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Biology of Reproduction 66, 1127-1133 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Cloning and Expression of Zebra Finch (Taeniopygia guttata) Steroidogenic Factor 1: Overlap with Hypothalamic but Not with Telencephalic Aromatase1

Robert J. Agatea, William R. Perlmana, and Arthur P. Arnold2,a

a Department of Physiological Science, Interdepartmental Program for Neuroscience and Laboratory of Neuroendocrinology of the Brain Research Institute, University of California, Los Angeles, California 90095-1606


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The zebra finch (Taeniopygia guttata) brain is highly sexually dimorphic. The organization and production of sex-specific song is considerably influenced by estrogens and androgens. Because the brain itself expresses several steroidogenic enzymes, the local production of sex steroids may contribute to sex differences in neural development. Sex steroid production in gonads is directed by a master regulatory factor, steroidogenic factor 1 (SF1). We have identified a cDNA encoding the homologue of SF1 in the zebra finch and utilized reverse transcription-polymerase chain reaction and in situ hybridization to examine early and late developmental expression of SF1 in brain and in early gonadal development. We found that SF1 is expressed early in embryonic development in the Rathke pouch, beginning at stage 15 and extending to at least stage 27 in both males and females. The earliest expression of SF1 in gonads was found at stage 17 for both males and females and extended to at least stage 27. In brain, we assessed SF1 mRNA expression in posthatch and adult telencephalon, and we compared SF1 and aromatase mRNA expression in adult hypothalamus. In the telencephalon and hippocampus, aromatase was expressed independently of SF1, whereas in the hypothalamus, aromatase and SF1 expression were more closely associated. Expression of SF1 and of aromatase overlapped in restricted regions of the hypothalamus, suggesting that SF1 may regulate aromatase expression in these regions. These findings suggest that steroidogenesis in the zebra finch brain may be regulated by both SF1-dependent and SF1-independent mechanisms. No sex differences were detected in SF1 expression in brain.

behavior, developmental biology, hypothalamus, neuroendocrinology, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In songbirds, sex steroids significantly influence brain development and expression of sex-specific song behavior. In zebra finches (Taeniopygia guttata), sex steroids are important for male-specific development of the neural song circuit that mediates song learning. For example, treatment of hatchling females with estrogen causes development of the song system that is morphologically more like that of a male than that of a female, so it is likely that estrogens normally play a role in masculine neural development and differentiation [1, 2]. Additionally, androgens are important for stabilizing song during a later phase of song acquisition and in modulating song expression during adulthood [3]. The presence of sex steroid receptors in and around telencephalic song nuclei supports these roles [4–7]. However, it is not known in detail how sex steroid production is regulated to affect song development and differentiation.

This may be because the levels of sex steroids acting in the brain are difficult to quantify, because the brain itself produces steroids and can regulate their local concentrations. In brain, the steroidogenic enzymes aromatase, 5{alpha}-reductase, and 5ß-reductase are widely expressed [8, 9]. Because each enzyme acts by metabolizing testosterone to different end products (estrogen, 5{alpha}-dihydrotestosterone [DHT], and 5ß-DHT, respectively), they are likely to influence local levels of active androgens and estrogens in brain and, in turn, to regulate the sex-specific actions of these steroids. This would suggest that these enzymes must be carefully regulated to ensure appropriate levels of sex steroids for normal brain development.

Aromatase in particular is known to be regulated in other systems by steroidogenic factor 1 (SF1, also known as ad4bp) [10]. An orphan member of the nuclear hormone-receptor superfamily and a homologue of the Drosophila fushi tarazu factor-1 protein, SF1 is a regulator of the fushi tarazu homeobox patterning gene [11]. It was originally identified in mammals as a tissue-specific transcriptional regulator of the cytochrome P450 hydroxylases, including aromatase [12, 13]. The SF1 transcripts in mice are expressed in early developing adrenal and gonadal tissues as well as in hypothalamus and pituitary, indicating that SF1 has an important role at multiple levels of the developing reproductive axis [14–19]. Mice with a null mutation in SF1 fail to develop adrenals and gonads, and they have pituitary deficiencies and abnormalities in hypothalamic organization [19], demonstrating that SF1 is part of the molecular cascade essential for the normal development of these tissues. Furthermore, the role of SF1 in reproductive development extends to sexual differentiation of the gonads, where expression becomes sexually dimorphic soon after gonadal differentiation because of its down-regulation in ovaries [14]. The significance of this down-regulation is not understood, but it may involve SF1's role in regulating the expression of müllerian-inhibiting substance [20], another component of the sexual differentiation pathway.

In chickens, SF1 expression is also sexually dimorphic during gonadal differentiation, but in contrast to mice, it becomes down-regulated in testes [21]. Because initial ovarian differentiation in early embryonic development is dependent on estrogens in chicks [22, 23], the greater expression of SF1 in developing ovaries may be necessary to maintain estrogen levels through its regulation of aromatase activity. To our knowledge, the expression of SF1 in other avian tissues, such as brain, has not been described previously. Based on the role of SF1 in steroidogenesis and gonadal differentiation as described above, we asked whether SF1 might play an important regulatory role during steroidogenesis in the zebra finch brain.

The zebra finch might be a particularly advantageous species for studying the role of SF1 in regulation of steroid synthesis in brain because of its unusually high levels of aromatase and other steroidogenic enzymes in the telencephalon [8, 24–29]. In particular, we asked whether SF1 is expressed in brain, and whether its expression overlaps with that of aromatase, the most abundant steroidogenic enzyme expressed in brain [30, 31]. Moreover, we asked if the expression of SF1 might also be sexually dimorphic in brain, which might suggest a role in sex-specific development of the song circuit, similar to what is thought to occur in gonads.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissues

Adult (>90 days of age) and Posthatch Day 1 (P1), P20, P25, and P30 zebra finches were obtained from our breeding colony at the University of California, Los Angeles (UCLA). Tissue for total RNA and poly(A)+ RNA isolation was collected by rapidly decapitating the animal, removing the telencephalon, freezing the tissues on dry ice, and then storing them at -80°C for future use. Total RNA was isolated from tissues using a Polytron homogenizer (Kinematica, Cincinnati, OH) and Trizol Reagent (Life Technologies, Rockville, MD) according to the manufacturers' instructions. The poly(A)+ RNA was obtained using the PolyATract mRNA Isolation System (Promega, Madison, WI). All use of animals was approved by the UCLA Institutional Animal Care & Use Committee.

Isolation of a Zebra Finch SF1 cDNA

A full-length mouse SF1 cDNA probe, generously provided by Dr. Keith Parker (University of Texas Southwestern Medical Center), was used to screen 1.5 x 105 plaques of an ovarian zebra finch cDNA library, from which we isolated two cDNAs (3.0 and 3.2 kilobases [kb]). The plaque-purified clones were excised from the {lambda}ZAP phage vector (Stratagene, Cedar Creek, TX) with the adjoining pBluescript SK plasmid vector. Preliminary sequencing of each cDNA revealed that the 3.0-kb cDNA was truncated at the 5' end relative to the 3.2-kb cDNA.

Sequencing

The 3.2-kb cDNA was designated zfSF1, linearized using XhoI and KpnI, and digested with endonuclease III to generate a series of nested deletions using the Erase-a-Base system (Promega). These deletions were sequenced in both directions using the Sequenase 2.0 system (U.S. Biochemical Corp., Cleveland, OH). All sequence analyses were done with the GCG sequence analysis package (Genetics Computer Group, San Diego, CA).

Northern Blot Analysis

The RNA was collected from four individuals of each sex for telencephalic mRNA and from 10 of each sex for gonadal and hypothalamic mRNA. The tissues were combined before mRNA extraction. The poly(A)+ RNA (4 µg/lane) was resolved by electrophoresis using a 1.2% agarose formaldehyde denaturing gel, transferred to a MagnaNT nylon membrane (Micron Separations, Westboro, MA), and immobilized by ultraviolet cross-linking. The 3.2-kb cDNA was random primed and labeled with [32P]dCTP, prehybridized (without probe) for 1 h (42°C), and hybridized for 12 h in 50% formamide/5x Denhardt/0.1% SDS/5x SSPE (single strength: 150 mM NaCl, 10 mM NaH2PO4, and 1 mM Na/EDTA)/150 µg/ml of low molecular weight DNA (42°C). Posthybridization washes were as follows; 1) 3x SSPE/0.1% SDS for 15 min at room temperature, 2) 1x SSPE/0.1% SDS for 15 min at 37°C, and 3) 0.1x SSPE/0.1% SDS/twice for 30 min at 55°C. The SF1-probed blot was exposed for 48 and 94 h on Kodak film (X-OMAT AR; Rochester, NY). The exposure for the loading control, cyclophilin, was for 24 h using a PhosphorImager (APBiotech, Piscataway, NJ).

Reverse Transcription-Polymerase Chain Reaction

Total RNA (3 µg) was reverse transcribed at 42°C using Moloney murine leukemia virus reverse transcriptase (Invitrogen Corp., Carlsbad, CA) and 1.0 pmol of primer. For polymerase chain reaction (PCR) amplification, a 50-µl reaction was assembled containing one-tenth the cDNA reaction (2 µl), 1.0 pmol of each primer, 1.0 mM MgCl2, 10 µM dNTPs, 20 mM Tris-HCL (pH 8.4), 50 mM KCL, and 2.5 U of Taq DNA polymerase (Invitrogen). The cycling conditions were as follows: 1) 3 min at 94°C for 1 cycle, 2) 1 min at 94°C, 1 min at 49°C, and 1 min at 72°C for 35 cycles, and 3) 5 min at 72°C for 1 cycle. The reverse transcription (RT) primer was 5'-AAGAGACAGAGAGACACAC-3', the forward primer was 5'-GCTGAGTGAGTGGGTAAGG-3', and the reverse primer was 5'-CAAAAAGAAGAAGGTGAAA-3'. Every experiment included negative-control reactions with water alone and reactions using RNA in the absence of reverse transcriptase. If the amplification was negative, the sample was tested for the synthesis of cDNA by amplifying the widely expressed TrkB receptor.

For PCR on P1, P20, P25, and P30, a different set of primers was used. The forward primer was 5'-AGGCTGAGGAGTACCTGTACCACA-3', and the reverse primer was 5'-TAAAACCACAGCCTGGAGGG-GT-3'. Total RNA (1 µg) was reverse transcribed using oligo dT as primer, and PCR was performed using Titanium Taq (Clontech, Palo Alto, CA). The PCR cycling conditions were as follows: 1) 5 min at 95°C for 1 cycle, 2) 45 sec at 95°C, 35 sec at 67°C, and 45 sec at 72°C for 35 cycles, and 3) 5 min at 72°C for 1 cycle.

Eggs and Embryos

Zebra finch eggs were removed from nest boxes on the day of laying and were incubated at 37.5°C with 55% humidity. Embryos were removed from eggs after 2.5–5 days of incubation, at which point they had the appearance of chick embryos at stages 13–27 of development [32]. At least 4 embryos of each sex at stages 14–16 were used for whole-mount in situ hybridization. Ten additional embryos were used to follow the expression pattern between stages 16 and 27.

Sexing of Embryos

In birds, the sex chromosomes are designated Z and W, with males as the homogametic sex (ZZ) and females as the heterogametic sex (ZW). The Z and W isoforms of the sex chromosome genes CHDZ and CHDW were amplified by PCR from genomic DNA [33]. The primers amplify both Z and W isoforms of this fragment, but the product from the W chromosome is larger (389 base pairs [bp]) than that from the Z chromosome (353 bp). The PCR cycling conditions were as follows: 1) 5 min at 94°C for 1 cycle, 2) 45 sec at 94°C, 1 min at 50°C, and 1 min at 72°C for 35 cycles, and 3) 5 min at 72°C for 1 cycle. The forward primer was 5'-YTKCCAAGRATGAGAAACTG-3' and the reverse primer was 5'-TCTGCATCACTAAAKCCTTT-3'.

Probe Synthesis

The zfSF1 (3.2 kb) and zf1a (3.2 kb; aromatase [34]) were used for in vitro transcription to generate sense and antisense RNA probes labeled with digoxygenin or [33P]uridine triphosphate (UTP) using the Boehringer Mannheim labeling kit (Indianapolis, IN). For in situ hybridization, the digoxygenin- and 33P-labeled riboprobes were alkaline hydrolyzed (0.2 N NaOH for 15 min on ice, neutralized with 1 N HCL and 10 mM Tris-HCl [pH 8.0]).

In Situ Hybridization

In situ hybridization was carried out as previously reported [5]. Adult brain tissue was collected from three male and two female zebra finches deeply anesthetized with Equithesin, perfused intracardially with PBS (100 mM NaPO4 [pH 7.2] and 0.15 M NaCl) followed by 4% paraformaldehyde. Tissue was postfixed for 3–5 days, cryoprotected using 20% sucrose in PBS overnight, and then embedded in optimal cutting temperature medium. A series of 10-µm sections, beginning caudally at the tuberal hypothalamic region and extending rostrally beyond the anterior commissure (AC), were mounted on Superfrost Plus microscope slides (Fisher Scientific, Tustin, CA) and hybridized with [33P]UTP-labeled RNA probes. Groups of five slides containing adjacent sections were used to compare zebra finch SF1 expression with zebra finch aromatase expression. Slides 1 and 2 were probed with zfSF1 sense and antisense, respectively, and slides 3 and 4 were probed with zf1A (aromatase [34]) antisense and sense, respectively. Thus, SF1 and aromatase probes were hybridized to cells approximately 10 µm from each other. Slide 5 was stained with thionin for recognition of anatomical structures in brain.

Whole-Mount In Situ Hybridization

Whole-mount in situ hybridization was performed using a protocol modified from that described by Riddle et al. [35]. Embryos were collected from eggs, and extraembryonic membranes were removed in PBS (pH 7.4) at room temperature. Embryos were fixed overnight in 4% paraformaldehyde in PBS at 4°C, washed in PBS/Tween-20, and then dehydrated and stored in 100% methanol at -20°C.

On the day of the experiment, embryos were treated as described by Riddle et al. [35] and incubated overnight in hybridization solution containing digoxygenin-labeled riboprobe at 1 µg/ml at 70°C. Following hybridization, embryos were washed to remove excess and nonspecific binding of probe. Embryos were then incubated in antidigoxygenin Fab alkaline phosphatase-conjugate antibody (Boehringer Mannheim) in 1% heat-inactivated sheep serum/Tris-buffered saline and Tween-20 overnight at 4°C. The antibody was preadsorbed with approximately 0.3% zebra finch embryo powder. Antibody detection was carried out by incubating embryos with 2% NBT/BCIP stock solution (Boehringer Mannheim) for 1–3 h at room temperature in the dark.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
zfSF1 cDNA Isolation

The zfSF1 cDNA (GenBank accession no. AF407573) consists of a 179-bp stretch of 5' untranslated region; a 1398-bp open reading frame (ORF) with a starting ATG at nucleotide 180, which corresponds to the start site in other species; and 1645 bp of 3' untranslated region (Fig. 1). Included in the 3' untranslated region is a putative consensus polyadenylation site 13 bp from the end. The predicted ORF codes for a 466-amino acid polypeptide, which shares the following overall percentage identities with SF1 of other species: chick, 93% (GenBank accession no. BAA76713); turtle, 89% (AAD01975); frog, 81% (BAA36789); mouse, 71% (A40716); and human, 70% (BAA34092).



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  		FIG. 1. Nucleotide sequence of zebra finch SF1 cDNA with presumed ORF translated. The zinc finger domain is enclosed by a solid box, and the P-box [47] within it is indicated. A putative ligand-binding domain is enclosed by dashed lines, and a putative polyadenylation signal is boxed

Northern Blot Analysis

The zfSF1 probe hybridized to a single distinct band at approximately 3.4 kb in adult ovary and testis (Fig. 2). A longer exposure revealed a faint band of similar size in male and female hypothalamus. In contrast, no message was detected in male and female telencephalon or liver (not shown). These results indicate that SF1 is expressed in a tissue-specific manner and encodes a single mRNA species. In addition, the relative intensity of bands in the Northern blot suggested that expression was much greater in adult ovaries than in adult testes (Fig. 2).



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  		FIG. 2. Northern blot shows higher expression of zfSF1 mRNA in adult ovary than in adult testes. Expression was also detected in adult female and male hypothalamus, whereas no mRNA was detected in adult female and male telencephalon. Cyclophilin-loading control indicates relatively equal loading

RT-PCR Analysis

Using RT-PCR, we detected SF1 mRNA in the adult male and female zebra finch hypothalamus, adrenal, ovary, and testis (Fig. 3), but not in the telencephalon (not shown). In addition, we used RT-PCR to examine the expression of SF1 in the telencephalon during development. As in adults, no SF1 transcripts were detected in males (three individuals) or females (three individuals) at P1, P20, P25, or P30 (not shown). Each negative RNA sample was tested for the presence of reverse-transcribed product by amplifying for the widely expressed TrkB receptor. Each positive RNA sample was tested for genomic DNA contamination by the absence of amplified product in RNA that was not reverse transcribed.



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  		FIG. 3. RT-PCR for zfSF1 from adult male and female hypothalamus, gonads, and adrenals. Lanes marked with a dash signify PCR run on an RNA template that had not been reverse transcribed (PCR product size, 525 bp)

Whole-Mount In Situ Hybridization

The SF1 was first detected in the Rathke pouch of zebra finch embryos of both sexes at developmental stage 15 (4 of 8 male embryos, 4 of 4 female embryos) (Fig. 4A). Expression was not detected in this region at either stage 13 (0 of 2 males, 0 of 3 females) or 14 (0 of 4 males, 0 of 5 females) or in embryos incubated with sense probes. Expression was observed in both sexes through at least stage 27 (15 of 15 males, 14 of 14 females, stages 16–27), which was the latest stage tested. Expression of SF1 was detected in the developing gonad/adrenal tissue of both males and females beginning at stage 17 (2 of 2 males, 4 of 4 females) (Fig. 4B) and continuing through at least stage 27 (5 of 5 males, 5 of 5 females, stages 18–27). Expression was not detected in this region at stage 16 (0 of 7 males, 0 of 5 females) or in embryos incubated with sense probes.



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  		FIG. 4. SF1 mRNA expression in zebra finch embryos as revealed by whole-mount in situ hybridization. A) Left, male stage 15 (antisense); right, male stage 15 (sense). B) Female stage 17 (antisense). G/Ad, Gonad/adrenal primordium; RP, Rathke pouch. Bar = 1.0 mm

In Situ Hybridization

In all sections examined, which ranged from the caudal hypothalamus to the preoptic area, we did not detect SF1 transcripts in the telencephalon or hippocampus of adult males or females. This examination included the caudal neostriatum, song nucleus HVC (higher vocal center), and song nucleus RA (robust nucleus of the archistriatum). In adult hypothalamus, SF1 transcripts were detected in both males and females beginning caudally at the level of the tuberal nucleus and extending rostrally to the preoptic area. No sex differences in SF1 expression were apparent. All sections showing SF1 and aromatase expression in Figure 5 are from a single adult female bird. We found cells expressing SF1 transcripts located in the ventromedial portion of the tuberal region ventral and lateral to the paraventricular organ (Fig. 5, A1). This expression continued rostrally, with some SF1-positive cells extending laterally along the supraoptic decussation (Fig. 5, B1). Near the AC, SF1-positive cells were again only seen ventromedially (Fig. 5, D1). These positive cells continued rostral to the AC, in the preoptic region (Fig. 5, E1), where SF1-expressing cells diminished and became undetectable. The higher level of background shown in Figure 5, E1, was not seen in other birds. Transcripts for aromatase were also detected at the level of the tuberal nuclei and extended rostrally to the AC. However, aromatase expression was generally found dorsal to SF1 expression, though at some levels (Fig. 5, B–D), aromatase expression clearly overlapped that of SF1.



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  		FIG. 5. In situ hybridization comparing the distribution of SF1 and aromatase mRNA expression in a single adult female zebra finch hypothalamus. Panel A is approximately 300 µm caudal to E. Each series (e.g., A1–A3) shows a 10-µm section hybridized with antisense SF1 probe (1) immediately adjacent to a section hybridized with the aromatase antisense probe (2). The thionin-stained section (3) was no more than 20 µm from 1. Although some aromatase-expressing cells clearly did not express SF1, fields of aromatase-expressing cells (see arrows in B2–D2) did overlap with fields of SF1-expressing cells (compare with B1–D1). Background in E1 was high (arrow indicates the only positive region for SF1 expression). The two half-sections shown on the left at each level are adjacent sections probed with sense probes for SF1 (left half) and aromatase (right half). AC, Anterior commissure; DS, supraoptic decussation; OM, occipitomesencephalic tract; POA, preoptic area; PVO, paraventricular organ; Tu, tuberal nucleus. Bar = 0.6 mm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We detected expression of SF1 mRNA in several endocrine tissues of adult male and female zebra finches, including the hypothalamus, gonads, and adrenals. In addition, SF1 transcripts were found early during embryonic development in pituitary, adrenal, and gonadal primordia. This temporal and spatial distribution parallels that found in previous studies in the mouse, rat, and chicken [14, 16, 17, 21, 36], and it suggests a conserved role for SF1 in reproductive endocrine functions and organogenesis of all these tissues in both birds and mammals. The highly similar protein sequence of SF1 in birds and mammals supports a conserved function, and it suggests that SF1 acts as a key regulator of steroidogenesis in birds, as has been demonstrated in mammals [12, 37].

The SF1 was not expressed, however, in much of the zebra finch brain, where there is extensive aromatase activity throughout brain development. For instance, aromatase is expressed widely in the telencephalon and hippocampus beginning at least by P5 in the telencephalon and P10 in the hippocampus [5] and continues into adulthood [38, 39], but we did not detect SF1 mRNA expression in either of these regions at any developmental period examined. The aromatase gene appears to be regulated by at least two promoters, one of which contains an SF1-binding consensus sequence. Although this is the minor promoter in brain, it suggests potential regulation of aromatase by SF1 [40]. The lack of SF1 and aromatase coexpression indicates that estrogen synthesis is not regulated by SF1 in the telencephalon. Importantly, the recently reported sex difference in estrogen synthesis in telencephalon, which may mediate masculine development of HVC and RA [2], cannot be induced by SF1. Because SF1 appears not to be expressed in areas of the zebra finch telencephalon where significant steroidogenesis occurs, steroidogenesis in the telencephalon must be regulated in a manner that is fundamentally different from that in the gonads and adrenals. Determining how steroidogenesis is regulated in these regions of the brain is likely to reveal novel signaling mechanisms that may have evolved to decouple sex-specific song development from sex-specific development of the hypothalamic-pituitary-gonadal axis.

We did detect SF1 expression in the hypothalamus ventrally and medially along the third ventricle, extending from the tuberal region caudally to the preoptic region rostrally [41]. This pattern was found in both sexes, with no apparent sex difference. We compared SF1 mRNA and aromatase mRNA expression in the hypothalamus to examine whether they might be expressed in the same cells. Although aromatase was expressed in cells located just dorsal to those expressing SF1 in this region, at some levels the expression of aromatase overlapped that of SF1, indicating that the two mRNAs are expressed in the same, or in closely adjacent, cells. This overlap differs from the situation in mice, in which SF1 is expressed in the dorsal medial region of the hypothalamic ventromedial nucleus (VMH) [18, 19]. Because aromatase expression is not observed in this region and its expression in SF1 knockout mice appears to be normal, regulation by SF1 is unlikely [18]. In the zebra finch, however, SF1 and aromatase may be coexpressed by single cells in some regions of the hypothalamus (Fig. 5, B1–D2), suggesting that SF1 may directly regulate aromatase in some cells.

Expression of SF1 in the zebra finch hypothalamus is not as regionally confined as it is in mammals. This may be because the VMH in this species is not as clearly delineated and appears to be more diffusely organized. If so, SF1 expression will provide a valuable marker for identifying the avian homologue of the VMH. SF1 may perform additional functions that differ from those in mammals. Studies in both mammals and birds, however, implicate a role for the VMH in similar behaviors, including reproductive behaviors [42, 43]. For example, bilateral lesions in the basomedial hypothalamus of White Leghorn cockerels results in loss of sexual activity subsequent to testicular and pituitary atrophy [43, 44]. In hens, similar lesions result in atrophy of the ovary, oviduct, and pituitary [45]. Our finding that the reproductively related genes, SF1 and aromatase, are expressed in the basomedial hypothalamus, along with the estrogen and androgen receptors [6, 46], is consistent with a role for SF1 in reproduction.


   ACKNOWLEDGMENTS
 
Thanks to Dr. Keith Parker (University of Texas Southwestern Medical Center) for the SF1 probe, Dr. Xianjie Yang (UCLA) for assistance with whole-mount in situ hybridization of avian embryos, and the UCLA Mental Retardation Research Center for construction of the ovarian cDNA library. Special thanks to Dr. Laura Carruth (UCLA) for helpful comments on the manuscript.


   FOOTNOTES
 
First decision: 25 September 2001.

1 Supported by National Institutes of Health grants DC00217 and HD07228. R.J.A. is an ARCS Foundation Scholar. Back

2 Correspondence: Arthur P. Arnold, Department of Physiological Science, UCLA, 621 Charles E. Young Drive South, Los Angeles, CA 90095-1606. FAX: 310 825 8081; arnold{at}ucla.edu Back

Accepted: November 12, 2001.

Received: August 20, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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R. J. Agate, W. Grisham, J. Wade, S. Mann, J. Wingfield, C. Schanen, A. Palotie, and A. P. Arnold
Neural, not gonadal, origin of brain sex differences in a gynandromorphic finch
PNAS, April 15, 2003; 100(8): 4873 - 4878.
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