TISSUE-SPECIFIC STEM CELLS
Sohlh2 Knockout Mice Are Male-Sterile Because of Degeneration of
Differentiating Type A Spermatogonia
JING HAO,a,b MIWAKO YAMAMOTO,a,b TIMOTHY E. RICHARDSON,a,b KAREN M. CHAPMAN,a,b,c
BRAY S. DENARD,a,b ROBERT E. HAMMER,a,d GUANG QUAN ZHAO,a,b F. KENT HAMRAa,b
a
Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of bPharmacology and
Biochemistry, and cHoward Hughes Medical Institute, University of Texas Southwestern Medical Center,
Dallas, Texas, USA
d
Key Words. Spermatogonial • Spermatocyte • Spermatogenesis • Germ cells • Germline • Embryonic stem cells • Cell death
ABSTRACT
The spermatogenesis and oogenesis-specific transcription
factor Sohlh2 is normally expressed only in premeiotic germ
cells. In this study, Sohlh2 and several other germ cell
transcripts were found to be induced in mouse embryonic
stem cells when cultured on a feeder cell line that overexpresses bone morphogenetic protein 4. To study the function
of Sohlh2 in germ cells, we generated mice harboring null
alleles of Sohlh2. Male Sohlh2-deficient mice were infertile
because of a block in spermatogenesis. Although normal
prior to birth, Sohlh2-null mice had reduced numbers of
intermediate and type B spermatogonia by postnatal day 7.
By day 10, development to the preleptotene spermatocyte
stage was severely disrupted, rendering seminiferous tubules with only Sertoli cells, undifferentiated spermatogonia, and degenerating colonies of differentiating spermatogonia. Degenerating cells resembled type A2 spermatogonia
and accumulated in M-phase prior to death. A similar phe-
notype was observed in Sohlh2-null mice on postnatal days
14, 21, 35, 49, 68, and 151. In adult Sohlh2-mutant mice, the
ratio of undifferentiated type A spermatogonia (DAZLⴙ/
PLZFⴙ) to differentiating type A spermatogonia (DAZLⴙ/
PLZFⴚ) was twice normal levels. In culture, undifferentiated type A spermatogonia isolated from Sohlh2-null mice
proliferated normally but linked the mutant phenotype to
aberrant cell surface expression of the receptor-tyrosine
kinase cKit. Thus, Sohlh2 is required for progression of
differentiating type A spermatogonia into type B spermatogonia. One conclusion originating from these studies would
be that testicular factors normally regulate the viability of
differentiating spermatogonia by signaling through Sohlh2.
This regulation would provide a crucial checkpoint to optimize the numbers of spermatocytes entering meiosis during
each cycle of spermatogenesis. STEM CELLS 2008;26:
1587–1597
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
To produce new gametes following each round of fertilization,
a small population of cells in the proximal epiblast is instructed
by bone morphogenetic proteins (BMP4 and BMP8b) to develop into primordial germ cells (PGCs) [1–3]. As the embryo
develops further, the PGCs increase in number as they migrate
to the developing ovaries or testes [4, 5]. In male rodents, after
PGCs reach the developing testes, they become enclosed within
the nascent seminiferous cords [6]. Subsequently, the gonocytes
proliferate for a couple of days and then become quiescent; it is
around this point that they are often referred to as gonocytes or
prespermatogonia [6 – 8]. Shortly after birth, gonocytes resume
mitotic activity and develop further into type A spermatogonia
that function as spermatogonial stem cells [6 –10]. During this
period, gonocytes also give rise to the first populations of
differentiating type A spermatogonia [8, 10]. Thus, some gonocytes bypass self-renewal and proceed directly into spermatogenesis to form the first generations of spermatozoa [8, 11].
Once formed, spermatogonial stem cells function throughout
adult life to renew large populations of developing gametes that
are constantly being released from the testes as mature spermatozoa.
In adult mammals, spermatogonial stem cells are thought
to reside as a subset of the undifferentiated type A spermatogonia that are located along the basement membrane of the
seminiferous tubules. One prominent hypothesis is that spermatogonia in the single cell state, termed A-single (As)
spermatogonia, function as spermatogonial stem cells in the
adult testis [12–14]. As such, they must be able to divide to
form new As spermatogonia, plus daughter cells that give rise
to “undifferentiated” chains of interconnected spermatogonia
[12–14]. Two-cell chains are termed A-paired (Apr) spermatogonia. Chains of 4 –32 cells are termed A-aligned (Aal)
spermatogonia. From the viewpoint of gene expression, undifferentiated As, Apr, and Aal spermatogonia express relatively low levels of the receptor-tyrosine kinase cKit [15–18]
but higher levels of other essential proteins, including Plzf
[19, 20], Ret, Gfra1 [21–23], Sox3 [24], and Ngn3 [25].
Consistent with this pattern of expression, most of the latter
molecules are required for maintenance of undifferentiated
spermatogonia in the testes [19 –24], whereas signaling pathways linked to cKit are well established as being required for
Correspondence: F. Kent Hamra, Ph.D., Department of Pharmacology; Cecil H. and Ida Green Center for Reproductive Biology Sciences,
University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390, USA. Telephone: 214-645-6279; Fax:
214-645-6276; e-mail: [email protected] Received June 26, 2007; accepted for publication March 4, 2008; first published
online in STEM CELLS EXPRESS March 13, 2008. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2007-0502
STEM CELLS 2008;26:1587–1597 www.StemCells.com
Sohlh2 Is Required for Spermatogenesis
1588
differentiating spermatogonia and spermatocytes to progress
through spermatogenesis [16, 26 –34].
Here, when we cultured mouse embryonic stem cells (ESCs)
on an epithelial cell line that stably expresses recombinant BMP4
[3], several germ cell-specific transcripts were upregulated in the
ESCs. Recombinant forms of BMPs were also recently reported to
induce germ cell development in cultures of human ESCs [35].
These results mirror the abilities of BMP4 and BMP8b to induce
germ cell fate in cells of the epiblast [1–3]. Interestingly, one of the
transcripts induced in mouse ESCs by the BMP4-expressing cell
line encoded the newly described transcription factor Sohlh2 [36].
Sohlh2 was named on the basis of its homology with the initially
described spermatogenesis and oogenesis-specific basic helix-loophelix (bHLH) transcription factor Sohlh1 [36, 37]. As their names
indicate, Sohlh1 and Sohlh2 are specifically expressed in male and
female germ cells [36 –38], and genetic studies in mice have shown
that Sohlh1 is required for oogenesis and spermatogenesis [37]. We
show that in addition to its induction in ESCs, Sohlh2 is critical for
proper development of differentiating spermatogonia into preleptotene spermatocytes, and we identify the receptor-tyrosine kinase
cKit as a potential downstream effector of Sohlh2 function. Because overproduction of preleptotene spermatocytes severely disrupts spermatogenesis and leads to male infertility [39 – 42], signaling through Sohlh2, and potentially Sohlh1, could allow the
seminiferous epithelium to regulate the numbers of differentiating
spermatogonia that enter meiosis.
MATERIALS
AND
METHODS
Protocols on generation of Sohlh2-deficient mice, embryonic stem
cell culture, immunohistochemistry, immunocytochemistry, transcript analyses, fractionation of mouse testis cells, flow cytometry,
and derivation of spermatogonial lines are given in supplemental
online Materials and Methods.
Histological Analysis of Mouse Testes
Spermatogenesis was evaluated in hematoxylin and eosin (H&E)stained, 5-␮m-thick histological sections prepared from testes of wildtype and Sohlh2-null littermates on embryonic day 17.5 post coitus
(E17.5) and on postnatal days 1, 7, 10, 14, 21, 35, 49, 68, and 151. Prior
to sectioning, the isolated testes were incubated overnight at 22°C–
24°C in Bouin’s fixative, washed thoroughly in 70% ethanol, and then
embedded in paraffin. The average numbers of Sertoli cells, gonocytes,
type A spermatogonia, intermediate to type B spermatogonia, preleptotene spermatocytes, leptotene to early pachytene spermatocytes, midpachytene to diplotene spermatocytes, round spermatids, and elongating spermatids were scored per tubular cross-section from mice on
E17.5 and postnatal day (D) 7, D10, D14, D21, and D151. At each age,
sections were prepared from duplicate animals from separate litters for
each genotype. Numbers of cells per tubular cross-section were
counted for each of the above categories by morphometric analysis
using the Simple-PCI software (C-Imaging Systems, Compix, Cranberry Township, PA, http://www.cimaging.net) in line with an AX70
light microscope (Olympus, New Hyde Park, NY, http://www.
olympus-global.com). Cells in cross-sections of 20 tubules (10 tubules
from each of two animals per genotype) from E17.5, D7, D10, D14,
D21, and D151 mice were counted in microscopic fields (16.8 mm2)
captured using a ⫻60 objective (supplemental online Table 1). Average
counts for each cell type were normalized per 1,000 Sertoli cells from
duplicate mice at each respective age (Fig. 2). Spermatogenic cell types
in wild-type mice were classified on the basis of their morphologies in
the H&E-stained sections and localization to specific stages of a seminiferous epithelial cycle [43– 45]. In Sohlh2 knockout mice, spermatogonia were classified as either undifferentiated or differentiated on the
basis of their patterns of H&E staining in comparison with wild-type
mice. This was due to our current inability to clearly identify distinct
spermatogenic stages in Sohlh2 knockout mice.
Histological Analysis of Spermatogonial Types
Spermatogonial types present in Sohlh2-deficient mice were analyzed in H&E-stained sections as described above (5 ␮m) and on
the basis of staining profiles of different spermatogonial types
present in adult wild-type mice. In wild-type mice, the numbers of
undifferentiated and differentiating spermatogonia per tubular
cross-section were first scored at stages II, III, V, VIII, X, and XII
of spermatogenesis to avoid scoring differentiating spermatogonia
in M-phase [44]. The spermatogenic stage of each tubule used to
count spermatogonia and Sertoli cells was verified in parallel crosssections (5 ␮m) stained by the periodic acid-Schiff method to
visualize steps of mouse spermiogenesis (supplemental online Fig.
1A) [43]. Then, average numbers of each spermatogonial type
scored per tubular segment (n ⫽ 5– 8 cross-sections per stage) were
normalized per 1,000 Sertoli cells (supplemental online Fig. 1B).
Undifferentiated spermatogonia were clearly distinguished from
differentiating types during each stage because of their relative lack
of nuclear staining (supplemental online Fig. 1C). As bright-field
images, undifferentiated spermatogonia were more lightly stained,
with a uniform pattern throughout the cytoplasm and nucleus,
compared with more differentiated types of spermatogonia. The
intensity of chromatin staining gradually increased as undifferentiated spermatogonia differentiated into type A1 spermatogonia (supplemental online Fig. 1C). Between stages VI and VIII, nucleoli of
Aal spermatogonia became more prominent, with a distinct “pinkish
halo morphology” as they differentiated into type A1 spermatogonia
(supplemental online Fig. 1C). In contrast, types A1 to B spermatogonia showed increasingly darker staining in distinct regions of
their chromatin as they differentiated. Types A1 and A2 spermatogonia often showed 1–3 “spots” of darkly stained chromatin situated
randomly in the nucleus, which sharply contrasted with lighter
staining chromatin throughout the rest of the nucleus. Nuclei of
types A1 and A2 spermatogonia were clearly distinct from nuclei of
undifferentiated spermatogonia and were four- to fivefold more
abundant than undifferentiated spermatogonia in their respective
stages (supplemental online Fig. 1B). Relative numbers of each
spermatogonial type scored per 1,000 Sertoli cells in adult wild-type
mice during distinct stages of a spermatogenic cycle are plotted in
supplemental online Figure 1B. These data are in close agreement
with the relative numbers of spermatogonial types scored per 1,000
Sertoli cells in whole mounts of mouse seminiferous tubules [13].
Types A4, intermediate, and B spermatogonia showed the darkest
overall patterns of chromatin staining, with increasing perinuclear
localization of the most intensely stained regions of chromatin.
Type A3 spermatogonia often appeared similar to type A2 spermatogonia, containing two to five small dark spots of chromatin, but
some also showed staining similar to type A4 spermatogonia, with
more darkly stained perinuclear regions of chromatin. Many A4 to
B spermatogonia also contained spots of dark chromatin, and these
spots often became large and more irregularly shaped in perinuclear
regions (supplemental online Fig. 1C).
Statistical Analyses
Results from wild-type and Sohlh2 mutant animal studies were
tested for statistical significance using the Student t test. Differences
were considered significant if p ⬍ .05.
RESULTS
Induction of Germ Cell Transcripts in Embryonic
Stem Cells
In experiments studying germ cell specification in culture, transcripts normally expressed only during germ cell development,
such as DAZL [46, 47], Stella [48], Fragilis [49], and Stra8 [50],
were selectively induced in mouse ESCs following their culture
on a COS-7 cell line that stably expresses BMP4 (Fig. 1A) [3].
This population of transcripts included one that encoded a
bHLH transcription factor (GenBank accession no. AI427377)
predicted by the Unigene Database (National Center for Bio-
Hao, Yamamoto, Richardson et al.
A 800
Control
BMP4
700
Microarray Signal
1589
600
500
400
300
200
100
B Postnatal Week 8 Testes
I4
Wild-Type
Sohlh2 Knockout
100
80
60
40
P < 0.05
-/-
Testis Weight (mg)
+/-
Sohlh2 Genotype
(S 273
oh 77
lh
2)
A
C 140
120
+/+
D
m
rt
1
A
ar
d
lis
Fr
ag
i
el
la
St
8
ra
St
D
A
ZL
Xl
r3
b
0
20
0
WT
KO
0 2 4 6 8 10 12 14 16 18
Postnatal Age (Weeks)
technology Information) to be expressed only in male and
female gonads. More recently, this protein was termed Sohlh2
[36]. In adult tissues, transcripts encoding Sohlh2 were specifically detected in the testes by reverse transcription (RT)-polymerase chain reaction (PCR) (supplemental online Fig. 2A). In
postnatal testes, the abundance of Sohlh2 transcripts gradually
increased in parallel with germ cell abundance from birth to
postnatal day 18 and then decreased slowly until postnatal day
29 (supplemental online Fig. 2B). In germ and somatic cell
fractions isolated from 19-day-old mice, Sohlh2 transcripts were
highly enriched in the germ cell fractions (supplemental online
Fig. 2B) [51]. These results were consistent with Sohlh2 being
expressed specifically in premeiotic germ cells in vivo [36] and
also show that Sohlh2 can be selectively induced in embryonic
stem cells by a COS-7 cell line that expresses BMP4 as a
physiological effector of germ cell specification [3].
Male Sohlh2-Null Mice Are Sterile
To investigate the function of Sohlh2 during germ cell development, we mutated the Sohlh2 gene in mouse ESCs. Clonal
ESC lines harboring a truncated Sohlh2 gene were derived and
then used to generate mutant mice deficient in functional
Sohlh2. The truncated gene was designed to encode for a form
of Sohlh2 protein that lacks the bHLH DNA binding domain
(supplemental online Fig. 3A). This genotype was confirmed by
genomic Southern blot and PCR analyses (supplemental online
Fig. 3B, 3C). In homozygous Sohlh2-deficient mice, no native
www.StemCells.com
Figure 1. Identification Sohlh2 and its effects on spermatogenesis. (A): Germ cellspecific transcripts, including Sohlh2, were
selectively induced in embryonic stem cells
by culturing them on the COS-pBMP4 cell
line, compared with embryonic stem cells
that were cultured on the COS-pIRES cell
line (control). (B): Sohlh2-null mice show a
large reduction in testis size. Top, testes
from WT (⫹/⫹) mice and testes of their
litter mates that were heterozygous (⫹/⫺) or
homozygous (⫺/⫺) for the mutant Sohlh2
allele. Testes were isolated on postnatal
week 8. Bottom, histological cross-sections
through testes from adult WT and Sohlh2null mice (KO) on postnatal day 56. Spermatogonia and spermatocytes were selectively labeled using an antibody to DAZL
(pink). Nuclei of all cells were labeled with
Hoechst 33342 dye (blue). Note the severe
reduction in the number of spermatogonia
and spermatocytes in seminiferous tubules
of Sohlh2-null mice (KO). (C): Early onset
of testicular atrophy in Sohlh-null mice.
Postnatal weight of testes from WT mice and
mice homozygous for the mutant Sohlh2 allele (Sohlh2 KO). A significant difference
between weights of testes from WT and
Sohlh2 KO mice was observed starting at 2
weeks of age. Abbreviations: BMP4, COSpBMP4 cell line; DAZL, deleted in
azoospermia-like protein; KO, knockout;
WT, wild-type.
Sohlh2 mRNA was detected in the testis or in spermatogonial
lines derived from the Sohlh2-mutant mice, on the basis of
analysis by RT-PCR (supplemental online Fig. 3D). Male mice
that were homozygous for the mutant Sohlh2 allele developed
into apparently normal adults except that they were infertile, and
their testes were clearly undersized (Fig. 1B). Interbreeding of
heterozygous mice yielded the Mendelian ratio (79:151:73) of
Sohlh2⫺/⫺, Sohlh2⫹/⫺, and Sohlh2⫹/⫹ F1 progeny. These
results indicated that there is no lethality caused by the Sohlh2
mutation. Immunohistochemical analysis with antibodies to the
deleted in azoospermia-like (DAZL) protein (Fig. 1B) and germ
cell nuclear antigen (GCNA) (supplemental online Fig. 4),
which label all stages of spermatogonia and spermatocytes [46,
47, 52, 53], revealed that the germ cell population was severely
depleted in adult testes. A significant difference in testis weight
was measured as early as postnatal day 14 and the difference in
testis weight increased over time (Fig. 1C). Testes from adult
Sohlh2⫺/⫺ males (8 –16 weeks old; n ⫽ 5) on average weighed
3 to 4 times less than testes from Sohlh2⫹/⫹ or Sohlh2⫹/⫺
mice (Fig. 1C). These results showed a clear defect in spermatogenesis in Sohlh2-null mice.
Type A Spermatogonia Degenerate in Testes of
Sohlh2-Null Mice
On the basis of the early onset of testicular atrophy (Fig. 1C), we
next analyzed postnatal germ cell development in testes of
Sohlh2 Is Required for Spermatogenesis
1590
1,250
4,000
500
250
PL
pe
e
G
on
oc
yt
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Day 7 Mice
pe
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Cells/1,000 Sertoli Cells
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E17.5 Mice
Cells/1,000 Sertoli Cells
Cells/1,000 Sertoli Cells
1,250
Sohlh2 Knockout
Cells/1,000 Sertoli Cells
Cells/1,000 Sertoli Cells
1,250
Cells/1,000 Sertoli Cells
Wild-Type
250
Day 21 Mice
1,000
750
500
250
S
R
PL
LEP
M
PD
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rt
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Ty l i
pe
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pe
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Month 5 Mice
3,000
2,000
1,000
0
Se
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Ty oli
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LEP
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Ty i
pe
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LEP
M
PD
R
S
ES
1,000
Day 14 Mice
Se
rt
ol
Ty i
pe
A
Ty
pe
B
Day 10 Mice
Figure 2. Morphometric analysis of spermatogenesis in Sohlh2-knockout mice. Spermatogenic cell types present in wild-type and Sohlh2 knockout
mice on E17.5; postnatal days 7, 10, 14, 21; and month 5. The numbers of Sertoli cells, gonocytes, Type A, Type B, PL, L-EP, MP-D, RS, and ES
were scored in duplicate animals at each age and genotype. Type A include undifferentiated to A4 spermatogonia. Average numbers of each cell type
per tubular cross-section were normalized per 1,000 Sertoli cells. Abbreviations: E, embryonic day; ES, elongating spermatids; L-EP, leptotene to
early pachytene spermatocytes; MP-D, midpachytene to diplotene spermatocytes; PL, preleptotene spermatocytes; RS, round spermatids; Type A, type
A spermatogonia; Type B, intermediate/type B spermatogonia.
Sohlh2-null mice. Examination of H&E-stained tubular crosssections on E17.5 revealed normal numbers of gonocytes and
Sertoli cells in Sohlh2-null mice (Fig. 2). However, by postnatal
day 7, Sohlh2-null mice had normal numbers of Sertoli cells and
type A spermatogonia but started to show reduced numbers of
intermediate/type B spermatogonia (Fig. 2). By day 10, development to the preleptotene spermatocyte stage was severely
disrupted in Sohlh2-null mice, rendering predominantly Sertoli
cells and type A spermatogonia in their seminiferous tubules
(Figs. 2, 3). In contrast, germ cells in testes from 10-day-old
wild-type mice had already developed into pachytene spermatocytes. Similar results were observed in testes of D14 and D21
Sohlh-null mice, where all spermatocyte stages from preleptotene to diplotene were severely depleted (Figs. 2, 3). However,
by D21, Sohlh-null mice also showed a twofold reduction in
their numbers of type A spermatogonia and greater than a
threefold reduction in their numbers of intermediate/type B
spermatogonia per Sertoli cell (Figs. 2, 3). This phenotype
progressed into adulthood, as 1-, 2-, and 5-month-old Sohlh2null mice (i.e., D35, D68 and D151 Sohlh2-null mice) showed
no spermatocytes or spermatids but did contain undifferentiated
and differentiating type A spermatogonia (Figs. 2, 3). Fivemonth-old Sohlh2-null mice (D151) contained ⬃60% as many
type A spermatogonia and ⬃15% as many intermediate/type B
spermatogonia as were counted in wild-type mice per Sertoli
cell (Figs. 2– 4).
The spermatogenic defect was widespread and was observed
in 100% of the tubular cross-sections examined in mice on D10,
D14, D21, and D151 (supplemental online Fig. 5A, 5C). Results
from the histological analyses indicated that disruption of
Sohlh2 caused a progressive degenerative defect in type A
spermatogonia (Fig. 4). Consistent with this hypothesis, we
observed increased numbers of abnormal differentiating type
A-like spermatogonia on the tubular basement membrane of
Sohlh2-null mice at all ages examined (Figs. 3, 4; supplemental
online Fig. 5B–5D). The abnormal cells displayed heterochro-
Hao, Yamamoto, Richardson et al.
Wild-Type Day 7
1591
Knockout Day 7
A2
Undif
Undif
A2
Mit
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Aal -A1
Mit
A3
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SC
Wild-Type Day 10
Sertoli
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Mit
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Mit
L-EP
Sertoli
Wild-Type Day 14
Knockout Day 14
A3
Mit
P
Undif
Mit
A2
Mit
A2
Undif
Undif
Figure 3. Spermatogenesis is disrupted early in Sohlh2 knockout mice.
Spermatogenic cells are shown in hematoxylin and eosin (H&E)-stained
histological cross-sections of seminiferous tubules from wild-type and
Sohlh2 knockout mice on postnatal days 7, 10 and 14. Sertoli, Undif,
Aal-A1, A2, A3, Mit, PL, L-EP, and P. Scale bars ⫽ 50 ␮m. Abbreviations: A2, A2 spermatogonia; A3, A3 spermatogonia; Aa1–A1, Aaligned spermatogonia transitioning into A1 spermatogonia; L-EP, leptotene-early pachytene spermatocytes; Mit, mitotic spermatogonia; P,
pachytene spermatocytes; PL, preleptotene spermatocytes; SC, Sertoli
cells; Undif, undifferentiated spermatogonia.
matin patterns most similar to that reported for degenerating
type A2–A4 spermatogonia in M-phase [15, 43, 54]. Mitotic
figures of spermatogonia expressing phospho-histone-3 were
still present in 2- and 5-month-old Sohlh2-null mice and appeared to accumulate in M-phase prior to death (supplemental
online Figs. 1D, 5B, 5D). In H&E-stained sections, the degenerating cells increased in volume as they approached metaphase;
thereafter, these cells displayed irregular spindle apparatus and
intensely stained pericellular regions of heterochromatin (supplemental online Fig. 5D). Studies examining the uptake of
bromodeoxyuridine in 2-month-old mice further indicated that
spermatogonia were still dividing in testes of the Sohlh2-mutant
mice and that the ratio of undifferentiated type A spermatogonia
(DAZL⫹/PLZF⫹) to differentiating type A spermatogonia
(DAZL⫹/PLZF⫺) in Sohlh-null mice was double normal levels
(supplemental online Fig. 6). These results show that Sohlh2null mice are infertile because of a defect that prevents differentiating type A spermatogonia from properly developing into
type B spermatogonia.
Undifferentiated Spermatogonia Appear Normal in
Sohlh2-Null Mice
We next analyzed the populations of undifferentiated spermatogonia in 10-day-old Sohlh2 null mice. Numerous segments in
seminiferous tubules from Sohlh2-null mice contained higher
concentrations of undifferentiated spermatogonia (DAZL⫹/
www.StemCells.com
PLZF⫹) relative to differentiating spermatogonia (DAZL⫹/
PLZF⫺) compared with wild-type mice (Fig. 5A). Also, on the
basis of their ability to bind to laminin, more DAZL⫹/PLZF⫹
spermatogonia were isolated from testes of Sohlh2-null mice
than from wild-type mice on D10 (Sohlh2-null, 7.2 ⫾ 0.7 ⫻ 104
laminin-binding cells per mouse; wild-type, 3.9 ⫾ 0.2 ⫻ 104
laminin-binding cells per mouse; n ⫽ 6 mice per genotype; p ⬍
.005). Because undifferentiated spermatogonia were able to
develop in testes of Sohlh2-null mice, we attempted to establish
Sohlh2-deficient spermatogonial lines from 10-day-old mice.
Spermatogonial lines were successfully derived from both wildtype and Sohlh2-kockout mice. Duplicate lines were derived for
each genotype, with a 100% success rate. After plating equal
numbers of germ cells per well from the fully derived spermatogonial lines, the growth rates of spermatogonia from Sohlh2-null
and wild-type mice were similar (Fig. 5B). After proliferating
for more than 20 passages in culture, spermatogonia from each
genotype increased ⬎500,000-fold in number over a 6-month
period and continually expressed high relative levels of both
DAZL and PLZF, which classified them as undifferentiated,
type A spermatogonia (Fig. 5C, 5D). Thus, Sohlh2 is not required for development of undifferentiated spermatogonia in
vivo, and loss of functional Sohlh2 did not affect the rate of
spermatogonial proliferation in culture.
Aberrant Expression of cKit on Spermatogonia from
Sohlh2-Null Mice
To identify genes that could be linked to the phenotype observed
in Sohlh2-null mice, we initially compared the relative abundance of transcripts expressed in the testes of mutant and wildtype mice by using Affymetrix 430 2.0 microarray chips (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). We
chose to extract RNA from the testes of 8-day-old mice because
of the substantially lower abundance of differentiating germ cell
types normally present at this young age compared with adult
mice [51]. Supplemental online Table 3 shows the relative
abundance of several transcripts important for spermatogonial
renewal, survival, and/or differentiation. Many of these transcripts were subsequently analyzed by RT-PCR using the same
pools of RNA (Fig. 6A). Of note, genes implicated in meiosis
and apoptosis, such as Mlh1, Bcl2, Bax, and Bcl2l2, were
expressed at relatively similar levels in Sohlh2-null and wildtype testes (supplemental online Table 3). However, some transcripts expressed by undifferentiated spermatogonia were more
abundant in Sohlh2-null testes compared with wild-type testes,
including Gfra1, Oct4, and Sox2 (Fig. 6A; supplemental online
Table 3). In contrast, some genes associated with oogonial and
spermatogonial differentiation appeared to be downregulated in
Sohlh2-null testes, including Lhx8, cKit, Crabp1, and Ngn3
(Fig. 6A; supplemental online Table 3).
Because transcripts encoding cKit were reduced in the testes
of 8-day-old mice, and because some mutations that disrupt cKit
signaling [27, 30, 34] result in a similar germ cell phenotype, as
observed in Sohlh2-null mice, we next examined the expression
of cKit on spermatogonia from wild-type and Sohlh2-null mice.
In seminiferous tubules from 10-day-old Sohlh2-null mice, tubular segments without cKit expression were common (Fig.
6B), whereas tubular segments expressing cKit were prevalent
in their wild-type litter mates (Fig. 6B). Consistent with these
results, the expression of cKit on spermatogonial lines from
Sohlh2-null mice was reduced more than threefold compared
with spermatogonial lines derived from wild-type mice (Fig. 6C;
supplemental online Fig. 7). Thus, Sohlh2 is required for spermatogenesis and can regulate the expression of cKit on spermatogonia.
Sohlh2 Is Required for Spermatogenesis
1592
A
5 Month Old Wild-Type Mouse
Stage VIII
Stage X
Undif
Ser
Stage XII
Undif
A2
A3
Undif
A1
PL
Stage II
Stage III
Stage V
Undif
Undif
A4
Undif
Int
B
B
5 Month Old Sohlh2 Knockout Mouse
Dif
Dif
Undif
Undif
Ser
Dif
Dif
Undif
Ser
Dif
Ser
WT
KO
300
250
200
150
100
50
Type B Spermatogonia
175
WT
KO
150
125
100
75
50
25
Here, we show that the spermatogenesis- and oogenesis-specific
bHLH transcription factor Sohlh2 is required for spermatogenesis. In Sohlh2-null mice, spermatogenesis is disrupted throughout postnatal life because of degeneration of differentiating type
A spermatogonia (Fig. 7). On the basis of comparisons with
wild-type mice, a dramatic loss of differentiating spermatogenic
cells in Sohlh2-null mice is evident by postnatal days 10 and 14,
and the defect persists into adulthood when the seminiferous
tubules contain predominantly Sertoli cells and type A spermatogonia. A requirement for survival of differentiating spermatogonia points to a potential physiological role of Sohlh2 in
the regulation of cell density during spermatogenesis [39, 40,
54, 55]. Selective loss of differentiating spermatogonia is well
documented in rodents, where it is primarily accounted for by
death of A2, A3, and A4 spermatogonia [45, 54 –56]. Normally,
21
M
on
th
5
ay
14
D
ay
10
DISCUSSION
D
ay
D
ay
21
M
on
th
5
ay
14
D
D
ay
10
7
ay
D
ay
7
0
0
D
200
Type A Spermatogonia
D
350
Cells/1,000 Sertoli Cells
Cells/1,000 Sertoli Cells
C
Undif
Figure 4. Development of differentiating
spermatogonia is disrupted in Sohlh2-null
mice. (A): Histological representation of
H&E-stained spermatogonia in a 5-monthold WT mouse. Shown are A1, A2, A3, A4,
Int, B, and PL in their respective stages of
spermatogenesis. Scale bars ⫽ 10 ␮m. (B):
Histological representation of H&E-stained
spermatogonia in a 5-month-old Sohlh2 KO
mouse. Shown are Ser, Undif, and Dif. Spermatogenic stages could not be established in
the KO animal because of severe disruption
of spermatogenesis. Scale bars ⫽ 10 ␮m.
(C): Relative numbers of type A (left) and
intermediate/type B (right) spermatogonia
scored in random (i.e., independent of stage)
tubular cross-sections form duplicate WT
and Sohlh2 KO animals at 5 months of age.
Average numbers of each cell type are normalized per 1,000 Sertoli cells. Abbreviations: A1, Aal–A1 spermatogonia; A2, A2
spermatogonia; A3, A3 spermatogonia; A4,
A4 spermatogonia; B, B spermatogonia; Dif,
differentiating A1-like, A2-like and A3-like
spermatogonia; Int, intermediate spermatogonia; KO, knockout; PL, preleptotene spermatocytes; Ser, Sertoli cells; Undif, undifferentiated spermatogonia; WT, wild-type.
numbers of A1 spermatogonia fluctuate by more than threefold
during different spermatogenic cycles and are frequently produced in excess [45, 55]. However, densities of type B spermatogonia and preleptotene spermatocytes remain constant in
different spermatogenic waves from cycle to cycle [45, 55]. This
shows that the density of spermatogonia entering meiosis is
tightly regulated by eliminating excess A2, A3, and A4 spermatogonia [45, 55]. The importance of this regulation is highlighted in transgenic mice, where death of differentiating spermatogonia is blocked either by overexpression of antiapoptotic
Bcl-2 family proteins (i.e., Bcl-2 and Bcl-XL) [39, 40] or by
inactivation of apoptosis-inducing Bcl-2 family members, such
as Bax, Bim, and Bik [41, 42, 57]. In these animals, a surplus of
type B spermatogonia and preleptotene spermatocytes is
formed, which leads to infertility due to massive germ cell death
during the early steps of meiosis [39 – 42, 57]. Because Sohlh2
is required for development of type B spermatogonia, this germline-specific bHLH family member could function to regulate
Hao, Yamamoto, Richardson et al.
Figure 5. Undifferentiated spermatogonia appear normal in Sohlh2
KO mice. (A): Immunolabeling of DAZL (green) and PLZF (red) in
germ cells within seminiferous tubules dissected from 10-day-old wildtype (top) and Sohlh2-KO (bottom) mice. Scale bars ⫽ 50 ␮m. (B):
Growth rates of spermatogonial lines following derivation from 10-dayold wild-type and Sohlh2-KO mice. After their initial plating on mouse
embryonic fibroblasts (MEFs), spermatogonia were harvested and then
passaged onto fresh MEFs every 12–16 days at 1–2 ⫻ 104 cells per cm2.
Data represent average cell counts obtained at each passage for duplicate
cell lines of each genotype. (C): Left, immunolabeling showing coexpression of DAZL (green) and PLZF (red) proteins in cultures of
spermatogonial lines established from 10-day-old wild-type and Sohlh2null (Sohlh2 KO) mice. Right, phase contrast microscope images of the
same cells shown in the left panels. Expression of DAZL was highly
localized to the cytoplasm of spermatogonia, whereas PLZF was highly
localized to spermatogonial nuclei. The spermatogonia depicted are
from passage 18 after proliferating for more than 200 days in culture.
(D): Left, immunolabeling of the same respective cultures shown in
Figure 5C with isotype control antibodies. Purified rabbit IgG (green)
and purified mouse IgG1 (red) showed background levels of immunoglobin binding to spermatogonial lines derived from wild-type and
Sohlh2 KO mice and to the MEF feeder cells within the cultures. Right,
phase-contrast microscope images of the same cells shown in the left
panels. Abbreviation: KO, knockout.
the numbers of spermatocytes that enter meiosis so as not to
disrupt spermatogenesis.
In addition to a reduction in numbers of differentiating
spermatogonia, we also observed higher concentrations of
undifferentiated spermatogonia in the testes of Sohlh2-null
mice compared with wild-type mice. On postnatal day 10, we
were able to isolate almost twice the number of lamininbinding, Plzf-positive cells from Sohlh2-null mice as from
wild-type mice. A block in the ability of spermatogonia to
differentiate during the initial cycles of spermatogenesis
www.StemCells.com
1593
would potentially result in increased numbers of spermatogonia that express Plzf in the testes. The accumulation of
undifferentiated spermatogonia at day 10 could also be explained by an increased rate of spermatogonial amplification
in Sohlh2-null mice. However, under culture conditions
where stem cell activity is maintained in response to glial cell
line-derived neurotrophic factor (GDNF) [58], spermatogonial lines derived from Sohlh2-null mice did not proliferate
faster than spermatogonial lines derived from wild-type mice
(Fig. 5B). Thus, it is possible that undifferentiated spermatogonia in young Sohlh2-null mice do not undergo a developmental inhibition as normally occurs in rodent testes [59, 60].
This negative regulatory mechanism is thought to be mediated by factors that are produced by differentiating spermatogonia, potentially intermediate and type B spermatogonia
[59, 60]. Accordingly, the relative lack of intermediate and
type B spermatogonia in Sohlh2 null mice could result in the
absence of factors that function to block amplification of
undifferentiated spermatogonia. Although such paracrine factors are normally produced in the testes, they may not be
present at effective concentrations in cultures of highly pure
undifferentiated spermatogonia or in testes of Sohlh2-null
mice. We recently reported that GDNF can function in synergy with another polypeptide, termed neuregulin-1, to stimulate cultures of undifferentiated spermatogonia to develop
into chains of spermatogonia at the 4-, 8-, 16-, and 32-cell
stages [61] (Fig. 7). In the presence of low neuregulin-1
concentrations, GDNF functioned only as a partial agonist to
stimulate formation of longer spermatogonial chains [61].
However, in the presence of higher neuregulin-1 concentrations, the effectiveness of GDNF for stimulating the formation of longer spermatogonial chains was dramatically increased [61]. Therefore, loss of differentiating spermatogonia
in Sohlh2-null mice could unmask cooperative effects of
GDNF and neuregulin-1 to amplify the numbers of A1 spermatogonia produced during a spermatogenic cycle (Fig. 7).
Ballow et al. initially reported the identification of Sohlh2
by Blast homology searches against Sohlh1; the two genes they
found shared high homology at the amino acid level within their
DNA-binding domains (⬎50%) [36]. Like Sohlh1, Sohlh2 is
specifically expressed in the male and female germ lines [36 –
38]. Sohlh2 transcripts are clearly expressed as early as E12.5 in
the gonads of both male and female mice [36]. In males, Sohlh2
protein was detected in undifferentiated and differentiating spermatogonia of mice at postnatal days 5, 21, and 49 [36], which
substantially overlaps the expression profile reported for Sohlh1
in developing gametes [37, 38]. In females, Sohlh2 protein is
expressed in developing germ cells of the ovary by E17.5 and in
oocytes of primordial and primary follicles in adults; Sohlh2
was not detected in secondary follicles [36]. Disruption of
Sohlh1 blocks follicle formation and is linked to downregulation
of genes that are essential for folliculogenesis [38]. Preliminary
experiments show that Sohlh2 is also required for oocyte development and fertility in females (J.H., unpublished data). In
addition to their overlapping expression profile in male and
female germ cells and their requirement for premeiotic stages of
male and female gametogenesis, both Sohlh1 and Sohlh2 regulate several of the same genes. Based on transcript analyses,
both Sohlh1 [37, 38] and Sohlh2 function to maintain the
relative abundance of Lhx8, Crabp1, Ngn3, and cKit in spermatogonia. The high degree of homology in their DNA binding
domains, their overlapping gene expression profiles, and their
remarkably similar effects on male and female germ cell development suggest that Sohlh1 and Sohlh2 could function as a
heterodimer to regulate analogous biochemical pathways required for development of male and female germ cells.
Sohlh2 Is Required for Spermatogenesis
1594
Figure 6. Expression of germ cell differentiation factors in Sohlh2 KO mice. (A): Gene expression during spermatogenesis was analyzed by reverse
transcription-polymerase chain reaction to help validate microarray data presented in supplemental online Table 3. Hprt was used as a standard. Total
RNA was purified from testes of 8-day-old wild-type (⫹/⫹) and homozygous mutant Sohlh2 (⫺/⫺) mice. (B): Immunolabeling of DAZL (green)
and cKit (red) in germ cells within seminiferous tubules that were dissected from 10-day-old wild-type (top) and Sohlh2-KO (bottom) mice. Scale
bar ⫽ 50 ␮m. (C): Analysis of cKit expression on spermatogonial lines derived from wild-type and Sohlh2-null (Sohlh2 KO) mice by flow cytometry.
Similar results were obtained using two separate spermatogonial lines derived from mice for each genotype. Fluorescence values representing binding
of the anti-cKit IgG above background levels on wild-type and KO lines from three separate passages were 58.1 ⫾ 11.0 and 15.4 ⫾ 5.2 (⫾ SEM),
respectively. Details on gating spermatogonia and mouse embryonic fibroblast populations are given in supplemental online Figure 7. Abbreviation:
KO, knockout.
Undifferentiated Spermatogonia
GDNF & Neuregulin-Dependent*
A single
A single
1M
A paired
A aligned: 4, 8, 16 -cell clones
1M to 4M
.
1M
G1 Transition
A1
.
cKit-Dependent**
.
1M
A2
1M
Differentiating
Spermatogonia
A3
1M
Sohlh2-Dependent***
A4
1M
Int
1M
B
1M
PL
Meiosis
The requirement of cKit and its ligand, stem cell factor
(SCF), for development of differentiating spermatogonia is
exemplified by naturally occurring mutations in mice [42, 62,
63]. Mutations in the Dominant White Spotting (W) locus,
Figure 7. Illustration showing steps of
spermatogonial development in rodents. The
model is based on the phenotype of Sohlh2mutant mice reported herein, plus phenotypes of W and Sl mutant mice and mice with
inactivating mutations in genes encoding
GDNF or its downstream effectors GFR␣1
and Ret, as reviewed by de Rooij [73]. ⴱ, It
should be noted that the effects of Neuregulin on spermatogonial development have
been reported only in tissue culture [61]. ⴱⴱ,
In addition to cKit, other molecules have
been reported to regulate transition of Aal
spermatogonia into A1 spermatogonia, including retinoic acid, testosterone, and Cyclin D [73]. ⴱⴱⴱ, As postulated here for
Sohlh2, Bcl-2 family proteins have been implicated in regulating the density of differentiating spermatogonia [73]. Abbreviations:
GDNF, glial cell line-derived neurotrophic
factor; PL, preleptotene spermatocytes.
encoding KIT, or the Steel (Sl) locus, encoding SCF, result in
sterility, hypoplastic anemia, and the depletion of mast cells
and melanocytes [64]. Mutations in either the W or Sl locus
can severely disrupt the ability of cKit to signal survival of
Hao, Yamamoto, Richardson et al.
proliferating PGCs, which results in markedly reduced numbers of spermatogonia in the postnatal testes [42, 62, 63]. In
postnatal mouse testes, the transmembrane form of cKit is
normally expressed on differentiating spermatogonia, spermatocytes, and Leydig cells [15–17, 26], whereas Sertoli
cells produce both soluble and transmembrane forms of SCF
[65– 68]. Spermatogonia formed in mice with Sl and W mutations are unable to undergo spermatogenesis due a block in
the ability of Aal spermatogonia to differentiate into A1
spermatogonia [15]. However, if spermatogonia from Sl mutant mice are transplanted into testes of W mutant mice where
SCF is produced, the mutant spermatogonia are able to differentiate [29] and develop into spermatozoa [69]. Likewise,
wild-type spermatogonia differentiate and develop normally
if transplanted into testes of W mice [70, 71]. In contrast, if
spermatogonia from normal mice are transplanted into testes
of Sl mice, or if spermatogonia from W mice are transplanted
into normal mice, colonies of proliferating undifferentiated
spermatogonia do develop, but they do not produce differentiating spermatogonia [28, 29]. Thus, Sl and W mutations
resulting in loss of cKit activity can disrupt the ability of Aal
spermatogonia to differentiate into A1 spermatogonia. In this
respect, it should be stressed that even though morphometric
analysis of Sohlh2-deficient mice shows a spermatogenic
block after differentiation into type A1 spermatogonia, it is
still possible that loss of Sohlh2 function alters spermatogonial staining patterns and that the developmental block actually occurs because of a defect in the ability of Aal spermatogonia to properly differentiate. Therefore, subsequent studies
are needed to verify the types of spermatogonia most affected
by loss of Sohlh2 function.
Interestingly, normal numbers of undifferentiated spermatogonia are able to develop from PGCs in mice with a point
mutation in the W locus that disrupts the phosphoinositide
3-kinase binding site on cKit (KitY719F); however, the subsequent formation of spermatocytes in these animals is severely
disrupted [27, 30]. Thus, cKit signaling pathways required for
survival and development of PGCs into gonocytes are distinct
from those required for differentiation of spermatogonia into
spermatocytes [32, 72]. More detailed morphometric studies
should be performed on testes of KitY719F mutant mice to
determine whether this point mutation blocks differentiation of
Aal spermatogonia into A1 spermatogonia or whether the defect
results in degeneration of differentiating type A spermatogonia,
as in Sohlh2 mice. However, in mutant mice with haploid levels
of fully functional cKit (Wlacz/⫹ mice), differentiating type A
spermatogonia are initially formed at normal numbers but show
a pronounced decrease in their numbers of type B spermatogonia produced after 1.5 months of age [34]. After this point, their
phenotype is similar to but not as severe as that shown here in
Sohlh2 mutant mice. At 5 months of age, Wlacz/⫹ mice produce
only 15% of the number of type B spermatogonia counted in
wild-type mice. However, unlike Sohlh2-null mice, which are
completely infertile, Wlacz/⫹ mice showed only a 15% decrease
in fertility [34]. Thus, haploid levels of cKit activity are sufficient and necessary for development of Aal spermatogonia into
differentiating type A spermatogonia, but they become limiting
for their subsequent development into type B spermatogonia in
older animals [34]. Biochemical studies using neutralizing antiserum and recombinant SCF have long predicted such a requirement for cKit by differentiating spermatogonia [26, 33].
However, because cKit is expressed abundantly by Leydig cells,
and because the lack of cKit activity blocks formation of A1
spermatogonia, a developmental requirement for the expression
of cKit on differentiating spermatogonia and early spermatocytes remains to be established genetically.
www.StemCells.com
1595
Although many genes related to apoptotic signaling in
germ cells were not affected by the lack of functional Sohlh2,
transcripts encoding cKit were less abundant in testes of
Sohlh2-null mice. The reduction in cKit expression on postnatal day 8 appeared modest; however, in a background
where cKit transcripts are predominantly produced by Leydig
cells [16, 26], it suggested that loss of Sohlh2 function
potentially had a more profound effect on cKit expression in
germ cells. Germ cells in testes of 10-day-old Sohlh2-null
mice did show lower levels of cKit expression at the protein
level, but this result likely reflects the lack of differentiating
spermatogonia and spermatocytes in tubular segments where
cKit was not detected. For example, in 10-day-old Sohlh2null mice, this would be possible in tubular segments during
stages IV–VI of a spermatogenic cycle, given their lack of
intermediate and type B spermatogonia (Figs. 3–5). Even
longer continuous segments enriched in PLZF-expressing
spermatogonia could occur within seminiferous tubules of
Sohlh2-null mice if PLZF is expressed in A1 spermatogonia
and if cKit is not upregulated during their transition from the
Aal stage (Fig. 6C). Because we show the expression of cKit
to be dependent on Sohlh2 in our spermatogonial lines, and
because the phenotypes of Sohlh1 and Sohlh2 mutant mice
are manifested by an inability of spermatogonia to differentiate into meiosis, the loss of cKit expression in Sohlh2deficient germ cells could contribute to their defects in spermatogenesis. If so, forced expression of a cKit transgene
selectively in spermatogonia and spermatocytes would theoretically rescue spermatogenesis in Sohlh2-null mice (Fig. 7).
SUMMARY
Male mice that lack Sohlh2 are infertile because of an early
block in spermatogenesis. The spermatogenic block is manifested as degeneration of type A spermatogonia, which results in
mice that lack meiotic and haploid germ cells in their testes
throughout adult life. More specifically, loss of Sohlh2 prevents
the normal progression of differentiating type A spermatogonia
into type B spermatogonia that would otherwise produce new
preleptotene spermatocytes to initiate meiosis. These results
shed light on potential models where testicular factors normally
regulate the viability of differentiating spermatogonia by signaling through Sohlh2 and/or its effectors. This would provide a
key checkpoint for optimizing the numbers of spermatozoan
progenitors that enter meiosis during each cycle of spermatogenesis. Future experiments should more precisely define
which types of differentiating spermatogonia (i.e., types A2,
A3, and A4) are most sensitive to loss of Sohlh2 activity and
their mechanism of death. This could lead to an understanding of physiological signaling pathways that maintain spermatocyte homeostasis by regulating spermatogonial density.
Because spermatocyte overgrowth can completely disrupt
spermatogenesis, such a mechanism would prove critical for
male fertility.
ACKNOWLEDGMENTS
We thank the University of Texas Southwestern Transgenic
Center for mouse chimera generation. We thank Tuyetanh
Nguyen and John Shelton for help with these experiments. We
are grateful to Dr. George Enders for kindly providing antiGCNA1 antibody. A portion of this work was performed in the
laboratory of the late David L. Garbers prior to his death. Drs.
Jing Hao and Miwako Yamamoto contributed equally to this
Sohlh2 Is Required for Spermatogenesis
1596
work. This work was supported in part by grants from the
National Institute of Child Health and Human Development (to
G.Q.Z.), the Howard Hughes Medical Institute (to David L.
Garbers), and the Cecil H. and Ida Green Center for Reproductive Biology Sciences.
REFERENCES
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Lawson KA, Dunn NR, Roelen BA et al. Bmp4 is required for the
generation of primordial germ cells in the mouse embryo. Genes Dev
1999;13:424 – 436.
Ying Y, Liu XM, Marble A et al. Requirement of Bmp8b for the
generation of primordial germ cells in the mouse. Mol Endocrinol
2000;14:1053–1063.
Ying Y, Qi X, Zhao GQ. Induction of primordial germ cells from murine
epiblasts by synergistic action of BMP4 and BMP8B signaling pathways.
Proc Natl Acad Sci U S A 2001;98:7858 –7862.
Tam PP, Snow MH. Proliferation and migration of primordial germ cells
during compensatory growth in mouse embryos. J Embryol Exp Morphol
1981;64:133–147.
Kemper CH, Peters PW. Migration and proliferation of primordial germ
cells in the rat. Teratology 1987;36:117–124.
Sapsford CS. Changes in the cells of the sex cords and seminiferous
tubules during the development of the testes of the rat and mouse. Aust
J Zool 1962;10:178 –192.
Huckins C, Clermont Y. Evolution of gonocytes in the rat testis during
late embryonic and early post-natal life. Arch Anat Histol Embryol
1968;51:341–354.
Kluin PM, de Rooij DG. A comparison between the morphology and cell
kinetics of gonocytes and adult type undifferentiated spermatogonia in
the mouse. Int J Androl 1981;4:475– 493.
Novi AM, Saba P. An electron microscopic study of the development of
rat testis in the first 10 postnatal days. Z Zellforsch Mikrosk Anat
1968;86:313–326.
Clermont Y, Perey B. Quantitative study of the cell population of the
seminiferous tubules in immature rats. Am J Anat 1957;100:241–267.
Yoshida S, Sukeno M, Nakagawa T et al. The first round of mouse
spermatogenesis is a distinctive program that lacks the self-renewing
spermatogonia stage. Development 2006;133:1495–1505.
Huckins C. The spermatogonial stem cell population in adult rats. I.
Their morphology, proliferation and maturation. Anat Rec 1971;169:
533–557.
de Rooij DG. Spermatogonial stem cell renewal in the mouse. I. Normal
situation. Cell Tissue Kinet 1973;6:281–287.
Oakberg EF. Spermatogonial stem-cell renewal in the mouse. Anat Rec
1971;169:515–531.
Schrans-Stassen BH, van de Kant HJ, de Rooij DG et al. Differential
expression of c-kit in mouse undifferentiated and differentiating type A
spermatogonia. Endocrinology 1999;140:5894 –5900.
Manova K, Nocka K, Besmer P et al. Gonadal expression of c-kit
encoded at the W locus of the mouse. Development 1990;110:
1057–1069.
Prabhu SM, Meistrich ML, McLaughlin EA et al. Expression of c-Kit
receptor mRNA and protein in the developing, adult and irradiated rodent
testis. Reproduction 2006;131:489 – 499.
Tajima Y, Sawada K, Morimoto T et al. Switching of mouse spermatogonial proliferation from the c-kit receptor-independent type to the
receptor-dependent type during differentiation. J Reprod Fertil 1994;102:
117–122.
Buaas FW, Kirsh AL, Sharma M et al. Plzf is required in adult male germ
cells for stem cell self-renewal. Nat Genet 2004;36:647– 652.
Costoya JA, Hobbs RM, Barna M et al. Essential role of Plzf in
maintenance of spermatogonial stem cells. Nat Genet 2004;36:653– 659.
Meng X, Lindahl M, Hyvonen ME et al. Regulation of cell fate
decision of undifferentiated spermatogonia by GDNF. Science 2000;
287:1489 –1493.
Tadokoro Y, Yomogida K, Ohta H et al. Homeostatic regulation of
germinal stem cell proliferation by the GDNF/FSH pathway. Mech Dev
2002;113:29 –39.
Naughton CK, Jain S, Strickland AM et al. Glial cell-line derived
neurotrophic factor-mediated RET signaling regulates spermatogonial
stem cell fate. Biol Reprod 2006;74:314 –321.
Raverot G, Weiss J, Park SY et al. Sox3 expression in undifferentiated
spermatogonia is required for the progression of spermatogenesis. Dev
Biol 2005;283:215–225.
Yoshida S, Takakura A, Ohbo K et al. Neurogenin3 delineates the
earliest stages of spermatogenesis in the mouse testis. Dev Biol 2004;
269:447– 458.
DISCLOSURE
OF POTENTIAL
OF INTEREST
CONFLICTS
The authors indicate no potential conflicts of interest.
26 Yoshinaga K, Nishikawa S, Ogawa M et al. Role of c-kit in mouse
spermatogenesis: Identification of spermatogonia as a specific site of
c-kit expression and function. Development 1991;113:689 – 699.
27 Blume-Jensen P, Jiang G, Hyman R et al. Kit/stem cell factor receptorinduced activation of phosphatidylinositol 3⬘-kinase is essential for male
fertility. Nat Genet 2000;24:157–162.
28 Ohta H, Tohda A, Nishimune Y. Proliferation and differentiation of
spermatogonial stem cells in the w/wv mutant mouse testis. Biol Reprod
2003;69:1815–1821.
29 Ohta H, Yomogida K, Dohmae K et al. Regulation of proliferation and
differentiation in spermatogonial stem cells: The role of c-kit and its
ligand SCF. Development 2000;127:2125–2131.
30 Kissel H, Timokhina I, Hardy MP et al. Point mutation in kit receptor
tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses. EMBO J 2000;
19:1312–1326.
31 Dym M, Jia MC, Dirami G et al. Expression of c-kit receptor and its
autophosphorylation in immature rat type A spermatogonia. Biol Reprod
1995;52:8 –19.
32 Feng LX, Ravindranath N, Dym M. Stem cell factor/c-kit up-regulates
cyclin D3 and promotes cell cycle progression via the phosphoinositide
3-kinase/p70 S6 kinase pathway in spermatogonia. J Biol Chem 2000;
275:25572–25576.
33 Packer AI, Besmer P, Bachvarova RF. Kit ligand mediates survival of
type A spermatogonia and dividing spermatocytes in postnatal mouse
testes. Mol Reprod Dev 1995;42:303–310.
34 Guerif F, Cadoret V, Rahal-Perola V et al. Apoptosis, onset and maintenance of spermatogenesis: Evidence for the involvement of Kit in
Kit-haplodeficient mice. Biol Reprod 2002;67:70 –79.
35 Kee K, Gonsalves JM, Clark AT et al. Bone morphogenetic proteins
induce germ cell differentiation from human embryonic stem cells. Stem
Cells Dev 2006;15:831– 837.
36 Ballow DJ, Xin Y, Choi Y et al. Sohlh2 is a germ cell-specific bHLH
transcription factor. Gene Expr Patterns 2006;6:1014 –1018.
37 Ballow D, Meistrich ML, Matzuk M et al. Sohlh1 is essential for
spermatogonial differentiation. Dev Biol 2006;294:161–167.
38 Pangas SA, Choi Y, Ballow DJ et al. Oogenesis requires germ cellspecific transcriptional regulators Sohlh1 and Lhx8. Proc Natl Acad Sci
U S A 2006;103:8090 – 8095.
39 Furuchi T, Masuko K, Nishimune Y et al. Inhibition of testicular germ
cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia. Development 1996;122:1703–1709.
40 Rodriguez I, Ody C, Araki K et al. An early and massive wave of
germinal cell apoptosis is required for the development of functional
spermatogenesis. EMBO J 1997;16:2262–2270.
41 Knudson CM, Tung KS, Tourtellotte WG et al. Bax-deficient mice with
lymphoid hyperplasia and male germ cell death. Science 1995;270:
96 –99.
42 Mintz B, Russell ES. Gene-induced embryological modifications of
primordial germ cells in the mouse. J Exp Zool 1957;134:207–237.
43 Oakberg EF. A description of spermiogenesis in the mouse and its use in
analysis of the cycle of the seminiferous epithelium and germ cell
renewal. Am J Anat 1956;99:391– 413.
44 Monesi V. Autoradiographic study of DNA synthesis and the cell cycle
in spermatogonia and spermatocytes of mouse testis using tritiated thymidine. J Cell Biol 1962;14:1–18.
45 Huckins C, Oakberg EF. Morphological and quantitative analysis of
spermatogonia in mouse testes using whole mounted seminiferous tubules, I. The normal testes. Anat Rec 1978;192:519 –528.
46 Cooke HJ, Lee M, Kerr S et al. A murine homologue of the human DAZ
gene is autosomal and expressed only in male and female gonads. Hum
Mol Genet 1996;5:513–516.
47 Reijo R, Seligman J, Dinulos MB et al. Mouse autosomal homolog of
DAZ, a candidate male sterility gene in humans, is expressed in male
germ cells before and after puberty. Genomics 1996;35:346 –352.
48 Saitou M, Barton SC, Surani MA. A molecular programme for the
specification of germ cell fate in mice. Nature 2002;418:293–300.
49 Tanaka SS, Matsui Y. Developmentally regulated expression of mil-1
and mil-2, mouse interferon-induced transmembrane protein like genes,
during formation and differentiation of primordial germ cells. Gene Expr
Patterns 2002;2:297–303.
50 Oulad-Abdelghani M, Bouillet P, Decimo D et al. Characterization of a
premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a
novel retinoic acid-responsive gene. J Cell Biol 1996;135:469 – 477.
Hao, Yamamoto, Richardson et al.
51 Schultz N, Hamra FK, Garbers DL. A multitude of genes expressed
solely in meiotic or postmeiotic spermatogenic cells offers a myriad of
contraceptive targets. Proc Natl Acad Sci U S A 2003;100:12201–12206.
52 Reijo RA, Dorfman DM, Slee R et al. DAZ family proteins exist
throughout male germ cell development and transit from nucleus to
cytoplasm at meiosis in humans and mice. Biol Reprod 2000;63:
1490 –1496.
53 Enders GC, May, JJ 2nd. Developmentally regulated expression of a
mouse germ cell nuclear antigen examined from embryonic day 11 to
adult in male and female mice. Dev Biol 1994;163:331–340.
54 Huckins C. The morphology and kinetics of spermatogonial degeneration
in normal adult rats: An analysis using a simplified classification of the
germinal epithelium. Anat Rec 1978;190:905–926.
55 De Rooij DG, Lok D. Regulation of the density of spermatogonia in the
seminiferous epithelium of the Chinese hamster: II. Differentiating spermatogonia. Anat Rec 1987;217:131–136.
56 Clermont Y. Quantitative analysis of spermatogenesis of the rat: A
revised model for the renewal of spermatogonia. Am J Anat 1962;111:
111–129.
57 Coultas L, Bouillet P, Loveland KL et al. Concomitant loss of proapoptotic BH3-only Bcl-2 antagonists Bik and Bim arrests spermatogenesis.
EMBO J 2005;24:3963–3973.
58 Kanatsu-Shinohara M, Ogonuki N, Inoue K et al. Long-term proliferation in culture and germline transmission of mouse male germline stem
cells. Biol Reprod 2003;69:612– 616.
59 de Rooij DG, Lok D, Weenk D. Feedback regulation of the proliferation
of the undifferentiated spermatogonia in the Chinese hamster by the
differentiating spermatogonia. Cell Tissue Kinet 1985;18:71– 81.
60 van Keulen CJ, de Rooij DG. Spermatogonial stem cell renewal in the
mouse. II. After cell loss. Cell Tissue Kinet 1973;6:337–345.
61 Hamra FK, Chapman KM, Nguyen D et al. Identification of neuregulin
as a factor required for formation of aligned spermatogonia. J Biol Chem
2007;282:721–730.
1597
62 Bennett D. Developmental analysis of a mutation with pleiotropic effects
in the mouse. J Morph 1956;98:199 –234.
63 McCoshen JA, McCallion DJ. A study of the primordial germ cells
during their migratory phase in Steel mutant mice. Experientia 1975;31:
589 –590.
64 Besmer P, Manova K, Duttlinger R et al. The kit-ligand (steel factor) and
its receptor c-kit/W: Pleiotropic roles in gametogenesis and melanogenesis. Dev Suppl 1993;125–137.
65 Tajima Y, Onoue H, Kitamura Y et al. Biologically active kit ligand
growth factor is produced by mouse Sertoli cells and is defective in SId
mutant mice. Development 1991;113:1031–1035.
66 Munsie M, Schlatt S, deKretser DM et al. Expression of stem cell factor
in the postnatal rat testis. Mol Reprod Dev 1997;47:19 –25.
67 Vincent S, Segretain D, Nishikawa S et al. Stage-specific expression of
the Kit receptor and its ligand (KL) during male gametogenesis in the
mouse: A Kit-KL interaction critical for meiosis. Development 1998;
125:4585– 4593.
68 Manova K, Huang EJ, Angeles M et al. The expression pattern of the
c-kit ligand in gonads of mice supports a role for the c-kit receptor in
oocyte growth and in proliferation of spermatogonia. Dev Biol 1993;
157:85–99.
69 Ogawa T, Dobrinski I, Avarbock MR et al. Transplantation of male germ
line stem cells restores fertility in infertile mice. Nat Med 2000;6:29 –34.
70 Brinster RL, Avarbock MR. Germline transmission of donor haplotype
following spermatogonial transplantation. Proc Natl Acad Sci U S A
1994;91:11303–11307.
71 Brinster RL, Zimmermann JW. Spermatogenesis following male germcell transplantation. Proc Natl Acad Sci U S A 1994;91:11298 –11302.
72 De Miguel MP, Cheng L, Holland EC et al. Dissection of the c-Kit
signaling pathway in mouse primordial germ cells by retroviral-mediated
gene transfer. Proc Natl Acad Sci U S A 2002;99:10458 –10463.
73 de Rooij DG. Proliferation and differentiation of spermatogonial stem
cells. Reproduction 2001;121:347–354.
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