7 research outputs found
Ovarian follicle growth and development: role of anti-Müllerian hormone
The central reproductive organ of the female is the ovary. In mammalian species, the
females have two ovaries, which are located in the abdomen near the kidneys. The ovary
has two lnajor functions. First of all, it produces the female gametes or oocytes, which can
develop inside the ovary until they reach the developmental stage at which they can be
fertilised by the male gametes. Second, the ovary produces steroid hormones, whicll are
important for the development of female characteristics and behaviour.
In the ovary the gametes are found in special structures, the so-called ovarian follicles.
Normal developnlent of the ovary and the ovarian follicles is very iInportant for female
fertility. This cl,apter starts with a description of gonad formation in the mammalian
female, on basis of observations on the development in female nlice, and focuses on the
most iInportant factors involved in gonad formation. Fl1l1hernlore, the process of ovarian
follicle development is described, again on basis of data generated mainly from studies
in the mouse, and also rat. Ovarian follicle developnlent is under tight control by luan)
hormones and growth factors, and therefore the action of only the most important regulatory
factors will be mentioned. This thesis focuses on the role of anti-Mullerian hormone
(AM H), one of the ovarian growth factors, in ovarian follicle development
Regulation of ovarian function: the role of anti-Mullerian hormone
Anti-Mullerian hormone (AMH), also known as Mullerian inhibiting
substance, is a member of the transforming growth factor beta superfamily
of growth and differentiation factors. In contrast to other members of the
family, which exert a broad range of functions in multiple tissues, the
principal function of AMH is to induce regression of the Mullerian ducts
during male sex differentiation. However, the patterns of expression of
AMH and its type II receptor in the postnatal ovary indicate that AMH may
play an important role in ovarian folliculogenesis. This review describes
several in vivo and in vitro studies showing that AMH participates in two
critical selection points of follicle development: it inhibits the
recruitment of primordial follicles into the pool of growing follicles and
also decreases the responsiveness of growing follicles to FSH
Control of primordial follicle recruitment by anti-Mullerian hormone in the mouse ovary
The dimeric glycoprotein anti-Mullerian hormone (AMH) is a member of the
transforming growth factor-beta superfamily of growth and differentiation
factors. During male fetal sex differentiation, AMH is produced by Sertoli
cells and induces degeneration of the Mullerian ducts, which form the
anlagen of part of the internal female genital system. In females, AMH is
produced by the ovary, but only postnatally. The function of AMH in the
ovary is, however, still unknown. Female AMH null mice were reported to be
fertile, with normal litter size, but this does not exclude a more subtle
function for ovarian AMH. To investigate the function of AMH in the ovary,
the complete follicle population was determined in AMH null mice, in mice
heterozygous for the AMH null mutation, and in wild-type mice of different
ages: 25 days, 4 months, and 13 months. In the present study we found that
ovaries of 25-day- and 4-month-old AMH null females, compared to those of
wild-type females, contain more preantral and small antral follicles. In
addition, in 4- and 13-month-old AMH null females, smaller numbers of
primordial follicles were found. Actually, in 13-month-old AMH null
females, almost no primordial follicles could be detected, coinciding with
a reduced number of preantral and small antral follicles in these females.
In almost all females heterozygous for the AMH null mutation the number of
follicles fell in between the numbers found in wild-type and AMH null
females. In 4-month-old AMH null females serum inhibin levels were higher
and FSH levels were lower compared to those in wild-type females. In
contrast, inhibin levels were lower in 13-month-old AMH null females, and
FSH levels were unchanged compared to those in wild-type females.
Furthermore, the weight of the ovaries was twice as high in the
4-month-old AMH null females as in age-matched wild-type females. We
conclude that AMH plays an important role in primordial follicle
recruitment, such that more primordial follicles are recruited in AMH null
mice than in wild-type mice; the mice heterozygous for the AMH null
mutation take an in-between position. Consequently, the ovaries of AMH
null females and those of females heterozygous for the AMH null mutation
will show a relatively early depletion of their stock of primordial
follicles. The female AMH null mouse may thus provide a useful model to
study regulation of primordial follicle recruitment and the relation
between follicular dynamics and ovarian aging
Temporal changes in inhibin subunit mRNAs during atresia of preovulatory follicles in the rat
This study aimed to investigate the time course of disappearance of the
mRNAs of the various subunits of inhibin in follicles which become
atretic. An animal model was used in which atresia of preovulatory
follicles could be studied in a chronological order. Injection of
gonadotrophin-releasing hormone (GnRH) antagonist (20 microg) at the
morning of pro-oestrus (P) blocked ovulation and the 10-12 preovulatory
follicles became gradually atretic. A second injection was given the next
day to prevent delayed ovulation. The rate of atresia could be delayed by
simultaneous administration of a subovulatory dose of human chorionic
gonadotrophin (hCG) (0.5 IU) and could be advanced by administration of a
fivefold larger amount of GnRH antagonist. Functional activity of
follicles becoming atretic was studied by measuring oestradiol production
after incubation of individual follicles for 4 h. Follicles isolated 24 h
after the first injection of GnRH antagonist (P+24) already secreted
significantly less oestradiol in vitro than follicles isolated at
pro-oestrus, although they were morphologically not different from
pro-oestrous follicles. Follicles isolated at P+24 from hCG-treated rats
secreted more oestradiol compared with follicles from rats not treated
with hCG. In contrast, follicles isolated at P+24 from rats that were
given a fivefold larger amount of GnRH antagonist secreted less
oestradiol. Once this model was validated, temporal changes in inhibin
subunit mRNAs in follicles undergoing atresia were measured by in situ
hybridization and RNase protection assay. In situ hybridization showed
abundant alpha- and betaA-subunit mRNA in the whole granulosa layer of
preovulatory follicles at P and P+24, while betaB-subunit mRNA was
restricted to the antral layer and cumulus. At P+48 the amount of alpha-
and betaA-subunit mRNA had declined and was restricted to the cumulus,
whereas betaB-subunit mRNA was absent. In the atretic follicles present at
P+72 and P+96, mRNAs of all three inhibin subunits were absent.
Administration of 0.5 IU hCG delayed the decline in the amount of alpha,
betaA and betaB mRNA in preovulatory follicles at P+48. RNase protection
assay of inhibin subunits in isolated follicles revealed no changes
between P and P+24. However, at P+48, the mRNAs of alpha- an
Anti-Mullerian hormone inhibits initiation of primordial follicle growth in the mouse ovary
Recruitment of primordial follicles is essential for female fertility;
however, the exact mechanisms regulating this process are largely unknown.
Earlier studies using anti-Mullerian hormone (AMH)-deficient mice
suggested that AMH is involved in the regulation of primordial follicle
recruitment. We tested this hypothesis in a neonatal ovary culture system,
in which ovaries from 2-d-old C57Bl/6J mice were cultured for 2 or 4 d in
the absence or presence of AMH. Ovaries from 2-d-old mice contain multiple
primordial follicles, some naked oocytes, and no follicles at later stages
of development. We observed that in the cultured ovaries, either
nontreated or AMH-treated, follicular development progressed to the same
extent as in in vivo ovaries of comparable age, confirming the validity of
our culture system. However, in the presence of AMH, cultured ovaries
contained 40% fewer growing follicles compared with control ovaries. A
similar reduction was found after 4 d of culture. Consistent with these
findings, we noted lower inhibin alpha-subunit expression in AMH-treated
ovaries compared with untreated ovaries. In contrast, expression of AMH
ligand type II receptor and the expression of oocyte markers growth and
differentiation factor 9 and zona pellucida protein 3 were not influenced
by AMH. Based on the results, we suggest that AMH inhibits initiation of
primordial follicle growth and therefore functions as an inhibitory growth
factor in the ovary during these early stages of folliculogenesis
Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the mouse ovary
Although ovarian follicle growth is under the influence of many growth
factors and hormones of which FSH remains one of the most prominent
regulators. Therefore, factors affecting the sensitivity of ovarian
follicles to FSH are also important for follicle growth. The aim of the
present study was to investigate whether anti-Mullerian hormone (AMH) has
an inhibitory effect on follicle growth by decreasing the sensitivity of
ovarian follicles to FSH. Furthermore, the combined action of AMH and FSH
on ovarian follicle development was examined. Three different experiments
were performed. Using an in vitro follicle culture system it was shown
that FSH-stimulated preantral follicle growth is attenuated in the
presence of AMH. This observation was confirmed by an in vivo experiment
showing that in immature AMH-deficient females, more follicles start to
grow under the influence of exogenous FSH than in their wild-type
littermates. In a third experiment, examination of the follicle population
of 4-month-old wild-type, FSH beta-, AMH-, and AMH-/FSH beta-deficient
females revealed that loss of FSH expression has no impact on the number
of primordial and preantral follicles, but the loss of inhibitory action
of AMH on the recruitment of primordial follicles in AMH-deficient mice is
increased in the absence of FSH. In conclusion, these studies show that
AMH inhibits FSH-stimulated follicle growth in the mouse, suggesting that
AMH is one of the factors determining the sensitivity of ovarian follicles
for FSH and that AMH is a dominant regulator of early follicle growth
Apoptotic and proliferative changes during induced atresia of pre-ovulatory follicles in the rat
Atresia, a degenerative process through which many follicles are removed
from the growing pool, involves apoptotic changes in the follicular
granulosa cells. To identify histochemical markers of early stages of
atresia, an in-vivo rat model was used which allowed the study of atresia
of pre-ovulatory follicles in a synchronized and chronological order. By
blocking the pre-ovulatory luteinizing hormone surge with a
gonadotrophin-releasing hormone (GnRH) antagonist, ovulation of the
pre-ovulatory follicles is prevented, after which these follicles became
atretic. The first morphological sign of atresia (pyknotic granulosa cell
nuclei) was found 27 h after injection of GnRH antagonist. Since the
pre-ovulatory follicles gradually become atretic in a synchronous fashion,
this model provided an opportunity to study and define markers of future
atresia in pre-ovulatory follicles. Atresia involves apoptosis of
granulosa cells, and therefore internucleosomal DNA fragmentation was
examined. Using the terminal deoxynucleotidyltransferase-mediated
dUTP-biotin nick end labelling (TUNEL) assay it was found that the first
sign of internucleosomal DNA fragmentation in granulosa cells of
pre-ovulatory follicles was detectable 24 h after GnRH antagonist
treatment. In order to find an upstream marker of atresia, the
5-bromo-deoxyuridine (BrdU) labelling index was used as a measure of
proliferation. Already at 14 h after GnRH antagonist treatment, when
morphological signs of atresia were not yet present, a clear decrease in
BrdU labelling index was found in the granulosa cells