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Department of Biology, Boston University, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Gloria V. Callard, Ph.D., Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215. E-mail: gvc{at}bio.bu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Teleost fish are ideal experimental models to investigate the importance of brain-formed (neuro-) estrogen in lifelong processes of neurodevelopment and neuroplasticity. Exaggerated levels of neural P450arom expression are found in all teleost species examined to date. For example, aromatase enzyme activity in goldfish HPOA is 100- to 1000-fold higher than in corresponding regions of adult or fetal rat, mouse, rabbit, and human brain and 10-fold higher than in goldfish ovary (6). In addition to HPOA, aromatase enzyme activity and immunolabeled neurons, tracts, fibers, and terminals are widely distributed in the retina, visual processing areas of the optic tectum, and identified sensory and premotor pathways of the telencephalon, midbrain, hindbrain, and spinal cord (6, 7, 8). It may be relevant that the brain of adult fish retains a remarkable potential for neurogenesis. Not only does the brain continue to grow throughout life, but connections are continually remodeled to accommodate the expansion, and functional regeneration can occur after neural damage, a property that has been especially well studied in the fish visual system (9). In contrast to the human cyp19 gene, which is believed to occur as a single copy in the haploid genome and has multiple tissue-specific promoters and first exons (2), the goldfish has at least two separate and distinct cyp19 loci (10). cyp19b/P450aromB is constitutively expressed at high levels in the brain and is further up-regulated by estrogen, which is the basis for 6- to 8-fold seasonal variations in enzyme protein and messenger RNA (mRNA) (6, 11, 12). cyp19a/P450aromA is expressed in ovary where enzyme levels and mRNA are relatively low (6, 10). If neuroestrogen functions as a neurotrophic factor, we reasoned that the neural P450aromB isoform would be expressed in early stages of CNS development when the rate of neuronal proliferation and differentiation are high. Although taxonomically related to the goldfish, the zebrafish (Danio rerio) has additional advantages as a developmental and genetic model system, and the morphology and regulation of the developing CNS has been especially well documented (13). As a first step in defining a role for neuroestrogen in brain development, we have isolated and characterized complementary DNAs (cDNAs) encoding the zebrafish orthologs of P450aromB and P450aromA; determined patterns of expression by tissue-type in adult fish and at defined developmental stages in embryos and larvae; and demonstrated that an estrogen response system, as measured by up-regulation of P450aromB (but not P450aromA mRNA), is functional as early as 24 h postfertilization (hpf).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sample collection and RNA extraction
Adult fish were killed by decapitation after anesthetizing in
0.02% 2-phenoxyethanol. For each RNA preparation, brain, eye, liver,
and muscle were pooled from ten adult fish of mixed sex; ovaries and
testes were pooled separately. Unfertilized eggs were obtained by
manually stripping gravid females and approximately 200 used for each
RNA extract. Embryos and larvae were killed by quick-freezing on dry
ice. For each RNA preparation, 200 embryos were collected at 1.5–2, 6,
and 12 hpf, 50 embryos at 24 and 48 hpf, 30 embryos at 72 hpf, 25
larvae at 96 hpf, and 20 larvae at 120 hpf and subsequent time points.
The Totally RNA kit (Ambion, Inc. Austin, TX) was used for
adult tissues and RNAqueous kit (Ambion, Inc.) for eggs,
embryos and larvae. Extracts from 12 hpf or older were treated with
DNase I (Promega Corp., Madison, WI).
Oligonucleotides
Oligonucleotides (Ransom Hill Bioscience, Ramona, CA) used as
PCR primers (nos. 1–10, 13, 14) and hybridization probes (nos. 11, 12,
15) are listed below. Nucleotide (nt) numbering is based on our
reported GenBank sequences (accession numbers AF226619 and AF226620 for
P450aromB and A, respectively) and AF025305 for zebrafishß-actin.
Oligonucleotides nos. 1 and 2 were degenerate primers used for both
brain and ovarian cDNAs and designed to target sequences in highly
conserved I-helix and heme-binding regions of previously identified
vertebrate P450arom sequences (10). Oligonucleotides nos.
3–15 were gene specific. IUB group codes were used for degenerate
primers (D = A+G+T; S = G+C; n = A+G+C+T; Y = C+T;
R = A+G; K = G+T; B = G+T+C; V = G+A+C; W =
A+T).
No. 1: nt 959–984 (A), 962–987 (B): 5'-CARTGYRTNYTNGARATGNTNATHGC-3'.
No. 2: nt 1382–1402 (A), 1385–1405 (B): 5'-CACCATNGCDATRWRYTTNCC 3-'.
No. 3: nt 1048–1075: 5'-TGCTGTGGAAGAGCAAATCGTACAGGAG- 3'.
No. 4: nt 1341–1367: 5'-CACAACCGAATGGCTGGAAGTAACGAC-3'.
No. 5: nt 1060–1081: 5'-GCAAATCGTACAGGAGATACAG-3'.
No. 6 nt 1250–1269: 5'-CGTCCAATGTTCAGGATTAG-3'.
No. 7: nt 1045–1062: 5'-AGATGTCGAGTTAAAGATCC-3'.
No. 8: nt 1135–1154: 5'-ACTCGTTGATAAAACTCTCC-3'.
No. 9: nt 1–25: 5'-ATCCGTTCTTATGGCAGGTGATCTG-3'.
No. 10: nt 1561–1585: 5'-TGGCGAATGAGTGTGTGTGGATCAG-3'.
No. 11: nt 1079–1102: 5'AGTGTTTTAGCTGGCCAGAGCCTCT-3'.
No. 12: nt 1217–1241: 5'ATTGATGGCTACCGGGTGGCAAAGG-3'.
No. 13: nt 142–161: 5'-GGTATGGGACAGAAAGACAG-3'.
No. 14: nt 452–471: 5'-AGAGTCCATCACGATACCAG-3'.
No. 15: nt 345–369: 5'-GGCCAACAGGGAAAAGATGACACAG-3'.
RT-PCR cloning of zebrafish P450arom cDNAs
cDNA was synthesized from 5 µg total RNA from adult brain and
ovary using oligo(dT) primers and SuperScript II reverse transcriptase
(Life Technologies, Inc., Gaithersburg, MD) according to
the manufacturer’s instructions. An aliquot (10%) of the first-strand
reaction was amplified with degenerate primers nos. 1 and 2 using a DNA
Thermal Cycler 480 (PE Applied Biosystems, Foster City,
CA). PCR was performed in 50-µl final volume containing 5 µl 10x
reaction buffer, 2 µM MgCl2, 200
µM deoxynucleotide triphosphate, 2 µM of
each primer, and 2.5 U Taq DNA Polymerase (Promega Corp.). The reaction mixture was heated at 94 C for 5 min, then
amplified for the first 5 cycles as follows: 1 min denaturing at 94 C,
2 min annealing at 37 C, and 3 min extension at 72 C. The remaining 30
cycles were carried out similarly except for an annealing temperature
of 50 C instead of 37 C. The amplified products were separated on a 2%
agarose gel, isolated from the gel using GLASSMILK (GENE-CLEAN III kit;
BIO 101, Vista, CA), and an aliquot (20%) of each was used as a
template for reamplification exactly as described above. The
reamplified products were purified and T-A cloned into pGEM-T Easy
vector (Promega Corp.).
The 5' and 3' ends of the brain-derived P450aromB cDNA were obtained by rapid amplification of cDNA ends (RACE) using the Marathon cDNA Amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA), and gene-specific primers nos. 3 (3' RACE) and 4 (5' RACE). The amplification of adaptor-ligated, oligo(dT)-primed double-stranded brain cDNA was done with Advantage cDNA polymerase mix containing a proof-reading polymerase (CLONTECH Laboratories, Inc.) and a touch-down program: denaturing at 94 C for 1 min followed by 5 cycles of 94 C for 30 sec, and 72 C for 4 min, 5 cycles of 94 C for 30 sec, and 70 C for 4 min, and 25 cycles of 94 C for 20 sec, and 68 C for 4 min. During the course of these experiments, an unpublished zebrafish ovary-derived P450arom sequence was deposited in the GenBank database by Bauer and Goetz (accession number AF004521). Because the sequence of our initial P450aromA cDNA fragment had 97% identity when compared with the GenBank sequence, the remaining coding sequence was obtained by end-to-end PCR using primers nos. 9 and 10, which corresponded to nucleotides in the 5' and 3'-untranslated (UTR) regions of the reported sequence. Amplification of double-stranded oligo (dT)-primed ovarian cDNA was performed under the same conditions used to obtain the initial cDNA fragments except that the high fidelity Advantage cDNA polymerase mix (CLONTECH Laboratories, Inc.) was substituted and amplification was done by denaturing at 94 C for 5 min followed by 30 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 4 min.
Sequencing and computer analysis
Sequencing of cDNA was done on both strands of double-stranded
DNA using the BigDye Terminator Cycle Sequencing Ready Reaction
(PE Applied Biosystems) and an automated sequencer (ABI
PRISM 377 DNA Sequencer, PE Applied Biosystems). The
complete sequences were obtained using Sp6, T7, PucM13 forward, and
PucM13 reverse primers (Promega Corp.), which were
complementary to the vector, and sequential internal primers. The
nucleotide and deduced amino acid sequences were analyzed using the WI
Package Version 9.0, Genetics Computer Group (GCG)
(Madison, WI). For phylogenetic analysis the deduced amino acid
sequences of zebrafish brain- and ovary-derived P450arom, together with
P450arom sequences reported for other vertebrate species, were aligned
by CLUSTAL W, version 1.6 (19). The aligned sequences were
used to construct phylogenetic trees by distance (Neighbor-Joining
algorithm) and maximum parsimony criteria PAUP (Phylogenetic Analysis
Using Parsimony, version 4.0, b2) (20). Only regions
without length variations (393 conserved characters of 544 total) were
used to construct phylogenetic trees. Trees were drawn with shark
P450arom (ovary-derived) as an outgroup (Wang, C., M. Betka, and
G. V. Callard, unpublished; GenBank accession number
AF203106). Deduced amino acid sequences also were analyzed by Motif on
the GenomeNet (The Supercomputer Laboratory, The Institute for Chemical
Research, Kyoto University, Kyoto, Japan).
Northern analysis
Total RNAs (20 µg) from adult brain, ovary, liver, and testis
were size-separated on a 1% formaldehyde agarose gel and transferred
to a nylon membrane (BrightStar-Plus; Ambion, Inc.). The
hybridization probe used to detect P450aromB mRNA was a 1.5 kb 5'-RACE
cDNA (nt 1–1367, see Fig. 2
) and random primed with
[
-32P]ATP using the Strip-EZ DNA kit
(Ambion, Inc.) according to the manufacturer’s protocol.
Hybridization was performed using the Northern Max Plus kit
(Ambion, Inc.) at 64 C for 2 h and a probe
concentration of 1 x 106 cpm/ml. The final
stringent wash was 0.1 x SSC, 0.1% SDS before exposure to film
(BioMax MR; Kodak, Rochester, NY). The membrane was
stripped according to the manufacturer’s protocol and rehybridized
with a 247-base P450aromA complementary RNA derived from the
corresponding cDNA fragment (nt 1104–1350). The plasmid was linearized
with SalI, transcribed, and labeled with
[32P]UTP using the Strip-EZ RNA kit
(Ambion, Inc.). The membrane was hybridized in ULTRAhyb
(Ambion, Inc.) containing 1 x
106 cpm/ml overnight at 68 C. The final stringent
wash was 0.1x SSC and 0.1% SDS. To control for RNA loading, the
membrane was rehybridized with a 330-base zebrafish ß-actin
complementary RNA, which was transcribed from a cloned cDNA obtained by
RT-PCR using liver RNA and primers nos. 13 and 14. Transcript sizes
were estimated using an RNA ladder (Life Technologies, Inc.).
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-32P]ATP (DuPont/NEN Life Science Products, Boston, MA) and T4
polynucleotide kinase (Promega Corp.) according to the
manufacturer’s protocol. Hybridization was carried out overnight at 60
C in hybridization solution (5x SSC, 0.1% SDS, and 100 µg/ml
sheared salmon sperm DNA, Ambion, Inc.) containing 1
x 106 cpm/ml radiolabeled oligonucleotide. After
each hybridization, the membrane was washed, 5 min each, in 6x SSC and
6x SSC/0.1% SDS at room temperature and then exposed to film for
3–24 h. Between hybridizations, the membrane was stripped in 0.1x
SSC/0.5% SDS at 95 C. The autoradiographs were scanned using Adobe
Photoshop software, and the integrated density of each amplified
fragment was determined from the digital image using Scion Image
software based on NIH Image, version ß3b (Scion Corp., Frederick,
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Although a second ATG was located
immediately downstream from the first (nt 77), only the first had a
nucleotide context (GAGGTGATGA) similar to the proposed
consensus sequence for initiation (21). A third, in-frame
ATG was located upstream of the first two at nt 41 but was discounted
as an initiation site due to an intervening stop codon at nt 47. The 5'
UTR of the zP450aromB cDNA was 73 bp long, a little shorter than the 5'
UTR of the goldfish P450aromB cDNA (109 bp) obtained by library
screening (12), but much longer than the reported sequence
of the catfish and tilapia P450aromA cDNAs (20 and 33 bp,
respectively) (22, 23). The 3' UTR of the zP450aromB was
2208 bp in length and contained three polyadenylation signals.
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). Similar to the other reported fish ovarian
aromatases (goldfish, 10; medaka, 24; tilapia, 23; trout, 25; catfish,
22), the zebrafish P450aromA had a second potential initiation site
30-bp downstream from the first, but neither of the two ATGs had a
nucleotide context that matched the proposed consensus sequence for
initiation of translation. The zebrafish P450aromB had a shortened N
terminus and an extended carboxy terminus relative to the P450aromA
isoform, features that distinguish the two goldfish P450arom forms
(10, 12).
Sequence comparisons
Figure 1 shows the deduced amino acid sequence of the two
zebrafish aromatases aligned with P450arom forms of representative
other vertebrates: goldfish ovary (10); goldfish brain
(12); medaka ovary (24); tilapia ovary
(23); trout ovary (25); catfish ovary
(22); chicken ovary (26); human placenta
(27). The two zebrafish aromatases shared only 61%
overall sequence identity, but each isoform was 88% identical with the
corresponding B- or A-isoform of goldfish. When compared with catfish,
medaka, tilapia, and trout aromatases (all ovary-derived), the
zebrafish P450aromA was more closely related (64–73% identity) than
P450aromB (58–62% identity). The two zebrafish isoforms had similar
degrees of relatedness to chicken and human aromatases (52–54%), but
the degree of conservation was higher in the putative functional
domains, including the I-helix (65–71% vs. chicken and
human), the aromatase-specific region (83–87% vs. chicken
and human), and the heme-binding region (79–71% vs.
chicken and human), and was lowest at the termini of the different
forms. Based on mutational analysis and molecular modeling, amino acids
known to be essential for catalytic functions in the human P450arom
(I133, E302, P308, D309, T310, R435, C437; for review, Ref.
28) were identical in both zebrafish forms.
Where the zebrafish aromatases overlap, there were a total of 194 amino
acid differences, of which 54% were nonconservative substitutions.
Many of the differences were clustered at the N terminus and in the
central part of the molecule, including regions of putative exons 5, 6,
and 8, and the N terminus of exon 1, indicating that the two
aromatases, like their goldfish counterparts, are more likely to be
derived from different genes than from alternative exon splicing. Also,
sequences within the two isolated cDNAs have been identified by PCR in
zebrafish genomic DNA (29). Some substitutions distinguish
the zebrafish B-isoform from conserved residues in all fish ovarian or
all nonneural forms, including chicken and human (Fig. 1). These were
located in putative functional domains: I-helix (D293E and D294N) and
the aromatase-specific conserved region (L393I) (based on numbering of
the zebrafish P450aromB). Of these, the first two were conservative
substitutions. Also, a histidine residue at position 128 of the human
P450arom, which may be involved in orientation of the substrate in the
active site (28), corresponded to a histidine in both
goldfish and zebrafish P450aromA, but a phenylalanine in the two fish
P450aromB forms. Further analysis with the Motif program revealed
consensus sequences that are common to all or most aromatases, and
other sites that are B- or A-isoform specific, an indication that these
sites have possible functional importance (see legend, Fig. 1).
Phylogenetic analysis
Phylogenetic analysis of the full-length zebrafish P450aromB and
P450aromA sequences together with other published full-length P450arom
sequences (including only regions without length variations) yielded
four most parsimonious trees. Figure 2
shows the strict consensus tree of these four trees (CI = 0.863).
P450aromB isoforms from zebrafish and goldfish formed a group (clade)
that branched separately from a second clade that included the
zebrafish and goldfish P450aromA together with all other teleostean
P450arom forms (all ovary-derived). Mammalian and avian P450arom
sequences formed a third distinct group (although no brain-derived
P450arom-encoding cDNAs have been reported to date). The unpublished
shark ovarian P450arom was the designated outgroup. The topology of
this tree was similar to that obtained by distance analysis
(Neighbor-Joining algorithm; not shown) and is consistent with
duplication of an ancestral gene early in the teleostean lineage,
resulting in the fish P450aromB and -A paralogs.
Tissue-specific expression
Northern blot analysis of zebrafish total RNA was carried out to
determine the size and number of P450aromB and P450aromA mRNAs (Fig. 3). A single 4.4-kb transcript was
identified in brain using a 1.36-kb cDNA probe derived from the
isolated P450aromB cDNA, but no signal of any size was detectable in
ovary, testis, or liver. Rehybridization of the membrane with cDNA
probes from different regions of the P450aromA sequence failed to
detect a signal even after long exposures, indicating a lower level of
expression than the P450aromB isoform. However, hybridization with a
P450aromA riboprobe (247 bases in length) followed by a 30-day exposure
revealed a single major transcript of approximately 2.1-kb in ovary. No
P450aromA-specific mRNA was detected in brain, testis, or liver, but
the same probe nonspecifically labeled 28 S ribosomal RNA in all lanes.
Both P450aromB and P450aromA transcripts were larger than the
PCR-derived cDNAs: respectively, 4.4 vs. 3.8 kb and 2.1
vs. 1.6 kb.
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). The two primer sets
generated products of different length when used in the same or
different amplification reactions (210-bp for P450aromB and 110-bp for
P450aromA) but amplification efficiencies of P450arom plasmids were
similar. Likewise, the 32P-labeled
oligonucleotide hybridization probes targeted internal sequences
specific to the P450aromB or P450aromA products. P450aromB mRNA was
highest in the brain, whereas P450aromA mRNA was highest in ovary. Both
P450arom forms were expressed in the eye (which includes retina, a site
of P450arom enzyme and mRNA expression in goldfish; 7, 10). Notably,
faint bands were seen in the ovary lane after hybridization with the
P450aromB probe, and in the brain lane after hybridization with the
P450aromA probe, indicating a low level of overlapping expression.
Neither P450aromB nor P450aromA mRNA was detectable in adult liver,
testis, or muscle
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shows the onset and
temporal pattern of expression of P450aromB and P450aromA in
unfertilized eggs and whole embryos as measured by RT-PCR/Southern
analysis. Developmental profiles are also plotted after scanning, with
the band areas at 1.5 hpf for each cDNA product series designated as
the arbitrary reference point (1.0) (Fig. 5B). Although
RT-PCR/Southern analysis is at best semiquantitative, comparison of
ethidium bromide stained bands and hybridization signals show clear
stage- and isoform-related differences. Both P450aromB and P450aromA
specific mRNAs were detectable in unfertilized eggs and at the
first time point in embryogenesis (1.5 hpf; 16 cell stage), but signal
intensity declined progressively at 6 and 12 hpf. This pattern is
consistent with transfer and degradation of maternally transcribed
mRNA. Despite a lower initial band intensity relative to the A-isoform,
the P450aromB product was less rapidly degraded. A secondary rise in
both isoforms was seen between 12 and 24 hpf, indicative of the onset
of embryonic transcription; thereafter, stage-related patterns
differed. An abrupt and dramatic approximately 11-fold increase in
relative signal intensity of P450aromB mRNA occurred between 24 and 48
hpf, and by 120 hpf (early larval period) the signal was about 25-fold
higher than at 24 hpf. Relative signal intensity of P450aromA mRNA
increased more gradually than that of P450aromB and the difference
between 24 and 120 hpf was 7-fold. Note that the P450aromB product
gives a more intense ethidium bromide signal than P450aromA product
band at each stage of development. Also, although the P450aromA product
was much greater than the B-product immediately after fertilization
(1.5 hpf), consistent with the predominant ovarian A-isoform in adult
fish, P450aromB signal intensity greatly exceeded that of P450aromA in
free swimming larvae (120 hpf), consistent with the high level of
P450aromB expression in adult fish (compare Fig. 5 to Figs. 3 and 4).
In contrast to progressive increases in relative P450arom levels during
the first 120 h of development, the two mRNA species changed
little or declined between 1 and 3 weeks of age (Fig. 6), possibly due to changes in the
proportion of P450arom-expressing and nonexpressing tissues in the
growing larvae. Although results shown are based on total RNA
equivalents (equal loading), developmental profiles were similar (but
even greater in magnitude) if expressed on a per embryo basis (see
legend to Fig. 5).
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, Exp. 1; Fig. 6B, Exp. 2) amplified and advanced the developmentally
programmed increase in P450aromB band intensity when compared with
stage-matched controls. The stimulatory effect of
E2 treatment between 2 and 48 hpf (not shown), or
between 2 and 72 hpf (Fig. 6B), persisted for 2 weeks after washout but
was no longer discernible after 3 weeks. The ER antagonist ICI.164.384
alone decreased P450aromB band intensity and the effect was reversed by
simultaneous addition of E2, implying blockade of
an endogenous estrogen/ER-mediated component of P450aromB expression.
In contrast to P450aromB, signal intensity of P450aromA was lower (Fig. 6, A and B) or unchanged after E2 treatment (Fig. 7, one of three experiments). Note that
the fold-increase in P450aromB band intensity with
E2 exposure in some experiments (e.g.
Fig. 7) was more modest than in other experiments (e.g. Fig. 6), which could be explained by differences in the degree of
developmental synchrony in different cohorts (unpublished
observations).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Present day fish have multiple loci for many single-copy mammalian genes, which is presumed to be the consequence of a genome duplication event in an early fish ancestor. One mechanism for preservation of gene duplicates is the subdivision of ancestral expression domains by complementary degenerative mutations in their regulatory regions (32). In support of this theory, Northern analysis in goldfish (10) and zebrafish shows that expression of P450aromB is restricted to brain, which has high levels of mRNA and enzyme activity, whereas P450aromA is expressed exclusively in ovary which has relatively low mRNA and activity. RT-PCR confirms differential expression in neural and nonneural tissues but also reveals a degree of overlap, which cannot be discounted as an evolutionary remnant. For example, although the B-isoform predominates in both brain and retina, the relative abundance of the A-isoform differs, which could be due to differential regulation of a single cell population or to differences in the relative numbers of isoform-specific cell types. Likewise, although only trace amounts of P450aromB are detected in adult fish ovary, the B isoform is higher relative to the predominant A isoform in unfertilized and newly fertilized eggs.
In diverse animal species, maternally inherited mRNAs and proteins are used to program the earliest stages of development but are degraded by the midblastula transition, allowing genetic control of development to pass to zygotically synthesized transcripts (33). The presence and temporal patterns of P450aromB- and -A mRNAs in unfertilized eggs and embryos (1.5–12 hpf) are consistent with maternal transfer and degradation. A possible source of maternally derived P450arom mRNAs is transcription by maturing or preovulatory oocytes. Although fish granulosa cells have high steady-state levels of P450arom mRNA (A-subtype) (23, 25), localization in oocytes has not been noted, nor has the B-isoform been characterized or used for mRNA analysis in these studies. Aromatase mRNA has been detected in some samples of isolated rat oocytes using a sensitive RT-PCR assay, but results were discounted as being due to contaminating somatic cells (34). Although we cannot rule out the possibility that residual follicle cells account for a fraction of the measured mRNAs in unfertilized zebrafish eggs, which were obtained by stripping, this explanation is unlikely to apply to early embryos, which were collected after natural spawning. An alternative possibility is that mRNAs transcribed in granulosa cells are transferred to oocytes by way of gap junction-like complexes that are present before ovulation or after hCG-induced follicle maturation in several fish species (35). It may be relevant here that a novel P450arom promoter switching event from the ovary-specific promoter II to the brain-specific promoter 1F is associated with HCG-induced ovulation in equine granulosa cells (36). A mechanism of this type, if present in zebrafish, would explain the presence of both A and B isoforms in adult ovary and reinforce the idea that follicle-oocyte transfer accounts for maternally derived aromatase mRNAs in eggs and early embryos. Interestingly, P450aromB degrades more slowly than P450aromA mRNA (1.5 vs. 6 hpf), which may be related to its larger size when compared with the ovarian transcript (4.4. vs. 2.1 kb, respectively). Because differential stabilization of transcripts in specific regions of the cytoplasm is one of several RNA localization mechanisms that play a role in patterning and cell-fate decisions (37), it will be important to determine the spatial arrangement of each P450arom species during the earliest stages of development and to determine whether P450arom transcripts are among the maternal mRNAs that are not specifically blocked from translation (33).
In zebrafish, transcription of the zygotic genome begins at about 3 hpf (38). Shortly thereafter, the onset of transcription of the two cyp19 genes is detectable as an increase in P450arom mRNAs (12–24 hpf; segmentation period). The segmentation period in zebrafish is characterized by major expansion, morphogenesis, and cellular differentiation of the developing CNS: e.g. the neural plate is sculpted into the three major brain divisions and primary neurons are first identifiable (13). At the same time, primordial germ cells complete their migration to their final destination in the lateral mesoderm where they form two clusters ventral to somites 3–5 (39). Consistent with continued expansion and development of the CNS and gonads, P450aromB and P450aromA mRNAs accumulate progressively through embryogenesis and the early larval period (24–120 hpf), but isoform-specific differences in relative abundance and temporal patterns are evident. P450aromB mRNA is characterized by an abrupt and dramatic 11-fold increase between 24 and 48 hpf and continues accumulating to a level about 15-fold higher by 120 hpf. The rise in P450aromA mRNA is more gradual, and at 120 hpf is 7-fold higher than at 24 hpf. We have not yet determined whether expression of the two P450arom isoforms in embryos is predominantly neural and gonadal, as in adult zebrafish, or whether there is an extraembryonic origin (e.g. yolk syncitial layer), as in mammals. However, a preliminary study shows that microinjection of fertilized eggs with a green fluorescent protein (GFP) construct driven by the goldfish cyp19b promoter results in labeling of neuron-like cells in the forebrain of 30–48 hpf embryos, a timing that is consistent with RT-PCR of endogenous mRNA (Tchoudakova, A., M. Kishida, E. Wood, and G. V. Callard, unpublished data). No labeling was seen after injection of the cyp19a-driven reporter, which could be due to lower expressed levels. The onset of P450arom mRNA and enzyme is at 6 days post coitus in porcine and equine blastocyts (40, 41). In these species, aromatase is localized mainly in extraembryonic trophectoderm, but a few labeled cells are identifiable in hypoblast, a subembryonic layer of primitive endoderm and the route of migration of primordial germ cells (42). The earliest onset of aromatase mRNA in dissected whole brain or diencephalon is at 14 days gestation in rats (43), 11 days gestation in mice (44), and stage 13 (approximately 10 days at 31 C in ovo) in turtles (45), whereas the earliest measured gonadal aromatase mRNA is at 17 days gestation in mice (46) and 50–55 days post hatching in flounder and trout (47, 48), and at stage 18 (approximately 21 days at 31 C in ovo) in adrenal/kidney/gonadal complex in turtles (45).
In this report, we present evidence that mechanisms regulating high constitutive expression and estrogen mediated up-regulation of P450aromB in the brain of adult fish are already operative in 24–48 hpf zebrafish embryos. In response to estrogen, the increase in B-specific mRNA in zebrafish embryos is 5- to 10-fold, depending on the duration of exposure (1–3 days), which is similar in magnitude to seasonal and estrogen-mediated changes in adult goldfish (12). Surprisingly, the estrogen effect persists in larvae up to 2 weeks after washout, which might be explained by sequestration of administered estrogen in yolk (16). Yolk is reported to contain substantial amounts of maternally derived estrogen and aromatizable androgen (17). Estrogen taken up directly from yolk, or in situ synthesis, would explain why ICI164.384 (an ER antagonist) is able to block a component of the developmentally programmed increase in P450aromB mRNA. Although radiolabeled tracer analysis is not sufficiently sensitive to detect estrogen biosynthesis in 72 hpf zebrafish embryos, addition of testosterone to embryo media between 2 and 48 hpf mimics estrogen-induced up-regulation of P450aromB (Kishida, M., unpublished data), implying that mRNA present in the embryo is translated into functional enzyme. In support of this conclusion, aromatase activity has been detected by tracer analysis in embryos and larvae of steelhead trout (22–30 dpf) (17) and Arctic char (28–62 dpf) (18). Whereas brain aromatase in fish and birds is induced by estrogen, brain aromatase expression in adult rats is up-regulated by androgen (4). It is noteworthy, however, that treatment of perinatal female rats with aromatizable androgen enhances the subsequent expression and androgen responsiveness of aromatase in the adult brain (49).
The early onset of P450arom expression in zebrafish embryos, and
differences in the developmental programming and estrogen
responsiveness of B- and A-isoforms, imply that estrogen probably has
multiple roles in development. It is well established that estrogen
synthesized by the mammalian preimplantation blastocyst, or secreted by
the maternal ovary, is essential for the successful establishment of
pregnancy. Although the adult female reproductive tract is viewed as
the main target of early estrogen action, ER mRNA has been identified
by RT-PCR in mouse oocytes and fertilized eggs and, after a period of
decline, reappears at the blastocyst stage (50). We have
identified three distinct ER subtypes (termed ER, ß and ) in
adult zebrafish liver by PCR cloning, and RT-PCR analysis of embryos
reveals a developmental profile similar to that in the mouse
(Akten, B., M. Kishida, and G. V. Callard, unpublished
data). This is consistent with the onset of estrogen responsiveness, as
measured by up-regulation of P450aromB at 24–48 hpf. Taken together,
these and earlier observations (for review, see Ref. 50)
support the conclusion that the embryo itself is a target of estrogen
action.
Because zebrafish are genetically manipulable and have many advantages for studying development (e.g. transparent embryos, numerous identified mutants), they are an excellent model system for investigating estrogen’s role as a developmental hormone. Moreover, the presence of two cyp19 genes with subdivided expression domains and distinct regulatory mechanisms should facilitate recognition of brain- and gonad-specific processes, even in the earliest stages of embryogenesis, and in future will allow clear cause-and-effect relationships to be established by gene-specific manipulation strategies.
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Acknowledgments |
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Footnotes |
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Received June 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase activities correlate significantly with
seasonal reproductive cycles in goldfish (Carassius
auratus). Endocrinology 122:1349–1356-hydroxylase and aromatase in embryonic cell layers of
elongating porcine blastocysts. Endocrinology 135:2248–2254-hydroxylase/C17–20
lyase, and P450 aromatase in fetal mouse gonads. Endocrinology 135:262–268[Abstract]
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