Epidermal Growth Factor Activates Reproductive Behavior Independent of Ovarian Steroids in Female Rodents

Ede Marie Apostolakis, Janos Garai, Jennifer E. Lohmann, James H. Clark and Bert W. O’Malley

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sex steroids exert profound influence on neural development and function through activation of intranuclear receptors. However, during sexual differentiation and at onset of puberty, intracerebral estrogen (E) availability is subsequent to these effects. The potent mitogen epidermal growth factor (EGF) activates estrogen receptor (ER)-dependent transcription in cultured cells in the absence of exogenous E. Since reproductive behavior in female rodents is the 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. Here we demonstrate that EGF can signal through the classical E receptor (ER{alpha}) to alter in vivo function in rodent central nervous system. EGF and EGF receptor ligands induced lordosis in a dose- and time-dependent manner in the absence of steroid treatment in ovariectomized rats and mice. Using antisense oligonucleotides, pharmacological and antibody blockade, and mutant mice, we also report that this behavioral responsiveness is mediated through ER{alpha} by specific stimulation of membrane-bound EGF receptors and EGF receptor-specific tyrosine kinase rather than by direct ligand activation of the ER{alpha}. Of biological significance, delayed onset of puberty and the absence of synchronization between reproductive behavior and ovulation was detected in intact mutant Wa-2 mice that express a naturally occurring point mutation in the EGF receptor. To our surprise, EGF-mediated behavior was independent of progesterone (P) and progesterone receptor (PR) since antiprogestins, PR antisense oligonucleotides, and targeted disruption of PR in ovariectomized transgenic mice failed to impede the display of lordosis after EGF. Finally, we also found that another growth factor, insulin-like growth factor-1, which provokes ER-dependent transcription in vitro, activates mating behavior in a similar E-independent manner. Thus, growth factor mediation of ER-targeted function may be a universal feature in the rodent central nervous system, raising critical questions about the role of growth factors in mediating ER-dependent processes in development and reproduction.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sex steroids exert profound influence on neural development and function (1 2 3 4 5 ). The ovarian steroid estrogen (E) stimulates differentiation of neural circuits known to be critical for reproduction and reproductive behavior (6 ). Biological signaling by E is transmitted through two different nuclear receptors: the classical estrogen receptor-{alpha} (ER{alpha}) and the more recently cloned ERß (7 ). Both receptors are members of the steroid/thyroid receptor gene superfamily of transcription factors. As such, both receptors serve as cis-acting DNA-binding transcription factors when bound to E, by dimerizing and binding specifically to consensus E-response elements within the regulatory region of E-responsive genes (8 9 ). Subsequent phosphorylation and interaction with basal transcription factors (10 ) and coactivators (11 ) result in formation of preinitiation complexes and regulation of gene transcription.

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{alpha} have been extensively investigated throughout the brain of multiple mammalian species, including rats and mice. Importantly, robust expression of ER{alpha}, 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{alpha} 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, 24–48 h. Here we show that rapid activation (within 1–4 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reproductive Behavioral Responses to EGF
Human recombinant EGF was injected into the third ventricle of ovariectomized (OVX) rats in the absence of estradiol benzoate (EB)-priming and the animals were subsequently observed. As expected, animals failed to display sexual receptivity (lordosis) before experimental treatment (Fig. 1Go, bar 1), and after treatment with vehicle (bar 2), EB-only (bar 3), and P-only (bar 4). Also expected, the administration of P, 44 h after EB-priming (Fig. 1Go, bar 5), resulted in high levels of lordosis. EGF doses of 2 ng (Fig. 1Go, bar 6) failed to induce significant reproductive behavior. Females exhibited receptivity after 10 ng microinjection of EGF (Fig. 1Go, bar 7) compared with pretreatment (bar 1; ANOVA, P = 0.05) and/or that of vehicle-, EB-, or P-treated animals (bars 2–4, respectively; ANOVA followed by Mann-Whitney U test). EGF doses of 75 ng (Fig. 1Go, bar 8) and 200 ng (data not shown) failed to induce behavior. For all females receiving 10 ng EGF (n = 18), 88% (n = 16) were mounted whereas 45% (n = 12) of all females receiving 2 ng EGF (n = 27) were mounted. Thus, EGF at the optimal dose of 10 ng induced reproductive behavior in the absence of both E and P. The optimal dose is within an estimated physiological range for stimulated synthesis of EGF within the rat brain (31 32 ). A time course for EGF induction revealed that receptivity was displayed within 1–4 h (data not shown). The data demonstrate that reproductive behavior in female rats is not absolutely dependent upon E-priming. More importantly, the findings present a novel mechanism for rapid activation of lordosis, which is EB-independent.



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Figure 1. EGF Activates Reproductive Behavior of OVX Female Rats in the Absence of E

Single icv microinjection of EGF induced mating behavior within 1–4 h. No EB was given when effect of EGF was tested. For effect of suboptimal doses of EB and EGF, EB was given sc 44 h before EGF. For effect of high dose of EB, behavioral testing was performed 44 h after sc EB. Sex behavior is expressed as Lordosis Quotient (LQ%; percent positive responses ± SEM). For each experiment, animals served as their own control since all animals were tested 1–3 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. Asterisk indicates a significant increase (P <= 0.05) in LQ compared with pretreatment response and/or response of control females.

 
In cultured cells, synergism between GFs and steroids has been reported previously (4 5 ). In the present study, when EB at behavioral ineffective concentration (Fig. 1Go, bar 3) and EGF (bar 9) were given 44 h apart to OVX females, receptivity was displayed at levels greater than those seen with EGF alone (Fig. 1Go, bar 7). The level of receptivity also was comparable with that induced by either EB subsequently followed by P (Fig. 1Go, bar 5) or a high dose of EB (Fig. 1Go, bar 10). This additive effect of EGF and EB on sex behavior is consistent with in vitro data and supports the hypothesis that, in the brain, EGF is acting through a convergent, ER-mediated pathway in a steroid-independent manner to affect reproductive behavior.

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. 2Go, bar 1) failed, EGF (bar 2) induced target behavior in control animals. Also, the antiestrogen ICI 164,384 (Fig. 3Go, bar 3) alone failed to stimulate lordosis. In contrast, antiestrogen pretreatment suppressed lordotic responses in females challenged with EGF (Fig. 2Go, bar 4), suggesting that EGF may regulate ER-dependent behavior.



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Figure 2. EGF Induction of Reproductive Behavior Is Mediated, in Part, by ER{alpha} in the Rodent Brain

Chronically cannulated rats were given the antiestrogen ICI164,384 icv 1 h before challenge with EGF. Phosphorothiolated oligonucleotides to ER{alpha} were microinjected 24 h before EGF challenge. Specificity for ER{alpha} has been shown previously (34 ).

 


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Figure 3. EGF Induces Lordosis in the Absence of PR

Antiprogestins RU486 and ZK98299 were administered icv 1 h before EGF challenge. Oligonucleotides for PR were injected 24 and 48 h before EGF. PR oligonucleotides blocked EB + P-induced behavior. Specificity for PR has been previously reported (29 ). Likewise, EGF induced sex behavior in wild-type and homozygote PRKO mice (data not shown).

 
To examine this further, animals primed with low-dose EB were given with ICI 164,384 icv and subsequently challenged with EGF. Control animals treated with low doses of either EB (Fig. 2Go, bar 5) or EGF (bar 6) failed to exhibit receptivity while control animals treated with a combination of low-dose EB and EGF (bar 7) displayed the behavior. In contrast to an optimal dose of EGF (Fig. 2Go, bar 2) or low doses of both EGF and EB (bar 7), ICI 164,384 again inhibited the target behavior (bar 8). Thus, the data support the hypothesis that EGF induces reproductive behavior by regulating ER activity in the brain. However, since ICI 164,384 shows selective affinity for both ER{alpha} and ERß (33 ), the data cannot discriminate which ER isoform is undergoing an effect by EGF.

To specifically analyze dependency of EGF on endogenous ER{alpha}, phosphorothiolated antisense (AS) oligonucleotides to the initiation region of the ER{alpha} 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. 2Go, bars 9–11, respectively). Significantly, EGF failed to induce lordosis in the presence of ER{alpha} antisense oligonucleotides (Fig. 2Go, 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{alpha} 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. 1Go, 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. 3Go, bars 1–3). Of significance, EGF induced lordosis in spite of RU486 pretreatment (Fig. 3Go, 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. 3Go, 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. 3Go, bars 7–9) 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. 3Go, 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-{alpha} (TGF{alpha}), or platelet-derived growth factor (PDGF) (45 ) were administered icv 2 h before EGF. As shown in Fig. 1Go (bar 7), Fig. 2Go (bar 2), and Fig. 3Go (bar 2), recombinant EGF induced mating behavior (Fig. 4AGo, bar 2). Antibodies alone failed to effect rat behavior (Fig. 4AGo, bar 3). No lordosis was displayed after EGF challenge with antibodies (Fig. 4AGo, bar 4), suggesting the rat EGF receptor is mediating the effect of recombinant human EGF.



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Figure 4. Membrane-Bound Receptors for EGF Mediate the Behavioral Effects of EGF

A, The EGF kinase antagonist A15 served as the inhibitor of EGF receptor-specific kinase activity and was given icv 30 min before EGF. Polyclonal antibodies to EGF were administered icv 2 h before challenge with EGF. Oligonucleotides were given icv 24 h before EGF. B, Specificity of the EGF receptor oligonucleotide is shown by Western blot analysis in a clearly defined experimental system using rat-2 embryonic fibroblast cells treated with varying amounts of AS or RS and/or vehicle, challenged with EGF and/or vehicle, and subsequently incubated in semistarved conditions for 72 h. Studies were performed in duplicate. C, Specificity of the oligonucleotide is also shown by immunohistochemistry in VMN using OVX rats treated icv with AS (representative finding, right panel) or RS (representative finding, left panel) and/or vehicle (data not shown) 48 h before tissue collection. Studies were performed in triplicate.

 
Because one of the initial consequences of EGF receptor binding to ligand is receptor autophosphorylation at discrete tyrosine sites in the carboxyl terminus (46 ), the EGF receptor-specific tyrosine kinase inhibitor A15 (47 ) was used to explore the specificity of recombinant EGF for endogenous EGF receptor-mediated intracellular events in the rat brain. Unlike animals receiving EGF (Fig. 4AGo, bar 2), animals receiving A15 icv alone or 30 min before EGF (Fig. 4AGo, bars 5–7) did not display significant lordosis, thus supporting the hypothesis that the EGF receptor was mediating, in part, the behavioral responsiveness in our paradigm.

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. 4AGo, bars 8 and 9). Also as expected, AS alone failed to induce an effect on behavior (Fig. 4AGo, 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. 4AGo, bar 11). To determine EGF receptor oligonucleotide specificity, rat fibroblast cells were treated in culture. EGF receptor was detected when treated with vehicle (Fig. 4BGo, lane 1) but not EGF (lane 2), as expected with EGF receptor degradation (42 ). Likewise, receptor was detected after treatment with RS (Fig. 4BGo, 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. 4BGo, lane 5) compared with that in vehicle-treated cells (Fig. 4BGo, lane 1). Again, no adverse effect of AS was observed since receptor degradation (Fig. 4BGo, 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. 4CGo, 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, 30–73 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 4–5 days after vaginal opening in mutant Wa-2 mice compared with its appearance at 1–2 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. 5Go, bars 1–6). Again, since all groups of OVX females exhibited mating behavior in response to exogenous EB + P (Fig. 5Go, bars 7–9), 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 10–12) 1 week before EGF and immediately before EGF treatment (bars 13–15). 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. 5Go, bars 16 and 17). Interestingly, mutant Wa-2 mice failed to display reproductive behavior (Fig. 5Go, 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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Figure 5. EGF Receptor Mutant Wa-2 Mice Fail To Exhibit EGF-Induced Mating Behavior

Wild-type (open bars), heterozygote (hatched bars), and homozygote (closed bars) mice were cannulated after five consecutive weeks of EB + P priming. Behavioral test for response to EB + P was recorded at the end of fifth week and results are noted in bars 1–9. One week later, EGF was given icv and animals were tested after 2–4 h. LQ% represents a total of 10 male mounts per female for a minimum of 6 females per group. Behavior test was repeated twice.

 
Specificity of GF-Induced Reproductive Behavior
Other EGF-like ligands such as amphiregulin and TGF{alpha} bind to the EGF receptor in vitro (36 ). When given icv amphiregulin, female rats exhibited sex behavior at a higher dose of 100 ng (Fig. 6Go, bars 3–5). Less frequent displays of lordosis were noted in response to microinjections of TGF{alpha} (Fig. 6Go, bars 6 and 7). Thus, EGFR ligands other than EGF appear to induce some mating behavior in the absence of E, supporting the hypothesis that stimulation of hypothalamic EGF receptor can mediate this critical behavior independent of the ovarian steroid E in females.



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Figure 6. Behavioral Effect of GF Is Specific to EGF Receptor and IGF-I Receptor but Not to PDGF Receptor

Other ligands (amphiregulin, TGFa) for the EGF receptor induce sex behavior in rats when given icv. Ligands were given 1–4 h before testing for behavior. As in cultured cells for ER-dependent transcription, both EGF and IGF-I but not PDGF induce mating behavior. All GFs were given icv 2–4 h before testing.

 
Like EGF, IGF-I induces DNA synthesis in neuroblastoma SK-N-BE cells stably expressing ER{alpha} (52 ) and in cotransfection studies using ER{alpha}-responsive target genes (4 5 12 ). To assess whether these in vitro results correlated with our in vivo model, animals were administered IGF-I icv in the absence of EB. Lordosis was exhibited within 2 h of IGF-I microinjection in a dose-dependent manner (Fig. 6Go, bars 8 and 9). Moreover, a degree of specificity for receptors of GFs is revealed by the failure of PDGF to elicit lordosis (Fig. 6Go, bars 10–11). The sum of our present in vivo findings is consistent with the ligand-independent model proposed in vitro; e.g. membrane-bound trophic receptors can signal the intracellular ER{alpha} to produce a biologically relevant alteration in function. Further, GF modulation of ER{alpha}-targeted transcription may be a general feature for brain cells in which receptors for GFs are colocalized with select steroid receptors.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Data in a variety of cultured cells support the existence of intracellular cross-talk between nuclear steroid receptors and membrane-bound GFs. However, it is critical to understand how individual components function within the context of the entire biological system; e.g. the living animal. The existence of ligand-independent regulation of steroid receptor-mediated functions and its physiological impact in the presence of feedback systems have been difficult to obtain in living animals. By using a well accepted behavioral assay, which is exquisitely dependent upon steroid receptor transcriptional activity and protein synthesis in the hypothalamus, we present evidence for nonclassical, steroid-independent regulation of ER{alpha} in rats and mutant mice.

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{alpha}) 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{alpha} to effect this behavioral change. The antiestrogen ICI 164,384 and ER{alpha} antisense oligonucleotide blocked EGF-induced lordosis. Taken together, the present data demonstrate the biological phenomenon of ligand-independent activation of brain ER{alpha} 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{alpha}-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 18–24 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 1–2 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{alpha} 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{alpha} 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{alpha} 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animal Preparation
OVX female rats (180–200 g; Sasco, Houston TX) were maintained in accordance with Federal guidelines and screened for steroid-dependent behaviors as described previously (30 44 ). Females displaying lordosis underwent stereotaxic implantation of third ventricle cannula guides (22 gauge, Plastics One, Roanoke VA) using Paxinos coordinates (65 ). Seven days after surgery, cannulated EB-primed females (10 µg sc at -44 h) were again screened at 3–4 h after receiving P (1 µg icv at 0 h) to verify the presence of inducible lordosis. Only those cannulated females exhibiting high levels of proceptivity and receptivity were used in experiments.

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 (0–200 ng) of recombinant human EGF in water (1 µl/animal) by icv injection 12–14 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 {surd} total number mounts by a series of 4 males] {surd} 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 2–4 h after treatment. For each experiment, animals served as their own control, since all animals were tested 1–3 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. Duncan’s 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 30–60 min of administration. None of the oligonucleotides show homology with other reported sequences in the GenBank. The oligonucleotide sequences for rat ER{alpha} (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 Ham’s F10 with 12% horse serum, washed with PBS, treated with varying amounts of AS or S (0–2 nmol/well) and/or vehicle. After a 6-h challenge with EGF, 50 ng/well and/or vehicle, cells were incubated in Ham’s 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 manufacturer’s 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 (20–30%, 4 C overnight), stored at -70 C until sectioned (5–7 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.


    ACKNOWLEDGMENTS
 
We wish to thank C. Owens, D. Zhou, and G. Curran for their technical assistance. We thank D. H. Polk (Northwestern University, Chicago, IL) for kindly providing EGF antibodies. We also wish to acknowledge Suzanne Krnacik and Jeffrey Rosen for helpful discussions during the course of this work.


    FOOTNOTES
 
Address requests for reprints to: Bert O’Malley, Chairman, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.

Preliminary findings were presented at the 10th International Congress on Endocrinology and the 78th Annual Meetings of The Endocrine Society on June 15–18, 1996, Abstract OR63–7.

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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