Differentiation-Dependent Prolactin Responsiveness and Stat (Signal Transducers and Activators of Transcription) Signaling in Rat Ovarian Cells
Darryl L. Russell and
JoAnne S. Richards
Baylor College of Medicine Department of Cell Biology
Houston, Texas 77030
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ABSTRACT
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PRL activates an important cytokine signaling
cascade that is obligatory for maintaining luteal cell function in the
rat ovary. To determine when specific components of this cascade are
expressed and can be activated by PRL, we analyzed the expression of
receptor subtypes (short, PRL-RS, and long,
PRL-RL), the presence and kinetics of Stat
(signal transducer and activator of transcription) activation using the
PRL-response element (PRL-RE) of the
2M (
2-macroglobulin) gene,
and the content and hormonal regulation of three specific modulators of
cytokine signaling; the tyrosine phosphatases (SHP-1 and SHP-2), and
the protein inhibitor of activated Stat3 (PIAS-3). These components
were analyzed in differentiating granulosa/luteal cells of
hypophysectomized (H) rats and in corpora lutea of pregnant rats.
Levels of PRL-R mRNAs increased as granulosa cells differentiated and
reached maximal levels in luteal cells of pregnant rats where levels of
PRL-RS approached those of
PRL-RL. The relative concentrations shifted
from a 27-fold excess of PRL-RL in preovulatory
granulosa cells to a 3.7-fold difference in luteal cells during
midgestation. Despite the increased PRL-RL
expression in differentiated granulosa cells, PRL did not stimulate
detectable activation of Stats. Rather PRL activation of Stat5,
principally Stat5b, occurred in association with luteinization. In
contrast, granulosa cells of untreated immature and H rats contained a
high level of DNA binding activity, which was shown to be comprised
entirely of activated, phosphorylated Stat3. Treatment with
estrogen and FSH reduced the amount of phosphorylated Stat3 and
abolished its ability to bind DNA, an effect temporally related to
increased PIAS-3. Expression of SHP-1 (but not SHP-2) was also
hormonally regulated; SHP-1 mRNA and protein were high in granulosa
cells of H rats, decreased by estrogen and FSH, and subsequently
increased dramatically with luteinization. Of particular note, SHP-1
was localized in cytoplasm of granulosa cells in atretic follicles but
was distinctly nuclear in luteal cells, indicative of different
functional roles. Collectively, these results indicate that Stat3 and
Stat5 are activated by distinct cytokine-signaling pathways modulated
through differentiation-dependent transcriptional regulation of
signaling pathway components and mediate distinct functional processes
in the rat ovary: early follicle growth and atresia vs.
luteinization.
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INTRODUCTION
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The mechanisms controlling the progression of granulosa cell fate
(growth vs. atresia) and terminal differentiation
(luteinization) are not well understood. During the ovarian cycle
numerous cytokines modulate gene expression and function (Ref. 1 for
review), while luteinization involves the acquisition of PRL-induced
Stat5 (signal transducer and activator of transcription 5)
responsiveness, a process requisite for maintenance of luteal cell
function. The specific modulation of cytokine-signaling mechanisms
through progression of follicle differentiation and acquisition of
PRL-responsive Stat5-mediated gene induction during luteinization
comprise the primary focus of the studies described herein. PRL is a
pleiotropic pituitary hormone long known for its luteotropic actions in
the rodent (2). PRL receptors (PRL-R) are expressed in the ovary (3, 4, 5)
and have been shown to regulate multiple genes and events in ovarian
follicular development and function, as evidenced by the recently
reported PRL-R null mutant mice (6). Female
PRL-R-/- mice never become pregnant and fail
to establish pseudopregnancy, indicating impaired function of corpora
lutea (CL). These PRL-R-/- mice also had
fewer primary follicles, fewer ovulations, delayed or mistimed oocyte
release, and impaired oocyte maturation, all signs of disruption in
follicular development and possibly atresia. Additionally, PRL has been
shown to increase vascular endothelial cell proliferation (7) and
leukocyte infiltration in luteinizing ovaries as well as ovaries
undergoing luteal regression (8). The pleiotropic effects of PRL in
ovarian function are related to the different patterns of pituitary PRL
secretion and release of placental lactogens (2), as well as the
involvement of diverse components of the PRL-signaling cascade
including: PRL-R subtypes (9), their associated Janus kinases (Jaks;
Ref. 10), and substrates for Jak2 such as tyrosine phosphatases (11, 12), and Stat1, Stat3, and primarily Stat5 (Refs. 13, 14 for
review).
Briefly, cytokine signaling occurs through ligand-induced dimerization
of receptors which associate with and are phosphorylated by Jak2.
Targets for Jak2 include Stats (13) and phosphotyrosine phosphatases
(PTPs) (11, 12). In the rat, PRL-R are expressed in two variant forms,
long (PRL-RL) and short (PRL-RS). As products
of differential mRNA splicing from a single gene, the ligand binding
and transmembrane regions as well as 44 amino acids of the cytoplasmic
domains are identical, but these receptor isoforms diverge over the
majority of the intracellular sequences (9, 14). Importantly, although
both forms associate with Jak2, the signaling events mediated by PRL-R
remain controversial and may be more complicated than originally
proposed (14, 15, 16, 17, 18, 19). In particular, PRL-RS appears not to
transduce Stat5-dependent induction of milk protein genes in mammary
cells in culture (15, 16, 20, 21). Thus, PRL-RL is thought
to be the only endogenous PRL-R form capable of transducing a
Stat5-dependent signal (22), while PRL-RS may act in a
dominant-negative fashion to impair the activity of PRL-RL
(23, 24).
Negative regulation of cytokine signaling downstream of Stat activation
can also occur through several families of modulators. Two SH-2 domain
containing PTPs have been shown to modulate PRL/GH-induced Stat
responses. The PTP SHP-2 is a substrate for Jak2 and is obligatory for
PRL/Stat5 induction of ß-casein (11). SHP-2 may bind P-Tyr residues
on PRL-R, Jak-2, and/or Stat5 via dual SH-2 domains which, along with
catalytic activity, are required to maintain the intact signaling
pathway (11). The highly homologous phosphatase SHP-1 binds activated
Stat5b catalyzing rapid dephosphorylation resulting in the transient
activation pattern characteristic of this family of transcription
factors (25). In addition, a recently described class of proteins can
bind and neutralize activated Stats. One of these, PIAS-3 (protein
inhibitor of activated Stat3), is a specific inhibitor of Stat3 DNA
binding and transactivation (26). Thus, regulated expression of SHP-1,
SHP-2, and PIAS-3 in the ovary and the manner in which they interact
with PRL-R/Stat signaling complexes can modulate cell-specific
responses to PRL and other cytokines.
Differential patterns of Stat activation and gene expression are also
determined by the manner in which PRL is secreted in female rats.
Pulsatile release of PRL from the pituitary occurs during early
pregnancy (days 19) but is replaced at midpregnancy by elevated and
chronic secretion of placental lactogenic hormones (rPL) until luteal
regression and parturition (2). Several genes are regulated by these
lactogenic hormones including the estrogen receptor (ER) subtype,
ER
, which confers autocrine effects of estrogen to synergize with
PRL and maintain luteal function and the progression of pregnancy to
term (27). This luteotropic complex maintains expression of several
genes in CL including P450arom (CYP19; Ref. 28) and LH receptor (29).
Inhibitory effects of PRL are also exerted at this time on
20
-hydroxysteroid dehydrogenase expression, thereby
preventing the metabolism of progesterone to its inactive metabolite
20
OH-progesterone (30). In addition, PRL and rat placental lactogen
(rPL) induce and regulate
2-macroglobulin (
2M) expression by
luteinized cells (31, 32). The initial increase in
2M occurring soon
after luteinization is followed by a secondary increase in pregnant CL
around day 10 stimulated by high, constitutive rPL release. We
subsequently demonstrated that in luteal cells, but not granulosa
cells, PRL activates Stat5b and Stat5a (33), which bind the
2M
promoter to induce expression (34, 35).
In this study, we have addressed the question of how a pleiotropic
factor such as PRL elicits specific effects in ovarian tissues at
different stages of differentiation. Specifically we addressed three
questions. How do the levels of PRL-R subtypes relate to the activation
of Stat5 or Stat3? Is the activation of Stat3 or Stat5 related to
specific stages of granulosa and luteal cell differentiation? Is the
activation of Stat5 or Stat3 modulated by specific Stat regulators
SHP-1, SHP-2, or PIAS? For this we have used two in vivo
models. Hypophysectomized (H) rats were treated with estradiol (E) and
FSH (HEF) to stimulate the growth of healthy preovulatory follicles
that would otherwise become atretic. Luteal cell responses to PRL were
analyzed in HEF rats treated with an ovulatory dose of hCG (HEF/hCG)
and in pregnant rats when fully functional CL are maintained by
endogenous pulsatile (day 19) or chronic (day 1021) exposure to
lactogenic hormones or are undergoing regression (day 21post partum).
Results of these studies provide the novel observation that expression
of PRL-RS is increased markedly in luteal cells, suggesting
a role other than to exert a dominant negative effect on Stat5
activation by PRL. Likewise, high levels of SHP-1 are present in luteal
cell nuclei, suggesting it may exert positive regulatory effects on
Stat activation/turnover. Lastly, high Stat3 DNA binding activity, as
well as SHP-1 expression, are present in granulosa cells of
unstimulated H rat ovaries. Thus, Stat3 and Stat5 are activated by
different signaling pathways in the rat ovary, modulated by changes in
positive and negative effectors, and associated with distinct stages of
granulosa cell differentiation and function.
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RESULTS
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PRL Receptor Long (PRL-RL) and Short
(PRL-RS) Isoforms
To determine whether changes in the response of ovarian cells to
PRL might be related to expression of specific forms of the PRL-R, the
acute regulation of mRNA levels encoding the short and long forms of
PRL-R was analyzed by specific semiquantitative, as well as
quantitative, RT-PCR assays (3). The quantitative assay specifically
amplifies each rat PRL-R isoform yielding single products of predicted
molecular size; 422-bp PRL-RL and 332-bp PRL-RS
(Fig. 1
). Product identities were further
confirmed by restriction digest analysis (data not shown). Truncated
cDNA clones of each PCR amplicon were introduced into the RT-PCR
reactions at titrated concentrations from 0.130 x
105 copies per tube. Competition of the cloned DNA with the
RNA-derived cDNA from reverse transcriptase reactions permitted
quantiative comparisons of the two receptor mRNAs (see Materials
and Methods).

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Figure 1. Regulated Expression of PRL-R Long and Short
Isoforms during Follicular Development and Luteinization
Competitive RT-PCR analysis of PRL-R isoforms using RNA from hormonally
stimulated H rats or pregnant rats. Specific primer sets were used to
amplify PRL-RL or PRL-RS mRNA-derived cDNA or
truncated competitor introduced at indicated concentrations. A,
Representative autoradiographs of amplified competitive RT-PCR products
resolved on nonreducing acrylamide gels. mRNA was analyzed from
immature/atretic (H), or preovulatory (HEF) granulosa cells or luteal
cells of newly formed (HEF/hCG) or midgestational CL (day 15). B,
Combined mean ± SEM data from three competitive
RT-PCR analyses performed on RNA from granulosa or luteal cells.
Quantitated results were calculated from phosphorimager analysis of
target vs. competitor amplification as described in
Materials and Methods. C, Semiquantitative RT-PCR
analysis of specific PRL-R isoforms in immature/atretic granulosa cells
(H), preovulatory granulosa cells (HEF), newly formed CL (HEF/hCG), or
mature functional CL at early (day 7), midgestation (day 15), or
regressing CL (day 22). PRL was administered as described in
Materials and Methods for 1 h, 24 h, or
24 h followed by a 1-h pulse as indicated. Results are expressed
as the ratio of each specific PRL-R isoform to the L-19 internal
control; mean ± SEM values are shown for three
repeated analyses.
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By quantitative RT-PCR analysis we found that granulosa cells
isolated from H rat ovaries contained near-undetectable levels of
PRL-RL (0.4 x 106 copies/µgRNA) and
PRL-RS (0.16 x 106 copies/µg RNA, Fig. 1
, A and B). In preovulatory granulosa cells of HEF rats, each PRL-R
isoform had increased 20-fold and 2-fold, respectively such that
expression of PRL-RL (7.7 x 106) was
27-fold greater than PRL-RS (0.28 x 106).
PRL treatment at this stage of differentiation resulted in a
time-dependent reduction of both PRL-R isoforms, making their
expression after 24 h similar to that in H granulosa cells (not
shown). However, 24 h of hCG treatment induced luteinization in
which PRL-RL increased 4-fold (30.5 x 105
copies/µg RNA), whereas PRL-RS increased 10-fold
(2.8 x 106 copies/µg RNA) above that in HEF
granulosa cells. In CL on day 15 of pregnancy, PRL-RS mRNA
had further increased, reaching 10.2 x 106
copies/µg, 3.7-fold lower than mRNA encoding PRL-RL,
which changed little compared with HEF/hCG CL. Similar quantities of
both isoforms were found on day 7 of gestation, but each had declined
dramatically on day 22 (not shown).
Semiquantitative RT-PCR analyses comparing specific PRL-R amplification
against that of the ribosomal proteins L-19 or S-16 (as internal
controls for equal RNA loading and reaction efficiency) produced highly
similar patterns of regulation to competitive PCR studies; however,
higher reaction efficiency using PRL-RS primer resulted in
overestimation of the relative PRL-RS/PRL-RL
ratio. Thus, comparison between the two different assays by
semiquantitative methods was impossible. Consistent with the fully
quantitative results, however, we found the most dramatic increase in
PRL-RL after E+FSH treatment of granulosa cells, which
decreased time dependently with PRL treatment (Fig. 1C
) but was
increased only 2-fold further after hCG-induced luteinization. The
transcript for PRL-RS, conversely, increased most
dramatically after luteinization and further increased in early and
midgestational CL, while both transcripts declined upon onset of
luteolysis (day 22, Fig. 1C
).
Collectively, these results demonstrate that the amount of PRL-R as
well as the ratio of PRL-RL/PRL-RS is altered
during ovarian cell differentiation. PRL-RL is most
predominant in granulosa cells of preovulatory ovaries. In contrast,
PRL-RS increases most during luteinization and attains
levels quantitatively similar to that of PRL-RL in CL of
pregnant rats. These changes are remarkably similar and ascribe
additional isoform-specific information to the observed changes in
125I-PRL binding in HEF, HEF/hCG, and pregnant rat ovaries
(36).
Stat-DNA Binding Activity during Folliculogenesis and
Luteinization
Changes in the expression of PRL-R were then compared with
the ability of PRL to induce specific DNA binding complexes. This was
analyzed in whole cell extracts (WCE) of hormonally treated granulosa
or luteal cells by electrophoretic mobility shift assays (EMSA) using
the
2M PRL-RE as a probe (33, 34). Extracts of unstimulated H rat
granulosa cells formed an intense band (Fig. 2A
, lane 1), which was specifically
competed by 10-fold excess unlabeled probe (Fig. 2B
, lane 2). Treatment
with E and FSH separately (not shown) or in sequential combination
(Fig. 2A
, lane 2) reduced the granulosa cell DNA binding activity
without inducing the formation of any new complexes either before or
after PRL stimulation (lanes 24). Extracts prepared from CL 1 day
after induction of luteinization of HEF/hCG-treated rats also showed
little specific DNA binding; however, 1 h after PRL treatment a
protein/DNA complex was induced (Fig. 2A
, lanes 5 and 6). This activity
was absent 24 h after PRL treatment (lane 7). However, intense DNA
binding activity was stimulated by acute injection of PRL to
HEF/hCG-PRL (24 h) rats (Fig. 2A
, lane 8). Supershift analysis
demonstrated that the DNA binding complex in H granulosa cells
contained exclusively Stat3, not Stat5a or Stat5b (Fig. 2B
left
panel), while the complex in luteal cells is composed
predominantly of Stat5b and some Stat5a but not Stat3 (Fig. 2B
, right panel). Both the Stat3 and Stat5 complexes were
competed by incubation with 10-fold excess unlabeled probe DNA (Fig. 2B
). Supershift analysis for Stat1 has repeatedly failed to detect any
Stat1 binding activity in any ovarian extract (not shown; Refs. 33, 34).

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Figure 2. Stat Binding Activity in Granulosa Cells and CL:
Shift from Stat3 to Stat5 with Luteinization
A, DNA binding activities in isolated immature/atretic (H) and
preovulatory (HEF) granulosa cells or CL (HEF/hCG). WCE prepared from
granulosa cells or luteal tissue of hypophysectomized (H) rats with or
without E, FSH, and PRL stimulation were incubated with labeled 2M
PRL-RE probe and analyzed by EMSA as described in Materials and
Methods. Animals were treated with PRL either for 24 h by
ip injection or with a 1-h pulse iv injection, or for 24 h
followed by a 1-h pulse as indicated. B, Supershift analysis of protein
complexes binding the 2M PRL-RE probe in extracts of H granulosa
cells and HEF/hCG+PRL 24+1 h-treated CL. Antisera to Stat3, Stat5a, or
Stat5b were incubated with protein extracts for 15 min before
incubation with PRL-RE probe and EMSA as for panel A.
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Extracts of CL isolated on days 49 of gestation contained no
constitutive PRL-RE binding complexes. However, within 5 min of
exogenous PRL administration on days 4, 7, and 9, an intense but
transient DNA binding activity was induced (Fig. 3A
). A more complete time course of PRL
treatment on day 7 of gestation demonstrated maximal binding after 5
min with decreasing amounts at 15 and 30 min and none by 60 min. These
rapid kinetics of Stat5 activation/deactivation differ from those
observed in the H rat model (data herein and Ref. 33) where maximal
activation of Stat5b occurred 1 h after PRL injection. On days
1115 of gestation a constitutive protein/DNA complex was formed, and
exogenous PRL administration had little or no influence on the
magnitude or duration of activity (Fig. 3A
). Supershift analysis of the
induced complex present on day 7 and the constitutive complex from day
15 CL confirmed that the activity observed was Stat5b (Fig. 3B
).
Stat3-specific antibody did not supershift any of either complex.

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Figure 3. Stat5b Is Transiently Activated in Response to PRL
in CL of Early Gestation but Constitutively Active in Midluteotropic
Phase of Gestation
A, EMSA of protein complexes binding labeled PRL-RE probe in extracts
of isolated CL from pregnant rats before or 5 min after iv injection of
10 µg PRL on days 4, 7, 9, 11, 12, and 15 of gestation. During early
(day 7) and mid (day 15) luteotropic phases of pregnancy, more complete
time courses of response to exogenous PRL were examined. B, Supershift
analysis of PRL inducible (day 7, left panel) and
constitutive (day 15, right panel) DNA binding complexes
from early or midgestation, respectively. Note exposure of the
right panel was 3 times longer than that in the
left panel, enabling supershifted bands to be more
easily visualized.
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Thus, four distinct phases of Stat-DNA binding activity were identified
during differentiation of ovarian cells. Constitutive Stat3 activity is
present in immature/atretic (H) granulosa cells, while no Stat activity
is detectable in preovulatory granulosa cells. In CL of nonpregnant
(HEF/hCG) rats or rats at early stages of gestation (days 49),
Stat5a/b are inducibly activated by PRL while after day 9 of gestation
Stat5b retains a low constitutive activity.
Regulation of Cytokine Signaling Pathways: Phosphorylation and
Expression of Stat3 and Stat5
To investigate whether Stat3 activation is unique to granulosa
cells of H rats or whether activated Stat3 is prevented from binding
DNA through additional changes in the hormonally differentiated
granulosa cells, we analyzed the levels of phospho-Stat3 (P-Stat3) in
extracts of granulosa cells at each stage of differentiation. As shown
by Western blot analyses using a specific P-Stat3 antibody, levels of
P-Stat3 were high in granulosa cells of H rats (Fig. 4A
, lane 1), affirming the Stat3 DNA
binding activity seen at this time. Despite the absence of DNA binding
activity in HEF extracts (Fig. 2A
), P-Stat3 remained present in
E+FSH-treated granulosa cells and hCG stimulated luteal cells (Fig. 4A
, lanes 26), albeit at 5060% reduced levels. PRL treatment had no
influence on the phosphorylation of Stat3 in granulosa cells at any
stage of differentiation (Fig. 4A
, lanes 3, 5, and 6). Protein extracts
of CL isolated from pregnant rats on days 7 and 15 of gestation had
lower amounts (1020%) of P-Stat3, which appeared slightly
induced 1 h after iv PRL administration (Fig. 4A
, right
panel). Total Stat3 content in these extracts remained consistent
except for a 60% reduction in HEF granulosa cells (Fig. 4A
, lower panel).

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Figure 4. Stat3 Phosphorylation and Stat Expression during
Stages of Follicle Differentiation and Luteinization
A, Immunoblot analysis using antibodies specific for Stat3
phosphorylated on tyrosine-705 (upper panel); after
stripping the same blot was reprobed for total Stat3 content of cells
(lower panel). Protein extracts from isolated granulosa
cells of immature/atretic (H) or preovulatory granulosa cells (HEF) or
luteal cells (HEF/hCG) were isolated before or after PRL treatment for
1 h or 24+1 h as indicated or from CL of pregnant rats before or
after 1 h PRL treatment on the indicated days of gestation.
Different blots were probed using specific antibodies for Stat5a (B,
upper panel), or Stat5b (B, lower panel); a
slower migrating Stat5b immunoreactive band was evident 1 h after
PRL treatment in CL extracts (arrow) corresponding to
phosphorylated Stat5b. Rats were treated with PRL for 1 h, 24
h, or 24+1 h as indicated.
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Western blot analyses using specific antibodies for the detection of
Stat5a and Stat5b were also performed. Stat5a protein was present and
showed little regulation throughout granulosa and luteal cell
differentiation (Fig. 4B
, upper panel). Stat5b protein was
also present in granulosa cell extracts and increased approximately
2-fold in luteal cell extracts of HEF/hCG rats. After 1 h PRL
treatment in extracts prepared from luteal cells of HEF/hCG rats, a
slower migrating band was observed (Fig. 4B
, lower panel,
arrow), representing a phosphorylated form of Stat5b and
supporting the observed Stat5b activation in these samples in EMSA
experiments (Fig. 2
). Concentrations of Stat5a/b in extracts of
pregnant CL were comparable to those in CL of HEF/hCG-treated rats and
remained constant throughout gestation (Fig. 4B
, right
panels). The phosphorylated form of Stat5b was evident in
PRL-treated extracts of day 7 CL and was constitutively present on day
15.
Stat Signaling Regulators
Downstream of ligand-receptor interaction several mechanisms
modulate cytokine signaling. One such regulator is PIAS-3. To determine
whether this inhibitor might be involved in the regulation of ovarian
Stat activity, specific primers for PIAS-3 were designed and used to
amplify a specific band of the predicted size (308 bp). This band was
cloned into the PCRII-topo vector and sequenced confirming the identity
of the amplicon as PIAS-3. RT-PCR analyses showed that PIAS-3 mRNA
expression was low in H rat granulosa cells but increased 4-fold in
response to treatment with E+FSH (Fig. 5
, lanes 1 and 2, respectively). In CL of HEF/hCG- treated rats, PIAS-3
expression declined slightly (lanes 58), was low in RNA from CL of
pregnant rats on days 7, and approached nondetectable levels on days 15
and 22 of gestation. Thus, the highest levels of PIAS-3 mRNA were
observed in the HEF granulosa cells that contained phosphorylated Stat3
(Fig. 4A
) but no DNA binding activity (Fig. 2
).

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Figure 5. PIAS-3 Expression Is Induced during Granulosa Cell
Differentiation
Semiquantitative RT-PCR analysis using specific primer sets for PIAS-3
and L-19 as internal control. Total RNA was isolated from
immature/atretic (H), or preovulatory granulosa cells (HEF) or luteal
cells (HEF/hCG) before or after PRL treatment for 1 h, 24 h,
or 24+1 h as indicated or from pregnant rat CL on the indicated days of
gestation (right panels). Lower panels
are mean ± SEM of phosphorimage analysis from three
separate RT-PCR assays.
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To investigate the importance of the SH-2 domain-containing
phosphatases, SHP-1 and SHP-2, as potential regulators of Stat
activation, specific RT-PCR assays for each of these phosphatases were
designed and used to analyze their expression in granulosa and luteal
cells. Single bands of predicted sizes were obtained in RT-PCR of
ovarian cell RNA for SHP-1 (443 bp) and SHP-2 (223 bp). Products from
all RT-PCRs showed the predicted restriction digest patterns,
confirming their specific identities (data not shown). SHP-2 was highly
expressed at relatively constant levels in granulosa and luteal cells
of H, HEF-, and HEF/hCG-treated rats with or without PRL treatment
(Fig. 6A
, left panel). Levels
of SHP-2 mRNA remained high through both luteotropic (days 7 and 15)
and luteolytic (d22) phases of pregnancy (Fig. 6A
, right
panel). Western blot also showed SHP-2 present and not regulated
throughout stages of follicle differentiation and luteal function (Fig. 6B
).

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Figure 6. Expression of SHP-2 Is Not Regulated during
Follicle Differentiation and Luteinization
A, Semiquantitative RT-PCR analysis using specific primer sets for
SHP-2 and L-19 and RNA from granulosa cells or CL of hormone-treated H
rats isolated either before or after PRL stimulation for 1 h,
24 h, or 24 h followed by 1 h pulse as indicated. CL of
pregnant rats were isolated on day 7, 15, and 22 of gestation as
indicated. Upper panel shows autoradiography from one
representative experiment; lower panel shows combined
mean ± SEM data from three repeated experiments
quantitated by phosphorimage analysis. B, Immunoblot analysis of SHP-2
protein in extracts from isolated granulosa cells or CL of H rats that
received the same treatments as in panel A, or pregnant rats on days 7,
15, or 22 of gestation before or after 1 h PRL treatment.
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In contrast, expression of SHP-1 showed dramatic changes with
hormonal treatments. Granulosa cells of H rats contained moderately
detectable SHP-1 mRNA, which declined to nondetectable concentrations
in HEF granulosa cells both with or without PRL treatment (Fig. 7A
). Expression increased 4-fold after
hCG treatment compared with HEF samples and increased 2-fold further
24 h after PRL treatment of HEF/hCG rats (Fig. 7A
). SHP-1 was also
expressed at elevated levels in CL of pregnant rats (Fig. 7A
, right panel) where the ratio of SHP-1/L-19 was 2-fold higher
than in HEF/hCG samples (Fig. 7A
, right panel; note that the
autoradiogram was exposed for less time than for HEF samples) and
remained elevated through the luteolytic phase (Fig. 7
). Western blot
analysis of whole cell protein extracts from granulosa and luteal cells
confirmed observations of mRNA regulation of SHP-1. Immunoreactive
SHP-1 was low but clearly detectable in H extracts and declined to
undetectable levels in preovulatory granulosa cells. After
luteinization, SHP-1 protein concentration increased and remained high
in luteal extracts of HEF/hCG-treated rats and throughout gestation
(Fig. 7B
).

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Figure 7. Expression of SHP-1 Is Hormonally Regulated during
Follicle Differentiation and Luteinization
A, Semiquantitative RT-PCR analysis using specific primer sets for
SHP-1 and L-19 internal control. Total RNA was from granulosa cells or
CL of hormone- treated H rats isolated either before or after PRL
stimulation for 1 h, 24 h, or 24 h followed by 1 h
pulse as indicated. CL of pregnant rats were isolated on day 7, 15, and
22 of gestation as indicated. Upper panel shows
autoradiography from one representative experiment; lower
panel shows combined mean ± SEM data from
three repeated experiments quantitated by phosphorimage analysis. B,
Immunoblot analysis of SHP-1 protein in extracts from isolated
granulosa cells or CL of H rats that recieved the same treatments as in
panel A or pregnant rats on days 7, 15, or 22 of gestation before or
after 1 h PRL treatment.
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Cellular and Subcellular Localization of SHP-1
Immunohistochemical localization of SHP-1 in ovaries from H,
HEF, and HEF/hCG-treated rats as well as pregnant rats confirmed the
results from RT-PCR and Western analysis and further revealed dynamic
changes in the cellular and subcellular localization of this protein in
granulosa/luteal cells at different stages of differentiation. In H and
HEF-treated ovaries, SHP-1 protein was localized in the cytoplasm of
granulosa cells in small follicles that had the appearance of being
atretic. Healthy follicles present in H and HEF ovaries had no
detectable SHP-1 (Fig. 8
, A, B, E, and
F). SHP-1 was detected in nuclei of a subpopulation of cells throughout
the ovarian stroma surrounding healthy follicles of H and HEF ovaries
(Fig. 8F
). The highest amount of immunoreactive SHP-1 was observed in
CL of HEF/hCG-treated rats (data not shown) and pregnant rats in early
(day 7) and middle (day 15) stages of gestation (Fig. 7
, C, D, G, and
H). In luteal cells, SHP-1 was selectively localized in nuclei
irrespective of PRL treatment or levels of endogenous PRL/rPL. The
distinct cellular and subcellular compartmentalization of SHP-1 in
atretic granulosa cells compared with luteal tissue suggests differing
functional roles possibly related to the specific activation of
Stat3 vs. Stat5 and atresia vs.
luteinization, respectively.

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|
Figure 8. Changes in Cellular and Subcellular
Immunolocalization of SHP-1 Protein in Immature, Preovulatory, or
Luteinized Ovaries
Ovaries from H rats (A and E), rats treated with E and FSH (B and F),
and pregnant rats on day 7 (C and G) or 15 (D and H) of gestation were
subjected to immunohistochemical detection of SHP-1 using nickel
chloride diaminobenzidine color enhancement (black
coloration) as described in Materials and
Methods. Shown at 50x (upper panels) and 150x
(lower panels) magnification. SHP-1 immunoreactivity was
present in cytoplasm of granulosa cells in atretic follicles
(arrowheads) but was not detected in healthy follicle
granulosa cells (gc). In CL, strong staining was detected in nuclei of
luteal cells on days 7 (C and G) and 15 (D and H) of gestation. Atretic
follicles in pregnant rat sections also showed intense cytoplasmic
staining (not shown).
|
|
 |
DISCUSSION
|
---|
In the ovary, follicles are predestined to undergo atresia and
degenerate by apoptotic processes unless rescued and induced to grow,
ovulate, and luteinize by hormonal stimuli. The transition from
immature follicles to terminally differentiated CL involves specific
reprogramming of cellular responsiveness to external signals (such as
gonadotropins and cytokines), through changing interactions of positive
and negative intracellular regulatory elements. Results of the studies
described herein indicate that the actions of the gonadotropins and
steroids are linked to specific changes in cytokine-signaling pathways
as ovarian cells differentiate. We document that increases in PRL-R
mRNAs, specifically a marked increase in PRL-RS, are
associated with luteinization and related to enhanced activation of
Stat5b during luteinization and in functional CL of pregnancy. Thus,
PRL-RS may not exert a dominant negative regulatory
mechanism in luteal cells. We also show for the first time that Stat3
is selectively activated in granulosa cells of small follicles by a
factor other than PRL, and that the DNA binding capacity of Stat3 is
reduced in growing follicles, possibly by the up-regulation of the
specific inhibitory protein PIAS-3. Lastly, the expression as well as
the cellular and subcellular localization of SHP-1 are selectively
regulated. SHP-1 was elevated and cytoplasmic in granulosa cells of
small atretic follicles, absent in healthy growing follicles, and then
expressed at elevated levels in luteal cells where it is specifically
nuclear.
The differentiation of granulosa cells by E+FSH to preovulatory stage
is associated with several changes in the PRL signaling cascade, most
notably the pronounced increase in the amount of PRL-RL
(but not PRL-RS) mRNA. Despite an approximately 27-fold
increase in preovulatory granulosa cells compared with small follicles
of H rats, no Stat activation occurred after PRL administration. The
increased PRL-RL expression in preovulatory (compared with
immature) granulosa cells suggests a role for PRL in mature follicles,
one of which is the time-dependent decrease in expression of its own
receptor. Whether such a negative feedback mechanism serves a
physiological function in preovulatory follicles is not yet clear, but
similar observations have been noted in other tissues (37, 38, 39, 40). Since
PRL does not activate Stat5b in granulosa cells, alternative signaling
pathways appear to be involved at this time (41, 42, 43), while clearly
factors other than PRL-RL are requisite for PRL-Stat5
signaling in ovarian cells.
PRL-RS prevalently increased (10-fold vs.
4-fold) after luteinization in HEF/hCG rats in which PRL-responsive
Stat5 activation was observed. PRL-RS further increased in
pregnant rat CL, attaining levels only 3.7-fold less than the long form
on day 15, which contains rPL-mediated constitutively active Stat5. The
up-regulation and relative abundance of PRL-RS in
lactogen-responsive luteal cells suggests that it may not exert potent
inhibitory effects on ovarian gene expression as demonstrated for milk
protein gene activation in cultured cells of mammary epithelial origin
cotransfected with PRL-RS and PRL-RL (23, 24).
Rather, the appearance of high PRL-RS is associated with
PRL-induced activation of Stat5b and induction of
2M gene expression
as well as other genes in the CL, including LH receptor (43, 44),
aromatase (30), and ER
(27). In support of our observations,
identical patterns of PRL binding were reported in similarly treated
granulosa and luteal cells (36). Highly quantitative RT-PCR analysis of
whole ovary mRNA (3) indicated that PRL-RL transcripts are
1 order of magnitude more abundant than PRL-RS in
proestrous ovaries, whereas PRL-RL and PRL-RS
mRNAs were essentially equal in diestrus-I ovaries containing newly
formed CL. Furthermore, in studies employing a different
semiquantitative RT-PCR analysis, both isoforms showed equal expression
in RT-PCR of RNA of rat CL day 15 of pregnancy (45), a time when PRL is
critical to functional CL maintenance. Thus, the acquisition and
maintenance of Stat5b activation and expression of PRL-responsive
luteal genes such as
2M correlates with a high
PRL-RS/PRL-RL ratio compared with that in Stat5
quiescent granulosa cells. That PRL-RS is directly involved
with acquisition of Stat5 responsiveness cannot be concluded, but its
relative abundance renders it unlikely that PRL-RS exerts
dominant negative influence on PRL signaling in luteal cells.
One potentially important difference between ovarian and mammary cells
is that the milk protein genes transcriptionally repressed by
PRL-RS are selectively Stat5a-induced genes (46, 47),
whereas we have found that ovarian
2M expression is Stat5b dependent
(34, 35) and PRL activated predominantly Stat5b in luteinized cells
despite the presence of high levels of Stat5a (present study).
Additionally, PRL-RS may antagonize milk protein gene
induction by mechanisms other than or in addition to changing Stat
phosphorylation, such as by modulating nuclear translocation (19).
Furthermore, in the ovary, PRL-RS has been shown to
specifically interact with the CL-specific
PRL-RS-associated phosphoprotein (48, 49), which has
recently been identified as 17ß-hydroxysteroid dehydrogenase-7 (50).
These associations link the PRL-signaling pathway not only to the
regulation of estrogen receptor (27) but also to the endogenous
production of estradiol within the CL from exogenous testosterone (51).
Estradiol is a potent inducer of protein kinase C
(52), which has
recently been shown to enhance PRL- regulated expression of relaxin
(53), a mediator of CL formation and function (54). Thus, luteal
PRL-RS may serve significant luteotropic functions that are
independent of PRL-RL and Stat5b.
The presence of distinct, active Stat/DNA complexes at defined stages
of granulosa cell differentiation indicates that Stat3 and Stat5b
control different cellular functions and are regulated by distinct
pathways. That activated Stat3 was observed in the granulosa cells of H
rats, while E or FSH treatment caused dramatic reductions in its DNA
binding activity, suggests that E and FSH may regulate expression of
the ligand or the receptor involved in activating Stat3. By analyzing
the phosphorylation of Stat3 using a specific phospho-Stat3 antibody,
we were able to demonstrate that although the amount of phospho-Stat3
in healthy growing follicles decreased, significant amounts
of phosphorylated Stat3 remained present in differentiated granulosa
cells. The absence of Stat3 binding to DNA in granulosa cells treated
with E+FSH was temporally associated with a 4-fold increase in PIAS-3
expression, a negative regulator of Stat3 DNA binding activity. These
results provide evidence that suppression of Stat3 function may be
requisite for steroid- and gonadotropin-supported stages of growth and
differentiation of follicles as opposed growth of primary follicles or
apoptosis. Since many of the follicles in immature and H rats are
undergoing atresia, it is possible that activated Stat3, as well as
SHP-1, are associated with the apoptosis of granulosa cells. SHP-1 may
mediate the atretogenic effect of angiotensin-II (55) through
suppression of FSH-mediated growth signals involving the inhibition of
extracellular regulated kinase activity (56). Thus, down-regulation of
SHP-1 and induction of PIAS-3 may be important events in rescue of
follicles from atresia. Although a selective role for Stat3 in small
follicles is not yet known, activated Stat3 has been linked in other
systems to both proapoptotic (57) and antiapoptotic cell survival
pathways (58). When the ligand activating Stat3 becomes known and its
targets identified, the role of Stat3 in early follicular cell function
will be clearer.
In contrast to Stat3, an essential role for Stat5 in ovarian cell
function is evidenced by the phenotypes of a line of Stat5b-specific
null mutant mice that prematurely abort litters between days 8 and 17
of gestation unless exogenous progesterone is administered to
substitute for CL function (59). Interestingly
Stat5a/b-/- mice fail to produce CL at all
(47), suggesting that Stat5a can act in a redundant or alternative but
requisite fashion at least in early stages of luteal function in the
absence of Stat5b, and that acquisition of PRL-inducible Stat5
activation and the resulting changes in gene expression outlined above
are essential steps in the transition from granulosa to luteal cells.
In this study we found that Stat5b is specifically induced in response
to an exogenous pulse of PRL administered in a transient fashion in
early gestation, while from day 11 constitutive Stat5b activation is
unresponsive to the same exogenous PRL dose. These two patterns of
activation probably result from the change in lactogen secretion in the
latter stages of gestation when the placenta replaces the
pituitary as the major source of lactogenic hormone secretion. Whether
these different patterns of Stat5b activation mediate changes in luteal
gene expression in early vs. late CL, as has been
demonstrated for signaling through Stat5 by interleukin vs.
erythropoietin (60), will be interesting to determine. It is known that
2M at this time undergoes a secondary phase increase in expression
(31, 32, 33), while progesterone production and luteal cell hypertrophy
also undergo incremental increases (2).
Activation of Stats is complex and involves many factors, and in some
situations gene regulation may be dependent on signal duration (25, 60, 61, 62), indicating that the deactivation of signals is as important as
the ligand- dependent activation in establishing and maintaining tissue
responsiveness. In this regard, protein tyrosine phosphatases have been
shown to play key roles in cytokine signaling and cell function. The
tyrosine phosphatase SHP-2 plays an essential mediatory role in
PRL-regulated milk protein gene induction (11) as well as in signal
transduction by several growth factors and interferon-
/ß
involving activation of Stats1 and 2 (63). Therefore, we initially
hypothesized that the acquisition of Stat5 responsiveness and/or
changes in rapid and transient vs. chronic Stat5b activation
in luteal cells at specific times during pregnancy might be related to
changes in the expression of SHP-2 mRNA and protein. However, SHP-2 was
present with little change in abundance throughout granulosa cell
differentiation, luteinization, and through to the onset of luteal
regression. Thus, although this phosphatase may be a necessary mediator
of PRL-regulated gene expression, its presence is not a limiting factor
in luteal cells.
In contrast, we did observe a positive correlation between the presence
of activated Stats and the expression of SHP-1, a tyrosine phosphatase
thought to be required for recycling of activated Stat5b and
maintaining responsiveness to pulsatile GH in hepatocytes (25, 61). In
the ovary, SHP-1 expression was detected in H granulosa cells when
Stat3-DNA binding is detected. As discussed above, it is suggested that
SHP-1 may be involved with the process of follicular atresia. SHP-1
expression (mRNA and protein) was undetectable in preovulatory
follicles but maximal in luteal cells, in a temporal pattern that
mimics the acquisition of PRL-responsive activation of Stat5b.
Immunoreactivity for SHP-1 was intensely localized in the nuclei of
luteal cells. Transient (<1 h) Stat5b activation in PRL-treated CL
during early gestation implicates the activity of a phosphatase such as
SHP-1 in rapid dephosphorylation of Stat5b in CL. SHP-1 was expressed
at its highest level and remained specifically nuclear in CL at day 15
of gestation, a time when Stat5b-DNA binding was constitutively
maintained. We propose that nuclear SHP-1 may continuously
dephosphorylate activated Stat5b, recycling it to the cytoplasm and
enabling reactivation through continuous rPL/PRL-R stimulation in
midpregnancy. Thus, nuclear SHP-1 may be requisite for mediating the
rapid turnover of activated Stat5b, resulting in a transient activation
profile during pulsatile PRL exposure and maintaining a pool of
inactive Stat5b able to respond to constant circulating lactogens in
midterm pregnant rats. While this manuscript was under review, it was
reported that a vanadate-sensitive factor, probably SHP-1, is involved
in the constant turnover of activated Stat5b in GH-treated liver cells.
Moreover, enhanced dephosphorylation of Stat5b (as well as Jak2) in
constant GH-exposed cells results in a lower continuous activation
similar to that seen in midpregnant luteal cells (64). In contrast to
GH signaling in female liver, constant rPL exposure in luteal cells and
the resulting low constitutive Stat5b activation have enhanced tropic
effects on midgestational CL function.
Attractive as this hypothesis for the role of SHP-1 might be, a
functional role in the corpus luteum remains to be verified. A
naturally occurring mutation in SHP-1 gene has been described in the
motheaten strain of mice, which are infertile and fail to form CL (65).
However, when ovaries of the mutant mice were transplanted to wild-type
recipients, fertility (normal ovarian function?) was rescued. These
results indicate that the defect may not be intrinsic to the ovary,
that factors present in the recipient regulate other SHP-1-like
activities in the mutant ovaries, or that SHP-1 is present and plays a
role in extraovarian (immune?) cells to regulate fertility and CL
formation. The presence of SHP-1 in the nuclei of luteal cells suggests
that there is an intrinsic role for this phosphatase in ovarian cells;
however, it should be noted that SHP-1 expression was present in
additional cells surrounding follicles (Fig. 6
, B and F). This
expression pattern mimicked that seen with macrophage-specific
antibodies (Ref. 66 and R. Robker and J. S. Richards,
unpublished). Since SHP-1 is a known modulator of immune cell function
and leukocytes have numerous important actions in the ovary (66),
immune defects, the predominant phenotype of motheaten mice, may
explain their ovarian failure.
In conclusion, we have demonstrated a dynamic transition in Stat
activation during the maturation of ovarian follicle cells. In
undifferentiated/atretic follicles deprived of gonadotropin, Stat3-DNA
binding is uniquely active possibly because a permissive milieu is
maintained through low PIAS-3 expression. Increased PIAS-3 mRNA in
granulosa cells of growing follicles suggests that suppression of
Stat3-DNA binding may be an important requirement for progression of
follicle growth and differentiation. Specific expression of SHP-1 and
localization to the cytoplasm in atretic granulosa cells may also
regulate Stat signaling and mediate apoptotic events, directing cells
toward atresia rather than growth and differentiation. Acquisition of
Stat5b responsiveness to PRL is initiated after luteinization and
associated with increased PRL-R, most strikingly increased
PRL-RS, providing evidence that the transition from
granulosa cells to luteal cells is not related to a dominant negative
effect of PRL-RS on Stat5b activation. Additionally, high
PRL-RL expression alone in granulosa cells appears
insufficient to activate Stat5 signaling. Lastly, our results indicate
that SHP-1 is temporally expressed in a pattern that mimics the
cellular activation of Stats. Atresia in granulosa cells involves
moderate expression of cytoplasmic SHP-1. Luteal cells express higher
levels of SHP-1 where it is localized to nuclei and may be an important
requirement for maintaining PRL responsiveness through Stat5
deactivation and recycling.
 |
MATERIALS AND METHODS
|
---|
Materials
17ß-Estradiol was purchased from Sigma (St Louis
MO), ovine FSH and PRL from National Hormone and Pituitary Program
(Baltimore MD), hCG from Organon special chemicals (West
Orange NJ) and [
-32P] from ICN Biochemicals, Inc. (Cleveland OH). BenchMark molecular size markers were from
Life Technologies, Inc.(Gaithersburg, MD), and the
enhanced chemiluminescence (ECL) detection system was from
Amersham Pharmacia Biotech (Arlington Heights IL). Kodak
X-Omat AR film was from Eastman Kodak Co. (Rochester, NY).
AMV-Reverse Transcriptase and Taq Polymerase were from
Promega Corp. (Madison WI). Anti-Stat5a and
Stat5b-specific antibodies, catalog nos. sc-1081 and sc-835,
respectively, were from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA ). Anti-Stat3 antiserum used for supershift analysis
was generously provided by Dr. David Levy (New York School of Medicine,
New York, NY). Stat3 Western blots employed an antibody purchased from
Santa Cruz Biotechnology, Inc. (catalog no. 7179), P-Stat3
antiserum was from New England Biolabs, Inc. (Beverly,
MA). Antibodies to SHP-1 and SHP-2 were from Transduction Laboratories, Inc. (Lexington KY).
Animals
Pregnant, hypophysectomized, and immature (day 26) rats were
purchased from Harlan Bioproducts for Science, Inc.
(Indianapolis, IN), provided food and water ad libitum, and
housed under a 16-h light, 8-h dark schedule. Animals were treated in
accordance with the NIH Guide for Care and Use of Laboratory Animals.
Protocols were approved by the Institutional Animal Care and Use
Committee, Baylor College of Medicine (Houston TX).
H Rats
Proliferation and differentiation of ovarian follicles were stimulated
in H rats by hormonal treatment as described previously (33). Follicles
of H rats inevitably become atretic and undergo apoptotic degeneration
unless rescued by E and FSH administration (67). Commencing 34 days
after hypophysectomy, H rats received sc injections of 17ß-estradiol
(1.5 mg) for 3 consecutive days, followed on the next 2 days with two
injections of FSH (1 µg) each day (HEF rats). Luteinization was
induced in HEF rats by ip injection of 10 IU hCG (HEF/hCG). At each
stage of follicular development, animals were killed either before or
1 h after tail vein injection of 10 µg PRL. Additional groups of
HEF- and HEF/hCG-treated rats were treated ip with PRL 24 h before
receiving a second iv PRL injection 1 h before ovaries were
collected. Each treatment group included 6 rats except for the H group
for which 16 animals were used. After removal, ovaries were divided at
random into two groups for RNA or WCE preparation.
Pregnant Rats
Day 1 of pregnancy was assigned as the day a sperm-positive vaginal
swab was observed. On selected days of pregnancy, ovaries were
collected from two rats either before or 5 min after iv PRL
administration. On days 7 and 15, representing the early (pulsatile
endogenous PRL) and mid (continuous rPL) luteotropic stages of
gestation, a time course of response to exogenous PRL was examined by
collecting ovaries 5, 15, 30, and 60 min after PRL injection. All PRL
treatments (10 µg) were given at approximately 1100 h.
WCE and RNA Isolation
Ovaries were extirpated and granulosa cells were isolated from
preovulatory ovaries by puncture with a 26-gauge needle, or CL were
dissected from luteinized ovaries. Granulosa cells isolated from
preovulatory ovaries were resuspended in 150200 µl of 10
mM Tris buffer containing 1 mM EDTA, 1
mM dithiothreitol, 10% glycerol, 400 mM
potassium chloride, 1 mM vanadate, and protease inhibitors
(WCE buffer; Ref. 68). Cells and nuclei were lysed by three rapid
freeze-thaw cycles and centrifuged at 12,000 x g, and
protein concentrations of soluble extracts were measured (Bradford
method, Bio-Rad Laboratories, Inc., Richmond CA). Isolated
CL were homogenized at 4 C in WCE buffer and then treated as for
granulosa cell extracts.
For RNA extraction, granulosa cells or CL were homogenized in 25
mM Tris buffer containing 1% Nonidet P-40. RNA was
extracted in phenol-chloroform, ethanol precipitated, and resuspended
in RNase free water.
EMSA
EMSA were performed as described previously (33). Briefly,
protein extracts (15 µg per lane) were incubated for 30 min at room
temperature with 50,000 cpm of end-labeled double-stranded
oligonucleotide probe and poly(deoxyinosinic-deoxycytidylic)acid in
a final buffer volume of 20 µl containing 15 mM Tris-HCl
(pH 7.5) 100 mM KCl, 5 mM dithiothreitol, 1
mM EDTA, 5 mM MgCl2, and 12%
glycerol. For supershift and competition experiments, antibodies or
10-fold excess of unlabeled competitor DNA were incubated with extracts
for 30 min on ice before labeled probe DNA was added. Bound probe-DNA
complexes were resolved on 5% acrylamide gel electrophoresis before
autoradiography. The sequences of oligonucleotides used for
2M
PRL-RE probe were as follows::
5'-TGGATCATCCTTCTGGGAATTCTGATATCCTTC-TGGGAATTCTG-3'
annealed to the reverse complimentary strand.
PRL-R Competitive PCR
Quantitative PCR assays for PRL-RL and
PRL-RS were developed using modifications of the method of
Nagano et al. (3). A sense PRL-R oligonucleotide primer
targeted to the conserved extracellular domain present in all rat PRL-R
isoforms used for amplification of both PRL-R isoforms was
5'-ATACTGGAGTAGATGGAGCCAGGAGAGTTC-3'. Specific antisense
primer to the divergent cytoplasmic domains of the long isoform
sequence was 5'-CTTCCGTGACCAGAGTCACTGTCGGGATCT-3', and the short
isoform was 5'-TCCTATTTGAGTCTGCAGCTTCAGTAGTCA-3'. These primer pairs
gave predicted amplification products of 422 bp and 332 bp from the
long and short PRL-R, respectively. Amplicons from long and short
PRL-R PCR with 100-bp deletions were cloned into the pCRII-topo vector
(Invitrogen, Carlsbad CA). Total RNA (350 ng) from each
sample was reverse transcribed in a 25 µl total volume containing 500
ng oligo-dT1218 and 1 IU AMV-RT at 42 C for 90 min; 4
µl of RT reaction mix was aliquoted into each of five tubes
containing titrated concentrations of competitor cDNA from
0.0110 x 106 cDNA copies and 15 µl PCR reaction
cocktail [20 mM Tris-HCl (pH 8.0), 2.5 mM
MgCl2 and 100 mM NaCl, 2.5 IU Taq
DNA polymerase, 1 µCi 32P-dCTP, and 40 pmol forward and
isoform-specific reverse primer]. PCR reactions were performed for 30
cycles at 95 C (2 min), 65 C (2 min), and 72 C (3 min). To confirm
equivalent amounts of each competitor construct and their efficiency in
PCR reactions, PCR was also performed for each competitor dilution
using T7 and SP6 oligonucleotide primers that flank the cloned inserts.
Reaction products were separated on 5% polyacrylamide gels, and
intensity of the competitor and target bands was analyzed as for
semiquantitative PCR and expressed as the log10 of the
target to competitor ratio. When plotted on a log scale against input
competitor concentration, parallel straight lines were obtained for the
titration of competitor against each RNA sample. The Y axis origin
represents the concentration at which competitor and target
amplification are equivalent, and thus the number of cDNA copies
present in the original RT-cDNA mix. This procedure was repeated a
minimum of three times for each RNA sample with highly reproducible
results, and PRL-RL and PRL-RS were analyzed in
parallel in each assay.
Semiquantitative RT-PCR Analysis
Primer pairs based on rat SHP-1 sequence were
5'-AGCCGTGTCATCGTCATGACCACCCGAGAG-3' and
5'-CATC-TGGATGGTCTTCTGGATGTCAATGTC-3'; for rat SHP-2 primer pairs
were 5'-AGCCAGAGCCACCCTGGGGACTTCGTCCTC-3' and
5'-AATACGAGTTGTGTTGAGGGGCTGTTTGAG-3'. Predicted products from SHP-1 and
SHP-2 amplification were 443 bp and 248 bp, respectively. Primers for
PIAS-3 were 5'-CAGATGAATGAGAAGAAGCCGACATGG-3' and
5'-TCTGATGAGCTTTCGATGGTCAAG-3' and generated a product of predicted
size 308 bp. Primer pairs for the internal control ribosomal protein
L-19 were as described previously (69); predicted product size for L-19
PCR amplification was 194 bp. Total RNA (350 ng) was reverse
transcribed using 500 ng oligo-dT1218 primer
(Pharmacia Biotech, Piscataway NJ) at 42 C for 90 min in a
20 µl reaction volume. To the RT reactions were added 80 µl
of buffer containing 20 mM Tris-HCl (pH 8.0), 2.5
mM MgCl2, and 100 mM NaCl, 2.5 IU
Taq DNA polymerase, [32P]dCTP (5 µCi of 3000
Ci/mmol), and specific oligonucleotide primer pairs (5080 pmol) for
each individual gene along with L-19 internal control. Twenty-cycle PCR
reactions (within the linear amplification range for input RNA) were
performed in a DNA Engine thermocycler (MJ Research, Inc.,
Watertown, MA) using conditions of 95 C (2 min) for denaturing, 65 C (2
min) annealing, and 72 C (3 min) extension. Reaction products were
separated on 5% polyacrylamide gels and exposed to Kodak
X-Omat AR x-ray film followed by quantitation of products using a
Storm860 PhosphorImager and ImageQuant version 2.1 software
(Molecular Dynamics, Inc., Sunnyvale, CA). Intensity of
signal for each sample was normalized to the L-19 internal control. All
PCR assays were performed three separate times from the same RNA
samples, and the mean ± SE for normalized results was
calculated. All genes were also analyzed in comparison to ribosomal
protein S-16 internal controls in separate PCR analyses with identical
results to L-19 obtained.
Immunoblot Analyses
Whole-cell protein extracts (50 µg) were resolved on 10%
acrylamide gels by reducing SDS-PAGE, followed by electrophoretic
transfer to polyvinylidine fluoride membrane (Immobilon-P,
Millipore Corp., Bedford, MA). Membranes were blocked by
incubation for 1 h at room temperature with 3% nonfat milk,
followed by 1 h incubation with specific primary antibodies in 3%
milk and washing in TBST [10 mM Tris (pH 7.5), 150
mM NaCl, and 0.05% Tween-20]. Blots were then incubated
with 1:10,000 of horseradish peroxidase-linked antirabbit or antimouse
IgG (Amersham Pharmacia Biotech) followed by six 5-min
washes with TBST. ECL detection was performed according to the
manufacturers specifications. Blots were stripped for subsequent
reanalysis by washing at 50 C for 30 min in 62.5 mM
Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS.
Quantitation from Western blots was performed using a Molecular Dynamics, Inc. densitometer and ImageQuant software.
Immunohistochemistry
SHP-1 tissue and cellular localization was analyzed by
immunostaining of 4% paraformaldehyde-fixed paraffin sections of
ovaries from each of the indicated treatment groups. Rehydrated
sections were boiled in 20 mM sodium citrate for 10 min,
and endogenous peroxidase activity was quenched by 10 min treatment
with 0.1% H2O2 followed by PBS wash.
Nonspecific antibody binding was blocked by 30 min incubation with 10%
nonimmune goat serum after which anti-SHP-1 IgG (Santa Cruz
Biotechnology, Inc.), 0.5 µg/ml in 10% goat serum, was incubated
with sections overnight at room temperature. After washing with PBS,
biotinylated antirabbit antiserum (Vector Laboratories, Inc., Burlingame, CA) was added for 30 min, slides were washed,
and streptavidin-conjugated horseradish peroxidase was applied for 30
min. After washing, sections were incubated with
diaminobenzidine substrate containing nickel chloride color
enhancement (Vector Laboratories, Inc.) for 2 min and then dehydrated
and mounted without counterstaining.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Darryl L. Russell, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.
Received for publication October 27, 1998.
Revision received July 9, 1999.
Accepted for publication August 24, 1999.
 |
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