HEFZIBAH EYAL-GILADI, NAOMI FEINSTEINZoology Department, Hebivw University of Jerusalem, Jerusalem, Israel
MICHAEL FRIEDLANDER AND DINA RAVEH*Biology Department, Ben Gurion University of the Segev, Beersheba, Israel
SUMMARY
The intracellular sites of glycogen degradation in the mid to late uterine chick embryo weredetermined by cytochemical localization of glucose-6-phosphatase at the ultrastructural level.Enzyme activity was found between the two membranes of the nuclear envelope, in the annulatelamellae and in specialized glycogen-containing membrane scrolls. Annulate lamellae and glycogenscrolls were most frequent during the stages of intensive glycogen degradation. Annulate lamellaeappear to be formed from the nuclear envelope. During the early post-laying stages annulatelamellae disappeared and were replaced by endoplasmic reticulum that appeared initially inscroll-like formations.
I INTRODUCTION
Glycogen appears to be the primary energy source of early embryonic develop-ment (e.g. see Gusseck & Hedrick, 1972; Vernier & Sire, 1976). In the chick,although the oocyte accumulates large quantities of glycogen during oogenesis(Greenfield, 1966; Paulson & Rosenberg, 1972), this glycogen is utilized completelyby the time the uncleaved egg reaches the magnum. There is, however, a secondaccumulation of glycogen based on yolk degradation, which begins before cleavage,reaching a maximum in the mid-uterine embryo (Eyal-Giladi, Raveh, Feinstein &Friedlander, 1979). This glycogen is synthesized in special cytoplasmic vesicles,which break down in mid-uterine embryos, releasing free clusters of glycogenparticles into the cytoplasm. Almost all this intracellular glycogen is utilized duringthe second half of uterine development, beginning at the time of symmetrization ofthe embryo. Glycogen utilization is first evident at the future posterior side of theblastoderm, from where it spreads anteriorly (Kochav, Ginsburg & Eyal-Giladi,1980). Degradation of glycogen takes place within glycogen scrolls and cytoplasmicvesicles that contain floccular material (FLOVs) (Eyal-Giladi et al. 1979).
Glucose-6-phosphatase (G6Pase) has been shown to be involved in utilization ofembryonic glycogen in the rat (Devos & Hers, 1974), and salmon embryo (Vernier
•Author for correspondence at: Department of Human Genetics, Yale University MedicalSchool, 333 Cedar Street, New Haven, CT 06510, U.S.A.
& Sire, 1976). G6Pase is strongly bound to the microsomal membranes in rathepatocytes (Hers, Berthet, Berthet & De Duve, 1951; Duttera, Byrne & Ganoza,1968). It has been localized in situ to the cisternae of the endoplasmic reticulum(ER), and the nuclear envelope of differentiating rat (Leskes, Siekevitz & Palade,1971a,6) and salmon embryo (Vernier & Sire, 1976) hepatocytes. However,although cells of the uterine chick embryo show very active glycogen metabolismthey do not contain ER. Furthermore, ER is very sparse in the early incubatedembryo before primitive streak formation (Raveh, Friedlander & Eyal-Giladi, 1971;Bellairs, Lorenz & Dunlap, 1978).
In the present study we show that G6Pase activity is located in the nuclearenvelope, the cytoplasmic annulate lamellae and in membranes of specializedscroll-like structures that contain glycogen. These membrane structures becomeparticularly abundant during the stages of intensive glycogen degradation.
MATERIALS AND MKTHODS
Uterine and early laid eggs were collected and processed for light and electron microscopy asdescribed by Eyal-Giladi et al. (1979). The uterine stages of the chick embryo are designated byRoman numerals referred to as E.G & K (Eyal-Giladi & Kochav, 1976). For cytochemicallocalization of G6Pase whole embryos were treated according to the method of Vernier & Sire(1976). Control embryos were treated as described by Yokoyama, Okada, Tokue & Aso (1975).
RKSU I.TS
Intracelhdar localization of glycogen
In mid to late uterine embryos (stages VI—VII E.G & K) glycogen particles werefrequently found enclosed in membranous scroll-like structures suspended by stalksinside FLOVs (Fig. 1; Eyal-Giladi et al. 1979). In addition, glycogen particles werefound in vesicles associated with stacks of annulate lamellae and within FLOVs (Fig.2). Occasionally glycogen particles and floccular material were found within theperinuclear lumen and sometimes a direct continuity between the intramembranouslumen of the nuclear envelope and FLOVs was found.
In the above mid to late uterine embryos (stages VI—VIII E.G & K) the nuclearenvelope, which was previously smooth, became extremely undulated and dilated.Stacks of annulate lamellae were found parallel to it, sometimes showing continuity
Fig. 1. Stage VII E.G & K mid-uterine embryo showing a cross-section through aglycogen scroll suspended by a stalk within a FLOV (/). Rows of glycogen particles areenclosed between two membranes. X20 000.Fig. 2. Stage VII E.G & K embryo as above showing a stack of annulate lamellae (al)between the nucleus and a FLOV (/). Cytoplasmic vesicles containing glycogen are indirect continuity with a FLOV that contains glycogen (g). ne, nuclear envelope.X24000.
Glycogen metabolism in chick embryonic tissue 401
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402 H. Eyal-Giladi and others
with protrusions of the external membrane of the nuclear envelope (Fig. 3). FLOVswere found in direct continuity with the terminal cisternae of stacks of annulatelamellae, from which they appear to arise (Fig. 4A,B).
Intracellular localization of G6Pase activity
Embryos at the above stages incubated with glucose 6-phosphate and lead ions forthe localization of G6Pase activity, showed the enzyme between the two membranesof the nuclear envelope (Figs 5A,B, 6), in glycogen scrolls suspended within FLOVs(Fig. 5A,B) and in stacks of annulate lamellae (Figs 6, 7). Control sections incubatedwith the reaction mixture in the presence of 10mM-NaF did not show any activityover any of these membranes (Fig. 8).
Temporal correlation between abundance of annulate lamellae and glycogendegradation
Stacks of annulate lamellae were encountered frequently in embryos of the abovestages (VI-VIII), during which glycogen was previously shown to be utilized(Eyal-Giladi et al. 1979; Kochav et al. 1980). At the time of laying (stage X E.G &K) the embryos no longer contained glycogen and the annulate lamellae wereexceedingly rare. The latter were seldom found in unincubated and early incubated(up to stage XII E.G & K) embryos and never found in embryos with a fullydeveloped hypoblast or at the primitive streak stage (Raveh et al. 1971).
Inverse relation between annulate lamellae and ER at different developmentalstages
We did not find ER in uterine embryos. After the disappearance of the glycogenduring the late uterine stages, the cells of the stage X E.G. & K embryo of a freshlylaid egg became packed with ribosomes and rough ER appeared for the first time.Characteristically, the early rough ER showed a scroll-like formation (Fig. 9); laterstage XII E.G & K embryos showed loose elements of rough ER, which appear tobe remnants of these scrolls.
DISCUSSION
We have localized G6Pase activity to the membrane structures that are associatedwith glycogen, i.e. the nuclear envelope, annulate lamellae and glycogen scrolls,indicating that these structures are involved in glycogen degradation. This is the firstlocalization of enzymic activity to the annulate lamellae. Further support for the roleof the annulate lamellae in glycogen degradation is indicated by the appearance ofvesicles filled with floccular material, which are in open communication with theannulate lamellae, as well as by the finding that the latter were especially abundant atthe stages of active glycogen degradation during the transformation of a non-polarstage VI E.G & K blastodisc into a stage X blastoderm with a fixed posterior-anterior polarity. These stages have previously been shown to be important in
Glycogen metabolism in chick embryonic tissue 403
Ifeft^V1'-- 4B 01Fig. 3. Stack of annulate lamellae associated with an evagination of the outer membraneof the nuclear envelope (we) showing a continuity at one point with the outer membrane(arrow). X24000.
Fig. 4. A. Stack of annulate lamellae showing formation of FLOVs (arrows) by dilationof the terminal cisternae. B. F L O V S of various sizes (arrows), the smallest being pinchedoff from the annulate lamellae. X20 000.
404 H. Eyal-Giladi and others
5A I - 7
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5B
i
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tFig. 5. A,n. Localization of G6Pase activity to the intramembrane cistern of the nuclearenvelope (lie), to a glycogen scroll (gs), and to the membrane of a FLOV (/). X20000.
Fig. 6. Annulated glycogen scroll sectioned transversely, heavily labelled with leadphosphate deposit indicative of G6Pase activity. X20000.
Fig. 8. Control blastoderm incubated in the reaction mixture in the presence oflOm.M-NaF. The nuclear envelope (ne) and the annulate lamellae are not labelled.X20000. Granular inclusions appear in the mitochondria of both control and treatedembryos but are not demonstrative of G6Pase activity.
Glycogen metabolism in chick embryonic tissue 405
Fig. 9. Longitudinal section of a scroll of ER found in an embryo of a freshly laid egg(stage XII E.G & K), which lacks glycogen particles and contains mitochondria andribosomes between the leaves of the scroll. Compare with earlier scroll shown in Fig. 1.X20000.
nucleolar ontogenesis and onset of embryonic ribosomal synthesis (Raveh,Friedlander & Eyal-Giladi, 1976).
Localization of G6Pase activity to the membranes of the glycogen scrolls found inthe FLOVs confirms our previous conclusions, which were based on morphologicalevidence, that these structures are involved in glycogen degradation (Eyal-Giladi etal. 1979).
The annulate lamellae may present a mechanism for increasing the nuclearmembrane surface and thus increasing some additional activities of this membrane.This could be particularly important in cells that are poor in ER, as it has beensuggested that the nuclear envelope can fulfil the minimal endomembrane functions(Franke, Scheer, Krohne & Jarasch, 1981). Our localization of G6Pase activity tothe annulate lamellae is thus consistent with the morphological evidence on theircontinuity with the nuclear envelope. There is no indication that the pore complexesare involved in glycogen degradation, as we found that glucose 6-phosphatase islocated between the pores in both the annulate lamellae and the nuclear envelope.Moreover, the enzymes that have been localized to the pore complexes seem to beinvolved in transport of macromolecules between the nucleus and the cytoplasm
406 H. Eyal-Giladi and others
(e.g. see Lam & Kasper, 1979; McDonald & Agutter, 1980; Baglia & Maul, 1983).In the mid-uterine chick embryo the nuclear envelope seems to be involved in
formation of annulate lamellae, as shown earlier in other organisms (e.g. seeSteinkamp & Hoefert, 1974; Smith & Berlin, 1977; Thomopoulos & Kastritsis,1983; reviewed by Kessel, 1983). It shows increased blebbing activity during thestages of annulate lamella generation, stacks of lamellae accumulate preferentiallyadjacent to the nuclear envelope and occasionally continuity is found between theouter nuclear membrane and the membrane of the annulate lamellae.
Our cytochemical results indicate a functional continuity between the nuclearenvelope and the annulate lamellae. In addition, gradual replacement of lamellae byER after the formation of functional embryonic nucleoli (Raveh el al. 1976) and thegeneration of the first rough ER as scroll-like formations in the blastoderm of afreshly laid egg suggest a transformation of glycogen scrolls, and possibly alsoannulate lamellae, into ER. Thus our data support the hypothesis of a membranecontinuum with interchangeability of the membranous elements (Porter, 1961;Franke et al. 1981). This idea is also supported by numerous reports of enzymeactivities common to both the nuclear membrane and the ER (Jarasch et al. 1979;reviewed by Franke et al. 1981), and by reports of a direct continuity betweenannulate lamellae and ER (Jasper, 1976; Kessel, 1983).
We thank Mr Gideon Raziel for skilful photographic reproductions.
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(Received 8 May 1984 -Accepted, in revised form, 18 September 1984)
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