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(Received for publication, October 19, 1995) From the
Müllerian duct regression in male embryos is
due to early production by fetal Sertoli cells of
anti-Müllerian hormone, a homodimeric protein of
the transforming growth factor-
Regression of Müllerian ducts, the primordia
for female genital ducts, is mediated in male embryos by
anti-Müllerian hormone (AMH), (
A second
screening, performed on 1.2
Figure 1:
Nucleotide
sequence of the chick AMH gene and predicted amino acid sequence. The
nucleotide sequence is displayed from the 5` to the 3` direction, with
1 corresponding to the main initiation site for transcription and 4200
to the site of polyadenylation. The coding regions are shown in uppercase and the untranslated regions in lowercase.
The polyadenylation signal is underlined. Nucleotide
variations between clones are indicated above the nucleotide sequence
and amino acid changes under the protein sequence. The site of signal
peptide cleavage, indicated by an arrow, was predicted
according to von Heijne(25) .
The 4200 bp of the ckAMH gene are organized in five exons (Fig. 1); the position of the introns, relative to the coding
sequence, is conserved by comparison with mammalian genes (Fig. 2). The complete cDNA contains 2834 nucleotides. As all
AMH cDNAs, ckAMH cDNA is rich in G and C. The overall GC content is
62.5%, rising to 68.5% in the part coding for the protein C-terminal
domain. As shown in Fig. 1, ten punctual nucleotide variations
were observed in cDNA, often in several independent clones. Three out
of the nine variations located in the coding sequence lead to amino
acid changes.
Figure 2:
Comparison of chick and mammalian AMH
sequences. Alignment of chick AMH protein sequence with human (45) , rat(46) , mouse(5) , and bovine (45) AMH sequences was performed using Clustal W (1.5) software
with default parameters(47) , and optimized manually. Numbering
of the amino acid residues relates to ckAMH. The position of the
introns is indicated by open triangles. Amino acids shared by
at least three proteins are shaded. Amino acids identical in
the five proteins are underlined. Conservative substitutions,
A=G=P= S=T; D=E=N=Q;
F=Y=W; H=R=K;
I=L=M=V(48) , are indicated by an asterisk. Cysteines conserved in all five proteins are
indicated by a closed circle, and the cysteine absent only in
mouse is indicated by an open circle. Potential N-glycosylation sites are marked by boxes. Mature
protein N terminus, known in human and bovine AMHs, is shown by a small arrow. Position of the cleavage site involved in
proteolytic processing of the hormones is indicated by a large
arrowhead.
A cleavage site for the signal sequence is
predicted between Ala-20 and Leu-21(25) . A consensus sequence
for monobasic cleavage(26) , 106 amino acids upstream from the
C terminus, is identical to the proteolytic site where full-length
human AMH dimers are cleaved into N- and C-terminal
domains(3) . Chick AMH has four potential N-linked
glycosylation sites. The glycosylation site common to the four
mammalian AMHs and effectively glycosylated in human and bovine
proteins(27) , Asn-416, is also conserved in ckAMH. Another
potential glycosylation site, Asn-537, just precedes the site of
cleavage between N- and C-terminal domains and is not found in
mammalian proteins.
Figure 3:
Southern blot hybridization of chick,
turtle, and human DNAs with a ckAMH cDNA probe. Genomic DNA from turtle (T), chick (C), and human (H), digested by HindIII or BamHI, was hybridized to a full-length
ckAMH cDNA
Northern blot analysis by hybridization of
gonadal RNA with a probe corresponding to parts of exons 4 and 5 is
shown on Fig. 4. Hybridization was repeated with a probe
corresponding to exons 2 and 3 with identical results (not shown). No
hybridization was observed with heart tissues. The main band
corresponds to an mRNA species of about 2.8 kb. This size implies the
existence of a very short poly(A) tail, since cDNA is already 2,834 bp
long. Two more slowly migrating minor bands, at 4.5 and 6.5 kb, have
intensities always correlated with that of the 2.8-kb band and may
correspond to aggregates. The size of these bands is too great to be
explained by differences in the length of poly(A) tails, as found for
rat AMH mRNA(28) .
Figure 4:
Expression of ckAMH mRNA in different
tissues, studied by Northern blot hybridization. Top,
hybridization of chick total RNA (20 µg per sample) with a
Chick AMH mRNA expression in the testis
peaks at 10 days of embryonic life and decreases thereafter; only a
relatively small amount is still present in adult life. In female
embryos, ckAMH mRNA is present in both gonads at much lower levels than
in males. Both ovaries express the same amount of transcript between 8
and 10 days, but thereafter levels are higher in the left gonad. The
maximum, reached on both sides at 17 days, is lower than in testicular
tissue at the same age. In the adult hen, the left ovary still
expresses AMH at a moderate level while the vestigial right ovary could
not be studied.
Figure 5:
Expression of AMH mRNA in chick embryo
testes and ovaries, studied by in situ hybridization. 8-day
gonads were fixed with 4% paraformaldehyde, and 17-day gonads were
fixed with Bouin's fluid. Hybridization was performed with an
antisense ckAMH DIG-labeled riboprobe. Alkaline phosphatase reaction
times were 4 h for testes and 14 h for ovaries. All controls with sense
probe were negative. A, 8-day testis (T); B,
17-day testis. At both stages, testicular cords (t.c.) are
labeled, whereas interstitial tissue and mesonephos (M) are
negative. C, detail of 17-day testicular cords. The cytoplasm
of Sertoli cells (s.c.) is labeled; germ cells (g.c.)
are negative. D, 8-day left ovary. Ovary (O) and
mesonephos (M) appear identically negative. E, 17-day
left ovary. AMH mRNA is localized in the dense region between cortex (c) and medulla (m), with clusters of strongly
labeled cells (shown by arrows). F, detail of a
17-day left ovary showing the cortex and two clusters of labeled cells.
The bar represents 25 µm in A, C, D, F and 50 µm in B and E.
Antibodies raised against a partially purified preparation of
chick AMH have allowed us to isolate the cDNA coding for chick AMH.
Although, as shown on Fig. 1and Fig. 2, the gross
structure of the gene and the amino acid sequence of the protein
bioactive domain are conserved between chick and mammals, the chick
gene and protein are longer and diverge significantly from mammalian
ones. Divergences affect essentially the untranslated regions, which
show no homology, and the N terminus (Table 2). The overall low
nucleotide conservation explains the absence of cross-hybridization by
Southern analysis and validates a posteriori the cloning
strategy, favoring expression cloning over hybridization with mammalian
probes. The evolutionary variation of AMH appears much greater than for
TGF- Sex determination mechanisms differ significantly between mammals
and birds. In mammals, the testis-determining gene, SRY(30) , present in the male heterogametic sex,
induces testicular development. In its absence, XX individuals develop
as females. In birds, the female is heterogametic, with males having
two copies of a large Z chromosome and females having one Z and one
smaller W sex chromosome(31) , devoid of any detectable
sex-specific SOX gene(32, 33) . Hormonal
manipulations such as left ovariectomy(34) , testicular
grafts(35) , or administration of aromatase inhibitors (36) result in complete sex reversal and spermatogenesis in
genetic females. These authors suggest that AMH, by inhibiting
aromatase transcription in embryonic gonads, might play a role in
physiological sex determination in birds, a hypothesis that will be
tested as soon as recombinant chick AMH becomes available. Study of
AMH expression sheds some light on the molecular basis of
Müllerian duct development in the chick embryo. In
males, bilateral regression of Müllerian ducts,
between 8 and 13 days, coincides with high expression of AMH by testes
during that period. In females, the right Müllerian
duct and ovary regress, but the left Müllerian duct
develops normally, despite the fact that the adjacent ovary exhibits
some anti-Müllerian activity prior to hatching when
measured using rat fetal Müllerian ducts (8) or ovaries (9) as target organs. At 8 days, the
time at which chick Müllerian ducts are sensitive
to AMH(10, 37) , Northern analysis (Fig. 4)
confirms the expression of AMH by both ovaries, albeit at lower levels
than in testes at similar ages. The surprising maintenance of the left
chick Müllerian duct in the face of early AMH
exposure has been attributed to protection by estrogens produced in
abundance by the chick embryonic ovary (38) and acting through
nuclear estrogen receptors, which are present in higher amounts on the
left side(39) . Estrogen pretreatment of mice (40) or
chick (41) embryos leads to Müllerian duct
insensitivity to AMH. The putative avian AMH receptor differs
physiologically from the mammalian one, since the latter responds to
both avian and mammalian AMHs while chick embryonic reproductive organs
respond only to the homospecific
hormone(9, 37, 42) . Mammalian AMH binds to a
serine-threonine kinase membrane receptor, belonging to the type II
TGF-
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s)
X89248[GenBank].
Volume 271,
Number 9,
Issue of March 1, 1996 pp. 4798-4804
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
superfamily. In mammals, both
female Müllerian ducts develop into the uterus and
Fallopian tubes, whereas in birds, the right oviduct does not develop.
To gain insight into sex differentiation in birds, we have cloned the
cDNA for chick anti-Müllerian hormone using
antibodies raised against the partially purified protein. Expression
cloning was required because of the lack of cross-hybridization between
mammalian and chick anti-Müllerian hormone DNA. The
chick DNA and protein are significantly longer, due to insertions that
abolish nucleotide homology, except in the cDNA coding for the
C-terminal, bioactive part of the protein. Nevertheless, the general
structure of the gene, sequenced from the transcription initiation to
the polyadenylation site, and the main features of the protein are
conserved between the chick and mammals. The chick
anti-Müllerian hormone gene is expressed at high
levels in Sertoli cells of the embryonic testes and in lower amounts in
both ovaries, higher levels being reached on the left side after 10
days of incubation.
)also
called Müllerian inhibiting substance, a member of
the transforming growth factor (TGF-)
family(1, 2) . Mammalian AMHs are homodimers formed of N- and O-glycosylated protein chains linked by
disulfide bonds. After removal of the signal peptide and dimerization,
AMH undergoes an activating peptide cleavage at the target tissue level (3) . The C-terminal fragment is the active moiety, but its
bioactivity is strongly enhanced by the presence of the N-terminal
fragment(4) . In mammals, AMH is synthesized by Sertoli cells
immediately after testicular differentiation, but AMH production by
granulosa cells begins only after birth(5) . Untoward exposure
of fetal mammalian female reproductive organs to AMH results in
Müllerian regression and severe ovarian
lesions(6, 7) . In birds, the situation is somewhat
different. Embryonic gonads of both sexes are endowed with
anti-Müllerian activity(8, 9) ,
but this does not affect the development of the female left
Müllerian duct; only the right one regresses in
female embryos. The role of AMH in avian sex differentiation has not
been investigated in depth because mammalian probes do not recognize
chick AMH and because human AMH, the only recombinant hormone available
at the present time, is inactive in the chicken(10) .
Purification of avian AMH from chick testicular tissue has been
reported some time ago (11) but has not led to further
molecular developments. To obtain tools appropriate for molecular
analysis, we have successfully cloned the chick AMH gene (ckAMH), using
an expression cloning approach.
Obtention of Anti-chick AMH Rabbit
Immunoglobulins
Chick AMH was obtained from 16-day-old White
Leghorn chick embryo testes maintained in organ culture for 3 days. The
hormone was partially purified from the culture medium by affinity
chromatography on a Lens culinaris lectin column (Sigma)
followed by a preparative polyacrylamide gel electrophoresis as
described(9) . Polyacrylamide gel fragments containing the AMH
band were crushed, mixed with adjuvant, and injected thrice at monthly
intervals to a female rabbit sacrificed 1 month after the last
injection. Immunoglobulins were purified by adsorption on a protein
A-Sepharose-4 Fast Flow column (Pharmacia Biotech Inc.).Construction of a
A cDNA library was constructed from 16-day-old chick
embryo testis RNA, according to Young and Davis(12) . Total RNA
was extracted from 120 mg of frozen tissue (approximately 100 testes)
as described(13) . Poly(A)gt11 cDNA Expression
Library
RNA was purified by
oligo(dT)-cellulose binding. cDNA was synthesized by murine Moloney
leukemia virus reverse transcriptase, using 4 µg of
poly(A)
RNA and oligo(dT)
as
first strand primer (Time Saver cDNA synthesis kit, Pharmacia). EcoRI-NotI cohesive-end adaptors were added to
double-stranded cDNA of a size above 1 kb. 67 ng of the resulting cDNA
were ligated to 1.3 µg of dephosphorylated EcoRI-digested
gt11 vector (Promega). Packaging of recombinant
DNA
(Gigapack II Gold packaging extract, Stratagene), infection of Y1090 Escherichia coli bacteria and plating were carried out as
recommended. The resulting library contained 4.5
10
recombinant clones. The amplified library was derived from
1.1
10 recombinant phages.Expression Screening of the cDNA Library
Screening
of the expression library with anti-AMH rabbit polyclonal antibodies
was performed according to a modification of the protocol of de Wet et al.(14) , using Vectastain Elite kit (Vector,
Burlingame, CA). Approximately 6 10
clones from the
nonamplified library were adsorbed onto Y1090 E. coli bacteria, plated at a density of 5 10
plaque-forming units per 150-mm diameter Petri dish, grown at 42
°C for 3 h, and induced with isopropyl--thiogalactoside-soaked
Hybond C nitrocellulose filters (Amersham), at 37 °C for 3 h, as
described(12) . Extensive washing of the filters with 1
Tris-buffered saline (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl) was performed between each incubation step. The membranes
were incubated at room temperature in the blocking solution (1
Tris-buffered saline, 2% glycine, 3% lowfat milk, 0.02% sodium azide)
for 30 min and overnight at 4 °C in a solution of anti-ckAMH rabbit
antibodies diluted 1:1000 (12.8 µg of protein/ml) in 1
Tris-buffered saline, 2% glycine, 3% bovine serum albumin, and 0.02%
sodium azide. After a second incubation in the blocking solution for 15
min and in biotinylated goat anti-rabbit IgG antibodies for 30 min in
the presence of an excess of normal goat serum, the filters were
incubated with avidin-biotinylated peroxidase complex for 30 min.
Peroxidase was revealed by reaction with oxygen peroxide and
4-chloro-1-naphtol (Vector). Positive clones are seen as fast appearing
purple spots or rings on a background of more slowly staining plaques.
Positive clones were further purified by three rounds of replating and
screening.Hybridization Screening with a Polynucleotide
Probe
Recombinant gt11 phages (1.2
10 clones) from the amplified library were adsorbed onto Y1090 E. coli bacteria, plated (3 10 plaque-forming units per 22-cm square plates), and grown for 3.5
h at 42 °C. Replicas of the plates on Hybond-N membranes (Amersham)
were treated successively with 0.5 M NaOH, 1.5 M NaCl
(2 min), 0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl (5 min),
and 3 SSC (5 min) and cross-linked with UV (0.6
kJ/cm
). Screening was performed by hybridization with a
[
-P]dCTP-labeled random hexamer-generated
probe (Megaprime DNA labeling system, Amersham) corresponding to the
306-bp 5`-terminal NcoI digestion fragment of cDNA clone 23
(position 571-1956), overnight at 42 °C in a 6
SSC, 5
Denhardt's solution, 10% polyethylene glycol 6000, 1%
SDS, 50% formamide solution, containing 0.1 mg/ml denatured sonicated
herring sperm DNA. Membranes were washed twice at 65 °C with 2
SSC, 0.1% SDS for 20 min and autoradiographed. A screening of
the positive crude phage suspensions was performed by selective
amplification of ckAMH inserts by PCR, using an antisense ckAMH primer
(primer a6, Table 1) and forward or reverse primers (0.25
µg each). 40 cycles of PCR amplification were performed as
described (15) using the DNA purified from the volume of the
first step phage suspension and 3.35 mM MgCl
.
Lambda clones containing the longest inserts were purified to
homogeneity.
Rapid Amplification of 5`-cDNA Ends (RACE) by
PCR
The 5`-end of ckAMH cDNA was amplified and cloned using a
modification of the RACE procedure of Frohman et
al.(16) . First strand cDNA was synthesized by reverse
transcription of 10 µg of total RNA from 16-day-old chick embryo
testes, using 15 pmol of oligonucleotide a3, located in exon 2, as
ckAMH-specific primer (Table 1) and 800 units of murine Moloney
leukemia virus reverse transcriptase (Life Technologies, Inc.), in the
buffer provided with the enzyme with addition of 10 mM dithiothreitol, 1 mM each of the four dNTPs, and 20 units
of RNasine (Promega), in a volume of 50 µl for 1 h at 42 °C.
The enzyme was inactivated by heating at 95 °C for 10 min. The RNA
was hydrolyzed with RNase A (5 µg) at 37 °C for 20 min and
freed from nucleotides and primers by centrifugation through a
Centricon-100 spin filter (Amicon). The following steps were performed
on half the volume of each sample. For poly(dG) tailing, cDNA was first
denatured at 65 °C for 5 min and placed on ice. Denatured DNA was
incubated at 37 °C for 15 min with 50 units of terminal
deoxyribonucleotide transferase (Boehringer Mannheim) in 0.2 M potassium cacodylate, 25 mM Tris-HCl at pH 6.6, 5 mM dGTP, 0.25 mg/ml bovine serum albumin, and 0.75 mM CoCl (20 µl, final volume). Subsequent
amplification of cDNAs was performed by 40 cycles of PCR as
described(15) , with primer a7 as nested ckAMH primer (50 pmol)
and 20 pmol of a poly(dC) containing anchor primer (Table 1),
using 3.35 or 4.47 mM MgCl. PCR products were
drop-dialyzed on VMWP membranes (Millipore, Marlborough, MA) floating
on distilled water and cloned into pGEM plasmid (TA-cloning system,
Promega). The clones of interest were screened by hybridization with P end-labeled oligonucleotide a8 (Table 1).
Cloning of the Introns
The position of introns was
determined by comparing the size of the products obtained by PCR
amplification of genomic DNA and cDNA. Intron 1 was amplified using
primer pairs s9/a3 or s11/a3 and introns 2-4 using primer pair
s5/a9 (Table 1). PCR fragments were filled in using the Klenow
fragment of E. coli DNA polymerase and cloned into SmaI-digested phosphatase-treated M13 mp18 phage as described
previously(15) .Sequencing
Recombinant gt11 DNA was purified
by the plate method(17) , and both ends of the inserts were
sequenced using CircumVent thermal cycle sequencing kit (New England
Biolabs, Beverly, MA), with 0.9 pmol of
P or
P 5`-labeled forward or reverse
primers (Promega)
and 12 fmol of recombinant
DNA for 25 cycles (95 °C, 30 s; 50
°C, 30 s; 72 °C, 1 min). For complete sequencing, cDNA inserts
were subcloned into Bluescript KSII(+) (Stratagene) at EcoRI or NotI sites. Plasmid DNA was purified using
Wizard minipreps (Promega) or CsCl gradient centrifugation and
sequenced by the dideoxynucleotide method (18) with Sequenase
2.0 (U. S. Biochemical Corp.) using the procedure indicated or the
denaturation method of Hsiao(19) . Whenever possible, at least
two independent overlapping cDNA clones were sequenced in both
directions. Regions of the sequence difficult to elucidate by
double-stranded DNA sequencing were PCR-amplified and subcloned into
phage M13 mp18; at least four clones were sequenced as single-stranded
DNA. Sequences were analyzed on 6% Hydrolink long ranger (Bioprobe
Systems, Montreuil, France), 8 M urea sequencing gels, with
0.6
TBE (54 mM Tris borate, 1.2 mM EDTA) as
running and gel buffers.Southern Blot Analysis
Southern blot analysis was
performed on genomic DNA extracted from chick liver, human blood, and
the blood of the European pond turtle, Emys orbicularis. DNA
(6 µg/lane), digested with 12 units of HindIII or BamHI restriction endonucleases, separated on 0.8% agarose
gel, and transferred to Hybond-N membrane (Amersham) was hybridized, as
described above, with a random-primed P-labeled probe
spanning all the cDNA. The probe was made from equimolecular amounts of
the inserts of a RACE clone (position 1-393) and cDNA clone 81
(position 331-4197). The membrane was washed at 65 °C in 5
SSC, 0.1% SDS for 25 min; 2 SSC, 0.1% SDS for 15 min; and twice
in 1 SSC, 0.1% SDS for 10 min.Northern Blot Analysis
Total RNA was isolated from
frozen chick embryo and adult heart, testis, and ovaries(13) .
RNA samples (20 µg) were analyzed by electrophoresis on 1.2%
agarose, 1% formaldehyde gels and blotted onto Hybond N membranes
(Amersham), as described(20) . Chick AMH cDNA probes were made
with the 306-bp 5`-terminal NcoI fragment of cDNA clone 23
(position 571-1956) or the 821-bp 5`-terminal AccI fragment of
cDNA clone 30 (position 2121-3108) and P labeled by
random hexamer priming. Hybridization was performed as described above.
The membranes were washed at 65 °C for 15 min with successively 1
SSC, 1% SDS; 0.5 SSC, 1% SDS; and 0.2 SSC, 1%
SDS and autoradiographed. The membranes were stripped and rehybridized
with a rabbit ribosomal P 5`-labeled oligonucleotide
probe(21) . A 0.24-9.5-kb RNA ladder (Life Technologies,
Inc.), hybridized with a
P-labeled oligonucleotide, was
used as size marker.
In Situ Hybridization
In situ hybridization with a ckAMH digoxigenin-labeled riboprobe (DIG-RNA)
was performed according to the procedure of Millar et
al.(22) . Testes and ovaries from 8- and 17-day-old chick
embryos were removed, fixed in 4% paraformaldehyde or in Bouin's
fluid (7.7% paraformaldehyde, 3.8% acetic acid (v/v) in saturated
picric acid solution) for 5 h, processed into paraffin wax, and cut at
6 µm. A Bluescript KSII(+) plasmid vector containing the
821-bp 5`-terminal AccI fragment of clone 30 (position
2121-3108) was used to produce the probes. The vector was
linearized by digestion with AccI or EcoRI and
transcribed with T7 or T3 RNA polymerase to synthesize sense or
antisense probes, respectively. Transcription was performed using the
RNA digoxigenin labeling kit (Boehringer) as indicated. The sections
were prehybridized at 55 °C for 2 h and hybridized with sense or
antisense digoxigenin-labeled probes overnight at 55 °C. DIG-RNA
was detected with anti-DIG antibody coupled to alkaline phosphatase
(Boehringer) and revealed by reaction with nitro blue tetrazolium salt,
5-bromo-4-chloro-3-indolyl phosphate (Boehringer), and levamisole
(Sigma) in the dark.
Cloning of Chicken AMH cDNA
18 positive clones
were detected by screening 6 10 independent clones
from a gt11 expression cDNA library of 16-day-old chick embryo
testicular tissue, using rabbit polyclonal antibodies raised against a
partially purified preparation of ckAMH; both extremities of their
inserts were sequenced. Comparison of the encoded protein sequences
with protein data banks, using the BLASTP program, allowed the
identification of one partial ckAMH cDNA clone, clone 13 (spanning
nucleotides 2103-4190). It was used as hybridization probe to
identify two additional ckAMH clones, clones 23 (spanning nucleotides
571-4179) and clone 30 (spanning nucleotides 2121-4200), which
ends with a 21-nucleotide-long poly(A) tail. The other immunologically
positive clones presented no homology with AMH; their selection may be
due to immunological cross-reaction or to contaminants present in the
ckAMH protein preparation used to raise antibodies.
10 clones of the
amplified gt11 library, using a 5`-terminal fragment of clone 23
as hybridization probe, recovered 120 positive clones. Clones 81
(spanning nucleotides 331-4197) and 165 (starting at nucleotide 185)
harbored the longest inserts but still lacked an ATG initiation codon
and a sequence coding for a signal peptide. The 5`-end of ckAMH cDNA
was obtained by the technique of RACE, using total RNA from 16-day-old
chick embryo testis. 47 positive clones were isolated, 19 had inserts
corresponding to a cDNA fragment of about 400 bp, and all other inserts
were smaller.
Nucleotide Sequence of the ckAMH Gene
The complete
nucleotide sequence of the ckAMH gene is shown in Fig. 1. The
sequence of the introns was determined on cloned PCR-amplified genomic
DNA fragments. The 5`-end of the cDNA was determined on clones from two
RACE experiments. The 19 large inserts started in a common region,
while smaller inserts started at various downstream positions. Several
5` termini were found, corresponding to alternative transcription
sites. These can be identified through the presence of a copy of the
cap G, present in some of the reverse-transcribed RACE clones but not
in DNA(23) . The main initiation site is designated A1; 10
clones out of 13 begin with an extra G. Two minor initiation sites were
also detected. One is located at G+2 (2 clones out of 3 have an
extra G). The second putative one is located 2 bases upstream from A1;
in 2 clones A1 is preceded by a GGC sequence and in 1 clone only by GC.
However, because genomic DNA has not been sequenced at that locus, it
is not possible to determine whether the first G represents a copy of
the cap G or whether it is an authentic part of the DNA. Multiple
initiation sites have also been found in human AMH gene(24) ,
rather more distally from the main initiation site than in the chicken
gene.
Predicted Protein Sequence
The open reading frame
encodes a protein of 644 amino acid residues with a calculated
molecular mass of 70,565 Da. Glycosylations are presumably responsible
for the difference with the value determined under denaturating
electrophoresis conditions for the mature protein (Table 2). Fig. 2shows the alignment of ckAMH with various mammalian
sequences. The four mammalian protein sequences are closer to each
other than they are to ckAMH. Overall, chick and mammalian AMH proteins
have 27% amino acid identity. The identity is 49% for the C terminus
and 23% for the N terminus. The homology, taking into account similar
residues, is 63% for the C terminus and 38% for the N terminus (Fig. 2). Despite this relatively low degree of amino acid
conservation, 11 of the 13 cysteine residues of ckAMH are also present
in mammalian AMHs.
Blot Hybridization
Southern blot analysis (Fig. 3) indicates the existence of a single ckAMH gene in the
chicken genome. With human or turtle (Emys orbicularis)
genomic DNAs, the ckAMH probe displayed no hybridization, even at low
stringency, indicating a low degree of homology in the AMH genes
between these species.
P-labeled probe. Exposure was for 2 days at
-70 °C. The ckAMH gene contains no BamHI site, and
only one hybridizable fragment is observed. Two HindIII sites
are present in the gene, at positions 730 and 4074, generating three
hybridizable fragments. The internal 3344-bp fragment corresponds to
the lower band.
P-labeled ckAMH cDNA probe (position 2121-3108).
Film exposure at -70 °C was 5 h for embryonic testes and 70 h
for ovaries, heart, and adult testis. Bottom, stripped blot,
rehybridized with a
P-labeled rabbit ribosomal
oligonucleotide probe (same exposure for all the
samples).
In Situ Hybridization
Using a digoxigenin-labeled
ckAMH riboprobe, chick AMH mRNA was detected by in situ hybridization in the testis at 8 and 17 days of embryonic life.
AMH expression was restricted to the cytoplasm of perigerminal cells,
the future Sertoli cells (Fig. 5). The mRNA was undetectable in
the left ovary at 8 days but was present at 17 days in the compact zone
located between the cortex and the medulla lacunary region, with some
clusters of strongly positive cells (Fig. 5). The right ovary
was negative at all times examined.
(29) . This suggests that selective pressure for
conservation of the sequence is much lower for AMH than for TGF-. receptor family(20, 43, 44) .
Cloning of the chick AMH receptor and study of its interaction with
recombinant chick AMH will be significant steps toward the
understanding of hormone-mediated sex differentiation in the chick.
)
-We thank Franck Louis, Isabelle Lamarre, and
Corinne Belville for technical assistance. We are grateful to Dr. R.
Cate for helpful discussion, to Dr. A. Leroux for competent advice in
RACE cloning, to Dr. Marc Girondot for help in sequence alignments and
the gift of Emys orbicularis DNA, and to Dr. P. Saunders and
M. Millar for helpful advice in DIG in situ hybridization.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 E. Shiraishi, N. Yoshinaga, T. Miura, H. Yokoi, Y. Wakamatsu, S.-I. Abe, and T. Kitano Mullerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes) Endocrinology, April 1, 2008; 149(4): 1813 - 1819. [Abstract] [Full Text] [PDF]
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 P.A Johnson, T.R Kent, M.E Urick, and J.R Giles Expression and Regulation of Anti-Mullerian Hormone in an Oviparous Species, the Hen Biol Reprod, January 1, 2008; 78(1): 13 - 19. [Abstract] [Full Text] [PDF]
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 T. Miura, C. Miura, Y. Konda, and K. Yamauchi Spermatogenesis-preventing substance in Japanese eel Development, January 6, 2002; 129(11): 2689 - 2697. [Abstract] [Full Text] [PDF]
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 J. Teixeira, S. Maheswaran, and P. K. Donahoe Mullerian Inhibiting Substance: An Instructive Developmental Hormone with Diagnostic and Possible Therapeutic Applications Endocr. Rev., October 1, 2001; 22(5): 657 - 674. [Abstract] [Full Text] [PDF]
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M. Dutertre, L. Gouedard, F. Xavier, W.-Q. Long, N. di Clemente, J.-Y. Picard, and R. Rey Ovarian Granulosa Cell Tumors Express a Functional Membrane Receptor for Anti-Mullerian Hormone in Transgenic Mice Endocrinology, September 1, 2001; 142(9): 4040 - 4046. [Abstract] [Full Text] [PDF]
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 K. Watanabe, T. R. Clarke, A. H. Lane, X. Wang, and P. K. Donahoe Endogenous expression of Mullerian inhibiting substance in early postnatal rat Sertoli cells requires multiple steroidogenic factor-1 and GATA-4-binding sites PNAS, February 15, 2000; 97(4): 1624 - 1629. [Abstract] [Full Text] [PDF]
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 P. De Santa Barbara, N. Bonneaud, B. Boizet, M. Desclozeaux, B. Moniot, P. Sudbeck, G. Scherer, F. Poulat, and P. Berta Direct Interaction of SRY-Related Protein SOX9 and Steroidogenic Factor 1 Regulates Transcription of the Human Anti-Mullerian Hormone Gene Mol. Cell. Biol., November 1, 1998; 18(11): 6653 - 6665. [Abstract] [Full Text]
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N. Pilon, R. Behdjani, I. Daneau, J. G. Lussier, and D. W. Silversides Porcine Steroidogenic Factor-1 Gene (pSF-1) Expression and Analysis of Embryonic Pig Gonads during Sexual Differentiation Endocrinology, September 1, 1998; 139(9): 3803 - 3812. [Abstract] [Full Text] [PDF]
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 C. M. HAQQ and P. K. DONAHOE Regulation of Sexual Dimorphism in Mammals Physiol Rev, January 1, 1998; 78(1): 1 - 33. [Abstract] [Full Text] [PDF]
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