Animals

Ovigerous female (body weight, 46–95 g) and male (body weight, 30–45 g) P. hypsinotus which were caught in the offshore waters of Ishikawa Peninsula, the Japan Sea were obtained from local fishermen in February 1999. They were kept in recirculating tanks of the Obama National Center for Stock Enhancement at 1–3°C under natural photoperiod conditions and were fed moist pel-lets. After the eggs of ovigerous females hatched larvae during February-March 1999, 103 female shrimp were taken for the sampling experiment.

Sampling

Sampling was carried out in March, April, May, June, August, October, and December 1999, and February, April, June, August, and November 2000. At each sampling, female shrimp (2–12 animals) were randomly chosen and sacrificed. Hemolymph samples were taken from the pericardial cavity using a syringe with a needle, and were stored at −80°C until analysis. Ovaries were dissected and weighed, and pieces of the midportion of the ovaries were fixed for histological observation. From some female shrimp, pieces of the ovaries were quickly frozen in liquid nitrogen and stored at −80°C until analysis. Gonadosomatic index (GSI) was calculated as the gonad weight (g) × 100/body weight (g). During the experiment period, March 1999-November 2000, 20 animals died.

Histological procedures

For light microscopy, the ovarian pieces were fixed with Bouin's solution. After dehydration with an ethanol and xylene series, the fixed samples were embedded in paraffin, and sectioned at 4 μm thickness. Sections were stained with hematoxylin-eosin or hematoxylin-periodic acid Schiff (PAS).

For transmission electron microscopy, the ovarian pieces were fixed with in ice-cold 2.25% paraformaldehyde, 2% glutaraldehyde, and 6.5% sucrose buffered with 0.1 M sodium cacodylate, pH 7.5. The fixed ovaries were post-fixed with 1% osmium tetroxide, and embedded in Epon 812 after dehydration in a series of ethanol, propylene oxide, and QY-1. Ultra-thin sections were stained with uranyl acetate and lead citrate. The sections were observed under a Hitachi H-7100 electron microscope at 75 kV or a JEOL JEM-100CX electron microscope at 80 kV.

Classification of developing stages of oocytes and ovary

According to criteria previously reported for the giant freshwater prawn, Macrobrachium rosenbergii (Okumura and Aida, 2000), the developmental stages of the oocyte were classified into four stages: previtellogenic oocyte (oocytes with homogeneously hematoxylin-stained cytoplasm), endogenous vitellogenic oocyte (oocytes with PAS-positive but eosin-negative vesicles in the cytoplasm), exogenous vitellogenic oocyte (oocytes with eosin-positive yolk globules in the cytoplasm), and maturing oocyte (nucleus is not observable in the center of the cytoplasm due to germinal vesicle break down (GVBD)). For naming cytoplasmic components, we followed the terminology for M. rosenbergii (Okumura and Aida, 2000) and the spiny lobster, Panulirus japonicus (Minagawa and Sano, 1997).

The ovarian developmental stage was determined according to the relative abundance of the most advanced type of oocyte in the ovary. We followed the terminology for the spiny lobster (Minagawa and Sano, 1997), and the ovarian developmental stages were classified into five stages: inactive stage (ovaries without exogenous vitellogenic oocytes), early developing stage (ovaries containing exogenous vitellogenic oocytes, GSI<2), late developing stage (ovaries containing exogenous vitellogenic oocytes, GSI≥2), ripe stage (ovaries containing maturing oocytes), and spawned stage (ovaries after oviposition).

Purification of vitellin

Vitellogenic blue/green ovaries from a female (GSI: 3.2) were homogenized in 0.02 M Tris-HCl buffer, pH 8.7, containing 0.15 M NaCl using a HG-30 homogenizer (Hitachi, Tokyo, Japan). The homogenate was centrifuged at 10000 g for 10 min at 4°C, and the interphase was carefully taken so as not to contaminate the upper lipid phase and the precipitate. The interphase was subjected to gel filtration on a HiPrep 16/60 Sephacryl S-300 HR column (1.6×60 cm, 120 ml in bed volume, Amersham Biosciences, Piscataway, NJ) and eluted by the same buffer at a flow rate of 12 ml/h. The absorbance at 280 nm was monitored during the elution, and the eluted fractions were collected every 2 ml. Molecular weight of the eluted peak was estimated using a HMW gel filtration calibration kit (Amersham Biosciences). Protein content of each fraction was determined using an Advanced Protein Assay Reagent (Cytoskeleton, Denver, CO) with bovine γ-globulin as a standard.

The major protein peak fractions were pooled and concentrated using the water-absorbing beads Ms. Btaury-kn (Atto, Tokyo, Japan). After desalting on a PD-10 column (Amersham Biosciences), the major protein fractions were subjected to ion-exchange chromatography on a HiPrep 16/10 DEAE FF column (1.6×10 cm, 20 ml in bed volume, Amersham Biosciences) for further separation. Elution was done at a flow rate of 5 ml/min with a linear gradient from 0.02 M Tris-HCl buffer, pH 8.7 to 0.02 M Tris-HCl buffer, pH 8.7, containing 0.5 M NaCl in 100 ml elution. Fractions were collected every 4 ml. The fraction with the highest protein content was taken as the purified vitellin.

Preparation of antiserum against vitellin

Polyvalent antiserum against the purified vitellin was raised in a rabbit. An emulsion of the purified vitellin fraction and Freund's complete adjuvant was injected intradermally into the back of the rabbit twice in two weeks. Blood was collected 8 days after the second injection. Antiserum was stored at −80°C.

Electrophoresis and immunoblotting

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the presence of 2-mercaptoethanol on precast 5–20% acrylamide gradient slab gels (Bio–Rad, Hercules, CA) using routine procedures. The gels were stained with Coomassie Brilliant Blue R-250. Molecular weights of bands were estimated using Bio-Rad broad molecular weight standards.

After SDS-PAGE, proteins were electroblotted onto a polyvinylidine fluoride (PVDF) membrane (immune-blot PVDF membrane, Bio-Rad) using a wet transfer apparatus (Mini trans-blot cell, BioRad). The blotted membrane was blocked with a blocking solution of Block Ace (Snow Brand Milk Products, Sapporo, Japan) for 1 h at room temperature. After washing the membrane in 0.01 M phosphate buffer, pH 7.4, containing 0.9% NaCl (PBS), the membrane was incubated in the raised anti-vitellin antiserum in a Block Ace solution (1:1000) for 1 h at room temperature. Immunoreactive bands were made visible by a Histofine Simple Stain PO (MULTI) (Nichirei, Tokyo, Japan) and a peroxidase substrate solution (0.02% 3,3′-diaminobenzidine, 0.06% H₂O₂ in 0.1 M phosphate buffer, pH 6.4).

Immunohistochemistry

After deparaffinization, sections of vitellogenic ovary were incubated for 5 min in 3% H₂O₂. After washing in PBS, sections were incubated for 10 min in a Block Ace solution, and further incubated in the raised anti-vitellin antiserum diluted in a Block Ace solution (1:5000) for 30 min. After washing in PBS, sections were incubated in a Histofine Simple Stain PO (MULTI) solution for 30 min, and developed in the substrate solution.

Eu-labeling of anti-vitellin immunoglobulin G

The anti-vitellin antiserum was desalted and eluted in 0.02 M phosphate buffer, pH 7 using a PD-10 column. The elution was subjected to a HiTrap rProtein A 1ml column (Amersham Biosciences), and anti-vitellin immunoglobulin G (IgG) was eluted in 0.1 M sodium citrate buffer, pH 3.6. After changing the buffer from 0.1 M sodium citrate buffer to 0.05 M sodium carbonate buffer, pH 9.0, the anti-vitellin IgG was labeled with isothiocyanatophenyl-EDTA-Eu chelate (Eu-labeling reagent) using a DELFIA Eu-labeling kit (PerkinElmer Life and Analytical Sciences, Boston, MA) according to the manufacturer's protocol. Briefly, 0.04 mg of Eu-labeling reagent was added to 0.05 ml of sodium carbonate buffer which contained 0.23 mg of IgG, and incubated for 27 h at room temperature. The Eu-labeled IgG was separated from free Eu-chelate and eluted in an elution buffer (0.05 M Tris-HCl buffer, pH 7.8, containing 0.9% NaCl and 0.1% NaN₃) using a PD-10 column. The labeling yield (Eu³⁺/IgG) was 15.

Vitellogenin time-resolved fluoroimmunoassay

Vitellogenin TR-FIA was carried out using a sandwich method. The wells of a DELFIA Strip Plate 8×12-well (PerkinElmer Life and Analytical Sciences) were coated with the protein A-purified anti-vitellin IgG by physical adsorption. The anti-vitellin IgG in sodium carbonate buffer, pH 9.0 (0.2 ml) was put into wells, and allowed to stand at room temperature overnight. After three washes with wash buffer (5 mM Tris-HCl buffer, pH 7.8, containing 0.05% Tween 20 and 0.1% NaN₃), the wells were blocked with 0.5% bovine serum albumin in 0.05 M Tris-HCl buffer, pH 7.8, which contained 0.9% NaCl and 0.1% NaN₃ at 4°C overnight. The blocking solution was discarded, and the plates were briefly dried before being stored at 4°C.

Hemolymph samples were diluted in assay buffer (0.05 M TrisHCl buffer, pH 7.8, containing 0.9% NaCl, 0.5% bovine serum albumin, 0.05% NaN₃, 0.01% Tween 40, and 0.02 mM diethylenetriaminepentaacetic acid) (1000–20000 fold dilutions). The purified vitellin was dissolved in assay buffer as standard (0.98–2000 pg/ml). The hemolymph samples or standards (0.2 ml) were put into the anti-vitellin IgG-coated wells, and incubated at 4°C overnight. After four washes with wash buffer, 0.2 ml of the Eu-labeled anti-vitellin IgG in assay buffer (250 ng/ml) was put into the wells, and incubated for 4 h at room temperature. After five washes with wash buffer, 0.2 ml of enhancement solution (0.1 M acetate-phthalate buffer, pH 3.2, containing 0.1% Triton X-100, 0.015 M 2-naphthoyltrifluoroacetone, and 0.05 mM tri-n-octylphosphine oxide, Perkin-Elmer Life and Analytical Sciences), and the plates were slowly shaken for 5 min at room temperature. The Eu fluorescence in the wells was measured by a time-resolved fluorometer (Wallac 1420 ARVOsx-d, PerkinElmer Life and Analytical Sciences).

Statistical analysis

The quantitative data are presented as mean±SD (standard deviation). Nonparametric statistics were used because the data exhibited non-normal distributions. For multiple comparisons, overall analysis was done by the Kruskal-Wallis test, followed by the Dunn test when overall significance was detected. The Spearman rank correlation test was done for correlation analysis. Probability values of P<0.05 were considered statistically significant.

Purification of vitellin

Six peaks were detected by gel filtration chromatography, and the third peak (65 ml-elution volume) was the major protein peak (Fig. 1). This peak was successively purified by DEAE ion-exchange column chromatography, and the major peak was taken as purified vitellin (Fig. 2). The molecular weight of the purified vitellin was estimated as 295 kDa on gel filtration chromatography.

Figure 1

Gel filtration chromatogram of vitellogenic ovarian extract of Pandalus hypsinotus on a HiPrep 16/60 Sephacryl S-300 HR column. Flow rate, 12 ml/h; fraction size, 2 ml. Arrows indicate the vitellin peak. Closed fractions were subjected to ion-exchange chromatography.

Figure 2

Ion-exchange chromatogram of gel-filtration fractions of Pandalus hypsinotus ovarian extract. Dotted line indicates NaCl gradient elution. Closed peak was taken as the purified vitellin.

The purified vitellin was separated into two bands of 86 and 98 kDa in SDS-PAGE (Fig. 3). These two bands were also observed as major bands in the gel-filtrated fraction and crude extracts of vitellogenic ovary and matured ovary (after GVBD), but the bands were not observed in the crude extract of immature ovary.

Figure 3

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (5–20% gradient gel) of ovarian extracts (lanes 1–3) and purified vitellin (lanes 4 and 5) of Pandalus hypsinotus. Lane 1, ovary at the inactive stage (GSI: 0.78); lane 2, ovary at the late developing stage (GSI: 3.23); lane 3, ovary at the ripe stage (GSI: 8.53); lane 4, fraction of gel-filtration chromatography; lane 5, fraction of ion-exchange chromatography.

Characterization of vitellin and vitellogenin

By Western blotting using the antibody raised against the purified vitellin, the 86 and 98 kDa bands were detected in extracts of vitellogenic ovaries as the major immunoreactive bands, but not detected in the extracts from immature ovaries (Fig. 4). This result indicates that the antibody is specific to the vitellin. In addition to the two major bands, minor immunoreactive bands were detected at 26, 38, and 57 kDa in extracts of vitellogenic ovaries. In the hemolymph of vitellogenic females, the 86 kDa band was detected as the major band, and 98 and 190 kDa bands were detected as minor bands (Fig. 4).

Figure 4

Western blotting analysis of hemolymph (lanes 1–8) and ovarian extracts (lanes 9–11) of Pandalus hypsinotus stained with anti-vitellin antiserum. Lane 1, male (GSI: 0.33); lane 2, immature female (GSI: 0.63); lanes 3–8, vitellogenic females (GSI: 2.92, 3.70, 4.12, 4.76, 6.51, and 9.76, respectively); lane 9, immature female (GSI: 0.63); lanes 10 and 11, vitellogenic females (GSI: 4.12 and 6.51), lane 12, molecular weight marker.

Validation of time-resolved fluoroimmunoassay

A standard curve for vitellogenin TR-FIA was obtained in the range of 0.98–2000 ng/ml (Fig. 5A). In this range, the coefficient of variation for each standard was less than 6%. A dilution series of hemolymph samples (700–89600 fold dilutions) were parallel to the standard curve (Fig. 5A). Analytical sensitivity was 0.5 ng/ml (0.1 ng/well), which was calculated as the value, 2 SD above the mean of blank measurement values.

Figure 5

Validation of time-resolved fluoroimmunoassay for Pandalus hypsinotus vitellogenin. A, parallelism between standard curve and hemolymph serial dilution; B, recoveries of standard vitellin added to hemolymph.

Intraassay and interassay variation was examined by measuring hemolymph samples of immature and mature females. Intraassay coefficients were 0.9–6.4% (6 replications for each sample) for the concentrations of 2.6–1000 ng/ml, and interassay coefficients were 1.4–5.1% (6 replicated assays for each sample) for the concentrations of 14.7, 65.5, 248, and 993 ng/ml.

Recovery was examined by measuring the hemolymph samples that were prepared by adding varying levels of standard vitellin (7.8–1000 ng/ml) to immature female hemolymph (Fig. 5B). Recoveries were in the range of 95–111% (102.2±5.1%, mean±SD, 4 replications for each sample).

Ovarian developmental stages

In the inactive stage of the ovary, the ovary contains oogonia, previtellogenic oocytes, and endogenous vitellogenic oocytes (Fig. 6A, B). The previtellogenic oocytes are present in the center, and the endogenous vitellogenic oocytes are at the periphery. The previtellogenic oocytes are 35–60 μm in diameter, and in electron microscopy observations, the ooplasm contains ribosomes and mitochondria (Fig. 7A). The endogenous vitellogenic oocytes are 60–280 μm in diameter, and larger oocytes (120–280 μm in diameter) are enveloped by follicle cells (Fig. 6B). There are unstained vesicles in the cytoplasm of the endogenous vitellogenic oocytes, which are considered to be lipid droplets. In electron microscopy observations (Fig. 7B,C), the ooplasm is filled with rough endoplasmic reticulum (0.5–3 μm in diameter) which contains relatively dense homogeneous material. The rough endoplasmic reticulum also contains small electron-dense granules at the periphery.

Figure 6

Cross sections of Pandalus hypsinotus ovaries. A, B, inactive stage after releasing the hatched larvae (GSI: 0.62); C, D, beginning of yolk accumulation at the early developing stage (GSI: 1.00); E, late developing stage (GSI: 6.12); F, ripe stage (GSI: 8.52); G, spawned stage (GSI: 0.28); H, regressing mature oocytes in ovary at the spawned stage (GSI: 3.05). A, C–H, hematoxylin-eosin staining; B, hematoxylin-periodic acid Schiff staining. Bars: 0.1 mm. En, endogenous vitellogenic oocytes; Ex, exogenous vitellogenic oocytes; FC, follicle cells; N, nucleus; Og, oogonia; Pr, previtellogenic oocytes; OT, ovulation traces; RO, regressing mature oocytes; VM, vitellin membrane; Y, yolk globules.

Figure 7

Transmission electron micrographs of Pandalus hypsinotus ovaries. A, previtellogenic oocyte (GSI: 0.60); B,C, endogenous vitellogenic oocyte (GSI: 0.55); D, exogenous vitellogenic oocyte and follicle cell (GSI: 1.17); E, F, endocytosis in the exogenous vitellogenic oocyte (GSI: 1.12); G, exogenous vitellogenic oocyte (GSI: 2.25); H, maturing oocyte (GSI: 8.52). Bars: 1 μm. EC, endocytosis; FC, follicle cells; L, lipid droplets; M, mitochondria; MV, microvilli; RER, rough endoplasmic reticulum; VM, vitellin membrane; Y, yolk globules.

In the early developing stage of the ovary, the exogenous vitellogenic oocytes are present at the periphery, and the endogenous vitellogenic oocytes are present in the center. At the beginning of this stage, small eosin-positive yolk globules are observed in the cytoplasm of the oocytes (300–340 μm in diameter, Fig. 6C, D), and at the end of this stage, yolk globules fill the ooplasm. The exogenous vitellogenic oocytes were positively stained by the anti-vitellin antibody, but the previtellogenic oocytes, endogenous vitellogenic oocytes, oogonia, and follicle cells were not stained (Fig. 8). In electron microscopy observations, the ooplasm contains electron-dense yolk globules (0.8–2.6 μm) in addition to mitochondria, lipid droplets and rough endoplasmic reticulum (Fig. 7D). The follicle cells have mitochondria but no developed rough endoplasmic reticulum in the cytoplasm. Active endocytosis was observed at the oocyte surface (Fig. 7E, F).

Figure 8

Immunohistochemistry of an ovarian section (GSI: 1.00) with anti-vitellin antiserum. En, endogenous vitellogenic oocyte; Ex, exogenous vitellogenic oocytes; FC, follicle cells; Og, oogonia.

In the late developing stage of the ovary, the exogenous vitellogenic oocytes become larger and are filled with yolk globules (Fig. 6E). Oogonia, the previtellogenic oocytes, and the endogenous vitellogenic oocytes are present in the center of the ovaries. The diameter of the oocytes reaches 875–950 μm at the end of this stage. In electron microscopy observations, yolk globules become larger (2–15 μm), and fill the ooplasm (Fig. 7G). Among the accumulated yolk globules, rough endoplasmic reticulum is present.

In the ripe stage of the ovary, the maturing oocytes are present at the periphery (Fig. 6F). The maturing oocytes (875–975 μm) are still enveloped by the follicle cells, and vitellin membrane develops on the oocyte surface. The vitellin membrane is 4 μm thick, and the oocytes project microvilli toward the follicle cells (Fig. 7H).

In the spawned stage of the ovary, the ovulation traces are present at the periphery of the ovary, and oogonia, the previtellogenic oocytes, and the endogenous vitellogenic oocytes are present in the center (Fig. 6G). The ovary often contains regressing mature oocytes (Fig. 6H).

Ovarian development and hemolymph vitellogenin levels

Seasonal changes in ovarian development and vitellogenin levels are shown in Figs. 9 and 10. In March 1999, all the sampled females had ovaries at the inactive stage, and GSI and hemolymph vitellogenin levels were low (0.55±0.04 and 0.010±0.011 mg/ml, respectively, mean±SD). In April and May 1999, females had ovaries at the early developing stage appeared, but most females had ovaries at the inactive stage. The number of females with ovaries at the early developing stage increased during June-October 1999. In October 1999, GSI became significantly higher (1.00±0.21, P<0.05) than in March 1999, but vitellogenin levels (0.25±0.52 mg/ml) were not significantly different. During December 1999-April 2000, all the sampled females had ovaries at the early or late developing stages, and GSI was high (2.0–3.2). Vitellogenin levels were significantly higher in February and April 2000 (1.30±1.40 and 2.82±0.62 mg/ml, respectively) than in March 1999. In June 2000, females with ovaries at the ripe stage appeared, and some females had spawned. GSI and vitellogenin levels were high but showed high variances (5.6±2.1 and 2.26±2.94 mg/ml, respectively). In August 2000, all the sampled females had ovaries at the spawned stage. Two to the four sampled females carried embryos on the abdomen, but the other two females did not carry the embryos, probably due to a failure to fertilize the eggs. In August 2000, GSI significantly declined (1.36±1.93, P<0.05) due to oviposition; on the other hand, vitellogenin levels highly varied (2.24±3.31 mg/ml) due to the occurrence of shrimp having unspawned mature oocytes. In November 2000, females brooded embryos on the abdomen and had ovaries at the inactive stage. GSI and vitellogenin levels were low (0.34±0.06 and 0.001±0.000 mg/ml, respectively).

Figure 9

Seasonal frequency of the ovarian developmental stages in Pandalus hypsinotus. In, inactive stage; De', early developing stage; De'', late developing stage; Ri, ripe stage; Sp, spawned stage.

Figure 10

Seasonal changes in GSI and hemolymph vitellogenin levels in Pandalus hypsinotus. Points and bars indicate the mean and SD. Points without common letters indicate significant differences (P<0.05).

All the female shrimp molted once during May-August 1999 (the first ecdysis). The second ecdysis occurred during November 1999-February 2000, although some female shrimp did not molt during this period. Before oviposition, female shrimp molted (the second or third ecdysis).

GSI and vitellogenin levels were compared between the ovarian developmental stages (Fig. 11). In the females with ovaries at the late developing stage, GSI was significantly higher (3.82±1.13, P<0.05) than at the inactive stage (0.63±0.14), and vitellogenin levels was significantly higher (2.66±0.93 mg/ml, P<0.05) than at the inactive stage (0.006±0.008 mg/ml). In the females with ovaries at the ripe stage, GSI peaked (8.40±0.19), but vitellogenin levels became low (0.040±0.012 mg/ml, not significant mainly due to the small number of samples). Vitellogenin levels and GSI (after logarithmic transformation) were significantly correlated (P<0.05, Fig. 11C).

Figure 11

GSI (A) and hemolymph vitellogenin levels (B) at ovarian developmental stages and relationship between GSI and hemolymph vitellogenin levels (C) in Pandalus hypsinotus. Bars indicate the mean and SD. Bars without common letters indicate significant differences (P<0.05). Numbers in parenthesis indicate the number of animals sampled for each ovarian developmental stage. Abbreviations are shown in Fig. 9.