Animals

Mature and immature female kuruma prawn, M. japonicus, were obtained from the Momoshima Station of the Japan Sea-Farming Association in Hiroshima Prefecture, Japan. Collected ovaries were immediately frozen in a liquid nitrogen and stored at −80°C until analysis.

Extraction and purification of CRP

A mature ovary from a kuruma prawn of body weight 98.1 g and gonadosomatic index (GSI) of 5.71 was homogenized in 20 mM Tris-HCI buffer, pH 8.7, containing 150 mM NaCI and 2 mM phenylmethylsulfonylfluoride using a Handy Sonic homogenizer UR-20P (Tomy Seiko, Tokyo, Japan) in the ice box. The homogenate was centrifuged at 10,000×g for 10 min at 4°C. The precipitate was extracted using the same buffer and centrifuged at 10,000×g for 10 min at 4°C. The supernatant was then subjected to gel filtration on a Sephadex G-75 column (2.5×75 cm; Bio-Rad, Hercules, CA, USA) using 0.05 M CH₃COONH₄ buffer, pH 7.0 at a flow rate of 23.8 ml/h. Fractions of 4.5 ml were collected and the absorbance at 280 nm of each fraction was measured. The second peak fractions were pooled and separated by high-performance liquid chromatography (HPLC; Hitachi, Tokyo, Japan) on a reversed-phase C18 column (Wakosil-II 5C 18HG, column size 0.46×25 cm) at 40°C at a flow rate of 1ml/min. Linear gradient elution was performed with acetonitrile containing 0.1% trifluoroacetic acid. Elution was monitored at 220 nm.

Electrophoresis

Fractions purified by gel filtration and HPLC were separated by SDS-PAGE under reducing (containing 2-mercaptoethanol) and non-reducing conditions using 15–25% and 4–20% SDS-PAGE gel (Daiichi Pure Chemicals, Tokyo, Japan) according to Laemmli (1970). Gels were stained with 0.1% Coomassie Brilliant Blue R250 (CBB) according to manufacturer's protocol. Six markers of known molecular weight, i.e., myosin (200 kDa), β-galactosidase (116 kDa), bovine serum albumin (66 kDa), aldolase (42 kDa), carbonic anhydrase (30 kDa) and myosin (17 kDa) were purchased from Daiichi Pure Chemicals.

Preparation of antiserum against the 28.6 kDa molecule

The 28.6 kDa molecule purified on HPLC was used as an antigen in the preparation of antiserum in rabbits. The antigen was emulsified with Freund's complete adjuvant and was injected into several points on the back of the animals once every two weeks. Blood was collected from the carotid artery of anesthetized animals 1 week after the last booster injection. Antiserum was stored at −80°C until analysis.

Histology and immunohistochemistry

Ovarian tissues (GSI 5.71) were fixed with Bouin's fixative for 24 hr at room temperature, dehydrated through an alcohol series, and embedded in paraplast (Oxford Labware, St. Louis, MO, USA) or paraffin according to conventional histological procedures. Hematoxylin-eosin (HE) staining and periodic acid-Schiff (PAS) reaction was performed after sectioning (4 μm).

Immunohistochemistry was performed by the avidin-biotin immunostaining method (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA). Sections (4 μm) were deparaffinized, rehydrated and rinsed in distilled water, and then incubated with 0.3% H₂O₂ in phosphate buffered saline (PBS) for 30 min at room temperature to deplete endogenous peroxidase activity. After 3 washes in phosphate buffered saline with Tween 20 (PBST:10 mM NaH₂PO₄·2H₂O, Na₂HPO₄·12H₂O, 0.9% NaCI, 0.05% Tween 20, pH 7.2), sections were blocked for 30 min with 10% normal goat serum solution in PBS. After washing, sections were incubated with rabbit antiserum raised against the 28.6 kDa molecule (1:50–1000) or Vt (1:1000) overnight at 37°C. After 3 washes, sections were then incubated with biotinylated goat antirabbit IgG (1:1000) for 1 hr at room temperature. Following another 3 washes, sections were lastly incubated with avidin-biotinylated horseradish peroxidase complex (1:1000) for 30 min at room temperature. After washing, substrate solution (0.025% 3,3′-diaminobenzidine (DAB; Sigma, St. Louis, MO, USA), 0.006% H₂O₂, in PBS, pH 7.2) was added, and development was conducted for 2 to 5 min at room temperature. Unless otherwise stated, PBST was used as a dilution buffer and in all washing steps. Sections were observed under a light microscope.

Western blotting analysis

Western blotting analysis was performed according to Towbin et al (1979). After SDS-PAGE, the proteins were transferred onto polyvinylidene-difluoride (PVDF) membranes (Immobilon Transfer membrane; Millipore, Billerica, MA, USA). Membranes were blocked with Block Ace (Yukijirushi Nyugyou, Tokyo, Japan) at room temperature for 30 min. Membranes were incubated with rabbit antisera raised against the 28.6 kDa molecule (1:10,000) or Vt of kuruma prawn (1:100,000) overnight at 4°C. Subsequently, membranes were incubated with biotinylated goat antirabbit IgG (1:5000)(Vector Laboratories) and avidin-biotinylated horseradish peroxidase complex (1:1000)(Vectastain ABC kit) for 30 min, respectively. PBST was used for dilutions and each washing. Finally, 0.1 M Tris-HCI buffer, pH 7.5 containing substrate solution (0.05% 3, 3′-DAB (Wako, Osaka, Japan) and 0.01% H₂O₂) was added to visualize immunoreaction on the membrane.

Analysis of carbohydrates

The 28.6 kDa and 30.5 kDa molecules purified by HPLC were subjected to SDS-PAGE, transferred to PVDF membranes and incubated with biotinylated concanavalin A complex (Con A) (1:1000) (Vector Laboratories) overnight. The subsequent procedures were carried out in the same manner as described above.

Partial amino acid sequence analysis

Fractions purified by HPLC were electroblotted onto PVDF membranes after separation by SDS-PAGE, and stained with CBB. The amino-terminal amino acid sequences of the 28.6 kDa and 30.5 kDa proteins were determined using a protein sequencer (Perkin-Elmer Applied Biosystems, Model 476A, USA) according to the methods of Matsudaira (1987).

Isolation of a DNA fragment encoding the N-terminal amino acid sequence of the 28.6 kDa and 30.5 kDa molecules

Total RNA was isolated from mature ovary using ISOGEN (Nippongene, Toyama, Japan) according to manufacturers protocol. First-strand cDNA was synthesized in a 20 μl solution containing 1 μg of total RNA, 10 nmol Oligo (dT), 375 μM dNTP, 10 mM dithiothreitol, 20 U of RNase inhibitor (Ribonuclease inhibitor, Human Placenta, Nippongene) and Superscript^TM II RNase H^– reverse transcriptase (Invitrogen, Carlsbad, CA, USA) at 50°C for 1 hr. The first-strand cDNA reaction was used as a template for the following polymerase chain reaction (PCR) protocol. Three degenerate primers, CRP-F01, F02 and R01 (Table 1) were designed based on the N-terminal amino acid sequence of the 28.6 kDa and 30.5 kDa molecules. PCR was carried out in a 20 μl solution containing 1 μl of cDNA, 0.25 mM dNTP (Takara, Shiga, Japan), 1×PCR buffer (Takara), 0.5 μM of degenerate primers (Table 1) and 0.5 U Taq DNA polymerase (Takara). PCR amplification was performed as follows: denaturation at 94°C for 3 min, followed by 40 amplification cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The final elongation step was performed at 72°C for 10 min. The PCR product was ligated into a pBluescript SK(–) vector (Stratagene, La Jolla, CA, USA). The plasmid DNA was purified and sequenced with T3 and T7 primer using a SQ-5500 DNA sequencer (Hitachi, Tokyo, Japan).

Table 1

Primers design

Rapid amplification of 3′ cDNA end (3′ RACE)

A primer, CRP-F03 (Table 1), was designed based on the nucleotide sequence of cDNA encoding the N-terminal amino acid sequence in order to amplify the 3′-end of the cDNA. The first PCR was carried out in a 20 μl solution containing 0.25 mM dNTP (Takara), 1×PCR buffer (Takara), 0.5 U Taq DNA polymerase (Takara), 0.5 μM of primer, CRP-F03 and adaptor (Table 1) and 1 μl of cDNA. PCR amplification was performed as follows: denaturation at 94°C for 3 min, followed by 40 amplification cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 2 min. The final elongation step was performed at 72°C for 10 min. Next, a nested PCR amplification was conducted with the same cDNA as a template and the CRP-F04 and CRP-R04 primers (Table 1) newly designed based on the nucleotide sequence obtained above and using the same conditions. The amplified DNA fragment was ligated into a pCR 2.1-TOPO II vector (Invitrogen) using a TOPO TA cloning kit and was sequenced.

Rapid amplification of 5′ cDNA end (5′ RACE)

The cDNA obtained from the above procedures was termed as CRP cDNA. After the determination of the nucleotide sequences of CRP cDNA encoding the 3′-region, a primer, CRP-R07 (Table 1) was designed for 5′ RACE. First-strand cDNA was synthesized in the same manner as described above containing 3 μg total RNA and 0.1 μM specific primer, CRP-R08 (Table 1). The resulting first-strand cDNA was tailed with poly (A)⁺ at the 3′-end using terminal deoxynucleotidyl transferase (Invitrogen). PCR was carried out in a 20 μl solution containing 0.5 μM of each primer dT-adaptor, adaptor and CRP-R07 (Table 1) and the poly (A)-tailed cDNA as a template. Initial denaturation at 94°C for 3 min, 5 amplification cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 3 min was followed by 35 amplification cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min. The PCR product was subcloned and sequenced as described above.

Identification of the cDNA sequence of cortical rod proteins

Four primers, F05, F06, R05 and R06 (Table 1) were designed based on the nucleotide sequences obtained from 5′ RACE and 3′ RACE in order to identify two clones of CRP. PCR and subcloning and sequencing of the resultant product were carried out as described above.

Purification of the 28.6 kDa and 30.5 kDa molecules from mature ovary of M. japonicus

Based on the results of SDS-PAGE analysis of ovarian extract at different developmental stages of M. japonicus, it was previously elucidated that four proteins having molecular weights of 30 kDa, 91 kDa, 128 kDa, 186 kDa increase remarkably with the progression of vitellogenesis (Fig. 1; Kawazoe et al., 2000). The three bands corresponding to 91 kDa, 128 kDa and 186 kDa proteins were further identified as vitellin subunits. However, the nature of the 30 kDa molecule had not been elucidated at this stage.

Fig. 1

SDS-PAGE (4–20%) of Marsupenaeus japonicus ovarian extracts. Gels were stained with Coomassie Brilliant Blue R250. Lane 1: previtellogenic ovary; lane 2: early exogeneous vitellogenic ovary; lane 3: late exogeneous vitellogenic ovary. Molecular weight markers: myosin (200 kDa), β-galatosidase (116 kDa), BSA (66 kDa), aldolase (42 kDa), carbonic anhydrase (30 kDa), and myoglobin (17 kDa).

In the present investigation, four peaks were obtained from gel filtration on a Sephadex G-75 column (Fig. 2a), with the second peak containing a large quantity of the 30 kDa molecule. The fractions corresponding to this peak were pooled in order to purify the 30 kDa molecule. This molecule was further separated into two peaks by reversed-phase HPLC (Fig. 2b). The A and B fractions of this peak were subjected to 15–25% SDS-PAGE under reducing and non-reducing conditions (Fig. 3a and b). On SDS-PAGE, fraction A separated into two bands corresponding to 28.6 kDa and 30.5 kDa. Fraction B contained the 28.6 kDa only. Under reducing conditions, fraction A separated into 34.5 kDa and 35.4 kDa bands, and fraction B contained the 34.5 kDa band only.

Fig. 2

(a) Gel filtration chromatography profile of mature ovarian extract on Sephadex G-75 (column size, 2.5×75 cm). Flow rate: 20 ml/h; fraction size, 5 ml. (b) Isolation a molecule of approximate molecular weight of 30 kDa using reversed-phase HPLC. Flow rate: 1 ml/min.

Fig. 3

SDS-PAGE (15∼25%) of fractions A and B obtained by reversed-phase HPLC under non-reducing (a) and reducing (b) conditions. Lane 1: fraction A; lane 2: fraction B.

Analysis of N-terminal amino acid sequence

Amino acid sequence analyses of the 28.6 kDa and 30.5 kDa proteins revealed the identity of 29 residues in the N-terminal region (Fig. 4). Homology of these sequences was examined using BLAST (Altschul et al., 1997). The amino acid sequence corresponding to residues 1–21 of the 28.6 kDa molecule and residues 9–29 of the 30.5 kDa molecule were identical. This sequence was similar to the N-terminal amino acid sequence of SOP in P. semisulcatus (Khayat et al., 2001).

Fig. 4

Comparison of N-terminal amino acid sequence of 28.6 kDa and 30.5 kDa molecules in M. japonicus with that of SOP in P. semisulcatus. Asterisks indicate unidentified amino acid residues.

Histology and immunohistochemistry

Fig. 5 shows the results of histology and immunohistochemistry on serial sections at the oocytic maturation stage using anti-28.6 kDa antiserum. CRs were observed around the periphery of mature oocytes after HE staining (Fig. 5A) and also showed positive signals with PAS reaction (Fig. 5B). CRs stained positively with the anti-28.6 kDa antiserum, although they were negative to the anti-Vt antiserum (Kawazoe et al., 2000) (Figs. 5C and D).

Fig. 5

Immunohistochemical identification of the 28.6 kDa molecule in mature oocytes. The CRs were observed at the cortical region of mature oocytes after HE staining (A) and showed positive signals with PAS staining (B). CRs showed positive immunoreactivity to anti-28.6 kDa antiserum (C) but reacted negatively with anti-Vt antiserum (D). Arrowheads indicate cortical rods. Bars=100 μm.

Western blotting analysis

Both the 28.6 kDa and 30.5 kDa molecules in fraction A isolated by HPLC were visualized using anti-28.6 kDa antiserum (data not shown). Western blotting analysis was performed using anti-28.6 kDa antiserum with ovarian extracts from ovaries having GSI of 0.32, 1.26, 3.67 and 5.71 under non-reducing and reducing conditions (Fig. 6A and B), compared with those using anti-28.6 kDa antiserum and anti-Vt antiserum under non-reducing conditions (Fig. 6C). No signal was observed in the previtellogenic ovary (GSI 0.32). Two bands corresponding to a 30.5 kDa protein and a protein of greater than 200 kDa were detected in the endogenous vitellogenic ovary (GSI 1.26), and three bands corresponding to proteins of 28.6 kDa, 30.5 kDa and greater than 200 kDa were observed in the early exogenous vitellogenic ovary (GSI 3.67). The results for maturation stage ovary (GSI 5.71) were identical to those of the endogenous vitellogenic ovary. All bands that showed positive reaction against the anti-28.6 kDa antiserum reacted negatively to the anti-Vt antiserum, while Vt corresponding to 91 kDa was stained (Fig. 6C). Under reducing conditions (Fig. 6B), no signal in the previtellogenic ovary, one 35.4 kDa band in the endogenous vitellogenic and maturation stage ovaries, and 35.4 kDa and 34.5 kDa bands in the early exogenous vitellogenic ovary were observed. When western blotting analysis was performed using a 10-fold concentrated sample, positive but weak reaction was observed in the previtellogenic ovary (data not shown).

Fig. 6

Western blotting analysis of ovarian extract using anti-28.6 kDa antiserum under non-reducing (A) and reducing (B) conditions and comparison of those using anti-28.6 kDa antiserum and anti-Vt antiserum under non-reducing conditions (C). Lane 1: previtellogenic ovary; lane 2: endogeneous vitellogenic ovary; lane 3: early exogeneous vitellogenic ovary; lane 4: maturation stage ovary.

Carbohydrate staining

In order to determine whether CRP is glycosylated, carbohydrate staining was performed using biotinylated Con A. Con A was found to bind to the 28.6 kDa and 30.5 kDa proteins, indicating that both molecules are glycosylated (Fig. 7).

Fig. 7

Carbohydrate staining with biotinylated concanavalin A (Con A). The 28.6 kDa and 30.5 kDa molecules stained with Con A.

Molecular cloning of CRP cDNA

Two cDNA fragments considered to encode the N-terminal amino acid sequence of the 28.6 kDa and/or 30.5 kDa molecules were isolated by PCR amplification using three degenerate primers, CRP-F01, F02 and R01. Subsequently, the 5′ and 3′-ends of these cDNAs were obtained by 5′ and 3′ RACE using gene-specific primers, respectively (Table 1). These cDNAs were termed as CRP1 and CRP2 cDNAs.

The nucleotide and deduced amino acid sequences of these cDNAs are shown in Fig. 9. Both CRP1 and CRP2 cDNA clones consisted of 966 bp, containing 15 bp for the 5′ untranslated region, 828 bp for the open reading frame and 120 bp for the 3′ untranslated region. The open reading frame encoded 276 amino acids (Fig. 8). Based on the N-terminal sequence of the two CRP cDNAs, we predicted that a stretch of 19 and 27 highly hydrophobic amino acids corresponded to the signal peptides of CRP1 and CRP2. The molecular masses of CRP1 and CRP2 were estimated to be 28,628 Da and 27,778 Da, respectively. The DNA sequences of CRP1 and CRP2 were found to be heterogeneous at 16 positions in the translated parts of the cDNA, resulting in 9 differing residues between the deduced amino acid sequences. There was one potential N-linked glycosylation site in CRP1 and CRP2.

Fig. 8

Nucleotide and deduced amino acid sequences of CRP cDNA. Positions of nucleotides and amino acids are indicated by numbers on the right-hand side of the figure. Differences between the nucleotide sequences of CRP1 and CRP2 clones are underlined. The resulting differences observed in the deduced CRP2 amino acid sequence are indicated below the nucleotide sequence. The amino acid residue indicated by netting (position 160) is a potential N-glycosylation site. The asterisk indicates the stop codon and the polyadenylation signal (AATAAA) is underlined. The nucleotide sequences have been submitted to the GenBank™/EMBL Data Bank with accession number  AB164639 and  AB164640.

The amino acid sequences of the CRP of M. japonicus and those of SOP in P. semisulcatus (Khayat et al., 2001) are shown in Fig. 9. CRP1 and CRP2 in M. japonicus respectively exhibited amino acid identity of 77 and 79% with SOP1 and SOP2, the two SOP clones obtained in P. semisulcatus.

Fig. 9

Comparison of the deduced amino acid sequences of CRP cDNA clones from M. japonicus and SOP from P. semisulcatus. Open enclosures indicate amino acid sequences common to all molecules. Black boxes indicate shared cysteine regions. Netting indicates a potential N-glycosylation site.