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Changes in follicle cell morphology, ovarian protein synthesis and ovarian DNA synthesis during oöcyte maturation in Leucophaea maderae: Role of juvenile hormone
J. Insect Ph.vsiol.. Vol. 21. No. 4,pp. Printed in Great Britain.
281-291.
1981.
0022-1910/81/040281-I 1 $02.00/O cl981 Pergumon Press Ltd.
CHANGES IN FOLLICLE CELL MORPHOLOGY, OVARIAN PROTEIN SYNTHESIS AND OVARIAN DNA SYNTHESIS DURING OijCYTE MATURATION IN LEUCOPHAEA MADERAE: ROLE OF JUVENILE HORMONE JOHN K. KOEPPE, FORIS
N. JARNAGIN and LAWRENCE N. BENNETT
Department of Zoology, University of North Carolina, Chapel Hill. North Carolina
27514, U.S.A.
(Received 4 August 1980; revised 7 November 1980)
Abstract--Changes
in follicle cell morphology were correlated with changes in rates of protein synthesis and DNA synthesis by the ovary during ovarian maturation in Leucophaea maderae. During the vitellogenic period of oiicyte development, which lasts approx. 15 days, morphological changes in the follicle cells are accompanied by moderate rates of ovarian protein synthesis and rapid rates of ovarian DNA synthesis. At approx. 15 days after mating. the shape of the follicle cells changes from cuboidal to squamous, ovarian DNA synthesis is arrested, and ovarian protein synthesis increases slightly. During the final period of o6cyte development, which lasts approx. two days, the interfollicular channels between the follicle cells have disappeared and the squamous follicle cells, which contain an extensive rough endoplasmic reticulum, deposit a chorion around the mature oiicyte. These morphological changes are accompanied by a radical increase in ovarian protein synthesis, while ovarian DNA synthesis remains arrested. Immediately before ovulation, ovarian protein synthesis starts to decline, reaching a minimal level 24 hr post-ovulation. Ovarian maturation is dependent on the presence ofjuvenile hormone (JH) only during the vitellogenic stage of oiicyte development. Decapitation of insects at any point during the first 10 days after mating arrests the synthesis of DNA and retards the synthesis of protein by the ovary, resulting in degeneration of the okyte. Subsequent injection of JH restores both events to normal levels within 72 hr. Decapitation on or after the tenth day following mating does not alter normal oiicyte development, chorion deposition, ovulation or egg case formation. Primary induction of protein synthesis in ovaries from virgin females can be achieved by either an in vivo or in vitro exposure of the tissue to JH, thus confirming a site of action for JH to be ovarian tissue. Electrophoretic analysis of the soluble proteins from JH-exposed ovaries in vivo reveals that JH stimulates general protein synthesis, rather than the synthesis of a specific major protein such as vitellogenin. Key Word Index: Follicle cell, Leucophaea maderae, juvenile hormone, DNA synthesis, protein synthesis, ovary
INTRODUCTION
OVARIAN maturation
in the ovoviviparous cockroach requires approx. 18 days and is characterized by numerous biochemical and morphological changes within the follicular epithelium. After mating, the oijcytes within the 20-22 terminal follicles of each panoistic ovary become vitellogenic, incorporate large quantities of macromolecules from the haemolymph and grow from 1.2 to 6.5 mm in length. At the end of the growth period, a chorion is deposited around each mature oijcyte by the follicular epithelium. Previous research has demonstrated that juvenile hormone (JH) influences various biochemical and morphological events that are processes associated with ovarian maturation in L. maderae. For example, it has been shown that JH induces morphological changes in the follicular epithelium of virgin females that are characteristic of S-day mated females, including the development of interfollicular spaces and channels, the enlargement of the follicle cell and its nucleus, and a limited increase in rough endoplasmic reticulum within the follicle cell (KOEPPE et al., 1980a). Further. it has been shown that JH stimulates vitellogenin synthesis by the fat body Leucophaea
maderae
281
(BROOKES, 1969; ENGELMANN, 1969; KOEPPE and OFENGAND, 1976a) and DNA synthesis by the ovary (KOEPPE and WELLMAN, 1980). More recently, it has been observed that changes in the rates of synthesis of vitellogenin by the fat body, and DNA by the ovary, coincide with fluctuations in the haemolymph titre of JH. The peaks in these two synthetic events and in the haemolymph JH-titre occur at approx. 12 days after mating, or when the oijcyte is between 5 and 5.5 mm in length (KOEPPE et al., 1980b; KOEPPE, 1981). Thus, these two biochemical events appear to be directly regulated by JH, although in decapitated, mated females we have been unable to restore rates of ovarian DNA synthesis to normal levels through injections of JH I (mixed isomer) (KOEPPE et al.. 1980a). With regard to protein synthesis in the ovaries, it has been reported that the rate of protein synthesis in ovaries from L. maderae at 10 days post-parturition was greater than at 5 days, that this increased rate of synthesis appeared to be JH-dependent and that the induced protein was vitellogenin (WYSSHUBER and LUSCHER, 1972). More recently, in Periplaneta americana, it has been demonstrated by autoradiography that JH stimulates the synthesis of non-vitellogenic proteins by the follicle cell. Those
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JOHNK. KOEPPE,FOREST N. JARNAGIN AND LAWRENCE N. BENNETT
proteins appear to be secreted into the interfollicular spaces and are postulated to aid the translocation of vitellogenin through the follicular epithelium (BELL and SAMS, 1974; SAMSand BELL, 1977). Thus, there is limited evidence that JH may regulate protein synthesis in the ovaries. The research reported in this communication was designed to enhance our understanding of terminal follicle maturation in L. maderae. In particular, attention has been focused on refining our knowledge of the rates of protein and DNA synthesis by the ovary, correlating these rates to morphological changes, and determining the extent to which JH regulates morphological and biochemical changes during ovarian maturation. MATERIALS
AND METHODS
A. Animals Colonies of Leucophaea maderae were maintained on Wayne Dog Chow and water, with 12 hr light and 12 hr dark, at 78°F. Animal staging and colony organization are described in a previous publication (KOEPPEand WELLMAN,1980). Females one day after adult ecdysis are referred to as V-l females. Under normal conditions newly-emerged adult males and females were maintained separately. On V-10, three-week-old males were added to the female cage for mating. After one day, the mated females were removed from the cage and maintained separately. Under the described conditions, egg case formation normally occurred between M-17 and M-19. B. Electron microscopy Animal preparation, tissue fixation, embedding procedures, sectioning and microscopy are described in a previous publication (KOEPPEet al., 1980a). C. Biochemical assays 1. Protein synthesis. (a) Tissue culture. Ovariole cultures with 250 ~1 of 3H-leucine medium were maintained for five hours at 31°C in a shaking waterbath. A time-course study revealed that incorporation of 3H-leucine into newly-synthesized TCA precipitable protein was linear from 0.5 to 10 hr, regardless of ovariole length, and thus five hr was selected as the standard incubation time. The number of ovarioles cultured was dependent on their size: 22 or fewer ovarioles if they were less than 2 mm in length, 10 or fewer ovarioles if they were 2-4 mm in length and 5 or fewer ovarioles if they were 4.0-6.5 mm in length. Unless otherwise noted, ovarian cultures refer to the culture of ovarioles, not whole ovaries with an ovarian sheath. The wells (maximum capacity of 2 ml) were maintained in a humid environment (without additional 0, or CO,) in large carrier trays. Each 250 ~1 culture contained 25 &i of 3H-leucine with a specific activity of 72.9 mCi/mmole or 34.5 pmole of leucine/culture. The medium was prepared by adding 1 ml of sterile L-(4,5-3H)-leucine (56 Ci/mmole) in water to 9 ml of M-20 medium with 7.5% foetal calf serum, and penicillin and streptomycin at a concentration of 1000 units/ml.
After incubation, the ovarioles were removed from the medium and homogenized in 1 ml of 0.4 M NaCl at 4°C with a ground glass homogenizer. Aliquots of the whole homogenate were quantified, as indicated below, for 3H-leucine-labelled proteins. In all trial assays less than 10% of the labelled protein was found to be secreted from the ovarioles, and unless otherwise noted, protein in the medium was not quantified. (b) Quantification of labelled proteins (protein assay). Sample aliquots were placed into 3 ml of 10% trichloroacetic acid (TCA) and placed in a boiling water bath for 15 min to precipitate the proteins and to destroy t-RNA with bound 3H-leucine. Labelled proteins were separated from free 3H-leucine by filtration on Gelman glass fibre filters, type A/E. Residual 3H-leucine was washed from the filter with three volumes of 5% TCA. TCA was removed with a final rinse of 50% ethanol and the filters were dried for 6 hr at 55°C. Macromolecule digestion and quantification of the labelled protein bound to the filter is described in a previous publication (KOEPPE and WELLMAN,1980). 2. Vitellogenin radioimmunoassay. Analysis of culture medium and of tissue extracts for the presence of radioactive vitellogenin was performed as described in a previous publication (KOEPPE and WELLMAN, 1980). The antiserum was made against 28s vitellin obtained from L. maderae and bound both precursor and processed vitellogenin (KOEPPEand OFENGAND, 1976b). 3. DNA synthesis. The assay for quantification of 3H-thymidine incorporation into ovarian DNA is described in a previous publication (KOEPPE and WELLMAN.1980). D. Hormonal stimulation in vivo Unless otherwise stated, the procedures for animal treatment and hormone preparation and administration are the same as reported in a previous publication (KOEPPEand WELLMAN,1980). E. Hormonal stimulation in vitro 1. Tissue preparation. Animals were anaesthetized by exposure to CO, for approx. 30 set, rinsed with 0.2% HgCl, and washed twice with sterile water (MARKS er al., 1972). The animals were then pinned to a sterile dissecting dish, ventral side up, and their abdominal sternites were removed. Ovaries were removed under sterile conditions and placed into 2.0 ml of modified M-20 medium consisting of 100 ml M-20 medium, 0.1 ml penicillin and streptomycin (100,000 units/ml) and 7.5 ml foetal calf serum. In the wash medium all extraneous tissue, except the ovarian sheath, was removed. 2. Tissue culture. An aliquot of juvenile hormone I (Roche) was weighed and M-20 medium was added to make a dilution of 1 mg/ml. This solution was sonicated on ice for 8-10 set at 2 mHz, using a Sonifier Sonicator. Control medium was sonicated with mineral oil. After sonication, an aliquot of the stock JH-medium was diluted lOO-fold, yielding a JH concentration of 10 pg/ml or 3.4 x 10v5M. Likewise, a dilution of the control medium was made. Stock incubation media were stored at - 15°C. Unless otherwise stated, the left ovary (experimental ovary) was cultured in 1 ml of JH-medium
2X3
Fig. 1. Electron micrographs of vitellogenic follicle ceils. Panel A is a cross-section of a follicle cell from a terminal follicle 3 mm in length obtained froni an M-8 female. Panel B is a cross-section of a follicle cell from a terminal follicle 5 mm in length obtained from an M-12 female. Tunica propria (TP). rough endoplasmic reticulum (RER). apparent glycogen (G).
284
Fig. 2. Electron micrograph of a transitional follicle cell. This is a cross-section terminal follicle 5.5 mm in length obtained from an M-15 female. Tunica propria reticulum (RER), apparent glycogen (G).
of a follicle cell from a (TP), rough endoplasmic
Fig. 3. Electron micrograph composite of a follicle cell from a terminal follicle 6 mm in length obtained from an M-18 female. Tunica propria (TP), exochorion (EX), endochorion (EN), nucleus(N), apparent glycogen CC).
2x7
Changes in follicle cell morphology while the other ovary (control ovary) was cultured for an identical time in mineral oil-medium. Media were changed every 24 hr. After culturing, the ovarian sheaths were removed from the ovaries and the separated ovarioles were assayed for rates of 3H-leucine incorporation into newly-synthesized protein. To determine if the rate of protein synthesis in an experimental ovary (minus the ovarian sheath) is altered by its exposure to JH, its rate of synthesis was compared to the rate of protein synthesis in a control ovary (minus the ovarian sheath) removed from the same female. The ratio of these two rates is referred to as an ovarian ratio. A ratio of 1.0 would indicate the rates of protein synthesis in each ovary are the same. F. Protein analysis-gel
electrophoresis
Techniques for non-denaturing gel electrophoresis are described in a previous publication (KOEPPE and GILBERT, 1973), as are the techniques for SDS slab gel electrophoresis and the techniques for quantification of labelled proteins in the gel (KOEPPE and OFENGAND, 1976a). G. Chemicals The juvenile hormones used in these experiments were obtained from Hoffman-LaRoche (JH I) and from Eco-Control Incorporated (JH I, JH III). 20-hydroxyecdysone was obtained from Calbiochem and was 99.8% pure. t_-[4,5-3H]-leucine with a specific activity of 56 Ci/mmole was purchased in sterile water from Amersham/Searle. 3H-amino acid mixture was a composite of 15 amino acids from Amersham/Searle (TRK440). M-20 medium, penicillin G, streptomycin and foetal calf serum were all obtained from Grand Island Biological Supply. Ultrapure acrylamide, TEMED (N,N,N’, N’-tetramethylethylenediamine), ammonium persulphate and BIS (N,N’-methylenebis-acrylamide), were all obtained from Bio-Rad Laboratories. Specially pure sodium lauryl sulphate (SDS) was obtained from BDH Chemicals Ltd. All other chemicals, including enzyme pure TRIS, were purchased from Sigma. Thin layer chromatography plates were obtained from Eastman Kodak. NCS was obtained from Amersham/Searle while all other scintillation chemicals were from Fisher.
RESULTS I. Maturation
of ovarian terminal follicles
A. Changes in follicle cell morphology. Crosssections of terminal follicles from selected stages of development were analyzed by electron microscopy. The electron micrographs in Fig. 1 illustrate typical morphology of follicle cells from the terminal follicles during vitellogenesis. In these cells the rough endoplasmic reticulum is located towards the haemolymph side of the cell, the nuclei are enlarged and the spaces between the follicle cells are large. In later stages of vitellogenesis. large accumulations of what appear to be glycogen granules are evident in the follicle cell (Fig. 1B). In the final stage of oiicyte development, the follicle cells are responsible for the synthesis and deposition of the chorion. This change in function is attended by a change in the structure of the follicle cell.
Figure 2 is an electron micrograph of a transitional follicle cell prior to chorion deposition (approx. 15 days after mating). This follicle cell and its nucleus are becoming squamous, and the accumulations of apparent glycogen granules are dispersing and appear to be migrating toward the oocyte side of the follicle cell. Figure 3 illustrates the completion of these changes. The follicle cell, which is in the process of chorion deposition (16-18 days after mating), contains an extensive rough endoplasmic reticulum and the apparent glycogen granules are dispersed in the area adjacent to the chorion (see insert, Fig. 3). B. Protein and DNA .synthe.ks hj, the ovary. The appearance of an extensive rough endoplasmic reticulum in the squamous follicle cells suggests a greatly enlarged capacity of the ovary to synthesize protein. This was confirmed by quantifying rates of protein synthesis by ovaries throughout terminal oocyte maturation. Rates of DNA synthesis, a JH-inducible process ( KOEPPE and WELLMAN. 1980). were also monitored. The results (Fig. 4) indicate that DNA synthesis is arrested during chorion deposition. In contrast, protein synthesis by the ovaries, while keeping pace with the enlargement of the follicle cell during vitellogenesis, rapidly accelerates and reaches a peak rate during chorion deposition. approx. 16-18 days after mating or approx. 4-6 days after the JH-haemolymph titre peaks. The results for rates of protein synthesis in Fig. 4 were obtained by measuring incorporation rates of leucine into newly synthesized proteins in culture. Identical patterns were obtained when a mixture of “H-labelled amino acids was substituted for 3H-leucine. II. Regulation OJ ovarian protein juvenile hormone
synthesis:
role qf
A. JH restoration of ovarian protein synthesis in decapitated ir7.srct.s. To determine whether JH regulates protein synthesis in the ovaries, females were maintained and treated as follows: a large population of M-6 females was divided into two groups; one group remained untreated, but was maintained without food or water, while the females in the second group were decapitated. Ovaries from both groups were immediately assayed for rates of DNA and protein synthesis. Three days later (M-9), ovaries from the two groups were again assayed for rates of DNA and protein synthesis. Thereafter the decapitated females were further divided into two subgroups; those in one group were injected with paraffin oil, while those in the other received an injection of JH I. Three days later, ovaries from females in each group were assayed for rates of DNA and protein synthesis. The results, illustrated in Fig. 5, demonstrate that decapitation arrests DNA synthesis and significantly retards protein synthesis in the ovary. Both processes were restored to levels equivalent to those in the control females through an injection of 2.5 pg of JH I (Eco-Control) suspended in 25 ~1 paraffin oil. Injections of 25 ~1 of paraffin oil neither stimulate nor further retard either process. B. In vivo stimulation of ovarian protein synthesis in virgin ,females. Virgin females, 24 hr post-ecdysis, were decapitated and injected with either 25 pg of JH I (Roche) suspended in 25 ~1 of mineral oil, or mineral oil alone. Thereafter, at 24-hr intervals,
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JOHN K. KOEPPE. FOREST N. JARNAG~N AND
LAWRENCE N. BENNETT
Mid
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TERMINAL
OOCYTE
LENGTH
(mm)
Fig. 4. Rates of 3H-thymidine incorporation into ovarian DNA ( x ---x ) and 3H-leucine incorporation into ovarian proteins (t-0) during maturation of the terminal follicle. To minimize the effects of animal variation the data points are illustrated as a function of terminal oijcyte length. Under normal rearing conditions terminal oiicytes 1.3-l .4 mm in length are obtained from virgin females, while larger oijcytes are obtained from mated females. Oijcytes 3.0 mm long are found in M-6 to M-9 females; oiicytes 4.0 mm long are found in M-8 to M-l 1 females; ocicytes 5.5 mm long are found in M-12 to M-16 females, and oiicytes 6.2 mm in length (depositing chorion) are found in M-16 to M-18 females. The data points represent the mean (+ S.D.) of the results from a minimum of 10 ovaries. Assays are described in the text. Chorion deposition was first apparent in terminal follicles 6 mm in length.
ovaries were removed from the females and assayed in vitro for rates of 3H-leucine incorporation into newly synthesized proteins. The results, depicted in Fig. 6, illustrate a 1.75-fold increase in the rate of protein synthesis by virgin ovaries when exposed to JH I. Similar results (not illustrated) have been obtained using JH I obtained from Eco-Control, although at one-tenth the concentration (2.5 pg). Data derived from injection of JH I (Roche) were plotted since they provide a comparison to the in vitro stimulation experiments (see Fig. 8). In this report, and in many previous experiments, JH I has been used to study the regulatory effects of JH on female reproductive development. Since JH III is the natural hormone found in L. maderae (KOEPPE et al., 1980b), we found it necessary to determine whether the different forms of the hormone (JH I and JH III) display different activities in stimulating protein synthesis by the ovaries. In these experiments the females were decapitated on V-2 and two days later were injected with varying concentrations of JH I or JH III, both from Eco-Control. Three days thereafter, the ovaries were removed and assayed in vitro for rates of 3H-leucine incorporation into newly synthesized proteins. The results in Fig. 7 illustrate that while JH I stimulates protein synthesis by the ovary at concentrations lower than those necessary for stimulation with JH III, both hormones stimulated protein synthesis to similar levels at higher concentrations.
The effect of 20-hydroxyecdysone on protein synthesis by the ovary was also monitored in V-l and V-12 females. No response (up to 72 hr post-injection) was obtained in either group of decapitated females with injected doses ranging from 0.001 to 10 pg (data not illustrated). C. Analysis of’JH-induced soluble proteins. Results from electrophoretic and radioimmunoassay studies demonstrate that JH-induction of protein synthesis by ovarioles is general rather than specific, and that vitellogenin is not a JH-inducible ovariole protein. In these experiments, decapitated virgin females, 24 hr post-ecdysis, were separated into two groups and injected with either 25 pg of JH I (Roche) suspended in mineral oil (25 ~1) or 25 ~1 of mineral oil. After 72 hr, the ovaries were removed, cultured for 5 hr in an 3H-leucine medium, and the ovarioles were then homogenized in 0.4 M NaCl (with 0.1 mg/ml of PMSF) at 0°C before centrifugation at 10,000 g. The supernatants revealed the concentration of the labelled proteins in the extract from the JH-exposed ovarioles to be 1.89 times greater than the extract from the mineral oil-exposed ovarioles. Subsequeni analysis of these extracts suggested JH induction of ovarian protein synthesis was general rather than specific. In one experiment, labelled proteins from the JH-exposed ovarioles were added to an extract of vitellin and analyzed by SDS-gel electrophoresis (see Materials and Methods). The results revealed that labelled protein from the
Changes in follicle cell morphology
6
12
9
I
I
9 DAYS
AFTER
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I2
MATING
Fig. 5. Analysis of DNA and protein synthesis in ovarian tissue from females decapitated 6 days after mating (terminal follicles about 2.4 mm in length) and thereafter injected with JH/paraffin oil. Data illustrated on both graphs were obtained from tissues from the same animals. Each point represents the mean (+ S.D.) of the results from 5 females. Injections were completed 3 days after decapitation of M-6 females. Analysis of the tissue was performed as described in the text. The graphs illustrate responses in starved, intact mated females (M), decapitated, JH-injected females (t+) and decapitated, paraffin oil-injected females (t---o). Panel A illustrates 3H-thymidine incorporation into newly synthesized ovarian DNA in vitro. Panel B illustrates 3H-leucine incorporation into newly synthesized ovarian proteins in vitro.
ovarioles did not migrate with the vitellin. Negative results from a vitellogenin radioimmunoassay of extracts from cultured JH-stimulated ovarioles and their culture medium confirmed these observations. Finally, quantification of label associated with each protein, separated by non-denaturing gel electrophoresis, revealed concentrations of label for each protein from the JH-exposed ovaries to be 1.98 times greater (+ 0.2. dependent on the digestion of gel) than the label associated with the corresponding separated protein from ovarioles not exposed to JH. These results indicate that JH stimulates the synthesis of all extractable soluble proteins. Thus vitellogenin synthesis was not found to be associated with the ovariole, although it was found to be associated with the whole ovaries, suggesting the ovarian sheath or the dispersed fat body within the sheath is responsible for such vitellogenin synthesis.
D. In vitro stimulation of ovarian protein synthesis. the protocol described in the Materials and Methods, protein synthesis was stimulated in vitro in ovaries from V-l to V-8 females. Results from V-l ovaries, illustrated in Fig. 8, demonstrate that 0.01 mg/ml of mixed isomer JH I (Roche) stimulates a 1.67-fold increase in ovarian protein synthesis in vitro. Similar results have been obtained using ovaries from V-2 to V-8 females. When a higher concentration of JH I (Roche) was used in the culture medium (0.1 mg/ml), an ovarian ratio of approx. 0.2 was obtained, indicating that 80% inhibition of ovarian protein synthesis occurs at this hormone concentration. At one-tenth the JH concentration (0.001 mg/ml), no observable effect was detected. In an effort to further localize the site of JH, we removed the ovarian sheath and cultured only isolated ovarioles obtained from I-2-day virgin or Using
JOHN K. KOEPPE,FORESTN. JARNAGINAND LAWRENCEN. BENNETT
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48
24
HOURS AFTER
JH INJECTION
Fig. 6. Time-response
curve for JH-stimulation of 3H-leucine incorporation into ovarian proteins. Females were decapitated 24 hr after their adult ecdysis. Two days after decapitation they were injected with 25 pg of JH I (Roche) and assayed at the times indicated in the figure. Each data point represents the mean (+ S.D.) of the results from 5 females. The ovarian sheath and any other tissue were removed prior to the 5 hr 3H-leucine culture. Similar results
Fig. 7. Dose-response curves for JH I and JH III stimulation of 3H-leucine incorporation into ovarian proteins. Females were decapitated 24 hr after their adult ecdysis, injected two days later and assayed 72 hr thereafter, as indicated in the
have been obtained with injections of 2.5 ng of JH I from EcoControl. JH-treated (O---U); mineral oil-treated (M).
text. Each point represents the mean (+ SD.) of the results from 10 females. JH III-injected females (O--_-O); JH Iinjected females (O----O).
6-day mated females. At the end of the culture period of 72 hr, the ovarioles in both the experimental and control cultures had degenerated and would not incorporate 3H-leucine into newly-synthesized protein during the 5 hr 3H-leucine culture time. Hence, it was concluded that these culture conditions for the ovarioles were too harsh (e.g. perhaps the constant shaking) and would have to be extensively modified.
analyses confirm that two distinct periods of development characterize the follicle cells during terminal oijcyte maturation in L. maderae. These two periods are associated, respectively, with vitellogenesis and with chorion deposition. Morphological differences clearly distinguish the two basic periods of development. The characteristics of the JH-treated follicle cell (KOEPPE et al., 198Oa) persist throughout the period of vitellogenesis. At approx. 15 days after mating, these morphological characteristics begin to change, as the cuboidal follicle cell becomes squamous. This transition is attended by the formation of an extensive rough endoplasmic
DISCUSSION Both morphological
observations
and biochemical
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50
60
70
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vim stimulation of protein synthesis in ovaries from virgin females. The data points represent the S.D.) of the ‘ovarian ratios’ obtained from 46 pairs of ovaries. The ‘ovarian ratio’ is described in and Methods, as are the culture and assay techniques. The bar graph in the centre of the graph a hypothetical ‘ovarian ratio’ of one, meaning the rates of protein synthesis in both the treated and ovary would be identical. Statistics using a matched pair t-test show ap -C 0.05 for the points at 45 and 51 hr and a p < 0.07 at 69 hr.
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Changes in follicle cell morphology reticulum within the follicle cell and the beginning of chorion deposition. The accumulation of apparent glycogen granules within the follicle cell during the later stages of vitellogenesis has not been reported previously in L. maderae. and the function of such granules is not known. The dispersion of these granules (Figs. 2,3) before chorion deposition suggests that they may serve as an energy source or as a source of carbohydrates for the synthesis of glycoproteins. Alternatively, it is possible that such granules may be translocated into the oiicyte for later embryonic use. The fact that they disperse immediately before chorion deposition indicates it is a regulated process that appears to be dependent on the regulator of chorion deposition. Major biochemical differences also distinguish the two basic periods of o6cyte maturation. A vitellogenic terminal follicle is characterized by a rapid rate of DNA synthesis (20-IOO-fold increase over a non-vitellogenic follicle) and a moderate rate of protein synthesis (24fold increase over an uninduced follicle). In contrast, during chorion deposition the terminal follicle is characterized by a radical increase (loo-fold increase over uninduced follicles) in the rate of protein synthesis, while the rate of DNA synthesis declines to an undetectable level. JH is present in the haemolymph during the vitellogenic period of development, but during chorion deposition it is either non-existent or at such a minimal level as to be undetectable (KOEPPE et al., 1980b). Evidence to date has revealed direct regulatory influences of JH during vitellogenesis, with perhaps an indirect regulatory role during chorion deposition. The most visible role of JH is its induction of morphological characteristics of the follicle cells during vitellogenesis (KOEPPE et al., 1980a). JH also is associated with the regulation of vitellogenin synthesis by the fat body and DNA synthesis by the ovary during the vitellogenic period (KOEPPE and WELLMAN, 1980). Data presented in this communication demonstrate that JH stimulates synthesis during this period ovarian protein of development. More specifically, the in vitro stimulation of the ovaries with JH demonstrates that this tissue is a site of JH action and that such stimulated protein synthesis is a JH-regulated event. The inadequacy of the culture medium to sustain normal DNA synthesis in ovarian tissue for periods of more than 180 min has prevented a direct demonstration that JH stimulates DNA synthesis by the ovary (KOEPPE and WELLMAN, 1980). However, the finding that JH injections restore DNA and protein synthesis by the ovary in decapitated insects is evidence that JH regulates both processes during vitellogenesis. In contrast, during the period ofchorion deposition, DNA synthesis is arrested, protein synthesis is accelerated, and the haemolymph JH-titre is declining. Further, the decapitation of M-10 females to eliminate all cephalic regulatory factors does not impede the normal maturation of the oiicyte, thus suggesting the critical time of JH influence to be during the
first 10 days of maturation. Finally, the introduction of JH into the haemolymph following completion of vitellogenesis does not impede chorion deposition (KOEPPE and JARNIGAN, unpublished observations), These findings demonstrate that JH plays no direct regulatory role, either positive or negative, during the period of chorion deposition. Acknon~ledgmlents~We thank Ms. WILMA HANTON for the electron microscopy, Ms. SUSAN WELLMAN for her technical assistance and Mr. RICHARI) H. ROBINSON for his editorial assistance. This work was supported by grants from the National Institute for Environmental Health Sciences (ESOl563) and from the North Carolina Science and Technology Committee (798).
REFERENCES BELL W. J. and SAMS G. R. (1974) Factors promoting vitellogenic competence and yolk deposition in the cockroach ovary: the post-ecdysis female. J. In.srct Physiol. 20, 2475-2485. BROOKEDV. J. (1969) The induction of yolk protein synthesis in the fat body of an insect, Leucophaea maderae by an analog of the juvenile hormone. Devl. Eiol. 20, 459-471. ENCELMANN F. (1969) Female specific protein: biosynthesis controlled by corpus allatum in Leucaphaea maderae. Science, N. Y. 165, 407-409. KOEPPE J. K. (1981) Juvenile hormone regulation of ovarian maturation in Leurophaea maderar. Symposium on “Regulation of Insect Development and Behavior” sponsored and published by the Technical University of Wroclaw, Poland, (in press). KOEPPE J. K. and GILBERT L. I. (1973) Immunochemical evidence for the transport of haemolymph protein into the cuticle of Manduca .re.yta. .I. Insect Physiol. 19, 6 15-634. KOEPPE J. K. and OFENGAND J. (1976a) Juvenile hormoneinduced biosynthesis of vitellogenin in Leucophaea maderae. Archs. Biochem. Biophys. 173, 100-l 13. KOEPPE J. K. and OFENGAND J. (197613) Juvenile hormoneinduced biosynthesis of vitellogenin in Leucophaea maderae from large precursor polypeptides. In: The Juvenile Hormones (Ed. by GILBERT L. I.). pp. 486-504. Plenum, New York. KOEPPE J. K. and WELLMAN S. E. (1980) Ovarian maturation in Leucophaea maderae; juvenile hormone regulation of thymidine uptake into follicle cell DNA. J. In.uc,c/ Ph,vsio/. 26, 219-228. KOEPPE J. K., HOBSON K. and WELLMAN S. E. (1980a) Juvenile hormone regulation of structural changes and DNA synthesis in the follicular epithelium of Leucophueu maderae. J. Insect Ph.vsiol. 26, 229-240. KOEPPE J. K.. BRANTLEY S. G. and NIJHOUT M. M. (1980b) Structure, haemolymph titre, and regulatory effects of juvenile hormone during ovarian maturation in Leucophaea maderae. J. Insect Physiol. 26, 749-753. MARKS E. P., ITTCHERIAH P. I. and LELOUP A. M. (1972) The effect of /&ecdysone on insect neurosecretion in vitro. J. Insect Physiol. 18, 847-850. SAMS G. R. and BELL W. J. (1977) Juvenile hormone initiation of yolk deposition in vitro in the ovary of the cockroach. Periplaneta americana. Ad. in Invert. Reprod. 1, 404--113. WYSS-HUBER M. and LUSCHER M. (1972) In vitro synthesis and release of proteins by fat body and ovarian tissue of Leucophaea maderae during the sexual cycle. J. Insect Physiol. 18, 689-710.
281-291.
1981.
0022-1910/81/040281-I 1 $02.00/O cl981 Pergumon Press Ltd.
CHANGES IN FOLLICLE CELL MORPHOLOGY, OVARIAN PROTEIN SYNTHESIS AND OVARIAN DNA SYNTHESIS DURING OijCYTE MATURATION IN LEUCOPHAEA MADERAE: ROLE OF JUVENILE HORMONE JOHN K. KOEPPE, FORIS
N. JARNAGIN and LAWRENCE N. BENNETT
Department of Zoology, University of North Carolina, Chapel Hill. North Carolina
27514, U.S.A.
(Received 4 August 1980; revised 7 November 1980)
Abstract--Changes
in follicle cell morphology were correlated with changes in rates of protein synthesis and DNA synthesis by the ovary during ovarian maturation in Leucophaea maderae. During the vitellogenic period of oiicyte development, which lasts approx. 15 days, morphological changes in the follicle cells are accompanied by moderate rates of ovarian protein synthesis and rapid rates of ovarian DNA synthesis. At approx. 15 days after mating. the shape of the follicle cells changes from cuboidal to squamous, ovarian DNA synthesis is arrested, and ovarian protein synthesis increases slightly. During the final period of o6cyte development, which lasts approx. two days, the interfollicular channels between the follicle cells have disappeared and the squamous follicle cells, which contain an extensive rough endoplasmic reticulum, deposit a chorion around the mature oiicyte. These morphological changes are accompanied by a radical increase in ovarian protein synthesis, while ovarian DNA synthesis remains arrested. Immediately before ovulation, ovarian protein synthesis starts to decline, reaching a minimal level 24 hr post-ovulation. Ovarian maturation is dependent on the presence ofjuvenile hormone (JH) only during the vitellogenic stage of oiicyte development. Decapitation of insects at any point during the first 10 days after mating arrests the synthesis of DNA and retards the synthesis of protein by the ovary, resulting in degeneration of the okyte. Subsequent injection of JH restores both events to normal levels within 72 hr. Decapitation on or after the tenth day following mating does not alter normal oiicyte development, chorion deposition, ovulation or egg case formation. Primary induction of protein synthesis in ovaries from virgin females can be achieved by either an in vivo or in vitro exposure of the tissue to JH, thus confirming a site of action for JH to be ovarian tissue. Electrophoretic analysis of the soluble proteins from JH-exposed ovaries in vivo reveals that JH stimulates general protein synthesis, rather than the synthesis of a specific major protein such as vitellogenin. Key Word Index: Follicle cell, Leucophaea maderae, juvenile hormone, DNA synthesis, protein synthesis, ovary
INTRODUCTION
OVARIAN maturation
in the ovoviviparous cockroach requires approx. 18 days and is characterized by numerous biochemical and morphological changes within the follicular epithelium. After mating, the oijcytes within the 20-22 terminal follicles of each panoistic ovary become vitellogenic, incorporate large quantities of macromolecules from the haemolymph and grow from 1.2 to 6.5 mm in length. At the end of the growth period, a chorion is deposited around each mature oijcyte by the follicular epithelium. Previous research has demonstrated that juvenile hormone (JH) influences various biochemical and morphological events that are processes associated with ovarian maturation in L. maderae. For example, it has been shown that JH induces morphological changes in the follicular epithelium of virgin females that are characteristic of S-day mated females, including the development of interfollicular spaces and channels, the enlargement of the follicle cell and its nucleus, and a limited increase in rough endoplasmic reticulum within the follicle cell (KOEPPE et al., 1980a). Further. it has been shown that JH stimulates vitellogenin synthesis by the fat body Leucophaea
maderae
281
(BROOKES, 1969; ENGELMANN, 1969; KOEPPE and OFENGAND, 1976a) and DNA synthesis by the ovary (KOEPPE and WELLMAN, 1980). More recently, it has been observed that changes in the rates of synthesis of vitellogenin by the fat body, and DNA by the ovary, coincide with fluctuations in the haemolymph titre of JH. The peaks in these two synthetic events and in the haemolymph JH-titre occur at approx. 12 days after mating, or when the oijcyte is between 5 and 5.5 mm in length (KOEPPE et al., 1980b; KOEPPE, 1981). Thus, these two biochemical events appear to be directly regulated by JH, although in decapitated, mated females we have been unable to restore rates of ovarian DNA synthesis to normal levels through injections of JH I (mixed isomer) (KOEPPE et al.. 1980a). With regard to protein synthesis in the ovaries, it has been reported that the rate of protein synthesis in ovaries from L. maderae at 10 days post-parturition was greater than at 5 days, that this increased rate of synthesis appeared to be JH-dependent and that the induced protein was vitellogenin (WYSSHUBER and LUSCHER, 1972). More recently, in Periplaneta americana, it has been demonstrated by autoradiography that JH stimulates the synthesis of non-vitellogenic proteins by the follicle cell. Those
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proteins appear to be secreted into the interfollicular spaces and are postulated to aid the translocation of vitellogenin through the follicular epithelium (BELL and SAMS, 1974; SAMSand BELL, 1977). Thus, there is limited evidence that JH may regulate protein synthesis in the ovaries. The research reported in this communication was designed to enhance our understanding of terminal follicle maturation in L. maderae. In particular, attention has been focused on refining our knowledge of the rates of protein and DNA synthesis by the ovary, correlating these rates to morphological changes, and determining the extent to which JH regulates morphological and biochemical changes during ovarian maturation. MATERIALS
AND METHODS
A. Animals Colonies of Leucophaea maderae were maintained on Wayne Dog Chow and water, with 12 hr light and 12 hr dark, at 78°F. Animal staging and colony organization are described in a previous publication (KOEPPEand WELLMAN,1980). Females one day after adult ecdysis are referred to as V-l females. Under normal conditions newly-emerged adult males and females were maintained separately. On V-10, three-week-old males were added to the female cage for mating. After one day, the mated females were removed from the cage and maintained separately. Under the described conditions, egg case formation normally occurred between M-17 and M-19. B. Electron microscopy Animal preparation, tissue fixation, embedding procedures, sectioning and microscopy are described in a previous publication (KOEPPEet al., 1980a). C. Biochemical assays 1. Protein synthesis. (a) Tissue culture. Ovariole cultures with 250 ~1 of 3H-leucine medium were maintained for five hours at 31°C in a shaking waterbath. A time-course study revealed that incorporation of 3H-leucine into newly-synthesized TCA precipitable protein was linear from 0.5 to 10 hr, regardless of ovariole length, and thus five hr was selected as the standard incubation time. The number of ovarioles cultured was dependent on their size: 22 or fewer ovarioles if they were less than 2 mm in length, 10 or fewer ovarioles if they were 2-4 mm in length and 5 or fewer ovarioles if they were 4.0-6.5 mm in length. Unless otherwise noted, ovarian cultures refer to the culture of ovarioles, not whole ovaries with an ovarian sheath. The wells (maximum capacity of 2 ml) were maintained in a humid environment (without additional 0, or CO,) in large carrier trays. Each 250 ~1 culture contained 25 &i of 3H-leucine with a specific activity of 72.9 mCi/mmole or 34.5 pmole of leucine/culture. The medium was prepared by adding 1 ml of sterile L-(4,5-3H)-leucine (56 Ci/mmole) in water to 9 ml of M-20 medium with 7.5% foetal calf serum, and penicillin and streptomycin at a concentration of 1000 units/ml.
After incubation, the ovarioles were removed from the medium and homogenized in 1 ml of 0.4 M NaCl at 4°C with a ground glass homogenizer. Aliquots of the whole homogenate were quantified, as indicated below, for 3H-leucine-labelled proteins. In all trial assays less than 10% of the labelled protein was found to be secreted from the ovarioles, and unless otherwise noted, protein in the medium was not quantified. (b) Quantification of labelled proteins (protein assay). Sample aliquots were placed into 3 ml of 10% trichloroacetic acid (TCA) and placed in a boiling water bath for 15 min to precipitate the proteins and to destroy t-RNA with bound 3H-leucine. Labelled proteins were separated from free 3H-leucine by filtration on Gelman glass fibre filters, type A/E. Residual 3H-leucine was washed from the filter with three volumes of 5% TCA. TCA was removed with a final rinse of 50% ethanol and the filters were dried for 6 hr at 55°C. Macromolecule digestion and quantification of the labelled protein bound to the filter is described in a previous publication (KOEPPE and WELLMAN,1980). 2. Vitellogenin radioimmunoassay. Analysis of culture medium and of tissue extracts for the presence of radioactive vitellogenin was performed as described in a previous publication (KOEPPE and WELLMAN, 1980). The antiserum was made against 28s vitellin obtained from L. maderae and bound both precursor and processed vitellogenin (KOEPPEand OFENGAND, 1976b). 3. DNA synthesis. The assay for quantification of 3H-thymidine incorporation into ovarian DNA is described in a previous publication (KOEPPE and WELLMAN.1980). D. Hormonal stimulation in vivo Unless otherwise stated, the procedures for animal treatment and hormone preparation and administration are the same as reported in a previous publication (KOEPPEand WELLMAN,1980). E. Hormonal stimulation in vitro 1. Tissue preparation. Animals were anaesthetized by exposure to CO, for approx. 30 set, rinsed with 0.2% HgCl, and washed twice with sterile water (MARKS er al., 1972). The animals were then pinned to a sterile dissecting dish, ventral side up, and their abdominal sternites were removed. Ovaries were removed under sterile conditions and placed into 2.0 ml of modified M-20 medium consisting of 100 ml M-20 medium, 0.1 ml penicillin and streptomycin (100,000 units/ml) and 7.5 ml foetal calf serum. In the wash medium all extraneous tissue, except the ovarian sheath, was removed. 2. Tissue culture. An aliquot of juvenile hormone I (Roche) was weighed and M-20 medium was added to make a dilution of 1 mg/ml. This solution was sonicated on ice for 8-10 set at 2 mHz, using a Sonifier Sonicator. Control medium was sonicated with mineral oil. After sonication, an aliquot of the stock JH-medium was diluted lOO-fold, yielding a JH concentration of 10 pg/ml or 3.4 x 10v5M. Likewise, a dilution of the control medium was made. Stock incubation media were stored at - 15°C. Unless otherwise stated, the left ovary (experimental ovary) was cultured in 1 ml of JH-medium
2X3
Fig. 1. Electron micrographs of vitellogenic follicle ceils. Panel A is a cross-section of a follicle cell from a terminal follicle 3 mm in length obtained froni an M-8 female. Panel B is a cross-section of a follicle cell from a terminal follicle 5 mm in length obtained from an M-12 female. Tunica propria (TP). rough endoplasmic reticulum (RER). apparent glycogen (G).
284
Fig. 2. Electron micrograph of a transitional follicle cell. This is a cross-section terminal follicle 5.5 mm in length obtained from an M-15 female. Tunica propria reticulum (RER), apparent glycogen (G).
of a follicle cell from a (TP), rough endoplasmic
Fig. 3. Electron micrograph composite of a follicle cell from a terminal follicle 6 mm in length obtained from an M-18 female. Tunica propria (TP), exochorion (EX), endochorion (EN), nucleus(N), apparent glycogen CC).
2x7
Changes in follicle cell morphology while the other ovary (control ovary) was cultured for an identical time in mineral oil-medium. Media were changed every 24 hr. After culturing, the ovarian sheaths were removed from the ovaries and the separated ovarioles were assayed for rates of 3H-leucine incorporation into newly-synthesized protein. To determine if the rate of protein synthesis in an experimental ovary (minus the ovarian sheath) is altered by its exposure to JH, its rate of synthesis was compared to the rate of protein synthesis in a control ovary (minus the ovarian sheath) removed from the same female. The ratio of these two rates is referred to as an ovarian ratio. A ratio of 1.0 would indicate the rates of protein synthesis in each ovary are the same. F. Protein analysis-gel
electrophoresis
Techniques for non-denaturing gel electrophoresis are described in a previous publication (KOEPPE and GILBERT, 1973), as are the techniques for SDS slab gel electrophoresis and the techniques for quantification of labelled proteins in the gel (KOEPPE and OFENGAND, 1976a). G. Chemicals The juvenile hormones used in these experiments were obtained from Hoffman-LaRoche (JH I) and from Eco-Control Incorporated (JH I, JH III). 20-hydroxyecdysone was obtained from Calbiochem and was 99.8% pure. t_-[4,5-3H]-leucine with a specific activity of 56 Ci/mmole was purchased in sterile water from Amersham/Searle. 3H-amino acid mixture was a composite of 15 amino acids from Amersham/Searle (TRK440). M-20 medium, penicillin G, streptomycin and foetal calf serum were all obtained from Grand Island Biological Supply. Ultrapure acrylamide, TEMED (N,N,N’, N’-tetramethylethylenediamine), ammonium persulphate and BIS (N,N’-methylenebis-acrylamide), were all obtained from Bio-Rad Laboratories. Specially pure sodium lauryl sulphate (SDS) was obtained from BDH Chemicals Ltd. All other chemicals, including enzyme pure TRIS, were purchased from Sigma. Thin layer chromatography plates were obtained from Eastman Kodak. NCS was obtained from Amersham/Searle while all other scintillation chemicals were from Fisher.
RESULTS I. Maturation
of ovarian terminal follicles
A. Changes in follicle cell morphology. Crosssections of terminal follicles from selected stages of development were analyzed by electron microscopy. The electron micrographs in Fig. 1 illustrate typical morphology of follicle cells from the terminal follicles during vitellogenesis. In these cells the rough endoplasmic reticulum is located towards the haemolymph side of the cell, the nuclei are enlarged and the spaces between the follicle cells are large. In later stages of vitellogenesis. large accumulations of what appear to be glycogen granules are evident in the follicle cell (Fig. 1B). In the final stage of oiicyte development, the follicle cells are responsible for the synthesis and deposition of the chorion. This change in function is attended by a change in the structure of the follicle cell.
Figure 2 is an electron micrograph of a transitional follicle cell prior to chorion deposition (approx. 15 days after mating). This follicle cell and its nucleus are becoming squamous, and the accumulations of apparent glycogen granules are dispersing and appear to be migrating toward the oocyte side of the follicle cell. Figure 3 illustrates the completion of these changes. The follicle cell, which is in the process of chorion deposition (16-18 days after mating), contains an extensive rough endoplasmic reticulum and the apparent glycogen granules are dispersed in the area adjacent to the chorion (see insert, Fig. 3). B. Protein and DNA .synthe.ks hj, the ovary. The appearance of an extensive rough endoplasmic reticulum in the squamous follicle cells suggests a greatly enlarged capacity of the ovary to synthesize protein. This was confirmed by quantifying rates of protein synthesis by ovaries throughout terminal oocyte maturation. Rates of DNA synthesis, a JH-inducible process ( KOEPPE and WELLMAN. 1980). were also monitored. The results (Fig. 4) indicate that DNA synthesis is arrested during chorion deposition. In contrast, protein synthesis by the ovaries, while keeping pace with the enlargement of the follicle cell during vitellogenesis, rapidly accelerates and reaches a peak rate during chorion deposition. approx. 16-18 days after mating or approx. 4-6 days after the JH-haemolymph titre peaks. The results for rates of protein synthesis in Fig. 4 were obtained by measuring incorporation rates of leucine into newly synthesized proteins in culture. Identical patterns were obtained when a mixture of “H-labelled amino acids was substituted for 3H-leucine. II. Regulation OJ ovarian protein juvenile hormone
synthesis:
role qf
A. JH restoration of ovarian protein synthesis in decapitated ir7.srct.s. To determine whether JH regulates protein synthesis in the ovaries, females were maintained and treated as follows: a large population of M-6 females was divided into two groups; one group remained untreated, but was maintained without food or water, while the females in the second group were decapitated. Ovaries from both groups were immediately assayed for rates of DNA and protein synthesis. Three days later (M-9), ovaries from the two groups were again assayed for rates of DNA and protein synthesis. Thereafter the decapitated females were further divided into two subgroups; those in one group were injected with paraffin oil, while those in the other received an injection of JH I. Three days later, ovaries from females in each group were assayed for rates of DNA and protein synthesis. The results, illustrated in Fig. 5, demonstrate that decapitation arrests DNA synthesis and significantly retards protein synthesis in the ovary. Both processes were restored to levels equivalent to those in the control females through an injection of 2.5 pg of JH I (Eco-Control) suspended in 25 ~1 paraffin oil. Injections of 25 ~1 of paraffin oil neither stimulate nor further retard either process. B. In vivo stimulation of ovarian protein synthesis in virgin ,females. Virgin females, 24 hr post-ecdysis, were decapitated and injected with either 25 pg of JH I (Roche) suspended in 25 ~1 of mineral oil, or mineral oil alone. Thereafter, at 24-hr intervals,
288
JOHN K. KOEPPE. FOREST N. JARNAG~N AND
LAWRENCE N. BENNETT
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TERMINAL
OOCYTE
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Fig. 4. Rates of 3H-thymidine incorporation into ovarian DNA ( x ---x ) and 3H-leucine incorporation into ovarian proteins (t-0) during maturation of the terminal follicle. To minimize the effects of animal variation the data points are illustrated as a function of terminal oijcyte length. Under normal rearing conditions terminal oiicytes 1.3-l .4 mm in length are obtained from virgin females, while larger oijcytes are obtained from mated females. Oijcytes 3.0 mm long are found in M-6 to M-9 females; oiicytes 4.0 mm long are found in M-8 to M-l 1 females; ocicytes 5.5 mm long are found in M-12 to M-16 females, and oiicytes 6.2 mm in length (depositing chorion) are found in M-16 to M-18 females. The data points represent the mean (+ S.D.) of the results from a minimum of 10 ovaries. Assays are described in the text. Chorion deposition was first apparent in terminal follicles 6 mm in length.
ovaries were removed from the females and assayed in vitro for rates of 3H-leucine incorporation into newly synthesized proteins. The results, depicted in Fig. 6, illustrate a 1.75-fold increase in the rate of protein synthesis by virgin ovaries when exposed to JH I. Similar results (not illustrated) have been obtained using JH I obtained from Eco-Control, although at one-tenth the concentration (2.5 pg). Data derived from injection of JH I (Roche) were plotted since they provide a comparison to the in vitro stimulation experiments (see Fig. 8). In this report, and in many previous experiments, JH I has been used to study the regulatory effects of JH on female reproductive development. Since JH III is the natural hormone found in L. maderae (KOEPPE et al., 1980b), we found it necessary to determine whether the different forms of the hormone (JH I and JH III) display different activities in stimulating protein synthesis by the ovaries. In these experiments the females were decapitated on V-2 and two days later were injected with varying concentrations of JH I or JH III, both from Eco-Control. Three days thereafter, the ovaries were removed and assayed in vitro for rates of 3H-leucine incorporation into newly synthesized proteins. The results in Fig. 7 illustrate that while JH I stimulates protein synthesis by the ovary at concentrations lower than those necessary for stimulation with JH III, both hormones stimulated protein synthesis to similar levels at higher concentrations.
The effect of 20-hydroxyecdysone on protein synthesis by the ovary was also monitored in V-l and V-12 females. No response (up to 72 hr post-injection) was obtained in either group of decapitated females with injected doses ranging from 0.001 to 10 pg (data not illustrated). C. Analysis of’JH-induced soluble proteins. Results from electrophoretic and radioimmunoassay studies demonstrate that JH-induction of protein synthesis by ovarioles is general rather than specific, and that vitellogenin is not a JH-inducible ovariole protein. In these experiments, decapitated virgin females, 24 hr post-ecdysis, were separated into two groups and injected with either 25 pg of JH I (Roche) suspended in mineral oil (25 ~1) or 25 ~1 of mineral oil. After 72 hr, the ovaries were removed, cultured for 5 hr in an 3H-leucine medium, and the ovarioles were then homogenized in 0.4 M NaCl (with 0.1 mg/ml of PMSF) at 0°C before centrifugation at 10,000 g. The supernatants revealed the concentration of the labelled proteins in the extract from the JH-exposed ovarioles to be 1.89 times greater than the extract from the mineral oil-exposed ovarioles. Subsequeni analysis of these extracts suggested JH induction of ovarian protein synthesis was general rather than specific. In one experiment, labelled proteins from the JH-exposed ovarioles were added to an extract of vitellin and analyzed by SDS-gel electrophoresis (see Materials and Methods). The results revealed that labelled protein from the
Changes in follicle cell morphology
6
12
9
I
I
9 DAYS
AFTER
289
I2
MATING
Fig. 5. Analysis of DNA and protein synthesis in ovarian tissue from females decapitated 6 days after mating (terminal follicles about 2.4 mm in length) and thereafter injected with JH/paraffin oil. Data illustrated on both graphs were obtained from tissues from the same animals. Each point represents the mean (+ S.D.) of the results from 5 females. Injections were completed 3 days after decapitation of M-6 females. Analysis of the tissue was performed as described in the text. The graphs illustrate responses in starved, intact mated females (M), decapitated, JH-injected females (t+) and decapitated, paraffin oil-injected females (t---o). Panel A illustrates 3H-thymidine incorporation into newly synthesized ovarian DNA in vitro. Panel B illustrates 3H-leucine incorporation into newly synthesized ovarian proteins in vitro.
ovarioles did not migrate with the vitellin. Negative results from a vitellogenin radioimmunoassay of extracts from cultured JH-stimulated ovarioles and their culture medium confirmed these observations. Finally, quantification of label associated with each protein, separated by non-denaturing gel electrophoresis, revealed concentrations of label for each protein from the JH-exposed ovaries to be 1.98 times greater (+ 0.2. dependent on the digestion of gel) than the label associated with the corresponding separated protein from ovarioles not exposed to JH. These results indicate that JH stimulates the synthesis of all extractable soluble proteins. Thus vitellogenin synthesis was not found to be associated with the ovariole, although it was found to be associated with the whole ovaries, suggesting the ovarian sheath or the dispersed fat body within the sheath is responsible for such vitellogenin synthesis.
D. In vitro stimulation of ovarian protein synthesis. the protocol described in the Materials and Methods, protein synthesis was stimulated in vitro in ovaries from V-l to V-8 females. Results from V-l ovaries, illustrated in Fig. 8, demonstrate that 0.01 mg/ml of mixed isomer JH I (Roche) stimulates a 1.67-fold increase in ovarian protein synthesis in vitro. Similar results have been obtained using ovaries from V-2 to V-8 females. When a higher concentration of JH I (Roche) was used in the culture medium (0.1 mg/ml), an ovarian ratio of approx. 0.2 was obtained, indicating that 80% inhibition of ovarian protein synthesis occurs at this hormone concentration. At one-tenth the JH concentration (0.001 mg/ml), no observable effect was detected. In an effort to further localize the site of JH, we removed the ovarian sheath and cultured only isolated ovarioles obtained from I-2-day virgin or Using
JOHN K. KOEPPE,FORESTN. JARNAGINAND LAWRENCEN. BENNETT
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JH INJECTION
Fig. 6. Time-response
curve for JH-stimulation of 3H-leucine incorporation into ovarian proteins. Females were decapitated 24 hr after their adult ecdysis. Two days after decapitation they were injected with 25 pg of JH I (Roche) and assayed at the times indicated in the figure. Each data point represents the mean (+ S.D.) of the results from 5 females. The ovarian sheath and any other tissue were removed prior to the 5 hr 3H-leucine culture. Similar results
Fig. 7. Dose-response curves for JH I and JH III stimulation of 3H-leucine incorporation into ovarian proteins. Females were decapitated 24 hr after their adult ecdysis, injected two days later and assayed 72 hr thereafter, as indicated in the
have been obtained with injections of 2.5 ng of JH I from EcoControl. JH-treated (O---U); mineral oil-treated (M).
text. Each point represents the mean (+ SD.) of the results from 10 females. JH III-injected females (O--_-O); JH Iinjected females (O----O).
6-day mated females. At the end of the culture period of 72 hr, the ovarioles in both the experimental and control cultures had degenerated and would not incorporate 3H-leucine into newly-synthesized protein during the 5 hr 3H-leucine culture time. Hence, it was concluded that these culture conditions for the ovarioles were too harsh (e.g. perhaps the constant shaking) and would have to be extensively modified.
analyses confirm that two distinct periods of development characterize the follicle cells during terminal oijcyte maturation in L. maderae. These two periods are associated, respectively, with vitellogenesis and with chorion deposition. Morphological differences clearly distinguish the two basic periods of development. The characteristics of the JH-treated follicle cell (KOEPPE et al., 198Oa) persist throughout the period of vitellogenesis. At approx. 15 days after mating, these morphological characteristics begin to change, as the cuboidal follicle cell becomes squamous. This transition is attended by the formation of an extensive rough endoplasmic
DISCUSSION Both morphological
observations
and biochemical
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70
in Hours
vim stimulation of protein synthesis in ovaries from virgin females. The data points represent the S.D.) of the ‘ovarian ratios’ obtained from 46 pairs of ovaries. The ‘ovarian ratio’ is described in and Methods, as are the culture and assay techniques. The bar graph in the centre of the graph a hypothetical ‘ovarian ratio’ of one, meaning the rates of protein synthesis in both the treated and ovary would be identical. Statistics using a matched pair t-test show ap -C 0.05 for the points at 45 and 51 hr and a p < 0.07 at 69 hr.
191
Changes in follicle cell morphology reticulum within the follicle cell and the beginning of chorion deposition. The accumulation of apparent glycogen granules within the follicle cell during the later stages of vitellogenesis has not been reported previously in L. maderae. and the function of such granules is not known. The dispersion of these granules (Figs. 2,3) before chorion deposition suggests that they may serve as an energy source or as a source of carbohydrates for the synthesis of glycoproteins. Alternatively, it is possible that such granules may be translocated into the oiicyte for later embryonic use. The fact that they disperse immediately before chorion deposition indicates it is a regulated process that appears to be dependent on the regulator of chorion deposition. Major biochemical differences also distinguish the two basic periods of o6cyte maturation. A vitellogenic terminal follicle is characterized by a rapid rate of DNA synthesis (20-IOO-fold increase over a non-vitellogenic follicle) and a moderate rate of protein synthesis (24fold increase over an uninduced follicle). In contrast, during chorion deposition the terminal follicle is characterized by a radical increase (loo-fold increase over uninduced follicles) in the rate of protein synthesis, while the rate of DNA synthesis declines to an undetectable level. JH is present in the haemolymph during the vitellogenic period of development, but during chorion deposition it is either non-existent or at such a minimal level as to be undetectable (KOEPPE et al., 1980b). Evidence to date has revealed direct regulatory influences of JH during vitellogenesis, with perhaps an indirect regulatory role during chorion deposition. The most visible role of JH is its induction of morphological characteristics of the follicle cells during vitellogenesis (KOEPPE et al., 1980a). JH also is associated with the regulation of vitellogenin synthesis by the fat body and DNA synthesis by the ovary during the vitellogenic period (KOEPPE and WELLMAN, 1980). Data presented in this communication demonstrate that JH stimulates synthesis during this period ovarian protein of development. More specifically, the in vitro stimulation of the ovaries with JH demonstrates that this tissue is a site of JH action and that such stimulated protein synthesis is a JH-regulated event. The inadequacy of the culture medium to sustain normal DNA synthesis in ovarian tissue for periods of more than 180 min has prevented a direct demonstration that JH stimulates DNA synthesis by the ovary (KOEPPE and WELLMAN, 1980). However, the finding that JH injections restore DNA and protein synthesis by the ovary in decapitated insects is evidence that JH regulates both processes during vitellogenesis. In contrast, during the period ofchorion deposition, DNA synthesis is arrested, protein synthesis is accelerated, and the haemolymph JH-titre is declining. Further, the decapitation of M-10 females to eliminate all cephalic regulatory factors does not impede the normal maturation of the oiicyte, thus suggesting the critical time of JH influence to be during the
first 10 days of maturation. Finally, the introduction of JH into the haemolymph following completion of vitellogenesis does not impede chorion deposition (KOEPPE and JARNIGAN, unpublished observations), These findings demonstrate that JH plays no direct regulatory role, either positive or negative, during the period of chorion deposition. Acknon~ledgmlents~We thank Ms. WILMA HANTON for the electron microscopy, Ms. SUSAN WELLMAN for her technical assistance and Mr. RICHARI) H. ROBINSON for his editorial assistance. This work was supported by grants from the National Institute for Environmental Health Sciences (ESOl563) and from the North Carolina Science and Technology Committee (798).
REFERENCES BELL W. J. and SAMS G. R. (1974) Factors promoting vitellogenic competence and yolk deposition in the cockroach ovary: the post-ecdysis female. J. In.srct Physiol. 20, 2475-2485. BROOKEDV. J. (1969) The induction of yolk protein synthesis in the fat body of an insect, Leucophaea maderae by an analog of the juvenile hormone. Devl. Eiol. 20, 459-471. ENCELMANN F. (1969) Female specific protein: biosynthesis controlled by corpus allatum in Leucaphaea maderae. Science, N. Y. 165, 407-409. KOEPPE J. K. (1981) Juvenile hormone regulation of ovarian maturation in Leurophaea maderar. Symposium on “Regulation of Insect Development and Behavior” sponsored and published by the Technical University of Wroclaw, Poland, (in press). KOEPPE J. K. and GILBERT L. I. (1973) Immunochemical evidence for the transport of haemolymph protein into the cuticle of Manduca .re.yta. .I. Insect Physiol. 19, 6 15-634. KOEPPE J. K. and OFENGAND J. (1976a) Juvenile hormoneinduced biosynthesis of vitellogenin in Leucophaea maderae. Archs. Biochem. Biophys. 173, 100-l 13. KOEPPE J. K. and OFENGAND J. (197613) Juvenile hormoneinduced biosynthesis of vitellogenin in Leucophaea maderae from large precursor polypeptides. In: The Juvenile Hormones (Ed. by GILBERT L. I.). pp. 486-504. Plenum, New York. KOEPPE J. K. and WELLMAN S. E. (1980) Ovarian maturation in Leucophaea maderae; juvenile hormone regulation of thymidine uptake into follicle cell DNA. J. In.uc,c/ Ph,vsio/. 26, 219-228. KOEPPE J. K., HOBSON K. and WELLMAN S. E. (1980a) Juvenile hormone regulation of structural changes and DNA synthesis in the follicular epithelium of Leucophueu maderae. J. Insect Ph.vsiol. 26, 229-240. KOEPPE J. K.. BRANTLEY S. G. and NIJHOUT M. M. (1980b) Structure, haemolymph titre, and regulatory effects of juvenile hormone during ovarian maturation in Leucophaea maderae. J. Insect Physiol. 26, 749-753. MARKS E. P., ITTCHERIAH P. I. and LELOUP A. M. (1972) The effect of /&ecdysone on insect neurosecretion in vitro. J. Insect Physiol. 18, 847-850. SAMS G. R. and BELL W. J. (1977) Juvenile hormone initiation of yolk deposition in vitro in the ovary of the cockroach. Periplaneta americana. Ad. in Invert. Reprod. 1, 404--113. WYSS-HUBER M. and LUSCHER M. (1972) In vitro synthesis and release of proteins by fat body and ovarian tissue of Leucophaea maderae during the sexual cycle. J. Insect Physiol. 18, 689-710.