- Email: [email protected]
Vitellogenic arrest in the cockroach, Blatta orientalis
J. Insect Physiol., 1975, Vol. 21, pp. 1203 to 1210.
VITELLOGENIC
Pergamon
Press.
Printed
in Great Britain.
ARREST IN THE COCKROACH,
BLATTA
ORIENTALIS
GARY R. SAMS Department
of Physiology and Cell Biology, University (Received
of Kansas, Lawrence,
14 October
Kansas 66045, U.S.A.
1974)
Abstract-Starvation stimulated vitellogenic arrest occurs in the cockroach Blat& orientalis after 5 days. This is characterized by cessation of yolk uptake and oijcyte growth. After 5 days of starvation, protein and RNA synthesis decrease, but some macromolecular synthesis continues during the entire starvation period. No oocyte resorption occurs for up to 15 days of starvation. In contrast to starvation, injection of actinomycin-D results in resorption within 8 hr. The results suggest that B. orientalis copes with starvation by maintaining arrested oijcytes as an alternative to immediate resorption.
INTRODUCTION REMOVAL of corpora allata or cessation of juvenile hormone (JH) secretion because of starvation results in vitellogenic arrest and oijcyte resorption in many insect species (ENGELMANN, 1970). Vitellogenic arrest is characterized by permanent or temporary curtailment of yolk deposition, whereas resorption entails irreversible breakdown of ovarian follicles. The following study was undertaken to elucidate cellular processes which occur during starvationinduced vitellogenic arrest in the cockroach Blattu orientalis, a species in which oScytes are retained in the arrested state for lengthy periods prior to resorption.
MATERL4LS
AND
hETI-IODS
Starved adult female cockroaches were maintained at 26”C, 60% r.h., and a 12 : 12 hr photoperiod, with access to water. Starvation was initiated on the day of oiitheca formation (BF) and the number of days of starvation was calculated from this point. Control females were maintained under similar conditions but were fed Purina Laboratory Chow ad lib. Starved females were injected with 10 PC 10 Ci/mM, New 3H-uridine (specific activity England Nuclear), 3H-histidine (specific activity 15 Ci/mM, New England Nuclear, or 3H-thymidine (specific activity 6.7 Ci/mM, New England Nuclear), using a micro-syringe. Animals were dissected 24 hr after isotope injection and the oacytes in each ovary measured. Oijcyte volume was calculated from length and width measurements according to the method of BELL (1969a). One ovary from each animal was fixed in Bouin’s for histology or autoradiography as described by BELL and SAMS (1975). The other was prepared for scintillation counting using methods similar to
those of MANS and NOWLLI (1961). Ovaries were homogenated in glass grinders and the homogenate pipetted to glass fibre discs. Discs prepared for 3H-uridine incorporation analysis were dropped into ice-cold 5% perchloric acid, washed four times with cold 5% perchloric acid and then twice each with ethanol and ether, dried, and transferred to scintillation vials. Discs prepared for SH-histidine incorporation analysis were rinsed four times in cold 5% TCA containing 5 mM non-labelled histidine and then twice each with ethanol and ether, dried, and transferred to scintillation vials containing 5 ml of liquid scintillation fluid (4 g/l PPO and 0.05 g/l POPOP). Vitellogenic activity was determined by injecting 10 /*l of sterile 1% trypan blue into female cockroaches at specific times after initiating starvation. The ovaries were dissected out 24 hr later and the extent of trypan blue uptake by the oacytes was observed. Incubations with sterile trypan blue were performed by placing dissected ovaries from females into saline containing sterile 0.1% trypan blue for 30 min. The ovaries were then washed several times with saline, and dye uptake was recorded photographically. To further analyse 3H-uridine incorporation, 1 pg/animal actinomycin-D (act-D) was injected into females beginning to form an oiitheca. At various times after injection, as noted in the text, the ovaries were dissected. One ovary was incubated in saline containing 10 &X/ml 3H-uridine for 2 hr and processed for scintillation counting; the other was incubated in 0.1% trypan blue as described above, then photographed.
RESULTS Vitellogenic
cycle
06cyte
growth
in
the
vitellogenic
cycle
of
1204
GARY
FL
SAMS
*...*** -
. ..* *...a*
SF
. ..*
/
.
.
5
10
.
15
DAYS
Fig. 1. Comparison of oijcytes volumes in fed (A) and starved (B) B. orientalis females. Each point represents mean values for 3 to 7 females. Basal oiicytes, 0; penultimate oiicytes, 0; ultimate oijcytes, 0.
B. orientalis is depicted
in Fig. 1-A. Basal oocytes are ovulated approximately every 5 to 6 days and penultimate oocytes begin growth shortly before the basals are ovulated. Thus the uptake of vitellogenins (female-specific blood proteins) by the oiicytes is continuous in that when the basal is ovulated, the penultimate is already engaged in yolk deposition. As shown in Fig. 1 -A, the largest growth increment occurs in penultimate oocytes 2 to 4 days after the basal oiicytes are ovulated. OGcytes of starved females (Fig. 1-B) continue to increase in volume until the fifth day of starvation (BF + 5). At this time, increase in volume of the basal oijcytes ceases. After BF + 5 no further growth occurs in the basal oiicyte, growth of the penultimate oocytes is not initiated, except in a few isolated instances, and ovulation does not occur. Basal oocytes from animals starved longer than 5 days remain at an intermediate size (0.6-l *5 mm3). They contain yolk but remain in vitellogenic arrest for up to 20 days of starvation.
Trypan blue uptake Trypan blue uptake can be used effectively to detect or quantitatively measure yolk uptake in oijcytes (ANDERSON, 1971; BOHM, 1972) and also provides an index of cellular integrity (BECK and LLOYD, 1963; BECK et al., 1967). Trypan blue
injected into fed females is taken up by vitellogenic oocytes, giving the oijcyte cortex a bluish hue which can be seen under the dissecting microscope. Trypan blue uptake occurs generally over the surface of vitellogenic basal and penultimate oijcytes (Fig. 2) and is especially intense at the poles
Fig. 2. Diagrammatic representation of oiicyte trypan blue uptake during transition from vitellogenesis to arrest. Dark oijcytes (trypan uptake) = vitellogenic; light oijcytes = non-vitellogenic; light oijcytes with thick border = chorionated, non-vitellogenic. a, basal; b, penultimate; c, ultimate oijcytes.
of the penultimate oocyte (see arrows, Fig. 5-A). No dye is taken up by the pre-vitellogenic or chorionated oocytes. As depicted in Fig. 2, trypan blue in starved animals is sequestered by the basal oijcyte during the first 5 days, though at stage BF+ 5 uptake is slight. No trypan blue was observed in either the basal or penultimate oijcytes after 5 days of starvation. These results, along with the changes in oijcyte volume discussed above, suggest that
1205
Fig. 3. Histological preparations of basal oijcytes. A, stained section of a vitellogenic oijcyte, Haemotoxylin stained and counterstained Rith eosin. B to D, incorporation of 3H-histidine in animals starved for different lengths of time: B, 1-5 days; C, S-10 days; D, lo-15 days. E and F, incorporation of 3H-uridine: E, starved 1-5 days; F, starved longer than 5 days. G and H, incorporation of 3H-thymidine: G, starved l-5 days: H, starved longer than 5 days. B, blood; FCE, follicle cell epithelium; OC, oiicyte cortex; YS, yolk sphere. Vertical lines represent boundaries of follicle cell epithelium.
Fig. 3. In vitro uptake of trypan blue following saline or Act-D injection in vow. A, saline injection. B to E, Act-D treatments for various periods: B, 4 hr; C, 4-6 hr (dark field photo) (arrows indicate patches of trypan blue impregnation); D, 6-8 hr; E, 8 hr (chorionated basal oiicyte in E; basal oacytes are not chorionated in A to D). B, basal o&ytes; P, penultimate ob;cytes; l-1, ultimate o&ytes.
Vitellogenic arrest in the cockroach
1207
DAYS OF STARVATION
Fig. 4. Incorporation of JH-histidine into TCA-precipitated material (-), and incorporation of 3H-uridine into PCA-insoluble material (- - - - -), after specific periods of starvation. Each point represents the mean value for single ovaries from 3 to 7 females. vitellogenesis is arrested as a result of starvation and that the oijcytes are retained intact in the ovaries. Protein, RNA,
and DNA
synthesis
To determine if a cessation or decrease in synthesis of follicle cell proteins in starved females is causally related to vitellogenic arrests, the following experiment was performed. Females were injected with 3H-histidine after specific periods of starvation and 24 hr later the ovaries were prepared for autoradiography. A stained section (Fig. 3-A) demonstrates the positions of different tissue components in the autoradiographs. Incorporation of 3H-histidine occurred in the follicle cells and oijcyte during the entire 15day starvation period. However, silver grains over yolk spheres were observed in fed females and starved females through BF+5 stage (Fig. 3-B), and were not extensively associated with yolk spheres in stages after BF + 5 (Fig. 3-C, D). 3H-Uridine incorporation was also observed during the entire starvation period. Though silver grains were found mainly over the follicle cells, grains were also associated with yolk spheres in the oijcyte cortex (Fig. 3-E, F). Whether these grains indicate RNA synthesis by follicle cells and transfer to the oScyte or endogenously synthesized oijcyte RNA is not known. Incorporation of 3H-thymidine was observed (Fig. 3-G, H) during the entire starvation period,
although it could not be determined if there were changes in the number of follicle cell nuclei undergoing mitotic divisions. Fig. 4 shows quantitative measurements of SH-uridine and SH-histidine incorporation by ovaries of starved females. Incorporation of SH-histidine decreased sharply after the BF+ 5 period and levelled off to values just above background. SH-Uridine incorporation peaked at BF + 5 and then decreased sharply until a slight increase occurred at stage BF+lO; the BF+lO peak was followed by a progressive decrease through Thus detectable protein and RNA BF+lS. synthesis occurred up to 10 days after vitellogenesis had ceased. Actinomycin-D
treatment
To determine if inhibition of follicle cell RNA synthesis promotes vitellogenic arrest, Act-D was injected into yolk-depositing females. At various times after injection, vitellogenic activity was measured using the trypan blue index (Fig. 5-B-E) and 3H-uridine incorporation was assessed by scintillation counting (Fig. 6). Figure 5-A demonstrates normal uptake of trypan blue (see discussion in previous section). Within 4 hr of Act-D treatment (Fig. S-B) heavy uptake of trypan blue occurred (Fig. S-B), but, rather than the homogeneous peripheral uptake seen in vitellogenic o8cytes, a stippled pattern of uptake was observed. After 4 to
1208
GARY R. SAMS
6 hr of Act-D treatment large patches of trypan blue impregnation were observed (Fig. S-C, see arrows) and by 6 to 8 hr the oocytes were completely permeable to trypan blue (Fig. S-D), indicating a Noncomplete loss of membrane integrity. vitellogenic oiicytes were unaffected by Act-D as indicated by trypan blue uptake (see previtellogenic oocyte and chorionated oijcyte in Fig. S-E). At the same time a considerable reduction in 3H-uridine incorporation occurred (Fig. 6). Thus, it seems that
appears that Act-D did not alter the yolk precursor supply. Recovery from vitellogenic arrest It was of interest to investigate recovery from starvation-induced vitellogenic arrest when feeding is resumed. Starved females, therefore, were re-fed and the time of oviposition for the next two cycles recorded. The results indicate that the longer a female is starved, the longer recovery takes. Females starved for short periods (5 to 6 days) and then fed, exhibited a delay of 3 days in ovulation of the first The second oviposition occurred on ootheca. schedule. On the other hand, females starved longer than 6 days exhibited a considerable delay (up to 16 days) before ovipositing the first ootheca. Oijthecae of the first ovulation from animals starved for long periods were abnormal in shape and smaller in size. DISCUSSION
Fig. 6. In vitro incorporation of 3H-uridine for 2 hr at specific periods after treatment with 1 fcg Act-D. Each point represents mean values for single ovaries from 2 or 3 females.
inhibition of RNA synthesis results in resorption of vitellogenic oocytes, although as shown in Fig. 4, not in the resorption of non-vitellogenic oiicytes. Perhaps a continuous programme of RNA synthesis is required to maintain yolk deposition and to prevent resorption in vitellogenic oijcytes. Because most Act-D-treated B. orientalis females died after several days, the reversibility of Act-D effects on the ovary could not be determined. A larger and more vigorous species, Perifilaneta americana, which has the same S-day continuous vitellogenic cycle as B. orientalis, was injected with various dosages of Act-D and the effect on vitellogenesis was assessed. Females injected on the day of ootheca formation with less than 5 pg of Act-D exhibited no oijcyte resorption when dissected 5 or 12 days after treatment. Those injected with more than 7 pg died within 6 days, Females administered with 5 pg of Act-D exhibited resorption in basal oiicytes after 5 days; penultimates were vitellogenic. No oijthecae were deposited, and after 12 days substantial yolk was deposited in penultimate oocytes. Basal oiicytes were eventually completely resorbed, subsequent vitellogenic cycles occurred normally, and the females showed no further indication of Act-D effects. Thus, although Act-D causes irreversible resorption of vitellogenic oocytes, the inhibitor does not prevent yolk deposition in previtellogenic oijcytes. Moreover, since vitellogenesis was initiated in penultimate oiicytes, it
The initial effect of starvation on vitellogenesis in B. orientalis is the cessation of yolk deposition which is in contrast to P. americana where oijcytes continue vitellogenesis for up to 10 days of starvation and then resorb (BELL, 1971). That curtailment of growth is linked to vitellogenic arrest is suggested by comparison of oiicyte volume measurements and trypan blue uptake analysis. Concomitant with vitellogenic arrest is a decrease in RNA and protein synthesis, although these processes continue at low levels during the entire starvation period. Whether the decrease in macromolecular synthesis is the cause of vitellogenic arrest is not clear, but it is known that follicle cell protein synthesis is necessary for yolk deposition (ANDERSON and TIXLFER,1969; ANDERSON,1971; CRUIKSHANK, 1971; BELL and SAMS, 1974) and thus reduced follicle cell protein synthesis might result in vitellogenic arrest. Another possible reason for arrest might be that lack of yolk precursors during starvation prevents yolk deposition. HIGHNAMet al. (1963) suggested such a situation in Schistocerca gregaria in which decreased availability of yolk precursors caused resorption. BELL (1971), however, showed that injecting yolk precursors into starved P. americana did not suffice to stimulate yolk deposition in starved females. Likewise BELL (1969b) and PRATT and DAVEY (1972) demonstrated in P. americana and Rhodniusprolixus that yolk precursor supply was not the limiting factor in vitellogenesis. Thus it would seem that other factors are probably involved in the control of vitellogenic arrest. In B. orient&is, lack of yolk precursors can be ruled out as a controlling agent in vitellogenic arrest since trypan blue, which mimics yolk precursors, was not sequestered during the period of starvation-induced vitellogenic
Vitellogenic arrest in the cockroach arrest. This evidence along with that mentioned above suggests that factors other than availability of yolk precursors are probably responsible for vitellogenic arrest. Resorption does not immediately follow vitellogenie arrest in B. orientalis. This is in contrast to P. americana in which oijcyte resorption is virtually completed after 12 days of starvation (BELL, 1971). Instead it seems that B. orientalis retains its oijcytes in a minimal metabolic state. This is suggested by the continued synthesis of RNA and protein in the follicles for up to 15 days of starvation without oocyte resorption. Perhaps the low level of RNA and protein synthesis is a mechanism by which the organism maintains oijcyte integrity whereas, in P. americana, mechanisms have been developed to resorb vitellogenic oijcytes. Instead of arrest, resorption followed Act-D treatment. The initial change is the appearance of a stippled pattern of trypan blue rather than the homogeneous peripheral veneer deposited in the cortex of vitellogenic oiicytes. Since intense permeability to trypan blue is an indication of cell death (BECK and GRIFFITHS, 1967), it can be suggested that the initial loci of trypan blue uptake might be the first sites of breakdown of the membranes in the follicle cells and oocyte. Following the stippled pattern, resorbing oijcytes exhibit larger patches of trypan blue, much like the ‘opaque patches’ which appear during the initiation of oocyte resorption in S. gregaria (LUSIS, 1963). LUSIS described these patches as the first characteristic by which resorbing oiicytes could be identified. In B. orientalis these sites may reflect the initiation of membrane breakdown which increases in area to encompass finally the entire oocyte. SAHOTA (1973) found that Act-D injected into Dendroctonus pseudotsugae resulted in vitellogenic arrest within 24 hr. After 96 hr, vitellogenesis was reinitiated, and resorption did not occur. It is interesting that in this organism Act-D treatment results in vitellogenic arrest rather than resorption, whereas in B. orientalis Act-D treatment stimulates resorption. Finally, P. americana, an organism which quickly resorbs its oocytes upon starvation, responds to Act-D by resorbing its vitellogenic oocytes. This suggests that different mechanisms might be involved in these different responses to Act-D. Recovery experiments suggest that B. orientalis females starved for short periods of time (up to 6 days) reinitiate yolk deposition in their arrested vitellogenic oiicytes ; an oiitheca of normal structure is laid 6 days after feeding is reinitiated. This is similar to the response obtained by SAHOTA(1973) in that D. pseudotsugae vitellogenic oijcytes which were arrested by Act-D reinitiated yolk deposition after 96 hr. B. orientalis females starved longer than 6 days require longer periods for oviposition to, resume. It is highly unlikely that basal oijcytes in
1209
this case are capable of resuming yolk deposition. Thus, arrested oiicytes must be eliminated before development of the next batch of oijcytes can occur. Once the bulk of oocyte substances has been resorbed, the remains are ovulated and packaged into shrunken, abnormally small oiitheca, permitting ovulation of the next batch of oiicytes. DJXLOOFand LAGASSE(1970) found residual portions of resorbed oiicytes which were expelled a few days after the completion of resorption. This expulsion might be analogous to the packaged material in shrunken cockroach oijthecae although the beetle eggs are not oviposited in egg cases. Once vitellogenesis has been reinitiated in B. orientalis, normal vitellogenic events occur and the next oviposition period follows on schedule with the oiithecae normally structured. In general, it seems that different insect species evolved different strategies to cope with periods of In one case, the oiicytes are quickly starvation. resorbed when starvation prompts curtailment of yolk deposition. This type of strategy is exemplified in most species studied (ENGELMANN, 1970). On the other hand, the strategy of B. orientalis is to arrest vitellogenesis and to maintain viable oiicytes for the longest period possible without initiating resorption. Arrested oijcytes retain abilities to reengage in yolk deposition for only about 1 to 2 days; however, longer periods of arrest reduce vitellogenic competence and the non-viable basal oocytes are ovulated as penultimate oijcytes become vitellogenic.
Acknowledgements-This research was funded by a University of Kansas Biomedical Sciences Support Grant (RR-07037). The author is grateful to Professor WILLIAM J. BELL for advice during this work and assistance in manuscript preparation. Professors KARL STOCKHAMMER and CHARLES WYTTENBACH kindly reviewed the manuscript.
REFERENCES ANDERSONL. M. (1971) Protein synthesis and uptake by isolated Cecropia oocytes. J. Cell Sri. 8, 735-750. ANDERSONL. M. and TELFER W. H. (1969) A follicle cell contribution to the yolk spheres of moth oiicytes. Tissue &f Cell 1, 633444. BECK F. and LLOYD J. B. (1963) The preparation and teratogenic properties of pure trypan blue and its common contaminates. J. Embryol. exp. Morph. 11, 175-184. BECK F., LLOYD J. B., and GRIFFITHS A. (1967) Lysosomal enzyme inhibition by trypan blue: a theory of teratogenesis. Science, Wash. 157, 1180-1181. BELL W. I. (1969a) Continuous and rhythmic reproductive cycle observed in Petiplaneta .americana. Biol. Bull., Woods Hole, 137, 239-249. BELL W. J. (1969b) Dual r6le of juvenile hormone in the control of yolk formation in Pertplaneta americana. 7. _ Insect Phvsiol. 15., 1279-1290. _. _ “\
,
1210
GARY R. SAMS
BELL W. J. (1971) Starvation-induced oijcyte resorption and yolk protein salvage in Periplaneta americana. J. Insect Physiol. 17, 1099-1111. BELL W. J. and SAMS G. R. (1975) Factors promoting vitellogenic competence and yolk deposition in the cockroach ovary: larval-adult transition. J. Insect Pkysiol. 21, 173-180. BELL W. J. and SAMSG. R. (1974) Factors promoting vitellogenic competence and yolk deposition in the cockroach ovary: the post-ecdysis female. J. Insect Physiol. 20, 2475-2485. BOHM M. (1972) Effects of environment and juvenile hormone on ovaries of the wasp, Polistes metricus. J. Insect Physiol 18, 18751884. CRUIK~HANKW. J. (1971) Follicle cell protein synthesis in moth oocytes. 3. Insect Physiol. 17, 217-232. DELOOF A. and L#GASSEA. (1970) Resorption of the terminal oiicyte in the allatectomized Colorado beetle, Leptinotarsa decemlineata. Proc. Kon. Ned. Akad. (C)3, 284-297.
ENGELMANNF. (1970) Tke Physiology of Insect Reproduction. Pergamon Press, Oxford. HIGHNAM K. C., LUSIS O., and HILL L. (1963) Factors affecting oacyte resorption in the desert locust, Sckistocerca gregaria (Forskal). J. Insect Physiol. 9, 827-837. Lusrs 0. (1963) The histology of development and resorption in the terminal oiicytes of the desert locust, Schistocerca gregaria. Quart. 3. micr. Sci. 104, 57-68. MANS R. J. and NOVELLI G. D. (1961) Measurements of the incorporation of radioactive amino acids into protein by a filter-paper disc method. Arch. Biochem. Biophys. 94, 48-53. PRATT G. E. and DAVEY K. G. (1972) The corpus allatum and oiigenesis in Rhodnius prolixus (St&l)-II. The effects of starvation. g. exp. BioE. 56, 215-221. SAHOTA T. S. (1973) Yolk deposition in Douglas-fir beetle o8cytes: possible role of RNA synthesis in the follicular epithelium. 3. Znsect Physiol. 19,1087-1096.
VITELLOGENIC
Pergamon
Press.
Printed
in Great Britain.
ARREST IN THE COCKROACH,
BLATTA
ORIENTALIS
GARY R. SAMS Department
of Physiology and Cell Biology, University (Received
of Kansas, Lawrence,
14 October
Kansas 66045, U.S.A.
1974)
Abstract-Starvation stimulated vitellogenic arrest occurs in the cockroach Blat& orientalis after 5 days. This is characterized by cessation of yolk uptake and oijcyte growth. After 5 days of starvation, protein and RNA synthesis decrease, but some macromolecular synthesis continues during the entire starvation period. No oocyte resorption occurs for up to 15 days of starvation. In contrast to starvation, injection of actinomycin-D results in resorption within 8 hr. The results suggest that B. orientalis copes with starvation by maintaining arrested oijcytes as an alternative to immediate resorption.
INTRODUCTION REMOVAL of corpora allata or cessation of juvenile hormone (JH) secretion because of starvation results in vitellogenic arrest and oijcyte resorption in many insect species (ENGELMANN, 1970). Vitellogenic arrest is characterized by permanent or temporary curtailment of yolk deposition, whereas resorption entails irreversible breakdown of ovarian follicles. The following study was undertaken to elucidate cellular processes which occur during starvationinduced vitellogenic arrest in the cockroach Blattu orientalis, a species in which oScytes are retained in the arrested state for lengthy periods prior to resorption.
MATERL4LS
AND
hETI-IODS
Starved adult female cockroaches were maintained at 26”C, 60% r.h., and a 12 : 12 hr photoperiod, with access to water. Starvation was initiated on the day of oiitheca formation (BF) and the number of days of starvation was calculated from this point. Control females were maintained under similar conditions but were fed Purina Laboratory Chow ad lib. Starved females were injected with 10 PC 10 Ci/mM, New 3H-uridine (specific activity England Nuclear), 3H-histidine (specific activity 15 Ci/mM, New England Nuclear, or 3H-thymidine (specific activity 6.7 Ci/mM, New England Nuclear), using a micro-syringe. Animals were dissected 24 hr after isotope injection and the oacytes in each ovary measured. Oijcyte volume was calculated from length and width measurements according to the method of BELL (1969a). One ovary from each animal was fixed in Bouin’s for histology or autoradiography as described by BELL and SAMS (1975). The other was prepared for scintillation counting using methods similar to
those of MANS and NOWLLI (1961). Ovaries were homogenated in glass grinders and the homogenate pipetted to glass fibre discs. Discs prepared for 3H-uridine incorporation analysis were dropped into ice-cold 5% perchloric acid, washed four times with cold 5% perchloric acid and then twice each with ethanol and ether, dried, and transferred to scintillation vials. Discs prepared for SH-histidine incorporation analysis were rinsed four times in cold 5% TCA containing 5 mM non-labelled histidine and then twice each with ethanol and ether, dried, and transferred to scintillation vials containing 5 ml of liquid scintillation fluid (4 g/l PPO and 0.05 g/l POPOP). Vitellogenic activity was determined by injecting 10 /*l of sterile 1% trypan blue into female cockroaches at specific times after initiating starvation. The ovaries were dissected out 24 hr later and the extent of trypan blue uptake by the oacytes was observed. Incubations with sterile trypan blue were performed by placing dissected ovaries from females into saline containing sterile 0.1% trypan blue for 30 min. The ovaries were then washed several times with saline, and dye uptake was recorded photographically. To further analyse 3H-uridine incorporation, 1 pg/animal actinomycin-D (act-D) was injected into females beginning to form an oiitheca. At various times after injection, as noted in the text, the ovaries were dissected. One ovary was incubated in saline containing 10 &X/ml 3H-uridine for 2 hr and processed for scintillation counting; the other was incubated in 0.1% trypan blue as described above, then photographed.
RESULTS Vitellogenic
cycle
06cyte
growth
in
the
vitellogenic
cycle
of
1204
GARY
FL
SAMS
*...*** -
. ..* *...a*
SF
. ..*
/
.
.
5
10
.
15
DAYS
Fig. 1. Comparison of oijcytes volumes in fed (A) and starved (B) B. orientalis females. Each point represents mean values for 3 to 7 females. Basal oiicytes, 0; penultimate oiicytes, 0; ultimate oijcytes, 0.
B. orientalis is depicted
in Fig. 1-A. Basal oocytes are ovulated approximately every 5 to 6 days and penultimate oocytes begin growth shortly before the basals are ovulated. Thus the uptake of vitellogenins (female-specific blood proteins) by the oiicytes is continuous in that when the basal is ovulated, the penultimate is already engaged in yolk deposition. As shown in Fig. 1 -A, the largest growth increment occurs in penultimate oocytes 2 to 4 days after the basal oiicytes are ovulated. OGcytes of starved females (Fig. 1-B) continue to increase in volume until the fifth day of starvation (BF + 5). At this time, increase in volume of the basal oijcytes ceases. After BF + 5 no further growth occurs in the basal oiicyte, growth of the penultimate oocytes is not initiated, except in a few isolated instances, and ovulation does not occur. Basal oocytes from animals starved longer than 5 days remain at an intermediate size (0.6-l *5 mm3). They contain yolk but remain in vitellogenic arrest for up to 20 days of starvation.
Trypan blue uptake Trypan blue uptake can be used effectively to detect or quantitatively measure yolk uptake in oijcytes (ANDERSON, 1971; BOHM, 1972) and also provides an index of cellular integrity (BECK and LLOYD, 1963; BECK et al., 1967). Trypan blue
injected into fed females is taken up by vitellogenic oocytes, giving the oijcyte cortex a bluish hue which can be seen under the dissecting microscope. Trypan blue uptake occurs generally over the surface of vitellogenic basal and penultimate oijcytes (Fig. 2) and is especially intense at the poles
Fig. 2. Diagrammatic representation of oiicyte trypan blue uptake during transition from vitellogenesis to arrest. Dark oijcytes (trypan uptake) = vitellogenic; light oijcytes = non-vitellogenic; light oijcytes with thick border = chorionated, non-vitellogenic. a, basal; b, penultimate; c, ultimate oijcytes.
of the penultimate oocyte (see arrows, Fig. 5-A). No dye is taken up by the pre-vitellogenic or chorionated oocytes. As depicted in Fig. 2, trypan blue in starved animals is sequestered by the basal oijcyte during the first 5 days, though at stage BF+ 5 uptake is slight. No trypan blue was observed in either the basal or penultimate oijcytes after 5 days of starvation. These results, along with the changes in oijcyte volume discussed above, suggest that
1205
Fig. 3. Histological preparations of basal oijcytes. A, stained section of a vitellogenic oijcyte, Haemotoxylin stained and counterstained Rith eosin. B to D, incorporation of 3H-histidine in animals starved for different lengths of time: B, 1-5 days; C, S-10 days; D, lo-15 days. E and F, incorporation of 3H-uridine: E, starved 1-5 days; F, starved longer than 5 days. G and H, incorporation of 3H-thymidine: G, starved l-5 days: H, starved longer than 5 days. B, blood; FCE, follicle cell epithelium; OC, oiicyte cortex; YS, yolk sphere. Vertical lines represent boundaries of follicle cell epithelium.
Fig. 3. In vitro uptake of trypan blue following saline or Act-D injection in vow. A, saline injection. B to E, Act-D treatments for various periods: B, 4 hr; C, 4-6 hr (dark field photo) (arrows indicate patches of trypan blue impregnation); D, 6-8 hr; E, 8 hr (chorionated basal oiicyte in E; basal oacytes are not chorionated in A to D). B, basal o&ytes; P, penultimate ob;cytes; l-1, ultimate o&ytes.
Vitellogenic arrest in the cockroach
1207
DAYS OF STARVATION
Fig. 4. Incorporation of JH-histidine into TCA-precipitated material (-), and incorporation of 3H-uridine into PCA-insoluble material (- - - - -), after specific periods of starvation. Each point represents the mean value for single ovaries from 3 to 7 females. vitellogenesis is arrested as a result of starvation and that the oijcytes are retained intact in the ovaries. Protein, RNA,
and DNA
synthesis
To determine if a cessation or decrease in synthesis of follicle cell proteins in starved females is causally related to vitellogenic arrests, the following experiment was performed. Females were injected with 3H-histidine after specific periods of starvation and 24 hr later the ovaries were prepared for autoradiography. A stained section (Fig. 3-A) demonstrates the positions of different tissue components in the autoradiographs. Incorporation of 3H-histidine occurred in the follicle cells and oijcyte during the entire 15day starvation period. However, silver grains over yolk spheres were observed in fed females and starved females through BF+5 stage (Fig. 3-B), and were not extensively associated with yolk spheres in stages after BF + 5 (Fig. 3-C, D). 3H-Uridine incorporation was also observed during the entire starvation period. Though silver grains were found mainly over the follicle cells, grains were also associated with yolk spheres in the oijcyte cortex (Fig. 3-E, F). Whether these grains indicate RNA synthesis by follicle cells and transfer to the oScyte or endogenously synthesized oijcyte RNA is not known. Incorporation of 3H-thymidine was observed (Fig. 3-G, H) during the entire starvation period,
although it could not be determined if there were changes in the number of follicle cell nuclei undergoing mitotic divisions. Fig. 4 shows quantitative measurements of SH-uridine and SH-histidine incorporation by ovaries of starved females. Incorporation of SH-histidine decreased sharply after the BF+ 5 period and levelled off to values just above background. SH-Uridine incorporation peaked at BF + 5 and then decreased sharply until a slight increase occurred at stage BF+lO; the BF+lO peak was followed by a progressive decrease through Thus detectable protein and RNA BF+lS. synthesis occurred up to 10 days after vitellogenesis had ceased. Actinomycin-D
treatment
To determine if inhibition of follicle cell RNA synthesis promotes vitellogenic arrest, Act-D was injected into yolk-depositing females. At various times after injection, vitellogenic activity was measured using the trypan blue index (Fig. 5-B-E) and 3H-uridine incorporation was assessed by scintillation counting (Fig. 6). Figure 5-A demonstrates normal uptake of trypan blue (see discussion in previous section). Within 4 hr of Act-D treatment (Fig. S-B) heavy uptake of trypan blue occurred (Fig. S-B), but, rather than the homogeneous peripheral uptake seen in vitellogenic o8cytes, a stippled pattern of uptake was observed. After 4 to
1208
GARY R. SAMS
6 hr of Act-D treatment large patches of trypan blue impregnation were observed (Fig. S-C, see arrows) and by 6 to 8 hr the oocytes were completely permeable to trypan blue (Fig. S-D), indicating a Noncomplete loss of membrane integrity. vitellogenic oiicytes were unaffected by Act-D as indicated by trypan blue uptake (see previtellogenic oocyte and chorionated oijcyte in Fig. S-E). At the same time a considerable reduction in 3H-uridine incorporation occurred (Fig. 6). Thus, it seems that
appears that Act-D did not alter the yolk precursor supply. Recovery from vitellogenic arrest It was of interest to investigate recovery from starvation-induced vitellogenic arrest when feeding is resumed. Starved females, therefore, were re-fed and the time of oviposition for the next two cycles recorded. The results indicate that the longer a female is starved, the longer recovery takes. Females starved for short periods (5 to 6 days) and then fed, exhibited a delay of 3 days in ovulation of the first The second oviposition occurred on ootheca. schedule. On the other hand, females starved longer than 6 days exhibited a considerable delay (up to 16 days) before ovipositing the first ootheca. Oijthecae of the first ovulation from animals starved for long periods were abnormal in shape and smaller in size. DISCUSSION
Fig. 6. In vitro incorporation of 3H-uridine for 2 hr at specific periods after treatment with 1 fcg Act-D. Each point represents mean values for single ovaries from 2 or 3 females.
inhibition of RNA synthesis results in resorption of vitellogenic oocytes, although as shown in Fig. 4, not in the resorption of non-vitellogenic oiicytes. Perhaps a continuous programme of RNA synthesis is required to maintain yolk deposition and to prevent resorption in vitellogenic oijcytes. Because most Act-D-treated B. orientalis females died after several days, the reversibility of Act-D effects on the ovary could not be determined. A larger and more vigorous species, Perifilaneta americana, which has the same S-day continuous vitellogenic cycle as B. orientalis, was injected with various dosages of Act-D and the effect on vitellogenesis was assessed. Females injected on the day of ootheca formation with less than 5 pg of Act-D exhibited no oijcyte resorption when dissected 5 or 12 days after treatment. Those injected with more than 7 pg died within 6 days, Females administered with 5 pg of Act-D exhibited resorption in basal oiicytes after 5 days; penultimates were vitellogenic. No oijthecae were deposited, and after 12 days substantial yolk was deposited in penultimate oocytes. Basal oiicytes were eventually completely resorbed, subsequent vitellogenic cycles occurred normally, and the females showed no further indication of Act-D effects. Thus, although Act-D causes irreversible resorption of vitellogenic oocytes, the inhibitor does not prevent yolk deposition in previtellogenic oijcytes. Moreover, since vitellogenesis was initiated in penultimate oiicytes, it
The initial effect of starvation on vitellogenesis in B. orientalis is the cessation of yolk deposition which is in contrast to P. americana where oijcytes continue vitellogenesis for up to 10 days of starvation and then resorb (BELL, 1971). That curtailment of growth is linked to vitellogenic arrest is suggested by comparison of oiicyte volume measurements and trypan blue uptake analysis. Concomitant with vitellogenic arrest is a decrease in RNA and protein synthesis, although these processes continue at low levels during the entire starvation period. Whether the decrease in macromolecular synthesis is the cause of vitellogenic arrest is not clear, but it is known that follicle cell protein synthesis is necessary for yolk deposition (ANDERSON and TIXLFER,1969; ANDERSON,1971; CRUIKSHANK, 1971; BELL and SAMS, 1974) and thus reduced follicle cell protein synthesis might result in vitellogenic arrest. Another possible reason for arrest might be that lack of yolk precursors during starvation prevents yolk deposition. HIGHNAMet al. (1963) suggested such a situation in Schistocerca gregaria in which decreased availability of yolk precursors caused resorption. BELL (1971), however, showed that injecting yolk precursors into starved P. americana did not suffice to stimulate yolk deposition in starved females. Likewise BELL (1969b) and PRATT and DAVEY (1972) demonstrated in P. americana and Rhodniusprolixus that yolk precursor supply was not the limiting factor in vitellogenesis. Thus it would seem that other factors are probably involved in the control of vitellogenic arrest. In B. orient&is, lack of yolk precursors can be ruled out as a controlling agent in vitellogenic arrest since trypan blue, which mimics yolk precursors, was not sequestered during the period of starvation-induced vitellogenic
Vitellogenic arrest in the cockroach arrest. This evidence along with that mentioned above suggests that factors other than availability of yolk precursors are probably responsible for vitellogenic arrest. Resorption does not immediately follow vitellogenie arrest in B. orientalis. This is in contrast to P. americana in which oijcyte resorption is virtually completed after 12 days of starvation (BELL, 1971). Instead it seems that B. orientalis retains its oijcytes in a minimal metabolic state. This is suggested by the continued synthesis of RNA and protein in the follicles for up to 15 days of starvation without oocyte resorption. Perhaps the low level of RNA and protein synthesis is a mechanism by which the organism maintains oijcyte integrity whereas, in P. americana, mechanisms have been developed to resorb vitellogenic oijcytes. Instead of arrest, resorption followed Act-D treatment. The initial change is the appearance of a stippled pattern of trypan blue rather than the homogeneous peripheral veneer deposited in the cortex of vitellogenic oiicytes. Since intense permeability to trypan blue is an indication of cell death (BECK and GRIFFITHS, 1967), it can be suggested that the initial loci of trypan blue uptake might be the first sites of breakdown of the membranes in the follicle cells and oocyte. Following the stippled pattern, resorbing oijcytes exhibit larger patches of trypan blue, much like the ‘opaque patches’ which appear during the initiation of oocyte resorption in S. gregaria (LUSIS, 1963). LUSIS described these patches as the first characteristic by which resorbing oiicytes could be identified. In B. orientalis these sites may reflect the initiation of membrane breakdown which increases in area to encompass finally the entire oocyte. SAHOTA (1973) found that Act-D injected into Dendroctonus pseudotsugae resulted in vitellogenic arrest within 24 hr. After 96 hr, vitellogenesis was reinitiated, and resorption did not occur. It is interesting that in this organism Act-D treatment results in vitellogenic arrest rather than resorption, whereas in B. orientalis Act-D treatment stimulates resorption. Finally, P. americana, an organism which quickly resorbs its oocytes upon starvation, responds to Act-D by resorbing its vitellogenic oocytes. This suggests that different mechanisms might be involved in these different responses to Act-D. Recovery experiments suggest that B. orientalis females starved for short periods of time (up to 6 days) reinitiate yolk deposition in their arrested vitellogenic oiicytes ; an oiitheca of normal structure is laid 6 days after feeding is reinitiated. This is similar to the response obtained by SAHOTA(1973) in that D. pseudotsugae vitellogenic oijcytes which were arrested by Act-D reinitiated yolk deposition after 96 hr. B. orientalis females starved longer than 6 days require longer periods for oviposition to, resume. It is highly unlikely that basal oijcytes in
1209
this case are capable of resuming yolk deposition. Thus, arrested oiicytes must be eliminated before development of the next batch of oijcytes can occur. Once the bulk of oocyte substances has been resorbed, the remains are ovulated and packaged into shrunken, abnormally small oiitheca, permitting ovulation of the next batch of oiicytes. DJXLOOFand LAGASSE(1970) found residual portions of resorbed oiicytes which were expelled a few days after the completion of resorption. This expulsion might be analogous to the packaged material in shrunken cockroach oijthecae although the beetle eggs are not oviposited in egg cases. Once vitellogenesis has been reinitiated in B. orientalis, normal vitellogenic events occur and the next oviposition period follows on schedule with the oiithecae normally structured. In general, it seems that different insect species evolved different strategies to cope with periods of In one case, the oiicytes are quickly starvation. resorbed when starvation prompts curtailment of yolk deposition. This type of strategy is exemplified in most species studied (ENGELMANN, 1970). On the other hand, the strategy of B. orientalis is to arrest vitellogenesis and to maintain viable oiicytes for the longest period possible without initiating resorption. Arrested oijcytes retain abilities to reengage in yolk deposition for only about 1 to 2 days; however, longer periods of arrest reduce vitellogenic competence and the non-viable basal oocytes are ovulated as penultimate oijcytes become vitellogenic.
Acknowledgements-This research was funded by a University of Kansas Biomedical Sciences Support Grant (RR-07037). The author is grateful to Professor WILLIAM J. BELL for advice during this work and assistance in manuscript preparation. Professors KARL STOCKHAMMER and CHARLES WYTTENBACH kindly reviewed the manuscript.
REFERENCES ANDERSONL. M. (1971) Protein synthesis and uptake by isolated Cecropia oocytes. J. Cell Sri. 8, 735-750. ANDERSONL. M. and TELFER W. H. (1969) A follicle cell contribution to the yolk spheres of moth oiicytes. Tissue &f Cell 1, 633444. BECK F. and LLOYD J. B. (1963) The preparation and teratogenic properties of pure trypan blue and its common contaminates. J. Embryol. exp. Morph. 11, 175-184. BECK F., LLOYD J. B., and GRIFFITHS A. (1967) Lysosomal enzyme inhibition by trypan blue: a theory of teratogenesis. Science, Wash. 157, 1180-1181. BELL W. I. (1969a) Continuous and rhythmic reproductive cycle observed in Petiplaneta .americana. Biol. Bull., Woods Hole, 137, 239-249. BELL W. J. (1969b) Dual r6le of juvenile hormone in the control of yolk formation in Pertplaneta americana. 7. _ Insect Phvsiol. 15., 1279-1290. _. _ “\
,
1210
GARY R. SAMS
BELL W. J. (1971) Starvation-induced oijcyte resorption and yolk protein salvage in Periplaneta americana. J. Insect Physiol. 17, 1099-1111. BELL W. J. and SAMS G. R. (1975) Factors promoting vitellogenic competence and yolk deposition in the cockroach ovary: larval-adult transition. J. Insect Pkysiol. 21, 173-180. BELL W. J. and SAMSG. R. (1974) Factors promoting vitellogenic competence and yolk deposition in the cockroach ovary: the post-ecdysis female. J. Insect Physiol. 20, 2475-2485. BOHM M. (1972) Effects of environment and juvenile hormone on ovaries of the wasp, Polistes metricus. J. Insect Physiol 18, 18751884. CRUIK~HANKW. J. (1971) Follicle cell protein synthesis in moth oocytes. 3. Insect Physiol. 17, 217-232. DELOOF A. and L#GASSEA. (1970) Resorption of the terminal oiicyte in the allatectomized Colorado beetle, Leptinotarsa decemlineata. Proc. Kon. Ned. Akad. (C)3, 284-297.
ENGELMANNF. (1970) Tke Physiology of Insect Reproduction. Pergamon Press, Oxford. HIGHNAM K. C., LUSIS O., and HILL L. (1963) Factors affecting oacyte resorption in the desert locust, Sckistocerca gregaria (Forskal). J. Insect Physiol. 9, 827-837. Lusrs 0. (1963) The histology of development and resorption in the terminal oiicytes of the desert locust, Schistocerca gregaria. Quart. 3. micr. Sci. 104, 57-68. MANS R. J. and NOVELLI G. D. (1961) Measurements of the incorporation of radioactive amino acids into protein by a filter-paper disc method. Arch. Biochem. Biophys. 94, 48-53. PRATT G. E. and DAVEY K. G. (1972) The corpus allatum and oiigenesis in Rhodnius prolixus (St&l)-II. The effects of starvation. g. exp. BioE. 56, 215-221. SAHOTA T. S. (1973) Yolk deposition in Douglas-fir beetle o8cytes: possible role of RNA synthesis in the follicular epithelium. 3. Znsect Physiol. 19,1087-1096.