MOLECULAR REPRODUCTION AND DEVELOPMENT 28:79-84 (1991)
Further Observations on the Phagocytosis of Candida aZbicans by Hamster and Human Oocytes JAMES K. KOEHLER,* JUDY M. CLARK,' AND W. DIANNE SMITH' 'Department of Biological Structure, University of Washington, Seattle, Washington; and 'Reproductive Assays Laboratory, Tacoma, Washington ~
ABSTRACT Pathogenic yeast, Cundidu ulbicans, were incubated with hamster and human oocytes for up to 21 hours in order to determine the nature and time course of phagocytosis of these organisms. Aliquotes of the interacting cells were taken at various time intervals for electron microscopic examination. Some specimens had their zona pellucidae enzymatically removed prior to incubation with yeast, and these specimens showed the most extensive interaction and phagocytosis of Cundidu. The zona pellucida appears to be an effective barrier to yeast, at least over the time span studied. The observations are consistent with the hypothesis of an initial attachment of yeast via a surface component to oocyte microvilli followed by phagocytic uptake into an endosome. There is no compelling evidence of lysosomal degradation of the yeast over the time course of this study; however, the oocytes appear to undergo some degenerative changes at long incubation times.
hyaluronidase treatment, and further brief incubation in trypsin was used to produce zona free oocytes. Both zona intact and zona free oocytes were used in the present study. The medium used for oocyte and yeast incubation was a modified Tyrodes, as described in Clark et al. (19861, and all incubations were carried out at 37°C in either 7% C 0 2 or air incubators. Human eggs were obtained from the Reproductive Assays Laboratory a s soon as possible after collection. This time varied from several hours to a full day. These oocytes were supernumerary cells obtained during GIFT (gamete intrafallopian transfer) procedures and were used for research purposes with the patients' informed consent. Some human oocytes were also treated with enzymes, as were the hamster oocytes above, to remove extraneous coats. Incubation with yeast was in the same medium and incubators mentioned above. Control oocytes (both human and hamster) were incubated in the same manner as above in the absence of Candida.
Key Words: Yeast, Ultrastructure, Oocyte infec-
Candida albicans Source and Treatment Wild type strains of Candida albicans were obtained from fresh cultures from women with vulvovaginal infections. Oocyte culture medium was used to make a suspension of yeast scraped from a 5%sheep blood agar plate, and 0.1 cc of this suspension was added to a droplet (0.3 cc) containing several eggs under mineral oil. A small aliquot of the final solution was taken to determine the yeast concentration by counting with a hemocytometer. The yeast concentration in the final oocyte culture averaged 3 to 4 x lo6 per cc. Incubation periods varied from 112 to 21 hours.
tion
INTRODUCTION An earlier report from our laboratory demonstrated the phagocytic uptake of yeast cells (presumptive Candida albicans) by human oocytes in vivo (Koehler et al., 1987). In that study, the oocytes were obtained from a patient undergoing in vitro fertilization procedures. To our knowledge, this was the first report of a n interaction (phagocytosis andlor infection) between yeast and oocytes. In order to study this process under more controlled conditions and to assess the time course of the phagocytosis and describe other parameters of the infective process, we have undertaken a series of experiments using hamster oocytes as well a s additional human oocytes in culture. MATERIALS AND METHODS Oocyte Collection and Handling OocYtes from mature female golden hamsters (Mesocricetus auratus) were Obtained by superovu1ation as described in Clark e t al. (1986) and Clark and Koehler (1988). The oocytes were freed from egg masses by brief
0 1991 WILEY-LISS, INC.
Electron Microscopy Individual oocytes were removed from the incubation dishes, rinsed several times in fresh medium to remove
Received May 22, 1990; accepted June 12, 1990. Address reprint requests to Dr. James K. Koehler, Department of Biological Structure, SM-20, University of Washington, Seattle, WA 98195.
80
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Figs. 1-3.
PHAGOCYTOSIS OF CANDIDA ALBICANS TABLE 1. Time Course of Cundidu Incubations With Hamster Oocytes Incubation time Within Within (hour) Attached Internalized phagosome cytosol 112-1 NA NA 2-4 + + +
+
15-1 9
+
+
-
+
loosely adherent yeast, and placed in 1.5% glutaraldehyde buffered with 0.1 M cacodylate for a t least 1hour. The oocytes were postfixed in 1% OsO, in cacodylate buffer for a n additional hour, dehydrated in ethyl alcohols and propylene oxide, and embedded in Epon 812 (Luft, 1961). Sections were cut with a diamond knife on a Sorvall MT-11 ultramicrotome and were viewed and photographed in a Philips EM 420 electron microscope. Approximately 250 hamster eggs and 25 human eggs were used in the study.
RESULTS The time course of the interaction between zona free hamster oocytes and Candida albicans is summarized in Table 1. For simplicity, the data for three time periods are pooled, since no differences could be seen in cells incubated for 112 to 1, 2 to 4, and 15 to 19 hours. Even a t the earliest time period at which samples were analyzed (112 to 1 hour), yeast cells were adherent to the oocyte surface via the tips of elongated microvilli (Fig. 1).It can be seen that the association between the yeast cell wall and the microvillous plasma membrane are numerous and intimate. No yeast could be detected as having been internalized by the oocyte prior to 2 hours of incubation. At intermediate periods of yeast incubation ranging between 2 and 4 hours, Candida were found not only attached to the surface, but also internalized in endosoma1 vesicles (Figs. 2,3). Figure 2 illustrates a n elongated yeast cell in the initial process of internalization or phagocytosis. The yeast is surrounded by continuous oocyte plasma membrane, which is closely applied to the yeast, except at the apical region where the endosome has expanded into a vesicle. Sometimes the endosomes containing yeast have complex membrane
Fig. 1. Section showing portions of two Candida cells surrounded by and attached to elongate oocyte microvilli (arrows).Zona-free hamster oocyte incubated with Candida albicans for 1 hour. x 30,000. Fig. 2. An elongated Candida in the process of being internalized into a zona-free hamster oocyte after 3 112 hours in culture. Note the extensive microvillar activity a t the free surface of the endosome and the enlarged vesicle (*I anterior to the entering yeast. x 24,000. Fig. 3. Also after 3 112 hours of co-culture with Candida, a yeast cell is seen in the cortex of a zona-free hamster oocyte. Complex membranous swirls are associated with the endosomal vesicle surrounding the yeast cell (*I. x 27,000.
81
swirls and vesicular enlargements associated with them, as shown in Figure 3. Long-term incubations of 15 to 19 hours continue to show extensive external attachment of yeast as well as internalized forms (Fig. 4). In most cases, however, the endosomal membranes are no longer present a s continuous, intact membrane systems. Some remnants of these membranes are sometimes seen (Fig. 41, but generally the yeast are in continuity with the cytoplasm of the oocyte (Fig. 5). Note that the cytoplasm of the oocytes at these long incubation periods has the flocculated and disrupted appearance suggestive of partial degeneration, whereas the yeast cells appear about the same as in earlier incubation periods. Control oocytes incubated for such long periods in the absence of yeast also show similar signs of partial degeneration. No yeast were found to have breached the hamster zona pellucida in zona intact incubations for the time periods used, and such samples showed no membraneattached or internalized yeast cells in the oocytes. Insufficient human oocytes have been available to carry out detailed timing experiments like those with the hamster material. However, specimens that have been viewed suggest a similar time course and mechanism of attachment and internalization, including the degenerative aspects associated with long-term incubations. It must be kept in mind that the human oocytes utilized in these experiments were several to 24 hours old before they were available to us. In cases where the cumulus mass was still partially intact, it could be seen that yeast were actively taken up by the follicle cells (Fig. 6). Occasionally, yeast were found in the perivitelline space in zona intact cells (Fig. 7); however, i t is not clear that such oocytes did not have tears or imperfections in the zonae that might have admitted yeast. Candida were found in the cytoplasm of human oocytes particularly after long-term incubations of 15 to 20 hours (Fig. 8). These yeast were generally no longer surrounded by continuous endosomal vesicle membranes but were in direct continuity with ooplasm.
DISCUSSION The capacity of Candida to adhere to various cell types and the correlation of this activity to infectivity has been well documented (Douglas, 1987; Critchley and Douglas, 1987a). Candida produces a mannoprotein-adhesin, which has the capacity of binding to cell surface glycosides (Critchley and Douglas, 1987a). Glycosides containing L-fucose of N-acetyl-D-glucosamine seem to function a s binding sites in epithelial cells, depending on the strain of Candida used (Critchley and Douglas, 1987b). We found very rapid attachment of the yeast to zonafree oocytes, even at the earliest time sampled (112 hour), which would be consistent with such a lectinlike interaction. Phagocytosis of the yeast was not noted at this early time interval but was observed in samples taken after 2 hours or more in contact with the
82
J.K. KOEHLER ET AL.
Fig. 4. After 19 hours of incubation with zona-free hamster oocytes, yeast are still seen both attached to the surface and in internalized forms. The endosomal membrane is disrupted and presumptive vesicular remnants can be seen (arrows). The ooplasm appears flocculent and degenerative. x 16,500.
Fig. 5. An internalized yeast cell deep within the ooplasm of a hamster oocyte after 19 hours of incubation. No evidence of an endosoma1 membrane remains (arrows) and the ooplasm shows degenerative changes. x 40,000.
Candida. No clear morphological evidence of lysosomal fusion or similar defensive activity was seen in these endosomes containing yeast, even after 20 hours in culture. This finding is consistent with our earlier report on the human oocyte (Koehler et al., 1987) and also the studies of Thompson and Zamboni (1973) on early mouse blastomeres. In view of the fact that eggs are invested with several protective layers, including the zona pellucida and the granulosa cells, it is probably superfluous for them to also have a well-developed lysosomal defense system. This point is demonstrated by the finding of extensive phagocytosis of Candida by human granulosa cells in several samples in the present study. The present observations, however, cannot rule out the presence of a primitive lysosomal system in these oocytes. Zona intact oocytes were not as prone to yeast interactions as the zona free cells. In the limited time period investigated, yeast appeared to be unable to penetrate the hamster zona pellucida. Candida albicans is capable of producing extracellular proteinases and thus enhance their penetrability into tissues (Shimizu et al.,
1987). However, the time course of our experiments may not have been sufficient to allow for this activity to be expressed. Our initial observations (Koehler et al., 1987) indicated that yeast were able to breach the zona of the human oocyte, but this cell may have been in contact with yeast in the in-vivo situation for a long period of time before sampling took place. Shimizu et al. (1987) also noted that the proteinase production is strain dependent, and we presently have no data on the
Fig. 6. Human follicle cells surrounding an oocyte (not shown) after 15 hours incubation with Candida. Extensive uptake of yeast (arrows) by the follicle cells has occurred. x 6,600.
Fig. 7. Human oocyte with intact zona showing yeast in the perivitelline space 14 hours after initiation of culture with Candida. x 16,500. Fig. 8. Human oocyte cocultured with yeast for 15 hours, showing Candida (arrow) deep within the ooplasm. Although not obvious at this magnification, this yeast is not surrounded by an endosomal membrane. x 7,700.
PHAGOCYTOSIS OF CANDIDA ALBICANS
Figs. M.
83
84
J.K. KOEHLER ET AL.
enzyme producing ability of the strain used in our experiments. One of the difficulties encountered in this work involved the tendency of oocytes to undergo degenerative changes during longer incubation periods. It was clear that hamster oocytes cultured under our conditions for longer than 10 or 12 hours underwent slow degeneration. The situation with human cells was even more difficult, since the oocytes were some hours old before being received. These older cultures showed yeast in the cytosol free of their surrounding vacuolar membranes. It is not clear if this is the result of yeast activity such as the release of proteolytic enzymes capable of breaking down the membrane or if it is due to general degenerative changes in the oocyte. No signs of lysosomal activity on the part of the oocytes were seen which might account for such changes and in contrast to the oocytes, the yeast in these older cultures appeared to be quite healthy. It is also possible that Candida is capable of breaching and entering a partially degenerated oocyte directly without a n intermediate phagocytic step, thus coming immediately and directly in contact with ooplasm. As reported previously (Koehler et al., 19871, nonpathogenic yeast (Saccharomyces cerevisea) were not taken up by, or invasive to, zona-free hamster oocytes, although attachments between the cells were noted. Taken together with the present experiments, this suggests that the interaction with Candida has some specificity. In summary, the present observations confirm that Candida albicans are capable of binding to and being taken up by zona free hamster and human oocytes. Their presence in the cells does not appear to trigger lysosomal activity, and eventually they gain access to the cytosolic compartment of the oocytes and concomitant degeneration of the oocyte takes place. This degeneration may be enhanced by the presence of the internalized yeast but also occurs spontaneously in the longer-term cultures. Zona and granulosa layer intact cells seem to be protected against yeast uptake, a t least in the limited time frame investigated in this study.
ACKNOWLEDGMENTS Portions of this research were supported by a GSRF award from the University of Washington and the National Science Foundation (PCM-8408773). Dr. Sharon L. Hillier, Department of Obstetrics and Gynecology, kindly provided Candida cultures and the use of incubators and other facilities in her laboratory. Word processing and editorial assistance were provided by Ms. Marianna Wright.
REFERENCES Clark JM, Koehler J K , Smith WD (1986): Freezefracture observations of unfertilized and fertilized hamster oocytes with special reference to the use of lipid probes. Gamete Res 14:129-147. Clark JM, Koehler J K (1988): Does phospholipase C inhibit in-vitro fertilization in the hamster? Gamete Res 19:339-348. Critchley IA, Douglas LJ (1987a): Role of glycosides as epithelial cell receptors for Candida albicans. J Gen Microbiol 133:637-643. Critchley IA, Douglas LJ (198713): Isolation and partial characterization of a n adhesin from Candida albicans. J Gen Microbiol 133:629-636. Douglas LJ (1987): Adhesion of Candida albicans species to epithelial surfaces. CRC Crit Rev Microbiol 15~27-43. Koehler J K , Smith WD, Ravnik S (1987): Phagocytosis of yeast by human oocytes: Fine structural observations. Gamete Res 17:237-244. Luft J H (1961): Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol9:409-414. Shimizu K, Kondoh Y, Tanaka K (1987): Proteinase production and pathogenicity of Candida albicans I. Invasion into chorioallantoic membrane by C. albicans strains of different activity. Microbiol Immunol 31: 1045-1060. Thompson RS, Zamboni L (1973): Phagocytosis of supernumerary spermatozoa by two cell mouse embryos. Anat. Rec. 178:3-14.
Further Observations on the Phagocytosis of Candida aZbicans by Hamster and Human Oocytes JAMES K. KOEHLER,* JUDY M. CLARK,' AND W. DIANNE SMITH' 'Department of Biological Structure, University of Washington, Seattle, Washington; and 'Reproductive Assays Laboratory, Tacoma, Washington ~
ABSTRACT Pathogenic yeast, Cundidu ulbicans, were incubated with hamster and human oocytes for up to 21 hours in order to determine the nature and time course of phagocytosis of these organisms. Aliquotes of the interacting cells were taken at various time intervals for electron microscopic examination. Some specimens had their zona pellucidae enzymatically removed prior to incubation with yeast, and these specimens showed the most extensive interaction and phagocytosis of Cundidu. The zona pellucida appears to be an effective barrier to yeast, at least over the time span studied. The observations are consistent with the hypothesis of an initial attachment of yeast via a surface component to oocyte microvilli followed by phagocytic uptake into an endosome. There is no compelling evidence of lysosomal degradation of the yeast over the time course of this study; however, the oocytes appear to undergo some degenerative changes at long incubation times.
hyaluronidase treatment, and further brief incubation in trypsin was used to produce zona free oocytes. Both zona intact and zona free oocytes were used in the present study. The medium used for oocyte and yeast incubation was a modified Tyrodes, as described in Clark et al. (19861, and all incubations were carried out at 37°C in either 7% C 0 2 or air incubators. Human eggs were obtained from the Reproductive Assays Laboratory a s soon as possible after collection. This time varied from several hours to a full day. These oocytes were supernumerary cells obtained during GIFT (gamete intrafallopian transfer) procedures and were used for research purposes with the patients' informed consent. Some human oocytes were also treated with enzymes, as were the hamster oocytes above, to remove extraneous coats. Incubation with yeast was in the same medium and incubators mentioned above. Control oocytes (both human and hamster) were incubated in the same manner as above in the absence of Candida.
Key Words: Yeast, Ultrastructure, Oocyte infec-
Candida albicans Source and Treatment Wild type strains of Candida albicans were obtained from fresh cultures from women with vulvovaginal infections. Oocyte culture medium was used to make a suspension of yeast scraped from a 5%sheep blood agar plate, and 0.1 cc of this suspension was added to a droplet (0.3 cc) containing several eggs under mineral oil. A small aliquot of the final solution was taken to determine the yeast concentration by counting with a hemocytometer. The yeast concentration in the final oocyte culture averaged 3 to 4 x lo6 per cc. Incubation periods varied from 112 to 21 hours.
tion
INTRODUCTION An earlier report from our laboratory demonstrated the phagocytic uptake of yeast cells (presumptive Candida albicans) by human oocytes in vivo (Koehler et al., 1987). In that study, the oocytes were obtained from a patient undergoing in vitro fertilization procedures. To our knowledge, this was the first report of a n interaction (phagocytosis andlor infection) between yeast and oocytes. In order to study this process under more controlled conditions and to assess the time course of the phagocytosis and describe other parameters of the infective process, we have undertaken a series of experiments using hamster oocytes as well a s additional human oocytes in culture. MATERIALS AND METHODS Oocyte Collection and Handling OocYtes from mature female golden hamsters (Mesocricetus auratus) were Obtained by superovu1ation as described in Clark e t al. (1986) and Clark and Koehler (1988). The oocytes were freed from egg masses by brief
0 1991 WILEY-LISS, INC.
Electron Microscopy Individual oocytes were removed from the incubation dishes, rinsed several times in fresh medium to remove
Received May 22, 1990; accepted June 12, 1990. Address reprint requests to Dr. James K. Koehler, Department of Biological Structure, SM-20, University of Washington, Seattle, WA 98195.
80
J.K. KOEHLER ET AL.
Figs. 1-3.
PHAGOCYTOSIS OF CANDIDA ALBICANS TABLE 1. Time Course of Cundidu Incubations With Hamster Oocytes Incubation time Within Within (hour) Attached Internalized phagosome cytosol 112-1 NA NA 2-4 + + +
+
15-1 9
+
+
-
+
loosely adherent yeast, and placed in 1.5% glutaraldehyde buffered with 0.1 M cacodylate for a t least 1hour. The oocytes were postfixed in 1% OsO, in cacodylate buffer for a n additional hour, dehydrated in ethyl alcohols and propylene oxide, and embedded in Epon 812 (Luft, 1961). Sections were cut with a diamond knife on a Sorvall MT-11 ultramicrotome and were viewed and photographed in a Philips EM 420 electron microscope. Approximately 250 hamster eggs and 25 human eggs were used in the study.
RESULTS The time course of the interaction between zona free hamster oocytes and Candida albicans is summarized in Table 1. For simplicity, the data for three time periods are pooled, since no differences could be seen in cells incubated for 112 to 1, 2 to 4, and 15 to 19 hours. Even a t the earliest time period at which samples were analyzed (112 to 1 hour), yeast cells were adherent to the oocyte surface via the tips of elongated microvilli (Fig. 1).It can be seen that the association between the yeast cell wall and the microvillous plasma membrane are numerous and intimate. No yeast could be detected as having been internalized by the oocyte prior to 2 hours of incubation. At intermediate periods of yeast incubation ranging between 2 and 4 hours, Candida were found not only attached to the surface, but also internalized in endosoma1 vesicles (Figs. 2,3). Figure 2 illustrates a n elongated yeast cell in the initial process of internalization or phagocytosis. The yeast is surrounded by continuous oocyte plasma membrane, which is closely applied to the yeast, except at the apical region where the endosome has expanded into a vesicle. Sometimes the endosomes containing yeast have complex membrane
Fig. 1. Section showing portions of two Candida cells surrounded by and attached to elongate oocyte microvilli (arrows).Zona-free hamster oocyte incubated with Candida albicans for 1 hour. x 30,000. Fig. 2. An elongated Candida in the process of being internalized into a zona-free hamster oocyte after 3 112 hours in culture. Note the extensive microvillar activity a t the free surface of the endosome and the enlarged vesicle (*I anterior to the entering yeast. x 24,000. Fig. 3. Also after 3 112 hours of co-culture with Candida, a yeast cell is seen in the cortex of a zona-free hamster oocyte. Complex membranous swirls are associated with the endosomal vesicle surrounding the yeast cell (*I. x 27,000.
81
swirls and vesicular enlargements associated with them, as shown in Figure 3. Long-term incubations of 15 to 19 hours continue to show extensive external attachment of yeast as well as internalized forms (Fig. 4). In most cases, however, the endosomal membranes are no longer present a s continuous, intact membrane systems. Some remnants of these membranes are sometimes seen (Fig. 41, but generally the yeast are in continuity with the cytoplasm of the oocyte (Fig. 5). Note that the cytoplasm of the oocytes at these long incubation periods has the flocculated and disrupted appearance suggestive of partial degeneration, whereas the yeast cells appear about the same as in earlier incubation periods. Control oocytes incubated for such long periods in the absence of yeast also show similar signs of partial degeneration. No yeast were found to have breached the hamster zona pellucida in zona intact incubations for the time periods used, and such samples showed no membraneattached or internalized yeast cells in the oocytes. Insufficient human oocytes have been available to carry out detailed timing experiments like those with the hamster material. However, specimens that have been viewed suggest a similar time course and mechanism of attachment and internalization, including the degenerative aspects associated with long-term incubations. It must be kept in mind that the human oocytes utilized in these experiments were several to 24 hours old before they were available to us. In cases where the cumulus mass was still partially intact, it could be seen that yeast were actively taken up by the follicle cells (Fig. 6). Occasionally, yeast were found in the perivitelline space in zona intact cells (Fig. 7); however, i t is not clear that such oocytes did not have tears or imperfections in the zonae that might have admitted yeast. Candida were found in the cytoplasm of human oocytes particularly after long-term incubations of 15 to 20 hours (Fig. 8). These yeast were generally no longer surrounded by continuous endosomal vesicle membranes but were in direct continuity with ooplasm.
DISCUSSION The capacity of Candida to adhere to various cell types and the correlation of this activity to infectivity has been well documented (Douglas, 1987; Critchley and Douglas, 1987a). Candida produces a mannoprotein-adhesin, which has the capacity of binding to cell surface glycosides (Critchley and Douglas, 1987a). Glycosides containing L-fucose of N-acetyl-D-glucosamine seem to function a s binding sites in epithelial cells, depending on the strain of Candida used (Critchley and Douglas, 1987b). We found very rapid attachment of the yeast to zonafree oocytes, even at the earliest time sampled (112 hour), which would be consistent with such a lectinlike interaction. Phagocytosis of the yeast was not noted at this early time interval but was observed in samples taken after 2 hours or more in contact with the
82
J.K. KOEHLER ET AL.
Fig. 4. After 19 hours of incubation with zona-free hamster oocytes, yeast are still seen both attached to the surface and in internalized forms. The endosomal membrane is disrupted and presumptive vesicular remnants can be seen (arrows). The ooplasm appears flocculent and degenerative. x 16,500.
Fig. 5. An internalized yeast cell deep within the ooplasm of a hamster oocyte after 19 hours of incubation. No evidence of an endosoma1 membrane remains (arrows) and the ooplasm shows degenerative changes. x 40,000.
Candida. No clear morphological evidence of lysosomal fusion or similar defensive activity was seen in these endosomes containing yeast, even after 20 hours in culture. This finding is consistent with our earlier report on the human oocyte (Koehler et al., 1987) and also the studies of Thompson and Zamboni (1973) on early mouse blastomeres. In view of the fact that eggs are invested with several protective layers, including the zona pellucida and the granulosa cells, it is probably superfluous for them to also have a well-developed lysosomal defense system. This point is demonstrated by the finding of extensive phagocytosis of Candida by human granulosa cells in several samples in the present study. The present observations, however, cannot rule out the presence of a primitive lysosomal system in these oocytes. Zona intact oocytes were not as prone to yeast interactions as the zona free cells. In the limited time period investigated, yeast appeared to be unable to penetrate the hamster zona pellucida. Candida albicans is capable of producing extracellular proteinases and thus enhance their penetrability into tissues (Shimizu et al.,
1987). However, the time course of our experiments may not have been sufficient to allow for this activity to be expressed. Our initial observations (Koehler et al., 1987) indicated that yeast were able to breach the zona of the human oocyte, but this cell may have been in contact with yeast in the in-vivo situation for a long period of time before sampling took place. Shimizu et al. (1987) also noted that the proteinase production is strain dependent, and we presently have no data on the
Fig. 6. Human follicle cells surrounding an oocyte (not shown) after 15 hours incubation with Candida. Extensive uptake of yeast (arrows) by the follicle cells has occurred. x 6,600.
Fig. 7. Human oocyte with intact zona showing yeast in the perivitelline space 14 hours after initiation of culture with Candida. x 16,500. Fig. 8. Human oocyte cocultured with yeast for 15 hours, showing Candida (arrow) deep within the ooplasm. Although not obvious at this magnification, this yeast is not surrounded by an endosomal membrane. x 7,700.
PHAGOCYTOSIS OF CANDIDA ALBICANS
Figs. M.
83
84
J.K. KOEHLER ET AL.
enzyme producing ability of the strain used in our experiments. One of the difficulties encountered in this work involved the tendency of oocytes to undergo degenerative changes during longer incubation periods. It was clear that hamster oocytes cultured under our conditions for longer than 10 or 12 hours underwent slow degeneration. The situation with human cells was even more difficult, since the oocytes were some hours old before being received. These older cultures showed yeast in the cytosol free of their surrounding vacuolar membranes. It is not clear if this is the result of yeast activity such as the release of proteolytic enzymes capable of breaking down the membrane or if it is due to general degenerative changes in the oocyte. No signs of lysosomal activity on the part of the oocytes were seen which might account for such changes and in contrast to the oocytes, the yeast in these older cultures appeared to be quite healthy. It is also possible that Candida is capable of breaching and entering a partially degenerated oocyte directly without a n intermediate phagocytic step, thus coming immediately and directly in contact with ooplasm. As reported previously (Koehler et al., 19871, nonpathogenic yeast (Saccharomyces cerevisea) were not taken up by, or invasive to, zona-free hamster oocytes, although attachments between the cells were noted. Taken together with the present experiments, this suggests that the interaction with Candida has some specificity. In summary, the present observations confirm that Candida albicans are capable of binding to and being taken up by zona free hamster and human oocytes. Their presence in the cells does not appear to trigger lysosomal activity, and eventually they gain access to the cytosolic compartment of the oocytes and concomitant degeneration of the oocyte takes place. This degeneration may be enhanced by the presence of the internalized yeast but also occurs spontaneously in the longer-term cultures. Zona and granulosa layer intact cells seem to be protected against yeast uptake, a t least in the limited time frame investigated in this study.
ACKNOWLEDGMENTS Portions of this research were supported by a GSRF award from the University of Washington and the National Science Foundation (PCM-8408773). Dr. Sharon L. Hillier, Department of Obstetrics and Gynecology, kindly provided Candida cultures and the use of incubators and other facilities in her laboratory. Word processing and editorial assistance were provided by Ms. Marianna Wright.
REFERENCES Clark JM, Koehler J K , Smith WD (1986): Freezefracture observations of unfertilized and fertilized hamster oocytes with special reference to the use of lipid probes. Gamete Res 14:129-147. Clark JM, Koehler J K (1988): Does phospholipase C inhibit in-vitro fertilization in the hamster? Gamete Res 19:339-348. Critchley IA, Douglas LJ (1987a): Role of glycosides as epithelial cell receptors for Candida albicans. J Gen Microbiol 133:637-643. Critchley IA, Douglas LJ (198713): Isolation and partial characterization of a n adhesin from Candida albicans. J Gen Microbiol 133:629-636. Douglas LJ (1987): Adhesion of Candida albicans species to epithelial surfaces. CRC Crit Rev Microbiol 15~27-43. Koehler J K , Smith WD, Ravnik S (1987): Phagocytosis of yeast by human oocytes: Fine structural observations. Gamete Res 17:237-244. Luft J H (1961): Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol9:409-414. Shimizu K, Kondoh Y, Tanaka K (1987): Proteinase production and pathogenicity of Candida albicans I. Invasion into chorioallantoic membrane by C. albicans strains of different activity. Microbiol Immunol 31: 1045-1060. Thompson RS, Zamboni L (1973): Phagocytosis of supernumerary spermatozoa by two cell mouse embryos. Anat. Rec. 178:3-14.
