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BOR - Papers in Press, published online ahead of print February 15, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.049403
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BIOLOGY OF REPRODUCTION 74, 1016–1025 (2006)
DOI: 10.1095/biolreprod.105.049403
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Tandem-Repeated Zebrafish zp3 Genes Possess Oocyte-Specific Promoters and Are Insensitive to Estrogen Induction1

Xingjun Liu , Hai Wang , and Zhiyuan Gong 2 

Department of Biological Sciences, National University of Singapore, Singapore 119260

ABSTRACT

Zona pellucida (Zp) proteins are glycoproteins in fish chorion and are encoded by multiple gene families, including zp1, zp2, zp3, and potentially other zp genes. Expression of zp genes in teleosts is either in the liver under the induction of estrogen or in the ovaries. In the present study, we isolated and characterized a zebrafish zp3 genomic clone and found three tandem-repeated zp3 genes with high sequence identities. We estimated that there were 10–15 zp2 and 17–21 zp3 genes in a haploid genome. We also found some variant zp2 and zp3 subfamilies, and each subfamily may have multiple members. These zp2 and zp3 genes are distributed in several different chromosomes. Ontogenetic expression of zp2 and zp3 mRNAs was first detected at 3 wk postfertilization, which was about 5 wk earlier than initial vtg1 expression, indicating that ovary development was earlier than vitellogenesis. Both zp2 and zp3 mRNAs were expressed specifically in early-growing oocytes and are insensitive to estrogen induction. Because zp3 genes are organized in tandem repeats, to investigate whether an individual zp3 promoter is capable of driving oocyte-specific expression, green fluorescence protein (gfp)-transgenic zebrafish were developed by using a 3.8-kb zp3 5' upstream region, and we found that the gfp reporter gene was faithfully expressed in developing oocytes in zp3:gfp transgenic females. Thus, the new transgenic line not only provided a convenient living marker for monitoring female gonad development, but also demonstrated that a single zp3 gene promoter is sufficient for oocyte-specific transcription.

developmental biology, estradiol, gene regulation, oocyte development, ovary

INTRODUCTION

Vitellogenins and zona pellucida proteins (Zps) are two major classes of female-specific glycoproteins related to reproduction in female vertebrates. Vitellogenins are yolk precursor proteins, whereas Zp proteins (or egg envelope proteins) are glycoproteins located in the inner layer of fish chorion or in the mammalian egg envelope. Vitellogenins are encoded by multiple gene families in essentially all characterized oviparous species, including zebrafish [1, 2], and are synthesized in the female liver in response to estrogen and transported to the ovary. Vertebrate Zp proteins are also encoded by multiple gene families, and there are three reported zp gene subfamilies, including zp1 (zpb), zp2 (zpa), and zp3 (zpc) [3–5]. Recently, two other zp genes, named zpd and zpx, have also been identified in the chicken and Xenopus [6–8]. In mammals, Zp3 proteins function as the primary sperm receptors and induce the acrosome reaction, whereas Zp2 proteins function as the secondary sperm receptors and Zp1 proteins provide structural integrity of the egg membrane [9, 10]. Unlike vertebrate vitellogenins, most Zp proteins are expressed only in the ovary, as in human and mouse [11]. However, hepatocyte expression of some zpb and zpc genes in the liver has also been found in some teleost fishes, such as medaka, Atalantic salmon, rainbow trout, and winter flounder [12–18].

In teleost fishes, multiple copies of zp genes have been reported in medaka [12] and carp [19, 20]. In zebrafish, we previously characterized two cDNA clones encoding Zp2 and Zp3; their expression was found only in the ovary and not in the liver by Northern blot analysis [21]. Another zebrafish zp3 homolog, zpc (named zp3b according to zebrafish unigene nomenclature), which shares only 47% identity with our reported zp3 cDNA, was also found to be transcribed specifically in developing oocytes and not in mature eggs [4]. More recently, a cluster of zp2 genes including zp2.2, zp2.3, and zp2.4 was found in tandem repeats in the zebrafish, with each gene containing eight exons and seven introns; expression of the three zp2 genes was also ovary-specific [22]. These studies demonstrated that zebrafish zp2 and zp3 genes have multiple copies in the genome and are expressed specifically in the ovary. However, the exact copy numbers of zp2 and zp3 have not been determined, and the mechanism of ovary-specific expression has not been characterized. In this study, we attempted to characterize the zebrafish zp3 gene family and the regulation of its expression. We found that zebrafish zp3 genes, like zp2 genes, were clustered as tandem repeats in the genome. Consistent with previous reports [4, 21, 22], we found that zp2 and zp3 genes were predominantly expressed in the ovary, but that their expression was insensitive to estrogen induction. To investigate whether the ovary-specific transcription of tandem-repeated zp3 genes is dependent on individual zp3 promoters, a zp3 promoter from one of the tandem-repeated genes was used to generate zp3:gfp (green fluorescence protein) transgenic zebrafish lines; we demonstrated faithful oocyte-specific GFP expression in the transgenic lines, and thus that an individual zp3 gene promoter contains sufficient regulatory elements for oocyte-specific expression.

MATERIALS AND METHODS

Zebrafish and Maintenance

Zebrafish were purchased from a local ornamental fish farm in Singapore and cultured in our departmental zebrafish aquarium. All animal experiments conducted in this project are in accordance with the guidelines of the National University of Singapore and Agriculture and Veterinary Authorities of Singapore.

Isolation of a Zebrafish zp3 Genomic Clone

A zebrafish zp3 genomic clone was isolated by a PCR-navigated approach [23]. Briefly, a zebrafish genomic DNA library was purchased from Clontech and amplified in 28 petri dishes. Each dish represented a sublibrary containing about 70 000 clones and was eluted as the template for PCR screening with a pair of primers based on the 5' sequence of the zebrafish zp3 cDNA clone [21]: zp3f (forward), 5'-AGGATGGAGTTCCGTCAAGG; zp3r (reverse), 5'-CCACCATCACATCAGGACTGA. Initially, five sublibraries were identified as positive for a zp3 gene. One of the positive sublibraries was further divided with fewer clones (<7000/dish) and screened by PCR again using the same set of primers. With further subdivisions, finally, a subdivided pool of about 80 clones was identified, and PCR was applied to individual clones. A genomic clone with an insert of ~13 kb was identified and isolated.

DNA Sequencing and Sequence Analysis

The zp3 genomic clone was sequenced from both ends by using ABI PRISM BigDye terminator Cycle Ready Reaction Kit with an automatic sequencer (ABI Prism 377; Perkin Elmer) and sequencing of the entire genomic clone was completed by primer walking. DNA sequences were analyzed and assembled by using DNAMAN V4.15 (Lynnon Biosoft). Exons and introns were identified by comparison with the zp3 cDNA sequence by the NCBI Splign program (http://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi). For sequence comparisons, additional zp2 and zp3 DNA sequences were collected by BLAST search of NCBI's Genbank and Unigene databases. Sequence alignment was carried out by using ClustalW online (http://www.ebi.ac.uk/ Clustalw). The genome location information was obtained by direct search and by BLAST search of the zp3 cDNA sequences in the zebrafish genome database (http://www.ensembl.org/Danio_rerio/).

Determination of Copy Numbers of zp2 and zp3 Genes

Genomic DNAs were prepared individually from three individual fish (one male and two females) by phenol/chloroform extraction and ethanol precipitation. Determination of zp2 and zp3 copies was carried out by real-time PCR using LightCycler Faststart DNA Master SYBR Green I Kit (Roche Applied Science) according to the manufacturer's instruction manual. The two primers for zp2 (forward, 5'-CAATGGCAACTAGCTGGTCT; reverse, 5'-CCACTGATACCAGGAGGTC) were designed to amplify the first exon (300 bp) of zp2 genes (zp2.2, zp2.3, and zp2.4). The primers for zp3 (forward, 5'-GATGACTGGTCCTATCAGAG; reverse, 5'-ACCCATGATTCTCAATGAAGG) were designed to amplify the fourth exon (178 bp) of zp3.2 and zp3.3 genes. Each real-time PCR reaction was prepared in a final volume of 10 µl, containing 5.8 µl of H2O, 1.2 µl of 4 mM MgCl2, 1 µl of primer mix (0.5 µM of each primer), 1 µl of DNA Master SYBR Green I, and 1 µl of genomic or plasmid DNA (1 ng/µl) in a LightCycler capillary tube. PCR was performed at 95°C for 10 min, followed by amplification for 40 cycles at 95°C for 10 sec, 55°C for 5 sec, and 72°C for 10 sec, and finally a melting curve analysis was performed as described by the manufacturer (Roche Applied Science). Serially diluted zp2 and zp3 plasmids of known concentrations were used to construct standard curves for estimation of copy numbers in the genome, which were calculated based on the molecular weight of a haploid zebrafish genome (1.7 x 109 bp) [24].

RT-PCR and Real-Time RT-PCR

Total RNAs were prepared from pools of at least 10 fish of 1–9 wk old or selected adult tissues by using Trizol reagent (Invitrogen) according to the manufacturer's instructions. RT-PCR was performed according to the protocol of the One-Step RT-PCR Kit (QIAGEN) with the following program: 50°C, 30 min; 95°C, 15 min; 25 cycles of 94°C (30 sec), 55°C (30 sec), and 72°C (1 min); and finally 72°C for 10 min. The PCR primers used are: 5'-GAAATGCAGTCACTGTCCAG (forward) and 5'-GAAACCTCAAACTGGCTGTCT (reverse) for zp2; 5'-TGTGGTGATGATGACTGA (forward) and 5'-GACCAGTCATCAGTCATGAG (reverse) for zp3; 5'-ATCATGAAGGATGTTGGCTTGG (forward) and 5'-CTTCCATCATTGCAGCACCA (reverse) for vtg1; 5'-AGGATCTGTCTCTGCATGAC (forward) and 5'-GACACACAAATTCCTCCAGC (reverse) for esr1 (previously known as ER{alpha}). Real-time RT-PCR was performed using the LightCycler-RNA Amplification Kit Master SYBR Green I (Roche Applied Science) as previously described [23]. Each real-time RT-PCR reaction mix was prepared to a final volume of 10 µl, including 4.4 µl of H2O, 0.65 µl of 3.25 mM Mn(OAc)2, 1.2 µl of primer mix (0.3 µM of each primer), 3.75 µl of Master SYBR Green I, and 1 µl of RNA sample (1 µg/µl). Reverse transcription was performed at 61°C for 20 min, followed by denaturation at 95°C for 30 sec and PCR amplification for 40 cycles with denaturation at 95°C (1 sec), annealing at 55°C (5 sec), and extension at 72°C (10 sec). RNA standard curves were generated by amplification of 10-fold serially-diluted ovary RNAs by the same set of primers and used to determine the relative levels of zp2 and zp3 mRNAs.

Estrogen Treatment

Stock solution (1 mg/ml) of 17ß-estradiol (E2; Sigma) was prepared by dissolving E2 in 100% ethanol. The solution was then sonicated for 30 min on ice and stored at 4°C. Both male and female zebrafish were treated for 3 days at a concentration of 5 µg/L of E2. Water was changed daily with fresh E2. The control zebrafish were maintained in a tank supplemented with equal volume of 100% ethanol instead of E2 solution.

Paraffin Section and In Situ Hybridization

Zebrafish ovary tissues were fixed in 4% (w/v) paraformaldehyde in PBS (pH 7.4) overnight, followed by dehydration in increasing gradients of ethanol (50%, 70%, 95%, and 100%). After being cleared in Histo-Clear II (National Diagnostics), tissues were embedded in molten paraffin (TissuePrep; Fisher Scientific). Sections 6–10 µm thick were made using a microtome (Reichert-Jung 2003). Antisense riboprobes of zp2 and zp3 were synthesized using the DIG RNA Labeling kit (Roche) based on the manufacturer's protocol. In situ hybridization of paraffin sections and whole mount in situ hybridization were performed essentially as previously described [25].

Generation of zp3:gfp Transgenic Zebrafish

A 3.8-kb 5'-upstream region of zp3.2 was amplified by PCR from the original zp3 genomic DNA clone and inserted into the gfp vector pEGFP-1 (Clontech) to construct a chimeric plasmid pZP3-EGFP. The pZP3-EGFP was lineralized by EcoR I and microinjected into zebrafish embryos at the 1–2 cell stage. These injected embryos were raised to adulthood and mated with wild-type fish. From each injected founder, at least 100 F1 embryos were collected for transgenic screening by PCR. After transgene-positive founders were identified, more F1 embryos were collected to mature, and fin clips were used to identify transgene-positive F1 fish, which were then used to establish zp3:gfp transgenic lines. For observation of GFP expression, zp3:gfp embryos (0–48 hours postfertilization [hpf]) were collected and observed under a Zeiss Axiovert 25 fluorescence microscope with a blue filter (450–490 nm). GFP-expressing ovaries were isolated and fixed in 4% paraformaldehyde before observation under a fluorescence microscope.

RESULTS

Genomic Structure of Zebrafish zp3 Genes

A zp3 genomic clone was isolated and completely sequenced. As shown in Figure 1A, the genomic clone is 12.5 kb long and contains three tandem-repeated zp3 genes, which were named zp3.1, zp3.2, and zp3.3. The complete zp3.2 (middle) contains all of the eight exons and seven introns, whereas zp3.1 and zp3.3 were truncated at the 5' and 3' ends respectively; zp3.1 started from the truncated fourth exon and zp3.3 ended at the fourth exon. Sequences of the three zp3 genes are highly identical in exons (94%–99% identity) as well as in introns (70%–98% identity; Fig. 1B). The highest sequence variation was found in the first introns of zp3.2 and zp3.3; the first intron of zp3.3 is more than 1 kb longer than that of zp3.2. Also, the 5' flanking region of zp3.3 is about 1 kb longer than that of zp3.2, but the proximal 208 bp upstream of the ATG start codons of the two genes are highly identical (87%; Fig. 1B). These observations indicate that these zebrafish zp3 genes are highly identical and exist as a cluster, like zp2 genes in the zebrafish genome [21]. By sequence comparison, we found that the three linked zp3 genes share 96%–98% identity with our previously reported zp3 cDNA [20] in the overlapping coding region. At the amino acid level, the predicted sequences from the three new zp3 genes are also highly conserved (95%–96% identity); their alignment is shown in Figure 2. Thus, the three linked zp3 genes are closely related to the previously reported zp3 cDNA, and they should belong to the same zp3 subfamily.


Figure 1
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  		FIG. 1. Schematic representation of tandem-repeated zebrafish zp3 genes. A) Linear representation of the zp3 genomic clone. Three zp3 genes exist as a cluster in a 12.5-kb zebrafish genomic clone and are named zp3.1, zp3.2, and zp3.3. The black boxes represent exons. The upper numbers indicate exon length (bp) and lower numbers intron length (bp). B) Alignment of the three tandem-repeated zp3 genes. Identity between corresponding introns and exons is indicated. The 208-bp proximal promoters of zp3.2 and zp3.3 share high sequence identity (87%) and are shown with thick lines.


Figure 2
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  		FIG. 2. Amino acid sequence alignment of four Zp3 proteins. The complete sequence of the original Zp3 (NCBI Accession No. NP_571406) is shown. Dashes represent identical residues and dots represent missing sequence information. An asterisk represents a gap insertion for maximal sequence alignment. The signal peptide sequence is underlined and the Zp domain region is highlighted with the Zp signature boxed.

Zebrafish zp2 and zp3 Gene Families

In previously published literature, there are four zp2 cDNA/genes (zp2, zp2.2, zp2.3, and zp2.4) and two zp3 (zp3 and zpc) cDNAs reported in zebrafish [4, 21, 22]. In the zebrafish unigene database (http://www.ncbi.nlm.nih.gov/, there are four zp2 unigene clusters, as listed above, and three zp3 unigene clusters, including zp3, zp3a, and zp3b (zpc). From the zebrafish genome sequence (http://www.ensembl.org/Danio_rerio/index.html), we found two additional divergent zp2 (LOC555180 and LOC559251, named zp2v1 and zp2v2 in the present study) and two additional divergent zp3 genes (LOC563179 and LOC555835, named zp3v1 and zp3v2 in the present study). Thus, both zp2 and zp3 genes have several subfamilies, and each subfamily may have multiple copies of the genes. Because the four zp2 genes (zp2, zp2.2, zp2.3, and zp2.4) have very high sequence identities (96%–98%), they can be considered to be the same subfamily. The two divergent zp2 genes, zp2v1 and zp2v2, share only 71%–78% sequence identity with the zp2 sequence and 57.5% identity between the two variant genes. Thus, there are at least three subfamilies of zp2 genes in zebrafish (zp2, zp2v1, and zp2v2). For the zp3 family, there are at least five subfamilies, including the three subfamilies as defined in the unigene clusters, zp3, zp3a, and zp3b, plus the two newly identified zp3 variants, zp3v1 and zp3v2. The cross-identities of the mRNA sequences between subfamilies are shown in Table 1. In predicted protein primary structure, all Zp proteins possess a conserved Zp domain (240–270 amino acids) in addition to a signal peptide (Fig. 3). Zp2 proteins have an additional repetitive domain and trefoil domain. The structures of two Zp2 variants (Zp2v1 and Zp2v2) are like that of ZP2 but have no repetitive domain. ZP3B has a repetitive domain but not the trefoil domain. The two ZP3 variants (ZP3v1 and ZP3v2) have the same structure as ZP3 protein.


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  		TABLE 1. Cross-sequence identity and chromosome locations of zp2 and zp3 subfamily genes.


Figure 3
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  		FIG. 3. Schematic representation of structures of zebrafish Zp2 and Zp3 proteins. Each protein represents a subfamily, and different domains are marked by different box patterns as indicated in the legend. The numbers on the right sides of the boxes indicate the lengths (numbers of amino acid residues) of the proteins.

By searching in the zebrafish ENSEMBLE genome database, we located our zp3 cluster on the reverse strand of chromosome 21 (the region from 3 324 864 to 3 355 523 bp). By carefully examining the sequence, we found that this region (~30.7 kb) includes five zp3 genes, and that three of them have high identity (93%–98%) with zp3.1 (ENSDARG00000044777), zp3.2 (GENSCAN00000024595), and zp3.3 (ENSDARG00000044780) in both the transcribed and the nontranscribed regions. Similarly, the originally reported zp3 sequence [21] was also located on chromosome 21 by BLAST search of the genome database. By searching the zebrafish genome sequence and unigene mapping, we also located all known zp genes in zebrafish chromosomes, as summarized in Table 1. In addition, there is one zpa gene sequence available (Genbank access number NM_212718) coding for a new Zpa domain-containing protein that belongs to the Zp2 family. However, there is no trefoil domain or repetitive domain in this Zpa domain-containing protein as there is in other Zp2 proteins (Fig. 3). In a previous phylogenetic analysis of the fish zp1/zpB (zpa/zp2) family, it was suggested that teleost zp2 genes are not the orthologs of mammalian zpa and that they may belong to a special class of zp genes named zpx [26]; in the present report, we continue to use the classical name zp2 to avoid confusion.

In chromosome 20, besides the zp2 gene cluster, we also found two zp3a genes (ENSDARG00000042129 and ENSDARG00000042130) as tandem repeats. These two zp3a genes have the same genomic structure as zp2 and zp3, with eight exons, and the transcripts of the two zp3a genes share 91% identity. In addition, two zpa genes (ENSDARG00000041527, and ENSDARG00000011436) were also found as tandem repeats on chromosome 20, and their transcripts share 99% identity. Thus, it is quite common that zp genes are organized as tandem repeats in the genome.

Because both zp2 and zp3 subfamily genes are organized in multiple tandem copies in the genome, in order to estimate copy numbers of these two gene families, real-time PCR was performed. As summarized in Table 2, male and female genomes contain about equal numbers of zp2 (12.6 ± 2.1) and zp3 (19.4 ± 1.6) genes. Our estimation should include most zp2 (zp2, zp2.2–zp2.4) and zp3 (zp3, zp3.1–zp3.3) subfamily genes, but not the other subfamilies. Conceptually, these numbers represented the minimal estimation, because our PCR primers may not amplify the divergent zp2 and zp3 genes. However, we believe that the numbers are close to the real numbers, because the primers we used perfectly or nearly perfectly (with one nucleotide mismatch) match essentially all known zp2 and zp3 sequences available from Genbank.


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  		TABLE 2. Copy numbers of zp2 and zp3 genes in a haploid genome of zebrafish.

Developmental Expression of zp2, zp3, and vtg1 in Zebrafish

Because Zp proteins and vitellogenins are two major classes of female-specific proteins and play important roles in reproduction, it is interesting to compare their ontogenetic expression during development. Thus, total RNA was prepared from zebrafish fry on a weekly interval from 1 to 9 wk, and zp2, zp3, and vitellogenin1 (vtg1) mRNAs were monitored by RT-PCR. As shown in Figure 4A, expressions of zp2 and zp3 mRNA were detected as early as 3 wk postfertilization (wpf), whereas vtg1 expression was detected only from 8 wpf. Thus, expression of zp genes is about 5 wk earlier than that of vtg genes, indicating that oocyte differentiation is much earlier than vitellogenesis in zebrafish.


Figure 4
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  		FIG. 4. Comparison of expression profiles of zp2, zp3, vtg1, and esr1 mRNAs. A) Ontogenetic activation of zp2, zp3, and vtg1. RT-PCR was performed on RNAs isolated from 1- to 9-wk-old zebrafish fry by using primers against zp2, zp3, and vtg1. To ensure the quality of RNA and successful RT-PCR reactions, ß-actin was used as control. B–E) Expression of zp2, zp3, vtg1, and esr1 mRNAs in the liver and ovary in response to E2. Expression levels were determined by real-time RT-PCR. The mRNA levels in ovary (for zp2 and zp3) or liver (for vtg1 and esr1) in control female tissue were arbitrarily defined as 100, and the remaining values are relative to the defined values. The actual values, in percentages, are indicated above each bar. The levels of zp2 and zp3 mRNAs in the liver and vtg1 mRNA in the ovary were low but significantly higher than detection background by real-time RT-PCR. White bars, control; black bars, E2-treated.

Insensitivity of zp2 and zp3 Expression to Estrogen Induction

It is well established that vitellogenins are produced in female liver in response to estrogen [2, 23]. Some Zp proteins, such as choriogenins, are also reported to be produced in liver in some teleosts in response to estrogen [12, 27]. Previously, by Northern blot hybridization, we showed that both zp2 and zp3 mRNAs are specifically produced in the ovary [21]. In the present study, to determine whether zebrafish zp2 and zp3 genes are responsive to estrogen, both male and female fish were treated with E2. We employed a more sensitive approach, real-time RT-PCR, to quantify the relative level of zp2 and zp3 mRNAs in various tissues after estrogen treatment. We found that expression of zp2 and zp3 mRNAs was predominantly in the ovary and that expression in the ovary was more than 103 times higher than that in the liver and other tissues (Fig. 4, B and C, and data not shown). Upon treatment of E2, a 2.5-fold increase of vtg1 mRNA was detected in female liver (Fig. 4D); however, no induction of zp2 or zp3 mRNA was observed in either ovary or female liver (Fig. 4, B and C). Similarly, no expression of zp2 and zp3 mRNA was observed in all male tissues examined (including liver, intestine, skin and testis) in either the presence or the absence of E2. Thus, in contrast to vtg genes, zebrafish zp2 and zp3 genes are rather insensitive to E2 induction.

It is well known that regulation of transcription by E2 is mediated by estrogen receptor (ER), which interacts with estrogen-responsive elements (EREs) in the promoters of target genes [28–30]. In the present study, the relative levels of esr1 mRNA in the liver and ovary was also measured by real-time RT-PCR. As shown in Figure 4E, the level of esr1 in the ovary was only about 17.5% of that in the liver. After E2 treatment, the level of esr1 mRNA in the liver was increased by at least 4-fold, whereas there was a nearly 50% reduction of the esr1 mRNA in the ovary. Thus, the ER-mediated transcriptional activation occurs predominantly in the liver and not in the ovary.

To further demonstrate the insensitivity of zp2 and zp3 genes to E2, ovaries from both control and E2-treated female fish were sectioned and in situ hybridization using antisense riboprobes of zp2 and zp3 was carried out. As shown in Figure 5, A and B, expression of both zp2 and zp3 mRNAs was detected in the ooplasm of stage I and II oocytes (primary growth stage) as strong and evenly distributed signals. It has been reported that the thickness of the zona pellucida layer is dramatically increased from the early phase of stage II (cortical alveolus stage) to late stage II, and to the maximum thickness in the early phase of stage III (vitellogenesis stage) [31]. Parallel to this phenomenon, the expressions of zp2 and zp3 mRNAs were high at early stage II, but gradually declined and became barely detectable in early stage III oocytes, which were characterized by the presence of yolk granules in ooplasm. In ovary of E2-treated fish, the expression patterns of zp2 and zp3 mRNAs are essentially identical to those of control fish, and there is no indication of induction of zp2 and zp3 mRNAs (Fig. 5, C and D), consistent with our data obtained by real-time RT-PCR experiments (Fig. 4, B and C).


Figure 5
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  		FIG. 5. Expression of zp2 and zp3 mRNAs in oocytes of control females (A and B) and E2 treated females (C and D) by in situ hybridization with antisense riboprobes of zp2 (A and C) and zp3 (B and D). Stages of developing oocytes are indicated by Roman letters I, II, and III. IIa and IIb indicate early and late stage II oocytes. Bar = 100 µm.

Oocyte-Specific Expression of GFP in zp3:gfp-Transgenic Zebrafish

Because both zp2 and zp3 genes are organized in tandem repeats in the genome, one outstanding question is whether each individual gene contains an oocyte-specific promoter or whether the whole gene cluster has a local control region (LCR) for oocyte expression, as documented in the globin gene cluster for erythrocyte lineage expression in mammals [32]. To address the question, the 5' flanking sequence of zp3.2 was amplified and inserted into a gfp vector to construct a pZP3-EGFP chimeric plasmid. In this region, several potential cis-elements that may be responsible for oocyte expression were identified, including 10 E-boxes for bHLH transcription factors such as Fig1a [33], 3 ovary-specific protein 1 (Osp1) binding sites [34], and 1 pou5f1 binding site [35] (Fig. 6, A and B). Consistent with the insensitivity of zp3 to estrogen, no ERE was found in this 5' flanking region.


Figure 6
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  		FIG. 6. A) Sequence of the 2.2-kb 5' flanking region of zp3.2. This region is the complete intragenic region between zp3.1 and zp3.2, and was used to make the gfp transgenic construct. The cis-elements for important transcription factors in ovary-specific transcription are labeled with different patterns, including 10 E-boxes (CANNTG), boxed; three Osp1 binding sites (G[G/A]T[G/A]A), highlighted; and one pou5f1 binding sites (ATGCAAAT), underlined and highlighted. The putative TATA box and the coding region are in bold and underlined. Numbers of nucleotides are indicated on the left, with the nucleotide A in the first ATG codon as +1. B) Schematic representation of the 2.2-kb zp3.2 promoter region that was included in the gfp transgenic construct. The important cis-elements are designated by different symbols, as indicated in the legend.

To test the promoter activity of zp3, linearized plasmid pZP3-EGFP was injected into embryos at the 1–2 cell stage. No GFP expression was observed in injected embryos or in fry or adult fish developed from these injected embryos. After extensive screening of F1 embryos by observation of maternal GFP expression as well as by PCR confirmation, two germline-transmitted female founders were found from 65 screened fish developed from injected embryos, and thus two stable zp3:gfp transgenic lines were established, as confirmed by typical Mendelian transmission rates from the F1 generation. So far, these transgenic lines are at their F2 and F3 generations. The transgenic fish from both transgenic lines showed the same constant ovary-specific GFP expression in female fish, and no GFP expression was found in any other tissue in female fish (Fig. 7C). There was also no detectable GFP expression in any tissues in male fish. The earliest ovary-specific GFP expression was in 3-wk-old juvenile fish (Fig. 7C). Consistent with this, endogenous zp3 mRNA was also first detected in the ovary of 3-wk-old fish (Fig. 7, A and B). In adult female ovary, stronger GFP fluorescence was observed in smaller developing oocytes (Fig. 7D). GFP expression was detected mainly in stages I (Fig. 7F), II (Fig. 7G) and early III (Fig. 7H), and weakly in later-stage oocytes (Fig. 7I), consistent with our earlier observation on zp3 mRNA detected by in situ hybridization (Fig. 4). Thus, transgenic GFP expression faithfully recapitulates the endogenous zp3 expression. In addition, these transgenic lines also provided a specific marker to trace female gonad development. It is interesting to note that strong GFP expression was observed in the germinal vesicles in late stage II and early stage III oocytes, in contrast to the cytoplasmic localization of zp3 mRNA (Fig. 4), implying a preferential nuclear accumulation of GFP in oocytes, probably caused by the accumulation of large amount of oily yolk in the cytoplasm. In addition, strong maternal GFP expression was also observed in all early embryos from transgenic females (Fig. 7, J–M). The maternal GFP lasted until 48 hpf, and disappeared gradually by 72 hpf. GFP expression in early embryos was not observed when a transgenic male fish was crossed with a wild-type female, consistent with the fact that GFP was produced from the maternal genome during oogenesis of the transgenic female rather than from the zygotic genome after fertilization.


Figure 7
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  		FIG. 7. Ovary expression of zp3 mRNAs and GFP expression pattern in zp3:gfp transgenic zebrafish. A) Initial expression of zp3 mRNA in the ovary (arrow) in a 3-wk-old juvenile zebrafish as detected by in situ hybridization. B) Cross cryosection of the in situ hybridized 3-wk-old fry to show the ovary tissue with oocytes stained for zp3 mRNA. C) GFP expression in a 3-wk-old juvenile fish (seen through the body). D and E) GFP expression in isolated ovaries of adult female transgenic zebrafish: control (D) and E2 treated (E). F–I) Stage I–IV oocytes from transgenic fish. Strong GFP fluorescence was detected in stage I (F) oocytes and in the germinal vesicles (GV) of stage II–III oocytes (G and H). Very weak GFP fluorescence existed in stage IV oocytes (I). J–M) Maternal GFP expression in developing embryos. 1–2 cell stage (J and K), shield stage (L), and 24 hpf (M).

To test estrogen inducibility on the zp3 promoter, zp3:gfp transgenic zebrafish were treated with 5 µg/L E2. As shown in Figure 7E, although there were generally more mature eggs after treatment, there was no visible increase of GFP fluorescence in early-growing oocytes after E2 treatment. There was also no induced GFP expression in any tissue in male fish treated with E2 (data not shown). These experiments further demonstrated that the zp3 promoter is not estrogen-inducible.

DISCUSSION

zp2 and zp3 Gene Families in Zebrafish

In the present study, we found that both zp2 and zp3 genes have several subfamilies and that many of the subfamilies have multiple members. Previously, it has been reported that some zp2 genes are organized in tandem repeats [22]. In this study, we found that zp3 genes were also organized in tandem repeats. In addition, based on available zebrafish genome sequences, we also found several other zp genes in tandem repeats in the genome. In particular, in zebrafish chromosome 20, three different classes of zp genes can be located, and all of them are organized as tandem repeats, including the zp2, zp3a, and zpa subfamilies. In a previous study of the zp2 family based on a BLAST search of the dbEST database, there are at least six or seven copies of zp2 genes in the carp, medaka, and zebrafish [26]. Using quantitative real-time PCR analyses, we estimated that zebrafish had about 10–15 copies of zp2 genes and about 17–21 copies of zp3 genes in a haploid genome. In the fugu (Takifugu rubripes) genome, it appears that there are only two zpa, three zp2, and nine zp3 homologs (http://www.ensembl.org/Fugu_rubripes/index.html); thus, it seems that the zebrafish genome contains many more zp2 and zp3 genes.

We also found several divergent members of zp2 and zp3 genes in the zebrafish genome. Despite the high sequence divergence (24%–62% identity at the amino acid level), the basic protein structure of these proteins in the Zp2 or Zp3 families is quite conserved; all of them have the same genomic structure, with eight exons and seven introns, and the encoded proteins share the conserved Zp domain. It is likely that the major zp-family genes were evolved before the divergence of fish and amphibians [36] and that the divergent subfamily genes on different chromosomes may result from whole-genome duplication, whereas the multiple tandem-repeated zp2 and zp3 genes may be caused by lineage-specific gene duplication events. However, whether these proteins within each zp2 and zp3 families have distinct or common functions remains an interesting question for future studies.

Expression of zp2 and zp3 Genes

Ontogenically, both zp2 and zp3 genes were activated around 3 wpf, which was about 5 wk earlier than the expression of vtg1 (8 wpf). This was expected, because vitellogenesis began in oocytes at developmental stage III, by which stage expression of zp genes was greatly decreased or completely suppressed. Our data indicated that development of the first batch of oocytes might begin at 3 wpf and the first vitellogenesis might occur at 8 wpf. However, the early expression of Zp protein genes at 3 wpf may indicate not sex differentiation but only the early hermaphroditism of the zebrafish, because the zebrafish has a juvenile hermaphroditism period during development [37].

In other teleost fish, zp genes are expressed either in liver or in oocytes [12, 16, 17, 27, 38–43]. In medaka, three Zp precursor protein genes (choriogeninH, choriogeninL and choriogeninHm), which are divergently related to the zp2 and zp3 family and have not been found in zebrafish, have been found to be expressed specifically in the female liver. Similarly, liver-specific expression of zp2/zpb genes has also been found in Atlantic salmon, rainbow trout, winter flounder, and sheepshead minnows [13–15, 27, 38–43]. In contrast, carp, goldfish, and zebrafish have only ovary-specific zp genes [4, 19–22, 26]. Thus, the expression pattern of zp genes varies among fish species. Recently, it has been reported that medaka and fugu have both liver-specific zp genes (choriogenins) and oocyte-specific zp genes [12]. However, it is not clear whether this is the case for all teleost species. Based on quantitative real-time RT-PCR analyses of choriogenins, we found that both zp2 and zp3 mRNAs are predominantly expressed in the ovary in zebrafish. Though both mRNAs were detected in the liver, the levels of these mRNAs are at least 103-fold lower than those in the ovary, So far, the zebrafish genome sequence is nearly complete, and there is no sign of the presence of homologous-to-medaka choriogenins, suggesting that likely the zebrafish genome does not have such orthologs. The reason for liver-specific Zp expression in some teleost fishes is unknown, but it might be related to an ancient duplication of zp genes for which expression patterns evolved further in liver, or to the loss of some zp genes that led to either liver or oocyte expression only [26].

Estrogen induction of liver-specific zp genes was found in medaka, Atlantic salmon, sheephead minnows, and rainbow trout [14, 27, 38–43]. In both medaka and salmonid species, both vitellogenin and choriogenin proteins can be induced by estrogen in the male liver, and zp genes were more sensitive to estrogen than vitellogenin genes [14, 23, 27, 40]. All these estrogen-responsive zp genes are liver-specific genes, like vitellogenin genes, that are also inducible by estrogen. It is likely that this group of zp genes shares the common transcriptional regulatory mechanism with the vitellogenin genes in some teleost fishes. In the present study, we found that both zebrafish zp2 and zp3 are specifically expressed in early oocytes and that there was no induction of zp2 and zp3 mRNAs after E2 treatment. Furthermore, there is no ERE in the zebrafish zp2 and zp3 gene promoters [22] and there is no induction of GFP expression in the zp3:gfp transgenic zebrafish. Thus, ovary-expressed zp2 and zp3 are insensitive to estrogen induction. Consistent with this, the E2 receptor gene, esr1, was induced by E2 in the liver but not in the ovary, suggesting that ovary tissue, unlike liver, is not sensitive to E2 in the zebrafish. Collectively, these observations suggest that the ovary-specific ZP genes and liver-specific vitellogenin genes have different regulating mechanisms in zebrafish.

Oocyte Specificity of an Individual zp3 Gene Promoter

Because zebrafish zp genes are frequently organized as tandem repeats in the genome, an interesting question regarding tissue-specific transcription is whether there is an LCR for the whole cluster or whether ovary-specific transcription is controlled by individual gene promoters. To test whether an individual zp3 promoter from the zp3 gene cluster is capable of driving oocyte-specific gene transcription, zp3:gfp transgenic zebrafish lines were established using the 5'-flanking region of the zp3.2 gene. We found that GFP expression faithfully recapitulated the expression pattern of endogenous zp3 genes, and thus that an individual zp3 promoter is sufficient to support oocyte-specific expression. This is in contrast to the well-known regulatory mechanism of the globin gene cluster, wherein a DNase I-sensitive LCR is critical to control the expression of the whole globin gene cluster in the erythrocyte lineage [32].

In our zp3:gfp transgenic line, the earliest GFP expression in the ovary was observed at 3 wpf, and strong GFP expression was detected in stage I–III oocytes in the ovary; thus, the pattern of transgene expression pattern highly mimicked endogeous zp3 gene expression. In addition, we also observed maternal persistence of GFP in early developing embryos up to 3 dpf. The overall expression pattern is very similar to that of a previously reported transgenic line, zpc0.5:GFP [44], wherein a 412-bp zpc (zp3b) promoter was used, but sequence comparison revealed no obvious sequence homology between the zpc promoter and the zp3.2 promoter we used. By searching the zebrafish genome data, we found that zpc is located in chromosome 2, and there is no indication that zpc is presented in multiple tandem copies in the genome. However, like the zp3.2 promoter, the zpc gene promoter (693 bp) also contains about four cis-elements for Osp1 and seven E-boxes for Fig1a factor [44]. Comparison of zp3.2 and zp3.3 promoters indicated that the homology between the two promoters is limited to only a 208-bp sequence of 5' upstream of the ATG start codon (87% identity). The zp3.3 promoter up to the 3.3-kb upstream region also contains three sites for Osp1, three sites for pou5f1, and 24 E-boxes (data not shown). Thus, the oocyte-specific expression is likely achieved by the presence of the binding sites of these ovary transcription factors.

Finally, the establishment of ovary-specific living color gfp transgenic lines provides a valuable experimental tool to monitor and investigate ovary development. These lines are also useful in investigation of sex differentiation, sex inversion, hormonal regulation, etc. The availability of the female germline-specific promoter will also be useful in genetic manipulation of fish germlines—for example, in the development of a germline transgene excision system by using the Cre-loxP recombination system [45]. Compared to a male germline transgenic excision, it is likely that the removal of the transgene is more complete because of the longer developmental duration of oocyte ontogeny.

FOOTNOTES

1 Supported by the Biomedical Research Council of Singapore and National University of Singapore Academic Research Fund. X.L. and H.W were recipients of NUS postgraduate scholarships. The genomic sequence of the zp3 cluster has been submitted to GenBank with the Genbank accession number DQ 285563. Back

2 Correspondence. FAX: 65 67792486; dbsgzy{at}nus.edu.sg Back

Received: 18 November 2005.

First decision: 13 December 2005.

Accepted: 6 February 2006.

REFERENCES

  1. Trichet V, Buisine N, Mouchel N, Moran P, Pendas AM, Le Pennec JP, Wolff J. Genomic analysis of the vitellogenin locus in rainbow trout (Oncorhynchus mykiss) reveals a complex history of gene amplification and retroposon activity. Mol Gen Genet 2000 263:828-837[CrossRef][Medline]
  2. Wang H, Tan JT, Emelyanov A, Korzh V, Gong Z, Hepatic and extrahepatic expression of vitellogenin genes in zebrafish, Danio rerio. Gene 2005 356:91-100[CrossRef][Medline]
  3. Dunbar BS, Avery S, Lee V, Prasad S, Schwahn D, Schwoebel E, Skinner S, Wilkins B, The mammalian zona pellucida: its biochemistry, immunochemistry, molecular biology, and developmental expression. Reprod Fertil Dev 1994 6:331-347[CrossRef][Medline]
  4. Del Giacco L, Diani S, Cotelli F, Identification and spatial distribution of the mRNA encoding an egg envelope component of the Cyprinid zebrafish Danio rerio, homologous to the mammalian ZP3 (ZPC). Dev Genes Evol 2000 210:41-46[CrossRef][Medline]
  5. Harris JD, Hibler DW, Fontenot GK, Hsu KT, Yuewicz EC, Sacco AG, Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families. DNA Seq 1994 4:361-393[Medline]
  6. Lindsay LL, Yang JC, Hedrick JL, Identification and characterization of a unique Xenopus laevis egg envelope component, ZPD. Dev Growth Differ 2002 44:205-212[CrossRef][Medline]
  7. Okumura H, Kohno Y, Iwata Y, Mori H, Aoki N, Sato C, Kitajima K, Nadano D, Matsuda T, A newly identified zona pellucida glycoprotein, ZPD, and dimeric ZP1 of chicken egg envelope are involved in sperm activation on sperm-egg interaction. Biochem J 2004 384:191-199[CrossRef][Medline]
  8. Smith J, Paton IR, Hughes DC, Burt DW, Isolation and mapping the chicken zona pellucida genes: an insight into the evolution of orthologous genes in different species. Mol Reprod Dev 2005 70:133-145[CrossRef][Medline]
  9. Dean J, Reassessing the molecular biology of sperm-egg recognition with mouse genetics. BioEssays 2003 26:29-38[Medline]
  10. Hoodbhoy T, Dean J, Insights into the molecular basis of sperm-egg recognition in mammals. Reproduction 2004 127:417-422[Abstract/Free Full Text]
  11. Wassarman PM, Jovine L, Litscher ES, Mouse zona pellucida genes and glycoproteins. Cytogenet Genome Res 2004 105:228-234[CrossRef][Medline]
  12. Kanamori A, Naruse K, Mitani H, Shima A, Hori H, Genomic organization of ZP domain containing egg envelope genes in medaka (Oryzias latipes). Gene 2003 305:35-45[CrossRef][Medline]
  13. Celius T, Walther BT, Oogenesis in Atlantic salmon (Salmo salar L.) occurs by zonagenesis preceding vitellogenesis in vivo and in vitro. J Endocrinol 1998 158:259-266[Abstract]
  14. Celius T, Matthews JB, Giesy JP, Zacharewski TR, Quantification of rainbow trout (Oncorhynchus mykiss) zona radiata and vitellogenin mRNA levels using real-time PCR after in vivo treatment with estradiol-17 beta or alpha-zearalenol. J Steroid Biochem Mol Biol 2000 75:109-119[Medline]
  15. Arukwe A, Kullman SW, Berg K, Goksoyr A, Hinton DE, Molecular cloning of rainbow trout (Oncorhynchus mykiss) eggshell zona radiata protein complementary DNA: mRNA expression in 17beta-estradiol- and nonylphenol-treated fish. Comp Biochem Physiol B Biochem Mol Biol 2002 132:315-326[CrossRef][Medline]
  16. Sugiyama H, Yasumas S, Murata K, Iuchi I, Yamagami K, The third egg envelope subunit in fish: cDNA cloning and analysis, and gene expression. Dev Growth Differ 1998 40:35-45[CrossRef][Medline]
  17. Sugiyama H, Iuchi I, Molecular structure and hardening of egg envelope in fish. Recent Res Dev Comp Biochem Physiol 2000 1:139-161
  18. Lyons CE, Payette KL, Price JL, Huang RC, Expression and structural analysis of a teleost homolog of a mammalian zona pellucida gene. J Biol Chem 1993 268:21351-21358[Abstract/Free Full Text]
  19. Chang YS, Hsu CC, Wang SC, Tsao CC, Huang FL, Molecular cloning, structural analysis and expression of Carp ZP2 gene. Mol Reprod Dev 1997 46:258-267[CrossRef][Medline]
  20. Chang YS, Wang SC, Tsao CC, Huang FL, Molecular cloning, structural analysis and expression of Carp ZP3 gene. Mol Reprod Dev 1996 44:295-304[CrossRef][Medline]
  21. Wang H, Gong Z, Characterization of two zebrafish cDNA clones encoding egg envelope proteins ZP2 and ZP3. Biochimi Biophys Acta 1999 1446:156-160[Medline]
  22. Mold DE, Kim IF, Tsai CM, Lee D, Chang CY, Huang RC, Cluster of genes encoding the major egg envelope protein of zebrafish. Mol Reprod Dev 2001 58:4-14[CrossRef][Medline]
  23. Tong Y, Shan T, Poh YK, Yan T, Wang H, Lam SH, Gong ZY, Molecular cloning of zebrafish and medaka vitellogenin genes and comparison of their expression in response to 17beta-estradiol. Gene 2004 328:25-36[CrossRef][Medline]
  24. Beier DR, Zebrafish: genomics on the fast track (review). Genome Res 1999 8:9-17[Medline]
  25. Gong Z, Ju B, Wang X, He J, Wan H, Sudha PM, Yan T, Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8. Dev Dyn 2002 223:204-215[CrossRef][Medline]
  26. Conner SJ, Hughes DC, Analysis of fish ZP1/ZPB homologous genes—evidence for both genome duplication and species-specific amplification models of evolution. Reproduction 2003 126:347-352[Abstract]
  27. Muruta K, Sasaki T, Yasumasu S, Iuchi I, Enami J, Yasuma I, Yamagami K, Cloning of cDNA and estrogen-induced hepatic gene expression for choriogenin H, a precursor protein of the fish egg envelope (chorion). Proc Natl Acad Sci U S A 1997 94:2050-2055[Abstract/Free Full Text]
  28. Wahli W, Evolution and expression of vitellogenin genes. Trends Genet 1988 4:227-232[CrossRef][Medline]
  29. Menuet A, Pellegrini E, Anglade I, Blaise O, Laudet V, Kah O, Pakdel F, Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions. Biol Reprod 2002 66:1881-1892[Abstract/Free Full Text]
  30. Ueno T, Yasumasu S, Hayashi S, Iuchi I, Identification of choriogenin cis-regulatory elements and production of estrogen-inducible, liver-specific transgenic Medaka. Mech Dev 2004 121:803-815[Medline]
  31. Selman K, Wallace RA, Sarka A, Qi X, Stages of oocyte development in the zebrafish, Brachydanio rerio. J Morphol 1993 218:203-224[CrossRef]
  32. Cao A, Moi P, Regulation of the globin genes. Pediatr Res 2002 51:415-421[Medline]
  33. Liang L, Soyal SM, Dean J, FIG-alpha, a germ cell specific transcription factor involved in the coordinate expression of the zona pellucida genes. Development 1997 124:4939-4947[Abstract]
  34. Schickler M, Lira SA, Kinloch RA, Wassarman PM, A mouse oocyte-specific protein that binds to a region of mZP3 promoter responsible for oocyte-specific mZP3 gene expression. Mol Cell Biol 1992 12:120-127[Abstract/Free Full Text]
  35. Scholer HR, Dressler GR, Balling R, Rohdewohld H, Gruss P, Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J 1990 9:2185-2195[Medline]
  36. Spargo SC, Hope RM, Evolution and nomenclature of the zona pellucida gene family. Biol Reprod 2003 68:358-362[Abstract/Free Full Text]
  37. Uchida D, Yamashita M, Kitano T, Iguchi T, Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. J Exp Biol 2002 205:711-718[Abstract/Free Full Text]
  38. Knoebl I, Hemmer MJ, Denslow ND, Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus). Mar Environ Res 2004 58:547-551[Medline]
  39. Arukwe A, Celius T, Walther BT, Goksoyr A, Effects of xenoestrogen treatment on zona radiata protein and vitellogenin expression in Atlantic salmon (Salmo salar). Aquat Toxicol 2000 49:159-170[CrossRef][Medline]
  40. Lee C, Jeon SH, Na JG, Choi YJ, Park K, Sensitivities of mRNA expression of vitellogenin, choriogenin and estrogen receptor by estrogenic chemicals in medaka, Oryzias latipes. J Health Sci 2002 48:441-445[CrossRef]
  41. Hyllner SJ, Westerlund L, Olsson PE, Schopen A, Cloning of rainbow trout egg envelope proteins: members of a unique group of structural proteins. Biol Reprod 2001 64:805-811[Abstract/Free Full Text]
  42. Larkin P, Folmar LC, Hemmer MJ, Poston AJ, Lee HS, Denslow ND, Array technology as a tool to monitor exposure of fish to xenoestrogens. Mar Environ Res 2002 54:395-399[CrossRef][Medline]
  43. Oppen-Berntsen DO, Arukwe A, Yadetie F, Lorens JB, Male R, Salmon eggshell protein expression: a marker for environmental estrogens. Mar Biotechnol (NY) 1999 1:252-260
  44. Onichtchouk D, Aduroja K, Belting HG, Gnugge L, Driever W, Transgene driving GFP expression from the promoter of the zona pellucida gene zpc is expressed in oocytes and provides an early marker for gonad differentiation in zebrafish. Dev Dyn 2003 228:3393-404[Medline]
  45. Lewandoski M, Wassarman KM, Martin GR, Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line. Curr Biol 1997 7:148-151[CrossRef][Medline]



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D. E. Mold, A. E. Dinitz, and D. R. Sambandan
Regulation of Zebrafish Zona Pellucida Gene Activity in Developing Oocytes
Biol Reprod, July 1, 2009; 81(1): 101 - 110.
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