Department of Cell Biology (E.M.A., J.E.L., J.H.C., B.W.O.)
Baylor College of Medicine Houston, Texas 77030-3498
Department of Pathophysiology (J.G.) University Medical
School of Pécs Pécs, Hungary
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ABSTRACT |
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INTRODUCTION |
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Alternatively, in a variety of transformed nonneuronal cell lines, growth factors (GFs) activate ER-dependent transcription of reporter genes with E-responsive elements in the absence of E (4 5 12 ). Membrane-bound receptors for GFs mediate in vitro activation of signal transduction cascades rather than acting directly as a ligand for ER. In cultured cells, ligand-independent transcription activities of the mouse ER stimulated by EGF were associated with phosphorylation (13 14 15 ).
The distribution and ligand-dependent regulation of the gene encoding
the classical ER have been extensively investigated
throughout the brain of multiple mammalian species, including rats and
mice. Importantly, robust expression of ER
, but not ERß , mRNA
takes place within specific nuclei associated with reproduction
including the ventromedial hypothalamus (VMN) and arcuate nucleus (16 ).
Receptors for GFs such as EGF (17 18 ), insulin-like growth factor I
(IGF-I) (19 ), and other GF ligands (20 21 ) are widely distributed in
the mammalian central nervous system (CNS) including the cerebral
cortex, hippocampus, substantia nigra, and specific hypothalamic nuclei
associated with reproduction. Many biological functions of GFs overlap
with those of E (22 23 24 25 26 ). Of significance, increases in circulating
levels of GFs precede intracerebral aromatization of androgens to E
during sexual differentiation and development (1 2 ) and changes in E
at time of the initiation of puberty (3 25 26 ). Indeed, the
initiation of puberty in females is dependent, in part, on activation
of hypothalamic membrane-bound GF receptors (18 ). Although activation
of intracellular signaling pathways by membrane-bound receptors (and
ion channels) is a key CNS feature, in vivo regulation of
ER-dependent brain function by GFs is unexplored.
Since reproductive behavior in female rodents is the exquisite result of E-dependent transcriptional activity and protein synthesis, lordosis serves as a well established in vivo model for probing cellular and molecular mechanisms of steroid receptor-dependent behavior. In the standard lordosis paradigm, low dose estradiol benzoate (EB) is administered subcutaneously and subsequently a single administration of progesterone (P) 44 h later for the induction of mating behavior [see review in Ref. 21 ]. It has been reported that this pathway is blocked by prior administration of RU486 and is dependent on intact progesterone receptors (PRs) (21 27 28 29 30 ). Importantly, it is also well known that high doses of EB alone can induce lordosis [see review in Ref. 21 ]. We considered the hypothesis that ER-dependent transcription may be activated in vivo in a ligand-independent manner by EGF to produce reproductive behavior.
We present here evidence that GFs can play a fundamental role in the
function of steroid receptors in the adult CNS. We have used mutant
Wa-2 mice with suppressed EGF receptor activity, transgenic mice with
targeted disruption of the PR (PRKO), and pharmacological and antisense
oligonucleotide treatment of rats to demonstrate ligand-independent
activation of reproductive function and ER by EGF and IGF-I in the
adult brain of intact animals. The data demonstrate a novel mechanism
for activation of lordosis, a behavior previously thought to be
dependent upon EB priming over, at least, 2448 h. Here we show
that rapid activation (within 14 h after intracerebroventricular
(icv) administration to the third ventricle) of reproductive behavior
by EGF is EGF receptor- and ER-dependent but not P- or PR-dependent. We
go on to show that, in the absence of EGF receptor activity in intact
mice, the initiation of puberty is delayed and estrus cyclicity and the
synchronization of mating behavior and estrous is disrupted. These
findings demonstrate a greater role for GFs in the adult brain than
previously believed, including the existence and functional utilization
of conserved cross-talk mechanisms between membrane receptors and
nuclear receptors within the hypothalamus.
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RESULTS |
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Females in estrous and OXV females primed with EB + P are known also to display self-initiated, proceptive behaviors including hopping, darting, ear wiggling, and approach toward the male (21 ). Here, animals receiving EGF (icv) in the absence of E exhibited only sporadic proceptive behaviors, unlike females treated with high does of EB (200 µg, sc) or females primed with EB (10 µg sc at -48 h) subsequently followed with P (1 µg sc at -4 h). Likewise, rejection behaviors such as kicking, biting, standing, rolling over, and running away were absent in all females receiving EGF. Males demonstrated interest in the females given an optimal EGF dose in spite of the absence of proceptive behaviors. The absence of proceptive behaviors in EGF-treated animals suggests some divergence in the neural circuits mediating the full repertoire of behaviors associated with reproduction.
EGF in Vivo Effects and Brain ERs
To ascertain whether the in vivo effects of EGF were
mediated, in part, by ERs in the brain, animals were given the
antiestrogen ICI 164,384 into the third ventricle (icv) 1 h before
EGF. As expected, whereas vehicle (Fig. 2, bar 1) failed, EGF (bar 2) induced
target behavior in control animals. Also, the antiestrogen ICI 164,384
(Fig. 3
, bar 3) alone failed to stimulate
lordosis. In contrast, antiestrogen pretreatment suppressed lordotic
responses in females challenged with EGF (Fig. 2
, bar 4), suggesting
that EGF may regulate ER-dependent behavior.
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To specifically analyze dependency of EGF on endogenous ER,
phosphorothiolated antisense (AS) oligonucleotides to the initiation
region of the ER
mRNA were administered 24 h before EGF
challenge. Specificity for these oligonucleotides has been shown
elsewhere (34 ). Nonspecific effects of oligonucleotide treatment were
not apparent in control animals treated with random sense sequence
oligonucleotide (RS) to the AS, RS + EGF, and AS (Fig. 2
, bars 911,
respectively). Significantly, EGF failed to induce lordosis in
the presence of ER
antisense oligonucleotides (Fig. 2
, bar 12). Consistent with the absence of ERß in the VMN
(16 ), oligonucleotides to ERß failed to have any effect on
EGF or EB+P induction of mating behavior (data not shown). Further,
while this manuscript was being prepared, Krege and colleagues (35 )
reported that transgenic mice lacking the ERß receptor display normal
sex behavior. Thus, the combined findings conclusively implicate
hypothalamic ER
as a mediator of EGF and functional behavioral
changes in OVX rats.
EGF in Vivo Function Independent of PR
In vitro studies have shown some ligand-independent
gene activation of the avian PR by EGF (36 ). We (29 30 37 38 ) and
others (27 39 ) have shown that lordosis is dependent, in part, upon
the unoccupied PR. Further, for at least 7 days after OVX, PR can be
detected in the female hypothalamus (40, Fig. 1, panel C) and
extrahypothalamic regions (41 42 ). To identify the functional
significance of PR in the above model, the antiprogestin RU486 was
given icv 1 h before EGF challenge. As expected in the control
animals treated with either vehicle, EGF, or RU486, EGF alone brought
about lordosis (Fig. 3
, bars 13). Of significance, EGF induced
lordosis in spite of RU486 pretreatment (Fig. 3
, bar 4). Next, the full
antiprogestin ZK98299, thought to inhibit DNA binding of PR (43 ), was
tested. Again, whereas ZK98299 alone had no behavioral effect (Fig. 3
, bar 5), lordosis was displayed after EGF challenge (bar 6). Since we
have reported previously that both RU-486 and ZK98299 are effective
inhibitors of PR-dependent reproductive behavior (33 ), the data suggest
the behavioral effect of EGF is not dependent upon unoccupied PR.
Antisense oligonucleotides are known to reduce PR content by more than
50% (38 ) in the portion of the hypothalamus mediating the expression
of steroid-dependent mating behavior, i.e. medial basal
hypothalamus (21 ). To confirm the absence of an EGF effect on
unoccupied PR, PR AS was given icv 44 h before EGF challenge. As
expected and reported elsewhere for E + P treatment (Refs. 29 30 44 ; data not shown), the oligonucleotides lacked a nonspecific effect
in control animals (Fig. 3, bars 79) compared with vehicle-only and
EGF treatment (bars 1 and 2, respectively). Consistent with the above
antiprogestin findings, PR AS failed to block the behavioral effect of
EGF (Fig. 3
, bar 10). In EB-treated animals also receiving PR AS,
lordosis was not induced by P, as expected (29 30 44 ). Finally, we
tested the hypothesis that EGF induces receptivity via a PR-independent
pathway using homozygous mice with targeted disruption of PR (37 ). EGF
induced mating behavior in both wild-type and homozygous PRKO mice
(data not shown), whereas P failed to induce lordosis after EB-priming
in homozygous but not wild-type PRKO mice [data not shown but
previously published (37 )]. Taken together, the findings indicate the
presence of a divergent ER-dependent pathway for activation of
reproductive behavior independent of hypothalamic P and PR in the
female rat.
EGF Receptor and EGF-Induced Behavior
Recombinant human EGF is thought to bind with EGF receptors in
rats. As a 170-kDa transmembrane glycoprotein receptor, EGF receptors
are widely distributed in the rat brain (17 ). To verify the behavioral
specificity of recombinant human EGF for the rat EGF receptor,
polyclonal EGF antibodies to human EGF having no cross-reactivity with
mouse EGF or human insulin, IGF-I, IGF-II, transforming growth
factor- (TGF
), or platelet-derived growth factor (PDGF) (45 ) were
administered icv 2 h before EGF. As shown in Fig. 1
(bar 7),
Fig. 2
(bar 2), and Fig. 3
(bar 2), recombinant EGF induced
mating behavior (Fig. 4A
, bar 2).
Antibodies alone failed to effect rat behavior (Fig. 4A
, bar 3). No
lordosis was displayed after EGF challenge with antibodies (Fig. 4A
, bar 4), suggesting the rat EGF receptor is mediating the effect of
recombinant human EGF.
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Finally, oligonucleotides to the EGF receptor were administered. As
expected, nonspecific oligonucleotide effects were not observed in
animals treated with random sense (RS) or RS + EGF (Fig. 4A, bars 8 and
9). Also as expected, AS alone failed to induce an effect on behavior
(Fig. 4A
, bar 10). However, when AS to rat EGF receptor was
administered 24 h prior to EGF, activation of sex behavior by EGF
was blocked (Fig. 4A
, bar 11). To determine EGF receptor
oligonucleotide specificity, rat fibroblast cells were treated in
culture. EGF receptor was detected when treated with vehicle (Fig. 4B
, lane 1) but not EGF (lane 2), as
expected with EGF receptor degradation (42 ). Likewise, receptor was
detected after treatment with RS (Fig. 4B
, lane 3) but not RS + EGF
(lane 4). Thus, the loss of EGF receptor after RS + EGF suggests that
receptor degradation in the presence of EGF is not altered by RS
treatment. Antisense oligonucleotides were specific for rat EGF
receptor, since Western blot analysis detected a 57% decrease in band
intensity when cells were treated with EGF receptor AS (Fig. 4B
, lane
5) compared with that in vehicle-treated cells (Fig. 4B
, lane 1).
Again, no adverse effect of AS was observed since receptor degradation
(Fig. 4B
, lanes 2 and 4) appears to be present after AS treatment (lane
6). To further test for oligo specificity, immunohistochemistry on rat
brain was performed. As with Western blotting, icv antisense (Fig. 4C
, right panel) but not random sequence (left panel)
oligonucleotide decreased immunoreactive EGF receptor in the
pericellular and nuclei of cells within the rat VMN, again
demonstrating the specificity of the oligonucleotide for rat EGF
receptor. The cellular localization of EGF receptors by
immunohistochemistry is consistent with previous reports (see review in
Ref. 48 ) for several species including humans (49 ).
Wa-2 Mice and EGF-Induced Behavior
To more definitively test for the physiological role of the EGF
receptor signaling, behavioral responsiveness of Wa-2 mice also was
tested. Wa-2 homozygous mice express a single point mutation at residue
743 of the EGF receptor that results in a significant reduction in
EGF-stimulated tyrosine kinase activity (up to >90% reduction) (50 ).
These mice are healthy and are distinguished by pronounced waviness of
whiskers and fur. Since the mutant female mice have infrequent litters
(four to five per year) compared with wild-type, time of onset of
puberty and ovulatory competency was accessed by daily inspection
(between 1300 and 1400 h) for vaginal opening and, once opened,
daily vaginal lavages with assessment of cytology. In rodents, vaginal
opening usually occurs the day after the first preovulatory surge of
gonadotropins (first estrus) and represents the most overt somatic
change associated with initiation of puberty (3 ). When compared with
that of wild-type (n = 30), vaginal opening of mutant mice (n
= 37) was delayed by 18.4 ± 2 days and widely varied (range,
3073 days). Vaginal opening in wild-type Wa-2 mice was observed at
34.5 ± 1 days. In most cases, first diestrus (a predominance of
leukocytes) was detected within 45 days after vaginal opening in
mutant Wa-2 mice compared with its appearance at 12 days in wild-type
mice. Also, unlike wild-type Wa-2 mice, estrus (predominance of
cornified cells) in mutant mice was irregular when vaginal lavages were
obtained daily over a 24-day period starting 1 month after vaginal
opening. For the mutant females, 66% of the estrus cycles were
extended over 2 days compared with >10% for wild-type females.
Collectively, the data support the results of Ojeda and colleagues (18 51 ) who found that EGF receptor signaling plays a critical role
in the initiation of puberty and ovulatory cycling.
Lordosis was displayed in association with 42 ± 10% of the total number of naturally occurring estrus cycles for all adult mutant female mice compared with 88 ± 4% for wild-type mice. However, when estrus was induced by exogenous steroids (34 ), all mutant intact Wa-2 mice displayed receptivity comparable with that of the wild-type mice. This is consistent with enhanced EGF receptor signaling seen in the tissue of mutant animals when given particularly high doses of EGF (50 ). It will be interesting to determine whether this effect of exogenous steroids in Wa-2 mice is due to E-dependent up-regulation of EGF receptors and/or induction of EGF synthesis. E is known to synthesize several members of the EGF receptor family (51 ). Collectively, the data suggest that EGF receptor activity in intact mice contributes to the synchronization of reproductive behavior with estrus and that the biological effect of EGF receptor stimulation can be masked by the administration of exogenous steroids.
Next, we tested Wa-2 mice after ovariectomy (34 ) and sc
injections of EB and P. As expected, all animals failed to display
receptivity after ovariectomy and/or vehicle treatment (Fig. 5, bars
16). Again, since all groups of OVX females exhibited mating behavior
in response to exogenous EB + P (Fig. 5
, bars 79), the neural circuit
for ER-mediated behavior was intact and does not require EGF receptors
for exogenous steroid elicitation of mating behavior. Note
that no mating was displayed after control treatments (bars 1012) 1
week before EGF and immediately before EGF treatment (bars 1315).
Finally, we tested whether EGF would induce behavior in OVX Wa-2 mice
in the absence of steroids. Both wild-type and heterozygous mice
exhibited lordosis in response to an optimal dose of EGF (Fig. 5
, bars 16 and 17). Interestingly, mutant Wa-2 mice failed
to display reproductive behavior (Fig. 5
, bar 18), demonstrating the
specificity of EGF for the EGF receptor and its activity as, in part, a
mediator of functional activity in the rodent brain. Collectively, the
results implicate, at least, the hypothalamic EGF receptor and
activation of its tyrosine kinase cascade in signal transduction for
synchronization of reproductive behavior with reproductive competence
in this in vivo model.
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DISCUSSION |
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We have found that, in the absence of E, OVX rats and mice exhibit
reproductive behavior 1 h after receiving icv EGF and other
ligands (amphiregulin, TGF) for the EGF receptor. This effect was
specific since it was blocked by pretreatment with an EGF
receptor-specific kinase inhibitor, EGF antibodies, or rat EGF receptor
antisense oligonucleotides. More conclusively, EGF induced behavior in
wild-type but not EGF receptor mutant Wa-2 mice. This is consistent
with our unpublished Western analysis using antibodies to EGF receptor
followed by stripping and antibodies to phosphorylated tyrosine kinase
(50 ); i.e. phosphorylated tyrosine kinase is enhanced in
wild-type but not mutant Wa-2 medial basal hypothalami treated with
EGF. In addition, we report several lines of evidence to show that
hypothalamic EGF receptor stimulation converges on ER
to effect this
behavioral change. The antiestrogen ICI 164,384 and ER
antisense
oligonucleotide blocked EGF-induced lordosis. Taken together, the
present data demonstrate the biological phenomenon of
ligand-independent activation of brain ER
by nonsteroidal GFs in
living animals.
It has long been thought that lordosis is E dependent and genomic in nature. Previous studies have shown that E induces PR in the hypothalamic VMN for the appearance of P-dependent feminine sex behavior in female but not male rodents (see Ref. 21 ). This induction is dependent on RNA and protein synthesis and morphological changes indicative of cellular growth, genomic activations, and either new synapse formation or rearrangement of existing synapses. Although neurochemicals such as serotonin, dopamine, and oxytocin have also been implicated in the control of sex behavior, all require EB priming and the presence of functional PR (see Ref. 21 ). Without EB priming, these neurochemicals evoke only nonsexual behaviors such as fighting.
It was somewhat surprising that the behavioral change induced by EGF occurred in the absence of EB priming and the endogenous ovarian steroid E. However, the present data demonstrate that it is ER, not the ovarian steroid E or another isoform of ER, that is critical for receptivity. These findings are consistent with recently published behavioral findings in ERKO mice lacking a functional ER, who are treated with EB + P (53 ). Likewise, we (27 44 ) and others (25 39 54 ) have established previously that ligand-dependent (EB + P) receptivity requires functional PR in the hypothalamus since icv PR antisense oligonucleotides blocked lordosis. Supporting this, EB-primed homozygote PR knockout mice failed to display lordosis after EB and P (38 ). However, for EGF-induced receptivity, sex behavior is independent of ovarian P and hypothalamic PR. This is consistent with the absence of ER-dependent induction of PR synthesis in the VMN since DNA synthesis and PR synthesis are absent in uteri from ERKO mice after EGF treatment (55 ). These results indicate that another parallel pathway exists for reproductive behavior that does not converge on PR.
Researchers have identified a putative neural circuit with serial
connectivity controlling the execution of lordosis (21 56 ). For this
response, E acts at certain point(s) along the circuit with
hierarchical control emanating from ER-expressing neurons of the VMN, a
subset of which project directly to the periaqueductal gray. Our
present data suggest that EGF may act on those points along the circuit
that are downstream to the VMN PR. Hypothetically, EGF-stimulated cells
could then signal ER-expressing cells in the VMN through reciprocal
projections between the periaqueductal gray, arcuate nucleus, and VMN,
which are known to facilitate lordosis. Alternately, a more sensitive
circuitry may be independent of the effects of ovarian P and
hypothalamic PR as a result of EGF- stimulated changes. That is, EGF
could be inducing the rapid synthesis and/or secretion of one or more
neuropeptides (17 ) which, in turn, may alter the sensitivity of the
neural circuit controlling sex behavior and eliminate the need for
ovarian P or hypothalamic PR.
Also of surprise is the present finding that EGF can elicit lordosis
1 h after its icv administration. In previous ligand-dependent (EB
+ P) studies, behavior appeared only with synthesis of PR at 1824 h
after EB-priming (21 ). Relative to the present study, hypothalamic
changes could have been induced by EGF since it is well accepted that
the intracellular effects include genomic (57 ), which are detected
within minutes and persist for hours. In vivo genomic
effects of EGF include the synthesis of PR (58 ). Our unpublished data
using an estrogen response element-driven Lac Z adenoviral
construct microinjected onto the VMN of female rats support the notion
that EGF can induce ER-dependent gene transcription within 12 h.
Further, it is also known that both EGF and E act by nongenomic
mechanisms to rapidly activate cAMP (12 ), phospholipase C (PLC), and
inositol phosphate (59 60 ) and mitogen-activating protein kinase (61 62 ). Interestingly, some rapid effects of E have been attributed to its
ability to act on ER located directly in the membrane (46 62 ).
Although the mechanisms essential for the present behavioral phenomenon
must still be elucidated, it is probable that genomic mechanisms are
essential since the effects of EGF on behavior were not observed during
the first hour after challenge. The present findings implicate
hypothalamic ER
and, at least, tyrosine kinase EGF receptors.
For the brain, the findings of this study may provide a clue into
several physiological observations that thus far have gone unexplained.
For example, signal cross-coupling may regulate the homeostatic
setpoint for hormone response threshold by steroid receptors whereby
low levels of biologically active neurosteroids could be
synergistically activated by GFs to produce gene responses. This would
be consistent with our observation that EGF enhanced the effect of
suboptimal EB, resulting in the exhibition of lordosis. Such
cross-coupling is reminiscent of onset of puberty when E concentrations
are low and the synthesis of EGF receptor ligands increase in the
median eminence. Ojeda and co-workers (18 ) have already shown that EGF
receptor stimulation by TGF contributes to the initiation of the
first preovulatory GnRH surge and the mechanism underlying precocious
puberty (63 ). Indeed, delayed onset of puberty was a finding in our
studies of the EFG receptor-defective Wa-2 mouse. That EGF receptor
stimulation initiates both sex behavior and onset of the GnRH surge
suggests that it may serve to coordinate the timing of behavioral
estrus with ovulation. In fact, we have shown that, in the intact
mutant Wa-2 mouse, there is a disruption in synchrony between mating
behavior and estrus. Finally, reminiscent of biological coupling
between receptors for GFs and E is a second, earlier developmental
period when initial intraneuronal enzymatic conversion by aromatization
of androgens to estradiol is limited (1 2 ). Thus, the
ligand-independent model of ER activation may be a common biological
feature within the rodent CNS.
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MATERIALS AND METHODS |
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Age-matched wild-type and mutant PRKO mice were generated and obtained from the breeding colony at Baylor College of Medicine (37 ). All were maintained in accordance with Federal guidelines on a 12-h light, 12-h dark cycle, and food and water were available ad libitum. Females were genotyped as previously described (37 ) and ovariectomized under anesthesia. Females were then primed every week for four consecutive weeks with EB (0.5 µg sc) followed by P (100 µg sc at + 40 h) and tested for sexual receptivity (at 46 h) in the presence of male mice of the same genotype. On the fifth week, stainless steel cannulae (26 gauge, Plastics One) were surgically implanted into the third ventricle using the stereotaxic coordinates (anteroposterior bregma, 1.9 mm; and dorsoventral, 5.7 mm) of Franklin and Paxinos (66 ). All animals were housed individually after surgery to prevent cannula disruption.
Breeding pairs of wild-type and mutant Wa-2 mice (ala Egfr wa2/+) were obtained from The Jackson Laboratory (Bar Harbor ME) and maintained as independent colonies at Baylor College of Medicine. A third colony of heterozygous Wa-2 mice were generated by prodigy testing of female offspring from breeding pairs of wild-type females and mutant males. This also provided equal numbers of Wa-2 homozygous and heterozygous (control) female littermates. It should be noted that mutant females are detectable by their curly whiskers, vibrissae, and guard hairs, a pleiotropic recessive effect of the EGF receptor point mutation (67 ).
Behavioral Testing for Lordosis
For each experiment with rats, females were treated with various
doses (0200 ng) of recombinant human EGF in water (1 µl/animal) by
icv injection 1214 days after screening described above. No EB was
given unless otherwise indicated. Sexual behavior was measured as
described previously (30 44 ); e.g. LQ = [total number
positive responses total number mounts by a series of 4 males]
100 when LQ represents lordosis quotient. Results for LQ are expressed
as percent positive responses for all females mounted by males ±
SEM. Thus, animals that failed to be mounted
during testing were excluded from calculations of LQ that are shown in
the figures. The per cent of females failing to be mounted is reported
in the text. With the exception of the time course experiment, animals
were observed for experimental effect between 24 h after treatment.
For each experiment, animals served as their own control, since all
animals were tested 13 h before experimental treatment and excluded
if positive responses were exhibited. Each experiment also included
control groups of EB + P and non-EB-primed animals receiving vehicle
only. Each group consisted of a minimum of six females and each
experiment was repeated two to three times.
For experiments with mice, females were treated and tested 7 days after cannula placement. Thirty minutes after icv injections, female mice were placed in the home cage of the male mice of proven sexual vigor and of the same strain as the female. The testing continued until the male had mounted (and displayed pelvic thrusting) the experimental female 10 times. The female was placed with a different male if the male failed to mount the female during the testing period. Lordosis was considered positive if the female exhibited a rigid posture with arching of the back, elevation of the hind quarters, and deviation of the tail to facilitate male mounting and intromission (68 ). Complete immobility with the hindquarters down was not considered a positive response. The observer was blind to individual animal treatment and genotype for PRKO mice. For Wa-2 mice, the observer was not blind because mutant Wa-2 have curly hair and whiskers. Each experiment consisted of wild-type and mutant Wa-2 mice under identical treatment conditions. Control groups received similar injections of EB + P and/or vehicle only. Each group consisted of a minimum of four females and each experiment was repeated three to four times.
Statistics
Statistical analysis was performed using one-way ANOVA followed
by Mann-Whitney U test for individual differences. Two-way
ANOVA with repeated measures was used to assess significant change in
reproductive behavior when animals served as their own control.
Duncans Multiple Range test was used for individual comparisons.
Compounds and Oligonucleotides
All injections were prepared immediately before administration
suspended in sterile distilled water unless otherwise stated. GFs
(Upstate Technology, Inc. Lake Placid, NY) and
amphiregulin (R & D Systems, Minneapolis, MN) were
aliquoted immediately after dissolving and stored at 4 C until use.
Whenever possible, doses were based on published studies for effective
concentrations or verified when appropriate. EGF antibody (1:4 stock
solution that detects 2 ng of EGF/ml) was kindly provided by D. H.
Polk (Northwestern University, Chicago, IL). Since we diluted the
antibodies 1:1000, the final concentration of native product that was
administered was 1:4000 of native product. Steroids and other drugs
were dissolved in sesame oil as reported previously (33 44 ).
Sense (5' to 3') and antisense (3' to 5') oligodeoxynucleotides were
designed to symmetrically cover the translation initiation sites of the
target sequences. Synthetic, phosphorothiolated lyophilized oligos were
dissolved in sterile distilled water within 3060 min of
administration. None of the oligonucleotides show homology with other
reported sequences in the GenBank. The oligonucleotide sequences for
rat ER (AS, 5''-CAT-GGT-CAT-GGT-CAG-3'; RS,
5'-ATC-GTG-GAT-CGT-CAC-3') have been reported and specificity has been
shown elsewhere (34 ). For rat PR, the AS sequence was
5'-GCT-CAT-GAG-CGG-GGA-CAA-CA-3', and S was
5'-TCT-TGT-CCC-CGC-TCA-TGA-GC-3'. Specificity for PR has been
previously reported (29 ).
Specificity of EGFR Oligonucleotides
For rat EGF receptor, phosphorthiolated oligonucleotides
(National Biosciences, Plymouth, MN) were designed against the start
site [(68 ); accession code M37394] and synthesized. EFG receptor AS
was 5'-AGG-GTC-GCA-TCC-CGG-CT-3', and S sequence was
5'-ACG-AGC-GAT-GCG-ACC-CT-3'. Specificity of the oligonucleotide for
EGF receptor and its mediated events first was shown by Western blot
analysis in a clearly defined experimental system. Rat-2 embryonic
fibroblast cells were grown in Hams F10 with 12% horse serum,
washed with PBS, treated with varying amounts of AS or S (02
nmol/well) and/or vehicle. After a 6-h challenge with EGF, 50 ng/well
and/or vehicle, cells were incubated in Hams F10 with 3.5% horse
serum + 0.5% FCS for 72 h as described elsewhere (33 44 ). After
harvesting, cells were processed for EGF receptor by Western analysis
using a polyclonal antibody that recognizes rat EGF receptor (1:500;
Upstate Biotechnology, Inc.) and rabbit antisheep antibody
(1:100; Zymed Laboratories, Inc., South San Francisco, CA)
conjugated to horseradish peroxidase per manufacturers
protocol. Studies were performed in duplicate and quantitated as
described above.
Next, the specificity of oligonucleotide was determined in rat VMN using immunohistochemistry. Cannulated, OVX females were treated icv with 2 nM AS or S and/or vehicle. They were deeply anesthetized 44 h later and perfused transcardially with heparinized PBS followed by fixation (4% paraformaldehyde in PBS at 4 C for 1 h). The tissue was then cryoprotected with buffered sucrose (2030%, 4 C overnight), stored at -70 C until sectioned (57 µm) using a cryostat. Mounted sections were incubated with 3% hydrogen peroxide in methanol at -20 C for 20 min followed by 1% Antigen Retrieval Solution (Vector Laboratories, Inc., Burlingame, CA) at 96 C for 10 min. To diminish nonspecific binding, sections were incubated in 5% normal rabbit serum for 30 min followed by overnight incubation at 4 C in a polyclonal sheep antihuman EGF receptor antibody (1:100; Upstate Biotechnology, Inc.). This antibody also recognizes rat and chicken EGF receptors. Sections were then incubated with biotinylated second antibody (2 h at room temperature), and antigen was visualized using the Elite Vectastain Kit (Vector Laboratories, Inc.). PBS was used to wash sections three times for a total of 15 min between all steps, and slides were counterstained with nuclear fast red. For each immunostaining, control sections were included in which the primary or secondary antibody was obtained.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Preliminary findings were presented at the 10th International Congress on Endocrinology and the 78th Annual Meetings of The Endocrine Society on June 1518, 1996, Abstract OR637.
This work was supported by NIH Grant NR-O6826 to E.M.A. and HD-07857 to B.O.
Received for publication August 17, 1999. Revision received February 25, 2000. Accepted for publication March 30, 2000.
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REFERENCES |
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