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Endocrinology, doi:10.1210/en.2006-0845
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Endocrinology Vol. 148, No. 7 3391-3401
Copyright © 2007 by The Endocrine Society

Focal Adhesion Kinase and Paxillin: Novel Regulators of Brain Sexual Differentiation?

Debra B. Speert, Anne T. M. Konkle, Susan L. Zup, Jaclyn M. Schwarz, Chaitanya Shiroor, Michael E. Taylor and Margaret M. McCarthy

Department of Physiology and Program in Neuroscience, University of Maryland, Baltimore, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Debra B. Speert, Department of Physiology and Program in Neuroscience, University of Maryland, Baltimore, Baltimore, Maryland 21201. E-mail: debspeert{at}gmail.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroid-mediated sexual differentiation of the brain is a developmental process that permanently organizes the brain into a male or female phenotype. Previous studies in the rodent have examined the steroid-mediated mechanisms of male brain development. In an effort to identify molecules involved in female brain development, a high-throughput proteomics approach called PowerBlot was used to identify signaling proteins differentially regulated in the neonatal male and female rat hypothalamus during the critical period for brain sexual differentiation. Focal adhesion kinase (FAK) and paxillin, both members of the focal adhesion complex family of proteins, were significantly elevated in the newborn female compared with the male hypothalamus. Sex differences in these proteins were not detected in brain regions that are not subject to substantial organizational effects of steroids. Estrogens, the aromatized products of testosterone in the male, can both masculinize and defeminize the male brain. Daily estradiol administration to neonatal females significantly reduced FAK and paxillin in the hypothalamus, and aromatase inhibition increased paxillin in males to levels comparable with females. Androgens also appear to modulate paxillin levels in combination with estrogen action. Across development, hypothalamic levels of FAK were significantly elevated in females compared with males on postnatal d 6. Synaptic circuits in the hypothalamus develop sex differences perinatally. Estradiol treatment of cultured hypothalamic neurons significantly enhanced axon branching (P < 0.01), consistent with the phenotype of FAK-deficient neurons. Together, these data implicate FAK and paxillin as regulators of sex differences in neuronal morphology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING A WELL-DEFINED critical period, the bipotential brain differentiates into a male or female phenotype, resulting in profound sex differences in neuron number, glial complexity, neurochemical expression, and synaptic connectivity (reviewed in Refs. 1 and 2). Gonadal hormones are the best-known mediators of this dynamic developmental switch. In the rodent, the testes develop on embryonic d 14 (E14), and secretion of the gonadal androgen testosterone peaks in the male on E18 (3) and exhibits a second surge at birth [postnatal d 0 (PN0)]. Once it reaches the brain, testosterone is converted to estradiol by the aromatase enzyme. Estrogens are well documented to both masculinize (induce the ability to express male sexual behavior in the adult) and defeminize (remove the ability to express female sexual behavior in the adult) the male rat brain (4 ; reviewed in Ref. 2). Androgens play a lesser and more poorly understood role in both processes. Circulating estrogens from the maternal ovaries and the placenta are blocked from masculinizing the rodent brain by the plasma-binding protein {alpha}-fetoprotein, allowing androgens access to the brain where they can be locally aromatized (5). To date, the only known mediator of brain feminization (the ability to express female sexual behavior in the adult) is the absence of estrogens or androgens in the perinatal brain, thereby making female the default brain phenotype. Nonetheless, this does not preclude the requirement for carefully orchestrated cellular processes to achieve the female endpoint.

There are sex differences in signal transduction in the neonatal rat brain. Expression of the immediate early gene, Fos, is elevated in the medial preoptic area (mPOA) of the male compared with the female during the first several days of life (6). On the day of birth (PN0), phosphorylation of the cAMP response element-binding protein (CREB) is elevated in the ventromedial nucleus of the hypothalamus and the POA of the male. Females administered estradiol on PN0 have increased phosphorylated CREB (pCREB) in these areas on PN1 relative to vehicle-treated controls (7). Muscimol, a GABAA receptor agonist and a major excitatory signal in the perinatal brain, increases pCREB in these brain regions in the male, but decreases pCREB in the same parts of the female brain (8). Thus, the same signal mediates opposite responses in the male and female, suggesting that different signaling pathways may be active in males vs. females during the critical period for sexual differentiation of the brain.

To identify other signaling molecules differentially regulated in the male and female hypothalamus during the hormonally sensitive critical period, we used a high-throughput proteomics approach called PowerBlot (BD Biosciences PharMingen, La Jolla, CA). We identified two signaling proteins elevated in the female hypothalamus on the day of birth: focal adhesion kinase (FAK) and paxillin. Neither of these proteins has previously been implicated in hormonal modulation of the developing rat brain. Both FAK and paxillin are members of the focal adhesion complex family of proteins and are active in cell adhesion, migration, and growth. FAK is a nonreceptor tyrosine kinase associated with integrin, epidermal growth factor, and ephrin receptors (reviewed in Ref. 9). FAK also associates with a number of intracellular adaptor proteins, including paxillin, which is implicated in cytoskeletal changes in response to environmental signals (reviewed in Ref. 10).

Since Raisman and Field’s landmark 1971 study (11), sex differences in synaptic patterning in the hypothalamus and POA have been well established. Toran-Allerand demonstrated in 1976 (12) that hypothalamic explants treated with estradiol showed enhanced neuritogenic outgrowth in vitro; however, the mechanism mediating the effects of estradiol on neurite extension remains unclear. Here, we describe a high-throughput assay that suggested FAK and paxillin may participate in this mechanism, and we characterize the regulation of these proteins by estradiol and androgens in the developing hypothalamus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were bred in our animal facility. Pregnancy was confirmed by the presence of sperm in the vaginal lavage. Birth was allowed to proceed normally, and the day of birth was designated PN0. All rats were kept on a 12-h light, 12-h dark cycle with lights on at 1100 h. Food and water were ad libitum. All procedures were approved by the University of Maryland Institutional Animal Care and Use Committee.

Experiment 1: survey of signaling proteins in the male and female hypothalamus during the critical period for sexual differentiation of the brain
Brains were removed from two male and two female pups on PN0 within 6 h of birth and placed in a Zivic Miller brain mold. A 3-mm section was cut that contained both the POA and medial basal hypothalamus (MBH) (13). The anterior commissure was used to guide both the dorsal and lateral borders of the dissection (14). The two MBH samples were pooled for each sex and homogenized with a polytron in boiling lysate buffer [10 mM Tris (pH 7.4), 1 mM sodium orthovanadate, 1% sodium dodecyl sulfate] supplemented with 1:1000 phosphatase inhibitor (cocktail I; Sigma-Aldrich, St. Louis, MO). All additional PowerBlot analysis was performed by BD Biosciences PharMingen. Briefly, protein concentration was determined by bicinchoninic acid protein assay (Pierce Biotechnology, Inc., Rockford, IL). Protein lysates (200 µg/sample) were separated on polyacrylamide gels with one wide lane. Samples were run in triplicate and transferred to membranes using standard protocol. Membranes were then clamped to physically isolate 39 lanes from top to bottom, and cocktails of primary antibodies were applied to each lane. Eighty primary antibodies were used in this PowerBlot PhosphoScreen to detect 30 proteins in the phosphorylated and unphosphorylated state (Table 1Go). All analysis was performed by BD Biosciences PharMingen and consisted of comparing each triplicate of the male sample to each triplicate of the female sample for a total of nine comparisons.


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  		TABLE 1. Proteins assayed for and detected by the PowerBlot analysis completed by BD Biosciences Pharmingen

 
Experiment 2: FAK and paxillin levels on PN0 in males and females in multiple brain regions
Untreated male and female pups were killed within 6 h of birth on PN0. Cortex, thalamus, and MBH were dissected using a Zivic Miller mold as previously described (13). The frontal cortex was removed in the brain mold. A sample of the thalamus was removed from the 2-mm brain section containing the MBH. Both the cortex and thalamus samples were removed from the same brains as the MBH. Groups contained four to seven rats from at least two litters. Samples were not pooled and were only used if enough protein was available from a single sample for Western analysis, causing the n to vary between experiments. Tissue was lysed and prepared for Western immunoblots for FAK and paxillin as described below.

Experiment 3: effect of estradiol treatment on PN0 and PN1 on FAK and paxillin levels on PN2
Female pups were treated with estradiol (100 µg estradiol benzoate in 0.1 ml sesame oil sc; n = 6) or vehicle (n = 5) and males with vehicle (n = 6) on PN0 within 6 h of birth and again on PN1. Pups were killed and brains collected 48 h later on PN2. The dose of estradiol benzoate was chosen to override the sequestering capacity of the steroid-binding protein {alpha}-fetoprotein (5) and is an established protocol for permanently masculinizing the female brain (14, 15). Western immunoblots for FAK and paxillin were performed as described below.

Experiment 4: effect of estradiol treatment on PN0 on FAK and paxillin levels on PN1
Female pups were treated with estradiol (100 µg estradiol benzoate in 0.1 ml sesame oil sc; n = 5 for FAK; n = 8 for paxillin) or vehicle (n = 4 for FAK; n = 8 for paxillin) and males with vehicle (n = 7 for FAK; n = 10 for paxillin) on PN0 within 6 h of birth. Pups were killed and brains collected 24 h later on PN1. Western immunoblots for FAK and paxillin were performed as described below.

Experiment 5: effect of 6 h estradiol treatment on FAK and paxillin levels on PN0
Pups were treated on PN0 and killed 6 h later. Females received 250 µg estradiol in 0.1 ml ethyl oleate vehicle (n = 5) or vehicle (n = 7), and males received vehicle (n = 8). The ethyl oleate vehicle facilitated faster delivery of estradiol into the bloodstream (16), and the dose was chosen to maximize exposure during the short treatment time. MBH was removed as described (13). Groups contained five to eight rats from at least two litters. Western immunoblots for FAK and paxillin were performed as described below.

Experiment 6: effect of treatment with an estrogen receptor antagonist or aromatase inhibitor on FAK and paxillin in the neonatal rat
Male and female rats were treated sc within 6 h of birth on PN0 and on PN1 with 200 µg of the aromatase inhibitor letrozol (generous gift from Novartis, Cambridge, MA; n = 6 females; n = 7 males) or 1 mg/kg of the estrogen receptor inhibitor tamoxifen (n = 6 males and females) in sesame oil or vehicle (n = 8 females; n = 5 males) and killed on PN2. Each group contained rats from three litters. Western immunoblots for FAK and paxillin were performed as described below.

Experiment 7: effect of androgen treatment on FAK or paxillin levels in neonatal hypothalamus
Female pups were treated sc with vehicle (n = 6), 100 µg testosterone (n = 5), 100 µg of the nonaromatizable androgen dihydrotestosterone (DHT; n = 5), or 100 µg each of estradiol and DHT (n = 5) in 0.1 ml sesame oil on PN0 and PN1 and killed on PN2. Males were treated with vehicle (n = 5). Treatment groups contained rats from at least two litters, and all treatments on PN0 were completed within 6 h of birth.

Experiment 8: developmental time course of FAK and paxillin in the postnatal male and female hypothalamus
Untreated male and female rats were killed and MBH removed on PN2 (n = 4 females; n = 6 males), PN4 (n = 5 females; n = 6 males), PN6 (n = 4 females; n = 5 males), PN8 (n = 6 females and males), and PN10 (n = 6 females and males) and prepared for Western blot for FAK and paxillin.

Western immunoblots
Tissue was homogenized and prepared for standard Western blot as previously described (15) with the following exceptions. Monoclonal anti-FAK and anti-paxillin antibodies (BD Transduction Laboratories, BD Biosciences, San Jose, CA) were incubated at a working concentration of 1:1000 in blocking buffer comprised of 2% nonfat milk powder (Bio-Rad Laboratories, Inc., Hercules, CA) in Tris-buffered saline with 0.1% Tween 20 (TTBS; Bio-Rad) for 3 h at room temperature or overnight at 4 C. Horseradish peroxidase-conjugated goat antimouse secondary antibody (Cell Signaling Technology, Inc., Danvers, MA) was incubated at a working concentration of 1:3000 in blocking buffer for 1 h at room temperature. Proteins were visualized using LumiGlo chemiluminescent reagent (Cell Signaling Technology) and Amersham Hyperfilm ECL film (GE Healthcare Biosciences Corp., Piscataway, NJ). FAK was represented by a band of approximately 125 kDa, and paxillin was represented by a complex of approximately 65–68 kDa, which was analyzed as a whole.

To control for differences in protein loading for each lane, membranes were either washed extensively in TTBS and stained for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein or incubated with Ponceau S solution (0.5% Ponceau S, 1% glacial acetic acid in deionized water) (6). For GAPDH protein, membranes were incubated with monoclonal anti-GAPDH antibody (Chemicon International, Inc., Temecula, CA) for 2 h and horseradish peroxidase-conjugated goat antimouse secondary antibody (Cell Signaling Technology) for 30 min at room temperature. The 38-kDa GAPDH band was then visualized as described above. For Ponceau S staining, membranes were incubated with Ponceau S solution for 5 min and washed twice with deionized water. Membranes were then dried and the 45- to 47-kDa complex was visualized and quantified as described below. Ponceau S staining was used to normalize protein levels for all experiments involving hormone treatment or hormone inhibition because unlike GAPDH (17), it is unaltered by exogenous treatment with estradiol treatment (6).

A lightbox and CCD camera were used to capture images from all films and Ponceau S-stained membranes. Grayscale integrative area density was quantified using NIH Image software calibrated with known values. Raw integrative density units were used to compare levels of FAK and paxillin in the male and female MBH in experiment 1 because levels of GAPDH did not differ between samples, and all samples fit on one 15-well gel. For all other experiments, protein levels were expressed as a ratio of FAK or paxillin to Ponceau S integrative density units. Because these experiments used more than 15 samples and therefore required more than one gel, each sample was normalized to common controls that were loaded on each gel to control for membrane-to-membrane differences in protein transfer or Lumiglo exposure.

Experiment 9: effect of estradiol on primary cultures of dispersed hypothalamic cells
Using sterile technique, brains were removed from E18 rat embryos of mixed sex. With the dorsal side of the brain face down, sterile razor blades were used to cut an approximately 1-mm section between the optic chiasm and mammillary bodies. The MBH was removed from the slice as described above for the PN0 brain. Cells were dissociated and plated as described (18) at a density of 75,000 cells per plate. Cultures were treated with 10 nM estradiol or dimethylsulfoxide vehicle in sterile phenol red-free, standard culture media (18) 1 h after plating. Dimethylsulfoxide treatment was limited to 1 µl/ml of culture media. Cultures were kept at 37 C and 5% CO2 for 20–24 h, and then fixed in 5% sucrose in 4% paraformaldehyde at 37 C for 30 min. Cultures were kept for less than 24 h to maximize the ability to measure axon outgrowth (19).

Immunocytochemistry (TUJ-1 immunoreactivity)
Neuronal class III ß-tubulin immunoreactivity was used to visualize neuronal morphology. Immunocytochemistry was performed as described (18) with a monoclonal antibody against neuronal class III ß-tubulin (TUJ-1; Covance Research Products, Inc., Berkeley, CA). Slides were examined using a Zeiss Axioskop, CCD camera, and NIH Image software. Neurons were identified by TUJ-1 staining and standard morphology; glia cells were not analyzed. The experimenter was blind to treatment group. For each neuron, the number of neurites, length of the longest neurite (µm), and the number of branches off the longest neurite were measured. Somal area was also measured and found not to differ between treatment groups, consistent with previous reports (data not shown) (18). Between four and 12 fields were sampled from 10 coverslips. A total of 86 estradiol-treated and 56 vehicle-treated neurons were quantified.

Statistical analysis
Data were analyzed by Statview software (SAS Institute, Cary, NC) using ANOVA followed by Fisher post hoc analysis or unpaired Student’s t test where appropriate. The criterion for significance was set at {alpha} < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
High-throughput Western blot analysis reveals elevated expression of focal adhesion complex proteins in the female hypothalamus on the day of birth
To identify novel signaling molecules mediating brain sexual differentiation in the rat, PowerBlot analysis was performed on male and female day of birth (PN0) hypothalamus including the medial preoptic area (mPOA) and MBH, regions dramatically regulated by steroids during the perinatal sensitive period for sexual differentiation. Eighty antibodies were used to examine 30 proteins in both the phosphorylated and unphosphorylated state. Table 1Go lists the proteins examined. For several proteins, multiple antibodies were used to probe specific phosphorylated moieties. For simplicity, the antibodies were collapsed into phosphorylated and unphosphorylated groups. The 37 proteins listed in bold were detected in either or both male and female samples. Of those, only four exhibited a sex difference (see Table 2Go). Although we expected sex differences in levels of phosphorylated signaling molecules indicating alterations in their activity, all of the sex differences detected were of total or unphosphorylated proteins. Two of these proteins, FAK and paxillin, were elevated in the female compared with the male hypothalamus. The remaining two proteins, the IIB regulatory subunit of the cAMP-dependent protein kinase (PKARIIB) and phospholipase C{gamma}1 (PLC{gamma}1) were elevated in the male compared with the female hypothalamus. Because there are no known signals mediating feminization of the brain, and FAK and paxillin are members of the same family of focal adhesion complex proteins, remaining studies focused on the regulation of these proteins.


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  		TABLE 2. Proteins differing in male and female hypothalamus in the PowerBlot analysis

 
The sex differences in FAK and paxillin expression is evident in the hypothalamus, but not in other brain regions examined
The mPOA is an important locus for the expression of male sexual behavior, whereas the ventromedial nucleus of the hypothalamus (VMN) is vital for the expression of female sexual behavior (2). Standard Western blot analysis was performed to confirm the sex differences in FAK and paxillin specifically in the MBH, which encompasses the VMN but excludes the mPOA. Consistent with the PowerBlot analysis, FAK expression was significantly elevated in the female compared with the male MBH on PN0 [Fig. 1AGo; t (9) = 3.59; P < 0.01]. Similarly, paxillin expression was also elevated in the female MBH compared with the male [Fig. 1CGo; t (8) = 2.48; P < 0.05]. In contrast, sex differences in FAK and paxillin expression were not apparent in the cortex [t (12) = 0.36 for FAK, Fig. 1BGo; t (12) = 0.54 for paxillin, Fig. 1DGo] or thalamus [t (10) = 0.23 for FAK, Fig. 1BGo; t (10) = 0.62 for paxillin, Fig. 1DGo). These data suggest that the sex difference in focal adhesion complex proteins may be confined to regions like the hypothalamus that are actively undergoing sexual differentiation.


Figure 1
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  		FIG. 1. Western blot analysis indicates significantly elevated FAK (A) (*, P < 0.01) and paxillin (C) (*, P < 0.05) protein expression in the female compared with the male MBH on PN0 (n = 4–7 per group). In contrast, no significant sex difference for either protein (B and D) is detected in the frontal cortex or thalamus (n = 5–8 per group). I.D.U., Integrative density units.

 
Estradiol regulates FAK expression in the hypothalamus on PN0 but not later in development
If FAK and paxillin are regulators of feminization, then estradiol, the major known signal mediating the opposite functions of brain masculinization and defeminization, should decrease their expression in the MBH. Given on PN0 and PN1, 100 µg estradiol benzoate permanently masculinizes the mPOA and MBH (15, 20). Western blot analysis did not show reduced FAK expression in the MBH on PN2 in female rats administered this standard masculinizing treatment of estradiol compared with vehicle-treated controls (Fig. 2AGo; F2,14 = 0.50). Similarly, rats administered estradiol on PN0 did not exhibit reduced FAK protein expression on PN1 (Fig. 2BGo; F2,13 = 0.38). In contrast, an increased dose of estradiol (250 µg) in a fast-release vehicle administered on PN0 significantly reduced FAK expression within 6 h compared with vehicle-treated controls (Fig. 2CGo; F2,17 = 8.60; P < 0.005 for a main effect; P < 0.05 for females given estradiol vs. vehicle-treated females). Furthermore, a significant sex difference in FAK protein was detected only on the day of birth (P < 0.001) and not on PN1 or PN2, suggesting that sex-specific hormonal regulation of FAK may occur within a very limited developmental window.


Figure 2
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  		FIG. 2. Estradiol regulates FAK expression on the day of birth but not thereafter. A, Representative image and quantification of Western blot analysis of FAK expression in the MBH on PN2 in vehicle-treated males (M) and females (F) and females administered 100 µg estradiol benzoate on PN0 and PN1 (F+E; n = 6–7 per group); B, representative image and quantification of Western blot analysis of FAK expression in the MBH on PN1 in vehicle-treated males and females and females administered 100 µg estradiol benzoate on PN0 (n = 4–7 per group); C, representative image and quantification of Western blot analysis of FAK expression in the MBH on PN0 in vehicle-treated males and females and females administered 250 µg estradiol in ethyl oleate vehicle for 6 h (*, P < 0.05; **, P < 0.001 vs. vehicle-treated females; n = 6–8 per group).

 
Estradiol regulates paxillin expression in the hypothalamus on PN2, but not earlier in development
A timeline of estradiol sensitivity was also examined for paxillin. In contrast to FAK, Western blot analysis demonstrated significant reduction in paxillin protein on PN2 after estradiol administration on PN0 and PN1 (Fig. 3AGo; F2,14 = 6.20; P < 0.05 for a main effect; P < 0.05 for females given estradiol vs. vehicle-treated females). Paxillin was not reduced, however, on PN1, 24 h after estradiol treatment (Fig. 3BGo; F2,21 = 1.5) or on PN0, 6 h after estradiol treatment (Fig. 3CGo; F2,18 = 0.24).


Figure 3
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  		FIG. 3. Estradiol treatment at birth does not regulate paxillin expression until PN2. A, Representative image and quantification of Western blot analysis of paxillin expression in the MBH on PN2 in vehicle-treated males (M) and females (F) and females administered 100 µg estradiol benzoate on PN0 and PN1 (F+E) (*, P < 0.05; **, P < 0.005 vs. vehicle-treated females; n = 5–6 per group); B, representative image and quantification of Western blot analysis of paxillin in the MBH on PN1 in vehicle-treated males and females and females administered 100 µg estradiol benzoate on PN0 (n = 8 per group); C, representative image and quantification of Western blot analysis of paxillin in the MBH on PN0 in vehicle-treated males and females and females administered 250 µg estradiol in ethyl oleate vehicle for 6 h (n = 6–8 per group).

 
Inhibition of endogenous estradiol reverses the low level of FAK and paxillin in the male relative to the female MBH
Because the timing of exogenous estradiol regulation of FAK and paxillin was restricted, we next tried blocking endogenous estradiol by inhibiting aromatase or antagonizing the estrogen receptor during the first 2 d of life. There was no effect of the aromatase inhibitor letrozol (200 mg) or the estrogen receptor inhibitor tamoxifen (1 mg/kg) on FAK or paxillin in females, consistent with a lack of endogenous estradiol action during female brain development. However, in males, there was a trend toward elevated FAK after treatment with letrozol or tamoxifen (Fig. 4AGo; F5,33 = 0.87; post hoc Fisher’s P = 0.07 and P = 0.10, respectively). For paxillin, there was a significant effect of treatment (Fig. 4BGo; ANOVA, F5,34 = 2.49; P < 0.05) and post hoc analysis indicated that letrozol treatment increased paxillin in males (P = 0.05). Moreover, there was no difference between letrozol-treated males and females, whereas vehicle-treated (P < 0.05) and tamoxifen-treated (P < 0.01) males were significantly different from females. Because aromatase inhibition, but not estrogen receptor inhibition, blocked the reduction of paxillin in the male rat, these data suggest that androgens may modulate the expression of paxillin.


Figure 4
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  		FIG. 4. Blocking endogenous estrogens increases FAK and paxillin in males. Quantification of Western blot for FAK (A) and paxillin (B) in the MBH on PN2 after treatment on PN0 and PN1 with vehicle, 200 mg of the aromatase inhibitor letrozol (Let), or 1 mg/kg of the estrogen receptor inhibitor tamoxifen (Tam). Tamoxifen and letrozol treatment did not alter FAK and paxillin in females. A, In males (M), letrozol (P = 0.07) and tamoxifen (P = 0.1) treatment induced trends toward increased FAK. B, In males, there was a significant effect of treatment (ANOVA, P < 0.05), and post hoc analysis indicated that letrozol treatment increased paxillin (P = 0.05) relative to vehicle-treated males. Moreover, there was no difference between letrozol-treated males and females, whereas vehicle-treated males (P < 0.05) and tamoxifen-treated males (P < 0.01) were significantly different from vehicle-treated females, indicating that aromatase inhibition, but not estrogen receptor blockade, prevented the reduction of paxillin in the male rat (*, P < 0.05; n = 6–8 per group).

 
Testosterone reduces paxillin in female MBH by PN2
Treatment with testosterone or the nonaromatizable androgen DHT did not alter FAK in the female (data not shown). However, testosterone treatment on PN0 and PN1 reduced paxillin on PN2 (Fig. 5Go; F4,21 = 1.35; P < 0.05). DHT did not significantly alter paxillin levels, suggesting that androgen receptors are not primarily involved in paxillin regulation. Given that testosterone can be aromatized to estradiol, these data suggest that a combination of androgen and estrogen action is optimal for paxillin regulation.


Figure 5
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  		FIG. 5. Testosterone reduces paxillin in females. Quantification of Western blot for paxillin in the MBH on PN2 after treatment with vehicle or 100 µg testosterone (T), DHT, and estradiol plus DHT. Testosterone reduces paxillin (P < 0.05), whereas the nonaromatizable androgen DHT does not. Although estradiol and DHT did not significantly reduce paxillin in females (F), these data suggest that estrogens and androgens may combine to regulate paxillin in males (M).

 
Levels of FAK are elevated in females relative to males on PN6, when paxillin levels are high in both males and females
Neurons form synaptic connections within the hypothalamus beginning perinatally and extending into the first 10 d of life (21). We hypothesized that expression of FAK and paxillin, which are important in axon extension and pathfinding, would be regulated during a similar time course. There was a profoundly different pattern of FAK expression in females compared with males across development. FAK levels in females progressively increased until PN6 before falling again, whereas in males, levels stayed relatively constant. As a result, FAK was significantly elevated in female compared with male MBH on PN6 (Fig. 6AGo; F9,40 = 1.12; P < 0.05). In contrast, there was no significant sex difference in paxillin, but paxillin was significantly elevated on PN6 in the female MBH relative to all other days during development (F9,44 = 2.66; P < 0.05).


Figure 6
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  		FIG. 6. On PN6, FAK is elevated in females (F) relative to males (M), and paxillin is elevated in females relative to other postnatal days. PN2 in the male and female is referred to as M2 and F2, respectively. A and B, Quantification of Western blots for FAK (A) and paxillin (B) on untreated males and females on PN2, PN4, PN6, PN8, and PN10. *, P < 0.05 for F6 vs. M6, F8, and F10 (A); *, P < 0.05 for PN6 vs. PN2, PN8, and PN10 (B).

 
Estradiol increases neurite branching in cultures of dispersed hypothalamic cells
Hippocampal neurons deficient for FAK expression exhibit enhanced axon length and branching in vitro (22). Because estradiol treatment reduced FAK and paxillin expression, albeit with different time courses, and FAK levels were different in males and females during synapse formation in vivo, we hypothesized that MBH neurons would exhibit enhanced axon growth in response to estradiol. Dissociated hypothalamic cells from mixed-sex E18 embryos were treated with 10 nM estradiol or vehicle at plating. Cells were fixed 20–24 h after plating, consistent with studies of axon outgrowth after changes in FAK expression (22, 23, 24). Neuronal morphology was visualized by neuronal class III ß-tubulin immunoreactivity, and the longest neurite was determined to be the axon. Neurites are neuronal projections extending off the cell soma within approximately the first 12 h in primary culture. Within 24 h, one neurite takes on the properties of the axon and extends further than others (25). Representative images of vehicle- and estradiol-treated cultures are shown in Fig. 7Go, A and B, respectively. The number of neurites was similar in vehicle- and estradiol-treated neurons [Fig. 7CGo; t (141) = 0.71]. The length of the longest neurite per neuron (axon) was also not altered by treatment [Fig. 7DGo; t (141) = 1.23]. In contrast, the number of branches per axon was elevated in estradiol-treated compared with the vehicle-treated neurons [Fig. 7EGo; t (141) = 2.41; P < 0.01]. Enhanced axonal branching is consistent with diminished FAK expression (22).


Figure 7
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  		FIG. 7. Estradiol enhances axonal branching in primary cultures of dispersed hypothalamic cells. Representative photomicrographs of neuronal class III ß-tubulin immunoreactivity in dispersed hypothalamic cells treated with vehicle (A) or 10 nM estradiol (B) and fixed 20 h after plating. Scale bar, approximately 25 µm. Estradiol treatment did not impact the number of neurites emanating from the soma (C) or the length of the longest neurite (D). The number of branches off the longest neurite (E) was, however, increased in estradiol-treated cultures of dispersed hypothalamic cells. *, P < 0.01 for branches; n = 56–87 cells per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We describe here the first use of a high-throughput proteomics technique to discover novel signaling molecules involved in sexual differentiation of the brain. Both PowerBlot and conventional Western blot analysis showed elevated expression of the novel candidate proteins FAK and paxillin on PN0 in the female compared with the male hypothalamus. Neither of these proteins exhibited sex differences in the cortex or thalamus, regions less affected by sexual differentiation of the brain. To our knowledge, this is the first report of a sex difference in the brain in which a regulatory protein is decreased in males relative to females and thereby suggests a role for FAK and paxillin, both members of the focal adhesion complex, in feminization of the brain.

Cytoskeletal changes are fundamental to neuronal development, and recent evidence strongly implicates the focal adhesion complex in this process. FAK expression is highly enriched in growth cones, and FAK expression in the brain peaks on PN0 (reviewed in Ref. 26). Similarly, paxillin is highly phosphorylated during embryonic development in the rat (27). FAK is necessary in netrin-mediated chemoattraction and chemorepulsion of axons (23, 24, 28), and it may be required for growth cone turning (29). Hippocampal neurons deficient for FAK demonstrate increased axon length and branching in vitro (22). Neuroblastoma cells overexpressing paxillin demonstrate enhanced cell spreading and growth factor-induced lamellopodia formation (30). Thus, FAK and paxillin are important regulators of neuronal process formation.

The observation that both FAK and paxillin were elevated in the female hypothalamus relative to the male on the day of birth led to the hypothesis that gonadal steroids reduce expression of these proteins in developing male rats. Treatment of females with a large dose of estradiol on PN0 down-regulated FAK expression within 6 h but had no effect on paxillin. Treatment of females with more conventional doses of estradiol did not impact FAK expression on PN1 or PN2 but did down-regulate paxillin to the level seen in males by PN2. Thus, PN0 may constitute an important developmental window for estradiol-mediated suppression of FAK, whereas estradiol-induced suppression of paxillin on PN2 may be an indirect result of earlier changes in FAK because FAK recruits paxillin to the focal adhesion complex (9).

This interpretation is potentially confounded by experimental variables that include injection stress. Initial observations of sex differences in FAK and paxillin observed in both the PowerBlot assay and conventional Western blot analysis involved animals that were not manipulated. All subsequent experiments included animals that received either hormone or vehicle injection, with the exception of the developmental time course. Stress associated with the injection may impair hormonal modulation or endogenous expression of FAK and paxillin expression. The expression of many signaling molecules is altered by handling stress. In the early postnatal rat, pCREB peaks within 15 min of the onset of stress (31), and handling increases levels of cAMP and cAMP-dependent protein kinase (PKA) activity in the postnatal rat (32). Importantly, PN0 rats injected with saline exhibit a reversal in the sex difference in CREB-binding protein, CBP, in the hypothalamus on PN1 (33). These observations suggest stress can impact or interfere with steroid-mediated sexual differentiation of the brain and highlights an important experimental limitation in mechanistic studies of sex differentiation.

Nonetheless, we found convincing evidence of hormonal modulation of FAK and paxillin in the developing hypothalamus. In the male rat, treatment with the aromatase inhibitor letrozol significantly increased paxillin and induced a trend toward increased FAK. The estrogen receptor inhibitor tamoxifen also induced a trend toward increased FAK but did not alter paxillin protein levels. These data suggest that endogenous estrogens regulate FAK expression. Given that estrogen receptor blockade did not alter paxillin expression, whereas aromatase inhibition did, androgens may also regulate paxillin expression in the male rat. In a subsequent experiment, treatment of females with testosterone significantly reduced paxillin by PN2, but the nonaromatizable androgen DHT had no significant effect. These data suggest that the combined actions of androgens and estrogens may be important in paxillin regulation.

Sexual differentiation of the brain establishes sexually dimorphic synaptic patterning. Sex differences in prevalence of synapse type have been reported in the VMN, POA, and arcuate nucleus of the hypothalamus (ARC) (34, 35, 36). Dendritic morphology is also sexually dimorphic, with increased dendritic branching in the male compared with female VMN and increased density of dendritic spines in the female ARC compared with the male (37). These sex differences in synapse type and dendritic complexity emerge as early as PN2 (35), on a timeline similar to the regulation of FAK and paxillin by gonadal hormones shown here. Little work has been done in vivo to study hypothalamic axons that are synapsing upon other hypothalamic nuclei or traveling to other regions of the brain during development. Although the targets of these axons have been well defined by tract-tracing studies (reviewed in Ref. 2), little is known about their development. On PN6, FAK expression was significantly elevated in female relative to male MBH, and paxillin was elevated in the female MBH relative to other postnatal time points. In the rat, PN6 is also when neurons from the ARC innervate the dorsomedial nucleus of the hypothalamus, and both the ARC and dorsomedial nucleus of the hypothalamus are found in the MBH (21). In mixed-sex primary cultures of hypothalamic neurons and glia, branching was enhanced from the longest neuron process (the presumed axon) as quickly as 24 h after estradiol treatment. These neurons were considerably younger than PN6 but demonstrate the capacity for estradiol to regulate the branching of extending axons and are consistent with studies on FAK-deficient neurons that show increased branching (22). Future studies will address whether reduction in FAK expression is necessary for estradiol-mediated enhancement of axon branching.

The initial rationale for investigating hormonal modulation of FAK and paxillin was the unusual observation that both of these proteins were elevated in newborn female hypothalamus compared with males, thereby implicating them as possible mediators of brain feminization. However, gonadal steroids mediate both masculinization and defeminization of the brain, whereas feminization occurs in the absence of steroid action (at least in the hypothalamus). It is currently theoretically impossible to distinguish between the importance of steroid-mediated reduction in FAK and paxillin in brain masculinization/defeminization and the importance of elevated FAK and paxillin in normal female brain development. The active suppression of FAK/paxillin by steroids is consistent with the active suppression of female brain development that occurs during defeminization of the rat brain. Because ovarian hormones act later in postnatal development to feminize the corpus callosum (reviewed in Ref. 38), an alternative hypothesis is that signaling events that mediate brain feminization occur later in development. However, we are not aware of any other examples in which a signaling molecule is elevated in the female diencephalon and suppressed by steroids during the critical perinatal period for sexual differentiation, making this one of the first cellular candidates for regulating rat brain feminization.


   Footnotes
 
This work was supported by National Institute of Mental Health Grant R01 MH052716 (to M.M.M.).

Disclosure Statement: The authors have nothing to declare.

First Published Online April 5, 2007

Abbreviations: ARC, Arcuate nucleus of the hypothalamus; CREB, cAMP response element-binding protein; DHT, dihydrotestosterone; E14, embryonic d 14; FAK, focal adhesion kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MBH, medial basal hypothalamus; mPOA, medial preoptic area; pCREB, phosphorylated CREB; PN0, postnatal d 0; TTBS, Tris-buffered saline with 0.1% Tween 20; VMN, ventromedial nucleus of the hypothalamus.

Received June 22, 2006.

Accepted for publication March 21, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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