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Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 3N5
Address all correspondence and requests for reprints to: Dr. Nancy Sherwood, Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 3N5. E-mail: nsherwoo{at}uvic.ca
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Abstract |
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A downstream promoter is used in the brain and gonads by salmon GnRH messenger RNA1 (mRNA1) and mRNA2, but mRNA2 also uses an upstream promoter only in the gonads. Two types of long mRNA2 transcripts in ovary and testis both use an alternative start site at position -323; one of these types also retains intron 1. This long 5'-untranslated region is a likely site for distinct regulation of mRNA in the gonad. Additional evidence for separate regulation is that a different expression pattern exists in brain and gonads for GnRH mRNAs during development and maturation. Gene duplication did not alter the encoded peptide, but changed the expression pattern and resulted in complete divergence of the promoter sequence from position -215. A comparison of the mammalian and trout GnRH genes reveals that the promoters are without sequence identity except for a few consensus sites in both regulatory regions. The duplicated trout genes provide a model to study a critical gene whose product controls reproduction in all vertebrates.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies show that GnRH is not made exclusively in neurons. The isolation and localization of GnRH and its receptor in various reproductive tissues support an extrapituitary autocrine/paracrine role for GnRH in reproduction. For example, mammalian GnRH (mGnRH) complementary DNA (cDNA) has been isolated or detected in the primate ovary and testis (2, 3) as well as placenta (3, 4). In situ hybridization studies in females localize mGnRH and its receptor primarily in granulosa cells (5, 6). In males, GnRH is present in Sertoli cells, and its receptor is found in Leydig cells (7).
GnRH is first expressed in the developing mouse brain at approximately 10 days after fertilization (8) and in the salmonid brain at 17 days after fertilization (9, 10). GnRH is then continuously expressed in the brain throughout the life of the animal (11, 12). For the ovary, the pattern of expression of GnRH is reported for the rat; GnRH is first observed at 18.5 days postcoitum, and the GnRH receptor mRNA is detected at 15.5 days postcoitum (13). Both receptor and GnRH mRNA are expressed in primary, secondary, and tertiary follicles in the ovary (5, 6). In the testis, the mRNA for both GnRH and its receptor are detected at 14.5 days postcoitum (13).
Data are not available currently on the transcription factors involved in activation of the GnRH gene in the gonads. However, one area of regulatory control at the posttranscriptional level could be the extended 5'-untranslated region present in the GnRH mRNA isolated from these tissues. An upstream promoter is used in GnRH mRNAs transcribed in the ovary and testis of human (2) and monkey (3), but not rodents (3).
Although GnRH has been demonstrated in vitro to have inhibitory and stimulatory influences on the developing and maturing ovary (14) and testis (15), the precise role of GnRH in the gonads is still not clear. To determine whether the processes governing GnRH regulation and expression are conserved in vertebrates, we have taken an evolutionary approach by studying GnRH in rainbow trout. Rainbow trout are an unusual example of the salmonid group because they mature early at 3 yr of age, spawn every year for a number of years, and spend their lives exclusively in fresh water. However, like other salmonids, rainbow trout are tetraploid (16); this means that comparison and analysis of the 5'-flanking regions of the two genes that encode salmon GnRH (sGnRH; gene1 and gene2) can be used to understand the changes that have occurred since duplication in the coding and regulatory regions of the two genes. In addition, this study investigates whether different splicing strategies are used by the two genes in the ovary and testis compared with those in the brain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation of RNA from ovary and testis
Between 5 months and 2 yr of age, fish were quickly killed by
decapitation. Ovarian or testicular tissue was removed, placed on dry
ice, and stored at -80 C. Total RNA was extracted in Trizol as
recommended by the manufacturer (BRL, Bethesda, MD).
cDNA synthesis and RT-PCR
First strand cDNA was synthesized from 2–5 µg total RNA from
tissue extracts using Superscript RT ribonuclease (RNase)
H- reverse transcriptase by methods recommended by the
manufacturer (Life Technologies, Inc., Gaithersburg, MD).
The RT reaction product (0.5–1.0 µl) was diluted to a final 50-µl
volume containing 1 x Promega Corp. buffer (50.0
mM KCl, 10.0 mM Tris-HCl, 1.5 mM
MgCl2, and 0.1% Triton X-100), 200 mM dNTPs,
2.5 U Taq DNA polymerase (Promega Corp.,
Madison, WI), and 30 pmol sense or antisense strand primers.
Amplification of cDNA was achieved with 40 cycles of 1 min at 94 C, 1
min at 50 C, and 1 min at 72 C using the sense strand primers A
(5'-GAAGCTTATGCACTAAGCAGG-3'), B (5'-TAGGAAGGAATACACAGAACGG-3'), F
(5'-AGGACATTTCTAAGTGACC-3'), and G (5'-CTACACTGTATTTCTGATC-3') in
combinations with antisense strand primers C
(5'-TTATTTATGGGGCATCCATTTC-3') and D (5'-CCAGGTAGCCAGCCATACGA-3'; as
shown in Fig. 1A
). The design of these
primers was based on the sockeye salmon sGnRH gene2 that we sequenced
earlier (17).
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The synthesis and amplification of sGnRH cDNA1 were achieved using RNA
derived from 1.5-yr-old rainbow trout ovary by the methods described
above (accession no. AF110992). The sense strand primer S
(5'-AGGAATAGACCGAACGGAC-3') was complementary to exon 1 (positions
12–30) in the rainbow trout sGnRH gene1 described below (Fig. 1A
). The
antisense strand primer T (5'-TTGAATGCTCCATCATCGC-3') was designed
against a consensus region for the 3'-UTR of both sockeye salmon (18)
and masou salmon (19) sGnRH cDNA1. The 3'-UTR region was used, as it is
distinct from its counterpart in the sGnRH gene2. Sense strand primer J
(5'-GGAGAAGGGATTCTAATCC-3') is complementary to a region located in
position -82 to -64 in the sGnRH gene1. The integrity of the cDNA for
each examined tissue was confirmed by separate PCRs using primers that
were specific for a salmon tubulin cDNA clone (bases 523–545 and
719–740) (20).
The PCR products were separated on a 1.5% agarose gel containing ethidium bromide and were retrieved by electroelution in dialysis tubing. The retrieved DNA was subcloned into pGEM-T vector (Promega Corp.), and recombinant plasmids were sequenced on both strands by the chain termination method (Sanger) using Sequenase version 2 kit (U.S. Biochemical Corp., Cleveland, OH) or Circumvent Thermal Cycle sequencing kit (New England Biolabs, Inc., Beverly, MA).
Primer extension
Rainbow trout ovaries, testes and brains were excised and
quickly frozen. Total RNA was isolated as discussed above. An
oligonucleotide, primer E (5'-TCTCCGTTCTGTGTATTCC-3'), was used in each
RT reaction for each tissue (Fig. 1A). The primer (500 ng) was 5'-end
labeled with [
-32P]ATP (DuPont NEN,
Boston, MA) by T4 polynucleotide kinase and purified to a specific
activity of 3 x 108 cpm/µg. A mixture of 3.0 ng
labeled primer and 20 µg ovarian, testicular, or brain total RNA from
rainbow trout was combined with 1 x first strand buffer
and heated to 90 C for 5 min. After primer hybridization at 50 C for
4.5 h in the presence of 50 U RNAguard RNase inhibitor (Pharmacia,
Dorval, Canada), the annealed primer was extended upon addition of 10
mM dithiothreitol, 0.5 mM of each deoxy-NTP,
and 4 U Superscript II reverse transcriptase (BRL) when incubated at 45
C for 2 h. The reaction was stopped by the addition of 1.0 µl
0.5 M EDTA (pH 8.0) and 1.0 µl deoxyribonuclease
(DNase)-free RNase (10 mg/ml). The mixture was extracted once with
phenol/chloroform and precipitated with 2.5 mM ammonium
acetate and 2.5 vol ethanol. The purified extended products were
dissolved in 4 µl Tris-EDTA buffer (pH 8.0) and 4 µl
sequencing buffer, then electrophoresed on a 6% polyacrylamide-8
M urea gel in parallel with sequencing products generated
from extension of primer E from a complementary site within a subclone
containing a 2833-bp fragment of sGnRH gene2 (17). The gel was dried
and exposed to Kodak Biomax film (Eastman Kodak Co.,
Rochester, NY) at -80 C for 24 h.
Characterization of the promoter regions for the trout sGnRH gene1
and gene2
Genomic DNA isolated from rainbow trout ovary was amplified by
PCR using a sense strand primer complementary to a 5'-flanking region
of the sockeye salmon sGnRH gene2 (position -583 to -565;
5'-GCACTCAAGCATCTTGTTCC-3') and primer D. Gel fractionation yielded two
amplification products of 1257 and 920 bp in length. Sequence analysis
revealed that the largest fragment represented the promoter for the
rainbow trout sGnRH gene1 (accession no. AF110533), and the shorter
fragment represented a portion of the promoter for the rainbow trout
sGnRH gene2 (accession no. AF110993).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Comparison of coding regions of cDNA1 and cDNA2
Amplification of sGnRH cDNA1 was achieved using primers designed
specifically to the 5'-untranslated region (UTR) and 3'-UTR to
differentiate the sGnRH cDNA1 from its sGnRH cDNA2 counterpart in both
gonads (Figs. 1 and 2). It can be deduced
that the two transcripts are each translated into preproprotein, which
includes the signal peptide, GnRH, and GnRH-associated peptide (exons
2–4). The preproprotein translated from the sGnRH mRNA2 differs by 15
aa in comparison to the sGnRH mRNA1 (Fig. 2). Although two nucleotide
(nt) changes occur in the region that encodes GnRH (aa codons 2 and 6),
the processed hormone is identical when translated from each template.
Thus, the 15-aa changes are restricted to the signal peptides (6-aa
changes) and the GnRH-associated peptide (9-aa changes).
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). In PCR analysis of
first strand cDNA made from ovarian or testicular total RNA using
primers A/C, we observed PCR products that were 629, 467, and 440 bp in
length (Fig. 3A) after gel fractionation.
PCRs with primers F/D resulted in products of 567, 405, and 378 bp in
length (Fig. 3B). Subsequent cloning and sequencing of the
A/C-amplified PCR products revealed that each amplification product
contained sequences upstream from exon 1 and that the larger
transcripts retained intron 1 (but not intron 2 or 3; Fig. 1B and Fig. 3, A and B). Primers B/C were used to identify the brain transcripts
(Fig. 3C). That the use of the upstream start site is specific for the
ovary and testis is shown in Fig. 3, A and B, by the lack of observable
bands for the brain with the A/C and F/D primer sets, but the
amplification of a PCR product in Fig. 3C with primer set B/C at
286 bp.
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). Third, the
smaller 440-bp product from the ovary (Fig. 3A) was isolated, cloned,
and sequenced to confirm that introns 1–3 were not retained and that
the start site originated upstream from that shown for the brain.
Modifying the PCR parameters failed to yield the larger intron
1-containing transcript observed for the rainbow trout testis (Fig. 3A). However, using the sense primer F in conjunction with antisense
primer D in PCRs of the same gonadal cDNA, we found amplification of
two bands; one (567 bp) retained intron 1, but the other (378 bp) did
not (Fig. 3B). These results were obtained even after DNase I treatment
of total RNA derived from rainbow trout ovary or testis before the
RT-PCRs were initiated. However, it should be noted that the ovary did
not consistently provide both products, but, rather, the larger or
smaller fragment alone, when primers F/D were used for PCR.
Definitive determination of the start site in the rainbow trout ovary
and testis by primer extension analysis (see below) permitted the
design of primer G. PCRs performed with primers G/D produced bands of
the expected sizes that were cloned and sequenced to confirm that each
transcript was generated from the upstream start site (Figs. 1B and 4). The full-length sGnRH cDNA2 and the
alternative splicing are detailed in Fig. 4. PCR products were not
produced with a sense primer (I) complementary to a region upstream of
the alternative start site (data not shown).
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). The largest extension was confirmed
by PCR and represents a transcript that contains 323 nt of 5'-UTR
upstream from the start site found in the brain transcript (Fig. 5A).
It is not clear whether the primer extension product that migrated to
position -81 indicates a site of termination of an extended sGnRH
cDNA2 because we did not isolate a cDNA of the appropriate size (Fig. 5B).
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). Sense strand primer J is complementary to a
region in the promoter of both gene1 and gene2 immediately downstream
from the potential site of termination at -81 (Fig. 5B). Amplification
products were not generated using A/T or J/T primer sets in separate
RT-PCRs of gonadal tissues of different developmental stages (data not
shown). Furthermore, although the possibility exists that primer E
could bind sGnRH cDNA1 (84% homology), the start site observed in Fig. 5C is specific for rainbow trout sGnRH cDNA2 and not sGnRH cDNA1, which
would be expected to terminate 3 nt further downstream (see below).
Organization and sequence of the promoter region for trout sGnRH
gene1 and gene2
The 5'-flanking region of the rainbow trout sGnRH gene1 is shown
in Fig. 6. The transcriptional start site
for this gene was deduced from the start site for sockeye (18) and
masou (19) salmon sGnRH cDNA1. The 5'-flanking region of the rainbow
trout sGnRH gene2 is shown in Fig. 7. The
promoter of the trout sGnRH gene1 is highly conserved in the 215-bp
proximal to the transcription start site compared with the sGnRH gene2
found in Atlantic salmon (21), sockeye salmon (17), and rainbow trout
(Fig. 8). However, the 400 bp of DNA
beyond -215 (-589 to -215) in the rainbow trout sGnRH gene1 diverge
completely from similar positions in the three other salmonid sGnRH
gene2 promoters that are known (Fig. 8). Thereafter, about 100 bp of
flanking region in the rainbow trout sGnRH gene1 (-708 to -589) has
high sequence identity to the sGnRH gene2 in rainbow trout (-557 to
-432), sockeye salmon (-550 to -425), and Atlantic salmon (-1689 to
-1565). This region of identity is found about 60 bp upstream from a
site in the sockeye salmon gene that appears to have lost 1152 bp of
DNA in comparison with the Atlantic salmon gene (17).
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). The upstream regions of both the
sGnRH and mGnRH genes appear to have consensus sequences that share the
potential to bind POU homeodomain regulators (Oct-1 and Brn-2). Also of
interest is the position of both AP-1 and estrogen regulatory elements
in close proximity to the alternative upstream start site in both the
mammalian (primate) (22) and salmonid (sGnRH gene2) promoters. Finally,
the strong conservation of the salmonid GnRH promoters in the proximal
200 bp might indicate that an element critical to GnRH regulation,
similar to the Sox recognition motif, is shared among these genes.
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). The Inr CAA+1TGT at -325 to -320
resembles the consensus sequences of Py-Py-A+1-N-T/A-Py-Py
or T-C-A+1-G/T-T-T/C for mammalian (26) or
Drosophila (24) genes. Three candidate DPEs that are
homologous to the consensus A/GGA/TCGTG
established for the Drosophila DPE are found in positions
-280 to -274 (GGACAAA), -248 to -242 (GGACATT), and -216 to -210
(GGTAGTG) (25).
In the rainbow trout gene2 between the upstream and downstream start
sites are two nonpalindromic estrogen response elements (EREs) and one
palindromic ERE. This is similar to the sockeye salmon and Atlantic
salmon proximal promoter regions. Also, the upstream start site is
centered between two palindromic EREs that are within 100 bp on each
side (Fig. 7). These two palindromic EREs have 100% identity to EREs
shown to bind human estrogen receptor in the Atlantic salmon GnRH gene
(27), although they lie in a downstream section of DNA in the opposite
orientation in the region examined here (17). Furthermore, sequences in
position -318 to -306 closely resemble a putative ERE found near to
the start site for GnRH expression in human and monkey reproductive
tissues (2, 3). The position of the start site in relation to these
EREs in all three salmonids could point to the involvement of estrogen
receptor in the responsiveness of the upstream promoter in gene2.
Developmental expression of sGnRH cDNA2 in the ovary and testis
Our original intention was to determine whether alternative
promoters are used in the ovary and testis during early development. In
studying the expression pattern, we observed an unusual pattern of
expression during development in the gonads compared with the brain.
Expression of sGnRH cDNA2 was examined extensively. However, cDNA1
expression was also detected in tissue during embryogenesis and at
adult stages (data not shown).
At 5–8 months after fertilization (May to August) the gonads were
immature, yet they expressed GnRH mRNA (Fig. 10A). Once the gonads were further
differentiated in the first year, expression of GnRH was limited to a
short period: September and October in the ovary and only October in
the testis. This coincided with the time of year in which they would
eventually mature and spawn in their third year and thereafter. GnRH
was not expressed in rainbow trout ovary and testis from November of
the first year through May of the second year (Fig. 10, A and B).
Expression of GnRH mRNA was not detected in juvenile tissue at any
stage in the second year except in December (Fig. 10B).
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). GnRH was expressed in each precocious tissue examined in June
through October except in ripe ovaries (August) and in jack testes
(October; Fig. 10C). The testes examined from June through October were
very large compared with their immature counterparts. The ovaries
examined were considered to be jills because they had well defined
features, indicating that they were maturing (visible and
orange-colored eggs, larger gonad size).
The immature gonadal tissue did not use the upstream promoter in June
(year 1), but did use it in August to generate GnRH mRNA2 (data not
shown; Fig. 10A). Both May and July gonadal tissue were examined for
expression of GnRH mRNA only with primer set B/C, which detects use of
the downstream promoter. The upstream promoter was used in all juvenile
fish in September to October (year 1) and again in December (year 2;
Fig. 10, A and B, and Fig. 11). The
same was true for the precocious fish examined in year 2 (Fig. 10C and
Fig. 11).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extended GnRH transcripts in gonads during maturation
In developing gonads, upstream promoters were used in late stages
of immature gonads and throughout the juvenile and precocious stages.
In the ovary and testis, the same promoter that is used in the
brain is used, as well as the more upstream alternative promoter, for
transcription of the sGnRH mRNA2 transcripts. This generates at least
three sGnRH mRNA2s; two transcribed from the upstream promoter (with or
without intron 1) and at least one from the more downstream promoter.
Testes transcripts also have a variation due to an extra 27 nt from the
alternative splice site between intron 1 and exon 2. A similar
requirement for long 5'-UTRs has been demonstrated for mGnRH mRNA in
the reproductive tissue of monkey (3) and human (2). Although the rat
ovary has been shown to express GnRH, neither rat nor mouse appears to
use upstream promoters for transcription of GnRH in reproductive
tissues (2, 3). Also, a comparison of transcript sizes from Northern
analysis indicates that the GnRH mRNA expressed in the gonads of
goldfish (28) and midshipman (29) are not generated from upstream
promoters. It therefore appears that the mechanism for control of GnRH
expression in the ovary and testis of rodents as well as in goldfish
and midshipman differs from that in primates and salmonids.
Function and origin of extended GnRH transcripts in gonads
It has been demonstrated in the mammalian brain that various
neurotransmitters and modulators regulate GnRH levels by modulating
mRNA stability (30). Perhaps the extended 5'-UTRs of sGnRH or mGnRH
transcripts found in the salmonid and mammalian gonads are required by
nonneuronal tissue to similarly modulate GnRH levels. Specific
sequences contained within a variety of mRNAs have been demonstrated to
influence the half-life of the mRNA (for a review, see Ref. 31). The
extended 5'-UTRs characterized here may contain regulatory sequences
that are recognized by stabilization or destabilization factors
associated with RNases in the ovary and testis. Alternatively, the
5'-UTR may inhibit or enhance GnRH translation (through facilitatory
binding proteins) at important periods during development and
maturation. These functional questions need to be addressed to
understand the processes that govern GnRH in the gonad.
To our knowledge, only mGnRH and sGnRH transcripts bearing these unusually long 5'-UTRs have been isolated from reproductive tissue. The finding that both mGnRH and sGnRH genes use two different promoters and generate alternative transcripts provides further support for the hypothesis that the gene encoding sGnRH arose from the ancient mammalian GnRH gene in bony fish (1). We have shown previously that mGnRH of identical structure is present in humans and an ancient bony fish, the sturgeon (32, 33). The salmon form of GnRH (sGnRH) arose as mGnRH disappeared in fish that evolved later than the sturgeon.
Start site and intron splicing in extended gonadal
transcripts
A series of potential AUG translational start sites is present in
the extended 5'-UTRs and in intron 1. However, these potential start
sites resemble neither the functional translational start sites
(CTC/TCCAUGG) nor the Kozak consensus sequence rule
(CCA/GCCAUGG) (34). Furthermore, when read in-frame, the
potential start sequences are always followed by termination codons. It
is doubtful that translation begins at any other site in the extended
transcripts except the same site that is used in the brain.
In human placental or breast tumor cell lines, the major promoter for mGnRH was shown to be the more upstream one (2). The sequences permitting tissue-specific retention of intron 1 may be present in the 5'-end of intron 1, where the fourth nt of the salmonid or the fifth nt of the human (4) GnRH gene is changed from the intron consensus 5'-GTAAGT-3' donor site. These changes may provide some flexibility or recognition by the spliceosome for intron retention or splicing. In rainbow trout testis, the mechanism for selection of the more upstream AG acceptor site (27 nt upstream from the acceptor used in the brain) is clear because both acceptor sites for intron splicing closely match the splice site consensus sequences found in vertebrates [(T/C)11NCAG] (35). Use of this alternative splice site in the 3'-end of intron 1 was also observed in preliminary studies on 1.5-yr-old sockeye salmon (23), and therefore may occur more frequently in salmonids than reported here.
Promoter changes in duplicated GnRH genes encoding identical
peptides
Recent evidence that zebrafish and possibly many ray-finned fish
are tetraploid has been suggested as an explanation for the
considerable diversity seen in fish (36). It is argued that the
duplicated genes may be expressed in different regions and at different
times in development, making them important indicators of new functions
in vertebrates. The rainbow trout genes reported here are an excellent
example of the changes in structure, regulation, and expression that
can occur in two gene copies after a tetraploidization event that
occurred over 27 million yr ago (37).
The promoters of the two genes characterized here have 94% sequence identity within the region proximal to the start site. Whatever alterations have occurred to the promoters after duplication of the ancestral sGnRH gene at the transition from a diploid to tetraploid fish, the proximal 215 bp have been conserved. This core promoter region is presumably important for binding of the basic transcription factors near the start site.
Remarkably, there are no large regions of similar sequences beyond the 215 nt at the start site that are shared between the salmonid GnRH promoters and any of the known mammalian GnRH promoters. However, small consensus sequences that resemble binding motifs for mammalian transcription regulators are present in the sGnRH gene2 of sockeye salmon (17) and rainbow trout. Also, the upstream A/T-rich block of DNA that is conserved among the salmonid sGnRH-encoding genes does possess recognition sequences that may be important for GnRH regulation in both the brain and the gonads. For example, consensus elements are present in each promoter that potentially could bind members of the POU homeodomain family of transcription regulators that are involved in morphogenesis and neurogenesis (38). Each promoter in this conserved region contains two elements that strongly resemble recognition sequences for mammalian Brn-2 (CATnTAAT) and at least one centrally positioned element that could be engaged by Oct-type factors (ATGCAAAT) (39). Importantly, members of this family of regulators have been isolated in both the mammalian brain and gonad (38).
The block of repeating DNA between position -457 to -294 in the sGnRH gene1 contains no apparent consensus sites for factors that direct GnRH transcription in mammals. However, this GC-rich region does contain four repeating blocks of DNA (each 41 bp in length) that each hold a potential ERE half-site. The Atlantic salmon sGnRH gene2 promoter contains six EREs, three that are palindromic and three that are nonpalindromic (17, 27). These TGTCC half-sites were shown to bind human estrogen receptor only if they were part of a complete palindromic element in footprinting assays and gel retardation experiments with the Atlantic salmon sGnRH gene2 (27). However, unlike the sGnRH gene2, complete palindromic EREs are not present in the sGnRH gene1 promoter, indicating that if estrogen is involved in activation of sGnRH gene1 expression in the gonads, it may not be through estrogen receptor binding to the half-site.
Also, the more proximal portions of the rainbow trout and sockeye salmon promoters are highly homologous to the Atlantic salmon sGnRH gene2 promoter (27). This is of some interest considering that the alternative upstream promoter of GnRH in salmonids as well as those in both human (2) and monkey (3) contain nonpalindromic EREs, which were demonstrated by Radovick to stimulate GnRH expression (22).
Regulation of GnRH transcription and function of GnRH in
gonads
Little is currently known about the regulation of GnRH
transcription in the gonad. We speculate that potential EREs in the
salmonid and primate GnRH promoters means that estrogen receptor or
other steroid receptors play a role in the transcription of GnRH in the
ovary and testis. Future analysis using promoter deletion and
electrophoretic mobility shift assays are needed to define the
regulatory factors and the specific recognition motifs that they
bind.
The intermittent expression of GnRH in juvenile gonads during the first 2 yr of life suggests a different function compared with that in the brain, where GnRH is continuously expressed (10, 11, 12). GnRH is expressed in juvenile gonads only in September and October of the first year and in December of the second year. Longer periods of expression are observed in precocious gonads in the second year. In females, there is a strong indication from several physiological studies that GnRH acts as a meiosis-stimulating factor in the oocytes of rat (40) and fish (goldfish and seabream) (41). In male rats, GnRH inhibits LH-stimulated testosterone secretion both in vitro and in vivo, and GnRH receptors are present on the Leydig cells (15). However, in males, the physiological importance of testicular GnRH, which is found in the Sertoli and spermatogenic cells, is not resolved (15). Another possible function of GnRH is related to its role in T cell proliferation (42), gonadotrope differentiation (43), and tunicate gonadogenesis (44). In this light, the expression of GnRH might be needed for growth and differentiation of a new wave of germ cells required by any vertebrate with repeating reproductive cycles, such as rainbow trout, which spawn once a year. The expression of GnRH in immature gonads suggests that the role of GnRH is considerably broader than presently understood.
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Acknowledgments |
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Footnotes |
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Received December 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tubulins from chum salmon
(Oncorhynchus keta). DNA Seq 3:257–262[Medline]
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T. Hamalainen, M. Poutanen, and I. Huhtaniemi Promoter Function of Different Lengths of the Murine Luteinizing Hormone Receptor Gene 5'-Flanking Region in Transfected Gonadal Cells and in Transgenic Mice Endocrinology, June 1, 2001; 142(6): 2427 - 2434. [Abstract] [Full Text] [PDF]
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