- Email: [email protected]
Oogenesis in Xenopus laevis (Daudin). III. Localization of negative charges on the surface of developing oocytes
JOURNAL OF ULTRASTRUCTURE RESEARCH 55, 4 - 1 6
(1976)
Oogenesis in Xenopus laevis (Daudin) III. Localization of Negative Charges on the Surface of Developing Oocytes ANNA RUTH BRUMMETT AND J A M E S N . DUMONT Oberlin College, Oberlin, Ohio 44074, and the Biology Division, Oak Ridge National Laboratory,1 Oak Ridge, Tennessee 37830 Received May 5, 1975, and in revised form, December 10, 1975 Developing oocytes and coelomic eggs of m a t u r e HCG-stimulated Xenopus females were dissected from the ovary, fixed in glutaraldehyde, and treated with a positively charged solution of colloidal iron. Electron micrographs reveal anionic sites preferentially localized in or on the p l a s m a l e m m a covering the distal portions of the numerous microvilli of Stage IV oocytes. Both prior to and subsequent to Stage IV (the peak of vitellogenic activity), the anionic sites on the oolemma are more randomly distributed. No iron particles appear in endocytotic pits of the oolemma a t any stage of development, but endocytotic pits of follicle cells are labeled. There are few if any anionic sites in the vitelline envelope. The assumption t h a t the negative charges are due to sialic acid moieties of surface mucopotysaccharides was borne out by t r e a t m e n t with neuraminidase. Oocytes subjected to the enzyme and subsequently fixed and treated with colloidal iron show a marked reduction in the amount of labeling. These results are discussed in the light of earlier evidence regarding the endocytosis of vitellogenin, a negatively charged molecule.
During its period of development within the ovary, the Xenopus oocyte increases in volume by a factor of 15 000 to 20 000. It has been amply demonstrated (8, 23, 26) that materials produced in the liver of the female are transported through the circulatory system to the ovary, where they are incorporated into the growing oocytes as storage materials which will later be utilized as a source of energy for the developing embryo. Although a layer of follicle cells and the vitelline envelope intervene between the oocyte and the thecal layer which contains the blood vessels, the final barrier to the passage of these materials from the circulation into the oocyte is the plasmalemma of the oocyte itself. The intense activity of the oocyte during this period of growth can be surmised from electron microscope studies of the cell surface. Numerous long, almost filamentous, microvilli greatly increase the surface area of the oocyte. Deep crypts or invaginations 1 Operated by the Union Carbide Corporation under contract with the U. S. Energy Research and Development Administration. Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
between microvilli and a large number of endocytotic (micropinocytotic) pits add additional surface area. The presence of these, and the even more numerous endocytotic vesicles, documents the great activity of the cell membrane at this time. Techniques of biochemistry and molecular biology have been used to good advantage in investigating uptake of materials by the oocyte in vivo (25, 26) and in vitro (12, 27-30). The objective of the experiments reported here was to investigate the role of the plasmalemma during oogenesis by characterizing its surface topography in terms of the charge carried by its surface molecules. From this we hoped to learn something about how the oocyte plasmalemma exercises selectivity in the uptake of molecules in its immediate environment. MATERIALS AND METHODS
Collection ofoocytes. Ovaries were removed from m a t u r e female Xenopus laevis approximately i week after a single injection of HCG ( h u m a n chorionic gonadotropin; 1000 units into the dorsal lymph sac). The excised ovaries were immediately placed in O-
NEGATIVE CHARGES ON XENOPUS OOCYTES R2 (a buffered balanced salt solution described by Wallace et al., 28). With the aid of a dissecting microscope and two pairs of sharpened watchmaker's forceps, individual oocytes at the desired stage of development (Stages II through VI, 7) were dissected from the ovary. The oocytes were fixed within an hour after dissection. (It has been demonstrated by Wallace et al., 28, that dissected Xenopus oocytes maintain their normal cytology for several hours in O-R2.) Coelomic eggs were collected from frogs which had been injected with 1000 units of HCG approximately 8 hr previously. Colloidal iron treatment. The oocytes were fixed for 2 hr in cold glutaraldehyde (3% in 0.05 M phosphate buffer, pH 7.4) and washed in 0.05 M phosphate buffer. Following a rinse in distilled water, the fixed oocytes were treated for 30 min at room temperature with a positively charged solution of M~iller's colloidal iron prepared according to the method of Mowry (17). The colloidal iron treatment was .followed by two brief washes in 12% acetic acid and one in distilled water. The oocytes were then postfixed for 2 hr in cold osmium tetroxide (1% in 0.05 M phosphate buffer), dehydrated through a series of alcohols followed by propylene oxide, and embedded in Epon. Neuraminidase treatment. The enzyme neuraminidase (Sigma Chemicals, neuraminidase from Cl. perfringens, Type VI, chromatographically purified) was dissolved in O-R2. Final enzyme concentration was 4.4 units/ml, incubation was accomplished at room temperature, and the length of the treatment was varied from 5-25 min. Following neuraminidase treatment, the oocytes were rinsed with O~R2, fixed in glutaraldehyde, and treated with colloidal iron as described above. Electron microscopy. Embedded oocytes were sectioned with a diamond knife mounted in a PorterBlum MT-2 ultramicrotome. Some of the grids were stained with uranyl acetate followed by lead citrate, some with lead citrate solution alone, and some were left unstained. All pictures were taken with a Hitachi 11E electron microscope operated at 75 kV. RESULTS The m a i n objective of these experiments was to investigate the localization of negative charges on the p l a s m a l e m m a of the oocyte. To allow access to the o o l e m m a by the colloidal iron, the i n v e s t i n g layers (see 7 for description) were carefully removed from living oocytes in various stages of development. Such individual oocytes, covered only with the vitelline envelope and a few a d h e r i n g follicle cells, were the subjects of our experiments.
S t a g e s H a n d IIIo Stage II oocytes h a v e a few microvilli in e a r l y stages of developm e n t a n d show some evidence of endocytotic activity. Such microvilli are relatively short a n d few in n u m b e r ; invaginations (or crypts) b e t w e e n t h e m are infreq u e n t a n d relatively shallow. Such oocytes, t r e a t e d w i t h positively charged colloidal iron, exhibited the g r e a t e s t concent r a t i o n of iron on the distal regions of the microvilli. The r e m a i n d e r of the cell surface was more lightly labeled except for the i n f r e q u e n t endocytotic pits, which showed no label a t all. The colloidal iron also labeled the p l a s m a l e m m a of the few a d h e r i n g follicle cells. Stage III oocytes exhibit longer a n d more n u m e r o u s microvilli, but the distribution of iron particles on their surfaces was very similar to t h a t of Stage II oocytes. S t a g e I V . We devoted most of our attention to the Stage IV oocytes because it is this stage which exhibits the g r e a t e s t a m o u n t of vitellogenic activity (7, 2 7 ) . The microvilli are n u m e r o u s and long, and there are large n u m b e r s of endocytotic pits and vesicles. I n all Stage IV oocytes examined, the colloidal iron was found to be selectivley localized on the distal regions of the microvilli. Very little iron appeared on the proximal portions of the microvilli or in the i n v a g i n a t i o n s of the oocyte surface between microvilli. Significantly, alt h o u g h iron particles appeared adjacent to endocytotic pits, t h e y are conspicuously absent from the pits themselves. F i g u r e s 1, 2, and 3 (see also Fig. 9) show the characteristic a p p e a r a n c e of these oocytes. Adh e r i n g follicle cells a p p e a r to be s o m e w h a t more heavily stained with iron on the surface distal to the oocyte; the proximal surface, including t h a t of the m a c r o v i l l i which extend t h r o u g h the vitelline envelope to come in close contact with the oocyte, is more lightly labeled. Endocytotic pits on the surface of the follicle cells proximal to the oocyte lacked iron, as did the pits in the oocyte surface. In contrast to this, however, endocytotic pits on the dis-
i,i!i!ii,~~
i: ¸
~i
~ii!iiii~ ~!ii~.
i ¸,
i! ~'i~ii~ ~ i~i ~ ....
~
•
i~ ¸~.
~,
A
"IA ~
:%
,k
711: !i! i ,!
~i ¸
'PT I
I
FIG. 1. Adhering follicle cell and microvilli of Stage IV oocyte dissected from the ovary, fixed, and treated with positively charged colloidal iron. The distal surface of the follicle cell is more heavily labeled than the proximal surface• Essentially no iron is present in the vitelline envelope. The distal portions of the microvilli are much more heavily labeled than the proximal portions and crypts. Section unstained. Scale, 0.5 ~m. × 31 000. 6
FIGS. 2, 3. Microvilli and endocytotic pits of S t a g e IV oocytes dissected from the ovary, fixed, and treated with positively charged colloidal iron. The distal portions of the microvilli are heavily labeled, b u t the endocytotic pits (arrows) are completely devoid of iron. Section in Fig. 2 is unstained; section in Fig. 3 is stained with lead citrate. Scale, 0.5 tLm. x 63 000. 7
8
BRUMMETT AND DUMONT
tal surface of follicle cells were frequently heavily labeled with iron. Stage V. By Stage V the oocyte has passed the peak of active uptake, and the morphology of the surface changes accordingly. Microvilli are less numerous and shorter and there are fewer endocytotic pits and vesicles. Concomitantly, the localization pattern of the colloidal iron became less specific. Iron still appeared to be localized predominantly on the distal portions of the microvilli but, particularly in late Stage V, the entire surface of the oocyte, including the invaginated regions, exhibited the presence of some iron particles. Endocytotic pits in the oocyte surface, however, continued to be devoid of the colloid. Stage VI. Stage VI oocytes have been described as postvitellogenic (7). These oocytes exhibit a marked reduction in the number and length of microvilli, and endocytotic pits are very few. Treated with the positively charged solution of colloidal iron, such oocytes exhibited a fairly even distribution of iron particles over the entire surface of the p l a s m a l e m m a - t h e few endocytotic pits again were a marked exception. Any adhering follicle cells showed heavy labeling on the surface distal to the oocyte, and on that surface endocytotic pits were also labeled. The surface proximal to the oocyte, including endocytotic pits and macrovilli extending into the vitelline envelope, was more lightly labeled (Fig. 4). Coelomic eggs. To complete the story of the localization of negative charges on the surface of the oocyte, a group of coelomic eggs was recovered from a female frog 8 hr after injection of 1000 units of HCG. These coelomic eggs were treated with the positively charged colloidal iron solution following the procedures described for dissected oocytes. Because c~elomic eggs are fully developed eggs which have ruptured from ovarian follicles in a normal fashion under the influence of gonadotropic hormones, they are naked of all ovarian in-
vestments, the plasmalemma being protected only by the vitelline envelope. Such eggs possess only a few surface irregularities. Microvilli have essentially disappeared; the few that remain are short and club-shaped, and there are no endocytotic pits. When the eggs were treated with the positively charged colloidal iron solution, the entire surface of the plasmalemma was coated and a fairly thick layer of the iron colloid was associated with the clubshaped microvilli. Essentially no iron remained attached to the vitelline envelope, however (Fig. 5).
Oocytes treated with neuraminidase. Negatively charged sites on the surface of cell membranes are generally attributed to the presence of terminal sialic acid moieties of surface glycoproteins (4, 32, 33). The enzyme neuraminidase has been shown to be specific for the selective removal of such terminal sialic acid molecules (11). In these experiments, neuraminidase was prepared in a concentration of 4.4 units/ml of O-R2, and freshly dissected oocytes were incubated in the enzyme solution at room temperature (approximately 22°C) for periods of 5, 10, 15, 20, and 25 min, rinsed, fixed, and treated with colloidal iron as described previously. Oocytes that had been subjected to the enzyme for progressively longer periods of time exhibited, as might be expected, a progressive reduction in the amount of iron present. Again, the iron was localized only on the distal portions of the microvilli; none w a s visible in the endocytotic pits. Even in the case of those oocytes treated for only 5 min, the amount of iron present on the surface of the microvilli appeared to be significantly reduced over that seen in the untreated oocytes (compare Figs. 1 and 6). It was also noted that the oocyte surface became progressively less contoured as the duration of neuraminidase treatment was increased. These results are summarized in Fig. 7.
NEGATIVE CHARGES ON XENOPUS OOCYTES
9
FIG. 4. Adhering follicle cell and microvilli of a Stage VI oocyte,,dissected from the ovary, fixed, and treated with positively charged colloidal iron. The distal surface of the follicle cell, including an endocytotic pit (arrow), is more heavily labeled than the proximal surface (including the macrovillus). The iron particles are fairly evenly distributed over the entire surface of the oolemma. Section unstained. Scale, 0.5 /~m. x 47 000. DISCUSSION Electrophoretic studies h a v e demonstrated t h a t most, if not all, cells exhibit a net negative surface charge (32). In recent y e a r s positively c h a r g e d solutions of coll0i: dal iron have been used to good a d v a n t a g e in d e m o n s t r a t i n g , at the u l t r a s t r u c t u r a l level, the localization of negativelY charged sites on the Surface of ~/variety of cells, e.g., intestinal e p i t h e l i u m (5), Ehr-
lich ascites t u m o r cells (34), m a m m a l i a n sperm (36, 37), bladder epithelium (19), and erythrocytes (14, 18, 35). The negative surface charge m a y be attributed to the presence of several molecular types in or on the cell surface, such as certain amino acid residues, nucleic acids, and sialic acid moieties of glycoproteins. Studies using techniques specific for the m a s k i n g or r e m o v a l of such molecules
VITELLINE
ELOPE
FIG. 5. A portion of the vitelline envelope and surface ofa coelomic egg, fixed and treated with positively charged colloidal iron. The microvilli are short and club-shaped. The iron is fairly evenly distributed over the entire surface of the oolemma. Essentially no iron remains attached to the vitelline envelope. Section unstained. Scale, 0.5 ~m. x 41 000. FIG. 6. A Stage IV oocyte treated with neuraminidase (4.4 units/ml, 22°C, 5 rain), fixed, and treatec] with positively charged colloidal iron. Under these conditions the enzyme has greatly reduced the number of negative sites on the oolemma. (Compare with Fig. 1.) Section stained with uranyl acetate and lead citrate. Scale, 0.5 t~m. x 46 000. 10
NEGATIVE CHARGES ON XENOPUS OOCYTES
11
EFFECT OF NEURAMINIDASE (4,4 units/ml of 22 °C) ON NEGATIVE CHARGE DISTRIBUTION ON Xenopus OOCYTES
CONTROL
Ii
l~I
JJ/I_ 15 MIN
10 MIN .~.. "
5 MIN
"'~<
20 MIN
25 MIN 0
Fro. 7. This series of diagrams shows the progressive reduction in the amount of positively charged
colloidal iron adhering to the surface of oocytes after they are treated with neuraminidase for progressively longer periods of time. The reduction in the number of negative sites is accompanied by a gradual change in the surface contour. All diagrams were traced from electron photomicrographs of identical magnification. h a v e convincingly d e m o n s t r a t e d , how- m o r e likely p e r h a p s , a n i n t e g r a l p a r t of ever, t h a t sialic acid is the m o s t i m p o r t a n t the p l a s m a l e m m a itself (3, 18). contributor, a c c o u n t i n g for a b o u t 80 to 85% In view of the e x p e r i m e n t a l results cited of the n e g a t i v e c h a r g e (18, 32). We feel a b o v e , our success in labeling n e g a t i v e t h a t our own e x p e r i m e n t s w i t h t h e e n z y m e sites on the surface of the Xenopus oocyte n e u r a m i n i d a s e offer a convincing demon- w i t h positively c h a r g e d colloidal iron w a s s t r a t i o n t h a t anionic sites on the surface of not unexpected. W h a t was unexpected, the oocyte p l a s m a l e m m a are predomi- however, w a s t h a t our initial e x p e r i m e n t n a n t l y the r e s u l t of the presence of sialic w i t h S t a g e IV oocytes would r e v e a l a patacid moieties. Such sialo-muco s u b s t a n c e s t e r n of p r e f e r e n t i a l localization of the posim a y be p a r t of a n e x t r a c e l l u l a r surface tive iron particles on the distal portion of' l a y e r (the "glycocalyx" of Bennett, 1) or, the microvilli a n d t h a t iron would be ab-
12
BRUMMETT AND DUMONT
sent in endocytotic pits in the plasmalemma. Further experiments confirmed this negative site localization pattern in the case of the Stage IV oocyte and, in addition, revealed that developmental changes occur in the pattern during the course of oogenesis. That the localization pattern of the iron in the Stage IV oocytes is real and not an artifact of differential penetration, is borne out by the following: (a) A more random distribution of iron, covering the entire surface of the oolemma, was obtained in the case of oocytes of earlier (Stage II) and later (Stage VI) oocytes. (b) The localization pattern remained the same on Stage IV oocytes which were shown to be essentially devoid of follicle cells and also to have large holes in the vitelline envelope. We feel that our experiments support the following conclusions: 1. Anionic sites (presumed to be primarily sialic acid moieties ofglycoproteins) are preferentially located in or on the plasmalemma covering the distal portions of the numerous microvilli of Stage IV oocytes of Xenopus (Figs. 1 and 9). 2. The distribution of anionic sites on the oocyte plasmalemma appears to change from one of randomness in early stages of development, to one of striking specificity at the peak of vitellogenesis, followed by a gradual change to randomness in coelomic eggs (Figs. 1, 4, and 5). 3. Endocytotic pits present in the plasmalemma ofXenopus oocytes at any stage of development are devoid of accessible anionic sites (Figs. 2, 3, and 9). 4. There are apparently few, if any, anionic sites in the vitelline envelope (Figs. 1, 2, 4, 5, and 9). 5. Follicle cells adhering to the surface of developing oocytes of Xenopus have a greater concentration of anionic sites on the surface distal to the oocyte (including endocytotic pits on that surface) than on the surface proximal to the oocyte (including macrovilli extending from that surface) (Figs. 1, 4, and 9).
@ ~ Vitellogeninmolecule (negatively charged) @s-i: -
-
-]I
'
i
@
-__ (.
-)}
-If "- I
,-
-_
(
(negatively Microvilli charged)
Endocyfoficpit,presumedsite of vifellogeninuptake(positive chargehypofhefical)
FIG. 8. Schematicrepresentation of a portion of the microvillous surface of a vitellogenic oocyte summarizing the distribution of colloidaliron binding. (See text for full explanation.) The possible significance of these conclusions with respect to vitellogenesis is discussed below.
The Microvilli The presence of a large number of microvilli at a time when the cell is in its most active period of uptake and growth suggests that a significant increase in surface area is an important factor in the kinetics of uptake of materials. Electron microscope studies, however, have shown t h a t the microvilli are not involved in endocytosis at all, for there are no endocytotic pits on the microvilli themselves (7). This is not to say, however, that the microvilli may not play an important role in the uptake of materials through the mechanisms of diffusion or active transport, neither of which involves the formation of endocytotic pits.
Negative Surface Charge Quinton and Philpott (20, p. 794) have suggested %hat ionized anions on the membrane set up repulsive surface patentials along the membrane surface which establish a minimum distance of approach between surfaces and prevent fusion of juxtaposed membranes. Such phenomena may be particularly crucial in certain
NEGATIVE CHARGES ON XENOPUS OOCYTES
13
v
Enaocyl"oticpit ~) ~')'~"~'Endocytoticvesicle Oocyte cytoplasm FIG. 9. Summarizing diagram of the Stage IV oocyte, dissected from the ovary, fixed, and treated with a solution of positively charged colloidal iron, pH 1.25. The upper (distal) surface of adhering follicle cells, including endocytotic pits, is heavily labeled with the iron, while the lower (proximal) surface is more lightly stained. Essentially no iron is present in the vitelline envelope. The distal portions of the microvilli show a high density of the iron particles, while the proximal portions and the crypts are more lightly labeled. Endocytotic pits in the surface of the oolemma are completely devoid of iron. m e m b r a n e configurations such as microvilli, lateral processes, or basal infoldings." Applying this hypothesis to the vitellogenic oocyte, one could imagine a situation such as t h a t d i a g r a m m e d in Fig. 8. Thus, the m a i n t e n a n c e of the microvilli as individual extensions of the cell surface m a y depend, in p a r t at least, on t h e i r mutual repulsion by the strongly negative surface charge. At the same time, channels are provided which m a y funnel and subsequently concentrate large n e g a t i v e l y charged molecules (e.g., vitellogenin) into the crypts and endocytotic pits which lie between the microvilli and which m a y carry a positive charge. Other studies (e.g., 16, 31) have presented evidence in support of the hypothesis t h a t negative surface charge m a y play an i m p o r t a n t role in m a i n t a i n i n g n o r m a l cell surface morphology. This suggests the possiblity t h a t the strongly negative sialic
acid moieties of surface glycoproteins m a y play an i m p o r t a n t role in m a i n t a i n i n g the microvillous morphology of the Stage IV oocyte. The observation t h a t oocyte s u r face contouring decreased with prolonged n e u r a m i n i d a s e t r e a t m e n t is consonant with this suggestion as is the report by Quinton and Philpott (20) t h a t the micro-. villi of gall bladder epithelium, exposed to cationic polymers which p r e s u m a b l y ad.sorb to anionic sites on the cell surface, lose t h e i r rigidity and sometimes collapse onto the cell surface.
Compartmentalization of Function O t h e r studies h a v e revealed a patterned, as opposed to a random, localization of negative sites on a v a r i e t y of cells, e.g., t r a c h e a l epithelial cells (15), erythrocytes (9, 14, 18), alveolar and endothelial cells of the r a t lung (2), Ehrlich ascites t u m o r cells (34), and m a m m a l i a n sperm
14
BRUMMETT AND DUMONT
(36, 37). None of these reports, however, provides insight into the possible functional significance of the observed differences in negative site distribution. We would like to be able to assign a function to the negatively charged sialic acid moieties which the colloidal iron labeling has demonstrated to be present. A number of studies have implicated negative surface charge in permeability and transport phenomena (6, 10, 13, 19, 20, 21), and we can assume a role for such phenomena in oogenesis. Wallace and Ho (29), for example, have shown that %uabain, which inhibits the membrane Na-, K-transport mechanism, produced a slight but significant depression in the oocyte's ability to incorporate protein." They conclude that ~micropinocytosis is facilitated by but not dependent on a functioning Na-, K-transport system." The localization of the negatively charged sialic acid moieties on the distal regions of the microvilli of the Xenopus oocytes, and their absence from endocytotic pits, may be simply a further example of ~division of labor" or compartmentalization of function in the cell membrane, for which there is a growing body of evidence. The microvilli with their negatively charged sialic acid moieties may be the sites of cation exchange (and perhaps small-molecule uptake?) - thus providing a mechanism whereby important ionic balance for the entire cell is maintained and small molecules important to cell metabolism are taken up. On the other hand, the uptake of the main yolk precursor, vitellogenin, is presumed to occur deeper in the crypts between microvilli through the mechanism of endocytosis. (Evidence that the latter actually occurs has been obtained in this laboratory in experiments using radioactively labeled vitellogenin followed by autoradiography and electron microscopy. These experiments will be reported in a subsequent paper.) Vitellogenin, a phosphoprotein, is a negatively charged molecule at physiological
pH (Wallace, personal communication) and has been shown to be sequestered 64 to 189 times more rapidly than several other purified proteins tested by Wallace and Bergink (24). Since this molecule is taken up by endocytosis, and since in our experiments the positively charged colloidaFiron particles consistently failed to label the endocytotic pits, it seems reasonable to assume that the oolemma of the endocytotic pits may carry a positive charge. Wallace and Ho (29) have presented biochemical evidence that the negatively charged colloid, trypan blue, essentially abolishes the ability of the Xenopus oocyte to incorporate labeled vitellogenin. It is suggested that this and other biochemical evidence ~supports a preliminary model whereby protein-binding sites reside within positively charged areas of the surface membrane" (29, p. 315). The failure of the endocytotic pits to stain with the positively charged colloidal iron is indirectly supportive of this preliminary model provided by Wallace and Ho. [It should be noted, however, that more recently Wallace and Jared (personal communication) have obrained evidence that the oocytes' selectivity for vitellogenin does not appear to involve either the size of the molecule or its negative charge.]
The Follicle Cells It is of interest to note that while the endocytotic pits on the surface of the oocyte are conspicuous in their lack of the positively charged colloid, those on the surface of adhering follicle cells are conspicuously labeled with the iron. This suggests that follicle cells may be actively involved in the uptake of positively charged molecules. Such an interpretation is consonant ~with the evidence presented by Wallace et al. (30) that follicle cells do not play a direct role during vitellogenin uptake by the oocytes and thus are apparently not involved in the uptake of negatively charged molecules. It is also of interest t h a t the follicle cells
NEGATIVE CHARGES ON X E N O P U S OOCYTES
exhibit an uneven distribution of negatively charged sites. The significance of the greater density of negative charges (sialic acid moieties) on the surface distal to the oocyte, as compared with the surface proximal to the oocyte (including the macrovilli), is almost certain to reside in a functional differentiation in these two surfaces of the follicle cells. Our experiments provide no further information regarding the role of the follicle cells during oogenesis in Xenopus, but these data raise some interesting questions which are deserving of further study. CONCLUSIONS
On the basis of the data presented in this paper, we are inclined to believe that as the oocyte grows and develops with time, its surface topography changes not only morphologically, with the gradual formation and subsequent regression of microvilli and endocytotic pits, but also with respect to the molecular makeup of the plasmalemma. More specifically, as the Xenopus oocyte undergoes normal development within the ovary, there is evidence of a concomitant developmental change in the distribution of sialic acidbearing glycoproteins at the cell surface. A random distribution of such components during the early stages of oogenesis is followed by a preferential localization of the negatively charged moieties on the distal regions of the microvilli. This specificity of localization is most striking at that stage when the oocyte exhibits its greatest vitellogenic activity and presumably is playing some functional role in that activity; as the oocyte passes this peak, a return to the earlier randomized situation occurs. Our interpretation of these experimental results appears to be compatible with recently proposed models of the cell membrane as a fluid mosaic, with the possibility of lateral translation of molecules composing the membrane (22). The evidence strongly indicates, however, that such topographical changes in the distribution of
15
molecules composing the cell membrane are part of a regulated pattern that almost certainly reflects functional differences important in the processes of oocyte development and differentiation. This work was u n d e r t a k e n and completed while Dr. B r u m m e t t was on sabbatical leave from Oberlin College, Oberlin, Ohio, a n d a faculty participant in the Great Lakes Colleges Association-Oak Ridge Science Semester. The research was sponsored by the U. S. Energy Research and Development Administration under contract with the Union Carbide Corporation. REFERENCES 1. BENNETT, H. S., J. Histochem. Cytochem. 11, 14 (1963). 2. CHRISTNER, A., SCHAAF, P., MEYER, C., LINSS, W., AND GEYER, G., Acta Histochem. (Jena) 38, 121 (1970). 3. COOK, G. M. W., Biol. Rev. 43, 363 (1968). 4. COOK, G, M., HEARD, D. H. AND SEAMAN, G. V., Nature (London) 191, 44 (1961). 4. CURRAN~ R. C., CLARK, A. E., AND LOVELL, D., J. Anat. 3, 427 (1965). 6. DIBONA, D. R., CIVAN, M. M., AND LEAF, A., J.
Membrane Biol. 1, 79 (1969). 7. DUMONT, J. N., J. Morphol. 136, 153 (1972). 8. FLICKINGER, R. A., AND ROUNDS, D. E.~ Biochim. Biophys. Acta 22, 38 (1956). 9. GEYER, G., LINSS, W., AND SCHAAF, P., Acta Histochem. (Jena) 42, 138 (1972). 10. GLICK, J. L., AND GITHENS III, S., Nature (London) 201, 88 (1965). 11. GOTTSCRALK,A., Biochim. Biophys. Acta 23, 645 (1957). 12. JARED, D. W., AND WALLACE, R. A., Exp. Cell Res., 57, 454 (1969). 13. LUBIN, M., Fed. Proc. 23, 994, (1964). 14. MARIKOVSKY, Y,, AND DANON, D., J. Cell Biol. 43, 1 (1969). 15. MEYER, C., CHRISTNER, A., LINSS, W., QUADE, R., AND GEYER, G., Acta Histochem. (Jena) 39, 176 (1971). 16. MICHAEL, A. R., BLAU, E., AND VERNIER, R. L., Lab. Invest. 32, 649 (1970). 17. MowRY, R. W., Lab. Invest. 7, 566 (1958). 18. NICOLSON, G. L., J. Cell Biol. 57~ 373 (1973). 19. PISAM, M., RIPOCHE, P., AND RAMSOURG, A., C. R. Acad. Sci., Ser. D 271, 105 (1970). 20. QUINTON, P. M., AND PHILPOTT, C. W., J. Cell Biol. 56, 787 (1973). 21. SCOTT, W. N., AND SAPIRSTEIN, V. S., Science 184, 797 (1974). 22. SINGER, S. J., AND NICOLSON, G. L., Science 175, 720 (1972). 23. SMITH, L. D., AND ECKER, R. E., Develop. Biol.
16
BRUMMETT AND DUMONT
25, 232 (1971). 24. WALLACE,R. A., AND BERGINK, E. W., Amer. Zool. 14:1159 (1974). 25. WALLACE,R. A., AND DUMONT, J. N., J. Cell. Physiol. 72, Suppl., 73 (1968). 26. WALLACE, R. A., AND JARED, D. W., Develop. Biol. 19, 498 (1969). 27. WALLACE,R. A., JA~ED, D. W., AND NELSON, B. L., J. Exp. Zool. 175, 259 (1970). 28. WALLACE,R. A., JARED, D. W., DUMONT,J. N., AND SEGA, M. W., J. Exp. Zool. 184, 321 (1973). 29. WALLACE,R. A., AND Ho, T., J. Exp. Zool. 181, 303 (1972). 30. WALLACE,R. A., Ho, T., SALTER, D, W., AND
JARED, D. W., Exp. Cell Res. 82, 287 (1973). WEISS, L., J. Cell Biol. 26, 735 (1965). WEISS, L., Int. Rev. Cytol. 26, 63 (1969). WEISS, L., J. Nat. Cancer Inst. 50, 3 (1973). WEIss, L., AND SUBJECK,J. R., J. Cell Sci. 14, 215 (1974). 35. WEISS, L., ZEmEL, R., JUNG, O. S., AND BROSS,I.' D. J., Exp: Cell Res. 70, 57 (1972). 36. YANA~IMACHI,R., NmHOLSON, G. L., Noda, Y. D., AND Fujimoto, M., J. Ultrastruct. Res. 43, 344 (1973). 37, YAIWAGIMACHI,R., NODA, Y. D., FUJIMOTO, M., AND NICOLSON,G. L., Amer. J. Anat. 135, 497 (1972).
31. 32. 33. 34.
(1976)
Oogenesis in Xenopus laevis (Daudin) III. Localization of Negative Charges on the Surface of Developing Oocytes ANNA RUTH BRUMMETT AND J A M E S N . DUMONT Oberlin College, Oberlin, Ohio 44074, and the Biology Division, Oak Ridge National Laboratory,1 Oak Ridge, Tennessee 37830 Received May 5, 1975, and in revised form, December 10, 1975 Developing oocytes and coelomic eggs of m a t u r e HCG-stimulated Xenopus females were dissected from the ovary, fixed in glutaraldehyde, and treated with a positively charged solution of colloidal iron. Electron micrographs reveal anionic sites preferentially localized in or on the p l a s m a l e m m a covering the distal portions of the numerous microvilli of Stage IV oocytes. Both prior to and subsequent to Stage IV (the peak of vitellogenic activity), the anionic sites on the oolemma are more randomly distributed. No iron particles appear in endocytotic pits of the oolemma a t any stage of development, but endocytotic pits of follicle cells are labeled. There are few if any anionic sites in the vitelline envelope. The assumption t h a t the negative charges are due to sialic acid moieties of surface mucopotysaccharides was borne out by t r e a t m e n t with neuraminidase. Oocytes subjected to the enzyme and subsequently fixed and treated with colloidal iron show a marked reduction in the amount of labeling. These results are discussed in the light of earlier evidence regarding the endocytosis of vitellogenin, a negatively charged molecule.
During its period of development within the ovary, the Xenopus oocyte increases in volume by a factor of 15 000 to 20 000. It has been amply demonstrated (8, 23, 26) that materials produced in the liver of the female are transported through the circulatory system to the ovary, where they are incorporated into the growing oocytes as storage materials which will later be utilized as a source of energy for the developing embryo. Although a layer of follicle cells and the vitelline envelope intervene between the oocyte and the thecal layer which contains the blood vessels, the final barrier to the passage of these materials from the circulation into the oocyte is the plasmalemma of the oocyte itself. The intense activity of the oocyte during this period of growth can be surmised from electron microscope studies of the cell surface. Numerous long, almost filamentous, microvilli greatly increase the surface area of the oocyte. Deep crypts or invaginations 1 Operated by the Union Carbide Corporation under contract with the U. S. Energy Research and Development Administration. Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
between microvilli and a large number of endocytotic (micropinocytotic) pits add additional surface area. The presence of these, and the even more numerous endocytotic vesicles, documents the great activity of the cell membrane at this time. Techniques of biochemistry and molecular biology have been used to good advantage in investigating uptake of materials by the oocyte in vivo (25, 26) and in vitro (12, 27-30). The objective of the experiments reported here was to investigate the role of the plasmalemma during oogenesis by characterizing its surface topography in terms of the charge carried by its surface molecules. From this we hoped to learn something about how the oocyte plasmalemma exercises selectivity in the uptake of molecules in its immediate environment. MATERIALS AND METHODS
Collection ofoocytes. Ovaries were removed from m a t u r e female Xenopus laevis approximately i week after a single injection of HCG ( h u m a n chorionic gonadotropin; 1000 units into the dorsal lymph sac). The excised ovaries were immediately placed in O-
NEGATIVE CHARGES ON XENOPUS OOCYTES R2 (a buffered balanced salt solution described by Wallace et al., 28). With the aid of a dissecting microscope and two pairs of sharpened watchmaker's forceps, individual oocytes at the desired stage of development (Stages II through VI, 7) were dissected from the ovary. The oocytes were fixed within an hour after dissection. (It has been demonstrated by Wallace et al., 28, that dissected Xenopus oocytes maintain their normal cytology for several hours in O-R2.) Coelomic eggs were collected from frogs which had been injected with 1000 units of HCG approximately 8 hr previously. Colloidal iron treatment. The oocytes were fixed for 2 hr in cold glutaraldehyde (3% in 0.05 M phosphate buffer, pH 7.4) and washed in 0.05 M phosphate buffer. Following a rinse in distilled water, the fixed oocytes were treated for 30 min at room temperature with a positively charged solution of M~iller's colloidal iron prepared according to the method of Mowry (17). The colloidal iron treatment was .followed by two brief washes in 12% acetic acid and one in distilled water. The oocytes were then postfixed for 2 hr in cold osmium tetroxide (1% in 0.05 M phosphate buffer), dehydrated through a series of alcohols followed by propylene oxide, and embedded in Epon. Neuraminidase treatment. The enzyme neuraminidase (Sigma Chemicals, neuraminidase from Cl. perfringens, Type VI, chromatographically purified) was dissolved in O-R2. Final enzyme concentration was 4.4 units/ml, incubation was accomplished at room temperature, and the length of the treatment was varied from 5-25 min. Following neuraminidase treatment, the oocytes were rinsed with O~R2, fixed in glutaraldehyde, and treated with colloidal iron as described above. Electron microscopy. Embedded oocytes were sectioned with a diamond knife mounted in a PorterBlum MT-2 ultramicrotome. Some of the grids were stained with uranyl acetate followed by lead citrate, some with lead citrate solution alone, and some were left unstained. All pictures were taken with a Hitachi 11E electron microscope operated at 75 kV. RESULTS The m a i n objective of these experiments was to investigate the localization of negative charges on the p l a s m a l e m m a of the oocyte. To allow access to the o o l e m m a by the colloidal iron, the i n v e s t i n g layers (see 7 for description) were carefully removed from living oocytes in various stages of development. Such individual oocytes, covered only with the vitelline envelope and a few a d h e r i n g follicle cells, were the subjects of our experiments.
S t a g e s H a n d IIIo Stage II oocytes h a v e a few microvilli in e a r l y stages of developm e n t a n d show some evidence of endocytotic activity. Such microvilli are relatively short a n d few in n u m b e r ; invaginations (or crypts) b e t w e e n t h e m are infreq u e n t a n d relatively shallow. Such oocytes, t r e a t e d w i t h positively charged colloidal iron, exhibited the g r e a t e s t concent r a t i o n of iron on the distal regions of the microvilli. The r e m a i n d e r of the cell surface was more lightly labeled except for the i n f r e q u e n t endocytotic pits, which showed no label a t all. The colloidal iron also labeled the p l a s m a l e m m a of the few a d h e r i n g follicle cells. Stage III oocytes exhibit longer a n d more n u m e r o u s microvilli, but the distribution of iron particles on their surfaces was very similar to t h a t of Stage II oocytes. S t a g e I V . We devoted most of our attention to the Stage IV oocytes because it is this stage which exhibits the g r e a t e s t a m o u n t of vitellogenic activity (7, 2 7 ) . The microvilli are n u m e r o u s and long, and there are large n u m b e r s of endocytotic pits and vesicles. I n all Stage IV oocytes examined, the colloidal iron was found to be selectivley localized on the distal regions of the microvilli. Very little iron appeared on the proximal portions of the microvilli or in the i n v a g i n a t i o n s of the oocyte surface between microvilli. Significantly, alt h o u g h iron particles appeared adjacent to endocytotic pits, t h e y are conspicuously absent from the pits themselves. F i g u r e s 1, 2, and 3 (see also Fig. 9) show the characteristic a p p e a r a n c e of these oocytes. Adh e r i n g follicle cells a p p e a r to be s o m e w h a t more heavily stained with iron on the surface distal to the oocyte; the proximal surface, including t h a t of the m a c r o v i l l i which extend t h r o u g h the vitelline envelope to come in close contact with the oocyte, is more lightly labeled. Endocytotic pits on the surface of the follicle cells proximal to the oocyte lacked iron, as did the pits in the oocyte surface. In contrast to this, however, endocytotic pits on the dis-
i,i!i!ii,~~
i: ¸
~i
~ii!iiii~ ~!ii~.
i ¸,
i! ~'i~ii~ ~ i~i ~ ....
~
•
i~ ¸~.
~,
A
"IA ~
:%
,k
711: !i! i ,!
~i ¸
'PT I
I
FIG. 1. Adhering follicle cell and microvilli of Stage IV oocyte dissected from the ovary, fixed, and treated with positively charged colloidal iron. The distal surface of the follicle cell is more heavily labeled than the proximal surface• Essentially no iron is present in the vitelline envelope. The distal portions of the microvilli are much more heavily labeled than the proximal portions and crypts. Section unstained. Scale, 0.5 ~m. × 31 000. 6
FIGS. 2, 3. Microvilli and endocytotic pits of S t a g e IV oocytes dissected from the ovary, fixed, and treated with positively charged colloidal iron. The distal portions of the microvilli are heavily labeled, b u t the endocytotic pits (arrows) are completely devoid of iron. Section in Fig. 2 is unstained; section in Fig. 3 is stained with lead citrate. Scale, 0.5 tLm. x 63 000. 7
8
BRUMMETT AND DUMONT
tal surface of follicle cells were frequently heavily labeled with iron. Stage V. By Stage V the oocyte has passed the peak of active uptake, and the morphology of the surface changes accordingly. Microvilli are less numerous and shorter and there are fewer endocytotic pits and vesicles. Concomitantly, the localization pattern of the colloidal iron became less specific. Iron still appeared to be localized predominantly on the distal portions of the microvilli but, particularly in late Stage V, the entire surface of the oocyte, including the invaginated regions, exhibited the presence of some iron particles. Endocytotic pits in the oocyte surface, however, continued to be devoid of the colloid. Stage VI. Stage VI oocytes have been described as postvitellogenic (7). These oocytes exhibit a marked reduction in the number and length of microvilli, and endocytotic pits are very few. Treated with the positively charged solution of colloidal iron, such oocytes exhibited a fairly even distribution of iron particles over the entire surface of the p l a s m a l e m m a - t h e few endocytotic pits again were a marked exception. Any adhering follicle cells showed heavy labeling on the surface distal to the oocyte, and on that surface endocytotic pits were also labeled. The surface proximal to the oocyte, including endocytotic pits and macrovilli extending into the vitelline envelope, was more lightly labeled (Fig. 4). Coelomic eggs. To complete the story of the localization of negative charges on the surface of the oocyte, a group of coelomic eggs was recovered from a female frog 8 hr after injection of 1000 units of HCG. These coelomic eggs were treated with the positively charged colloidal iron solution following the procedures described for dissected oocytes. Because c~elomic eggs are fully developed eggs which have ruptured from ovarian follicles in a normal fashion under the influence of gonadotropic hormones, they are naked of all ovarian in-
vestments, the plasmalemma being protected only by the vitelline envelope. Such eggs possess only a few surface irregularities. Microvilli have essentially disappeared; the few that remain are short and club-shaped, and there are no endocytotic pits. When the eggs were treated with the positively charged colloidal iron solution, the entire surface of the plasmalemma was coated and a fairly thick layer of the iron colloid was associated with the clubshaped microvilli. Essentially no iron remained attached to the vitelline envelope, however (Fig. 5).
Oocytes treated with neuraminidase. Negatively charged sites on the surface of cell membranes are generally attributed to the presence of terminal sialic acid moieties of surface glycoproteins (4, 32, 33). The enzyme neuraminidase has been shown to be specific for the selective removal of such terminal sialic acid molecules (11). In these experiments, neuraminidase was prepared in a concentration of 4.4 units/ml of O-R2, and freshly dissected oocytes were incubated in the enzyme solution at room temperature (approximately 22°C) for periods of 5, 10, 15, 20, and 25 min, rinsed, fixed, and treated with colloidal iron as described previously. Oocytes that had been subjected to the enzyme for progressively longer periods of time exhibited, as might be expected, a progressive reduction in the amount of iron present. Again, the iron was localized only on the distal portions of the microvilli; none w a s visible in the endocytotic pits. Even in the case of those oocytes treated for only 5 min, the amount of iron present on the surface of the microvilli appeared to be significantly reduced over that seen in the untreated oocytes (compare Figs. 1 and 6). It was also noted that the oocyte surface became progressively less contoured as the duration of neuraminidase treatment was increased. These results are summarized in Fig. 7.
NEGATIVE CHARGES ON XENOPUS OOCYTES
9
FIG. 4. Adhering follicle cell and microvilli of a Stage VI oocyte,,dissected from the ovary, fixed, and treated with positively charged colloidal iron. The distal surface of the follicle cell, including an endocytotic pit (arrow), is more heavily labeled than the proximal surface (including the macrovillus). The iron particles are fairly evenly distributed over the entire surface of the oolemma. Section unstained. Scale, 0.5 /~m. x 47 000. DISCUSSION Electrophoretic studies h a v e demonstrated t h a t most, if not all, cells exhibit a net negative surface charge (32). In recent y e a r s positively c h a r g e d solutions of coll0i: dal iron have been used to good a d v a n t a g e in d e m o n s t r a t i n g , at the u l t r a s t r u c t u r a l level, the localization of negativelY charged sites on the Surface of ~/variety of cells, e.g., intestinal e p i t h e l i u m (5), Ehr-
lich ascites t u m o r cells (34), m a m m a l i a n sperm (36, 37), bladder epithelium (19), and erythrocytes (14, 18, 35). The negative surface charge m a y be attributed to the presence of several molecular types in or on the cell surface, such as certain amino acid residues, nucleic acids, and sialic acid moieties of glycoproteins. Studies using techniques specific for the m a s k i n g or r e m o v a l of such molecules
VITELLINE
ELOPE
FIG. 5. A portion of the vitelline envelope and surface ofa coelomic egg, fixed and treated with positively charged colloidal iron. The microvilli are short and club-shaped. The iron is fairly evenly distributed over the entire surface of the oolemma. Essentially no iron remains attached to the vitelline envelope. Section unstained. Scale, 0.5 ~m. x 41 000. FIG. 6. A Stage IV oocyte treated with neuraminidase (4.4 units/ml, 22°C, 5 rain), fixed, and treatec] with positively charged colloidal iron. Under these conditions the enzyme has greatly reduced the number of negative sites on the oolemma. (Compare with Fig. 1.) Section stained with uranyl acetate and lead citrate. Scale, 0.5 t~m. x 46 000. 10
NEGATIVE CHARGES ON XENOPUS OOCYTES
11
EFFECT OF NEURAMINIDASE (4,4 units/ml of 22 °C) ON NEGATIVE CHARGE DISTRIBUTION ON Xenopus OOCYTES
CONTROL
Ii
l~I
JJ/I_ 15 MIN
10 MIN .~.. "
5 MIN
"'~<
20 MIN
25 MIN 0
Fro. 7. This series of diagrams shows the progressive reduction in the amount of positively charged
colloidal iron adhering to the surface of oocytes after they are treated with neuraminidase for progressively longer periods of time. The reduction in the number of negative sites is accompanied by a gradual change in the surface contour. All diagrams were traced from electron photomicrographs of identical magnification. h a v e convincingly d e m o n s t r a t e d , how- m o r e likely p e r h a p s , a n i n t e g r a l p a r t of ever, t h a t sialic acid is the m o s t i m p o r t a n t the p l a s m a l e m m a itself (3, 18). contributor, a c c o u n t i n g for a b o u t 80 to 85% In view of the e x p e r i m e n t a l results cited of the n e g a t i v e c h a r g e (18, 32). We feel a b o v e , our success in labeling n e g a t i v e t h a t our own e x p e r i m e n t s w i t h t h e e n z y m e sites on the surface of the Xenopus oocyte n e u r a m i n i d a s e offer a convincing demon- w i t h positively c h a r g e d colloidal iron w a s s t r a t i o n t h a t anionic sites on the surface of not unexpected. W h a t was unexpected, the oocyte p l a s m a l e m m a are predomi- however, w a s t h a t our initial e x p e r i m e n t n a n t l y the r e s u l t of the presence of sialic w i t h S t a g e IV oocytes would r e v e a l a patacid moieties. Such sialo-muco s u b s t a n c e s t e r n of p r e f e r e n t i a l localization of the posim a y be p a r t of a n e x t r a c e l l u l a r surface tive iron particles on the distal portion of' l a y e r (the "glycocalyx" of Bennett, 1) or, the microvilli a n d t h a t iron would be ab-
12
BRUMMETT AND DUMONT
sent in endocytotic pits in the plasmalemma. Further experiments confirmed this negative site localization pattern in the case of the Stage IV oocyte and, in addition, revealed that developmental changes occur in the pattern during the course of oogenesis. That the localization pattern of the iron in the Stage IV oocytes is real and not an artifact of differential penetration, is borne out by the following: (a) A more random distribution of iron, covering the entire surface of the oolemma, was obtained in the case of oocytes of earlier (Stage II) and later (Stage VI) oocytes. (b) The localization pattern remained the same on Stage IV oocytes which were shown to be essentially devoid of follicle cells and also to have large holes in the vitelline envelope. We feel that our experiments support the following conclusions: 1. Anionic sites (presumed to be primarily sialic acid moieties ofglycoproteins) are preferentially located in or on the plasmalemma covering the distal portions of the numerous microvilli of Stage IV oocytes of Xenopus (Figs. 1 and 9). 2. The distribution of anionic sites on the oocyte plasmalemma appears to change from one of randomness in early stages of development, to one of striking specificity at the peak of vitellogenesis, followed by a gradual change to randomness in coelomic eggs (Figs. 1, 4, and 5). 3. Endocytotic pits present in the plasmalemma ofXenopus oocytes at any stage of development are devoid of accessible anionic sites (Figs. 2, 3, and 9). 4. There are apparently few, if any, anionic sites in the vitelline envelope (Figs. 1, 2, 4, 5, and 9). 5. Follicle cells adhering to the surface of developing oocytes of Xenopus have a greater concentration of anionic sites on the surface distal to the oocyte (including endocytotic pits on that surface) than on the surface proximal to the oocyte (including macrovilli extending from that surface) (Figs. 1, 4, and 9).
@ ~ Vitellogeninmolecule (negatively charged) @s-i: -
-
-]I
'
i
@
-__ (.
-)}
-If "- I
,-
-_
(
(negatively Microvilli charged)
Endocyfoficpit,presumedsite of vifellogeninuptake(positive chargehypofhefical)
FIG. 8. Schematicrepresentation of a portion of the microvillous surface of a vitellogenic oocyte summarizing the distribution of colloidaliron binding. (See text for full explanation.) The possible significance of these conclusions with respect to vitellogenesis is discussed below.
The Microvilli The presence of a large number of microvilli at a time when the cell is in its most active period of uptake and growth suggests that a significant increase in surface area is an important factor in the kinetics of uptake of materials. Electron microscope studies, however, have shown t h a t the microvilli are not involved in endocytosis at all, for there are no endocytotic pits on the microvilli themselves (7). This is not to say, however, that the microvilli may not play an important role in the uptake of materials through the mechanisms of diffusion or active transport, neither of which involves the formation of endocytotic pits.
Negative Surface Charge Quinton and Philpott (20, p. 794) have suggested %hat ionized anions on the membrane set up repulsive surface patentials along the membrane surface which establish a minimum distance of approach between surfaces and prevent fusion of juxtaposed membranes. Such phenomena may be particularly crucial in certain
NEGATIVE CHARGES ON XENOPUS OOCYTES
13
v
Enaocyl"oticpit ~) ~')'~"~'Endocytoticvesicle Oocyte cytoplasm FIG. 9. Summarizing diagram of the Stage IV oocyte, dissected from the ovary, fixed, and treated with a solution of positively charged colloidal iron, pH 1.25. The upper (distal) surface of adhering follicle cells, including endocytotic pits, is heavily labeled with the iron, while the lower (proximal) surface is more lightly stained. Essentially no iron is present in the vitelline envelope. The distal portions of the microvilli show a high density of the iron particles, while the proximal portions and the crypts are more lightly labeled. Endocytotic pits in the surface of the oolemma are completely devoid of iron. m e m b r a n e configurations such as microvilli, lateral processes, or basal infoldings." Applying this hypothesis to the vitellogenic oocyte, one could imagine a situation such as t h a t d i a g r a m m e d in Fig. 8. Thus, the m a i n t e n a n c e of the microvilli as individual extensions of the cell surface m a y depend, in p a r t at least, on t h e i r mutual repulsion by the strongly negative surface charge. At the same time, channels are provided which m a y funnel and subsequently concentrate large n e g a t i v e l y charged molecules (e.g., vitellogenin) into the crypts and endocytotic pits which lie between the microvilli and which m a y carry a positive charge. Other studies (e.g., 16, 31) have presented evidence in support of the hypothesis t h a t negative surface charge m a y play an i m p o r t a n t role in m a i n t a i n i n g n o r m a l cell surface morphology. This suggests the possiblity t h a t the strongly negative sialic
acid moieties of surface glycoproteins m a y play an i m p o r t a n t role in m a i n t a i n i n g the microvillous morphology of the Stage IV oocyte. The observation t h a t oocyte s u r face contouring decreased with prolonged n e u r a m i n i d a s e t r e a t m e n t is consonant with this suggestion as is the report by Quinton and Philpott (20) t h a t the micro-. villi of gall bladder epithelium, exposed to cationic polymers which p r e s u m a b l y ad.sorb to anionic sites on the cell surface, lose t h e i r rigidity and sometimes collapse onto the cell surface.
Compartmentalization of Function O t h e r studies h a v e revealed a patterned, as opposed to a random, localization of negative sites on a v a r i e t y of cells, e.g., t r a c h e a l epithelial cells (15), erythrocytes (9, 14, 18), alveolar and endothelial cells of the r a t lung (2), Ehrlich ascites t u m o r cells (34), and m a m m a l i a n sperm
14
BRUMMETT AND DUMONT
(36, 37). None of these reports, however, provides insight into the possible functional significance of the observed differences in negative site distribution. We would like to be able to assign a function to the negatively charged sialic acid moieties which the colloidal iron labeling has demonstrated to be present. A number of studies have implicated negative surface charge in permeability and transport phenomena (6, 10, 13, 19, 20, 21), and we can assume a role for such phenomena in oogenesis. Wallace and Ho (29), for example, have shown that %uabain, which inhibits the membrane Na-, K-transport mechanism, produced a slight but significant depression in the oocyte's ability to incorporate protein." They conclude that ~micropinocytosis is facilitated by but not dependent on a functioning Na-, K-transport system." The localization of the negatively charged sialic acid moieties on the distal regions of the microvilli of the Xenopus oocytes, and their absence from endocytotic pits, may be simply a further example of ~division of labor" or compartmentalization of function in the cell membrane, for which there is a growing body of evidence. The microvilli with their negatively charged sialic acid moieties may be the sites of cation exchange (and perhaps small-molecule uptake?) - thus providing a mechanism whereby important ionic balance for the entire cell is maintained and small molecules important to cell metabolism are taken up. On the other hand, the uptake of the main yolk precursor, vitellogenin, is presumed to occur deeper in the crypts between microvilli through the mechanism of endocytosis. (Evidence that the latter actually occurs has been obtained in this laboratory in experiments using radioactively labeled vitellogenin followed by autoradiography and electron microscopy. These experiments will be reported in a subsequent paper.) Vitellogenin, a phosphoprotein, is a negatively charged molecule at physiological
pH (Wallace, personal communication) and has been shown to be sequestered 64 to 189 times more rapidly than several other purified proteins tested by Wallace and Bergink (24). Since this molecule is taken up by endocytosis, and since in our experiments the positively charged colloidaFiron particles consistently failed to label the endocytotic pits, it seems reasonable to assume that the oolemma of the endocytotic pits may carry a positive charge. Wallace and Ho (29) have presented biochemical evidence that the negatively charged colloid, trypan blue, essentially abolishes the ability of the Xenopus oocyte to incorporate labeled vitellogenin. It is suggested that this and other biochemical evidence ~supports a preliminary model whereby protein-binding sites reside within positively charged areas of the surface membrane" (29, p. 315). The failure of the endocytotic pits to stain with the positively charged colloidal iron is indirectly supportive of this preliminary model provided by Wallace and Ho. [It should be noted, however, that more recently Wallace and Jared (personal communication) have obrained evidence that the oocytes' selectivity for vitellogenin does not appear to involve either the size of the molecule or its negative charge.]
The Follicle Cells It is of interest to note that while the endocytotic pits on the surface of the oocyte are conspicuous in their lack of the positively charged colloid, those on the surface of adhering follicle cells are conspicuously labeled with the iron. This suggests that follicle cells may be actively involved in the uptake of positively charged molecules. Such an interpretation is consonant ~with the evidence presented by Wallace et al. (30) that follicle cells do not play a direct role during vitellogenin uptake by the oocytes and thus are apparently not involved in the uptake of negatively charged molecules. It is also of interest t h a t the follicle cells
NEGATIVE CHARGES ON X E N O P U S OOCYTES
exhibit an uneven distribution of negatively charged sites. The significance of the greater density of negative charges (sialic acid moieties) on the surface distal to the oocyte, as compared with the surface proximal to the oocyte (including the macrovilli), is almost certain to reside in a functional differentiation in these two surfaces of the follicle cells. Our experiments provide no further information regarding the role of the follicle cells during oogenesis in Xenopus, but these data raise some interesting questions which are deserving of further study. CONCLUSIONS
On the basis of the data presented in this paper, we are inclined to believe that as the oocyte grows and develops with time, its surface topography changes not only morphologically, with the gradual formation and subsequent regression of microvilli and endocytotic pits, but also with respect to the molecular makeup of the plasmalemma. More specifically, as the Xenopus oocyte undergoes normal development within the ovary, there is evidence of a concomitant developmental change in the distribution of sialic acidbearing glycoproteins at the cell surface. A random distribution of such components during the early stages of oogenesis is followed by a preferential localization of the negatively charged moieties on the distal regions of the microvilli. This specificity of localization is most striking at that stage when the oocyte exhibits its greatest vitellogenic activity and presumably is playing some functional role in that activity; as the oocyte passes this peak, a return to the earlier randomized situation occurs. Our interpretation of these experimental results appears to be compatible with recently proposed models of the cell membrane as a fluid mosaic, with the possibility of lateral translation of molecules composing the membrane (22). The evidence strongly indicates, however, that such topographical changes in the distribution of
15
molecules composing the cell membrane are part of a regulated pattern that almost certainly reflects functional differences important in the processes of oocyte development and differentiation. This work was u n d e r t a k e n and completed while Dr. B r u m m e t t was on sabbatical leave from Oberlin College, Oberlin, Ohio, a n d a faculty participant in the Great Lakes Colleges Association-Oak Ridge Science Semester. The research was sponsored by the U. S. Energy Research and Development Administration under contract with the Union Carbide Corporation. REFERENCES 1. BENNETT, H. S., J. Histochem. Cytochem. 11, 14 (1963). 2. CHRISTNER, A., SCHAAF, P., MEYER, C., LINSS, W., AND GEYER, G., Acta Histochem. (Jena) 38, 121 (1970). 3. COOK, G. M. W., Biol. Rev. 43, 363 (1968). 4. COOK, G, M., HEARD, D. H. AND SEAMAN, G. V., Nature (London) 191, 44 (1961). 4. CURRAN~ R. C., CLARK, A. E., AND LOVELL, D., J. Anat. 3, 427 (1965). 6. DIBONA, D. R., CIVAN, M. M., AND LEAF, A., J.
Membrane Biol. 1, 79 (1969). 7. DUMONT, J. N., J. Morphol. 136, 153 (1972). 8. FLICKINGER, R. A., AND ROUNDS, D. E.~ Biochim. Biophys. Acta 22, 38 (1956). 9. GEYER, G., LINSS, W., AND SCHAAF, P., Acta Histochem. (Jena) 42, 138 (1972). 10. GLICK, J. L., AND GITHENS III, S., Nature (London) 201, 88 (1965). 11. GOTTSCRALK,A., Biochim. Biophys. Acta 23, 645 (1957). 12. JARED, D. W., AND WALLACE, R. A., Exp. Cell Res., 57, 454 (1969). 13. LUBIN, M., Fed. Proc. 23, 994, (1964). 14. MARIKOVSKY, Y,, AND DANON, D., J. Cell Biol. 43, 1 (1969). 15. MEYER, C., CHRISTNER, A., LINSS, W., QUADE, R., AND GEYER, G., Acta Histochem. (Jena) 39, 176 (1971). 16. MICHAEL, A. R., BLAU, E., AND VERNIER, R. L., Lab. Invest. 32, 649 (1970). 17. MowRY, R. W., Lab. Invest. 7, 566 (1958). 18. NICOLSON, G. L., J. Cell Biol. 57~ 373 (1973). 19. PISAM, M., RIPOCHE, P., AND RAMSOURG, A., C. R. Acad. Sci., Ser. D 271, 105 (1970). 20. QUINTON, P. M., AND PHILPOTT, C. W., J. Cell Biol. 56, 787 (1973). 21. SCOTT, W. N., AND SAPIRSTEIN, V. S., Science 184, 797 (1974). 22. SINGER, S. J., AND NICOLSON, G. L., Science 175, 720 (1972). 23. SMITH, L. D., AND ECKER, R. E., Develop. Biol.
16
BRUMMETT AND DUMONT
25, 232 (1971). 24. WALLACE,R. A., AND BERGINK, E. W., Amer. Zool. 14:1159 (1974). 25. WALLACE,R. A., AND DUMONT, J. N., J. Cell. Physiol. 72, Suppl., 73 (1968). 26. WALLACE, R. A., AND JARED, D. W., Develop. Biol. 19, 498 (1969). 27. WALLACE,R. A., JA~ED, D. W., AND NELSON, B. L., J. Exp. Zool. 175, 259 (1970). 28. WALLACE,R. A., JARED, D. W., DUMONT,J. N., AND SEGA, M. W., J. Exp. Zool. 184, 321 (1973). 29. WALLACE,R. A., AND Ho, T., J. Exp. Zool. 181, 303 (1972). 30. WALLACE,R. A., Ho, T., SALTER, D, W., AND
JARED, D. W., Exp. Cell Res. 82, 287 (1973). WEISS, L., J. Cell Biol. 26, 735 (1965). WEISS, L., Int. Rev. Cytol. 26, 63 (1969). WEISS, L., J. Nat. Cancer Inst. 50, 3 (1973). WEIss, L., AND SUBJECK,J. R., J. Cell Sci. 14, 215 (1974). 35. WEISS, L., ZEmEL, R., JUNG, O. S., AND BROSS,I.' D. J., Exp: Cell Res. 70, 57 (1972). 36. YANA~IMACHI,R., NmHOLSON, G. L., Noda, Y. D., AND Fujimoto, M., J. Ultrastruct. Res. 43, 344 (1973). 37, YAIWAGIMACHI,R., NODA, Y. D., FUJIMOTO, M., AND NICOLSON,G. L., Amer. J. Anat. 135, 497 (1972).
31. 32. 33. 34.