Molecular Human Reproduction, Vol.18, No.11 pp. 523–534, 2012
Advanced Access publication on August 16, 2012 doi:10.1093/molehr/gas030
ORIGINAL RESEARCH
Analysis of meiosis regulators in
human gonads: a sexually dimorphic
spatio-temporal expression pattern
suggests involvement of DMRT1 in
meiotic entry
Anne Jørgensen1, John E. Nielsen 1, Martin Blomberg Jensen 1,
Niels Græm 2, and Ewa Rajpert-De Meyts 1,*
1
Department of Growth and Reproduction, University of Copenhagen, Blegdamsvej 9, Copenhagen DK-2100, Denmark 2Department of
Pathology, Rigshospitalet, Blegdamsvej 9, Copenhagen DK-2100, Denmark
*Correspondence address. E-mail: [email protected]
Submitted on April 3, 2012; resubmitted on June 13, 2012; accepted on July 10, 2012
abstract: The mitosis–meiosis switch is a key event in the differentiation of germ cells. In humans, meiosis is initiated in fetal ovaries,
whereas in testes meiotic entry is inhibited until puberty. The purpose of this study was to examine the expression pattern of meiosis regulators in human gonads and to investigate a possible role of DMRT1 in the regulation of meiotic entry. The expression pattern of DMRT1,
STRA8, SCP3, DMC1, NANOS3, CYP26B1 and NANOS2 was investigated by RT –PCR and immunohistochemistry in a series of human
testis samples from fetal life to adulthood, and in fetal ovaries. DMRT1 was expressed in testes throughout development but with marked
spatio-temporal changes. At the early fetal period of 8–20 gestational weeks (GW) and at infantile mini-puberty, DMRT1 was predominantly
expressed in Sertoli cells, whereas at later stages of gestation (22 –40 GW), during childhood and in post-pubertal testes, DMRT1 was most
abundant in spermatogonia, except in the A-dark type. In fetal ovaries, DMRT1 was detected in oogonia and oocytes until 20 GW, but was
completely down-regulated following meiotic entry. STRA8, SCP3 and DMC1 were expressed mainly in oocytes and spermatogonia in accordance with their role in initiation and progression of meiosis. The putative meiosis inhibitors, CYP26B1 and NANOS2, were primarily
expressed in Leydig cells and spermatocytes, respectively. In conclusion, the expression pattern of the investigated meiotic regulators is
largely conserved in the human gonads compared with rodents, but with some minor differences, such as a stable expression of
CYP26B1 in human fetal ovaries. The sexually dimorphic expression pattern of DMRT1 indicates a similar role in the mitosis–meiosis
switch in human gonads as previously demonstrated in mice. The biological importance of the changes in expression of DMRT1 in
Sertoli cells remains to be established, but it is consistent with DMRT1 reinforcing the inhibition of meiosis in the testis.
Key words: CYP26B1 / gonadal development / SCP3 / Sertoli cells / STRA8
Introduction
The mitosis–meiosis switch is a unique feature of germ cell development and is one of the first manifestations of sex differentiation in the
developing gonads. In human fetal ovaries, meiosis is induced in the
first trimester of gestation, whereas in testes the entry of germ cells
into meiosis is inhibited until puberty and the onset of adult spermatogenesis. The current understanding of regulation of meiosis is derived
primarily from studies in mice (Bowles et al., 2006; Koubova et al.,
2006; Suzuki and Saga, 2008; Matson et al., 2010). It has been demonstrated that murine fetal germ cells acquire meiotic competence
through the involvement of Dazl in both XX and XY embryos (Lin
et al., 2008; Kee et al., 2009). Hereafter, the murine germ cells are
directed towards the alternative pathways of oogenesis and spermatogenesis mainly by the action of somatic cells that mediate the response
to the retinoic acid (RA) produced by the neighbouring mesonephros
of both sexes. In fetal ovaries, RA was shown to induce Stra8 expression followed by initiation of meiosis, while in the developing testes
this is prevented by Cyp26b1, the RA-degrading enzyme which is
expressed in Sertoli cells from 12.5 days post coitum (dpc) (Bowles
et al., 2006; Koubova et al., 2006). The model of RA-induced
meiosis was recently challenged by the findings of Kumar et al.
(2011) in Raldh22/2 mice lacking RA synthesis. These authors suggested that RA signalling is not required for induction of Stra8
& The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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524
expression in the fetal ovary and that the Cyp26b1-mediated prevention of meiosis initiation in fetal testes does not involve degradation of
RA (Kumar et al., 2011). The implications of these findings are discussed in detail in a recent review (Griswold et al., 2012).
Instead of meiosis, fetal male germ cells enter a state of mitotic quiescence (G0/G1 arrest) (Bowles and Koopman, 2010; Guerquin et al.,
2010) and the inhibition of meiosis is subsequently maintained by
Nanos2, which was proposed to take over the inhibition of Stra8 expression following Cyp26b1 (Suzuki and Saga, 2008; Bowles and
Koopman, 2010). However, very recently, Nodal, a member of the
transforming growth factor-b superfamily, was suggested to be an additional inhibitor of Stra8 and meiosis in fetal testes of mice (Souquet
et al., 2012). Furthermore, precocious meiotic entry was recently
reported in male mice with targeted loss of Dmrt1, and it was demonstrated that Dmrt1 restricts RA responsiveness in spermatogonia, directly represses Stra8 transcription and activates transcription of the
spermatogonial differentiation factor Sohlh1 (Matson et al., 2010). In
female mice, Dmrt1 appears to have the opposite effect as it transcriptionally activates Stra8 and is required for initiation of a normal
meiotic prophase thereby ensuring formation of a normal number of
ovarian follicles (Krentz et al., 2011). In accordance with this role,
Dmrt1 is only transiently expressed in murine ovaries and disappears
from germ cells by 15.5 dpc (Krentz et al., 2011).
In humans only few studies have investigated meiosis in fetal gonads,
primarily in females. Two recent studies of human fetal ovaries have
shown that initiation of meiosis takes place from around 12 weeks
of gestation (GW) by responding to the presence of RA (Le Bouffant
et al., 2010; Childs et al., 2011). Subsequently, STRA8 is up-regulated
and this is followed by the induction of SCP3 and DMC1 that are
markers of meiotic progression. Also, NANOS3 is implicated in
meiosis of human fetal ovaries based on the timing and level of expression (Childs et al., 2011). The RA-synthesizing enzyme ALDH1A also
seems to be involved in the regulation of meiosis in human female
germ cells as meiosis is prevented when ALDH is inhibited in cultured
fetal ovaries (Le Bouffant et al., 2010). In fetal testes, meiosis is inhibited and from around 12 to 15 GW gonocytes gradually mature into
pre-spermatogonia, which continue to divide by mitosis. The knowledge about the signalling responsible for inhibition of meiosis
remains limited. In contrast to mice, the expression pattern and
level of CYP26B1 appears to be relatively similar in human fetal
testes and ovaries. This suggests that CYP26B1-mediated degradation
of RA may not be the primary mechanism of meiosis inhibition in the
human fetal testes (Le Bouffant et al., 2010; Childs et al., 2011). In
contrast, the role of NANOS2 appears to be more conserved as it is
developmentally regulated and sexually dimorphically expressed also
in human gonads (Childs et al., 2011). Interestingly, DMRT1 has
been implicated in human sexual differentiation on the basis of the observation of male-to-female sex reversal in some patients with a deletion of at least one DMRT1 loci on chromosome 9p (Bennett et al.,
1993; reviewed in Matson and Zarkower, 2012) and of its malespecific expression in gonadal ridges and embryonic Sertoli cells
(Moniot et al., 2000). DMRT1 has also been localized to spermatogonia in the adult human testis (Looijenga et al., 2006), suggesting involvement in spermatogenesis, but the precise biological function
remains to be elucidated.
The aim of this study was to establish the expression pattern of
meiosis regulators in human germ cells, with particular emphasis on
Jørgensen et al.
DMRT1. The data show, for the first time, the pattern of expression
of meiosis regulators at protein level in the human gonads, including a
dynamic developmental pattern of expression of DMRT1, indicating a
key function of this gene in the sexually dimorphic regulation of the
mitosis–meiosis switch, as previously demonstrated functionally in
mice.
Materials and Methods
Tissue
A regional Committee for Medical Research Ethics in Denmark approved
the use of human tissues stored in the tissue archives of Copenhagen University Hospital. The tissue samples from adult men (n ¼ 25) were
obtained after orchidectomy for testicular cancer and only preserved fragments of testis parenchyma with complete spermatogenesis were used for
this study. Fetal tissues were obtained from planned induced abortions or
miscarriages, and only those without any signs of developmental disorders
on autopsy were selected for this study (n ¼ 26). The pre-pubertal testis
tissue samples (n ¼ 5) were from diagnostic biopsies to monitor for
relapses of acute leukemia and only samples without pathological
changes were selected. Details regarding the gestational age of fetal
gonads and pre-pubertal testis samples are listed in Table I. Tissue
samples were either fixed overnight in 4% buffered formalin or Stieve fixative (VWR, Leuven, Belgium) or snap-frozen in liquid nitrogen and stored
at 2808C. In addition, a few RNA samples that were collected in Sheffield
from abortion material for a previous study (Sonne et al., 2009) were used
for RT– PCR.
Reverse transcription – polymerase chain
reaction
Total RNA was extracted from a representative sample of the frozen specimens and isolated with NucleoSpin RNA II purification kit as described
by the manufacturer (Macherey-Nagel, Düren, Germany). cDNA was
synthesized using a dT20 primer and random hexamers. Specific primers
targeting each mRNA were designed (Table II) to span intron – exon
boundaries. RPS20 was used as an internal control. PCR conditions
were: 1 cycle of 5 min at 958C; 40 cycles of 30 s at 958C, 1 min at
628C, 1 min at 728C and 1 cycle of 5 min at 728C. Representative
bands from each primer combination were excised and sequenced for
verification (Eurofins MWG, Ebersberg, Germany).
Immunohistochemistry
Primary antibodies used in this study, dilutions and retrieval buffers
are listed in Table III. DMC1, STRA8 and NANOS2 were not investigated
in fetal and pre-pubertal samples because of an incompatibility of the
antibodies with the fixation method of these tissues. All primary antibodies
were tested for specificity by a western blot using normal adult testis
tissue with only one band detected for each antibody (data not shown).
Immunohistochemical staining was performed as previously described
(Blomberg Jensen et al., 2010). In brief, paraffin sections were deparaffinized and rehydrated. Antigen retrieval was accomplished by microwaving
the sections for 15 min in retrieval buffer. Then sections were incubated
with 2% non-immune goat serum (Zymed Histostain Kit, Life Technologies, San Francisco, CA, USA) or 0.5% skimmed milk powder diluted
in Tris-buffered saline (TBS) to minimize cross-reactivity. Primary antibody
was added and incubated for 16 h at 48C, and then the sections
were incubated with biotinylated goat anti-rabbit immunoglobulin G
(IgG) or biotinylated goat anti-mouse IgG, before a peroxidase-conjugated
streptavidin complex was used as a tertiary layer (Zymed Histostain Kit).
525
Regulation of meiotic entry in human gonads
Table I Expression of DMRT1 and meiosis regulators in fetal ovaries, fetal testes and pre-pubertal testes.
Tissue
Age
DMRT1
SCP3
CYP26B1
NANOS3
Nuclear
Cytoplasmic, nuclear*
Cytoplasmic
Perinuclear
...............................
..................................
............................
...............................
.............................................................................................................................................................................................
Fetal ovaries
8 GW
++/+
Oog
10 GW
All other cells
+
Oog
+1
Oog/Ooc
+2
Ooc
+
All other cells
+1
Oog
++/+
Ooc
+ 2*
Oog
+
All other cells
+1
Ooc
++/2 *
Oog
+
All other cells
+1
Oog
+
1
Ooc
++/2*
Ooca
+1
Oog
+1
All other cells
+1
Ooc
++/2*
Oog
+
All other cells
+1
Ooc
++/2*
Oog
+
All other cells
+1
Ooc
++/2*
Oog
+
All other cells
+1
Ooc
++/2*
Oog
+
All other cells
+1
Ooc
+/++*
All other cells
+
Ooc
+/++
All other cells
+
Ooc
+/++
All other cells
+
Ooc
+/++
All other cells
+
All cells
+1
a
Ooc
12 GW
Ooc
14 GW
Ooc
Ooc
Ooc
Ooc
24 GW
–
40 GW
14 GW
15 GW
17 GW
18 GW
++/+
–
27 GW
14 GW
++/2
–
26 GW
10 GW
+
1
++/+
Oog
a
Fetal testes
++/+
++/2
Oog
a
20 GW
++/+
+1
Oog
a
19 GW
++/+
++/+
Oog
a
17 GW
1
++/+
Oog
a
13 GW
+2
++/+
a
11 GW
Oog/Ooc
Ooc
a
–
Sertoli
++/+
Gon
+2
Sertoli
++/+
Gon
+2
Sertoli
Oog/Ooc
+2
Oog/Ooc
++
Oog/Ooc
+/2
Oog/Ooc
++
Oog/Ooc
+2
Oog/Ooc
++/+
Oog/Ooc
+/2
Oog/Ooc
++/2
Oog/Ooc
+/2
Oog/Ooc
++/+
Oog/Ooc
+2
Oog/Ooc
++/+
Oog/Ooc
+1
Oog/Ooc
++/+
Oog/Ooc
+2
Oog/Ooc
++/+
Oog/Ooc
+1
Oog/Ooc
++/+
Oog/Ooc
+2
Oog/Ooc
++/+
Oog/Ooc
+/2
Oog/Ooc
++/+
Oog/Ooc
+2
Oog/Ooc
++/+
Oog/Ooc
+2
Oog/Ooc
++/+
Leydig
++/+
Gon
++
Sertoli
+/2
++
Gon
+1
Gon
++
Gon
++
Gon
++
Gon
++/+
1
1
1
1
1
1
1
1
1
1
1
All cells
+/++
Leydig
Sertoli
+/2
++/+
Leydig
+/++
Leydig
++/+
Gon
+2
All other cells
+1
Sertoli
+/2
Sertoli
+2
All other cells
+ / ++
Leydig
+1
Gon
+
Leydig
+
1
Sertoli
+/2
Sertoli
++/+
All other cells
+1
Leydig
++/+
Gon
+/2
Leydig
+/++
Sertoli
2/+
Peritubular
2/+
Sertoli
++/+
All other cells
+
Leydig
+1
Gon
2/+
Leydig
+/++
Sertli
2/+
2
1
Continued
526
Jørgensen et al.
Table I Continued
Tissue
Age
DMRT1
SCP3
CYP26B1
NANOS3
Nuclear
Cytoplasmic, nuclear*
Cytoplasmic
Perinuclear
...............................
..................................
............................
...............................
.............................................................................................................................................................................................
20 GW
22 GW
24 GW
25 GW
33 GW
39 GW
41 GW
Childhood testes
2 month
7 years
10 years
1412 years
1512 years
Sertoli
++/+
Gon
+
Leydig
+/++
Sertoli
2/+
Sertoli
++/2
All other cells
+/++
Leydig
+1
Gon
++/2
Leydig
+/++
Sertoli
2/+
Sertoli
+2
All other cells
+/++
Leydig
++/2
Gon/pre-spg
++/+
Leydig
++/2
Sertoli
++/2
Sertoli
+/2
All other cells
2/+
Leydig
++/+
Pre-spg
++/+
Leydig
++/2
Sertoli
2/+
Sertoli
+/2
All other cells
+1
Leydig
+1
Pre-spg
++/+
Leydig
++/2
Sertoli
2/+
Sertoli
+2
All other cells
++/2
Leydig
++/+
Pre-spg
++/+
Leydig
++/2
Sertoli
++/2
Sertoli
+2
All other cells
+1
Leydig
+1
Pre-spg
++/+
Leydig
++/2
Sertoli
+
Pre-spg
–
All cells
+2
Leydig
Sertoli
++/+
Spg
++/2
All cells
+/2
All cells
+/2
2
Sertoli
–
Spg
+/2
All other cells
+1
Sertoli
+/2
Spg
++/2
Spg
++/2
Spg
++/2*
++/2
+1
Gon
++/–
Gon/pre-spg
++/+
Pre-spg
++
Pre-spg
+1
Pre-spg
++/+
Pre-spg
+1
+/2
Pre-spg
+2
Leydig
–
Spg
++/2
Leydig
–
Spg
+2
Leydig
+2
Spg
++/2
Leydig
+/2
Spg
++/2
1
b
Sertoli
+
All cells
+/2
Spg
++/2
Spg
++/2*
Sertoli
+1
All cells
+/2
1
Gon
Leydig
The expression is listed with the most prevalent staining listed first and intensity assessed using an arbitrary score: + +, strong staining in all cells of a given type in the sample; + +/+,
strong staining prevalent, but some weakly stained also visible; + +/2, strong staining present but negative cells also present; +/+ +/2, heterogeneous pattern with a mixture of
strongly positive, weakly stained and negative cells; +/+ +, majority of cells weakly stained, but some strong staining present; +, 1weak staining overall or 2strong staining in a small
number of cells; +/2, weak staining in limited areas; 2/+,weak staining in single cells; –, no staining.
GW, gestational week; Sertoli, Sertoli cells; Gon, gonocytes; pre-spg, pre-spermatogonia; Spg, spermatogonia; Leydig, Leydig cells; Oog/ooc, oogonia/oocytes; ND, not determined.
a
Expression detected only in a subpopulation of oocytes.
b
Only five positive cells were observed in the section. One sample for each developmental time-point was investigated.
*Nuclear SCP3 staining.
Visualization was performed with amino ethyl carbasole (Zymed Histostain Kit) yielding a deep red colour. Between incubation steps, the slides
were washed in TBS. For negative controls, serial sections were processed
with the primary antibody replaced by the dilution buffer alone. None of
the control slides showed any staining (data not shown). For each antibody
at least three independent sections of the same histology were investigated. Counterstaining was performed with Meyer’s haematoxylin. Serial
sections were stained with maturation markers to identify gonocytes
(OCT4), pre-spermatogonia (MAGE-A4), oocytes/oogonia (OCT4 and
MAGE-A4), immature Sertoli cells (AMH) and A-dark spermatogonia
(OCT2). Two investigators evaluated all stainings independently. Double
immunohistochemical staining was performed according to the manufacturer’s manual, Dako K5361 EnvisionTM G/2 double stain system,
rabbit/mouse DAB+/Permanent Red (Dako, Glostrup, Denmark),
except that incubation with DMRT1 and OCT2 or STRA8 antibodies
was overnight (O/N) at 48C for each antibody. In double-stainings
DMRT1 is brown, whereas OCT2 and STRA8 are dark pink. All sections
were first investigated manually on a Nikon Microphot-FXA microscope,
then scanned on a NanoZoomer (Hamamatsu Photonics, Herrsching
am Ammersee, Germany) and analysed using the software NDPview
(Hamamatsu Photonics). Intensity of staining was classified according to
an arbitrary semi-quantitative reference scale: ++, strong staining in all
cells of a given type in the sample; ++/+, strong staining prevalent,
but some weakly stained also visible; ++/2, strong staining present
but negative cells also present; +/+ +/2, heterogeneous pattern with
a mixture of strongly positive, weakly stained and negative cells;
+/++, majority of cells weakly stained, but some strong staining
present; +, weak staining overall1 or strong staining in a small number
of cells2; +/2, weak staining in limited areas; 2/+, weak staining in
single cells; 2, no staining. To visualize potential co-localization of
DMRT1 and OCT2, double staining was performed by immunofluorescence. Primary antibodies against DMRT1 (1:800) and OCT2 (1:50)
were applied O/N at 48C and for 1 h at RT, followed by incubation
with the secondary antibodies FITC goat anti-rabbit (Alexa Flour 488, Invitrogen) and TRITC donkey anti-mouse (Alexa Flour 568, Invitrogen) both
1:600 for 45 min at RT. Finally, the sections were counterstained with
527
Regulation of meiotic entry in human gonads
Table II RT –PCR primers.
Gene
Forward primer
Reverse primer
Amplicon size
.............................................................................................................................................................................................
DMRT1
GGAGGAATTGGGTATCAGCCA
GGATGACCATACGTCCCTCTG
173
STRA8
CTGTGGCAGAACCTCTCGG
GAACCTCACTTTTGTCCAGGAA
238
SYCP3
TCAAAGGCAGAAGCTTAACCAA
CTTGCTGCTGAGTTTCCATCA
320
DMC1
TACCCTCTGTGTGACAGCTCA
GAAGATGCCAGCTTCTTCATG
250
CYP26B1
GAGCTGATCTTTGCGGCCTAT
GCGCATGACCTCCTTGATGA
216
NANOS2
GAGTCCCGCCACGTCTACT
TAGGGACTTGGGTGGGATG
270
NANOS3
GACGCTTCTGCCCACTTACTG
GAAGGCGAAGGCTCAGACTTC
194
ALDH1A1
CTGCATCAAAACATTGCGCTAC
AACCAACGGGAAATTCCAAGG
148
ALDH1A2
AACAACGAGTGGCAGAACTCA
TCTGAAGCATCCATCCTTCTCC
176
ALDH1A3
GAAGTGGAAGAAGGAGATAAGCC
TCTGAGGGTTCTAATACAGCCC
243
RPS20
AGACTTTGAGAATCACTACAAGA
ATCTGCAATGGTGACTTCCAC
179
All primers shown in 5′ –3′ direction.
Table III Antibodies and conditions used for IHC.
Antibody
Species
Dilution IHC
Dilution IHC (fetal)
Retrieval buffer
Company
.............................................................................................................................................................................................
DMRT1
Rabbit
1:1200
1:800
Citrate
Sigma HPA027850
SCP3
Rabbit
1:1500
1:800
TEG
Novus NB300-232
STRA8
Rabbit
1:400
—
Citrate
Biosite AP11436B
DMC1
Rabbit
1:40
—
Citrate
Sigma HPA001232
CYP26B1
Rabbit
1:75
1:75
TEG
Sigma HPA012567
NANOS2
Rabbit
1:40
—
Citrate
Biosite AP2730B
NANOS3
Rabbit
1:400
1:400
Citrate
Abcam ab70001
OCT4
Mouse
1:50
1:50
Urea
Santa Cruz sc-5279
AMH
Mouse
—
1:100
Citrate
Gift from R. Cate
MAGE-A4
Mouse
1:1000
1:250
Citrate
Gift from G. Spagnoli
OCT2
Mouse
1:50
—
Citrate
Novo Castra Oct 207
4(6-diamidino-2-phenylindole) before they were mounted with Vectrashield. Fluorescence microscopy was conducted on an Olympus BX63
microscope using the cellSens Dimension Software (Olympus, Hamburg,
Germany).
Results
Expression pattern of meiosis regulators in
fetal gonads and pre-pubertal testis
Expression of DMRT1, SCP3, STRA8, DMC1, CYP26B1, NANOS2,
NANOS3 and ALDH1A1 was first determined at transcriptional level
in representative samples of fetal testis and ovary (age 11–12 GW)
with and without the presence of mesonephros in the samples
(Fig. 1A). DMRT1, CYP26B1, ALDH1A1, NANOS2 and NANOS3 were
expressed in all samples, whereas SCP3 and STRA8 were not detected
in fetal testes. DMC1 was not expressed in any of the samples at this
developmental stage.
The expression patterns of DMRT1, SCP3, CYP26B1 and
NANOS3 were then investigated at protein level in fetal gonads of
both sexes and pre-pubertal testes, the details of which are summarized in Table I. In order to identify the developmental stage of the
investigated germ cells, serial sections were stained with germ cell
markers OCT4 and MAGE-A4. In fetal testis, OCT4 staining is specific
to gonocytes, which can be found until around 20 –24 GW (and in
very few cells thereafter until post-natal infantile ‘mini-puberty’),
whereas MAGE-A4 is expressed in pre-spermatogonia from around
GW 17–19 but not in gonocytes (Aubry et al., 2001; Gaskell et al.,
2004; Honecker et al., 2004; Rajpert-De Meyts et al., 2004). In contrast, simultaneous expression of OCT4 and MAGE-A4 in fetal
ovaries with expression of both OCT4 and MAGE-A4 was detected
in the majority of oogonia, in accordance with previous studies
(Gjerstorff et al., 2007; Hoei-Hansen et al., 2007; Nelson et al.,
2007; Byskov et al., 2011).
In fetal and infantile testes, DMRT1 was detected both in germ cells
and in Sertoli cells, with changes in the relative expression pattern
between the two cell types observed at several developmental
stages (Figs 2, 3 and 5, Table I). In the earliest fetal testis samples
investigated in this study (10-20 GW), the most pronounced staining
528
Figure 1 (A) RT – PCR analysis of mRNA expression of meiosis
regulators in fetal testes and ovaries with or without mesonephros
(M) included in the sample. All samples are from 11 to 12 GW.
RPS20 is included as loading control. (B) RT – PCR analysis of
mRNA expression of meiosis regulators in normal adult testes and
ovaries. RPS20 was included as loading control.
was observed in Sertoli cells, while from 24 GW the most intense
staining was found in pre-spermatogonia (Figs 2 and 3). In a prepubertal testis sample from mini-puberty (2 months postnatally) an
intense staining of DMRT1 was observed in Sertoli cells with low or
no DMRT1 expression in pre-spermatogonia. In the older prepubertal testes (7 –1512 years), DMRT1 was primarily expressed in
spermatogonia with less pronounced staining in Sertoli cells. In fetal
ovaries, DMRT1 was germ-cell specific and highly expressed in
nuclei of oogonia and in a subset of oocytes until 20 GW (Fig. 2).
From this developmental time-point until 40 GW, expression of
DMRT1 was not detected in any of the investigated fetal ovaries
(Table I).
SCP3 was transiently expressed in nuclei of oocytes in fetal ovaries
around 12–20 GW. Generally, nuclei of oocytes were intensely
stained while oogonia were negative or with a faint staining (Fig. 4).
Subsequently, the SCP3 staining decreased in dictyate oocytes (in
the meiotic prophase). In fetal testes, no nuclear expression of
SCP3 was detected, whereas in pre-pubertal testis samples aged
10– 1512 years, nuclear staining of SCP3 was observed in some spermatogonia (Table I). In fetal gonads and pre-pubertal testis samples, a
weak cytoplasmic SCP3 staining was observed in both somatic cells
and germ cells, likely an unspecific reaction of the antibody, which
was not observed in adult testis samples. NANOS3 was expressed
in the cytoplasm of germ cells with a distinct perinuclear staining
Jørgensen et al.
Figure 2 Immunohistochemical expression and localization of
OCT4, DMRT1 and MAGE-A4 in serial sections from human fetal
testes (age 10 and 24 GW) and ovaries (age 12 and 20 GW).
OCT4 is included as a marker of gonocytes and oogonia, whereas
MAGE-A4 is a marker of pre-spermatogonia and oogonia. Arrows indicate DMRT1-stained gonocytes and pre-spermatogonia. Arrowheads represent DMRT1-stained Sertoli cells. Asterisks indicate
oogonia. Scale bar: 25 mm.
and such a staining pattern was observed in fetal testes, ovaries and
in pre-pubertal testes (Fig. 4).
CYP26B1 was expressed in fetal and pre-pubertal testes, primarily
in cytoplasm of Leydig cells with occasional faint expression in immature Sertoli cells. In fetal ovaries, CYP26B1 was faintly expressed in
cytoplasm of germ cells. At protein level, expression of CY26B1
was stronger in fetal testes compared with fetal ovaries. Interestingly,
in oocytes the CYP26B1 expression did not appear to be mutually
exclusive with SCP3.
Expression pattern of meiosis regulators in
adult testis
Expression of DMRT1, SCP3, STRA8, DMC1, CYP26B1, NANOS2,
NANOS3, ALDH1A1, ALDH1A2 and ALDH1A3 was first investigated
by RT –PCR in samples from normal adult testes and ovary
(Fig. 1B). Generally, all investigated meiosis regulators were expressed
in normal testes, whereas most were not expressed in adult ovary.
The gene expression pattern was in accordance with the immunohistochemistry (IHC) results when both gene and protein expression
were investigated in testis samples.
529
Regulation of meiotic entry in human gonads
The expression patterns of DMRT1, SCP3, STRA8, DMC1,
CYP26B1, NANOS2 and NANOS3 were then investigated at
protein level in adult testes with full spermatogenesis as summarized
in Table IV. DMRT1 was primarily expressed in nuclei of spermatogonia but was also detected in Sertoli cells. DMRT1 was absent in
A-dark spermatogonia, discriminated from A-pale cells by morphological features, especially the presence of a large nucleolus (von
Figure 3 Immunohistochemical expression and localization of
AMH and DMRT1 in serial sections from human fetal testes aged
10 and 24 GW. AMH is included as a marker of fetal Sertoli cells
Arrows indicate gonocytes and pre-spermatogonia, while asterisks
mark Sertoli cells. Scale bar corresponds to 25 mm.
Kopylow et al., 2012). This was confirmed by double-staining with
OCT2, which in the testis is a specific A-dark spermatogonia
marker (Lim et al., 2011), with no observation of co-expression
(Fig. 5). In contrast, DMRT1 was strongly positive in A-pale spermatogonia, and especially an intense nuclear staining was observed in Bspermatogonia and possibly also preleptotene spermatocytes but
not in the leptotene stage. No expression of DMRT1 was detected
in the pachytene and post-meiotic spermatocytes, spermatids,
Leydig cells and peritubular cells.
Expression of the pre-meiosis marker STRA8 and the two meiosis
markers SCP3 and DMC1 in adult testis is shown in Fig. 6. In normal
seminiferous tubules, STRA8 was weakly expressed in the nuclei of a
subpopulation of spermatogonia and spermatocytes. Co-expression of
STRA8 and DMRT1 in a subset of spermatogonia was confirmed by
double-staining (Fig. 5). SCP3 and DMC1 were predominantly
expressed in the nuclei of spermatocytes and in addition a faint staining
was observed in a small subpopulation of spermatogonia (Fig. 6). SCP3
was also occasionally weakly expressed in a subpopulation of round
spermatids. NANOS3 was highly expressed in a perinuclear ring in
spermatogonia and spermatocytes in normal testes (Fig. 6). In
normal adult testes, the RA-degrading enzyme CYP26B1 was intensely
stained in the cytoplasm of Leydig cells, but a weak staining was also
detected in the cytoplasm of some spermatocytes and peritubular
cells (Fig. 6). NANOS2 was moderately expressed in the nuclei of
spermatogonia and spermatocytes and faintly in some round spermatids (Fig. 6).
Unexpectedly, a weak-to-moderate cytoplasmic staining of
NANOS2, STRA8, SCP3 and DMC1 was observed in Leydig cells.
For DMC1 and STRA8, this expression was occasionally strong in a
small subset of the Leydig cells. This is most likely an unspecific reaction, since these markers of meiosis have not previously been
reported in Leydig cells. Because of the high content of peptides
and lipids, Leydig cells often show unspecific immunohistochemical reaction with various antibodies.
Figure 4 Immunohistochemical expression and localization of SCP3, CYP26B1 and NANOS3 in serial sections from human fetal testes (age
14 GW) and ovaries (age 12 GW). Arrows mark perinuclear staining of gonocytes. Asterisks represent oocytes. Scale bar: 25 mm.
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Jørgensen et al.
Figure 5 Immunohistochemical expression and localization of DMRT1. Serial sections of a post-natal infantile testes (2 month of age, corresponding
to mini-puberty) were stained with MAGE-A4, DMRT1 and OCT2. Arrows mark pre-spermatogonia stained with MAGE-A4 but negative for DMRT1.
Asterisks represent DMRT1-positive Sertoli cells. Scale bar: 25 mm. Adult testes with full spermatogenesis stained with DMRT1 (red staining). Followed by double-staining of DMRT1 (brown) and OCT2 or STRA8 (red), respectively. Arrows mark DMRT1-stained spermatogonia (A-pale or
B). Arrowheads represent OCT2-stained A-dark spermatogonia or STRA8-stained B-spermatogonia. Asterisks indicate DMRT1-stained Sertoli
cells. Scale bar corresponds to 50 mm. Immunofluorescence in adult testis with full spermatogenesis stained with DMRT1 (green), OCT2 (red)
and DAPI (blue) as nuclear counterstaining. DIC, differential interference contrast. Scale bar corresponds to 10 mm.
Discussion
In this study, we examined the developmental pattern of expression of
several known and putative meiosis regulators and provide novel findings that implicate DMRT1 as a key factor involved in the regulation of
meiosis in human gonads, in analogy to the recently reported data in
mice (Matson et al., 2010; Krentz et al., 2011). We show that DMRT1
is expressed in human testes throughout development and that it is
found in germ cells and Sertoli cells, but with dynamic and very specific
spatio-temporal changes (summarized in Fig. 7). Interestingly, a switch
in DMRT1 expression from predominantly Sertoli cells to germ cells
was observed in fetal testis samples, around 20 –22 GW, coinciding
with the acceleration of the differentiation of gonocytes into prespermatogonia. One previous study investigated DMRT1 expression
in human embryonic gonads by in situ hybridization and found abundant transcripts in a male genital ridge at 6 GW and in Sertoli cells
of a 7-GW fetal testis, with no expression in gonocytes (Moniot
et al., 2000). This could indicate that expression of DMRT1 in gonocytes is first initiated when the sexual cords are fully formed
between 8 and 10 GW. The functional importance of the strong
DMRT1 expression in immature Sertoli cells, which are in direct
contact with gonocytes, remains to be established, but the lack of
DMRT1 expression in granulosa cells that are the ovarian counterparts
of Sertoli cells indicates a role in masculinization of the testicular
somatic cells, as recently shown in mice (Matson et al., 2011;
Matson and Zarkower, 2012). The observation that there is a transient up-regulation of DMRT1 in Sertoli cells at infantile mini-puberty is
less certain (based on one sample) and more difficult to explain, but
during this early post-natal period, the last gonocytes are thought to
lose expression of pluripotency genes and settle in the contact with
peritubular cells thus maturing to infantile spermatogonia. Therefore,
we suggest that DMRT1 in Sertoli cells may be involved in indirectly
reinforcing the inhibition of meiosis during this milestone of germ
cell differentiation.
In adult testes, a distinct expression pattern of DMRT1 was found;
with DMRT1 completely absent in A-dark spermatogonia that constitute a self-renewing reserve population of spermatogonial stem cells,
which in contrast to A-pale and B-spermatogonia do not mature
during spermatogenesis. After a transient strong up-regulation of
DMRT1 expression in proliferating A-pale and especially in B-
531
Regulation of meiotic entry in human gonads
Table IV Expression of DMRT1 and meiosis regulators in normal adult testes.
Tissue/antibody
DMRT1
(n 5 22)
SCP3
(n 5 12)
DMC1
(n 5 7)
STRA8
(n 5 8)
CYP26B1
(n 5 8)
NANOS2
(n 5 8)
NANOS3
(n 5 7)
.............................................................................................................................................................................................
Normal testis
Spermatogonia
++/2 a
N
2/+
N
2/+
N
+2
N
–
Spermatocytes
+
N
++/2
N
++/2
N
++/2
N
2/+
Spermatids
–
N
–
–
–
Sertoli cells
++/+
–
–
–
Leydig cells
–
2/+
Peritubular cells
–
–
2
N
+2
2
+2
–
2
–
C
+/++
–
C
+/++
–
C
C
+2
N
++/2
PN
+2
N
++/2
PN
++/+
C
++/2
2/+
C
–
C
–
–
The expression intensity is presented as an arbitrary score, and the sub-cellular localization is marked as N (nuclear), C (cytoplasmic) or PN (perinuclear). Intensity of staining was
assessed using an arbitrary code: + +, strong staining in all cells of a given type in the sample; + +/+, strong staining prevalent, but some weakly stained also visible; + +/2, strong
staining present but negative cells also present; +/+ +/2, heterogeneous pattern with a mixture of strongly positive, weakly stained, and negative cells; +/+ +, majority of cells
weakly stained, but some strong staining present; +, 1weak staining overall or 2strong staining in a small number of cells; +/2, weak staining in limited areas; 2/+,weak staining in
single cells; –, no staining.
a
All A-dark spermatogonia are negative. n, number of samples.
Figure 6 Immunohistochemical expression and localization of SCP3, STRA8, DMC1, CYP26B1, NANOS2 and NANOS3 in normal human testes
with full spermatogenesis. Scale bar: 50 mm.
spermatogonia, DMRT1 was subsequently completely down-regulated
in the zygotene and pachytene stages, in accordance with previous
human studies (Looijenga et al., 2006; von Kopylow et al., 2012).
This expression pattern is slightly different compared with mice,
where DMRT1 is highly expressed in all A-type spermatogonia, but expression decreases already in B-spermatogonia and disappears with
initiation of meiosis (Matson et al., 2010). Interestingly, we observed
an overlap in expression of DMRT1 and STRA8 in a small subset of
B-spermatogonia in adult human testes. This is in contrast to results
from mice, where Dmrt1 and Stra8 are mutually exclusive in Bspermatogonia (Matson et al., 2010). Despite these small differences,
the overall similarity in expression pattern between mice and humans
indicates a conserved function of DMRT1.
In this study, we also established the expression pattern of DMRT1
in human fetal ovaries, never studied before at protein level around
the female meiosis entry. We found DMRT1 weakly expressed in a
subpopulation of oogonia and a strong DMRT1 expression in
oocytes only in a short developmental period from 8 to around
20 GW, coinciding with the meiotic entry of germ cells (summarized
in Fig. 7). Consistent with our findings, no DMRT1 expression was
detected at mRNA level in 6– 7 GW ovaries (Moniot et al., 2000).
These results suggest that expression of DMRT1 in human fetal
ovaries is initiated between 7 and 8 GW. The expression patterns
of DMRT1 in human and murine ovaries resemble one another,
except that we did not observe a switch to cytoplasmic staining
prior to complete down-regulation of DMRT1 as previously reported
in mice (Lei et al., 2007). We noticed that in human fetal ovary the
strongest DMRT1 expression is just prior to initiation of meiosis and
this resembles the strong DMRT1 expression in human Bspermatogonia immediately before meiosis in the adult testes. We
532
Jørgensen et al.
Figure 7 An overview of the human DMRT1 expression pattern. In fetal ovaries, DMRT1 is germ-cell specific with strong expression in oogonia and
a complete down-regulation coinciding with meiotic entry. In the fetal testis, DMRT1 is expressed in gonocytes and is strongly expressed during the
transition from gonocytes to pre-spermatogonia and in pre-spermatogonia. Furthermore, DMRT1 is expressed in fetal Sertoli cells with a switch in
expression level around 20– 22 GW, where expression becomes less pronounced. During mini-puberty around 2 – 3 month postnatal, a strong
DMRT1 expression is found in Sertoli cells, whereas DMRT1 is down-regulated in pre-spermatogonia. After pubertal onset of spermatogenesis,
DMRT1 remains expressed in Sertoli cells and in germ cells where DMRT1 is expressed proliferating A-pale and B-spermatogonia. After initiation
of meiosis, DMRT1 is completely down-regulated in spermatocytes. The very early expression pattern (before 8 GW) is uncertain and is therefore
indicated as a dotted line. Pre-spg, pre-spermatogonia; Spg, spermatogonia; Spc, spermatocytes; GW, gestational week.
therefore speculate that DMRT1 in adult human testes may be
involved in inducing meiosis rather than blocking it, as commonly
believed. The opposing roles of DMRT1 in fetal testes and ovaries
are intriguing and the currently available data suggest that part of
this is mediated through different effects on STRA8 expression.
However, the precise mechanism is not yet elucidated and new important players in the regulation of meiosis continue to emerge,
thereby indicating that we have just begun to understand in detail
which signalling factors are involved, how they are regulated and the
pathways they activate.
In addition to DMRT1, several other proteins are involved in the
regulation of meiosis. In mice, Cyp26b1 and Nanos2 are expressed
in a sexually dimorphic manner, with higher expression in testes
where they are considered meiosis inhibitors (Bowles et al., 2006;
Koubova et al., 2006; Suzuki and Saga, 2008; Kashimada et al.,
2011). In this study, CYP26B1 was primarily expressed in Leydig
cells and we found no difference in the transcript levels of CYP26B1
between human fetal testes and ovaries at 12 GW, when meiosis is
initiated in the latter. This is in accordance with a recent study by
Childs et al. (2011), who found no difference in expression between
human fetal testes and ovaries, except at GW 14 –16, where the
level of CYP26B1 was unexpectedly significantly higher in fetal
ovaries. Taken together, these data indicate that the expression
pattern of CYP26B1 in human samples might be different from that
reported in mice.
We found a similar expression of NANOS2 in fetal testes and
ovaries, which differs somewhat from previous results showing a
stronger expression of NANOS2 at 14 –20 GW in fetal testis
samples (Childs et al., 2011). Both human studies are in contrast to
mice, where Nanos2 expression is restricted to male germ cells
(Tsuda et al., 2003). On the basis of the currently available data on
NANOS2 expression in human fetal gonads, it is not possible to conclude whether or not this protein represses meiosis in human fetal
testes, as previously demonstrated in mice (Suzuki and Saga, 2008).
Another plausible candidate to be involved in meiosis regulation is a
NANOS2-related gene, NANOS3, as suggested by Childs et al.
(2011). However, we did not find a clear sexually dimorphic expression of NANOS3 in fetal gonads at mRNA level most likely because
of differences in the developmental time-point investigated, different
primers used and the more sensitive quantitative method used by
Childs et al. (2011). We also investigated NANOS3 expression by
IHC during fetal development (10 –40 GW) and did not detect any
clear sex-specific difference in expression level or pattern.
Transcripts of the meiosis markers STRA8 and SCP3 were detected
at 12 GW in fetal ovaries, whereas no expression of DMC1 was found
at this time-point. This is in accordance with previous human studies
showing STRA8 expression human fetal ovaries around 12 GW
(Houmard et al., 2009; Le Bouffant et al., 2010; Childs et al., 2011)
and DMC1 expression from 15 weeks post fertilization (13 GW)
(Le Bouffant et al., 2010). We observed nuclear SCP3 staining in a
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Regulation of meiotic entry in human gonads
few single oocytes already from 11 GW by IHC, which corroborates
the observed mRNA expression in 12 GW fetal ovaries. However, we
did consistently detect a cytoplasmic SCP3 staining in all cells in fetal
gonads and pre-pubertal testis, which could be an unspecific reaction,
probably due to the fact that the samples were primarily obtained
from autopsies. However, SCP3 expression not associated with chromatin was previously described in male germ cells in mice and cattle
(Di Carlo et al., 2000; Pfeifer et al., 2003). Therefore, the cytoplasmic
SCP3 staining in male germ cells detected in this study could reflect
the preparatory stage to meiosis-inducing signals. Together these
results imply that several aspects of meiosis initiation are conserved
between mice and humans. An interesting difference is that in mice
RA is produced in mesonephros and diffuses to the gonads,
whereas two recent studies in humans indicate that the gonads also
possess an intrinsic capacity to synthesize RA based on the expression
of the RA-synthesizing enzyme ALDH1A (Le Bouffant et al., 2010;
Childs et al., 2011). The transcriptional expression of ALDH1A1 in
fetal testes and ovaries was confirmed in this study, with a similar expression level in samples with and without mesonephros. Le Bouffant
et al. (2010) suggested that ALDH1A1 may play a role in the initiation
of meiosis as the increase in its expression level coincided with initiation of meiosis. In contrast, Childs et al. (2011) did not find any increase in expression of ALDH1A13 with initiation of meiosis of fetal
ovaries. Thus, the precise role of ALDH1A in human fetal gonads
needs to be investigated further.
In the adult testes, expression of STRA8, SCP3 and DMC1 was
found predominantly in spermatocytes, which is expected, given
their role in initiation and progression of meiosis. The strong STRA8
expression in a subpopulation of B-spermatogonia was in accordance
with a previous report (Oulad-Abdelghani et al., 1996). The putative
inhibitor of meiosis, NANOS2, was also present in a subpopulation
of spermatogonia and spermatocytes, in accordance with a previous
study that found NANOS2 expressed in a perinuclear ring of spermatogonia, spermatocytes and round spermatids (Kusz et al., 2009).
Interestingly, in our study NANOS3, rather than NANOS2, was localized in a well-defined perinuclear ring around germ cells throughout
development in testes and in fetal ovaries. A recent study also
found NANOS3 in human germ cells, except it was localized in
nuclei (Julaton and Reijo Pera, 2011). Interestingly, they also demonstrated functionally that NANOS3 is required for pluripotency as
well as meiotic initiation and progression (Julaton and Reijo Pera,
2011), thereby indicating that the role of NANOS3 in meiosis
should be investigated in more detail in future studies.
In conclusion, this study extends previous observations in mice and
provides novel information describing the spatio-temporal expression
pattern of proteins involved in the regulation of meiosis in human
testes and ovaries, demonstrating similarities but also a few differences
compared with the findings in mice. The overall human expression
pattern of DMRT1, summarized in Fig. 7, indicates a conserved role
of DMRT1 in regulating the mitosis –meiosis switch presumably
through regulation of STRA8 expression. However, the sex-specific
difference in the function of DMRT1 needs further studies in order
to establish the exact mechanism. The strong expression of DMRT1
in Sertoli cells, especially during the early fetal period and minipuberty, which has not previously been reported, remains unexplained
but could suggest a role for DMRT1 in maturation of fetal Sertoli cells
or in reinforcing meiosis inhibition during these important
developmental time-points. Taken together, the expression pattern
of meiosis regulators in human gonads constitutes a reference for
further studies of the mitosis– meiosis switch in cancer and in disorders of sexual development.
Acknowledgements
We thank Dr L. Ruban and Prof. H. Moore (University of Sheffield) for
frozen fetal samples, Prof. R. Cate (Biogen) for the AMH antibody and
Prof. G. Spagnoli (University of Basel) for the MAGE-A4 antibody. We
are grateful to Prof. Niels Erik Skakkebaek for helpful discussions,
Dorte L. Egeberg for help with immunofluorescence and Ana Ricci
Nielsen, Brian Vendelbo Hansen and Betina F. Nielsen for excellent
technical assistance.
Authors’ roles
A.J. was involved in conception and design of the study, collection and
assembly of data, data analysis and interpretation, and drafted the
manuscript. J.E.N. was involved in collection and assembly of data,
data analysis and interpretation. M.B.J. was involved in data analysis
and interpretation. N.G. took part in provision of study materials.
E.R.D.M. was involved in conception and design of the study, provision
of study materials, data analysis and interpretation and manuscript
writing. All the authors contributed to writing and approved the
final version of the manuscript.
Funding
This work was supported by the VKR Foundation, the Lundbeck Foundation and Danish Cancer Society.
Conflict of interest
None declared.
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