Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes

We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved.     

Isolation and some properties of low choriolytic enzyme (LCE), a component of the hatching enzyme of the teleost, Oryzias latipes

One of the two component proteases of the hatching enzyme of the fish, Oryzias latipes, low choriolytic enzyme (LCE), was isolated from the hatching liquid and partly characterized. The enzyme was a basic protein with molecular weight of about 25.5 kDa. Like high choriolytic enzyme (HCE), the other component of the O. latipes hatching enzyme [Yasumasu, S. et al. (1989) J. Biochem. 105, 204-211], LCE was considered to be a zinc-protease from the results of inhibitor studies and metal analyses. However, LCE was found to be distinct from HCE not only in some biochemical characteristics such as molecular weight, amino acid composition, and isoelectric point, but also in some enzymological properties such as substrate specificity, heat stability, and mode of action toward their natural substrate, chorion (egg envelope). Although LCE was almost incapable of digesting the inner layer of intact chorion, it very efficiently digested the inner layer of chorion that had been swollen previously by the action of HCE. Taking account of the fact that HCE swells the inner layer of intact chorion by partial proteolysis but does not efficiently digest the swollen chorion any more [Yasumasu, S. et al. (1989) J. Biochem. 105, 204-211], the present results demonstrated an essential role of LCE in choriolysis, in cooperation with HCE.     

Analysis of the exon-intron structures of fish, amphibian, bird and mammalian hatching enzyme genes, with special reference to the intron loss evolution of hatching enzyme genes in Teleostei

Using gene cloning and in silico cloning, we analyzed the structures of hatching enzyme gene orthologs of vertebrates. Comparison led to a hypothesis that hatching enzyme genes of Japanese eel conserve an ancestral structure of the genes of fishes, amphibians, birds and mammals. However, the exon-intron structure of the genes was different from species to species in Teleostei: Japanese eel hatching enzyme genes were 9-exon-8-intron genes, and zebrafish genes were 5-exon-4-intron genes. In the present study, we further analyzed the gene structures of fishes belonging to Acanthopterygii. In the species of Teleostei we examined, diversification of hatching enzyme gene into two paralogous genes for HCE (high choriolytic enzyme) and LCE (low choriolytic enzyme) was found only in the acanthopterygian fishes such as medaka Oryzias latipes, Fundulus heteroclitus, Takifugu rubripes and Tetraodon nigroviridis. In addition, the HCE gene had no intron, while the LCE gene consisted of 8 exons and 7 introns. Phylogenetic analysis revealed that HCE and LCE genes were paralogous to each other, and diverged during the evolutionary lineage to Acanthopterygii. Analysis of gene synteny and cluster structure showed that the syntenic genes around the HCE and LCE genes were highly conserved between medaka and Teraodon, but such synteny was not found around the zebrafish hatching enzyme genes. We hypothesize that the zebrafish hatching enzyme genes were translocated from chromosome to chromosome, and lost some of their introns during evolution.     

Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii- environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene

The hatching enzyme of oviparous euteleostean fishes consists of two metalloproteases: high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They cooperatively digest the egg envelope (chorion) at the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling activity, HCE-like activity, was found in the ovarian fluid carrying the embryos immediately before the hatching stage. Two kinds of HCE were partially purified from the fluid, and the relative molecular masses of them matched well with those deduced from two HCE cDNAs, respectively, by MALDI-TOF MS analysis. On the other hand, LCE cDNAs were cloned; however, the ORF was not complete. These results suggest that the hatching enzyme is also present in ovoviviparous fish, but is composed of only HCE, which is different from the situation in other oviparous euteleostean fishes. The expression of the HCE gene was quite weak when compared with that of the other teleostean fishes. Considering that the black rockfish chorion is thin and fragile, such a small amount of enzyme would be enough to digest the chorion. The black rockfish hatching enzyme is considered to be well adapted to the natural hatching environment of black rockfish embryos. In addition, five aberrant spliced LCE cDNAs were cloned. Several nucleotide substitutions were found in the splice site consensus sequences of the LCE gene, suggesting that the products alternatively spliced from the LCE gene are generated by the mutations in intronic regions responsible for splicing.     

Structure and developmental expression of hatching enzyme genes of the Japanese eel<Emphasis Type="Italic"> Anguilla japonica</Emphasis>: an aspect of the evolution of fish hatching enzyme gene

We isolated seven cDNA clones from embryos of the Japanese eel Anguilla japonica. Each deduced amino acid sequence consisted of a signal peptide, a propeptide and a mature enzyme portion belonging to the astacin protease family. A phylogenetic analysis showed that the eel enzymes resembled the high choriolytic enzyme (HCE) of medaka Oryzias latipes, and the hatching enzymes of the zebra fish Danio rerio and masu salmon Oncorhynchus masou. Hatching enzymes of these teleosts belonged to the group of the medaka HCE, and not the medaka low choriolytic enzyme (LCE), another hatching enzyme of medaka. Southern blot analysis showed that the genes of the eel hatching enzymes were multicopy genes like the medaka HCE genes. However, one of the eel hatching enzyme genes comprised eight exons and seven introns, and the exon-intron organization was similar to the medaka LCE gene, which is a single-copy gene. The molecular evolution of the fish hatching enzyme genes is discussed. In addition, whole-mount in situ hybridization and immunocytochemistry showed that the eel hatching enzyme was first expressed in the pillow anterior to the forebrain of early neurula, and finally in the cell mass on the yolk sac of later stage embryos. The early differentiation profile of eel hatching gland cells was similar to that of medaka, masu salmon and zebrafish, whereas the final location of the gland cells was different among fishes.Edited by N. Satoh     

Purification and partial characterization of high choriolytic enzyme (HCE), a component of the hatching enzyme of the teleost, Oryzias latipes

The hatching enzyme is an embryo-secreted enzyme(s) which digests the egg envelope, allowing the embryo to emerge at the time of hatching. The hatching enzyme of the fish, Oryzias latipes, has recently been found to consist of two kinds of proteases which may digest the inner layer of chorion (egg envelope) cooperatively [Yasumasu, S. et al. (1988) Zool. Sci. 5, 191-195]. In the present study, one of them, high choriolytic (egg envelope digesting) enzyme (HCE) was purified and some biochemical and enzymological properties were examined. The enzyme was a basic protein with a molecular weight of about 24 kDa, and exhibited choriolytic activity as well as proteolytic (caseinolytic) activity. The results of inhibitor studies and metal analyses strongly suggested that it was a zinc-protease. The purified HCE consisted of two probable isomers, HCE-1 and HCE-2. Both of them were markedly similar in amino acid composition, specific activities of choriolysis and proteolysis, and substrate specificity as determined using MCA-peptides. Moreover, they were not separable on SDS-PAGE, electrofocusing PAGE, or ultracentrifugal analysis, but were discriminated only on HPLC with a CM-300 column. Thus, the mixture of HCE-1 and HCE-2 could be regarded as almost a single enzyme, HCE. When it acted on an intact chorion, the purified HCE caused a remarkable swelling of its inner layer with concomitant release of peptides from it. Once the inner layer of chorion was swollen, the enzyme hardly digested it.     

Hatching enzyme of Japanese eel Anguilla japonica and the possible evolution of the egg envelope digestion mechanism

We purified eel hatching enzyme (EHE) from the hatching liquid of Japanese eel Anguilla japonica belonging to Elopomorpha to a single band on SDS/PAGE. TOF-MS analysis revealed that the purified EHE contained several isozymes with similar molecular masses. Comparison of the egg envelope digestion specificities of the purified EHE and of recombinant EHE4, one of the EHE isozymes, suggested that the isozymes contained in the purified EHE were functionally the same in terms of egg envelope digestion. By electron microscopy, the egg envelope became swollen after treatment with the purified EHE. The EHE cleavage sites on the zona pellucida (ZP) protein of the egg envelope were located in the N-terminal repeat regions. In previous phylogenetic analysis, we suggested that fishes included in Elopomorpha, as basal teleosts, possess a single type of hatching enzyme genes, and that fishes in Otocephala and Euteleostei gain two types of hatching enzyme genes called clade I and II genes by duplication. Further, the clade I enzymes, zebrafish hatching enzyme (ZHE1) and medaka high choriolytic enzyme (HCE), swell the egg envelope by cleaving the N-terminal regions of ZP proteins, while the clade II enzyme, medaka low choriolytic enzyme (LCE), solubilizes the swollen envelope by cleaving the site at the middle region on the ZP domain. In this evolutionary scenario, our findings support that hatching of Japanese eel conserves the ancestral mechanism of fish egg envelope digestion. The clade I enzymes inherit the ancestral enzyme function, and the clade II enzymes gain a new function during evolution to Otocephala and Euteleostei.     

Two constituent proteases of a teleostean hatching enzyme: concurrent syntheses and packaging in the same secretory granules in discrete arrangement.

Formation, accumulation, and storage of two components of the Oryzias latipes hatching enzyme, high and low choriolytic enzymes (HCE and LCE), were examined by immunocytochemical and immunoblotting methods. Both of the enzymes were found to be formed specifically in the hatching gland cells at the stages of lens formation to eye pigmentation and their accumulation proceeded markedly and concurrently up to Day 5.5 embryos (the stage just before hatching). The amount of HCE formed was more abundant than that of LCE. In the hatching gland cells, HCE and LCE were found to be packaged in the same secretory granules but in distinct arrangement; HCE is localized to the inside of granules whereas LCE is situated at the periphery of the same granules. Their segregated arrangement is compatible with their relative quantities formed per embryo. The results provide not only the cellular and developmental basis for a view that this hatching enzyme is an enzyme system composed of HCE and LCE but also a clue to the regulatory mechanism of concurrent syntheses of two different specific proteins in the same embryonic cell.     

A unique proteolytic action of HCE, a constituent protease of a fish hatching enzyme: tight binding to its natural substrate, egg envelope

High choriolytic enzyme (HCE), a constituent protease of the hatching enzyme of the teleost, Oryzias latipes, swells its natural substrate, egg envelope (chorion) by hydrolyzing it partially. This enzyme was found to be bound tightly to the chorion when it exerted catalytic action. This was evidenced by the experimental results showing (i) that the turnover of this enzyme seemed to be hindered by the chorion, (ii) that the enzyme bound to the chorion could be recovered by washing with an alkaline medium, and (iii) that the bound enzyme could be quantified by radioimmunological estimation. The bound enzyme sustained its original activity and the binding between the enzyme and the chorion seems to be stoichiometric.     

Species-dependent migration of fish hatching gland cells that commonly express astacin-like proteases in common

Two constituent proteases of the hatching enzyme of the medaka ( Oryzias latipes ), choriolysin H (HCE) and choriolysin L (LCE), belong to the astacin protease family. Astacin family proteases have a consensus amino acid sequence of HExxHxxGFxHExxRxDR motif in their active site region. In addition, HCE and LCE have a consensus sequence, SIMHYGR, in the downstream of the active site. Oligonucleotide primers were constructed that corresponded to the above-mentioned amino acid sequences and polymerase chain reactions were performed in zebrafish ( Brachydanio rerio ) and masu salmon ( Oncorynchus masou ) embryos. Using the amplified fragments as probes, two full-length cDNA were isolated from each cDNA library of the zebrafish and the masu salmon. The predicted amino acid sequences of the cDNA were similar to that of the medaka enzymes, more similar to HCE than to LCE, and it was conjectured that hatching enzymes of zebrafish and masu salmon also belonged to the astacin protease family. The final location of hatching gland cells in the three fish species: medaka, zebrafish and masu salmon, is different. The hatching gland cells of medaka are finally located in the epithelium of the pharyngeal cavity, those of zebrafish are in the epidermis of the yolk sac, and those of masu salmon are both in the epithelium of the pharyngeal cavity and the lateral epidermis of the head. However, in the present study, it was found that the hatching gland cells of zebrafish and masu salmon originated from the anterior end of the hypoblast, the Polster, as did those of medaka by in situ hybridization. It was clarified, therefore, that such difference in the final location of hatching gland cells among these species resulted from the difference in the migratory route of the hatching gland cells after the Polster region.     

Structure and enzymatic removal of the chorion of embryos of the Nile tilapia

Dechorionation or partial digestion of the egg chorion is necessary for introducing embryonic stem cells into blastulas for chimera production and for harvesting blastulas. Several methods to digest the Nile tilapia Oreochromis niloticus chorion were tried and it was found that the chorions of most clutches of eggs were digested in <3 h using hatching medium [produced by allowing embryos to hatch in Hanks balanced salt solution (HBSS) in an incubator], or 2 mg ml⁻¹ pronase P6911 in 10% Ca/Mg free HBSS. The chorion of Nile tilapia possesses multiple lamellae as found in most teleost species that have been studied. It was found to be thinner than that of medaka Oryzias latipes and thicker than that of zebrafish Danio rerio . During natural hatching the chorion was digested from the inner surface, and tail movements helped to break the remaining chorion; however, chorion digestion has to be complete for experimental dechorionation, because digestion starts at the external surface. The zona radiata externa remained intact after experimental digestion with hatching media but was disrupted by pronase. Embryos dechorionated at the cleavage or blastula stage only survived for 2 or 3 days, but some dechorionated at the gastrula stage or early segmentation stage developed until the natural hatching time. If the chorion was partially digested at the cleavage or blastula stage, some embryos survived to hatch.     

Properties of the hatching enzyme from Xenopus laevis.

Using an anti-(glutathione S-transferase-UVS.2 cDNA) Ig and uterine egg vitelline envelope (UEVE) protein of Xenopus laevis as probes, the hatching enzyme (HE) from Xenopus was solubilized in hatching medium and purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties. The hatching medium solubilized the UEVE and contained molecules reactive to the anti-(GST UVS.2) Ig against Xenopus HE. It was found that the HE had a molecular mass of 60 kDa, and often preparations also contained a 40-kDa form. The 60-kDa HE had a high hydrolytic and UEVE-solubilizing activity, and its activities against Boc-Leu-Gly-Arg-7-amino-4-methylcoumarin (-NH-Mec) and UEVE were inhibited by anti-(GST UVS.2) Ig in a dose-dependent manner. The 60-kDa form was easily autodigested into a 40-kDa form. The 40-kDa molecule alone had no detectable UEVE-solubilizing activity, even it still had high hydrolytic activity. It probably represents the main protease domain of the 60-kDa form after loss of two CUB repeats during autodigestion or digestion. The autodigestion of the 60-kDa molecule into 40-kDa molecule is probably a congenital behavior for successfully dissolving the embryo envelope during the hatching process. The two molecules may play different roles at different stages of the hatching process, during which they co-ordinate with each other to achieve complete solubilization of the embryo envelope, similar to the high and low choriolytic enzymes in medaka (Oryzias latipes). Their hydrolytic activity against Boc-Leu-Gly-Arg-NH-Mec was optimal at pH of 7.4, and with an apparent Km value of 200 micromol.L-1 at 30 degrees C. The HE is very sensitive to trypsin-specific inhibitors such as leupeptin, (4-amidino-phenyl)methane sulfonyl fluoride, diisopropyl fluorophosphate (DFP) and N-alpha-tosyl-L-lysylchloromethane (Tos-Lys-CH2Cl), indicates that it is a trypsin-type protease. The results on EDTA and some metal ions, combined with the occurrence of a astacin family metalloprotease-specific 'HExHxxGFxHE' sequence in the deduced HE amino-acid sequence, indicates that this HE is a Zn2+ metalloprotease.     

Purification and characterization of hatching enzyme from shrimp Penaeus chinensis

By using Penaeus chorion as a specific substrate, the hatching enzyme (HE) from Penaeus chinensis was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that the molecular weight of Penaeus HE is about 43.0 kDa in SDS-PAGE. The Penaeus HE had obvious choriolytic activity, which was optimal at pH 6.0 and temperature of 40 degrees C, respectively. The Km value of the HE for casein was 7.47 mg ml(-1). The HE activity was almost completely inhibited by SBTI, p-APMSF, bestatin, and NEM, greatly inhibited by ovomucoid, TLCK, IAM, chymostatin, and PMSF, and slightly inhibited by pepstatin A, TPCK, LBTI, and leupeptin. These results indicate that the HE is most probably a trypsin-type serine protease. Besides of these, the HE was extremely sensitive to EDTA, Zn2+, Ca2+, Mg2+, and Cu2+. Combined with the results that the EDTA-pretreated HE activity could be perfectly recovered by Zn2+, it is indicated that shrimp HE is most probably a kind of Zn-metalloprotease.     

Methylmercury teratogenesis in the killifish, Fundulus heteroclitus.

Exposure of developing eggs of the killifish, Fundulus heteroclitus, to 0.03 or 0.04 mg/l of methylmercuric chloride resulted in a variety of abnormalities. Percentage of axis formation was reduced somewhat, and many embryos developed cyclopia or intermediate conditions leading to cyclopia, reflecting interference with induction of the forebrain. Defects in the cardiovascular system also appeared in the form of failure of the heart to differentiate properly into chambers. The heart was a thin, feebly beating tube, incapable of causing the blood to circulate. Other tissues, however, continued developing fairly normally, and embryos showed spontaneous movement comparable to controls. Embryos with severe cardiovascular or optic defects did not hatch. Upon hatching, some embryos which had previously appeared normal were found to have skeletal malformations in the form of vertebral bends or the inability to uncurl from the position which they had while still inside the chorion. Exposure to the toxicant for shorter periods of time (6, 12, or 24 hours) reduced the incidence of abnormalities. The second day of development was found to be the most sensitive period.     

Taking the plunge: California Grunion embryos emerge rapidly with environmentally cued hatching

The process of hatching has been well studied in some model species of teleosts: the medaka Oryzias latipes, the mummichog Fundulus heteroclitus, and the zebrafish Danio rerio. These models are compared to the California Grunion, Leuresthes tenuis that has some unique features of reproduction related to tidal synchrony of spawning and environmentally cued hatching (ECH). During oviposition at spring tides, this marine teleost spawns out of water to bury its clutches on sandy beaches in the high intertidal zone. After embryos of L. tenuis reach hatching competence, hatching can be triggered at any time. Incubation above the water line inhibits hatching until ECH is triggered by rising tides during the following lunar phase, and hatching occurs within a few seconds. We review the embryo's response to environmental cues at hatching and the effects of the surrounding medium on the chorionase and chorion for this form of ECH. Leuresthes tenuis shares some similarities as well as some important differences with the model species. Comparison of hatching across teleostean taxa indicates great variability in stage at hatching and in duration of incubation that suggest hatching plasticity in response to environmental cues may be more widespread than currently appreciated.     

Early estrogen exposure induces abnormal development of Fundulus heteroclitus

Many chemicals released into the environment exhibit estrogenic activity, having the potential to disrupt development and the functioning of the endocrine system. In order to establish a model system to study the effects of such environmental chemicals on aquatic animals, we examined the effects of a natural estrogen, 17 beta-estradiol (E(2)), on early development of Fundulus heteroclitus. Embryos of F. heteroclitus were reared in seawater containing 10(-10), 10(-8), and 10(-6) M E(2) throughout the experiment. Hatching and survival rates decreased in a dose-dependent manner, and fry treated with 10(-6) M E(2) and 10(-8) M E(2) were dead by two weeks and 12 weeks after hatching, respectively. More than 85% of fry treated with 10(-8) M E(2) showed malformations: i.e., eye extrusion, crooked vertebral column, faded lateral-stripe pattern eight weeks after hatching. Body weight and head and body lengths were significantly reduced in E(2)-treated fry when compared to controls. Ossification was not completed in vertebrae, cranial bones, and other bones in fry treated with 10(-8) M E(2) even 12 weeks after hatching. Sex ratio of control fry was 57% male and 43% female, whereas fry treated with 10(-8) M E(2) were 100% female eight weeks after hatching. The present results demonstrate that exogenous estrogen induced death of embryos and fry, malformations, sex reversal, and incomplete ossification of vertebrae and cranial bones, which would result in shorter body and head lengths and in malformed vertebrae leading to a hunchback condition.     

Evolution of teleostean hatching enzyme genes and their paralogous genes

We isolated genes for hatching enzymes and their paralogs having two cysteine residues at their N-terminal regions in addition to four cysteines conserved in all the astacin family proteases. Genes for such six-cysteine-containing astacin proteases (C6AST) were searched out in the medaka genome database. Five genes for MC6AST1 to 5 were found in addition to embryo-specific hatching enzyme genes. RT-PCR and whole-mount in situ hybridization evidenced that MC6AST1 was expressed in embryos and epidermis of almost all adult tissues examined, while MC6AST2 and 3 were in mesenterium, intestine, and testis. MC6AST4 and 5 were specifically expressed in jaw. In addition, we cloned C6AST cDNA homologs from zebrafish, ayu, and fugu. The MC6AST1 to 5 genes were classified into three groups in the phylogenetic positions, and the expression patterns and hatching enzymes were clearly discriminated from other C6ASTs. Analysis of the exon–intron structures clarified that genes for hatching enzymes MHCE and MAHCE were intron-less, while other MC6AST genes were basically the same as the gene for another hatching enzyme MLCE. In the basal Teleost, the C6AST genes having the ancestral exon–intron structure (nine exon/eight intron structure) first appeared by duplication and chromosomal translocation. Thereafter, maintaining such ancestral exon–intron structure, the LCE gene was newly diversified in Euteleostei, and the MC6AST1 to 5 gene orthologs were duplicated and diversified independently in respective fish lineages. The HCE gene lost all introns in Euteleostei, whereas in the lineage to zebrafish, it was translocated from chromosome to chromosome and lost some of its introns.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.The nucleotide sequence data reported in the present paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with accession numbers from AB256940 to AB256952.     

Developmental genetics of the esterase isozymes of Fundulus heteroclitus

The ontogeny of the esterase isozymes of the teleost, Fundulus heteroclitus, has been investigated. One group of esterase isozymes is present at all stages of development, whereas other esterase isozymes only very gradually appear at later stages of development, or abruptly appear at such dramatic developmental events as hatching. The ontogeny of these isozyme patterns is interpreted as the expression of differential regulation of separate esterase genes. The general pattern of teleost esterase gene activation is similar to that reported for birds and mammals. Allelic variation was detected at two of the esterase loci. On the basis of electrophoretic mobility, substrate specificity, inhibitor specificity, genetic variation, and ontogeny of esterases, there appear to be at least 15 different esterase isozymes, which constitute 6–8 groups, each of which is probably encoded in one or more genetic loci.This study was supported by NSF Grant GB 544OX to Professor C. L. Markert and an NSF Graduate Fellowship to G. S. Whitt.     

Induction of cytochrome P-450-dependent monooxygenase systems in embryos and eleutheroembryos of the killfish Fundulus heteroclitus

Microsomal aryl hydrocarbon hydroxylase (AHH) activity was highly inducible by polychlorinated biphenyls (PCBs) in Fundulus embryos, and stages prior to the appearance of the liver rudiment were competent to respond to these inducers. Consistent with previous observations, basal AHH activity in whole eleutheroembryo microsomes was shown to increase about 9-fold within 24 h of hatching. Aminopyrine N-demethylase (APD) activity also increased with time after hatching. However, the increase in APD activity was much less than that of AHH activity, suggesting a post-hatching change in basal cytochrome P-450 isozyme composition. Also associated with hatching was an increase in the sensitivity to PCBs as inducers of AHH activity. The ED50 for induction of AHH activity in eleutheroembryos was estimated to be only one-third to one-fourth that in embryos. This developmental increase in the sensitivity to PCBs was not due to a redistribution of PCBs between the yolk and tissues with yolk absorption, and was not simply age-dependent, but appeared to require hatching. An additional change in the monooxygenase system associated with hatching was that microsomal NADPH-cytochrome c reductase activity was not inducible by PCBs prior to hatching, but was modestly inducible after hatching. High performance liquid chromatographic (HPLC) analysis of benzo[a]-pyrene (BP) metabolites formed by microsomes from control and PCB-treated eleutheroembryos demonstrated production of dihydrodiols in the 7,8- and 9,10-positions of the benzo-ring. The formation of these metabolites was completely inhibited by the epoxide hydrolase (EH) inhibitor, trichloropropene oxide, indicating the presence of EH in Fundulus eleutheroembryos. Furthermore, these results indicate the Fundulus eleutheroembryos probably can activate BP to its ultimate carcinogenic form, the 7,8-dihydrodiol-9,10-epoxide, and induction of AHH activity by PCBs is likely to increase the rate of formation of activated metabolites from BP and related compounds. However, during the most active period of organogenesis, prior to hatching, basal AHH activity was low, and prehatching stages were relatively insensitive to cytochrome P-450 inducers. The combination of these effects may help to protect these stages from damage from activated metabolites.