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Regular Article |
a Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103
b Scandinavian QC Laboratories, Medicinaregatan 3A, SE-413 46 Göteborg, Sweden
c Department of Cell and Molecular Biology, Unit of Physiology, Umeå University, SE-901 87 Umeå, Sweden
d Roche Institute of Molecular Biology, Hoffman-LaRoche, Nutley, New Jersey 07110
e Gemini Consulting, D-61352 Bad Homburg, Germany
ABSTRACT
All vertebrate eggs are surrounded by an extracellular envelope that protects the egg and is vital for a successful fertilization. The terminology and functions of the egg envelope vary in different vertebrate groups, but the envelope itself is consistently composed of a few major proteins that are deposited around the oocyte during oocyte growth. Here, we describe the deduced amino acid sequences and tissue expression patterns of the three major egg envelope proteins for rainbow trout (Oncorhynchus mykiss). All three vitelline envelope proteins (VEPs) are expressed in the livers of both male and female fish, with higher expression in females. In addition, VEP
mRNA is also detected in the female gonads. To our knowledge, this is the first time that expression of a VEP protein gene has been demonstrated to occur in more than one organ. Sequence comparison reveals that all three VEP proteins share distinct homology with their amphibian, avian, and mammalian counterparts. Whereas mammalian zona pellucida protein 3 isoforms contain two conserved serines needed for sperm binding, these are not conserved in teleost species, in which sperm entry is restricted to the micropyle. Besides the difference in VEP sperm-binding function, the high sequence homology suggests that the egg envelope proteins from these distinct vertebrate groups share a common ancestry and form a unique group of structural proteins.
embryo, estradiol, gamete, biology, gene regulation, oocyte development
INTRODUCTION
During growth of the ovarian follicle, all vertebrate oocytes become surrounded by an acellular coat, an egg envelope [1]. This envelope is a relatively thick, proteinaceous, extracellular matrix that protects the egg and the developing embryo. The egg envelope also plays a significant role during fertilization and in prevention of polyspermy [2]. The terminology used for describing the envelope is quite confusing, because it varies between vertebrate groups. Commonly used terms include zona pellucida, vitelline membrane, chorion, egg shell, zona radiata, and vitelline envelope. Egg envelope or vitelline envelope are the most neutral of the descriptive terms, and for simplicity, we use the term vitelline envelope in this article.
In several vertebrate groups, the envelope is composed of different layers, but the innermost part of the egg envelope is that formed during the growth phase of the oocyte [1, 3]. This is a significant developmental characteristic that suggests the inner egg envelope of vertebrate eggs are homologous structures. Furthermore, the filamentous ultrastructure and chemical composition (basically proteins and carbohydrates) of the envelope are other characteristics that are shared by mammals, birds, amphibians, and teleost fish. In these diverge vertebrate groups, the relatively large vitelline envelope is composed of two to four major proteins [4–7]. The relationship of the envelope constituents to other proteins is not clear, but circumstantial evidence suggests that the vitelline envelope proteins (VEPs) belong to a separate class of structural proteins [8].
The egg envelope (i.e., zona pellucida) of mouse eggs is composed of three major glycoproteins, called ZP1, ZP2, and ZP3, with molecular masses of approximately 200, 120, and 83 kDa, respectively [9, 10]. The nucleotide sequence of the gene encoding ZP3 [11] and the ZP2 gene transcript [12] and ZP1 [13] have been characterized. The zona pellucida glycoproteins have been suggested to possibly represent a separate class of extracellular proteins different from laminin, fibronectin, entactin, and collagen IV [2, 14–16]. In rainbow trout (Onchorynchus mykiss) the vitelline envelope consists of three major proteins, named VEP
, VEPß, and VEP [5]. All three proteins jointly form the protective egg shell.
To date, teleost VEP nucleotide sequences and deduced protein sequences have been obtained from winter flounder [17], Japanese medaka [18–20], carp [21, 22], goldfish [22], Atlantic salmon [23], and zebrafish [24]. So far, characterization of all the VEP isoforms, which correspond to the mammalian ZP1, ZP2, and ZP3, has only been reported for one teleost species, the Japanese medaka. Therefore, in the present study, we aimed to clone VEP cDNAs from rainbow trout, to determine the site of synthesis of the different isoforms, and to investigate the conservation of VEPs in different vertebrates to determine if the VEPs represent a unique group of extracellular structural proteins.
MATERIALS AND METHODS
Construction of a cDNA Library from Estradiol-17ß-Induced Rainbow Trout Liver
Juvenile rainbow trout of both sexes were treated with estradiol-17ß (E2) as previously described [25]. One week after treatment, the fish were killed, and the livers were dissected out and immediately frozen on dry ice and kept at -80°C until used. Liver tissue samples (0.5 g) from two males and two females were pooled, and total RNA was isolated with RNAzol (Biotec Laboratories, Houston, TX) according to the protocol provided by the supplier. Five micrograms of poly(A)+ RNA were isolated with the PolyATtract mRNA isolation system (Promega, Madison, WI) and used to create an oligo(dT)-primed, directionally cloned cDNA expression library in ZAP Express vector arms (Stratagene, La Jolla, CA). The library was packaged with the Gigapack II Gold extract (Stratagene).
Amino Acid Sequencing
The vitelline envelopes from rainbow trout were isolated and solubilized as previously described [5]. The solubilized proteins were electrophoresed on 9% w/v SDS-polyacrylamide gels and stained with Coomassie brilliant blue. Following a brief destaining and several washes with water, each of the three VEPs was excised, briefly lyophilized, and applied on a 15% w/v polyacrylamide gel. The slices were overlaid with 10 µl of sample buffer [26] and 1 µg of endoproteinase Glu-C (Sigma, St. Louis, MO). The proteins were electrophoresed and subsequently electrotransferred onto a polyvinylidene fluoride membrane according to published procedures [26]. Polypeptides of approximately 20 kDa were excised and sequenced using a Gas-Phase Applied Biosystems Sequenator (Perkin-Elmer Biosystems, Foster City, CA). The following sequences were obtained: VEP, NRMSSSYVVGNGPF; VEPß, VAYTSYYTEADY; and VEP, MQYFHVPL.
Screening of the E2-Induced Rainbow Trout Liver cDNA Library
Screening was performed using oligonucleotides designed according to the obtained amino acid sequences as follows: VEP, 5'-TAIGTIGTIGGIAAIGGICCITTC-3'; VEPß, 5'-TACTACACIGAIGCIGACTACCC-3'; and VEP, 5'-ATGCAGTACTTCCAIGTICCICT-3'. These radioactive-labeled oligonucleotides were used as probes to screen the library by plaque hybridization, and positive phages were isolated according to the protocol provided by the manufacturer (Stratagene). As a supplement, the picoBlue immunoscreening kit (Stratagene) was employed for antibody screening of the cDNA library using specific antisera directed against the VEPs [5]. In total, 30 positive clones were isolated. These clones were used for in vitro translation of polypeptides.
In Vitro Translation of Polypeptides
Each identified clone was transformed into Escherichia coli, and the lac promoter on the vector was used to induce polypeptide expression from the inserted cDNA by isopropylthiogalactose treatment. The resulting recombinant proteins were subjected to Western blot analysis [27] using specific antisera directed against VEPs. Thereafter, six clones (two per VEP) were chosen for nucleic acid sequencing. Besides immunoreactivity, the molecular mass of the recombinant protein and the size of the inserted cDNA were used as selection criteria.
Nucleic Acid Sequencing
Plasmids were purified using the QIAprep Spin Plasmid Kit (Qiagen, Chatsworth, CA). Both strands were sequenced using T3 and T7 primers as well as specific internal oligonucleotide primers with dye terminators (373 DNA ABI Sequencer; Perkin-Elmer Biosystems). Sequence comparison and translation was performed using the Genetic Computer Group (Madison, WI) program. The accession numbers of the cDNA sequences in GeneBank are as follows: VEP, AF231706; VEPß, AF231707; and VEP, AF231708.
Sequence Alignments
The amino acid sequence alignments were made using sequences obtained from the sequence data banks with the following accession numbers: winter flounder ZP1, U03674; Japanese medaka ZP1, D89609; Japanese medaka ZP2, AB025967; Japanese medaka ZP3, D38630; goldfish ZP3, S52845; Xenopus ZPB, U44950; Xenopus ZPC, U44952; chicken ZPC, AB025428; mouse ZP1, U20448; mouse ZP2, M34148; mouse ZP3, M20026; human ZPB, U05781; human ZP2, M90366; and human ZP3, M60504. The sequence alignment was performed using ClustalW multiple alignment with the SeqPup program (D.G. Gilbert, Indiana University, Bloomington, IN). Amino acids identical to those found in the novel rainbow trout sequences were highlighted in black boxes.
Northern Blot Analysis
Two female and two male rainbow trout with an average body weight of 447.5 and 255 g, respectively, were used for Northern blot analysis. The fish was killed by a blow on the head, and the liver, gonads, kidney, and muscle tissue were removed. All samples were frozen in liquid nitrogen and stored at -80°C until RNA extraction. Total RNA was extracted using the guanidium thiocyanate (4 M guanidium thiocyanate, 25 mM sodium citrate [pH 7.0], 0.5% w/v laurolylsarcosyl, and 0.1 M 2-mercaptoethanol) method [28]. Ten micrograms of total RNA were loaded onto a 1% w/v agarose gel containing formaldehyde. The gel electrophoresis was performed at 150 V for approximately 1–2 h. The RNA was blotted onto a nylon membrane (Hybond-NX; Amersham, Little Chalfont, UK). The membrane was prehybridized at 68°C for 3 h in a 50% v/v formamide hybridization buffer. The digoxigenin (DIG)-labeled cRNA probe was then added and the hybridization allowed to continue for 12 h. The membrane was washed twice for 5 min each time in 2x SSC (single-strength SSC: 0.15 M sodium chloride and 0.015 M sodium citrate) and 0.1% w/v SDS at room temperature, twice for 15 min each time in 0.5x SSC and 0.1% w/v SDS at 68°C, and twice for 15 min each time in 0.2x SSC and 0.1% w/v SDS at 68°C. The detection was performed using Boehringer's (Mannheim, Germany) CSPD method.
Western Blot Analysis
To investigate the estrogen induction of VEPs, juvenile rainbow trout (average body weight, 64 g) were injected i.p. with 10 mg/kg of E2. As a control, non-E2-induced juvenile rainbow trout were used. After 72 h, blood samples were taken from the caudal vein. The plasma was immediately purified by centrifugation, and the samples were stored at -80°C until analysis. The fish was killed by a blow to the head, and the sex of the fish was determined by dissection. A total of 0.6 µl of plasma was subjected to Western blot analysis. The samples were loaded onto a discontinuous polyacrylamide gel (i.e., SDS-PAGE). A 4% w/v stacking gel and a 9% w/v separating gel were used, and the electrophoresis was performed at 25 mA/gel. The proteins were blotted onto a nitrocellulose membrane (Hybond-ECL; Amersham Pharmacia Biotech, Uppsala, Sweden) using a semidry electrophoretic transfer cell (trans-blot SD; Bio-Rad Laboratories AB, Sundbyberg, Sweden). The transfer was performed at 10–15 V for 20 min. Before immune detection, the membranes were incubated for 1 h in Tris-buffered saline (TBS) with 0.1% v/v Tween (TBST) to block nonspecific binding. The membranes were incubated for 1 h at room temperature with the primary antibodies diluted 1:3000 v/v in TBST. The primary antibody was directed against rainbow trout VEPs. The membranes were washed twice for 10 min each time in TBST and incubated for 1 h with the secondary antibody (hrp-conjugated anti-rabbit Ig; Dako A/S, Glostrup, Denmark) diluted 1:5000 v/v in TBST. The membranes were washed twice for 10 min in TBST and 10 min in TBS each time. The detection was performed using the ECL detection system (Amersham Pharmacia Biotech).
RESULTS
An advantage used to prepare cDNA clones complementary to the VEP mRNAs of rainbow trout is that these mRNAs are predominantly expressed in the liver of most estrogen-treated fish [5, 17, 19]. Hence, liver tissue from estrogen-treated juvenile rainbow trout were used to construct a cDNA expression library. Internal amino acid sequences were obtained for each of the three rainbow trout VEPs (i.e., VEP, VEPß, and VEP) [5]. Based on these sequences, specific oligonucleotides were designed and used together with specific antisera [5] to screen the cDNA library. Positive clones were subjected to in vitro translation of polypeptides, and the molecular mass and immunoreactivity of the translation products were examined by Western blot analysis [27]. The majority of the immunoreactive polypeptides had molecular masses of approximately 60, 55, or 50 kDa, which correspond to the molecular masses of VEP, VEPß, and VEP, respectively [5]. Six clones (two per VEP) were chosen for nucleic acid sequencing, which showed three different cDNA sequences: clone 24 contained a 1787-base pair (bp) insert, clone 2 contained a 1732-bp insert, and clone 25 contained a 1415-bp insert. Sequence analysis further revealed that each clone contained one long, open-reading frame.
To verify that the sequenced clones were complementary to the mRNAs that encode for VEPs, we employed primarily four criteria: 1) the molecular mass of the deduced amino acid sequence of each clone, 2) the amino acid composition of each clone, 3) the immunoreactivity of each clone with the specific antisera, and 4) the presence of the sequence obtained from amino acid sequencing.
The molecular mass of the deduced amino acid sequence of each clone corresponded well with the molecular mass of the translation product and the respective VEP. Clones 24, 2, and 25 had deduced molecular masses of 62.9, 58.0, and 50.0 kDa, respectively. Subtracting the possible signal sequence gave somewhat lower molecular masses of 60.6 (clone 24), 55.8 (clone 2), and 47.7 (clone 25) kDa. These values correspond well with the molecular masses of VEP (60 kDa), VEPß (55 kDa), and VEP (50 kDa).
The amino acid composition of the deduced amino acid sequences of the three clones corresponded exactly with the previous published amino acid composition of VEP, VEPß, and VEP [5]. The translation product of each isolated clone immunoreacted with the specific antisera (data not shown). The deduced amino acid sequence of clones 24, 2, and 25 contained the internal amino acid sequence obtained for VEP (amino acids 315–328), VEPß (354–365), and VEP (263–270), respectively. From this information, clones 24, 2, and 25 clearly correspond to VEP, VEPß, and VEP, respectively.
Alignment of the newly cloned VEP proteins with previously published sequences revealed a high degree of homology between the VEP and VEPß proteins from rainbow trout and the ZP1 and ZP2 homologues of amphibians and mammals (Fig. 1). Eight cysteines are conserved between these species, and an additional 12 cysteines are common to both rainbow trout VEPs. Similarly, a high degree of conservation exists between members of the VEP/ZP3 protein family (Fig. 2). Eleven cysteines are conserved in this group of proteins, and one N-linked glycosylation site is conserved in the VEP protein (Fig. 2). Comparison of the region containing the sperm-recognition sequence in mammals revealed a major difference between viviparous and oviparous species. In the viviparous species, two serines are important for sperm recognition [29], and mutation of either serine in mouse completely inhibits sperm-binding. Thus, it appears that the sperm-recognition function of ZP3 is not an universal feature of ZP/VEP proteins.
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To determine the transcription site of the VEP mRNA, total RNA was extracted from two potential sites of synthesis (i.e., the liver and gonads) and two control organs (i.e., the brain and kidney). We further determined if the VEP protein synthesis was restricted to female rainbow trout. The results showed that all three VEP proteins are synthesized in the liver of rainbow trout, and that the mRNA can be detected in both sexes (Fig. 3). Interestingly, in addition to the signals detected in liver, a weak VEP signal was detected in the ovaries, but not in the testes (Fig. 3C).
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The estrogen responsiveness of the three VEPs was determined by injecting 10 mg/kg of E2 into male and female rainbow trout. Using homologous rainbow trout VEP antibodies, we observed that all three VEPs were induced by E2 in both male and female fish (Fig. 4). Because the analysis was made using plasma samples, these results also demonstrated that all three VEPs are transported in the plasma from the liver to the gonads following induction.
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DISCUSSION
In the present study, we have cloned and characterized the three major constituents of the vitelline envelope from rainbow trout. Using specific antisera directed against the individual VEP isoforms, we have confirmed that the identities of the translation products correspond to the three previously identified VEP proteins (i.e., VEP, VEPß, and VEP) from rainbow trout [27]. Furthermore, the amino acid sequences that were used to construct oligonucleotide primers were present in the cDNA clones. Considered together, these results demonstrate that the isolated clones correspond to the VEP proteins identified earlier by Western blot analysis of plasma samples [27].
Alignment of the VEP proteins with egg shell proteins from other vertebrate species indicates that these proteins are highly conserved in evolution. Furthermore, they can be divided into two groups: the VEP/VEPß family of proteins, corresponding to ZP1/ZP2; and the VEP family, corresponding to ZP3. Thus, VEP and VEPß proteins show high homology with each other and with members of the ZP1 and ZP2 protein families from other teleosts, Xenopus, and mammals, as shown in Figure 1. The VEP protein, on the other hand, is more similar to the ZP3 proteins found in other teleosts, Xenopus, chickens, and mammals, as shown in Figure 2. In rainbow trout, the protein is glycosylated and belongs to the N-linked glycoprotein family [30]. Similar results were also obtained by Iuchi and Yamagami [31] in the same species; in addition, the and ß proteins were indicated as not being glycoproteins. Our results confirm that one N-linked glycosylation site is conserved in the VEP sequence (Fig. 2), whereas no such sites are found in either VEP or VEPß (Fig. 1). The high general sequence similarity between VEP proteins from different vertebrate species indicates that the function of these proteins as structural components of the vitelline envelope is highly conserved. This idea is also supported by a recent study showing that injection of mouse ZP1–3 into the stage VI oocyte of Xenopus gives a transient expression and translation of the ZP proteins, followed by an incorporation of the expressed mouse ZPs into the Xenopus vitelline envelope [32]. Thus, these highly specialized proteins can be considered as being evolutionarily conserved and forming a unique group of structural proteins.
Whereas these proteins appear to be highly conserved to serve as structural components of the egg shell in different species, their function in sperm recognition does not seem to be conserved. In mammalian ZP3 proteins, a sperm-recognition region is in the C-terminal part [29]. In the mouse ZP3 sequence, two serines, located at positions 332 and 334, are instrumental for sperm recognition, and mutation of either one completely abolishes sperm binding. These serines are conserved in other mammals, but they are absent from teleost, amphibian, and avian ZP3 homologues (Fig. 2). Therefore, whether this function is conserved in the ZP3 homologues of oviparous organisms is unclear. Whereas sperm can penetrate the egg shell anywhere on mammalian eggs, this is not the case in teleosts, in which sperm must enter through the micropyle. This indicates that the ZP3 proteins, which are evenly distributed across the egg shell, may not be optimal for sperm binding, and that other proteins may be needed for this function. In teleosts, a sperm-guiding component is present in a vestibular region of the micropyle, indicating that a localized molecule is needed to guide sperm to the micropyle. In herring, it has been suggested that a sperm motility initiation factor is involved in sperm recognition [33, 34]. This component has been identified as a 105-kDa protein located in the micropyle region [33, 34]. In herring, this protein also appears to function as a motility initiation factor [33]. However, it has been indicated that the medaka ZI3, the ZP3 homologue, has a higher sperm-binding affinity than ZI1 or ZI2 [35], and the authors concluded that ZI3 may be important as a sperm-guiding component of the medaka egg shell. Thus, information regarding the involvement of the teleost ZP3 homologues in sperm recognition is conflicting. However, the alignment of ZP3 homologues (Fig. 2) shows that the sperm-binding region is not conserved, and that the two serines are absent in oviparous species. This suggests that the sperm-recognition function of ZP3 may have evolved in mammals.
The VEPs are primarily regulated by estrogens, but testosterone also up-regulates VEPs in rainbow trout [36]. They can be induced by exogenous administration of estrogen (Fig. 4), confirming the earlier observation that both female and male fish can synthesize VEPs [5]. In male fish, however, VEPs have no known function. In rainbow trout, all three VEPs are produced in the liver and transported in the circulation to the ovaries, as demonstrated by the detection of VEPs using Western blot analysis (Fig. 4). The synthesis sites of the VEPs, however, are not evolutionarily conserved. The egg shell components are synthesized in the liver of salmonids [5, 37], which is in contrast to chickens, mice, Xenopus, and carp, in which they are synthesized in the gonads. In chickens, synthesis occurs in the granulosa cells [7, 38], whereas in mice and Xenopus, it occurs in the oocyte [9, 13]. In carp [21, 22], synthesis of VEPs takes place in the ovary. In all species so far studied, it has been observed that all VEPs are generally synthesized in the same tissue, so the reason for the species differences in tissue expression of VEPs is not clear.
In the present study, we found that VEP mRNA can be detected in both the liver and ovaries (Fig. 3). In zebrafish, ZP has been indicated to be expressed in both ovary and liver [39]. The reason for this dual site of synthesis to occur in some but not in other fish species is unknown. Therefore, it will be interesting to characterize the promoter regions of the rainbow trout and zebrafish VEPs to identify the mechanisms for tissue-specific regulation. It should be noted that the expression of VEP in the ovary was much lower than that observed in the liver. Using VEP antisera, we have so far been unable to confirm that VEP mRNA is translated in the ovary of rainbow trout.
In conclusion, we have characterized three VEP cDNA from rainbow trout and shown that they code for the three major vitelline envelope constituents. We have also shown that VEP mRNAs are transcribed in the liver of both sexes, confirming the results of earlier protein studies that showed the presence of VEPs in liver [37]. The VEPs identified in rainbow trout show high homology with ZP proteins from other vertebrates, except for the sperm-recognition sequence, which is only found in mammals. This suggests that these proteins constitute a unique group of extracellular structural proteins, even though all their functions are not evolutionarily conserved.
ACKNOWLEDGMENTS
We thank Dr. Steve McCormick (S.O. Conte Anadromous Fish Research Laboratory, U.S. Department of the Interior) for kindly providing the experimental facilities at Turner Falls, MA; Robert J. Connelly and Kurt Hollfelder for the sequencing; and Drs. Sylvia Christakos, Colin L. Steward, and Emily B. Cullinan for helpful support and advice.
FOOTNOTES
1 Supported by the Swedish Council for Forestry and Agricultural Research (S.J.H.), Boehringer Ingelheim Fonds (A.S.), and the Centre for Environmental Research, Umeå, Sweden (P.-E.O.). 
2 Correspondence. FAX: 46 90 786 6691; per-erik.olsson{at}biology.umu.se
Accepted: October 13, 2000.
Received: March 22, 2000.
REFERENCES
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