© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 4298-4310 doi:10.1242/dev.112888
RESEARCH ARTICLE
The zinc-finger protein basonuclin 2 is required for proper mitotic
arrest, prevention of premature meiotic initiation and meiotic
progression in mouse male germ cells
Amandine Vanhoutteghem1, Sé bastien Messiaen2, Françoise Hervé 1, Brigitte Delhomme1, Delphine Moison2,
Jean-Maurice Petit3, Virginie Rouiller-Fabre2, Gabriel Livera2 and Philippe Djian1, *
Absence of mitosis and meiosis are distinguishing properties of
male germ cells during late fetal and early neonatal periods.
Repressors of male germ cell meiosis have been identified, but
mitotic repressors are largely unknown, and no protein repressing
both meiosis and mitosis is known. We demonstrate here that the
zinc-finger protein BNC2 is present in male but not in female germ
cells. In testis, BNC2 exists as several spliced isoforms and
presumably binds to DNA. Within the male germ cell lineage,
BNC2 is restricted to prospermatogonia and undifferentiated
spermatogonia. Fetal prospermatogonia that lack BNC2 multiply
excessively on embryonic day (E)14.5 and reenter the cell cycle
prematurely. Mutant prospermatogonia also engage in abnormal
meiosis; on E17.5, Bnc2−/− prospermatogonia start synthesizing
the synaptonemal protein SYCP3, and by the time of birth,
many Bnc2−/− prospermatogonia have accumulated large
amounts of nonfilamentous SYCP3, thus appearing to be blocked
at leptonema. Bnc2−/− prospermatogonia do not undergo proper
male differentiation, as they lack almost all the mRNA for the malespecific methylation protein DNMT3L and have increased levels of
mRNAs that encode meiotic proteins, including STRA8. Bnc2−/−
prospermatogonia can produce spermatogonia, but these enter
meiosis prematurely and undergo massive apoptotic death during
meiotic prophase. This study identifies BNC2 as a major regulator of
male germ stem cells, which is required for repression of meiosis
and mitosis in prospermatogonia, and for meiosis progression
during spermatogenesis. In view of the extreme evolutionary
conservation of BNC2, the findings described here are likely to
apply to many species.
KEY WORDS: BNC2, Gonocytes, Mouse, Spermatogonia,
Transcription
INTRODUCTION
In mammals, primordial germ cells remain sexually bipotential
during their migration across the embryo; they become sexually
differentiated upon reaching the fetal gonad. Sexual differentiation
is correlated with the timing of entry into meiosis. In mice, female
1
Laboratoire de physiologie cé ré brale, Centre National de la Recherche
2
Scientifique, Université Paris Descartes, UMR 8118, Paris, France. Laboratoire de
dé veloppement des gonades, Université Paris Diderot, Sorbonne Paris Cité ,
INSERM U967, CEA/DSV/iRCM/SCSR, Fontenay-aux-Roses F-92265, France.
3
Service central de microscopie, Centre Universitaire des Saints-Pères, Université
Paris Descartes, Paris, France.
*Author for correspondence ( [email protected])
Received 16 May 2014; Accepted 11 September 2014
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germ cells enter meiosis shortly after they reach the fetal gonad,
whereas male germ cells become quiescent in the fetal gonad and
initiate meiosis after birth, when spermatogonia start forming
spermatocytes. Transplantation studies have shown that bipotential
germ cells are intrinsically programmed to differentiate following
the female pathway and to undergo early meiosis (McLaren and
Southee, 1997). Prevention of meiosis in fetal testis is achieved
through synthesis in male Sertoli cells of CYP26B1, an enzyme that
degrades the retinoic acid necessary for meiosis induction (Bowles
et al., 2006; Koubova et al., 2006). When the level of testicular
CYP26B1 decreases, prevention of meiosis is taken over by
NANOS2, a protein that is synthesized by fetal male germ cells in
the absence of retinoic acid signaling (Suzuki and Saga, 2008).
During spermatogenesis, spermatogonia undergo meiosis, in
the first division of which homologous chromosomes pair,
recombine and synapse. Disturbance of any of these processes
leads to massive apoptosis-mediated death of the spermatocytes at
a specific check point in stage IV of the seminiferous epithelial
cycle (Hamer et al., 2008).
Basonuclin 2 (BNC2) is an extremely conserved zinc-finger
protein (Romano et al., 2004; Vanhoutteghem and Djian, 2004). It is
orthologous to BNC1, a protein that is essential for germ cells
(Mahoney et al., 1998; Tseng and Green, 1992; Yang et al., 1997;
Zhang et al., 2012). Mice with a homozygous disruption of Bnc2 die
shortly after birth from a cleft palate (Hervé et al., 2012;
Vanhoutteghem et al., 2011, 2009).
Because of its abundance in testis (Vanhoutteghem and Djian,
2004), we thought that BNC2 might have a function in male
reproduction in addition to its function in palatal mesenchymal
cells. Direct evidence for a function of BNC2 as a DNA-binding
protein that regulates meiosis and mitosis in male germ cells is
described below.
RESULTS
In mouse testis, BNC2 is presumably a DNA-binding protein
and exists as several splicing isoforms
In this study, we have used the previously reported Ayu21:18 mouse
line, the Bnc2 gene of which is disrupted by a gene-trap insertion.
The disrupted allele is likely to be null because all major
Bnc2 mRNA isoforms are absent from the homozygous mutant
(Vanhoutteghem et al., 2009).
We have previously described, in human keratinocytes, an
insoluble form of human BNC2, which colocalizes with the splicing
factor SC35 in nuclear speckles and has a presumed function in
nuclear RNA processing. The apparent molecular mass of insoluble
BNC2 is 145 kDa (Vanhoutteghem and Djian, 2006). We also
found that, in human keratinocytes, there exists a second BNC2
isoform with a molecular mass of 160 kDa and a homogenous
DEVELOPMENT
ABSTRACT
distribution throughout the nucleus. The two human isoforms result
from alternative splicing of the transcript.
As shown in Fig. 1A, when the testes of newborn mice were
analyzed by western blotting with an antibody against amino acid
residues 722-849 of BNC2 (herein referred to as BNC2722-849),
three protein bands in the range of 120-160 kDa were detected in
Bnc2+/+ and Bnc2+/− testis, but not in Bnc2−/− testis. These proteins
were present in the nuclear extract and absent from the cytoplasm.
The major protein ran at ∼160 kDa, and we thought it probable that
it was the mouse equivalent of human soluble BNC2. The identity
of the 160-kDa protein was confirmed by its staining with another
antibody against BNC2 (supplementary material Fig. S1A).
Testicular BNC2 was found in the soluble nuclear extract and in
association with chromatin. No BNC2 was detected in either the
insoluble nuclear fraction or the cytoplasm (Fig. 1B). We conclude
that in mouse testis, BNC2 is a largely soluble nuclear protein with
an apparent molecular mass of ∼160 kDa.
Both of the antibodies against BNC2722-849 and amino acid
residues 661-782 of BNC2 produced homogenous nuclear staining
in newborn testis (supplementary material Fig. S1B-E). Such a
diffuse nuclear distribution is in keeping with the presence of
BNC2 in the soluble nuclear extract and its association with
chromatin. This suggests that BNC2 is a DNA-binding protein
in testis.
Development (2014) 141, 4298-4310 doi:10.1242/dev.112888
Because alternative promoters and alternative splicing are
prominent features of the human BNC2 gene, we searched mouse
testis for alternative Bnc2 promoters and alternatively spliced Bnc2
transcripts. For promoter mapping, the 5′ end of the mRNA was
amplified by 5′ rapid amplification of cDNA ends (RACE), resolved
by using agarose gel electrophoresis and then sequenced. A single
transcription start site in exon 1 was found (supplementary material
Fig. S2A,B). Alternative exons were identified by using RT-PCR
with primers in either exons 1 and 3, or 4 and 6. The unfractionated
products were cloned and sequenced. Because a large number of
clones were sequenced, we could obtain an estimate of the relative
abundance of each Bnc2 mRNA isoform. We found a total of 11
exons, of which five were constitutive (exons 1, 3, 4, 5 and 6) and six
were alternative (1a, 2, 2a, 2c, 5cS, 5cL and 5b) (supplementary
material Fig. S2C). The 5′ alternative exons (1a, 2, 2a and 2c) were
all in frame with the rest of the coding region and caused variations
in the N-terminal part of the protein, whereas the 3′ alternative exons
(5cS, 5cL and 5b) resulted in either premature termination and
suppression of the last three zinc-fingers or disruption of finger 4
with conservation of fingers 5 and 6. As a result, testis contained a
family of BNC2 proteins with a constant central sequence that
included the three N-terminal zinc-fingers and the nuclear
localization signal, but variable N-terminal regions and variable
numbers of C-terminal zinc-fingers (Fig. 1C).
Fig. 1. BNC2 exists as multiple isoforms, the major of which is soluble and associated with chromatin. (A,B) Western blot analysis of newborn testis.
(A) BNC2 runs as a 160-kDa protein in nuclear extracts (NE) of Bnc2+/+ and Bnc2+/− testes (arrows). The protein is neither found in cytoplasm (Cyt) nor in the
mutant. Two weaker bands with an electrophoretic mobility of ∼140 and ∼120 kDa might correspond to splicing or degradation products of BNC2. Proteins with an
electrophoretic mobility of 100 kDa or less are stained nonspecifically, as they are found in Bnc2−/− testis. (B) Subnuclear fractionation. BNC2 (arrows) is found in
nuclear (NE) and chromatin (Chr) extracts; it is absent from the insoluble nuclear fraction (Nins) and cytoplasm (Cyt). (C) Map of the Bnc2 gene and the splicing
isoforms detected by using RT-PCR. Exons are represented by green boxes and designated by Arabic numerals. Intron sizes (kb) are shown in between the
green boxes. The arrowhead indicates the most abundant RNA isoform. BNC2 protein isoforms resulting from alternative splicing are shown in the schematic to
the right. Black and red vertical bars represent zinc-fingers and nuclear localization signal, respectively. Protein isoforms vary by their N-terminal regions and/or
number of C-terminal zinc-fingers. (D) Agarose gel electrophoresis of RT-PCR products. Using primers in exons 1 and 5 (1+5), the most abundant isoform
contains exons 1, 2, 2a, 3, 4 and 5. Using primers in exons 5 and 6 (5+6), the most abundant isoform contains exon 6 directly spliced to exon 5. Therefore, the
major isoform contains exons 1, 2, 2a and 3-6. (E) Western blot analysis of nuclear extract from HeLa cells that had been transduced with lentivirus encoding
the 1, 2, 2a, 3-6 isoform of BNC2 fused to c-myc. In transduced cells that had been stained with an antibody against myc (+LVmyc), myc-tagged BNC2 runs at
160 kDa (arrow). It is not detected in nontransduced cells (−LVmyc). Staining of transduced cells with an antibody against amino acid residues 1-34 of
BNC2 (+LVBNC2) confirms that the 160-kDa protein is BNC2 (arrow).
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DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
The most abundant RNA isoform was composed of exons 1, 2,
2a, 3-6 (Fig. 1D) and encoded a 1099-amino acid protein. All other
isoforms were much less abundant. The RNA comprising exons 1,
2a and 3-6, which is fairly abundant in human keratinocytes
(Vanhoutteghem and Djian, 2007) and encodes the speckleassociated BNC2, was not detected, thus explaining the absence
of speckle-associated BNC2 in mouse testis. Lentiviral expression
in HeLa cells of the 1, 2, 2a, 3-6 isoform fused to c-myc resulted in
the production of soluble BNC2 that had an apparent molecular
mass undistinguishable from that of the testicular protein (Fig. 1E).
BNC2 is found in prospermatogonia and undifferentiated
spermatogonia. It is absent from Sertoli and Leydig cells
To define the cell types expressing Bnc2 in fetal testis,
paraffin-embedded sections of Bnc2+/+ testes were stained
by using immunohistochemistry and the antibody against
BNC2722-849 (subsequently used in all immunofluorescence
and immunohistochemistry analyses). We first examined testes
on embryonic day (E)14.5, when germ cells undergo male
differentiation. As shown in Fig. 2A, abundant nuclear BNC2
was detected in prospermatogonia, identified by their central
position within the testicular cords and by the presence of
VASA (DDX4 – Mouse Genome Informatics) (Castrillon et al.,
2000). Sertoli cells, which entirely form the tubular epithelium
until postnatal day (P)6, did not possess detectable levels of
BNC2. This was confirmed by double staining for BNC2 and
anti-Müllerian hormone. Some interstitial cells possessed
smaller amounts of BNC2 (Fig. 2B). To determine whether
Development (2014) 141, 4298-4310 doi:10.1242/dev.112888
these interstitial cells were Leydig cells, we double-stained
testis for BNC2 and for 3-β-hydroxysteroid dehydrogenase
(Majdic et al., 1998). No BNC2 was detected in Leydig cells
(Fig. 2C). On E18.5, BNC2 was found in the nuclei of
prospermatogonia at a lower level than that on E14.5. There was
also some protein in interstitial cells. Neither Sertoli nor Leydig
cells possessed detectable BNC2 (Fig. 2E,F).
Postnatal distribution of BNC2 was investigated by indirect
immunofluorescence staining. At birth, BNC2 was found to occur in
prospermatogonia and interstitial cells (Fig. 3A) but was absent
from both Sertoli (Fig. 3B) and Leydig cells (Fig. 3C). On P8,
prospermatogonia had been replaced by spermatogonia, located in
the basal layer of the tubular epithelium, whereas spermatocytes
had started to form suprabasally. BNC2 was confined to some
basal cells, identified as undifferentiated spermatogonia by the
presence of the promyelocytic leukemia zinc-finger protein (PLZF;
ZBTB16 – Mouse Genome Informatics), a marker of spermatogonial
progenitor cells (Buaas et al., 2004; Costoya et al., 2004). BNC2 was
absent from both differentiated spermatogonia and spermatocytes
(Fig. 3D-F). On postnatal day 30, BNC2 was found in a few cells
within the tubules and in some cells outside of the tubules. Within the
tubules, BNC2-positive cells were invariably basal; they were
identified as undifferentiated spermatogonia by double staining for
BNC2 and PLZF; all tubular BNC2-containing cells possessed PLZF
and vice versa. Differentiating spermatogonia, spermatocytes and
spermatids lacked both BNC2 and PLZF (Fig. 3G-I). The BNC2containing cells located outside of the tubules (cells stained red in
Fig. 3G,I) probably corresponded to the nonsteroidogenic interstitial
DEVELOPMENT
Fig. 2. Cellular distribution of BNC2 in fetal testis. Paraffin
sections of fetal testis double-stained for BNC2 and markers of
prospermatogonia, Sertoli and Leydig cells. (A-C) E14.5.
(A) Prospermatogonia identified by the presence of cytosolic
VASA contain an abundant amount of nuclear BNC2. Sertoli ‘S’
cells (arrows) lack BNC2. (B) Double staining for BNC2 and antiMü llerian hormone (AMH), a Sertoli cell marker. Sertoli cells
contain abundant AMH but no BNC2. Some interstitial cells
contain BNC2 at a lower level than that in prospermatogonia.
(C) Double staining for BNC2 and Leydig cell-specific 3-βhydroxysteroid dehydrogenase (3βHSD). Leydig cells lack BNC2
(arrowheads). Prospermatogonia contain BNC2, but Sertoli cells
do not. (D-F) E18.5. Results are similar to those at E14.5, except
that BNC2 level in prospermatogonia is lower. In B,C,E,F, whole
testis is shown in the upper images, a higher magnification is
shown in the lower images. i, nonsteroidogenic interstitial cells; p,
prospermatogonia. Scale bars: 25 μm.
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Development (2014) 141, 4298-4310 doi:10.1242/dev.112888
Fig. 3. Postnatal BNC2 testicular distribution. Frozen sections
double-stained by using indirect immunofluorescence for BNC2
and various germ and somatic cell markers. DNA is stained blue
with DAPI. (A-C) BNC2 distribution on P0. Insets show details of
main frames. (A) BNC2 and VASA double staining. BNC2 is present
in VASA-containing prospermatogonia (arrowheads) and interstitial
cells, and absent from Sertoli cells ‘S’. (B) BNC2 and AMH staining.
Sertoli cells contain AMH but no BNC2 (arrowheads). (C) BNC2
and 3βHSD staining. BNC2 is absent from Leydig cells that were
identified by the presence of 3βHSD (arrowheads). (D-F) BNC2 and
PLZF staining at P8 to assess BNC2 distribution during incipient
spermatogenesis. BNC2 is strictly colocalized with PLZF; therefore,
it is specific to undifferentiated spermatogonia and absent from
differentiated spermatogonia and spermatocytes. D,E show the
single stains, F shows the merged images. (G-I) BNC2 and PLZF
staining at P30. BNC2 and PLZF are colocalized in undifferentiated
spermatogonia (arrowheads), which are very sparse at this age.
Neither BNC2 nor PLZF is detected in the progeny of
undifferentiated spermatogonia. Some interstitial cells contain
BNC2 (arrow). G,H show the single stains, I shows the merged
images. i, nonsteroidogenic interstitial cells; p, prospermatogonia.
Scale bars: 50 μm (A-C); 25 μm (D-I).
Fetal Bnc2−/− prospermatogonia multiply excessively on
E14.5 and reenter the cell cycle prematurely
In view of the abundance of BNC2 in prospermatogonia, we thought
that the protein might have a function in these cells. The fact that
BNC2 has been implicated in cell multiplication (Vanhoutteghem
et al., 2009) prompted us to compare the mitotic status of the
prospermatogonia of the Bnc2 −/− mice with that of their wild-type
littermates. During the fetal period, prospermatogonia divide
actively from around E12 and then gradually become quiescent
between E14 and E16, depending on the mouse strain (Vergouwen
et al., 1991). We analyzed fetal germ cell proliferation by using
immunohistochemical staining of testicular sections for VASA and
Ki-67 (MKI67 – Mouse Genome Informatics), a nuclear marker of
multiplying cells (Gerdes et al., 1983). On E14.5, ∼40% of Bnc2+/+
germ cells contained Ki-67, whereas almost all Bnc2−/− germ cells
expressed Ki-67 (Fig. 4A-C). Real-time quantitative PCR (RTqPCR) analyses have indicated that Lefty and Nodal, two genes
known to be expressed in mitotically active prospermatogonia
(Souquet et al., 2012), are overexpressed in Bnc2−/− testis at E14.5.
Expression of neither Nanos2, a marker of male differentiation, nor
Oct4 (Pou5f1 – Mouse Genome Informatics), a germ cell marker,
was affected (supplementary material Fig. S3). On both E17.5 and
E18.5, all prospermatogonia in both Bnc2−/− and Bnc2+/+ testes
lacked Ki-67 (Fig. 4D-G). Therefore, we conclude that fetal
prospermatogonia lacking BNC2 multiply excessively on E14.5
but do reach mitotic quiescence by E17.5.
We then examined newborn testes by staining paraffin-embedded
sections with hematoxylin & eosin (HE). In Bnc2+/+ newborns,
prospermatogonia were evident at the center of the cords as large
round cells with a spherical nucleus and dispersed homogenous
chromatin; no mitotic prospermatogonia were seen (Fig. 4H).
In Bnc2−/− mice, many prospermatogonia were similar to those of
wild-type littermates, but 20-30% showed condensed chromosomes
and appeared engaged in either mitosis or meiosis (Fig. 4I). To
determine whether prospermatogonia with condensed chromosomes
were engaged in mitosis, we double-stained newborn testes for Ki-67
and VASA. In the wild type, no prospermatogonia stained positively
for Ki-67; all cells containing Ki-67 were somatic cells, as they
lacked VASA (Fig. 4J). In the Bnc2−/− newborns, approximately onethird of the prospermatogonia contained Ki-67 and therefore were
engaged in the mitotic cycle (Fig. 4K).
Bnc2−/− prospermatogonia are engaged in an incomplete
form of meiosis
We then tested whether Bnc2−/− prospermatogonia could also be
engaged in meiosis. Testes were stained for synaptonemal complex
protein 3 (SYCP3), a marker of early meiosis (Lammers et al.,
1994). On E17.5, wild-type testis did not contain any SYCP3positive cells (Fig. 5A), whereas in the Bnc2−/− littermates, ∼10%
of the prospermatogonia contained foci of SYCP3 (Fig. 5B). By P0,
Bnc2+/+ prospermatogonia still stained negatively for SYCP3
(Fig. 5C), whereas in the Bnc2−/− testis, the proportion of
SYCP3-positive prospermatogonia had increased to 25-30%.
SYCP3 was mostly in the form of large aggregates (Fig. 5D) but
sometimes in threads typical of zygotene (Fig. 5E). The presence of
SYCP3 in Bnc2−/− prospermatogonia was confirmed using another
antibody against SYCP3. Because a nonfilamentous distribution of
SYCP3 is typical of cells in the preleptotene and leptotene stages
that have not yet formed synaptonemal complexes, we hypothesized
that most Bnc2−/− prospermatogonia that had entered meiosis had
remained thereafter blocked in preleptotene or leptotene. The
presence of the premeiotic-specific protein stimulated by retinoic
acid 8 (STRA8) (Koubova et al., 2006; Mark et al., 2008; OuladAbdelghani et al., 1996) confirmed the engagement of Bnc2−/−
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DEVELOPMENT
cells that had been identified at earlier ages. Therefore, in the germ
cell lineage, BNC2 was restricted to the most primitive cells –
prospermatogonia and undifferentiated spermatogonia.
RESEARCH ARTICLE
Development (2014) 141, 4298-4310 doi:10.1242/dev.112888
prospermatogonia in meiosis. STRA8 was not detectable in wildtype testis (Fig. 5F,G). In order to determine whether the
prospermatogonia that had abnormally engaged in meiosis were
undergoing apoptosis, we stained testes at E17.5, E18.5 and P0 for
activated caspase 3, which is associated with apoptotic cell death
(Nicholson et al., 1995). Almost no cleaved caspase 3-containing
cells were detected in either the Bnc2−/− mice or their wild-type
littermates (supplementary material Fig. S4A,B). Double staining
for SYCP3 and cleaved caspase 3 was performed on P0 testis; none
of the numerous SYCP3-containing prospermatogonia stained
positive for the caspase (supplementary material Fig. S4C,D).
We finally sought to determine whether the prospermatogonia
engaged in the mitotic cycle and containing Ki-67 (Fig. 4K)
were the same ones that aberrantly produced SYCP3. We triplestained newborn Bnc2−/− testes for VASA, SYCP3 and Ki-67;
prospermatogonia contained either Ki-67 or SYCP3, but never the
two proteins together (Fig. 5H,I).
BNC2 is required for expression of Dnmt3l and repression of
meiotic proteins in prospermatogonia
To identify the genes of which the expression was affected by a lack
of BNC2, we used massive parallel sequencing (RNA-Seq). To
achieve this, we prepared total RNA from the testes of six Bnc2−/−
and four Bnc2+/+ newborn mice. We first examined the ribosomal
(r)RNA gene transcripts. BNC1 is known to be a transcription factor
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for RNA polymerase I (Iuchi and Green, 1999; Tian et al., 2001).
The first pair of zinc-fingers of BNC2 is very similar to that of
BNC1 and has been shown to bind to the rRNA gene promoter
in vitro (Romano et al., 2004). We did not detect any decrease in the
number of 18S and 28S RNA molecules in the Bnc2−/− testes.
On the contrary, we observed a slight increase (supplementary
material Table S1). We conclude that BNC2 is not a transcription
factor for RNA polymerase I.
A list of genes specifically expressed in each testicular cell
type is given in the supplementary material Table S2. The
expression of the genes encoding DNMT3L and albumin showed
the greatest changes. Both genes are associated with male germ
cell differentiation and showed over a tenfold decrease in
expression. DNMT3L is a zinc-finger protein that resembles
cytosine-5-methyltransferase 3, but lacks its catalytic domains.
Dnmt3l is expressed from E14.5 until the perinatal period in
prospermatogonia, but not in oocytes; it is required for
retrotransposon inactivation (Aapola et al., 2000; Bourc’his and
Bestor, 2004; Bourc’his et al., 2001; Shovlin et al., 2007). The
albumin gene is specifically expressed in prospermatogonia of
newborn testis and is not expressed in the ovary (Gelly et al.,
1994; McLeod and Cooke, 1989). We also observed an increase
in the premeiotic-specific mRNAs encoding STRA8, MSX1 and
MSX2 (Le Bouffant et al., 2011), all of which are normally
specific to female germ cells during fetal life.
DEVELOPMENT
Fig. 4. Mitotic cycling of Bnc2−/− prospermatogonia in fetal and
neonatal testis. Sections of Bnc2+/+ and Bnc2−/− testis doublestained for VASA and Ki-67 or with HE. (A,B) E14.5. Paraffinembedded sections stained by using immunohistochemistry. (A) In
a substantial number of cords of wild-type testis, prospermatogonia
identified by the presence of VASA do not contain Ki-67.
(B) In Bnc2−/− testis, nearly all prospermatogonia possess Ki-67.
(C) The number of prospermatogonia engaged in the cell cycle is
significantly higher in homozygous mutant than in wild-type
embryos. At least 200 cells counted per testis. n=3, ***P<0.001,
unpaired Student’s t-test. Error bars show mean±s.d. (D,E) E17.5.
Frozen sections of whole testis stained by using indirect
immunofluorescence. All Bnc2+/+ and Bnc2−/− prospermatogonia
lack Ki-67 and therefore are quiescent. Many somatic cells contain
Ki-67. Single prospermatogonia lacking Ki-67 are shown to the right
at higher magnification. (F,G) Similar results at E18.5 were found on
paraffin sections stained by using immunohistochemistry.
(H,I) HE-stained paraffin sections of newborn testes. Single
prospermatogonia are shown to the right at higher magnification.
(H) In wild-type mouse, numerous prospermatogonia are present in
the center of testicular cords; they possess round homogenous
nuclei (arrowheads). (I) In mutant testis, many prospermatogonia
are engaged in cell division as shown by the lack of nuclear
membrane, chromosome condensation and symmetrical
distribution of chromosomes (arrowheads). (J,K) Frozen sections
of newborn testis examined by immunofluorescence staining. (J) In
wild-type testis, all prospermatogonia lack Ki-67 and therefore are
not engaged in mitosis. Only Ki-67-positive cells are somatic cells
(arrowheads). Prospermatogonia lacking Ki-67 and a somatic cell
lacking VASA but containing Ki-67 are shown at higher
magnification to the right. (K) In Bnc2−/− testis, numerous
prospermatogonia contain Ki-67 (arrowheads) and possess
condensed chromosomes typical of mitosis. Prospermatogonia
containing Ki-67 and engaged in mitosis are shown at higher
magnification to the right. Scale bars: 25 μm.
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Development (2014) 141, 4298-4310 doi:10.1242/dev.112888
Fig. 5. Abnormal meiosis in fetal and neonatal Bnc2−/−
prospermatogonia. Immunofluorescence staining of Bnc2+/+
and Bnc2−/− testis for the indicated combinations of VASA and
the meiotic proteins SYCP3, STRA8 and Ki-67. DNA is
stained with DAPI (blue). (A,B) E.17.5. (A) Wild-type
prospermatogonia contain VASA and no SYCP3. (B) Some
Bnc2−/− prospermatogonia contain SYCP3, which forms nuclear
foci (arrowheads). (C-I) P0. (C) Wild-type prospermatogonia do
not contain SYCP3. (D) 25-30% of Bnc2−/− prospermatogonia
contain aggregated SYCP3 (arrowheads). (E) A few Bnc2−/−
prospermatogonia contain threads of SYCP3, typical of zygotene
(arrowheads). (F) Neither STRA8 nor SYCP3 is detected in wildtype testis. (G) Both proteins coexist in some Bnc2−/−
prospermatogonia (arrowhead). (H) Black and white DAPI
staining, nuclei of prospermatogonia are clearly recognizable
(arrow and arrowheads). (I) Bnc2−/− prospermatogonia contain
VASA and either SYCP3 (arrow) or Ki-67 (arrowheads), but
not SYCP3 and Ki-67 together. Scale bars: 25 μm (A-D,F-I);
12.5 μm (E).
Large numbers of leptotene-blocked spermatocytes
accumulate in prepubertal Bnc2−/− mice
Even though the Bnc2 mutation is overwhelmingly lethal
(Vanhoutteghem et al., 2009), we obtained in the course of this
study six Bnc2−/− males that survived the perinatal period because
their palates closed. Because BNC2 is found in undifferentiated
spermatogonia, we thought that it might have a function in
spermatogenesis.
We first examined 9-day-old males. At this age, the germ cell
population comprises spermatogonia and spermatocytes at the
initial stages of meiosis (Bellvé et al., 1977; Goetz et al., 1984).
Undifferentiated spermatogonia were identified by double staining
for BNC2 and PLZF, and spermatocytes by the presence of SYCP3.
In 9-day-old wild-type mice, numerous PLZF-containing
undifferentiated spermatogonia were visible in the basal layer of
the tubules (Fig. 6A-C). The number of these spermatogonia was
not markedly different in the Bnc2−/− littermates (Fig. 6D-F). In
contrast to the similarity of the PLZF staining, the SYCP3 staining
differed between Bnc2−/− and wild-type testis. In the latter, only
∼12% of the tubules had started producing spermatocytes, in which
SYCP3 had mostly formed threads typical of the zygotene stage
(Scherthan et al., 1996) (Fig. 6G). In Bnc2−/− testes, over 80% of the
tubules contained vast numbers of SYCP3-positive cells, most of
which showed large aggregates of SYCP3; no zygotene
spermatocytes were observed (Fig. 6H). These aggregates were
similar to those seen in Bnc2−/− prospermatogonia (see Fig. 5B,D).
We thought it probable that cells with large aggregates had
accumulated SYCP3 that could not disperse in synaptonemal
complexes because the cells had not progressed to zygotene. These
spermatocytes did not contain activated caspase 3 and therefore
were not apoptotic (supplementary material Fig. S4E,F). We
conclude that in the 9-day-old Bnc2−/− mouse, the entry of
spermatogonia into meiosis is greatly accelerated, but the cells are
then blocked or delayed at the leptotene-zygotene transition.
BNC2 is required for meiotic progression of spermatocytes
We then compared the Bnc2−/− and Bnc2+/− testes of 1-month-old
mice. Staining with HE showed that all Bnc2+/− tubules contained
basal spermatogonia, several layers of spermatocytes and groups of
spermatids towards the lumen (Fig. 7A). By contrast, the Bnc2−/−
tubules could be divided into type I tubules that contained only
basal cells on most of their circumference (Fig. 7B), and type II
tubules that were stratified and sometimes contained typical
pachytene spermatocytes, often located in the center of the tubule
instead of in their normal peripheral location (Fig. 7C,D). No
spermatids were seen in any of the Bnc2−/− tubules. Type I tubules
represented 46% of total tubes (n=100).
In order to clarify the nature of the cells present in Bnc2−/−
tubules, we first characterized undifferentiated spermatogonia by
using PLZF staining. Heterozygous testes contained an average of
1.3 PLZF-positive spermatogonia per tubule (Fig. 7E); a similar
number was found in Bnc2−/− tubules (Fig. 7F). We then identified
mitotic and meiotic cells by double staining for Ki-67 and SYCP3.
In the Bnc2+/− testis, tubules generally possessed a small number of
basal cells that were positive for Ki-67 and a suprabasal layer of
meiotic cells with filamentous SYCP3 (Fig. 7G). In the Bnc2−/−
testis, tubules were smaller and could be divided into two types,
which were identified by using HE staining. Type I tubules
contained only basal cells, virtually all of which had nuclear Ki-67
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The RNA-Seq results for Dnmt3l and Stra8 were confirmed by
using RT-qPCR. On E18.5, the level of the methylase-like RNA
was approximately fourfold lower in Bnc2−/− testis than in that of
wild type. This difference reached tenfold on P0. On E18.5 and P0,
the levels of the Stra8 mRNA were ∼15- and 3.5-fold higher in
Bnc2−/− embryos than in wild-type embryos, respectively,
(supplementary material Fig. S5). Therefore, BNC2 is necessary
for proper male differentiation.
Fig. 6. Prepubertal Bnc2−/− mice possess spermatogonial stem cells but
accumulate large numbers of abnormal leptotene spermatocytes.
Immunofluorescence staining of testes of 9-day-old Bnc2+/+ and Bnc2−/− mice
for BNC2, PLZF and SYCP3. DNA is stained with DAPI (blue). (A-F) Double
exposure of BNC2 and PLZF. (A-C) In wild-type testis, undifferentiated
spermatogonia contain both BNC2 and PLZF (arrowheads). A,B show single
colors, C shows the merged image. (D-F) In Bnc2−/− testis, undifferentiated
spermatogonia contain PLZF (arrowheads) but no BNC2. Their number is not
obviously different from that of wild-type testis. D,E show single colors, F
shows the merged image. (G,H) Staining for SYCP3. (G) In wild-type testis, a
few tubules have formed meiotic cells, identified by the presence of SYCP3.
The inset shows that most spermatocytes are in zygotene. (H) Bnc2−/− testis
is smaller than wild-type testis (in keeping with the reduced overall size of
Bnc2−/− mice) and contains a large numbers of cells with staining of SYCP3,
mostly in the form of large aggregates (inset), presumably spermatocytes
arrested at leptonema. Scale bars: 25 μm.
and no SYCP3 and therefore were dividing. The situation was
reversed for type II Bnc2−/− tubules, which contained very few Ki67-positive cells, but numerous basal and suprabasal SYCP3positive cells (Fig. 7H).
The stage of meiosis of Bnc2+/− and Bnc2−/− cells was
determined by double staining for SYCP3 and phosphorylated
histone H2AX (γ-H2AX). Phosphorylation of H2AX is required for
the formation of γ-H2AX foci at meiotic double-strand breaks
(Mahadevaiah et al., 2001). γ-H2AX produces a weak diffuse
nuclear staining in type A spermatogonia (Hamer et al., 2003),
forms multiple foci in leptotene and zygotene spermatocytes and
concentrates in a unique XY body in pachytene spermatocytes.
Bnc2+/− testis contained numerous pachytene spermatocytes,
located suprabasally; in some tubules, leptotene and zygotene
spermatocytes were detected in the basal layer (Fig. 7I). In type I
tubules of Bnc2−/− testes, most of the cells showed weak diffuse
nuclear γ-H2AX staining and no SYCP3 staining, and therefore
were type A spermatogonia (Fig. 7J). The majority of these
spermatogonia were differentiating because they contained c-KIT
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(Fig. 7K), a marker of differentiating spermatogonia (Yoshinaga
et al., 1991). The differentiating Bnc2−/− spermatogonia were nearly
all engaged in the mitotic cycle as they contained both nuclear Ki-67
and histone H3 phosphorylated on serine residue 10, a protein that is
strictly associated with the chromosomal condensation that occurs
during mitosis and meiosis (Goto et al., 1999; Gurley et al., 1978)
(Fig. 7L). Bnc2−/− type II tubules showed intense staining of
SYCP3 and γ-H2AX over several suprabasal layers. Both SYCP3
and γ-H2AX had a distribution similar to that of leptotene-zygotene
spermatocytes, except that the staining was much more intense
(compare Bnc2−/− spermatocytes in Fig. 7M,N with normal
leptotene-zygotene spermatocytes indicated by the arrowhead in
Fig. 7I). We presumed that these cells were abnormal spermatocytes
blocked in leptotene-zygotene (Liebe et al., 2006; Mahadevaiah
et al., 2001). In other tubules, the cells in the luminal region
showed filamentous SYCP3 and XY bodies typical of pachytene
spermatocytes (Fig. 7N,O).
We were puzzled by the existence of two kinds of tubules in the
mutant testis, and we questioned whether, in type I tubules,
spermatogonia had been unable to form spermatocytes or whether
they had formed spermatocytes that subsequently underwent
apoptotic death. To investigate apoptosis, TdT-mediated dUTPbiotin end labeling (TUNEL) was used. A low level of apoptotic
death was observed in heterozygous testis (approximately one cell
per five tubules; see Fig. 8A). By contrast, ∼15% of the mutant
littermate tubules contained many apoptotic cells (Fig. 8B) that
accumulated in the suprabasal layers (Fig. 8C). Staining for
activated caspase 3 confirmed the results of the TUNEL assay
(Fig. 8D,E). We interpret the findings as follows: during
spermatogenesis in Bnc2−/− mice, spermatocytes enter meiosis
but undergo apoptotic death sometime during meiotic prophase.
Death of the spermatocytes during stages I-V of the cycle of the
seminiferous epithelium (Oakberg, 1956) results in type I tubules,
which contain only spermatogonia until stage VI, when new
preleptotene spermatocytes are formed that enter leptotene in stage
VIII (Fig. 7M) and pachytene at the end of stage XII (Fig. 7N), thus
generating type II tubules. The situation observed here is analogous
to that of the SYCP3 mutant, in which tubules containing only
Sertoli cells and spermatogonia form through massive apoptotic
death during meiosis prophase (Yuan et al., 2000).
BNC2 is necessary to maintain the pool of PLZF-positive
spermatogonia
Finally, we examined 2-month-old Bnc2−/− mice. In the wild-type
testis, all tubules contained densely packed germ cells at all stages of
differentiation (Fig. 9A). Undifferentiated spermatogonia were
detected by the presence of PLZF (Fig. 9B). In the Bnc2−/− testis,
some seminiferous tubules lacked germ cells altogether, whereas
others retained a homogenous population of cells that appeared to be
germ cells. Neither spermatocytes nor spermatids were observed
(Fig. 9C). No PLZF-positive cells were detected among the 300
Bnc2−/− tubules that we examined (Fig. 9D). The presumed germ
cells of Bnc2−/− mice did not look like tumor cells, and how
these cells could have persisted in the absence of PLZF-positive
spermatogonia in 2-month-old mice is unclear. Younger Bnc2−/−
mice still retain spermatogonial stem cells, and the presumed
Bnc2−/− germ cells may have been produced from these earlier
spermatogonial stem cells before their pool had been exhausted.
Testicular somatic cell development does not require BNC2
Because BNC2 was found in nonsteroidogenic interstitial cells
(Fig. 3C), we examined these cells in the Bnc2 –/– mice. We detected
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Development (2014) 141, 4298-4310 doi:10.1242/dev.112888
Fig. 7. Abnormal meiotic progression in 1-month-old Bnc2−/− males. (A-D) HE-stained sections of Bnc2+/− and Bnc2−/− testis. (A) A typical seminiferous
tubule in Bnc2+/− mouse, spermatogonia, primary spermatocytes and spermatids are visible. (B-D) Tubules of Bnc2−/− testis. (B) Type I tubule with mostly basal
cells. (C) Stratified type II tubule with no typical meiotic spermatocytes. (D) Stratified tubule with pachytene spermatocytes in the luminal space instead of in their
normal suprabasal position. Spermatids are absent from all Bnc2−/− tubules. (E-O) Immunofluorescence staining. (E,F) Staining for PLZF. Undifferentiated
spermatogonia (arrowheads) are present in similar numbers in Bnc2+/− (E) and Bnc2−/− tubules (F). (G,H) Double staining for SYCP3 and Ki-67. (G) In Bnc2+/−
testis, tubules generally contain a small number of cycling cells in basal layer (Ki-67), and suprabasal meiotic cells (SYCP3). (H) In Bnc2−/− mouse, tubules are
smaller. Type I tubules contain a large proportion of Ki-67-positive cycling cells and no cells with SYCP3, whereas type II tubules possess very few cycling
cells and many meiotic cells. (I) Bnc2+/− testis stained for SYCP3 and γ-H2AX showing a typical tubule with suprabasal pachytene spermatocytes, recognized
by the presence of filamentous SYCP3 and the concentration of γ-H2AX in XY bodies; part of an adjacent tubule with leptotene spermatocytes in the basal layer is
visible (arrowhead). (J-O) Bnc2−/− tubules. (J-L) Type I Bnc2−/− tubules. (J) Cells do not contain SYCP3 and show weak diffuse nuclear staining for γ-H2AX,
characteristic of spermatogonia. (K) Such spermatogonia are identified as differentiating by the presence of c-KIT. (L) Nearly all differentiating spermatogonia
are multiplying as they contain Ki-67 and histone H3 phosphorylated on serine residue 10. DNA is not stained in J-L. (M-O) Type II Bnc2−/− tubules. (M) In
most type II tubules, an abundance of diffuse SYCP3 and γ-H2AX identifies cells as spermatocytes that are blocked in leptotene. (N) Other type II Bnc2−/− tubules
have a ring of abnormal leptotene spermatocytes and a luminal space containing pachytene spermatocytes. (O) Some type II tubules possess simple epithelium
comprising spermatogonia surrounding pachytene spermatocytes. Scale bars: 25 μm (A-F,I-O); 100 μm (G,H).
Bnc2 –/– mice. The mRNA for the androgen receptor was not
significantly affected by the absence of BNC2 (supplementary
material Table S2). Therefore, lack of BNC2 appeared to affect
predominantly, if not exclusively, the germ cell lineage.
Germ cell BNC2 is male specific
We finally asked whether Bnc2 was expressed in female germ cells.
We first studied postmigratory germ cells at the time of their sexual
determination. Male and female primordial germ cells were purified
on E12.5 and E13.5 by using magnetic-activated cell sorting and
stage-specific embryonic antigen 1 (FUT4 – Mouse Genome
Informatics) (Solter and Knowles, 1978). Microarray analysis
showed that the Bnc2 mRNA was much more abundant in male
than in female germ cells. The difference in Bnc2 expression was
comparable to that of Nanos2 (Fig. 11A), a marker of male
primordial germ cells (Tsuda et al., 2003).
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no abnormalities of interstitial cells in either Bnc2−/− fetuses or
newborns. We also examined Sertoli cells by using both
immunostaining of anti-Müllerian hormone in paraffin-embedded
sections and RT-qPCR analysis of the Sertoli cell markers Cyp26b1,
Fgf9 and Sox9. We detected no overt defect in Bnc2−/− Sertoli cells
on either E14.5 (Fig. 10A-C) or E18.5 (Fig. 10D-F). We found no
abnormalities in the number or the morphology of Leydig cells that
we identified by histological staining for 3-β-hydroxysteroid
dehydrogenase. RT-qPCR analysis of Leydig cell markers
detected a decrease in insulin-like 3 mRNA on E14.5 and inhibin
βA mRNA on E18.5. The other Leydig cell-specific mRNAs tested
(Cyp11a1, Star and Notch) were unaffected (Fig. 10G-L).
In the RNA-Seq analysis, markers specific for Sertoli cells
showed almost no difference between Bnc2−/− and Bnc2+/+ newborn
mice. Myoid cell markers were also relatively unaffected, except for
actin α2. Leydig cell-specific mRNAs tended to be increased in the
Fig. 8. Apoptosis of spermatocytes in the testis of 1-month-old Bnc2−/−
mice. (A-C) Immunofluorescent staining using the TUNEL assay. (A) Very little
apoptotic death is seen in heterozygous mouse. (B) A mutant littermate
possesses a much smaller testis with massive apoptotic death in some
seminiferous tubules. (C) TUNEL-positive cells are suprabasal. (D,E) Staining
for activated caspase 3. (D) No caspase 3-containing cells are detected in
heterozygous mice. (E) Apoptotic cells accumulate in ∼15% of Bnc2−/−
tubules. Scale bars: 50 μm.
We then examined fetal ovaries by immunostaining for BNC2
and VASA. On E14.5, BNC2 was undetectable in ovarian germ
cells (Fig. 11B), whereas it was abundant in male germ cells
(Fig. 11C). On P0, double staining for BNC2 and SYCP3 showed
that oocytes did not contain detectable levels of BNC2 and that
entry into meiosis had not been affected by lack of the protein
(Fig. 11D-H).
DISCUSSION
BNC2 as a DNA-binding protein with variable numbers of
zinc-fingers
The mouse Bnc2 transcript is subject to alternative splicing. As a
result, the mouse, like the human (Vanhoutteghem and Djian,
2007), possesses a major BNC2 isoform with six zinc-fingers, and
less abundant isoforms with different N-terminal sequences and
variable numbers of zinc-fingers. Alternative-splicing events
conserved between human and mouse are likely to be of primary
biological importance (Yeo et al., 2005). Therefore, both the more
and the less abundant mouse BNC2 isoforms that are shared with
the human are likely to be functionally important. The
pleiomorphism of the BNC2 effect might be explained by
the existence of multiple isoforms of the protein, as well as by the
presence of multiple zinc-finger pairs that each potentially binds to a
different target sequence. In mouse testis, BNC2 is found in the
soluble nuclear extract and associated with chromatin. Such a
distribution certainly suggests that BNC2 binds to DNA through its
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Development (2014) 141, 4298-4310 doi:10.1242/dev.112888
Fig. 9. Absence of spermatogonial stem cells in 2-month-old Bnc2−/−
mice. (A,C) HE-stained sections. (B,D) Fluorescence staining for PLZF and
DNA. (A) Tubules in wild-type mouse contain spermatogonia, spermatocytes,
round spermatids and elongated spermatids. (B) Immunostaining for PLZF
identifies undifferentiated spermatogonia in the basal layer (arrowheads).
(C) Bnc2−/− tubules contain a homogenous population of germ cells with
intermediate-size nuclei and chromatin granules. (D) No PLZF-containing
spermatogonia are detected in Bnc2−/− mice. Scale bars: 25 μm.
zinc-fingers, but whether BNC2 is a transcription factor remains to
be determined.
Does BNC2 act directly on male germ cells?
In testis, BNC2 is synthesized by germ cells and nonsteroidogenic
interstitial cells. This raises the question of whether BNC2 acts on
germ cells directly or through interstitial cells. We did not find any
evidence that the number or morphology of interstitial cells was
markedly affected by a lack of BNC2. Two somatic cell types that
could affect germ cells are Leydig and Sertoli cells, but BNC2 was
undetectable in both. Neither of the two main sertolian regulators of
germ cell commitment, Fgf9 and Cyp26b1, was affected by the lack of
BNC2, and RNA-Seq analysis did not detect meaningful alterations
in the number of Leydig or Sertoli cell-specific transcripts in mice
that lacked BNC2. The very strong expression of Bnc2 in
prospermatogonia, particularly at the time of sexual differentiation
on E14.5, and the precise regulation of its expression during
spermatogenesis further support the notion of a cell-autonomous
phenotype.
The nature of the nonsteroidogenic interstitial cells that
synthesize BNC2 is not clear. Nonsteroidogenic interstitial cells
include macrophages, myoid cells and mesenchymal cells (Skinner,
1991). Because BNC2 has been found in several kinds of
mesenchymal cells, it is possible that BNC2 also resides in
mesenchymal cells in the testis, where it might have a function
in cell multiplication, as it does in the developing palate and urethra
(Bhoj et al., 2011; Vanhoutteghem et al., 2009).
The function of BNC2 in meiosis and mitosis of male germ
cells
BNC2 appears to have functions in both prospermatogonia and
spermatogonia, and these functions are all related to mitosis
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Development (2014) 141, 4298-4310 doi:10.1242/dev.112888
Fig. 10. Absence of an alteration of Sertoli and Leydig
cells in Bnc2−/− fetal testis. Immunohistochemistry
(A-E,G-K) and RT-qPCR analyses (C,F,I,L) of fetal testes
for somatic cell markers. (A-F) Sertoli cells. (G-L) Leydig
cells. (A-C,G-I) E14.5, (D-F,J-L) E18.5. (A) On E14.5,
wild-type testis shows an abundance of BNC2 in
prospermatogonia but no BNC2 in Sertoli cells that were
identified by the presence of AMH. (B) At E14.5, Bnc2−/−
testis shows prospermatogonia with no BNC2. Sertoli cells
are not visibly affected. (C) RT-qPCR analysis of Sertoli
cell markers shows no alteration in the expression of these
markers in Bnc2−/− testes. (D-F) Similar results are
obtained at E18.5. (G) Wild-type Leydig cells possess
3βHSD but no BNC2. (H) Bnc2−/− Leydig cells are not
obviously affected. (I) The level of insulin-like 3 (Insl3)
mRNA is decreased in Bnc2−/− Leydig cells but not those
of inhibin βA (Inhba), Star or Notch RNAs. (J-L) Similar
results are obtained at E18.5, except that Inhba instead
of Insl3 mRNA is decreased in Bnc2−/− Leydig cells.
Error bars show means±s.d., aP<0.06; *P<0.05.
Scale bars: 25 μm.
MATERIALS AND METHODS
Subcellular fractionation and western blotting
Testes were frozen in liquid nitrogen and homogenized on ice in a tissue
grinder using ten strokes. Cytoplasmic and nuclear extracts were prepared
using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce). We
used the Subcellular Protein Fractionation Kit for Tissues (Pierce) for
subcellular fractionation. The residual pellet was thoroughly mixed with
Laemmli buffer (Laemmli, 1970). The protein concentration was
determined using a Bio-Rad protein assay. Proteins were then resolved by
electrophoresis and electroblotted onto nitrocellulose membranes. The
membranes were blocked for 1 h at room temperature, incubated for 1 h in
the presence of the indicated antibody and then overnight at 4°C.
Membranes were washed and incubated for 1 h at room temperature with
a horseradish peroxidase-conjugated secondary antibody. The membranes
were then washed and the blots developed by using chemiluminescence
(SuperSignal West Femto Maximum Sensitivity Substrate, Pierce). Further
detail is given in the supplementary material methods and antibodies are
listed in supplementary material Table S4.
RT-PCR
For RNA preparation, testes were pulverized under liquid nitrogen. RNA
was extracted using the Tripure isolation reagent (Roche). Reverse
transcriptase (RT)-PCR was performed using the superscript first-strand
synthesis system for RT-PCR (Life Technologies). First strand synthesis
was initiated from total RNA (5 μg) with either oligo(dT) or a Bnc2-specific
primer (supplementary material Table S3). The cDNAs were then subjected
to 30 cycles of amplification (95°C for 1 min, 57°C for 1 min and 72°C for
1 min) using various combinations of primers (supplementary material
Table S3). Sequencing was performed by Eurofins MWG Operon. RT-PCR
was also used to generate the lentiviral expression vectors, detailed
information is given in the supplementary material methods.
RT-qPCR
Gonads were frozen in RLT buffer (Qiagen), and total RNA was extracted
using the RNeasy Mini-Kit (Qiagen). RNA (200 ng) was reverse transcribed
using the High Capacity cDNA Reverse Transcription Kit (Applied
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and meiosis. In contrast to Bnc2−/− spermatocytes, Bnc2−/− meiotic
prospermatogonia do not show evidence of apoptotic cell death,
possibly because prospermatogonia are not part of a cycling
seminiferous epithelium. Apoptosis of abnormal meiotic cells is
thought to result from a stage IV paracrine signal that is derived from
Sertoli cells. Such a signal is unlikely to exist in the non-cycling
epithelium of fetal and neonatal testis. Many defects in either DNA
repair or meiotic pairing cause apoptosis at a checkpoint in stage IV
of the epithelial cycle (Hamer et al., 2008). This raises the question
of a possible function of BNC2 in meiotic pairing, recombination or
synapsis. Such a function would have to be indirect because BNC2
is absent from meiotic cells.
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Development (2014) 141, 4298-4310 doi:10.1242/dev.112888
Fig. 11. Sexual dimorphism of Bnc2 expression in fetal
germ cells, and the absence of an ovarian phenotype in
Bnc2−/− newborn mice. (A) The relative Bnc2 and Nanos2
mRNA levels in male and female germ cells of wild-type
mice during the period of germ cell determination. Sorted
germ cells were analyzed by using microarray
hybridization. White and black columns correspond to
female and male germ cells, respectively. Data are
expressed in arbitrary units (A.U.) as a function of a
common external reference composed of male and female
gonads (equal to zero). Bnc2 is differentially expressed in
male germ cells on E12.5, and this differential expression
becomes more pronounced on E13.5. Differential Bnc2
expression is comparable to that of Nanos2. Error bars
show means±s.d., n=4, **P<0.01, ***P<0.001, Student’s
t-test. (B) Double staining of wild-type paraffin sections of
ovary and testis at E14.5 for BNC2 and VASA shows an
absence of detectable BNC2 in oocytes. (C) Abundant
nuclear BNC2 protein is detected in prospermatogonia.
(D-F) Double staining of wild-type newborn ovary for BNC2
and SYCP3 demonstrates that primordial oocytes,
identified by the presence of filamentous SYCP3, do not
possess BNC2. Only somatic cells contain BNC2.
D,E show single colors, F shows the merged image.
(G,H) Staining of wild-type and Bnc2−/− newborn ovary for
SYCP3. (G) Wild-type ovary possesses numerous
peripheral oocytes with filamentous SYCP3 that is typical
of pachytene. (H) Bnc2−/− oocytes have also engaged
normally in meiosis. Scale bars: 25 μm.
RNA-Seq
Total RNA was prepared as described under RT-PCR. Sequencing was
carried out by Eurofins MWG Operon.
Histological analysis
For HE staining, samples were either embedded in paraffin or frozen in
optimal cutting temperature (OCT) compound. For paraffin embedding,
samples were fixed in 4% paraformaldehyde, washed in PBS, embedded in
paraffin and sectioned at 7 µm. For frozen sections, samples were
successively incubated in 4% paraformaldehyde for 1 h, PBS containing
15% sucrose for 3 h and PBS containing 30% sucrose overnight, and snapfrozen in OCT (Miles).
Indirect immunofluorescence was performed as previously described
(Vanhoutteghem and Djian, 2007) with some modifications, described in
the supplementary material methods. Briefly, frozen sections (6 μm) were
fixed in 4% formaldehyde for 5 min on ice and permeabilized using 0.2%
Triton X-100, or fixed in acetone without permeabilization. For primary
polyclonal antibodies, 5% BSA in PBS was used for blocking. For mouse
monoclonal antibodies, MOM (Vector Laboratories) was used for blocking.
Incubations with primary antibodies were performed overnight at 4°C,
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except for the anti-γ-H2AX antibody, which was incubated for 2 h at room
temperature. Secondary antibodies were incubated for 1 h at room
temperature.
Immunohistochemical analyses were performed as previously described
(Messiaen et al., 2013). Antibodies are listed in supplementary material
Table S4.
In situ analysis of DNA fragmentation was performed using the ApopTag
Fluorescein In Situ Apoptosis Detection Kit (Chemicon International),
according to the manufacturer’s recommendations.
Acquisition of images by using microscopy is described in the
supplementary material methods.
Acknowledgements
We are very grateful to one of the reviewers for insights into the interpretation of the
−/−
different types of tubules found in the adult Bnc2 mice.
Competing interests
The authors declare no competing financial interests.
Author contributions
A.V., S.M., F.H., B.D., D.M. and P.D. performed experiments. A.V., F.H., V.R.-F., G.L.
and P.D. prepared the manuscript. J.-M.P. prepared the figures.
Funding
This study was funded by the Association pour la Recherche sur le Cancer, the
Ligue contre le Cancer, Centre national de la recherche scientifique (CNRS) and
Institut national de la santé et de la recherche mé dicale (INSERM).
DEVELOPMENT
Biosystems) and amplified by using RT-qPCR as previously described
(Souquet et al., 2012). Either β-actin or Vasa mRNA were included as an
endogenous reporter. Results are presented as a fraction of the maximum.
The final concentration of all primers was 400 nM. Samples were run in
duplicate.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.112888/-/DC1
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