Animals

Six-month-old juveniles of P. depressus, hatched and reared at the Fukuoka Prefectural Fish Farming Center, were transferred to the Coastal Station of the National Research Institute of Aquaculture, maintained in 1,000-liter tanks supplied with sand-filtered sea water at 30 l/min, and reared on kelp, Eisenia bicyclis. Two- to three-year-old immature adults were used in the study.

Isolation of RNA

After removing the peristomial membrane, coelomic fluid was collected with Pasteur pipettes and centrifuged at 600 g for 5 min to obtain the coelomocytes. The esophagus, stomach, intestine, rectum and gonad were excised and their total RNA was isolated with the TRIZOL reagent (GIBCO BRL, USA) according to the manufacturer's instructions. Small specimens of gonads were fixed in Bouin's solution for histological observation. Paraffin sections 6 μm thick were prepared and stained with haematoxylin and eosin.

Reverse transcription-polymerase chain reaction (RT-PCR)

Reverse transcription was carried out by the Superscript II preamplification system for first strand cDNA (GIBCO BRL, USA) using the oligo(dT) primer according to the manufacturer's instructions. The primers used for PCR amplification are listed in Table 1. Primers F1 and R3 were used for fragment 2, F2 and R4 for fragment 3 (Fig. 1). Thirty cycles of amplification were carried out under the following conditions: 95°C for 0.5 min, 50°C for 0.5 min, and 72°C for 1 (fragment 2) and 3 (fragment 3) min. Fragment 2 was subcloned into the pCR II plasmid with the TA cloning kit (Invitrogen, USA) and used as a template to generate the digoxigenin-labeled DNA probe. The nucleotide sequence of the fragments was determined with the BigDye Terminator Cycle Sequencing FS Ready Reaction kit and the ABI PRISM 377 DNA sequencer (PE Biosystems, USA). For the RT-PCR analysis of Vg mRNA expression, primers F1′ and R3′ were used. Thirty cycles of amplification were carried out under the following conditions: 95°C for 0.5 min, 60°C for 0.5 min, and 72°C for 1 min.

Table 1

Sequences of the primers

Fig. 1

Cloning and sequencing strategy for Vg cDNA of P. depressus. Primers used and PCR fragments obtained are shown. Numbers in the parentheses indicate their base pair positions.

5′RACE and 3′RACE

Rapid amplification of the 5′cDNA and 3′cDNA ends was carried out by the 5′ RACE and 3′ RACE system for rapid amplification of cDNA ends (GIBCO BRL, USA) according to the manufacturer's instructions. In 5′RACE, primers R3, R2 and R1 were used for reverse transcription, the first PCR and the nested PCR, respectively. Thirty cycles of amplification were carried out under the following conditions: 95°C for 0.5 min, 60°C for 0.5 min and 72°C for 1 min. In 3′RACE, primers F3 and F4 were used for the first PCR and the nested PCR, respectively. Thirty cycles of amplification were carried out under the following conditions: 95°C for 0.5 min, 62°C for 0.5 min and 72°C for 3 min. The nucleotide sequence of the fragments was determined as described above.

Isolation and amino acid sequence analysis of MYP

MYP used for amino acid sequence analysis was purified from the immature gonad according to the purification method for MYP from the egg (Unuma et al.,1998) with some slight modifications. One gram of the immature gonad was homogenized with 15 ml of 10 mM Tris-HCl buffer containing 10 mM NaCl (pH 8.0; TBS) using Polytron (Kinematica, Switzerland). The homogenate was centrifuged at 25,000 g for 20 min at 4°C, and the supernatant was applied to a HiLoad 16/10 Q Sepharose Fast Flow column (Amersham Pharmacia Biotech, England) equilibrated with TBS. After washing with 60 ml of the same buffer, the retained proteins were eluted with a NaCl linear gradient from 10 mM to 1 M (300 ml in total) using FPLC (Amersham Pharmacia Biotech) at a flow rate of 3.0 ml/min. The protein eluted with 240 mM NaCl was collected, concentrated threefold using Molcut LGC (Millipore Corp., USA), and applied to a HiLoad 16/60 Superdex 200 column (Amersham Pharmacia Biotech) equilibrated with 10 mM TrisHCl buffer containing 150 mM NaCl (pH 8.0). The proteins were eluted with the same buffer at a flow rate of 1.0 ml/min. The MYP eluted at an elution volume of 50 ml was collected, incubated with 8M urea for 2 h at 37°C, and then digested with lysyl endopeptidase (Wako Pure Chemical Industries, Ltd., Japan) in 0.1M N-ethylmorpholine (pH 9.0) at 37°C overnight. The digests were then separated by reverse-phase HPLC using a Cosmosil 5C18 AR-II column (2.0×150 mm; Nacalai Tesque, Japan) with a 60 min linear gradient of 0–60% acetonitrile in 0.05% trifluoroacetic acid (TFA) at a flow rate of 0.2 ml/min. The amino acid sequence of the peptide fragment eluted at 31.1 min was determined by the G1005A Protein-Sequencing system (Hewlett-Packard, USA) in the pulse-liquid mode.

Northern blot analysis

The digoxigenin-labeled cDNA probe, 415 bp, was prepared using the PCR DIG probe synthesis kit (Loche Diagnostic, Germany) with primers F1′ and R3′. Thirty cycles of amplification were carried out under the following conditions: 95°C for 0.5 min, 60°C for 0.5 min, and 72°C for 1 min. The total RNA (5 μg) extracted was electrophoresed on a 1% formaldehyde-agarose gel and blotted onto nylon membranes. The blotted membrane was hybridized with the probe dissolved in Easy Hyb (Loche Diagnostic) at 60°C for 20 hr and washed with 0.1×SSC/0.1%SDS at 65°C for 40 min. The hybridized probe was visualized with the DIG nucleic acid detection kit (Loche Diagnostic) according to the manufacturer's instructions.

In situ hybridization

In situ hybridization of the immature gonad was done with the In situ hybridization starting kit (Nippon Gene Co., Ltd., Japan) and oligonucleotide probes. Four sense and four antisense oligonucleotide probes (60 bp each) were synthesized and labeled with digoxigenin using the DIG-tailing kit (Loche Diagnostic) according to the manufacturer's instructions. The positions of the oligonucleotide probes are shown in Fig. 2. The immature gonad was excised and fixed with 4% paraformaldehyde in PBS (10 mM phosphate buffer, 0.1M NaCl, pH 7.4). Tissues were dehydrated in ethanol, embedded in paraffin, and cut into 6-μm-thick sections. After removing the paraffin, the sections were digested with proteinase K (2 μg/ml) in PBS for 4 min, and then acetylated with 0.1 M triethanolamine (pH 8.0) containing 0.25% (v/v) acetic anhydride for 10 min. The four antisense or sense oligonucleotide probes (0.025 μg/ml each, 0.1 μg/ml total) were mixed with the hybridization buffer (50% formamide, 2×SSC, 1 μg/μl tRNA, 1 μg/μl salmon sperm DNA, 1 μg/μl BSA, and 10% dextran sulfate). After 30 min of prehybridization in 50% formamide/2×SSC at 42°C, hybridization was performed for 16 hr at 42°C. The slides were washed with 50% formamide/2×SSC for 60 min at 42°C and then with 0.1×SSC for 120 min at 42°C. The hybridized probes were visualized with the DIG nucleic acid detection kit (Loche Diagnostic) according to the manufacturer's instructions.

Fig. 2

Nucleotide and deduced amino acid sequences of P. depressus Vg cDNA. Numbers on the right margin indicate nucleotide positions from the first nucleotide. Amino acid numbers from the first methionine are indicated on the left margin. The termination signal of translation is indicated by an asterisk. The underlined region of the deduced amino acid sequence has about 25% homology to the vertebrate transferrin family. Double underline represents the result of amino acid sequence analysis. Arrows represent primers F1′ and R3′, which were used for the synthesis of the DIG-labeled DNA probe and for the analysis of tissue expression by RT-PCR. The underlined nucleotide sequences represent the position of DIG-labeled oligonucleotide probes used for in situ hybridization.

Isolation of the cDNA encoding Vg

In a preliminary Northern blot analysis with the S. purpuratus Vg probe (Shyu et al., 1986) provided by Dr. Blumenthal, Vg mRNA expression was confirmed in the immature gonad of P. depressus. Thus the Vg cDNA was isolated from the immature gonad.

The strategy for cloning Vg cDNA is shown in Fig. 1. As a first step, a set of degenerate primers (F1 and R3) was designed based on the S. purpuratus Vg cDNA (Shyu et al. 1987), then RT-PCR was conducted with these primers and the total RNA from the immature gonad as a template. A PCR fragment of the expected size (416bp; fragment 2) was isolated, subcloned and sequenced. The nucleotide sequence showed 85% homology to the corresponding region of S. purpuratus Vg.

To obtain information on the internal amino acid sequence, MYP was purified from the immature gonad, digested with lysyl endopeptidase, and the amino acid sequence of the peptide fragment was analysed. A degenerate primer (R4) was designed based on the sequence, LSNSYPNFDAVR (Fig. 2), then RT-PCR was conducted with this primer and another primer (F2) which was designed based on the nucleotide sequence of fragment 2. A PCR product of 2066 bp (fragment 3) was isolated and sequenced.

Primers F3, F4, R1, R2 and R3′ were designed based on the nucleotide sequences of fragments 2 and 3 and used in conducting 5′RACE and 3′RACE. PCR products of 430 bp (fragment 1) and 2508 bp (fragment 4) were isolated and sequenced.

Characterization of cDNA encoding Vg

The nucleotide and deduced amino acid sequences of P. depressus Vg cDNA are shown in Fig. 2. The cDNA had an open reading frame that encodes a 1349-amino-acid protein with a predicted molecular mass of 153.6 kDa. The prediction of protein sorting signals (PSORT) program ( http://psort.ims.utokyo.ac.jp) predicted the first 15 amino acids as a signal sequence of the protein. Twenty-one potential N-linked glycosylation sites were found in the deduced amino acid sequence (positions not shown). The amino acid sequence was compared with that of other proteins in the database by FASTA ( http://fasta.genome.jp), and P. depressus Vg was found to have little homology to Vg of other animals other than that to S. purpuratus Vg; instead, it showed about 25% homology in its 132- to 510- amino-acid region to the vertebrate transferrin family.

Distribution of Vg mRNA.

Total RNA was extracted from the esophagus, stomach, intestine, rectum, immature gonad and coelomocytes of the immature adult P. depressus, and the tissue specific expression of Vg mRNA in these organs was analysed by Northern blotting (Fig. 3). Vg mRNA was detected in the gonad, stomach, intestine and rectum, but not in the esophagus or coelomocytes. The immature gonad showed the most intense signal. The probe hybridized to a single band of approximately 5 kb in each tissue. The size of the mRNA was in agreement with that of the Vg cDNA obtained in this study.

Fig. 3

Northern blot analysis of tissue-specific Vg mRNA expression. Total RNA (5 μg/lane) extracted from the esophagus, stomach, intestine, rectum, gonad, and coelomocytes of immature P. depressus was electrophoresed, blotted onto a nylon membrane, and probed with digoxigenin-labeled Vg cDNA which correspond to nucleotides 100–514. The positions of the four molecular weight markers (10, 6, 4, 3 kb) are indicated on the left margin. Ribosomal RNA visualized with ethidium bromide is shown as a loading control in the lower panel.

The Vg mRNA expression not detected in the esophagus or coelomocytes by Northern blotting was revealed by RT-PCR using primers F1′ and R3′ (Fig. 4). The PCR products of the expected size (416 bp), detected in both the esophagus and coelomocytes as well as in the immature gonad, were not derived from genomic DNA contamination since no product was detected when reverse transcriptase was excluded from the RT-PCR protocol (- RT).

Fig. 4

RT-PCR analysis of Vg mRNA expression. Total RNA extracted from the esophagus, coelomocytes, and gonad was reverse transcribed and amplified by PCR using primer F1′ and R3′. The products were not derived from the genomic DNA contamination since no product was detected when reverse transcriptase was excluded from the RT-PCR protocol (-RT).

A comparison of the Vg mRNA expression in female and male gonads and the histological features of the immature ovary (Fig. 5A) and testis (Fig. 5B) are shown in Fig. 5. The gonadal lumina of both sexes were filled with nutritive phagocytes; in the ovary, small oocytes were observed at the periphery of the acini; in the testis, however, male germ cells were difficult to detect by light microscopy. Instead, testicular nutritive phagocytes often contained haematoxylin-stained dark-blue amorphous speckles derived from phagocytized residual spermatozoa. The immature gonads with these characteristics were sexed, and their Vg mRNA expression was compared by Northern blot analysis using two individuals of each sex (Fig. 5C). Similar amount of transcripts was detected in both the immature ovaries and testes.

Fig. 5

Histological features of the immature ovary and testis, and their Vg mRNA expression by Northern blot analysis. (A) The immature ovary stained with haematoxylin and eosin. Gonadal lumina are filled with nutritive phagocytes. Small oocytes (a) are observed at the periphery of the acini. (B) The immature testis stained with haematoxylin and eosin. Gonadal lumina are filled with nutritive phagocytes, as in the ovary. Male germ cells are not observed. Nutritive phagocytes contain dark-blue amorphous speckles (b), derived from phagocytized residual spermatozoa. The bar represents 100 μm. (C) Total RNA (5 μg/lane) extracted from the immature gonads was subjected to Northern blot analysis as described in Fig. 3. Two individuals of each sex were analysed.

In situ hybridization was carried out in the immature ovary and testis using antisense and sense oligonucleotide probes. Hybridization with antisense probes in the immature ovary revealed Vg mRNA signal in nutritive phgocytes (Fig. 6A); hybridization with a sense control probe produced no significant signal (Fig. 6B). In the immature testis, a Vg mRNA signal was also detected in nutritive phagocytes, as in the ovary (data not shown).

Fig. 6

In situ hybridization of Vg mRNA. (A) Antisense oligonucleotide probes were hybridized with a section of the immature ovary; nutritive phagocytes expressed Vg mRNA. (C) Sense oligonucleotide probes were hybridized with a section. No significant signal is observed. The bar represents 100 μm.