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An outer layer protein of carp fertilization envelope (FE), FEO-1, was purified from carp oocytes. The cDNAs encoding FEO-1 were cloned. The mature protein of FEO-1 is 21 kDa in molecular weight and contains 177 amino acid residues whose sequence has 58% identity to the outer layer protein of chick vitelline membrane. In situ hybridization and immunocytochemistry show that FEO-1 is expressed in oocytes and liver. In oocytes, FEO-1 is stored in the cortical granules. During cortical reaction, it is exocytosed to the perivitelline space and then gradually added to the outer layer of FE (FE(o)). FEO-1 first appears as discrete deposits along FE(o), then merges to form a continuous layer. The thickness of FE(o) increases as cortical reaction proceeds. In addition to FEO-1, FE(o) contains cystatin, fibroin-like substance (FLS), and cathepsin-like substance (CLS) as well. They are stored in the cortical granules and are exocytosed to FE(o) simultaneously with FEO-1 during cortical reaction. In FE(o), FEO-1 is present in monomer form and can be completely extracted by sodium dodecyl sulfate (SDS)-mercaptoethanol (MSH). On the other hand, the cystatin, FLS, and CLS present in FE(o)are cross-linked together. They are partially extracted by SDS-MSH but can be completely extracted by guanidium thiocyanate-lauroylsarcosine., (Copyright 1999 Wiley-Liss, Inc.)

Carp hyosophorin (HSP) is purified from oocytes. It is a highly glycosylated protein (10% protein and 90% carbohydrate) of high molecular weight (>100 kDa) and is localized in the cortical granules of oocytes. During cortical reaction carp HSP is exocytosed into the perivitelline space and is rapidly cleaved to the low-molecular-weight forms of 20 to 30 kDa. The major part of carp HSP cDNA is composed of tandem repeats, the repetitive domain. A repeat is 36 base pairs (bp) in length, which encodes 12 amino acid residues. The sequences of repeats vary within a given cDNA and among different cDNAs. The predominant sequences of repeats are DDGSGSNATTTQ. In addition, the length of the repetitive domain is highly variable among different genes and cDNAs, and ranges from 170 to 1,010 bp. Transcription of carp HSP is restricted in oocytes and starts very early during oogenesis. Carp HSP is highly species-specific. The RNA of goldfish ovary shows no positive signals when probed by carp HSP cDNA.

Two types of cDNAs coding for a major component of carp egg membrane were clones from a carp ovarian cDNA library. They encode polypeptides of 422-424 amino acid residues whose sequences are homologous to those of medaka and mammalian ZP3. Similar to the mammalian ZP3 genes, carp ZP3 gene also consists of eight exons and seven introns. Carp ZP3 genes are 2.9 kb in length and present in multiple forms. Carp ZP3 is a glycoprotein of 45 kDa. It was transcribed and translated exclusively in oocytes, in contrast with medaka ZP3, which was synthesized in liver. The transcription of carp ZP3 starts very early in oogenesis, but translation occurs during vitellogenesis, as it is present in vitellogenic but not in previtellogenic oocytes. ZP3 content in oocytes increases as vitellogenesis proceeds.

There are two genes encoding the alpha subunit of carp gonadotropin (alpha 1 and alpha 2 subunits). Our previous data have demonstrated that both alpha 1 and alpha 2 subunits expressed in insect cells are able to associate with the beta subunit, but only the alpha 1/beta heterodimer displays biological activity. In the mature protein, there are only four amino-acid residues different between the alpha 1 and alpha 2 subunits. In this study we used site-directed mutagenesis and expressed different mutant alpha subunits in insect cells to identify which residue might be important for biological activity of the alpha subunit. Our results suggested that the change of Arg-71 to Gln-71 affected the activity of the carp gonadotropin alpha subunit.

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.

The primary structures of growth hormone (GH) of three cyprinid species, bighead carp, silver carp, and grass carp, were determined by a chemical method and/or by molecular cloning. The bighead carp GH was extracted from pituitary tissue by use of an alkaline medium, then successively purified by gel filtration, hydrophobic interaction column chromatography, and reverse-phase high-pressure liquid chromatography. The primary structure of bighead carp GH determined chemically is identical to that deduced from the cDNA nucleotide sequence. By molecular cloning, the primary structures of silver carp and grass carp GHs were also determined. The GHs of these three cyprinid species all contain 188 amino acid residues and their sequences are identical. When four of the five cysteines of cyprinid GHs were arranged to match the same positions of cysteines of other vertebrate GHs, a maximally matched alignment was achieved. Among fishes, GHs are relatively conserved within the same order (82 to 100% identity) but they are more diversified between orders (49 to 68% identity). In further comparison, fish GHs are even more different from tetrapod GHs (37 to 58% identity). Although the primary structures of vertebrate GHs are relatively variable, four homologous sequences, notably one located at the C-terminal, are found.

Using the cDNA encoding the beta subunit of carp gonadotropin (cGTH-beta) as a probe, 14 clones containing cGTH-beta gene have been isolated from a carp genomic library. Nucleotide sequence analysis indicated that the transcriptional unit of the cGTH-beta gene is 1.2 Kb. Similar to mammalian GTH-beta genes, cGTH-beta gene contains three exons and two introns. The locations of the exon/intron junctions also correspond to those of mammalian GTH-beta gene. Using the primer extension assay, the start site of transcription was determined to be 35 or 37 bp upstream from the translation initiation codon. The TATAA box is present in the 5' flanking region of the gene, 21 bp upstream from the start site of transcription. Three polyadenylation signals, AATAAA, are located in the 3' noncoding region, 111, 430, and 442 bp downstream from the stop codon of translation, respectively.

We have used PCR to amplify and align the sequence of two genes encoding cGTH alpha. Both genes comprise four exons and three introns. The organization of cGTH alpha genes is very similar to that of mammalian GTH alpha genes. However, the cGTH alpha genes only span a region of 1.2 kb which is much smaller than those mammalian GTH alpha genes.

The alpha and beta subunit of silver carp gonadotropin (scGTH-alpha and scGTH-beta) were isolated by high-performance liquid chromatography. Heterogeneity of N-terminal amino acid sequence was observed in scGTH-alpha but not in scGTH-beta. For determining the complete primary structures of scGTH-alpha and scGTH-beta, their cDNAs were cloned. Combining the data of N- and C-terminal sequences determined from proteins and the amino acid sequences deduced from cDNAs, we infer that scGTH-alpha consists of 95 and/or 93 residues and scGTH-beta consists of 115 residues. Both scGTH-alpha and scGTH-beta are glycoprotein. Their carbohydrate content is about 20 g per 100 g protein. The molecular weights of scGTH-alpha and scGTH-beta were calculated to be 12,700 and 15,700 Da, respectively. The amino acid sequences of scGTH-alpha and scGTH-beta are very similar to those of the corresponding subunit of carp GTH, different in only 2 and 4 residues, respectively. In addition, a high extent of homology (70%) was also observed between the alpha subunits of fish and mammalian GTHs. In the case of beta subunit, homology among various species of fish (75 to 98%) is much higher than that between fish and mammal (40%). These data suggest that the alpha subunit is conserved while the beta subunit is diversified during the molecular evolution of vertebrate GTH.

The gonadotropin of silver carp (scGTH) was purified from the pituitary glands in a yield of 8.4 mg/g through extraction with 6% ammonium acetate (pH 5.1)-40% ethanol followed by 80% ethanol precipitation, DEAE-cellulose chromatography and gel filtration. The molecular weight of scGTH is about 32,000 as estimated by gel filtration. Leucine and serine were the two N-termini of scGTH. The amino acid composition of scGTH is rich in aspartic acid/asparagine, glutamic acid/glutamine, valine, proline and half cystine. Its carbohydrate content is 22.2%. Biologically, scGTH significantly stimulated the steroidogenesis in carp testis and ovary in vitro and induced the ovulation in loaches. It also stimulated the 32P-phosphate uptake into 1-day-old chicken testis. Immunologically, scGTH could cross-react with the antiserum against cGTH and vice versa. In both cases parallel displacement curves were displayed.