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Reproductive Technology |
Department of Zoology,3 The University of Hong Kong, Hong Kong
State Key Laboratory for Biological Control,4 School of Life Sciences, Zhongshan University, Guangzhou, People's Republic of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene regulation, oocyte development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The site of vitellogenin synthesis in crustacean has been investigated by many different approaches. Three tissues are thought to be possible sites of synthesis: subepidermal adipose tissue, hepatopancreas, and ovary [4]. Because different methodologies and different species of crustacean have been used, no conclusive evidence currently exists regarding the site of vitellogenin production. Ovarian synthesis of yolk protein was reported in Penaeus monodon [5], P. japonicus [6], P. vannamei [7], and P. semisulcatus [8]. The hepatopancreas was reported as a site of vitellogenesis in P. monodon [9, 10]. As determined by the molecular approach, vitellogenin is exclusively produced by the hepatopancreas in the freshwater shrimp Macrobrachium rosenbergii [11]. However, in the marine shrimp Penaeus japonicus, both the ovary and the hepatopancreas are sites of vitellogenin synthesis [12]. Similarly, we have previously reported cloning of the MeVg1 gene and provided evidence for expression of MeVg1 in both the hepatopancreas and ovary [13].
Previous studies using Northern blot analysis and/or real-time polymerase chain reaction (PCR) to study the expression pattern of the vitellogenin gene have been reported [10–12]. Those studies were performed under the assumption that only one vitellogenin gene existed in that species. However, based on the organization of vitellogenin gene of Metapenaeus ensis [13] and the increasing evidence for multiple vitellogenin genes in shrimp, a need exists to re-examine the expression pattern of the vitellogenin gene during the reproduction cycle. Furthermore, because of the complicated nature of the expression pattern for these genes, a thorough study regarding the total number of vitellogenin genes in each species is important to the understanding of vitellogenesis in crustacean.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both genomic DNA library screening and a PCR-based primer walking approach were used to clone the full-length shrimp vitellogenin gene (MeVg2). For library screening, the hepatopancreas-specific vitellogenin gene (MeVg2) was isolated from one of the lambda clones (
8.3) of the initial library screening for the MeVg1 gene [13]. Potential positive clones from agar plates were plugged and eluted for second- and third-round screenings. The genomic DNA fragments containing the shrimp vitellogenin gene were cloned into a pBluescript II KS(+) vector (Stratagene, La Jolla, CA). The DNA sequences for the inserts were determined, and the introns/exons were initially identified based on the vitellogenin coding sequence of the MeVg1 gene [13]. For the cloning of the MeVg2 full-length cDNA, selected primers designed for sequencing of the 8.3 clone were used in reverse transcription (RT)-PCR to amplify cDNA from the hepatopancreas of maturing females. Additionally, a rapid amplification of cDNA ends (RACE) cloning kit (Invitrogen, Carlsbad, CA) was used for the cloning of the missing 3' and 5' ends of the cDNA. The PCR products were subcloned into a PCR TA-cloning kit (Promega, Madison, WI), and DNA sequences were determined. The full-length cDNA for MeVg2 was reconstructed from overlapping cDNA clones.
The library screening approach was performed to isolate clones of smaller vitellogenin cDNAs. The template used for the synthesis of probe was derived from the coding sequence corresponding to AA2200–AA2435 of the MeVg2 cDNA. The probe was labeled by a High Prime Labeling Kit (Roche, Indianapolis, IN) according to the manufacturer's instructions. Blots were prehybridized in 5x SSC (1x SSC: 0.15 sodium chloride and 0.015 M sodium citrate), 0.1% SDS, and 5x Denhardt solution at 65°C overnight. Blots were washed twice in 2x SSC and 0.1% SDS for 20 min each at 55°C. The dry membrane was exposed to x-ray film at –80°C. For sequence alignment, the deduced amino acid sequence of MeVg2 was aligned and compared with vitellogenins of different organisms. The full-length amino acid sequences of these vitellogenin cDNAs were used in the construction of the phylogenetic trees. In the phylogenetic analysis, the vitellogenins for the nematode Caenorhabditis elegans, the mosquito Acedes aegytica, the zebrafish Danio rerio, and Xenopus were used. Phylogenetic analysis and tree construction were performed using the GeneWorks software (IntelliGenetics, Mountain View, CA).
Tissue-Specific Expression of MeVg2
Northern blot analysis and RT-PCR were used in the study of tissue-specific expression of MeVg2 gene. The RNA from different tissues (eyestalk, hepatopancreas, ovary, testis, and subepidermal adipose tissue) were first analyzed by 1% formaldehyde agarose gel and transferred onto a nylon membrane. Total RNA (1 µg) was used in the RT reaction for the synthesis of first-strand cDNA. The RT was performed in a final concentration of 1x transcription buffer (50 mM Tris-HCl, 8 mM MgCl2, 30 mM KCl, 2 mM each of dNTP, and 10 mM dithiothreitol), 2 pmol of oligo(dT)17 primer, and 1 U of reverse transcriptase (Promega). The reaction mixture was incubated at 42°C for 2 h. To investigate the tissue-specific expression for vitellogenin, we designed PCR primers based on the sequence alignment information from the MeVg1 and MeVg2 clones. The final PCR mix (30 µl/reaction) consisted of 10 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 50 mM KCl, 0.5 pmol primer, and 1.0 µl of reaction mix from the RT as described above. The PCR conditions included 1 min at 95°C for one cycle, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min. At the end of the last cycle, the PCR mix was incubated at 72°C for another 10 min for the completion of DNA synthesis. The PCR products were analyzed on 1.0% agarose gel to determine the presence of specific DNA amplification.
Immunohistochemical Analysis of the Vitellogenin in the Hepatopancreas
Hepatopancreas were cut from shrimp and fixed in 10% paraformaldehyde at room temperature for 20 h and then dehydrated and embedded in paraffin. Paraffin sections (thickness, 7 µm) were mounted onto the slide and dried at 37°C overnight. Tissue sections were dewaxed in xylene solution (twice for 5 min each) and dehydrated through increasing percentages of ethanol. The sections were blocked in a blocking buffer (0.5% BSA, 0.5% gelatin, and 0.1% NaN3 in PBS) at room temperature for 30 min and then rinsed briefly with PBS. The sections were blocked again in a blocking buffer containing a 1:40 dilution of normal goat serum at room temperature for another hour. After the removal of blocking buffer, the sections were incubated in the first antibody (1:50 000 dilution in blocking buffer) overnight. The control sections were incubated in preimmunized rabbit serum (1:1000 dilution). The slides were washed in PBS buffer twice for 10 min each time and then incubated in a second antibody (1: 5000 dilution of goat anti-rabbit immunoglobulin [Ig] G-alkaline phosphate [AP; 0.1 M Tris of pH 9.5, 0.1 M NaCl, and 50 mM MgCl2] conjugate) at room temperature for 1 h. For color detection, the slides were washed with PBS twice for 10 min each time and then with AP detection buffer twice for 5 min each. Signals were visualized by adding 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) in AP detection buffer, and color development was terminated with running tap water. The sections were counterstained with eosin and dehydrated through an increasing strength of ethanol. The sections were cleaned with xylene and mounted with coverslips in DPX mountant (Sigma, St. Louis, MO).
Characterization of the Shrimp MeVg2 Precursor
To analyze the vitellogenin subunit produced by the hepatopancreas of the reproductive female, either the whole hepatopancreas (1 g) or small pieces (0.25 g) of hepatopancreas from individual female were incubated in a M199 culture medium (Sigma) at a 1:10 ratio of tissue to medium. At different time intervals, aliquots (0.5 ml) of the culture medium were removed, and the proteins were precipitated (4°C) by 0.2 volume of 100% trichloroacetic acid. The precipitated protein was washed with acetone twice for 5 min each, dried, and resuspended in a SDS-PAGE sample buffer. The polypeptides in the medium were analyzed by Western blot. After electrophoresis, the proteins on the SDS-PAGE gel were transferred onto a nitrocellulose membrane at 4°C for 2 h. The membranes were rinsed once with a washing buffer (0.3% Tween 20 in 1x PBS). The membrane was blocked in a blocking buffer (0.3% BSA, 0.5% gelatin, and 0.1% NaN3 in PBS) at room temperature for 1 h and then rinsed briefly with 0.05% Tween 20 in 1x PBS. After removal of the blocking buffer, the membrane was incubated in the first antibody (1:4000 dilution in 0.05% Tween 20 in 1x PBS) overnight. The membrane was rinsed and washed three times in washing buffer (0.05% Tween 20 in 1x PBS) for 20 min each time and then incubated in a second antibody (1:5000 dilution of goat anti-rabbit IgG-AP conjugate) at room temperature for 1 h. For color detection, the membrane was washed twice with 0.3% Tween 20 in 1x PBS for 10 min each time and then with AP detection buffer. Signals were visualized by adding BCIP and NBT in AP detection buffer, and color development was terminated with running tap water. In the analysis of the vitellogenin precursor processing, proteins from the ovary and hepatopancreas were analyzed by Western blot as described before. Polypeptides that corresponded to those identified from the Western blot analysis were transferred to a polyvinylidene fluoride (PVDF). The membrane was stained by Coomassie blue to locate the vitellogenin-specific bands, which were excised, and N-terminal peptide sequence analysis was performed using a Hewlett-Packard model G1000A protein sequencer (Boise, ID). Approximately 15–20 sequencing cycles were performed to obtain a partial sequence of those polypeptides.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During cloning of the MeVg1 gene, we have isolated another lambda clone (8.3) by the MeVg1 partial cDNA probe. Subsequently, the DNA sequence of the whole 8.3 clone was determined. The 8.3 clone carries the coding sequence of a gene (MeVg2) that shows high similarity to the MeVg1 gene. By DNA sequence determination, amino acid sequence alignment, and cDNA sequence information (see below), only 13 exons and 12 introns were identified in the MeVg2 gene (Fig. 1). The intron-exon boundary of this MeVg2 gene also conforms to the consensus splice junction GT-AG rule [14]. The boundary of the reconstructed MeVg2 gene was later confirmed by the reconstructed full-length cDNA from RT-PCR and RACE cloning (see below). Like the MeVg1 gene, all the introns of the MeVg2 gene are small (<300 bp). Unlike the MeVg1 gene, the average exon size of the MeVg2 gene is larger. In comparison to the organization of the MeVg1 gene, the hepatopancreas-specific vitellogenin gene consists of 13 exons. This may be caused, in part, by the fusion of exons 2 and 3 and of exons 9 and 10 of the original MeVg1 before evolution to form the MeVg2 gene (Fig. 1).
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The full-length cDNA for MeVg2 was obtained from the RT-PCR and RACE cloning (Fig. 2a). The 3' and 5' ends of the cDNA were obtained by RACE. The MeVg2 cDNA consists of 7677 nucleotides, with the longest open-reading frame encoding a protein of 2559 amino acid residues (Fig. 2). The MeVg2 cDNA encoded for a precursor of 2559 amino acid residues with a predicted molecular mass of 287 kDa (http://us.expasy.org/cgi-bin/pi_tool). Based on the Kyte-Doolittle method (http://bioinformatics.weizmann.ac.il/hydroph/hydroph_help.html) for the calculation of protein hydrophilicity, the first 18 amino acid residues are most likely the signal peptides of the vitellogenin precursor. The MeVg2 precursor also contained several consensus sequences of RXXR or (R/K)X(R/K)R (Fig. 2). This consensus dibasic sequence represents a potential cleavage site for the serine protease of the subtilsin-family endopeptidase [15, 16]. Unlike the MeVg1 gene, which consists of only a few potential cleavage sites, at least 8–10 potential cleavage sites are known for the subtilsin-convertase endopeptidase. This suggested that the MeVg2 precursor may be cleaved into much smaller subunits compared to the three major subunits predicted in the MeVg1 precursor (i.e., 100, 78, and 157 kDa). However, MeVg1 precursor is similar to MeVg2 precursor, because it also lacks a phosvitin/polyserine domain, which is present in the vitellogenins of other animals. The polyadenylation signal is approximately 100 base pairs 3' to the translation stop signal (TAA). Both MeVg1 and MeVg2 share an overall 54.2% amino acid sequence identity with each other. When the amino acid sequence of MeVg2 was compared to the vitellogenins of the shrimp P. semisulcatus, the freshwater shrimp Macrobrachium rosenbergii, and the crayfish Cherax quadricarinatus, they shared 49.8%, 35.9%, and 40.7% of identity, respectively. This suggests that the shrimp MeVg2 is more closely related to the vitellogenin of the shrimp P. semisulcatus. However, the result from phylogenetic tree analysis of those vitellogenin sequences (data not shown) indicated that MeVg1 and Vtg of P. semisulcatus clustered together as a group, whereas the MeVg2 sequence formed a more closely related group with the crayfish C. quadricarinatus and freshwater shrimp M. rosenbergii. Moreover, MeVg2 may be considered as a recently evolved protein derived from MeVg1.
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Expression of MeVg2 Precursor
Using a MeVg2 partial 3'-end cDNA probe, strong hybridization signals were detected in hepatopancreas, but no hybridization was detected in other tissues (Fig. 3a). Although the Northern blot looked smeary, these signals did not represent degrading mRNA transcripts, because the 8.0-kilobase (kb) vitellogenin transcript appeared to be intact in a shorter exposure of the film. Moreover, when the blots were deprobed and rehybridized to a Metapenaeus ensis-specific actin probe, RNA transcripts corresponded to the intact actin mRNA, suggesting that the integrity of the mRNA was preserved. In a shorter exposure of the Northern blot, several major RNA transcripts were labeled by the probe. These transcripts included the 8.0-, 2.5-, and 1.4-kb RNA. The results from RNA Northern blot and RT-PCR also indicated that the MeVg2 gene is expressed exclusively in the hepatopancreas. To study the expression pattern of the MeVg2 gene during the reproductive cycle, total RNA from the ovary or hepatopancreas of female shrimp of different gonadosomatic index were used for Northern blot analysis. The results indicated that MeVg2 expression was slight in the immature female, increased slightly at the initial phase of vitellogenesis, reached a maximum at stage III, and remained high toward the end of the reproductive cycle. The relative abundance of the transcripts, however, varied. For example, the 8.0-kb, full-length RNA transcript is maximally expressed in stage II and early stage III (Fig. 3b). The transcript, however, becomes weakly detectable at stage IV and stage V. Late in the reproductive cycle (stages IV and V), most of the transcripts are approximately 2–4 kb. This indicates that the smaller transcripts represent the major transcription product of the MeVg2 gene.
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In addition to Northern blot analysis, we also performed RT-PCR using a pair of gene-specific primers for MeVg2 and obtained similar results (Fig. 3c). To further confirm the presence of smaller MeVg2 transcripts, we performed hepatopancreas cDNA library screening with a probe synthesized from the 3' end of the MeVg2 gene and obtained two smaller cDNAs of 2.3 and 1.5 kb. The 2.5-kb cDNA carries a coding sequence that is overlapping and identical with exons 10–13 of the MeVg2 gene. Furthermore, the 1.5-kb cDNA carries a coding sequence of exons 11–13 identical to that of the MeVg2 gene (Fig. 2). Translation of these mRNAs would result in the production of polypeptides with predicted sizes close to 34 and 28 kDa.
Contribution of the Hepatopancreas-Specific MeVg2 Gene to Shrimp Vitellogenesis
To localize vitellogenin in the hepatopancreas, we performed immunohistochemical analysis using vitellin antibody that we had generated earlier (unpublished results). Unlike in the ovary, positive signals were observed in the extracellular space of the hepatopancreas (Fig. 4, b–d), and only a trace amount of vitellin-immunopositive signal was identified inside the hepatopancreas cells. By Western blot analysis, only a few polypeptides showed immunopositive signals for the vitellin antibody. In the ovary, polypeptides of 97 and 157 kDa were reactive with the vitellin antibody. Moreover, intense immunopositive signals can also be detected for smaller peptides (<34 kDa). Again unlike in the ovary, large polypeptides (>100 kDa) were not detected in the hepatopancreas tissue fraction, suggesting that these polypeptides are not actively synthesized. Furthermore, only smaller polypeptides (34 and 28 kDa) were immunopositive with the vitellin antibody (Fig. 5a).
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Because only a minor quantity of small vitellogenin subunits were detected in the hepatopancreas tissue, a short-term in vitro hepatopancreas incubation was performed to analyze the polypeptide secreted from the hepatopancreas. Using whole hepatopancreas incubated in a nutrient medium (M199), only small polypeptides (<76 kDa) were identified, and only polypeptides of small size (76, 35, and 28 kDa) were immunopositive for the vitellin antibody (Fig. 5b). The sequence of these polypeptides was determined by sequencing after transfer onto a PVDF membrane. The results show that the 34-kDa polypeptides contained a sequence identical to a portion of the MeVg2 precursor (Fig. 5b). For example, in the sequence determination of a 34-kDa protein from the ovary, a polypeptide with the sequence APYGESTECP could be identified from the N-terminal amino acid sequencing; this sequence corresponded to AA19 of the MeVg2 precursor. A similar sequence could also be identified in the ovarian protein. We speculate that this 35-kDa protein may be derived from the 76-kDa subunit, because an identical N-terminal sequence was also obtained from the 76-kDa protein and a potential cleavage site was present. Thus, it is logical to suggest that the large subunit (76 kDa) is processed into a smaller polypeptide and then secreted into the hemolymph to be taken up by the ovary.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The sizes of both MeVg1 and MeVg2 genes are approximately 10 kb, with an average coding sequence of 2560 amino acids. However, there seems to be no direct relationship between the size of the vitellogenin gene and the size of the vitellogenin precursor in different animals. Although the number of amino acid residues for the vertebrate and nematode vitellogenins are similar (as they also are in insect and shrimp) and the vertebrate and invertebrate vitellogenin gene may have a common ancestor [16], the sizes of the vitellogenin gene and the number of exons/introns of the gene vary greatly. This is because the vitellogenin genes are interspersed by larger introns, and these introns increase the sizes of these genes in the vertebrates and insect. For example, the size of the vitellogenin gene in gypsy moth is 11.96 kb, with coding sequencing for 2584 amino acid residues [20]. The size of the nematode vitellogenin (vit-5) is approximately 5 kb, and it carries a coding sequence for 1603 amino acid residues [21]. However, in the chicken, the vitellogenin gene is relatively large, spanning 20.3 kb, with a coding sequence for only 1850 amino acids residues [22]. Another characteristic of vertebrate vitellogenin genes is that they are interrupted by larger number of introns and their exons encode short stretches of amino acid residues [23–25]. However, in insect, shrimp, and nematode vitellogenin genes, the numbers and sizes of the introns are relatively small, and the sizes of the exons are relatively larger.
The site for vitellogenin synthesis in crustacean has been a subject of debate for many years. Recent studies using the molecular approach have demonstrated that the hepatopancreas is the major site of vitellogenin synthesis. For example, using the molecular cloning technique, the hepatopancreas has been established as the major site of vitellogenin synthesis in Macrobrachium rosenbergii, P. japonicus, and C. quadricarinatus [11, 12, 13]. However, in those studies, the conclusion was based on the assumption that a single vitellogenin gene was expressed or that the vitellogenin expressed was highly similar in those species. However, the expression study using cDNA (partial or full length) will be obscured, because the total number of genes and their similarities have not been clarified. If multiple vitellogenin genes also exist in these shrimp, then the analysis of vitellogenin gene expression patterns by either Northern blot or RT-PCR will not reflect the true expression level of the vitellogenin gene under investigation. In the present study, we have used the less homologous, 3'-end MeVg2 cDNA probe for Northern blot analysis. The probe should be able to distinguish the type of vitellogenins expressed. Because vitellogenins are large structural/nutritive molecules that allow a much higher rate of mutation throughout evolution, it is reasonable to predict that the yet-to-be-identified vitellogenins in other crustacean species may also show a lower degree of homology. Thus, it is still too early to conclude that the hepatopancreas in crustacean is the major synthesis site and sole source of vitellogenin.
Expression of the Smaller Vitellogenin Transcripts
In reviewing the expression study of vitellogenin in most other crustaceans, only a single size (8 kb) of transcript was reported. For example, in P. semisulcatus, Averre et al. [19] used a cDNA probe derived from nucleotides 84–164 of Vtg and obtained a major RNA band of 8 kb that hybridized to the probe. If the shrimp express other smaller transcripts from the 3' end, this probe will not hybridize to those smaller transcripts. In our study of the MeVg2 gene, smaller RNA transcripts were consistently detected in the Northern blots using much larger cDNA probes at the N-terminal end (spanning AA300–600) and C-terminal end (spanning AA2300–2461). Regardless of the region of MeVg2 cDNA probe used in the Northern blot analysis, these smaller RNA transcripts consistently constituted greater than 80% of the overall vitellogenin hybridized signal, whereas the full-length, 8-kb transcript constituted less than 10%. We have also demonstrated that smaller-sized transcripts for MeVg2 gene exist. Thus, the hepatopancreas likely expressed these smaller MeVg2 transcript, and translation of these smaller transcripts (<1 kb) would result in the production of smaller MeVg2 subunits of 300–400 amino acid residues. In one study, Khayat et al. [17] reported the presence of a 1-kb RNA transcript as the major vitellogenin in P. semisulcatus. However, in that study, the sequence for that mRNA was not known. In summary, the findings from the Northern blot analysis and in vitro incubation study suggested that the small transcripts are important for the contribution of vitellin biosynthesis in Metapenaeus ensis.
To study the localization of vitellogenins, we performed immunohistochemical staining of hepatopancreatic sections with antibody generated from vitellin of M. ensis (Fig. 4). Compared with the ovarian sections (unpublished results), a much weaker immunopositive signal was detected for hepatopancreatic sections using the vitellin antibody. This may result from the use of vitellin purified from the ovary and from the hepatopancreas-derived vitellogenin subunits not being processed at their final stage. The current view for the processing of vitellogenin is that the vitellogenin precursor is cleaved into several subunits before secretion to the hemolymph and subsequent uptake into the oocytes by receptor-mediated endocytosis. Inside the oocyte, vitellogenin subunits will be processed by accumulating carbohydrate, lipid, and carotenoid to form the final lipoglycocarotene product, vitellin. Because the major function of vitellin is to supply the embryo and early larvae with nutrients, such as amino acid and lipid, a large vitellin molecule would be an efficient means to provide for those needs [26]. An 8.0-kb transcript will produce a translated precursor of 280 kDa, which is one of the largest vitellogenin precursors recorded so far. As demonstrated from the Western blot of ovarian polypeptide, most of the immunopositive signals concentrated in the lower-molecular-weight fraction (Fig. 5b). It is possible that the vitellin consists of many smaller subunits contributed from the vitellogenin precursor protein. Analysis of the MeVg1 and MeVg2 precursors revealed the presence of three to four potential cleavage sites on MeVg1, whereas six to eight potential cleavage sites have been identified in MeVg2 precursor. Because of the appearance of small hepatopancreatic polypeptides in the tissue extracts as well as in the secreted protein, we speculate that MeVg2 may be processed into several smaller subunits of 30–40 kDa. Because MeVg2 is expressed only in the hepatopancreas, MeVg2 subunits should be identified in the tissues (or the medium fraction of the explant). From the analysis of hepatopacreatic proteins, low levels of polypeptides were usually revealed by SDS-PAGE. Using the approach of incubation of hepatopancreas explant, we have identified two polypeptides (76 and 34 kDa from Western blot analysis) from the medium that show an amino acid sequence identical to the AA19– AA25 of the MeVg2. Because polypeptide with identical N-terminal sequence was also identified in the ovarian extract and MeVg2 is not expressed in the ovary, it is logical to speculate that the large (76-kDa) polypeptide has been processed into a smaller subunit by the hepatopancreas before it is secreted into the hemolymph.
Together with the lack of larger MeVg2 subunits (157 and 200 kDa) in the hepatopancreas and the small percentage of 8.0-kb mRNA transcripts in the ovary, it is logical to predict that the hepatopancreas expresses most of the smaller transcripts for vitellogenins (MeVg2), the translational products of which may be further processed into smaller subunits before uptake by the ovary. In the study of the processing of vitellogenin [17], there is no mention of multiple vitellogenin, and the sequence information for additional vitellogenins is lacking. Therefore, it is too early to predict any similarity of vitellogenin processing in M. ensis with that in other shrimps.
Posttranslation modification of the larger precursor to smaller subunits would require enzyme(s) to cleave the precursors; the presence of such enzyme is a prerequisite for posttranslation processing. So far, little information is available regarding the identification of such an endopeptidase in crustacean. Another strategy for vitellogenin processing may be attributed to the posttranscriptional regulation. From an efficiency standpoint, it is possible that the shrimp express small vitellogenin transcript that is translated into smaller polypeptides, which are more readily taken up by the oocytes. At present, we have estimated that M. ensis has at least three to four vitellogenin genes. Multiple vitellogenin genes have also been reported in the nematode, insect, and fish [23–25]. Based on these data, we predict that multiple vitellogenin genes also exist in other decapods, such as the shrimp, crab, crayfish, and lobster. Consequently, some of these genes may also express a large form of vitellogenin precursors, and in the ovary as well. Thus, it is too early to conclude that the hepatopancreas is the major source of vitellogenesis in crustacean. Furthermore, the process of vitellogenin synthesis can only be studied when the expression patterns of different vitellogenin genes are known. In conclusion, the findings in the present study from Northern blot analysis, immunochemical analysis, and in vitro incubation suggest that the hepatopancreas produces MeVg2 small transcripts and MeVg2 subunits that are important for the contribution of vitellin biosynthesis in M. ensis.
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FOOTNOTES |
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2 Correspondence: Siu-Ming Chan, Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong. FAX: 852 2857 4672; chansm{at}hkucc.hku.hk
Received: 6 October 2003.
First decision: 31 October 2003.
Accepted: 21 April 2004.
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