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Reproductive Isolation in Hybrid Mice Due to Spermatogenesis Defects at Three
Meiotic Stages
Ayako Oka*§, Akihiko Mita§, Yuki Takada**, Haruhiko Koseki** and Toshihiko Shiroishi§
*Transdsciplinary Research Integration Center, Research Organization of Information
and Systems, Toranomon, Tokyo, Japan 105-0001
§Mammalian Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka,
Japan 411-8540
**RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan
230-0045
Short running title (>35 characters): meiotic disruptions in hybrid males
Key words: mouse, male sterility, speciation, reproductive isolation, meiosis
Corresponding author:
Name: Toshihiko Shiroishi,
Mailing address: Mammalian Genetics Laboratory, National Institute of Genetics, Yata
1111, Mishima, Shizuoka, Japan 411-8540
Tel: +81-55-981-6818, Fax: +81-55-981-6817
Email: [email protected]
Genetics: Published Articles Ahead of Print, published on August 9, 2010 as 10.1534/genetics.110.118976
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ABSTRACT
Early in the process of speciation, reproductive failures occur in hybrid animals between
genetically diverged populations. The sterile hybrid animals are often males in
mammals and they exhibit spermatogenic disruptions, resulting in decreased number
and/or malformation of mature sperms. Despite the generality of this phenomenon,
comparative study of phenotypes in hybrid males from various crosses have not been
done, and thereby comprehensive genetic basis of the disruption is still elusive. In this
study, we characterized the spermatogenic phenotype especially during meiosis in four
different cases of reproductive isolation: B6-ChrXMSM, PGN-ChrXMSM, (B6 x Mus
musculus musculus-NJL/Ms) F1 and (B6 x Mus spretus) F1. The first two are consomic
strains, both bearing the X chromosome of M. m. molossinus; in B6-ChrXMSM, the
genetic background is the laboratory strain C57BL/6J (predominantly M. m.
domesticus), while in PGN-ChrXMSM the background is the PGN2/Ms strain purely
derived from wild M. m. domesticus. The last two cases are F1 hybrids between mouse
subspecies or species. Each of the hybrid males exhibited cell-cycle arrest and/or
apoptosis at either one or two of three distinct meiotic stages: premeiotic stage,
zygotene-to-pachytene stage of prophase I and metaphase I. This study shows that the
sterility in hybrid males is caused by spermatogenic disruptions at multiple stages,
suggesting that the responsible genes function in different cellular processes.
Furthermore, the stages with disruptions are not correlated with the genetic distance
between the respective parental strains.
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INTRODUCTION
When animals from genetically diverged populations hybridize, complete or partial
sterility is often observed in the F1 hybrids or in their descendants. This phenomenon
belonging to postzygotic reproductive isolation accelerates irreversible genetic
divergence by preventing free gene flow across the two diverging populations, and
thereby plays a pivotal role in speciation. Sexual dimorphism is a general feature of
reproductive isolation (WU and DAVIS 1993; LAURIE 1997; ORR 1997; KULATHINAL and
SINGH 2008). In mammals, impairment is much more severe in males than in females,
and in general the heterogametic sex is more sensitive to interspecific and
intersubspecific genetic incompatibility. This phenomenon is well known as Haldane’s
rule (HALDANE 1922; LAURIE 1997; ORR 1997).
In many animals, the reproductive isolation is caused by spermatogenic
disruptions characterized by reduced number of germ cells and small testis size. These
animals include Drosophila (JOLY et al. 1997), stickleback fish Pungitius (TAKAHASHI
et al. 2005), caviomorph rodent Thrichomys (BORODIN et al. 2006), house musk shrew
Suncus (BORODIN et al. 1998), wallaby Petrogale (CLOSE et al. 1996) and genus Mus
(FOREJT and IVÁNYI 1974; MATSUDA et al. 1992; YOSHIKI et al. 1993; HALE et al. 1993;
KAKU et al. 1995; GREGOROVÁ and FOREJT 2000; ELLIOTT et al. 2001; ELLIOTT et al.
2004; GOOD et al. 2008). Although reproductive isolation by spermatogenic impairment
is a well-known phenomenon, its underlying genetic mechanism and molecular basis
have remained elusive. Dobzhansky-Muller model, which infers that hybrid sterility or
inviability is caused by deleterious epistatic interactions between nuclear genes derived
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from their respective parent species or subspecies (DOBZHANSKY 1936; MULLER 1942),
is widely accepted in animals and plants, and is also applicable to the sterility of hybrid
animals in F2 or backcross generations, so-called hybrid breakdown, in which the genes
causing postzygotic reproductive isolation are partially recessive (ORR 2005).
The genetic incompatibility between house mouse subspecies is an ideal
animal model for studying the early stage of speciation. Two subspecies of mouse, Mus
musculus domesticus and M. m. musculus, diverged from their common ancestor 0.3-1.0
million years ago (YONEKAWA et al. 1980; MORIWAKI 1994; BONHOMME and GUÉNET
1996; BOURSOT et al. 1996; DIN et al. 1996). M. m. domesticus ranges across western
Europe and the Middle East, whereas M. m. musculus ranges throughout eastern Europe
and northern Asia (BONHOMME and GUÉNET 1996). The two subspecies meet in a
narrow hybrid zone, which is most likely maintained by a balance between dispersal
and selection against hybrids (HUNT and SELANDER 1973; BONHOMME and GUÉNET
1996; PAYSEUR et al. 2004). M. m. domesticus also displays reproductive isolation from
the Japanese wild mouse, M. m. molossinus, which originated from hybridization of M.
m. castaneus and M. m. musculus, and its nuclear genome is predominantly derived
from M. m. musculus (YONEKAWA et al. 1980; YONEKAWA et al. 1988; MORIWAKI 1994;
SAKAI et al. 2005). To investigate the reproductive isolation between M. m. domesticus
and M. m. molossinus, we previously constructed a consomic strain B6-ChrXMSM (OKA
et al. 2004). This strain has the X chromosome from the MSM/Ms strain, which is
derived from M. m. molossinus, in the genetic background of the laboratory strain
C57BL/6J (B6), which is predominantly derived from M. m. domesticus (MORIWAKI
1994). F1 hybrid animals between B6 and MSM/Ms strains are fully fertile. On the
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contrary, B6-ChrXMSM shows male-specific sterility characterized by a reduced sperm
number and dysfunction of the sperm, including abnormal morphology and low motility,
indicating that B6-ChrXMSM is a model of hybrid breakdown in animals (OKA et al.
2004; OKA et al. 2007). Our previous study indicated that the abnormal morphology of
the sperm head results from the genetic incompatibility between MSM/Ms-derived X-
linked genes and B6 genes on autosomes including chromosomes 1 and 11 (OKA et al.
2007).
In this study, to understand the genetic mechanism of reproductive isolation in
mice, we first undertook in-depth characterization of phenotype for each B6-ChrXMSM
male especially during meiosis. Meiosis is a special cell division that produces four
haploid cells after one round of chromosome replication and two rounds of chromosome
segregation. During meiosis, homologous chromosomes pair, synapse, undergo
crossing-over, and achieve bipolar attachment to the spindle to segregate one set of
chromosomes to each daughter cell. Homologous recombination is initiated during the
leptotene stage of meiotic prophase I with the formation of DNA double-strand breaks
(DSBs), which are repaired immediately during the zygotene stage or after crossing-
over of homologous chromosomes during the pachytene stage (ROEDER 1997;
TARSOUNAS and MOENS 2001).
During the first wave of spermatogenesis, most mitotic spermatogonia in the
B6-ChrXMSM testes fail to initiate meiotic DNA replication. Some proportion of those
spermatogonia that enter into meiosis are again arrested and eliminated by apoptosis at
the pachytene stage, resulting in the production of small number of sperms. We
extended the same analysis to three other cases of reproductive isolation. Another
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consomic strain PGN-ChrXMSM has an MSM/Ms-derived X chromosome in the genetic
background of the PGN2/Ms strain derived from wild mice (M. m. domesticus). PGN-
ChrXMSM males produce a small number of dysfunctional sperms as was the case with
B6-ChrXMSM males, but the former males show apoptosis mainly at metaphase of
meiosis I. Furthermore, we examined F1 hybrid males from intersubspecific cross of (B6
x M. m. musculus-NJL/Ms) and interspecific cross of (B6 x M. spretus). These F1 hybrid
males exhibited apoptosis at metaphase I and at the zygotene-to-pachytene stage of
prophase I. As a whole, the postzygotic reproductive isolation in mice is caused by
disruptions at a minimum of three different spermatogenic stages.
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MATERIALS AND METHODS
Mouse strains: Construction of the consomic strain B6-ChrXMSM was described in our
previous report (OKA et al. 2004). The strain is maintained in the animal facility of the
National Institute of Genetics (NIG). For maintenance of B6-ChrXMSM, females that
have the MSM-derived X chromosome heterozygously are selected by genotyping with
twelve X-linked microsatellite markers and backcrossed to B6 males at each backcross
generation. From these backcrosses, males with the MSM-derived X chromosome and
males with the B6-derived X chromosome are obtained at a frequency of 12.5% each.
Because the reproductive phenotype of males with the B6-derived X chromosome (B6-
ChrXB6) is almost identical to that of B6 males (OKA et al. 2004), we used the B6-
ChrXB6 males as controls in this study.
The proximal and distal microsatellite markers used for the maintenance are
DXMit89 at 10.0 Mb and DXMi160 at 162.5 Mb, respectively. Thus, B6-ChrXMSM strain
possibly has B6 genome in the proximal side beyond DXMit89 and the distal side
beyond DXMit160 (Figure S1). To clarify the origin of the pseudoautosomal region,
which starts from the middle of Mid1 gene at 165.2 Mb toward telomere, in B6-
ChrXMSM strain, we designed several primer sets for PCR, which can distinguish
between B6 and MSM/Ms alleles. The primers are: X163466600 Forward (F) primer
5’-CAAGCTGTGCTGAATATCTGGTG-3’ and reverse (R) primer 5’-
GCAGCCAGGCTTAAGGTAGGACC-3’); X164174029 F-primer 5’-
CGTCATCTCTCAAAGCTCTTCAC-3’ and R-primer 5’-
GAAGATCTGTAATGTTTTACTGGG-3’); X164783477 F-primer 5’-
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CACTTGAGGGAGAAATGTATTGTC-3’ and R-primer 5’-
CCCTAGAGGTCTATATGAAGCC-3’); X165318225 F-primer 5’-
GTCCTGTAATCCACCTTCACTGG-3’ and R-primer 5’-
CATTCACAAAATATCTAGTGCTCC-3’); X165319677 F-primer 5’-
AAATGAGTATGGAATGACCCAGC-3’ and R-primer 5’-
CACAGCCTAGGAACCATGCTGGCC-3’. Genotyping of B6-ChrXMSM showed that
the MSM genome remained only at marker X163466600, and the other regions were
completely substituted by B6 genome, indicating that the pseudoautosomal region of
B6-ChrXMSM strain is derived from the B6 strain (Figure S1).
M. spretus strain (BRC No. RBRC00208) was provided by RIKEN BRC,
which is supported by the National Bio-Resource Project of MEXT, Japan. The NJL/Ms
inbred strain was derived from wild M. m. musculus mice trapped in Northern Jutland,
Denmark and maintained by NIG. PGN-ChrXMSM strain was constructed by the same
strategy used in the construction of B6-ChrXMSM. The inbred strain PGN2/Ms was
derived from M. m. domesticus mice trapped in Pigeon, Canada and maintained by NIG.
Histology and detection of apoptosis: Testes were placed in Bouin’s fixative and then
embedded in paraffin. Sections (6 μm) were stained with hematoxylin and eosin (HE).
Apoptotic cells were visualized by TUNEL assay (In Situ Cell Death Detection Kit, AP;
Roche) following the manufacturer’s protocol. To examine the progression of the first-
wave spermatogenesis, 50 tubules were randomly chosen from a section of each male,
and were classified based on the cell types in the innermost layer of epithelium. Germ
cells in the epithelium were classified according to the book by RUSSELL et al
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(RUSSELL et al. 1990). Three individuals from each strain were used for the evaluation.
Immunohistochemistry: For immunohistochemistry, testes were fixed in Mildform
20NM (Wako) and embedded in paraffin. After deparaffinization, antigen retrieval was
performed by boiling the sections in 0.01 M citrate buffer, pH 6.0, in a microwave for 5
min. After blocking with 10% fetal calf serum (FCS) in phosphate-buffered saline
(PBS), the sections were incubated with primary antibody for 1 h at room temperature
(RT). After washing, the sections were incubated with Alexa-Fluor-488 conjugated
secondary antibody at RT for 30 min. Then, after washing, the sections were counter-
stained with Hoechst 33258 in PBS. Primary antibodies used were rabbit polyclonal
anti-γH2AX (1:50; Upstate), rabbit polyclonal anti-SYCP3 (1:50; Novus Biologicals),
mouse monoclonal anti-PLZF (1:50, Santa Cruz), and rat monoclonal anti-CBX1/HP1β
(1:50, Abcam).
For bromodeoxyuridine (BrdU) staining, we injected mice intraperitoneally
with 40 mg of BrdU (Sigma) per kilogram body weight. After 1 h, testes were fixed in
Mildform 20NM and processed in paraffin. We detected BrdU incorporation with
monoclonal antibody to BrdU (Sigma), incubated samples with biotinylated secondary
antibody and streptavidin-HRP and then detected with diaminobenzidine.
Testicular cell spread preparations: For spermatocyte preparations, 18- or 19-day-old
mice were used. After removal of tunica albuginea, testes were minced in 10%
FCS/PBS and large tissues were removed with 70-μm nylon mesh. Collected cells were
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washed twice with 10% FCS/PBS and then fixed for 1 min in a solution of 1.85%
formaldehyde and 0.05 M sucrose in PBS. After washing with 10% FCS/PBS, cells
were pelleted and resuspended in a small volume of 10% FCS/PBS (<20 μl/testis). Cells
were then smeared on APS-coated glass slides (Matsunami) and air-dried. Specimens
were used for immunostaining immediately after the preparation.
For immunocytochemistry, slides were immersed in 0.5% Triton-X 100/PBS
for 10 min, then washed with PBS and incubated with 10% FCS/PBS for 1 h at RT to
block non-specific binding. Primary antibodies were applied and incubated either
overnight at 4°C or 1 h at RT. After washing with 0.1% Triton-X 100 in PBS (PBST),
slides were incubated with Alexa-Fluor-conjugated secondary antibodies for 30 min at
RT. Following PBST washes, slides were mounted in Vectashield medium (Vector
Laboratories). Staging of prophase spermatocytes was based on SYCP3 staining on
autosomes and the sex chromosomes (ASHLEY et al. 2004; TURNER et al. 2004). The
substaging of pachytene spermatocytes was based on H1t staining (YANG et al. 2008).
Fluorescent signals were detected under a Carl Zeiss laser scanning confocal
microscope (LSM510). Images of 50-80 cells for each male were captured for
quantitative analysis. Primary antibodies used were rabbit polyclonal anti-SYCP3
(1:200; Novus Biologicals), goat polyclonal anti-ATR (1:20; Santa Cruz), rabbit
polyclonal anti-γH2AX (1:200; Upstate), mouse monoclonal anti-RAD51 (1:20,
Abcam), mouse monoclonal anti-MLH1 (1:20; BD Pharmingen), guinea pig polyclonal
anti-H1t (1:1000), which was generously provided by Dr. Handel (INSELMAN et al.
2003), and mouse monoclonal anti-XLR (1:300), which was generously provided by Dr.
Garchon (ESCALIER and GARCHON 2000). For the immunocytochemistry of γH2AX, we
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used anti-SYCP3 antibody labeled by Zenon rabbit IgG labeling kits (Molecular
Probes).
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RESULTS
Failure to initiate meiotic S phase in B6-ChrXMSM testes: We previously reported
variation in the degree of testicular defects across seminiferous tubules within
individual adult B6-ChrXMSM males, ranging from relatively normal spermatogenesis to
loss of all germ cells except spermatogonia (OKA et al. 2004). To learn when the
disruption of spermatogenesis starts, we examined testicular histology of B6-ChrXMSM
males at various stages in the first wave of spermatogenesis, which progresses
synchronously in mice. After the several rounds of mitosis, type B spermatogonia enter
the meiotic preleptotene stage, during which meiotic DNA replication occurs, at around
10 days post partum (dpp) (ELLIS et al. 2004). Then, meiotic spermatocytes enter the
prolonged prophase, which takes 10-12 days (ELLIS et al. 2004). The first meiotic
prophase is divided into five stages: leptotene, zygotene, pachytene, diplotene and
diakinesis stages. Pachytene stage is the longest one, continuing for 5 to 7 days, and the
first pachytene spermatocytes are observed at around 14 dpp (ELLIS et al. 2004; COHEN
et al. 2006).
Histological examination of B6-ChrXMSM testes by light microscopy revealed
no detectable difference from those of control littermates at 8 dpp (data not shown).
Perceptible changes in the constitution of germ cells within the tubules in B6-ChrXMSM
testes became visible at 10 dpp (Figure 1A). At 14 dpp, a clear difference in histology
was noted between B6-ChrXMSM and control testes: meiotic spermatocytes in the
seminiferous tubules were abundant in control animals, but rarely seen in the B6-
ChrXMSM animals (Figure 1A). At 19 dpp, many tubules in the B6-ChrXMSM testes still
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contained no cells engaged in meiosis (Figure 1A). The disruption of spermatogenesis
became more severe in the B6-ChrXMSM testes at 32 dpp. Some tubules contained
surviving spermatocytes and round spermatids, but none of the mature sperm cells that
were observed in most tubules of the control testes (Figure 1A). These results show that
spermatogenesis of B6-ChrXMSM mice is affected in the meiotic phase. We quantified
the progression of spermatogenic development by counting tubules containing germ
cells of the various developmental stages in the innermost layer of seminiferous
epithelium. As summarized in Figure 1B, delayed timing of emergence of developing
germ cells was detected in the B6-ChrXMSM testes at all ages tested.
To examine whether mitotic spermatogonia are affected in B6-ChrXMSM, we
performed immunohistochemical analysis of the testicular sections with antibodies
against PLZF and CBX1/HP1β, which are localized to nuclei of undifferentiated type A
and type B spermatogonia, respectively (HOYER-FENDER et al. 2000; BUAAS et al.
2004; COSTOYA et al. 2004). The antibodies used for immunostaining in this study are
listed in Table 1. Control and B6-ChrXMSM testes showed no obvious differences in the
number of undifferentiated type A and type B spermatogonia at 14 dpp (Figure S2).
We further investigated proliferation of spermatogonia and spermatocytes in
the testes at 19 dpp by analyzing the incorporation of BrdU. We distinguished
proliferative spermatogonia from spermatocytes based on size and number of BrdU-
positive nuclei in the seminiferous tubules: tubules containing preleptotene
spermatocytes were recognized as those with a larger number of small positive nuclei.
In the control testes, BrdU incorporation was detected in both spermatogonia and
preleptotene spermatocytes (Figure 2A). In contrast, in the B6-ChrXMSM testes, BrdU
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signals were detected mainly in spermatogonia, and rarely in the preleptotene
spermatocytes (Figure 2B). Although most of tubules in the B6-ChrXMSM testes were
aberrant, the frequency of tubules containing BrdU-positive germ cells was not
significantly different between control and the B6-ChrXMSM testes (31.8 and 27.0% for
the two control males; 36.1 and 28.9% for the two B6-ChrXMSM males), suggesting
relatively normal proliferation of spermatogonia in B6-ChrXMSM.
We next investigated whether B6-ChrXMSM germ cells enter meiosis.
Immunohistochemistry was performed on testis sections at 14 dpp with antibody against
the phosphorylated form of histone H2AX, γH2AX. Since γ H2AX is observed in
chromatin immediately after the induction of DSBs at the leptotene and zygotene stages
and on the sex chromosomes at the pachytene stage (MAHADEVAIAH et al. 2001), we
used γH2AX as a marker of early spermatocytes. Almost all tubules of the control testes
contained spermatocytes at the leptotene-to-zygotene stage or the pachytene stage
(Figure 2C). By contrast, in the B6-ChrXMSM testes, a small proportion of tubules
showed γH2AX-positive spermatocytes at the leptotene-to-zygotene stage but very few
tubules showed spermatocytes at the pachytene stage (Figure 2D). We then quantified
frequency of early spermatocytes by detecting SYCP3-positive cells. SYCP3 is a
component of the synaptonemal complex that physically tethers homologous
chromosomes, and is localized in different manners from leptotene stage to metaphase I.
SYCP3 therefore serves as a marker of early spermatocytes. We performed
immunocytochemical analysis with SYCP3 antibody for cell spreads from B6-ChrXMSM
and control testes at 18 or 19 dpp, and quantified the ratio of SYCP3-positive early
spermatocytes to all testicular cells stained by Hoechst 33258. As shown in Figure 2E,
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almost half (46.9%) of cells in the control testes were SYCP3-positive, whereas only a
small proportion (6.4%) of cells in the B6-ChrXMSM testes were SYCP3-positive. All
these data indicate that in the B6-ChrXMSM testes, proliferation and differentiation are
normal prior to type B spermatogonia, but initiation of meiotic DNA replication is
prevented in the majority of type B spermatogonia, allowing only a small proportion of
the spermatogonia to enter into meiosis.
Synaptic errors and apoptosis at pachytene stage in the B6-ChrXMSM
spermatocytes: We next characterized the early meiotic spermatocytes found in the B6-
ChrXMSM testes. During prophase I, synapsis of the homologous chromosomes can be
monitored by localization of SYCP3 (COHEN et al. 2006; HAMER et al. 2006). The
lateral axes of synaptonemal complexes are formed during the leptotene stage, and
synapsis between homologous chromosomes occurs during the zygotene stage and
becomes complete prior to the pachytene stage (COHEN et al. 2006). Unsynapsed and
synapsed chromosomes can be distinguished by fine vs. thick fibers of SYCP3. We
observed that the pattern of SYCP3 signals in B6-ChrXMSM was similar to that in the
control spermatocytes during leptotene to zygotene (Figure S3A). Because pachynema
is prolonged stage, we divided it into two substages (early and mid-to-late) based on the
testis-specific H1 variant H1t, which replaces somatic H1 on chromatin at mid-
pachytene and thus serves as a marker of mid-to-late pachytene spermatocytes (MOENS
1995; COBB et al. 1999). In most of early pachytene spermatocytes in B6-ChrXMSM
males, SYCP3 signals were observed as thick fibers as observed in control males, but
B6-ChrXMSM spermatocytes also frequently exhibited aggregation of SYCP3 (Figure
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3A). This SYCP3 aggregation was also found in mid-to-late pachytene B6-ChrXMSM
spermatocytes, though less frequently (Figure 3C). We then immunostained the
pachytene spermatocytes with antibody against the kinase ATR, which during pachytene
stage localizes on the sex chromosomes, but it also persists on unrepaired DSBs
specifically on asynaptic autosomes (KEEGAN et al. 1996; MOENS et al. 1999). Control
pachytene spermatocytes showed ATR only on the sex chromosomes, whereas some
proportion of B6-ChrXMSM pachytene spermatocytes showed ATR on the asynaptic
autosomes (Figure 3B). Such spermatocytes with asynapsis included both
spermatocytes with and without signals on the sex chromosomes. Frequencies of
spermatocytes with SYCP3 aggregation and asynapsis were significantly higher in B6-
ChrXMSM than in the control, though the variance between individuals was relatively
large (Figure 3C). Those aberrant chromosomes were prominent during early pachytene
substage. We then estimated the proportion of spermatocytes in the two pachytene
substages. Compared with the control B6-ChrXB6, B6-ChrXMSM had a significantly
reduced proportion of the mid-to-late pachytene spermatocytes (Figure 3D). This
suggests that cell-cycle arrest occurred during the early pachytene substage in the B6-
ChrXMSM males.
To examine whether DSBs and recombination nodules are properly formed in
B6-ChrXMSM males, we immunostained the early spermatocytes with antibodies against
γH2AX and RAD51 as markers of DSBs and early recombination nodules, respectively.
We found no obvious differences in the patterns of γH2AX and RAD51 signals between
control and the B6-ChrXMSM testes in leptotene, zygotene and pachytene spermatocytes
(Figure S3A and S3B). We next immunostained the pachytene spermatocytes with
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antibody against MLH1, a protein that functions in meiotic crossing-over and DNA
mismatch repair in the late recombination nodules from the mid-pachytene substage
(ASHLEY et al. 2004). Again, we found no significant difference in the pattern of MLH1
foci between control males and B6-ChrXMSM (Figure S3A and S3B).
During pachytene stage, a specialized meiotic chromatin domain named the
XY (sex) body is formed, in which X and Y chromosomes are transcriptionally silenced.
To evaluate the proper development of pachytene spermatocytes, we examined the XY
body formation with antibodies against three marker proteins, ATR, γH2AX and XLR,
which during pachytene stage all accumulate exclusively in the XY body (CALENDA et
al. 1994; ESCALIER and GARCHON 2000; REYNARD et al. 2007). Most of the
spermatocytes of the B6-ChrXMSM males properly formed an XY body, although the
marker proteins were also frequently detected in autosomal regions (Figure S4A and
S4B).
Next, we examined whether the spermatogonia arrested prior to the meiotic
entry and spermatocytes arrested at pachytene stage are subject to apoptosis by using
the TUNEL assay on testicular sections at 15 and 19 dpp. At 15 dpp, occasional
TUNEL-positive cells were present in some tubules of both control and B6-ChrXMSM
testes (Figure 4A). This suggests that most of the arrested B6-ChrXMSM spermatogonia
at the premeiotic stage did not induce apoptosis. At 19 dpp, numerous apoptotic cells
were detected in the tubules in the B6-ChrXMSM testes, but not in control testes (Figure
4A). Apoptosis was rarely observed in severely defective tubules lacking meiotic
spermatocytes (Figure S5A). Most of the apoptotic spermatocytes were found in tubules
at epithelial stage IV, as judged by the presence of mitotic intermediate spermatogonia
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and early type B spermatogonia (Figure S5B). We compared numbers of tubules
containing apoptotic cells in sections of testes at various ages stained with hematoxylin
and eosin (HE). The frequency of tubules containing apoptotic cells became higher in
the B6-ChrXMSM testes than in the control testes starting at 19 dpp during first-wave
spermatogenesis (Figure 4B).
Meiotic cell cycle arrest in other cases of reproductive isolation: We further
investigated the meiotic phenotypes in three other cases of reproductive isolation. The
first case is males of a second X chromosomal consomic strain, PGN-ChrXMSM. Because
the classical laboratory inbred strains, including B6, have contributions of multiple
subspecies to their genome, it was uncertain whether meiotic defects of the B6-ChrXMSM
males indeed reflect the reproductive isolation between M. m. domesticus and M. m.
molossinus. Thus, we constructed the consomic stain PGN-ChrXMSM with the X
chromosome from the MSM/Ms strain in the genetic background of the PGN2/Ms strain,
which is derived purely from wild mice (M. m. domesticus). PGN-ChrXMSM males
showed phenotypes similar to those of B6-ChrXMSM males: sterility with reduced testis
weight and abnormal morphology of mature sperm (Table S1, S2, Figure S6). This
result indicated that PGN-ChrXMSM males show reproductive isolation like that of B6-
ChrXMSM males. Immunohistochemical analysis with anti-SYCP3 antibody revealed that
the number of SYCP3-positive early spermatocytes was almost identical in PGN-
ChrXMSM testes and in control PGN-ChrXPGN and B6-ChrXB6 testes (Figure 5A). This
suggests that early spermatocytes in PGN-ChrXMSM, unlike those in B6-ChrXMSM, are
not arrested or eliminated. On the other hand, histological analysis of HE- and TUNEL-
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stained testes showed that the PGN-ChrXMSM testes have increased apoptosis in
spermatocytes at epithelial stage XII, which is equivalent to metaphase I, rather than
stage IV (Figure 5B).
The second case of reproductive isolation is F1 hybrid males from an
intersubspecific cross of B6 and the NJL/Ms strain derived from east European wild
mice (M. m. musculus). These hybrids show male sterility with massive loss of germ
cells during meiosis (KAKU et al. 1995). Their testicular histology revealed extensive
apoptosis in spermatocytes at the zygotene and pachytene stages (Figure 6A).
Immunohistochemical analysis showed that the (B6 x NJL/Ms) F1 hybrid males had a
significantly decreased proportion of SYCP3-positive early spermatocytes (Figure 6B).
We observed a smaller proportion of pachytene spermatocytes relative to zygotene
spermatocytes in the (B6 x NJL/Ms) F1 testes compared to control testes (data not
shown). Since these testes contained only a small number of H1t-positive mid-to-late
pachytene spermatocytes, we examined the marker proteins only at the early pachytene
substage. Immunostaining with anti-ATR and anti-γH2AX antibodies showed that these
markers of XY bodies rarely accumulated in the F1 hybrid spermatocytes (Figure 6C).
Furthermore, their pachytene spermatocytes showed a significantly elevated frequency
of persistent RAD51 foci and a lack of MLH1 foci on asynaptic axes (Figure 6C). These
results suggest that DSBs were not repaired and homologous crossing-over was not
completed in nearly all zygotene-to-pachytene spermatocytes in (B6 x NJL/Ms) F1
hybrid males.
The last case of reproductive isolation is F1 hybrid males from the
interspecific cross of B6 and M. spretus. It was reported that (B6 x M. spretus) F1 hybrid
20
males are sterile, showing spermatogenic breakdown at the first meiotic metaphase and
only a small number of sperm, which are dysfunctional (MATSUDA et al. 1991;
MATSUDA et al. 1992). Histological analysis of the F1 hybrid testes revealed a
significant increase of apoptosis in spermatocytes, mainly at epithelial stage XII,
equivalent to metaphase I (Figure 6A). We further carried out immunohistochemical
analysis of (B6 x M. spretus) F1 hybrid males with antibodies directed against several
meiotic marker proteins. The proportion of SYCP3-positive early spermatocytes in (B6
x M. spretus) F1 hybrid males was not significantly different from that of control B6-
ChrXB6 males (Figure 6B). Immunostaining with ATR and γH2AX antibodies on cell
spreads from the F1 hybrid testes showed a slight but significant decrease in the
frequency of XY body formation, especially at the early pachytene substage (Figure 6C).
Furthermore, the axes of (B6 x M. spretus) F1 hybrid pachytene spermatocytes
displayed persistent RAD51 foci and lack of MLH1 foci (Figure 6C). This indicates that
DSBs were not repaired and homologous crossing-over was not completed in the small
population of spermatocytes in the F1 hybrid, though the spermatocytes were mainly
impaired at metaphase I.
21
DISCUSSION
Figure 7 summarizes the spermatogenic stages at which four different cases of
reproductive isolation undergo cell-cycle arrest and apoptosis.
For B6-ChrXMSM, we showed that the number of type B spermatogonia is
unchanged, but most of them fail to initiate meiotic DNA replication, suggesting that
cell-cycle arrest occurs at the stage prior to the meiotic entry. Premeiotic cell-cycle
arrest in spermatogonia has seldom been reported. One report showed that the knockout
mutant of Cdk inhibitor p27, which negatively regulates the G1/S transition, has a small
proportion of spermatogonia that fail to undergo meiotic entry (BEUMER et al. 1999).
Thus, B6-ChrXMSM is the first example in which spermatogonia are predominantly
subject to cell-cycle arrest at the premeiotic stage. In this study, during the first wave of
spermatogenesis, the number of meiotic spermatocytes in B6-ChrXMSM males was
reduced to less than one-sevenths of those in control males at 18 and 19 dpp. However,
we previously observed that the number of mature sperms in adult B6-ChrXMSM males
was approximately one-thirds of control males (OKA et al. 2004). Thus, the premeiotic
cell-cycle arrest in adult B6-ChrXMSM males may be recovered partially.
Three of the types of hybrid males, B6-ChrXMSM, (B6 x NJL/Ms) F1 and (B6 x
M. spretus) F1, exhibited arrest at the zygotene-to-pachytene stage of meiosis I. A most
prominent cell-cycle arrest at this stage occurs in the testes of intersubspecific (B6 x
NJL/Ms) F1 hybrids. They show extensive arrest and apoptosis at the late zygotene and
early pachytene stages. Early studies of budding yeast, Saccharomyces cerevisiae,
revealed that persistent unrepaired DSBs or asynapsis triggers meiotic arrest at the
22
pachytene stage (ROEDER and BAILIS 2000). This response to the meiotic defects, the
pachytene checkpoint, is known to function in many animals including mice. In various
mouse meiotic mutants, cell-cycle arrest and apoptosis at zygotene-to-pachytene are
associated with unrepaired DSBs, asynapsis and possibly lack of the XY body,
suggesting that the pachytene checkpoint monitors these defects (PITTMAN et al. 1998;
DE VRIES et al. 1999; YUAN et al. 2000; TURNER et al. 2004; DE VRIES et al. 2005).
Thus, it is likely that zygotene and pachytene spermatocytes in (B6 x NJL/Ms) F1 males
are subject to the pachytene checkpoint-dependent apoptosis, because the spermatocytes
showed severe asynapsis and unrepaired DSBs, which were represented by the
extensive RAD51 foci that persisted throughout the pachytene stage. On the other hand,
pachytene spermatocytes in B6-ChrXMSM males showed relatively mild synaptic defect
and no persistence of unrepaired DSBs. Thus, cell-cycle arrest and apoptosis at
pachytene stage in this strain less likely resulted from pachytene checkpoint response,
and are rather attributable to the incompetence of meiosis; the early pachytene
spermatocytes cannot complete cellular events due to the genetic incompatibility and
may be hard to reach the mid-to-late pachytene stage.
A recent study demonstrated that one of the genes responsible for the
reproductive isolation between the M. m. musculus-derived PWD strain and B6 is the
histone H3 lysine 4 trimethyltransferase Prdm9 (MIHOLA et al. 2009). Both (PWD x
B6) F1 spermatocytes and Prdm9 knockout spermatocytes fail to form XY bodies and
undergo apoptosis at pachytene stage (MIHOLA et al. 2009), similar to the phenotype we
observed in the (B6 x NJL/Ms) F1 hybrids. Epigenetic regulation induced by the genetic
incompatibility was shown to cause the meiotic defects. It will be interesting to see
23
whether checkpoint responses are involved in these meiotic defects.
The third stage at which spermatocytes of the hybrid males are arrested is the
first meiotic metaphase. Apoptosis at this stage occurs in spermatocytes in PGN-
ChrXMSM and, to a much greater extent, in interspecific (B6 x M. spretus) F1 hybrids.
During both mitotic and meiotic metaphase, the faithful separation of chromosomes
requires accurate connections between chromosomes and microtubules, with
kinetochores playing the most significant roles. Some of the spindle checkpoint proteins,
which monitor the attachment of microtubules to kinetochores in mitosis, are known to
function in meiosis (YIN et al. 2008). Moreover, cell-cycle arrest and apoptosis at
metaphase of meiosis I have been observed in mutant mice having meiotic impairments
such as lack of chiasmata and dissociation of homologous autosomes and XY
chromosomes (BAKER et al. 1996; LIPKIN et al. 2002; MARK et al. 2008). Previous
studies revealed that genetic divergence between the parental species in the
pseudoautosomal region of the XY chromosomes causes dissociation of these
chromosomes in (B6 x M. spretus) F1 spermatocytes (MATSUDA et al. 1991; MATSUDA
et al. 1992; HALE et al. 1993). This dissociation is caused by asynapsis between sex
chromosomes during pachytene (although X and Y chromosomes are still close to each
other), and continues through metaphase I (MATSUDA et al. 1991; MATSUDA et al. 1992;
HALE et al. 1993). In this study, we observed that (B6 x M. spretus) F1 pachytene
spermatocytes successfully form XY bodies that appear to contain normal amounts of
ATR and γH2AX. This suggests that the dissociated XY chromosomes can be packaged
into XY bodies, and are protected from pachytene checkpoint monitoring. Thus, we
infer that the XY chromosome dissociation finally triggers the spindle checkpoint, and
24
the spermatocytes are eventually eliminated by apoptosis.
This study showed that male sterility in hybrid mice from various crosses is
caused by disruptions at a minimum of three stages during meiosis. In addition to
meiotic disruptions, consomic strains such as B6-ChrXMSM and PGN-ChrXMSM show
impairments during spermiogenesis, terminal differentiation stage during
spermatogenesis, resulting in the malformation of sperm heads. These results indicate
that genes responsible for reproductive isolation do not function in a particular specific
process, rather in different multiple processes. In many cases of mouse reproductive
isolation, meiotic defects are predominantly observed (MATSUDA et al. 1991; KAKU et
al. 1995; FOREJT 1996). This is possibly due to the high intricacy of meiosis, which
includes many cellular events such as the induction of DSBs, homologous
recombination, repair of DSBs and segregation of homologous chromosomes.
Notably, the genetic distance between the two parental strains was not
explicitly correlated with the stages at which cell-cycle arrest and apoptosis occur. For
instance, intersubspecific incompatibility in B6-ChrXMSM causes arrest both at the
premeiotic stage and the pachytene stage, but similar intersubspecific incompatibility in
PGN-ChrXMSM causes extensive apoptosis at metaphase I. In the case of another
consomic stain B6-XPWD, which has an X chromosome of wild M. m. musculus-derived
PWD strain in the genetic background of B6 strain, it was reported that meiotic
disruption occurs mainly at the pachytene stage (STORCHOVÁ 2004). Interspecific
incompatibility in the (B6 x M. spretus) F1 hybrid causes partial arrest and apoptosis at
the pachytene stage, but extensive apoptosis was observed at metaphase I as well. In a
substantially long period of time for speciation, it is conceivable that mutations
25
responsible for the reproductive isolation have continuously accumulated in the
diverging populations, which may stochastically increase the chance that mutations
responsible for reproductive defects at early meiotic stages, i.e. the premeiotic stage,
occurs. Thus, the absence of obvious correlation between the genetic distance and the
stages of the meiotic defects, which was observed in this study, may imply that these
house mice are in early stage of speciation.
26
ACKNOWLEDGMENTS
We thank M. A. Handel for kindly providing H1t antibody, and H. J. Garchon for XLR
antibody. We thank J. Turner for providing useful advice on experimental design and
methodology, and D. G. de Rooij for helpful comments on histology. We thank
members of the Shiroishi lab for providing critical comments on early versions of the
manuscript. We are grateful to A. Yamakage, F. Kobayashi, H. Nakazawa, I. Okagaki
and the staff of the animal facility of National Institute of Genetics for assistance with
mouse husbandry. This study was supported in part by a Grant-in-Aid for Scientific
Research on Priority Areas (‘‘Genome Science’’) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan. This study was also supported in part
by the Transdisciplinary Research Integration Center, Research Organization of
Information and Systems. This article is contribution no. 2514 from the National
Institute of Genetics, Mishima, Japan.
27
FIGURE LEGENDS
FIGURE 1
Testicular histology and spermatogenic development in B6-ChrXMSM testes during first-
wave spermatogenesis. (A) Hematoxylin and eosin (HE) staining of cross sections of
testes from 10, 14, 19, and 32 dpp from control B6-ChrXB6 males (left panels) and B6-
ChrXMSM males (right panels). The B6-ChrXMSM testes showed few cells engaged in
meiosis and vacuolation in the seminiferous tubules at ages after 14 dpp. Scale bar: 100
μm. (B) The proportion of developing germ cells in the innermost layer of seminiferous
tubules in control (left) and B6-ChrXMSM (right) males during first-wave
spermatogenesis. Leptotene and zygotene spermatocytes were grouped together as pre-
pachytene spermatocytes.
FIGURE 2
Failure in initiation of meiotic S-phase and reduced proportion of meiotic spermatocytes
in B6-ChrXMSM testes. (A, B) BrdU incorporation into control (A) and B6-ChrXMSM (B)
testes at 19 dpp was detected by anti-BrdU antibody. In control testis, BrdU signals
were detected in type A spermatogonia, intermediate spermatogonia (In), type B
spermatogonia (SB) and preleptotene spermatocytes (preL). In B6-ChrXMSM testis, anti-
BrdU antibody detected type A and B spermatogonia but not preleptotene spermatocytes.
(C, D) Meiotic spermatocytes were indicated by γH2AX signals in testes at 14 dpp. In
control testis (C), leptotene to zygotene spermatocytes (LZ) and pachytene
spermatocytes with XY bodies (P) were detected. In contrast, a few leptotene to
28
zygotene spermatocytes were detected in B6-ChrXMSM testis (D). Signals are γH2AX
(green) and Hoechst (gray). Scale bar: 200 μm. (E) Cell spreads from control (upper
panel) and B6-ChrXMSM (bottom panel) testis at 19 dpp. Anti-SYCP3 antibody (red)
stained spermatocytes at meiotic prophase I. All nuclei were stained by Hoechst (blue).
Only a few cells were SYCP3-positive in B6-ChrXMSM testis. Frequency of
spermatocytes at prophase I in control and B6-ChrXMSM testes at 18-19 dpp (right graph).
We counted 1731 and 1473 cells from four control and B6-ChrXMSM males, respectively.
The difference in frequency was significant by t-test.
FIGURE 3
Synaptic errors and cell-cycle arrest at early pachytene stage in B6-ChrXMSM
spermatocytes. (A) SYCP3 and H1t double immunostaining of control and B6-ChrXMSM
spermatocytes at early and late pachytene substages. The early pachytene spermatocyte
from B6-ChrXMSM showed aggregation of SYCP3 (arrows). (B) SYCP3 and ATR double
immunostaining of control and B6-ChrXMSM early pachytene spermatocytes. ATR
localized to the sex chromosomes in the control spermatocyte. Note the dotted ATR
signals along asynaptic regions on autosomes in the B6-ChrXMSM spermatocyte. (C)
Frequencies of aggregation and asynapsis at each substage. We counted 1035 pachytene
spermatocytes from nine control testes and 1008 from seven B6-ChrXMSM testes. (D)
Proportion of early pachytene and mid-to-late pachytene spermatocytes, which were
subtyped by staining with anti-H1t antibody. We counted 1463 pachytene spermatocytes
from seven control males and 1334 from five B6-ChrXMSM males. All p-values were
evaluated by t-test.
29
FIGURE 4
Increased apoptosis at the pachytene stage in B6-ChrXMSM testes. (A) Incidence of
apoptosis in testes at 15 dpp and 19 dpp. Occasional apoptosis was observed in both
control and B6-ChrXMSM testes. In B6-ChrXMSM testis at 19 dpp, extensive apoptosis was
observed in the tubules with meiotic spermatocytes. Scale bars: 100 μ m. (B) The
frequency of seminiferous tubules with apoptotic cells during first-wave
spermatogenesis. Fifty tubules for each of three males were counted at each age tested.
Apoptotic cells were judged by the condensed staining of nuclei by HE staining.
FIGURE 5
Increased apoptotic spermatocytes at metaphase I in PGN-ChrXMSM testis. (A, left
panel) Cell spreads from PGN-ChrXMSM testis at 19 dpp were immunostained with anti-
SYCP3 antibody (red) and counterstained by Hoechst (blue). (A, right graph)
Frequency of SYCP3-positive spermatocytes in control and PGN-ChrXMSM testes at 18-
19 dpp. The difference was not significant by t-test. (B) Testicular histology of control
PGN-ChrXPGN (upper left panel) and PGN-ChrXMSM (upper right panel) males at 24 dpp.
TUNEL staining of adult control PGN-ChrXPGN (bottom left panel) and PGN-ChrXMSM
(bottom right panel) testes. Increased apoptosis of metaphase I spermatocytes was
observed at epithelial stage XII in PGN-ChrXMSM testis (arrow heads in upper right
panel and positive nuclei in bottom right panel). Scale bars: 200 μm.
FIGURE 6
30
Meiotic phenotypes of inter-subspecific and inter-specific F1 hybrid males. (A) HE-
stained testicular histology of (B6 x NJL/Ms) F1 (upper left panel) and (B6 x M.
spretus) F1 (upper right panel) at 19 dpp. (B6 x NJL/Ms) F1 testis contained tubules with
apoptotic zygotene spermatocytes (ap) and tubules lacking spermatocytes (*). Increased
apoptosis of metaphase I was observed at epithelial stage XII in (B6 x M. spretus) F1
testis. TUNEL stained testicular histology of (B6 x NJL/Ms) F1 (bottom left panel) and
(B6 x M. spretus) F1 (bottom right panel) at 24 dpp. Scale bars: 50 μm. (B) Ratio of
SYCP3-positive meiotic spermatocytes to all testicular cells for each male at 18-19 dpp.
Reduction of SYCP3-positive spermatocytes in (B6 x NJL/Ms) F1 testes was significant
by t-test. (C) Immunostaining of control, (B6 x M. spretus) F1 and (B6 x NJL/Ms) F1
pachytene spermatocytes with anti-ATR, γ H2AX, RAD51 and MLH1 antibodies
(indicated in green). All spermatocytes were double-stained with anti-SYCP3 antibody
(red). Graphs at the side indicate the quantification of signal-positive spermatocytes at
early and mid-to-late pachytene stages. * indicates significant difference by t-test
(P<0.05).
FIGURE 7
Summary of meiotic arrest in four different cases of reproductive isolation. Horizontal
arrows indicate the progression of spermatogenic development. Downward arrows
(blue) indicate points of disruptions (arrest and/or apoptosis). Orange horizontal lines
indicate defective spermiogenesis. Thickness of blue and orange lines represents the
proportion of spermatocytes subjected to arrests and/or apoptosis.
31
TABLE 1
MARKER PROTEINS USED FOR IMMUNOASSAYS
Protein Feature Marked
PLZF Undifferentiated type A spermatogonia
CBX/HP1β Type B spermatogonia
γH2AX DSBs (leptotene-zygotene); XY body (pachytene)
SYCP3 Synaptonemal complex (leptotene-metaphase I)
ATR XY body (pachytene); unrepaired DSBs on unsynapsed
autosomes (pachytene)
XLR XY body (pachytene)
H1t Mid-to-late pachytene
RAD51 Early recombination nodules on DSBs (leptotene-pachytene)
MLH1 Late recombination nodules on DSBs (mid-to-late
pachytene)
32
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B6-ChrXB6(control) B6-ChrXMSM
A
14 dpp
19 dpp
32 dpp
B
10 dpp
10 14 19 26 3210 14 19 26 32age (day)
0
100
20
40
60
80
B6-ChrXB6(control) B6-ChrXMSM
prop
ortio
n of
dev
elop
ing
cells
age (day)
spermatogoniapre-pachytene spermatocytepachytene spermatocyte
round spermatidelongated spermatidsperm
0
100
20
40
60
80
(%) (%)
Figure 1. Testicular histology and spermatogenic development in B6-ChrXMSM
testes during first-wave spermatogenesis. (A) Hematoxylin and eosin (HE) staining of cross sections of testes from 10, 14, 19, and 32 dpp from control B6-ChrXB6 males (left panels) and B6-ChrXMSM males (right panels). The B6-ChrXMSM testes showed few cells engaged in meiosis and vacuolation in the seminiferous tubules at ages after 14 dpp. Scale bar: 100 μm. (B) The proportion of developing germ cells in the innermost layer of seminiferous tubules in control (left) and B6-ChrXMSM (right) males during first-wave spermatogenesis. Leptotene and zygotene spermatocytes were grouped together as pre-pachytene spermatocytes.
0
20
40
60
B6-ChrXB6(control) B6-ChrXMSM
BASB
preL
SB
In
SB
SB
SY
CP
3-p
ositi
ve s
perm
atoc
ytes
B6-ChrXB6
(control)B6-ChrXMSM
P<0.01(%)
E
D’C
PP P
P
P
LZ
LZ
LZ
D
LZ
LZLZ
preL
B6-ChrXB6 (control)
B6-ChrXMSM
Figure 2. Failure in initiation of meiotic S-phase and reduced proportion of meiotic spermatocytes in B6-ChrXMSM testes. (A, B) BrdU incorporation into control (A) and B6-ChrXMSM (B) testes at 19 dpp was detected by anti-BrdU antibody. In control testis, BrdU signals were detected in type A spermatogonia, intermediate spermatogonia (In), type B spermatogonia (SB) and preleptotene spermatocytes (preL). In B6-ChrXMSM testis, anti-BrdU antibody detected type A and B spermatogonia but not preleptotene spermatocytes. (C, D) Meiotic spermatocytes were indicated by γH2AX signals in testes at 14 dpp. In control testis (C), leptotene to zygotene spermatocytes (LZ) and pachytene spermatocytes with XY bodies (P) were detected. In contrast, a few leptotene to zygotene spermatocytes were detected in B6-ChrXMSM testis (D). Signals are γH2AX (green) and Hoechst (gray). Scale bar: 200 μm. (E) Cell spreads from control (upper panel) and B6-ChrXMSM (bottom panel) testis at 19 dpp. Anti-SYCP3 antibody (red) stained spermatocytes at meiotic prophase I. All nuclei were stained by Hoechst (blue). Only a few cells were SYCP3-positive in B6-ChrXMSM testis. Frequency of spermatocytes at prophase I in control and B6-ChrXMSM testes at 18-19 dpp (right graph). We counted 1731 and 1473 cells from four control and B6-ChrXMSM males, respectively. The difference in frequency was significant by t-test.
A
C
SYCP3 H1t merge
earlypachytene
latepachytene
SYCP3 ATR merge
B
0
20
40
60
80
100D
B6-ChrXB6 B6-ChrXMSM
prop
ortio
n of
pac
hyte
ne s
ubst
ages P<0.01
(%)
B6-ChrXB6(control)
SYCP3 H1t merge
B6-ChrXMSM
SYCP3 ATR merge
H1t(-) H1t(+)
0
20
40
60
80(%) P<0.01
0
20
40
60
80
1 2
P=0.03
P=0.05
Aggregation Asynapsis
P<0.01
(%)
B6-ChrXB6 B6-ChrXMSM B6-ChrXB6 B6-ChrXMSM
Ht1(-) = early pachyteneH1t(+) = mid-to-late pachytene
Ht1(-)H1t(+)
B6-ChrXB6 B6-ChrXMSM
Figure 3. Synaptic errors and cell-cycle arrest at early pachytene stage in B6-ChrXMSM
spermatocytes. (A) SYCP3 and H1t double immunostaining of control and B6-ChrXMSM
spermatocytes at early and late pachytene substages. The early pachytene spermatocyte from B6-ChrXMSM showed aggregation of SYCP3 (arrows). (B) SYCP3 and ATR double immunostaining of control and B6-ChrXMSM early pachytene spermatocytes. ATR localized to the sex chromosomes in the control spermatocyte. Note the dotted ATR signals along asynaptic regions on autosomes in the B6-ChrXMSM spermatocyte. (C) Frequencies of aggregation and asynapsis at each substage. We counted 1035 pachytene spermatocytes from nine control testes and 1008 from seven B6-ChrXMSM
testes. (D) Proportion of early pachytene and mid-to-late pachytene spermatocytes, which were subtyped by staining with anti-H1t antibody. We counted 1463 pachytene spermatocytes from seven control males and 1334 from five B6-ChrXMSM males. All p-values were evaluated by t-test.
B
0
10
20
30
10 14 19 26 32
tubu
les
with
apo
ptot
ic c
ells (%)
B6-ChrXB6
B6-ChrXMSM
A
dpp
B6-ChrXB6(control) B6-ChrXMSM
15 dpp
19 dpp
Figure 4. Increased apoptosis at the pachytene stage in B6-ChrXMSM testes. (A) Incidence of apoptosis in testes at 15 dpp and 19 dpp. Occasional apoptosis was observed in both control and B6-ChrXMSM testes. In B6-ChrXMSM testis at 19 dpp, extensive apoptosis was observed in the tubules with meiotic spermatocytes. Scale bars: 100 μm. (B) The frequency of seminiferous tubules with apoptotic cells during first-wave spermatogenesis. Fifty tubules for each of three males were counted at each age tested. Apoptotic cells were judged by the condensed staining of nuclei by HE staining.
0
20
40
60
PGN-ChrXPGN (control) PGN-ChrXMSM
B
B6-ChrXB6
(control)PGN-ChrXMSM
SC
P3-
posi
tive
sper
mat
ocyt
es
(%)A
HE
TU
NE
L
PGN-ChrXPGN (control)
PGN-ChrXMSM
PGN(control)
Figure 5. Increased apoptotic spermatocytes at metaphase I in PGN-ChrXMSM testis. (A, left panel) Cell spreads from PGN-ChrXMSM testis at 19 dpp were immunostained with anti-SYCP3 antibody (red) and counterstained by Hoechst (blue). (A, right graph) Frequency of SYCP3-positive spermatocytes in control and PGN-ChrXMSM testes at 18-19 dpp. The difference was not significant by t-test. (B) Testicular histology of control PGN-ChrXPGN (upper left panel) and PGN-ChrXMSM
(upper right panel) males at 24 dpp. TUNEL staining of adult control PGN-ChrXPGN (bottom left panel) and PGN-ChrXMSM
(bottom right panel) testes. Increased apoptosis of metaphase I spermatocytes was observed at epithelial stage XII in PGN-ChrXMSM
testis (arrow heads in upper right panel and positive nuclei in bottom right panel). Scale bars: 200 μm.
0
50
100
0
50
100
0
50
100
0
50
100
0
20
40
60
A
B
prop
ortio
n of
mei
otic
spe
rmat
ocyt
es
*
(%)
B6-XB6
(control)(B6xNJL)F1 (B6xSpr)F1
C
B6-ChrXB6
ATR ATR+SYCP3
γH2AX γH2AX+SYCP3
RAD51 RAD51+SYCP3
(B6 x NJL)F1
MLH1 MLH1+SYCP3
*
*
*
*
*
B6-XB6 (B6xSpr)F1(B6xNJL)F1
H1t (-) (+) (-) (-) (+)
*
*
*
*
(B6 x M. spretus)F1
ATR
γH2AX
RAD51
MLH1
(B6 x M. spretus)F1
HE
TU
NE
L
**
ap
ap
(B6 x NJL)F1
NJL(control)
% o
f cel
ls w
ith A
TR
-po
sitiv
e X
Y b
ody
% o
f cel
ls w
ith γ
H2A
X-
posi
tive
XY
bod
y%
of c
ells
with
>20
RA
D51
fo
ci%
of c
ells
with
MLH
1 fo
ci
Figure 6. Meiotic phenotypes of inter-subspecific and inter-specific F1 hybrid males. (A) HE-stained testicular histology of (B6 x NJL/Ms) F1 (upper left panel) and (B6 x M. spretus) F1 (upper right panel) at 19 dpp. (B6 x NJL/Ms) F1 testis contained tubules with apoptotic zygotene spermatocytes (ap) and tubules lacking spermatocytes (*). Increased apoptosis of metaphase I was observed at epithelial stage XII in (B6 x M. spretus) F1 testis. TUNEL stained testicular histology of (B6 x NJL/Ms) F1 (bottom left panel) and (B6 x M. spretus) F1 (bottom right panel) at 24 dpp. Scale bars: 50 μm. (B) Ratio of SYCP3-positive meiotic spermatocytes to all testicular cells for each male at 18-19 dpp. Reduction of SYCP3-positive spermatocytes in (B6 x NJL/Ms) F1 testes was significant by t-test. (C) Immunostaining of control, (B6 x M. spretus) F1 and (B6 x NJL/Ms) F1 pachytene spermatocytes with anti-ATR, γH2AX, RAD51 and MLH1 antibodies (indicated in green). All spermatocytes were double-stained with anti-SYCP3 antibody (red). Graphs at the side indicate the quantification of signal-positive spermatocytes at early and mid-to-late pachytene stages. * indicates significant difference by t-test (P<0.05).
Wild type normal sperm
no sperm
(B6 x M. spretus)F1inter-specific F1
no sperm
B6-ChrXMSM
inter-subspecific consomicabnormal sperm
PGN-ChrXMSM
inter-subspecific consomicabnormal sperm
InterphaseInterphase Meiosis IMeiosis I
PremeioticPremeioticZygotene
to pachyteneZygotene
to pachyteneMetaphase IMetaphase I
mitosis spermiogenesisMeiosis IIMeiosis II
arrestapoptosis
apoptosis
apoptosisapoptosis?
(B6 x NJL/Ms)F1inter-subspecific F1
apoptosis
Figure 7. Summary of meiotic arrest in four different cases of reproductive isolation. Horizontal arrows indicate the progression of spermatogenic development. Downward arrows (blue) indicate points of disruptions (arrest and/or apoptosis). Orange horizontal lines indicate defective spermiogenesis. Thickness of blue and orange lines represents the proportion of spermatocytes subjected to arrests and/or apoptosis.
