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Departments of Endocrinology and Reproduction (A.L.L.D., M.J.G.G., P.K., B.K., F.H.J., J.T.J.U., J.A.G., A.P.N.T.) and Internal Medicine (F.H.J.), Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, Rotterdam, The Netherlands 3000; Departments of Pathology (T.R.K., M.M.M.,), Molecular and Cell Biology (T.R.K., M.M.M.), and Molecular and Human Genetics (M.M.M.), Baylor College of Medicine, Houston, Texas 77030; and N.V. Organon, Department of Pharmacology (U.M.R.), Oss 5340 BH, The Netherlands
Address all correspondence and requests for reprints to: Axel P. N. Themmen, Department of Endocrinology and Reproduction, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: themmen{at}endov.fgg.eur.nl
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During the estrous cycle, FSH sensitivity of ovarian follicles seems to be important during the process of cyclic recruitment (5), a process by which some large preantral and small antral follicles are selected to grow to the preovulatory follicle stage, and the nonselected follicles will undergo atresia and disappear through apoptosis of the granulosa cells (6). Experiments have shown that the level of serum FSH is very important in this process. Elevated level of serum FSH at estrus prevents some, but not all, follicles to become atretic so that the former follicles are able to continue growth (7). In this process of selection, the sensitivity of the individual follicle to FSH appears to be of central importance. Studies in bovines show that increased FSH sensitivity could be a result of enhanced FSH receptor expression (8, 9).
The development of ovarian follicles, however, is not regulated by FSH alone. Other intraovarian factors, such as E and growth factors, also are involved. Activins, inhibins, and growth and differentiation factor 9 (GDF9), all members of the TGFß family of growth and differentiation factors, can influence follicular development (10, 11, 12). Using anti-Müllerian hormone-deficient female mice, we showed recently that anti-Müllerian hormone (AMH), another member of the TGF-ß family, inhibits the initiation of primordial follicle growth (13). In the same study, we found that AMH-deficient (AMHKO) female mice have an increased number of growing follicles in spite of a lower serum FSH level, compared with their wild-type littermates. Together with previous studies showing inhibitory effects of AMH on FSH-stimulated actions of cultured granulosa cells (14, 15, 16), this would suggest that AMH inhibits FSH-stimulated follicle growth. This hypothesis was studied using three different experimental approaches.
In an in vitro study, FSH-stimulated growth of preantral follicles was studied in the presence or absence of AMH. Subsequently, immature AMHKO females and their wild-type littermates were treated for several days with GnRH antagonist or with GnRH antagonist in combination with FSH after which follicle growth was examined. Finally, follicle growth was studied in 4-month-old AMH-/FSHß-deficient (FAKO) females, by comparing the follicle population of the FAKO females with the follicle populations in AMHKO, FSHßKO, and wild-type females of the same age.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Exp 1, F1 female offspring (B6CBA) from C57Bl/6J females and CBA/J males from Harlan Winkelmann GmbH (Bohren, Germany) were used to collect 21- to 23-d-old ovaries. The mice were anesthetized with ether, and blood was collected by eye extraction. After clotting, blood was centrifuged for 15 min at 4,000 x g, and serum was collected and stored at -20 C until it was used in the follicle culture medium. The ovaries were used to isolate preantral follicles (17).
For Exp 2, wild-type and AMHKO female mice on a C57Bl/6J background
were generated as described previously (13). Female mice
of 25 d of age were divided into four groups, each group
containing six wild-type and six AMHKO animals. The different treatment
schedules are shown in Fig. 2A
. All injections were given twice daily
at 0900 h and at 2100 h. The animals were killed by
decapitation 12 h after receiving the last injection. Group A was
treated with saline from d 25 to d 28. Group B received during the same
period 200 µg GnRH antagonist per injection (Org 30276; NV
Organon, Oss, The Netherlands). Two additional groups of
mice received GnRH antagonist from d 25 to d 31. In addition to GnRH
antagonist, one of these two groups received saline (group C), and the
other group (group D) received 2.5 IU recombinant human FSH
(Puregon; NV Organon) per injection from d 29
to d 31.
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In Exp 3, female and male mice heterozygous for both FSHß and AMH null allele were used to obtain AMH-/FAKO females, FSHß-deficient (FSHßKO), AMHKO, and wild-type female mice. Male FSHßKO mice that were used in the breedings were originally developed by Kumar et al. (1). FAKO females 4 months of age and their wild-type, FSHßKO and AMHKO littermates were killed by decapitation at 1600 h. Wild-type and AMHKO females were killed on the day of estrus because this day of the cycle can be recognized by female behavior (13). Every group contained five animals.
In Exp 2 and 3, after decapitation ovaries were removed, weighed, and fixed overnight in Bouin’s fluid. For histological examination of the follicle population, the fixed ovaries were embedded in paraffin, and after routine histological procedures, 8-µm sections were mounted on slides and stained with hematoxylin and eosin. In addition, uteri of these mice were also weighed.
Determination of mouse AMH and FSH genotype
The genotype of the mice used in Exp 2 and 3, were determined by
PCR reactions. The AMH genotyping was described previously
(13), but a different method for isolation of genomic DNA
was used (18).
Primers FSHß-FOR (5'-TTCAGCTTTCCCCAGAAGAG-3') and FSHß-REV (5'-CTGCTGACAAAGAGTCTATG-3') were used to determine the presence of the FSHß allele. Primer FSHß-FOR anneals to nucleotide sequence 32–51 located in exon 1 of the FSHß gene (numbering according to GenBank sequence, accession number U12932), whereas primer FSHß-REV anneals to antisense sequence 278–259 located in intron 1 of the FSHß gene, resulting in a PCR product of 247 bp. In animals carrying the FSHß null allele, exons 1 and 2 and most of exon 3 are replaced by the PGK-Hprt expression cassette. Primer HPRT-FOR (5'-CCTGCTGGATTACAT-TAAAGCACT-3') and HPRT-REV (5'GTCAAGGCA-TATCCAACAACAAA-3') were used to determine the presence of the FSHß null allele, i.e. the PGK-hprt expression cassette (1). Primer HPRT-FOR anneals to the nucleotide sequence 318–341, whereas primer HPRT-REV anneals to the antisense sequence 669–646 in the PGK-hprt expression cassette, resulting in a PCR product of 352 bp.
For the PCR reactions, 25 pmol of all primers were used, and the PCR reactions were executed as described previously (18). An annealing temperature of 45 C for the wild-type allele and 55 C for the FSHß null allele was used. The products were electrophoresed on a 1.5% agarose gel.
Ovarian histology and follicle counting
Serial 8-µm sections of the ovaries were used for follicle
counting, which was performed as described previously
(13). In Exp 2, both ovaries were used to determine the
follicle population. With the exception of primordial follicles
(diameter < 20 µm), both nonatretic and atretic follicles were
included in the study. Atresia was defined according to the description
of Osman (6). The follicles were divided into four groups
on the basis of their mean diameter, which was determined by measuring
two perpendicular diameters in the section in which the nucleolus of
the oocyte was present: small preantral follicles (20–170 µm), large
preantral follicles (171–220 µm), small antral follicles (221–310
µm), and large antral follicles (311–370 µm).
In Exp 3 the right ovary of each animal was used for follicle counting because we did not observe a difference in the composition of the follicle populations of the right and left ovaries of the same female at the day of estrus (unpublished observations). In this study, all follicles, both nonatretic and atretic, were counted, including primordial follicles. The follicles were divided into three groups on the basis of their mean diameter (micrometers); primordial follicles (diameter < 20 µm), small follicles (20–310 µm), and large follicles (diameter > 310 µm). The number of atretic oocytes was also determined (13).
AMH preparation
In the follicle culture experiment (Exp 1) recombinant rat AMH
was used. Human embryonic kidney 293 (HEK293) cells were stably
transfected with a cDNA encoding His-tagged rat AMH inserted in the
pRc/CMV expression vector. The AMH cDNA contained an optimized cleavage
site to ensure maximal amounts of cleaved mature AMH (19, 20). HEK293 cells were cultured in DMEM/F-12 (Life Technologies, Inc., Paisley, Scotland, UK) supplemented with
10% FCS, penicillin (400 IU/ml), streptomycin (0.4 mg/ml), and
neomycin G418 (0.4 mg/ml). At a cell confluence of about 80–90%, the
medium was replaced by medium without FCS. After 4 d the medium
was collected.
His-tagged rat AMH was purified from the concentrated medium in a three-step procedure. First, proteins with a molecular mass above 10 kDa (including AMH) were concentrated approximately 35-fold using an Amicon filter system (Amicon/Millipore Corp., Bedford, MA). Next, the concentrated medium was run over a Ni-column (NiNTA Superflow, QIAGEN GmbH, Hilden, Germany) to separate the His-tagged AMH from other proteins; subsequently the His-tagged rat AMH was eluted from the Ni-column by imidazole (Sigma, St. Louis, MO). Finally, imidazole and AMH were separated using size chromatography (PD10 column, Amersham Pharmacia Biotech, Buckinghamshire, UK).
The amount of AMH was measured by ELISA, using the TMB peroxidase ElA substrate kit (Bio-Rad Laboratories, Inc., Hercules, CA). The primary antibody, pentaHis monoclonal antibody (QIAGEN), was used at 100 ng/ml. The secondary antibody, a goat-antimouse IgG peroxidase conjugate (Sigma), was used in a 1:1000 dilution. The amount of AMH was calibrated using the same standard preparation of His-tagged AMH in every ELISA and was expressed in arbitrary units. The concentrated supernatant of nontransfected HEK293 cells was used as control medium.
The presence of AMH in the media used in the experiments was investigated by Western blotting using a primary polyclonal antibody to AMH (C-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in a 1:2000 dilution and a secondary peroxidase-conjugated mouse-antigoat/sheep antibody (Sigma) in a dilution of 1:10000 (data not shown).
The AMH preparations (800 U/ml) were shown to be bioactive using a Müllerian duct regression assay (19) (data not shown).
Follicle collection and follicle culture
For each culture, the ovaries of at least four animals were
collected aseptically and transferred into L-15 Leibovitz
medium (Life Technologies, Inc.) supplemented with 0.3%
BSA (Sigma), 5 µg/ml insulin (Sigma), 2
mM glutamine (Life Technologies, Inc.), 10
µg/ml transferrin (Sigma), 50 µg/ml
L-ascorbic acid (Sigma), and 2 ng/ml selenium
(Sigma) at 37 C. Preantral follicles with a diameter of
135–210 µm were isolated by needle dissection (29-gauge 1/2
in.) and collected in 
MEM medium (Life Technologies, Inc.) supplemented with 0.3% BSA, 5 µg/ml insulin, 2
mM glutamine, 10 µg/ml transferrin, 50 µg/ml
L-ascorbic acid, and 2 ng/ml selenium. Follicles from
different ovaries were pooled in the collection medium and incubated in
a humidified incubator, gassed with 5% CO2 in
air at 37 C.
Isolated follicles with a normal morphological appearance
(i.e., a central spherical oocyte, high density of granulosa
cells, and a theca cell layer enclosing the entire follicle) were
divided into two groups with a diameter of 135–165 µm or 165–210
µm and were subsequently individually cultured in Millicel-CM culture
plate inserts (Millipore Corp.) with 250 µl MEM
culture medium supplemented with 5% immature mouse serum in a
humidified incubator gassed with 5% CO2 in air
at 37 C. Half of the number of follicles was cultured in the presence
of purified rat AMH (800 U/ml). To induce follicle growth, 100 mIU/ml
(10 ng/ml) recombinant human FSH (Puregon; NV
Organon) was added to the culture medium after the first
24 h of culture. Culture medium was exchanged on culture d 1 and
4. The diameter of the follicles was measured on d 0, 1, 4, and 5 using
100x magnification and a calibrated micrometer. In addition, on the
same days, the survival rate of the follicles was checked by evaluation
of degeneration (blackening of the follicle) and bursting (loss of
oocyte). Three individual culture experiments were performed.
Hormone analyses
In Exp 2 serum FSH was determined by RIA using rat FSH as ligand
and antibodies against ovine FSH (21). All results are
expressed in terms of NIDDK rat FSH RP-2. The intraassay variation was
7.2%, and all samples were measured in one assay.
Inhibin A and B were measured using kits purchased from Serotec Limited (Oxford, UK), using the human standards provided with the kits. Suitability of the Serotec assays for measuring serum mouse inhibin A and B dimers was investigated previously (22). The intraassay variation of the inhibin A assay was 9% and of inhibin B assay 15%. All samples were measured in one assay.
Statistical analysis
Results are presented as the mean ± SEM. The
data were evaluated for statistical differences either by one-way
ANOVA, followed by Duncan’s new multiple range test or by independent
samples t test using SPSS, Inc. 9 (SPSS, Inc.,
Chicago, IL) computer software. Serum inhibin A and B levels were
tested by a nonparametric test (Mann-Whitney test). Differences were
considered significant at P 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In both follicle size groups, addition of exogenous AMH caused
inhibition of FSH-stimulated preantral follicle growth in a
time-dependent manner, indicated by the significantly smaller diameter
of follicles cultured in the presence of AMH at d 4 and 5 of culture
(Fig. 1). During the follicle culture,
the increase in diameter was mainly the result of an increase in the
number of granulosa cells (data not shown). The follicles with the
larger size at the start of the culture maintained a larger diameter
during the culture than the follicles with a smaller starting size.
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Exp 2: FSH-stimulated follicle growth in AMHKO females vs.
wild-type females
The effect of AMH on FSH-stimulated follicle growth was also
determined in vivo. Therefore, immature wild-type and AMHKO
female mice were treated with GnRH antagonist to induce low-serum FSH
levels or with GnRH antagonist in combination with FSH to obtain high
levels of serum FSH. The treatment schedule is shown in Fig. 2A
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Serum FSH level
Serum FSH level was measured in groups A, B, and C to confirm the
inhibitory effect of GnRH antagonist treatment on endogenous serum FSH
level and to compare this level between wild-type and AMHKO females.
The serum FSH level of group D could not be determined because the
antibody used in the RIA partially cross-reacts with the exogenous FSH
(23).
As expected, GnRH antagonist treatment resulted in a significantly
reduced serum FSH level in mice of both genotypes (Table 1). Treatment with GnRH antagonist for
7 d (group C) instead of 4 d (group B) did not significantly
lower the serum FSH level further. No significant difference in serum
FSH level was found between wild-type and AMHKO females in the three
treatment groups.
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). Within the treatment groups, a
significant difference between the two genotypes was found only for the
ovarian weight and only when a low serum level of FSH was present
(groups B and C). In these groups the ovarian weight was significantly
higher in AMHKO females, compared with the ovarian weight of their
wild-type littermates.
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. In mice treated with saline from d 25 to d 28 (group A), the follicles reached the large antral stage in both genotypes, and no significant difference was found in the number of large antral follicles between wild-type and AMHKO females. In contrast, the number of large preantral and small antral follicles was significantly higher in AMHKO females than in wild-type females, whereas no significant difference was found between the two genotypes for the number of small preantral follicles. The decrease in serum FSH level in group B resulted in the expected decrease in the number of antral follicles in both genotypes. However, in AMHKO females a large increase in the number of small preantral follicles was found. An extended period of low-serum FSH level (group C) caused a significant decrease in the number of large preantral follicles in wild-type females, but this number was not affected in AMHKO females. Notwithstanding the low serum FSH level, the number of small antral follicles was increased in AMHKO ovaries, but it was not affected in wild-type females. Administration of FSH (group D) resulted in the presence of large antral follicles and in an increase in the number of small antral follicles in both genotypes. However, this increase in follicle number and size was more pronounced in AMHKO mice.
Serum inhibin A and B levels
To determine whether the different treatments affected serum
inhibin A and B levels, serum levels of both hormones were determined
in all treatment groups.
In all groups, serum inhibin B level was higher than the serum inhibin
A level (Table 3). Treatment of the
animals with GnRH antagonist resulted in a decrease of both serum
inhibin A and B levels in both genotypes, although for inhibin B, this
decrease was not significant in AMHKO mice. Treatment with GnRH
antagonist together with FSH resulted in an increase of both
inhibins.
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Exp 3: Comparison of follicle growth in 4-month-old wild-type,
AMHKO and FSHßKO, and FAKO females
The long-term effects of the absence of FSH and/or AMH on the
ovarian follicle population were investigated in vivo in
4-month-old AMHKO, FSHßKO, and FAKO mice.
Ovarian and uterine weight
In FSHßKO mice ovarian weight was significantly decreased, but
ovarian weight was increased in AMHKO mice, compared with wild-type
mice (Fig. 3A). In FAKO females, the
effect of the absence of AMH on ovarian weight was further augmented,
resulting in a slight but significantly larger ovarian weight than in
AMHKO females.
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). The absence of only AMH did not affect
uterine weight.
Ovarian morphology and follicle counts
The ovaries of all genotypes contained primordial, preantral,
small antral follicles, both nonatretic and atretic, and atretic
oocytes (Fig. 4, A–D). As expected, no
large antral follicles were found in FSHßKO and FAKO females (Fig. 4, C and D) because of the lack of FSH in these animals, but in wild-type
and AMHKO females, large follicles were absent (Fig. 4, A and B)
because at estrus these follicles have not yet developed in mice with a
normal estrous cycle. In wild-type and AMHKO females, numerous fresh
and old corpora lutea were present (Fig. 4A). The ovaries of FSHßKO
females were small owing to the lack of corpora lutea but contained
follicles that had progressed to the small antral stage (Fig. 4C). In
contrast, despite the lack of corpora lutea, ovaries of FAKO females
were large and contained many small preantral follicles and small
antral follicles (Fig. 4D). Furthermore, in these ovaries many remnants
of atretic follicles, also referred to as atretic oocytes, were found
(Fig. 4, E and F).
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shows the effects of the absence
of AMH and/or FSH on the number of follicles in the different follicle
classes. Two-fold more nonatretic small follicles were found in the
absence of AMH (AMHKO), and this effect was strongly increased in the
additional absence of FSH (FSHßKO) (Fig. 5B). Absence of FSH alone
did not affect the number of nonatretic small follicles. In all
genotypes the changes in the number of atretic small follicles and
atretic oocytes were similar to the changes in the number of nonatretic
small follicles (Fig. 5, C and D). The increase in the number of small
follicles in AMHKO and FAKO females is reflected by a concomitant
decrease in the number of primordial follicles (Fig. 5A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have studied the effect of AMH on the FSH sensitivity of growing follicles more in detail. In an in vitro follicle culture, we found that AMH inhibits FSH-stimulated growth of mouse preantral follicles. However, in another recent in vitro study (24), it was reported that AMH enhanced FSH-stimulated growth of rat preantral follicles. Two possible explanations for these contradictory results can be put forward. Animal age (prepubertal vs. adult) could be the cause, as was demonstrated by its influence on the effect of activin A and TGFß, like AMH members of the TGFß superfamily, on preantral follicle growth. Although activin A stimulates growth of preantral follicles from immature mice and not from adult mice, TGFß stimulates growth of preantral follicles derived from adult mice and not from immature mice (25, 26). Besides age, the developmental stage (e.g., number of granulosa cell layers) of the follicle can also be of importance for the outcome of follicle culture experiments because we found that growth of preantral follicles with a diameter between 165 and 210 µm derived from immature mice was stimulated by GDF9, but smaller follicles (135–165 µm) were unaffected by GDF9 (our unpublished results). However, because in both our study and the study of McGee et al. (24) preantral follicles with seven to nine layers of granulosa cells from immature animals were used, a difference in age or developmental stage of the follicle cannot readily be used to explain the difference in outcome. The difference, however, could be the result of the use of mouse serum in the culture medium in our study, suggesting that the serum might contain factors influencing the action of AMH.
The inhibitory effect of AMH on preantral follicle growth in vitro is in agreement with previous studies in cultured granulosa cells in which AMH was shown to negatively influence FSH-induced processes, such as a decrease of the aromatase activity and LH receptor number and the inhibition of progesterone synthesis (14, 15, 16). In addition, the result of our in vitro study is in accordance with the result of our in vivo study, in which follicle growth in the presence of high or low serum FSH level was compared between wild-type and AMHKO females. Growth of large preantral and small antral follicles in AMHKO mice is less affected by longtime exposure to low serum FSH level than in wild-type females (group C vs. group B). Furthermore, in the presence of high serum FSH level (group D), stimulation of follicle growth was more pronounced in AMHKO females than in wild-type females both in terms of numbers and of developmental stage. These findings indicate that in the absence of AMH, follicle growth is more sensitive to stimulation by FSH.
Both in vivo studies show that ovarian as well as uterine weight can indicate changes in the follicle population. Although in adult mice the ovarian weight is mainly determined by the mass of corpora lutea, as is clearly illustrated by the 5-fold decrease in weight of the anovulatory ovaries of FSHßKO females, compared with the weight of wild-type ovaries (1) (and present results), a previous study on AMHKO females (13) and this study showed that a high number of preantral follicles can significantly increase ovarian weight. This observation is supported by the ovarian weight of FAKO females, which is even further augmented by the very high number of small follicles. Uterine weight can be informative because the uterine epithelium is strongly stimulated by E2 (27), produced mainly by large antral and preovulatory follicles, although small antral and even preantral follicles are able to produce small amounts of E as well. In both in vivo studies, the changes in uterine weight clearly support the histological observations in the ovaries.
In addition to the ovarian and uterine weights, serum inhibin can also be used to register changes in the ovarian follicle population. Inhibin A level was high in mice having large antral follicles in their ovaries, but inhibin B level was high in mice predominantly having preantral and small antral follicles. These results are in accordance with follicle culture experiments using immature mouse follicles showing that inhibin A is predominantly produced by large antral follicles and inhibin B by preantral and small antral follicles (28).
Going back to the respective roles and interaction of AMH and FSH in the ovary, the results of cross-breeding FSHßKO and AMHKO animals (Exp 3) clearly indicate that AMH is a dominant regulator of early follicle growth. FSH does not appear to be essential in preantral follicle growth because in both AMHKO and FAKO females, less primordial and more growing follicles were found. Indeed, even more growing follicles are found in ovaries of FAKO females, probably as a result of accumulation of these follicles because the absence of FSH causes a block of follicular growth at the large preantral/small antral follicle stage. It is known that FSH is not necessary for preantral follicle growth, but because preantral follicles are sensitive to FSH, one might expect that a complete loss of FSH production in mice would lead to some effects on preantral follicle growth. However, comparison of the follicle population between wild-type and FSHßKO females revealed that preantral follicle growth occurs to the same extent in both genotypes. In addition, no effect was seen in the absence of FSH on the primordial follicle pool. This is in contrast to several studies in which the pool of primordial and growing follicles was influenced by decreasing or increasing gonadotropin levels, either by hypophysectomy in adult mice or unilateral ovariectomy in aged rats (29, 30). The only difference between these experimental animal models and the model used in this study, the FSHßKO mouse model, is that in the present model, only FSH production is absent, but in the other models both FSH and LH and/or other hormones or growth factors are affected. In particular, it has been shown that LH can stimulate primordial follicle recruitment because in mice overexpressing LH increases the outgrowth of primordial follicles (31). Until now, no data are available about other factors from the pituitary gland, which might exert an effect on recruitment of primordial follicles.
The question of by which mechanisms AMH inhibits the stimulatory effect of FSH on follicle growth remains. Unlike other members of the TGFß superfamily, little is known about the AMH signaling pathway. Members of this family signal via complexes containing type I and II receptors. So far only an AMH type II receptor has been identified (32). More insight into the signaling pathway of AMH will be gained after identification of the AMH type I receptor. Only recently the activin receptor-like kinase 2 (33, 34) and 6 (35) have been identified as candidate AMH type I receptors.
The inhibitory mechanism could involve an effect of AMH on FSH receptor expression. A change in the expression of the FSH receptor may change the sensitivity of a follicle to FSH, as was demonstrated by studies in the bovine (8, 9). However, several studies have shown that, besides FSH effects on the ovary, AMH can also inhibit similar effects induced by cAMP, which is the second messenger of FSH (14, 36, 37). This would suggest that the molecular target site of AMH action is downstream of the FSH receptor.
This study provides evidence for an inhibitory role of AMH on FSH-stimulated preantral follicle growth. Regulation of the sensitivity of large preantral follicles to FSH by AMH could be important during cyclic recruitment because FSH is a crucial regulator of this process. A possible role of AMH in cyclic recruitment is indicated by the presence at estrus of a group of nonatretic large preantral/small antral follicles with high AMH mRNA expression and a group with much lower AMH mRNA expression, whereas these follicles were otherwise indistinguishable (38). The increased sensitivity to FSH of the follicles with low AMH mRNA expression may allow these follicles to be selected for continued growth and ovulation in the next estrous cycle. The inhibitory action of AMH on growth stimulation by FSH can also be relevant for small preantral follicles, even though these follicles do not depend on FSH for their growth. Besides factors that stimulate preantral follicle growth, like FSH (3, 4), stem cell factor (39), and GDF9 (40), other inhibitory factors, such as AMH, are important to make preantral follicle growth a well-balanced process.
Taken altogether, ovarian follicle growth is under the influence of many growth regulatory factors, and from the present study, it can be concluded that AMH is one of these regulatory factors. Besides inhibiting the outgrowth of primordial follicles, AMH is also able to inhibit FSH-stimulated follicle growth, by diminishing the sensitivity of the follicle for FSH. Furthermore, AMH proves to be a more dominant regulator of early follicle growth than FSH.
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Acknowledgments |
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Footnotes |
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Received March 16, 2001.
Accepted for publication July 19, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit promotor/simian
virus 40 T-antigen fusion gene: evidence for negative autoregulation of
the inhibin -subunit gene. Mol Endocrinol 10:1667–1677[Abstract]
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T.-H. Lee, C.-H. Liu, C.-C. Huang, Y.-L. Wu, Y.-T. Shih, H.-N. Ho, Y.-S. Yang, and M.-S. Lee Serum anti-mullerian hormone and estradiol levels as predictors of ovarian hyperstimulation syndrome in assisted reproduction technology cycles Hum. Reprod., January 1, 2008; 23(1): 160 - 167. [Abstract] [Full Text] [PDF]
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 F. J Diaz, K. Sugiura, and J. J Eppig Regulation of Pcsk6 Expression During the Preantral to Antral Follicle Transition in Mice: Opposing Roles of FSH and Oocytes Biol Reprod, January 1, 2008; 78(1): 176 - 183. [Abstract] [Full Text] [PDF]
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E. Arbo, D.V. Vetori, M.F. Jimenez, F.M. Freitas, N. Lemos, and J.S. Cunha-Filho Serum anti-mullerian hormone levels and follicular cohort characteristics after pituitary suppression in the late luteal phase with oral contraceptive pills Hum. Reprod., December 1, 2007; 22(12): 3192 - 3196. [Abstract] [Full Text] [PDF]
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S. Catteau-Jonard, P. Pigny, A.-C. Reyss, C. Decanter, E. Poncelet, and D. Dewailly Changes in Serum Anti-Mullerian Hormone Level during Low-Dose Recombinant Follicular-Stimulating Hormone Therapy for Anovulation in Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., November 1, 2007; 92(11): 4138 - 4143. [Abstract] [Full Text] [PDF]
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L. J. Moran, M. Noakes, P. M. Clifton, and R. J. Norman The Use of Anti-Mullerian Hormone in Predicting Menstrual Response after Weight Loss in Overweight Women with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., October 1, 2007; 92(10): 3796 - 3802. [Abstract] [Full Text] [PDF]
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S. M. Nelson, R. W. Yates, and R. Fleming Serum anti-Mullerian hormone and FSH: prediction of live birth and extremes of response in stimulated cycles implications for individualization of therapy Hum. Reprod., September 1, 2007; 22(9): 2414 - 2421. [Abstract] [Full Text] [PDF]
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M. E. Kevenaar, A. P.N. Themmen, F. Rivadeneira, A. G. Uitterlinden, J. S.E. Laven, N. M. van Schoor, P. Lips, H. A.P. Pols, and J. A. Visser A polymorphism in the AMH type II receptor gene is associated with age at menopause in interaction with parity Hum. Reprod., September 1, 2007; 22(9): 2382 - 2388. [Abstract] [Full Text] [PDF]
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S. Tsepelidis, F. Devreker, I. Demeestere, A. Flahaut, Ch. Gervy, and Y. Englert Stable serum levels of anti-Mullerian hormone during the menstrual cycle: a prospective study in normo-ovulatory women Hum. Reprod., July 1, 2007; 22(7): 1837 - 1840. [Abstract] [Full Text] [PDF]
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M. E. Kevenaar, A. P.N. Themmen, J. S.E. Laven, B. Sonntag, S. L. Fong, A. G. Uitterlinden, F. H. de Jong, H. A.P. Pols, M. Simoni, and J. A. Visser Anti-Mullerian hormone and anti-Mullerian hormone type II receptor polymorphisms are associated with follicular phase estradiol levels in normo-ovulatory women Hum. Reprod., June 1, 2007; 22(6): 1547 - 1554. [Abstract] [Full Text] [PDF]
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