submicroscopic localization of glycogen in mouse blastocysts developedin vivo and in blastocysts...

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Acta histochem. 86, 143-150 (1989) VEB Gustav Fischer Verlag Jena Department of Histology and Embryology (Head: Prof. MUDr. M. DVOIl.AK, DrSc.), Medical Faculty, J. E. University, Bmo, Czechoslovakia Submicroscopic localization of glycogen in mouse blastocysts developed in vivo and in blastocysts developed in vitro from two-cell embryos By JITKA SfASTNA and SVATOPLUK CECH With 6 Figures (Received November 23, 1988) Summary Ultrahistochemical method according to THIERY (1967) was used to determine the occurrence and localization of glycogen in blastocysts developed in vivo and in blastocysts developed from 2-cell embryos of the mouse for 62 to 64 h in in vitro culture. The presence of glycogen was found in blastocysts of both experimental groups. Glycogen had a monoparticulate character, i.e. the form of localized above all in the ground cytoplasm of cells. Their size varied from 10 to 30 nm. In the blastocysts developed in the physiological uterine environment the glycogen content was relatively low, trophoblast cells containing regularly a higher amount of glycogen particles than embryoblast cells. In the blastocysts developed in the culture medium in the presence of currently used energy sources the distribution and content of glycogen were clearly graded according to the cell types. Compared with the in vivo-blastocysts, an abnormally high amount of glycogen was observed in the cytoplasm of trophoblast cells, a medium amount in the prospective endoderm cells and the minimum amount in the prospective ectoderm cells. The authors are of the opinion that differences in the accumulation of glycogen and its occurrence in the individual cells are in connection with their position in the blastocyst and with their relation to the surrounding microenvironment. It can be judged from the findings ofglycogen deposits inside autophagic vacuoles and multivesicular bodies as well as inside extracellular located sacs that simultaneously with glycogen accumulation there also proceeds its partial degradation in lysosomal structures of blastocyst cells. 1. Introduction Synthesis and cumulation of glycogen by preimplantation mouse embryos during development in vivo and in vitro culture has been the subject of considerable histochemical, biochemical, and autoradiographic studies. Characteristic of cleaving mouse ova developing in vivo is low glycogen content which is further decreased in the course of blastocyst development (THOMSON and BRINSTER 1966; STERN and BIGGERS 1968; OZIAS and STERN 1973). By contrast, the glycogen content of embryos cultured in vitro in the presence of glucose is maintained at a higher level and increases considerably with the development of the blastocyst (OZIAS and STERN 1973; PIKE and WALES 1982; EDIRISINGHE et al. 1984a, b). The basic ultrahistochemical data about the occurrence and submicroscopic distribution of glycogen particles in preimplantation mouse embryos developed in vivo using THIERY'S (1967) method were published by CECH (1980, 1985). The corresponding studies in embryos developing under in vitro conditions have been missing so far. This fact as well as the results of the above biochemical and autoradiographic observations have stimulated us to verify ultrastructurally the differences in the occurrence and distribution of glycogen between blastocysts obtained under in vivo conditions from the uterine cavity and those developed from 2-cell mouse embryos after 62 to 64 h culture. .

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Page 1: Submicroscopic localization of glycogen in mouse blastocysts developedin vivo and in blastocysts developed in vitro from two-cell embryos

Acta histochem. 86, 143-150 (1989)VEB Gustav Fischer Verlag Jena

Department of Histology and Embryology (Head: Prof. MUDr. M. DVOIl.AK, DrSc.), Medical Faculty,J. E. PuRKYN~ University, Bmo, Czechoslovakia

Submicroscopic localization of glycogen in mouse blastocysts developedin vivo and in blastocysts developed in vitro from two-cell embryos

By JITKA SfASTNA and SVATOPLUK CECH

With 6 Figures

(Received November 23, 1988)

Summary

Ultrahistochemical method according to THIERY (1967) was used to determine the occurrence and localization ofglycogen in blastocysts developed in vivo and in blastocysts developed from 2-cell embryos of the mouse for 62 to 64 h inin vitro culture. The presence of glycogen was found in blastocysts of both experimental groups. Glycogen had amonoparticulate character, i.e. the form of ~-granules, localized above all in the ground cytoplasm of cells. Their sizevaried from 10 to 30 nm.

In the blastocysts developed in the physiological uterine environment the glycogen content was relatively low,trophoblast cells containing regularly a higher amount of glycogen particles than embryoblast cells.

In the blastocysts developed in the culture medium in the presence ofcurrently used energy sources the distributionand content of glycogen were clearly graded according to the cell types. Compared with the in vivo-blastocysts, anabnormally high amount of glycogen was observed in the cytoplasm of trophoblast cells, a medium amount in theprospective endoderm cells and the minimum amount in the prospective ectoderm cells. The authors are of the opinionthat differences in the accumulation of glycogen and its occurrence in the individual cells are in connection with theirposition in the blastocyst and with their relation to the surrounding microenvironment. It can be judged from the findingsofglycogen deposits inside autophagic vacuoles and multivesicular bodies as well as insideextracellular located sacs thatsimultaneously with glycogen accumulation there also proceeds its partial degradation in lysosomal structures ofblastocyst cells.

1. Introduction

Synthesis and cumulation ofglycogen by preimplantation mouse embryos during development invivo and in vitro culture has been the subject of considerable histochemical, biochemical, andautoradiographic studies. Characteristic of cleaving mouse ova developing in vivo is low glycogencontent which is further decreased in the course ofblastocyst development (THOMSON and BRINSTER1966; STERN and BIGGERS 1968; OZIAS and STERN 1973). By contrast, the glycogen content ofembryos cultured in vitro in the presence of glucose is maintained at a higher level and increasesconsiderably with the development ofthe blastocyst (OZIAS and STERN 1973; PIKE and WALES 1982;EDIRISINGHE et al. 1984a, b).

The basic ultrahistochemical data about the occurrence and submicroscopic distribution ofglycogen particles in preimplantation mouse embryos developed in vivo using THIERY'S (1967)method were published by CECH (1980, 1985). The corresponding studies in embryos developingunder in vitro conditions have been missing so far. This fact as well as the results of the abovebiochemical and autoradiographic observations have stimulated us to verify ultrastructurally thedifferences in the occurrence and distribution ofglycogen between blastocysts obtained under in vivoconditions from the uterine cavity and those developed from 2-cell mouse embryos after 62 to 64 hculture. .

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144 J. SfASTNA and S. CECH

2. Material and methods

Blastocysts developed in vivo were obtained by flushing the uterine horns of hybrid mouse females (C57B II10 x CBA) Fl' Altogether 30 blastocysts were processed for electron microscopy immediately after taking them (seebelow).

Two-cell embryos obtained by flushing the oviducts of hybrid female mice (C57B 1110 x CBA) F) 32 to 35 h aftermating with (C57B 1/10 x CBA) F) male mice were cultured in a drop of medium (0.1 ml) covered with purified andsterile liquid paraffin oil in plastic Petri dishes or on watch glasses in a culture box with a 5 % O2+5 % CO2+90 % N2atmosphere at 37°C and 100% humidity. For flushing out and culture WHITTINGHAM'S (1971) medium modified bySfASTNA and TRAVNfK (1984) was used. From the total n = 100 two-cell embryos, 92 reached the stage of expandedblastocyst 62 to 64 h after starting cultivation. Expanded blastocysts were processed for electron microscopy.

The blastocysts of both groups were fixed 90 min with glutaraldehyde solution (300 mmolll) in cacodylate buffer(100 mrnolll). After washing in cacodylate buffer (100 mrnolll), they were postfixed 30 min with OS04 solution(40 mrnolll) in cacodylate buffer (100 mrn01ll). Finally, they were dehydrated and embedded in Durcupan ACM.

Ultrathin sections were cut on an Ultrotome ill LKB ultramicrotome and stained for the demonstration ofglycogenin the mannerprescribed by THI~RY (1967). The sections were not counterstained. The validity ofresults was verified byomission ofthe periodic acid oxidation step and/orby blocking the secondary aldehyde groups with 2 % Na2S03 (Merck)for 15 min.

Ultrathin sections were all examined and photographed in a Testa BS 500 electron microscope.

3. Results

The application ofthe THIERY method has confmned that all examined blastocysts contained onlythe monoparticulate form of glycogen. But the size, the same as the density and distribution ofglycogen granules was diametrally different in blastocysts developed in the natural uterineenvironment and in those obtained by the culture of 2-cell embryos.

3.1. Blastocysts developed in vivo: Glycogen granules are sparsely and more or less regularlyscattered in their cells.

In trophoblast cells, glycogen particles reached the size of about 25 nm. They were distributedabove all in the ground cytoplasm, in isolated cases also in the regions of fibrillar and crystalloidinclusions. Fine aggregations of granules contained autophagic vacuoles and/or multivesicularbodies (Fig. 1).

In embryoblast cells, glycogen granules had the size of no more than 10 to 15 nm, being boundpredominantly only to the ground cytoplasm. In comparison with trophoblast cells, their number wasalways lower. Regularly small deposits of glycogen were found inside autophagic vacuoles. Therewere no significant differences in the occurrence and distribution of glycogen particles between theindividual embryoblast cells (Fig. 2).

Vesicles and sacs with granular material corresponding by the size and electron density ofparticlesto the cytoplasmic glycogen were also observed in the perivitelline space and among embryolast cells.

3.2. Blastocysts developed in vitro: In blastocysts developed under in vitro-conditions the storeof glycogen was, particularly in trophoblast cells, regularly higher than in them developed in vivo(Figs. 3, 4).

Glycogen granules in trophoblast cells had the average size of20 to 30 nm, being often present insuch high amount as to form extensive deposits taking up a considerable part ofthe cytoplasm (Figs. 3,4). As a rule, they were missing near the cell surface. Autophagic vacuoles with glycogen frequentlyreached considerable dimensions, being found regularly in most cells (Fig. 4). In trophoblast cellscontaining an anusually high amount of glycogen, even the vacuolization of ground cytoplasm wasobserved (Figs. 3, 4), and sometimes further signs of degeneration.

Fig. I. Trophoblast cells of a blastocyst developed in vivo. Zona pellucida (ZP), blastocyst cavity (BC). x 37,800.Inset: Autophagic vacuole with glycogen particles (AV). x 32,500.

Fig. 2. Blastocyst developed in vivo - parts of 2 embryoblast cells. Nuclei (N), autophagic vacuoles (A V). x 23,000.

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Submicroscopic localization of glycogen 145

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146 J. SfASTNA and S. tECH

As for embryoblast cells, they contained - unlike trophoblast cells - always an essentially lowernumber of glycogen particles (Figs. 3 to 6). In comparison with blastocysts developed in vivo,embryoblast cells were, however, characterized by certain differences in their glycogen content.

A layer of flat or cubic cells adjacent to the blastocyst cavity (presumptive endoderm cells)differed regularly from the remaining embryoblast cells (presumptive ectoderm cells) by a somewhathigher occurrence of glycogen particles (Figs. 3, 5, 6). Their size varied between 20 and 30 nm.Particles were distributed in the cell cytoplasm more or less regularly, and/or formed clusters ofsmallsize (Figs. 3, 5, 6). A variable content of glycogen was observed in autophagic vacuoles (Figs. 5, 6).

Presumptive ectoderm cells contained relatively few glycogen granules, the particles beingsmaller (15 to 25 nm) and found only in ground cytoplasm. The presence of glycogen in autophagicvacuoles was observed in some cases (Fig. 5).

Besides polysaccharide distributed in the cytoplasm, glycogen particles were also found invesicles and sacs located in intercellular spaces, most frequently in the blastocyst cavity (Fig. 6).Occasionally, solitary glycogen granules were present in both, intercellular spaces and blastocystcavity.

4. Discussion

The method according to THIERY (1967) for the visualization of glycogen and other substances with apolysaccharide component in ultrathin sections is characterized by great reliability and reproducibility of results(ANDERSON 1972; THORNELL et al. 1977). The presence ofglycogen was established both in blastocysts obtained fromuterine horns and in blastocysts developed after 62 to 64 h culture from 2-cell mouse embryos. Glycogen was locatedabove all in the ground cytoplasm ofcells, occurring in the monoparticulate form, ~-granules, whose size varied within arelatively broad range from 10 to 30 nm.

According to some biochemical studies, 2 forms of glycogen are synthesized in the course of the preimplantationdevelopment ofmouse embryos. Early embryos synthesize acid-insoluble glycogen which may act as core or nucleus forthe subsequent synthesis ofacid-soluble glycogen, present above all at the morula and blastocyst stages (PiKE and WALES1982; EDIRISINGHE et al. 1984b). A reflection ofthis fact might be the size heterogeneity ofglycogen particles, observedby the THIERY method, corresponding to different stages of polysaccharide synthesis. In early cleaving embryos, onlygranules of small size were found in the blastomeres (tECH 1980, 1983, 1985; GEUSKENS and ALEXANl,)RE 1984).

Besides glycogen particles in the ground cytoplasm of blastocyst cells developed under both in vivo and in vitroconditions, glycogen deposits were also found inside autophagic vacuoles, and/or multivesicular bodies. The number ofglycogen granules in autophagic vacuoles correlated with overall glycogen content in the individual cells. It is probablethat both in the course of the in vivo (tECH 1983, 1985) and in vitro development a certain part ofglycogen is degraded bylysosomal digestion.

Sacs and vesicles of different sizes with the content of glycogen or material similar to glycogen observed mostfrequently in the blastocyst cavity of blastocysts of both experimental groups originate probably by constriction andsubsequent fall-off of cytoplasmic protrusions with cumulated glycogen. It is possible that in this way glucose orglycogen macromolecules are transported into the blastocoele fluid (tECH 1983, 1985).

Fig. 3. Blastocyst developed in vitro with trophoblast (7), presumtive ectoderm (Ec) and endoderm (En) cells. Noteglycogen fields, vacuoles (V) and a large autophagic vacuole (AV) in the cytoplasm of trophoblast cell. Zona pellucida(ZP), blastocyst cavity (BC). x 14,400.

Fig. 4. Blastocystdeveloped in vitro. Note differences in glycogen content and distribution between trophoblast (7) andpresumptive ectoderm (Ec) cells. Autophagic vacuole (AV), vacuole (V), zona pellucida (ZP). x 14,400.

Fig. 5. Blastocyst developed in vitro - part of the embryoblast consisting from presumptive endoderm (En) andectoderm (Ec) cells. Autophagic vacuoles (AV), blastocyst cavity (BC). x 16,800.

Fig. 6. Blastocyst developed in vitro - presumptive endoderm cell (En) containing a great number of autophagicvacuoles with glycogen (A V). In the blastocyst cavity (BC), a sac with glycogen particles is present (x). Trophoblast cell(7). x 23,200. Inset: A large sac containing glycogen particles in the blastocyst cavity. x 14,000.

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148 J. StASTNA and S. tECH

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Submicroscopic localization of glycogen 149

The application of the THIERY method has unequivocally confirmed significant differences in glycogen contentbetween mouse blastocysts developed in vivo and those in vitro, found earlier by biochemical, histochemical andautoradiographic procedures (OZIAS and STERN 1973; PIKE and WALES 1982; EDIRISINGHE et al. 1984a, b). Inblastocysts obtained by culturing 2-cell mouse embryos in vitro, the amount ofpolysaccharide prevailed many times overits occurrence in blastocysts developed in the physiological uterine environment. The submicroscopic distribution ofglycogen in the both cases was dependent above all on the cell type. Under in vivo-conditions, as well as in vitro,trophoblast cells always contained considerably higher amount of glycogen, compared with embryoblast cells. Thisdifference was particularly conspicuous in blastocysts developed in vitro, whose trophoblast cells regularly accumulatedglycogen in enormous amounts. From among embryoblast cells, presumptive endoderm cells showed always higherstore of glycogen than presumptive ectoderm cells.

Differences in the accumulation of glycogen by the individual cell types seem to be closely connected with theirposition in the blastocyst and theirrelation to the microenvironment. A high content ofglycogen is particularly typical oftrophoblast cells which are formed in the course of the preimplantation development from the outer morula cells(TARKOWSKI and WROBLEWSKA 1967). During the blastulation and compaction of the embryoblast, taking place lateron, primitive endoderm cells get also inlo immediate contact with the external environment - the blastocyst fluid(ENDERS et al. 1978). Glycogen content observed in those cells was evidently higher than in primitive ectoderm cellswhich never have an immediate relation to the external environment. A different occurrence ofglycogen in the individualcell types of the blastocysts studied is considered to be further sign of their morphological, physiological, andbiochemical differentiation which is peculiar for this stage of development [for review see ADAMSON and GARDNER(1979), JOHNSON (1979)].

The reasons ofan abnormally cumulation ofglycogen above all in trophoblast cells ofbiastocysts developed under invitro-conditions are not exactly known so far. Due to the fact that glucose serves as a primary precursor for glycogensynthesis in late preimplantation embryos (OZIAS and STERN 1973; EDIRISINGHE et al. 1984a, b; LEESE and BARTON1984), one of the possible explanations can be seen in its high concentration in the culture medium. In culturing theembryos, the currently used glucose concentrations of 5.56 mmolll (BRINSTER 1965; WHITTINGHAM 1971 ; OZIAS andSTERN 1973) exceed its level in ulerine fluid (1.0 mmol/l) more than 5 times (EDIRISINGHE and WALES 1985).

It is remarkable that a high cumulation of glycogen in mouse morulae and blastocysts also occurs in substantiallylower glucose concentrations (0.28 mmol/l) in the culture medium (PIKE and WALES 1982; EDlRIDlNGHE and WALES1985). Itcan bejudged from that fact that glycogen content in blastocysts developed in vivo is controlled by further factorsrather than by the amount of glucose and other substrates in the uterine secretion (OZIAS and WEITLAUF 1971; SNYDERet al. 1971; OZIAS and STERN 1973; CECH 1984; EDIRISINGHE and WALES 1984, 1985; EDIRISINGHE et al. 1984a, b).

References

ADAMSON, E. D., and GARDNER, R. L., Control of early development. Brit. Med. Bull. 35, 113-119 (1979).ANDERSON, W. G., Methods for electron microscopic localization ofglycogen. In: GLICK, D., and ROSENBAUM, R. M.

(eds.), Techniques of Biochemical and Biophysical Morphology. 1. Wiley-Interscience, New York-London­Sydney-Toronto 1972, pp. 1-23.

BRINSTER, R. L., Studies on the development of mouse embryos in vitro. II. The effect of energy source. J. expo Zool.158, 59-68 (1985).

CECH, S., Differences in the glycogen content of cleaving mammalian ova. Folia morphol. (Prague) 28,373-375(1980).Stored materials in the course of developmenl of the mammalian ovum. Thesis, J. E. Purkyne Univ. Brno 1983[Czech].Stored materials during cleavage of mouse ova. In: DvoHK, M. (ed.), Differentiation of Preimplantation MouseEmhryos. Acla Fac. Med. Univ. Brunensis 90. 128-132 (1985).Histochemical demonstration of glycogen synthase in preimplantation mouse embryos. Scripta med. 57, 3-6(1984).

EDIRISINGHE, W. R., and WALES, R. G., Effect of parenteral administration of oestrogen and progesterone on theglycogen metabolism of mouse morulae-early blastocysts in vivo. J. Reprod. Fert. 72, 67 -73 (1984).- Influence of environmental factors on the metabolism of glucose by preimplantation mouse embryos in vitro.Austral. J. BioI. Sci. 38. 411-420 (1985).- and PIKE, I. L., Studies of the distribution of glycogen between the inner cell mass and trophoblast cells of mouseembryos. J. Reprod. Fert. 71, 533-538 (l984a).- - Degradation of biochemical pools labelled with e4C) glucose during culture of 8-cell and morula-earlyblastocyst-stage mouse embryos in vitro and in vivo. J. Reprod. Fert. 72, 59-65 (l984b).

II Acta histochem., Bd. 86.2

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Authors' address: Doc. MUDr. JITKA SfASTNA, CSc., Department of Histology and Embryology, MedicalFaculty, J. E. Purkyne University, Ti'. Obnincu miru 10, CSSR - 66243 Bmo.