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Original Studies |
Departments of Growth and Reproduction (E.R.-D.M., N.J., J.M., N.E.S.) and Pathology (N.G.), Copenhagen University Hospital (Rigshospitalet), DK-2100 Copenhagen, Denmark; and the Department of Biological Research, Biogen (R.C.), Cambridge, Massachusetts 02142
Address all correspondence and requests for reprints to: Ewa Rajpert-De Meyts, M.D., Ph.D., Department of Growth and Reproduction, Section GR 5064, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. E-mail: erm{at}rh.dk
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Abstract |
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Introduction |
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As first demonstrated by studies in mice (11) and recently in sheep (12), AMH expression is switched on when the indifferent gonad begins to differentiate into the testis. In humans, the initiation of AMH transcription was shown by in situ hybridization at approximately 8 weeks gestation (13). In all species examined to date, the production of AMH continues at a high level during the fetal and postnatal periods of testicular development (12, 14, 15, 16). In several species AMH expression rapidly decreases to trace amounts when Sertoli cells reach maturity (16, 17, 18). In humans, a similar decrease in the concentration of circulating MIS is observed at puberty (19, 20, 21). Although the down-regulation of AMH expression correlates with the onset of meiosis in germ cells (11, 22, 23), it is not known whether the presence of meiotic cells is required to switch off AMH expression.
Previous studies of the expression of AMH in the ovary reported that it is switched on soon after birth and concluded that there is no detectable ovarian production during normal fetal development (11, 24). In mature ovaries, AMH is expressed in the subpopulation of granulosa cells in correlation with ovarian cycle and follicular development (25, 26).
To understand better the regulation of AMH expression during fetal, neonatal, and postnatal development, we decided to investigate the AMH expression patterns in the gonads of normal humans and to compare them to the expression profiles in individuals with disorders of sexual differentiation and gonadal development. Specifically, we have addressed the question of how AMH expression is down-regulated in the male gonad by investigating the immunohistochemical expression of AMH in a series of testicular biopsies from adolescent boys with acute lymphoblastic leukemia (ALL), some of whom lacked germ cells because of treatment. In addition, we examined AMH expression in biopsies from young subjects with various abnormalities of gonadal differentiation and spermatogenesis, in particular those with germ cell aplasia (manifested as the Sertoli cell-only pattern), spermatogenic arrest, or mixed patterns with the simultaneous presence of normal meiosis and lack of it in the same testis. We also examined the expression patterns of AMH within human fetal ovarian specimens, which have not been reported previously.
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Materials and Methods |
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Tissue samples used in this study were from tissue archives of
the Department of Growth and Reproduction and the Departments of
Pathology of Copenhagen University Hospital and Herlev University
Hospital. Normal fetal specimens were isolated from induced or
spontaneous abortions and stillborn fetuses that did not show any signs
of developmental disorders on autopsy. The developmental age of fetuses
was calculated as the duration of gestation in weeks minus 2 weeks
(from the date of the last menstrual bleeding). The genetic sex of the
early gonads was determined by in situ hybridization with
sex chromosome-specific probes. This material was previously used for
immunohistochemical studies of germ cell tumor markers and was
described in detail previously (27). The study was approved by a local
ethical committee. The fetal gonads from the intersex subjects were
also isolated from aborted tissue. Elective abortions were performed
after genetic counseling when chromosomal aberrations or inherited gene
mutations were found. The gonads were subsequently isolated, fixed, and
evaluated for the presence of preinvasive neoplastic changes (carcinoma
in situ cells). Several perinatal and postnatal specimens of
normal testes and ovaries were isolated from individuals who died
because of perinatal complications, sudden infant death syndrome,
accidents, or other conditions presumed not to affect testicular
development or function. In addition, normal testicular biopsies from
boys with ALL who underwent this procedure to monitor testicular
relapses were included in this study. Most of the postnatal and adult
pathological testicular specimens were obtained as surgical biopsies
performed because of suspicion of carcinoma in situ or due
to azoospermia or severe oligozoospermia during andrological work-up of
infertility. A few samples were obtained at the time of gonadectomy,
as, for example, in cases of the androgen insensitivity syndrome (AIS)
in individuals with the female phenotype. All samples and their
clinical description are listed in Table 1
(normal testicular samples), Table 2 (normal ovaries), and Table 3 (pathological specimens).
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All tissue samples were fixed in Stieve’s fluid, Bouin’s fluid, or Lillie’s fluid (4% buffered formalin), and paraffin embedded. Sections cut at 4 µm on SuperFrost Plus microscopic glass slides were used (Menzel-Gläser, Germany). After dewaxing and rehydration, the sections were pretreated by heating in a microwave oven to unmask the antigen under the following conditions: 1 min at full power (1000 watts) followed by four 5-min treatments at 40% power in 5% urea buffer. After cooling for 15 min at room temperature, the sections were incubated for 30 min in 2% H2O2 in methanol to eliminate the activity of endogenous peroxidases. Subsequently, the sections were coated with diluted nonimmune goat serum (Zymed Laboratories, Inc.) to decrease unspecific staining. The incubation with a primary antibody was carried out overnight at 4 C. The antibody used was a monoclonal mouse antihuman AMH (MAb 10.6) from Biogen (Cambridge, MA). The incubation with second link antibody (biotinylated goat antimouse IgG, Zymed Laboratories, Inc., San Francisco, CA) and the color development (streptavidin-peroxidase complex, followed by acethylcarbazol and H2O2) were performed at room temperature, according to the manufacturer’s instructions. As a negative control, a parallel serial section from each specimen was processed with a dilution buffer substituted for the primary antibody. The positive reaction was manifested as an intense reddish orange staining.
The sections were examined under the light microscope, and the staining was assessed using an arbitrary code: -, no staining; -/+, weak staining visible only in single tubules; +, weak staining overall or strong staining in a small number of cells; +/-, weak staining in limited areas: +/++, majority of tubules weakly stained, but some strong staining present; ++/+, strongly stained tubules prevalent, but some weakly stained also visible; +/++/-, heterogeneous pattern with a mixture of strongly positive, weakly stained, and negative tubules; and ++, strong staining in all tubules in the sample.
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Results |
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The results are summarized in Table 1
. In all positive specimens
the staining was present only in Sertoli cells. Reaction was in most
cases intense and confined to the cytoplasm. The youngest fetal gonad
(8 weeks gestation or 6 weeks of development) did not demonstrate any
AMH expression. The indifferent gonad was not yet fully formed, and
several germ cells could be seen on their migration route toward
genital ridges (not shown). The male sex of this specimen was
determined by in situ hybridization. The expression of AMH
was first seen in the 8.5-week-old specimen (10.5 weeks gestation),
when very early Sertoli cells group around germ cells and sex cords
began to form, but their architecture is still chaotic (Fig. 1A). From that point and thereafter, all
fetal and prepubertal specimens were strongly positive for AMH (Fig. 1, B and C,) with one exception (specimen 17 in Table 1), which was
obtained at autopsy after intrauterine death at 31 weeks gestation.
This sample demonstrated normal testicular histology but rather poor
morphological detail. Therefore, the negative result was probably
caused by a partial degradation of the tissue, although a genuine lack
of expression cannot be excluded. In peripubertal samples, around the
time when the first spermatocytes could be seen, the expression of AMH
was dramatically decreased, even in tubules without the presence of
spermatocytes. In the tubules with spermatocytes or later stages of
spermatogenic maturation, the expression of AMH was uniformly
undetectable. In adjacent tubules in which spermatogenesis has not yet
progressed to the spermatocyte stage, weak AMH staining was seen,
although the staining was never as intense as in prepubertal testes
(Fig. 2A). In general, the AMH expression
correlated with age or morphological appearance of Sertoli cells. In
several patients with ALL, who exhibited an absence of germ cells due
to cytotoxic therapy, there was prolonged expression of AMH. However,
in boys older than 14 yr, the expression of AMH decreased, even in
tubules without germ cells (Fig. 2, B and C). In all specimens from
adult men with normal spermatogenesis up to the stage of late
spermatids, no trace of AMH staining was detected.
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Ovarian samples from early fetal specimens that contained only
primordial and primary follicles did not show expression of AMH
(samples 1–20 in Table 2). Faintly AMH-positive granulosa cells were
first observed in a sample from the 36th week of gestation. In that
specimen, only one secondary follicle with several layers of rounded or
cuboid granulosa cells surrounding a growing oocyte was observed; only
the granulosa cells in that one follicle were AMH positive. In ovarian
samples subsequent to this stage of development, strong AMH positivity
was seen in granulosa cells of secondary, tertiary, and preantral
follicles (Fig. 1D). The expression in larger antral follicles was
variable, which is in agreement with earlier reports in the rat ovary
(28). In samples of adult ovaries, granulosa cells in luteal and
atretic follicles were AMH negative.
In a 14-month-old intersex case with an ovotestis and XX genotype (case
10, Table 3), in which both Sertoli and granulosa cells were present
ipsilaterally, granulosa cells were negative for AMH. This was
consistent with the undifferentiated stage of flat granulosa cells
surrounding primordial follicles, but was in marked contrast to
strongly AMH-positive Sertoli cells in closely adjacent
seminiferous tubules (Fig. 1E).
Expression of AMH in disorders of sexual and gonadal differentiation
There were no marked changes in the expression of AMH in the
Sertoli cells of fetal and infantile testes from subjects with various
disorders of testicular development. It was not possible, however, to
evaluate the timing of the onset of AMH in these disorders, because no
material from 6–15 weeks gestation was available. One 16-week-old
fetal specimen with an XXY karyotype (Table 3, subject 2) demonstrated
a few seminiferous tubules with very low expression of AMH, but an
artifact of staining cannot be excluded. In all other cases, the
expression was strong and not different from that in normal testis.
Lower than expected expression of AMH was noted in a 7-yr-old subject
with adrenogenital syndrome, consistent with his precocious puberty and
very early onset of spermatogenesis (single primary spermatocytes were
observed in the biopsy). In testicular biopsies of two patients with
Klinefelter’s syndrome of the same age (14 yr), no germ cells were
visible. One of them had morphologically immature Sertoli cells, which
were weakly stained for AMH, and the other had completely hyalinized
tubules; thus, no Sertoli cells and no AMH expression could be seen. In
general, in patients older than 14 yr, the expression of AMH was rarely
seen, with the exception of the AIS. Two specimens in our series, from
20- and 22-yr-old phenotypic females with the complete form of AIS
demonstrated prepubertal histology of the testis. Despite the adult
age, Sertoli cells from the younger of the patients were strongly
positive for AMH (Fig. 2E). In several fields, a heterogeneity of
staining was observed, with weaker staining observed in tubules with
greater diameter. The 22-yr-old patient demonstrated very weak AMH
expression, and in large areas the staining was completely absent. It
was consistent with the somewhat larger diameter of tubules; moreover,
at closer examination of several sections, a tubule with single primary
spermatocytes was found (Fig. 2F).
In a 25-yr-old patient with Klinefelter’s syndrome, weak AMH
expression could be seen in only a few tubules with small immature
Sertoli cells, whereas in other tubules with larger and more
differentiated Sertoli cells there was no detectable AMH (Fig. 1F).
This pattern of two types of tubules was previously described in adult
patients with Klinefelter’s syndrome (29). No germ cells were seen in
the biopsy of the patient; however, the presence of partial
spermatogenesis in another area of the testis cannot be excluded.
In a few patients biopsied because of azoospermia, who had either
complete aplasia of germ cells (the so-called Sertoli cell-only
pattern) or an abnormality of spermatogenesis, characterized by lack of
maturation beyond the stage of spermatocytes (the so-called
spermatocytic arrest), weak expression of AMH was detected (Fig. 1D).
The AMH-positive cases included a 38-yr-old man with azoospermia, who
underwent bilateral orchidopexy in early childhood. However, in
patients with severe oligozoospermia and partial preservation of normal
spermatogenesis, AMH expression was not detected. This study included
only a representative group of infertile patients. A larger group was
examined in an unpublished preliminary study (Rajpert-De Meyts, E., and
N. E. Skakkebæk, presented at the Ninth European Testis Workshop,
Geilo, Norway).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It was assumed that the AMH was switched on in the human ovary some time after birth, as is the case in other species, except chicken (10, 11, 33, 34). In our study, the onset of AMH expression in the developing ovary was demonstrated for the first time to occur at the very end of fetal life, at approximately 36–38 weeks gestation. In four cases in our series, who died perinatally (36–43 weeks gestation), AMH was consistently positive in developing follicles that passed the stage of secondary follicles. Primordial and primary follicles were always AMH negative. The expression of AMH in adult ovaries with follicles in various stages of development was variable; in very large mature follicles and atretic follicles AMH expression was again down-regulated to levels undetectable by immunohistochemistry. Interestingly, in the individual with XX hermaphroditism, the ovotestis demonstrated a normal strong expression of AMH in Sertoli cells, whereas closely adjacent immature follicles exhibited a complete absence of AMH. Germ cells in both ovarian and testicular compartments appeared normal, but there was no sign of follicle development beyond the primordial stage. Moreover, despite the strong AMH expression in adjacent cords, the oocytes appeared to be in the meiotic prophase. This observation indicates that AMH is not able to destroy primordial oocytes or block meiotic entry unless the onset of AMH expression in the testicular compartment in this case was delayed and occurred after the oocytes in the ovarian compartment had entered meiosis (35).
In the testis from individuals with various disorders of sexual and gonadal differentiation, AMH expression appeared not to be markedly changed in fetal life, whereas at puberty prolonged expression was noted in some cases. The observation of strong AMH expression in fetal cases with XY/X0 gonadal dysgenesis confirmed the results reported previously by Rey and colleagues (22). The expression of AMH in fetal Sertoli cells in cases of Klinefelter’s syndrome will need to be explored further. In the postpubertal individuals with Klinefelter’s syndrome, AMH expression was, in general, weak, but with clear differences in intensity, which roughly correlated with two stages of differentiation of seminiferous tubules described previously by Skakkebæk (29).
AMH expression in the fetal testis of a case with complete AIS appeared normal at 20 weeks of gestational age. However, it cannot be excluded that in some cases the onset of AMH expression in AIS may be delayed beyond the critical period. Most cases with AIS exhibit normal regression of Müllerian structures; however, a few clinical observations reported a partial lack of regression in some patients with the complete form of AIS (36, 37, 38). The regression of Müllerian ducts takes place at 9–12 weeks gestation (39, 40, 41), during the so-called critical period of sensitivity (42). After this relatively short time window, Müllerian ducts quickly become insensitive to AMH, as was demonstrated using isolated gonadal ridges (43). Apparently, either the lack of functional androgen receptors or an excess of testosterone in AIS may interfere at least in some cases with the proper timing of Müllerian duct regression. The early fetal reproductive system in several mammalian species hardly expresses any androgen receptors (12, 44), suggesting that a direct androgen receptor-mediated effect is unlikely. A temporal asynchrony of Müllerian duct formation was recently proposed as an explanation of the persistence of duct remnants in fetal mice exposed to diethylstilbestrol (45). Both in AIS and after diethylstilbestrol exposure, fetal AMH production appear to be unchanged, but the levels of estrogens are relatively high; thus, it is possible that the estrogens, rather than androgens, may be responsible for the aberrant Müllerian regression in both conditions (36).
Several previous investigations in children with AIS and precocious puberty demonstrated an inverse relationship between serum AMH and testosterone levels (20, 46). This was supported by the high and prolonged expression of AMH in adolescent and adult subjects with AIS (46). It was initially suggested that the decrease in AMH secretion is mediated directly by androgens via the androgen receptor of the Sertoli cells. However, elegant studies in animal models of androgen-insensitive Tfm mice and XXSxrb mice, in which germ cells degenerate before meiosis, have demonstrated that the production of AMH at puberty is probably inhibited by a synergistic effect of androgens and meiotic entry (23). Our observations in this study are generally in agreement with those reports. The strong AMH expression was seen in all subjects below 14 yr of age, with the exception of a 7-yr-old subject with precocious puberty and onset of meiosis caused by the adrenogenital syndrome. Another example confirming the importance of meiotic entry was the observed difference in the expression of AMH between two XY females with AIS (20 and 22 yr old). A difference in the underlying molecular defect could perhaps further explain the heterogeneity of AMH expression. However, in both cases, the defect in androgen signaling is unknown.
The most interesting results of this study were obtained from investigations of biopsies of adolescent and young adult subjects with either germ cell aplasia or a complete or partial failure of meiosis. Especially illustrative were adolescent boys with acute leukemia. Most cases had normal spermatogenesis, but a few had germ cells eradicated in the course of cytotoxic treatment before they reached the onset of meiosis. The latter had a tendency to retain AMH expression longer than the boys entering spermatogenesis; however, even those eventually ceased to produce AMH. A similar phenomenon was observed in young adult men with azoospermia or severe oligospermia. Among azoospermic patients, a weak expression of AMH was retained in approximately 30–40% of cases (Rajpert-De Meyts, E., and N. E. Skakkebæk, unpublished observations). This is in agreement with a study of infertile adult men published while our investigations were in progress, which showed that AMH expression was retained in seminiferous tubules with the Sertoli cell-only pattern (47). However, in our study, we could not detect AMH in the oligospermic patients with mixed histological pattern who had tubules with the Sertoli cell-only pattern adjacent to normal tubules with visible spermatozoa. Moreover, a majority of the subjects who probably never experienced meiosis, did not demonstrate detectable expression of AMH. These observations clearly suggest that the down-regulation of AMH expression in the testis can occur without the presence of meiotic spermatocytes. Thus, testosterone and meiotic entry are only two elements in the regulatory puzzle of AMH expression, which remains to be solved. We conclude, therefore, that the decrease in testicular AMH expression at puberty reflects the terminal maturation of Sertoli cells, which in most cases follow their internal differentiation program.
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Acknowledgments |
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Footnotes |
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Received April 14, 1999.
Revised June 22, 1999.
Accepted June 29, 1999.
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