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Keywords:

  • fish;
  • germ line;
  • medaka;
  • primordial germ cell migration;
  • vasa

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

The medaka homolog of the Drosophila vasa gene, olvas (Oryzias latipes vas) was obtained using polymerase chain reaction of medaka cDNA from the testis and ovary. The spatio-temporal expression pattern of olvas transcripts was observed by in situ hybridization on gonads and embryos. The transcripts for olvas were exclusively detected in the cytoplasm of germ cells in the testis and ovary, not in gonadal somatic cells. In the early developmental stages, each blastomere possessed the maternal transcripts of olvas, which disappeared during gastrula stages. At the late gastrula stage, specific expression of olvas was observed only in germline cells located at the posterior shield. Embryos after the hybridization were examined histologically, and the distribution and migration path of primordial germ cells (PGC) during early stages of embryonic-body formation were revealed using the olvas gene as a germline cell marker. The PGC were translocated from the posterior shield to both sides of the embryonic body via the inner embryonic body in the medaka.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

The origin and fate of germ cells, which have the unique role of providing continuity of life between generations, have been widely studied. Although it is generally accepted that primordial germ cells (PGC) segregate from somatic cells early during embryogenesis, there are only a few species in which the germ cell lineage has been clarified throughout embryogenesis. In Drosophila melanogaster, the special cytoplasm localized in the posterior region of the egg, called polar plasm, has been well investigated. Only cells containing the polar plasm can develop into progenitor cells of germ cells, pole cells ( Illmensee & Mahowald 1974). Several factors required for pole cell formation have been revealed by genetic research (reviewed in Rongo & Lehmann 1996). The vasa (vas) gene, which is known to be required for posterior segment specification and germ cell formation, is one of such genes. It encodes a DEAD-box family protein, is expressed during oogenesis, and is retained in oocytes as a maternal factor. The product of the vasa gene is localized in polar granules ( Hay et al. 1988 ; Lasko & Ashburner 1988; Liang et al. 1994 ).

In vertebrates, specific cytoplasm similar to the polar plasm in Drosophila is found only in anurans, and is designated as the germ plasm (reviewed in Ikenishi 1998). Because of the presence of the germ plasm, germline cells are distinguishable from somatic cells throughout their embryonic stages, starting at that of the fertilized egg. In mammals and birds, PGC at early developmental stages have been identified by their high alkaline phosphatase activity or by the presence of a periodic acid Schiff reaction (PAS)-positive substance in their cytoplasm. By these characteristics as germ cell markers, we cannot trace the PGC back to earlier stages than the gastrula stage.

In teleosts, a number of investigations have also been conducted regarding the origin of PGC from a morphological perspective (reviewed in Johnston 1951). The common morphological characteristics of PGC include large cell size, large nucleus, definite nuclear and cellular membranes, darkly stained nucleoli, and hyaline cytoplasm. In most fish, the cells with these characteristics were identified after gastrula stages, and as yet no consensus has been reached about the location and number of these cells in the early stages. In the medaka, Oryzias latipes, the origin and migration path of PGC have been studied by Gamo (1961) by light microscopy and by Hamaguchi (1982) using electron microscopy. Gamo (1961) identified the progenitor cells of PGC by their large size at various sites of three germ layers of the gastrula. However, Hamaguchi (1982), by applying an ultrastructural approach, could not trace back earlier than the late neurula stage. More definitive markers of the germline cells are indispensable to a further understanding of the fate of the germline in early developmental stages.

Recently, homologs or genes related to Drosophila vasa have been introduced as a specific marker of the germline cells in several vertebrates. These genes have been demonstrated to be expressed specifically in the germline cells of Xenopus ( Komiya et al. 1994 ), mice ( Fujiwara et al. 1994 ), rats ( Komiya & Tanigawa 1995), and zebrafish ( Olsen et al. 1997 ; Yoon et al. 1997 ). In zebrafish, Yoon et al. (1997) demonstrated that transcripts for the vasa homolog, vas, were localized in primordial germ cells, and that cells with the vas messenger ribonucleic acid (mRNA) could be traced back to several cells in cleavage stages. However, it is controversial whether such a specific localization of vas transcripts in the blastomeres is applicable to other teleost fish.

In the present study, we isolated a vasa-like cDNA of the medaka, olvas (Oryzias latipes vas), by polymerase chain reaction (PCR) cloning, and elucidated their expression pattern in the gonads and embryos by in situ hybridization.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Animals

Orange–red variety of the medaka (O. latipes) was used for cDNA cloning, and the Hd-rR inbred strain was used in the analysis of in situ hybridization. The fish were maintained under an artificial photoperiod of 16L:8D, in an ambient temperature of 27 ± 2°C. Spontaneously spawned eggs were collected, and incubated in an ambient temperature of 26 ± 0.5°C. Developmental stages of the embryos were determined according to criteria set out by Iwamatsu (1994).

cDNA cloning of medaka vasa homolog

Two sets of degenerated primers were designed to amplify vasa-like cDNA fragments from medaka gonads. Locations of the primers:

A1:5′-ATGGCNTG(T/C)GCNCA(G/A)ACNGG-3′;

A2:5′-CATNCG(A/G)TCNGC(T/C)GC(A/G)TCNAGNAC-3′;

B1:5′-ATGGCNTG(T/C)GCNCA(A/G)ACNG-3′;

B2:5′-(A/G)AANCCCAT(A/G)TC(T/C)AACAT-3′, are indicated by underlining in Fig. 1.

image

Figure 1. Predicted amino acid sequences of olvas. olvas encoded 617 amino acids. Eight conserved regions are indicated in black boxes. Amino acids on which the degenerate primer sets are indicated; by solid lines (primer set A) or broken lines (primer set B).

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Total RNA was extracted from the adult testis and ovary of the medaka using Isogen (Nippon Gene, Osaka, Japan). Poly(A)+ RNA was purified by Oligo(dT)-Latex beads (Oligotex-dT30; Takara Shuzo Co., Kyoto, Japan) and used to synthesize single-stranded cDNA fragments by reverse transcription using oligo-dT primers. The cDNA fragments were used as a template for reverse transcription– polymerase chain reaction (RT-PCR) to amplify vasa-like cDNA fragments with primer set A. The reaction proceeded as follows: 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and finally an additional 72°C for 10 min. Primer set B was employed for amplification of vasa-like cDNA fragments directly from the medaka testis or ovary cDNA library: 94°C for 10 min, followed by 39 cycles of 94°C for 30 s, 55°C for 1 min, 72°C for 1 min. The resultant cDNA fragments from both primer sets were cloned into a plasmid vector pGEM T-easy (Promega, Madison, WI, USA) and subjected to sequencing by an ABI model 377 DNA sequencer (Perkin-Elmer, Oak Brook, IL, USA).

We screened the medaka testis and ovary cDNA libraries to isolate the full-length cDNA using vasa-like cDNA fragments labeled with digoxigenin (DIG DNA labeling kit; Boehringer Mannheim, Mannheim, Germany) as a probe. After three rounds of screening, positive λZAPII phages were subjected to in vivo excision according to the manufacturer’s recommendation and sequenced by an ABI model 377 DNA sequencer (Perkin-Elmer, xxx).

In situ hybridization analyses

In situ hybridization on paraffin sections of young and adult gonads of the medaka was carried out according to Shimizu et al. (1997) . Sense and antisense digoxigenin-labeled RNA probes were synthesized from the clone of an olvas fragment with a DIG RNA labeling kit (Boehringer Mannheim). Developmental stages of oocytes were determined according to criteria set out by Iwamatsu et al. (1988) .

Sense and antisense digoxigenin-labeled RNA probes for whole-mount in situ hybridization were synthesized from the clone of full length of olvas with a DIG RNA labeling kit (Boehringer Mannheim). The synthesized RNA probes were hydrolyzed into fragments with an average length of 350 bases.

For whole-mount in situ hybridization, medaka embryos at various stages of development were collected. To facilitate penetration of the fixatives through the chorion, embryos were placed and rolled gently on sheets of paper towel and sand paper (no. 120). Embryos were fixed for 4 h at room temperature in 4% paraformaldehyde phosphate-buffered saline containing 0.1% Tween 20 (PBT; 0.1% Tween-20 in 0.85 × phosphate-buffered saline (PBS)). Chorions were removed from the embryos by forceps under a stereomicroscope and refixed overnight at 4°C. The fixed embryos were washed twice in PBT and dehydrated through a methanol series, stored in methanol at – 20°C until use.

The embryos were rehydrated with 50% methanol in PBT and washed with PBT. The embryos were then treated with Proteinase K in PBS for 10 min at 37°C; at 2 μg/mL for 2-cell to 10-somite stage embryos (stage 22), at 5 μg/mL for embryos older than the 10-somite stage to somite completion stage (stage 32), and at 10 μg/mL for embryos older than somite completion stage; then washed twice with PBT. The embryos were refixed for 1 h at room temperature. After two washes with PBT, they were put into the prehybridization buffer (50% formamide, Boehringer Mannheim; 0.1% Tween-20, 5 mg/mL Torula Yeast RNA type VI, Sigma Chemical Co., St Louis, MO, USA; 50 μg/mL heparin, Sigma Chemical Co., 5 × standard sodium citrate (SSC)), prehybridized for 1 h at 65°C, and hybridized overnight with digoxigenin-labeled RNA probe (approximately 1 μg/mL) in prehybridized buffer at 65°C. The embryos were washed once each with 5 × SSC for 15 min, with 2 × SSC for 15 min, and with 50% formamide in 2 × SSC for 1 h at 65°C, then three times with 2 × SSC for 10 min at 45°C. The embryos were treated with 15 μg/mL RNase (Takara Shuzo Co.) in 2 × SSC for 30 min and washed three times with 2 × SSC for 10 min, once with 50% formamide in 2 × SSC for 1 h, three times with 2 × SSC for 10 min, and twice with 0.2 × SSC for 10 min at 45°C. Following a wash with solution 1 (0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5, 0.1% Tween-20) at room temperature, the embryos were preblocked by incubating with 10% sheep serum (Sigma Chemical Co.) in solution 2 (2% blocking reagent; Boehringer Mannheim; in solution 1) for 1 h at room temperature. After the blocking, the embryos were incubated overnight at 4°C with 1/8000-diluted antidigoxigenin antibody coupled with alkaline phosphatase (Boehringer Mannheim) in solution 2 containing 10% sheep serum. Following washes with solution 1, six times for 10 min and three times for 30 min, the embryos were washed with solution 3 (0.1 M NaCl, 50 m M MgCl2, 0.1 M Tris-HCl, pH 9.5) for 10 min. Then the embryos were incubated with 20 μL/mL 4-nitroblue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl-phosphate mix (Boehringer Mannheim) in solution 3 in the dark. When the color was developed to the desired extent, the embryos were washed with PBT and refixed with fixing solution overnight at 4°C. The refixed embryos were stored at 4°C in fixing solution and then observed with a light microscope.

For the purpose of identifying cells with signals, following in situ hybridization, the embryos were dehydrated through an ethanol series, cleared with xylene and embedded in Paraplast Plus (Oxford Labware, MO, USA). Serial transverse sections were cut at a thickness of 8 μm, and served for histologic observations.

Reverse transcription–polymerase chain reaction

Oocytes within follicles at various stages ( Iwamatsu et al. 1988 ) and unfertilized eggs were dissected out from adult ovaries using forceps under a stereomicroscope, and staged embryos ( Iwamatsu 1994) were collected. Total RNA from oocytes, unfertilized eggs, and embryos was isolated using ISOGEN (Nippon Gene), and single-stranded cDNA was synthesized by ReverTra Ace (Toyobo, Osaka, Japan) using Oligo (dT)15 Primer (Promega, Madison, WI, USA). Polymerase chain reaction amplification was carried out using the primers, olvas F and RV, to amplify the olvas cDNA fragment; sequences of these primers are, olvas F:5′-CGC ATG TTG GAC ATG GGC TTC-3′ and olvas RV: 5′-GTT CTC CCG ATG CGG TGG AC-3′. The PCR programs used were 94°C for 5 min, followed by 35 cycles of 94degC for 1 min, 66degC for 1 min, 74°C for 1 min, and finally an additional 72°C for 2 min. The PCR products were separated by electrophoresis on 1% agarose gels.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Isolation of the medaka vasa homolog cDNA

We amplified vasa-like cDNA fragments with two primer sets, A and B, from ovary or testis cDNA and directly from the testis or ovary cDNA libraries. The cDNA fragments from both strategies included DNA fragments showing high similarities to vasa nucleotide sequences of other species. Using the cDNA fragments as a probe, we isolated six positive phages from the testis cDNA library and four from the ovary cDNA library. The longest clone, T9-1, which originated from the testis cDNA library, was isolated and sequenced. The T9-1 has a DNA insert 2187 bp long that is predicted to encode the 617 amino acid polypeptide ( Fig. 1). The amino acid sequence contained all eight DEAD- box motifs generally found in DEAD-box helicase proteins ( Linder et al. 1989 ), suggesting that T9-1 is a member of the DEAD-box helicase family. The similarities of conserved domain of the T9-1 polypeptide (amino acids 225–525) to that of the VASA homologs of other species are 56, 73, 73, 66, and 83% identical to the Drosophila (M23560), mouse (Q61496), rat (Q64060), Xenopus (I51235) and zebrafish (BAA22535) VASA alignments, respectively (M. Tanaka et al., unpubl. data). We designated the clone isolated in the present study as the medaka vasa homolog, olvas (O. latipes vas). We also determined the nucleotide sequence of inserts of positive phages from the ovary, and found that there was no discrepancy between the testis and the ovary cDNA at the nucleotide level.

Spatial expression pattern of olvas in gametogenesis

To define the cellular localization of olvas transcripts in the testis and the ovary, in situ hybridization on paraffin sections of these organs using sense and antisense digoxigenin-labeled RNA probes corresponding to the cDNA of the olvas fragment was performed.

We examined testes from four fish. The testis of the medaka was composed of spermatogonia and cysts that contained germ cells at various stages of spermatogenesis. Spermatogonia were located at the most peripheral region. Spermatogenesis proceeded synchronously within each cyst, and cysts containing germ cells at progressively later stages of development were located closer to the efferent duct, which was situated in the central region ( Fig. 2A). Hybridization signals were restricted in germ cells, and not detected in testicular somatic cells ( Fig. 2B). Spermatogonia and spermatocytes at early stages were strongly stained, but spermatocytes at later stages were only slightly stained. In spermatids and spermatozoa, no signals were observed ( Fig. 2B).

image

Figure 2. olvas expression in the testis (A, B) and the ovary (C, D). Hematoxylin-eosin (H&E) staining (A) and in situ hybridization with the antisense strand (B) to sections from an adult testis. In situ hybridization with the antisense strand to ovary sections from premature fish (C) and from adult fish (D). Asterisk, efferent duct. Sg, spermatogonia; Ce, spermatocytes at early stage; Cl, spermatocytes at later stage; St, spermatids; Sz, spermatozoa; Og, oogonia; Op, oocytes in pachytene; I, II, III, IV, V, VI, oocytes in diplotene at stage I, II, III, IV, V, VI, respectively. Bar, 20 μm (A–C), 100 μm (D).

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We examined three ovaries from adult fish by means of in situ hybridization. Adult ovaries are composed mainly of follicles at later stages of oogenesis, and only a small number of oogonia and early oocytes could simultaneously be observed. To observe a number of oogonia and early oocytes, in situ hybridization of two ovaries from premature fish was also performed. Hybridization signals were restricted in germ cells, and not detected in ovarian somatic cells ( Fig. 2C,D). Oogonia were weakly stained, with small patches of slightly stronger signals in their cytoplasm ( Fig. 2C). In the cytoplasm of previtellogenic oocytes, up to stage V, signals were noted ( Fig. 2C,D). Signals were not uniformly distributed in the cytoplasm of these oocytes, but small patches with strong signals were scattered in the weakly stained cytoplasm. The strongest signals were observed in oocytes at stages II and III. As oocyte growth proceeded, the signal became weaker and almost undetectable in oocytes at stages later than late stage V, the early vitellogenic phase ( Fig. 2D).

Spatial expression pattern of olvas during embryogenesis

The expression pattern of olvas during embryogenesis, from the 2-cell stage to hatching stage, was analyzed by whole-mount in situ hybridization using a digoxigenin-labeled RNA probe synthesized from the clone of the full length of olvas. Histologic analysis on the whole-mount in situ hybridization samples was performed to identify the precise location of the olvas-positive cells.

From the cleavage stage to the blastula stage, each blastomere was strongly stained ( Fig. 3A,B). We could not detect signals in the samples when the sense strand was used as a probe ( Fig. 3C). As epiboly progressed, signals became weaker ( Fig. 3D,E). The signals were evenly distributed in their cytoplasm, and their nuclei could be distinguished as rather transparent areas in these cells. The diminution of signals does not occur uniformly among all cells, and slightly strong-stained cells and weakly stained cells were randomly mixed ( Fig. 3D,E). Although the embryonic shield and germ ring of the embryo seemed to be darker than the rest of the embryo ( Fig. 3D,E), this difference is supposed to result from differences in the thickness of the cell layer, not from the strength of the signals that these cells were expressing.

image

Figure 3. Expression of olvas transcripts from cleavage to somite formation stage by whole-mount in situ hybridization. Embryos were hybridized with the sense strand (C) and the antisense strand (except C). (A–C) Top views of embryos. Embryos in E,Fa,Ga,Ha and Ia are oriented such that the anterior side is to the top and dorsal side is to the front. Following whole-mount in situ hybridization, 8 μm of transverse sections (Fb,Fc,Gb,Hb,Ib) were prepared. Arrowheads indicate the cell expressing olvas. (A) Eight-cell stage; (B,C) stage 11 (late blastula stage); (D) stage 14 (premid gastrula stage, about 35% epiboly); (E) early phase of stage 16 (late gastrula stage); (F) late phase of stage 16; (G) stage 17 (early neurula stage); (H) stage 19 (2-somite stage); (I) stage 21 (6-somite stage). yk, yolk; ek, embryonic keel; md, mesoderm; ed, endoderm; nc, neural cord; no, notochord. (Δ) Midline of the embryonic body (Fb,Fc,Gb). Bar, 50 μm (Fb,Fc), 20 μm (Gb,Hb,Ib).

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At later stages of the gastrula (stage 16), when the embryonic body becomes more clearly visible as a narrow streak, signals became barely detectable except for some stained cells ( Fig. 3Fa, arrowheads). These olvas-positive cells, scattered in the posterior one-third of the embryonic shield, were distributed on both sides of the embryonic axis, and the distance from the midline of the embryonic body to these olvas-positive cells varied. The number of these cells per embryo was estimated at approximately 10–25. Serial transverse sections through an embryo at late stage 16 showed that olvas-expressing cells were located in cells of the embryonic shield ( Fig. 3Fb,Fc). At early neurula stage (stage 17), the olvas-expressing cells were gathered near the central axis of the embryonic body ( Fig. 3Ga). Based on the histologic observation of serial sections, we did not notice any specific distribution of the olvas-positive cells for the dorso- ventral or right–left axis in the embryonic body ( Fig. 3Fb,Fc,Gb).

With the onset of somite formation, the distribution of olvas-expressing cells tended bilaterally toward a rather distant site from the central axis of the embryo ( Fig. 3Ha, Ia). At stages 18–19, most of these cells were located in the distal region of the mesoderm–endoderm area, while a small number of these cells were placed in the proximal region ( Fig. 3Hb). By the 4-somite stage (stage 20), olvas-expressing cells were lined along the anterior–posterior axis on both sides of the embryonic body ( Fig. 3Ia). None were found in the proximal region. Serial transverse sections at this stage showed that they were situated at the distal region and underneath the endodermal layer ( Fig. 3Ib). From stage 16 to 21, olvas signals in the cells became more obvious.

At the 16-somite stage (stage 24), olvas-expressing cells were located bilaterally on the ventral side ( Fig. 4Aa,Ab). The signals of olvas were very strong. Serial transverse sections at this stage showed that olvas-positive cells were situated between the lateral plate and the periblast, and located alongside the digestive tract ( Fig. 4Ac). Based on the morphology of olvas-expressing cells in histologic observation, we could distinguish these cells from the other cells by their larger cell size and round form, whose features coincide with the morphological characteristics of PGC reported by Gamo (1961) and Hamaguchi (1982). At stage 34, olvas-expressing cells became one cluster, which was located at the ventral side of the embryo ( Fig. 4Ba,Bb). Transverse sections at this stage showed that the relevant cells were situated in the coelomic epithelium between the pronephroi and the digestive tract ( Fig. 4Bc). At stage 35, olvas-expressing cells formed two rows of cells dorsolateral to the digestive tract ( Fig. 4Ca,Cb). Serial transverse sections at this stage showed that these cells were located in the newly formed gonadal anlage ( Fig. 4Cc).

image

Figure 4. Expression of olvas transcripts during segmentation stage by whole-mount in situ hybridization. Embryos were hybridized with the antisense strand. Embryos in Aa, Ba, and Ca are oriented such that the anterior side is to the left. Ab, Bb, and Cb show higher magnification of Aa (dorsal view), Ba, and Ca (ventral views), respectively. Ac,Bc,Cc are 8 μm transverse sections following whole-mount in situ hybridization. Arrowhead indicates the area expressing olvas. (A) stage 24 (16-somite stage); (B) stage 34; (C) stage 35. yk, yolk; no, notochord; nt, neural tube; lp, lateral plate; gu, gut; pn, pronephric duct. Bar, 20 μm.

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Expression of olvas in grown oocytes and early embryos

Although the signals of the olvas transcripts were undetectable in grown oocytes of the paraffin sections ( Fig. 2D), we detected the signals in whole-mount embryos at the cleavage stage ( Fig. 3A). To determine whether the maternal transcripts of olvas were present in embryos at an early developmental stage, we examined the existence of olvas mRNA in grown oocytes (stages VIII, IX), unfertilized eggs (stage X) and embryos at the 1-cell stage and stage 9 (late morula) by RT-PCR. The sites of the primers used for the RT-PCR in the olvas cDNA are shown in Fig. 5A.

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Figure 5. (A) Schematic structure of the olvas cDNA clone, and the sites of primers used for reverse transcription–polymerase chain reaction (RT-PCR). A white box indicates the coding region and black boxes indicate eight conserved motifs of DEAD-box proteins. (B) One percent agarose gel electrophoresis of the products of RT-PCR for olvas. Lanes VIII,IX,X, grown oocytes and fertilized eggs (stage X); lane 1-cell, stage 9, embryos at 1-cell or stage 9; M, molecular size markers.

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Total RNA from oocytes at stages VIII, IX, unfertilized eggs and embryos at the 1-cell stage and stage 9 were isolated, and served for the experiments of cDNA synthesis and PCR. The olvas cDNA fragments were amplified from all samples of oocytes, unfertilized eggs, and embryos ( Fig. 5B, upper panel). No specific DNA fragments were amplified when total RNA of each sample was used as a template ( Fig. 5B, lower panel).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

The vasa gene is one of the members of Drosophila maternal genes that are required for pole cell formation (reviewed in Rongo & Lehmann 1996) and encoded DEAD-box family protein of putative adenosine triphosphate (ATP)-dependent RNA helicases that are specifically expressed in germline cells ( Hay et al. 1988 ; Lasko & Ashburner 1988; Liang et al. 1994 ). The vasa-related genes have been isolated in several species of vertebrates, including Xenopus ( Komiya et al. 1994 ), mice ( Fujiwara et al. 1994 ), rats ( Komiya & Tanigawa 1995) and zebrafish ( Olsen et al. 1997 ; Yoon et al. 1997 ). In these species, germ-cell specific expression of the vasa-related genes has been reported.

In the present study, we isolated a medaka cDNA olvas, and examined the expression pattern of olvas during gametogenesis and embryogenesis. The predicted amino acid sequence of OLVAS was found to contain the eight highly conserved regions characteristic of DEAD-box family proteins ( Pause & Sonenberg 1992; Pause et al. 1993 ), suggesting that OLVAS has ATP-dependent RNA helicase activity. The alignment shows that OLVAS exhibits higher similarities in the conserved region to VASA homologs in other species than to other groups of DEAD-box family proteins, such as elF-4A in Drosophila and mouse, p68 and PL10 in mouse and zebrafish (M. Tanaka et al., unpubl. data). In addition, expression of the olvas was exclusively restricted in germline cells, from PGC to spermatogenetic/oogenetic germ cells. These results imply that olvas is a homolog of the vasa gene of Drosophila and that olvas transcripts can be used as a marker for germline cells in the medaka.

Stage-specific expression patterns of olvas in gametogenesis

In our findings, the strength of signals of olvas varied according to the stages of germ cell differentiation. In the testis samples, olvas transcripts were observed in the cytoplasm of spermatogonia and spermatocytes. The signals became weaker as the spermatogenesis proceeded, and were hardly detected in spermatids and spermatozoa. In the ovary, the signals were observed in the cytoplasm from the oogonia to the oocytes until the early vitellogenic phase (stage V); signals weakened as oocyte growth proceeded, and could scarcely be detected in the oocytes after the late phase of stage V.

In spermatogenetic cells in Xenopus, the proteins of the vasa homolog, XVLG1, were immunologically detected strongly in spermatogonia and spermatocytes and weakly in spermatids ( Komiya et al. 1994 ). In mouse, transcripts and proteins of the mouse vasa homolog, Mvh, were also localized in spermatogenetic cells from premeiotic spermatocytes to spermatids ( Fujiwara et al. 1994 ). The results presented herein agree with those results. Fujiwara et al. (1994) observed the intracellular localization of Mvh protein in spermatocytes, and pointed out the correspondence between the intercellular localization of Mvh and the chromatoid body, which is identified in mammalian spermatogenetic cells by means of electron microscopy ( Eddy 1975). The germ cells of the medaka have a similar electron-dense structure (nuage), and their morphological changes during germ cell differentiation have been previously reported ( Hamaguchi 1985, 1987). Nuages are also observed in spermatogenetic cells from spermatogonia to spermatocytes, and have been shown to be diminished in spermatids ( Hamaguchi 1987). The parallelism of the occurrence between nuages and olvas expression is interesting, and ultrastructural study on the relationship between nuages and the olvas gene is now in progress.

In the present study, the signals of the olvas were absent in well-grown oocytes of paraffin sections. By using the RT-PCR method, the existence of the olvas mRNA was shown in grown oocytes, unfertilized eggs, and embryos at the 1-cell stage and late morula stage. It means that the olvas mRNA is supplied to eggs as a maternal substance. The reason why signals in later stages of oocytes could hardly be detected is obscure, but it is likely that the volume of the oocytes increased as oogenesis progressed, and the concentration of signals in an oocyte came under the critical level to be detected by in situ hybridization on sections.

olvas mRNA is expressed in primordial germ cells

During embryogenesis, olvas transcripts were detected in each cell of the blastomere at early developmental stages. Cell-specific expression of olvas has not been recognized before the late gastrula stage (stage 16). The cells that expressed olvas from stages 20–23 were localized at the distal and underneath region of the endodermal layer, which corresponds to the distribution of PGC remarked on in the report by Hamaguchi (1982). The distribution and migration path of olvas-expressing cells after stage 24 were also in agreement with those of PGC ( Hamaguchi 1982), and histologic observation revealed that these cells were large sized and round, which are typical morphologic characteristics of PGC. From stages 16–20, the distribution and number of olvas-expressing cells changed continuously, and we can safely conclude that the olvas-expressing cells at stage 16 are PGC.

Gamo (1961) described the ‘PGC’ in embryos after the early gastrula stage (stage 14) as large-sized cells with large distinctly outlined nuclei. The detailed morphological characters of olvas-expressing cells during stages 16–20 in the present study were not revealed by the histologic observation, and it is unclear whether these cells are the same type of cells as the PGC described by Gamo (1961). Although the distribution of the olvas-expressing cells in whole-mount embryos was similar to that of the PGC observed by Gamo (1961), histologic observation revealed that olvas-expressing cells were not located in the ectoderm at stages 18–19, unlike the description by Gamo. In addition, the total numbers of PGC by Gamo (1961), which varied dramatically among embryos, were overall fewer than the numbers of olvas-expressing cells found in the present study. These results suggest that olvas-expressing cells are not entirely coincidental with the PGC observed by Gamo (1961). The PGC at early developmental stages are difficult to distinguish from other types of cells solely on the basis of their morphologic characters.

The olvas-positive PGC at stage 16 were not assembled but scattered in the deep cells, and the positions of the respective cells varied among individuals. These results suggest that the PGC differentiated at a stage earlier than stage 16, and they began to express olvas at stage 16. The regulation mechanism of olvas expression is of interest.

Migration path of PGC at the early developmental stage

Although morphologic identification of PGC before somite formation stage in the medaka has been difficult, the olvas gene demonstrated the distribution and migration path of germline cells in morphogenetic movement during the formation of the embryonic body. Schematic summary of the distributions of PGC are shown in Fig. 6. The olvas-positive cells were scattered in the posterior one-third of the embryonic shield at the late gastrula stage (stage 16), and then moved in the embryonic body at early neurula stage (stage 17). In teleosts, it is known that the embryonic body is formed by the convergence of deep cells during the gastrula stages ( Trinkaus & Erickson 1983; Trinkaus et al. 1992 ). The migration path of olvas-positive cells from the posterior of the embryonic shield into the embryonic body seems to be coincident with the deep cell movement at these stages. At stages 18–19, the embryonic keel was formed, and the neural cord and notochord were differentiated by stage 20. During these stages, somites were also formed. From these histologic observations, PGC seems to be set aside from these morphogenetic cell movements. As a result of the primordia formations, PGC could be thrust aside from the inner region of the embryonic body to the bilaterally peripheral region of the germ layer. The translocation of the olvas-positive PGC from the late gastrula stage throughout the early somite formation stage could be interpreted without any active- movement of PGC.

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Figure 6. Schematic illustration of the location of medaka primordial germ cells at late gastrula stage (stage 16); early neurula stage (stage 17); late neurula to 2-somite stage (stages 18–19); and the 4-somite stage (stage 20). Schematic drawing of olvas-expressing cells in medaka embryos as determined by whole-mount in situ hybridization and histologic observation for in situ hybridized embryos. Arrows indicate the movement of deep cells to form the embryonic body (stage 16). The dot indicates a primordial germ cell (PGC).

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It is noticeable that the distribution and migration path of PGC during these stages were somewhat different from those of vas-positive cells in zebrafish ( Yoon et al. 1997 ; Weidinger et al. 1999 ). In the medaka, the location of the olvas-positive cells changed their position from the posterior shield to both sides of the embryonic body via the inner embryonic body. During the epiboly stage, olvas or vas-positive cells both in the medaka (in the present study) and zebrafish ( Weidinger et al. 1999 ) translocated toward the embryonic axis, which seems to be the result of the convergence of deep cells to form the embryonic body. At the later epiboly stage in zebrafish, four clusters of vas-positive cells, two of which were located very close to dorsal of the embryonic axis and two others located at positions on the ventral side, all migrated toward both sides of the embryonic body from each position ( Weidinger et al. 1999 ). Some steps of the migration path of vas-positive cells in zebrafish may be unexplainable except by the active movement of these cells.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References
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