Glycogen Metabolism in the Prelaid Chick Embryo HEFZIBAH EYAL-GILADI,I DINA RAVEH,' NOAMI FEINSTEIN AND MICHAEL FRIEDLANDER ' 'Department of Zoology, The Hebrew Uniuersity of Jerusalem and Department of Biology, Ben-Gurion University of the Negeu, Beer-Sheua, Israel
ABSTRACT
Glycogen metabolism has been studied during the development of the early chick embryo, a t the cytochemical and ultrastructural levels. TWO waves of glycogen synthesis and breakdown have been found. In the first, free clusters of glycogen particles are synthesized a t late oogenesis. These clusters are found later in invaginations of the membrane of vesicles containing a floccular material (FLOV). The glycogen clusters are degraded there during ovulation and the first hours in the oviduct. The second wave of glycogen synthesis begins before cleavage, reaching a maximum a t mid-uterine age. This second wave occurs in another type of vesicle (GLYV), which eventually disintegrates releasing free clusters of glycogen granules. This glycogen is degraded in membranous structures containing a floccular material, as in the first wave of degradation. The degradation ends a t the late uterine stages, and a t the same time numerous ribosomes are formed. This period corresponds to area pellucida formation, which probably depends on the energy liberated during the second wave of glycogen degradation.
Although glycogen appears to be the primary source of energy during early embryonic development (e.g., Gussek and Hedrick, '721, little has been published on glycogen metabolism during oogenesis and early chick embryogenesis (Gipson, '74). In different animal systematic groups, glyconeogenesis occurs mainly during the last stages of oogenesis (Bergami et al., '68; Huebner et al., '75; Ando, '60; Yurowitzky and Milman, '72; Brachet e t al., '70) and stops a t egg maturation while intensive glycogen consumption begins either a t ovulation or a t fertilization (Yurowitzky and Milman, '72). Numerous biochemical, histochemical and ultrastructural studies on glycogen metabolism in both embryonic (e.g., Benzo et al., '75; Sasse, '75) and adult tissues (e.g., Corvaja et al., '71; Takeuchi et al., '75; Davidowitz et al., '75) suggest a causal relationship between glycogen metabolism and the evolution of diverse membranous structures, in which the enzymes involved in this process may be localized. However, no scheme concerning this relationship has gained a general acceptance. The present work is a systematic cytochemical and ultrastructural study of glycogen metabolism in the chick during early embryonic development, from ovulation through the deJ. MORPH. (1979) 161: 23-38
scent of the fertilized egg in the oviduct and uterus until laying. A special emphasis was given to the relationship between membranes and glycogen particles. Subsequent stages through formation of the hypoblast were studied previously (Raveh et al., '71). MATERIALS AND METHODS
Early uncleaved eggs were collected from the coelomic cavity, infundibulum, and isthmus (fig. 1) of dissected hens. Later stages were extracted a t will by pressing the abdomen of the hens. The uterine and post uterine stages are enumerated with Roman numerals, according to Eyal-Giladi and Kochav ('76) (fig. 2). A parallel series of unfertilized eggs from ovulation until laying was collected from virgin hens. For light microscopy, germs were fixed with Sanfelice's fluid (Humason, '671, which caused the glycogen to aggregate into a conspicuous cap a t the lower portion of the cells. This facilitated estimation of the relative amount of glycogen a t the different stages. The germs were embedded in Epon, sectioned into slices 1-2 thick and stained for glycogen with the PAS technique. Control slides were treated with amylase before staining. Additional slides were stained with toluidine blue and
23
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EYAL-GILADI, RAVEH, FEINSTEIN AND FRIEDLANDER
.inf undibulurn
Fig. 1 Scheme of the genital tract of the hen.
these slides were used for the micrographs, as toluidine blue gives t h e same pattern as t h e PAS reaction but better contrast. To avoid distortion of t h e germs during preparation for electron-microscopy, t h e entire yolk with the germ in place, was immersed in 1.5% glutaraldehyde in 0.08M buffer phosphate pH 7.2 for three to four minutes. After hardening, t h e germ was removed from the yolk with the attached vitelline membrane, fixed for two hours in fresh fixative, washed overnight at room temperature in fresh phosphate buffer, post-fixed for one hour at 4" in 2%OsO, in veronal buffer a t pH 7.4, containing 0 . 0 4 ~sucrose, dehydrated and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a n electron microscope. The presence of glycogen was confirmed a t t h e ultrastructural level by t h e method of Thiery ('67). RESULTS
Light microscopy Stages I and I1 stain negatively for glycogen (fig. 3). Stage I11 gives the first positive reaction for glycogen (fig. 4).From stage 111 there is a gradual accumulation of glycogen, which
reaches i t s maximum at stages V and VI (9-11 hours uterine age) (figs. 5, 6). From stage VI, there is a steady reduction of the amount of glycogen, and a t stage VII some of the cells stain negatively for glycogen (fig. 7). From stage XI (unincubated laid eggs) no more glycogen caps are found (fig. 8 ) . At stage XII, t h e latest stage included here, we found no glycogen-rich cells. The gonocytes which stain positively for glycogen, appear only after the hypoblast has been completely formed (unpublished). Electron microscopy Preuterine fertilized eggs (before cleavage) The ovulated egg found in t h e body cavity just before entering t h e oviduct contains three different arrangements of glycogen particles: (A) scattered, unbound compact clusters (fig. 9); (B) short chains dispersed within a relatively small number of vesicles of sharply contrasted unit membrane (GLYV) (fig. 10); (C) compactly packed particles within vesicles or pockets, which protrude into the lumen of larger vesicles containing floccular material (fig. 9). Additional vesicles containing floccular material (FLOW abound in the cytoplasm; however, many lack glycogen pockets (figs. 9, 10). Besides these, numerous yolk platelets, lipid droplets and mitochondria are present in t h e cytoplasm. The general structure of t h e uncleaved egg found in t h e magnum is similar to t h a t of the newly ovulated egg described above, with two main differences: In t h e egg found in the magnum, the amount of free glycogen is smaller, and the pockets protruding into t h e FLOV are frequently devoid of glycogen. Uterine fertilized eggs (cleaving eggs) Cells at stages I-IV (1-5 hours uterine age) contain little free glycogen; most of t h e pockets protruding into t h e FLOVs are empty and are smaller t h a n at t h e previous stages; the free vesicles containing glycogen (GLYV) are more abundant and contain more glycogen than at earlier stages (fig. 11).Cells a t stage V (8-9 hours uterine age) have numerous FLOVs and GLYVs containing abundant particles and chains of glycogen. The membrane of the GLYV is less conspicuous t h a n at earlier stages (fig. 12). At stage VI (10-11 hours uterine age), t h e cytoplasm contains many FLOVs and many clusters of glycogen particles, lacking any delimiting membrane (figs.
I
IA
111
II
26
EYAL-GILADI, RAVEH, FEINSTEIN AND FRIEDLANDER
14-16), and a very elaborated system of cisterns, which sometimes appear connected to the FLOVs. Often a part of a glycogen cluster is found within an invagination of the membrane of a FLOV, forming glycogen pockets protruding into the lumen of the FLOV (fig. 17).Some of the cisterns connected to the FLOVs are either close to the glycogen clusters (fig. 15) or surrounded by the clusters (figs. 16, 17). At stage VII (12-14 hours uterine age), one or more glycogen pockets protrude into the lumen of the FLOV, but remain attached to the cytoplasm by a narrow stalk. Several of these pockets form rudimentary scrolls within the FLOVs. Similar figures are formed by the cytoplasmic cisterns which spiralize within the glycogen aggregates (figs. 18, 19). At stage VIII (15-17 hours uterine age), the cells contain little free glycogen. Most of the glycogen particles are arranged like strings of beads between the membranes of the scrolls (fig. 20). The space between the double membranes of the scrolls contains floccular material similar to t h a t of the FLOV. At stages IX-X (17-20 hours uterine age), the scrolls contain a small amount of glycogen or are even completely devoid of glycogen (fig. 21). Ribosome-like particles are found in the membranes of the scrolls, which may appear surrounded by FLOVs or mitochondria (fig. 22). Many free ribosomes are found in the cytoplasm. Post-layed eggs. At stages XI-XII, the cells contain many ribosomes but no glycogen particles. There are one or two strips of rough endoplasmic reticulum in every section of the cells. The strips form scrolls, varying from tight scrolls to open ones resembling question marks in transverse section (fig. 23). No rough endoplasmic reticulum was found a t the earlier uterine stages. Unfertilized eggs The ultrastructure of the preuterine unfertilized eggs is identical to that of the comparable fertilized eggs. Uterine unfertilized eggs do not cleave and only occasionally form abortive furrows. At stage IV-V (7 hours uterine age), the unfertilized eggs have an ultrastructure similar to that found in comparable fertilized eggs. The cytoplasm is filled with glycogen-containing vesicles (GLYV) and vacuoles with floccular material (FLOW, but is poor in free glycogen particles. After stage V, no further changes were found in unfertilized
eggs, and after normal laying (more than 20 hours in uterus) they still contain many GLYVs and FLOVs with intact membranes (fig. 13). Only occasional glycogen pockets were found within the FLOVs. No scrolls are formed, but the FLOVs and GLYVs enlarge during the second part of the uterine period having a t laying a diameter 3-4 times larger than a t stage IV. DISCUSSION
The scheme of glycogen metabolism during early chick embryogenesis, which stems mainly from our data, is as following. There seem to be two waves of glycogen synthesis and degradation. In the first wave, which occurs in the oocyte before ovulation, a large number of compact clusters of glycogen granules are synthesized (unpublished).During ovulation and the first hours in the oviduct these granules are rapidly degraded within glycogen pockets formed in invaginations of the membrane of large vesicles containing a floccular material (FLOVs). After glycogen has been degraded, numerous empty pockets protruding into the FLOVs are found in the uncleaved eggs taken from the final portion of the oviduct. In the cleaving egg that enters the uterus, almost all the glycogen granules have disappeared and the PAS reaction for glycogen is negative. This wave of glycogen degradation is triggered probably by the ovulation itself, but is independent of fertilization, as the same pattern of glycogen degradation was found in the unfertilized egg. The second wave of glycogen synthesis begins before cleavage, within a different type of vesicle (GLYV). These vesicles are very rare in the freshly ovulated egg, but their number increases gradually after ovulation. The synthesis begins with the formation of short chains of glycogen particles resembling the “rosary chains” of glycogen particles reported by Takeuchi et al. (’75).The synthesis reaches a maximum a t uterine stage V, as indicated by the strong positive PAS reaction. At stage IV the membrane of the GLYVs disintegrates, and the degradation of these granules commences. As in the first wave, the degradation occurs within membranous structures containing a floccular material. At the beginning, glycogen pockets appear within the FLOVs; later, these pockets spiralize. Additional spirals are formed by the cytoplasmic cisterns within the bulk of free glycogen deposits; finally the spirals develop into elaborate
GLYCOGEN METABOLISM IN THE PRELAID CHICKEN
scrolls. The degradation ends a t stages XI-XII, a t which time no more glycogen granules could be found in the scrolls and the PAS reaction was negative. This second wave of glycogen degradation depends on fertilization; as in the unfertilized eggs the membrane of the glycogen containing vesicles (GLYVs) remains intact, no naked glycogen clusters are formed. Consequently, the glycogen granules could not be trapped within the membranous structures involved in their degradation. It is possible that the second wave of glycogen degradation is regulated by specific information produced in the normal diploid nucleus. The membranous structure involved in this process are probably similar to the glycogen-membrane complexes described in normal and pathological adult tissues (Flaks, '68; Grant, '68), for which a glycolytic role has been proposed (Davidowitz, '75). The formation of the area pellucida, the first morphogenetic event in the chick embryo, occurs at stages VI-X, when the second wave of glycogen degradation takes place. This correlation might indicate that the formation of the area pellucida involves enhanced metabolic activity and that the energy for this is supplied by the second wave of glycogen degradation. The onset of nucleolar activity also occurs a t these stages (Raveh et al., '76) and is probably correlated with this morphogenetic event as well. The uterine and early laid stages through formation of the hypoblast are poor in rough ER (Raveh et al., '71). After the scrolls have finished their role in glycogen degradation, they unwind and apparently transform into rough endoplasmic reticulum, as this cellular component now appears for the first time. ACKNOWLEDGMENTS
Thanks are due to Mr. A. Niv for the skillful printing of photographs. This study was supported by a grant of the Israel Commission for basic research. LITERATURE CITED Ando, S. 1960 Physiological study on egg formation of the fish. I. Accumulation of carbohydrates and proteins during oogenesis. Embryologia, 5: 239-246.
27
Benzo, C. A,, L. D. De Gennaro and S. B. Stearns 1975 Glycogen metabolism in t h e developing chick glycogen body: functional significance of the direct oxidative pathway. J. Exp. Zool., 193: 161-166. Bergami, M., T. E. Mansour and E. Scarano 1968 Properties of glycogen phosphorylase before and after fertilization in the sea urchin eggs. Exp. Cell Res., 49: 650-655. Brachet, J., F. Hanocq and P. Van Gansen 1970 A cytochemical and ultrastructural analysis of in uitro maturation in amphibian oocytes. Devel. Biol., 21: 157-195. Corvaja, N., P. C. Megherini and 0. Pompeiano 1971 Ultrastructure of glycogen-membrane complexes in sensory nerve fibres of cat muscle spindles. Z. Zellforsch., 121: 199-217. Davidowitz, J., G. H. Phillips, B. R. Pachter and G. M. Breinin 1975 Cisternal distention in membrane-glycogen complexes of rabbit extraocular muscle. J. Ultrastruct. Res., 51: 307-313. Eyal-Giladi, H., and S. Kochav 1976 From cleavage to primitive streak formation: a complementary normal table and a new look a t t h e first stages of t h e development of the chick. I. General morphology. Devel. Biol., 49: 321-337. Flaks, B. 1968 Formation of membrane-glycogen arrays in r a t hepatoma cells. J. Cell Biol., 36: 410-414. Gipson, I. 1974 Electron microscopy of early cleavage furrows in t h e chick blastodisc. J. Ultrastruct. Res., 49: 331-347. Grant, Ph. R. 1968 Glycogen-membrane complexes within mouse striated muscle cells. J. Cell Biol., 36: 648-653. Gussek, D. J., and J. L. Hedrick 1972 The enzymatic characteristics and t h e control of glycogen phosphorylase during early amphibian development. J. Biol. Chem., 247: 6603-6609. Huebner, E., S. S. Tobe and K. G. Davey 1975 Structural and functional dynamics of oogenesis in Glossina austenz: vitellogenesis with special reference to t h e follicular epithelium. Tissue and Cell, 7: 535-558. Humason, G. L. 1967 Animal Tissue Techniques. W. H. Freeman & Co., San Francisco. Raveh, D., M. Friedlander and H. Eyal-Giladi 1971 Organelle differentiation in the chick blastoderm during hypoblast formation. Wilhelm Roux' Arch., 166: 287-299. 1976 Nucleolar ontogenesis in the uterine chick germ correlated with morphogenetic events. Exp. Cell Res., 100: 195-203. Sasse, D. 1975 Dynamics of liver glycogen. The topochemistry of glycogen synthesis, glycogen content and glycogenolysis under the experimental conditions of glycogen accumulation and depletion. Histochem., 45: 237-254. Takeuchi, T., M. Sasaki, H. Miyayama, M. Ohyumi and H. Miyajima 1975 Intracellular localization and size of glycogen particles in glycogen synthesized under histochemical conditions. J. Histochem. Cytochem., 23: 945-956. Thiery, J. P. 1967 Mise en evidence de plysaccharides sur coupes fines en microscopie electronique. J. Microscop., 6: 987-1018. Yurowitzky, Yu. G., and L. S. Milman 1972 Changes i n enzyme metabolism during oocyte maturation in a teleost, M i s g u m u s fossilis L. Wilhelm Roux' Arch., 171: 48-54.
Abbreviations C, Cisterna F, Cleavage furrow E, Epiblast EP, Empty pocket FL, Floccular material FLOV, Vesicles with floccular matrix GC, Glycogen cap
GP, Glycogen pocket GS, Glycogen scroll GLYV, Glycogen containing vesicle H, Hypoblast L, Lipid droplet RE, Rough endoplasmic reticulum RS, Scroll containing ribosomes Y, Yolk platelet
PLATE 1 EXPLANATION OF FIGURES
Figures 3-8 Evolution of glycogen caps produced by the Sanfelice fixative i n uterine and early incubated eggs. Stage 1-11 (uterine) shows no glycogen caps. x 100. Stage I11 (uterine). Glycogen caps are found in centrally located cells. x 100. Stage IV (uterine). Glycogen caps are present in all the cells.
X
500.
Stage V (uterine). The deepest staining of the glycogen caps occurs a t this stage. x 500. Stage VII (uterine). The glycogen caps are stained less than in the previous stage. x 500. Stage XI1 (post-layed, incubated for 10 hours). Glycogen caps are absent. E, epiblast; H, hypoblast. x 500.
28
GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi, Raveh, Feinstein and Friedlander
PLATE 1
29
PLATE 2 EXPLANATION OF FIGURES
9 J u s t ovulated, uncleaved germ, showing a glycogen-containing vesicle (GLYV), a vesicle with floccular material (FLOW, a glycogen pocket (GP) protruding into a FLOV and loose glycogen particles. x 12,000. 10 Uncleaved germ from the upper part of the oviduct showing a GLYV containing dispersed chains of glycogen particles. x 12,000.
11 Uncleaved germ from the magnum showing increased amounts of glycogen chains in the GLYVs and a FLOV with a n empty pocket. x 12,000.
30
GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi. Raveh, Feinstein and Friedlander
PLATE 2
31
PLATE 3 EXPLANATION OF FIGURES
12 Stage IV (uterine) showing numerous GLYVs filled with glycogen granules. The
GLYV membrane is very thin (cf. fig. 11).
X
12,000.
13 Post-layed, unfertilized egg showing enlarged GLYVs and empty FLOVs. x 6,000.
32
GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi, Raveh, Feinstein and Friedlander
PLATE 3
33
PLATE 4 EXPLANATION OF FIGURES
14 Stage VI (uterine) showing naked clusters of glycogen granules close to distended cisterns (0.x 12,000.
15 Stage VI (uterine) showing FLOVs or enlarged cisterns within the glycogen deposits. x 12,000.
16 Stage VII (uterine) showing a cistern of FLOV containing floccular material which spiralizes close to clusters of glycogen granules. X 12,000.
GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi, Raveh, Feinstein and Friedlander
PLATE 4
35
PLATE 5 EXPLANATION OF FIGURES
17 Stage VI (uterine) showing FLOVs with glycogen pockets and cisterns containing floccular material which appear to be connected with the FLOVs. X 24,000. 18 Stage VI showing a spiral cistern within a cluster of glycogen granules. x 24,000.
19 Stage VII (uterine) showing a cistern forming a scroll within a cluster of glycogen granules which appear close to the membranes. x 24,000. 20 Stage VIII (uterine) showing a well differentiated scroll with the glycogen particles arranged in a single row between the membranes. Golgi-like cisterns are close to t h e scroll. x 24,000.
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GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi, Raveh, Feinstein and Friedlander
PLATE 5
37
GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi. Raveh. Feinsteln and Friedlander
EXPLANATION OF FIGURES
21 Stage VIII (uterine) showing a scroll after degradation of t h e glycogen granules. Ribosomes appear in the scroll and in the surrounding cytoplasm. x 24,000. 22 Stage X (uterine) showing a scroll which continues into rough endoplasmic reticulum (RE). Note t h e absence of FLOV or cisterns containing floccular material connected with the scroll and the presence of ribosomes between the membranes. X 24,000. 23 Stage XI1 (post-layed) showing a step of unwinding of a scroll into RE. x 24,000.
38
PLATE 6
ABSTRACT
Glycogen metabolism has been studied during the development of the early chick embryo, a t the cytochemical and ultrastructural levels. TWO waves of glycogen synthesis and breakdown have been found. In the first, free clusters of glycogen particles are synthesized a t late oogenesis. These clusters are found later in invaginations of the membrane of vesicles containing a floccular material (FLOV). The glycogen clusters are degraded there during ovulation and the first hours in the oviduct. The second wave of glycogen synthesis begins before cleavage, reaching a maximum a t mid-uterine age. This second wave occurs in another type of vesicle (GLYV), which eventually disintegrates releasing free clusters of glycogen granules. This glycogen is degraded in membranous structures containing a floccular material, as in the first wave of degradation. The degradation ends a t the late uterine stages, and a t the same time numerous ribosomes are formed. This period corresponds to area pellucida formation, which probably depends on the energy liberated during the second wave of glycogen degradation.
Although glycogen appears to be the primary source of energy during early embryonic development (e.g., Gussek and Hedrick, '721, little has been published on glycogen metabolism during oogenesis and early chick embryogenesis (Gipson, '74). In different animal systematic groups, glyconeogenesis occurs mainly during the last stages of oogenesis (Bergami et al., '68; Huebner et al., '75; Ando, '60; Yurowitzky and Milman, '72; Brachet e t al., '70) and stops a t egg maturation while intensive glycogen consumption begins either a t ovulation or a t fertilization (Yurowitzky and Milman, '72). Numerous biochemical, histochemical and ultrastructural studies on glycogen metabolism in both embryonic (e.g., Benzo et al., '75; Sasse, '75) and adult tissues (e.g., Corvaja et al., '71; Takeuchi et al., '75; Davidowitz et al., '75) suggest a causal relationship between glycogen metabolism and the evolution of diverse membranous structures, in which the enzymes involved in this process may be localized. However, no scheme concerning this relationship has gained a general acceptance. The present work is a systematic cytochemical and ultrastructural study of glycogen metabolism in the chick during early embryonic development, from ovulation through the deJ. MORPH. (1979) 161: 23-38
scent of the fertilized egg in the oviduct and uterus until laying. A special emphasis was given to the relationship between membranes and glycogen particles. Subsequent stages through formation of the hypoblast were studied previously (Raveh et al., '71). MATERIALS AND METHODS
Early uncleaved eggs were collected from the coelomic cavity, infundibulum, and isthmus (fig. 1) of dissected hens. Later stages were extracted a t will by pressing the abdomen of the hens. The uterine and post uterine stages are enumerated with Roman numerals, according to Eyal-Giladi and Kochav ('76) (fig. 2). A parallel series of unfertilized eggs from ovulation until laying was collected from virgin hens. For light microscopy, germs were fixed with Sanfelice's fluid (Humason, '671, which caused the glycogen to aggregate into a conspicuous cap a t the lower portion of the cells. This facilitated estimation of the relative amount of glycogen a t the different stages. The germs were embedded in Epon, sectioned into slices 1-2 thick and stained for glycogen with the PAS technique. Control slides were treated with amylase before staining. Additional slides were stained with toluidine blue and
23
24
EYAL-GILADI, RAVEH, FEINSTEIN AND FRIEDLANDER
.inf undibulurn
Fig. 1 Scheme of the genital tract of the hen.
these slides were used for the micrographs, as toluidine blue gives t h e same pattern as t h e PAS reaction but better contrast. To avoid distortion of t h e germs during preparation for electron-microscopy, t h e entire yolk with the germ in place, was immersed in 1.5% glutaraldehyde in 0.08M buffer phosphate pH 7.2 for three to four minutes. After hardening, t h e germ was removed from the yolk with the attached vitelline membrane, fixed for two hours in fresh fixative, washed overnight at room temperature in fresh phosphate buffer, post-fixed for one hour at 4" in 2%OsO, in veronal buffer a t pH 7.4, containing 0 . 0 4 ~sucrose, dehydrated and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a n electron microscope. The presence of glycogen was confirmed a t t h e ultrastructural level by t h e method of Thiery ('67). RESULTS
Light microscopy Stages I and I1 stain negatively for glycogen (fig. 3). Stage I11 gives the first positive reaction for glycogen (fig. 4).From stage 111 there is a gradual accumulation of glycogen, which
reaches i t s maximum at stages V and VI (9-11 hours uterine age) (figs. 5, 6). From stage VI, there is a steady reduction of the amount of glycogen, and a t stage VII some of the cells stain negatively for glycogen (fig. 7). From stage XI (unincubated laid eggs) no more glycogen caps are found (fig. 8 ) . At stage XII, t h e latest stage included here, we found no glycogen-rich cells. The gonocytes which stain positively for glycogen, appear only after the hypoblast has been completely formed (unpublished). Electron microscopy Preuterine fertilized eggs (before cleavage) The ovulated egg found in t h e body cavity just before entering t h e oviduct contains three different arrangements of glycogen particles: (A) scattered, unbound compact clusters (fig. 9); (B) short chains dispersed within a relatively small number of vesicles of sharply contrasted unit membrane (GLYV) (fig. 10); (C) compactly packed particles within vesicles or pockets, which protrude into the lumen of larger vesicles containing floccular material (fig. 9). Additional vesicles containing floccular material (FLOW abound in the cytoplasm; however, many lack glycogen pockets (figs. 9, 10). Besides these, numerous yolk platelets, lipid droplets and mitochondria are present in t h e cytoplasm. The general structure of t h e uncleaved egg found in t h e magnum is similar to t h a t of the newly ovulated egg described above, with two main differences: In t h e egg found in the magnum, the amount of free glycogen is smaller, and the pockets protruding into t h e FLOV are frequently devoid of glycogen. Uterine fertilized eggs (cleaving eggs) Cells at stages I-IV (1-5 hours uterine age) contain little free glycogen; most of t h e pockets protruding into t h e FLOVs are empty and are smaller t h a n at t h e previous stages; the free vesicles containing glycogen (GLYV) are more abundant and contain more glycogen than at earlier stages (fig. 11).Cells a t stage V (8-9 hours uterine age) have numerous FLOVs and GLYVs containing abundant particles and chains of glycogen. The membrane of the GLYV is less conspicuous t h a n at earlier stages (fig. 12). At stage VI (10-11 hours uterine age), t h e cytoplasm contains many FLOVs and many clusters of glycogen particles, lacking any delimiting membrane (figs.
I
IA
111
II
26
EYAL-GILADI, RAVEH, FEINSTEIN AND FRIEDLANDER
14-16), and a very elaborated system of cisterns, which sometimes appear connected to the FLOVs. Often a part of a glycogen cluster is found within an invagination of the membrane of a FLOV, forming glycogen pockets protruding into the lumen of the FLOV (fig. 17).Some of the cisterns connected to the FLOVs are either close to the glycogen clusters (fig. 15) or surrounded by the clusters (figs. 16, 17). At stage VII (12-14 hours uterine age), one or more glycogen pockets protrude into the lumen of the FLOV, but remain attached to the cytoplasm by a narrow stalk. Several of these pockets form rudimentary scrolls within the FLOVs. Similar figures are formed by the cytoplasmic cisterns which spiralize within the glycogen aggregates (figs. 18, 19). At stage VIII (15-17 hours uterine age), the cells contain little free glycogen. Most of the glycogen particles are arranged like strings of beads between the membranes of the scrolls (fig. 20). The space between the double membranes of the scrolls contains floccular material similar to t h a t of the FLOV. At stages IX-X (17-20 hours uterine age), the scrolls contain a small amount of glycogen or are even completely devoid of glycogen (fig. 21). Ribosome-like particles are found in the membranes of the scrolls, which may appear surrounded by FLOVs or mitochondria (fig. 22). Many free ribosomes are found in the cytoplasm. Post-layed eggs. At stages XI-XII, the cells contain many ribosomes but no glycogen particles. There are one or two strips of rough endoplasmic reticulum in every section of the cells. The strips form scrolls, varying from tight scrolls to open ones resembling question marks in transverse section (fig. 23). No rough endoplasmic reticulum was found a t the earlier uterine stages. Unfertilized eggs The ultrastructure of the preuterine unfertilized eggs is identical to that of the comparable fertilized eggs. Uterine unfertilized eggs do not cleave and only occasionally form abortive furrows. At stage IV-V (7 hours uterine age), the unfertilized eggs have an ultrastructure similar to that found in comparable fertilized eggs. The cytoplasm is filled with glycogen-containing vesicles (GLYV) and vacuoles with floccular material (FLOW, but is poor in free glycogen particles. After stage V, no further changes were found in unfertilized
eggs, and after normal laying (more than 20 hours in uterus) they still contain many GLYVs and FLOVs with intact membranes (fig. 13). Only occasional glycogen pockets were found within the FLOVs. No scrolls are formed, but the FLOVs and GLYVs enlarge during the second part of the uterine period having a t laying a diameter 3-4 times larger than a t stage IV. DISCUSSION
The scheme of glycogen metabolism during early chick embryogenesis, which stems mainly from our data, is as following. There seem to be two waves of glycogen synthesis and degradation. In the first wave, which occurs in the oocyte before ovulation, a large number of compact clusters of glycogen granules are synthesized (unpublished).During ovulation and the first hours in the oviduct these granules are rapidly degraded within glycogen pockets formed in invaginations of the membrane of large vesicles containing a floccular material (FLOVs). After glycogen has been degraded, numerous empty pockets protruding into the FLOVs are found in the uncleaved eggs taken from the final portion of the oviduct. In the cleaving egg that enters the uterus, almost all the glycogen granules have disappeared and the PAS reaction for glycogen is negative. This wave of glycogen degradation is triggered probably by the ovulation itself, but is independent of fertilization, as the same pattern of glycogen degradation was found in the unfertilized egg. The second wave of glycogen synthesis begins before cleavage, within a different type of vesicle (GLYV). These vesicles are very rare in the freshly ovulated egg, but their number increases gradually after ovulation. The synthesis begins with the formation of short chains of glycogen particles resembling the “rosary chains” of glycogen particles reported by Takeuchi et al. (’75).The synthesis reaches a maximum a t uterine stage V, as indicated by the strong positive PAS reaction. At stage IV the membrane of the GLYVs disintegrates, and the degradation of these granules commences. As in the first wave, the degradation occurs within membranous structures containing a floccular material. At the beginning, glycogen pockets appear within the FLOVs; later, these pockets spiralize. Additional spirals are formed by the cytoplasmic cisterns within the bulk of free glycogen deposits; finally the spirals develop into elaborate
GLYCOGEN METABOLISM IN THE PRELAID CHICKEN
scrolls. The degradation ends a t stages XI-XII, a t which time no more glycogen granules could be found in the scrolls and the PAS reaction was negative. This second wave of glycogen degradation depends on fertilization; as in the unfertilized eggs the membrane of the glycogen containing vesicles (GLYVs) remains intact, no naked glycogen clusters are formed. Consequently, the glycogen granules could not be trapped within the membranous structures involved in their degradation. It is possible that the second wave of glycogen degradation is regulated by specific information produced in the normal diploid nucleus. The membranous structure involved in this process are probably similar to the glycogen-membrane complexes described in normal and pathological adult tissues (Flaks, '68; Grant, '68), for which a glycolytic role has been proposed (Davidowitz, '75). The formation of the area pellucida, the first morphogenetic event in the chick embryo, occurs at stages VI-X, when the second wave of glycogen degradation takes place. This correlation might indicate that the formation of the area pellucida involves enhanced metabolic activity and that the energy for this is supplied by the second wave of glycogen degradation. The onset of nucleolar activity also occurs a t these stages (Raveh et al., '76) and is probably correlated with this morphogenetic event as well. The uterine and early laid stages through formation of the hypoblast are poor in rough ER (Raveh et al., '71). After the scrolls have finished their role in glycogen degradation, they unwind and apparently transform into rough endoplasmic reticulum, as this cellular component now appears for the first time. ACKNOWLEDGMENTS
Thanks are due to Mr. A. Niv for the skillful printing of photographs. This study was supported by a grant of the Israel Commission for basic research. LITERATURE CITED Ando, S. 1960 Physiological study on egg formation of the fish. I. Accumulation of carbohydrates and proteins during oogenesis. Embryologia, 5: 239-246.
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Benzo, C. A,, L. D. De Gennaro and S. B. Stearns 1975 Glycogen metabolism in t h e developing chick glycogen body: functional significance of the direct oxidative pathway. J. Exp. Zool., 193: 161-166. Bergami, M., T. E. Mansour and E. Scarano 1968 Properties of glycogen phosphorylase before and after fertilization in the sea urchin eggs. Exp. Cell Res., 49: 650-655. Brachet, J., F. Hanocq and P. Van Gansen 1970 A cytochemical and ultrastructural analysis of in uitro maturation in amphibian oocytes. Devel. Biol., 21: 157-195. Corvaja, N., P. C. Megherini and 0. Pompeiano 1971 Ultrastructure of glycogen-membrane complexes in sensory nerve fibres of cat muscle spindles. Z. Zellforsch., 121: 199-217. Davidowitz, J., G. H. Phillips, B. R. Pachter and G. M. Breinin 1975 Cisternal distention in membrane-glycogen complexes of rabbit extraocular muscle. J. Ultrastruct. Res., 51: 307-313. Eyal-Giladi, H., and S. Kochav 1976 From cleavage to primitive streak formation: a complementary normal table and a new look a t t h e first stages of t h e development of the chick. I. General morphology. Devel. Biol., 49: 321-337. Flaks, B. 1968 Formation of membrane-glycogen arrays in r a t hepatoma cells. J. Cell Biol., 36: 410-414. Gipson, I. 1974 Electron microscopy of early cleavage furrows in t h e chick blastodisc. J. Ultrastruct. Res., 49: 331-347. Grant, Ph. R. 1968 Glycogen-membrane complexes within mouse striated muscle cells. J. Cell Biol., 36: 648-653. Gussek, D. J., and J. L. Hedrick 1972 The enzymatic characteristics and t h e control of glycogen phosphorylase during early amphibian development. J. Biol. Chem., 247: 6603-6609. Huebner, E., S. S. Tobe and K. G. Davey 1975 Structural and functional dynamics of oogenesis in Glossina austenz: vitellogenesis with special reference to t h e follicular epithelium. Tissue and Cell, 7: 535-558. Humason, G. L. 1967 Animal Tissue Techniques. W. H. Freeman & Co., San Francisco. Raveh, D., M. Friedlander and H. Eyal-Giladi 1971 Organelle differentiation in the chick blastoderm during hypoblast formation. Wilhelm Roux' Arch., 166: 287-299. 1976 Nucleolar ontogenesis in the uterine chick germ correlated with morphogenetic events. Exp. Cell Res., 100: 195-203. Sasse, D. 1975 Dynamics of liver glycogen. The topochemistry of glycogen synthesis, glycogen content and glycogenolysis under the experimental conditions of glycogen accumulation and depletion. Histochem., 45: 237-254. Takeuchi, T., M. Sasaki, H. Miyayama, M. Ohyumi and H. Miyajima 1975 Intracellular localization and size of glycogen particles in glycogen synthesized under histochemical conditions. J. Histochem. Cytochem., 23: 945-956. Thiery, J. P. 1967 Mise en evidence de plysaccharides sur coupes fines en microscopie electronique. J. Microscop., 6: 987-1018. Yurowitzky, Yu. G., and L. S. Milman 1972 Changes i n enzyme metabolism during oocyte maturation in a teleost, M i s g u m u s fossilis L. Wilhelm Roux' Arch., 171: 48-54.
Abbreviations C, Cisterna F, Cleavage furrow E, Epiblast EP, Empty pocket FL, Floccular material FLOV, Vesicles with floccular matrix GC, Glycogen cap
GP, Glycogen pocket GS, Glycogen scroll GLYV, Glycogen containing vesicle H, Hypoblast L, Lipid droplet RE, Rough endoplasmic reticulum RS, Scroll containing ribosomes Y, Yolk platelet
PLATE 1 EXPLANATION OF FIGURES
Figures 3-8 Evolution of glycogen caps produced by the Sanfelice fixative i n uterine and early incubated eggs. Stage 1-11 (uterine) shows no glycogen caps. x 100. Stage I11 (uterine). Glycogen caps are found in centrally located cells. x 100. Stage IV (uterine). Glycogen caps are present in all the cells.
X
500.
Stage V (uterine). The deepest staining of the glycogen caps occurs a t this stage. x 500. Stage VII (uterine). The glycogen caps are stained less than in the previous stage. x 500. Stage XI1 (post-layed, incubated for 10 hours). Glycogen caps are absent. E, epiblast; H, hypoblast. x 500.
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GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi, Raveh, Feinstein and Friedlander
PLATE 1
29
PLATE 2 EXPLANATION OF FIGURES
9 J u s t ovulated, uncleaved germ, showing a glycogen-containing vesicle (GLYV), a vesicle with floccular material (FLOW, a glycogen pocket (GP) protruding into a FLOV and loose glycogen particles. x 12,000. 10 Uncleaved germ from the upper part of the oviduct showing a GLYV containing dispersed chains of glycogen particles. x 12,000.
11 Uncleaved germ from the magnum showing increased amounts of glycogen chains in the GLYVs and a FLOV with a n empty pocket. x 12,000.
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GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi. Raveh, Feinstein and Friedlander
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
12 Stage IV (uterine) showing numerous GLYVs filled with glycogen granules. The
GLYV membrane is very thin (cf. fig. 11).
X
12,000.
13 Post-layed, unfertilized egg showing enlarged GLYVs and empty FLOVs. x 6,000.
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GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi, Raveh, Feinstein and Friedlander
PLATE 3
33
PLATE 4 EXPLANATION OF FIGURES
14 Stage VI (uterine) showing naked clusters of glycogen granules close to distended cisterns (0.x 12,000.
15 Stage VI (uterine) showing FLOVs or enlarged cisterns within the glycogen deposits. x 12,000.
16 Stage VII (uterine) showing a cistern of FLOV containing floccular material which spiralizes close to clusters of glycogen granules. X 12,000.
GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi, Raveh, Feinstein and Friedlander
PLATE 4
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PLATE 5 EXPLANATION OF FIGURES
17 Stage VI (uterine) showing FLOVs with glycogen pockets and cisterns containing floccular material which appear to be connected with the FLOVs. X 24,000. 18 Stage VI showing a spiral cistern within a cluster of glycogen granules. x 24,000.
19 Stage VII (uterine) showing a cistern forming a scroll within a cluster of glycogen granules which appear close to the membranes. x 24,000. 20 Stage VIII (uterine) showing a well differentiated scroll with the glycogen particles arranged in a single row between the membranes. Golgi-like cisterns are close to t h e scroll. x 24,000.
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GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi, Raveh, Feinstein and Friedlander
PLATE 5
37
GLYCOGEN METABOLISM IN THE PRELAID CHICKEN Eyal-Giladi. Raveh. Feinsteln and Friedlander
EXPLANATION OF FIGURES
21 Stage VIII (uterine) showing a scroll after degradation of t h e glycogen granules. Ribosomes appear in the scroll and in the surrounding cytoplasm. x 24,000. 22 Stage X (uterine) showing a scroll which continues into rough endoplasmic reticulum (RE). Note t h e absence of FLOV or cisterns containing floccular material connected with the scroll and the presence of ribosomes between the membranes. X 24,000. 23 Stage XI1 (post-layed) showing a step of unwinding of a scroll into RE. x 24,000.
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PLATE 6
