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BOR - Papers in Press, published online ahead of print April 2, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.011627
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BIOLOGY OF REPRODUCTION 69, 355–364 (2003)
DOI: 10.1095/biolreprod.102.011627
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Relationship Between Vitellogenin and Vitellin in a Marine Shrimp (Penaeus semisulcatus) and Molecular Characterization of Vitellogenin Complementary DNAs1

Jean-Christophe Avarre4, Regina Michelis3,4, Alisa Tietz5, and Esther Lubzens2,4

Israel Oceanographic and Limnological Research,4 Haifa 31080, Israel Department of Neurobiochemistry,5 Tel Aviv University, Tel-Aviv 66978, Israel


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The source of yolk proteins in crustacean ovaries has been the subject of controversy for several decades, and both extraovarian and intraovarian synthesized proteins have been implicated. To offer a new insight into the relationship of vitellogenin (VTG) and vitellin (VT), a comparison of extraovarian VTG and ovarian VT of the marine shrimp Penaeus semisulcatus was performed at the protein and cDNA levels. Two cDNAs (7920 and 2068 nucleotides [nt]) were sequenced for VTG from the ovary and one cDNA (7920 nt) was sequenced from the hepatopancreas. VTG cDNA from hepatopancreas was similar to VTG cDNA from ovary. Although a VTG gene was also found in the males, ~7.8-kilobase transcripts were only detected in the ovary and hepatopancreas of females. The mRNA expression pattern was related to the stage of ovarian development and to the molt cycle, as determined by real-time polymerase chain reaction assay. VTG and VT apoproteins were composed of two and three major subunits, respectively, as shown by SDS-PAGE. N-terminal sequences of these subunits revealed the presence of a cleavage site at a consensus motif for a subtilisin-like endoprotease in VTG and VT and an additional cleavage site in VT revealed by an unidentified endoprotease. These results indicate that penaeid shrimps constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.

oocyte development, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The major egg yolk protein, vitellin (VT), is a high-density lipoglycoprotein frequently associated with carotenoid pigments. In 1969, Kerr [1] demonstrated in the blue crab (Callinectes sapidus) the occurrence of a blood-borne protein present only in females with developing oocytes. This lipoprotein turned out to be serologically identical to the oocyte VT and was named vitellogenin (VTG). VTG found in the hemolymph of most crustacean species studied to date is immunologically identical to VT [2–8]. Therefore, various researchers have suggested that, as in other organisms producing yolk-laden eggs, the crustacean VTG is synthesized by extraovarian tissues and then transported via the hemolymph to the developing oocytes, where it is sequestered and subsequently forms the VT [3, 8–12].

In most oviparous animals, VTG is synthesized by the liver (nonmamalian vertebrates), fat body (insects), or intestine (nematodes). Most insect VTGs are derived from cleavage of a single precursor protein in the fat body and after extensive processing are secreted into the hemolymph. VTGs are sequestered by specific receptors on the ovarian surface and stored in intracellular deposits known as yolk platelets. Once inside the oocyte, the VTG of nonmamalian vertebrates is processed into smaller yolk proteins consisting of lipovitellins (LV1 and LV2), phosvitins, and phosvettes, which may in turn be degraded into even smaller cleavage products [13, 14].

The source of yolk proteins in crustacean ovaries has been a subject of controversy for many years. Whereas the hepatopancreas was considered the main site for VTG synthesis [15–17], substantial intraovarian de novo synthesis of yolk protein has been demonstrated for several species [18–25], including Penaeus semisulcatus [23]. The origin of VTG in the hemolymph of vitellogenic females has remained elusive, with a suspected contribution from the hepatopancreas and ovaries [26–28], thus challenging to some extent the original definition coined for VTG [29].

Although the understanding of gene structure and regulation of synthesis of VTG in oviparous vertebrates and insects has increased dramatically over the last two decades [reviewed in 13, 30, 31], information about VTG in crustaceans is still scarce and often speculative. Even though many studies have been dedicated to the apoprotein structure of crustacean VTG and VT over the past 30 years, many contradictory results have been obtained, even within a single species [32–34]. Recent use of molecular biology techniques have brought new insights into this debate. Four cDNA encoding crustacean VTG were recently obtained from the shrimps Penaeus japonicus [35] and Metapenaeus ensis (GenBank accession AF548363, 2002), the crayfish Cherax quadricarinatus (accession AF306784, 2000) [36], and the caridean prawn Macrobrachium rosenbergii [37]. Tsukimura [8] and Tseng et al. [38] also obtained a cDNA fragment encoding VTG from Sicyonia ingentis and Penaeus monodon, respectively. These studies showed that expression of VTG and VT in crustaceans occurs in the hepatopancreas and the ovary. However, the exact contribution of each of those tissues to the final form of ovarian VT remains unclear. Likewise, the exact relation between VTG and VT has yet to be resolved. Because species-specific differences have been suggested to account for variable and confusing results [39], there is a need for studies from various species to establish more coherent model(s) for crustacean vitellogenesis.

In the present study, we compared the apoprotein profiles of VT from the ovary and VTG from the hemolymph of the shrimp P. semisulcatus. For the sake of clarity, here we define VTG as the extraovarian protein isolated from hemolymph and VT as the intraovarian protein. The genes, cDNAs, and mRNAs from hepatopancreas and ovary are referred to as VTG. Our results show that 1) the protein profile of hemolymph VTG differs from that of ovarian VT; 2) the VTG cDNAs from the hepatopancreas and from the ovary are probably products of the same gene; 3) VTG mRNA is expressed in both the hepatopancreas and ovary of females but not in the male hepatopancreas, although a VTG gene is present in males; and 4) VTG mRNA expression varies during the cycle of ovarian development and during the molt cycle. Our results support the hypothesis that penaeid shrimps constitute a unique model for vitellogenesis.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Shrimps (P. semisulcatus de Haan) were collected in the Haifa Bay (Israel) and kept in 3000-L circular aerated tanks as described previously [28]. Four distinct categories of females were distinguished according to their stage of ovarian development and molt: previtellogenic immediately following a molt (postmolt, average oocyte diameter [AOD] {approx} 100 µm), previtellogenic (AOD < 100 µm), vitellogenic (AOD = 100–300 µm), and late vitellogenic (AOD > 300 µm) during the intermolt period.

Isolation of VTG and VT

Hemolymph was withdrawn and the ovary was removed from a vitellogenic female (AOD ~ 370 µm). VTG from hemolymph and VT from ovary were isolated by NaBr (density = 1.22 g/ml) followed by sucrose gradient (5–25%) ultracentrifugations according to the procedure described by Lubzens et al. [40]. Protease inhibitors (Complete, Roche Molecular Biochemicals, Mannheim, Germany) were present during all the isolation steps.

Characterization of VTG and VT Subunits

VTG and VT fractions isolated by ultracentrifugation from hemolymph and ovary, respectively, were subjected to 7.5% SDS-PAGE under reducing conditions. Proteins were either stained with Coomassie brilliant blue R-250 or transferred onto nitrocellulose membranes for Western blot analysis using an antiserum raised against the native form of VT protein [23]. Membranes were incubated with an alkaline phosphatase-conjugated antibody (Sigma, St. Louis, MO) and bands were revealed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Sigma).

N-Terminal Amino Acid Sequence Analysis

The VTG and VT were transferred onto polyvinylidene difluoride membranes (0.45 µm pore size). Bands were excised from the blot and sent for amino acid sequence analysis (Unité de Microséquençage, IBGC, Université Bordeaux II, Bordeaux, France).

Molecular Cloning of VTG cDNA from Ovary

Construction of an ovarian expression cDNA library Because it is difficult to separate oocytes from the connective tissues in ovaries and there was no clear indication of the ovarian tissue involved in VT mRNA expression, a cDNA library was constructed from the whole organ. Total RNA was extracted from a vitellogenic ovary (AOD ~300µm) with QuickPrep Total RNA Extraction kit (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Poly(A+) RNA was purified from the total RNA with a PolyATtract kit (Promega, Madison, WI) according to manufacturer's directions. A cDNA library was constructed in {lambda}-ZAP vector (Uni-ZAP XR) using a Stratagene (La Jolla, CA) kit, according to the manufacturer's instructions.

Screening of the library The library was screened using an affinity-purified serum raised against an ~80-kDa subunit of P. semisulcatus VT. After three rounds of screening, three clones of 2068, 1757, and 890 base pairs (bp) were isolated. Their sequences (Sequencing Unit, Tel Aviv University, Israel) revealed that they all shared an identical 3' end. Comparison of their deduced amino acid sequence with those in GenBank showed that they aligned with the carboxy-terminal of the deduced amino acid sequence of VTG from P. japonicus [35]. Attempts to obtain additional clones from this library, related to VTG, by screening with a probe designed from the obtained sequence were not successful.

Reverse transcription Total RNA from a late-vitellogenic ovary (AOD ~550 µm) was extracted as described above. For reverse transcription (RT), 10 µg of total RNA was mixed with 0.1 µg of oligo-dT (Promega) and adjusted to 17 µl with water. RNA was denatured at 70°C for 5 min and immediately cooled on ice. Five microliters of Moloney murine leukemia virus (MMLV) 5x buffer (Promega) was added with 500 µM dNTPs, 25 U of rRNasin (Promega), and 200 U of MMLV Reverse Transcriptase (Promega), to reach a final volume of 25 µl. Samples were mixed and incubated for 1 h at 42°C. Seventy-five microliters of water was added to stop the reaction, and cDNA was stored at -20°C.

Obtaining the full-length VTG cDNA by the "gene walking" strategy As a first step, a set of degenerated primers (sense, 5'-gcnccntggggngcnga-3'; antisense, 5'-ctcgggytggtaggcca-3') was designed from an N-terminal amino acid sequence previously obtained from an ~80-kDa subunit of P. semisulcatus VT (unpublished data) and from an N-terminal amino acid sequence obtained from P. japonicus [35]. A polymerase chain reaction (PCR) with this primer pair was carried out in a 25-µl reaction mixture containing 1.5 mM MgCl2, 1x PCR buffer (Promega), 2 µM of each primer, 200 µM of each dNTP, 0.5 µl ovarian cDNA, and 1 U of Taq DNA polymerase (Promega) under the following conditions: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 52°C for 1 min, and extension at 72°C for 1 min, and a final elongation at 72°C for 10 min. PCR amplification produced an 81-bp product whose sequence aligned with the 5' initial sequence of VTG cDNA from P. japonicus. Two exact primers were subsequently designed for further hot-start PCR: a forward primer according to the newly obtained 5' sequence (corresponding to nucleotides 84–101; accession AY051318, GenBank/EBI, 2001) and a reverse primer according to the 5' extremity of the 2068-nucleotide (nt) clone 1 sequence (corresponding to nt 6104–6124). The PCR mixture consisted of 5 µl 10x buffer (Qiagen, Alameda, CA), 200 µM dNTPs, 2 µM each primer, 1 µl cDNA, and 2.5 U of Taq DNA polymerase (Qiagen) in a final volume of 50 µl. A PCR was run under the following conditions: initial denaturation at 94°C for 2 min, followed by 10 cycles of denaturation at 94°C for 10 sec, annealing at 56°C for 1 min, and extension at 72°C for 6 min, and then another 25 cycles of denaturation at 94°C for 10 sec, annealing at 56°C for 1 min, and extension at 72°C for 6 min, with 10 sec added at each cycle. A PCR product of the expected length (6041 bp) was sequenced at both ends, and from these sequences new primers were designed and used for a new round of PCR. A shorter product was then obtained, its extremities were sequenced, and again new primers were designed and new rounds of PCR performed. The final 3' region (section VI, Fig. 1), corresponding to clone 1, was also sequenced by a series of PCRs using primers designed according to the clone 1 sequence.



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  		FIG. 1. Cloning strategy and schmematic view of P. semisulcatus VTG from ovary. The obtained cDNA is represented by the thick black line, and positions of the start codon (30 ATG) and the stop codon (7791 TAG) are indicated. A polyclonal antibody was used to screen the cDNA library, and three clones (C1, C2, and C3) were obtained. A PCR with ovarian cDNA, a forward primer designed from an N-terminal amino acid sequence previously obtained, and a reverse primer generated according to the 5' extremity of the clone 1 produced a 6041-bp product. From partial sequences of this product, the full sequence was obtained after seven rounds (I–VII) of PCR. The 5' extremity of VTG cDNA was obtained by 5'-RACE

5' RACE The 5' extremity was obtained with the 5' RACE system (rapid amplification of cDNA ends) version 2 (Gibco BRL, Life Technologies Ltd., Paisley, U.K.), according to the manufacturer's instructions. First strand cDNA synthesis was performed at 50°C with a reverse primer corresponding to nt 841–859 (5'-gaagggatgccgctcatgg-3'), and PCR was carried out as described above with an abridged anchor primer (5'-ggccacgcgtcgactagtacgggiigggiigggiig-3') and a nested primer corresponding to nt 148–164 (5'-gtcgggttggtaggcca-3').

Sequencing of VTG cDNA from Hepatopancreas

Total RNA was extracted from the hepatopancreas of a vitellogenic female (AOD ~300 µm), and RT was performed as described above. Using specific primers designed according to the cDNA sequence of ovarian VTG, several PCRs were carried out under the same conditions as described above. PCR products were sequenced, and the sequence corresponding to nt 1–7920 of VTG cDNA was obtained.

Sequence Analysis

Gene Runner version 3.00 (1994, Hastings Software, Inc., Hudson, NY) was used to analyze the obtained sequences. Search for sequence similarities was performed with the Basic Local Alignment Search Tool (BLAST) algorithm (http://www.ncbi.nlm.nih.gov/blast).

Northern Blot Analysis

Total RNA was extracted with Trizol (Gibco BRL) from 50-mg tissue fragments collected from a vitellogenic female (AOD ~275 µm) and a male. Ten micrograms of RNA from each sample was loaded on 1% agarose gels containing formaldehyde and transferred onto Hybond N membranes (Amersham). Northern blotting was performed with a VTG insert of 81 bp corresponding to nt 84–164 of the cDNA and with an 18S rDNA insert from Penaeus aztacus (provided by Dr. Trisha Spears, Florida State University, Tallahassee, FL). Inserts were labeled with {alpha}-32P-dCTP by random priming with the Neblot kit (New England BioLabs, Beverly, MA) and used as probes. Hybridizations were carried out overnight at 65°C for rDNA in a buffer consisting of 5x saline sodium citrate (SSC), 5x Denhardt, and 0.5% SDS and at 42°C for VTG in a buffer containing 50% formamide, 6x SSC, 1x Denhardt, and 0.4% SDS. Membranes were exposed to x-rays for several hours to several days.

VTG in Males

DNA from hepatopancreas of males and females was extracted with Genomic Prep Cells and Tissue DNA Exctraction kit (Amersham) according to the manufacturer's instruction. A PCR was performed using a pair of primers corresponding to nt 5033–5051 and 6104–6124 on the sequence of VTG cDNA. PCR products were electrophoresed on a 1% agarose gel containing ethidium bromide and were sequenced to confirm that they corresponded to VTG.

Real-Time PCR

To compare VTG gene expression in different samples, the relative abundance of VTG mRNA was normalized to the amount of an endogenous reference, the 18S subunit of rRNA, by the comparative threshold cycle (CT) method. The relative amount of VTG mRNA was calculated by the formula 2-{Delta}CT, where {Delta}CT corresponds to the difference between the CT measured for VTG and the CT measured for 18S rRNA. To validate this method, serial dilutions were prepared from an ovarian cDNA sample (1, 0.5, 0.2, 0.1, 0.05, 0.02, and 0.01), and the efficiencies of VTG amplification and 18S rRNA amplification were compared by ploting {Delta}CT versus log(template dilutions), according to the method described by PE Applied Biosystems (Perkin Elmer, Foster City, CA). Linear regressions of the plots showed the following R2 values and slope, respectively: 0.99 and -3.17 for 18S rRNA and 0.99 and -3.15 for VTG. The slope difference was 0.02, which further validated the use of the {Delta}CT method, because the amplification efficiency of VTG and 18S rRNA could be considered similar.

Total RNA was isolated from ovary and hepatopancreas with the RNeasy mini kit (Qiagen). RT was carried out as described previously, with an oligo-dT primer for VTG and an antisense primer for 18S rRNA (2 µM). For VTG, primers for PCR were designed to the 3' end of VTG cDNA, so that potential partial cDNAs (resulting from incomplete RT) were also taken into consideration in the quantification. These primers amplified a 201-bp product and corresponded to nt 6874–6893 (5'-gagaaggcaacggcattgaa-3') and 7051–7074 (5'-gcacgtccgttatgtagtgtggta-3') (accession AY051318). Primers for 18S rRNA (sense, 5'-cgctagtcagcatcgtttaaggt-3'; antisense, 5'-tcgtgcatggaatgatggaa-3') amplified a 251-bp product. The PCR mixture consisted of 1 µl of cDNA sample, 300 nM of each primer, and 12.5 µl of Syber green Master mix (PE Applied Biosystems) in a final volume of 25 µl. Amplification was carried out in a GenAmp 5700 thermocycler (Perkin Elmer) under the following conditions: initial denaturation at 94°C for 10 min, followed by 40 cycles of denaturation at 94°C for 15 sec and annealing-extension at 60°C for 1 min, and then a final extension at 60°C for 20 min. Amplification of VTG and 18S rRNA cDNAs was performed simultaneously in separate tubes and in triplicate, and results were analyzed with the ABI Prism 7700 Sequence Detection System version 1.6 software (PE Applied Biosystems).

Statistical Analysis

Statistical analyses were performed using the Student t-test and Tukey test for variability (SAS Institute, Cary, NC). Values were significantly different at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Hemolymph VTG and Ovarian VT

The VTG and VT fractions isolated from hemolymph and ovary of a vitellogenic female, respectively, were subjected to SDS-PAGE under reducing conditions. Coomassie blue staining (Fig. 2A) showed different profiles for the hemolymph and ovary: two protein bands of ~74 and ~199 kDa were visible in hemolymph, whereas three major bands of ~72, ~79, and ~100 kDa and a faint band of ~207 kDa were present in ovary. An antibody raised against the native form of VT recognized four bands in the ovary, corresponding to those observed by protein staining (Fig. 2B). The antibody recognized a weak band of ~105 kDa in the hemolymph VTG fraction (Fig. 2B) that was absent from the Coomassie blue-stained gel (Fig. 2A). N-terminal amino acid sequences were determined for each Coomassie blue-stained band (bands 1–6, Fig. 2A), and these are presented in Table 1. Band 1 from hemolymph and band 3 from ovary displayed an identical sequence (APWGADL). Three bands, band 2 from hemolymph and bands 5 and 6 from ovary, displayed a similar N-terminal sequence corresponding to SIDSSV. Band 4 from the ovary showed one major signal (VQYTVAEK) and a minor signal (MNHPVLPK). The identity of the N-terminal sequence of the weak band revealed by the antibody in hemolymph (Fig. 2B) could not be determined because of its low abundance.



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  		FIG. 2. Subunit composition of P. semisulcatus hemolymph VTG and ovarian VT. Fractions corresponding to VTG and VT were electrophoresed on 7.5% SDS-polyacrylamide gels under reducing conditions and stained with Coomassie blue (A) or electrotransferred and probed by an antibody raised against the native form of VT (B). Lane 1: hemolymph fraction; lane 2: ovarian fraction. Numbers 1–6 indicate subunit bands whose N-terminal sequences are presented in Table 1. The sizes (kDa) of the molecular standards (Bio-Rad, Richmond, CA) are indicated on the left


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  		TABLE 1. N-terminal sequences and apparent molecular mass of bands 1–6 from Figure 2

Molecular Cloning and Characterization of VTG cDNA

The cDNA sequence of VTG from ovary obtained by RT-PCR, consisted of 7920 nt (GenBank/EBI accession AY051318, 2001). It included a 5' untranslated region (UTR) of 29 nt and a 7764-nt open reading frame (Fig. 3). The coding region translates into a protein of 2587 amino acids, starting with a predicted signal peptide of 18 amino acid residues (-18 to -1) followed by an N-terminal sequence determined by amino acid sequencing (Fig. 3B). The predicted molecular mass of the mature protein is 283 352 daltons with a pI of 6.44. The protein sequence displays one asparagine glycosylation site situated at position 1112, and the most prominent amino acids are alanine (11.40%), glutamate (8.81%), valine (8.70%), and isoleucine (7.38%) (Fig. 3B). The best alignment scores of the deduced amino acid sequence of P. semisulcatus were obtained with P. japonicus VTG (79% identity), C. quadricarinatus VTG (43% identity), and M. rosenbergii VTG (36% identity). The 2068-nt sequence of clone 1 obtained by library screening (Fig. 1; GenBank/EBI accession AY137466, 2002) included a polyadenylation signal and a poly(A) tail (Fig. 3C).



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  		FIG. 3. Deduced amino acid sequence and 5' and 3' UTRs of P. semisulcatus ovarian VTG cDNA. A) Nucleotides 1–122 of VTG cDNA, containing the 5' UTR (nt 1–29) and the beginning of the coding region (nt 30–122) with its deduced amino acid sequence (-18 to 13). B) The deduced amino acid sequence of VTG cDNA. The putative signal sequence is in italics. Sequences confirmed by peptide sequencing are underlined. The unique putative N-glycosylation site (1112) is underlined and in bold. A consensus cleavage motif for endoproteases of the subtilisin family is shown in a black box. The arrow indicates the beginning of the amino acid sequence deduced from clone 1 cDNA, which aligns with the presented sequence. C) Nucleotides 7740–7920 of VTG cDNA obtained by PCR and nt 7912–7956 of VTG cDNA obtained from clone 1, containing the end of the coding region (nt 7740–7793) with its deduced amino acid sequence, including the stop codon, and the 3' UTR (nt 7794–7956). The stop codon is in bold, and the polyadenylation signal is underlined

The cDNA sequence of VTG from hepatopancreas also consisted of 7920 nt. It differed from the sequence of VTG cDNA from ovary by 23 nt spread all along the sequence, representing a difference of 0.29%. Considering the potential error that may result from the polymerase elongation and from the sequencing reaction on single strand, this observed error is in an acceptable range [41, 42], and it is thus very likely that the two cDNAs from ovary and hepatopancreas are identical.

Protein Structure of VTG and VT

The N-terminal sequences (Table 1) of VTG and VT isolated from hemolymph and ovary, respectively, were located on the deduced amino acid sequence presented in Figure 3. A cleavage site between amino acid residues 710 and 711 is suggested from the N-terminal sequence of bands 2, 5, and 6 (SIDSSV). This sequence is preceded by a paired basic motif RTRR (Fig. 3), which corresponds to a consensus motif (RX[K/R]R) recognized by subtilisin-like endoproteases [43, 44]. The N-terminal sequence of band 4 indicates an additional cleavage site between nt 1795 and nt 1796 in the VT isolated from ovary. The full amino acid sequence is accounted by the proteins bands 1 and 2 visualized in hemolymph and the protein bands 3, 4, and 5 visualized in the ovary.

Expression of VTG

VTG transcripts (~7.8 kilobases) were detected by Northern blot analyses in the ovary and hepatopancreas from females (Fig. 4, lanes 4 and 5, respectively) but not in the heart, muscle, or male hepatopancreas (Fig. 4, lanes 1–3).



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  		FIG. 4. Expression of VTG mRNA in various P. semisulcatus tissues. Northern blot analysis was performed with total RNA, and the membrane was succesively hybridized with a VTG probe (A) and an rDNA probe (B). Lane 1: heart; lane 2: muscle; lane 3: male hepatopancreas; lane 4: ovary; lane 5: hepatopancreas. Molecular mass markers are indicated on the left

VTG in Males

PCR using genomic DNA as a template indicated that male P. semisulcatus contains a gene encoding VTG (Fig. 5). The genomic fragment obtained from male hepatopancreas was sequenced (data not shown) and aligned with the region of VTG cDNA corresponding to nt 5058–6092. This region also included an intron of ~120 bp located between nt 5614 and nt 5615.



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  		FIG. 5. PCR amplification of P. semisulcatus genomic DNA. Genomic DNA was extracted from hepatopancreas of a female and a male, and PCRs were performed with a pair of specific primers designed according to the ovarian VTG cDNA sequence. Lane 1: female DNA; lane 2: male DNA; lane 3: negative control (water). Approximate size of amplification products is indicated on the left

Quantitative Estimation of VTG Transcripts During Vitellogenesis

The occurrence of VTG transcripts was determined by real-time PCR for ovaries and hepatopancreas removed from females at various stages of ovarian development (Fig. 6). The quantity of VTG mRNA was significantly lower (P = 0.0316) in previtellogenic ovaries removed from females immediately after molt than in ovaries from vitellogenic, late vitellogenic, and previtellogenic females during intermolt. In hepatopancreas, the amount of VTG mRNA was extremely low in postmolt previtellogenic females, high in vitellogenic and late-vitellogenic females, and reduced in intermolt previtellogenic females. Significant differences in mRNA levels were found for hepatopancreas removed from vitellogenic, late-vitellogenic, and previtellogenic females (P = 0.0411). In intermolt previtellogenic females, the relative level of mRNA in ovaries was six times higher than that of mRNA in hepatopancreas (P = 0.0336). However, the relative amount of transcripts was nearly the same in the ovaries and hepatopancreas from vitellogenic and late-vitellogenic females.



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  		FIG. 6. VTG mRNA levels in P. semisulcatus ovary and hepatopancreas during a full cycle of ovarian development. Ten micrograms of total RNA was reverse transcribed, and a 1/100 fraction of the reaction was used for real-time PCR. The relative abundance of VTG mRNA was normalized to the amount of the 18S subunit of rRNA by the CT cycle method, where 2-{Delta}CT reflects the relative amount of VTG transcripts. Results are the average ± SD of four to seven animals at each stage. *Significant difference (t-test, P ≤ 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The cloning and sequencing of VTG cDNAs from the hepatopancreas and the ovary in a single species enabled the first detailed comparison of the protein products from these two tissues. Comparison of the deduced amino acid sequences with the N-terminal sequences assisted in elucidating the subunit composition of VTG and VT and in clarifying the relationship between them.

Although the primary structure of VTG products in hepatopancreas and ovary seems to be identical, thereby explaining the serological identity between VTG and VT always reported for crustacean species [6], the major difference between VTG and VT seems to lie in their subunit composition. The hemolymph VTG is composed of two main subunits (~199 and 74 kDa), whereas the ovarian VT is composed of three major subunits (~100, 79, and 72 kDa) and a minor one (~207 kDa). Western blot analysis revealed the presence in hemolymph of a weak band (~105 kDa) that was not visualized by Coomassie blue staining. Resorbtion of oocytes was described as a common phenomenon during every cycle of ovarian development in captive penaeid females [45]. Therefore, this weak band may have originated from resorbing oocytes. Differences in the profiles of hemolymph VTG and ovarian VT also have been reported for several penaeid species [32, 34, 46–48]. The subunit molecular masses observed on the Coomassie blue-stained gel were in the range of those calculated from the deduced amino acid sequence (Fig. 7A).



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  		FIG. 7. Schematic representation of the structure of VTG and VT and proposed model for vitellogenesis in penaeid species. A) VTG is composed of two subunits, whereas VT is composed of three subunits. Calculated molecular masses from the deduced amino acid sequence of the resulting subunits are indicated and compared with those of the corresponding subunits observed on the Coomassie blue-stained gel in Figure 2A. B) A gene encoding VTG is transcribed in the hepatopancreas and the ovary, and the corresponding mRNA is translated into the precursor protein. In hepatopancreas, the precursor protein is cleaved between positions 710 and 711 to give rise to two subunits that are excreted into the hemolymph; whether hemolymph VTG is taken up by the oocytes and cleaved a second time is still unknown. In ovary, there are two possible pathways: 1) the precursor protein undergoes two cleavages in the follicle cells and is directly transferred to the oocytes; and 2) the precursor protein undergoes a first cleavage between positions 710 and 711 in the follicle cells and a second cleavage between positions 1795 and 1796 in the oocytes. Whether ovarian VTG is first released into the hemolymph to be further taken up by the oocytes and cleaved a second time also remains unknown

Because the full-length protein was never detected and the N-terminal sequence APWGADLP was only found in bands 1 and 3, the cleavage occurring between positions 710 and 711 is likely to take place immediately after the translation of VTG mRNA by a subtilisin-like endoprotease that must be present in the hepatopancreas and in the ovary. In contrast, the sequence VQYTVAEK corresponding to position 1796 was only present in the ovarian protein, suggesting that the cleavage between positions 1795 and 1796 occurs inside the ovary and that the enzyme responsible for this cleavage is not present, or at least not active, in hepatopancreas or hemolymph. The weak band at position 4 (Fig. 2A) with an N-terminal sequence consisting of MNHPVLPK may also indicate that the enzyme responsible for this second cleavage recognizes a broader motif. The presence of band 6 in the ovary, although weak, perhaps indicates that this cleavage does not take place immediately after the translation of VTG mRNA. Previous Western blot analyses performed with the same antibody used in this study have already revealed that band 6 is always present, although it is weaker than other bands, in vitellogenic and late-vitellogenic ovaries [40, 49]. In the present study, conditions were optimized for separating band 3 from band 4, which previously appeared as one protein band, explaining the weaker appearance of band 6 compared with previous studies.

For the freshwater prawn M. rosenbergii, Okuno et al. [37] showed that VTG is composed of two subunits in females at early stages of vitellogenesis and of four subunits in females at late stages of vitellogenesis. VTG is cleaved at the RXRR consensus cleavage site (between positions 710 and 711) for a subtilisin-like endoprotease to form two subunits, and this cleavage also seems to occur immediately after translation of VTG mRNA or at least before its excretion to the hemolymph. A second cleavage is assumed to take place in hemolymph by an enzyme of unknown identity to yield three subunits that are presumably sequestered by the ovary, resulting in three yolk proteins. This second cleavage yields a protein with an N-terminal sequence (RREEQKVTGTVELDI) aligning with subunit 4 (——-GT-ELDI, Fig. 3) but located three amino acid residues downstream of the cleavage site of P. semisulcatus. Okuno et al. [37], however, did not think that the second cleavage takes place in the ovary nor did they consider the occurrence of an endogenous source of ovarian VTG, as was recently suggested [50]. In M. rosenbergii, there are two additional paired basic motifs for subtilisin-like endoproteases, at positions 1742–1745 and 2162–2165. In P. japonicus, a second paired basic motif, RSRR, is located at position 1094–1097. However, no cleavage occurs at these positions, and these motifs were not found in the sequence of P. semisulcatus.

Information on VTG expression during ovarian development in crustaceans is confusing and incomplete. For P. japonicus, Tsutsui et al. [35] detected by Northern blot analysis VTG transcripts in the hepatopancreas and ovary, and VTG mRNA levels along a cycle of ovarian development were always higher in the hepatopancreas than in the ovary. These results, however, were not related to the molting stage. They also showed, by in situ hybridization, that ovarian VTG was exclusively expressed in the follicle cells and hepatopancreatic VTG was expressed in the "parenchyme" cells (of unspecified type). For the freshwater prawn M. rosenbergii, Yang et al. [51] found by Northern blot analysis that VTG mRNA was only expressed in the female hepatopancreas, although they detected a weak signal in ovaries by RT-PCR. In the same species, Chan [50] later claimed that in contrast to previous reports, ovary was also expressing VTG mRNA. Our experience indicates that the results obtained by Northern blot analysis are difficult to interprete, because the signal of VTG mRNA does not always appear as one clear band (Fig. 4) (for illustration in other animal groups, see [52, 53]). The use of real-time PCR for expression studies enabled this problem to be overcome and permitted quantitative evaluation of VTG transcripts in a more reliable and accurate way.

In P. semisulcatus, the hepatopancreas and ovary are involved in the expression of VTG mRNA (Fig. 6). In previtellogenic females, VTG mRNA levels in hepatopancreas were significantly different from VTG mRNA levels in ovary. Moreover, VTG mRNA levels in ovary and hepatopancreas of previtellogenic females with ovaries of the same diameter differed significantly according to the molting stage. The levels of VTG in the hemolymph reflected a similar pattern [54]. Considering that P. semisulcatus spawns two or three times within a single molt cycle [45], these results suggest upregulation of VTG after a molt and differential modulation of its expression during the intermolt period, possibly by one or more hormonal systems [55]. The regulation of VTG expression remains, however, totally unknown.

In most species studied to date, including vertebrates, insects, and nematodes, VTGs are synthesized in an extraovarian tissue. One of the better known exceptions is the higher Diptera, including Drosophila melanogaster, where the yolk proteins are synthesized in the fat body and the follicular cells [13, 56–58]. However, these yolk proteins are evolutionarily unrelated to VTGs [57, 59]. Additional exceptions have been reported for other insect species [60]. However, the uptake of VTGs and of the yolk precursor protein of Drosophila [61, 62] into growing oocytes follows a similar pathway in most species studied to date. If the mosquito is taken as a model species, VTG enters between the follicle cells and through pores of the VT envelope, binds its receptor located on the oocyte membrane, and is internalized into the cytoplasm [14]. Studies on P. japonicus indicate that the pathway for penaeid VTG is probably similar, except that VTG may originate either from the hemolymph or from the follicle cells, where it seems to be synthesized [35, 63]. A VTG-specific receptor has been found for several crustacean species: M. rosenbergii [64], Homarus americanus [65], Orconectes limosus [66], and the crab Scylla serrata [12].

VTGs of invertebrates are phosphorylated to a lesser degree than are those of vertebrates; the polyserine, or phosvitin domain, is generally smaller or absent [31]. Sequences of crustacean VTGs show that they lack this polyserine domain. Postendocytotic phosphorylation may occur in ovaries of some insect groups, suggesting that phosphorylation does not seem to be directly linked to the site of VTG synthesis [13, 67–69]. Vertebrate VTGs are processed by cathepsins D after internalization by oocytes [70, 71]. Comparison of known cleavage sites of vertebrate VTGs indicates that surrounding sequences are not similar and do not contain a paired basic motif [31] that is characteristic of the cleavage of insect VTGs occurring in the fat body. Our results suggest that penaeid species, and possibly crustaceans in general [50], appear as another exception in the model for synthesis of yolk proteins. Unlike dipteran yolk proteins, crustacean VTGs seem to be related to the VTG gene family (unpublished results).

Although it was not expressed in male hepatopancreas, VTG is present in males (Fig. 5). This feature is common to all oviparous vertebrates, and it may explain the results obtained by Wilder et al. [72] for M. rosenbergii. The presence of VTG in crustacean males may be extremely useful for future studies on vitellogenesis regulation, which has yet to be resolved for crustacean species. VTG in male crustaceans may also serve as a biomarker for xenobiotic compounds in the environments, as is the case in some fish species [73, 74].

In the light of the results presented here, a simple model for synthesis and processing pathways of the main egg yolk lipoprotein in penaeid species is proposed (Fig. 7B). A gene encoding VTG is transcribed in the hepatopancreas and the ovary, and the corresponding mRNA is translated into the precursor protein. This precursor protein first undergoes a cleavage between positions 710 and 711, just after translation of its mRNA, by a subtilisin-like endoprotease that must be present in both organs. At this stage, the protein can be referred to as VTG. Whereas VTG from hepatopancreas is released into the hemolymph and remains in this form, VTG in ovary undergoes a second cleavage between positions 1795 and 1796, which probably occurs with a certain delay. At this stage, there still are two possibilities: 1) the second cleavage occurs in the producing cells (i.e., the follicle cells [35, 63]) and VT is transferred to the oocytes; or 2) the second cleavage occurs in the oocytes and VTG is transferred to the oocytes to be later cleaved into VT. Whether ovarian VTG is first released into the hemolymph to be further taken up by the oocytes and then cleaved into VT remains unknown. Penaeid shrimps thus constitute a unique model for vitellogenesis because of their intraovarian expression and synthesis of VTG and because plasmatic VTG is not definitely the precursor of VT.


   ACKNOWLEDGMENTS
 
We thank Prof. H. Nagasawa for extensive discussions on the topic of the manuscript and for initial results on the N-terminal amino acid sequence of VT peptides, Mrs. Fidi Kopel for the statistical analyses, and Dr. T. Spears (Department of Biological Sciences, Florida State University, Tallahassee, FL) for a generous gift of rDNA. We also thank Prof. P. Babin (Génomique et Physiologie des Poissons, USC INRA, Université Bordeaux 1, Talence, France) for critical reading of the manuscript and his comments and suggestions and Prof. R.A. Wallace for his advice on the use of the term VTG in the manuscript. This publication is dedicated to Prof. R. Keller, on his official retirement, in recognition of his compelling contribution to the study of crustacean neuropeptides.


   FOOTNOTES
 
1 This study was supported by a European Union project (FAIR CT-97-3660). Back

2 Correspondence: Esther Lubzens, Israel Oceanographic and Limnological Research, Tel-Shikmona, P.O. Box 8030, Haifa 31090, Israel. FAX: 972 4 511 911; esther{at}ocean.org.il Back

3 Current address: Eliachar Research Laboratory, Western Galilee Hospital, Nahariya 22100, Israel Back

Received: 26 September 2002.

First decision: 28 October 2002.

Accepted: 10 March 2003.


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 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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