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Mechanisms of Development 99 (2000) 139±142
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Gene expression pattern
Differential expression of vasa homologue gene in the germ cells during
oogenesis and spermatogenesis in a teleost ®sh, tilapia,
Oreochromis niloticus
Tohru Kobayashi, Hiroko Kajiura-Kobayashi, Yoshitaka Nagahama*
Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
Received 17 March 2000; received in revised form 4 August 2000; accepted 1 September 2000
Abstract
Vas (a Drosophila vasa homologue) gene expression pattern in germ cells during oogenesis and spermatogenesis was examined using all
genetic females and males of a teleost ®sh, tilapia. Primordial germ cells (PGC) reach the gonadal anlagen 3 days after hatching (7 days after
fertilization), the time when the gonadal anlagen was ®rst formed. Prior to meiosis, no differences in vas RNA are observed in male and
female germ cells. In the ovary, vas is expressed strongly in oogonia to diplotene oocytes and becomes localized as patches in auxocytes and
then strong signals are uniformly distributed in the cytoplasm of previtellogenic oocytes, followed by a decrease from vitellogenic to
postvitellogenic oocytes. In the testis, vas signals are strong in spermatogonia and decrease in early primary spermatocytes. No vas RNA
expression is evident in either diplotene primary spermatocytes, secondary spermatocytes, spermatids or spermatozoa. The observed
differences in vas RNA expression suggest a differential function of vas in the regulation of meiotic progression of female and male
germ cells. q 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Germ cells; Vasa homologue (vas) RNA; Differential expression; In situ hybridization; Oogenesis; Spermatogenesis
The gene vasa encodes a DEAD (Asp-Glu-Ala-Asp)
family of putative RNA helicase and is present in both the
polar granules at the posterior end of the oocyte and the
nuage structure in the germ cells in Drosophila. It has
been shown that zygotic expression is also restricted to
the germ lineage (Lasko and Ashburner, 1988; Hay et al.,
1990, 1998; Linder et al., 1989; Ephrussi and Lehmann,
1992; Liang et al., 1994). Vasa-like homologues have
been cloned in frogs (Komiya et al., 1994), mice (Fujiwara
et al., 1994), rats (Komiya and Tanigawa, 1995) and zebra®sh (Yoon et al., 1997; Olsen et al., 1997). In these animals,
the vasa homologues were found to be expressed speci®cally in the germ line of older individuals. The localization
of vasa homologue in early embryos has been reported in
frogs (Ikenishi, 1998) and zebra®sh (Yoon et al., 1997;
Olsen et al., 1997). However, there have been no studies
in any vertebrate species describing the expression pattern
of vasa homologue genes during oogenesis and spermatogenesis. Tilapia, Oreochromis niloticus, is a gonochoristic
teleost ®sh and has been extensively used to examine
morphological gonadal sex differentiation (Nakamura et
* Corresponding author. Tel.: 181-564-55-7550; fax: 181-564-55-7556.
E-mail address: [email protected] (Y. Nagahama).
al., 1998). Using all genetic males and females of tilapia,
we examined the expression patterns of vasa homologue
genes during oogenesis and spermatogenesis.
1. Results and discussion
1.1. Isolation and characterization of tilapia vas cDNA
We obtained a clone containing a full-length open reading frame which was highly homologous to the vasa genes
previously reported in other animals. This cDNA encodes a
protein of 645 amino acids. The predicted amino acid
sequence of tilapia vasa is 70.0% identical to that of zebra®sh, 56.2% to Xenopus, 60.0% to rat, 61.2% to mouse and
48.1% to Drosophila. Furthermore, this tilapia gene shows a
higher degree of similarity to zebra®sh vasa gene (70%)
than to zebra®sh PL10 homologue (45%), another member
of the DEAD protein family (Olsen et al., 1997). From these
®ndings, we concluded that the clone we isolated encodes
the tilapia homologue of vasa and thus was designated the
tilapia vasa homologue, vas (DDBJ accession number;
AB032467).
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0925-477 3(00)00464-0
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T. Kobayashi et al. / Mechanisms of Development 99 (2000) 139±142
1.2. Expression pattern of tilapia vas during gametogenesis
RT-PCR was used to determine the tissue distribution of
tilapia vas mRNA. An ampli®ed product of tilapia vas was
seen only when testis and ovary RNAs were used. No ampli®ed products were detected in either brain, spleen, liver,
heart or kidney (data not shown).
In situ hybridization was used to determine the pattern of
tilapia vas expression in germ cells during gonadogenesis
and gametogenesis of both sexes (fry 0±100 days after
hatching, dah). All genetic females and males were used.
To identify primordial germ cells (PGC), we also stained
germ cells immunohistochemically with SGSA-1 antibody
which stains gonial type germ cells speci®cally (Kobayashi
et al., 1998). Immediately after hatching, vasa RNA was
already present, localized only in SGSA-1-positive PGC
which were distinguishable morphologically from somatic
cells by their large size and location at the outer layer of the
lateral plate mesoderm (Fig. 1a,b,d,e). PGC ®rst reached the
gonadal anlagen 3 dah and contained vas RNA (Fig. 1c,f).
During these stages, vas RNA was found only in germ cells.
In females of tilapia, oogenesis is initiated at 20±25 dah,
at which time oogonial proliferation begins to occur. Meiotic germ cells ®rst appear in ovaries of fry 25±30 dah and at
these stages auxocytes are also seen. Up to and during these
stages, strong vas RNA expression was observed throughout
the cytoplasm of oogonia and small oocytes (Fig. 2a,c). No
vas RNA was observed in nuclei of germ cells at any stages.
There were marked changes in the localization pattern of
vas RNA during the early stages of oogenesis (Fig. 3a±f).
Patches of strong vas signals were evident in the cytoplasm
of early pre-vitellogenic oocytes (Fig. 3a,b,d). As the size of
oocytes increased, the signals became much stronger, eventually occupying the entire cytoplasm (Fig. 3d). Vas RNA
signals became weaker with the progression of vitellogenesis, probably due to dispersion of RNA throughout the
enlarged oocytes (Fig. 3c). Northern blotting analysis
Fig. 2. Vas expression in fry gonads. Ovary at 45 dah stained with hematoxylin (a) or hybridized with DIG-labeled vas antisense RNA (c). Auxocytes have strong signals for vas. Testis at 35 dah stained with hematoxylin
(b) or hybridized with DIG-labeled vas antisence RNA (d). Strong vas
signals are seen in spermatogonia. BV, blood vessel; ED, intratesticular
efferent duct; OC, ovarian cavity; GC, spermatogonia. Scale bar, 20 mm.
showed that vasa RNA was present even in full-grown
oocytes, ovulated eggs and embryos at the 4-cell stage
(Fig. 4). This continuous presence of vas RNA suggests
that vas transcript can be supplied maternally.
In male tilapia, A-type spermatogonia including stem
type cells are the only germ cells present in gonads during
testicular differentiation. Active spermatogonial proliferation is not observed until 20±35 dah. At these stages, vas
RNA signals were seen in spermatogonia (Fig. 2b,d). The
initiation of spermatogenesis does not occur until 70 dah, at
which time active spermatogonial proliferation takes place.
Spermatozoa ®rst appear in testes of tilapia about 100 dah.
Male germ cells were positive for vas RNA, at stages from
spermatogonia to early primary spermatocytes (Fig. 5).
However, the signals were weaker in primary spermatocytes, compared to spermatogonia, and were absent in diplotene spermatocytes (Figs. 5b,c,e,f). No vas RNA was
observed in secondary spermatocytes, spermatids or spermatozoa (Fig. 5d).
Fig. 1. Vas expression in germ cells. Immunohistochemistry using anti-SGSA-1 antibody (a±c) and in situ hybridization using DIG-labeled antisense RNA (d±
f) were performed on larval sections ®xed at 0 dah (a,d), 2 dah (b,e) and 3 dah (c,f). Arrows indicate the vas expressing cells. PGC, primordial germ cells; LPM,
lateral plate mesoderm; G, gut; ND, nephric duct; CC, coelomic cavity. Scale bar, 20 mm.
T. Kobayashi et al. / Mechanisms of Development 99 (2000) 139±142
141
2. Experimental procedures
2.1. Animals
Tilapia, Oreochromis niloticus, were kept in re-circulating freshwater tanks with a capacity of 500 l at 268C until
use. To obtain fry, arti®cial fertilization was performed by
mixing sperm suspension and eggs. All genetic females
(XX) and males (XY) were obtained by arti®cial fertilization of eggs from a normal female (XX) and sperm from a
sex-reversed male (XX), and eggs from a normal female
(XX) and sperm from a super male (YY), respectively.
Fertilized eggs were cultured in re-circulating water at 268C.
2.2. cDNA cloning of tilapia vasa homologue and Northern
blot analysis
Fig. 3. Distribution of vas RNA during oogenesis. (a±d) Antisense probe;
(e,f) Sense probe. (a,b,d) Previtellogenic oocytes; (c,e,f) Mid-vitellogenic
oocytes. Patches of strong vas signals are evident in the cytoplasm of early
pre-vitellogenic oocytes (b, arrows). As the size of oocytes increases, the
signals become much stronger, eventually occupying the whole cytoplasm.
Signals become localized in the cortical region of mid-vitellogenic oocytes
(c, arrow heads). Og, oogonia. Sections (a) and (d) are of identical magni®cation. Sections (b,c,e,f) are the same magni®cation. Scale bar, 50 mm.
In summary, the results presented in this study clearly
showed the difference in vas RNA pattern in male and
female germ cells during gemetogenesis. These results
suggest that vas has a differential role in translational regulation during meiotic progression from zygotene to diplotene stages of both sexes. Further investigations are
necessary to determine the differences in vas function
during spermatogenesis and oogenesis.
A vasa cDNA fragment was PCR-ampli®ed with degenerate primers corresponding to the conserved amino acid
sequences of vasa MACAQTG and VLDEADRM. To
obtain a full length clone, an adult tilapia ovarian cDNA
library (Chang et al., 1997) was screened according to a
previous report (Kajiura et al., 1993). Ten micrograms of
total RNA was used for Northern blot analysis according to
a previous report (Kajiura et al., 1993).
2.3. In situ hybridization
Gonads of fry 0±10 dah were dissected attached to the
trunk and ®xed in 4% paraformaldehyde in 0.1 M phosphate
buffer (PB) (pH 7.4) (4% PFA) at 48C overnight. Testes and
ovaries from 10 to 100 dah were removed and ®xed in 4%
PFA. After ®xation, gonads were embedded into paraf®n.
Cross-sections were cut at 5 mm. Probes of sense and antisense digoxigenin (DIG)-labeled RNA strands were transcribed in vitro from linearized tilapia vas cDNA plasmid,
using an RNA labeling kit (Boehringer). In situ hybridization was performed as follows: Sections were deparaf®nized, hydrated and treated with proteinase K (Boehringer,
10 mg/ml) and then hybridized using sense or antisense
DIG-labeled RNA probe at 608C for 18±24 h. Hybridization
signals were then detected by using alkaline phosphataseconjugated anti-DIG antibody (Boehringer) and NBT as the
chromogen.
Acknowledgements
Fig. 4. Northern blot analysis of vas RNA during oogenesis and after
fertilization. LV, late vitellogenic oocytes (full grown oocytes); 4-cells,
4-cell stage after fertilization.
This work was supported in part by Grant-in-Aid for
Research for the Future (JSPS-RFTF 96 L00401) from the
Japan Society for the Promotion of Science, and Bio Design
from the Ministry of Agriculture, Forestry and Fisheries,
Japan to Y.N. and Grant-in-Aid for Research from the
Ministry of Education, Science, Sports and Culture of
Japan to T.K. (09740619).
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T. Kobayashi et al. / Mechanisms of Development 99 (2000) 139±142
Fig. 5. Vas expression during spermatogenesis. Testis at 100 dah. (a,b,d) In situ hybridization; (c,e,f) hematoxylin staining. Sections (e,f) are high magni®cation
of section (c). Strong signals for vas are seen in germ cells from A-type spermatogonia to primary spermatocytes but not from secondary spermatocytes to
spermatozoa. In section d, vas RNA positive and negative primary spermatocytes are seen. Section (b) is adjacent to section c. Note a marked decrease in vas
transcripts in primary spermatocytes with the progression of meiosis. Large arrows, vas-expressing primary spermatocytes (pachytene spermatocytes). Small
arrows, non-vas expressing primary spermatocytes (pachytene to diplotene spermatoytes). ED, intratesticular efferent duct; a±g, A-type spermatogonia; b±g, Btype spermatogonia; SC, primary spermatocytes; St, spermatids; Lc, Leydig cells. Scale bar, 50 mm (a±c); 20 mm (d±f).
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