RESEARCH ARTICLE 1495
Development 133, 1495-1505 (2006) doi:10.1242/dev.02316
The first round of mouse spermatogenesis is a distinctive
program that lacks the self-renewing spermatogonia stage
Shosei Yoshida1,2,*, Mamiko Sukeno1, Toshinori Nakagawa1, Kazuyuki Ohbo3, Go Nagamatsu3, Toshio Suda3
and Yo-ichi Nabeshima1
Mammalian spermatogenesis is maintained by a continuous supply of differentiating cells from self-renewing stem cells. The stem
cell activity resides in a small subset of primitive germ cells, the undifferentiated spermatogonia. However, the relationship
between the establishment of this population and the initiation of differentiation in the developing testes remains unclear. In this
study, we have investigated this issue by using the unique expression of Ngn3, which is expressed specifically in the undifferentiated
spermatogonia, but not in the differentiating spermatogonia or their progenitors, the gonocytes. Our lineage analyses
demonstrate that the first round of mouse spermatogenesis initiates directly from gonocytes, without passing through the Ngn3expressing stage (Ngn3– lineage). By contrast, the subsequent rounds of spermatogenesis are derived from Ngn3-positive
undifferentiated spermatogonia, which are also immediate descendents of the gonocytes and represent the stem cell function
(Ngn3+ lineage). Thus, in mouse spermatogenesis, the state of the undifferentiated spermatogonia is not an inevitable step but is a
developmental option that ensures continuous sperm production. In addition, the segregation of gonocytes into undifferentiated
spermatogonia (Ngn3+ lineage) or differentiating spermatogonia (Ngn3– lineage) is topographically related to the establishment of
the seminiferous epithelial cycle, thus suggesting a role of somatic components in the establishment of stem cells.
INTRODUCTION
In the mammalian testes, numerous spermatozoa are continuously
produced throughout the reproductive period. The continuity of
spermatogenesis is dependent on stem cells, defined by their selfrenewing and differentiating activities. During adult spermatogenesis
in mice, the stem cell activity resides in a small, primitive set of
spermatogonia referred as the undifferentiated spermatogonia, which
correspond to Asingle, Apaired, and Aaligned spermatogonia (de Rooij,
2001; de Rooij and Russell, 2000; Shinohara et al., 2000; Nishimune
et al., 1978). Besides self-renewing as a population, undifferentiated
spermatogonia generate differentiating spermatogonia [A1 to A4,
Intermediate (In), and B spermatogonia], which then differentiate
into meiotic spermatocytes, haploid spermatids and spermatozoa. In
the seminiferous tubules, all types of spermatogonia (Asingle to B) are
localized on the peripheral basement membrane, and the subsequent
cell types are arranged in a sequential order towards the lumen
(Russell et al., 1990).
The initiation of spermatogenesis and the establishment of stem
cells in the developing testes have been a focus of interest. It is
commonly accepted that spermatogonia with matured morphology
corresponding to adult In and B spermatogonia appear by the
beginning of the second week after birth; this is followed by the
sequential appearance of the differentiating cells (Bellve et al., 1977;
de Rooij, 1998; Kluin et al., 1982). By contrast, controversy exists
with regard to the process occurring in the first week. During this
period, gonocytes – the immediate precursors of spermatogonia –
migrate from their original central position in the seminiferous
1
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto
University, Kyoto, Japan. 2Recognition and Formation, PRESTO, JST, Saitama, Japan.
3
The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio
University, Tokyo, Japan.
*Author for correspondence (e-mail: [email protected])
Accepted 8 February 2006
tubules toward the periphery, and transform into spermatogonia on
the basement membrane around postnatal day (P) 3 to P6 (Bellve et
al., 1977). These nascent spermatogonia have primitive
morphological features similar to type A spermatogonia in adults.
Detailed morphological observation strongly suggests the
heterogeneous nature of these spermatogonia. However, their
characteristics are not sufficiently evident to gain a general
consensus with regard to their functions. Some researchers propose
that adult-type differentiating spermatogonia originate directly from
gonocytes (de Rooij, 1998; Kluin and de Rooij, 1981), whereas
others claim that gonocytes give rise to a special type of cell, termed
prespermatogonia, and that these cells subsequently generate adulttype spermatogonia (Bellve et al., 1977; Hilscher et al., 1974;
Huckins and Clermont, 1968). In addition, the establishment of stem
cells remains unclear because of the difficulties involved in
morphologically identifying undifferentiated spermatogonia. It has
been shown that transplantable stem cell activity appears around P2
to P4 (McLean et al., 2003). However, the nature of the cells that
represent this activity is unknown.
In mature testes, spermatogenesis progresses in a topographically
well-coordinated manner, known as the spermatogenic wave
(Leblond and Clermont, 1952; Russell et al., 1990). This is a
recapitulation of the seminiferous epithelial cycle in the linear layout
along the length of the seminiferous tubule. Based on the expression
patterns of the seminiferous stage-specific genes in the perinatal
immature Sertoli cells, Timmons et al. suggested that the
seminiferous epithelial cycle might be pre-patterned in Sertoli cells
from the embryonic stage (Timmons et al., 2002). However, the
relationship between this presumptive pre-pattern of the cycle and
spermatogenesis initiation has been barely investigated. Moreover,
it is well known that the first round of spermatogenesis during
puberty is less efficient than that of adults, and that it exhibits
massive apoptosis (Kluin et al., 1982; Mori et al., 1997). It is also
suspected that the first round of spermatogenesis may not produce
fertile spermatozoa.
DEVELOPMENT
KEY WORDS: Spermatogenesis, Mouse, Stem cells, Undifferentiated spermatogonia, Seminiferous epithelial cycle, Ngn3, Kit, Galectin 1
1496 RESEARCH ARTICLE
MATERIALS AND METHODS
Animals
Ngn3/Cre and CAG-CAT-Z transgenic mice have been described
previously (Yoshida et al., 2004; Araki et al., 1995). Ngn3/CreERTM
transgenic mice were generated by injecting a DNA construct of
CreERTM flanked by the 6.7-kb mouse Ngn3 upstream sequence (Gu et
al., 2002) into the pronucleus of a C57BL/6 fertilized egg (Oriental BioService). Ngn3/Cre and Ngn3/CreERTM mice were maintained on a
C57BL/6 background, whereas the genetic background of CAG-CAT-Z
mice was mixed but immunologically compatible with C57BL/6. The
C57BL/6 mice used as the wild type were purchased from Japan SLC and
Charles River Japan, and the WBB6F1W/Wv (W/Wv) mice were
purchased from Japan SLC. All the animals were maintained and
sacrificed in accordance with the animal experiment guidelines of Kyoto
University.
RT-PCR
Poly(A) RNA was prepared from C57BL/6 mouse testes by using a ␮MACS
mRNA isolation kit (Miltenyi). Reverse transcription (RT), with a random
primer, and polymerase chain reaction (PCR) were performed using Super
Script III (Invitrogen) and LA Taq (Takara) enzymes in accordance with the
manufacturers’ recommendations. Primers used for Ngn3, Ret, Oct4 have
been described previously (Yoshida et al., 2004) and are as follows: 5⬘ACATACACGTGCAGCAACAG-3⬘ and 5⬘-TCAGAATGCAGCCATGTACC-3⬘ for Kit; 5⬘-CTCTTTGATGTCACGCACGACGATTTC-3⬘ and 5⬘GTGGGCCGCCTCTAGGCACCAA-3⬘ for ␤-actin.
In situ hybridization (ISH) on sections
Under anesthesia with avertin, mice were perfusion fixed with 4%
paraformaldehyde (Nakalai) in PBS, and their testes were excised. Mice less
than 2 weeks of age were anaesthetized with isoflurane, and their testes were
excised without perfusion. After removal of the tunica albuginea, the testes
were immersed overnight in the same fixative, embedded in paraffin wax and
sectioned. ISH was performed as described previously (Yoshida et al., 2001);
the detailed protocol is available upon request. For double-staining ISH, a
fluorescein-labeled probe (synthesized using a Fluorescein RNA Labeling
mix) was hybridized simultaneously with a digoxigenin (DIG)-labeled
probe. DIG was visualized by using an AP-conjugated anti-DIG antibody
and BM purple substrate, and fluorescein was visualized by using an APconjugated anti-Fluorescein antibody and the HNPP Fluorescent Detection
Set. The second colorization was performed after detection of the first label
and inactivation of AP in PBS at 75°C for 30 minutes. For Ngn3 (DIG) and
Kit (fluorescein) double staining, fluorescein was visualized first; for Ngn3
or Kit (DIG) and galectin 1 (fluorescein) staining, DIG was detected first.
All the reagents were obtained from Roche. Specimens were counterstained
with Nuclear Fast Red, Hoechst 33258, or propidium iodide (PI), as
appropriate. Galectin 1 probes were prepared from the EST clone IMAGE
5712148 (Invitrogen). Templates for other probes have been described
previously (Yoshida et al., 2001; Yoshida et al., 2004). None of the sense
probes yielded any signal. The specimens were photographed using a
DMRBE fluorescence microscope (Leica) equipped with an Axiocam digital
camera (Zeis Vision), and the images were processed with Adobe
PhotoShop.
Whole-mount ISH
Whole-mount ISH was performed on untangled P4 seminiferous tubules
attached to APS-coated glass slides (Matsunami). Slides were then fixed
with 4% paraformaldehyde in PBS (4°C, 2 hours) and dehydratedrehydrated through a methanol series. The subsequent hybridization
procedure and reagents used were based on those described previously
(Hogan et al., 1994), with modifications adapted for samples on slides;
hybridization buffer was 50% formamide, 5⫻SSC (pH 4.5), 1% SDS, 50
ug/ml tRNA and 50ug/ml heparin. The detailed protocol is available upon
request. The antibody detection and colorization was carried out as on
sections, using an AP-conjugated anti-DIG antibody and BM purple AP
substrate (Roche).
Scoring Kit- and Ngn3-positve spermatogonia in the seminiferous
tubule segments with various galectin 1 mRNA levels
Testicular sections of young mice were double stained for Kit or Ngn3 and
galectin 1. All the tubule cross sections were photographed under a
fluorescence microscope and categorized according to their galectin 1 signal
strength (high, medium or low). The same specimens were independently
examined under bright-field illumination for the number of Kit- or Ngn3positive spermatogonia contained in individual tubule sections. Then,
expected positive cell numbers were calculated assuming their non-biased
distributions, and the ‘preferences’ (actual positive cell number/expected
positive cell number) were determined; these were statistically evaluated by
␹2 test between the actual and expected cell numbers within or outside of the
particular categories of the segments. For each data point, more than 600
seminiferous tubule cross-sections in five or six testicular slices were
examined.
The data were then analyzed to determine whether the Ngn3- and Kitpositive spermatogonia distributions have some correlation with the
categories based on the galectin 1 mRNA levels in seminiferous tubule
segments. Statistical evaluations were performed as explained below, using
the data of the Ngn3-positive cells at P5 as an example (see Tables S2-S6 in
the supplementary material).
The five P5 testes specimens used for analyses contained 741 crosssections of the seminiferous tubules in total, and 137 Ngn3-positive cells
were observed (see Table S2 in the supplementary material). These 137 cells
were classified according to the galectin 1 level of the seminiferous tubules
where they located. Numbers of the tubule cross-sections were also
summarized according to their galectin 1 levels. Then, the expected numbers
of Ngn3-positive cells in each category of tubule section were calculated on
the basis of the null hypothesis: the Ngn3-positive spermatogonia evenly
DEVELOPMENT
Gene expression and cell fate analyses may be advantageous
regarding the lineage relationship and/or the differentiation of the
cells observed. However, such analyses have not been conducted
because of the lack of an appropriate set of marker genes. On the
one hand, it has been established that the transition of
undifferentiated spermatogonia into differentiating spermatogonia
coincides with the gain of Kit (also known as c-Kit) expression, a
receptor tyrosine kinase. Kit continues to be expressed until meiosis
and play essential roles in the survival of the Kit-expressing cells
(Schrans-Stassen et al., 1999; Yoshinaga et al., 1991). On the other
hand, several genes have been described to be expressed in
undifferentiated spermatogonia or similar populations in adult
testes, including Ret, Gfr␣1, Oct4 (Pou5fl – Mouse Genome
Informatics), and Plzf (Zbtb16 – Mouse Genome Informatics),
some of which play crucial roles in the establishment and/or
maintenance of stem cell activities (Buaas et al., 2004; Costoya et
al., 2004; Meng et al., 2000; Pesce et al., 1998). However, these
genes are also expressed in gonocytes, and thus cannot differentiate
these two populations. Recently, we have identified that neurogenin
3 (Ngn3; Neurog3 – Mouse Genome Informatics), a basic helixloop-helix transcription factor, is specifically expressed in
undifferentiated spermatogonia, and not in gonocytes (Yoshida et
al., 2004). The Ngn3-expressing cells fulfill the criteria for the
undifferentiated spermatogonia, including their presence
throughout the seminiferous epithelial cycle with low frequency,
connection of the small number of 2n cells, survival in cryptoorchid
testes and Kit negativity. Upon transition into differentiating
spermatogonia, Ngn3 expression is downregulated. Thus, this
provides a unique tool with which to elucidate the ontogeny of
undifferentiated spermatogonia.
In this study, we have investigated the initial steps of
spermatogenesis by lineage analyses using an inducible and
constitutive Cre recombinase-loxP system, after a detailed profiling
of the Ngn3 and Kit expression patterns. Particular attention was
paid to the relationship between the gonocytes and the adult-type
spermatogonia, the origin and differentiation property of the first
round of spermatogenesis, and the establishment of the seminiferous
epithelial cycle.
Development 133 (8)
distribute among the three categories of tubule segments without bias. The
expected numbers of Ngn3-positive spermatogonia were obtained by
multiplying the total number of Ngn3-positive cells by the percentage of
tubule sections in the category. The preferences of positive cells in each
category were calculated as the ratio of the observed number of positive cells
in each category to the expected number in the same category. Thus, a
preference of 1 indicates that the actual number of Ngn3-positive cells is the
same as expected, i.e. a non-biased distribution. Values greater or smaller
than 1 suggest preference or avoidance, respectively.
The differences between the observed and expected numbers of Ngn3positive spermatogonia were statistically evaluated by ␹2 tests using Abacus
StatView software; these are summarized in Table S3 in the supplementary
material. The P-value (0.0004) was small enough to reject the null
hypothesis and indicated a non-random distribution of Ngn3-positive
spermatogonia. This was also true for Kit-positive spermatogonia at P3, P4
and P5 (data not shown). Deviations from the expected numbers were then
tested for individual categories. For example, the distribution of Ngn3positive spermatogonia to the galectin 1-high segments (a preference of
0.63) was tested after modifying Table S3 so that categories other than
galectin 1-high were combined (see Table S4 in the supplementary material).
A ␹2 test was applied to this two by two table and significant deviation was
supported by a P-value of 0.0125. Therefore, it was concluded that, at P5,
Ngn3-positive spermatogonia demonstrate significant avoidance from the
galectin 1-high seminiferous tubule segments. The same procedures were
applied for the other categories (see Tables S5, S6 in the supplementary
material); Ngn3-positive spermatogonia showed significant preference to the
galectin 1-medium segments, while avoidance from the galectin 1-low
segments was statistically non-significant. Table S7 in the supplementary
material is a summary of the preferences and corresponding P-values for all
the data points analyzed (Fig. 3I is based on this table).
Pulse labeling of Ngn3-positive spermatogonia and
transplantation
Tamoxifen [40 mg/kg; Calbiochem, solublized in Sesame oil (Nakalai) at 10
mg/ml] was administered intraperitoneally at P5 and P6 to double-transgenic
male mice obtained from crossing Ngn3/CreERTM males with CAG-CAT-Z
females. The control injection did not contain tamoxifen. For
transplantation, double-transgenic males injected with tamoxifen at P5 and
P6 were sacrificed at P8. A single-cell suspension was prepared from their
testes, and was transplanted into the seminiferous tubules of W/Wv mice
through the efferent ductules as described (Ogawa et al., 1997). The wholemount detection of ␤-galactosidase (␤-gal) has been described previously
(Yoshida et al., 2004). After being photographed, specimens were re-fixed
with 10% formalin, embedded in paraffin wax, sectioned, and stained with
Nuclear Fast Red or Hematoxylin and Eosin.
Analysis of offspring from Ngn3/Cre;CAG-CAT-Z male mice
The Ngn3/Cre;CAG-CAT-Z double transgenic male mice were weaned
around P28 and housed with C57BL/6 female mice in isolated cages for
natural mating. Offspring were analyzed after birth or after the dissection of
the pregnant females. The date of fertilization was determined based on the
date of birth or the developmental stage of the embryos. Using tail or limbs,
the offspring were genotyped by PCR using the primers described previously
(Araki et al., 1995). The presence of the CAG-CAT-Z reporter gene was
tested with Z1 and Z2 primers, and approximately 50% of the offspring
tested were positive. Intact and recombined transgenes were distinguished
using AG2 and Z3, which flank the CAT gene and loxP sequences. All
offspring demonstrated the presence of either the intact or the recombined
transgene. Consistent with this, samples showing the recombined pattern
never retained the CAT gene (using CAT2 and CAT3).
RESULTS
Appearance of Ngn3- and Kit-positive
spermatogonia in early spermatogenesis
As a first step to evaluate spermatogenesis initiation, the expression
of Ngn3 and Kit was examined by RT-PCR and in situ hybridization
(ISH) during the first postnatal week (Fig. 1). In neonates, when germ
cells are in the gonocyte stage, Ngn3 expression is below the
RESEARCH ARTICLE 1497
detection level of ISH and is barely detectable by RT-PCR. Detection
of Ngn3 expression by ISH starts around P3 to P4 in a tiny portion of
nascent spermatogonia located on the basement membrane, followed
by an increase in the number of Ngn3-positive spermatogonia
through P5 to P7 (Fig. 1B, parts a-f). This profile is reflected by the
results from RT-PCR using whole testis RNA (Fig. 1A). This is in
contrast to Ret and Oct4, which also exhibit limited expression in
undifferentiated spermatogonia, or similar populations, in the adult
testes (Meng et al., 2000; Pesce et al., 1998). These genes are readily
detected in neonatal gonocytes (P1-P2) and nascent spermatogonia
(around P3-P4), and they are then downregulated in subsets of
spermatogonia, although expression remains persistent in another
subset up until P7. RT-PCR gives consistent results (Fig. 1A,B, parts
m-x). Kit is not expressed in gonocytes at P1, although interstitial
cells express this gene throughout (Manova et al., 1990) (red
arrowheads, Fig. 1B, parts g-l). The germ line expression of Kit starts
at a similar or slightly (approximately 1 day) earlier time when
compared with that of Ngn3 (blue arrowheads). The number of Kitpositive spermatogonia was constantly greater than that of Ngn3positive spermatogonia (Fig. 1B, parts a-l). Both interstitial and germ
cell Kit expression contribute to the RT-PCR result.
Spatial separation of the newly born Ngn3- and
Kit-positive spermatogonia
As is apparent in Fig. 1B, Ngn3- or Kit-positive spermatogonia
showed a highly biased localization within the seminiferous tubule
segments; some segments contain many positive cells, whereas
others are devoid of these cells. Double-staining ISH (Fig. 2A-D)
clarified that Ngn3 and Kit signals are not only found in distinct cells
but are also spatially separated in different seminiferous tubule
segments. A comparison between adjacent specimens revealed that
the Ngn3 and Kit signals rarely overlap within a single seminiferous
tubule segment (Fig. 2A-D,E-G; see also Table S1 in the
supplementary material). These data indicate that the Ngn3-positive
spermatogonia and Kit-positive spermatogonia are generated in a
spatially separated manner along the length of the seminiferous
tubules at this early stage.
Emergence of the Ngn3- and Kit-positive
spermatogonia, and the seminiferous epithelial
cycle pre-pattern
In mature testes, the seminiferous epithelium repeats a cyclical
program, known as the seminiferous epithelial cycle (Leblond and
Clermont, 1952; Russell et al., 1990). In mice, an entire cycle takes
~8.6 days and is divided into stages I to XII, defined by particular
combinations of the different stages of spermatogenic cells, such as
spermatids and spermatocytes. Areas of seminiferous epithelium at
any stage occupy a cylindrical segment, and such segments are
arranged along the tubule length in their chronological order (I, II,
III, IV...). As a result, mouse spermatogenesis exhibits a wave-like
progression along the tubule length, which is known as the
spermatogenic wave.
Although the seminiferous epithelial cycle is defined by the
differentiation steps of germ cells, several genes expressed in Sertoli
cells also represent stage specificities, indicating that germ cells and
Sertoli cells are well coordinated in the seminiferous epithelial cycle.
Timmons et al. demonstrated that immature Sertoli cells of perinatal
stages have a similar gene expression profile to that observed in adult
seminiferous tubules (Timmons et al., 2002). Among a number of
genes examined, galectin 1 (Lgals1-Mouse Genome Informatics)
mRNA was the most representative and showed the clearest pattern
from the early stages. In adults, galectin 1 transcription appears to
DEVELOPMENT
Initiation of mouse spermatogenesis
1498 RESEARCH ARTICLE
Development 133 (8)
occur as a single pulse in each cycle; it initiates between stages VIII
and IX, has the highest accumulation of transcripts at stages X-XII,
and then becomes weaker before regaining strong expression at
stage IX (Timmons et al., 2002). These authors also demonstrated
that the cyclic expression of galectin 1 and other genes can be
observed in the XXSxra testes, which are devoid of germ cells. Based
on these observations, they suggested that, in the perinatal testis
cord, Sertoli cells possess an intrinsic cyclic program, which may be
a seminiferous epithelial cycle pre-pattern, and subsequently
become coordinated with the differentiation of germ cells.
To further evaluate pre-pubertal galectin 1 mRNA expression, we
investigated its topographical pattern by whole-mount ISH (see Fig.
S1A in the supplementary material). In P4 seminiferous tubules,
galectin 1 mRNA was detected in an essentially identical pattern to
that observed in the adult; segmental expressions of high, medium
and low level of galectin 1 mRNA are arranged along the tubule
length in this order. As is shown in Fig. S1B,C in the supplementary
material, galectin 1 expression was also variable among the tubule
segments at P14 and P21. At these immature stages, the relationship
of galectin 1 levels with the types of spermatogenic cells included
in each segment was identical to that observed in the mature testes.
Thus, seminiferous stage-specific galectin1 expression in mature
testes can be traced back to pre-pubertal stages. It has also been
suggested that the galectin 1 expression domain moves so that it is
always associated with a particular stage of spermatogenic cells.
This is in agreement with the interpretation by Timmons et al.
(Timmons et al., 2002) that pre-pubertal galectin 1 expression is a
pre-pattern of the adult cycle. We also examined the adult testes of
W/Wv mice and cryptorchid testes, both of which lack
differentiating spermatogenic cells. In these testes, galectin 1
mRNA in Sertoli cells represented variable levels among the tubule
sections, similar to as in normal adult testes (see Fig. S1D-F in the
supplementary material), supporting the idea that the galectin 1
cycle can occur independently of the synchronous spermatogenic
differentiation (Timmons et al., 2002).
We were, therefore, prompted to investigate the possible link
between the emergence of Ngn3- and Kit-positive spermatogonia
and the presumptive seminiferous epithelial cycle pre-pattern
represented by galectin 1 mRNA (Fig. 3). The intensities of the
galectin 1 signals in the Sertoli cell cytoplasm at the center of the
tubules largely differ among segments (Fig. 3A-H). At P3 and P4,
when Kit-positive spermatogonia begin to be detected, they
DEVELOPMENT
Fig. 1. Appearance of
Ngn3-positive
spermatogonia and Kitpositive spermatogonia
during the first postnatal
week. (A) RT-PCR of whole
testis mRNA at P1 to P7;
amplification for Ngn3, Kit,
Ret, and Oct4. No bands are
observed in control reactions
without reverse transcriptase;
no template, amplification
product without cDNA. ␤actin was also assayed as a
control. (B) ISH of sections
from testes at P1 to P7,
probed for the same gene set
described in A. Green and
blue arrowheads indicate the
Ngn3 and Kit signals in
spermatogonia, respectively;
red arrowheads indicate Kit
expression in the interstitial
cells. Scale bar: 100 ␮m.
Initiation of mouse spermatogenesis
RESEARCH ARTICLE 1499
showed a strong preference for segments with a high level of
galectin 1 (Fig. 3A,B,I; Table S2 in the supplementary material).
This preference continues as the number of Kit-positive
spermatogonia increases at P5, when they become detectable also
in segments with a medium level of galectin 1 (Fig. 3E,F,I).
Assuming the wave-like progression of the seminiferous epithelial
cycle, these data suggest that Kit-positive spermatogonia are born
at stages with a high level of galectin 1, and, as the cycle
progresses, their distribution extends to regions with a lower level
of galectin 1 (schematically represented in Fig. 3J). Ngn3-positive
spermatogonia prefer segments with medium level of galectin 1
(Fig. 3C,D,G-J).
Altogether, it is reasonable to suppose that a particular subset of
gonocytes that localizes to the galectin 1-high segments of the
seminiferous tubules directly gives rise to Kit-positive
spermatogonia without Ngn3 expression. This is in contrast to the
situation in adults, where essentially all of the Kit-positive
spermatogonia are descendants of Ngn3-positive spermatogonia
(Yoshida et al., 2004). Ngn3-positive spermatogonia preferentially
appear at segments with medium levels of galectin 1.
in a tamoxifen-dependent manner (Fig. 4E,F). Three months after
labeling, many patches of labeled cells with complete
spermatogenesis were observed (Fig. 4G,H). Given that the
completion of the spermatogenic process requires approximately 1
month (Russell et al., 1990) and that the activity of tamoxifen does
not persist beyond several days after its administration (T.N. and
S.Y., unpublished) (Gu et al., 2002), this result indicates that the
cells that expressed Ngn3 at the time of tamoxifen administration
continue to self-renew, as well as to generate differentiating cells.
In addition, when the labeled pre-pubertal testes were dissociated
and transplanted into germ cell-depleted seminiferous tubules
(Brinster, 2002), a number of spermatogenic colonies of labeled
cells were generated after 3 months (Fig. 4G-I), indicating a
repopulating activity of the pre-pubertal Ngn3-positive
spermatogonia. Thus, the pre-pubertal Ngn3-positive
spermatogonia already possess the essential traits of the adult-type
undifferentiated spermatogonia. It is noteworthy that the emergence
of Ngn3-positive spermatogonia parallels the appearance of
transplantable stem cell activity at around P2-P4 (McLean et al.,
2003).
Self-renewing stem cell activities of nascent
Ngn3-positive spermatogonia
We then analyzed the fates of the pre-pubertal spermatogonia
subpopulations to evaluate their differentiation characteristics. To
determine the fate of Ngn3-positive spermatogonia, transgenic mice
expressing tamoxifen-inducible Cre recombinase (CreERTM)
(Hayashi and McMahon, 2002) in Ngn3-positive spermatogonia
were generated using the Ngn3 regulatory sequence (Fig. 4B,C).
The CreERTM protein is activated transiently after tamoxifen
administration and recombines the target loxP sites in expressing
cells (Fig. 4A). In double-transgenic mice possessing the CAGCAT-Z reporter (Araki et al., 1995), Ngn3-positive spermatogonia
were successfully labeled for ␤-gal expression (encoded by lacZ)
Contribution of spermatogonia that express or do
not express Ngn3 to spermatogenesis
As discussed earlier, a particular subset of gonocytes appeared to
develop into Kit-positive spermatogonia without passing through the
Ngn3-positive undifferentiated spermatogonia stage. Ohbo et al.
reported that the prepubertal Kit-positive spermatogonia barely
show stem cell activity (Ohbo et al., 2003). However, their other
characteristics are as yet unknown; they might either differentiate
further or degenerate. Therefore, we developed a transgenic system
in which cells that have undergone a Ngn3-positive stage (Ngn3+
lineage) and those that have never expressed this gene (Ngn3–
lineage) can be identified by differential labeling (Fig. 5A). For this
purpose, we used transgenic mice that express constitutively active
DEVELOPMENT
Fig. 2. Distribution of Ngn3-positive spermatogonia and Kit-positive spermatogonia in the P5 testes. (A-D) Double-staining ISH for Ngn3
(purple, A) and Kit (red, B), with overlay images of A and B (C), and of B with DNA staining (blue, D). Purple arrows indicate Ngn3 in
spermatogonia; short and long pink and red arrows indicate Kit in spermatogonia and interstitial cells, respectively. Note that the Ngn3 and Kit
signals are observed in distinct cells in separate seminiferous tubule cross-sections. Scale bar: 100 ␮m. (E-G) ISH for Ngn3 and Kit on adjacent
sections, under low magnification. The distribution of Ngn3 (E) and Kit (F) germ cell signals are compared and represented in G, where seminiferous
tubule cross-sections containing Ngn3 (green), Kit (blue), or both signals (red) are indicated. For statistical evaluation see Table S1 in the
supplementary material. Scale bars: 100 ␮m, in A for A-D, in F for E,F.
Cre recombinase controlled by the Ngn3 regulatory sequence
(Ngn3/Cre) (Yoshida et al., 2004). In double-transgenic mice
possessing the CAG-CAT-Z reporter (Araki et al., 1995), the Ngn3+
lineage can be labeled for ␤-gal expression as a result of an
irreversible recombination, whereas the Ngn3– lineage retains the
intact form of the reporter gene and expresses the CAT
(chloramphenicol acetyltransferase) gene instead. In these mice, ␤gal is not detected in neonatal testes, reflecting the absence of Ngn3
expression in the germ line until birth (Yoshida et al., 2004). Active
Cre recombinase begins to be expressed in Ngn3-positive cells as
early as P3, and promptly causes the recombination that allows the
expression of lacZ (Fig. 5B-D). In P5 testes, more Kit-positive
spermatogonia were detected than lacZ-positive spermatogonia (Fig.
5E,F), indicating that the majority of these prepubertal Kit-positive
spermatogonia were devoid of lacZ expression, which is in
agreement with the direct generation of Kit-positive spermatogonia
without Ngn3 expression.
Development 133 (8)
Furthermore, the contributions of the Ngn3– (lacZ-negative/CATpositive) and Ngn3+ (lacZ-positive/CAT-negative) lineages of cells
were examined during the maturation of the testes. At P21, when
round spermatids first appear, the germ cells of the Ngn3– lineage are
localized almost exclusively at the center of the seminiferous tubules,
i.e. in the most advanced cells (Fig. 5G-I). At P28, the germ cells of
the Ngn3– lineage contribute to the innermost layer of the
spermatogenic cells (Fig. 5J-L). At this age, the degree of maturation
varies among the seminiferous tubule segments. The most advanced
segments contain elongated spermatids, which consist mostly of cells
of the Ngn3– lineage (e.g. tubule 1 in Fig. 5K). The contribution of the
Ngn3– lineage is prominent in the second advanced group of segments
(e.g. tubule 2). In the least matured segments, essentially no cells of
the Ngn3– lineage were observed (see tubule 3). Thus, cells of Ngn3–
lineage specifically contribute to the leading edge of pubertal
spermatogenesis, which gives rise to the first spermatozoa that are
released around P35 (Kluin et al., 1982). In the fully mature testes,
Fig. 3. Distribution of the newlyborn Ngn3- and Kit-positive
spermatogonia, and the
seminiferous epithelial cycle
prepattern. (A-H) Double-staining
ISH of testes sections at the
indicated ages with the indicated
genes. Overlaid images of Kit or
Ngn3 (purple, bright field) and
galectin 1 (red fluorescence) are
shown in A,C,E,G; galectin 1 signals
in the same field are shown in
B,D,F,H. Arrows in A and C indicate
the Kit- and Ngn3-positive
spermatogonia, respectively. E,F and
G,H are adjacent sections. Note the
variable intensities of galectin 1
signals within the seminiferous
tubule cross-sections and their
relationship with Kit- or Ngn3positive spermatogonia (see Results).
Asterisks (E-H) represent typical
segments with a high galectin 1
mRNA signal; Kit-positive, but not
Ngn3-positive, spermatogonia
preferentially localize to these
segments. Segments marked by
hearts show lower galectin 1 signals
and exhibit a preference for Ngn3positive spermatogonia. Scale bars:
100 ␮m, in D for A-D, in H for E-H.
(I) Quantification of Kit- or Ngn3positive spermatogonia localized in
tubule segments categorized by
different levels of galectin 1 mRNA
(high, medium or low). Tubule crosssections marked with H, M or L in B
and D are examples of segments
with high, medium and low levels of
galectin 1 mRNA, respectively. Data are represented as ‘preference’ to each category of tubule segment. a and b represent significant deviation with
P-values of <0.0001 and <0.05, respectively. See Materials and methods and Tables S2-S7 in the supplementary material. The number of Ngn3positive spermatogonia at P3 and P4 was too low for statistical analyses. (J) Model for the generation of spermatogonia subpopulations. One area
of seminiferous tubules is represented to align tubules with increasing age. The galectin 1 mRNA level is shown by the gradient: black, highest;
white, lowest. As galectin 1 expression increases and reduces in cycles (black to gray to white to black, etc.), its expression domain shifts leftward in
a wave-like manner. Kit-positive spermatogonia (red ovals) appear specifically at the galectin 1-high segments (red bands). By contrast, Ngn3positive spermatogonia (green ovals) are generated separately from Kit-positive spermatogonia around the segments of medium level of galectin 1
expression (green bands).
DEVELOPMENT
1500 RESEARCH ARTICLE
germ cells of the Ngn3– lineage are absent, and spermatogenesis is
completely dependent on germ cells of the Ngn3+ lineage, while
somatic cells, including Sertoli, myoid and interstitial cells, remain
CAT positive (arrows) (Fig. 5M,N) (Yoshida et al., 2004).
It is theoretically possible that the CAT-positive/␤-gal-negative
cells are the result of incomplete recombination in cells with weak
and short Ngn3 expression. However, in our double-transgenic mice,
the recombination by Cre is particularly efficient, as determined by
the quick and accurate recombination (Fig. 5B-D), and by the
essentially complete recombination in the matured testes (Fig.
5M,N, Fig. 6) (Yoshida et al., 2004). The pattern of the CAT-positive
cell contribution is highly reproducible among individuals from
independent Ngn3/Cre transgenic lines. Therefore, considering the
tight relationship between the pre-pubertal Kit-positive (Ngn3negative) spermatogonia subpopulation and the seminiferous
epithelial cycle, it is probable that at least a major part of the CATpositive cells found in the first round of spermatogeneis represents
a particular subpopulation of germ cells: those produced from a
distinct program that lacks the Ngn3-positive, undifferentiated
spermatogonia stage.
Fertilizing ability of the Ngn3– lineage of
spermatogenesis
The pubertal, so-called first wave of spermatogenesis includes
massive apoptosis (Kluin et al., 1982; Mori et al., 1997) and it is
sometimes suspected to not produce fertile spermatozoa. We
attempted to elucidate whether this inefficiency is related to the
Ngn3– and Ngn3+ lineages. As shown in Fig. 5J-L, the germ cells
RESEARCH ARTICLE 1501
of the Ngn3– lineage do survive and differentiate into
morphologically mature sperm. We also found that germ cells of
both the Ngn3– and Ngn3+ lineages exhibit cell death with no
clear preferences (data not shown). Next, the fertility of the
spermatozoa that were generated in the first round of
spermatogenesis was tested as follows: Ngn3/Cre;CAG-CAT-Z
male double-transgenic mice were mated with non-transgenic
females, and each of the offspring were tested to determine
whether they carried the recombined or the intact form of the
reporter gene in tissues without Ngn3 expression (Fig. 6A).
Offspring with an intact reporter gene are derived by fertilization
with spermatozoa of the Ngn3– lineage, whereas those with the
recombined reporter gene are derived from the Ngn3+ lineage. In
our experiment, the youngest paternal age at fertilization was P40.
Spermatozoa with an intact reporter gene exclusively contributed
to the offspring obtained from the mating at P40 and P41 (Fig.
6B), reflecting fertilization by the first-released spermatozoa after
epididymal passage. By contrast, offspring obtained by mating
older mice derived from the recombined spermatozoa. It is
noteworthy that males that produced offspring with the intact
reporter gene at their first matings, later produced offspring with
only the recombined reporter gene. This shows that, the first round
of spermatogenesis can produce functional spermatozoa
regardless of the distinct differentiation program. This is in
agreement with the observation that full-term embryonic
development can be supported by at least some of the round
spermatids in the first round of spermatogenesis after
microinsemination by injecting into oocytes (Miki et al., 2004).
Fig. 4. Pulse labeling of Ngn3positive spermatogonia using
tamoxifen-inducible Cre transgenic
mice, and their stem cell activities.
(A) Scheme for the tamoxifendependent recombination by CreERTM,
resulting in the labeling of cells with ␤gal expression. (B) The experiment
schedule. (C,D) ISH on adjacent
sections from a P8 Ngn3/CreERTM
transgenic mouse testis detecting Ngn3
and CreERTM expression and indicating
their overlapped expression
(arrowheads). (E,F) Whole-mount X-Gal
staining of a seminiferous tubule of a
P8 Ngn3/CreERTM; CAG-CAT-Z doubletransgenic mouse with (E) and without
(F) tamoxifen administration (tam).
Inset (E) is at higher magnification.
Note the tamoxifen-dependent
appearance of ␤-gal-positive
spermatogonia. Mice with only the
CAG-CAT-Z transgene do not exhibit
positive staining after tamoxifen
administration (data not shown).
(G) Double-transgenic mice were
injected with tamoxifen at P5 and P6,
and their seminiferous tubules were
subjected to X-gal staining at the age
of 3 months. Many ␤-gal-positive cells persist as distinct segments (arrowheads). (H) Cross section of a ␤-gal-positive segment containing a
complete set of spermatogenic cells stained with ␤-gal (nuclear counterstaining in red). (I-K) Stem cell activity of Ngn3-positive spermatogonia after
transplantation. Tamoxifen was administered to double-transgenic mice according to the same schedule. Their testicular cells were transplanted into
the seminiferous tubules of W/Wv mice at P8 and subjected to whole-mount X-Gal staining (blue) 3 months later. A number of blue spermatogenic
colonies were detected (I, arrowheads). (J) A typical section of ␤-gal-positive colonies (nuclear counterstaining in red); (K) Hematoxylin and Eosin
staining of the adjacent section. Scale bars: 100 ␮m in D,F,H,K; 1 mm in G,I.
DEVELOPMENT
Initiation of mouse spermatogenesis
Development 133 (8)
Fig. 5. Chase of cells of Ngn3+ and Ngn3–
lineages during the maturation of testes
using Ngn3/Cre;CAG-CAT-Z doubletransgenic mice. (A) Experimental design. In
Ngn3-positive cells of Ngn3/Cre;CAG-CAT-Z
mice, the reporter gene is irreversibly
recombined between loxP sites by Cre
recombinase driven by the Ngn3 regulatory
sequence, thereby labeling their progenies with
␤-gal (encoded by lacZ), while CAT expression is
lost (Ngn3+ lineage). The reporter gene remains
intact in cells that have never expressed Ngn3;
these cells express CAT, but not lacZ (Ngn3–
lineage). (B-D) Serial sections of P3 testes
probed for Ngn3, Cre and lacZ, representing the
overlapping expression of these genes in a
single seminiferous tubule (arrowheads). (E,F) A
pair of adjacent sections of a P5 testis, which
were hybridized for Kit and lacZ expression. The
Kit-positive spermatogonia apparently
outnumber the lacZ-positive ones. (G-I) P21
testis probed for CAT. H shows a higher
magnification of a part of G; I shows a PIstained fluorescence image of H. CAT signals are
preferentially detected at the center of the
seminiferous tubules (arrowheads). Arrows
indicate CAT signals in the interstitial cells.
(J-L) P28 testis probed for CAT and lacZ. (K)
Higher magnification of a part of J; (L) the same
location but in the next section. CAT-positive
and lacZ-negative (Ngn3– lineage)
spermatogenic cells are preferentially found in
the innermost layer (arrowheads). Seminiferous
tubules marked 1-3 are examples of the
different degrees of maturation (see Results).
(M,N) P56 testis probed for CAT and lacZ. All
germ cells are CAT negative and lacZ positive.
Arrows indicate somatic cells positive for CAT
(Sertoli and interstitial cells). Scale bars: 100 ␮m.
Fig. 6. Contribution of cells of the Ngn3+ and Ngn3–
lineages to functional spermatozoa. (A) Experimental
design. Ngn3/Cre;CAG-CAT-Z double-transgenic male mice
were mated with non-transgenic females, and the offspring
genotyped to determine whether the contributed spermatozoa
carried an intact (CAG-CAT-Z) or recombined (CAG-Z) form of
the reporter gene. (B) Frequency of offspring with the intact
(gray) and recombined (white) forms of the reporter gene,
classified according to the paternal age at fertilization.
DEVELOPMENT
1502 RESEARCH ARTICLE
The fragility of pubertal spermatogenesis may be a result of cellextrinsic factors, such as hormonal conditions, as described
previously (Russell et al., 1987).
DISCUSSION
On the basis of our study, we have proposed a model for the
initiation of spermatogenesis (Fig. 7). In the pre-pubertal testis, a
particular subset of gonocytes directly gives rise to Kit-positive
differentiating spermatogonia in the seminiferous tubule segments
with a high level of galectin 1 mRNA; these spermatogonia have not
passed through the Ngn3-positive undifferentiated stage. This
population (Ngn3– lineage) differentiates promptly in the first round
of spermatogenesis, resulting in the production of fertile
spermatozoa. Contrastingly, another set of gonocytes become Ngn3positive spermatogonia in distinct segments with a medium level of
galectin 1. This population (Ngn3+ lineage) is capable of selfrenewal and the generation of differentiating cells, and supports the
continuity of steady-state spermatogenesis. In Ngn3-expressing
undifferentiated spermatogonia, the expression of Plzf, Ret or Oct4,
genes that also characterize the PGC and/or gonocytes, is retained.
Gain of Ngn3 expression thus reflects a cell-type switch from
precursors to self-renewing cells.
The fact that the first round of spermatogenesis produces fertile
spermatozoa even though they are not derived from self-renewing
undifferentiated spermatogonia supports the idea that the stem cell
step is not ‘the gateway’ that all progenitor cells must enter and from
which all differentiating cells must originate. Rather, as shown in
Fig. 7, the stem cell step can be considered to be ‘a developmental
option’ that branches out of the process of spermatogenic
differentiation and ensures continuous spermatozoa production. This
idea might also be true for other stem cell systems. It has been
recently shown that, in the Drosophila germ line, some primordial
germ cells do not develop into self-renewing stem cells but directly
enter into the differentiation process (Asaoka and Lin, 2004). In
RESEARCH ARTICLE 1503
mammalian yolk sac hematopoiesis, although the first appearance
of the hematopoietic stem cells is not completely understood, the
first round of differentiation of blood cells occurs prior to the
appearance of detectable hematopoietic stem cell activity (Palis and
Yoder, 2001).
The determination of the mechanisms underlying the emergence
of the two spermatogonial subpopulations from gonocytes at
distinct segments of seminiferous tubule is an important issue.
Although this study still leaves this question unsolved, several
possibilities can be discussed. If we simply extend the interpretation
of Timmons et al. (Timmons et al., 2002) that the somatic
components have their intrinsic pre-pattern of the seminiferous
epithelial cycle, germ cell differentiation would be under their
downstream control. Therefore, one possibility is that gonocytes
might be a homogeneous population in which individual cells are
bipotential and select their fates in response to the local controls by
somatic cells upon transition into spermatogonia. A second
possibility is that gonocytes might be heterogeneous and that a
particular subpopulation is committed to contributing to the selfrenewing component. Compatible with this idea, Orwig et al.
(Orwig et al., 2002) reported that neonatal rat gonocytes exhibit a
morphological heterogeneity that is closely related to their
transplantable stem cell activities. At a molecular level, neonatal
mouse gonocytes represent some heterogeneity in terms of Oct4
expression level (Ohmura et al., 2004), raising the possibility that
Oct4 high-expressers might be committed to self-renewal. Other
possibilities include that commitment might occur under the control
of somatic cells at the gonocyte stage or earlier. However, we
cannot rule out the possibility that germ cells might decide their
own fate independently from the somatic cells, and that
coordination is brought about by germ-soma interactions, including
tuning of the Sertoli cell cycle by germ cells, or sorting of the germ
cell subpopulations according to somatic cell-derived
environments.
Fig. 7. Model for the two lineages in
the mouse spermatogenesis. In the first
postnatal week, gonocytes directly give
rise to Kit-positive differentiating
spermatogonia and Ngn3-positive
undifferentiated spermatogonia in
parallel. This process is closely related to
the presumptive seminiferous epithelial
cycle pre-pattern, which initiates before
birth. Kit-positive spermatogonia are
specifically generated in the galectin 1high segments (pink arrow 1). These cells
do not pass through a Ngn3-positive,
undifferentiated spermatogonia stage and
differentiate in the first round of
spermatogenesis, resulting in the
formation of fertile spermatozoa
(Ngn3– lineage). By contrast, Ngn3positive undifferentiated
spermatogonia are generated preferably
at galectin 1-medium segments (green
arrow). They subsequently act as a selfrenewing stem cell population, while also
providing cells that transform into
differentiating spermatogonia. Thus, these
cells support steady-state spermatogenesis
following the first round of spermatogenesis (Ngn3+ lineage). The transformation of undifferentiated spermatogonia into differentiating
spermatogonia is tightly related to the seminiferous epithelial cycle, and Kit-positive differentiating spermatogonia are established in stages of
high galectin 1 expression (stage IX–X), indicated by pink arrow 2. See Results for more details. PGC, primordial germ cells.
DEVELOPMENT
Initiation of mouse spermatogenesis
It is noteworthy that the direct generation of differentiating
spermatogonia from gonocytes occurs in the seminiferous
segments with a high level of galectin 1 mRNA (Fig. 7), because
this is likely to reflect the common features of pre-pubertal and
adult spermatogenesis, and, therefore, their continuity. In the adult,
Kit-positive differentiating spermatogonia are established around
stages IX-X (Schrans-Stassen et al., 1999). These stages show the
highest level of galectin 1 (Timmons et al., 2002) (Fig. 7). We
believe this coincidence would strengthen the assumption by
Timmons et al. that the seminiferous epithelial cycle is pre-figured
already in the pre-pubertal stage (Timmons et al., 2002). As a
result, the establishment of the spermatogenic wave may
accompany the completion of the first round of spermatogenesis,
thus ensuring a constant release of the mature spermatozoa
following puberty.
The direct derivation of the first differentiating spermatogonia
from gonocytes is in agreement with the hypothesis of de Rooij
and colleagues, which was based on morphology, and on the
backwards extrapolation of the progressive appearance of
differentiating cell types (de Rooij, 1998; Kluin and de Rooij,
1981). The present study not only provides evidence for their
hypothesis by means of gene expression and lineage analyses, but
also extends it with regard to the fate of the first differentiating
spermatogonia. Although previous studies could not define the fate
of the first differentiating spermatogonia because of the fragility
of pubertal spermatogenesis, our study provides evidence that the
first differentiating spermatogonia that appear give rise to
functional spermatozoa. We have also demonstrated that,
immediately after the gonocyte-spermatogonia transition, adulttype undifferentiated spermatogonia appear as Ngn3-positive cells,
which already exhibit stem cell characteristics (i.e. self-renewal
and the generation of differentiating cells). Accordingly, we do not
believe it necessary to consider the existence of the special type of
‘pre-spermatogonia’ between gonocytes and adult-type
spermatogonia (Bellve et al., 1977; Hilscher et al., 1974; Huckins
and Clermont, 1968). Finally, this study has shown the close
relationship between the genesis of the spermatogonia
subpopulations and the establishment of the seminiferous
epithelial cycle.
We thank Dr Douglas Melton for the Ngn3-cre-ERTM construct, Dr Jun-ichi
Miyazaki for the CAG-CAT-Z transgenic mice, Dr Takashi Shinohara for the
instructions on transplantation, Dr Hirohide Takebayashi for suggestions on
tamoxifen administration, and Mr Tsutomu Obata for preparation of the
sections. We also thank Dr Toshihiko Fujimori, Dr Takehiko Ogawa and Dr
Naoko Yoshida for their discussions and critical reading of the manuscript. This
work was partly supported by Grants-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and Technology, Japan.
References
Araki, K., Araki, M., Miyazaki, J. and Vassalli, P. (1995). Site-specific
recombination of a transgene in fertilized eggs by transient expression of Cre
recombinase. Proc. Natl. Acad. Sci. USA 92, 160-164.
Asaoka, M. and Lin, H. (2004). Germline stem cells in the Drosophila ovary
descend from pole cells in the anterior region of the embryonic gonad.
Development 131, 5079-5089.
Bellve, A. R., Cavicchia, J. C., Millette, C. F., O’Brien, D. A., Bhatnagar, Y. M.
and Dym, M. (1977). Spermatogenic cells of the prepuberal mouse. Isolation
and morphological characterization. J. Cell Biol. 74, 68-85.
Brinster, R. L. (2002). Germline stem cell transplantation and transgenesis. Science
296, 2174-2176.
Buaas, F. W., Kirsh, A. L., Sharma, M., McLean, D. J., Morris, J. L., Griswold,
M. D., de Rooij, D. G. and Braun, R. E. (2004). Plzf is required in adult male
germ cells for stem cell self-renewal. Nat. Genet. 36, 647-652.
Costoya, J. A., Hobbs, R. M., Barna, M., Cattoretti, G., Manova, K.,
Sukhwani, M., Orwig, K. E., Wolgemuth, D. J. and Pandolfi, P. P. (2004).
Essential role of Plzf in maintenance of spermatogonial stem cells. Nat. Genet.
36, 653-659.
Development 133 (8)
de Rooij, D. G. (1998). Stem cells in the testis. Int. J. Exp. Pathol. 79, 67-80.
de Rooij, D. G. (2001). Proliferation and differentiation of spermatogonial stem
cells. Reproduction 121, 347-354.
de Rooij, D. G. and Grootegoed, J. A. (1998). Spermatogonial stem cells. Curr.
Opin. Cell Biol. 10, 694-701.
de Rooij, D. G. and Russell, L. D. (2000). All you wanted to know about
spermatogonia but were afraid to ask. J. Androl. 21, 776-798.
Gu, G., Dubauskaite, J. and Melton, D. A. (2002). Direct evidence for the
pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct
progenitors. Development 129, 2447-2457.
Hayashi, S. and McMahon, A. P. (2002). Efficient recombination in diverse
tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated
gene activation/inactivation in the mouse. Dev. Biol. 244, 305-318.
Hilscher, B., Hilscher, W., Bulthoff-Ohnolz, B., Kramer, U., Birke, A.,
Pelzer, H. and Gauss, G. (1974). Kinetics of gametogenesis. I. Comparative
histological and autoradiographic studies of oocytes and transitional
prospermatogonia during oogenesis and prespermatogenesis. Cell Tissue
Res. 154, 443-470.
Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994). Manipulating the
Mouse Embryo: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor
Laboratory Press.
Huckins, C. and Clermont, Y. (1968). Evolution of gonocytes in the rat testis
during late embryonic and early post-natal life. Arch. Anat. Histol. Embryol. 51,
341-354.
Kluin, P. M. and de Rooij, D. G. (1981). A comparison between the morphology
and cell kinetics of gonocytes and adult type undifferentiated spermatogonia in
the mouse. Int. J. Androl. 4, 475-493.
Kluin, P. M., Kramer, M. F. and de Rooij, D. G. (1982). Spermatogenesis in the
immature mouse proceeds faster than in the adult. Int. J. Androl. 5, 282-294.
Leblond, C. P. and Clermont, Y. (1952). Definition of the stages of the cycle of
the seminiferous epithelium in the rat. Ann. New York Acad. Sci. 55, 548-573.
Manova, K., Nocka, K., Besmer, P. and Bachvarova, R. F. (1990). Gonadal
expression of c-kit encoded at the W locus of the mouse. Development 110,
1057-1069.
McLean, D. J., Friel, P. J., Johnston, D. S. and Griswold, M. D. (2003).
Characterization of spermatogonial stem cell maturation and differentiation in
neonatal mice. Biol. Reprod. 69, 2085-2091.
Meng, X., Lindahl, M., Hyvonen, M. E., Parvinen, M., de Rooij, D. G., Hess,
M. W., Raatikainen-Ahokas, A., Sainio, K., Rauvala, H., Lakso, M. et al.
(2000). Regulation of cell fate decision of undifferentiated spermatogonia by
GDNF. Science 287, 1489-1493.
Miki, H., Lee, J., Inoue, K., Ogonuki, N., Noguchi, Y., Mochida, K., Kohda, T.,
Nagashima, H., Ishino, F. and Ogura, A. (2004). Microinsemination with
first-wave round spermatids from immature male mice. J. Reprod. Dev. 50,
131-137.
Mori, C., Nakamura, N., Dix, D. J., Fujioka, M., Nakagawa, S., Shiota, K. and
Eddy, E. M. (1997). Morphological analysis of germ cell apoptosis during
postnatal testis development in normal and Hsp 70-2 knockout mice. Dev. Dyn.
208, 125-136.
Nishimune, Y., Aizawa, S. and Komatsu, T. (1978). Testicular germ cell
differentiation in vivo. Fertil. Steril. 29, 95-102.
Ogawa, T., Arechaga, J. M., Avarbock, M. R. and Brinster, R. L. (1997).
Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J.
Dev. Biol. 41, 111-122.
Ohbo, K., Yoshida, S., Ohmura, M., Ohneda, O., Ogawa, T., Tsuchiya, H.,
Kuwana, T., Kehler, J., Abe, K., Schoeler, H. R. et al. (2003). Identification
and characterization of stem cells in pre-pubertal spermatogenesis in mice. Dev.
Biol. 258, 209-225.
Ohmura, M., Yoshida, S., Ide, Y., Nagamatsu, G., Suda, T. and Ohbo, K.
(2004). Spatial analysis of germ stem cell development in Oct-4/EGFP transgenic
mice. Arch. Histol. Cytol. 67, 285-296.
Orwig, K. E., Ryu, B. Y., Avarbock, M. R. and Brinster, R. L. (2002). Male germline stem cell potential is predicted by morphology of cells in neonatal rat testes.
Proc. Natl. Acad. Sci. USA 99, 11706-11711.
Palis, J. and Yoder, M. C. (2001). Yolk-sac hematopoiesis: the first blood cells of
mouse and man. Exp. Hematol. 29, 927-936.
Pesce, M., Wang, X., Wolgemuth, D. J. and Scholer, H. (1998). Differential
expression of the Oct-4 transcription factor during mouse germ cell
differentiation. Mech. Dev. 71, 89-98.
Russell, L. D., Alger, L. E. and Nequin, L. G. (1987). Hormonal control of
pubertal spermatogenesis. Endocrinology 120, 1615-1632.
Russell, L., Ettlin, R., Sinha Hikim, A. and Clegg, E. (1990). Histological and
histopathological evaluation of the testis. Clearwater, Fl: Cache River Press.
Schrans-Stassen, B. H., van de Kant, H. J., de Rooij, D. G. and van Pelt, A. M.
(1999). Differential expression of c-kit in mouse undifferentiated and
differentiating type A spermatogonia. Endocrinology 140, 5894-5900.
Shinohara, T., Orwig, K. E., Avarbock, M. R. and Brinster, R. L. (2000).
Spermatogonial stem cell enrichment by multiparameter selection of mouse
testis cells. Proc. Natl. Acad. Sci. USA 97, 8346-8351.
Timmons, P. M., Rigby, P. W. and Poirier, F. (2002). The murine seminiferous
DEVELOPMENT
1504 RESEARCH ARTICLE
epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis.
Development 129, 635-647.
Yoshida, S., Ohbo, K., Takakura, A., Takebayashi, H., Okada, T., Abe, K. and
Nabeshima, Y. (2001). Sgn1, a basic helix-loop-helix transcription factor
delineates the salivary gland duct cell lineage in mice. Dev. Biol. 240, 517-530.
Yoshida, S., Takakura, A., Ohbo, K., Abe, K., Wakabayashi, J., Yamamoto,
RESEARCH ARTICLE 1505
M., Suda, T. and Nabeshima, Y. (2004). Neurogenin3 delineates the earliest
stages of spermatogenesis in the mouse testis. Dev. Biol. 269, 447-458.
Yoshinaga, K., Nishikawa, S., Ogawa, M., Hayashi, S., Kunisada, T. and
Fujimoto, T. (1991). Role of c-kit in mouse spermatogenesis: identification of
spermatogonia as a specific site of c-kit expression and function. Development
113, 689-699.
DEVELOPMENT
Initiation of mouse spermatogenesis