Compromised Reproductive Function in Adult Female Mice Selectively Expressing Mutant ErbB-1 Tyrosine Kinase Receptors in Astroglia
Biao Li,
Zhihui Yang,
Jingwen Hou,
April McCracken,
M. Anita Jennings and
Mark Y. J. Ma
Center for Human Molecular Genetics, Munroe-Meyer Institute, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska 68198
Address all correspondence and requests for reprints to: Mark Y. J. Ma, Ph.D., Center for Human Molecular Genetics, Munroe-Meyer Institute and Department of Genetics, Cell Biology and Anatomy, 985455 University of Nebraska Medical Center, Omaha, Nebraska 68198-5455. E-mail: yma{at}unmc.edu.
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ABSTRACT
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The ErbB-1 tyrosine kinase receptor plays critical roles in regulating physiological functions. This receptor-mediated signaling in astroglia has been implicated in controlling female sexual development via activating neurons that release LH-releasing hormone (LHRH), the neuropeptide required for the secretion of LH. It remains unknown whether astroglial ErbB-1 receptors are necessary for maintaining normal adult reproductive function. Here we provide genetic evidence that astroglia-specific and time-controlled disruption of ErbB-1 receptor signaling by expressing mutant ErbB-1 receptors leads to compromised reproduction due to alteration in LHRH neuron-controlled secretion of LH in adult female mice. Therefore, astroglial ErbB-1 receptors are required for controlling LHRH neuronal function and thus maintaining adult reproduction, suggesting that compromised astroglial ErbB-1 signaling may also contribute to reproductive abnormalities in aging females.
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INTRODUCTION
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ASTROGLIA ARE NOW viewed as active partners with neurons in the central nervous system (CNS) (1, 2). Evidence shows that astroglia are necessary for controlling functional plasticity of synapses via the release of signaling molecules (3, 4, 5, 6, 7). That astroglia are also important for other physiological function is indicated by the likely astroglia-dependent control of female sexual development (8). For example, ligand-induced activation of the ErbB-1 receptors (also known as epidermal growth factor receptor) in isolated hypothalamic astroglia leads to production of bioactive molecules that directly target LH-releasing hormone (LHRH) neurons resulting in LHRH release (9). This, in turn, stimulates LH secretion that may initiate the onset of puberty (10, 11). In contrast, blockade of the ErbB-1 receptor-mediated signal transduction in the hypothalamus delays the initiation of puberty due to the disruption of LHRH/LH secretion (10, 11). Thus, ErbB-1 receptor signaling system is involved in the regulation of mammalian sexual maturation. However, the conclusion related to the astroglial influence on female sexual maturation remains to be further demonstrated in animal models. It is currently unknown whether astroglia-specific ErbB-1 receptors are required for regulating adult female reproduction, although ErbB-1 receptors in the hypothalamus have been implicated to play a regulatory role in adult female reproductive function (12). The present study demonstrates that selectively targeted disruption of ErbB-1 receptor signaling in astroglia of adult female animals leads to abnormal reproduction. The in vivo astroglia-specific ErbB-1 receptor targeting was accomplished by expressing human ErbB-1 mutant receptors (hErbB-1 mutant) lacking the intracellular domain, but retaining the ability to dimerize with normal ErbB-1 receptors and thus blocking signal transduction (13, 14, 15) mediated by the normal receptors.
The astroglia-specific mutant receptor expression was accomplished via the ecdysone inducible expression system (16, 17, 18, 19), which was modified by 1) replacing the original cytomegalovirus promoter in the pVgRXR plasmid with the mouse GFAP (glial fibrillary acidic protein) gene promoter (PGFAP-pVgRXR, Fig. 1
, A, top); and 2) cloning a mutant gene, which encodes a human ErbB-1 receptor lacking the entire intracellular domain, into the pIND plasmid (pIND-hErbB-1 mutant, Fig. 1A
, bottom). The molecular principle of this expression system is that (Fig. 1A
), upon administering ecdysone (an insect molting hormone) or a synthetic steroid analog, ponasterone A (Pon A), this steroid binds to ecdysone receptors (EcR), which are the expressed products driven by the GFAP promoter, resulting in the formation of a heterodimer composed of the ecdysone-retinoid receptor complex, EcR-RXR. The DNA binding domain of this heterodimer will bind to the E/GREX5 hybrid response element, leading to the expression of a gene of interest. Because mammalian cells are not responsive to Pon A due to the absence of the ecdysone receptor (20), transgene expression is highly specific and inducible. The GFAP promoter is able to specifically direct transgene expression in astroglial cells (21, 22, 23, 24, 25). Hence, the EcR expression would be restricted in astroglia, and thus the hErbB-1 mutant receptors should be expressed in the same cell population at a selected time. Two transgenic lines [PGFAP-pVgRXR (G+/+/E-/-) and pIND-hErbB-1 mutant (G-/-/E+/+)] (Fig. 1B
) are required for producing bitransgenic animals (G+/+/E+/+) in which the endogenous astroglial ErbB-1-mediated signal transduction can be disrupted (Fig. 1B
). Selectively expressing hErbB-1 mutants in astroglia can be initiated in G+/+/E+/+ animals by administering Pon A that binds to EcR, an insect-specific component required for expressing hErbB-1 mutant (Fig. 1A
). Using this animal model, the expression of hErbB-1 mutant receptors in astroglial cells can be initiated in young adult female mice. The present study demonstrates that selectively disrupting astroglial ErbB-1 receptor-mediated signaling events lead to compromised reproduction in female animals, which phenotypically show abnormal estrous cycle and anovulation. This negative impact is specifically coupled to reduction in LHRH neuron-controlled LH release, but not FSH secretion. Clearly, the astroglial ErbB-1 receptor-mediated signal transduction is required for maintaining normal adult reproduction by controlling LHRH neurons that govern the release of LH.

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Fig. 1. Generation of Transgenic Animals Carrying Astroglial-Restricted Ecdysone Expression System
A, A schematic map of two constructs used for producing transgenic animals (Line-1, PGFAP-pVgRXR; Line-2, pIND-hErbB-1mut). In line-1, the GFAP promoter directs the expression of ecdysone receptor (EcR) only in astroglial cells. Upon administering ponasterone A (Pon A), EcR binds to Pon A and dimerizes with retinoid X receptor (RXR). The DNA binding domains of this heterodimer bind to the responsive elements (5XE/GRE) leading to the expression of hErbB-1mut (line-2) which encodes a human ErbB-1 mutant receptor. B, A simplified diagram depicting the principle of the astroglial-restricted and temporally controlled gene expression system. Transgenic line-1 [PGFAP-pVgRXR (G+/+/E-/-)] and line-2 [pIND-hErbB-1mut (G-/-/E+/+)] are required for producing bitransgenic animals (G+/+/E+/+). The homozygous G+/+/E+/+ animals are produced by crossing the homozygous line-1 and line-2. Upon administering Pon A, the mutant hErbB-1 receptors are expressed in astroglial cells in the CNS as depicted by a black-filled circle.
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RESULTS
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Generation of Transgenic Animals Carrying the Astroglial-Specific and Time-Controlled Ecdysone Inducible Expression System
To disrupt ErbB-1 receptor signaling in astroglia during a desired period of adulthood, we generated transgenic mice carrying the PGFAP-pVgRXR and pIND-hErbB-1 mutant gene that encodes a human ErbB-1 receptor lacking the entire intracellular domain. The GFAP promoter is able to specifically direct transgene expression in astroglial cells (21, 22, 23, 24, 25). The ability of the hErbB-1 mutant to block endogenous ErbB-1 receptor signaling has been documented in both cells and animals (13, 14, 15, 26). Four independent founders of transgenic line-1 [PGFAP-pVgRXR (G+/+/E-/-) or line-2 pIND-hErbB-1 mutant (G-/-/E+/+)] (Fig. 1B
) were produced. All founders were able to transmit the transgene to their offspring in a Mendelian fashion. Both line-1 and line-2 homozygous animals were used for producing homozygous bitransgenic animals (G+/+/E+/+) carrying both PGFAP-pVgRXR and pIND-hErbB-1 mutant transgenes.
Characterization of Transgenic Animals
1) EcR is expressed in astroglia-positive tissues.
To determine whether the GFAP promoter is able to direct the expression of EcR, tissues from wild-type (WT) and G+/+/E-/- mice were subjected to Western blot analysis. EcR expression was evident in the brains of G+/+/E-/- animals (Fig. 2A
). A weaker expression of EcR was found in the stomach due to the presence of fewer astroglia in the enteric nervous system located in the gastrointestinal track (27). No EcR was detected in any examined tissue from WT (Fig. 2A
). Observation of EcR immunofluorescence-positive signals in the brain of G+/+/E-/- animal, but not in that of WT (Fig. 2B
) further confirms that this GFAP promoter drives EcR expression in astroglia-positive tissues.

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Fig. 2. Detection of the EcR Expression in the G+/+/E-/- Animals
A, Detected by Western blot, the EcR is predominantly expressed in the brains from three different founder-produced G+/+/E-/- transgenic animals. In contrast, no expression in the hearts, livers, and ovaries was observed. A very weak expression of EcR was observed in the stomach. No detectable EcR in any examined tissue was found in WT animals. B, Immunofluorescence detection of EcR-positive cells (arrows) in the hypothalamus from a G+/+/E-/-, but not from WT animal. Bar, 50 µm.
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2) Expression of hErbB-1 mutant receptors is restricted in the brain and astroglia.
To examine whether the hErbB-1 mutant receptors are expressed in the CNS, adult transgenic animals were injected (sc or third ventricle) with Pon A. Expression of the mutant receptor was detected in the brains from G+/+/E+/+ animals treated with Pon A (Fig. 3A
). The highest mutant receptor expression was observed at 10 h after Pon A injection into the third ventricle of the brain (Fig. 3A
). This result is consistent with a previous report (19). No hErbB-1 mutant expression was detected in the stomach of G+/+/E+/+ animals by sc injection with Pon A (Fig. 3A
), possibly due to much less astroglia located in this organ. As expected, because of the absence of EcR expression in the liver from the G+/+/E+/+ animal, no detectable hErbB-1 mutant was found in this tissue (Fig. 3A
). These results demonstrate the high specificity of the GFAP promoter-driven hErbB-1 mutant expression system in the transgenic animals. Thus, the potential for expression outside of the brain is minimal. To further examine whether expression of the mutant receptor was indeed restricted in astroglia in the CNS, an antibody recognizing only the extracellular domain of human ErbB-1 was used to colocalize hErbB-1 mutants with GFAP-positive astroglial cells. As expected, all the mutant receptor immunofluorescence-positive signals were detected only in the GFAP-reactive astroglia from G+/+/E+/+ animals treated with Pon A (Fig. 3B
). This result demonstrates that the GFAP promoter-controlled hErbB-1 mutant expression system is spatially and temporally specific.

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Fig. 3. Tissue-Specific Expression of hErbB-1 Mutant Receptors in Transgenic Mice
A, upper panel, Detection of hErbB-1 mutant receptor in the brains of homozygous G+/+/E+/+ bitransgenic animals via Western blot analysis. The mutant ErbB-1 was detected in the brains of G+/+/E+/+ animals treated with Pon A injection for 515 h. A higher level was observed at 10 h post sc injection. The highest expression was found in G+/+/E+/+ animals by stereotaxically delivering Pon A into the brain third ventricle for 10 h. Neither was the mutant ErbB-1 detected in G+/+/E+/+ animals without Pon A (nt) or vehicle-treated (v), nor in the G+/+/E-/- or G-/-/E+/+ animals treated with Pon A. (lower panel) Absence of hErbB-1 mutant expression was observed in the stomach and liver from WT, G-/-/E+/+ and G+/+/E+/+ animals with or without treatments. B, Immunofluorescence-positive hErbB-1 mutant signals (representative arrowheads) were detected only in the GFAP-reactive cells (arrows) in the brains of G+/+/E+/+ mice stereotaxically injected with Pon A directly into the third ventricle for 10 h. No hErbB-1 mutant immunoreactivity was found in the WT animal injected with Pon A. V, Third ventricle; bar, 50 µm.
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3) Expressed hErbB-1 mutants are able to completely inhibit endogenous mouse ErbB-1, but not ErbB-2, receptor phosphorylation in astroglia.
It is important to test whether astroglial hErbB-1 mutants are able to block endogenous ErbB-1 receptor phosphorylation, an event required for initiating signal transduction (28), primary astroglial cultures derived from the brains of either G+/+/E+/+ or G-/-/E+/+ neonatal animals were used for detecting phosphorylated endogenous ErbB-1 receptors. Pon A-induced expression of hErbB-1 mutant receptors dramatically reduces phosphorylation of the endogenous ErbB-1 receptor in the astroglia of G+/+/E+/+ animals (Fig. 4
). No such effect was found in the cells of G-/-/E+/+ animals unable to express the hErbB-1 mutant receptors. Similarly, no inhibitory effect was evident in the astroglia of G+/+/E+/+ mice without Pon A-treatment (Fig. 4
). Thus, the hErbB-1 mutant receptors are able to disrupt endogenous ErbB-1 receptor-mediated signal transduction in astroglia. Because ErbB-1 receptors are able to interact with ErbB-2 receptors in many cellular systems (29, 30), but do not collaborate with ErbB-4 receptors in hypothalamic astroglia (31, 32), it is thus important to examine whether the expressed ErbB-1 mutant receptors could also inhibit the endogenous ErbB-2 receptor phosphorylation. The present findings show that activation of ErbB-1 by epidermal growth factor (EGF) does not stimulate phosphorylation of ErbB-2 receptors, suggesting no obvious interaction between ligand-activated ErbB-1 and ErbB-2 in astroglia (Fig. 4
). Intriguingly, expression of ErbB-1 mutant seems to reduce the basal phosphorylation level of ErbB-2 receptor (Fig. 4
).

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Fig. 4. Blockade of Ligand-Activated ErbB-1, But Not ErbB-2, Receptor Phosphorylation by hErbB-1 Mutant Receptors in Astroglial Cells
The upper panel shows Western blot detection of phosphorylated endogenous ErbB-1 and ErbB-2 receptors. The lower panel depicts the quantitative analysis of the image showed in the upper panel. Each bar represents the arbitrary OD value determined by densitometric analysis. The ligand-induced phosphorylation of ErbB-1 was completely abolished only in Pon A (p)-treated astroglial cells derived from the brains of G+/+/E+/+ animals. In contrast, no inhibitory effect was observed from all control astroglial cells. The total content of endogenous mouse ErbB-1 receptors was detected to serve as loading control for each sample. pErbB-1, Phosphorylated ErbB-1; tErbB-1, total ErbB-1; -p, no Pon A; +p, plus Pon A.
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Astroglial ErbB-1 Receptor-Mediated Signaling Events Are Required for Maintaining Adult Female Reproductive Function
The physiological impact of disrupting astroglial ErbB-1 receptor signaling on adult female reproduction was evaluated on the 2-month-old female animals consisting of WT, G-/-/E+/+ and G+/+/E+/+ genotypes and displaying at least two consecutive estrous cycles. As depicted in Fig. 5
, WT (both Pon A- or no Pon A treated), G+/+/E+/+ (vehicle-treated) and G-/-/E+/+ (Pon A-treated) animals, which were lacking the expression of hErbB-1 mutants, continued to cycle normally. In contrast, Pon A-treated G+/+/E+/+ females, which expressed the mutant receptors, quickly displayed irregular cyclicity, an index of compromised reproductive competence (33, 34). These animals showed a prolonged diestrous state. The results are consistent with our previous findings in female animals in which the ErbB-1 receptor signaling was pharmacologically (but not cell type specifically) inhibited (12). Moreover, the disrupted estrous cycle induced by the expression of hErbB-1 mutant receptors was prevented by the treatment of human chorionic gonadotropin (hCG; Fig. 5
). The effect of acutely induced expression of the mutant receptors on the estrous cycle was reversible, as the regular cyclicity reoccurred in Pon A-treated G+/+/E+/+ animals (Fig. 5
). This is due to a rapid clearance rate of Pon A (18, 19), an inducer that dictates the expression level and duration of the hErbB-1 mutant receptors. Taking the advantage of temporally controlled mutant expression in this model, the same animals were resubjected to the identical experimental treatments. The same phenotypes were produced (Fig. 5
). Furthermore, the expression of hErbB-1 mutant receptors in astroglial cells also disrupted ovarian ovulation as no corpus lutea were found in the ovaries of G+/+/E+/+ animals treated with Pon A (Fig. 6
). In contrast, multiple corpus lutea were identified from diestrous WT (+ Pon A) and G+/+/E+/+ animals without Pon A (Fig. 6
).

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Fig. 5. Disruption of Estrous Cyclicity in Transgenic Female Mice
In experiment 1, no changes in estrous cyclicity were detected in WT [no injection (inj) or p inj (1 mg/140 µl/animal, sc)], G+/+/E+/+ (V inj), G-/-/E+/+ (P inj) and G+/+/E+/+ [P + hCG inj, 10 U in 0.1 ml of 0.9% NaCl per animal, ip] animals. In contrast, alteration in estrous cyclicity was found in G+/+/E+/+ mice sc-injected with Pon A. In the experiment 2, the same animals after restoring normal cycles were re-subjected to the identical treatments. The same phenotypes were produced as illustrated. Each arrow and arrowhead represent one injection of Pon A and hCG, respectively, at a specific time of a defined cycle stage (for details, see Materials and Methods). The same Pon A treatment was applied for the following experiments. The estrous cycle was monitored by daily vaginal cytology.
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Fig. 6. Histological Ilustration of Hematoxylin-Eosin Stained Ovaries from Diestrous WT (+ Pon A, n = 2) and G+/+/E+/+ Mice Treated with (n = 2) or without Pon A (n = 2)
Notice the lack of corpora lutea in the ovary of G+/+/E+/+ mouse as compared with multiple corpora lutea (arrows, x50) presented in both ovaries of WT (+ Pon A) or G+/+/E+/+ mice without the Pon A treatment.
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Alteration in LH, But Not FSH, Release by Disrupting ErbB-1 Receptor Signaling Capacity in Astroglia Contributes to Abnormal Estrous Cyclicity
Maintenance of normal estrous cyclicity depends upon the preovulatory LH surge, which occurs on the afternoon of each proestrous day (34). We hypothesize that functionally compromised astroglial ErbB-1 receptors contribute to alteration in the preovulatory LH release, thus leading to the disrupted estrous cyclicity. To test this hypothesis, serum LH levels were determined from female mice. A significant reduction in LH release was observed in G+/+/E+/+ animals injected with Pon A as compared with all the controls among which no difference was found (Fig. 7
). Intriguingly, no significant difference in serum level of FSH was detected in all experimental groups (Fig. 7
). Serum level of estradiol or progesterone was similar among groups during 1700 h (Fig. 7
). These findings suggest that disruption of astroglial ErbB-1-mediated signal transduction via expression of the hErbB-1 mutants specifically alters the release of LH. This alteration consequently affects the estrous cyclicity and thus normal reproductive function in female animals. We further detected that expression of hErbB-1 mutant receptors in astroglial cells did not influence the transcriptional activity of LHRH neurons as no difference in LHRH mRNA level was found among experimental animals (Fig. 8
). This result suggests that compromised LH secretion is most likely due to abnormal release of LHRH.

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Fig. 7. Serum Estradiol, Progesterone, LH, and FSH Levels Detected in WT and Transgenic Mice
A significant reduction in plasma LH level was evident in the G+/+/E+/+ animals treated with Pon A as compared with the WT and other control groups lacking hErbB-1 mutant expression. No significant changes in serum level of FSH, estradiol, and progesterone were observed among all experimental animals at 1700 h. No difference in the level of LH, FSH, estradiol, and progesterone was detected among all groups at 1000 h [open bars, n = 15 (3/phenotype/treatment)]. Each filled bar equals the mean ± SEM of four to five independent observations. **, P < 0.01 vs. all other groups during proestrus at 1700 h.
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Fig. 8. LHRH mRNA Level Detected by Semiquantitative RT-PCR in WT and Transgenic Female Mice
Top panel, Result of one representative experiment (gel image) detecting the expression of LHRH mRNA in the POA of the hypothalamus. Cyclophilin (p1B15) mRNA was also determined for internal control and used for normalization of LHRH mRNA levels. Lower panel, Quantitative analysis of all assay results. Each bar represents the mean ± SEM of four to five POAs from proestrous mice at 1700 h, except G+/+/E+/+ mice treated with Pon A (at the diestrus).
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DISCUSSION
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The ErbB-1 tyrosine kinase receptor-mediated signaling events possibly in astroglial cells have been implicated in the control of female sexual maturation via activation of LHRH neurons. It remains unclear whether this astroglia-to-neuron cellular interaction is also essential for the maintenance of normal adult female reproductive function. To address this question, we generated a transgenic animal model carrying the astroglia-specific ecdysone inducible system able to selectively express mutant ErbB-1 receptors in the astroglial cells. Thus, the expressed mutant receptors dimerize with the endogenous ErbB-1 receptors upon ligand binding and block the receptor-mediated signal transduction in astroglial cells. The present study provides evidence that astroglial cells actively engage in the neuroendocrine control of reproductive function by controlling LH secretion via regulatory inputs mediated by ErbB-1 receptors located on astroglial cells.
Gene deletion of ErbB-1 receptors results in a lethal phenotype during different stages of development depending on the animal genetic background (35). It is thus impossible to examine the role of ErbB-1 receptors in regulating adult endocrine function by using conventional gene targeting. To overcome this problem, we employed a modified ecdysone inducible expression system and created a transgenic animal model that enables us to specifically disrupt the signaling capacity of ErbB-1 receptors in astroglial cells in a time-controlled manner. The specific cell targeting was accomplished by incorporating an astroglia-specific promoter PGFAP into the expression system. As demonstrated in this study, the PGFAP-driven EcR receptor expression, which defines the cellular site of hErbB-1 mutant receptor expression, was found only in the glial-positive tissues from G+/+/E-/- animals such as brain and stomach. No endogenous EcR was detected in any examined tissue from the C57BL/6XDBA2(F1) WT animals. Immunofluorescence detection of EcR-positive nonneuronal-like cells in the brains from G+/+/E-/- animals but not in that of WT further implicates that the EcR expression is likely restricted in the astroglial cells. One key issue is whether the hErbB-1 mutant receptors are expressed in the brains and, importantly, in the astroglial cells of transgenic animals. Indeed, the mutant receptor is expressed in the brains and localized in astroglia-positive cells. Our study demonstrates that the GFAP promoter used in our transgenic animals is able to direct transgene expression in astroglia, and this observation is consistent with other reported studies (21, 22, 23, 24, 25). Based on our findings, the level of mutant gene expression is dictated by the delivery route and duration of the inducer (Pon A) required for activating the expression system. Direct injection of Pon A into the brain third ventricle of the transgenic animals induced a higher level of expression of the hErbB-1 mutant receptor as compared with that of Pon A-injected sc for 10 h. This difference is obviously due to higher concentration of Pon A acting on responsive cells in the brain. Moreover, the duration of Pon A action is also important for achieving higher level of mutant gene expression in transgenic animals. Clearly, Pon A induces higher mutant expression in the brain of transgenic animals 10 h after administration. This time frame is also comparable with a previous report (19). Therefore, our studies in the established transgenic animal model demonstrate that the GFAP promoter-controlled ecdysone inducible expression system is tightly regulated in an astroglia-specific manner. Importantly, the conditionally expressed hErbB-1 mutant receptor is capable of disrupting functional signaling capacity of endogenous ErbB-1 receptor by inhibiting its phosphorylation, an event required for initiating downstream cellular signal transduction (28). Furthermore, because ErbB-1 receptors are able to transphosphorylate ErbB-2 receptors in several cellular systems (29, 30) but do not collaborate with ErbB-4 receptors in hypothalamic astroglia (31, 32), it is important to address whether the expressed ErbB-1 mutant receptors in astroglia can inhibit phosphorylation of endogenous ErbB-2 receptors. Our observation clearly shows that ligand-induced activation of ErbB-1 does not stimulate the phosphorylation of ErbB-2 receptors in astroglial cells, suggesting no obvious interaction between ligand-activated ErbB-1 and ErbB-2 in astroglia. Intriguingly, expression of ErbB-1 mutant seems to reduce the basal phosphorylation level of ErbB-2 receptor in astroglial cells.
A series of studies have demonstrated that ErbB-1 receptor-mediated signaling events are linked to female sexual development via the activation of LHRH release (10, 11, 36, 37). Recently, we have provided evidence implicating that ErbB-1 receptors in the hypothalamus also play an important role in regulating adult female reproductive function, and conversely, a non-cell type-specific blockade of ErbB-1 receptor signaling capacity may contribute to abnormalities associated with reproductive aging (12). However, it remains inconclusive whether the astroglial ErbB-1 receptors are required for maintaining normal adult reproduction. Employing the established transgenic animal model in the present study, we are able to address this issue by demonstrating that selectively disrupting signal transduction mediated by ErbB-1 receptors in astroglia leads to estrous acyclicity, an index of abnormal reproduction (34). The transgenic animals displayed a prolonged diestrus, a state associated with aged female animals (34). The normal cyclicity was soon restored in these acutely ErbB-1 receptor-disrupted animals possibly due to a timely regulated hErbB-1 mutant expression. As described, the bioactivity of Pon A determines the transgene expression in our transgenic animals. Because Pon A is a synthetic ecdysone-like steroid and has the nature of the lipophilic feature, short half-life and high clearance rate (19), the mutant receptor expression will be paused once Pon A is metabolized. Thus the normal ErbB-1 receptor signaling process could be resumed. Taking the advantage of this tight expression control of inducibility, the same phenotype was reproduced using the identical groups of transgenic animals after they were recovered from the previous experiment and resubjected to the same experimental manipulation. Currently, we are in the process of chronically disrupting ErbB-1 receptor signaling to examine whether there are additional variations in phenotype. It is important to point out that the effect of expressed ErbB-1 mutant receptor on reproductive function is via the disruption of the endogenous ErbB-1 receptor-mediated signaling events. This view is supported by the evidence that induced expression of mutant ErbB-1 receptor exerts physiological impacts on cellular signaling directly related to the receptor signaling in cells (13, 14, 26) and animals (15).
Intriguingly, the negative impact on estrous cyclicity is only associated with a significant reduction in LH release, an LHRH-dependent event required for triggering the ovulation. This alteration appears not to be due to disruption of the transcriptional activity of LHRH neurons because no difference in the level of LHRH mRNA is evident among all experimental animals. These results indicate that the disruption of estrous cyclicity may be caused by the alteration in the LHRH/LH secretion at the hypothalamo-pituitary level. Indeed, a time-controlled administration of hCG is able to prevent the disrupted estrous cycle. Therefore, the astroglial ErbB-1 receptor-mediated signaling events are required for controlling LH secretion most likely via modulation of LHRH release. This view is further supported by other findings that activation of ErbB-1 receptors in astroglial cells leads to the stimulation of LHRH secretion (but not the synthesis) (9, 10, 38, 39). Moreover, no notable changes in FSH secretion from the transgenic animals also support above view and suggest that LH and FSH release are differentially regulated by the astroglia-associated signaling molecules in vivo. The specific mechanisms associated with the differential astroglia control on LH and FSH secretion requires further study.
In summary, the present study demonstrates that the astroglia-specific and temporally controlled disruption of ErbB-1 receptor signaling compromises reproductive competence by altering LH release in female mice. The ability of the hErbB-1 mutant receptors to block endogenous ErbB-1 receptor signaling in astroglia, which affects the LHRH neuronal activity related to the control of LH secretion, supports a novel role of astroglial ErbB-1 receptors on regulating adult female reproductive function. Importantly, these results may shed new light on the molecular mechanism by which compromised ErbB-1 receptor signaling capacity in astroglia may contribute, at least in part, to reproductive abnormalities associated with aging. Our findings further broaden the concept that astroglial cells are actively engaged not only in the control of functional plasticity of neuronal synapses, but also in reproductive neuroendocine function via cellular interactions between astroglia and neurons.
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MATERIALS AND METHODS
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Generation and Identification of Transgenic Animals
Transgenic line-1 (PGFAP-pVgRXR, the GFAP promoter was a gift from Dr. Lennart Mucke, University of California, San Francisco, CA) and line-2 (pIND-hErbB-1mut, the mutant construct was provided by Dr. Axel Ullrich, Max-Planck Institute, Martinsried, Germany) were produced by the Transgenic Animal Facility at Oregon Health Sciences University (Portland, OR). In brief, the individual expression cassette (Fig. 1A
) was isolated and injected into the pronuclei of fertilized mouse eggs [obtained from C57BL/6XDBA2(F1) mice]. Thereafter, the eggs were transferred to the uteri of surrogate pseudopregnant mice, allowing them to develop to term. Genotyping was performed using PCR with primer PGFAP-S (5'-GTCCAACCCGTTCCTCCATAAA-3'); VgEcR-A2 (5'-CGTCTAAGTGGAGTTCGT-3'); Ecdy-F-S (5'-CTGAATACTTTCAAC AAGTTA-3'); and hErbB-1 (5'-CAAACTTTCTTTTCCTCCAGA-3'). PGFAP-S and VgEcR-A2 amplify a 258 bp DNA fragment encompassing the 3'-end of the mouse GFAP promoter and 5'-end of the ecdysone receptor. Ecdy-F-S and hErbB-1 amplify a portion of transgene fragment (360 bp) encircling part of pIND vector and extracellular domain of the human ErbB-1 receptor. PCR was carried out on genomic DNA (100 ng) derived from tail biopsies for 35 cycles of 94 EC, 15 sec; 67 C, 1 min; 72 C, 2 min in a 25-µl reaction volume containing: 20 pmol of each primer, 2.5 µl of 10x PCR buffer (Pwo, Roche, Germany), 1 µl of 25 mM MgSO4, 2 µl of 10 mM deoxynucleotide triphosphates (dNTPs) (dATP, dCTP, dGTP, and dTTP), 0.125 µl of Pwo:Tsg (3:1, Tsg was purchased from Bio Basic Inc., Ontario, Canada) polymerases mixture. The founder PCR genotyping results were further confirmed by Southern blot analysis using a procedure as reported (37).
Four independent founders of each transgenic line were identified. The PGFAP-pVgRXR and pIND-hErbB-1mut-positive founders were mated with C57BL/6XDBA2(F1) WT mice to establish the transgenic line-1 and line-2, respectively. No general morphological or physiological alteration has been observed in nontreated animals housed four per cage and maintained under a controlled temperature (2325 C) and photoperiod environment (14-h light, 10-h dark cycle). The bitransgenic hemizygous animals (G+/-/E+/-) were produced by crossing the line-1 (G+/+/E-/-) and line-2 (G-/-/E+/+) homozygous animals. The bitransgenic homozygous breeding animals (G+/+/E+/+) were generated by mating bitransgenic brothers and sisters, and confirmed by backcrossing with WT animals, which every progeny should be positive for both transgenes. The homozygous animals used for experiments were then produced by the proven homozygous parents. Animal care and use protocols were approved by University of Nebraska Medical Center according to the institutional guidelines.
Characterization of Transgenic Animals
1) Detection of EcR expression in transgenic animals.
Tissues from 2-month-old WT and G+/+/E-/- mice were collected and homogenized. The total protein concentration of each lysate was determined using a Bradford assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Lysates with equal amount of protein (3 mg) were then immunoprecipitated overnight at 4 C with an EcR antibody (15C3, 1:50 dilution, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). The immunoprecipitated proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel, transferred to nitrocellulose membranes and probed with an EcR antibody (10F1, 1:500, Hybridoma Bank). The reactions were developed using the enhanced chemiluminescence system (Pierce Chemical Co., New York, NY). For immunofluorescence detection, the same genotype animals were cardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Immunohistochemistry was performed using 15-µm-thick frozen sections. Antigen retrieval was performed using 1 mg/ml Protease XXV (Neomarkers) for 10 min at 37 C. Nonspecific protein binding was blocked with 10% normal goat serum (Zymed Laboratory, South San Francisco, CA) at room temperature for 30 min. EcR antibody (AG10.2, a gift from Dr. Thummel, Howard Hughes Medical Institute, University of Utah) was used at 1:20 dilution and incubated overnight at 4 C. Antibody detection was performed using Molecular Probe (Eugene, OR) Alexa Fluor 546 goat antimouse IgG incubated 1 h at room temperature, rinsed and viewed under an appropriate filter on a Leica fluorescent microscope.
2) Detection of mutant hErbB-1 expression in transgenic animals.
Two-month-old animals with various genotypes (WT, G+/+/E-/-, G-/-/E+/+, G+/+/E+/+) were treated with Pon A [p, for sc injection: 1 mg/140 µl/mouse (15 µl of 100% ethanol, 25 µl of dimethylsulfoxide and 100 µl of corn oil); for brain injection (third ventricle): 0.5 mg/15 µl/mouse (7.5 µl of ethanol and 7.5 µl of dimethylsulfoxide)]. Tissues were collected 5 to 20 h post injection. Each tissue lysate containing 3 mg of protein was used for immunoprecipitation overnight at 4 C with a human-specific ErbB-1 antibody (EGFR-Ab-13, 1:50, Labvision, Fremont, CA). The hErbB-1 mutant receptor was detected with another human-specific ErbB-1 antibody (EGFR-Ab-14, 1:500, Labvision). Some treated animals were perfused and brain tissues were prepared for immunohistochemistry using the similar procedure described above. Sections were incubated overnight at 4 C with a human-specific ErbB-1 (EGFR-Ab-10, 1:100, Labvision) and a GFAP-Ab-4 (1:10, Labvision) antibodies.
3) Detection of endogenous phosphorylated mouse ErbB-1 receptor in astroglial cells of transgenic animals.
Primary astroglia culture was carried out using the brains from 2-d-old animals employing a reported procedure (39). Each cell mix was maintained in a T-75 flask until reaching 95% of confluence. After mechanically removal of neuronal cells, the astroglial cells were seeded at 1 x 106 cells per 10-cm culture dish. The cells were treated with nt (nontreated), v (vehicle), or 10 µM of p for 24 h, followed by exposing to EGF (100 ng/ml, R&D Systems, Minneapolis, MN) for 5 min at room temperature. The treated cell lysates were immunoprecipitated with a mouse-specific ErbB-1 antibody [EGFR-SC-03 (1005), 1:100, Santa Cruz Biotechnology, Santa Cruz, CA]. The phosphorylated mouse ErbB-1 receptor was detected with PY20 (1:500, Santa Cruz Biotechnology) via Western blot. Thereafter, the total content of mouse ErbB-1 receptor was detected with EGFR-SC-03 antibody on the same membrane stripped with 0.2 N NaOH. After the similar procedure, endogenous phosphorylated ErbB-2 receptors were first immunoprecipated with a mouse reactive antibody [c-erbB-2, Ab-9 (1:100), MS-326-P, Labvision] and detected with PY20 (1:500). The total content of mouse ErbB-2 was detected with a mouse reactive antibody (c-erbB-2 Ab-1, RB-103-P0, 1:500, Labvision).
Impact on the Estrous Cycle by Pon A-Induced Expression of hErbB-1 Mutant
Two-month-old WT, G+/+/E+/+ and G-/-/E+/+ female animals displaying at least two consecutive estrous cycles were sc-injected with Pon A (same dosage as above); first injection at 1000 h of diestrous d 2; second injection on the following day at the same time]. Injection of hCG (Sigma catalog no. C8554; 10 U in 0.1 ml of 0.9% NaCl per animal, ip) was also performed. After injection, the estrous cycle was monitored by daily vaginal cytology. Once all the animals were recovered from experiment 1, they were resubjected to the identical experimental treatments.
Ovarian Histology
Ovaries were fixed in 10% formalin and embedded in paraffin for serial sectioning at 10 µm. The sections were stained with hematoxylin-eosin.
Measurements of Serum Estradiol, Progesterone, LH, and FSH in Transgenic Animals
Selected 2-month-old animals include: 1) WT (nt); 2) G-/-/E+/+ [Pon A-injected (p)]; 3) G+/+/E+/+ (nt); 4) G+/+/E+/+ [vehicle-injected (v)]; and 5) G+/+/E+/+ (p). Two injections (v or p) were conducted on each animal at the1700 h of the first and second diestrous day before proestrus. The trunk blood samples were collected at 24 h after the second injection (all control mice were at proestrus). The 1700-h time point was selected because the LH surge occurs at this time in the proestrous animals in this colony. The plasma levels of LH and FSH were measured using LH and FSH EIA kits (Amersham, Piscataway, NJ). Circulating estradiol and progesterone concentrations were determined by employing enzyme immunoassay detection kits as previously reported (40).
Determination of LHRH mRNA Level by RT-PCR Assay
Animals.
The preoptic areas (POA) from each group of animals used for gonadotropin and steroid determination were collected and used for RNA preparation according to the procedure reported previously (12). Because previous works showed that the highest level of LHRH mRNA was found at time when LH surge occurred (10, 41) in female rats housed under a 14-h light, 10-h dark photoperiod, we characterized the level of LHRH mRNA during the time when LH surge occurred in our mouse colony.
Oligodeoxynucleotides
All oligodeoxynucleotides used for the PCR were synthesized by MWG-BIOTECH, Inc. (High Point, NC). An oligonucleotide containing a 15-oligomer polydeoxythymidine sequence (purchased from Promega, Madison, WI) was used for reverse transcription of poly-A cellular mRNA. The LHRH mRNA levels were determined by using a sense (5'-AAGGCTGCTCCAGCCAGCACT-3') and an antisense primers (5'-TACATCTTCTTCTGCCCAGCT-3') to amplify a 223-bp cDNA fragment encompassing part of the signal peptide-, entire LHRH decapeptide- and part of the GnRH-associated peptide-encoding sequences (42). Individual sources of variability were accounted for by coamplifying cyclophilin mRNA, which is constitutively expressed in brain (43). The cyclophilin primers were 5'-GGCAAGTCCATCTACGGA-3' (corresponding to nucleotides 265282) and 5'-ACATGCTTGCCATCCAGC-3' (complementary to nucleotides 405422) (43).
RT-PCR Procedures
The procedures have been described previously in detail (12). In brief, RT reaction was carried out for 2 h at 37 C in a 20-µl volume. Each reaction mixture contained ribonuclease-free deoxyribonuclease-treated 200 ng of total RNA, 1x RT buffer, 0.01 M dithiothreitol, 0.5 mM of each dNTPs, 20 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD). PCR was performed in a 25-µl total volume consisted of 2 µl of diluted (1 µl RT:1 µl sH2O) RT mixture, 2.5 µl of 10x PCR buffer, 4 µl of 25 mM MgCl2, and 1 µl of 10 mM dNTPs, 20 pmol of each specific gene primer set including both 5' and 3' primers, 5 pmol of each 5'- and 3'-end cyclophilin primers and 0.625 units of Taq polymerase (Promega). After samples were treated at 94 C for 4 min to inactivate the RT transcriptase, PCR consisted of 35 cycles of denaturing (95 C, 15 sec), annealing (55 C, 1 min), and extension (72 C, 2 min), and a final extension of 7 min at 72 C.
Quantitative Analysis
After electrophoresis, 20 µl of each PCR sample in a 3% agarose gel containing ethidium bromide (0.1 µg/ml), PCR amplified cDNAs were captured by photographing on 555 Polaroid films (Cambridge MA). The photos were scanned for densitometric analysis using Hewlett Packard ScanJet 6200C flat bed scanner and the computer image program written by Dr. Wayne Rasband (National Institutes of Health, Bethesda, MD). We used a background subtracted-mean OD to measure each amplified cDNA signal. The mean OD values were normalized according to the coamplified cyclophilin cDNA value detected in each sample.
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ACKNOWLEDGMENTS
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We are grateful to Drs. Claudia Kappen, Robert Norgren, and Irvine Zucker for their critical review of this paper.
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FOOTNOTES
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This work was supported by NIH Grant AG-18078. Both the construction of the transgenes and the production of transgenic founders were carried out in Dr. Sergio Ojedas laboratory using NIH Grant HD-25123.
Abbreviations: CNS, Central nervous system; dNTP, deoxynucleotide triphosphate; EcR, ecdysone receptors; EGF, epidermal growth factor; GFAP, glial fibrillary acidic protein; hErbB-1, human ErbB-1; hCG, human chorionic gonadotropin; LHRH, LH-releasing hormone; nt, not treated; POA, preoptic area; Pon A, ponasterone A; v, vehicle; WT, wild-type.
Received for publication January 22, 2003.
Accepted for publication July 30, 2003.
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REFERENCES
|
---|
- Haydon PG 2001 GLIA: listening and talking to the synapse. Nat Rev Neurosci 2:185193[CrossRef][Medline]
- Bezzi P, Volterra A 2001 A neuron-glia signalling network in the active brain. Curr Opin Neurobiol 11:387394[CrossRef][Medline]
- Pfrieger FW, Barres BA 1997 Synaptic efficacy enhanced by glial cells in vitro. Science 277:16841687[Abstract/Free Full Text]
- Ullian EM, Sapperstein SK, Christopherson KS, Barres BA 2001 Control of synapse number by glia. Science 291:657661[Abstract/Free Full Text]
- Nagler K, Mauch DH, Pfrieger FW 2001 Glia-derived signals induce synapse formation in neurones of the rat central nervous system. J Physiol 533:665679[Abstract/Free Full Text]
- Mauch DH, Nagler K, Schumacher S, Goritz C, Muller EC, Otto A, Pfrieger FW 2001 CNS synaptogenesis promoted by glia-derived cholesterol. Science 294:13541357[Abstract/Free Full Text]
- Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha BK, Von Zastrow M, Beattie MS, Malenka RC 2002 Control of synaptic strength by glial TNF
. Science 295:22822285[Abstract/Free Full Text]
- Ojeda SR, Ma YJ 1999 Glial-neuronal interactions in the neuroendocrine control of mammalian puberty: facilitatory effects of gonadal steroids. J Neurobiol 40:528540[CrossRef][Medline]
- Ma YJ, Berg-von der Emde K, Rage F, Wetsel WC, Ojeda SR 1997 Hypothalamic astrocytes respond to transforming growth factor
with secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone. Endocrinology 138:1925[Abstract/Free Full Text]
- Ma YJ, Junier M-P, Costa ME, Ojeda SR 1992 Transforming growth factor alpha (TGF
) gene expression in the hypothalamus is developmentally regulated and linked to sexual maturation. Neuron 9:657670[Medline]
- Ma YJ, Dissen GA, Merlino G, Coquelin A, Ojeda SR 1994 Overexpression of a human transforming growth factor alpha (TGF
) transgene reveals a dual antagonistic role of TGF
in female sexual development. Endocrinology 135:13921400[Abstract]
- Hou J, Li B, Yang Z, Fager N, Ma MY 2002 Altered gene activity of epidermal growth factor receptor (ErbB-1) in the hypothalamus of aging female rat is linked to abnormal estrous cycles. Endocrinology 143:577586[Abstract/Free Full Text]
- Kashles O, Yarden Y, Fischer R, Ullrich A, Schlessinger J 1991 A dominant negative mutation suppresses the function of normal epidermal growth factor receptors by heterodimerization. Mol Cell Biol 11:14541463[Medline]
- Redemann N, Holzmann B, von Rüden T, Wagner EF, Schlessinger J, Ullrich A 1997 Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants. Mol Cell Biol 12:491498
- Wu J-X, Spencer-Dene B, Adamson ED 1998 Transgenic mice expressing a dominant negative epidermal growth factor receptor have neural and other defects and remain viable by low expression. Transgenics 2:287298
- No D, Yao T-P, Evans RM 1996 Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci USA 93:33463351[Abstract/Free Full Text]
- Reick M, Garcia JA, Dudley C, McKnight SL 2001 NPAS2: an analog of clock operative in the mammalian forebrain. Science 293:506509[Abstract/Free Full Text]
- Albanese C, Reutens AT, Bouzahzah B, Fu M, DAmico M, Link T, Nicholson R, Depinho RA, Pestell RG 2000 Sustained mammary gland-directed, ponasterone A-inducible expression in transgenic mice. FASEB J 14:877884[Abstract/Free Full Text]
- Saez E, Nelson MC, Eshelman B, Banayo E, Koder A, Cho GJ, Evans RM 2000 Identification of ligands and coligands for the ecdysone-regulated gene switch. Proc Natl Acad Sci USA 97:1451214517[Abstract/Free Full Text]
- Yao T-P, Forman BM, Jiang Z, Cherbas L, Chen J-D, McKeown M, Cherbas P, Evans RM 1993 Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature 366:476479[CrossRef][Medline]
- Johnson WB, Ruppe MD, Rockenstein EM, Price J, Sarthy VP, Verderber LC, Mucke L 1995 Indicator expression directed by regulatory sequences of the glial fibrillary acidic protein (GFAP) gene: in vivo comparison of distinct GFAP-LacZ transgenes. Glia 13:174184[Medline]
- Mucke L, Yu GQ, McConlogue L, Rockenstein EM, Abraham CR, Masliah E 2000 Astroglial expression of human
(1)-antichymotrypsin enhances alzheimer-like pathology in amyloid protein precursor transgenic mice. Am J Pathol 157:20032010[Abstract/Free Full Text]
- Smith-Arica JR, Morelli AE, Larregina AT, Smith J, Lowenstein PR, Castro MG 2000 Cell-type-specific and regulatable transgenesis in the adult brain: adenovirus-encoded combined transcriptional targeting and inducible transgene expression. Mol Ther 2:579587[CrossRef][Medline]
- Morelli AE, Larregina AT, Smith-Arica J, Dewey RA, Southgate TD, Ambar B, Fontana A, Castro MG, Lowenstein PR 1999 Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity. J Gen Virol 80(Pt 3):571583
- Kordower JH, Chen EY, Winkler C, Fricker R, Charles V, Messing A, Mufson EJ, Wong SC, Rosenstein JM, Bjorklund A, Emerich DF, Hammang J, Carpenter MK 1997 Grafts of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice: trophic and tropic effects in a rodent model of Huntingtons disease. J Comp Neurol 387:96113[CrossRef][Medline]
- Livneh E, Prywes R, Kashles O, Reiss N, Sasson I, Mory Y, Ullrich A, Schlessinger J 1996 Reconstitution of human epidermal growth factor receptors and deletion mutants in cultured hamster cells. J Biol Chem 261:1249012497
- Jessen KR, Mirsky R 1983 Astrocyte-like glia in the peripheral nervous system: an immunohistochemical study of enteric glia. J Neurosci 3:22062218[Abstract]
- Carpenter G 1987 Receptors for epidermal growth factor and other polypeptide mitogens. Annu Rev Biochem 56:881914[CrossRef][Medline]
- Yarden Y, Ullrich A 1988 Growth factor receptor tyrosine kinases. Annu Rev Biochem 57:443478[CrossRef][Medline]
- Burden S, Yarden Y 1997 Neuregulins and their receptors: a versatile signaling module in organogenesis and oncogenesis. Neuron 18:847855[Medline]
- Ma YJ, Hill DF, Creswick KE, Costa ME, Cornea A, Lioubin MN, Plowman GD, Ojeda SR 1999 Neuregulins signaling via a glial erbB-2-erbB-4 receptor complex contribute to the neuroendocrine control of mammalian sexual development. J Neurosci 19:99139927[Abstract/Free Full Text]
- Prevot V, Rio C, Cho GJ, Lomniczi A, Heger S, Neville CM, Rosenthal NA, Ojeda SR, Corfas G 2003 Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes. J Neurosci 23:230239[Abstract/Free Full Text]
- Ojeda SR, Urbanski HF 1994 Puberty in the rat. In: Knobil E, Neill JD, eds. The physiology of reproduction, 2nd ed. Vol 2. New York: Raven Press; 363409
- Wise PM, Smith MJ, Dubal DB, Wilson ME, Krajnak KM, Rosewell KL 1999 Neuroendocrine influences and repercussions of the menopause. Endocr Rev 20:243248[Abstract/Free Full Text]
- Threadgill DW, Dlugosz AA, Hansen LA, Tennenbaum T, Lichti U, Yee D, LaMantia C, Mourton T, Herrup K, Harris RC, Barnard JA, Yuspa SH, Coffey RJ, Magnuson T 1995 Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 269:230234[Medline]
- Ma YJ, Hill DF, Junier M-P, Costa ME, Felder SE, Ojeda SR 1994 Expression of epidermal growth factor receptor changes in the hypothalamus during the onset of female puberty. Mol Cell Neurosci 5:246262[CrossRef][Medline]
- Ma YJ, Costa ME, Ojeda SR 1994 Developmental expression of the genes encoding transforming growth factor
(TGF
) and its receptor in the hypothalamus of female rhesus macaques. Neuroendocrinology 60:346359[Medline]
- Junier M-P, Wolff A, Hoffman GE, Ma YJ, Ojeda SR 1992 Effect of hypothalamic lesions that induce precocious puberty on the morphological and functional maturation of the luteinizing hormone-releasing hormone neuronal system. Endocrinology 131:787798[Abstract]
- Ma YJ, Berg-von der Emde K, Moholt-Siebert M, Hill DF, Ojeda SR 1994 Region-specific regulation of transforming growth factor
(TGF
) gene expression in astrocytes of the neuroendocrine brain. J Neurosci 14:56445651[Abstract]
- Hou J, Li B, Yang Z, Fager N, Ma MY 2002 Functional integrity of ErbB-4/-2 tyrosine kinase receptor complex in the hypothalamus is required for maintaining normal reproduction in young adult female rats. Endocrinology 143:19011912[Abstract/Free Full Text]
- Park OK, Gugneja S, Mayo KE 1990 Gonadotropin-releasing hormone gene expression during the rat estrous cycle: effects of pentobarbital and ovarian steroids. Endocrinology 127:365372[Abstract]
- Adelman JP, Mason AJ, Hayflick JS, Seeburg PH 1986 Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat. Proc Natl Acad Sci USA 83:179183[Abstract]
- Danielson PE, Forss-Petter S, Brow MA, Calavetta L, Douglass J, Milner RJ, Sutcliffe JG 1988 p1B15: A cDNA clone of the rat mRNA encoding cyclophilin. DNA 7:261267[Medline]