During growth of the ovarian follicle, the teleost oocyte becomes surrounded by an acellular coat, the vitelline envelope. The nature, origin and number of the vitelline envelope proteins in fish appear to vary with species. In this work, polyclonal antibodies directed against vitelline envelope proteins from rainbow trout, brown trout and turbot were used to show that oestradiol-17 beta induces the major vitelline envelope proteins in juveniles, both males and females, from different species. The fact that males can synthesize vitelline envelope constituents shows that the origin of these proteins is not confined to the ovary. The vitelline envelope of rainbow trout eggs consists of three major proteins, designated alpha (60 kDa), beta (55 kDa) and gamma (50 kDa). The amino acid composition of each of the three proteins indicated that the three proteins are alike and the suggestion that these proteins represent a separate class of structural proteins is sustained.

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Eggshell zona radiata proteins (zr-proteins) were found to occur normally in plasma of sexually mature female (but not male) Atlantic salmon (Salmon salar). In order to ascertain the physiological relevance of these findings, we developed a specific enzyme-linked immunoassay for zr-proteins and screened plasma from sexually maturing Atlantic salmon females throughout an annual reproductive cycle. While zr-proteins were detectable at low levels in female adult salmon plasma prior to sexual maturation, zr-protein levels increased dramatically as sexual maturation proceeded. The strong correlation between gonado-somatic index (GSI) and plasma zr-proteins indicates a reproductive role for blood borne zr-proteins. During the vitellogenic phase, levels of plasma zr-proteins were positively correlated with GSI and with plasma levels of gonadotropin I (GtH I) and estradiol-l7p, as determined by radioimmuno-assays. However, during final sexual maturation, only the plasma level of gonadotropin I1 (GtH 11) was positively correlated to GSI. In contrast, zr-proteins and estradiol-17P were both negatively correlated to plasma level of GtH I1 during this period. In view of estradiol-17f3-induced hepatic synthesis and secretion of zr-proteins (Oppen-Berntsen et al.:Journal of Endocrinology 135: 293-302,1992a) and the established role of gonadotropins in regulating ovarian estradiol synthesis, we interpret the observed correlations among plasma levels of GtH I, estradiol-17P and zr-proteins in Atlantic salmon to signify that GtH I regulates ovarian estradiol-17P synthesis, which in turn regulates hepatic synthesis and secretion of both vitellogenin and zr-proteins during oogenesis. o

The present study delineates the origin of the three major proteins constituting the rainbow trout (Oncorhynchus mykiss) zona radiata. Intraperitoneal administration of oestradiol-17 beta induced the appearance in the blood from juvenile fish (both sexes) of proteins immunoreactive to rabbit antisera against zona radiata proteins (zr proteins). These proteins had similar molecular weights to the zr proteins (alpha, 60 kDa; beta, 55 kDa; and gamma, 50 kDa). Primary hepatocyte cultures from fish treated with oestradiol-17 beta incorporated radioactive [35S] methionine into four major proteins with molecular weights of 160, 60, 55 and 50 kDa. Only the latter three proteins were specifically immunoprecipitated with antibodies to zr proteins. Furthermore, our data demonstrate that in such cultures the biosynthetic mole ratios of these secreted proteins (60, 55 and 50 kDa) are close to one. Control cultures from fish that had not been treated with oestradiol-17 beta failed to produce immunoreactive proteins. The data support the hypothesis that zr proteins are synthesized in a concerted manner in the liver during teleostean oogenesis and transported by the blood for deposition in ovarian follicles.

: A liver complementary DNA expression library from Atlantic salmon (Salmo salar) pretreated with estradiol-17beta (E2) was constructed and screened with antibodies raised against salmon eggshell (zona radiata) proteins. Two clones, SalZr2_19 and SalZr2_23 were sequenced and shown to encode proteins of approximately 50 kDa. SalZr2_23 contains 12 octamer sequence lpqr/kpa/vq repeats also found in SalZr2_19, but only twice. Alignment reveals that the two salmon sequences are similar to piscine zona radiata proteins and mammalian zona pellucida proteins. Several transcripts ranging from 2.3 to 12 kb appeared in liver extracts of E2-treated fish. Juvenile fish treated with E2 or 4-nonylphenol showed strong induction of zona radiata protein. The use of egg envelope transcriptional and translational induction in male or juvenile fish as a biological marker of environmental estrogens is discussed.