Developmental Biology 400 (2015) 43–58

Contents lists available at ScienceDirect

Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology

Gene expression ontogeny of spermatogenesis in the marmoset
uncovers primate characteristics during testicular development
Zachary Yu-Ching Lin a, Takamasa Hirano b, Shinsuke Shibata a, Naomi M. Seki b,c,
Ryunosuke Kitajima d, Ayako Sedohara e, Mikiko C. Siomi b,c, Erika Sasaki a,e,f,Haruhiko Siomi b, Masanori Imamura a,d,n, Hideyuki Okano a,nnaDepartment of Physiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Department of Molecular Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japanc
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japand
Molecular Biology Section, Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japane
Department of Applied Developmental Biology, Central Institute for Experimental Animals, 3-25-12 Tonomachi, Kawasaki 210-0821, Japanf
PRESTO Japan Science and Technology Agency, Japanb

art ic l e i nf o

a b s t r a c t

Article history:
Received 15 July 2014
Received in revised form
14 January 2015
Accepted 16 January 2015
Available online 23 January 2015

Mammalian spermatogenesis has been investigated extensively in rodents and a strictly controlled
developmental process has been deﬁned at cellular and molecular levels. In comparison, primate
spermatogenesis has been far less well characterized. However, important differences between primate
and rodent spermatogenesis are emerging so it is not always accurate to extrapolate ﬁndings in rodents
to primate systems. Here, we performed an extensive immunoﬂuorescence study of spermatogenesis inneonatal, juvenile, and adult testes in the common marmoset (Callithrix jacchus) to determine primatespeciﬁc patterns of gene expression that underpin primate germ cell development. Initially wecharacterized adult spermatogonia into two main classes; mitotically active C-KIT þ Ki67 þ cells andmitotically quiescent SALL4 þ PLZF þ LIN28 þ DPPA4 þ cells. We then explored the expression of a set ofmarkers, including PIWIL1/MARWI, VASA, DAZL, CLGN, RanBPM, SYCP1 and HAPRIN, during germ celldifferentiation from early spermatocytes through round and elongating spermatids, and a clear programof gene expression changes was determined as development proceeded. We then examined the juvenile
marmoset testis. Markers of gonocytes demonstrated two populations; one that migrates to the basal
membrane where they form the SALL4 þ or C-KIT þ spermatogonia, and another that remains in thelumen of the seminiferous tubule. This later population, historically identiﬁed as pre-spermatogonia, expressed
meiotic and apoptotic markers and were eliminated because they appear to have failed to correctly migrate.
Our ﬁndings provide the ﬁrst platform of gene expression dynamics in adult and developing germ cells of thecommon marmoset. Although we have characterized a limited number of genes, these results will facilitate
primate spermatogenesis research and understanding of human reproduction.
& 2015 Elsevier Inc. All rights reserved.

Keywords:
Spermatogenesis
Common marmoset
Spermatogonia
Spermatocyte
Gonocyte
Germ cell

Introduction
Mammalian spermatogenesis is a strictly organized developmental process involved in the passage of genetic material.
Spermatogenesis originates from a population of spermatogonia
called spermatogonial stem cells that undergo the sequential
processes of mitotic and meiotic spermiogenesis to producenCorrespondence to: Molecular Biology Section, Department of Cellular andMolecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi
484-8506, Japan. Fax: +81 568 63 0085.
nn
Corresponding author. Fax: +81 333575445.
E-mail addresses: imamura.masanori.2m@kyoto-u.ac.jp (M. Imamura),hidokano@a2.keio.jp (H. Okano).

http://dx.doi.org/10.1016/j.ydbio.2015.01.014
0012-1606/& 2015 Elsevier Inc. All rights reserved.

spermatozoa. In rodents, many aspects of spermatogenesis
have been analyzed including cell morphology, organization, andgene expression (Hermo et al., 2010). These studies have led tothe identiﬁcation of numerous molecular signatures to discriminate each stage of spermatogenesis. By contrast, owing to lessaccessibility to tissue samples and ethical and legal issues, there
are fewer such comparative studies on primate spermatogenesis.
An increasing number of studies have revealed that primatesand rodents have a different developmental program for spermatogenesis (YDBIO|6681Hermann et al., 2010; McKinnell et al.,2013; Schlatt and Ehmcke, 2014). For example, the stem cellsystem underlying spermatogenesis differs between primatesand rodents. In humans there are two distinct types of undifferentiated spermatogonia; quiescent Adark spermatogonia, which are44

Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

believed to serve as reserve stem cells, and renewing Apale
spermatogonia,
which
proliferate
regularly(YDBIO|
6681Clermont, 1963, 1966, 1969, 1972). This categorization is also
the case in New-World (YDBIO|6681Eildermann et al., 2012; Millar
et al., 2000) and Old-World monkeys (YDBIO|6681Ehmcke et al.,2006; Hermann et al., 2010), but is not incorporated into rodent
spermatogenesis that does not enclose reserve stem cells (Schlatt
and Ehmcke, 2014).
Differences in the molecular proﬁle are also evident between
primate and rodent spermatogenesis. In our previous study, we
compared the molecular signatures of spermatogenic cells in mouseand common marmoset (Callithrix jacchus) with regard to DNA
methylation status, transcripts, and protein expression (Lin et al.,2012). We revealed that in marmosets the NANOG and OCT4
promoters exhibit DNA hypomethylation and hypermethylation,
respectively, unlike mouse homologs, which display an opposite
DNA methylation pattern (YDBIO|6681Imamura et al., 2006;
Western et al., 2010). Additionally, we found that marmoset
spermatogonia express DAZL but not VASA protein, while bothproteins are expressed in mouse spermatogonia. These observations
reveal that simple extrapolation of rodent insights to primates is not
appropriate. Hence, investigation of primates should be considered.
However, very little is known about the molecular dynamics ofprimate spermatogenesis. There is, therefore, a fundamental need fora suitable non-human primate model.
As a model animal to investigate primate spermatogenesis,
we have focused on a New-World monkey, the common marmosetthat is native to the Atlantic coastal forests in northeastern Brazil.
Among primates this species has attracted much attention in recent
years as it is an excellent experimental animal for biomedical
research. It has a small body size (20–30 cm height; 350–400 g
weight in adulthood), fast sexual maturation (around 1.5 years old),
breeds throughout the year with a proliﬁcacy of 40–80 offspring fora female life, and is physiologically similar to humans (YDBIO|
6681Kishi et al., 2014; Mansﬁeld, 2003; Okano et al., 2012). In
addition, our group has succeeded in producing offspring withassisted reproduction technologies (YDBIO|6681Takahashi et al.,2014, Tomioka et al., 2012), and creating a transgenic marmosetwith germline transmission (Sasaki et al., 2009), expanding theirpotential for genetic research. Furthermore, compared with othernon-human primates, marmosets and humans share a similarorganization of spermatogenesis and testicular development(YDBIO|6681Millar et al., 2000; Mitchell et al., 2008; Saunders etal., 1996; Wistuba et al., 2003) as both species retain gonocytes in
the postnatal testes (YDBIO|6681Albert et al., 2010; Sharpe et al.,2003). To date, some studies have examined gene expression of
marmoset germ cells but only focused on either a single or a few
genes (e.g. OCT4, SALL4, and VASA) at a particular developmental
stage (YDBIO|6681Eildermann et al., 2012; Lin et al., 2012;
McKinnell et al., 2013). A comprehensive gene expression atlas ofmarmoset spermatogenesis remains to be deﬁned.
In this study, to develop the atlas of gene expression dynamics inthe course of marmoset spermatogenesis, we dissected a sequenceof gene expression associated with spermatogenic processes. Extensive immunoﬂuorescent microscopy of neonatal, juvenile, and adulttestes elucidated not only gene expression benchmarks but also aunique developmental event occurring in the juvenile period. We
believe that our ﬁndings could serve as the ﬁrst molecular platform
to facilitate primate spermatogenesis research, and be a ﬁrst stepfor unveiling primate-speciﬁc developmental programs.

Materials and methods
Animals and tissue collection
All the common marmosets used in this study were housed incages with dimensions of 82  61  160 cm3. This study wasapproved by Institutional Animal Care and Use Committee of theCentral for Experimental Animals (CIEA) and was performed inaccordance with CIEA guidelines that agree with the Guidelines for
Proper Conduct of Animal Experiments by the Science Council ofJapan (2006).
RT-PCR
Total RNA prepared with TRIzol reagent was isolated using the
RNeasy Mini kit (Qiagen, 74106) according to the manufacturer's
instructions. First-strand cDNA was synthesized with SuperScriptIII reverse transcriptase (Invitrogen, 18080-044) and oligo-dT18
primer. The primers were designed based on sequences in the
marmoset genome database (http://genome.wustl.edu/tools/blast/index) as described in Supplementary Table S1. Subsequent PCR
analysis was performed with Ex Taq Hot Start Version (TaKaRa,
RR006A). All experiments were performed semiquantitatively atthree different escalation cycles, and only representative images
are shown in the result.
Western blot
Marmoset tissues were homogenized in lysis buffer containing
20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA (pH 8.0) and
1% Nonidet-P40 (NP-40). After centrifugation, the supernatants
were diluted with sample buffer and boiled. Ten microgram of
total proteins per lean was separated on 7.5% SDS-PAGE andtransferred nitrocellulose (Whatman, 10 401 196) or PVDF (GEHealthcare, RPN303F) membranes. The membranes were blockedat room temperature with 5% (w/v) skim milk for 30 min. After
ﬁve washes with 0.1% PBS-T, the membranes were incubated withprimary antibodies at room temperature for 1 h or overnight at
4 1C. The following primary antibodies were used: culture supernatant of anti-MARWI hybridoma cells (1:1, 2D9) (Hirano et al.,2014), anti-GST (1:1000, Santa Cruz Biotechnology, sc-138), antiFLAG (M2) (1:5000, Sigma, F3165), anti-GAPDH (14C10) (1:3000,
Cell Signaling, 2118) antibodies. HRP-conjugated anti-mouse IgG(1:5000, MP BIOMEDICALS, 55558) or anti-rabbit IgG (1:5000,
Immuno-Biological Laboratories, 17502) were used as secondary
antibodies. Blots were developed using ECL detection reagents(Amersham Biosciences, RPN2109).
Immuno-electron microscopy
Frozen sections from adult marmoset testes were incubatedwith anti-MARWI antibody (1:200, 2D9-1H6) for 4 days at 4 1C
followed by incubation with nanogold conjugated anti-mousesecondary antibody (1:100, Invitrogen, A-24921) for 1 day at
4 1C. After ﬁxation in 2.5% glutaraldehyde for 10 min and enhancement with HQ-Silver enhancement kit (Nanoprobes Inc., 2012) for
12 min at 25 1C in a dark room, sections were post-ﬁxed with 0.5%
osmium tetroxide, dehydrated through ethanol, acetone, and QY1,
and embedded in Epon. Ultrathin sections (70 nm thickness)were prepared and stained with 2% uranyl acetate and 80 mM
lead citrate (Reynolds methods) for 15 and 10 min respectively.
The sections were observed under a transmission electron microscope (JEOL model 1230), while images were taken with a Digital
Micrograph 3.3 (Gatan Inc.).

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Immunoﬂuorescence microscopy of marmoset testis
Testes were isolated from neonatal (1-day-old), juvenile (10month-old), and adult marmosets (2, 3, 4, 5, 9, 10-year-old). Eachtestis was ﬁxed in 4% (w/v) paraformaldehyde, dehydrated,
embedded in parafﬁn wax and sectioned at 5 μm thickness.
The sections were de-waxed with xylene, rehydrated in a gradedalcohol series, and washed with distilled water. For immunoﬂuorescence analyses, the sections were subjected to antigen retrieval
by autoclave at 105 1C for at least 5 min in 1  Target Retrieval
Solution (Dako, S1699). The sections were blocked at room
temperature with 5% (w/v) skim milk for 30 min and incubatedwith the primary antibody at 4 1C overnight in a humidiﬁed
chamber. The following primary antibodies were used: rabbit
anti-SALL4 (1:100, Abcam, ab29112), mouse anti-SALL4 (1:100,
Abcam, ab57577), rabbit anti-Ki67 (1:100, Leica Biosystems,
NCL-Ki67p), mouse anti-Ki67 (1:100, Leica Biosystems, NCLL-Ki67-MM1), rabbit anti-C-KIT (1:100, Abcam, ab32363), mouseanti-PLZF (1:100, Abcam, ab104854), goat anti-LIN28A (1:100, R&D
systems, AF3757), rabbit anti-DPPA4 (1:50, Abcam, ab154642),
mouse anti-MARWI (1:50, 2D9-1H6), rabbit anti-DDX4 (1:50,
Abcam, ab13840), mouse anti-DDX4 (1:50, Abcam, ab27591),
rabbit anti-DAZL (1:100, Abcam, ab34139), rabbit anti-Calmegin(1:100, Abcam, ab171971), rabbit anti-RanBPM (1:100, Abcam,
ab78127), rabbit anti-SYCP1 (1:100, Abcam, ab15087), goat antiRNF98 (1:100, Abcam, ab4836), mouse anti-phospho-histone H2A.
X (ser139) (1:100, Millipore, 05-636), rabbit anti-active Caspase 3(1:100, Abcam, ab13847), and rabbit anti-PARP1(Asp214) (1:100,
Cell Signaling, 9541S) antibodies. Secondary antibodies included
Alexa Fluor 488 goat anti-mouse IgG (1:500; Invitrogen, A-11029),
Alexa Flour 488 donkey anti-mouse IgG (1:500, Invitrogen,
A-21202), Alexa Flour 488 donkey anti-goat IgG (1:500, Invitrogen,
A-11055), Alexa Fluor 555 goat anti-rabbit IgG (1:500, Invitrogen,A-21429), Alexa Flour 555 donkey anti-rabbit IgG (1:500, Invitrogen, A-31572), and Alexa Flour 555 donkey anti-goat IgG (1:500,
Invitrogen, A-21432). Nuclei were stained with 10 ng/ml Hoechst
33342. For each experiment, a negative control was included inwhich the primary antibody had been omitted. Images were
captured using a Zeiss confocal microscope system (LSM700).

Results
Molecular subtyping of spermatogonia in adult marmoset testis
The common marmoset has two subtypes of undifferentiatedA spermatogonia; one is mitotically quiescent Adark spermatogonia, and the other is mitotically active Apale spermatogonia (YDBIO|
6681Hermann et al., 2010; Millar et al., 2000). These two types ofspermatogonia have been classically recognized by their characteristic nuclear staining patterns and morphological criteria, but
their molecular signatures remain unclear. To discriminate marmoset spermatogonia subtypes by gene expression, we carried out
immunoﬂuorescence analyses of adult marmoset testes, referring
to known spermatogonia markers in other mammalian species.
First, we focused on the Ki67 antigen, an indicator for dividing
cells (Gerdes et al., 1984), because the major cytological difference
between Adark and Apale spermatogonia is their mitotic activity(YDBIO|6681Dym et al., 2009; Ehmcke and Schlatt, 2006). The
distribution of Ki67 þ cells in the seminiferous tubules wascompared with cells expressing pluripotency transcription factor
SALL4, which is known to be a type A spermatogonium marker(Eildermann et al., 2012). Although both SALL4 and Ki67 weredetected in marmoset spermatogonia, the SALL4 þ and Ki67 þ
spermatogonia clearly constituted different populations (Fig. 1A
and Supplementary Fig. S1).

45

We next examined the expression of C-KIT protein, which isalso known to mark spermatogonia in marmosets (von Schonfeldt
et al., 1999) and rhesus macaques (Hermann et al., 2009). C-KIT
protein was mostly expressed in the Ki67 þ spermatogonia, albeit apart of C-KIT þ spermatogonia did not display Ki67 staining (Fig. 1B
and Supplementary Fig. S2). By contrast, the expression of SALL4
and C-KIT proteins seemed to be mutually exclusive (Fig. 1C) whileother spermatogonial markers, PLZF (YDBIO|6681Hermann et al.,2007; Hermann et al., 2009) and LIN28 (Aeckerle et al., 2012)proteins, were basically co-expressed with SALL4 protein (Fig. 1D
and E), which is similar to the case of mouse homologs (YDBIO|
6681Gassei and Orwig, 2013; Hobbs et al., 2012). DPPA4(Maldonado-Saldivia et al., 2007) protein was also speciﬁed in
the SALL4 þ spermatogonia (Fig. 1F). Taken together, it seems that
marmoset spermatogonia can be classiﬁed into two major subtypes, which are SALL4 þ PLZF þ LIN28 þ DPPA4 þ mitotically quiescent cells, and C-KIT þ Ki67 þ mitotically active cells, possibly
suggesting Adark and Apale spermatogonia respectively.
Marmoset PIWIL1, a component of chromatoid body
Next, we were interested in identifying gene expression of the
spermatocytes and spermatids in adult marmoset testis. We havealready found that germ cell-speciﬁc RNA helicase VASA isexpressed from primary spermatocytes to round spermatids(Lin et al., 2012); hence, we intended to examine the expressionof other marker genes. We particularly focused on the PIWI-family
proteins, which associate with PIWI-interacting RNAs (piRNAs) toprotect genomic integrity from transposable elements and, therefore, are expected to be conserved across mammalian species(Siomi et al., 2011). RT-PCR analysis of marmoset tissues identiﬁed
the expression of all four PIWI genes (PIWIL1, 2, 3, and 4) in testis
together with the related genes (Supplementary Fig. S3), as
reported in humans (Sasaki et al., 2003). Among them, PIWIL1
was subjected to detailed investigation because mouse PIWIL1/MIWI is expressed from pachytene spermatocytes to the elongating spermatids (Deng and Lin, 2002) and interacts with VASA
protein (Kuramochi-Miyagawa et al., 2004).
To examine the expression of marmoset homologue of PIWIL1,
which we designated as MARWI, we utilized an anti-MARWI
monoclonal antibody derived from a hybridoma line 2D9 (Supplementary Fig. S4) (Hirano et al., 2014). In mice, PIWIL1/MIWI
protein is concentrated in the chromatoid body, a germ cellspeciﬁc cloud-shaped granule, together with VASA protein in thecytoplasm of pachytene spermatocytes and spermatids (Kotaja andSassone-Corsi, 2007). We carried out immunoﬂuorescence analysis of marmoset testis and found that the subcellular localizationof MARWI protein corresponded to that of VASA protein (Fig. 2A),
in accordance with our recent report (Hirano et al., 2014). Notably,large granular staining of MARWI and VASA proteins was clearlyobserved in the round spermatids. The localization of MARWI
protein was further examined by immuno-electron microscopythat identiﬁed marmoset chromatoid bodies with high electron
density, in which MARWI protein was incorporated (Fig. 2B).
Molecular staging of marmoset spermatogenesis from spermatocytesto spermatids
To characterize developmental stage-speciﬁc gene expression inmarmoset spermatocytes and spermatids, we examined MARWI
protein in combination with other germ cell-related genes.
As reported previously (YDBIO|6681Lin et al., 2012; Ruggiu et al.,2000), an RNA-binding protein DAZL was expressed in marmoset
spermatogonia and spermatocytes (Fig. 3A). By contrast, MARWI
protein appeared at the pachytene spermatocyte stage and theexpression continued throughout the round and elongating

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Ki67

SALL4

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Ki67

C-KIT

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SALL4

C-KIT

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PLZF

SALL4

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LIN28

SALL4

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SALL4

DPPA4

Merge

Fig. 1. Spermatogonial subtypes in the adult marmoset testis. Confocal images of adult marmoset testis sections co-stained with antibodies against spermatogonial markers,
including (A) anti-Ki67 and anti-SALL4 antibodies, (B) anti-Ki67 and anti-C-KIT antibodies, (C) anti-SALL4 and anti-C-KIT antibodies, (D) anti-PLZF and anti-SALL4 antibodies,(E) anti-LIN28 and anti-SALL4 antibodies, and (F) anti-SALL4 and anti-DPPA4 antibodies. In the image of (B), arrowheads indicate C-KIT þ Ki67- spermatogonia. Nuclei werecounterstained with Hoechst 33342. Scale bar, 50 μm.

Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

MARWI

VASA

50μm

50μm

2μm

47

Merge

50μm

0.5μm

Fig. 2. Subcellular localization of marmoset PIWIL1 protein in chromatoid body. (A) Confocal images of adult testis frozen sections co-stained with anti-MARWI and antiVASA antibodies. Arrowheads indicate chromatoid body-like cytoplasmic granules consisted of condensed MARWI and VASA proteins. Nuclei were counterstained with
Hoechst 33342. Scale bar, 50 μm. (B) Immuno-electron microscopic analysis of adult testis frozen section stained with anti-MARWI antibody. MARWI proteins were detectedin chromatoid bodies with high electron density in the cytoplasm.

spermatid stages; therefore only the pachytene spermatocytes
expressed both proteins. During mouse spermatogenesis, a testisspeciﬁc chaperone protein Calmegin (CLGN) is expressed in thepachytene spermatocytes to the elongating spermatids, and disappears in the mature spermatids (Yoshinaga et al., 1999). Marmoset CLGN protein ﬁrst appeared in the early spermatocytes
modestly, and then culminated at the pachytene spermatocyte
stage in accordance with the onset of MARWI expression (Fig. 3B).
The expression was also observed in the round spermatids but notin the elongating spermatids.
The expression of VASA and MARWI proteins mostly overlapped during the course of marmoset spermatogenesis as bothproteins were expressed in the pachytene spermatocytes to theround spermatids (Fig. 3C). However, in the elongating spermatids,
MARWI, but not VASA, was detected. RanBPM, a scaffold proteinthat is known to interact with VASA protein (YDBIO|6681Puverel
et al., 2011; Shibata et al., 2004), also exhibited the expression
pattern similar to VASA in adult marmoset testis. The nucleocytoplasmic expression of marmoset RanBPM was observed in
MARWI þ spermatocytes and round spermatids but not in theelongating spermatids (Fig. 3D). On the other hand, a synaptonemal complex protein SYCP1 and a haploid germ cell-speciﬁc RING
ﬁnger protein HAPRIN (YDBIO|6681Kitamura et al., 2005;
Kitamura et al., 2003) displayed more restricted expression. Like
cynomolgus monkeys and humans (YDBIO|6681Pousette et al.,
1997; Yamauchi et al., 2009), marmoset SYCP1 was preferentiallyexpressed in pachytene spermatocytes (Fig. 3E). HAPRIN was
detected in the acrosomal region of spermatids following theinitiation of spermiogenesis (Fig. 3F).

Meiosis-associated nuclear structures of marmoset germ cells during
spermatogenesis
Meiotic male germ cells can be characterized by their uniquechromosome structures, such as the XY body (de Vries et al., 2012)and synaptonemal complex assembly (Page and Hawley, 2004), atmeiosis prophase I. The former is a chromatin domain composed ofcondensed sex chromosomes while the latter is involved in pairing
homologous chromosomes. These structures can be marked byphosphorylated histone γH2AX and SYCP1 proteins respectively,
and are organized predominantly in the pachytene spermatocytesof mice (YDBIO|6681Mahadevaiah et al., 2001; Monesi, 1965) andhumans (YDBIO|6681de Vries et al., 2012; Pousette et al., 1997).
Similarly, in marmoset testis, the dot-shaped single staining ofγH2AX was detectable in the nuclei of DAZL þ VASA þ pachytene
spermatocytes while it was widely diffuse in the nuclei at earlier
stages (Fig. 4A and B). The cells with a pronounced γH2AX focus also
expressed SYCP1 protein (Fig. 4C and D). Thus, during marmoset
spermatogenesis the nuclear localization of γH2AX and SYCP1proteins clearly identiﬁed the formation of the XY body andsynaptonemal complex in pachytene spermatocytes, but not in theearly spermatocytes or the round spermatids.
Juvenile marmoset testis contains the unique germ cells expressing
meiosis-associated genes
One of the unique features of primate reproduction is theexistence of long pre-pubertal and pubertal periods before reaching sexual maturity (YDBIO|6681Chemes, 2001; Kelnar et al., 2002;
Rey et al., 1993). At the juvenile phase in marmosets, we found aunique group of germ cells located in the luminal region ofseminiferous tubules (Lin et al., 2012). Historically, these cells
had been identiﬁed as ‘pre-spermatogonia’ that exhibit the morphological features corresponding to those of human gonocytes

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MARWI

VASA

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MARWI

RanBPM

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MARWI

SYCP1

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MARWI

HAPRIN

Merge

Fig. 3. Stage-speciﬁc expression of spermatogenesis-related proteins in adult marmoset testis. Confocal images of adult marmoset testis sections co-stained with antibodies
against spermatogenesis-related proteins, including (A) anti-MARWI and anti-DAZL antibodies, (B) anti-MARWI and anti-CLGN antibodies, (C) anti-MARWI and anti-VASA
antibodies, (D) anti-MARWI and anti-RanBPM antibodies, (E) anti-MARWI and anti-SYCP1 antibodies, and (F) anti-MARWI and anti-HAPRIN antibodies. Nuclei werecounterstained with Hoechst 33342. Scale bar, 50 μm.

Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

γH2AX

DAZL

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γH2AX

VASA

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γH2AX

SYCP1

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γH2AX

SYCP1

Merge

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Fig. 4. Meiosis-associated formation of XY body and synaptonemal complex in the adult spermatocytes. Confocal images of adult marmoset testis sections co-stained with
antibodies against germ cell and meiotic nuclear markers, including (A) anti-γH2AX and anti-DAZL antibodies, (B) anti-γH2AX and anti-VASA antibodies, and (C, D) antiγH2AX and anti-SYCP1 antibodies. (D) A higher magniﬁcation of (C) clearly displays the formation of the XY body and synaptonemal complex in the nuclei of pachytene
spermatocytes. Arrowheads indicate the XY body with conﬁned sublocalization of γH2AX protein. In the image of (A), an arrow indicates DAZL þ γH2AX  spermatogonia.
Nuclei were counterstained with Hoechst 33342. Scale bar, 50 μm.

but are consistently degenerated in the central part of seminiferous tubules (Li et al., 2005). Therefore, we next moved on tocharacterize the juvenile marmoset testis, particularly focusing on
the pre-spermatogonia. According to our previous study, DAZL and
VASA proteins were expressed in the pre-spermatogonia in 10month juvenile marmoset testes whereas spermatogonia adjacentto the basement membrane expressed DAZL but not VASA protein(Fig. 5A and B). Surprisingly, the pre-spermatogonia also expressed
MARWI protein, which in the adult was observed in the pachytene
spermatocytes and onward (Fig. 3). Neither quiescent (SALL4) nor
dividing spermatogonia (Ki67) markers were expressed in the
MARWI þ pre-spermatogonia (Fig. 5C and D). Nevertheless, wesometimes found the pre-spermatogonia expressing SALL4 and
C-KIT proteins (Fig. 5E) but they were negative for MARWI protein(Fig. 5C). These observations implied that the pre-spermatogonia
might be a cell population that escapes from the normal commitment to a spermatogonial cell fate.
Then, we examined the expression of other meiosis-associatedgenes in the pre-spermatogonia. The MARWI þ pre-spermatogonia
indeed expressed CLGN and RanBPM proteins (Fig. 6A and B), but thesubcellular localization of RanBPM protein was conﬁned to thecytoplasm unlike the nucleocytoplasmic localization observed in adults(Fig. 3D). We further investigated the meiotic nuclear structures basedon the expression of γH2AX and SYCP1 proteins. Again, γH2AX waspresent in the VASA þ pre-spermatogonia (Fig. 6C–E). The nuclear
distribution was primarily punctate (Fig. 6C) and only a few prespermatogonia exhibited the formation of an XY body (Fig. 6D). This
was also the case in another juvenile marmoset testis with a slightly

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C-KIT

Merge

Fig. 5. Premature expression of MARWI protein in the pre-spermatogonia of juvenile marmosets. Confocal images of juvenile marmoset testis sections co-stained with(A) anti-MARWI and anti-DAZL antibodies, (B) anti-MARWI and anti-VASA antibodies, (C) anti-MARWI and anti-SALL4 antibodies, (D) anti-MARWI and anti-Ki67 antibodies,and (E) anti-SALL4 and anti-C-KIT antibodies. (C) SALL4 protein was observed in some pre-spermatogonia as well as spermatogonia population present on the basement
membrane. The SALL4 þ pre-spermatogonia were negative for MARWI (arrow) and vice versa (arrowhead). (E) A few pre-spermatogonia expressed both SALL4 and C-KIT
proteins (arrow), while, in spermatogonia, these proteins were expressed in exclusive cell populations. Nuclei were counterstained with Hoechst 33342. Scale bar, 50 μm.

Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

CLGN

Merge

MARWI

RanBPM

Merge

γH2AX

VASA

Merge

γH2AX

VASA

Merge

γH2AX

VASA

Merge

MARWI

51

A

Fig. 6. Expression of meiotic markers in the pre-spermatogonia of juvenile marmosets. Confocal images of juvenile marmoset testis sections co-stained with antibodies
against meiosis-associated proteins, including (A) anti-MARWI and anti-CLGN antibodies, (B) anti-MARWI and anti-RanBPM antibodies, and (C–E) anti-γH2AX and anti-VASA
antibodies. (D) In a few seminiferous tubules, the VASA þ pre-spermatogonia exhibited a γH2AX signal in the form of the XY body (arrowhead). (E) In the slightly mature
juvenile testis, very few pre-spermatogonia remained, expressing VASA protein and γH2AX with the punctate nuclear distribution (arrow). The newly formed spermatocytes
also expressed VASA protein and γH2AX but in the form of the XY body (arrowhead). Nuclei were counterstained with Hoechst 33342. Scale bar, 50 μm.

52Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

mature appearance, in which the number of the pre-spermatogonia
had decreased (Fig. 6E). In the nuclei of the pre-spermatogonia, SYCP1protein was also detectable in the form of a synaptonemal complex(Fig. 7A and B) but the abundance seemed much lower comparedwith adult spermatocytes (Fig. 4C and D). Additionally, in the juvenile
testis of another marmoset with a slightly mature appearance, thesynaptonemal complex seemed to be deformed in association withabnormal cellular and nuclear morphologies (Fig. 7C and D).

The pre-spermatogonia in juvenile marmoset testis undergo
apoptosis
Given the observation of the degrading nuclear structure and cellmorphology, we assumed that the pre-spermatogonia in juvenile
testis might result in apoptosis. We therefore investigated apoptosis
markers in the pre-spermatogonia, and found that activated Caspase3(Said et al., 2004) was present in the pre-spermatogonia (Fig. 8A) andbecame predominant according to testicular development (Fig. 8B).
The activated caspase3 was also observed in the spermatocytes
derived from spermatogonia (Fig. 8B). We subsequently examinedthe cleavage form of PARP1, a target protein of activated Caspase3 in

MARWI

SYCP1

Merge

MARWI

SYCP1

Merge

MARWI

SYCP1

Merge

MARWI

SYCP1

Merge

Fig. 7. Degeneration of synaptonemal complex in the pre-spermatogonia of juvenile marmosets. Confocal images of juvenile marmoset testis sections co-stained with antiMARWI and anti-SYCP1 antibodies. (A, B) Immature juvenile testis. (B) A higher magniﬁcation of (A). Synaptonemal complex-like structures could be seen in the nucleus of
the MARWI þ pre-spermatogonia (arrowhead) although the signal intensity was weaker than that observed in adult spermatocytes. (C, D) Juvenile marmoset testis withslightly mature appearance. (D) A higher magniﬁcation of (C). Some pre-spermatogonia formed synaptonemal complexes (arrowhead) while the others exhibited
degenerated structures (arrow). Nuclei were counterstained with Hoechst 33342. Scale bar, 50 μm.

Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

MARWI

Activated Caspase 3

Merge

MARWI

Activated Caspase 3

Merge

MARWI

Cleavage PARP1

Merge

MARWI

Cleavage PARP1

Merge

53

Fig. 8. Apoptotic markers in the pre-spermatogonia of juvenile marmosets. Confocal images of juvenile marmoset testis sections co-stained with antibodies against germ celland apoptotic markers, including (A, B) anti-MARWI and anti-activated Caspase3 antibodies, and (C, D) anti-MARWI and anti-cleavage PARP1 antibodies. (A) In immature
juvenile testis, a few pre-spermatogonia exhibited an activated Caspase3 signal (arrowhead). (B) Almost all of the pre-spermatogonia became positive for activated Caspase3
in juvenile testis with a slightly mature appearance. (C) In the immature juvenile testes, cleavage PARP1 was present in very few pre-spermatogonia. (D) In the juvenile testis
with a slightly mature appearance, cleavage PARP1 was present in almost all of the pre-spermatogonia. Nuclei were counterstained with Hoechst 33342. Scale bar, 50 μm.

an apoptotic pathway (YDBIO|6681Agarwal et al., 2009; D’Amours etal., 2001). A small number of pre-spermatogonia were positive forcleavage PARP1 in the less mature juvenile testis, similar to the case ofactivated caspase3 (Fig. 8C). In contrast, in the juvenile testis with aslightly mature appearance, cleavage PARP1 was frequently observedin the pre-spermatogonia but not in the spermatocytes (Fig. 8D). These
data demonstrated that the pre-spermatogonia expressing meiosis-

associated genes would be destined to undergo apoptotic cell deatheventually.

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Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

SALL4

DAZL

Merge

VASA

SALL4

Merge

SALL4

C-KIT

Merge

PLZF

SALL4

Merge

SALL4

Ki67

Merge

VASA

Ki67

Merge

Fig. 9. Expression of germ cell markers in the neonatal gonocytes. Confocal images of neonatal marmoset testis sections co-stained with antibodies against germ cellmarkers, including (A) anti-SALL4 and anti-DAZL antibodies, (B) anti-VASA and anti-SALL4 antibodies, (C) anti-SALL4 and anti-C-KIT antibodies, (D) anti-PLZF and anti-SALL4
antibodies, (E) anti-SALL4 and anti-Ki67 antibodies, and (F) anti-VASA and anti-Ki67 antibodies. Nuclei were counterstained with Hoechst 33342. Scale bar, 50 μm.

Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

The gonocyte origin of the pre-spermatogonia in juvenile marmoset
testis
To identify whether the pre-spermatogonia in the juvenile
testis originate from gonocytes or spermatogonia, we characterized
the molecular signature of gonocytes in neonatal marmoset testis.
Our previous study revealed that DAZL and VASA proteins wereexpressed in the marmoset gonocytes (Lin et al., 2012), similar tothe pre-spermatogonia in juveniles. We found that the DAZL þVASA þ gonocytes also expressed SALL4 protein (Fig. 9A and B)
unlike the quiescent spermatogonia expressing SALL4 and DAZL
but not VASA protein (YDBIO|6681Figs. 1 and 3) (Lin et al., 2012).
Furthermore, in contrast to the spermatogonia, SALL4 and C-KIT
proteins were co-expressed in the gonocytes (Fig. 9C) whereas thequiescent spermatogonia marker PLZF was absent (Fig. 9D), as is
the case in mice (Hobbs et al., 2012). In addition, the SALL4 þ VASA þ gonocytes were Ki67-negative (Fig. 9E and F) as theneonatal gonocytes are mitotically arrested (YDBIO|6681Culty,
2009, 2013). Taken together, gene expression of gonocytes wasvery similar to that of the MARWI-negative population of prespermatogonia in juveniles (Fig. 5C and E). Considering the present
results, the pre-spermatogonia most likely came from the gonocytes, but not spermatogonia, left over in the luminal region ofseminiferous tubules during postnatal testicular development, andeventually disappear by apoptosis via a meiosis-like event.

Discussion
Developmental stages of primate spermatogenesis have been
deﬁned by classical cellular and nuclear morphologies and testis
organization (YDBIO|6681Chemes, 2001; Dreef et al., 2007;
Haruyama et al., 2012; Jackson and Edmunds, 1984; Millar etal., 2000; Schlatt and Ehmcke, 2014; Wistuba et al., 2003),
while, in mice, the identiﬁcation of numerous stage-speciﬁcmarker genes has enabled each developmental process to be
identiﬁed at a molecular level. By contrast only a limited
number of molecular markers are available for primates. To
develop our understanding of primate reproductive biology, it isfundamental and indispensable to expand molecular insights ofprimate spermatogenesis. In this study, we carried out sequential immunoﬂuorescence analyses during postnatal testiculardevelopment in the common marmoset to depict a basic
molecular atlas of spermatogenesis. Taken all together, weformulated the scenario of gene expression dynamics in germ
cells during marmoset testicular development (Fig. 10). We
believe that this insight could be essential for understanding
primate germ cell development and cell culturing (YDBIO|
6681Imamura et al., 2014, 2012).
According to Clermont's histological classiﬁcation, primate
undifferentiated spermatogonia are deﬁned as two subtypes,
mitotically quiescent (reserve) Adark and actively dividing (renewing) Apale spermatogonia (YDBIO|6681Clermont and Antar, 1973;
Dym et al., 2009; Ehmcke and Schlatt, 2006; Millar et al., 2000). In
the adult marmoset testis, we clearly identiﬁed two major
mutually exclusive populations of spermatogonia, SALL4 þ PLZF þ LIN28 þ
DPPA4 þ DAZL þ , and C-KIT þ Ki67 þ DAZL þ (YDBIO|6681Figs. 1, 10,
and Supplementary Fig. S1). Considering that Ki67 is an indicatorformitotic
division,itisconceivable
that
SALL4 þ PLZF þ LIN28 þ DPPA4 þ
DAZL þ quiescent cells would represent Adark spermatogonia while
C-KIT þ Ki67 þ DAZL þ active cells would represent Apale spermatogonia, as deﬁned by differential BrdU (YDBIO|6681Ehmcke et al.,2005a, 2005bSimorangkir et al., 2009) or 3H-thymidine incorporation (Clermont, 1969). This scenario ﬁts with the ﬁnding of human55

spermatogonia that FGFR3 þ UTF1 þ Adark and C-KIT þ Ki67 þ Apale
spermatogonia can be detected in a mutually exclusive manner(von Kopylow et al., 2012). However, the relationship between the
Adark/Apale classiﬁcation and our molecular discrimination should
be carefully considered because the only faithful biological basis of
the Adark/Apale classiﬁcation is due to their mitotic activity. Classical discrimination of spermatogonial subtypes by hematoxylin
staining largely relies on researchers' proﬁciency, hence, is methodologically inadequate for accurate validation and data comparison among laboratories (Ehmcke and Schlatt, 2006). The
conclusion can differ depending on the protocols, equipment,
and even observers from each experiment. Of course, there is no
doubt that this classical classiﬁcation provided an informative
guideline to advance developmental biology of the primate spermatogenesis. However, this strategy has become insufﬁcient tofulﬁll the current diagnostic criteria in the late-breaking stem cellbiology for specifying a particular cell type. We determined thespermatogonial subtypes with easily accessible but speciﬁc immunoﬂuorescence by focusing on their mitotic activity. This brings
out unequivocal, reliable identiﬁcation of spermatogonia in thecommon marmoset, leading to further molecular dissections.
On the other hand, the molecular character of differentiated B
spermatogonia remains to be determined. In the current study,
we found that C-KIT þ spermatogonia are comprised of Ki67 þ andKi67  cell subpopulations (Fig. 1B and Supplementary Fig. S2A).
Furthermore, a part of C-KIT þ spermatogonia exhibits thenucleus-diffuse, punctate γH2AX staining, as seen in the prepachytene spermatocytes (Supplementary Fig. S2B). Given that
γH2AX protein is predominantly enriched in spermatocytes (Fig. 4)
and the nucleus-diffuse γH2AX protein is also present in mouse B
spermatogonia (Hamer et al., 2003), C-KIT þ Ki67  DAZL þ γH2AX þ
spermatogonia might represent marmoset B spermatogonia.
At the onset of spermatogenesis, it seems that gene expression
undergoes the sequential processes as follows (Fig. 10). The earlyspermatocytes derived from spermatogonia can be recognized as
DAZL þ γH2AX þ CLGNweak þ cells, in which the γH2AX protein is
widely distributed in the whole nucleus in a punctate manner.
At the pachytene stage, spermatocytes become DAZL þ γH2AX þ
CLGN þ VASA þ RanBPM þ SYCP1 þ MARWI þ . In their nuclei, the γH2AX
protein becomes conﬁned to the XY body while SYCP1 protein
localizes at the synaptonemal complexes. Then, round spermatids
lose DAZL, γH2AX, and SYCP1 proteins but still express CLGN, VASA,RanBPM, and MARWI proteins. Finally, MARWI þ HAPRIN þ elongating
spermatids and spermatozoa appear. Thus, we describe the basic
scheme of gene expression dynamics during marmoset spermatogenesis, but the information is still limited to a subset of genes. Nevertheless greater exploration of primate spermatogenesis is needed.
In addition to adult spermatogenesis, we also investigated gene
expression dynamics in the sexually immature period before
puberty. In the neonatal testis, gonocytes express SALL4 and CKIT as well as DAZL and VASA proteins (Fig. 9) (Lin et al., 2012).
During juvenile development, the gonocytes migrate to the basal
membrane (McKinnell et al., 2013) where they form the SALL4 þ or
C-KIT þ spermatogonia. It is uncertain whether both typesof spermatogonia are directly derived from the gonocytes.
Considering gene expression in the adult spermatogonia, SALL4 þ C-KIT þ DAZL þ VASA þ gonocytes would turn to SALL4 þ DAZL þ
spermatogonia while losing C-KIT and VASA expression. The
SALL4 þ DAZL þ spermatogonia would subsequently give rise to CKIT þ DAZL þ spermatogonia.
On the other hand, some DAZL þ VASA þ germ cells remain in
the lumen of the juvenile seminiferous tubule (Fig. 5) (Lin et al.,2012). Previously, two types of germ cells could be observed in
prepubertal marmoset testis; one in a central tubular position andthe other at the basement membrane (Li et al., 2005). The identityof the centrally located germ cells, named pre-spermatogonia, had

56

Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

Juvenile

Neonate
C-KIT
NANOG
OCT4
VASA

CLGN

SALL4

C-KIT

DAZL
RanBPM
MARWI

Luminal

VASA

germ cells

DAZL
SALL4

Ki67

Ki67
SYCP1
Apoptosis(Caspase3, PARP1)

γH2AX

Gonocytes

γH2AX (XY body)

Spermatogonia

Adult
LIN28

γH2AX

Spermatocytes

RanBPM
VASA
C-KIT

Spermatids

Ki67

Lumen

SALL4

HAPRIN

DAZL

MARWI

PLZF

SYCP1

Spermatogonia

CLGN

γH2AX (XY body)
DPPA4
Adult spermatogenesis

Cell types

SALL4 PLZF LIN28 DPPA4 Ki67 C-KIT DAZL γH2AX CLGN VASA RanBPM SYCP1 MARWI HAPRIN

Quiescent sg
Active sg
Early sc

WN

Pachytene sc

XY

Round sd
Elongated sd
Fig. 10. A scheme of gene expression dynamics in the marmoset spermatogenesis. Schematic representation of spermatogenic development from neonate to adult in thecommon marmoset. In the seminiferous cords of neonatal testis, gonocytes express SALL4, C-KIT, DAZL, and VASA proteins. Pluripotency factors OCT4 and NANOG can be also
observed in some of the gonocytes (Lin et al., 2012). Ki67 is present in non-germ cells, possibly Sertoli cell precursors. In the juvenile period, gonocytes relocated to thebasement membrane turn to the member of the spermatogonial population. However, some gonocytes are left in the luminal region of seminiferous tubules. They lose thegonocyte gene expression and exhibit the spermatocyte-like expression, including DAZL, γH2AX, CLGN, VASA, RanBPM (cytoplasmic), SYCP1, MARWI proteins as well as an
apoptosis marker (activated Caspase3, cleavage PARP1). In adult testis, SALL4 þ PLZF þ LIN28 þ DPPA4 þ DAZL þ quiescent spermatogonia (Quiescent sg) and
C-KIT þ Ki67 þ DAZL þ active spermatogonia (Active sg) can be seen on the basement membrane. Upon differentiation, early spermatocytes (early sc) are recognized as
DAZL þ γH2AX þ CLGNweak þ cells. At this stage, the distribution of γH2AX protein is in the whole nucleus (WN). Then, pachytene spermatocytes (pachytene sc) turn to be
DAZL þ γH2AX þ CLGN þ VASA þ RanBPM þ SYCP1 þ MARWI þ . The γH2AX protein is conﬁned to the XY body (XY). Round spermatids (round sd) still express CLGN, VASA,RanBPM, and MARWI proteins. Finally, MARWI þ HAPRIN þ elongating and elongated spermatids (elongated sd) and spermatozoa appear.

not been elucidated, but it turned out that these cells exhibited
meiosis-associated gene expression. Nevertheless, the meiotic
process seemed irregular, and the pre-spermatogonia result in
apoptosis. In general, during development, germ cells should be
recruited to the appropriate niches. If primordial germ cells are
misdirected from the normal path, they undergo apoptosis (Tres etal., 2004). Likewise, migration failure to the basement membrane
induces apoptosis in mouse gonocytes (YDBIO|6681Orth et al.,
2000; Tres and Kierszenbaum, 2005). Taking this into account, we
speculate that a part of the marmoset gonocytes might fail tomigrate to the basement membrane and be eliminated by apoptosis through a meiosis-like process in the juvenile period. This
scenario is also supported by cytoplasmic localization of RanBPM
protein observed in pre-spermatogonia, which indicates the activation of an apoptotic pathway (Atabakhsh et al., 2009). However,
we can still not exclude another possibility that the prespermatogonia might be abnormal spermatocytes derived fromthe start-up spermatogonia. In this assumption, the prespermatogonia (spermatocytes) might be located ectopically inthe luminal region of the seminiferous tubules because of

Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

immaturity of testicular niche and hormonal regulation (Li et al.,2005), which cannot support meiosis properly. In order to determine the origin of the pre-spermatogonia, further comprehensive,
serial dissection of gene expression dynamics focusing on the
neonatal-juvenile period is needed.
Following testicular development, juvenile spermatogonia start
producing spermatocytes (YDBIO|6681Figs. 6 and 7), most ofwhich are positive for activated Caspase3 (Fig. 8). Since the ﬁrst
wave of spermatogenesis mobilized after birth is accompanied bymassive apoptosis of spermatocytes in rodents (Rodriguez et al.,1997), this could also occur in the common marmoset. On theother hand, unlike the pre-spermatogonia, cleavage PARP1 was notobserved in the spermatocytes (Fig. 8). This could be explained by
the different time course of apoptosis between pre-spermatogonia
and spermatocytes. The pre-spermatogonia appeared earlier than
the spermatocytes did during testicular development. Indeed,
many activated caspase3- and cleavage PARP1-negative pre-spermatogonia existed in the less mature juvenile testis, in which thespermatocytes did not appear (Fig. 8). As the cleavage of PARP1 is
implemented by the activated caspase3 (YDBIO|6681Agarwal et al.,2009; D’Amours et al., 2001; Said et al., 2004), the spermatocytes
may become positive for cleavage PARP1 in later development.
In conclusion, we investigated molecular signatures of germ
cells during postnatal testicular development in the common
marmoset, which deﬁned gene expression involved with developmental transitions. This also clariﬁed, at least partly, thecharacteristics of primate germ cells, especially in the juvenile
period. We expect that this fundamental ontogeny promotes
better understanding of the primate germ cells and reproduction
thereafter.

Acknowledgments
We thank Orie Hikabe, Kimie Ohta, Kazumichi M. Nishida,
Miharu K. Kamatani, and Hirotaka James Okano for technicalassistance, scientiﬁc discussions, and other support. This work
was supported by Grants from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (MEXT); the Ministry of
Health, Labour and Welfare; the Japan Society for the Promotion of
Science (JSPS); the National Institute of Biomedical Innovation; theStrategic Research Program for Brain Sciences and Leading Projectfor Realization of Regenerative Medicine, MEXT; the Project forRealization of Regenerative Medicine, MEXT; the Funding Program
for World-Leading Innovative R&D in Science and Technology(FIRST); a Keio University Grant-in-Aid for the Encouragement ofYoung Medical Scientists; Leave a Nest Grant Life Technologies
Japan Award; Interuniversity Bio-Backup Project for Basic Biology;
and Otsuka Toshimi Foundation.

Appendix A. Supplementary materials
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ydbio.2015.01.014.
References
Aeckerle, N., Eildermann, K., Drummer, C., Ehmcke, J., Schweyer, S., Lerchl, A.,
Bergmann, M., Kliesch, S., Gromoll, J., Schlatt, S., Behr, R., 2012. The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial
stem cells? Mol. Hum. Reprod. 18, 477–488.
Agarwal, A., Mahfouz, R.Z., Sharma, R.K., Sarkar, O., Mangrola, D., Mathur, P.P., 2009.
Potential biological role of poly (ADP-ribose) polymerase (PARP) in malegametes. Reprod. Biol. Endocrinol. 7, 143.
Albert, S., Ehmcke, J., Wistuba, J., Eildermann, K., Behr, R., Schlatt, S., Gromoll, J.,
2010. Germ cell dynamics in the testis of the postnatal common marmosetmonkey (Callithrix jacchus). Reproduction 140, 733–742.

57

Atabakhsh, E., Bryce, D.M., Lefebvre, K.J., Schild-Poulter, C., 2009. RanBPM hasproapoptotic activities that regulate cell death pathways in response to DNA
damage. Mol. Cancer Res. 7, 1962–1972.
Chemes, H.E., 2001. Infancy is not a quiescent period of testicular development. Int.
J. Androl. 24, 2–7.
Clermont, Y., 1963. The cycle of the seminiferous epithelium in man. Am. J. Anat.
112, 35–51.
Clermont, Y., 1966. Renewal of spermatogonia in man. Am. J. Anat. 118, 509–524.
Clermont, Y., 1969. Two classes of spermatogonial stem cells in the monkey(Cercopithecus aethiops). Am. J. Anat. 126, 57–71.
Clermont, Y., 1972. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol. Rev. 52, 198–236.
Clermont, Y., Antar, M., 1973. Duration of the cycle of the seminiferous epithelium
and the spermatogonial renewal in the monkey Macaca arctoides. Am. J. Anat.
136, 153–165.
Culty, M., 2009. Gonocytes, the forgotten cells of the germ cell lineage. Birth Defects
Res. C Embryo Today 87, 1–26.
Culty, M., 2013. Gonocytes, from the ﬁfties to the present: is there a reason tochange the name? Biol. Reprod. 89, 46.
D’Amours, D., Sallmann, F.R., Dixit, V.M., Poirier, G.G., 2001. Gain-of-function of poly
(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications
for apoptosis. J. Cell Sci. 114, 3771–3778.
de Vries, M., Vosters, S., Merkx, G., D’Hauwers, K., Wansink, D.G., Ramos, L., de Boer,P., 2012. Human male meiotic sex chromosome inactivation. PLoS One 7, e31485.
Deng, W., Lin, H., 2002. miwi, a murine homolog of piwi, encodes a cytoplasmic
protein essential for spermatogenesis. Dev. Cell 2, 819–830.
Dreef, H.C., Van Esch, E., De Rijk, E.P., 2007. Spermatogenesis in the cynomolgus
monkey (Macaca fascicularis): a practical guide for routine morphologicalstaging. Toxicol. Pathol. 35, 395–404.
Dym, M., Kokkinaki, M., He, Z., 2009. Spermatogonial stem cells: mouse and human
comparisons. Birth Defects Res. C Embryo Today 87, 27–34.
Ehmcke, J., Luetjens, C.M., Schlatt, S., 2005a. Clonal organization of proliferating
spermatogonial stem cells in adult males of two species of non-humanprimates, Macaca mulatta and Callithrix jacchus. Biol. Reprod. 72, 293–300.
Ehmcke, J., Schlatt, S., 2006. A revised model for spermatogonial expansion in man:lessons from non-human primates. Reproduction 132, 673–680.
Ehmcke, J., Simorangkir, D.R., Schlatt, S., 2005b. Identiﬁcation of the starting point
for spermatogenesis and characterization of the testicular stem cell in adultmale rhesus monkeys. Hum. Reprod. 20, 1185–1193.
Ehmcke, J., Wistuba, J., Schlatt, S., 2006. Spermatogonial stem cells: questions,models and perspectives. Hum. Reprod. Update 12, 275–282.
Eildermann, K., Aeckerle, N., Debowski, K., Godmann, M., Christiansen, H.,
Heistermann, M., Schweyer, S., Bergmann, M., Kliesch, S., Gromoll, J., Ehmcke,J., Schlatt, S., Behr, R., 2012. Developmental expression of the pluripotency
factor sal-like protein 4 in the monkey, human and mouse testis: restriction topremeiotic germ cells. Cells Tissues Organs 196, 206–220.
Gassei, K., Orwig, K.E., 2013. SALL4 expression in gonocytes and spermatogonialclones of postnatal mouse testes. PLoS One 8, e53976.
Gerdes, J., Lemke, H., Baisch, H., Wacker, H.H., Schwab, U., Stein, H., 1984. Cell cycle
analysis of a cell proliferation-associated human nuclear antigen deﬁned by the
monoclonal antibody Ki-67. J. Immunol. 133, 1710–1715.
Hamer, G., Roepers-Gajadien, H.L., van Duyn-Goedhart, A., Gademan, I.S., Kal, H.B.,
van Buul, P.P., de Rooij, D.G., 2003. DNA double-strand breaks and gammaH2AX signaling in the testis. Biol. Reprod. 68, 628–634.
Haruyama, E., Suda, M., Ayukawa, Y., Kamura, K., Mizutamari, M., Ooshima, Y.,
Tanimoto, A., 2012. Testicular development in cynomolgus monkeys. Toxicol.
Pathol. 40, 935–942.
Hermann, B.P., Sukhwani, M., Hansel, M.C., Orwig, K.E., 2010. Spermatogonial stem
cells in higher primates: are there differences from those in rodents? Reproduction 139, 479–493.
Hermann, B.P., Sukhwani, M., Lin, C.C., Sheng, Y., Tomko, J., Rodriguez, M., Shuttleworth, J.J., McFarland, D., Hobbs, R.M., Pandolﬁ, P.P., Schatten, G.P., Orwig, K.E.,
2007. Characterization, cryopreservation, and ablation of spermatogonial stem
cells in adult rhesus macaques. Stem Cells 25, 2330–2338.
Hermann, B.P., Sukhwani, M., Simorangkir, D.R., Chu, T., Plant, T.M., Orwig, K.E.,
2009. Molecular dissection of the male germ cell lineage identiﬁes
putative spermatogonial stem cells in rhesus macaques. Hum. Reprod. 24,
1704–1716.
Hermo, L., Pelletier, R.M., Cyr, D.G., Smith, C.E., 2010. Surﬁng the wave, cycle, life
history, and genes/proteins expressed by testicular germ cells. Part 1: background to spermatogenesis, spermatogonia, and spermatocytes. Microsc. Res.
Tech. 73, 241–278.
Hirano, T., Iwasaki, Y.W., Lin, Z.Y., Imamura, M., Seki, N.M., Sasaki, E., Saito, K.,
Okano, H., Siomi, M.C., Siomi, H., 2014. Small RNA proﬁling and characterizationof piRNA clusters in the adult testes of the common marmoset, a modelprimate. RNA.
Hobbs, R.M., Fagoonee, S., Papa, A., Webster, K., Altruda, F., Nishinakamura, R., Chai, L.,
Pandolﬁ, P.P., 2012. Functional antagonism between Sall4 and Plzf deﬁnes
germline progenitors. Cell Stem Cell 10, 284–298.
Imamura, M., Hikabe, O., Lin, Z.Y., Okano, H., 2014. Generation of germ cells in vitro
in the era of induced pluripotent stem cells. Mol. Reprod. Dev. 81, 2–19.
Imamura, M., Miura, K., Iwabuchi, K., Ichisaka, T., Nakagawa, M., Lee, J., KanatsuShinohara, M., Shinohara, T., Yamanaka, S., 2006. Transcriptional repression andDNA hypermethylation of a small set of ES cell marker genes in male germline
stem cells. BMC Dev. Biol. 6, 34.

58

Z.Y.-C. Lin et al. / Developmental Biology 400 (2015) 43–58

Imamura, M., Okuno, H., Tomioka, I., Kawamura, Y., Lin, Z.Y., Nakajima, R.,
Akamatsu, W., Okano, H.J., Matsuzaki, Y., Sasaki, E., Okano, H., 2012. Derivationof induced pluripotent stem cells by retroviral gene transduction in mammalian species. Methods Mol. Biol. 925, 21–48.
Jackson, M.R., Edmunds, J.G., 1984. Morphological assessment of testicular maturity
in marmosets (Callithrix jacchus). Lab. Anim. 18, 173–178.
Kelnar, C.J., McKinnell, C., Walker, M., Morris, K.D., Wallace, W.H., Saunders, P.T.,
Fraser, H.M., Sharpe, R.M., 2002. Testicular changes during infantile ‘quiescence’in the marmoset and their gonadotrophin dependence: a model for investigating susceptibility of the prepubertal human testis to cancer therapy? Hum.
Reprod. 17, 1367–1378.
Kishi, N., Sato, K., Sasaki, E., Okano, H., 2014. Common marmoset as a new model
animal for neuroscience research and genome editing technology. Dev. GrowthDiffer. 56, 53–62.
Kitamura, K., Nishimura, H., Nishimune, Y., Tanaka, H., 2005. Identiﬁcation ofhuman HAPRIN potentially involved in the acrosome reaction. J. Androl. 26,
511–518.
Kitamura, K., Tanaka, H., Nishimune, Y., 2003. Haprin, a novel haploid germ cellspeciﬁc RING ﬁnger protein involved in the acrosome reaction. J. Biol. Chem.
278, 44417–44423.
Kotaja, N., Sassone-Corsi, P., 2007. The chromatoid body: a germ-cell-speciﬁc
RNA-processing centre. Nat. Rev. Mol. Cell Biol. 8, 85–90.
Kuramochi-Miyagawa, S., Kimura, T., Ijiri, T.W., Isobe, T., Asada, N., Fujita, Y., Ikawa, M.,
Iwai, N., Okabe, M., Deng, W., Lin, H., Matsuda, Y., Nakano, T., 2004. Mili, amammalian member of piwi family gene, is essential for spermatogenesis.
Development 131, 839–849.
Li, L.H., Donald, J.M., Golub, M.S., 2005. Review on testicular development,
structure, function, and regulation in common marmoset. Birth Defects Res. B
Dev. Reprod. Toxicol. 74, 450–469.
Lin, Z.Y., Imamura, M., Sano, C., Nakajima, R., Suzuki, T., Yamadera, R., Takehara, Y.,
Okano, H.J., Sasaki, E., Okano, H., 2012. Molecular signatures to deﬁnespermatogenic cells in common marmoset (Callithrix jacchus). Reproduction
143, 597–609.
Mahadevaiah, S.K., Turner, J.M., Baudat, F., Rogakou, E.P., de Boer, P., BlancoRodriguez, J., Jasin, M., Keeney, S., Bonner, W.M., Burgoyne, P.S., 2001.
Recombinational DNA double-strand breaks in mice precede synapsis. Nat.
Genet. 27, 271–276.
Maldonado-Saldivia, J., van den Bergen, J., Krouskos, M., Gilchrist, M., Lee, C., Li, R.,
Sinclair, A.H., Surani, M.A., Western, P.S., 2007. Dppa2 and Dppa4 are closely
linked SAP motif genes restricted to pluripotent cells and the germ line. StemCells 25, 19–28.
Mansﬁeld, K., 2003. Marmoset models commonly used in biomedical research.
Comp. Med. 53, 383–392.
McKinnell, C., Mitchell, R.T., Morris, K., Anderson, R.A., Kelnar, C.J., Wallace, W.H.,
Sharpe, R.M., 2013. Perinatal germ cell development and differentiation in themale marmoset (Callithrix jacchus): similarities with the human and differencesfrom the rat. Hum. Reprod. 28, 886–896.
Millar, M.R., Sharpe, R.M., Weinbauer, G.F., Fraser, H.M., Saunders, P.T., 2000.
Marmoset spermatogenesis: organizational similarities to the human. Int. J.
Androl. 23, 266–277.
Mitchell, R.T., Cowan, G., Morris, K.D., Anderson, R.A., Fraser, H.M., McKenzie, K.J.,
Wallace, W.H., Kelnar, C.J., Saunders, P.T., Sharpe, R.M., 2008. Germ celldifferentiation in the marmoset (Callithrix jacchus) during fetal and neonatal
life closely parallels that in the human. Hum. Reprod. 23, 2755–2765.
Monesi, V., 1965. Synthetic activities during spermatogenesis in the mouse RNA
and protein. Exp. Cell Res. 39, 197–224.
Okano, H., Hikishima, K., Iriki, A., Sasaki, E., 2012. The common marmoset as a novel
animal model system for biomedical and neuroscience research applications.
Semin Fetal Neonatal Med. 17, 336–340.
Orth, J.M., Jester, W.F., Li, L.H., Laslett, A.L., 2000. Gonocyte-Sertoli cell interactions
during development of the neonatal rodent testis. Curr. Top. Dev. Biol. 50,
103–124.
Page, S.L., Hawley, R.S., 2004. The genetics and molecular biology of the synaptonemal complex. Annu. Rev. Cell Dev. Biol. 20, 525–558.
Pousette, A., Leijonhufvud, P., Arver, S., Kvist, U., Pelttari, J., Hoog, C., 1997. Presenceof synaptonemal complex protein 1 transversal ﬁlament-like protein in humanprimary spermatocytes. Hum. Reprod. 12, 2414–2417.
Puverel, S., Barrick, C., Dolci, S., Coppola, V., Tessarollo, L., 2011. RanBPM is essential
for mouse spermatogenesis and oogenesis. Development 138, 2511–2521.

Rey, R.A., Campo, S.M., Bedecarras, P., Nagle, C.A., Chemes, H.E., 1993. Is infancy aquiescent period of testicular development? Histological, morphometric, andfunctional study of the seminiferous tubules of the cebus monkey from birth tothe end of puberty. J. Clin. Endocrinol. Metab. 76, 1325–1331.
Rodriguez, I., Ody, C., Araki, K., Garcia, I., Vassalli, P., 1997. An early and massive
wave of germinal cell apoptosis is required for the development of functional
spermatogenesis. EMBO J. 16, 2262–2270.
Ruggiu, M., Saunders, P.T., Cooke, H.J., 2000. Dynamic subcellular distribution of the
DAZL protein is conﬁned to primate male germ cells. J. Androl. 21, 470–477.
Said, T.M., Paasch, U., Glander, H.J., Agarwal, A., 2004. Role of caspases in maleinfertility. Hum. Reprod. Update 10, 39–51.
Sasaki, E., Suemizu, H., Shimada, A., Hanazawa, K., Oiwa, R., Kamioka, M., Tomioka, I.,
Sotomaru, Y., Hirakawa, R., Eto, T., Shiozawa, S., Maeda, T., Ito, M., Ito, R., Kito, C.,Yagihashi, C., Kawai, K., Miyoshi, H., Tanioka, Y., Tamaoki, N., Habu, S., Okano, H.,
Nomura, T., 2009. Generation of transgenic non-human primates with germline
transmission. Nature 459, 523–527.
Sasaki, T., Shiohama, A., Minoshima, S., Shimizu, N., 2003. Identiﬁcation of eight
members of the Argonaute family in the human genome. Genomics 82,
323–330.
Saunders, P.T., Gaughan, J., Saxty, B.A., Kerr, L.E., Millar, M.R., 1996. Expression ofprotamine P2 in the testis of the common marmoset and man visualized using
non-radioactive in-situ hybridization. Int. J. Androl. 19, 212–219.
Schlatt, S., Ehmcke, J., 2014. Regulation of spermatogenesis: an evolutionary
biologist's perspective. Semin. Cell Dev. Biol. 29C, 2–16.
Sharpe, R.M., Fraser, H.M., Brougham, M.F., McKinnell, C., Morris, K.D., Kelnar, C.J.,
Wallace, W.H., Walker, M., 2003. Role of the neonatal period of pituitarytesticular activity in germ cell proliferation and differentiation in the primate
testis. Hum. Reprod. 18, 2110–2117.
Shibata, N., Tsunekawa, N., Okamoto-Ito, S., Akasu, R., Tokumasu, A., Noce, T., 2004.
Mouse RanBPM is a partner gene to a germline speciﬁc RNA helicase, mousevasa homolog protein. Mol. Reprod. Dev. 67, 1–7.
Simorangkir, D.R., Marshall, G.R., Plant, T.M., 2009. A re-examination of proliferation and differentiation of type A spermatogonia in the adult rhesus monkey(Macaca mulatta). Hum. Reprod. 24, 1596–1604.
Siomi, M.C., Sato, K., Pezic, D., Aravin, A.A., 2011. PIWI-interacting small RNAs: thevanguard of genome defence. Nat. Rev. Mol. Cell Biol. 12, 246–258.
Takahashi, T., Hanazawa, K., Inoue, T., Sato, K., Sedohara, A., Okahara, J., Suemizu, H.,
Yagihashi, C., Yamamoto, M., Eto, T., Konno, Y., Okano, H., Suematsu, M., Sasaki, E.,
2014. Birth of healthy offspring following icsi in in vitro-matured common
marmoset (Callithrix jacchus) oocytes. PLoS One 9, e95560.
Tomioka, I., Takahashi, T., Shimada, A., Yoshioka, K., Sasaki, E., 2012. Birth ofcommon marmoset (Callithrix jacchus) offspring derived from in vitro-matured
oocytes in chemically deﬁned medium. Theriogenology 78, 1487–1493.
Tres, L.L., Kierszenbaum, A.L., 2005. The ADAM-integrin-tetraspanin complex infetal and postnatal testicular cords. Birth Defects Res. C Embryo Today 75,
130–141.
Tres, L.L., Rosselot, C., Kierszenbaum, A.L., 2004. Primordial germ cells: what does ittake to be alive? Mol. Reprod. Dev. 68, 1–4.
von Kopylow, K., Staege, H., Schulze, W., Will, H., Kirchhoff, C., 2012. Fibroblast
growth factor receptor 3 is highly expressed in rarely dividing human type A
spermatogonia. Histochem. Cell Biol. 138, 759–772.
von Schonfeldt, V., Krishnamurthy, H., Foppiani, L., Schlatt, S., 1999. Magnetic cellsorting is a fast and effective method of enriching viable spermatogonia from
Djungarian hamster, mouse, and marmoset monkey testes. Biol. Reprod. 61,
582–589.
Western, P.S., van den Bergen, J.A., Miles, D.C., Sinclair, A.H., 2010. Male fetal germ
cell differentiation involves complex repression of the regulatory network
controlling pluripotency. FASEB J. 24, 3026–3035.
Wistuba, J., Schrod, A., Greve, B., Hodges, J.K., Aslam, H., Weinbauer, G.F., Luetjens, C.M.,
2003. Organization of seminiferous epithelium in primates: relationship tospermatogenic efﬁciency, phylogeny, and mating system. Biol. Reprod. 69,
582–591.
Yamauchi, K., Hasegawa, K., Chuma, S., Nakatsuji, N., Suemori, H., 2009. In vitro
germ cell differentiation from cynomolgus monkey embryonic stem cells. PLoS
One 4, e5338.
Yoshinaga, K., Tanii, I., Toshimori, K., 1999. Molecular chaperone calmegin localization to the endoplasmic reticulum of meiotic and post-meiotic germ cells in themouse testis. Arch. Histol. Cytol. 62, 283–293.