Vitellogenic : Endocrine Regulated Vitellogenic proteins () synthesized by extraovarian tissues are known from animals phylogenetically as far apart as birds, amphibians, reptiles, crustaceans, and insects. All produce yolky eggs within short time intervals. Several aspects in the machin- ery of precursor synthesis are common to most of these animals, a fact that makes a comparative study a fruitful approach. Among these are synthesis of the yolk precursor exclusively by the female of the species in most cases, and the involvement of in the specific synthesis. Sex specificity finds its expression in the term female specific protein, often used and quite appropriate. However, there are a few exceptions to this and consequently the term has been coined to describe a class of proteins which is preferentially taken up by the oocytes against a concentration gradient. This term Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 is also not quite accurate, since many of the blood proteins contribute to the yolk protein pool as well, evea though only to a minor extent. Yet the term vitellogenin is conveniently used. The specificity of the vitellogenic proteins, immunologically identifiable, provides a useful tool for a detailed analysis of induction of a protein and the mode of action on several levels in this process. The vitellogenic systems are potential model systems for the studies of hormonal control of protein biosynthesis. In recent years, considerable progress has been made on several aspects of vitellogenin synthesis in divers animals. These include the isolation and identification of the specific protein, hormonal involvement, and mode of hormone action. This symposium was therefore organized to bring together our present day knowledge on vitellogenin synth- esis in several diverse species of animals and point to the evolutionary convergence of a system that appears to be advantageous to the species. The symposium was held on December 29, 1973, in Houston, Texas, as part of the meetings of the American Society of Zoologists and was sponsored by the Division of Comparative Endocrinology. FRANZ ENGELMANN Department of Biology, University of California, Los Angeles, California 90024 CONTENTS ROBIN A. WALLACE AND Amphibian vitellogenin: Properties, hor- E. WlLLEM BERGINK monal regulation of hepatic synthesis and ovarian uptake, and conversion to yolk proteins 1159 E. WILLEMBERGINK, -induced synthesis of yolk proteins ROBIN A. WALLACE, in roosters 1177 JOHAN A. VAN DE BERG, EBO S. BOS, MAX GRUBER, AND GEERT AB

FRANZ ENGELMANN induction of the insect yolk protein precursor 1195 H. H. HAGEDORN The control of in the , Aedes aegypti 1207 YVON CROISILLE, The female-specific protein (vitellogenic HENRIETTE JUNERA, protein) in Crustacea with particular re- JEAN-JACQUES MEUSY, AND ference to Orchestia gammarella HELENE CHARNIAUX-COTTON (Amphipoda) 1219 WOLF ENGELS Occurrence and significance of vitellogenins in female castes of social Hymenoptera . 1229 M. L. PAN AND Cecropia vitellogenin: Isolation and R. W. WALLACE characterization 1239 AMER ZOOL., 14:1159-1175 (1974).

Amphibian Vitellogenin: Properties, Hormonal Regulation of Hepatic Synthesis and Ovarian Uptake, and Conversion to Yolk Proteins

ROBIN A. WALLACE AND E. WILLEM BERGINK

Biology Division, Oak Ridge National Laboratory, and The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37830

SYNOPSIS Amphibian vitellogenin is a sex-limited protein found in the serum of normal Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 females but not that of males. Its appearance can be induced in males, females, and hypophysectomized animals by , particularly estradiol-17/3. The natural source of estradiol-17/3 is the ovary, and ovarian synthesis is markedly stimulated by gonadotropin(s). The pituitary is the natural source of gonadotropin(s), and hypophysectomy invariably leads to ovarian regression. Pituitary secretion in turn appears to depend primarily upon food supply rather than other environmental factors. The is the site of vitellogenin synthesis, and its induction by estrogen may involve both transcriptional and translational control mechanisms. Estrogen induction of unprimed liver (in which no vitellogenin synthesis occurs) may involve a cell differentiation or division phenomenon and has not yet been achieved in vitro. Estrogen induction of primed liver (in which a basal level of vitellogenin synthesis takes place) appears to be primarily a modulation phenomenon and can be dupli- cated to some extent in vitro. The secretion of vitellogenin by liver parenchymal cell is as yet poorly understood. Available evidence indicates that synthesis, phosphorylation, and addition are closely linked events, and that approximately 2 hr are required for the newly synthesized peptide chain to be secreted from the liver. Serum vitellogenin is sequestered by the ovary, and specifically by the vitellogenic oocyte, by a micropinocytotic process. The uptake of vitellogenin is highly selective, is stimulated by gonadotropin(s), and may involve attachment to receptor sites on the oocyte membrane. Once incorporated into the oocyte, vitellogenin is converted within yolk platelets into the yolk proteins lipovitellin and phosvi- tin, for which it seems to be the sole source. The conversion process appears to involve a proteolytic cleavage of the vitellogenin peptides into those that make up lipovitellin and phosvitin.

INTRODUCTION cently, the principal proteins present in hen's egg yolk have been purified and clas- Some 80 years ago, Klemperer (1893) ob- sified as and phosphoproteins served that antibody-like activity could be (Cook, 1961; Cook and Martin, 1969), and found in the "yolk" derived from the eggs a specific yolk phosphoprotein, phosvitin, of hens previously immunized with specific has been isolated from the serum of both . Immunological (Jukes and Kay, estrogen-treated and laying hens (Mok et 1932; Roepke and Bushnell, 1936; Abe et al., 1961; Heald and McLachlan, 1963). al., 1958) and biochemical (Laskowski, Heald and McLachlan (1965) have further 1936; McCullyetal., 1959; Williams, 1962) demonstrated with isotopic procedures similarities between certain serum proteins that liver slices from the laying hen can of the laying hen and hen's egg-yolk pro- synthesize and release labeled phosvitin into teins have also long been noted. More re- the medium in vitro. These latter observa- tions have thus served to reinforce a long- „ ,, . standing conviction among; workers in E. W. B. was a Postdoctoral Investigator supported • • • , • i i • L by subcontract no. 3322 from the Biology Division of avian reproductive biology that JUSt about Oak Ridge National Laboratory to the University of all macromolecular materials of the de- Tennessee. Present address: Department of veloping oocyte, including the yolk pro- Biochemistry, University of Rochester School of teins, are derived from the maternal blood- Medicine and Dentistry, Rochester, New York 14642. f Oak Ridge National Laboratory is operated by Union ' . . , , , Carbide Corporation for the U.S. Atomic Energy Sucn a conviction has not been held Commission. among those investigating amphibian re- 1159 1160 ROBIN A. WALLACE AND E. WILLEM BERGINK

production, mainly because considerable REPRODUCTIVE ACTIVITY IN FEMALES UNDER cytological work indicated diverse NATURAL AND CAPTIVE CONDITIONS mechanisms of yolk formation within the oocyte (for reviews see Ward, 1962; Where documented, ovarian growth in Busson-Mabillot, 1969). Indeed, the in- amphibians under natural conditions has tense ribonuclease-sensitive basophilia ob- been found to show a marked seasonal var- served in the early amphibian oocyte, fol- iation. Data forX. laevis have been supplied lowed by a later period of protein deposi- by Shapiro and Shapiro (1934) and Gitlin tion, was the circumstantial evidence which (1939) and indicate that ovarian enlarge- Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 led Brachet (1942) to be among the first to ment and oocyte growth occur only from formulate the valuable hypothesis that May to July in its native South Africa. From RNA was intimately involved in protein July until September, breeding takes place: synthesis. Another reason for the general yolk-filled eggs are laid and the ovaries of belief that yolk was synthesized within the spent females then contain only previtel- oocyte is that no one had ever provided logenic oocytes. An extensive series of ex- convincing evidence for the alternate pos- periments has indicated that the primary sibility. Laskowski (1936) observed that cause of ovarian growth in X. laevis is the plasma phosphoprotein appeared during seasonal availability of food supply rather the breeding season of a number of female than the light cycle or other environmental oviparous , but his data are in- factors (Alexander and Bellerby, 1935, complete for amphibians. Aside from an 1938; Bellerby, 1938). In unpublished ex- older study indicating a seasonal correla- periments, we have also observed that food tion of serum calcium levels with ovarian intake rather than light cycle or ambient weight in Xenopus laevis (Zwarenstein and temperature primarily regulates oocyte Shapiro, 1933), there are essentially no rec- growth in captive animals. A similar situa- ords on seasonal variation of plasma com- tion seems to exist in certain species of fish ponents in amphibians under natural con- (De Vlaming, 1971). ditions. The maintenance of X. laevis in numer- In 1959, Glass demonstrated that pro- ous laboratories throughout the world has teins from the maternal bloodstream could now taken place for more than 30 years. apparently be incorporated antigenically Such animals are routinely fed an abundant intact into developing amphibian oocytes. diet of chopped meat, and as a result The significance of these findings was in- ovarian development is no longer syn- creased when Urist and Schjeide (1961) chronous. Rather, at any time of the year demonstrated that an amphibian re- captive females can be obtained whose sponded to estrogen treatment in much the ovaries contain oocytes in every conceivable same way as birds: components with stage (Dumont, 1972). Oocyte develop- biochemical and ultracentrifugal proper- ment seems to be slow but continuous: ties similar to yolk proteins appeared in the "fully developed" oocytes accumulate and, serum. Encouraged by these results, we if not ovulated, eventually become overripe began to explore in more detail the "yolk- and regress. One can prevent this oocyte like" components present in the serum of growth only by curtailing food (Holland captive X. laevis, and we were joined in this and Dumont, 1973) or by hypophysectomy effort by several groups from England. (Bellerby and Hogben, 1938). Apparently, The net results of our findings to date are an adequate diet assures the ..continuous that a glycolipophosphoprotein, vitellogen- secretion by the pituitary of those hor- in, is secreted by the liver under estrogenic mones necessary for oocyte growth and control and, under the influence of yolk deposition. The main point we wish to gonadotropins, is selectively taken up by make is that captive, well-fed females are in growing oocytes and converted into the a continuous state of reproductive activity yolk proteins lipovitellin and phosvitin. A and so we might expect that they would be more detailed description of these events continuously synthesizing yolk protein to follows. some extent. AMPHIBIAN VITELLOGENIN 1161

APPEARANCE AND TURNOVER OF A SERUM induced components to some extent (Fol- LIPOPHOSPHOPROTEIN lett et al., 1968). In 1968, Follet and Redshaw published At the same time, we independently de- data indicating that implantation of veloped a chromatographic system for estradiol-17/3 in female X. laevis caused a examining the macromolecular compo- remarkable increase in the concentration of nents of serum and found that a variety of serum constituents (Fig. 1). estradiol-17/3. induced the appearance in the serum of a single, new lipophosphopro- After 40 days, the protein concentration Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 had increased over threefold to the point tein, which we designated SLPP (Fig. 2a, b) where the serum approached the consist- (Wallace and Jared, 1968a). Its presence in ency of a sludge. Total calcium increased the serum could be strictly correlated with 13-fold, protein-bound phosphorus protein phosphorus levels, and both the 30-fold, and phospholipid some 40-fold. rate of its synthesis and secretion and the These increases were ascribed to the amount that accumulated in the serum emergence of a new plasma protein or pro- were dependent on the amount of estrogen teins with high phosphorus, calcium, and injected (Follett and Redshaw, 1968; Wal- lipid content. A similar effect was noted in lace and Jared, 19686; Redshaw et al., estrogen-implanted males and hypophy- 1969). We further noted that SLPP was sectomized females, so the response was not normally present to some extent in the sex-specific or dependent on pituitary serum of captive females (Fig. 2c) and that function. In fact, the pituitary gonadotro- its rate of synthesis and secretion but not its pin FSH (follicle-stimulating hormone) was accumulation could be stimulated by found to depress the levels of the estrogen- gonadotropins such as HCG (human chorionic gonadotropin) (Fig. 2d) (Wallace and Jared, 19686; Wallace and Jared, 1969). The observations made by Follett 1000: 0 and Redshaw (1968) could thus be equated CALCIUM^__^--^' with our own on SLPP: a single component appeared to be responsible for the various biochemical changes observed after es- - o trogen treatment. Evidence was also ob- tained by Munday et al. (1968) that the - PROTEIN-PHOSPHORUS -j changes in serum calcium levels observed 1 after estrogen treatment were also due to SLPP. E 100- o : o o As a further step in defining the role of estrogens and gonadotropins in the dispo- /, / sition of SLPP, two groups of females were < injected with estradiol-17/3 and HCG, re- spectively. Protein phosphorus and 32P-labeled protein, as specific markers for / PROTEIN (-10"3) / SLPP (see Fig. 2), were then determined at ' I various times in the serum in order to indi- 1 cate the course of SLPP synthesis and turn- / -i / over (Fig. 3). Estrogen-treated females

-, (group 1 in Fig. 3) immediately began to synthesize SLPP, and this component ap- 0 10 20 30 40 peared to accumulate until around day 12 32 DAYS AFTER IMPLANTATION (Fig. 3a). When Pt was injected into FIG. 1. Effects of estradiol-17/3 implantation on group-1 females on day 5, the amount of plasma protein, phosphorus, calcium, and phos- labeled SLPP observed in the serum during pholipid as a function of time in X. laevis females. Semilog plot of data from Follettand Redshaw (1968). the following week was constant (Fig. Sb), 1162 ROBIN A. WALLACE AND E. WILLEM BERGINK

females on day 13, they ovulated their ma- ture oocytes within 24 hr; subsequently, all values for protein phosphorus, protein labeling, and specific activity decreased with time. The decline in the amount of labeled protein indicates that SLPP was re- moved from the circulation as a response to b ESTROGEN-TREATED MALE f

HCG administration, whereas the decline Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 in specific activity indicates that additional, unlabeled SLPP appeared in the blood- stream. The decline in the protein phos- phorus values suggests that even though additional SLPP was added to the serum compartment it was removed from this compartment at a faster rate. For comparison, females in group 2 were given HCG on day 0, after which they ovu-

FIG. 2. Chromatography on TEAE-cellulose of pooled and dialyzed sera derived from (a) normal males, (b) estrogen-treated males (0.2 mg estradiol- 17/3/10 g body weight injected 7 days previous to bleed- ing), (c) normal females, and (d) HCG-treated females (1000 units injected per animal 10 days previous to bleeding). 14C-leucine (1.25 fxCi/10 g body weight and 32P,) (0.25 /xCi/10 g body weight) were injected 20 hr prior to bleeding. The absorbancy of the effluent is indicated by a solid line, and effluent fractions were analyzed for protein phosphorus (•—•) and for pro- tein labeling associated with 14C (A—A) and 32P (O O). From Wallace and Jared (1969). 8 10 12 11 16 18 20 22 TIME (days) thus indicating that essentially no SLPP was FIG. 3. Patterns of serum phosphoprotein synthesis being removed from the circulation. Dur- and turnover as a function of time in two groups ofX. ing the same period, the specific activity of Uievts females as indicated by (a) the presence of alkali- the protein phosphorus dropped to some labile protein phosphorus in.the serum, (b) the amount of labeled protein present after the injection of 32FJ, extent (Fig. 3c) because of a dilution of and (c) the specific activity of the serum protein phos- labeled phosphoprotein by additionally phorus. Group-1 females (•—•) were injected with synthesized, unlabeled phosphoprotein. By estradiol-17/3 (0.1 mg/10 g body weight) on day 0, 32P, 12 days after the administration of es- (0.1 /xCillO g body weight) on day 5, and HCG (1000 trogen, SLPP synthesis ended and both its units/animal) on day 13. Group-2 females (O O) absolute amount in the serum and its were injected with HCG (1000 units/animal) on day 0 and 32P, (0.1 ftCi/10 g body weight) on day 8. Each specific activity reached a constant level. point represents the average value derived from three When HCG was then given to group-1 animals. From Wallace and Jared (1969). AMPHIBIAN VITELLOGENIN 1163 lated and began a period of enhanced vitel- (Redshaw et al., 1969), thus simply induce logenesis which normally lasts 3 to 4 weeks the appearance of SLPP rather than its (Wallace and Jared, 19686; Wallace et al., turnover. In fact, there is some evidence 1970). The total amount of protein phos- that excessive amounts of estrogen will phorus in the serum rose only slightly after suppress removal of SLPP from the circula- HCG administration (Fig. 3a), and when tion, presumably by depressing pituitary 32Pi was injected on day 8 the SLPP subse- secretion of gonadotropin (Follett et al., quently labeled was found to disappear 1908; Holland and Dumont, 1973). The from the serum at a rate of tip =1.8 days normal source of estrogens in X. laevis has Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 (Fig. 3b). The specific activity of SLPP been shown to be the ovary, and injected dropped at an identical rate (Fig. 3c), indi- gonadotropin also markedly enhances the cating that during the period examined a rate of estrogen synthesis (Redshaw and steady state existed in which the amount of Nicholls, 1971). SLPP entering the circulation was exactly balanced by the amount leaving the circula- tion. This amount can be calculated to be CHARACTERIZATION OF THE SERUM LIPOPHOS- about 7.7 mg/day (Wallace and Jared, PHOPROTEIN (VITELLOGENIN) 1969). The estrogen-induced lipophosphopro- We have also measured the physiological tein can be readily isolated from serum half-life of SLPP induced by estrogen in either by chromatography on TEAE- males and females and found that it is cellulose (Wallace and Jared, 1968a; Wal- around 40 days. It is also around 40 days in lace, 1970), preparative ultracentrifugation HCG-treated females subsequently (Redshaw and Follett, 1971), or precipita- ovariectomized (Wallace and Jared, 19686). tion with dimethylformamide (Munday et The ovary thus seems to be primarily re- al., 1968; Ansari et al., 1971). Chromatog- sponsible for removing SLPP from the cir- raphy appears to yield the most purified culation and appears to be the target organ preparations but suffers the disadvantages for gonadotropins (see also Follett et al., that calcium normally associated with the 1968; Redshaw, 1972). Gonadotropins protein is lost and much of the lipid can also given to males or previously ovariec- be lost unless one takes care to "precondi- tomized females do not induce the appear- tion" the column (Wallace, 1965). Prepara- ance of SLPP (Nicholls et al., 1968; as well tive ultracentrifugation apparently pre- as our own unpublished observations). Es- serves most of the associated calcium. Di- trogens, and particularly estradiol-17/3 methylformamide precipitation is the simp-

TABLE 1. Summary of chemical and physical measurements on vitellogenin from Xenopus laevis.

Data from: Ansari Redshaw Wallace et al. and Follett (1970) (1971) (1971) Lipid (%) 12 11.9 12.3 Phospholipid (%) — — 10.0 Neutral lipid (%) — — 3.0 Protein-phosphorus (%) 1.23 1.65 1.35 Calcium (%) 0.85 1.60 Carbohydrate (%) — 1.35 0.7 v (ml/g) 0.75 — 0.70a *&.»(S) 13.6 — 17.7 7 Dl,wx 10 (cmVsec) 2.75 — — 5 Mol. wt. X 10 (from s&,u and D^,w) 4.7 — 5.4 Mol. wt. (from sedimentation equilibrium) 4.5 — — Mol. wt. (from gel filtration) 7.0" 5.9-6.7 — "Estimated value. "From Wallace and Jared (1968a). 1164 ROBIN A. WALLACE AND E. WILLEM BERGINK

lest method and preserves about half of the released into the medium labeled protein associated calcium. Preparations obtained which associated with carrier phosvitin by the latter two procedures contain some (Rudack and Wallace, 1968). At the same contaminants; these can be removed by gel time, Munday et al. (1968) observed that filtration, but any associated calcium is then hepatectomized animals failed to respond lost (Ansari et al., 1971). to estrogen, so that it became fairly clear The results of chemical and physical that the liver was responsible for the syn- measurements which have been made on thesis of circulating vitellogenin. the isolated protein are provided in Table A more direct demonstration of hepatic Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 1. Gel filtration gave molecular weight val- synthesis was obtained by using an im- ues which were somewhat high and proba- proved, complete medium for culturing bly spurious since the protein eluted near small pieces of X. laevis liver (Wallace and the void volume. We consider a value of Jared, 1969) together with 3H-leucine and 32 about 460,000 to be the best estimate since Pt. Under these conditions labeled pro- it is derived from the most complete set of tein was continuously secreted into the data. Several interesting features about the medium over a 4-day period, and liver protein are that its greenish color appears slices from HCG-treated females or to be due to biliverdin bound noncovalently estrogen-treated males were the most active (Redshaw et al., 1971) and that, in the in this respect. To provide an indication of serum, one atom of calcium appears to be which labeled proteins were being secreted, associated with every protein-phosphate we chromatographed the culture medium group (Wallace, 1970). after 4 days of incubation on TEAE- We have postulated (Wallace and Dum- cellulose together with carrier serum from ont, 1968; Wallace, 1970) and gathered estrogen-treated males. The chromato- evidence (Wallace and Jared, 1969; Wallace grams (Fig. 4) indicated that liver pieces et al., 1972; Jared et al., 1973) that the from different types of animals synthesized estrogen-induced lipophosphoprotein and released labeled serum proteins at ap- found in serum acts as the direct precursor proximately the same rate, and that the to the yolk proteins lipovitellin and phosvi- main reason for the different amounts of tin. I n 1969, Pan et al. proposed the generic radioactive protein released during the term ' "vitellogenin" for circulating mac- time-course experiments was the relative romolecular precursors to yolk proteins, amount of labeled protein which eluted in and we (Wallace, 1970) as well as others the position of vitellogenin. None of this (Ansari et al., 1971; Redshaw and Follett, component was released into the medium 1971) have now adopted this term for the by liver from normal males. These results, estrogen-induced lipophosphoprotein. taken together with the observed incorpo- ration of isotopic precursors into the vari- ous proteins of the serum in vivo, indicate HEPATIC SYNTHESIS AND SECRETION OF VITEL- that vitellogenin and almost all the other LOGENIN IN VITRO protein inX. laevis serum are derived from As an initial attempt to indicate the the liver, and that the rate of synthesis and source of circulating vitellogenin, we cul- secretion of the normal serum proteins is tured tissue slices from different organs of relatively independent of the rate of vitel- X. laevis in a simple salt solution together logenin synthesis and secretion. with 14C-Na2CO3. After several hours, the Over the past few years we have made culture medium was subjected to an isola- numerous attempts to induce vitellogenin tion procedure specific for phosvitin, since, synthesis in vitro. These attempts have in- at the time, we believed that the secreted cluded the incubation of male liver slices in product would include free phosvitin. Al- a complete medium supplemented with though the incorporation observed was "normal" serum or serum from hormone- very low, the experiments nevertheless in- treated males and females and containing dicated that only the liver from HCG- estradiol either alone or in combination treated females or estrogen-treated males with other steroids and gonadotropins. We AMPHIBIAN VITELLOGENIN 1165

E06-| cultured as much as 6 months after the original injection of estrogen, a small amount of phosphoprotein was found to be released into the medium with time (Fig. 5a). This had never been observed in liver slices from unprimed males. Furthermore, in the presence of estrogen a distinct in- crease in the release of labeled phospho- protein occurred after about 40 hr (Fig. 5b). Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 In both cases, the secretion of 3H-labeled proteins was essentially linear with time, indicating the continuous secretion of serum proteins and an apparently healthy culture. Electrophoresis of samples of the culture media on SDS-polyacrylamide gels indicated that a small amount of vitelloge- nin polypeptide had been secreted in the absence of estrogen (Fig. be) and a much larger amount had been secreted in the presence of estrogen (Fig. bd). In both cases the relative amounts of vitellogenin sec- reted were very small, as indicated by the relative incorporation of 3H-leucine into vitellogenin as compared with that into the albumin polypeptide. Also, the lengthy lag period for the burst of, secretion observed in the presence of estrogen probably indi- cates that our culture procedures were still somewhat inadequate or that other neces- 0 0.2 0.4 0.6 0 8 1.0 sary hormonal factors were missing. ELUTION POSITION GRADIENT STARTED It is interesting that the rates of vitello- I— SAMPLE PLACED ON COLUMN genin in synthesis and secretion that we FIG. 4. Chromatography of culture medium to- gether with 1.0 ml carrier serum from estrogen-treat- have observed in liver slices from HCG- ed males. The medium originally contained 5 /i.Ci treated females are consistently greater 3H-leucineand 1 /xCi 32I^/mland was incubated for 96 than that found for liver from males given hr with liver slices derived from different animals. The estrogen for the first time (Fig. 4), even typical absorbancy trace observed in all cases is indi- though the amount of estrogen synthesized cated in (a). Samples of the effluent fractions from the various chromatograms were analyzed for 3H (A—A) after gonadotropin injection to females and 32P (O—O) (b-e). From Wallace and Jared (1969). would appear to be much less than the amount administered to males. Putting monitored the medium over at least a 3-day these observations together with the dis- period, and in no case did we observe the covery of a "memory effect" in estrogen- appearance of significant amounts of primed liver of roosters (Bergink, 1973; phosphoprotein. Within the past year we Bergink et al., 1973), it would appear that have achieved a modest success in this ef- estrogen has several types of action: (i) in fort by changing to liver slices from males unprimed liver (males) which apparently given estrogen at least once previously. We synthesize no vitellogenin (Wallace and had found that when an adult male was Jared, 1969; Wittliff and Kenney, 1972) a given 1 mg estradiol, vitellogenin synthesis differentiation ("commitment") step is and secretion into the bloodstream was in- made whereby the liver cells become more duced for about 18 days, after which it ap- sensitive to the presence of estrogen; and peared to end (Wallace and Jared, 19686). (ii) once committed, a basal level of vitel- Yet if liver slices from such an animal were logenin synthesis takes place which can be 1166 ROBIN A. WALLACE AND E. WILLEM BERGINK

increase to a level threefold above the con- b .' CONTROL trol values, and it remained at that level • ESTRADIOL-17/9 ^' 12 j 5 until about 9 hr; pulse-labeled RNA ob- 10 £ tained at that time was particularly high in uridylic acid content (Wittliff et al., 1972). D A second burst in RNA synthesis began by 06^ z 12 hr, when the base composition of pulse- 04 ¥ labeled RNA returned to the mixed pattern seen in control animals, most likely reflect- Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 O2 5 ing a secondary increase in synthesis of ribosomal RNA. Increased labeling of 20 40 60 80 .20 4 0 60 i80 TIME(hr) cytoplasmic ribosomal RNA was not ob- served until 12 hr, in agreement with this suggestion. No significant changes in leucyl-, methionyl-, or seryl-tRNA were detected, and phosphoserine was not found in seryl-tRNA formed either in vivo or in vitro. The effect of a single dose of 20 10 estradiol-17/3 on the rate of synthesis of GEL SLICE NUMBER hepatic vitellogenin is shown in Figure 6. FIG. 5. Secretion of labeled proteins into the culture medium by estrogen-primed liver slices (approxi- Using an isotopic-immunochemical proce- mately 10 mg/ml) in the absence (a, c) and presence (b, dure, Wittliff and Kenney (1972) could not d) of estradiol-17/3 (10 /xM). The liver was obtained detect a significant level of labeling of vitel- from a male which had been injected with estradiol- logenin in any of the control animals ex- 17/3 (0.2 mg/10 g body weight) 6 months previously, amined. Estrogen had no effect on vitel- and slices were incubated in the presence of 3H-leu- cine (33 /i.Ci/ml) and 32ti (333 ^Ci/ml). The serum logenin synthesis up to and including the used in this culture was obtained from an estrogen- 9-hr point, but the rate of synthesis of this treated male. Samples of the culture medium were protein increased sharply between 9 and 12 analyzed at various times for the presence of3 H-label- hr after hormone administration. This was ed and 32P-labeled protein (a, b). After 76 hr, 100 /tl of culture media was denatured and subjected to elec- trophoresis on SDS-polyacrylamide gels, and gel slices were analyzed for the presence of3 H-labeled and 32P- labeled protein (c, d). Under the conditions of elec- trophoresis, the vitellogenin peptide peak was at slice No. 7 and the albumin peak was at slice No. 18. Some proteolytic degradation of the vitellogenin peptide has occurred in (d). (Bergink, unpublished). profoundly modulated by the presence of estrogen. The commitment step (possibly involving a cell cycle phenomenon or the synthesis of estrogen receptors?) cannot as yet be induced in vitro, whereas the mod- ulation can to some extent.

BIOCHEMICAL AND CYTOLOGICAL CHANGES IN 0 6 12 THE LIVER AFTER ESTROGEN TREATMENT TIME POST-ESTROGEN (h) FIG. 6. Changes in the rate of synthesis of soluble Some of the molecular changes which protein and vitellogenin in the of X. laevis males take place in the liver of unprimed males in injected at 0 time with 0.1 mg estradiol-17/3 per ani- mal. One hour prior to sacrifice, all animals were in- vivo after the injection of estradiol-17/3 jected with 20 /i.Ci 14C-leucine. Each point represents have recently received some attention. Be- the mean of determinations from three animals and tween 3 and 6 hr after estrogen injection, the vertical bars indicate the standard errors of the the rate of RNA synthesis was found to mean. From Wittliff and Kenney (1972). AMPHIBIAN VITELLOGENIN 1167

at a time when the stimulation of soluble existence of translational mechanisms con- protein synthesis in the liver had reached a trolling hormone-induced vitellogenin maximum (Fig. 6). Vitellogenin synthesis synthesis. These results are obscured then continued to increase in a linear fash- somewhat by the use of poly(U) as a mes- ion until at least 24 hr after estrogen treat- senger in some of the experiments and by ment. Cytological changes in liver paren- the lack of any rigorous characterization of chymal cells can be correlated with these the polypeptide products when endogen biochemical events. Immediately after in- ous mRNA was translated. Nevertheless, it jection of estradiol-17/3, Nicholls et al. would appear that important translational Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 (1968) observed an increase in the size of control mechanisms also come into opera- the nucleolus and a considerable prolifera- tion when estrogen acts on unprimed male tion of the rough . liver. It will be interesting to see whether Presumably much of the early protein the same transcriptional and translational synthesis observed by Wittliff and Kenney mechanisms also operate in the primed (1972) reflects the appearance of ribosomal liver or, rather, the modulation of vitel- protein. logenin synthesis by estrogen is entirely under transcriptional control. REGULATION OF VITELLOGENIN SYNTHESIS Wittliff et al. (1972) found that stimula- VITELLOGENIN SECRETION tion of synthesis of both the total soluble Although our understanding of the protein fraction and vitellogenin was al- events during vitellogenin synthesis is still most completely blocked by actinomycin D samewhat primitive, even less is known given simultaneously with the hormone, about vitellogenin secretion. Zelson and implying the requirement for synthesis of Wittliff (1973) have found by im- RNA in this response. If actinomycin was munochemical methods that vitellogenin given more than 6 hr after hormone treat- appears within the serum by 12 hr after ment, inhibition was only partial. These re- administration of estradiol-17)8. Since Witt- sults thus imply that relatively stable RNA liff and Kenney (1972) were unable to de- species are formed during the initial (3 to 6 tect vitellogenin within the liver by 9 hr but hr) burst of RNA synthesis, and that es- could by 12 hr, these data indicate that the trogen regulation of vitellogenin synthesis secretion of vitellogenin into the blood occurs at a transcriptional level. stream occurs within 3 hr after it is synthe- Clemens and Tata (1973), on the other sized in the liver. hand, found recently that translation of Dolphin et al. (1971) have found that poly(U) by hepatic ribosomes was stimu- labeled vitellogenin appears in the incuba- lated following estrogen treatment. This tion medium within about 2 hr after stimulation was maintained when the ribo- isotopic precursors are supplied to liver somes were stripped of endogenous mRNA slices from estrogen-treated animals. Ex- by preincubation in vitro. Preincubation actly the same progress curves were ob- with puromycin and washing with 0.5 M KC1 served for incorporation of 3H-leucine and also were without effect, indicating the un- 32 Pi into the protein moiety and 14C-acetate likelihood that the change in activity after into the lipid moiety of serum vitellogenin, estrogen treatment is associated with either leading those authors to conclude that the the presence of nascent peptides remaining attachment of phosphate and to vitel- attached to the ribosomes after preincuba- logenin occurs at an early stage in its biosyn- tion or the attachment or possible super- thesis and about 2 hr before its release into natant factors. Also, they found that cell sap the incubation medium. Pulse-labeling of taken from the liver of estrogen-treated liver slices for 30 min (Fig. 7) also indicated animals supported an increased amino acid that the protein which was synthesized re- incorporation (relative to control cell sap) mained in the liver for several hours; be- by ribosomes from control or treated ani- tween 2 and 3 hr, approximately 60% of the mals in vitro. They thus argued for the labeled protein was then lost from the liver, 1168 ROBIN A. WALLACE AND E. WILLEM BERGINK

pulie amounts of two serum protein fractions (indicated in Fig. 2) and vitellogenin, all of which had been previously labeled with 3H-leucine, into three groups of HCG- treated females under the same conditions (Wallace and Jared, 1969). Labeled protein was monitored in the blood and ovary, and the results (Fig. 8) indicated that vitellogen- in was rapidly removed from the circula- Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 tion and became associated with ovarian protein, whereas the other two serum pro- tein fractions did so to a much lesser extent. In the beginning of the experiment, when 0 2 4 6 the three components were present in the Incubation time (h) 3 serum in equivalent amounts, the incorpo- FIG. 7. Incorporation of H-leucine into total tissue ration of vitellogenin into ovarian protein protein and vitellogenin after pulse-labeling for 30 min. All values are expressed as a percentage of that appeared to be about 50 times more rapid for total tissue protein at 2 hr, which was 5.0 x 103 than the incorporation of either of the dpm/100 mg wet weight of tissue. The mean ± SD for serum protein fractions. Cytological studies six experiments is shown. •, Incorporation into total have indicated that serum proteins are di- tissue protein; A, incorporation into secreted vitel- logenin. From Merry et al. (1973). rectly taken up by vitellogenic oocytes within the ovary by a micropinocytotic pro- corresponding to the appearance of labeled cess (Wallace and Dumont, 1968; Dumont, vitellogenin in the serum (Merry et al., 1972). 1973). These results are obscured some- We have recently developed a culture what by the use of buffer solutions rather procedure for isolated oocytes (Jared and than a complete medium for incubation. Wallace, 1969) in order to investigate some However, relatively high concentrations of of these events in vitro. Oocytes of various liver tissue were used, and an electropho- sizes isolated from normal females have retic analysis of the incubation medium in- been found to incorporate small amounts dicated a considerable amount of vitelloge- of protein when placed in medium contain- nin and albumin (Dolphin etal., 1971). Pre- ing labeled vitellogenin (Fig. 9a). When sumably this was derived from previously females were injected with HCG, many of synthesized serum entrained within the the very largest oocytes were ovulated and liver slices, so that adequate serum factors protein uptake by those which were vitel- and amino acids may have been present for logenic (i.e., producing yolk platelets) was the short-term periods studied. A more de- found to be greatly stimulated (Fig. %). finite conclusion concerning the addition of phosphorus and lipid to the vitellogenin o peptide must await a study which utilizes 2000- SERUM

more frequent time points and considers J SI •^VITELLOGENIN 1500- the precursor pools. Detailed cytological ,' studies on vitellogenin synthesis, transport, and secretion have yet to be done. 1000- E S^VITELLOGENIN 200 g 500- UPTAKE OF VITELLOGENIN BY OVARY AND OOCYTES 5O 75 0 25 50 75 We have previously mentioned that TIME AFTER [3H]PR0TEIN INJECTION (hr) gonadotropins promoted the removal of FIG. 8. Loss of labeled protein from the serum (a) vitellogenin from the bloodstream, an ef- and its incorporation into the ovary (6) following the injection of equivalent amounts of 3H-vitellogenin fect which was abolished by ovariectomy. 3 (O—O) or serum protein fractions H-S1 (• •) or More direct proof for the role of the ovary 3H-S2 (A-—A) into HCG-treated females. From Wal- was obtained by injecting equivalent lace and Jared (1969). AMPHIBIAN VITELLOGENIN 1169

< UJ CONTROL 200-

< O-L •• •»t»« Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 HCG-STIMULATED CM 800- og o -E o o ^ 600- CL •o -=- 400-

o o I '••s *v. O-i_ora 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 OOCYTE DIAMETER (mm) FIG. 9. Extent ofprotein incorporation as a function labeled protein is expressed both as dpm/oocyte (O) of oocyte size in oocytes from (a) normal females and and dpm/oocyte divided by the surface area of the (b) HCG-treated females. The incorporation of oocyte (•). From Wallace et al. (1970). Vitellogenin incorporation in vitro was logenin by the oocyte appears to be a highly linear with time for at least 6 days, and selective process (Fig. 8). This selectivity enhanced uptake was found to persist for at persists in isolated oocytes: vitellogenin is least 3 to 4 weeks after the initial injection of sequestered 64 to 189 times more rapidly gonadotropin (Wallace et al., 1970). At- than several other purified proteins which tempts to promote vitellogenin uptake in we tested (Table 2). Brief exposure of iso- vitro by various gonadotropins and pitui- lated oocytes to calcium-free solution tary extracts have thus far been unsuccess- causes a reversible loss of sequestering abil- ful. ity, an effect which we have ascribed to the As previously seen, the uptake of vitel- temporary loss of receptor sites for vitel-

TABLE 2. Uptake of various purified labeled proteins by isolated Xenopus laevis oocytes.a Concentration Specific Uptake in medium activity 8 2 (mg/ml) (cpm/Mg) (cpm/mm ) (ng/mm ) Ferritin (equine) 3.3 130 1.8 14 Hemoglobin (bovine) 2.9 767 3.6 5 Serum albumin (bovine) 2.7 1590 9.0 6 Vitellogenin (X. laevis) 2.75 2530 2241 886 "Proteins were labeled by reductive alkylation (Rice and Means, 1971). Oocytes were incubated with labeled protein for 18 hr. Each value for uptake represents the average value for 12 oocytes. All oocyte.s were taken from the same HCG-treated female and were approximately 1.0 mm diameter. (Wallace and Jared, unpublished.) 1170 ROBIN A. WALLACE AND E. WILLEM BERGINK logenin from the oocyte surface (Wallace et the more dense yolk platelets are also al., 1973). Pulse-chase experiments involv- labeled and only a minor peak appears in ing 3H, 32P-vitellogenin incorporation have the upper part of the gradient (Fig. 10c). also been performed, in which homoge- The terminus for incorporated vitellogenin nates of incubated oocytes were prepared thus seems to be structures with the same at various times and centrifuged on 20 to density as yolk platelets. 60% sucrose gradients (Jared et al., 1973). The results of these experiments have indi-

TRANSFORMATION OF VITELLOGENIN INTO Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 cated that immediately after a 10-min pulse, essentially all the radioactivity is in LIPOVITELLIN AND PHOSVITIN the upper half of the gradient associated Vitellogenin has never been isolated as with various membrane fractions (Fig. 10a). such from the ovary or oocytes. Rather, 80 Forty-five minutes later, labeled material in to 90% of the total protein in the full-grown part of the membrane region disappears oocyte is found in yolk platelets, crystalline and considerable isotope is found in the structures containing almost exclusively the upper part of the region containing yolk proteins lipovitellin and phosvitin platelets (Fig. 10b). Eighteen hours later, (Karasaki, 1963; Wallace, 1963; Wallace and Dumont, 1968). Lipovitellin monomer

6- has a molecular weight in its native state of about 200,000 and is comprised of about 20% lipid and about 0.5% protein-phos- phorus. Phosvitin has a molecular weight of 35,000 to 40,000 and a protein-phos- phorus content of about 10%. Vitellogenin has been found to have essentially the same amino acid content as the combined yolk platelet proteins (Redshaw and Follett, 1971), and it cross-reacts with an antibody preparation against lipovitellin (Wittliff and Kenney, 1972). In order to explore the dynamic relation- ship between vitellogenin and the yolk pro- teins, double-labeled (with 3H-leucine and 32 P() vitellogenin was injected into HCG- treated females. At various time intervals thereafter, the ovaries were removed and extracted with strong salt and the extracts were chromatographed on TEAE-cellu- lose. A typical chromatogram (Fig. 116) of such ovarian extracts indicates that most of the protein is represented by yolk platelet components (compare with Fig. lla). The labeling found in an extract several hours after injection of 3H, 32P-vitellogenin indi- 10 20 30 40 cated the presence of a single component, FRACTION NO. eluting between 0.50 and 0.60, which had a FIG. 10. Distribution of radioactivity in centrifuged 3H/3ZP ratio similar to the injected 3H, sucrose gradients derived from 15 oocytes which were 32 3 32 P-vitellogenin (Fig. 1 lc). Eight hours incubated in the presence of H, P-vitellogenin for 3 32 10 min and homogenized immediately (a) or after after the injection of H, P-vitellogenin further incubation in unlabeled medium for either 45 (Fig. lid) the labeling pattern appeared min (6) or 18 hr (c). Yolk platelets were distributed rather heterogeneous, with most of the 3H between fractions 12-25, mitochondria around 35, 32 and various membranous structures were found above eluting in position 0.40 and most of the P fraction 40. From Jared et al. (1973). eluting at position 0.72. By 20 hr (Fig. 1 le), AMPHIBIAN VITELLOGENIN 1171

3 32 a YOLK PLATELETS in the presence of H, P-vitellogenin for 1 E 0.8 hr and followed the fate of incorporated g Q6 •600 label at various times (Wallace et al., 1972).

-4O0 After the first hour, no incorporated vitel- logenin was yet converted into lipovitellin -ZOO or phosvitin. This would be at a time when -0 b OVARY EXTRACT some of the labeled vitellogenin would al- A ready have been transferred to the yolk platelets (Fig. 10b). An hour after the pulse, Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 conversion to lipovitellin and phosvitin was observed, so this process must take place E C OVARY EXTRACT - 2 HOURS within the yolk platelet rather than at the 200 c" cell surface or within transport particles. 0 \ d OVARY EXTRACT-8 HOURS •400 £ During the conversion process, the amino o acid pools remained essentially unlabeled, 200 indicating a macromolecular restructuring O rather than a process involving breakdown

OVARY EXTRACT - 20 HOURS and resynthesis. Control experiments 1200 3 32 1000 utilizing H-leucine and Pi in the medium indicated that the yolk platelets never be- 800 came labeled (Jared et al., 1972), nor was 6O0 any specific synthesis or phosphorylation of 400 lipovitellin or phosvitin detected within the 200 oocyte (Wallace et al., 1972). We were able

04 06 to conclude, therefore, that at least 99% of ELUTION POSITION the protein present in the yolk platelet •—SAMPLE PLACED ON COLUMN crystal is derived from a source outside the FIG. 11. Chromatography and labeling of yolk oocyte. platelet and ovarian extracts after injection of various precursors into intact females, a, Yolk platelets (220 We have recently analyzed vitellogenin mg) isolated from the ovary of an HCG-treated female and the yolk proteins on polyacrylamide (1000 units HCG given 13 days) and 0.06 /i.Ci/10 g gels in the presence of SDS and mercap- body weight injected 24 hr previous to isolation, b-e, Approximately 180 mg protein extracted with 1.5 M toethanol in order to indicate the relation- NaCI from the ovaries of HCG-treated females (given ship of component peptide chains (Bergink 1000 units HCG per animal 9 days prior to 3H, 32P-vi- and Wallace, 1974). Vitellogenin isolated tellogenin injection). Ovarian extracts were made 2 (b, 3 32 by our usual chromatographic procedures c), 8 (d), and 20 («) hr after the injection of H, P-vitel- was found to consist of a variety of peptides logenin. The absorbancy (a, b) is indicated by a solid line, 3JP-protein labeling (a, c-e) by open circles (Fig. 12a), but when it was isolated in the (O O) and 3H-protein labeling (c-e) by open trianr presence of proteolytic inhibitors and gles (A A). From Wallace and Jared (1969). chromatographed with sterile buffers, generally only one peptide was observed, with a molecular weight of about 200,000 the amounts of 3H label in position 0.40 and (Figs. 126, 14a). Lipovitellin was found to of 32P label in position 0.72, corresponding consist of two peptides with molecular to the positions for lipovitellin and phosvi- weights of 120,000 and 31,000 and present in a 1:1 molar ratio (Fig. 12c). When pro- tin, respectively, were even more pro- 32 nounced (lipovitellin contains 98% of the teins previously labeled with P were leucine in the yolk platelet crystal and analyzed on gels, it was discovered that the phosvitin contains 79% of the protein- smaller peptide contained essentially all of phosphorus) (Wallace and Jared, 1969). A the protein-phosphorus associated with similar conversion of 32P-vitellogenin into lipovitellin (Fig. 13a). Phosvitin formed two yolk platelet proteins in vivo has been ob- labeled bands, one corresponding to a served by Follett et al. (1968). molecular weight of 35,000 and the other, We have also incubated isolated oocytes migrating with the dye front, probably con- 1172 ROBIN A. WALLACE AND E. WILLEM BERGINK

VITELLOGENIN 120,000) similar to that of the large (degraded) lipovitellin peptide and of phosphopep- tides in a molecular-weight range of 30,000 to 40,000 (Fig. 146, c). Phosphopeptides migrating with the dye front were also seen. A high-molecular-weight (about 90,000) phosphoprotein was formed during the chymotrypsin treatment (Fig. 14c). This in- teresting peptide was an intermediate pro- Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 duct since it rapidly disappeared upon further incubation: Its appearance (along with that of a nonphosphopeptide with a molecular weight of 120,000) during pro- teolysis of vitellogenin is an indication that the phosphoproteins from the platelets, i.e., phosvitin and the small lipovitellin subunit, are clustered together on one side

8- TRACKING LIPOVITELLIN DYE 6-

LIPOVITELLIN 4-

2-

0 40- PHOSVITIN

20-

0 MIGRATION DISTANCE 100- FIG. 12. Spectrophotometric scan of polyacrylamide YOLK PLATELET gels (5% acrylamide, 0.1% SDS) stained with coomas- 80- PROTEINS sie brilliant blue, c, Vitellogenin degraded during the isolation procedure, b, Vitellogenin in which most of the predominant peptide (Mol wt = 200,000) was not 60- degraded, c, Lipovitellin, showing two peptides with a molecular weight of 110,000 and 31,000. From Ber- 40- gink and Wallace (1974). 20- sisting of small phosphopeptides (Fig. \%). Electrophoresis of the total yolk platelet 0 —i 1 1 1 1 1— proteins indicated that the three phospho- 70 rus-containing peptide fractions found in 0 10 20 30 40 50 60 MIGRATION DISTANCE (mm) lipovitellin and phosvitin were the only FIG. 13. Electrophoresis of 32P-labeled proteins on a ones present (Fig. 13c). Brief treatment of polyacrylamide gel (5% acrylamide, 0.1% SDS). a, 32P- undegraded 3H, 32P-vitellogenin (Fig. 14a) labeled, delipidated lipovitellin. 6, 32P-labeled phosvi- with dilute solutions of either trypsin or tin. c, 32P-labeled yolk platelet proteins (the isolated, intact platelets were dissolved in 1% SDS—0.1 M chymotrypsin was found to cause rapidly mercaptoethanol—0.01 M phosphate buffer, pH 6.8, the appearance both of a nonphosphopep- prior to electrophoresis). For further details, see Ber- tide with a molecular weight (110,000 to gink and Wallace (1974). AMPHIBIAN VITELLOGENIN 1173

100 radioactive isotope, similarity between vitellin and -40 lipovitellin. Proc. Soc. Exp. Biol. Med. 98: 703-707. Alexander, S. S., and C. W. Bellerby. 1935. The effect of captivity upon the reproductive cycle of the South African clawed toad (Xenopus laevis). J. Exp. Biol. 12: 306-314. Alexander, S. S., and C. W. Bellerby. 1938. Experi- mental studies on the sexual cycle of the South Afri- can clawed load {Xenopus laevis) I. J. Exp. Biol. 15:

74-81. Downloaded from https://academic.oup.com/icb/article/14/4/1159/2069954 by guest on 28 September 2021 Ansari, A. Q., P. J. Dolphin, C. B. Lazier, K. A. Mun- day, and M. Akhtar. 1971. Chemical composition of an estrogen-induced calcium-binding glyco- lipophosphoprotein in Xenopus laevis. Bio- chem.J. 122: 107-113. Bellerby, C. W. 1938. Experimental studies on the sexual cycle of the South African clawed toad (Xenopus laevis) II. J. Exp. Biol. 15: 82-90. Bellerby, C. W., and L. Hogben. 1938. Experimental studies on the sexual cycle of the South African clawed toad (Xenopus laevis) III. J. Exp. Biol. 15: 91-100. Bergink, E. W. 1973. Synthese van dooiereiwitten door de haan. Ph.D. Thesis, University of Groningen. Bergink, E. W., H. J. Kloosterboer, M. Gruber, and G. AB. 1973. Estrogen-induced phosphoprotein 0 20 40 60 80 synthesis in roosters. Kinetics of induction. Biochim. MIGRATION DISTANCE (mm) 3 32 Biophys. Acta 294: 497-506. FIG. 14. Electrophoresis of H, P-vitellogenin on Bergink, E. W., and R. A. Wallace. 1974. Precursor- polyacrylamide gels (5% acrylamide, 0.1% SDS). a, product relationship between amphibian vitelloge- Delipidated vitellogenin. b, Vitellogenin incubated for nin and the yolk proteins lipovitellin and phosvitin. 5 min with trypsin at an enzyme:substrate ratio of J. Biol. Chem. 249: 2897-2903. 1:10,000. c, Vitellogenin incubated for 5 min with Brachet, J. 1942. La localisation des acides chymotrypsin at an enzyme:substrate ratio of 3 pentosenucleiques dans les tissus animaux et les 1:10,000. H-labeled protein in gel slices is indicated oeufs d'Amphibiens en voie de developpement. by a dashed line ( ) while 32P-labeIed protein is Arch. Biol. 53: 207-257. indicated by a solid line ( ). From Bergink and Busson-Mabillot, S. 1969. Donnees recentes sur la Wallace (1974). vitellogenese. Ann. Biol. 8: 199-228. Clemens, J. J., and J. R. Tata. 1973. An analysis of the of the vitellogenin polypeptide chain and effects of oestrogen treatment in vivo on the pro- are not interrupted by the large lipovitellin tein-synthetic activity of male Xenopus liver cell-free subunit. systems. Eur. J. Biochem. 33: 71-80. Cook, W. H. 1961. Proteins of hen's egg yolk. We have thus obtained some evidence (London) 190: 1173-1175. that vitellogenin consists of at least two Cook, W. H., and W. G. Martin. 1969. Egg lipopro- polypeptide chains with a molecular weight teins, p. 579-615. In E. Tria and A. M. Scanu [ed.], of about 200,000, and that conversion of Structural and functional aspects of lipoproteins in these peptide chains into the peptides that living systems. Academic Press, New York. De Vlaming, V. L. 1971. The effects of food depriva- make up the yolk platelet proteins most tion and salinity changes on reproductive function likely occurs by specific proteolytic splitting in the estuarine gobiid fish, Gillichlhys mirabtlis. Biol. in regions which are also susceptible to pro- Bull. 141: 458-471. teolytic attack by trypsin and chymotrypsin. Dolphin, P. J., A. Q. Ansari, C. B. Lazier, K. A. Mun- The identity and source of a hypothetic day, and M. Akhtar. 1971. Studies on the induction and biosynthesis of vitellogenin, an oestrogen in- proteolytic enzyme which converts vitel- duced glycolipophosphoprotein. Biochem. J. 124: logenin into lipovitellin and phosvitin 751-758. within the forming yolk platelets remains to Dumont, J. N. 1972. Oogenesis in Xenopus laevis be established. (Daudin) I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136: REFERENCES 153-164. Abe, T., Y. Tanabe, T. Kaneko, K. Mogi, and T. Follett, B. K., T. J. Nicholls, and M. R. Redshaw. 1968. Hosoda. 1958. Immunochemical studies with The vitellogenic response in the South African 1174 ROBIN A. WALLACE AND E. WILLEM BERGINK

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