Oogenesis in Atlantic Salmon (Salmo Salar L.) Occurs by Zonagenesis Preceding Vitellogenesis in Vivo and in Vitro

259 Oogenesis in Atlantic salmon (Salmo salar L.) occurs by zonagenesis preceding vitellogenesis in vivo and in vitro T Celius and B T Walther Department of Molecular Biology, University of Bergen, HIB, N-5020 Bergen, Norway (Requests for offprints should be addressed to T Celius) Abstract Fish oogenesis represents pleiotropic cytodifferentiative injected with high E2 concentrations (0·4 mg or programs including hepatic synthesis of the molecular 1·1 mg/kg bw). After E2 treatment, hepatocytes isolated components for both the eggshell and the oocytic energy from male fish synthesized zr-proteins and vitellogenin deposits. Both hepatic processes are directly controlled by in vitro. However, zr-proteins were secreted into the plasma levels of estradiol (E2), and injected E2 induces both medium two days before vitellogenin, as measured by biogenetic processes in prepubertal fish of both sexes. This ELISA. The data indicate a preferential induction of work compares the temporal pattern of E2-induced bio- zr-proteins compared with vitellogenin, both with regard synthesis of zona radiata proteins (zr-proteins) and vitello- to E2 sensitivity and response time to E2 treatment. These genin in Atlantic salmon (Salmo salar L.) in vivo and in vitro. findings suggest an obligate sequence in salmon oogenesis. We monitored the presence of plasma zr-proteins and During sexual maturation low E2 levels at first induce only vitellogenin, using homologous polyclonal antiserum to zonagenesis, while increasing levels of E2 subsequently zr-proteins and a monoclonal antibody to vitellogenin. induce both zonagenesis and vitellogenesis. In nature, the Zr-proteins were induced by all E2 concentrations interval between zonagenesis and vitellogenesis may, (0·001–1·1 mg/kg body weight (bw)) within one week of therefore, be considerable. The data suggest new control exposure while vitellogenin was not induced until two mechanisms in fish oogenesis. weeks post-injection and then only in plasma from fish Journal of Endocrinology (1998) 158, 259–266 Introduction genin in teleosts is interrelated because both processes are controlled by estradiol-17â (E2) (Wallace 1985, Hyllner Sexual maturation in female fish has been extensively et al. 1991, Murata et al. 1991, Oppen-Berntsen et al. reviewed (Guraya 1986). The rapid growth of the oocyte 1992a,b, 1994). E2 synthesis is, in turn, regulated by prior to ovulation in most oviparous vertebrates is mainly gonadotropins (GtHs) secreted by the pituitary (Xiong et due to the incorporation of the yolk protein precursor al. 1994). Pituitary secretion is triggered by seasonal signals vitellogenin (Wallace 1985). Vitellogenin is synthesized by such as light and temperature, and complex neuro-sensory the liver (Chen 1983, Bidwell & Carlson 1995) and pathways ending in the hypothalamus, which transmit transported to the ovaries where it is sequestered to serve as environmental cues to initiate pituitary secretion an energy reserve for the developing embryo. Another (Sherwood et al. 1994). feature of oocyte development is the formation of an In Atlantic salmon (Salmo salar L.) zona radiata is extracellular eggshell, or vitelline envelope (Dumont & composed of three protein monomers named á, â and ã Brummet 1985). Zona radiata constitutes the major part of zr-proteins (Oppen-Berntsen et al. 1994). Zr-proteins are the fish eggshell, with the outer zona pellucida as the minor synthesized in extra-ovarian tissues in response to E2 part (Lønning 1972, Oppen-Berntsen et al. 1992b). During in both rainbow trout (Hyllner et al. 1991) and cod embryonic development the eggshell provides protection (Oppen-Berntsen et al. 1992b). Oppen-Berntsen et al. for the embryo. It is shed when hatching is induced by (1992a) reported that hepatocytes from E2-treated fish developmental and environmental signals (DiMichele & synthesized zr-proteins when further treated with E2 Powers 1984, Yamagami 1988, Helvik & Walther 1992). in vitro. Olsen & Walther (1993) reported that plasma The teleostean eggshell consists of two to four protein levels of zr-proteins correlated with activity of pituitary monomers (zona radiata (zr)-proteins) (Begovac & Wallace gonadotropic cells and pituitary contents of GtH I. Salmon 1989, Oppen-Berntsen et al. 1990, Brivio et al. 1991, plasma levels of zr-proteins are broadly correlated to Hyllner et al. 1991, Iuchi et al. 1991, Masuda et al. 1991, plasma levels of both E2 and GtH I (but not GtH II) during Hyllner et al. 1994). Synthesis of zr-proteins and vitello- sexual maturation (Oppen-Berntsen et al. 1994). Journal of Endocrinology (1998) 158, 259–266 1998 Society for Endocrinology Printed in Great Britain 0022–0795/98/0158–0259 $08.00/0 Downloaded from Bioscientifica.com at 09/25/2021 02:59:45AM via free access 260 T CELIUS and B T WALTHER · Oogenetic zonagenesis precedes vitellogenesis Sumpter et al. (1984) reported that significant increases (St Louis, MO, USA). Other chemicals for Western in plasma levels of GtH, vitellogenin, E2, and testosterone blotting and ELISA analysis were from Bio-Rad occurred at least one year before ovulation in trout. Laboratories (Hercules, CA, USA). Diethyl ether and 3 Formation of yolk vesicles in oocytes (termed endogenous heptane were from Merck (Darmstadt, Germany). [ H]E2, vitellogenesis) began a full year before ovulation, while for determination of recovery in E2 extraction, was ovarian uptake of liver-derived vitellogenin (exogenous obtained from Amersham International (Amersham, vitellogenesis) started three months later, or about ten Bucks, UK), and soybean oil was from Idun (Oslo, months before ovulation. Hyllner & Haux (1992) reported Norway). Monoclonal antibody against vitellogenin that the amount of vitelline envelope proteins in the (KB-1) was from Biosense (Bergen, Norway). plasma of maturing rainbow trout varied during the year, AutoDELFIA Estradiol kit was from Wallac Oy in a manner that was similar to vitellogenin. In Atlantic (Gaithersburg, MD, USA). Dulbecco’s modified Eagle’s salmon, Lindhom (1997) observed parallel changes in medium (DMEM), fetal bovine serum (FBS), glutamine plasma levels of zr-proteins and vitellogenin during a and antibiotic/antimycotic solution (AAS) were ob- 21-month period before ovulation. Both data sets were tained from GIBCO BRL (Life Technologies Ltd, interpreted in terms of a similar hormonal regulation of Paisley, Renfrewshire, UK). Secondary antibodies for liver synthesis of vitelline envelope proteins and vitello- Western blotting and ELISA analysis were purchased genin. However, Hyllner et al. (1994) later reported that from Bio-Rad Laboratories. Primaria plates for cell zr-proteins were detected in plasma nearly a year before cultures were from Falcon, Becton Dickinson Labware, vitellogenin, and that the initial formation of the eggshell Oxnard, CA, USA. Other chemicals were of the highest started before the active uptake of vitellogenin. They also commercial purity. stressed that the low pre-vitellogenic plasma levels of E2 in females are of physiological significance. These obser- vations in rainbow trout appear to differ from the findings Fish and treatments of Lindhom (1997) in Atlantic salmon, where monthly Juvenile Atlantic salmon were obtained from Salar Arlvik sampling of plasma did not distinguish the chronology of (Arlvik, Norway), belonging to the Mowi salmon strain appearance of zr-proteins and vitellogenin during sexual (Bergen, Norway). A total of 36 individuals (an even mix maturation. The reason for this seeming discrepancy is not of both sexes and weighing 89&12 g) was used for the known. experiment. Fish were kept in continuously running sea Selective uptake of vitellogenin by trout oocytes is water at a constant temperature of 8·5 )C(&0·5 )C) at the thought to occur by receptor-mediated endocytosis in a Industrial Laboratory (ILAB), HIB, Bergen. The exper- process involving specific cell surface receptors (Tyler et al. iment was performed in January; six groups of six fish each 1988, 1990). Tyler et al. (1991) reported that in rainbow were injected once with a single intraperitoneal dose of trout, receptor-mediated uptake of vitellogenin is devel- various amounts of E2 (0·001, 0·01, 0·1, 0·4, and 1·1 opmentally regulated and that oocytes must reach a certain mg/kg body weight (bw)) and subsequently maintained in size before they are able to sequester vitellogenin. How- separate tanks for two weeks. E2 was dispersed in soybean ever, Lancaster & Tyler (1994) demonstrated that vitello- oil and sonicated for 10 min (50% duty cycle and an output genin receptors are expressed at the oocyte surface in of 60 W) with an Ultrasonic Homogenizer (Cole Palmer follicles as small as 0·3 mm, although they were not shown Instrument Co, Chicago, IL, USA) just prior to injection. to be active. Any presence of vitellogenin in the oocyte The experiment also contained a control group of fish would, therefore, seem to suggest both its synthesis by the injected with soybean oil only. The sex of all fish was liver and its uptake by the oocyte. determined at the end of the experiment. In order to resolve the apparent discrepancies in the literature, we investigated the E2 induction of salmon zr-proteins and vitellogenin, in vivo and in vitro. Specifi- Collagenase perfusion of liver cally, we compared the inducibility of biosynthesis of Hepatocytes were isolated from prepubertal Atlantic zr-proteins and vitellogenin, both in terms of E2 concen- salmon (male, ]300 g) by a two-step perfusion method of tration, and in terms of the lag time needed for E2 to Berry and Friend (1969) as modified for fish by Andersson induce biosynthesis of zr-proteins and of vitellogenin. et al. (1983). The liver was perfused using a peristaltic pump at a flow rate of 22 ml/min. First, the liver was perfused in situ for 10 min with a calcium-free solution Materials and Methods containing: NaCl (7·14 g/l); KCl (0·36 g/l); MgSO4 (0·15 g/l); Na HPO (1·6 g/l); NaH PO (0·4 g/l); Chemicals 2 4 2 4 NaHCO3 (0·31 g/l) and EGTA (20 mg/l) at ambient Estradiol-17â (E2), 4-chloro-1-naphthol tablets, O- temperature, until all blood was washed out.

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