- Email: [email protected]
Reduction of anionic sites on the rabbit uterine surface during the preimplantation period
Animal Reproduction Science, 7 (1984) 555--562
555
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
REDUCTION OF ANIONIC SITES ON THE RABBIT UTERINE SURFACE DURING THE PREIMPLANTATION PERIOD
D. LESCOAT 1 and Y. CHAMBON
Laboratoire d 'Histologie et d 'Embryologie, U.E.R. M~dicales et Pharmaceutiques, 2 Avenue Professeur Ldon Bernard, 35043 Rennes Cedex (France) To w h o m reprint requests should be addressed (Accepted 26 March 1984)
ABSTRACT Lescoat, D. and Chambon, Y., 1984. Reduction of anionic sites on the rabbit uterine surface during the preimplantation period. Anim. Reprod. Sci., 7: 555--562. Studies have been carried out to analyze distribution of anionic sites on the uterine epithelium of the rabbit, using cationized ferritin as a label. A negatively charged glycocalyx was demonstrated by transmission electron microscopy on the luminal cell surface during estrus and days 5 - - 7 o f pregnancy. There was a general reduction of labeling from estrus and day 5 to 7 of pregnancy. At estrus and on day 5 and 6 of pregnancy, the results were similar on the meso- and antimesometrial sides of uterine horns and at or between egg recovery sites. At day 7, anionic sites were no longer detected antimesometrially facing the eggs. These results suggested that the progressive loss of anionic sites during the preimplantation period was due to the combined actions of uterus and egg and that this loss might play a role in blastocyst antimesometrial implantation.
INTRODUCTION
The first surface encountered by the trophoblastic cells at the initiation phase of blastocyst attachment to the endometrium is the apical surface of the uterine luminal epithelial cells. As most animal cells are covered by a surface coat composed chiefly of glycoproteins (Luft, 1976) and as adhesiveness appears to be a property of glycoproteins of the cell membrane (Moscona, 1971), they would be expected to play a role in the first cellular contacts. Surface charge of the cells has also often been correlated with cell ability to form contact (Weiss, 1965; Oppenheimer, 1978). Since most mammalian cells carry a net negative charge (Weiss, 1969; Weiss and Ziegel, 1971) and because of the importance of negative charge repulsion in regulating cell surface interactions, a negative charge is thought to be important in many aspects of reproduction, particularly in feto-maternal interactions. When negative charge is decreased, cell adhesion is enhanced (Vicker and
0378-4320/84/$03.00
© 1984 Elsevier Science Publishers B.V.
556
Edwards, 1972), and therefore blastocyst adhesion to the uterus would be expected to be facilitated. Therefore, the presence and distribution of cell anionic sites on the endometrial epithelium have been studied in various species; for example, ferret (Enders and Schlafke, 1972), mouse (Enders and Schlafke, 1974), rat (Hewitt et al., 1979; Enders et al., 1980), ewe (Guillomot et al., 1982) and rabbit (Anderson, 1982). In the rabbit, however, no ultrastructural histochemical data are available concerning pregnancy. In the present investigation using transmission electron microscopy, relative anionic site density was compared on rabbit uterine epithelium at estrus and days 5--7 of pregnancy. Possible changes that might play a role in blastocyst implantation were analyzed. MATERIALS AND METHODS
Animals A common rabbit strain was used. Adult females were caged individually in a controlled environment with 14 h light/24 h. They were mated with two fertile males. Times were expressed as days elapsed after coitus, the day of coitus being denoted as zero time. Endometrial tissues of animals on days 5, 6 and 7 of pregnancy (nine animals) and of unmated estrous females (two animals) were obtained from meso- and antimesometrial sides of the horn and from and between sites of egg recovery.
Tissue preparation The presence of negatively charged groups within the surface coat of uterine epithelial cells from rabbits in estrus and during the preimplantation period was demonstrated by the cationized ferritin technique.
Cationized ferritin technique. Prefixation was initiated in situ by perfusion via the abdominal aorta with 1% glutaraldehyde in 0.15 M cacodylate buffer, pH 7.2. Endometrial tissues rinsed with cacodylate buffer were immediately immersed for 30--60 min in the same fixative and they were washed for 1 h in buffer. Tissues were then incubated for 45 min in a cationized ferritin solution (CF; 0.4 mg/ml buffer) for localizing anionic sites (Danon et al., 1972), rinsed three times in buffer (10 min each batch), post-fixed for 1 h in 1% osmium tetroxide in 0.15 M cacodylate buffer, and rinsed in cacodylate buffer. Controls. After glutaraldehyde prefixation and rinse in buffer, control specimens were preincubated for 1 h in trypsin (2 mg/ml buffer), at 37°C, in a moist atmosphere. Then they were rinsed in buffer, incubated in the cationized ferritin solution (CF) and processed as described above.
557
Transmission electron microscopy. All samples were dehydrated in acetone and e m b e d d e d in epon-araldite. Ultrathin sections were examined after brief staining with uranyl acetate and lead citrate in a Jeol JEM 7 A or a Philips 100 CX electron microscope. RESULTS
Estrus At estrus, a thick surface coat lined the apex of the uterine epithelial cells suggesting the presence of a negatively charged glycocalyx. CF b o u n d extensively and uniformly to the luminal surface of all microvillous cells, equally on the meso- and antimesometrial sides of the horn (Fig. 1). Ferritin particles formed a thick layer on the outer surface of the cells. Binding was thicker on the tips of the microviUi, as more clearly observed at a higher resolution (Fig. 2). Ciliated cells appeared n o t to bind CF at all. The effect of preincubation with trypsin on CF binding was marked, leading to a reduction of staining.
Day 5 The pattern of CF labeling was similar to that found at estrus. It was dense and the distribution of anionic sites was relatively regular on the plasma membranes both of the microvilli and cell body. Labeling with CF was heavier on the tips of some microvilli. Binding was similar on the meso- and antimesometrial poles of the uterine horn and also in and between egg recovery sites (Figs. 3 and 4). It should also be pointed o u t that no electron-dense material penetrated the apical junctional complex (Fig. 3). No binding was observed on ciliated cells. After incubation with trypsin before incubation with CF, ferritin binding was greatly reduced or was abolished (Fig. 5).
Day 6 At this time, less binding was observed than on day 5, and ferritin particles formed a regular thinner layer on plasma membranes of microvilli and the cell body, stopping at the apical junctional complex (Figs. 6--8). CF b o u n d uniformly at the tips and sides of the microvilli. No difference in staining was observed on the two sides of the horn or in and between the sites of egg recovery. Ciliated cells appeared n o t to bind CF. After trypsin preincubation, labeling was greatly reduced or was abolished {Fig. 9).
558
Labeling of the endometrial epithelium by cationized ferritin (CF). Estrus. Fig. 1. CF bound extensively and uniformly to the plasma membranes of microvilli and cell body; x 16 000. Fig. 2. Binding was thicker at the tips of the microvilli; x 36 000.
559
Uterine epithelium incubated with cationized ferritin (CF). Day 6 of gestation. Figs. 6--8. Ferritin particles formed a regular, thinner layer than at day 5 on plasma membranes, stopping at the apical junctional complex (j). CF bound similarly on the two sides of the horn, in (Figs. 6 and 8) and between (Fig. 7) egg recovery sites; x 16 000. Fig. 9. Control tissue preincubated with trypsin. No electron dense material was observed on the surface; x 16 000.
Incubation of uterine epithelium in cationized ferritin (CF). Day 5 of gestation. Figs. 3--4. CF labeling was dense and regular. It was similar on the meso- (Fig. 3) and antimesometrial (Fig. 4) poles of uterus, in (Fig. 3) and between (Fig. 4) egg recovery sites. No electron dense material penetrated the apical junctional complex (j); x 16 000. Fig. 5. Control tissue preincubated with trypsin. Ferritin binding was abolished; x 20 000.
560
Day 7 On day 7 post coitus, labeling had thinned out considerably. In uterine regions occupied by the eggs, no labeling was observed on the antimesometrial side of the horn (Fig. 10), and no or heavily reduced labeling on
C a t i o n i z e d f e r r i t i n (CF) labeling o f t h e u t e r i n e e p i t h e l i u m . D a y 7 o f gestation. Fig. 10. N o f e r r i t i n b i n d i n g was o b s e r v e d o n t h e a n t i m e s o m e t r i a l side o f t h e h o r n in egg site r e c o v e r y ; X 16 000. Figs. 11--12. A heavily r e d u c e d labeling was p r e s e n t o n t h e surface in t h e m e s o m e t r i a l side o f t h e h o r n in f r o n t o f a n egg (Fig. 11) a n d a very little C F b i n d i n g was still o b s e r v e d o n t h e a n t i m e s o m e t r i a l side w i t h o u t egg (Fig. 12); x 16 000. Fig. 13. C o n t r o l tissue p r e i n c u b a t e d w i t h t r y p s i n . M e s o m e t r i a l side o f t h e h o r n , facing a n egg. No r e a c t i o n p r o d u c t was p r e s e n t ; x 16 000.
561
the mesometrial side (Fig. 11). In uterine regions without eggs, there was still a very little CF binding (Fig. 12). Ciliated cells never bound particles of CF. After trypsin treatment, binding of CF was completely abolished (Fig. 13). DISCUSSION
Our results clearly demonstrate the presence of a surface coat on uterine epithelial cells of rabbits. Anionic sites detected by the cationized ferritin technique progressively disappear from the endometrial epithelium during the preimplantation period. Effectively, at estrus and at day 5 of pregnancy, the surface coat is very thick, at day 6 it is thinner, and at day 7 it has thinned out considerably and is almost gone, especially antimesometrially, facing the eggs. The reduction or abolition of CF binding brought about by treatment with trypsin, a proteolytic enzyme, implies that the carbohydrates bearing the negative groups are bound to proteins. Unmasking of deeper anionic sites by trypsin could explain the fact that binding is sometimes only reduced rather than totally abolished after the proteolytic treatment (Guillomot et al., 1982). Similarly, there is a general reduction of anionic sites from days 2--6 of pregnancy or of pseudopregnancy in the rat uterus (Hewitt et al., 1979). In rabbits, there is also a progressive reduction of anionic sites during early pseudopregnancy (Anderson, 1982), CF binding appearing mostly on the tips of microvilli at day 4 and disappearing at day 6. Thus, the decrease in anionic sites from day 4 to day 6 does not occur in the same way in pregnant and pseudopregnant rabbits, as it does in pregnant and pseudopregnant rats. Moreover, in the rabbit, the loss of anionic sites is more important in uterine regions localized near the eggs at day 7. The loss of anionic sites from pregnant rabbit uterine epithelium is not a general uterine response, indicating that the effect is not solely the result of uterine activity but is due to the combined actions of uterus and egg. The disappearance of negatively charged residues at the antimesometrial pole of the uterus facing the eggs at day 7, coinciding with the beginning of antimesometrial blastocyst attachment to the uterine tissues, probably makes uterine cells attractive to the trophoblastic cells and may facilitate implantation. ACKNOWLEDGEMENTS
The authors thank M.T. Lavault and D. Quernee for their technical assistance, S. Lorre for typing the manuscript and R. Primault for photographic assistance.
562 REFERENCES
Anderson, T.L., 1982. Changes in the uterine luminal cell surface during early pseudopregnancy in rabbits. Anat. Rec., 202: 7A. Danon, D., Goldstein, L., Marikovsky, Y. and Skutelsky, E., 1972. Use of cationized ferritin as a label of negative charges on cell surfaces. J. Ultrastruct. Res., 38: 500-510. Enders, A.C. and Schlafke, S., 1972. Implantation in the ferret: epithelial penetration. Am. J. Anat., 133: 291--316. Enders, A.C. and Schlafke, S., 1974. Surface coats of the mouse blastocyst and uterus during the preimplantation period. Anat. Rec., 180: 31--46. Enders, A.C., Schlafke, S. and Welsh, A.O., 1980. Trophoblastic and uterine luminal epithelial surfaces at the time o f blastocyst adhesion in the rat. Am. J. Anat., 159: 59--72. Guillomot, M., Fl~chon, J.E. and Wintenberger-Torres, S., 1982. Cytochemical studies of uterine and trophoblastic surface coats during blastocyst attachment in the ewe. J. Reprod. Fertil., 65: 1--8. Hewitt, K., Beer, A.E. and Grinnell, F., 1979. Disappearance of anionic sites from the surface of the rat endometrial epithelium at the time of blastocyst implantation. Biol. Reprod., 2 1 : 691--707. Luft, J.H., 1976. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat. Rec., 171: 347--368. Moscona, A.A., 1971. Embryonic and neoplastic cell surfaces: availability of receptors for concanavalin A and wheat germ agglutinin. Science (N.Y.), 171 : 905--907. Oppenheimer, S.B., 1978. Cell surface carbohydrates in adhesion and migration. Am. Zool., 18: 13--23. Vicker, M.G. and Edwards, J.G., 1972. The effect of neuraminidase on the aggregation of BHK21 cells and BHK21 cells transformed by polyoma virus. J. Cell. Sci., 10: 759--768. Weiss, L., 1965. Studies on cell deformability. I. Effect of surface charge. J. Cell. Biol., 26: 735--739. Weiss, L., 1969. The cell periphery. Int. Rev. Cytol., 26: 63--105. Weiss, L. and Zeigel, R., 1971. Cell surface negativity and the binding of positively charged particles. J. Cell Physiol., 77: 179--186.
555
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
REDUCTION OF ANIONIC SITES ON THE RABBIT UTERINE SURFACE DURING THE PREIMPLANTATION PERIOD
D. LESCOAT 1 and Y. CHAMBON
Laboratoire d 'Histologie et d 'Embryologie, U.E.R. M~dicales et Pharmaceutiques, 2 Avenue Professeur Ldon Bernard, 35043 Rennes Cedex (France) To w h o m reprint requests should be addressed (Accepted 26 March 1984)
ABSTRACT Lescoat, D. and Chambon, Y., 1984. Reduction of anionic sites on the rabbit uterine surface during the preimplantation period. Anim. Reprod. Sci., 7: 555--562. Studies have been carried out to analyze distribution of anionic sites on the uterine epithelium of the rabbit, using cationized ferritin as a label. A negatively charged glycocalyx was demonstrated by transmission electron microscopy on the luminal cell surface during estrus and days 5 - - 7 o f pregnancy. There was a general reduction of labeling from estrus and day 5 to 7 of pregnancy. At estrus and on day 5 and 6 of pregnancy, the results were similar on the meso- and antimesometrial sides of uterine horns and at or between egg recovery sites. At day 7, anionic sites were no longer detected antimesometrially facing the eggs. These results suggested that the progressive loss of anionic sites during the preimplantation period was due to the combined actions of uterus and egg and that this loss might play a role in blastocyst antimesometrial implantation.
INTRODUCTION
The first surface encountered by the trophoblastic cells at the initiation phase of blastocyst attachment to the endometrium is the apical surface of the uterine luminal epithelial cells. As most animal cells are covered by a surface coat composed chiefly of glycoproteins (Luft, 1976) and as adhesiveness appears to be a property of glycoproteins of the cell membrane (Moscona, 1971), they would be expected to play a role in the first cellular contacts. Surface charge of the cells has also often been correlated with cell ability to form contact (Weiss, 1965; Oppenheimer, 1978). Since most mammalian cells carry a net negative charge (Weiss, 1969; Weiss and Ziegel, 1971) and because of the importance of negative charge repulsion in regulating cell surface interactions, a negative charge is thought to be important in many aspects of reproduction, particularly in feto-maternal interactions. When negative charge is decreased, cell adhesion is enhanced (Vicker and
0378-4320/84/$03.00
© 1984 Elsevier Science Publishers B.V.
556
Edwards, 1972), and therefore blastocyst adhesion to the uterus would be expected to be facilitated. Therefore, the presence and distribution of cell anionic sites on the endometrial epithelium have been studied in various species; for example, ferret (Enders and Schlafke, 1972), mouse (Enders and Schlafke, 1974), rat (Hewitt et al., 1979; Enders et al., 1980), ewe (Guillomot et al., 1982) and rabbit (Anderson, 1982). In the rabbit, however, no ultrastructural histochemical data are available concerning pregnancy. In the present investigation using transmission electron microscopy, relative anionic site density was compared on rabbit uterine epithelium at estrus and days 5--7 of pregnancy. Possible changes that might play a role in blastocyst implantation were analyzed. MATERIALS AND METHODS
Animals A common rabbit strain was used. Adult females were caged individually in a controlled environment with 14 h light/24 h. They were mated with two fertile males. Times were expressed as days elapsed after coitus, the day of coitus being denoted as zero time. Endometrial tissues of animals on days 5, 6 and 7 of pregnancy (nine animals) and of unmated estrous females (two animals) were obtained from meso- and antimesometrial sides of the horn and from and between sites of egg recovery.
Tissue preparation The presence of negatively charged groups within the surface coat of uterine epithelial cells from rabbits in estrus and during the preimplantation period was demonstrated by the cationized ferritin technique.
Cationized ferritin technique. Prefixation was initiated in situ by perfusion via the abdominal aorta with 1% glutaraldehyde in 0.15 M cacodylate buffer, pH 7.2. Endometrial tissues rinsed with cacodylate buffer were immediately immersed for 30--60 min in the same fixative and they were washed for 1 h in buffer. Tissues were then incubated for 45 min in a cationized ferritin solution (CF; 0.4 mg/ml buffer) for localizing anionic sites (Danon et al., 1972), rinsed three times in buffer (10 min each batch), post-fixed for 1 h in 1% osmium tetroxide in 0.15 M cacodylate buffer, and rinsed in cacodylate buffer. Controls. After glutaraldehyde prefixation and rinse in buffer, control specimens were preincubated for 1 h in trypsin (2 mg/ml buffer), at 37°C, in a moist atmosphere. Then they were rinsed in buffer, incubated in the cationized ferritin solution (CF) and processed as described above.
557
Transmission electron microscopy. All samples were dehydrated in acetone and e m b e d d e d in epon-araldite. Ultrathin sections were examined after brief staining with uranyl acetate and lead citrate in a Jeol JEM 7 A or a Philips 100 CX electron microscope. RESULTS
Estrus At estrus, a thick surface coat lined the apex of the uterine epithelial cells suggesting the presence of a negatively charged glycocalyx. CF b o u n d extensively and uniformly to the luminal surface of all microvillous cells, equally on the meso- and antimesometrial sides of the horn (Fig. 1). Ferritin particles formed a thick layer on the outer surface of the cells. Binding was thicker on the tips of the microviUi, as more clearly observed at a higher resolution (Fig. 2). Ciliated cells appeared n o t to bind CF at all. The effect of preincubation with trypsin on CF binding was marked, leading to a reduction of staining.
Day 5 The pattern of CF labeling was similar to that found at estrus. It was dense and the distribution of anionic sites was relatively regular on the plasma membranes both of the microvilli and cell body. Labeling with CF was heavier on the tips of some microvilli. Binding was similar on the meso- and antimesometrial poles of the uterine horn and also in and between egg recovery sites (Figs. 3 and 4). It should also be pointed o u t that no electron-dense material penetrated the apical junctional complex (Fig. 3). No binding was observed on ciliated cells. After incubation with trypsin before incubation with CF, ferritin binding was greatly reduced or was abolished (Fig. 5).
Day 6 At this time, less binding was observed than on day 5, and ferritin particles formed a regular thinner layer on plasma membranes of microvilli and the cell body, stopping at the apical junctional complex (Figs. 6--8). CF b o u n d uniformly at the tips and sides of the microvilli. No difference in staining was observed on the two sides of the horn or in and between the sites of egg recovery. Ciliated cells appeared n o t to bind CF. After trypsin preincubation, labeling was greatly reduced or was abolished {Fig. 9).
558
Labeling of the endometrial epithelium by cationized ferritin (CF). Estrus. Fig. 1. CF bound extensively and uniformly to the plasma membranes of microvilli and cell body; x 16 000. Fig. 2. Binding was thicker at the tips of the microvilli; x 36 000.
559
Uterine epithelium incubated with cationized ferritin (CF). Day 6 of gestation. Figs. 6--8. Ferritin particles formed a regular, thinner layer than at day 5 on plasma membranes, stopping at the apical junctional complex (j). CF bound similarly on the two sides of the horn, in (Figs. 6 and 8) and between (Fig. 7) egg recovery sites; x 16 000. Fig. 9. Control tissue preincubated with trypsin. No electron dense material was observed on the surface; x 16 000.
Incubation of uterine epithelium in cationized ferritin (CF). Day 5 of gestation. Figs. 3--4. CF labeling was dense and regular. It was similar on the meso- (Fig. 3) and antimesometrial (Fig. 4) poles of uterus, in (Fig. 3) and between (Fig. 4) egg recovery sites. No electron dense material penetrated the apical junctional complex (j); x 16 000. Fig. 5. Control tissue preincubated with trypsin. Ferritin binding was abolished; x 20 000.
560
Day 7 On day 7 post coitus, labeling had thinned out considerably. In uterine regions occupied by the eggs, no labeling was observed on the antimesometrial side of the horn (Fig. 10), and no or heavily reduced labeling on
C a t i o n i z e d f e r r i t i n (CF) labeling o f t h e u t e r i n e e p i t h e l i u m . D a y 7 o f gestation. Fig. 10. N o f e r r i t i n b i n d i n g was o b s e r v e d o n t h e a n t i m e s o m e t r i a l side o f t h e h o r n in egg site r e c o v e r y ; X 16 000. Figs. 11--12. A heavily r e d u c e d labeling was p r e s e n t o n t h e surface in t h e m e s o m e t r i a l side o f t h e h o r n in f r o n t o f a n egg (Fig. 11) a n d a very little C F b i n d i n g was still o b s e r v e d o n t h e a n t i m e s o m e t r i a l side w i t h o u t egg (Fig. 12); x 16 000. Fig. 13. C o n t r o l tissue p r e i n c u b a t e d w i t h t r y p s i n . M e s o m e t r i a l side o f t h e h o r n , facing a n egg. No r e a c t i o n p r o d u c t was p r e s e n t ; x 16 000.
561
the mesometrial side (Fig. 11). In uterine regions without eggs, there was still a very little CF binding (Fig. 12). Ciliated cells never bound particles of CF. After trypsin treatment, binding of CF was completely abolished (Fig. 13). DISCUSSION
Our results clearly demonstrate the presence of a surface coat on uterine epithelial cells of rabbits. Anionic sites detected by the cationized ferritin technique progressively disappear from the endometrial epithelium during the preimplantation period. Effectively, at estrus and at day 5 of pregnancy, the surface coat is very thick, at day 6 it is thinner, and at day 7 it has thinned out considerably and is almost gone, especially antimesometrially, facing the eggs. The reduction or abolition of CF binding brought about by treatment with trypsin, a proteolytic enzyme, implies that the carbohydrates bearing the negative groups are bound to proteins. Unmasking of deeper anionic sites by trypsin could explain the fact that binding is sometimes only reduced rather than totally abolished after the proteolytic treatment (Guillomot et al., 1982). Similarly, there is a general reduction of anionic sites from days 2--6 of pregnancy or of pseudopregnancy in the rat uterus (Hewitt et al., 1979). In rabbits, there is also a progressive reduction of anionic sites during early pseudopregnancy (Anderson, 1982), CF binding appearing mostly on the tips of microvilli at day 4 and disappearing at day 6. Thus, the decrease in anionic sites from day 4 to day 6 does not occur in the same way in pregnant and pseudopregnant rabbits, as it does in pregnant and pseudopregnant rats. Moreover, in the rabbit, the loss of anionic sites is more important in uterine regions localized near the eggs at day 7. The loss of anionic sites from pregnant rabbit uterine epithelium is not a general uterine response, indicating that the effect is not solely the result of uterine activity but is due to the combined actions of uterus and egg. The disappearance of negatively charged residues at the antimesometrial pole of the uterus facing the eggs at day 7, coinciding with the beginning of antimesometrial blastocyst attachment to the uterine tissues, probably makes uterine cells attractive to the trophoblastic cells and may facilitate implantation. ACKNOWLEDGEMENTS
The authors thank M.T. Lavault and D. Quernee for their technical assistance, S. Lorre for typing the manuscript and R. Primault for photographic assistance.
562 REFERENCES
Anderson, T.L., 1982. Changes in the uterine luminal cell surface during early pseudopregnancy in rabbits. Anat. Rec., 202: 7A. Danon, D., Goldstein, L., Marikovsky, Y. and Skutelsky, E., 1972. Use of cationized ferritin as a label of negative charges on cell surfaces. J. Ultrastruct. Res., 38: 500-510. Enders, A.C. and Schlafke, S., 1972. Implantation in the ferret: epithelial penetration. Am. J. Anat., 133: 291--316. Enders, A.C. and Schlafke, S., 1974. Surface coats of the mouse blastocyst and uterus during the preimplantation period. Anat. Rec., 180: 31--46. Enders, A.C., Schlafke, S. and Welsh, A.O., 1980. Trophoblastic and uterine luminal epithelial surfaces at the time o f blastocyst adhesion in the rat. Am. J. Anat., 159: 59--72. Guillomot, M., Fl~chon, J.E. and Wintenberger-Torres, S., 1982. Cytochemical studies of uterine and trophoblastic surface coats during blastocyst attachment in the ewe. J. Reprod. Fertil., 65: 1--8. Hewitt, K., Beer, A.E. and Grinnell, F., 1979. Disappearance of anionic sites from the surface of the rat endometrial epithelium at the time of blastocyst implantation. Biol. Reprod., 2 1 : 691--707. Luft, J.H., 1976. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat. Rec., 171: 347--368. Moscona, A.A., 1971. Embryonic and neoplastic cell surfaces: availability of receptors for concanavalin A and wheat germ agglutinin. Science (N.Y.), 171 : 905--907. Oppenheimer, S.B., 1978. Cell surface carbohydrates in adhesion and migration. Am. Zool., 18: 13--23. Vicker, M.G. and Edwards, J.G., 1972. The effect of neuraminidase on the aggregation of BHK21 cells and BHK21 cells transformed by polyoma virus. J. Cell. Sci., 10: 759--768. Weiss, L., 1965. Studies on cell deformability. I. Effect of surface charge. J. Cell. Biol., 26: 735--739. Weiss, L., 1969. The cell periphery. Int. Rev. Cytol., 26: 63--105. Weiss, L. and Zeigel, R., 1971. Cell surface negativity and the binding of positively charged particles. J. Cell Physiol., 77: 179--186.