Develop. Growth Differ.

(1999) 41, 463–471

cDNA cloning of a stage-specific gene expressed

during HCG-induced spermatogenesis in the
Japanese eel
Chiemi Miura,1 Takeshi Miura,1,2* Nozomi Kudo,1 Masakane Yamashita3
and Kohei Yamauchi1
1
Department of Biology, Faculty of Fisheries, Hokkaido University, Hakodate 041, 2‘Unit Process and
Combined Circuit’ PRESTO Japan Science and Technology Corporation and 3Division of Biological
Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan.

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature
Japanese eel (Anguilla japonica) testes consisting of only premitotic spermatogonia. Proliferation of spermato-
gonia, meiosis and spermiogenesis begin on 3, 12 and 18 days after HCG injection, respectively. To isolate the
genes responsible for regulating the initiation of meiosis, differential mRNA display using poly (A)+ RNA extracted
from testes of eels at different times after HCG treatment was carried out. Five cDNA clones in which expres-
sion was initiated before the onset of meiosis were obtained. Northern blot analysis showed that one clone,
which encoded activin bB subunit, was expressed in the initial phase of spermatogenesis (1–6 days after HCG
treatment), in agreement with the previous suggestion that activin B induces the initiation of spermatogenesis
in the Japanese eel. The remaining four were expressed in the testes during the following time frames:
3–18 days (two clones), 6–18 days (one clone) and 9–18 days (one clone) after HCG treatment. One of the two
clones expressed on day 3 exhibited strong expression on days 12 and 15, just at the initiation period of
meiosis. This clone was selected as a candidate gene responsible for initiating meiosis, and its full-length cDNA
isolated. The cDNA contained an open reading frame of 1571 nucleotides encoding a protein of 260 amino
acid residues, which showed high homology with the proliferating cell nuclear antigen (PCNA) of human, mouse
and Xenopus. Northern blot analysis using eel PCNA cDNA showed that a 1.6 kb transcript first appeared on
day 3 and became abundant, reaching maximum levels on days 12–15. In situ hybridization analysis revealed
that PCNA mRNA was expressed strongly in late type B spermatogonia before the sixth mitotic division. It has
already been shown that spermatogonia have a regulatory point to enter meiosis between the fifth and sixth
mitotic division. The coincidence of PCNA expression and this regulatory point suggests an involvement of
PCNA in the progression of mitotic germ cells into meiosis during HCG-induced spermatogenesis in the eel.

Key words: differential mRNA display technique, meiosis, proliferating cell nuclear antigen.

Introduction replication, is essential for producing the haploid

Gametogenesis is a complex process in which many
factors are involved. It begins with the mitotic prolifer-
ation of germ cells (oogonium and spermatogonium),
proceeds through meiosis, and results in the produc-
tion of two kinds of haploid cells (gametes), a large
cell containing nutrient reserves is necessary for early
development (the egg) and a small, flagellated cell
highly specialized in carrying male genome to the egg
(the spermatozoon). Meiosis, which consists of two
successive cell divisions following one round of DNA
*Author to whom all correspondence should be addressed at:
Department of Biology, Faculty of Fisheries, Hokkaido University,
Hakodate 041, Japan.
E-mail: takeshi@pop.fish.hokudai.ac.jp
Received 14 December 1998; revised 5 February 1999;
accepted 10 February 1999.
cells. Molecular mechanisms regulating the reinitiation
of meiosis from the first prophase, including the activ-
ation mechanisms of the cdc2/cyclin B complex (a
key regulator of meiosis as well as mitosis), have been
revealed in recent years (for review, see Masui 1992;
Nagahama et al. 1995; Taieb et al. 1997). However,
the mechanisms involved in the onset of meiosis are
still completely unknown in both female and male
gametogenesis (Gondos et al. 1996). This can be
mainly attributed to the absence of experimental
systems suitable for investigating the control mech-
anisms of the initiation of meiosis. For example, unlike
the natural arrest at the first meiotic prophase in
oogenesis, it is difficult to arrest gametogenesis just
before entry into meiosis. In addition, asynchronous
development of gametes, which is prominent especi-
ally in mammals, disturbs definite analyses of mech-
anisms controlling gametogenesis.
464 C. Miura et al.
Cultivated male Japanese eels, Anguilla japonica,
have only immature testes that do not enter the sper-
matogenic process. However, the entire process of
spermatogenesis, including meiosis, can be induced
in vivo by the injection of human chorionic gonado-
tropin (HCG), and in vitro by treatment with HCG or
11-ketotestosterone (a major androgen in the male
eel). Proliferation of spermatogonia, meiosis and
spermiogenesis in the testes occur at definite times:
3, 12 and 18 days after HCG injection, respectively
(Miura et al. 1991a,b,c, 1996). The eel testis therefore
provides an excellent experimental system for investi-
gating the control mechanisms of spermatogenesis
(see also review by Nagahama 1994).
We have recently found that in some cases HCG
injection fails to induce complete spermatogenesis
(Miura et al. 1997). Morphological observations indi-
cated that the majority of germ cells in testes of those
non-responding individuals contain late type B sper-
matogonia. In these testes, meiosis was not induced
although the proliferation of spermatogonia through
mitosis did occur. Furthermore, morphometric analy-
ses have demonstrated that the most advanced late
type B spermatogonia in these premeiotic testes have
undergone the fifth or sixth, but not the seventh mitotic
division (Miura et al. 1997). Eel spermatogonia enter
meiosis after the 10th mitotic division (Miura et al.
1991a). These results strongly suggest the presence
of a regulatory point between the fifth and sixth mitotic
division, when the germ cells of the Japanese eel are
being prepared to enter meiosis. This prompts us to
speculate that some factors are required for the cells
to cross this regulatory point and divide meiotically. To
identify the unknown factors controlling the initiation of
meiosis at the molecular level, we isolated cDNA
clones for genes expressed stage-specifically during
spermatogenesis by the differential mRNA display
technique using poly (A)+ RNA extracted from the
testes 0, 1, 3, 6, 9, 12, 15 and 18 days after HCG
treatment. We found that the proliferating cell nuclear
antigen (PCNA) was expressed strongly in late
type B spermatogonia before the sixth mitotic division,
coincident with their acquisition of meiotic compe-
tence.
Materials and Methods
Animals
Cultivated male Japanese eels (170–250 g in body-
weight) were purchased from a commercial eel sup-
plier, and kept in 500 L freshwater tanks at 23°C
without feed throughout the experimental period. Eels
received a single intramuscular injection of HCG dis-
solved in saline solution (150 mM NaCl) at a dose of
5 IU/g bodyweight. Fish were killed on 0, 1, 3, 6, 9, 12,
15 and 18 days after HCG injection and testes col-
lected for the extraction of RNA.
Differential display of mRNA
A modification of the differential mRNA display tech-
nique (Liang et al. 1992; 1993) was applied to the
study. Oligonucleotide primers were synthesized by
an Applied Biosystems DNA Synthesizer model 391
(Applied Biosystems, Norwalk, CT, USA). The primers
used in the study were four dTMX primers, dT12
(AGC)A, dT12 (AGC)G, dT12 (AGC)C, dT12 (AGC)T,
and nine short arbitrary primers, 59-TACAACGA-
GG-39, 59-TGGATTGGTC-39, 59-CTTTCTACCC-39,
59-CTGCTTGATG-39, 59-GATCTGACTG-39, 59-TGT-
CAAAGGG-39, 59-GTTGCGATCC-39, 59-CAAACGT-
CGG-39, 59-AGGTGACCGT-39. The arbitrary primers
were selected from the modified sequence list of
Bauer et al. (1993). Total RNA were prepared from
HCG-injected eel testes using ISOGEN (Nippongene,
Tokyo, Japan), and poly (A)+ RNA was purified with
Oligo(dT)-Latex beads (Oligotex-dT30; Takara, Ootu,
Japan). After reverse transcription of poly (A)+ RNA
(0.5 µg each) with the four kinds of dTMX primers, the
resulting cDNA were amplified by polymerase chain
reaction (PCR) reaction with one of the four dTMX
primers and one of the nine arbitrary primers. The
cycling parameters of PCR were as follows: one cycle
of 94°C for 30 s, 40°C for 5 min, 72°C for 5 min, and
35 cycles of 94°C for 30 s, 40°C for 2 min, 72°C for
30 s and lastly one cycle of 72°C for 5 min. The ampli-
fied cDNA were then separated on a 6% DNA
sequencing gel, recovered according to the method
of Liang et al. (1993), and reamplified using the same
primer set and PCR conditions as used in the mRNA
display. Reamplified PCR fragments were run on
1–2% agarose gel and purified using the Qiaex Kit
(Qiagen GmbH, Hilden, Germany).
Cloning and sequencing of full-length cDNA clone
Reamplified cDNA fragments were ligated into the
pCRII vector using the TA cloning kit (Invitrogen,
San Diego, CA, USA), and used as a probe for north-
ern blot analysis to detect differentially expressed
genes. Full-length cDNA clones for differentially
expressed genes were isolated by screening a cDNA
library constructed from the eel testes 15 days after
HCG injection, as follows: poly (A)+ RNA (5 µg) taken
from the testes on day 15 was reverse transcribed
to cDNA by Superscript Reverse Transcriptase II
(Gibco BRL, Gaithersburg, MD, USA). The cDNA was
 Stage-specific gene in eel spermatogenesis 465

size-fractionated on a Sepharose CL4B column with a eel PCNA (positions 317–794; cf. Fig. 3) using DIG-
lower cutoff value of 500 bp and inserted into lgt 10 labeled UTP (Boehringer, Mannheim, Germany) and
arms following addition of EcoRI adaptors which have SP6 or T7 RNA polymerase. The testicular fragments
EcoRI, BamHI, KpnI and NcoI restriction enzyme were fixed in 4% paraformaldehyde in 0.1 M phos-
cleavage sites (Amersham International Plc., Bucking- phate buffer at pH 7.4 for 18 h, embedded in paraffin,
hamshire, UK). Approximately 2 3 105 plaques were and cut into 5 µm serial sections. Deparaffinized sec-
blotted onto nylon membranes and hybridized with 1 tions were treated with proteinase K and acetylated.
3 107 c.p.m. of [a-32P]dCTP-labeled cDNA probes The sections were incubated with a hybridization
(Random Primer Plus Extension kit, NEN Research mixture of 200 mg/mL tRNA, 10 mM Tris-HCl (pH 7.4),
Products Inc., Boston, MA, USA) for 18 h at 65°C in 1 3 Denhardt’s solution, 600 Mm NaCl, 50% forma-
hybridization solution (10 3 Denhardt’s solution, 6 3 mide, 0.25% SDS and 500 ng/mL probe. After hybrid-
standard sodium citrate (SSC), 1% sodium dodecyl- ization at 50°C overnight, the sections were washed
sulfate (SDS) and 100 mg/mL herring sperm DNA).
The membranes were washed twice with 1 3 SSC/1%
(w/v) SDS at 65°C for 1 h.
The cDNA inserts were isolated from positively
hybridizing clones and digested with KpnI. The
digested fragments were then ligated into pBluescript
II KS (2) and overlapping subclones were generated
according to the stepwise deletion method (Henikoff
1984). Sequencing reactions were carried out by the
dye terminator method (a protocol supplied by
Applied Biosystems Japan) using a DNA thermal
cycler, followed by electrophoresis and analysis
using a 373 DNA Sequencer (Applied Biosystems).
Sequences were analyzed and compared using DNA-
SIS software (Hitachi, Tokyo, Japan). Similarity search
of the predicted amino acid sequence of the obtained
cDNA was carried out using the Swiss Protein
Sequence Data Bank.

Northern blot analysis

Poly (A)+ RNA (1 µg) isolated from the testes 0, 1, 3, 6,
9, 12, 15 and 18 days after HCG injection was elec-
trophoresed on a 1% (w/v) agarose denatured gel and
blotted onto nylon membranes. The membrane was
washed briefly and then baked at 80°C for 1 h. The
uniformity of sample loading and transfer were moni-
tored by staining the blotted RNA with methylene blue.
Following hybridization with 1 3 106 c.p.m. of [a-
32
P]dCTP-labeled fragment in hybridization solution at
65°C for 18 h, membranes were washed with 1 3
SSC/0.1% SDS solution at 65°C for 1 h. Finally, mem-
branes were exposed to an imaging plate for 18 h and
analyzed using a BAS 2000 Bio-Image Analyzer (Fuji
Photo Film; Fuji, Tokyo, Japan).

In situ hybridization
For in situ hybridization, a non-radioactive method
using a digoxigenin (DIG)-labeled RNA probe was
employed. Sense and antisense RNA probes were
transcribed in vitro from the 477 bp cDNA fragment of
Fig. 1. Differential display of mRNA from the testes 0, 1, 3, 6,
9, 12, 15 and 18 days after human chorionic gonadotropin (HCG)
treatment. Arrowhead indicates the fingerprint product (A30-3-
1-2) used for isolating eel proliferating cell nuclear antigen
(PCNA) cDNA.
466 C. Miura et al.
as follows: (i) with 2 3 SSC/50% formamide at 50°C for
30 min; (ii) with 2 3 SSC at 50°C for 30 min; (iii) with
RNase A treatment (5 µg/mL) at 45°C for 30 min; (iv)
twice with 2 3 SSC at 50°C for 20 min; and (v) twice
with 0.2 3 SSC at 50°C for 20 min. Hybridized DIG-
labeled probes were immunologically detected with a
Nucleic Acid Detection kit (Boehringer Mannheim) fol-
lowing the manufacturer’s instructions. The sections
were incubated with the Fab fragment of an anti-DIG-
alkaline phosphatase-conjugated antibody (Boeh-
ringer Mannheim). After the nitroblue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate color reaction,
Fig. 2. Nucleotide and predicted
amino acid sequence of eel pro-
liferating cell nuclear antigen
(PCNA). The AATAAA polyadenyl-
ation signal is underlined.
 Stage-specific gene in eel spermatogenesis
the slides were rinsed with 10 mM Tris-HCI (pH 8.0)
containing 1 mM ethylenediamine tetraacetic acid
(EDTA) then mounted.
Results
Characterization of differentially expressed mRNA
A single injection of HCG can induce complete sper-
matogenesis in immature eel testes consisting of only
premitotic spermatogonia. Proliferation of spermato-
gonia, meiosis and spermiogenesis occur on 3, 12
and 18 days after HCG injection, respectively (Miura
et al. 1991a). Each step of spermatogenesis is
thought to be regulated by several key genes, the
expression of which is controlled by HCG. To identify
such genes, we carried out differential mRNA display
using poly (A)+ RNA extracted from testes 0, 1, 3, 6, 9,
12, 15 and 18 days after HCG treatment. We obtained
28 fingerprint products of which expression changed
during spermatogenesis (Fig. 1).
After reamplification and cloning, the products were
used as probes for northern blot analysis, and we
obtained five clones expressed differentially during
spermatogenesis. One (C27-1-1-A) was expressed on
days 1–6, two (C12-12-2-9 and A30-3-1-2) on days
3–18, one (G27-15-1) on days 6–18, and one (C27-9-
1-3A) on days 9–18. Sequencing of C27-1-1-A has
shown that it was part of a cDNA encoding activin bB
subunit. The finding that the activin bB subunit was
Fig. 3. Comparison of proliferat-
ing cell nuclear antigen (PCNA)
protein sequences among eel,
human, mouse, Xenopus, Droso-
phila and Catharanthus. Dots
indicate conserved positions
throughout sequences.
467
expressed at the initial phase of spermatogenesis
(1–6 days after HCG injection) was in accord with our
previous suggestion that activin bB induces the initia-
tion of spermatogenesis in the Japanese eel (Miura et
al. 1995a,b). This result also confirms that differential
mRNA display is a powerful tool for identifying the
genes responsible for controlling spermatogenesis.
Isolation of cDNA for the gene responsible for
initiating meiosis
Among the cDNA fragments obtained in the present
study, A30-3-1–2 displayed strong expression in the
testes 9–15 days after HCG injection during which the
initiation of meiosis occurred. We therefore selected
A30-3-1-2 as a candidate gene responsible for initiat-
ing meiosis in eel spermatogenesis. Using A30-3-1-2
cDNA as a probe, we screened ∼ 2 3 105 plaques
from an eel 15 days postinjection testis cDNA library
and obtained 19 positive clones. The longest cDNA
clone was chosen and sequenced. The nucleotide
sequence contained an open reading frame of 1571
bp, starting from the first ATG codon at position 126
and terminating at a TAA stop codon at position 906,
which was predicted to encode 260 amino acid
residues (Fig. 2). The deduced amino acid sequence
showed high similarity to PCNA (Fig. 3). We therefore
concluded that the cDNA encoded eel PCNA. At the
amino acid sequence level, eel PCNA was 89, 87, 87,
66, and 64% homologous to that of human (Almendral
468 C. Miura et al.

et al. 1987; Travali et al. 1989), mouse (Shipman-

Appasamy et al. 1991), Xenopus (Leibovici et al.
1990), Drosophila (Yamaguchi et al. 1990) and the
plant Catharanthus (Kodama et al. 1991), respec-
tively.

Expression and localization of eel PCNA mRNA in

Fig. 4. Eel proliferating cell nuclear antigen (PCNA) mRNA
expression in developing testes. Northern blot analysis was per-
formed using poly (A)+ RNA extracted from testes 0, 1, 3, 6, 9,
12, 15 and 18 days after human chorionic gonadotropin (HCG)
treatment.
spermatogenesis
Using eel PCNA cDNA, northern blot analysis was
performed to evaluate the changes in PCNA tran-
scripts during spermatogenesis (Fig. 4). No signal
was detected in the testes from untreated eels or in
testes from eels collected 1 day after HCG injection,
while a transcript of ∼ 1.6 kb was detected in the
testes from eels collected 3–18 days after HCG injec-
tion. The signal was weak on day 3, became gradually
stronger, and reached a maximum on days 12–15. As
control, the same blots were reprobed with a labeled
b-actin cDNA. The level of actin transcripts was con-
stant throughout the experimental periods (data not

Fig. 5. Cellular localization of eel proliferating cell nuclear antigen (PCNA) mRNA in eel testes 18 days after human chorionic gonado-
tropin (HCG) injection by in situ hybridization. In situ hybridization was performed using digoxigenin (DIG)-labeled antisense (A,D)
and sense (B). The sections were stained with hematoxylin and eosin (C,E). Arrowheads in (E) indicate cysts of type B spermatogo-
nia expressing PCNA mRNA. GA, type A spermatogonia; GB, type B spermatogonia; C, spermatocytes; T, spermatids; Z, spermato-
zoa. Scale, 50 µm (A–C); 10 µm (D,E).
 Stage-specific gene in eel spermatogenesis
shown), confirming that the observed changes in eel
PCNA mRNA expression were not artificial.
To determine the distribution of eel PCNA mRNA
expression in testis, we performed in situ hybridization
using DIG-labeled sense and antisense RNA probes
(Fig. 5). A strong signal was detected in small cysts
consisting of late type B spermatogonia. In 200 cysts,
we counted all late type B spermatogonia and
checked for PCNA signals. The presence of about 14
late type B spermatogonia indicated 25 divisions while
about 22 indicated 26 divisions (Miura et al. 1997).
Strong PCNA signals were observed in 39 out of 39
cysts with 25 cells (100%) and in 75 out of 81 cysts
with 26 cells (93%), while no signal was detected in
the 80 cysts with more than 27 late type B spermato-
gonia. The signal was neither detectable in cysts con-
taining cells with more than 27 divisions nor in other
testicular cell types including type A, early type B,
spermatocytes, spermatids and spermatozoa and
somatic cells. Therefore, it is concluded that mRNA
expression of eel PCNA was confined to late type B
spermatogonia before the sixth mitotic division.
Discussion
When HCG is injected into cultivated Japanese eel,
complete spermatogenesis can be induced (Miura et
al. 1991a). As a first step to identify the genes that
control spermatogenesis, we started by investigating
the genes whose expression changes during the
spermatogenic process using the differential mRNA
display technique. We obtained five cDNA clones for
stage-specifically expressed genes. One of them,
expressed only in the early phase of spermatogenesis
(1–6 days after HCG injection), encoded the activin bB
subunit. This confirms our previous finding that activin
B induces the initiation of spermatogenesis in the
Japanese eel (Miura et al. 1995a,b). In addition, this
finding reinforces the usefulness of the differential dis-
play technique in identifying the genes responsible for
controlling spermatogenesis in the Japanese eel. We
have already isolated 12 cDNA clones by differential
mRNA display and by differential screening of a sub-
traction library constructed from testes of HCG-
injected and untreated eels, and tentatively named
these clones eel spermatogenesis-related substance
(eSRS) 1–12. The eSRS 1 and 9 encode activin bB
subunit and PCNA, respectively. Characterization of
the remaining cDNA clones is in progress.
In HCG-induced eel spermatogenesis, it is esti-
mated that a type A spermatogonium enters meiosis
following 10 mitotic divisions (Miura et al. 1991a).
Recently, we suggested that prior to entering meiosis,
a regulatory point exists around the fifth mitotic divi-
sion of spermatogonia, and that only late type B sper-
469
matogonia that have passed this point can enter
meiosis and proceed to the later stages of spermato-
genesis (Miura et al. 1997). It is highly likely that some
factors are required for late type B spermatogonia
to cross this regulatory point and enter meiosis.
However, the molecular nature of these factors is com-
pletely unknown. The present study has been under-
taken to identify genes responsible for the progression
of mitotic germ cells into meiosis in the Japanese eel
testis. Among the cDNA clones obtained by differen-
tial mRNA display, we focussed on the cDNA clone
with the strongest expression during the period when
the preparation to enter meiosis occurs (9–15 days
after HCG treatment). Sequencing of this cDNA has
revealed that it encodes eel PCNA. In situ hybridiza-
tion analysis has demonstrated that eel PCNA mRNA
is expressed strongly in late type B spermatogonia
that are below the sixth mitotic division. Expression
was not detected in advanced stages of late type B
spermatogonia, or in other germ cells or somatic cells
by this in situ hybridization. We found that late type B
spermatogonia numbers decrease 18 days after
injection (Miura et al. 1991a). The present results are
in agreement with the previous finding as northern
hybridization revealed that PCNA expression is lower
in the 18-day sample in comparison with the 15-day
sample. The close correlation between the timing of
PCNA expression and the preparation to enter meio-
sis suggest that PCNA is involved in the progression
of mitotic germ cells into meiosis in Japanese eel
spermatogenesis.
The presence of PCNA in male germ cells has been
demonstrated in several species. In the mouse testis,
PCNA protein is present in mitotically dividing sper-
matogonia (intermediate and type B spermatogonia)
and spermatocytes except for the preleptotene
spermatocytes (Chapman & Wolgemuth 1994), in
accordance with the activity of DNA polymerase
b responsible for DNA repair (Grippo et al. 1978). In
adult bovine seminiferous ephithelium, PCNA protein
is present in spermatogonia and primary spermato-
cytes (abundant in the nuclei of preleptotene primary
spermatocytes but scarce in diplotene spermato-
cytes), but absent in secondary spermatocytes and
spermatids (Wrobel et al. 1996). These results sug-
gest that PCNA is involved in the recombination
process via DNA excision repair. In contrast to this
suggestion, we demonstrate here that PCNA mRNA is
expressed only in late type B spermatogonia before
the sixth mitotic division, long before the entrance into
meiosis when recombination takes place (germ cells
must undergo at least four more mitotic divisions
before entering meiosis). Although we must await
information on the expression of PCNA protein during
eel spermatogenesis, the restricted expression of
470 C. Miura et al.
PCNA mRNA prior to meiosis suggests a role for
PCNA other than DNA recombination.
The PCNA is a DNA polymerase-d auxiliary protein
that enables this DNA polymerase to efficiently utilize
template/primers containing long stretches of single-
strand template (Mathews et al. 1984; Bravo et al.
1987; Prelich et al. 1987a,b; Krishna et al. 1994).
Therefore, the principal role of PCNA is in DNA repli-
cation and repair (Waga & Stillman 1994). It has also
been demonstrated, however, that PCNA directly
interacts with cell cycle regulating proteins such as
cyclins, cyclin-dependent kinases (cdk), and cdk
inhibitors (Xiong et al. 1992; Waga et al. 1994), sug-
gesting that PCNA functions not only as a construc-
tional element of the DNA replication/repair machinery
but also as an intermediary agent that links the cell
cycle to DNA replication. Our present finding that in
the Japanese eel testis, PCNA is expressed for a def-
inite period of mitotic proliferation prior to meiosis
(corresponding to the commitment period of sper-
matogonia to enter meiosis) also suggests a new role
for PCNA, namely that of involvement in the progres-
sion of mitotic germ cells into meiosis. Further studies
will clarify the actual role of PCNA during the early
stages of spermatogenesis.
Acknowledgements
We thank Mr M. N. Raif for reading the manuscript.
This research was supported by fellowships from the
Inoue Foundation for Science, a Grant-in-Aid for
Scientific Research from the Ministry of Education,
Sciences and Culture and the Ministry of Agriculture,
Forestry and Fisheries of Japan (BDP 98-IV-2-1).
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