Insect Biochem. Vol. 15, No. 6, pp. 735-747, 1985 0020-1790/85 $3.00+ 0.00 Printed in Great Britain. All rights reserved Copyright © 1985Pergamon Press Ltd BINDING OF VITELLOGENIN TO SPECIFIC RECEPTORS IN OOCYTE MEMBRANE PREPARATIONS OF THE OVOVIVIPAROUS COCKROACH NAUPHOETA CINEREA ROLF KONIG and BEATRICELANZREIN Division of Animal Physiology, University of Berne, Erlachstrasse 9a, CH-3012 Berne, Switzerland (Received 15 November 1984; revised and accepted 15 May 1985) Abstract--An in vitro binding assay was developed to investigate the presence of specific binding sites for vitellogenin in oocyte membrane preparations of a cockroach. Vitellogenin binding reached equilibrium within 1 hr at both 4 and 26°C and was shown to be pH-dependent, specific, saturable and tissue specific. Analysis of saturation isotherms revealed a concentration of receptors of 120 fmol per oocyte near the middle of the first vitellogenic cycle. The amount of vitellogenin bound per oocyte increased progressively during oocyte maturation. Scatchard analysis revealed a K a of 5 x 10 -7 M, and determination of the rate constants for association and dissociation yielded a similar Kd-value. Oocyte membrane preparations of Nauphoeta cinerea did not bind vitellogenin of the closely related cockroach, Leucophaea maderae, whereas oocyte membranes of the latter species did bind the vitellogenin of Nauphoeta cinerea. Key Word Index: Cockroach, vitellogenin, oocytes, specific binding, receptor-mediated endocytosis, kinetic analysis INTRODUCTION Vitellogenins are the predominant yolk protein pre- cursors in egg-laying animals. Their synthesis is hor- monally controlled and takes place in the vertebrate liver and the insect fat body. Vitellogenins are se- creted into the blood and sequestered by the maturing oocytes against a concentration gradient (for reviews see Wallace, 1978; Engelmann, 1979, 1983; Hagedorn and Kunkel, 1979). In most insect species the sesqui- terpenoid juvenile hormone induces vitellogenin syn- thesis, and indirect evidence suggests that the uptake of vitellogenin is also influenced by juvenile hormone (Bell and Barth, 1971; Wilhelm and Liischer, 1974; Sams and Bell, 1977; Giorgi, 1979; Postlethwait and Handler, 1979; Davey, 1981). Animal cells sequester nutritional and regulatory proteins from extracellular fluids by receptor- mediated endocytosis. According to a model devel- oped for the uptake of low density lipoprotein by human fibroblasts, this process involves the binding of the protein molecules to membrane-associated receptors and the internalization of the receptor-ligand complexes by micropinoeytosis (Brown and Goldstein, 1979). A similar mechanism is proposed for the incorporation of immunoglobulins and yolk proteins by chicken oocytes (Roth et aL, 1976; Yusko and Roth, 1976), asialoglycoproteins by hepatocytes (Kolb-Bachofen, 1981), yolk proteins by oocytes of Xenopus (Opresko et aL, 1980) and Aedes aegypti (Roth and Porter, 1964; Roth et al., 1976), as well as for the uptake of several protein hormones by their respective target tissues (for reviews see Gold- stein et aL, 1979; Kaplan, 1981). The presence of coated pits and coated vesicles has been demon- strated in Aedes aegypti using electron microscopy (Roth and Porter, 1964) and chicken oocyte mem- branes have been shown to bind vitellogenin specifically (Yusko and Roth, 1976). To study the role of juvenile hormone in the regulation of the uptake of vitellogenin, the mechanism of vitellogenin recognition and incorporation has first to be eluci- dated. The oocyte structure in our experimental insect, the ovoviviparous cockroach, Nauphoeta cinerea, has been studied in detail (W/Jest, 1979) and the endocrine control of oocyte maturation (Wilhelm and L/Jscher, 1974; B/jhlmann, 1976; Lanzrein et al., 1978), and the competence of oocytes to incorporate vitellogenin (Buschor et al., 1984) have been in- vestigated. Furthermore, vitellogenin and vitellin have been isolated (Buschor and Lanzrein, 1983) and characterized (Imboden et al., 1986) in this species. In the present paper we describe a vitellogenin binding assay and show vitellogenin binding to oocyte mem- brane preparations to be specific, saturable and tissue specific. The kinetic and equilibrium properties of vitellogenin binding to membrane preparations of maturing oocytes are characterized. In addition we demonstrate that oocyte membrane preparations of Nauphoeta cinerea do not bind vitellogenin of the related species Leucophaea maderae, whereas oocyte membrane preparations of the latter bind vitellogenin of the former. MATERIALSAND METHODS Insects Nauphoeta cinerea and Leucophaea maderae were kept at 26°C and 60% r.h. on dog flakes and water at a photoperiod of 12 hr. Under these conditions, oocyte maturation in N. cinerea was 12-13 days and in L. maderae 22-26 days. Gestation was 40-42 days in the former and approx. 60 days in the latter. Chemicals All chemicals were of analytical grade and were pur- chased from Merck and Co. if not otherwise stated. Sub- 735