Human Reproduction, Vol.25, No.10 pp. 2579–2590, 2010
Advanced Access publication on July 29, 2010 doi:10.1093/humrep/deq195
ORIGINAL ARTICLE Reproductive biology
Meiosis initiation in the human ovary
requires intrinsic retinoic acid synthesis
R. Le Bouffant 1,2,3,7,†, M.J. Guerquin 1,2,3,7,†, C. Duquenne 1,2,3,
N. Frydman 4,5,6, H. Coffigny 1,2,3, V. Rouiller-Fabre 1,2,3,
R. Frydman 4,5,6, R. Habert 1,2,3, and G. Livera 1,2,3,*
1
CEA, DSV/DRR/SEGG/LDRG, Laboratory of Differentiation and Radiobiology of the Gonads, Unit of Gametogenesis and Genotoxicity, 18
route du panorama, F-92265 Fontenay aux Roses, France 2University Paris 7-Denis Diderot, U.F.R of Biology, UMR-967, F-92265 Fontenay
aux Roses, France 3INSERM, U967, F-92265 Fontenay aux Roses, France 4Univ Paris-Sud, Clamart F-92140, France 5AP-HP, Service de
Gynécologie-Obstétrique et Médecine de la Reproduction, Hôpital Antoine Béclère, F-92140 Clamart, France 6INSERM, U782, F-92140
Clamart, France 7Present address: UPMC, CNRS, Laboratoire de Biologie du Développement UMR 7622, 9 quai Saint-Bernard, 75005
Paris, France
*Correspondence address. Tel: +33-1-46-54-99-36; Fax: +33-1-46-54-99-06; E-mail: [email protected]
Submitted on November 6, 2009; resubmitted on June 25, 2010; accepted on July 2, 2010
background: The initiation of meiosis is crucial to fertility. While extensive studies in rodents have enhanced our understanding of this
process, studies in human fetal ovary are lacking.
methods: We used RT –PCR and immunohistochemistry to investigate expression of meiotic factors in human fetal ovaries from 6 to 15
weeks post fertilization (wpf) and developed an organ culture model to study the initiation of human meiosis.
results: We observed the first meiotic cells at 11 wpf, when STRA8, SPO11 and DMC1 are first expressed. In culture, meiosis initiation is
observed in 10 and 11 wpf ovaries and meiosis is maintained by addition of fetal calf serum. Meiosis is stimulated, compared with control, by
retinoic acid (RA) (P , 0.05). No major change occurred in mRNA for CYP26B1, the RA-degrading enzyme proposed to control the timing
of meiosis in mice. We did, however, observe increased mRNA levels for ALDH1A1 in human ovary when meiosis began, and evidence for a
requirement to synthesize RA and thus sustain meiosis. Indeed, ALDH inhibition by citral prevented the appearance of meiotic cells. Finally, 8
wpf ovaries (and earlier stages) were unable to initiate meiosis whatever the length of culture, even in the presence of RA and serum.
However, when human germ cells from 8 wpf ovaries were placed in a mouse ovarian environment, some did initiate meiosis.
conclusions: Our data indicate that meiosis initiation in the human ovary relies partially on RA, but that the progression and regulation
of this process appears to differ in many aspects from that described in mice.
Key words: human fetal ovary / meiosis / retinoic acid / proliferation / germ cells
Introduction
The development and function of mammalian gonads has been the
subject of extensive research over the past few decades. This has
allowed the distinction to be made in both human and mouse
between somatic sex determination by chromosomal constitution
(XX or XY) (Wilhelm et al., 2007), and germ cell sex determination
for which the gonadal environment plays a major role (Kocer et al.,
2009), however, the pathways involved in the latter remain poorly
defined. Germ cell sex determination is characterized by a difference
in the timing of entry into meiosis (McLaren, 1995; Kimble and Page,
2007). During fetal life, germ cells in the ovary enter into meiosis thus
committing themselves to oogenesis while those in the fetal testis do
†
not; male meiosis being initiated later at puberty. Several recent findings in mammals suggest a key role for retinoic acid (RA) in this
sex-specific timing of meiosis (Bowles and Koopman, 2007).
However, no role for RA in initiating meiosis in humans has been
demonstrated so far.
Much of our knowledge regarding germ cell differentiation in
mammals originates from studies in rodents and very little is known
about germ cell development in human fetal gonads. In the human
fetal gonad, somatic sexual differentiation occurs around 6 weeks
post fertilization (wpf) (Pelliniemi et al., 1993; George and Wilson,
1994). In the human testis, germ cells undergo several rounds of
mitotic replication without any apparent arrest (Bendsen et al.,
2003; O’Shaughnessy et al., 2007). Germ cells in the human fetal
These authors contributed equally to this work.
& The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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ovary, termed oogonia, initially display a similar mitotic activity
(Bendsen et al., 2006) until around 10 wpf when the first signs of
meiosis initiation are apparent (Kurilo, 1981; Gondos et al., 1986).
At this early stage, meiosis initiation appears strikingly asynchronous,
as while more and more germ cells initiate meiosis some oogonia
expressing stem cell markers go on proliferating until at least 16 wpf
(Baker, 1963; Skrzypczak et al., 1981; Kerr et al., 2008).
In rodents, the differentiation of the bipotential gonad begins about
11.5 days post-coitus (dpc) in the mouse and 13.5 dpc in the rat
(Magre and Jost, 1991; Wilhelm et al., 2007). The first morphological
evidence of sex-specific germ cell development is seen when female
germ cells stop proliferating and enter prophase of the first meiotic
division in the developing ovary. This occurs at around 13.5 dpc in
the mouse and 16.5 dpc in the rat (Merchant, 1975; Bullejos and
Koopman, 2004; Trautmann et al., 2008). Germ cells then progress
rapidly through leptonema, zygonema, pachynema and diplonema.
At around the time of birth, they then enter a prolonged arrest
stage termed dictyate. At about the same age as meiosis initiates in
the ovary, germ cells in the testis arrest in G0/G1 of the cell cycle,
resuming mitosis after birth (Hilscher et al., 1974; McLaren, 1984;
Boulogne et al., 1999;Trautmann et al., 2008; Western et al., 2008).
In rodents, several pieces of data implicate RA signaling in the induction of germ cell meiosis in the developing ovaries. The addition of
exogenous RA has been shown to speed up the spontaneous initiation
of meiosis that occurs in organ culture of mouse and rat fetal ovaries
(Livera et al., 2000a; Koubova et al., 2006) and vitamin A deficiency in
vivo has been reported to prevent meiosis initiation in the fetal rat
ovary (Li and Clagett-Dame, 2009). One master gene controlling
meiosis induction in mouse ovaries is Stra8 (Stimulated by retinoic
acid 8). Expression of this gene appears at 12.5 dpc, shortly before
meiosis induction in the mouse ovary and is required for meiosis to
occur (Baltus et al., 2006), with germ cells in Stra8 –/– ovaries
remaining blocked at the pre-meiotic stage. Recently, Stra8 expression
was reported in the human ovary (Houmard et al., 2009). All-trans-RA
is mostly synthesized by three aldehyde dehydrogenases: Aldh1a1,
Aldh1a2 or Aldh1a3 (Ross et al., 2000). In vivo, in mouse, RA is
mostly produced in the mesonephros by Aldh1a2 before diffusing
into the adjacent gonad (Bowles et al., 2006). In the undifferentiated
gonad, meiosis initiation is inhibited by a RA degrading enzyme
Cyp26b1 (Cytochrome P450, family 26, subfamily b1) that is later
up-regulated in male somatic cells and down-regulated in the female
gonad (Bowles et al., 2006). Interestingly, several studies suggest a
conserved role of the meiotic inducer for RA in many species of vertebrate (Smith et al., 2008; Wallacides et al., 2009).
Most studies on the human fetal ovary have centered on events
occurring during meiosis prophase I and consequently very little data
exist on the process controlling human meiosis initiation. Since the
timing of these events differs strikingly in the developing ovaries of
mice and humans, we investigated whether similar signaling pathways
are involved in meiosis induction in the two species. We set up an
organ culture model similar to the one previously used to study the
human developing testis (Lambrot et al., 2006, 2007). In contrast to
observations made in rodents, here we report that meiosis initiation
in the human fetal ovary does not occur spontaneously in organ
culture. In addition, in humans the process requires RA production
from the fetal ovary itself. The key enzyme that seems to regulate RA
level in the human gonad is not CYP26B1 but ALDH1A1.
Le Bouffant et al.
Materials and Methods
Collection of human fetal gonads
Human fetal gonads were harvested from material available following
legally induced abortions in the first trimester of pregnancy and therapeutical termination of pregnancy in the second trimester, i.e. from the 6th until
the 15th week post conception, in the Department of Obstetrics and
Gynecology at the Antoine Béclère Hospital, Clamart (France) as previously described (Lambrot et al., 2006, 2007;Guerquin et al., 2009).
None of the terminations were for reasons of fetal abnormality and all
fetuses appeared morphologically normal. The Antoine Béclère Ethics
Committee approved this study and all women gave their informed
consent. The sex of the fetus was determined from the morphology of
the gonads and confirmed by histological analysis of the gonad. Ovaries
were thinner and more closely associated with the mesonephros than
testes. The fetal age was evaluated by measuring the length of limbs and
feet (Evtouchenko et al., 1996).
Mice
Naval Medical Research Institute mice were housed in controlled photoperiod conditions (lights on from 08:00 to 20:00) and were supplied
with commercial food and tap water ad libitum. Females were caged
with males overnight. All those in which a vaginal plug could be detected
the following morning were defined as being 0.5 dpc (days post conception). All animal studies were conducted in accordance with guidelines
for the care and use of laboratory animals from the French Ministry of
Agriculture.
Organ culture
All tissues were cultured on Millicell-CM Biopore membranes (pore size
0.4 mm; Millipore, Billerica, MA, USA) in 0.3 ml of Ham’s F12/Dulbecco’s
modified Eagle’s medium (1:1) containing 80 mg/ml gentamicin in a
humidified atmosphere of 95% air– 5% CO2. The medium was changed
every 48 h. Human gonads were cut into small pieces before being
placed separately on two or four Millicell membranes as required. Some
were used as controls and the others were cultured in the presence of
fetal calf serum (FCS, 10%) and/or RA (1 mM, Sigma-Aldrich, St Louis,
MO, USA). The vehicle (dimethyl sulfoxide) was also included in the
culture medium of the paired control. Ketoconazole (KET) (2 mM,
Sigma), a known CYP26B1 inhibitor, and citral (55 mM, Sigma), an inhibitor
of RA synthesizing enzyme, were added to some cultures with or without
RA and FCS. The pan RA receptor (RAR) antagonist BMS-189453 (BMS,
10 mM) was generously provided by Brystol-Myers Squibb and added to
some cultures with or without FCS. The efficiency of KET treatment
was confirmed by the observed up-regulation of typical RA-target genes,
such as CYP26A1 and RARB2, while BMS treatment down-regulated
these genes (n ¼ 1, data not shown).
Histology and germ cell counting
Gonads were fixed in Bouin’s fluid immediately after dissection or at the
end of the culture. The fixed gonads were dehydrated, embedded in paraffin and cut into 5-mm-thick sections. For all tissues, 1 in every 10 serial
sections were mounted onto glass slides. These sections were dewaxed,
rehydrated and stained with hematoxylin and eosin (H&E). Germ cells
on each section were identified on the basis of their large, spherical
nuclei and clearly visible cytoplasmic membrane, and counted. Normalization was achieved by dividing the number of germ cells in each fragment by
the area, to obtain a germ cell density. Meiotic stages were recognized on
the basis of their histological features. Meiotic cells displayed markedly
condensed chromatin forming distinct fine threads with a beaded
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appearance at the leptotene stage, and a nucleus showing a characteristic
criss-cross of coiled chromosome threads at the zygotene stage. Histolab
analysis software (Microvision Instruments, Evry, France) was used.
Immunohistochemistry
Immunohistochemistry, based on peroxidase activity, was performed using
commercially available primary antibodies: anti-SSEA1 (stage-specific
embryonic antigen 1, 1/10, monoclonal antibody, DSHB, IA, USA);
anti-cleaved caspase-3 Asp 175 (1/100, Cell Signaling); anti-DDX4
(DEAD box polypeptide 4/human VASA homolog 1/500, Abcam, Cambridge, UK). Five tissue sections per gonad were mounted onto glass
slides, dewaxed and boiled for 10 min in 10 mM Tris, pH 10.6, for immunostaining of cleaved caspase-3, or in 10 mM citrate pH6.0 for immunostaining of DDX4. Endogenous peroxidase activity was blocked with 3%
hydrogen peroxide for 10 min. The sections were washed with phosphatebuffered saline (PBS) (for cleaved caspase-3 and DDX4), and blocked for
30 min with 5% normal goat serum (NGS) and 10% bovine serum albumin
(BSA). Slides were incubated overnight at 48C with the primary antibody
and 5% NGS. We detected bound primary antibody with a biotinylated
goat anti-rabbit secondary antibody in 2% NGS and the avidin– biotin –
peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories,
Burlingame, CA, USA). Peroxidase activity was visualized using
3,3′ -diaminobenzidine (DAB, brown) or Vector VIP (purple) as a substrate. Phosphorylation of the histone H2A variant H2AX ( gH2AX) was
used to reveal DNA double-strand breaks, a hallmark of meiosis,
and detected by immunohistochemistry using an anti-gH2AX antibody
(1/500, Euromedex, Mundolsheim, France) as previously described
(Trautmann et al., 2008).
Measurement of cell proliferation
The percentage of cells in S-phase was evaluated by immunohistochemical
methods to measure BrdU (5-bromo-2’-deoxyuridine) incorporation,
using the Cell Proliferation Kit (GE Healthcare, Buckinghamshire, UK)
according to the manufacturer’s recommendations. BrdU (1%) was
added at the end of the culture period for 3 h before tissue fixation.
Briefly, three to five randomly selected sections were mounted, rehydrated and incubated for 1 h with anti-BrdU antibody and with a
peroxidase-linked anti-mouse immunoglobulin (Ig)G. Peroxidase activity
was then detected by DAB. The BrdU incorporation index was determined by the counting (blinded to experimental group) of at least 500
stained and unstained germ cells.
Aggregate cultures
Two to three gonads from 7 to 8 wpf human fetuses were cultured for 1 –
3 days in control medium. Gonads were digested first in 0.25%
trypsin-0.02% EDTA (Trypsin/EDTA solution, Sigma-Aldrich, St. Louis,
MO, USA) for 5 min, at 378C.Trypsin digestion was stopped by adding
fetal bovine serum to 10% and samples were centrifuged (500g for
10 min). The samples were then further digested with 2 mg/ml collagenase and 0.02 mg/ml DNase I in Hanks balanced salt solution for 10 min at
378C. Dispersed cells were then incubated with anti-SSEA1 antibody (1/5)
in PBS, 0.5% BSA (PB) for 20 min at 48C. At the end of the incubation, the
cells were centrifuged and washed once with 1 ml of PB. The cells were
then incubated with 20 ml of microbead-linked donkey anti-mouse IgM
antibody (Miltenyi Biotec, Germany) in 300 ml PB with 2 mM EDTA
(PBE) for 15 min at room temperature before being rinsed once with
PBE and applied onto an MS+ column (Miltenyi). The column was
rinsed three times with 500 ml PBE to wash out unbound cells, which represented the SSEA1-negative cell fraction. After removal from the magnet,
the column was flushed with 1 ml PB allowing the collection of the SSEA1-
positive cell fraction. Similar procedures were performed to obtain SSEA1negative cells from 50 mouse 13.5 dpc ovaries.
Human SSEA1-positive germ cells were mixed with SSEA1-negative
(all somatic cells) either from the same human gonads or from 13.5 dpc
mouse ovaries (about 30 000 SSEA1 + cells with 500 000 somatic cells).
The cell mixtures were centrifuged at 1200g to remove the aggregates,
which were then cultured on Millicell filters for 7 days. On the second
day of culture, 10% FCS was added to the culture media. To ascertain
the origin of SSEA1-positive germ cells from human gonads, BrdU was
added to fetal ovaries in organ culture prior to magnetic cell sorting.
One day later, gonads were removed and used for SSEA1 purification.
Real-time quantitative RT – PCR
At the end of the culture period, total RNA was extracted using the
RNeasy mini-kit (Qiagen) and reverse transcription carried out with the
Omniscript kit (Qiagen), according to the manufacturer’s instructions.
The ABI Prism 7000 system (Applied Biosystems) and SYBR-green labeling
were used for quantitative RT– PCR (QRT – PCR). Each RNA sample was
analyzed in triplicate. All primers were used at a concentration of 400 nM.
Valid primers for Synaptonemal complex protein 1 (SYCP1), Recombination
protein Rec8 homolog/Cohesin Rec8p (REC8), DDX4, Dosage suppressor of
mck1 homolog (DMC1), STRA8 and Sporulation protein 11 (SPO11) were
purchased from SuperArray (SABiosciences). Two different primer sets
were used to analyze DMC1 and REC8 expression patterns. Other
primers used were (forward, reverse): CYP26B1: 5′ -TCGAGCTTGAT
GGTTTCCAGA-3′ , 5′ -TGCTATACATGACACTCCAGCCTT-3′ ; ALDH1
A1: 5′ -ATCTCCTCTGCTCTGCAGGC-3′ , 5′ -CACGCCATAGCAATTC
ACCC-3′ ; ALDH1A2: 5′ -CAGACTTGGTGGAACGGGAC-3′ , 5′ -TTAGG
GATTCCATGGTTGCAA-3′ ; REC8: 5′ -TGCTGATCGCAGAGGAAG
AA-3′ , 5′ -GGAGCCGCGGGATTTC-3′ ; DMC1: 5′ -GGGAGACTGTGG
GTACGAGG-3′ , 5′ -AAAGTGGGCAACAGAAAAATAATCA-3′ and
b-ACTIN: 5′ -TGACCCAGATCATGTTTGAGA-3′ , 5′ -TACGGCCAGA
GGCGTACAGG-3′ . All efficiencies were between 95 and 100%.
Expression levels were normalized to b-ACTIN expression.
Statistical analysis
Each data point represents the mean + SEM of at least three independent
cultures. Images shown in the figures are representative of at least three
experiments. Data were analyzed using paired Student’s t-test when a
single treatment was performed (i.e. in Figs. 2 and 3). Other mean
values were compared using one-way or two-way analysis of variance
with GraphPad Instat 3.0. Statistical significance was set at P , 0.05.
Results
Analysis of meiosis markers
First, to better characterize meiosis initiation, we analyzed the
expression of DDX4 and of several meiotic markers: STRA8, REC8,
SPO11, SYCP1, DMC1 in human fetal ovaries ranging from 6 to 15 wpf
(Fig. 1A). The mRNAs for STRA8, SPO11 and SYCP1 were first detected
at 11 wpf, coinciding with a sharp increase in DDX4 expression. DMC1
mRNA was detected as early as 6 wpf and increased sharply later, at 15
wpf. REC8 mRNA level showed no major change though it tended to
increase from the 11th week onwards.
We next investigated the presence of the meiotic cell marker
gH2AX by immunostaining sections of human fetal ovaries from 9
to 15 wpf (Fig. 1B). gH2AX was first detected in very few germ
cells at 11 wpf. At 15 wpf, more cells were stained for gH2AX
though the majority still remained gH2AX-negative. The appearance
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Le Bouffant et al.
Figure 1 Meiotic markers appear at around 11 weeks post fertilization (wpf). Meiotic marker expression was assessed by measuring mRNA levels
using QRT – PCR in human fetal testes (T), ovaries (O) and mesonephroi (M) (A). The histograms show the relative expression of each marker, normalized to the maximum, using beta-actin as a reporter. Each column represents a single measure (6, 7, 10, 11, 12, 15 wpf ovaries) or the mean of two
measures at similar stages (testes, mesonephroi, 8 and 9 wpf ovaries). STRA8, stimulated by RA 8; SPO11, sporulation protein 11; SYCP1, synaptonemal complex protein 1; REC8, recombinase 8; DMC1, dosage suppressor of MCk1; DDX4, human VASA homolog. gH2AX (histone H2A variant)
was detected by immunohistochemistry in sections from human fetal ovaries ranging from 9 to 15 wpf (B). Black arrows point to germ cells negative
for gH2AX and open arrows to positive germ cells for gH2AX. The bar represents 20 mm.
of gH2AX correlated with the identification of meiotic cells in H&E
stained sections by the presence of condensed, thread-like chromatin
(data not shown).
Similar partitioning of meiotic stages and percentage of meiotic cells
was observed in a 15 wpf ovary.
RA induces meiosis
Serum sustains meiosis initiation in vitro
We next set up an in vitro model to study human meiosis. Twelve
human fetal ovaries, ranging from 8 to 11 wpf, were divided into
three categories ‘8– 9 wpf’, ‘10 wpf’ and ‘11 wpf’ according to their
predicted age, and cultured in control medium for 14 days (Fig. 2).
Germ cell density was maintained at high levels throughout the
culture period with an average of 3000 germ cells/mm2. No change
was observed in germ cell density (2374 + 185 on Day 0 versus
2964 + 343 on Day 14). At the end of the culture, meiotic cells
were quantified. Hardly any meiotic cells were found in the cultures
except for a small number in 11 wpf ovaries which already contained
a few meiotic cells at explantation. The addition of 10% FCS did not
alter germ cell density (3196 + 685 without serum versus 3721 +
504 with serum). The FCS, did however, significantly increase the percentage of meiotic cells observed in 10 and 11 wpf ovaries but did not
induce meiosis in 8 –9 wpf ovaries (Fig. 2A and B). Meiotic stages were
mostly zygotene with few leptotene and pre-leptotene oocytes.
We next tested the effect of RA on meiosis initiation. About 9–10
wpf fetal ovaries were cultured with or without RA. The addition of
RA (1 mM) strongly increased the rate of meiosis after a 14-day
culture (Fig. 3A) and increased the number of gH2AXpositive cells
(Fig. 3B). RA also stimulated meiotic initiation in the presence of
FCS. FCS alone did not stimulate meiotic initiation in this set of
experiments. In 9–10 wpf fetal ovaries in culture, serum neither
stimulated meiosis nor enhanced RA-induced meiosis. Indeed,
though the addition of both RA plus serum produced the highest
number of meiotic cells, this difference was not significant when
compared with RA treatment alone. The presence of meiotic cells
was further confirmed by the detection of meiotic markers by
QRT–PCR (Fig. 3C). RA plus FCS up-regulated mRNA levels for
all meiotic markers tested, strongly increasing STRA8, REC8 and
SPO11 expression and increasing DDX4 expression to a lesser
extent. While not significant, DMC1 and SYCP1 expression tended
toward an augmentation.
Human meiosis in vitro
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Figure 2 FCS sustains meiosis initiation. Human fetal ovaries ranging from 8 to 11 wpf were cultured without (open columns) or with fetal calf
serum (FCS, 10%, grey columns) for 14 days. At the end of the culture, tissues were fixed and the percentage of meiotic germ cells determined
(A). Mean + SEM of four different gonads are presented; **P , 0.01, ***P , 0.001 versus control in the paired Student’s ‘t’ test. Meiotic germ
cells were identified by the characteristic features of their chromatin (B). Black arrows point out oogonia and open arrows zygotene oocytes. The
bar represents 20 mm. The mean age in each of the categories 8 – 11 wpf was, respectively, 63.5, 71 and 79 dpc.
RA increases germ cell proliferation and
differentiation
Whatever the stage (wpf) analyzed, RA increased BrdU incorporation
into germ cells (Fig. 4A). This increase in germ cell proliferation was
repeatedly observed, even in early stages when RA did not induce
meiosis. This suggests that RA not only increases the percentage of
pre-meiotic S-phase cells but also effectively promotes germ cell
mitotic activity. Moreover, FCS addition had no effect on BrdU incorporation while, at some stages, it did stimulate meiosis.
RA also increased germ cell apoptosis, identified as large cells
stained for cleaved caspase-3 (Fig. 4B). RA alone also decreased
germ cell density by 30.7% (2164 versus 1498 germ cells/mm2;
n ¼ 6, P ¼ 0.047 in the paired Student’s t-test) This RA-increased
apoptosis was reversed by the addition of FCS, though serum alone
had no effect on the basal rate of germ cell apoptosis at the same
stages.
Finally, we investigated germ cell differentiation in response to RA
treatment by measuring the loss of SSEA1 protein, SSEA1 being a
marker of undifferentiated cells (Fig. 5). Whatever the stage, in the
absence of FCS, RA decreased the percentage of germ cells labeled
by SSEA1 antibodies. This decrease was proportional to the observed
increase of DDX4-stained germ cells. In the presence of serum, RA
treatment had no further effect on the proportion of SSEA1 or
DDX4-positive cells.
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Le Bouffant et al.
Figure 3 Retinoic acid (RA) promotes meiosis initiation. Human fetal ovaries ranging from 9 to 10 wpf (n ¼ 4) were cultured with or without RA for 14 days
in the absence [control (CTR)] or presence of serum (FCS, 10%). The histogram shows the percentage of meiotic germ cells (A). Different letters represent
significantly different values in analysis of variance comparisons, with P , 0.05. Black arrows point out oogonia and open arrows zygotene oocytes in hematoxylin/eosin stained sections. gH2AX was detected by immunohistochemistry in sections of the human fetal ovaries (B). Black arrows point to negative and
open arrows to positive germ cells for gH2AX. As described in Fig. 1, the expression of meiotic markers was measured by QRT–PCR in human fetal ovaries
cultured in control medium (CTR) or in the presence of FCS and RA (C). The histograms show the mean + SEM of six different experiments (i.e. six different
gonads). The bar represents 20 mm (A and B). *P , 0.05, **P , 0.01 in the paired Student’s ‘t’ test.
ALDH but not CYP26B1 regulates
meiosis initiation
We next wished to further investigate RA signaling in the developing
human ovary (Fig. 6). No major change in CYP26B1 mRNA level
from the 6th to the 15th wpf in the fetal ovary was observed
(Fig. 6A) though mRNA level increased about 2-fold from the 6th
to 10th wpf and later decreased. CYP26B1 expression observed in
the ovary also appeared slightly higher than that observed in the
human fetal testis or mesonephros. Interestingly, the RA-synthesizing
enzymes ALDH1A 1 and 2 were expressed in both the mesonephros
and the testis, and ALDH1A1 mRNA levels sharply increased in the
ovary at 11 wpf (i.e. when the first signs of meiosis are observed).
To investigate whether the expression of these enzymes corresponds to a potential role in meiosis initiation, we inhibited CYP or
Human meiosis in vitro
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Figure 4 RA increases germ cell proliferation and apoptosis. Human fetal ovaries ranging from 8 to 11 wpf were cultured with or without FCS and/
or RA for 14 days. Incorporation of 5-bromo-2’-deoxyuridine (BrdU) (A) and cleaved-caspase3 (Casp3, B) were detected by immunohistochemistry.
Each column is the mean + SEM of six cultures. Different letters represent significantly different values in ANOVA comparisons, with P , 0.05. Black
arrows point out negative and open arrows positive germ cells. The bar represents 20 mm.
ALDH enzymes in 9–10 wpf ovaries in organ culture over 14 days
(Fig. 6B and C). The addition of KET, a known CYP26B1 inhibitor,
alone (data not shown) or in the presence of serum alone or with
RA did not alter the percentage of meiotic cells (Fig. 6B). However,
BMS, a pan-RARs antagonist, and citral, an inhibitor of RA synthesizing
enzyme, did partially prevent the initiation of meiosis (Fig. 6C) though
not fully as a few meiotic cells could be retrieved in all cultures (n ¼ 4,
each).
In order to test whether CYP26B1 may be responsible for the prevention of meiosis in earlier stages, 7 –8 wpf ovaries were cultured
with or without KET (Fig. 6B, right panel). None of FCS, RA or
KET alone or in combination were able to induce meiosis at this stage.
Development of the human fetal ovary in
long-term cultures
Human fetal ovaries from 8 wpf (n ¼ 2) were cultured for 4 weeks in
control medium or in the presence of FCS with and without RA. At
the end of culture, hardly any meiotic cells were retrieved whatever
the condition used (data not shown). Analysis using chi-square test
(pooling with and without serum groups) indicated no effect of RA
treatment (P ¼ 0.39). Similarly, very few meiotic stages (,1%) were
observed in 9–10 wpf ovaries cultured for 4 or 5 weeks (Fig. 7A) in
the control medium without supplementation. Most cells displayed
the typical features of oogonia with the presence of prominent
nucleoli. However, when 9–10 wpf ovaries were cultured in the presence of FCS plus RA, about one-third of the germ cells entered
meiosis after 4 weeks of culture and half presented a condensed chromatin, typical of zygotene stages, after 6 weeks. After 7 weeks of
culture in the presence of both RA and FCS, the germ cell density
markedly decreased. In all long-term cultures analyzed, very few
pachytene stages were observed (,1%). Out of six cultures performed in the presence of both FCS and RA for 6 or 7 weeks, folliclelike structures appeared in four (Fig. 7C). These structures displayed
large oocytes (27 –33 mm in diameter) with a nucleolus and fine chromatin resembling that observed at diplotene stage surrounded by a
few flattened somatic cells. Interestingly, large numbers of oogonia
were retrieved in all long-term cultures; these tended to progressively
display a rather cortical-like localization after 4 weeks in culture containing FCS.
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Le Bouffant et al.
Figure 5 RA speeds up germ cell differentiation. Human fetal ovaries were cultured as described in Fig. 3. At the end of the culture, SSEA1 and
DEAD box polypeptide 4/human VASA homolog (DDX4) were detected by immunohistochemistry. The histograms show the mean + SEM of the
percentage of positive germ cells in four independent cultures (A). Different letters represent significantly different values in ANOVA comparisons,
with P , 0.05. DDX4 is stained brown and SSEA1 purple. The bar represents 20 mm.
Early female germ cells have the
competence to initiate meiosis
Last, we investigated whether human fetal germ cells from 7 to 8 wpf
ovaries were able to initiate meiosis when placed in a different somatic
environment. SSEA1-positive human cells were purified by magnetic
cell sorting and aggregated with SSEA1-negative cells either from the
same human ovaries or from mouse 13.5 dpc ovaries (Fig. 8).
These aggregates were then further cultured for a week in the presence of FCS. In aggregates containing only mouse SSEA1-negative
cells, hardly any germ cells were retrieved (data not shown). In
those containing human SSEA1-negative plus human SSEA1-positive
cells, hardly any germ cells initiated meiosis, as was observed in
organ culture. On the contrary, obvious meiotic stages (leptonema
and zygonema) were observed in the aggregates containing human
SSEA1-positive and mouse SSEA1-negative cells. In order to confirm
the human origin of these meiotic cells, human ovaries were cultured
for 24 h with BrdU to trace cells prior to cell sorting. The mouse
gonad was not exposed to BrdU. Preliminary experiments indicated
that BrdU was incorporated in about a quarter of the germ cells
after 24 h in organ culture. Though not all meiotic cells in the aggregates were labeled with BrdU, the fact that some were shows that
these meiotic cells came from the human gonad.
Discussion
We have provided the first model to study human meiosis initiation
in vitro. This organ culture system has confirmed the similar requirement of RA for human meiosis as in rodents, and highlighted several
Human meiosis in vitro
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Figure 6 Ovarian RA synthesis is required for meiosis initiation. The expression of RA synthesizing and degrading enzyme and meiotic markers was
measured in fresh (not cultured) fetal testes (T), ovaries (O) and mesonephroi (M) (A). Tissues are the same as described in Fig. 1A. The histograms
show the relative expression of each gene normalized to the maximum using beta-actin as a reporter. ALDH1A1 and 2, alcohol dehydrogenase 1 a1
and a2; CYP26B1, cytochrome P450 type 26 b1. The effect of KET, a CYP26B1 inhibitor, was analyzed in gonads at 9 – 10 wpf (n ¼ 4) and at 7 – 8 wpf
(n ¼ 3) (B). Gonads were cultured for 14 days in the absence or presence of FCS, or FCS plus RA. The mean age within the category 7 – 8 and 9 – 10
wpf was, respectively, 53 and 69 dpc. BMS189453 (BMS), a pan-RAR inhibitor, and citral (CIT), an inhibitor of RA synthesis, were also added to organ
cultures of 9 – 10 wpf ovaries (n ¼ 4) (C). The histograms show the percentage of meiotic germ cells. Different letters represent significantly different
values in ANOVA comparisons, with P , 0.05. Black arrows point out oogonia and open arrows zygotene oocytes. The bar represents 20 mm.
aspects of the process which seems to be different in the human
embryonic ovary.
We observed that meiotic cells appear in the human fetal ovary
from 11 wpf. This was further confirmed by the detection of
meiotic markers by QRT–PCR and immunostaining. This is in agreement with previous reports that also placed meiotic initiation between
9 and 11 wpf (Kurilo, 1981; Gondos et al., 1986). While we are unable
to exclude the existence of a small number of meiotic cells from 9 wpf
as we did not perform an exhaustive study of all sections, we believe
their presence to be extremely rare prior to 11 wpf. Interestingly, our
data indicating that STRA8 expression occurs from 11 wpf is in accordance with recent findings of M. Griswold and co. who also described
STRA8 expression in the human ovary at this stage (Houmard et al.,
2009). An intriguing observation made here is the considerable lack
of synchronicity in the expression of the meiotic markers studied,
with SPO11 and SCYP1 being expressed first, DMC1 up-regulated
later and no considerable change observed for REC8. While in the
mouse fetal ovary all these factors are expressed in close synchrony
from 13.5 dpc onwards, in the human ovary their expression is staggered in time, perhaps indicating their regulation by different mechanisms. Alternatively, such changes may be difficult to observe in the
mouse because of the narrow temporal window for meiotic initiation
in the mouse fetal ovary while meiotic initiation spans a longer time
period in the human fetal ovary.
We and others have previously demonstrated that RA and various
growth factors (including serum) can promote meiosis in the germ
cells of rodent fetal ovaries (Livera et al., 2000a; Bowles et al.,
2006). In this study, we observed that these factors also promote
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Figure 7 Long-term organ culture of human fetal ovaries. Human
fetal ovaries ranging from 9 to 10 wpf were cultured for five (A),
six (B) and seven (C) weeks. About 5-week cultures (n ¼ 2) were
performed with control, and FCS plus RA supplemented, media.
Six and seven weeks cultures (n ¼ 6) were performed in the presence
of FCS and RA. Black arrows point out oogonia, open arrows zygotene oocytes and asterisks point out follicle-like structures. The bar
represents 20 mm in (A) and (C), and 100 mm in (B).
human meiosis. However, isolated fetal ovaries from rodents can
sustain meiotic initiation even without the mesonephros. Indeed,
meiotic cells spontaneously appear in both mouse (personal observation) and rat (Livera et al., 2000a) fetal ovaries harvested at the
oogonial stage (11.5 dpc mouse, and 14.5 dpc rat) and cultured for
a few days even in a defined culture medium (i.e. with no serum or
retinoids for 3 or 4 days). We demonstrated here that this is not
the case for early human fetal ovaries (up to 9 wpf). This observation
is in accordance with the pioneering report of Gapienko (1975). Since
meiosis is not spontaneous in the human ovary it most probably
Le Bouffant et al.
requires the action of extra-gonadal factor(s). These factors remain
to be identified.
Here, we have demonstrated that RA is a possible intra-ovarian
factor involved in meiosis initiation in the human ovary. This argument
is supported by three main pieces of evidence. First, RA induced
meiosis as defined by cytological criteria and molecular markers.
Second, inhibiting RA synthesis or signaling, by inhibiting ALDH activity
with citral and RAR signaling with an antagonist (BMS), decreased
meiosis initiation. Last, ALDH1A1 was the only RA-regulating enzyme
that showed a change in mRNA levels, being up-regulated from the
11th wpf onwards coinciding with the increase in STRA8 expression
and meiosis initiation. Although one cannot exclude that the small
decrease in CYP26B1 mRNA level reported here may also be
involved, we believe this to be quite unlikely as KET treatment did
not induce more meiotic germ cells in the human fetal ovary.
Surprisingly, our work proposed that the expression of the RA
synthesizing enzyme ALDH1A1 rises during the development of the
human ovary and that the expression of the RA-degrading enzyme,
CYP26B1, is poorly affected during meiotic initiation. These data
differ from those previously described in the mouse. In mice,
RA-synthesizing enzymes are not expressed in the fetal ovary, RA is
thought to be produced by the adjacent mesonephros and Cyp26b1
expression sharply decreases when meiosis initiates (Bowles et al.,
2006). It seems therefore that though RA remains a key factor in
meiosis initiation in mammals, the manner in which it is regulated in
the embryonic gonad may vary in mice and humans. We cannot
exclude the possibility that this difference simply reflects a compressed
temporal sequence in the mouse, making it more difficult to observe
that change detected during human development. In mouse, RA status
is determined by a decrease in its degradation, whereas in human it is
its intra-ovarian synthesis. Furthermore, RA degradation appears
poorly regulated in human ovaries, as confirmed by the absence of
effect of the CYP26B1 inhibitor (KET). Aldh1a1 expression was
recently reported in the developing gonads from mouse and chicken
embryos though, surprisingly, it has been described in the male
gonad (Bowles et al., 2009). Here, an interesting hypothesis is that
ALDH1A1 could be a central regulator of RA in human fetal ovary
and replace the role of CYP26B1 in mouse.
Interestingly, we also report that RA stimulation, even in the presence of FCS, is not sufficient to induce a complete meiosis prophase I
in human female germ cells. Indeed in our organ culture experiments,
many cells (about half of the germ cells) never initiated meiosis, even
following several weeks of culture in the presence of a high dose of
RA. Moreover, the vast majority of the germ cells remained arrested
(or degenerated) at the zygotene stage. This represents another major
difference with rodents. Indeed, germ cells in organ culture of fetal rat
and mice ovaries can easily go through all the stages of meiosis prophase I and follicles are formed in about 10 days (McLaren and
Buehr, 1990; Livera et al., 2000a). This provides further evidence
for the requirement of non-retinoid extra-ovarian actors for meiosis
I completion in the human fetal ovary.
Finally, we observed that although germ cells in the early fetal
human ovary (i.e. ,9 wpf) do not initiate meiosis in response to
RA, they are able to initiate meiosis when placed in a ‘meiosis permissive environment’ i.e. with mouse ovarian somatic cells. While further
investigations are first needed to understand this finding, it does
strongly suggest that a meiotic inhibitor, unrelated to RA, exists in
2589
Human meiosis in vitro
Figure 8 Mouse somatic cells induce meiosis in early human germ cells human fetal ovaries from 7 to 8 wpf were cultured for 2 days and BrdU was
added to the culture media. Germ cells and somatic cells from human ovaries and mouse fetal ovaries at 13.5 dpc were sorted by magnetic cell sorting
directed against SSEA1. Human SSEA1-positive cells were associated with either human or mouse SSEA1-negative cells, forming aggregates. These
aggregates were cultured for 8 days in the presence of 10% FCS. At the end of the culture, BrdU incorporation was detected in order to trace
the human cells. Black arrows point out BrdU-labeled oogonia, open arrows BrdU-labeled oocytes, and arrowheads BrdU-negative oocytes. Enlargement of oogonia (left) and leptotene stage oocyte (right) are presented. The bar represents 20 mm.
the early human ovary. The existence of such an inhibitor could
explain why later (9– 11 wpf), not all germ cells initiate meiosis in
response to RA. It may also explain the long oogonial proliferating
phase and the occurrence of an asynchronous meiosis initiation in
the human ovary. Another argument that suggests the existence of
additional mechanisms independent from retinoids in the control of
meiotic initiation is that in the fetal testis the addition of RA does
not always induce meiosis. Indeed in our previous experiments,
though we reported that RA clearly induces meiosis in the mouse
fetal XY gonad (Trautmann et al., 2008), we could not detect any
meiotic cells in the human (6–10 wpf) or in the rat (14.5 dpc) fetal
testis treated with RA (Livera et al., 2000b; Lambrot et al., 2006;
and personal observation). Nevertheless, one should remain cautious
about these in vitro observations as we also reported an increased
germ cell apoptosis and thus we cannot exclude the chance that
some cells would have initiated meiosis before undergoing apoptosis.
In conclusion, our study demonstrates that the development of fetal
germ cells, and most particularly the decision to initiate meiosis in the
human ovary, relies partially on mechanisms shared with rodents but
also, and importantly, may involve specific regulatory pathways: these
can only be observed in the human developing ovary and require an
efficient model such as the organ culture system described in the
present study. Human oocytes, because of their precious nature
with regard to fertility, remain understudied. Understanding the physiological requirements of human fetal germ cells is therefore crucial if
we are to envisage developing new resources to allow further investigations of the biology of human oocytes.
Authors’ roles
H.C., V.R.-F., R.F., R.H. and G.L. designed the study. H.C. and G.L.
retrieved the gonads and sat up the organ cultures. M.J.G. and C.D.
performed the immunohistological studies. N.F. and R.L.B. analyzed
the expression of genes involved in the ovarian development. All
authors participated to the discussion of the data and helped creating
the manuscript.
Acknowledgements
We thank the staff within the Department of Obstetrics and Gynecology of the Antoine Béclère Hospital (Clamart, France). We are grateful to Brystol-Myers Squibb for providing the BMS-189453. We also
thank A. Gouret for her skillful secretarial assistance and E. Witty
(Angloscribe, Clarensac, France) for editing the English.
Conflict of interest: the authors have nothing to disclose.
Funding
This work was supported by the Université Paris Diderot-Paris 7,
CEA, INSERM, the Agence Française de Sécurité Sanitaire de l’Environnement et du Travail (AFFSET) and Electricité de France (EDF).
M.J.G. holds fellowships from the Ministère de l’Education Nationale
de la Recherche et de la Technologie.
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