Microperfusion fixation of embryos for ultrastructural studies

Copyright © 1972 by Academic Press, Inc. All rights of reproduction in any form reserved

176

J. ULTRASTRUCTURE RESEARCH

41, 176-188 (1972)

Microperfusion Fixation of Embryos for Ultrastructural Studies' RuI ABRUNHOSA2, s

Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark Received May 9, 1972 A method for in situ microperfusion of embryos has been developed to overcome the difficulties in preserving embryonic structures for ultrastructural studies. Laparotomy was performed on pregnant mice on days 10, 11, and 12 of gestation, and an opening was dissected through the uterine wall. A micropipette was introduced into the atrium of the embryonic heart and retracted, thus leaving an outflow opening. The tip of the pipette was then placed in the lumen of the ventricle and the embryo was fixed by vascular perfusion. Organ primordia and tissues were observed by light and electron microscopy, and compared with similar structures from embryos fixed by immersion only. The results show that tissues from embryos fixed in situ by vascular microperfusion, in comparison with tissues fixed by immersion fixation, have less distortion of cells, more constant intercellular spaces, more uniform preservation of mitochondria and other cytoplasmic structures, as well as absence of irregular cytoplasmic swellings, or cytoplasmic extrusions. It has been a c o m m o n experience that cells and tissues of developing structures, in embryos, fetuses, and later stages, are more difficult to preserve for ultrastructural studies than those of the adult. Examples of this general problem m a y be f o u n d in m a n y studies. It is specifically pointed out in those on neural tube (11), heart (10), lens (9), branchial pouches (2), n o t o c h o r d (11), somite cells (13), basal membranes (9), kidney (8, 17), thyroid (12), liver (25), cerebral cortex (7), lateral geniculate nucleus (14), and retina (21). Developing tissues are often f o u n d to show shrunken or swollen cells or cell organelles and frequently exhibit irregularities in intercellular relationships. The possibility was considered that this inadequate preservation m a y be due, in part, to the procedure of fixation used in these earlier studies of embryonic material z Supported by Research Grants Nos. 512-265 and 512-727 from the Danish Medical Research Council. The author was the recipient of a research grant from the Calouste Gulbenkian Foundation. 3 Present address: Instituto de Anatomia, Faculdade de Medieima, Porto, Portugal.

MICROPERFUSION FIXATION OF EMBRYOS

177

FIG. 1. Diagram of microperfusion fixation through the embryonic heart. The pipette is first introduced through enveloping membranes and precardiac wall into the atrium (1). It is then immediately retracted, thus leaving an outflow opening. In the second step, the tip of the pipette is forwarded into the lumen of the ventricle (2), and the perfusion is started.

as well as in m o s t of those of later stages, t h a t is, b y simple immersion, since this m e t h o d does n o t ensure a u n i f o r m distribution of the fixative within the tissue. T h e present r e p o r t describes some results o b t a i n e d f r o m the use of a m e t h o d d e v e l o p e d specifically for e m b r y o s a n d which involves in vivo vascular m i c r o p e r f u sion with fixative solutions. A brief a c c o u n t of p a r t of this study has previously been presented (1).

MATERIAL AND METHODS

Animals. Mouse embryos on days 10, 11, or 12 of gestation were studied. The pregnant mouse was anesthetized by intraperitoneal injection of 30 mg/kg body weight sodium pentobarbital. Perfusion method. The animal was then laparotomized and the uterus was exposed. Under a stereomicroscope, an opening which showed the yolk sac was dissected in the uterine wall. In doing this, care was taken not to disturb the circulations of the conceptus. The heart of the embryo was observed and the experiment was continued only if it was beating normally. A micropipette with a tip diameter of 25-50 # was thereafter inserted through the membranes of the conceptus and the embryonic precardiac membrane (thoracic wall) into the atrium of the heart as illustrated in Fig. 1. The micropipette was then immediatelyretracred, thus leaving an opening in the wall of the atrium through which the blood could flow out.

178

ABRUNHOSA

The tip of the pipette was then placed in the lumen of the ventricle and the perfusion of the fixative started. When the initial phase of the perfusion was considered successful (using criteria described under Results) the perfusion was continued for 0.5-2 minutes. The embryo with its enveloping membrane was then dissected free and immersion fixed for about 2 hours in the same fixative as used for perfusion. During the immersion fixation a large opening was made in the membranes of the conceptus. In some experiments, with embryo older than 10.5 days, the outflow opening was made in the ventricle, and the fixative was injected through the right chamber of the atrium. Dissection of organ primordia was carried out at the end of the immersion fixation. Fixation, embedding, microscopy. The fixative solution used routinely for perfusion was 1% or 2 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and containing 2 To PVP (polyvinylpyrrolidone) (5). In some experiments the concentration of glutaraldehyde or buffer was varied or the fixative solution was used without PVP. Postfixation was carried out in 1 To osmium tetroxide in sodium Veronal acetate buffer (pH 7.2) made isotonic with salts (26). After postfixation some specimens were stained en bloc in 0.5 % uranyl acetate in Veronal acetate buffer at p H 5.0 (16). The tissues were dehydrated in alcohol, starting with 70 %, and embedded in Epon (18) or dehydrated in acetone, starting with 70 To and embedded in Vestopal W (24). Sections for light microscopy were cut at 1/~ and stained with toluidine blue (27), or basic fuchsin (3). Ultrathin sections were collected on carbon-coated Formvar films and stained by lead citrate (23). When block staining in uranyl acetate was not used the sections were stained first with uranyl acetate (28) at 60 ° (6) and then with lead citrate. The sections were observed in a Siemens Elmiskop 1A, at an accelerating voltage of 80 kV, and with a 50 ,u molybdenum objective aperture. Control experiments. Tissues fixed only by immersion fixation were used as controls to tissues fixed by perfusion. The embryos were either immersed intact in the fixative or the organs were rapidly dissected free and immersed separately. In some experiments embryos were perfused with the glutaraldehyde fixative containing 1% Alcian blue (4) to indicate the degree of spreading of the fixative through the embryos during the perfusion. Subsequently these embryos were immersed in fixative without Alcian blue.

RESULTS

General observations I m m e d i a t e l y at the beginning of the perfusion the e m b r y o was seen to bleach a n d thereafter it b e c a m e r a p i d l y yellowish. T h e r a p i d i t y of fixation was d e m o n s t r a t e d by perfusion with the A l c i a n b l u e - m a r k e d fixative which showed t h a t all the capillaries h a d been p e n e t r a t e d with fixative within the first few seconds. Light m i c r o s c o p y of thick sections f r o m such e m b r y o s showed blue colored capillary walls t h r o u g h o u t . FIG. 2. Thymus anlage of day 10 embryo fixed by immersion. The intercellular space (IS) is prominent and irregular. Intracellular preservation is poor, and many mitochondria (M) show an empty matrix and apparent absence of inner membranes. The basement membrane (BM) is interrupted. x 12 000. FIG. 3. Thymus anlage of day 10 embryo fixed by perfusion. The cells show a uniform intracellular preservation, x 12 000.

180

ABRUNHOSA

The start of perfusion was also accompanied by the escape of blood through the outflow opening in the atrium. Failure of the embryo to change in color and the absence of blood flow through the atrium indicated that the perfusion was unsuccessful. Perfusion t h r o u g h the right chamber of the atrium gave the best bleaching of the hepatic primordium as well as some parts of the yolk sac. The embryonic vascular bed can withstand the perfusion pressure quite well if this is accomplished anterograde through the ventricle. In this situation the ventricle apparently functions as a pressure reducer system. The use of a micropipette of sufficiently small bore was also important in restricting the flow of fixative, thus preventing excess pressure effects f r o m this source. Perfusions carried out t h r o u g h the right chamber of the atrium in a retrograde direction were more harmful. Thin veins were sometimes disrupted, and this was apparently caused by excess pressure resulting f r o m the perfusion in this direction. However, when attention to this was paid, it was possible to perfuse without disruption of vessels. E m b r y o s fixed by immersion fixation usually showed asymmetries and irregularities of mechanical origin in the shape of the body, whereas following perfusion fixation the shape of the embryo was symmetrical and well preserved. The shape of internal organ primordia was very variable following immersion fixation and signs of traumatic injury during dissection occurred frequently. After perfusion fixation the shape of the organs was well preserved, and they became quite easy to dissect.

Intercellular relationships After immersion fixation, the cellular relationships were quite variable (Figs. 2, 4, 6, 8, and 10). Thus, in the pancreas primordium on day 11 of gestation (Figs. 4 and 6) FIG. 4. Pancreas anlage of day 11 embryo fixed by immersion. Notice a distortion of the epithelium with irregular appearances of the basal cell borders (arrows). The general intracellular preservation is poor and the nuclei irregular in shape, x 10 000. FIG. 5. Pancreatic anlage of day 11 embryo fixed by perfusion. The plasma membranes of different cells are separated by a space of rather constant width. The nuclei are rounded and have smooth contours, and cytoplasmic organelles are well preserved, x 9 000. FIo. 6. Basal border of pancreatic cells of day 11 embryo fixed by immersion. The plasma membranes are wavy and in places disrupted (arrow). The mitochondria (M) and the basement membrane (BM) is poorly preserved, x 30 000. FIG. 7. Basal border of pancreatic cells of day 11 embryo fixed by perfusion. The cell membranes are continuous and smooth. The basement membrane (BM) is rich in stainable material and cytoplasmic structures are well preserved, x 30 000. FIG. 8. Pancreatic cell of llth embryo fixed by immersion. The fixation is poor with "pop corn"shaped mitochondria and an irregular width of the space between the perinuclear membranes. x 30 000. Fro. 9. Pancreatic cells of day 11 embryo fixed by perfusion. Good preservation of cytoplasmic and organelles. Plasma and nuclear membranes are smooth and continuous. The width of the intercellular space is rather constant and the perinuclear space is filled with a moderately electron dense material. Perfusion fixation. × 30 000.

iI

184

ABRUNHOSA

the basement membrane and apposed epithelial cells were frequently infolded, irregular, and separated from the mesenchymal cells by very irregular spaces. The epithelial cells close to the basement membrane were often separated. However, following perfusion fixation (Figs. 3, 5, 7, 9, 11, and 12) epithelial cells showed no infoldings and the intercellular spaces in epithelial tissues had a rather constant width (Figs. 5, 9, and 11). The basement membrane varied considerably with different conditions of fixation. It contained more stainable material after perfusion fixation (Fig. 7) than after immersion fixation (Fig. 6), being in some material even interrupted and in places absent, after the later procedure (Fig. 2).

Ceil shape When immersion fixation was used, cells in different tissues (Figs. 2 and 4) as well as their nuclei (Figs. 2, 4, and 6) very often became distorted and showed irregular outlines. Disruption of plasma membranes was also frequently observed after immersion fixation and in some cases artifactual cytoplasmic connections between neighbouring cells were noted (Fig. 10). After perfusion fixation the shape of the cells was regular and the contours of plasma membranes (Figs. 5 and 11) and of the nuclear membranes (Figs. 5 and 9) were smooth.

Intracellular structures Several differences were noticed between cytoplasmic structures in material fixed by the two different methods. A swollen or "washed out" cytoplasm was a rather constant finding with immersion fixation whereas the cytoplasm had a uniform distribution and density after perfusion fixation (Figs. 3, 5, and 9). Mitochondria were almost always modified when the immersion procedure was used. An irregular "pop corn" shape (Figs. 6 and 8), an empty matrix (Figs. 2 and 4), and the apparent absence of inner membranes (Fig. 2) were the most consistent artifacts observed using this technique. However, after perfusion fixation the shape and structural appearance of the mitochondria was quite normal (Figs. 3, 5, 7, and 9). The endoplasmic reticulum, the Golgi apparatus, and other organelles had a consistent and well-preserved appearance after perfusion fixation (Fig. 12). In contrast, a disrupted and irregular Golgi apparatus was frequently observed after the immersion FIG. ]0. Mantle layer of mesencephalon in neurotube of day 11 embryo fixed by immersion. The cytoplasm has a "washed out" appearance and plasma membranes are broken and wide "intercellular bridges" are artifactually formed (arrows). x 30 000. FIo. 11. Mantle layer of mesencephalon in neurotube of day 11 embryo fixed by perfusion. The preservation of the cytoplasm is homogeneous. The plasma membranes are intact, and the width of intercellular spaces is constant. × 30 000.

lO ~,

186

ABRUNnOSA

procedure. Moreover, intracellular membranes in general showed an irregular wavy shape (Figs. 6 and 8) while they were smooth after perfusion fixation. The perinuclear membranes were also smooth and separated by a constant distance following perfusion fixation and the interspace between their membranes contained a moderately electron dense material (Figs. 3, 5, and 9). After immersion fixation (Figs. 6 and 8) the perinuclear membranes were very irregular and enclosed an irregular space which was devoid of stainable material.

Influence of different fixatives Variations in the composition of the fixative solution caused considerable differences in the appearance of the cells and tissues. Thus, cells in many tissues showed pronounced cytoplasmic swelling and exhibited irregular cytoplasmic protrusions when the buffer concentration of the fixative was decreased despite the maintenance of the same osmotic concentration by the use of higher concentrations of glutaraldehyde. When the buffer osmotic concentration was maintained at about 190 milliosmoles and the glutaraldehyde concentration was increased, the cells showed evidence of shrinkage and exhibited dense cytoplasm. It appeared that the sensitivity of embryonic cells to changes in the fixative concentration decreased as the age of the embryo increased. Fixative solutions containing PVP gave better preservation of perivascular intercellular relationships in that omission of PVP was seen to cause perivascular edema. Furthermore, the relationships between mesenchymal cells surrounding epithelial organ primordia seemed more regular around vessels when PVP was used.

DISCUSSION Ultrastructural studies of embryonic tissues have previously been carried out only after immersion fixation or intraparenchymal injection of fixative. The present observations demonstrate that the ultrastructure of embryonic cells and tissues is preserved better after perfusion fixation. These results extend those already obtained with adult tissues which have been shown to be better preserved using the technique of perfusion rather than immersion (15, 19, 22). The main reason for the improvement after perfusion fixation of embryonic as well as adult tissues appears to be the uniform and rapid penetration of the fixative through the tissue. During immersion fixation, on the other hand, a fixative gradient is created in the tissue which results in variable preservation between the surface and the center of the tissue block in such a way that the cells and their organelles vary in appearance in different parts of the tissue (20).

M I C R O P E R F U S I O N F I X A T I O N OF EMBRYOS

187

FIG. 12. Cell in the distal tip of posterior pancreatic anlage of day 11 embryo fixed by perfusion. The overall preservation is good. Triple-layered membranes are observed in all organelles, and microtubules are present throughout, x 60 000. Perfusion of a mouse embryo may be carried out after the 28 somite stage (day 10 of gestation) at which time the circulation is fully established and the heart can be clearly observed through the yolk sac, amniotic membrane and embryonic precardiae wall. The cardiac puncture can be done without interference with placental or vitellin circulations. An advantage with embryonic tissues is the absence of vasoconstriction of the developing vessels which may hinder successful perfusion in the adult. The fixative used routinely in this study, 1-2 % glutaraldehyde in 0.1 M cacodylate buffer, gave a satisfactory fixation of all tissue studied provided the fixative was adequately perfused. However, the variability in tissue preservation when the composition of the fixative was changed demonstrates that embryonic tissues, like adult tissues (19) are very sensitive to alterations in the fixative. It therefore appears necessary to explore in detail the most suitable fixative for a particular embryonic tissue, and indeed for each developmental stage before a detailed ultrastructural analysis is carried out.

18 8

ABRUNHOSA

It is concluded that perfusion fixation ensures a uniform and rapid distribution of the fixative, which makes it possible to fix embryonic tissue in situ and in vivo and avoids mechanical manipulations of soft and sensitive structures before fixation, This technique should be useful in ultrastructural studies of differentiating cells and tissues during embryogenesis. I wish to express my gratitude to Professor Arvid B. Maunsbach for his kind invitation to work in his department and for his support and criticism during the progress of this work. It is a pleasure to acknowledge the skillful technical assistance of Mrs. Marianne Ellegaard. I wish to express my indebtedness to "Caloust Gulbenkian Foundation". REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

ABRUNHOSA,R., J. Ultrastruct. Res. 38, 188 (1972). -Unpublished observations. ACKERMAN,G. A. and HOSTETLER,J. R., Lab. Invest. 18, 387 (1968). BEHNKE,0. and ZELANDER,T., J. Ultrastruct. Res. 31, 424 (1970). BOHMAN,S.-O. and MAUNSBACH,A. B., J. Ultrastruet. Res. 30, 195 (1970). BRODY,I., J. Ultrastruet. Res. 2, 482 (1959). CALEY,D. W. and MAXWELL,D. S., J. Comp. Neurol. 138, 3l (1970). CLARK, S. L., JR., ar. Biophys. Biochem. Cytol. 3, 349 (1957). COHEN,A. I., Develop. Biol. 3, 297 (1961). DEHAAN, R. L., in DEHAAN, R. L. and URSPRUNO, H. (Eds.), Organogenesis, p. 377. Holt, Rinehart and Winston, New York, 1965. DUNCAN, D., Tex. Rep. Biol. Med. 15, 367 (1957). FELDMAN,J. D., VAZQUEZ,J. J. and KURTZ, S. M., J. Biophys. Bioehem. Cytol. 11, 365 (1961). KALT, M. R. and TANDLER,B., J. Ultrastruct. Res. 36, 633 (1971). KARLSSON,U., J. Ultrastruet. Res. 17, 158 (1967). KARLSSON,U. and SCHULTZ, R., or. Ultrastruet. Res. 12, 160 (1965). KELLENBERGER,E., RYTER, A. and S~C~AUD, J., or. Biophys. Biochem. Cytol. 4, 671 (1958). LARSSON,L. In preparation. LUET, J., J. Biophys. Bioehem. Cytol. 9, 409 (1961). MAUNSBACH,A. B., J. Ultrastruet. Res. 15, 242 (1966). MAUNSBAC~,A. B., MADDEN, S. C. and LATrA, H., or. Ultrastruct, Res. 6, 511 (1962). NmssoN, S. E. G., or. Ultrastruet. Res. 11, 581 (1964). PALAY,S. L., McGEE RUSSELL, S. M., GORDON, S. and GRILLO, M. A., J. Cell Biol. 12, 385 (1962). REYNOLDS,E. S., or. Cell Biol. 17, 208 (1963). RYTER, A. and KELLENBEROER,E., J. Ultrastruet. Res. 2, 200 (1958). SANDSTROM,B., Lab. Invest. 23, 71 (1970). SJ6STRAND, F. S., Electron Microscopy of Cells and Tissues. Vol. 1. Academic Press, New York. 1967. TRUMI', B. F., SMUCKLER,E. A. and BENDITT,E. P., or. Ultrastruet. Res. 5, 343 (1961). WATSON, M. L., J. Biophys. Bioehem. Cytol. 4, 475 (1958).