Cell and Tissue Research

Cell Tissue Res. 203, 241-247 (1979)

9 by Springer-Verlag 1979

In vitro Induced Pinocytotic Activity by a Juvenile Hormone Analogue in Oocytes of Drosophila melanogaster Franco Giorgi Istituto di Istologia e Embriologia,Universitfidi Pisa, Pisa, Italy

Summary. Pinocytotic activity has been analyzed in D r o s o p h i l a oocytes following either in vivo or in vitro exposure to horseradish peroxidase. The enzyme tracer gains access to the yolk spheres only when supplied to the oocyte in vivo. In oocytes cultured in vitro, peroxidase remains restricted to the residual coated vesicles and to the tubular profiles formed in excess in the cortical ooplasm. In an attempt to induce peroxidase uptake by oocytes cultured in vitro, various incubations were tested. Among these, hemolymph from both sexes is capable of promoting peroxidase uptake up to a level comparable to that detectable in vivo. On the other hand, fat body extracts fail to promote such cellular activity. Finally, the juvenile hormone analogue ZR-515 is shown to be the only factor required to promote pinocytotic activity under the experimental conditions tested. The observations are interpreted to indicate that vitellogenin has no inductive role on pinocytosis but simply acts by adhering to the forming coated vesicles which in turn are produced by the oolemma in response to the action of juvenile hormone. Key words: Pinocytotic activity - Juvenile hormone -

Drosophila -

Oocytes.

Formation of yolk spheres in insect oocytes occurs by pinocytotic uptake of a soluble yolk precursor known as vitellogenin (Engelmann, 1970). For this process to occur, the two basic conditions are the presence of vitellogenin in the hemolymph, and the competence of the oocyte to engage in pinocytotic activity (Kambysellis, 1977). Apparently, both these conditions are controlled by the same factor, i.e., juvenile hormone, although ecdysone may also be required at some stage in the process (Spielman et al., 1971; Went, 1978; Handler and Postlethwait, 1978). The evidence presently available is in fact consistent with the view that juvenile hormone may induce vitellogenin synthesis in the fat body (Pan et al., 1969) and may induce the oocyte to incorporate and process vitellogenin (Handler and Postlethwait, Send offprint requests to: Dr. F. Giorgi, Istituto di Istologia e Embriologia, Universit~idi Pisa, Via A. Volta 4, 1-56100Pisa, Italy

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1977). T h e e n d o c r i n e c o n t r o l o f vitellogenesis in Drosophila has been studied b y e m p l o y i n g defective m u t a n t s (Postlethwait a n d Weiser, 1973; G a v i n a n d W i l l i a m s o n , 1976; K a m b y s e l l i s a n d C r a d d o c k , 1976; P o s t l e t h w a i t a n d H a n d l e r , 1978) as well as wild type altered flies (Postlethwait et al., 1976). In b o t h these instances, vitellogenesis c o u l d be a c c o m p l i s h e d only following t o p i c a l a p p l i c a t i o n o f a juvenile h o r m o n e analogue. T h e picture emerging f r o m these findings is in line with the generally held view t h a t vitellogenin, t h o u g h necessary ( H a u s m a n et al., 1971), is n o t by itself sufficient to result in its u p t a k e by the oocyte. W h i l e the h o r m o n a l c o n t r o l over vitellogenin synthesis in in vitro systems has been extensively studied in a n u m b e r o f species ( E n g e l m a n n et al., 1971); K o e p p e a n d O f e n g a n d , 1976; P a n a n d W y a t t , 1976), no k n o w l e d g e has so far been gained r e g a r d i n g p i n o c y t o t i c activity o f oocytes. T h e present study was u n d e r t a k e n to d e t e r m i n e w h e t h e r Drosophila oocytes m a i n t a i n e d in vitro are c a p a b l e o f p i n o c y t o t i c activity, and, if not, w h e t h e r a h o r m o n a l factor is able to restore such activity u n d e r these conditions. Materials and Methods

Drosophilamelanogaster(Or-K strain) were reared at 25~ on a standard Drosophila-diet.Female flies, 2-3 days old, were dissected in Drosophila-Ringerand ovaries were cultured for periods ranging from 30 rain to 2 h. In a preliminary series of experiments, ovaries were incubated in the presence of either female or male hemolymph or fat body extracts. Subsequently various culture media were tested and of these Schneider's (Gibco) gave the best ultrastructural preservation. Two sets of culturing conditions were assayed in this study: in each experimental group 1 ml of culture medium contained 1 ~tl of a 5 % solution of a juvenile hormone analogue - either ZR-515 (Zoecon) or farnesyl methyl ester (Ciba Geigy) - in acetone. Control ovaries were incubated for an equivalent length of time in the same culture medium with only acetone added. To detect pinocytotic activity in vitellogenic oocytes cultured as specified above, peroxidase was added to the culture medium as a cytochemical marker at a concentration of 0.1 mg/ml. For ultrastructural characterization of pinocytotic activity under in vivo conditions, several female flies were injected with peroxidase at 0.1 mg/ml concentration in Drosophila-Ringer,and ovaries were allowed to incorporate it for periods of time equivalent to those of the in vitro experiments. At the end of each culture period, ovaries were fixed for two hrs in 5 ~ glutaraldehyde in 0.1 M caeodylate buffer at pH 7.2. Followinga prolonged wash in cacodylate buffer, tissues were incubated for 15 min in a solution containing 10 mg of diamino benzidine-HC1 in 10 ml of 0.1 M TRIS-HCI buffer at pH 7.6 with 0.1 ml of 1% hydrogen peroxide added. Subsequently, the ovaries were post-fixed for 4 hrs in 1% OsO4 in 0.1 M cacodylate buffer at pH 7.2 at 4~ and embedded in an Epon-Araldite mixture which was left to polymerize at 60~C for three days. Silver-to-pale gold sections were stained in uranyl acetate and lead citrate and observed in a Siemens Elmiskop 101 electron microscope working at 60 Kv. Results

Vitellogenic oocytes show n u m e r o u s microvilli along the o o l e m m a , a n d pits a n d c o a t e d vesicles between o r i m m e d i a t e l y b e l o w them. L a r g e r vesicles a n d a limited n u m b e r o f t u b u l a r profiles are o b s e r v e d in deeper regions o f the cortical o o p l a s m , a n d some o f the vesicles m a y be identified with ease as f o r m i n g y o l k spheres. F o l l o w i n g in vivo e x p o s u r e to peroxidase, these oocytes show all o f the a b o v e m e n t i o n e d structures, i.e., pits, c o a t e d vesicles, tubules, a n d f o r m i n g y o l k spheres, labelled with p e r o x i d a s e r e a c t i o n p r o d u c t (Fig. 1). Vitellogenic oocytes fixed following in vitro e x p o s u r e to peroxidase, in either Drosophila R i n g e r o r Schneider's culture m e d i u m , a p p e a r r e m a r k a b l y different f r o m those e x p o s e d to the tracer in vivo. M a j o r u l t r a s t r u c t u r a l a l t e r a t i o n s include a

Fig. 1. Cortical ooplasm of stage 10 ovarian chamber exposed for 15 min to peroxidase in vivo. Note reaction product along limiting membrane of yolk spheres (arrows) and in some coated vesicles (cv) underneath oolemma (oo/). Section unstained. Vm vitelline membrane; rnb main body of yolk sphere; L lipid droplet. • 15,000 Fig. 2. Cortical ooplasm of stage 10 ovarian chamber exposed for 15 min to peroxidase in vitro: no yolk spheres present; an excessive number of tubular profiles (t) formed during exposure to tracer, x 12,000 Fig. 3. Cortical ooplasm of stage 10 ovarian chamber exposed for 30 min to peroxidase in vitro. Several fused tubular profiles (t) containing tracer, x 12,000

Fig. 4. Cortical ooplasm from stage 10 ovarian chamber exposed for 15 min to peroxidase in presence of juvenile hormone analogue ZR-515. Note several forming yolk spheres labelled with peroxidase reaction products, x 10,000 Fig. 5, Follicle-oocyte border of stage 8 ovarian chamber exposed for 15 min to peroxidase in presence of juvenile hormone analogue ZR-515. Peroxidase reaction products adhere to oolemma (oo/), to vesicles (v) in cortical ooplasm and to limiting membrane of forming yolk spheres (arrows). FC follicle cells. x 6000 Fig. 6, Cortical ooplasm of stage 10 ovarian chamber exposed for 30 min to peroxidase in presence of juvenile hormone analogue farnesyl methyl ester. Atypical vesicles and tubules formed in this region. Vm vitelline membrane, x t4,000

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reduction in the number of microvilli and of the pits and coated vesicles which become labelled with peroxidase during the culture period. These alterations are accompanied by an increase in the length and number of the tubular profiles in the cortical ooplasm and by the absence of peroxidase transfer to the forming yolk spheres (Figs. 2, 3). In ovaries cultured for the same periods in the presence of either female or male hemolymph, pinocytosis occurs as it does under in vivo conditions. On the contrary, the presence of fat body per se is not sufficient to elicit peroxidase uptake into the forming yolk spheres. The easiest way to interpret these results is to consider the presence of vitellogenin not necessary and not effective for pinocytosis to occur. Vitellogenin, a sex-limited protein, is present only in the female hemolymph and fat body. The experiments mentioned indicate that a factor common to male and female hemolymph is sufficient to promote pinocytosis even in the absence of vitellogenin. This factor is juvenile hormone, as shown by the result of incubation of ovaries with a juvenile hormone analogue ZR-515 (Figs. 4, 5). Farnesyl methyl ester may be considered as unspecific for the promotion of pinocytosis in Drosophila oocytes (Fig. 6). Discussion

The evidence presented in this study indicates that yolk spheres in in vitro cultured oocytes become labelled with peroxidase only when the juvenile hormone analogue ZR-515 is present in the culture medium. Thus juvenile hormone appears to be the only factor required to promote such activity under in vivo conditions (see also Lender and Laverdure, 1967; Ittycheriah and Stephanos, 1969; Adams and Eide, 1972; Laverdure, 1975). Recently, Srdic and Jacobs-Lorena (1978) have shown that male abdomens of Drosophila provide a suitable melieu for oocytes to undergo vitellogenic maturation. These observations along with those by Bell (1972) are taken to mean that male hemolymph may either possess a hormone controlling oogenesis or may be induced to produce it following ovarian implantation. However, it should be noted that hormonal stimulation of pinocytosis in oocytes does not necessarily imply that vitellogenesis may occur in the absence of viteUogenin. On the contrary, the evidence provided in this study is consistent with the view that the yolk precursor or vitellogenin molecules bind to portions of the oolemma forming pits and coated vesicles, and that the juvenile hormone presumably acts by controlling the oolemma in its modulation during pinocytosis. Pinocytosis is in fact thought of as comprising (a) attachment to the cell surface as a first step towards the intake process, and (b) invagination of the loaded membrane as a second one (Jacques, 1969). The fact that these two steps are not causally related to each other in insect oocytes differentiates the latter from both somatic cells and amphibian oocytes. A large body of evidence has demonstrated that protein uptake by somatic cells is proportional to the actual protein concentration in the medium (Cohn and Benson, 1965; Davis et al., 1973; Steinman and Cohn, 1974; Contractor and Krakauer, 1976). This indicates that exogenous proteins in somatic cells have a dual role: that of attachment to the cell surface and, in doing so, that of controlling the rate of membrane invagination (Schellens et al., 1976). In amphibian oocytes, growth in vitro is simply a function of vitellogenin concentration in the culture medium (Wallace et al., 1978). On the other hand, absence of vitellogenesis in

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insects, in spite of the presence of vitellogenin in the hemolymph, suggests independent control of pinocytosis such as that provided by the juvenile hormone (Bell, 1969; Bell and Barth, 1971; Giorgi, 1979). As previously shown, the cortical ooplasm of juvenile hormone deprived oocytes contains an excessive number of tubular profiles. This observation may provide a clue as to the actual mechanism by which pinocytosis is hormonally controlled. A crucial step in this control could be sought in the transfer of the membrane-bound vitellogenin from newly formed coated vesicles to forming yolk spheres. This interpretation is substantiated by the evidence that no such transfer occurs in the absence o f juvenile hormone. If pinocytosis is impeded at this juncture, membrane turnover could be affected in phases either preceding or following it. Accumulation of membraneous fragments in the form of tubules could then be explained as resulting from saturation of the membrane movement into the cell prior to exhaustion of the coated vesicle reservoir. According to this view, tubules would constitute a physiological alternative for direct transfer of coated vesicles to the yolk spheres and, in case this transfer is blocked, the only possible way for the oocyte to get rid of the temporary intake of excess membrane. On the basis of the present evidence several questions are now open. In particular, it would be of interest to know whether an inducibility threshold exists for pinocytosis in Drosophila oocytes, as shown for instance in Bombyx mori (Legay et al., 1976), or if instead this cellular activity is causally related to the hormonal inducer in a dose-response manner. It should be finally checked whether oocyte competence to respond to juvenile hormone is somehow related to the presence of ecdysone as suggested for other insect species (Hagedorn et al., 1977; Lagueux et al., 1977).

References Adams, T.S., Eide, P.E.: A method for the in vitro stimation of house fly egg development with a juvenile hormone analog. Gen. Comp. Endocrinol. 18, 12-21 (1972) Bell, W.I.: Dual role of juvenile hormone in the control of yolk formation in Periplaneta americana. J. Insect Physiol. 15, 1279-1290 (1969) Bell, WJ.: Yolk formation by transplanted' cockroach oocytes. J. Exp. Zool. 181, 4148 (1972) Bell, W.J., Barth, R.H Jr.: Initiation of yolk deposition by juvenile hormone. Nature New Biology. 230, 220-221 (1971) Cohn, Z.A., Benson, B.: The in vitro differentiation of mononuclear phacocytes. II. The influence of serum on granules formation, hydrolases production and pinocytosis. J. Exp. Med. 121, 835-848 (1965) Contractor, S.F., Krakauer, K.: Pinocytosis and intracellular digestion of 12Si.labelled haemoglobin by trophoblastic cells in tissue culture in the presence and absence of serum. J. Cell Sci. 21, 595-607 (1976) Davis, P., Allison, A.C., Haswell, A.D.: The quantitative estimation of pinocytosis using radioactive colloidal gold. Biochem. Biophys. Res. Commun. 52, 627-634 (1973) Engelmann, F.: The physiology of insect reproduction. Oxford: Pergamon Press (1970) Engelmann, F., Hill, L., Wilkens, J.L.: Juvenile hormone control of specific female protein synthesis in Leucophaea maderae, Schistocerca vaga and Sarcophaga bullata. J. Insect. Physiol. 17, 2179-2191 (1971) Gavin, J.A., Williamson, J.H.: Juvenile hormone induced vitellogenesis in apterous 4, a non-vitellogenic mutant in Drosophila melanogaster. J. Insect Physiol. 22, 1737-1742 (1976)

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Giorgi, G.: Coated vesicles in the oocyte. In: Coated Vesicles. (Whyte A. and Ockleford, C.D. eds.) Cambridge University Press (1979) Hagedorn, H.H., Turner, S., Hagedorn, E~., Pontecorvo, D., Greenbaum, P., Pferffer, D., Wheelock, G., Flanagan, T.R.: Postemergence growth of the ovarian follicles ofAedes aegypti. J. Insect Physiol. 23, 203-206 (1977) Handler, A.M., Postlethwait, J.H.: Endocrine control of vitellogenesis in Drosophila melanogaster: Effects of the brain and Corpus allatum. J. Exp. Zool. 202, 389-402 (1977) Handler, A.M., Postlethwait, J.H.: Regulation of vitellogeninsynthesis in Drosophila by ecdysterone and juvenile hormone. J. Exp. Zool. 206, 247-254 (1978) Hausman, S.J., Anderson, L.M., Teller, W.H.: The dependence of yolk formation in vitro on specific blood proteins. J. Cell Biol. 48, 303-313 (1971) Ittycheriah, P.I., Stephanos, S.: In vitro culture of ovary of the plant bug, lphita limbata Stal. Indian J. Exp. Biol. 7, 17-19 (1969) Jacques, J.P.: Endocytosis. In: Lysosome in Biology and Pathology (Dingle J.T., Fell H.B. eds.), I, pp. 395-420. New York and London: Wiley 1969 Kambysellis, M.P.: Genetic and hormonal regulation of vitellogenesis in Drosophila. Am. Zool. 17, 535-549 (1977) Kambysellis, M.P., Craddock, E.M.: Genetic analysis of vitellogenesis in Drosophila. Genetics 183, s38 (1976) Koeppe, J., Ofengand, J.: Juvenile hormone induced biosynthesis of vitellogeninin Leucophaea maderae. Arch. Biochem. Biophys. 173, 100-113 (1976) Lagueux, M., Hirn, M., Hoffmann, J.A.: Ecdysone during ovarian development in Locusta migratoria. J. Insect Physiol. 23, 109-119 (1977) Laverdure, A.M.: Culture in vitro des ovaries de Tenebrio molitor, hormone juvenile, vitellogenrse et suvie des jeunes oocytes. J. Insect Physiol. 21, 33-38 (1975) Legay, J.M., Calvez, B., Hirn, M., De Reggi, M.L.: Ecdysone and oocyte morhogenesis in Bombyx mori. Nature 262, 489-490 (1976) Lender, T., Laverdure, A.M.: Culture in vitro des ovaries de Tenebrio molitor (Colroptrre). Croissance et vitellogenrse, C.R. Acad. Sci., Paris, 265, 451-454 (1967) Pan, M.L., Wyatt, G.R.: Control ofvitellogeninsynthesis in the monarch butterfly by juvenile hormone. Dev. Biol. 54, 127-134 (1976) Pan, M.L., Bell, W.L., Telfer, W.H.: Vitellogenic blood protein synthesis by insect fat body. Science 165, 393-394 (1969) Postlethwait, J.H., Handler, A.M.: Non vitellogenetic female sterile mutants and the regulation of vitellogenesis in Drosophila melanogaster. Dev. Biol. 67, 225-236 (1978) Postlethwait, J.H., Weiser, K.: Vitellogenesis induced by juvenile hormone in the female sterile mutant apterous four in Drosophila melanogaster. Nature 244, 284-285 (1973) Postlethwait, J.H., Handler, A.M., Gray, P.W.: A genetic approach to the study of juvenile hormone control of vitellogenesis in Drosophila melanogaster. In: The Juvenile Hormones (Gilbert, L.I. ed.) pp. 449-469. New York: Plenum publishing Corp. 1976 Schellens, J.P.M., Brunk, U.T., Lindgren, A.: Influence on ruffling activity, pinocytosis and proliferation of in vitro cultivated human glia cells. Cytobiologie 13, 93-106 (1976) Spielman, A., Gwadz, R.W., Anderson, W.A.: Ecdysone-initiated ovarian development in mosquitoes. J. Insect Physiol. 17, 1807-1814 (1971) Srdic, Z., Jacobs-Lorena, M.: Drosophila egg chambers develop to mature eggs when cultured in vivo. Science 202, 641~43 (1978) Steinman, R.M., Cohn, Z.A.: Pinocytosis in flbroblasts. Quantitative studies in vitro. J. Cell Biol. 63, 949-969 (1974) Wallace, RA., Misulovin, Z., Jared, D.W., Wiley, H.S.: Development of a culture medium for growing Xenopus laevis oocytes. Gamete Research 1, 269-280 (1978) Went, D.F.: Ecdysone stimulates and juvenile hormone inhibits follicle formation in a gall midge ovary in vitro. J. Insect Physiol. 24, 53-59 (1978) Accepted July 27, 1979