Variations in the glycogen content of fat body, ovary, and embryo during the reproductive cycle of Leucophaea maderae

3. InsectPhysiol.,1967, Vol. 13, pp. 587 to 594. PerganwnPressLtd. Printedin Great Britain

VARIATIONS IN THE GLYCOGEN CONTENT OF FAT BODY, OVARY, AND EMBRYO DURING THE REPRODUCTIVE CYCLE OF LEUCOPHAEA MADERAE” A. WAYNE

WIENSI_

and LAWRENCE

Department of Biological Sciences, Northwestern

I. GILBERT

University, Evanston, Illinois

(Reckved 9 September 1966) Abstract-Changes in the fat body glycogen reserves of female Leucophaea maderae during the reproductive cycle follow a biphasic curve during oiigenesis and a large single phase of synthesis and degradation during embryogenesis. Glycogen in the ovary and developing embryos increases slowly except for a short-term rapid build-up in the last half of embryogenesis, which is rapidly depleted just prior to and following parturition. These changes are related to several other physiological events in the reproductive cycle and suggest a basis for further studies of the control of carbohydrate metabolism by hormonal or metabolic mechanisms. INTRODUCTION THE hormonal or metabolic control of carbohydrate metabolism is often reflected in quantitative alterations in glycogen, the major carbohydrate reserve. Marked changes in glycogen content in insects accompany moulting in Periplaneta americana (LIPKEet aA, 1965), flight in Phomzia regina (CLEGG and EVANS, 1961), adult development in HyaZophora cecropia (DOMROESE and GILBERT, 1964) and entry into embryonic diapause in Bombyx mori (CHINO, 1958). The association of temporal events in the reproductive cycle with specific periods of glycogen degradation and synthesis in particular tissues may yield information on the release and site of action of regulating molecules that control carbohydrate metabolism. In addition, they may provide some insight into the role carbohydrates play in oijgenesis and embryogenesis, and suggest approaches to a definitive analysis of these control mechanisms. Therefore, the glycogen content of the fat body, ovary, and developing embryos was analysed during the reproductive cycle of the ovoviviparous roach, Leucophaea maderae, prior to our studies on the possible endocrine control of carbohydrate metabolism in this insect (WIJXNS and GILBERT, 1967). * Supported by grant AM-02818 from NIAMD of the National Institutes of Health. This work was conducted at the Zoologisches Institiit, Universitat, Bern, Switzerland while A. W. W. was an NSF Graduate Fellow and L. I. G. was an NSF Senior Postdoctoral Fellow. We thank Professor Martin Ltischer and his associates for their hospitality and assistance. t Present address: Department of Biology, Bethel College, North Newton, Kansas. 587

588

A. WAYNEWIENSANDLAWRENCE I. GILBERT MATERIALS

AND METHODS

The cockroaches were raised at 26°C and 8.5 per cent r.h. on water and commercial dog biscuits. The adult females used were in the second reproductive cycle which requires about 85 to 90 days from the onset of oijgenesis until parturition. During the cycle, 1 to 6 females and 1 male shared a glass container and were fed ad libitum. The stage of the animals in the reproductive cycle was determined by the number of days from the termination of the first cycle and the length of the terminal oocyte (L~SCPIERand WYSS-HUBER, 1964). Females at appropriate stages of the cycle were sacrificed by freezing on solid carbon dioxide and stored at - 20°C until analysed. The tissues were thawed and dissected under buffered Ringer’s solution. The pooled fat body from a given stage was homogenized and the lipid extracted with chloroform-methanol (2 : 1) (FOLCII et al., 1957). After filtration and drying, the non-lipid residue (designated as lean dry weight-LDW) was weighed, pulverized, and extracted twice with about 1 ml 5% trichloroacetic acid/100 mg residue at 85°C for 20 min. The glycogen in the supernatant solution after centrifugation was precipitated with 2 vol. of 95% ethanol containing a trace of NasSO,. The solution was warmed to 70°C and left overnight at 3°C. The glycogen was resuspended, washed, dehydrated in absolute ethanol and ether, and weighed. Radiotracer procedures were as described in WIENS and GILBERT(1967). RESULTS Fat body One hundred and .eleven animals are included in this analysis, about half of which were sacrificed during the 20 day period of oijgenesis. The data are expressed as the mean weight of glycogen/unit LDW to compensate for weight variations in the experimental animals. The glycogen reserves appear to be mobile in terms of breakdown and rapid resynthesis (Fig. 1). During the period of oogenesis, changes in fat body glycogen take the shape of a biphasic curve. Maxima occur at the beginning of the cycle and at about 12 days thereafter, while minima are at 7 days after the cycle begins and again at about 19 days when oiigenesis is completed. Following ovulation, the fat body, now depleted of glycogen, builds up reserve carbohydrate again, reaching a maximum value about 1 month prior to parturition whereupon it decreases slowly until parturition. The net synthesis of glycogen/fat body, from ovulation to the 50th day of the cycle, is about 25 mg. Glycogen constitutes between O-5 and 10 per cent of the wet wt. and up to 25 per cent of the LDW of the fat body. The wet wt. of the fat body during the reproductive cycle (Table 1) follows a pattern similar to that of the glycogen content, with a maximum at about the twelfth day of oiigenesis. There is a steady increase from ovulation to the middle of embryogenesis and a decline thereafter. By subtracting the weight of glycogen from the LDW, we obtain the weight of the chloroform : methanol and TCAinsoluble fraction that probably includes protein and urates as its major components.

MBTABOLICRELATIONSHIPS

589

IN THE COCKROACH

DAY OF REPRODUCTIVE

CYCLE

FIG. 1. Variations in the glycogen content of the fat body from L. maderaeduring the reproductive cycle. Data are expressed as the mean glycogen content of the number of animals sacrificed at each stage (see Table 1). TABLE

~-CHANGES

IN FAT BODY WRIGHT, LDW, AND GLYCOGEN CONTENT SECONDREPRODIJCTIVRCYCLE OFFRMALE L.maderae

Day of reproductive cycle 1 4 5 6 7 8 1; 12 14 16 19 20 23 25 34 44 52 65 77 79 86 13-week-old castrates

No. of animals 13 10 5 4 4 8 9 3 5 5 4 4 4 3 5 4 2 3 4 5 4 3 5

Wet wt/animal (mg) (mean) 1.53 184 164 12.5 158 155 208 313 248 158 173 78 135 130 156 160 260 250 265 186 170 160 286

LDW/animal (mg) (mean) 37 46 44 36 30 40 38 54 53 33 38 23 32 33 39 44 78 77 83 60 54 47 56

DURING

Glycogen/anirnal (mg) (mean) 6.5 7.5 2.2 2.9 0.9 3.4 4.8 7.9 5.4 2.4 1.8 0.3 1.6 3.9 4.0 7.9 25.8 25.8 23.2 10.6 9.6 8.0 12.5

THE

A. WAYNE WIENS ANDLAWRENCE

590

I. GILBERT

The pattern of weight change of this fraction during the cycle is similar to that of the wet wt. Thus there appear to be a number of components of the fat body that undergo substantial concentration changes at about the same time during the reproductive cycle. The quantity of glycogen in the fat body of females ovariectomized as lastinstar larvae and analysed 13 weeks after the adult moult is about half the maximum amount found in normal females during the cycle. The wet wt. of the fat body, on the other hand, is as great as that from females at any stage in the cycle. Therefore, glycogen accumulation is not the reason for fat-body hypertrophy in castrated females. Ovary and embryos Sixty-nine animals were used in this analysis and represented 17 different days in the cycle. The glycogen content increased to about 1 mg/pair of ovaries during oiigenesis (Fig. 2). The largest increase occurred during the last half of embryo-

DAY OF

REPRODUCTIVE

CYCLE

FIG. 2. Variations in the glycogen content of the ovaries and embryos of L. maderae during the reproductive cycle. O- 0 = ovaries; @- @ = embryos; l = newly hatched larvae.

genesis, when a maximum of 4.6 mg of embryonic glycogen/female was observed at the seventy-fifth to eightieth day of the cycle. Toward the end of embryogenesis the glycogen level dropped sharply. Freshly hatched larvae that were sacrificed prior to tanning of the cuticle and were not fed had a considerably lower glycogen content than the late-stage embryos. Thus, the glycogen sequestered during embryogenesis is almost entirely consumed during the period immediately prior to and after the birth of the young cockroaches. The wet wt. of the two ovaries (Table 2) increased most rapidly during the last half of o6genesis. The wet wt. of the developing embryos increased steadily during embryogenesis, but at a lower rate. The lean dry wt. also rapidly increased during oijgenesis and its pattern is similar to that observed with the wet wt. However, the

591

METABOLIC RELATIONSHIPS IN THE COCKROACH

LDW remained fairly constant or decreased gradually during embryogenesis. Since the total lipid content also decreases between ovulation and parturition (GILBERT, 1967), the difference between the patterns of change of the wet wt. and LDW during this period must reflect water uptake. TABLE ~-CHANGES

IN OVARY AND EGG MASS (EMBRYO) WET WEIGHT, LDW, DURING THE SECOND REPRODUCTIVECYCLE

Day of reproductive cycle 5 8 9 11 12 14 16 19 20 23 34 44 52 65 77 79 86

Freshly hatched larvae

No. of animals 5 8 5 3 5 5 4 3 4 3 4 2 3 4 4 4 3 3*

AND GLYCOGEN

Glycogen/ 2 ovaries or 1 egg mass

Wet wtj animal

LDW] animal

Glycogenf mg LDW

(mg) (mean)

(mg) (mean)

kg) (mean)

(mg) (mean)

44 39 54 173 114 162 258 273 228 280 263 435 480 375 543 585 490 -

7.6 10.2 11.3 43.8 29.6 24.8 66.6 47.6 62.5 86.6 85.2 96.8 65.0 63.8 69.9 88.3 64.5 76.8

18.5 20.9 16.0 28.2 15-6 18.7 14.6 17.5 15.6 22.0 25.2 17.0 12~6 57.5 58.0 52.5 37.0 12.0

0.14 0.21 0.18 1.23 0.46 0.46 0.98 0.83 0.98 I.90 2.15 1.65 0.83 3.68 4.05 4.63 2.39 0.90

* Young of 3 females. When glycogen is expressed in terms of embryonic LDW (Table 2), the relative glycogen content appears constant through the first 55 days of the cycle, whereupon it increases by a factor of 3 to 4 during the next 15 to 20 days. This implies that the increase in glycogen at this time is distinct from the general increase in embryonic mass. The increase in embryonic glycogen during development may have resulted from conversion of other materials in the embryo or from substrates transported from the maternal tissues. To examine the latter suggestion, glucoseU-C?* was injected into the body cavity of pregnant females at day 33 rf:7 of the reproductive cycle. The embryonic glycogen was isolated 0 to 48 hr after injection, and the radioactivity determined. Incorporation of over 300 counts/min per mg of twice-precipitated glycogen was observed in the embryos 48 hr after injection, while 3, 6, and 24 hr after injection, the radioactivity in the glycogen was not significantly higher than at zero-time (27 counts/min per mg). Palmitate-l-Cl4

592

A. WAYNEWIENSAND LAWRENCE I. GILBERT

injected under the same conditions is more rapidly incorporated into embryonic lipid than glucose is into embryonic glycogen (GILBERT, 1967). This suggests that carbohydrate and lipid of maternal origin may enter the developing embryos and that retention of the embryos in the uterus (brood sac) during embryogenesis serves protective and nutritive functions. DISCUSSION The few known physiological events in the reproductive cycle of L. made‘erae suggest only partial explanations for the marked changes in fat body glycogen during this period. The decrease in glycogen between the middle and end of oijgenesis corresponds with the time of most rapid increase in lipid (GILBERT, 1967) and lean dry wt. of the oiicytes. The total oxygen consumption of L. maderae females during the reproductive cycle is also greatest during this period (S~~GESSER, 1960). These observations suggest that fat body glycogen is mobilized at this time to supply energy for the synthesis and transfer of material to the o&ytes. The immediate build-up of fat body glycogen following ovulation supports this supposition. The glycogen that is sequestered in the fat body during the first 30 days of embryogenesis is expended during the terminal phases of embryogenesis and the initial days of oijgenesis in the subsequent cycle. Glycogen that accumulated in the ovary during oijgenesis and in the embryo during the later stages of embryogenesis appears to be rapidly consumed in the few days prior to and immediately after parturition. A similar pattern exists in foetal and neonatal guinea-pig liver (KORNFIELDand BROWN, 1962), where glycogen accumulates rapidly in the final 10 days of foetal life and is metabolized during the first day or so of post-natal life. Hormones of the pituitary and adrenal have been implicated in the control of glycogen accumulation during late foetal development of the rat and rabbit (JOST, 1961). Whether similar mechanisms exist in insect embryos is not known. It is clear, however, that the sites of glycogen storage examined herein have dissimilar concentration patterns during the reproductive cycle. This suggests that the separate sites may be regulated independently by metabolic or hormonal means. The concomitant decline in fat body glycogen with an increase in embryonic glycogen during the fifty-fifth to seventy-fifth day of the cycle suggest carbohydrate transfer from the fat body to the embryo. However, this would only partially account for the decrease in fat body glycogen since the net decrease is more than 6 times the increase in glycogen in embryos of a single female. The quantitative changes in fat body, oiicyte, or embryonic glycogen content are not simply related to patterns of food consumption. ENGELMANNand RAU (1965) have shown that female L. maderae have a peak food consumption (60 mg/day) around the tenth day of the cycle. This then decreases to less than one-sixth the peak level during most of embryogenesis. Thus, the greatest increases in fat body glycogen occur during the period of the reproductive cycle marked by a lessened rate of food consumption. Since the lipid content of the developing embryos decreases rapidly during embryogenesis (by about 50 per cent) whereas that of the fat body decreases only slightly (GILBERT, 1967), it is felt that lipid is-the major substrate for embryogenesis

METABOLIC RELATIONSHIPS IN THECOCKROACH in

593

It is only toward the termination of embryogenesis that embryonic

glycogen is drastically reduced and this may be a result of cuticle synthesis (see LIPKE et al., 1965).

The decrease in fat body glycogen during the last month of

the reproductive cycle is most likely caused by the utilization of this substrate to support the many energy-requiring activities of the adult female as well as the possible transfer of substrate to the embryos. It thus appears that carbohydrates are the main source of energy for the adult femaie, and lipid for the developing embryos during this period. Apparently food consumed during pregnancy that is not oxidized immediately is selectively incorporated into glycogen rather than into lipid reserves.

VAN HANDEL and LEA (1965)

have postulated a mechanism in

female Aedes taeniorhynchus that regulates the quantity of glucose converted to either triglyceride or glycogen. Removal of the medial neurosecretory cells of the brain increased the content of glycogen while decreasing the rate of conversion to triglyceride.

Thus,

the brain appears to restrict the synthesis of glycogen and

facilitate the synthesis of triglycerides from glucose. Since we know that L. maderae can convert glucose to lipid (GILBERT, 1967), their hypothesis allows the prediction that in viva conditions for the preferential storage of glycogen should include a low level of the pertinent hormone or nervous stimulus, either of which would be eliminated by the removal of the neurosecretory cells. Our data from the normal reproductive cycle, demonstrating selective storage of fat body glycogen while the amount of lipid decreased, suggest the possibility of such a mechanism in LeucoIt is clear that the degradation of glycogen is hormonally controlled in some

phaea.

insects (STEELE, 1963; aspects

of glycogen

WIENS and GILBERT, 1965). synthesis may

(HASEGAWA and YAMASHITA, 1965).

also be

Recent studies indicate that

hormonally

regulated in insects

The changes in glycogen content discussed

in the present paper suggest that Leucophaea

may be a valuable experimental

animal for future studies on the endocrine control of carbohydrate synthesis in insects. REFERENCES CHINOH. (1958) Carbohydrate

metabolism in the diapause egg of the silkworm, Bombyx mori-II. Conversion of glycogen into sorbitol and glycerol during diapause. J. Insect Physiol. 2, 1-12. CLEGGJ. S. and EVANS D. R. (1961) The physiology of blood trehalose and its function during flight in the blowfly. r, exp. Biol. 38, 771-792. DOMROESE K. A. and GILBERTL. I. (1964) The role of lipid in adult development and flight-muscle metabolism in Hyalophora cecropia. J. exp. Biol. 41,573-590. ENGELMANN F. and RAU I. (1965) A correlation between feeding and the sexual cycle in Leucophaea maderae (Blattaria). J. lizsect Physiol. 11, 53-64. FOLCHJ., LEESiU., and STANLEYG. Ii. (1957) A simple method for the isolation and purification of total lipids from animal tissue. 3. biol. Chem. 226, 497-509. GILBERTL. I. (1967) Changes in lipid content during the reproductive cycle of Leucophaea maderae and effects of the juvenile hormone on lipid metabolism in vitro. Camp. Biochem. PhysioL To be published. HASEGAWA K. and YAMASHITA 0. (1965) Studies on the mode of action of the diapause hormone in the silkworm, Bombyx mwi L.-VI. The target organ of the diapause homone. J. exp. Viol. 43, 271-277.

594

A. WAYNE WIENS AND LAWRENCEI. GILBERT

JOST A. (1961) The role of fetal hormones in prenatal development. In Harvey Lectures, 1959-1960, pp. 201-226. Academic Press, New York. KORNFIELDR. and BROWND. H. (1962) Th e activity of some enzymes of glycogen metabolism in fetal and neonatal guinea pig liver. r. biol. Chem. 238, 1604-1607. LIPKE H., GRAVESB., and LETO S. (1965) Polysaccharide and glycoprotein formation in into bound carbohydrate. J. biol. the cockroach-II. Incorporation of D-glUCOSd?* Chem. 240, 601-608. LUSCHERM. and WYSS-HUBER M. (1964) Die Adenosin-Nukleotide im Fettkijrper des adulten Weibchens von Leucophaea maderae im Laufe des Sexualzyklus. Rev. suisse zooz. 71, 183-194. S~~GESSER H. (1960) uber die Wirkung der Corpora allata auf den Sauerstoff Verbrauch bei der Schabe Leucophaea maderae. J. Insect Physiol. 5, 264-285. STEELEJ. E. (1963) The site of action of insect hyperglycemic hormone. Gen. compar. Endom. 3,46-52. VAN HANDELE. and LEA A. (1965) Medial neurosecretory cells as regulators of glycogen and triglyceride synthesis. Science, N. Y. 149, 298-300. WIENS A. W. and GILBERTL. I. (1965) Regulation of cockroach fat-body metabolism by the corpus cardiacum in vitro. Science, N. Y. 150,614-616. WIENS A. W. and GILBERT L. I. (1967) Regulation of carbohydrate mobilization and utilization in Leucophaea madwae. J. Insect Physiol. In press