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Department of Molecular and Cellular Biology, Baylor College of Medicine (T.N.A., I.J.G.R., J.S.R.), Houston, Texas 77030; and the Department of Molecular and Cell Biology, University of California (P.B., G.L.F.), Berkeley, California 94720
Address all correspondence and requests for reprints to: Dr. JoAnne S. Richards, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: joanner{at}bcm.tmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since the initial report that sgk transcripts were present in RNA prepared from whole rat ovary (20), we have conducted additional experiments to determine whether sgk expression was localized to specific ovarian cell types and whether it was regulated by specific hormones during follicular or luteal development. Using well characterized cultures of rat granulosa cells, we have shown that sgk is induced in a biphasic pattern by FSH or forskolin, agonists that increase intracellular cAMP (26). In response to these agonists, sgk messenger RNA (mRNA) and protein increase rapidly by 2 h, decrease at 6 h, and then reach maximal levels at 48 h (26). This pattern of expression closely mirrors nuclear levels of the A-kinase C-subunit (4, 27), indicating that transcriptional regulation of the sgk gene is mediated in part by A-kinase. Using deletional and site-specific mutants of the sgk promoter, we have shown that the A-kinase-inducible expression of the sgk gene in ovarian cells is dependent in part on an Sp1/Sp3 binding region within the proximal promoter (26).
More recently, we have determined, using affinity-purified antibodies,
that the subcellular localization of Sgk protein is dependent on the
stage of granulosa cell function. Immature granulosa cells cultured in
defined medium exhibit little or no immunoreactive Sgk (27). When these
cells are exposed to FSH for 2 h, sgk is induced and is
localized to granulosa cell nuclei (27). In contrast, as granulosa
cells differentiate in culture in response to FSH/T, Sgk becomes
exclusively localized to a perinuclear region of the cytoplasm (27). In
a similar manner, Sgk protein was localized to nuclei of mammary
epithelial cells during S and G2/M phases of the cell cycle, but was
cytoplasmic during the G1 transition or in cells
arrested in G1 as a consequence of hormone
stimulation (28). These intriguing observations in ovarian granulosa
cells and mammary tumor cells indicate that Sgk may have dual functions
relating to cell cycle progression vs. terminal
differentiation when cells exit from the cell cycle (27, 28). Recent
results have also determined that Sgk is a substrate for and can be
activated by 3-phosphoinositide-dependent kinase, PDK1 (29). PDK1
phosphorylates threonine 256 in the activation loop of Sgk, a position
similar to that of other kinases phosphorylated by PDK1, such as
PKB
, p70s6k, protein kinase C, and A-kinase
(29). This places Sgk in a kinase cascade downstream of the PDK1, PI
3-kinase, and growth factor stimulation. Although specific substrates
for Sgk are not yet known, some preferred targets have been identified
using a synthetic peptide screening assay (29). That the ovarian cells
contain an abundance of Sgk, that it is hormonally regulated and
differentially localized to the nucleus or cytoplasm indicates that it
is likely to have more than one specific function in this tissue.
The forgoing studies have all been performed using primary cultures of rat ovarian cells (26, 27) or cell lines (20, 21, 28, 29) in which the direct and specific effects of hormones and other agonists on mRNA, protein, and promoter activity could be analyzed. Culture systems often, but not always, mimic events occurring within a physiological context. Therefore, in this study we sought to determine whether the pattern of sgk expression (mRNA and protein) as well as the subcellular localization of Sgk protein were hormonally regulated and dependent on the stage of follicular development and luteinization. Two in vivo model systems were used: a hypophysectomized rat system in which the effects of individual hormones could be assessed at specific developmental stages (30, 31, 32), and pregnant rats in which corpora lutea (CL) are fully functional (16, 19). In each system, the expression of sgk was analyzed by in situ hybridization, Northern and Western blotting, and immunohistochemistry. The collective advantages afforded by each of these models and approaches provide a detailed representation of the in vivo pattern of sgk expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hypophysectomized rats. Female Holtzman Sprague Dawley (Harlan, Indianapolis, IN) rats were hypophysectomized (H) on day 26 of age and were either left untreated or were injected once daily for 3 days with estradiol (E; 1.5 mg/0.2 ml propylene glycol; HE) to stimulate the growth of large preantral follicles. Some HE rats were additionally treated twice daily for up to 2 days with sc injections of FSH (1.0 µg/0.1 ml PBS; HEF) to stimulate the development of preovulatory follicles. The HEF rats were injected with an ip injection of 10 IU hCG to stimulate ovulation and luteinization. Twenty-four hours later, half the rats were left untreated; half were injected with PRL (10 µg in 0.2 ml PBS-10% polyvinyl pyrrolidone PBS-10% PVP twice daily for 2 days). Ovaries were harvested at the designated times (HEF/hCG plus PRL). The effects of E, FSH, and LH on ovarian cell gene expression in this model system have been well characterized (1, 2, 30, 31). In selected experiments, FSH (10 µg/0.1 ml PBS) was administered iv to H rats or HE rats, and E (1 mg/0.1 ml, iv) was administered to H rats to analyze the acute effects of these hormones on Sgk protein. In each experiment, ovaries were isolated for in situ hybridization as well as for preparation of RNA or protein.
Pregnant rats. Timed pregnant rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) were obtained on day 4 of gestation (the day of sperm positive was designated day 1). Corpora lutea and residual ovarian tissue (containing follicles and interstitial tissue) were isolated from ovaries on the designated days of gestation. The tissues were fixed for in situ hybridization, snap-frozen in liquid nitrogen for RNA or whole cell extracts (WCE) or homogenized in boiling SDS buffer (as described below) for total cell protein extracts.
In situ hybridization
Ovaries from hormonally primed H rats and pregnant rats were
fixed immediately in 4% paraformaldehyde in PBS overnight at 4 C
before dehydration and paraffin embedding. Sections (6 µm) were baked
at 42 C overnight onto 3-amino-propyltriethoxysilane-coated slides.
Slides were prehybridized, hybridized, washed, exposed, and developed
as previously described (10, 33). The 35S-labeled
riboprobes were also produced as previously described (10, 33).
Sgk sense and antisense probes were produced by
transcription from the T3 and T7 promoters, respectively, on the
NheI-digested pBS-sgk vector. Each slide was
incubated in 80 µl hybridization solution containing 5 million counts
of the appropriate probe overnight at 55 C in a humid chamber. After
washing, slides were exposed to X-OMAT-AR film to determine the
approximate NTB-2 emulsion exposure time. For most experiments, a 3-day
exposure was sufficient. For each in situ hybridization
analysis, slides containing ovaries in each treatment group were
included to permit direct comparisons of the relative amount of
sgk mRNA signal during follicular development and
luteinization.
RNA isolation and Northern analysis
RNA was isolated (34) from granulosa cells of H rats using a RNA
extraction buffer [140 mM NaCl, 5 mM KCl, 3
mM MgCl2, 25 mM Tris-HCl
(pH 7.5), and 1% Nonidet P-40] at 4 C followed by centrifugation,
phenol extraction in the presence of 1% SDS, and two subsequent
extractions with phenol/chloroform (1:1) and chloroform. RNA was
ethanol precipitated and resuspended in water previously treated with
diethyl pyrocarbonate and quantified by OD at 260 nm.
RNA was isolated from CL and residual tissue of HEF-hCG rats and
pregnant rats using a guanidine-isothiocyanate method (19). For
Northern analysis, RNA samples (20 µg) were resuspended in 45%
formamide-5.4% formaldehyde and denatured at 55 C for 15 min. After
the addition of 4 x RNA tracking dye (50 mM HEPES,
50% glycerol, and 0.25% bromophenol blue), RNA was resolved by
electrophoresis in 18% formaldehyde-1.2% agarose gels at room
temperature. Acridine orange (10 mg/ml) staining allowed assessment of
RNA ladder migration and confirmation of equal sample loading by the UV
intensity of 28S and 18S ribosomal RNA bands. After the RNA was
transferred to a nylon membrane in 20 x SSC (standard saline
citrate), the blot was baked for 1 h at 80 C, prehybridized, and
hybridized under standard conditions with 1 x
106 cpm/ml sgk complementary DNA
(cDNA) probe labeled as previously described using random primers and
[-32P]deoxy-CTP (35). Blots were washed
according to ICN specifications (ICN Pharmaceuticals, Inc., Costa Mesa, CA) and exposed to x-ray film at -70 C.
Results were quantified using a Betascope analyzer (Betagen Corp.,
Mountain View, CA).
Protein preparation and Western analysis
Protein was isolated from granulosa cells and luteal cells by
homogenization in WCE buffer (10 mM Tris, 1 mM
EDTA, 1 mM DTT, 400 mM KCl, 10% glycerol, 1
mM PMSF, 1 mM vanadate, 1 mM
diethyldithio- carbamic acid, and 0.1 mg/ml aprotinin)
followed by centrifugation (1 min in microfuge) to isolate soluble
protein (18, 36). The concentrations of soluble protein in each sample
were determined by Bradford assay (reagents from Bio-Rad Laboratories, Inc., Hercules, CA). Western blots were run using
30 µg WCE protein.
One-dimensional SDS-PAGE with 4.5% stacking and 10% separating acrylamide gels was used to resolve proteins. Proteins were electrophoretically transferred to 0.45-mm Immobilon membranes and blocked for 1 h in PBS containing 5% milk and 0.1% Tween-20. After one 20-min incubation in wash solution (1% milk in PBS and 0.1% Tween-20), filters were incubated for 1 h with appropriate dilutions of Sgk antibody (1:7500) (27) or Sp1 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After three washes (10 min each), blots were incubated with 1:5000 antirabbit horseradish peroxidase, washed as before, and detected using the enhanced chemiluminescence assay system ECL (Amersham Pharmacia Biotech, Arlington Heights, IL). Immunoreactive signals were analyzed and quantified using an AlphaImager 2000 (3.3, Alpha Innotech Corp., San Leandro, CA)
Immunohistochemical analyses
The cellular and subcellular localizations of Sgk and Sp1 were
analyzed by immunostaining 4% paraformaldehyde-fixed and
paraffin-embedded ovaries as described for in situ
hybridization. Sections (6 µm) were processed according to routine
procedures. Briefly, rehydrated sections were boiled in 0.1% hydrogen
peroxide followed by PBS washes. Sections were then incubated with 10%
nonimmune goat serum to block nonspecific sites followed by incubation
with affinity-purified Sgk antiserum (27, 28) or Sp1 antiserum
(Santa Cruz Biotechnology, Inc.) diluted 1:50 in 10% goat
serum overnight at room temperature. After washing in PBS, biotinylated
antirabbit antiserum (Vector, Burlingame, CA) was added for 30 min,
slides were washed, and streptavidin-conjugated horseradish peroxidase
was applied for 30 min. Sections were incubated with
diaminobenzidene substrate for 2 min, dehydrated without
counterstaining, and mounted.
Statistical analyses
Where indicated, statistical analyses were performed by ANOVA.
Values represent the mean ± SEM for at least three
separate experiments and were considered significantly different if
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Stimulation with estradiol (E) for 3 days
results in granulosa cell proliferation (10, 11, 30) and growth of
preantral follicles (HE) (31). Intravenous injections of FSH (1 µg)
were given to HE rats to rapidly increase granulosa cell production of
cAMP (37) and to examine the immediate early expression of
sgk mRNA. As shown, FSH did not markedly change the
histological appearance of the HE ovaries (Fig. 1, HE, FSH, 2 and
8 h). However, sc injections of FSH (1 µg twice daily for 2
days) stimulated the growth of large antral, preovulatory follicles
(HEF, 48 h), at which time injection with hCG can cause ovulation
and the subsequent formation of CL (HEF/hCG).
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, H). In
ovaries of HE rats, Sgk mRNA remained low in granulosa cells
of growing preantral follicles and was markedly reduced in the
interstitial cells (Fig. 1, HE). Sgk mRNA was induced in
granulosa cells within 2 h after a single iv injection of FSH, and
the intensity of the sgk signal increased further at 8
h (Fig. 1, FSH, 2 and 8 h). The sgk mRNA was also
induced in granulosa cells of HE rats after a regimen of FSH treatment
that stimulates the development of preovulatory follicles (Fig. 1, HEF,
48 h). In these preovulatory follicles sgk exhibits a
gradient pattern, with highest levels in the most differentiated, least
proliferative, mural granulosa cells (Fig. 1, HEF, 48 h). Corpora
lutea isolated 48 h after a bolus injection of hCG also expressed
high levels of Sgk mRNA (Fig. 1, HEF/hCG, 48 h).
Hybridization with the sense Sgk cDNA as a control showed no
signal (Fig. 1, HEF/hCG, sense). The rapid induction of sgk
mRNA by FSH in proliferating granulosa cells as well as its expression
in the more differentiated, mural granulosa cells and luteal cells
suggest that Sgk may regulate specific targets in proliferating
vs. differentiating granulosa cells.
To further examine the regulation of sgk expression in the
CL, ovaries from HEF rats that had been stimulated with an ovulatory
dose of hCG for 4, 12, 24, and 48 h were isolated for in
situ hybridization. The sgk mRNA was readily apparent
in granulosa cells of preovulatory follicles (Fig. 2. HEF, 48 h; also see Fig. 1). Four
hours after injection of hCG, sgk mRNA expression was
reduced in the ovulatory follicle (Fig. 2, HEF hCG, 4 h). However,
within 12 h, sgk mRNA was again increased in the
ovulatory follicle (not shown), and by 24–48 h of hCG stimulation,
sgk mRNA levels were markedly elevated in cells of the newly
formed CL.
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). An ovulatory dose of hCG caused
sgk mRNA levels to decline by 2 h and remain low at
8 h. However, in association with the formation of the CL at
24–48 h after hCG, sgk mRNA increased dramatically. Thus,
the highest levels of sgk mRNA are present in granulosa
cells that have terminally differentiated into nondividing luteal
cells.
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, H). E treatment for 3 days
had little or no effect (Fig. 4A, HE). However, a single iv injection
of FSH stimulated a rapid, 7-fold increase in Sgk protein at 2 h,
which remained elevated at 4 and 8 h (Fig. 4A; FSH, 2, 4, and
8 h). Similarly, two sc injections of FSH stimulated a 7.9-fold
increase in Sgk protein at 24 h (Fig. 4A, HEF, 24 h). In
these same samples, the levels of Sp1 (38), a transcription factor that
regulates sgk expression in granulosa cells (26), were high
and remained unchanged by hormone treatment (Fig. 4A).
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). In
this experiment, changes in the expression of Sgk during follicular
growth were compared with that following hCG-induced luteinization. As
described above, the level of Sgk was low in granulosa cells of H rats
and was increased 1.3-fold by E, 8.2-fold by acute FSH stimulation at
2 h (HEF, 2 h), and 17-fold by sc injections of FSH for
48 h (HEF, 48 h). An iv injection of hCG (10 IU) stimulated
only a slight increase in Sgk protein at 2 h (HEF/hCG, 2 h)
which was decreased 25% at 4 h. Sgk protein then increased
progressively at 12, 24, and 48 h after hCG to achieve levels 22-,
28-, and 35-fold greater than those in granulosa cells of H rats. When
levels of Sp1 were examined in these same samples, Sp1 remained
essentially unchanged, with the exception of a decrease 48 h after
hCG (Fig. 4B).
In many samples, there are multiple immunoreactive Sgk bands. As Sgk
has recently been shown to be a phosphoprotein (29), and the
phosphorylation of specific sites is requisite for its own kinase
activity (29) the slower migrating, immunoreactive bands are presumed
to represent phosphorylated states of Sgk. Note in particular the three
bands in the HEF, 2 h sample (Fig. 4B) and in the HEF/hCG samples
(Fig. 4B). Note also what appear to be smaller bands in the HEF/hCG 24
and 48 h samples (Fig. 4B). These may represent partially degraded
forms of Sgk. Whatever their identity, they represent a small
proportion of the total immunoreactive material.
To determine whether the induction and expression of Sgk protein in
response to the acute iv injection of FSH were dependent on the
exposure of granulosa cells to E, additional experiments were performed
in which either FSH (10 µg) or E (1 mg) was injected iv to H rats. As
shown in Fig. 5, Sgk was low in granulosa
cells of H rats. Exposure to FSH caused a rapid, 7.6-fold increase in
Sgk protein (Fig. 5), a response similar to that observed when HE rats
were stimulated with a similar dose of FSH (Fig. 4, A and B; HEF,
2 h). However, in the H rats the levels of Sgk protein in
granulosa cells declined 50% by 12 h. Note also that there were
multiple immunoreactive bands present in the H/FSH, 2 h sample,
some of which appeared to be phospho-Sgk, whereas others appeared to be
smaller, degraded fragments (Fig. 5). These data also show that E can
evoke a rapid, 3.7-fold increase in Sgk protein at 2 h that does
not exhibit lower mol wt forms (Fig. 5). However, the effect of E is
not sustained, and the levels of Sgk protein in granulosa cells return
to those in the H rats by 24 h (Fig. 5) as observed after 3 days
of E treatment (Fig. 4). Thus, although the rapid induction of Sgk
protein by FSH does not require prior exposure of granulosa cells to E,
E-mediated differentiation of granulosa cells appears to facilitate and
prolong Sgk expression in granulosa cells.
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). Interestingly, the
interstitial tissue of ovaries collected on days 7, 15, and 22 of
gestation also exhibited significant levels of sgk mRNA.
These in situ hybridization data indicate that
sgk expression is maintained in luteal cells throughout
pregnancy as well as in cells present in the interstitial
compartment.
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). Sgk mRNA was present in the
mature, functional CL on days 7 and 15 of gestation as well as on day
21, a time when luteal cells begin to undergo regression. Levels of
sgk mRNA in the residual compartment of pregnant rat ovaries
on days 7, 15, and 21 of gestation were similar to those observed in
luteal cells on these same days.
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). Sgk protein remained elevated on
days 13 (not shown), 15, and 22 of gestation and had decreased 25% in
CL obtained 1 day postpartum (Fig. 8). Despite the high levels of
sgk mRNA in the residual ovarian component (Fig. 7), Sgk
protein in this tissue was lower than that in the luteal cells (Fig. 8). For example, the ratios of Sgk protein in the CL compared with
residual tissue on days 7, 15, and 22 of gestation and postpartum day 1
were 4.9-, 4.0-, 1.8-, and 7.3-fold, respectively. Thus, the only time
during gestation when the residual tissue approximated that of the
luteal cells was on day 22, a time when the level in luteal cells
declined and that in the residual tissue had increased (i.e.
in day 22 residual tissue, Sgk was 2-fold higher than that on day 7).
Levels of Sgk in the ovary of a nonpregnant rat were similar to those
of CL in the postpartum rat (not shown). There seems to be no easy
explanation to account for why there is less Sgk protein in the
residual compartment than would be predicted based on the in
situ hybridization data and Northern blot results. The apparent
discrepancy is not related to the method of extraction, as similar
results were obtained when proteins were prepared in boiling SDS buffer
(data not shown). It is possible that Sgk protein is less stable than
the mRNA in the interstitial cells. However, we did not observe any
increase in lower mol wt immunoreactive bands. The transcriptional
regulation of the sgk gene is complex, and virtually nothing
is known for the interstitial cells. Likewise, it is becoming clear
that the functional roles of Sgk as well as its activation are complex.
When more is known about this kinase, the answer may be
forthcoming.
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), Sgk protein was present in
granulosa cells of preovulatory follicles (HEF) but decreased slightly
(20%) after exposure to PRL (1 µg/0.1 ml, iv) alone for 24 h.
In contrast, levels of Sgk protein increased (3-fold) in CL of
HEF-hCG-treated rats and were further elevated (1.6-fold) by exposure
to PRL for 24 h. Residual tissue exhibited a similar response; Sgk
protein increased (1.7-fold) in response to PRL in the HEF-hCG-treated
ovaries, but not in the HEF ovaries. In situ hybridization
(not shown), confirmed that sgk mRNA was elevated in CL of
HEF-hCG (24-h)-treated rats exposed to PRL for 6 and 24 h. Thus,
during the LH-induced luteinization process, sgk expression
is increased, and PRL further modulates the levels of sgk
mRNA and protein approximately 1.5-fold.
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(A–C),
immunoreactive Sgk was detected in large antral follicles (Fig. 10A)
with localization to both the granulosa cells (Fig. 10B, asterisk) as well as to specific cells within the thecal
layer (arrows). In the proliferative, but differentiating,
granulosa cells, immunostaining was varied among cells, but was
localized predominantly to the nuclear compartment (Fig. 10B, stained
cell next to the asterisk). Likewise, Sgk appeared nuclear
in the immunopositive cells within the thecal layer (Fig. 10C).
Although these Sgk-immunopositive cells appear to be thecal endocrine
cells, it is also possible that they represent proliferating
fibroblasts or immune cells. Intense immunostaining of Sgk was also
detected in the oocytes of primordial follicles (Fig. 10, D and E,
arrows) residing beneath the surface epithelium. In these
primordial oocytes, immunoreactive Sgk was localized primarily to a
perinuclear region of the cell. In contrast, immunoreactive Sgk was low
or absent in the oocytes, granulosa cells, and thecal cells of small
growing follicles, but was present in adjacent interstitial cells,
where it was localized to the cytoplasm (Fig. 10F). Intense
immunostaining of Sgk protein was also observed in CL of pregnant rats,
where it was localized to a perinuclear region of the cytoplasm (Fig. 10, G and H, arrows). Sgk protein was also present at high
levels in certain clusters of cells within the interstitial compartment
of the pregnant rat ovary. In these clusters of interstitial cells, as
in the luteal cells, Sgk was localized to the cytoplasmic region of the
cells, not the nucleus. Although the identification of the cells within
the interstitial region is not yet known, their size and morphology
indicate that they may be part of the steroidogenic component of the
interstitium. No immunostaining was observed in the absence of the
primary Sgk antibody (Fig. 10I). The transcription factor, Sp1 (and
Sp3, not shown), which is known to regulate Sgk expression in ovarian
cells was localized exclusively to the nuclei of granulosa, thecal,
luteal, and interstitial cells (Fig. 10, J–L). Collectively, these
data indicate that the subcellular localization of Sgk is dependent on
the stage of granulosa and luteal cell differentiation, whereas Sp1 is
always nuclear.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of sgk by FSH in granulosa cells is rapid, a pattern consistent with immediate early induction of this gene in other cells that respond to agonists such as serum and glucocorticoids (20, 21), brain injury (22), and cAMP (Ref. 26 and present study). The induction of sgk mRNA in granulosa cells is also associated with specific stages of granulosa cell proliferation. The level of sgk mRNA was low (Northern blot) or undetected (in situ hybridization) in granulosa cells of preantral follicles where the cells are proliferating at an extremely slow rate (H rat) (30, 32). Treatment of H rats with E markedly increased granulosa cell proliferation, as indicated by previous studies in which tritiated thymidine labeling was used to determine the labeling index (30) as well as by more recent studies in which the induction of the cell cycle activator, cyclin D2, has been analyzed (10, 11). Despite the marked effect of E on granulosa cell proliferation, sgk mRNA after 3 days of E treatment was only marginally affected by this steroid as indicated by both in situ hybridization and Northern blot analyses. However, when short time intervals were analyzed, the results show that E alone can increase Sgk within 2 h. However, the most dramatic increases in sgk expression were induced by FSH in granulosa cells of H rats or HE rats, where a 7- to 8-fold induction was seen within 2 h. The rapid induction of sgk expression by FSH in the HE rats coincides temporally with a secondary burst of proliferative activity that occurs in granulosa cells of large preantral follicles as they become preovulatory follicles (30). Both the labeling index (30) as well as the expression of cyclin D2 (10, 11) are increased by FSH in granulosa cells of HE rats. These results indicate that in the ovary, sgk expression is associated with but not strictly related to all stages of granulosa cell proliferation. Rather, sgk expression is stimulated in these cells by FSH/cAMP and, in this way, may synergize with events stimulated by other factors, such as E. These results in vivo are similar to the biphasic induction of sgk mRNA and protein observed throughout differentiation of immature primary cell cultures to the preovulatory phenotype (26, 27).
FSH initiates a cascade of cellular and biochemical signals in
granulosa cells that leads to changes in the way the cells respond to
those signals. For example, although Sgk, as well as specific kinases
(cdks) controlling progression through the cell cycle are immediate
targets of FSH action (10, 11, 26, 27), extended exposure of cells to
FSH is required for the expression of other genes, including aromatase
(resulting in elevated levels of estradiol) (1), the LH receptor (3),
and inhibin (39). Collectively, these events and other cellular
changes produce a differentiated granulosa cell phenotype. The
secondary induction of sgk after prolonged FSH exposure, therefore,
appears more a result of the unique differentiated state than a direct
response to a singular rapid effect of FSH. Within a preovulatory
follicle, the granulosa cells comprise a heterogeneous population,
because the progression of granulosa cell differentiation through the
follicle occurs in a gradient pattern. Granulosa cells adjacent to the
antrum of the follicle are still proliferative at this time (10, 11, 32). However, the mural granulosa cells adjacent to the basement
membrane are less mitotic and are the first to express markers of
granulosa cell differentiation such as the LH receptor (40, 41).
Reflecting the relationship between the differentiated state and
sgk expression is the observation that sgk mRNA
is also expressed in a gradient in the preovulatory follicle, with the
highest levels seen in the most differentiated mural granulosa
cells.
The LH surge rapidly initiates the terminal differentiation of
granulosa cells to luteal cells. Beginning within 4 h and complete
by 12 h of exposure to LH, granulosa cells cease to divide, as
indicated by the absence of cells showing positive staining for
bromodeoxyuridine (11). The cessation of cell division is associated
with the rapid loss of cyclin D2 and the increased expression of cell
cycle inhibitors, p21CIP1 and
p27KIP1 (10, 11). During this time, granulosa
cells are completely reprogrammed to become luteal cells (42, 43). They
acquire and maintain a stable luteal cell phenotype in vivo
and in vitro, as characterized by the constitutively
elevated expression of genes such as cholesterol side-chain cleavage
cytochrome P450, P450scc, even in the absence of FSH and LH (42, 43).
Although sgk expression is rapidly reduced by the LH surge,
this decrease is transient. The expressions of sgk mRNA and
protein are increased as the cells begin to luteinize (within 12 h
post-hCG) in vivo (results herein) and as we have recently
shown in vitro (27). The levels continue to rise as the
mature CL is formed (24–48 h post-hCG) until they reach maximal levels
during midgestation (day 15 of pregnancy). At this time the CL also
express maximal levels of mRNA encoding other proteins (15): P450scc
(44), aromatase (45), LH receptor (3),
2-macroglobulin (19, 46), and relaxin (47).
During pregnancy the expression of genes encoding these proteins is
regulated principally by the sequential action of pituitary PRL and rat
placental lactogens in conjunction with steroid hormones and other
factors. Therefore, maximal sgk expression concurs with
highest levels of steroidogenic activity and PRL/rat placental lactogen
secretion.
Because of the enhanced expression of sgk mRNA and protein
in the CL as well as the known luteotropic actions of PRL/rPL on
regulating gene expression in luteal cells (16, 17, 18, 19), we sought to
determine whether PRL might induce or regulate sgk
expression. As shown herein, PRL did not markedly alter Sgk expression
in preovulatory granulosa cells. This was not unexpected, as most of
the known effects of PRL in the rat ovary are mediated at the level of
the CL or interstitial cells. In this regard, PRL augmented, but did
not induce, the elevated expression of sgk in luteal cells.
This pattern of expression is similar to that of P450scc, which is
modulated, but not induced, by PRL (45). In contrast, PRL activation of
the Jak/Stat signaling pathway is obligatory for luteal cell expression
of 2-microglobulin (17, 18, 19). Thus,
sgk, like many, but not all, genes in the ovary, is a
PRL-regulated, but not PRL-inducible, gene.
The presence of Sgk protein in oocytes of primordial follicles indicates that Sgk has a function in the female germ cell as well as in somatic cells. As oocytes in primordial follicles are arrested in meiotic prophase, Sgk may be critical for ensuring arrest at this specific stage of the meiotic process. However, in growing follicles in which the oocytes have resumed or completed growth (but not meiosis), the level of Sgk protein was low or absent, indicating that Sgk may exert functions in oocytes of primordial follicles in addition to or other than meiotic arrest.
The expression of sgk in proliferating granulosa cells as well as in terminally differentiated luteal cells and resting oocytes suggests that Sgk may have multiple functions in controlling cell cycle progression and differentiation. This hypothesis is supported by the observations herein, which show that Sgk protein localizes to nuclei of granulosa cells but is clearly cytoplasmic and largely excluded from nuclei of luteal cells and oocytes. These results in vivo support and extend our recent observations in granulosa and luteal cells in culture (27) as well as studies of the expression and localization of Sgk during the cell cycle in mammary epithelial tumor cells (28). Nuclear import and export mechanisms are complex and involve many different control mechanisms (48, 49, 50, 51, 52). As Sgk protein contains both a putative nuclear localization signals (NLS) as well as a putative nuclear export signal (NES), the subcellular distribution of Sgk may be regulated by the changes in functional activity (phosphorylation?) of either one or both of these trafficking signals. Recent observations indicate that Sgk is a phosphoprotein and thus is probably a specific substrate of a specific cellular kinase cascade(s) (29). Cytoplasmic localization could indicate that function of the NLS is blocked, whereas the NES is active. Conversely, nuclear localization may occur when NLS, but not NES, is active. Anchoring of Sgk to specific docking sites (27, 28) may also occur in a fashion analogous to that in other kinases (53, 54). As Sgk is a kinase, it is tempting to speculate that restriction of Sgk to the nucleus in proliferating granulosa cells allows specific nuclear substrates to be phosphorylated, whereas exclusion of Sgk from the nucleus in luteal cells favors phosphorylation of cytoplasmic substrates that maintain a terminally differentiated state of luteal cells or the arrested state within oocytes.
In summary, these studies in vivo provide the first evidence that Sgk is expressed in several ovarian cell types, including the oocytes of primordial follicles, and that Sgk exhibits distinct subcellular localization depending on the hormonal stimulation and the stage of cell differentiation.
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Received August 11, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mol Endocrinol 10:171–1842-macroglobulin (2M) gene in rat ovarian
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