California 95616. E-mail: jlturgeon{at}ucdavis.edu
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Introduction |
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One approach to establishing the basis for the role of progesterone in the female reproductive cycle is the use of PR knockout (PRKO) mice, which are anovulatory (8). Recent in vivo studies from Levine’s group (9, 10) have shown that PRKO females lack endogenous or E2-induced preovulatory gonadotropin surges, do not respond to male mouse odor with a gonadotropin surge as normally occurs in wild-type (WT) female mice, and do not exhibit GnRH self-priming. This work clearly demonstrates a critical role for the PR in the normal progression of ovulatory cyclicity in general and in E2-induced signals in particular, but the specific impairments at hypothalamic and pituitary targets in PRKO females remain to be determined.
To define the impairments to anterior pituitary sites in PRKO mice requires comparison data for WT mouse pituitary cells. Although there are several in vivo studies implicating progesterone in mouse anterior pituitary function (e.g. Refs. 11, 12), to our knowledge there are no reports establishing the presence and regulation of the PR in mouse pituitary cells or defining LH secretion and its modulation by steroids for mouse pituitary cells cultured in vitro. To address this, using an in vitro model, we recently characterized the LH secretory response from WT mouse pituitary cells in culture and showed that GnRH self-priming can be elicited from mouse gonadotropes in an E2-dependent manner (13). For rat gonadotrope cells in this in vitro model system, we have established a role for progesterone-independent activation of the PR in GnRH involving cross-talk between the GnRH-receptor and the PR (4, 14). This is in addition to the well-established functions for progesterone-dependent activation of the PR in these cells (15, 16, 17). As predicted by our hypothesis on the basis of rat gonadotropes, cultured mouse PRKO pituitary cells showed a greatly attenuated GnRH self-priming response (13). This suggests that PRKO pituitary cells could serve as a model for identifying pathway components between GnRH-receptor activation and PR activation as well as downstream events leading to augmented LH secretion.
Implicit in previous studies of PRKO mouse LH secretion deficiencies is the assumption that gonadotrope cell lineage during development is unaffected by deletion of the PR. Further, implicit in the interpretation of LH secretion data from WT mice is the assumption that PRs are induced by E2 and localize exclusively to the gonadotrope, as is well documented for the rat and monkey (18, 19, 20, 21). Therefore, the aims of this study were to: 1) establish the proportion of LH-containing cells for PRKO mouse pituitary cells in culture, compared with WT mice as an indication of gonadotrope development and 2) establish which cell types contain PR in WT pituitaries and determine the regulation of mouse pituitary PR by E2 and progesterone.
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Materials and Methods |
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Pituitary cell culture and experimental protocols
Protocols employed in these experiments were reviewed and
approved by the University of California Davis Animal Use and Care
Administrative Advisory Committee. Adult, female WT and PRKO mice
(C57/6/129sv hybrid as described in Ref. 8) and rats
(Sprague Dawley, Simonsen Laboratories, Gilroy, CA) were
maintained in controlled light conditions (14 h light, 10 h dark).
Animals were ovariectomized (ovx) under either tribromoethanol [mice
(24)] or ether (rats) anesthesia and maintained for 2 wk
before use. Pituitary glands were removed following
CO2 narcosis and decapitation. Anterior pituitary
tissue was enzymatically dispersed and prepared for cell culture as
described (22). Cells were plated at 3 x
104 on Matrigel-coated 12-mm glass coverslips
placed in 22-mm multiwell plates. Wells were flooded with MEM
containing d-valine and supplemented with 200
µM kanamycin sulfate, 10% FBS that had been
charcoal treated to remove endogenous steroids, and ±0.2
nM E2. Residual steroid concentrations in the
charcoal-treated serum were 3 pM for progesterone
and less than 1 pM for E2 as determined by RIA.
Cells were maintained in a humidified atmosphere (37 C) of
5% CO2 in air; on 3, media were replenished
(day of plating = d 1).
On 4, the cells were changed to serum-free MEM containing 1 mg BSA/ml (MEM/BSA) with or without continued E2 as appropriate. For some experiments the medium also contained 200 nM progesterone. Incubation was continued for 6 h (±E2-only groups) or for 3 or 10 h (E2 + progesterone groups). For all groups, termination was accomplished by rinsing the cells in PBS at room temperature followed by fixation with precooled methanol:acetone (1:1) for 7 min at -20 C; fixed cells were stored in PBS at 4 C until subjected to immunofluorescence staining. The fixation conditions were chosen to optimize PR detection; the optimal fixation condition for identification of the pituitary hormones was not specifically tested (25).
Immunocytochemistry
Immunofluorescence staining was carried out at room temperature
by successive incubation of the coverslips in the following solutions:
1) 5% NGS, 1 h; 2) anti-PR (1:750) and either anti-LH
(1:500), anti-PRL (1:100), or anti-GH (1:40,000), 2 h; 3)
PBS-0.1%, BSA-0.1%, Tween 20 (PBSA) washes; 4) 5% NGS, 30 min;
5) TRITC-conjugated goat anti-mouse (1:160) or antimonkey (1:200)
IgG, 1 h; 6) PBSA washes; 7) FITC-labeled goat
antirabbit IgG (1:160) and 2.4 nM TOTO-3 nuclear DNA stain,
1 h; 8) PBSA washes; and 9) distilled water wash and
mount using Vectashield mounting medium for fluorescence. As controls,
either or both of the primary antibodies were omitted for some
coverslip incubations. All antibodies were diluted in 5% NGS.
Confocal microscopy
Microscopy data were obtained with an LSM 510 laser scanning
confocal microscope (Carl Zeiss, Inc., Thornwood, NY)
using a 40x/1.3 N.A. lens. Fluorescence data were collected in three
separate channels: channel 1 was set to collect fluorescence of the
nuclear stain TOTO-3 (excitation: 633 nm; emission: 650-nm long pass
filter); channel 2 collected FITC fluorescence for PR immunoreactivity
(PR-IR) (excitation: 488 nm; emission: 505- to 530-nm band pass
filter); channel 3 collected TRITC fluorescence for LH, PRL, or GH
immunoreactivity (excitation: 543 nm; emission: 560- to 615-nm band
pass filter). For each channel and for all collections, the pinhole was
set to 250 µm, giving an optical slice of approximately 3 µm.
Additionally, a transmitted light image was obtained in channel 4.
Fluorescence intensity analysis
For groups in which fluorescence intensity for nuclear PR-IR was
to be analyzed, overlapping optical slices (22–28; step size
approximately 1 µm) were obtained through the entire cell. Each slice
was 1024 x 1024 pixels and had a pixel depth of 8 bits.
Subsequently, from the data set of each cell, a single optical slice
was selected for analysis. The optical slices for each cell were
examined and the slice containing the maximum nuclear diameter was used
for analysis of PR-IR. The selected images were exported in TIFF format
and analyzed using ImageJ (available from NIH:
http://rsb.info.nih.gov/ij/applet/). Each exported TIFF file
was converted to an 8-bit RGB stack. Using the RGB stack the net mean
nuclear PR-IR was determined as the gray-scale intensity in the G
(green; channel 2) channel minus the corresponding background value
(relative intensity units).
Statistical analysis
Data are presented as the mean ± SEM. Each
experiment represents a separate pool of dispersed pituitary cells; n
refers to the number of separate experiments and reflects a minimum of
three separate cell dispersions per experimental group. At least three
fields were analyzed on each coverslip and the data averaged to
represent one experiment. For the evaluation of cell type proportion,
the number of cells counted per experimental group ranged from 174 to
609. For the quantification of PR-IR intensity, the number of cells
analyzed per experimental group ranged from 15 to 31 for gonadotropes
and 76 to 138 for lactotropes. All statistical analyses were done using
SigmaStat (SPSS, Inc., Chicago, IL). For multiple
comparisons, differences among groups were determined by ANOVA and the
Student-Neuman-Keuls’ method in which differences are indicated as
being significant, P < 0.05.
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Results |
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, a and b, and Table 1). The presence or absence of E2 during
culture did not significantly affect the proportion of LH-positive
cells. For ovxPRKO mouse anterior pituitary cells in primary culture,
the proportion of LH immunoreactive cells is identical to that for
ovxWT cells (Fig. 1d and Table 1). For ovxWT cells cultured in E2, all
LH-positive cells showed nuclear PR-IR. For ovxWT cells cultured in the
absence of E2 for 3 d, PR-IR was scored as negative (Fig. 1, a and
a'). Subsequent quantitative intensity analysis of zero-E2 gonadotropes
in ovxWT showed that low-level nuclear PR-IR could be detected at less
than 10% of the signal in E2-treated ovxWT cells (see Fig. 3). For
ovxPRKO cells, no PR-IR could be detected (Fig. 1, d and d'), and this
was confirmed by subsequent intensity analysis (data not shown).
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; Fig. 1, b and b').
This is in sharp contrast to cultured rat pituitary cells in which PR
exclusively colocates with gonadotropes as we reported earlier
(19) and as shown in the example of rat cells in Fig. 1, c
and c'. Because this expression of PR in mouse ovxWT cells was E2
dependent (see absence of nuclear PR-IR in Fig. 1, a and a'), we asked
whether nuclear PR localizes to lactotropes.
PR-IR in other pituitary cell types
As presented in Table 2, about
one-half of mouse pituitary cells are PRL positive with no significant
difference between ovxWT and ovxPRKO cultures. In the presence of E2,
there was a significant increase in the number of cells scored as
positive for PRL, both for ovxWT and ovxPRKO. The observed differences
attributable to E2 are due, at least in part, to a lower intensity of
the prolactin immunofluorescence in cells cultured in the absence of
E2, which complicated the subjective scoring of cells as being PRL
positive. Regarding PR-IR, as depicted in Fig. 2, a and a', all ovxWT PRL-positive cells
also showed nuclear PR-IR of varying intensity that was E2 dependent.
Fig. 2, a and a', includes a presumptive gonadotrope with nuclear PR-IR
but negative for PRL. Again, no PR-IR was detected in ovxPRKO cells
(Fig. 2, b and b').
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, c and c'). The cells in the
field shown in Fig. 2, c and c', are somewhat flattened and spread out,
which may contribute to the punctate distribution of the PRL
immunofluorescence. The field representing the rat cells also includes
a presumptive gonadotrope showing nuclear PR-IR.
Because LH-positive and PRL-positive cells combined account for about
65% of the cell types in ovx mouse pituitary cultures, the expression
of PR in gonadotropes and in lactotropes essentially could account for
all the nuclear PR-IR reported in Table 1 (67 ± 3% of total
cells positive for PR). As a further check, we examined for nuclear
PR-IR in somatotropes, which share lineage with lactotropes.
As shown in Table 2 and Fig. 2, d and d', less than 30% of ovx mouse
pituitary cells in culture are GH positive, and this proportion is not
significantly affected by PRKO status or E2. Although the majority of
somatotropes were negative for PR, a small percentage showed nuclear
PR-IR (7 of a total of 107 GH-positive cells from three separate
experiments cultured in E2). The possibility that these cells are
mixed-function somatolactotropes or somatogonadotropes was not
determined.
PR down-regulation
Because we had shown for rat pituitary cells that progesterone
leads to a rapid and extensive reduction in nuclear PR protein in
gonadotropes (26), we next determined whether similar
downregulation occurred in ovx mouse pituitary cells. For this study we
used quantitative confocal microscopy to analyze the intensity of
nuclear PR-IR signal in cells identified either as LH or PRL positive.
In addition, this approach allows the quantification of low-level
fluorescence as distinct from background and thus provides an objective
measure to examine for PR in the absence of E2. As presented in Fig. 3 for gonadotropes cultured in the
absence of E2, low-level nuclear PR-IR can be detected by quantitative
fluorescence intensity analysis. This type of low-level fluorescence,
which is <10% of that found in E2-treated cells, was scored as
negative in the studies shown in Table 1. When E2-treated cultures are
exposed to 200 nM progesterone for 3 h, no significant
effect on gonadotrope PR-IR was observed. However, by 10 h of
progesterone exposure, there was a significant decrease
(P < 0.05) in nuclear PR-IR to about 60% of control
(Fig. 3). For lactotropes the overall pattern for PR up- and
downregulation is similar to that for gonadotropes except the level of
nuclear PR-IR tended to be less in E2-treated lactotropes than in
similarly treated gonadotropes (P < 0.01; compare
Figs. 3 and 4). For lactotropes cultured
in the absence of E2, nuclear PR-IR could be detected as very low-level
fluorescence above background that was <5% of that found in
E2-treated cells (Fig. 4). Progesterone exposure for 10 h but not
3 h resulted in a significant decrease (P < 0.05)
in PR-IR in E2-treated lactotropes to about 70% of control (Fig. 4).
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The proportion of LH-, PRL-, and GH-positive cells in this in vitro model did not differ between ovx WT and PRKO mice. At 3 d in culture, less than 10% of the cells were LH-containing gonadotropes, which is comparable to what we reported for rat pituitary cells in the same experimental protocol (19). In relating these data to other published studies, we found that differences among protocols (e.g., duration of gonadectomy and length of time in culture) preclude specific detailed comparisons. However, the percent of LH positive cells in our in vitro model is comparable to that reported for acutely dispersed anterior pituitary cells from intact female rats (27, 28) but approximately half that reported for the 1-month ovx rat (27).
We found that about 50% of ovx mouse pituitary cells in culture were lactotropes and approximately 30% were somatotropes. This distribution is similar to that for freshly dispersed intact female rat anterior pituitary cells, although the reported proportion of lactotropes ranges from 25–50% (27, 28) and even less (about 15%) when the cells are taken from 1-month ovx rats (27). In agreement with this implied steroid dependence, in our study using cells taken from short-term (2-wk) ovx mice, we found that the addition of E2 to either WT or PRKO cultures led to an increase in the PRL immunofluorescence intensity with the result being an increase in the proportion of cells easily identified as lactotropes.
E2 treatment in vitro had no effect on the proportion of ovx WT or PRKO mouse cells classified as either gonadotropes or somatotropes. Although hormone immunofluorescence intensity was not quantitated, LH- and GH-positive cells could be identified with ease whether the culture had been exposed to the 3-d E2 regimen. For the more complex in vivo environment in intact female rats, Childs (29) has shown that pituitaries taken from specific cycle stages differ in the proportion of LH- or GH-positive cells and express different levels of GH mRNA. Similar studies testing for in vivo effects of reproductive cycle hormones on the proportions of LH and GH cells have yet to be carried out in the mouse, but under the in vitro conditions of our experiments E2 alone did not affect the proportion of LH- and GH-positive cells.
In contrast to the overall similarity between mouse and rat in the proportion of lactotropes, somatotropes, and LH-positive gonadotropes, we found a major departure in the expression of nuclear PR. Unlike the rat (18, 19) and the monkey (20, 21) in which PR localizes exclusively to gonadotropes, PR-IR was found in about two-thirds of ovxWT mouse pituitary cells under our experimental conditions. All ovxWT mouse LH-positive gonadotropes show nuclear PR-IR that is E2 dependent, similar to what we had reported for ovx rat pituitary cells in culture (26), but in the mouse this expression in gonadotropes accounted for only a small percentage of the PR-IR. The specificity of the nuclear PR-IR in the mouse cultures was indicated by the dependence on in vitro E2 treatment in ovxWT cells and the complete absence in PRKO pituitary cells. Because of the E2 dependence, a logical candidate was lactotropes, and indeed we found all PRL-containing cells to also exhibit nuclear PR-IR in the presence of E2. In addition, PR-IR colocalized with less than 10% of GH-positive cells. In the rat, GH-expressing cells have been shown to be a particularly dynamic population consisting of monohormonal somatotropes as well as varying proportions of multihormonal somatolactotropes and somatogonadotropes depending on physiological conditions (29). Although not tested directly, we speculate that the small proportion of mouse GH-positive cells that were also PR positive is likely part of the multihormonal population. In consideration of this, we conclude that gonadotropes and lactotropes can account for essentially all of the PR-IR in E2-treated ovxWT mouse cell cultures.
Activation of the PR in rat gonadotropes has multiple consequences,
both stimulatory and inhibitory. In addition to a chronic negative
effect on gonadotropin secretion, the acute action of progesterone in
rats is to cause a several-fold augmentation of the LH release action
of GnRH, which is apparent within an hour and begins to gradually
dissipate after about 6 h of progesterone exposure (1, 16, 22, 30, 31). We have shown that cessation of the acute
progesterone augmentation action can be explained in large part by a
progesterone-induced downregulation of PR protein in rat gonadotropes
with a time course consistent with the short-lived stimulatory role of
progesterone during the preovulatory LH surge, possibly leading to
termination of the surge (26). Using a similar approach,
we report here that progesterone can downregulate the PR in mouse
gonadotropes within a similar time frame, and we speculate that
downregulation of the PR may have a role in termination of the mouse LH
surge as well. However, the progesterone-induced decrease in PR after
9–10 h is much less dramatic in mouse gonadotropes than in rat cells
as determined in identical protocols [40% loss (Fig. 3)
vs. 75% loss (Ref. 26)]. For rat
gonadotropes, we showed that the decrease in PR involved a
ubiquitinproteasome pathway (26). Whether the
difference between mouse and rat gonadotrope PR downregulation
involves, for example, variations in proteasome regulation or different
ratios of PR-A and PR-B isoforms resulting in differences in substrate
stability, remains to be determined.
The function of the PR in mouse lactotropes is unknown. PRL has primary or modulatory roles in the reproductive cycles of female mammals, and progesterone has been implicated in the feedback regulation of PRL (32, 33, 34). In rats and mice, PRL serves a luteotrophic role following mating to enhance progesterone production, and therefore a feedback role for progesterone on the PRL regulatory system is not unexpected. Although there are reports to suggest an indirect action for progesterone in lactotrope function via actions on gonadotropes (34), studies of the site of progesterone action in modifying PRL secretion primarily have focused on the hypothalamus. For example, progesterone has been shown to affect PRL secretion through it actions on the tuberoinfundibular dopamine system (35, 36) in rats and monkeys, both of which lack PR in lactotropes (18, 19, 20, 21). The significance of the hypothalamic action of progesterone in the regulation of PRL is shown by the demonstrated ability of progesterone to advance or prolong the E2-induced PRL surge in rats (37, 38, 39). For the mouse, much less is known about the specific strategies that have been adapted for the regulation of PRL by progesterone. Interestingly, for in vivo studies in PRKO female mice, Chappell et al. (9) report that PRL levels are elevated over WT controls in the presence of similar levels of serum E2 and progesterone. On the basis of our results, this could be explained, at least in part, by progesterone acting directly on the lactotrope in WT mice to diminish either PRL production or secretion. Although this remains a subject for future study, our demonstration that the PR in ovx mouse lactotropes is up-regulated by E2 and down-regulated by progesterone supports a physiological role for the receptor in lactotrope function.
In summary, we show that the absence of a PR has no effect on the proportion of gonadotropes, lactotropes, and somatotropes in adult female ovxPRKO mice, compared with identically treated ovxWT mice, and the proportions are similar to that reported for identically treated female rats. In this in vitro model, all LH-gonadotropes of ovxWT female mice contain nuclear PRs that are up-regulated by E2 and down-regulated by progesterone, although the progesterone-induced downregulation is much less robust than in rat gonadotropes under the same experimental conditions. In striking contrast to rats and monkeys, E2-dependent PR also is present in lactotropes of ovxWT mice. This divergence underscores the risks in assuming identical cell biology between rats and mice; however, the different strategies that have been adapted by these animal models provide excellent experimental tools with which to study function.
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Acknowledgments |
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Footnotes |
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Abbreviations: FITC, Fluorescein isothiocyanate; NGS, normal goat serum; ovx, ovariectomized; PBSA, PBS-0.1%, BSA-0.1%, Tween 20; PR-IR, PR immunoreactivity; PRKO, PR knockout; TRITC, tetramethyl rhodamine isothiocyanate; WT, wild-type.
Received April 16, 2001.
Accepted for publication May 6, 2001.
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