(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- 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.
Regression of Müllerian ducts, the primordia
for female genital ducts, is mediated in male embryos by
anti-Müllerian hormone (AMH), ()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.
A second
screening, performed on 1.2 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.
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 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.
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 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).
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- (29) . This suggests that selective pressure for
conservation of the sequence is much lower for AMH 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- 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89248[GenBank].