Uptake of vitellogenin into developing oöcytes of Locusta migratoria

J. Insect. Physiol.. 1978, Vol. 24, pp. 273 lo 278. @ Pergamon Press ttd. Printed in Great Britain.

0022-l9lO/78/04Ol-0273/$02.00/O

UPTAKE OF VITELLOGENIN OOCYTES OF LOCUSTA

INTO DEVELOPING MIGRATORIA

HANS-J~RG FERENZ Technische Hochschule. Fachbereich Biologie (Zoologie), SchnittspahnstraBe 10. D-6100 Darmstadt. Federal Republic of Germany (Received

8 August

1977: revised 29 November

1977)

Abstract-The selective incorporation of vitellogenin into developing locust oBcytes was studied using 1~51-vitellin. Vitellogenin incorporation does not start before the oBcytes are I .5 mm in length. It increases rapidly up to a maximum at 4.7 mm oiicyte length and decreases steadily until the eggs are fully developed (6.5 mm). Concentrations of serum proteins and vitellogenin in the haemolymph show parallel changes, vitellogenin titre reaching a maximum of 7.5mg/ml. Incorporation rates for vitellogenin increase from I .5 pglhrloiicyte (2.2 mm) up to 13.8 pglhrloiicyte (4.7 mm). In this range incorporation per unit surface area increases 4-fold. While the vitelline and chorionic membranes are being

formed,

decrease

incorporation rates as well as the protein concentrations in the haemolymph the second gonotrophic cycle starts. The hormonal basis for oiigenesis and for selective uptake of locust vitellogenin are discussed. the

steadily until

the mechanism

INTRODUCTION IN LOCUSTS, vitellogenesis about 8 days (GOLTZENB,

is completed within 1977). In this short time remarkable amounts of protein, as well as lipids and other substances, are deposited as yolk in developing oiicytes. Yolk proteins consist mainly of the specific female vitellin. This glyco-lipo-protein has been characterized (YAMASAKI, 1974; GELLISSEN et al., 1976; CHEN et al., 1976; MCGREGOR and LOUGHTON. 1977). Vitellogenin from haemolymph and vitellin from eggs are identical (CHEN et al., 1976). Synthesis of vitellogenin in the fat body is controlled by juvenile hormones (CHEN et al., 1976). It is secreted into the haemolymph, subsequently incorporated by developing &ytes and concentrated. Uptake occurs by means of pinocytosis, but the mechanism of selective incorporation is not yet understood. In this paper data on the in viva-uptake of vitellogenin are presented. A description of vitellogenin flow into oiicytes was previously attempted by BAKKER-GRUNWALD and APPLEBAUM (1977). This paper describes the changing dynamics of vitellogenesis, which are in close agreement with morphological and functional changes in the oiicyte-follicle tissue. The study was performed as a basis for further in vitro-experiments in order to identify the mechanism of selective incorporation of vitellogenin. MATERIALS

AND METHODS

Locusts were reared in crowded conditions under photoperiods of 12 hr of light per day at 35°C ambient temperature. Fresh wheat shoots were fed twice daily. These breeding conditions are similar to those used by GOLTZEN~ (1977). Vitellin was * Present address: Fachbereich burg, Ammerllnder D-2900 Oldenburg,

IV. Universitit

Olden-

Heerstrat3e 67-99. Postfach 25 03. Oldb. Federal Republic of Germany.

isolated from eggs or from well developed oiicytes (at least 5 mm long) using the method of GEL.LISSEN et al. (1976). Taking the oBcyte as a cylinder with a half sphere at each end, the obcyte surface and volume can be calculated. As described by WAJC et al. (1977) the ticyte diameter, d, is one fifth of oijcyte length. 1. Thus oiicyte oijcyte

surface,

volume,

v=7r

s = n d = 0.628 I’ (:,‘(I-f)=O.O*93

1”

In most reactions phosphate buffered saline (PBS, 82 mM NaCl, 10 mM NaH,PO,, 49 mM Na,HPO,). pH 7.6 was used with 10 mM phenylmethylsulfonylfluoride. Protein estimations were done with the Folin reagent (LOWRY et al.. 1951) after precipitation with 10% TCA. For estimations of pure vitellin solutions the molar extinction coefficient at 280 nm was used (GELLISSEN et al., 1976). Crossed immunoelectrophoresis was performed according to WEEKE (1973). Antibodies against vitellin were obtained by immunization of a goat against other antigens of rabbits. Iodine- 125 (> 350 mCi/ml) was purchased from New England Nuclear Chemicals. Iodination was performed using the lactoperoxidase method according to KROHN and WELCH (1974). Iodinated vitellin was separated from the reagents by gel filtration on Sephadex G-25. Samples were counted in y-vials in a liquid scintillation counter. For radioimmunoassay 5 ~1 haemolymph were diluted with 100 ~1 PBS and stored at -20°C. After thawing and centrifugation (8OOOg, 4 min) three aliquots of 25 pl were diluted with 50 yl PBS each and incubated with 50 yl serum against vitellin. Incubation took place at room temperature for I hr and an additional 19 hr at 4°C. Thereafter samples were centrifuged as mentioned and the pellet washed twice with 200 pl PBS. The precipitated vitellogenin 273

HANS-JBRG FERENZ

274 n=l3

14 7

2

3

5

4

2

2

4

6

I

3

-

2

-

%

-

i

2 x

3

-I

x

2

I

E”

3

4 mm

5

6

I

7

Fig. 3. Vitellogenin content of haemolymph expressed as percentage of total haemolymph proteins (calculated from mean values of Figs. I and 2).

*I

mm

Fig. I. Haemolymph protein concentration of female locusts in relation to size of terminal oocytes (mean values with standard deviation: n. number of animals. 2 aliquots per animal were measured). was counted

(a)

(Fig. 2) calculated from

and its amount

a standard curve. RESULTS protein concentration

Haemolymph

During adult development the protein concentration of the haemolymph increases steadily, reaching a maximum of about 60 mglml. When vitellogenesis is finished and chorion formation starts the protein concentration decreases to the level of the previtellogenic stage (Fig. I). The concentration at agiven stage can vary considerably and the data are not corrected for size and weight of animals. haemolymph volume or other factors.

Vitellogenin concentration

of

haemolymph

The vitellogenin content of the haemolymph obtained from the same animals increases during vitellogenesis from0 mg/ml at I.5 mm oocyte length up to 7.5 mg/ml at 4.2 mm (Fig. 2). Before oviposition n= IO

7

4

5

I

2

(b)

3

4472

,..-

,

I

ICI

..&!L a-046

,

I

I

I

I

I

I

2

3

4

5

6

7

J 6

Fig. 2. Vitellogenin content of haemolymph during the first cycle of oogenesis (n, number of animals; 2 aliquots per animal were measured).

Fig. 4. Crossed immunoelectrophoresis in which female haemolymph reacted with yolk antiserum (a), yolk with female haemolymph antiserum (b) and with yolk antiserum (c).

Uptake of vitellogenin in locust oijcytes 70

l r

A

275

24hr 3hr

2

I

3

4

6

5

7

8

mm Fig.

5. Percentage

radioactivity

recovered from ovaries after 3 hr in vivo-incubation.

injection

of 1z51-vitellin after

24 and

the amount of vitellogenin decreases. Since the second generation of oocytes begin growth when the terminal oocytes are enveloped by the chorion, vitellogenin concentration does not drop below l-2 mglml. The rate of incorporation of vitellogenin is much greater than the increase in total haemolymph protein concentration. The percentage of vitellogenin as a fraction of the total haemolymph proteins rises from 0 to 25% (Fig. 3). This means that during vitellogenesis not only a general stimulation of protein synthesis occurs but also a specific one for vitellogenin. The vitellin content of I ml oocyte volume was

tween haemolymph and yolk (Fig. 4). With antibodies against yolk at least 10 immunologically different proteins are found in yolk. Except for vitellin, most proteins are present in rather small quantities only. Therefore the titre of the complementary antibodies is low. Serum against yolk reacted with more than 18 haemolymph proteins. In contrast, serum against female haemolymph precipitates only a few proteins in the yolk solution including vitellin. Again, this is due to the low concentration of some proteins and their antibodies.

found to be about 190 mg. The ratio of the concentration of vitellin in the oiicyte to that of vitellogenin in the haemolymph provides a concentration factor for vitellogenin in the oocytes. This concentration factor varies according to the stage of oocyte development as follows: 44 at 2.2 mm oocyte length, 25 at 4.2 mm, 29at 5.2 mm, and 63 at 5.7 mm.

The yield of incorporation of injected, iodinated vitellin (7.5, 34 or 66 pg) depends upon oocyte size. It increases proportional to oocyte length (Fig. 5). No more than 65% of the injected activity can be incorporated by the ovary within 24 hr. lncorporation does not start before 1.5 mm oocyte length and stops at 6.5 mm shortly before the eggs are released into the oviduct. The same experiment terminated after 3 hr shows that within this time about 50% of the activity detectable in the ovaries after 24 hr incubation is incorporated. Assuming a lOO%-

Immunofogical

comparisons

By means of qualitative crossed immunoelectrophoresis, similarities could be demonstrated bel =n2

6 4

2 13;

I

2

h

4 2

In vivo-incorporation

of L251-vitellin

6

7

-

Ih g

-

IO-

x L

_

c

I

1

2

3

4 mm

5

6

Fig. 6. Total uptake of vitellogenin by ovaries within hr t@) and 3 hr (m). Amounts of BSA (W) and 1gG incorporated within 24 hr are also shown.

7

24

(X )

3

2 I 12

I

5

5

4 mm

c IO

mm2

15

7

6 I

20

I 25

/ 30

Fig. 7. VitUogenin incorporation as a function of oocyte length (data from 24 hr (0) and 3 hr (A) incubations are shown together: open symbols represent values calculated from Figs. 2 and 4). Data are also displayed as uptake per mmr and hour TV.

HANS-J~~RG FERENZ

276 70 60

r

. %SP, . IgG

clearly demonstrates the highly selective uptake of vitellogenin (Fig. 6).

t DISCUSSION During oiigenesis a large amount of protein is deposited together with other substances in the developing oiicytes. These proteins enter the oiicytes from the haemolymph, as demonstrated by immunoelectrophoresis. Most serum proteins are also present in the oBcytes, although vitellin represents by far the largest fraction, while other serum proteins occur in very small quantities. Locust vitellin is a glyco-lipo-protein as are most insect vitellins that have been described. As in other insects, locust mm vitellogenin is synthesised in the fat body and secreted into the haemolymph. Haemolymph vitelloFig. 8. Per cent radioactivity recovered from ovaries 24 genin and egg vitellin are at least immunologically hr after injection of ““I-BSA and ““I-IgG. and electrophoretically identical (CHEN et al., 1976). There are no indications for differences between the incorporation of injected vitellin, the turnover rates two functional stages of this protein. vary from 90 (2 mm) to I 1(6 mm) hr. However, part The synchrony of the processes involved in the of the ‘?“I-vitellin is lost. development of oiicytes is remarkable: increase of Calculations on in vivo-incorporation of vitellogenin protein synthesis and onset of vitellogenin production as well as vitellogenin incorporation can be Since the relationship of vitellogenin content of observed from I.5 mm long oticytes on; the maxithe haemolymph to oiicyte size is known (Fig. 2) the mum is reached at 4.5-5.0 mm; thereafter protein dilution factor for injected ‘251-vitellin can be production and its incorporation strongly decrease estimated. By multiplying this factor by the incoruntil the eggs are mature and the second gonotrophic porated amount of iodinated vitellin the total cycle starts. amount of incorporated vitellogenin can be calcuWhen previtellogenesis is finished in the terminal lated (all calculations are based on mean values and oiicytes at 1.2-1.5 mm the protein content of the on the basis of 1.0 ml haemolymph per locust). haemolymph doubles within a short time up to 60 Figure 6 demonstrates that noticeable vitellogenin incorporation starts after I.7 mm oiicyte length, mglml and decreases beyond 4.7 mm oijcyte length. Nearly identical vaIues have been found by GOLTreaching a maximum at 4.7 mm. Comparing the vitellogenin incorporation at 3 and ZENB (1977), while MINKS (1967) found an increase 24 hr (Fig. 7) it is clear that after 3 hr two thirds of up to 90 mg/ml. In the locust, a similar relationship between protein concentration of the haemolymph the vitellogenin that can be demonstrated by this and oiicyte development is described for the grasstracer method after 24 hr has been incorporated: thus 50% is taken up after 2.25 hr. During vitelhopper Melanoplus sanguinipes (ELLIOT and GILLOT, 1977). Vitellogenin concentration inlogenesis in the locust the number of developing ticytes decreases from about 70 to some 38 (WAJC creases in parallel to a total protein content from et al., 1977). which is in close agreement with our 0 to 7.5 mg/ml. When the eggs are released into the oviduct, the vitellogenin concentration in the observations in the same species. Taking this into haemolymph is about 1.0 mg/ml. At this time the account. the rate of incorporation per oacyte can be second generation of oiicytes is already needing estimated (Fig. 7). The rate of vitellogenin incorporation is proporvitellogenin. BAR-ZEV et al. (1975) found an increase in glycoprotein content of haemolymph of tional to the increase in surface area of the oacytes and reaches a maximum of 13.8 pglhrloiicyte at 4.7 3.5. BAKKER-GRUNWALD and APPLEBAUM (1977) mm oijcyte length (I). Vitellogenin incorporation calculated a concentration of 8 mg glycoprotein per per unit oiicyte surface area (neglecting microvilli) ml after pooling the haemolymph from reproductive is not constant but rather increases from 0.1 females. pg/hr/mm’ (I = I .7 mm) to 0.8 gg/hr/mm2 (I = 4.7 The percentage vitellogenin of total haemolymph mm) (Fig. 7). proteins is not constant but increases to 25%; i.e. more vitellogenin is produced than is sequestered. In vivo-incorporation of 9-IgG and T-BSA At 5.0-5.5 mm oiicyte length the increase is someInjected 1251-IgG (rabbit, 9.3 pg) and ‘251-BSA what greater than expected. This may be explained (6.8 ag) are also incorporated by developingoiicytes by the sudden decrease of vitellogenin incorporation. (Fig. 8). Measurable amounts of these proteins are after 4.7 mm. A small contribution from the retaken up after 2 mm oiicyte length; i.e. when vitsorbing oiicytes cannot be excluded, although durellogenin is incorporated at a high rate. Although ing resorption an enzymatic degradation of the injected in smaller quantities more BSA than IgG is vitellin molecules is possible. incerporated. The uptake of both proteins can only Electron microscope investigations show that be compared with the incorporation of the vitellin after 4.0-5.0 mm the oiicytes and follicle cells after correction of the injected vitellin for the dilurespectively are active in producing a vitelline tion by endogenous vitellogenin. This calculation membrane and chorionic material (GOLTZENB.

Uptake of vitellogenin in locust oiicytes 1977). At this stage the main site of protein synthesis changes from the fat body to the follicle cells, and the growing membranes inhibit protein incorporation into the oiicytes. In locusts, juvenile hormone is essential for vitellogenesis, for production of vitellogenin and probably also for the incorporation of vitellogenin. Vitellogenin production increases after the administration of juvenile hormone to allatectomized females in viva, as well as after the application to female fat body. in vitro (CHEN eta/.. 1976). In locusts juvenile hormone titre tends to increase during the first cycle of vitellogenesis (JOLY and JOLY, 1974). This increase in juvenile hormone concentration corresponds to a similar increase in the relative proportion of vitellogenin in haemolymph during the same phase of development. The meaning of the apparent correlation between juvenile hormone and vitellogenin concentration warrants furoiicyte ther Allatectomy inhibits study. development but not the increase of haemolymph protein concentration (MINKS, 1%7; GOLTZEN~, 1977). Whether juvenile hormone regulates oiicyte development completely or whether certain steps such as chorion formation are controlled by the pars intercerebralis is still unknown. Together with the synthesis of vitellin and chorion membrane, large amounts of ecdysone are synthesized by the follicle cells during the last phase of vitellogenesis (LAGUEUX et al.. 1977). However, this hormone is stored by the oijcytes and has apparently no function in vitellogenesis. Vitellogenin may be sequestered preferentially by oiicytes from the haemolymph. Different haemolymph proteins are taken up in small amounts. The experiments including the injection of BSA and IgG demonstrate the ability of the oiicytes to incorporate non-vitellogenins probably by adventitious engulfment during pinocytosis. It should be noted that BSA is preferentially incorporated. Using iodine125labelled vitellin the incorporation of vitellogenin into developing oiicytes could be quantified. A quantitative description of vitellogenesis in locusts was previously attempted by BAKKER-GRUNWALD and APPLEBAUM (1977). Since their estimations are based on some generalized measurements neglecting, for example, the change of vitellogenin concentration, there are some differences with respect to our results. Labelled vitellin is rapidly incorporated: 50% of the injected radioactivity that can be incorporated within 24 hr has entered the ovary after some 3 hr. This is in good agreement with the value of about 2 hr in the cockroach Blatefla germanica (KUNKEL and PAN, 1976). Injected vitellin is diluted to different degrees by endogenous vitellogenin. Taking this into account the total vitellogenin incorporation can be estimated. Uptake into the oacytes shows a parabolic correlation with oiicyte length and a linear correlation with oijcyte surface (up to 4.7 mm). The incorporation rate rises from 100 gg/hr at 2.2 mm to 570 pg/hr at 4.7 mm per ovary and decreases continuously until oijcyte development is completed. Thus, incorporation rates are closely correlated with protein production and the activity of the follicle cells.

277

One locust egg contains about I .4 mg of vitellin (FERENZ, unpublished) and an ovary with fully developed eggs, some SO-55 mg. The same values may be calculated from the measured incorporation rates, allowing 8 days for vitellogenesis. Since all calculations are based on the assumption of 1 ml haemolymph per female they must be corrected for the true haemolymph volume: 0.5-0.8 ml (WAJC et al., 1977). Thus, a value of 30-40 mg vitellin per ovary can be derived. This lower value might be due to a slight change in the vitellin molecule caused by iodination or to not yet recognized differences between vitellin and vitellogenin. This last aspect, in particular, must be investigated in detail because this might give some indication for the mechanisms of selective uptake of vitellogenin. The growth of oiicytes is not proportional to time. Previtellogenesis and the beginning of vitellogenesis is a period of relatively slow growth. Thereafter. until chorion formation the rate of growth increases dramatically (GOLTZENB, 1977). In this last phase incorporation rates of vitellogenin are changing almost hourly. It is interesting that the incorporation rate per unit surface area increases by a factor of four during vitellogenesis. Since during the main period of vitellogenesis the vitellogenin concentration per unit surface area is nearly constant (taking the reabsorption of some oScytes into account), an increasing activation of pinocytosis may occur. The mechanism which is activated and causes the selective uptake of vitellogenin in locusts is still unknown. In the silkmoth Hyalophora cecropiu a follicle cell product is postulated tochange the vitellogenin in a way that allows it to be selectively incorporated by pinocytosis (ANDERSON and TELFER, 1970; BAST and TELFER. 1976). On the other hand receptors may be involved. In the clawed toad Xenopus laevis selectivity of vitellogenin incorporation is explained by the binding of the negatively charged vitellogenin molecule to positively charged areas on the oiicyte surface ( BRUMMET and DUMONT, 1976). Which properties of the vitellogenin and which mechanisms in the oiicyte-follicle tissue enable the selective uptake of vitellogenin will be investigated with in vitro-experiments. [Note at correction: Using improved methods. GELLISSF~~ and EMMERICH (1978. in preparation) demonstrated a somewhat higher vitellogenin titre in locust haemolymph than in the present study. reaching a peak at about 3.5 mm oiicyte length. Thus a vitellin accumulation near the expected value of 50 mg per ovary can be calculated However. in general their data suhTtantiate the present study.) Acknowledgements-The work was supported by a grant from the Volkswagen-Stiftung. Hannover. Federal Republic of Germany.

REFERENCES ANDERSON L. M. and TELFER W. H. (1970) Extracellular concentrating proteins in the cecropia moth follicle. J. Cell Physiol. 76, 37-54. BAKKER-GRUNWALDT. and APPLEBAUM S. f 19771 Aquantitative description of vitellogenesis in Locus?o mipmtoria migratorioides. J. Insect Physiol. 23. 259-263. BAR-ZEV A.. WAJC E.. COHEN E.. SAPIR L.. APPL.ERAUM S. and EMMERICH H. (1975) Vitellogenin accumulation

278

HANS-J~RG FERENZ

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KUNKEL J. G. and PAN M. L. (1976) Selectivity of yolk protein uptake: comparison of vitellogenins of two insects. J. Insect Physiol. 22, 809418. KROHN K. A. and WELCH M. J. (1974) Studies of radioiodinated fibrinogen--H. Lactoperoxidase iodination of fibrinogen and model compounds. Int. J. Appl. Rad. Isotop. 25, 3 15-323. LAGUEUX M., HIRN M. and HOFFMAN J. A. (1977) Ecdysone during ovarian development in Locusta migratoria. J. insect Physiol. 23, 109-l 19. LOWRY 0. H., ROSENBROUGH N. J., FARR A. L. and RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MINKS A. K. (1967) Biochemical aspects of juvenile hormone action in the adult Locusta migratoria. Arch. Nt!erl. Zool. 17, 175-257. MCGREGOR D. A. and LOUGHTON B. G. (19771 Aminoacid composition. degradation and utilization of locust vitellogenin during embryogenesis. Wilhelm Roux’ Archiv. 181, 113-122. WAJC E., BAKKER-GRUNWALD T. and APPLEBAUM S. (1977) Binding and uptake of trypan blue by developing oocytes of Locusta migratoria migratorioides. 1. Embryol. exp. Morph. 37, l-l I. WEEKE B. (1973) Crossed Immunoelectrophoresis. In Quantitative Immunoelectrophoresis (Ed. by AXELSEN N. H., KROLL J. and WEEKE B.). Universitetsforlaget. Oslo. YAMASAKI K. (1974) Yolk protein from eggs of Locusta migratoria and glycopeptides from the protein. Insect Biochem. 4.41 l-422.