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Endocrinology Vol. 143, No. 5 1901-1912
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Functional Integrity of ErbB-4/-2 Tyrosine Kinase Receptor Complex in the Hypothalamus Is Required for Maintaining Normal Reproduction in Young Adult Female Rats

Jingwen Hou, Biao Li, Zhihui Yang, Nicole Fager and Mark Y. J. Ma

Center for Human Molecular Genetics and Department of Cell Biology & Anatomy, University of Nebraska Medical Center, Omaha, Nebraska 68198-5455

Address all correspondence and requests for reprints to: Mark Y. J. Ma, Center for Human Molecular Genetics and Department of Cell Biology & Anatomy, 985455 University of Nebraska Medical Center, Omaha, Nebraska 68198-5455. E-mail: . yma{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ErbB-1 tyrosine kinase receptors are necessary for maintaining female reproduction by modulating the release of LH-releasing hormone (LHRH). Changes in ErbB-1 signaling capacity in aging rats are linked to compromised reproduction. The interactive and synergistic nature of different members of ErbB receptors in mediating signal transduction exists in many cellular systems. Particularly, the interactions among ErbB-1 and ErbB-2 or ErbB-4 and ErbB-2 are known to be involved in the stimulation of LHRH secretion during sexual maturation. Thus, ErbB-4/-2 receptors may also play a role in maintaining reproduction during adulthood, and consequently, alteration in ErbB-4/-2 signaling capacity may contribute to compromised reproductive competence during aging. By in situ hybridization histochemistry, ErbB-4/-2 mRNAs were detected in the preoptic area (POA) and arcuate nucleus, which are important areas involved in the control of LHRH neuronal activity. RT-PCR analyses showed that levels of ErbB-4/-2 mRNA increased to a maximal value in the POA of young adult animals before the LH surge. However, no such increase was found in middle-aged female rats. The timing of the decrease in ErbB-4 mRNA in the median eminence-arcuate nucleus of middle-aged rats was delayed compared with that in young adult animals. Disruption of functional ErbB-4/-2 receptor complex by blocking ErbB-2 receptor synthesis in the hypothalamus via an infusion of ErbB-2 antisense oligodeoxynucleotide resulted in an estrous acyclicity in young adult rats. These results indicate that changes in ErbB-4/-2 gene expression and functional integrity of this ErbB-4/-2 receptor complex in the hypothalamus of middle-aged female animals may lead to an altered preovulatory LH release. Thus, the ErbB-4/-2 receptor complex is a physiological component necessary for maintaining female reproduction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FEMALE REPRODUCTION IS regulated by a group of neurons that release LH-releasing hormone (LHRH), a neuropeptide that controls the release of gonadotropins (LH and FSH), which regulate ovarian function. The modulatory process of LHRH secretion is exceedingly complex as it requires an integral synchronization of excitatory and inhibitory inputs from regulatory neurons and astroglial cells (1, 2, 3, 4, 5, 6). Desynchronization of these regulatory components results in an abnormality in LHRH secretion leading to delayed sexual maturation (2, 6, 7) and compromised reproduction during aging (8, 9, 10, 11, 12, 13).

Evidence exists to support the concept that trophic factors mediated by receptor tyrosine kinases also play a pivotal role in controlling the LHRH neuronal system (6, 14, 15, 16, 17). Studies using immature female rats have demonstrated that glial growth factor induced-activation of ErbB-1 and ErbB-4/-2 receptor complex is involved in regulating LHRH release during the onset of puberty (6). That this ErbB signaling system may be functionally linked to controlling LHRH neuronal activity during adulthood is supported by recent findings that ErbB-1 receptors are important for maintaining reproductive function. Alteration in ErbB-1 receptor signaling capacity is associated with changes in LHRH neuronal activity in aging female rats, and thus contributes to compromised reproductive competence (18).

ErbB receptor-mediated signal transduction involves multiple receptor members. Ligand recognition by a specific ErbB receptor not only results in homodimerization of the same receptor, but also in recruitment of related receptors forming heterodimers via combinatorial protein interactions (19, 20). The family of ErbB receptors consists of ErbB-1 (21), ErbB-2 (22), ErbB-3 (23, 24), and ErbB-4, which has four isoforms (25, 26). ErbB-1 binds to epidermal growth factor (EGF), TGF{alpha}, and other EGF-related ligands (27). While ErbB-3 and ErbB-4 interact with a large group of structurally related, EGF-like peptides called neuregulins (NRGs) (20, 28, 29, 30, 31), a ligand for ErbB-2 has not yet been found (19). However, ErbB-2 appears to function as a coreceptor (32) necessary for forming receptor complexes with ligand-activated ErbB-1, ErbB-3, and ErbB-4 receptors (32, 33, 34, 35, 36, 37, 38). Because ErbB-2 acts as a coreceptor, heterodimers of ErbB-2 with ErbB-1, ErbB-4, or ErbB-3 are the preferred signaling complexes, which exist in many cellular systems (19, 20). We recently demonstrated that ErbB-2 and ErbB-4 gene expression within the hypothalamus occurs mostly in astrocytes (39). Furthermore, expression of these two genes is selectively increased in the hypothalamus during the onset of female puberty (39). Activation of ErbB-4/-2 receptor complexes in hypothalamic astrocytes stimulates LHRH release through a mechanism involving enhancement of astrocytic PGE2 secretion and facilitation of ErbB-1-mediated signaling events (39). Disruption of hypothalamic ErbB-2 synthesis using an antisense oligodeoxynucleotide prevents PGE2 release and the ability of astrocytes to stimulate LHRH release (39). Consequently, ovulation is blocked and the animals display constant estrus, a state associated with female animals undergoing reproductive aging. These observations indicate that, in addition to ErbB-1-mediated signaling, involvement of the ErbB-4/-2 signaling component is also essential for activation and maintenance of LHRH neuronal activity. Therefore, the interactive and synergistic nature exhibited by multiple members of the ErbB family strongly suggests that additional ErbB members are involved in controlling LHRH neurons and thus, reproduction during adulthood. The present study was undertaken to examine the hypothesis that ErbB-4/-2 receptor complexes are required for maintaining normal reproduction in adult female rats and to determine if altered ErbB-4/-2 receptor expression occurs during the onset of reproductive aging. We report here that: 1) cellular expression of ErbB-4/-2 is predominant in the hypothalamic areas related to regulating LHRH release; 2) selective increases in hypothalamic ErbB-4/-2 mRNA levels before and during the preovulatory LH surge occurs in young adult female rats, but this expression pattern is both quantitatively and temporally altered in middle-aged animals; and 3) in vivo disruption of ErbB-4/-2 signaling components by infusion of an ErbB-2 antisense oligodeoxynucleotide in the hypothalamus results in estrous acyclicity in young adult female rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Young adult (YA, 4–5 months old, nonpregnant proven breeder) and middle-aged (MA, 9–10 months old, retired breeder) female rats were purchased from Harlan (Indianapolis, IN). YA females (2 months old) were also purchased to be used for the infusion of an ErbB-2 antisense oligodeoxynucleotide. Animals were maintained two per cage in a room under a controlled temperature (23-25 C) and photoperiod environment (14-h light, 10-h dark, lights on at 0500 h) with free access for food and water. Estrous cyclicity was determined by daily monitoring vaginal cytology. The proestrous state was determined by: 1) vaginal cytology; and 2) animals with a uterine weight of at least 200 mg and a uterus ballooned with fluid. YA rats displaying at least two to three consecutive regular 4- to 5-d estrous cycles and MA rats also exhibiting two to three regular but prolonged (6–8 d) estrous cycles were killed during proestrus from the early morning to late afternoon (0600, 0800, 1000, 1200, 1500, and 1800 h). This time frame was chosen because it covers major neuroendocrine events during the proestrous phase. Seventy percent of the YA animals were 4-d cyclers. In the MA group, 60% of rats were 6-d cyclers.

Every experiment was performed in compliance with Guidelines on the Use of Laboratory and Experimental Animals. The protocols used were approved by our Institutional Animal Care and Use Committee at University of Nebraska Medical Center (protocol 99-055-00).

Tissue dissection and RNA isolation
Immediately after decapitation, brains were removed and two major parts were dissected from the hypothalamus as described previously (18). The preoptic area (POA) was collected by a coronal cut just posterior to the entry point of the optic chiasm and two cuts converging from the lateral edges of the optic chiasm to a point rostral to the decussation of the optic nervers. The median eminence-arcuate region (ME-ARC) was dissected by a coronal cut at the rostral border of the mammillary body and one sagittal cut halfway between the ME and the hypothalamic sulci on each side. A site on the anterior region of the cortex (CTX) was also collected to serve as a control tissue. All collected tissues were immediately stored at -80 C until RNA extraction. Total RNA was isolated by the acid-phenol method (40). Trunk bloods were collected, allowed to clot for overnight at 4 C followed by centrifugation. The supernatant was then collected and stored at -20 C until hormone assays.

RT-PCR assay
Oligodeoxynucleotides.
All oligodeoxynucleotides used for PCR were synthesized by MWG-BIOTECH, Inc. (High Point, NC). An oligonucleotide containing a 15-oligomer polydeoxythymidine sequence (purchased from Promega Corp., Madison, WI) was used for RT of poly-A cellular mRNA. Rat ErbB-2 (322 bp) and ErbB-4 (240 bp) cDNAs were amplified from total RNA derived from the POA, ME-ARC, or CTX. The sense primer (5'-CAGTGTGTCAACTGCAGTCA-3') used to amplify ErbB-2 corresponds to nucleotides (nt) 1610–1629 in the rat ErbB-2 mRNA sequence (22). The antisense primer (5'-CAGGAGTGGGTGCAGTTGAT-3') is complementary to nt 1913–1932. The 20-oligomer sense primer of ErbB-4 (5'-AACTGCACCCAGGGGTGTAA-3') corresponds to nt 1819–1910 in the rat ErbB-4 mRNA sequence (GenBank accession number AF041838). The antisense primer (5'-GGTTAAGGGCTCGACTAACT-3') is complementary to nt 2111–2130. Individual sources of variability were accounted for by coamplifying cyclophilin mRNA, which is constitutively expressed (41). In addition, it has been shown that no significant differences in cyclophilin mRNA levels are detectable regardless of reproductive status or age (13, 18, 42). Therefore, cyclophilin was used as an internal control. The cyclophilin primers are 5'-GGCAAGTCCATCTACGGA-3' (corresponding to nt 265–282) and 5'-ACATGCTTGCCATCCAGC-3' (complementary to nt 405–422) (41).

RT-PCR procedures.
The procedures have been described previously in detail (18, 43) with minor modifications. In brief, RT reaction was carried out for 2 h at 37 C in a 20-µl volume. Each reaction mixture contained 200 ng of total RNA, which was RNase-free DNase treated, 1x RT buffer, 0.01 M dithiothreitol, 0.5 mM of each deoxynucleotide triphosphate, and 20 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD). PCR was performed in a 25-µl total volume consisting 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, 1 µl of 10 mM deoxynucleotide triphosphate, 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 U of Taq polymerase (Promega Corp.). 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), extension (72 C, 2 min), and a final extension of 7 min at 72 C.

Quantitative analysis.
Amplified cDNAs were visualized by electrophoresis where 20 microliters of each PCR reaction sample were run on a 3% agarose gel containing ethidium bromide (0.1 µg/ml). The images were captured by photographing on 555 Polaroid film (Cambridge, MA). The photos were scanned for densitometric analysis using Hewlett-Packard Co. 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. Mean OD values were normalized according to the coamplified cyclophilin cDNA value detected in each sample. Authenticity of each PCR amplified product was confirmed by sequencing analysis (44).

In situ hybridization histochemistry.
Animals were cardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were dissected out and further fixed with the same fixative for 24 h at 4 C. Thereafter, the fixative was replaced with cold (4 C) 25% sucrose in 0.1 M phosphate butter (pH 7.4). After 24 h, the brains were transferred into new sucrose solution for an additional 1 or 2 d at 4 C. The brains were then blocked and stored at -80 C until being coronally sectioned at 20 µm using a freeze sliding microtome. The sections were then mounted on gelatin coated Superfrost glass slides (Fisher Scientific, Pittsburgh, PA) and allowed to dry overnight in a vacuum hood. The dried slides were stored in slide boxes at -85 C before hybridization.

Probe preparation.
To prepare the 35S-UTP-labeled ErbB-2 or ErbB-4 riboprobes, specific DNA template corresponding to sequences contained in the coding region of ErbB-2 or ErbB-4 mRNA was used as previously reported (39). All the cRNA probes were synthesized by in vitro transcription using the appropriate RNA polymerases.

Prehybridization, hybridization, and posthybridization.
The procedures were followed as described (45). In short, each slide was overlaid with 180 µl of hybridization solution containing 50% deionized formamide, 4x sodium chloride sodium citrate, 1x Denhardt’s solution, 0.5 mg/ml salmon sperm DNA, 0.25 mg/ml tRNA, 10% Dextran sulfate, and the riboprobe of interest (5–10 x 106 cpm/ml). The slides were hybridized in a slide moat (model 240000, Boekel Scientific, Feasterville, PA) for 16–20 h at 56 C. Posthybridization procedures were performed as reported (45). Following dehydration in graded alcohols, the slides were dipped in NTB3 emulsion (catalog no. 1654441, Kodak, Rochester, NY) and developed after 2 wk of exposure. All the slides were stained with Toluidine Blue O (Fisher Scientific, catalog no. T-161). Control sections were hybridized with a sense RNA probe.

Hormone assays
Plasma levels of LH were measured using enzymeimmunoassay (EIA) detection kit (code RPN 2562) purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Sensitivity of the assay was 0.1 ng/ml. The intraassay and interassay coefficients of variation were 7.6% and 5.1%, respectively. Circulating E2 and progesterone (P4) concentrations were determined by EIA detection kits [(catalog no. DE2000 (E2), R & D Systems, Inc., Minneapolis, MN; 07B70102A (P4), ICN Pharmaceuticals, Inc., Orangeburg, NY)]. The lowest detectable levels of E2 and P4 were 1.9 pg/ml and 0.05 ng/ml, respectively. The intraassay and interassay coefficients of variation: E2, 7.6% and 5.1%; P4, 10.2% and 7.5%, respectively.

Hypothalamic infusion of an ErbB-2 antisense oligodeoxynucleotide (ODN)
To determine the physiological role of ErbB-4/-2 receptor complexes on the maintenance of female reproduction, in vivo blockade of ErbB-2-mediated signal transduction was performed. An ErbB-2 antisense ODN (5'-CATGATGATCATTGCGGCTCC-3') encompassing the translation initiation codon of rat ErbB-2 mRNA (46) was infused into the preoptic area [George Paxinos & Charles Watson coordinates: Bregma -0.4 mm; depth from brain surface 8.0 mm (47)] or medial basal hypothalamus (Bregma -2.80 mm; depth from brain surface 9.0 mm) via a sterotaxically implanted cannula (Plastic One, Roanoke, VA) connected to a sc implanted Alzet mini-osmotic pump (model 2002, Alzet Corp., Palo Alto, CA). The pump has a constant flow rate of 0.5 µl/h for 14 d at 37 C and a capacity of 200 µl. Each pump was fully loaded with artificial CSF (48) containing either the ErbB-2 ODN or the scrambled sequence at 5 µg/µl. Before connecting to the infusion devise, the loaded pumps were incubated at 37 C for up to 4 h, which is required for pump activation. The whole infusion assembly was implanted into 2-month-old female intact rats displaying normal estrous cycles. Estrous cyclicity was determined before, during and after the infusion by daily monitoring vaginal cytology. At the end of the infusion experiment, all treated animals were killed for accessing cannula placement and exhaustion of the infusion solution. It is important to note that this ErbB-2 ODN has been shown to be effective to inhibit ligand-induced phosphorylation of ErbB-2 receptors in hypothalamic astrocytes and disrupt the normal process of female sexual maturation via blockade of LHRH release (39).

Statistical analyses
Comparisons of changes in ErbB-4/2 mRNA and all hormonal levels were analyzed by a two-way ANOVA, followed by the Student’s-Neuman-Keuls multiple comparison test for unequal replications. In all cases, significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene expression of hypothalamic ErbB-4/-2 receptor is altered in aging female rats during proestrus
To determine if changes in the hypothalamic gene expression of ErbB-4/-2 may occur during proestrus (a key stage before ovulation) and in association with reproductive aging, the relative tissue content of ErbB-4/-2 mRNA in this brain region was measured by semiquantitative RT-PCR assays as previously reported (18). These assays were also technically validated as shown in Fig. 1Go, which demonstrates that the amounts of RT reaction mix and the PCR cycle numbers used are in the best part of each curve under this procedure.



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  		Figure 1. Analyses of ErbB-4/-2 and cyclophilin (p1B15) mRNAs amplified by semiquantitative RT-PCR. On the left (A, C), each top panel illustrates the PCR results used for determining the linearity of ErbB-2 (A) or ErbB-4 (C) along with p1B15 cDNA in relation to the number of amplification cycles. A partial p1B15 mRNA fragment was simultaneously amplified with ErbB-4/-2 mRNA for the purpose of data normalization. The lower panels show the quantitative data of bands depicted in the top corresponding panel. On the right (B, D), each top panel presents the results of an RT-PCR amplified ErbB-2 (B) or ErbB-4 (D) along with p1B15 from various amounts [in volume (Vol)] of RT reaction. The lower panels illustrate the analysis of quantitative changes in bands depicted from the top corresponding panel. dil, Dilution.

 
Expression of ErbB-4 mRNA in the POA is low for both YA and MA rats in early morning, from 0600–0800 h of the proestrous day (Fig. 2AGo). ErbB-4 gene expression significantly increases in YA rats at 1000 h and reaches a maximal level at 1200 h, and interestingly, this high level of ErbB-4 mRNA is maintained between 1500 h and 1800 h (Fig. 2AGo). In contrast, no initial elevation at 1000 h is found in MA animals (Fig. 2AGo). Although a small increase in the level of ErbB-4 mRNA at 1200 h in MA rats is detected, it is not statistically significant and it then further decreases to the lowest levels from 1500–1800 h (Fig. 2AGo). In YA animals, ErbB-4 mRNA contents in the ME-ARC are high during early morning of the proestrous day and reach a maximal value at 0800 h, then decreased to low levels during the afternoon (Fig. 2BGo). In comparison to YA rats, the peak level of ErbB-4 mRNA in MA animals is observed 4 h later (Fig. 2BGo). It then sharply decreases to the lowest value from 1500 h.



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  		Figure 2. Changes in ErbB-4 mRNA levels in the hypothalamus of YA and MA female rats at different times during the proestrous day as determined by RT-PCR assay. A, Results of one representative experiment (gel picture) and the analyzed data of all assays using POA tissues. B, in the same format as panel A, demonstrates changes in ErbB-4 mRNA levels in the ME-ARC. Each point represents the mean ± SEM of 3–5 animals. *, P < 0.05; or **, P < 0.01 vs. 0600 and 0800 h in YA animals or vs. 0600–1200 h in MA rats. aa, P < 0.01 vs. the corresponding time point of either YA or MA animals.

 
It is also important to determine ErbB-2 gene activity as this receptor is required for forming ErbB-4/-2 receptor signaling complexes by dimerizing with ligand activated-ErbB-4 receptors. Expression of ErbB-2 mRNA in the POA of YA animals is remarkably similar to that of ErbB-4, which shows an initial increase at 1000 h and is maintained at a high level through out the rest of time points during proestrus (Fig. 3AGo). In a striking contrast, no increase in the ErbB-2 mRNA levels is found in the POA of MA female rats. Surprisingly, ErbB-2 mRNA levels are undetectable (Fig. 3AGo). In the ME-ARC on the other hand, levels of ErbB-2 mRNA in YA animals appear to be relatively constant during the proestrous day although higher levels are detected during 1000 h and 1200 h (Fig. 3BGo). No difference in ErbB-2 mRNA levels from 0600–1000 h is observed between YA and MA animals. However, ErbB-2 mRNA contents are significantly reduced during the late afternoon in MA animals (Fig. 3BGo). The gene expression pattern in YA animals and the differences in ErbB-4/-2 receptor expression between YA and MA female rats appear to be limited to these neuroendocrine brain regions because no such changes and differences were observed in the cerebral cortex (not shown), a brain area that has no known involvement in neuroendocrine function.



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  		Figure 3. Detection of changes in ErbB-2 mRNA levels in the hypothalamus of YA and MA female rats at different times during the proestrous day as determined by RT-PCR assay. A, Results of one representative experiment (gel picture) and the analyzed data of all assays using the POA tissues, in which individual brain regions from different animals at each time point were combined. B, in the same format as panel A, demonstrates changes in ErbB-2 mRNA levels in the ME-ARC. Each point represents the mean ± SEM of three to five animals. *, P < 0.05; or **, P < 0.01 vs. 0600 and 0800 h in YA animals or vs. 1000 h in MA rats. b, P < 0.05 vs. 1200 h time point of MA animals.

 
Localization of ErbB-4/-2 mRNA in the hypothalami of YA and MA female rats by hybridization histochemistry
A 35S-UTP-labeled ErbB-4 cRNA probe was used to determine the cellular expression of ErbB-4 mRNA in the hypothalami of YA and MA female rats during proestrus at 1200 h. In the POA of YA rats, the most abundant ErbB-4 mRNA labeled cells are located around the organum vasculosum of the lamina terminalis (OVLT) (Fig. 4AGo). In striking contrast, fewer labeled cells are detected in the matched areas of MA animals (Fig. 4BGo, Table 1Go). These observations further confirm the results detected by RT-PCR (Fig. 2AGo). In the medial basal hypothalamus (MBH), ErbB-4 mRNA positive cells are found in the arcuate nucleus in both YA and MA animals (Fig. 4Go, C, c, and D, d). However, the number of positive cells identified in this region from MA rats is significantly higher than that from YA animals (Fig. 4Go, c, d; Table 1Go).



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  		Figure 4. Detection of ErbB-4 mRNA in the hypothalamus of YA and MA female rats at 1200 h of the proestrous day by in situ hybridization histochemistry. Representative results derived from a group of four animals are depicted (in bright field). A, Abundant ErbB-4 mRNA labeled cells around the OVLT region (arrowheads) of a YA female rat. B, In contrast, fewer labeled cells are found around the OVLT area (arrowheads) of a MA rat. C and D show that ErbB-4 mRNA positive cells are located in the area of arcuate nucleus (ova frame) of either a YA (C) or MA (D) rat. ErbB-4 mRNA positive cells rectangularly framed in C or D are shown in higher magnification in c and d, respectively. Panel c represents a less number of ErbB-4 mRNA labeled cells in the arcuate nucleus of a YA animal. In contrast, panel d depicts a greater number of ErbB-4 labeled cells in the same area of a MA animal. 3V, 3rd ventricle. Each bar, 50 µm.

 

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  		Table 1. Estimated total number of ErbB-4/-2 mRNA labeled cells (mean ± SEM, n = 4) detected by in situ hybridization

 
Because ErbB-2 receptors interact with ErbB-4 to form functional receptor complexes that are important for mediating LHRH release in the hypothalamus (39), cellular gene expression of ErbB-2 is also characterized in the same hypothalami of YA and MA animals used for detection of ErbB-4 mRNA. Similar to ErbB-4 mRNA expression in the POA, ErbB-2 mRNA is highly expressed in cells around the OVLT of YA rats (Fig. 5AGo). However, virtually no detectable positive cells in the OVLT are found in MA animals (Fig. 5BGo). In the MBH, ErbB-2 mRNA is expressed in the area of the arcuate nucleus from both YA and MA rats (Fig. 5Go, C and D). However, the number of ErbB-2 mRNA-labeled cells is greater in YA vs. MA animals (Fig. 5Go, c, d; Table 1Go). It is important to note that the cellular expression of ErbB-2 mRNA is also consistent with the expression profile (at 1200 h) detected by RT-PCR (Fig. 3Go).



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  		Figure 5. Detection of ErbB-2 mRNA in the hypothalamus of YA and MA female rats at 1200 h of the proestrous day by in situ hybridization histochemistry. Representative results derived from a group of four animals are depicted (in bright field). A, Abundant ErbB-2 mRNA labeled cells around the OVLT region (arrowheads) of a YA female rat. B, In contrast, virtually no labeled cells are found around the same area of a MA rat. C and D exhibit that ErbB-2 mRNA positive cells are located in the area of arcuate nucleus (ova frame) of either a YA (C) or MA (D) rat. ErbB-2 mRNA positive cells (arrows) boxed in C or D are shown in higher magnification in c and d, respectively. Panel c represents a greater number of ErbB-2 mRNA labeled cells in the arcuate nucleus of a YA animal. In contrast, panel d depicts a less number of ErbB-2-labeled cells in the same area of a MA animal. Each bar, 50 µm.

 
Changes in LH secretion from YA and MA female rats during proestrus
Because an altered ErbB-4/-2 gene expression is detected in the hypothalamus of proestrous MA female rats before the preovulatory LH surge (an index of LHRH secretion), it is important to determine whether this alteration is temporally associated with the changes in LH release. There is no significant difference in the circulating concentration of secreted LH between YA and MA female rats from 0600–1500 h of proestrus. However, an expected preovulatory LH surge is detected at 1800 h in YA but not MA animals (Fig. 6Go). Furthermore, this difference in LH secretion is not likely due to differential ovarian function because similar levels of E2 and progesterone are observed between these two age groups (Fig. 6Go), whereas it is currently unknown why the levels of progesterone at 0800 h are high.



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  		Figure 6. Serum levels of LH, E2 and progesterone during proestrus in YA and MA female rats as measured by EIA. Each bar equals the mean ± SEM of four to seven independent observations. For LH: *, P < 0.05 vs. 1800 h; and **, P < 0.01 vs. 0600, 1000–1500 h in YA animals. aa, P < 0.01 vs. YA rats at 1800 h. For progesterone: *, P < 0.05; and **, P < 0.01 vs. 0600, 1000–1500 h in either YA or MA animals.

 
In vivo blockade of ErbB-2 receptor synthesis via central infusion of an anti-ErbB-2 ODN disrupts the estrous cycle of YA female rats
ErbB-2 serves as an essential coreceptor that interacts with ErbB-4 to form a functional receptor complex necessary for controlling LHRH release in the hypothalamus (39). Targeted disruption of ErbB-2 receptor synthesis via central infusion of an anti-ErbB-2 ODN results in a delay of sexual maturation due to blockade of LHRH secretion (39). To determine if ErbB-4/-2 receptor complex-mediated signal transduction is also required for maintaining normal reproductive function, the same anti-ErbB-2 ODN was chronically infused into the preoptic area or medial basal hypothalamus via a sterotaxically implanted cannula connected to a sc implanted Alzet mini-osmotic pump. Both control groups of animals (intact and scrambled ODN infused) exhibited normal estrous cycles before, during and after the infusion period (Fig. 7Go). In contrast, the estrous cycle was disrupted in animals during infusing with anti-ErbB-2 ODN into the POA or ME-ARC until the end of the infusion period (Fig. 7Go). The antisense ODN infused animals showed a prolonged diestrous phenotype (approximately 11 d) during a 14-d-infusion (P < 0.01 vs. both control groups; Fig. 7Go, lower panel). Consequently, the number of either proestrous or estrous day displayed by these animals is significantly lower compared with the control animals (Fig. 7Go, lower panel). The result also shows that normal cyclicity is immediately restored upon termination of the treatment. This disrupted effect on estrous cyclicity by anti-ErbB-2 ODN suggests that functional integrity of ErbB-4/-2 receptor signaling complex in the hypothalamus is required for maintaining normal reproductive function.



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  		Figure 7. Analysis of the disruption of estrous cyclicity in young adult cycling female rats by the blockade of ErbB-2 receptor synthesis via central infusion of anti-ErbB-2 ODN. The antisense or scrambled ODN were delivered into the POA or MBH using a stainless steel cannula that was connected to an sc implanted Alzet osmotic minipump. Animals were infused with the ODN at the rate of 2.5 µg/h for 14 d. For detailed procedures, see Materials and Methods. The top panel shows estrous cyclicity from one representative animal of each experimental group. Notice that the antisense ODN-treated animals show a prolonged diestrous phenotype compared with all the controls which exhibit regular cycles during the infusion time. D, Diestrus; P, proestrus; E, estrus. The bottom panel depicts that the antisense-treated animals displayed a significantly greater number of diestrous days compared with both intact and scrambled ODN-infused animals. Consequently, the anti-ErbB-2- infused animals showed a lesser number of either proestrous or estrous days. Numbers in parentheses are number of animals per group. *, P < 0.05 and **, P < 0.01 vs. intact and scramble controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous findings demonstrated that ligand-induced activation of ErbB-4 receptors results in production of ErbB-4/-2 receptor complexes in the hypothalamus, and importantly, these receptor complexes are intrinsic signaling components of the cell-cell interaction underlying the neuroendocrine control of sexual development (39). Little is known concerning these signaling complexes in regulating reproductive function during adulthood. The involvement of ErbB-4/-2 receptors in the process of female sexual maturation raises a strong possibility that cell-cell communications mediated by these receptors in the neuroendocrine brain are also necessary for maintaining reproduction during adulthood. This view is supported by our recent observations demonstrating that ErbB-1 receptors, also known to facilitate initiation of sexual maturation (6, 42), play an important role in regulating reproductive activity in adult female rats (18). It is conceivable that if ErbB-4/-2 receptors are involved in regulating adult female reproduction, alteration in the receptor signaling capacity may also contribute to compromised reproductive function in aging females. The present study provides initial evidence that supports this possibility.

Cellular localization of ErbB-4/-2 mRNA in YA and MA female rats was determined via in situ hybridization histochemistry. Similar to ErbB-1 mRNA expression profiles reported in our previous study (18), numerous ErbB-4/-2 mRNA labeled cells in YA female rats are observed around the OVLT area, a site scattered with LHRH cell bodies (49). This anatomical feature indicates that signal transduction mediated by ErbB-4/-2 receptors may be functionally linked to the regulation of LHRH neuronal activity in this region. It is important to note that differences in cellular ErbB-4/-2 mRNA expression in the POA of YA and MA female rats at 1200 h of proestrus were also examined. This time point was selected because ErbB-4/-2 mRNA expression levels detected by RT-PCR (see below) were found to be significantly different between the two age groups. Similar to the RT-PCR results, MA female rats have significantly fewer ErbB-4 mRNA labeled cells in the OVLT compared with that in the matched areas of YA animals. More strikingly, virtually no ErbB-2 mRNA positive cells are found in the OVLT of MA rats, which confirms the undetectable gene expression level in the POA by RT-PCR. Both ErbB-4/-2 mRNA positive cells are detected in the medial basal hypothalamus, predominantly in the medial- and ventral-lateral portion of the arcuate nucleus (ARC), which is known to be involved in regulating LHRH release (3, 50). This suggests that expression of these receptors in the ARC may be linked to the control of LHRH secretion. This view is supported by the evidence that activation of ErbB-4/-2 receptor complexes in the hypothalamus contributes to neuroendocrine control of sexual maturation via stimulation of LHRH release (39). ErbB-4/-2 mRNA expression in the ARC found in this study overlaps receptor expression in prepubertal rats (39). The difference in total ErbB-4/-2 positive cells in the ARC between YA and MA animals is compatible with the expression profiles detected by RT-PCR (see below). It is worth mentioning that the majority of cellular signal is associated with small, dark nuclei that are characteristics of astroglial cells, suggesting that ErbB-4/-2 receptors are expressed in astroglia. Indeed, this nonneuronal expression of ErbB receptors in the hypothalamus has been detected in younger female rats (39, 44). This dominant glial expression of ErbB-4/-2 receptors in the hypothalamus further supports the concept that control of neuroendocrine function by ErbB receptors in both prepubertal and adult females is dependent, at least in part, on glial intermediacy.

An increase in hypothalamic ErbB-4/-2 gene expression is linked to the initiation of puberty in female rats (39) via stimulating the release of LHRH. It is then possible that normal ErbB-4/-2 receptor gene activity may dictate, at least in part, the normalcy of reproductive function during adulthood. Thus, alterations in ErbB-4/-2 gene activity may contribute to changes in LHRH neuronal function, which, in turn, leads to altered gonadotropin release found in aging females (51, 52). The results of the present study indicate that alterations in hypothalamic ErbB-4/-2 gene expression occur in MA aging female rats compared with that in YA animals, and importantly these alterations may be linked to compromised gonadotropin secretion associated with the onset of reproductive aging. Specifically, the expression profiles of ErbB-4 and ErbB-2 mRNA are remarkably similar in the POA of YA animals. Both mRNA levels start to increase during the late morning when serum levels of E2 are high. They then reach and maintain a maximal value before and at the time when the preovulatory LH surge occurs. In a striking contrast, no such increase is found in either ErbB-4 or ErbB-2 mRNA levels in MA female rats during this time period. This difference in ErbB-4/-2 mRNA levels detected by RT-PCR is also consistent with a significantly greater number of ErbB-4/-2-labeled cells detected by in situ hybridization histochemistry in the POA of YA vs. MA animals. Importantly, these MA animals exhibit no preovulatory-like LH surge at 1800 h during proestrus compared with YA rats, suggesting an attenuated or delayed LH secretion in these animals. It is well documented that the preovulatory LH surge is either delayed or reduced in MA female rats (53). Whether this altered ErbB-4/-2 gene expression activity is causally associated with a decreased release of gonadotropins in MA rats (51, 52) requires further studies. Recent findings (39) demonstrate that an increase in ErbB-4/-2 receptor gene expression and ligand-induced activation of these receptor complexes result in stimulation of LHRH release. Conversely, disruption of the ErbB-4/-2 signaling system leads to a reduction of LHRH secretion. These results suggest that an increase in ErbB-4/-2 gene expression is also necessary for induction of the preovulatory LH surge. It is important to note that the synergistic interaction between ErbB-4/-2- and ErbB-1-mediated signal transduction in enhancing LHRH neuronal activity is well-documented (39). Also, increased ErbB-1 gene activity during proestrus is a part of regulatory events that are important for maintaining normal reproduction (18). Therefore, the interactive nature of ErbB receptor-mediated signal transduction may be necessary for inducing a maximal level of gonadotropin secretion.

Interestingly, while a similar high level of ErbB-4 mRNA in the ME-ARC is found in both proestrous YA and MA animals during the early morning hours, the timing of the decrease in ErbB-4 mRNA level was delayed in MA animals. This ErbB-4 expression pattern in the ME-ARC of both YA and MA animals is consistent with that of ErbB-1 (18), further suggesting functionally synchronized activation of these two signaling systems. Surprisingly, while the expression profiles of ErbB-4 and ErbB-2 mRNA are remarkably similar in the POA of YA animals, the ErbB-2 mRNA expression pattern is different from that of ErbB-4 in the ME-ARC. ErbB-2 mRNA levels in this brain area are relatively constant through out the proestestrous day in YA rats. These results indicate that a relatively fixed level of ErbB-2 (as coreceptors) in the ME-ARC may be necessary for ensuring a full capacity of ErbB-4/-2- and ErbB-1-mediated signaling activity in the ME-ARC. This view is strongly supported by evidence that ErbB-2 appears to function as an auxiliary component recruited by ligand-activated ErbB-1, ErbB-3, and ErbB-4 receptors (32, 34, 36, 37, 38). In fact, heterodimers of ErbB-2 with ErbB-1, ErbB-3, and ErbB-4 are the preferred signaling complexes that exist in many cellular systems (19, 20). An intriguing finding in this study is that ErbB-4 expression in the POA of YA animals is temporally different compared with that in the ME-ARC. Specifically, expression levels in the POA start to increase at 1000 h and reach maximal levels at 1200 h. It is then maintained during the rest of the afternoon period before and during the LH surge. However, ErbB-4 expression levels in the ME-ARC reach the highest value at 0800 h and decrease thereafter. The expression pattern of ErbB-4 is similar to the ErbB-1 expression profiles reported in our previous study (18). It thus suggests that differential expression of ErbB-4 gene activity in the POA and ME-ARC may be required, as one of the regulatory components, for contributing to the complexity of temporal and regional control of LHRH neuronal activity. Desynchronization of this component would result in disruption of an integral control of LHRH/LH release, which leads to compromised female reproduction during aging.

The functional role of ErbB-4/-2 receptor heterodimers in controlling reproduction during adulthood is demonstrated by the disruption of normal estrous cyclicity. The acyclicity was induced by central inactivation of ErbB-2 receptors via infusion of an anti-ErbB-2 ODN into the POA or MBH. The antisense ODN-treated animals displayed a prolonged diestrous phenotype during the 2-wk infusion period. During the ODN infusion, animals showed no obvious abnormality of biology, and the regular cyclicity was resumed after termination of the infusion. It thus suggests that the disrupted cyclicity clearly is not caused by pharmacological toxicity of the ODN. Furthermore, acyclicity caused by the disruption of hypothalamic ErbB-2 synthesis is specific because no such effect was found in the animals infused with a scrambled ODN, which consists of the same nucleotides as the antisense, but with a randomized sequence which shows no significant matches with any known gene sequences in current data bases. It is important to emphasize that the anti-ErbB-2 ODN is the same one used in a previous study in which the authors reported that infusion of ODN into the hypothalamus results in a delayed onset of puberty due to inhibitory effects on LHRH release (39). This ODN was also tested in hypothalamic astrocytes, which are known to express high levels of ErbB-4/-2 receptors (39). The test results show that it is able to specifically inhibit ligand-induced ErbB-2 receptor phosphorylation without affecting other tyrosine kinase receptors such as ErbB-4 or basic fibroblast growth factor receptor. One issue needs to be further discussed is that all antisense treated animals started cycling 1–2 d before the scheduled termination of the infusion. This is likely due to a decrease in the efficacy of ErbB-2 antisense oligonucleotide and/or the pumping efficiency of the mini-osmotic pump during the last 1–2 d infusion period. Nonetheless, it is clear that ErbB-4/-2 receptor complex is also a required signaling component which contributes to a part of the integral regulatory system for maintaining normal reproduction during adulthood. Moreover, it appears that multigrowth factor induced-activation of tyrosine kinase receptors in the rodent central nervous system is a common feature related to the control of reproductive function. This view is strongly supported by the findings that ErbB-1 (16, 18), insulin receptor (17), and IGF (16) are necessary for maintaining normal reproduction. Obviously, synchronization of these multiarray signaling systems in the control of neuroendocrine function is one of the essential processes to bring about the onset of puberty and maintenance of normal reproduction. This view is supported by the evidence that hypothalamic circadian clock governs the timing of initiation of puberty and normal estrous cyclicity in female mice via its downstream signaling events mediated by ErbB receptors (unpublished observations).

Taken together, the current study provides evidence that alteration in ErbB-4/-2 gene expression in the hypothalamus of aging female rats may be linked to changes in LHRH neuronal activities that leads to attenuated or delayed preovulatory LH secretion during reproductive aging. This study also demonstrates that blockade of ErbB-4/-2 receptor-mediated signal transduction results in disruption of the estrous cycle. Therefore, the ErbB-4/-2 signaling system is a physiological component necessary for maintaining female reproductive function.


   Footnotes
 
This work was supported by NIH Grant RO1-AG18078.

Abbreviations: ARC, Arcuate nucleus; CTX, cortex; EGF, epidermal growth factor; EIA, enzymeimmunoassay; LHRH, LH-releasing hormone; MA, middle aged; MBH, medial basal hypothalamus; ME-ARC, median eminence-arcuate region; NRG, neuregulins; nt, nucleotides; ODN, oligodeoxynucleotide; OVLT, organum vasculosum of the lamina terminalis; POA, preoptic area; YA, young adult.

Received November 21, 2001.

Accepted for publication January 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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