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Immunofluorescent Localisation of Cathepsin D in Trophoblastic Cells in Tissue Culture
Beitr. Path. Bd. I58, 445-449 (!976)
Short Communications
Charing Cross Hospital Medical School, Department of Obstetrics and Gynaecology, West London Hospital, London W6, Great Britain
Immunofluorescent Localisation of Cathepsin D in Trophoblastic Cells in Tissue Culture Immunofluoreszenzlokalisation von Kathepsin D in Trophoblastenzellen in der Gewebekultur S. F. CONTRACTOR and K. KRAKAUER1) With 1 Figure· Received Januar
22,
1976. Accepted in revised form March 15, 1976
Key words: Immunofluorescent localisation - Cathepsin D - Trophoblastic Cells - Tissue culture - Lyosomes
Summary Cathepsin D was localised by immunofluorescence within four different types of cells derived from human trophoblast in monolayer culture. Cells were stained with freshly reduced Fab' fragments of s,heep anti-(human cathepsin D) antiserum followed by FITC conjugate of pig anti-(sheep Fab') Fab' fragments. The green fluorescence seen in cytoplasmic droplets were characteristic of the appearance of lysosomes in other cells.
Abundant microvilli and pinocytic vesicles at the maternal surface of the syncytiotrophoblast indicate that human placenta is well adapted for endocytic transport. Lysosomal digestion of pinocytosed molecules is an important adjunct to the endocytic-vacuolar apparatus in a wide variety of cells. It was, therefore, thought possible that placental lysosomes may have an important role in digestion of endocytosed molecules for their utilisation by the placenta or foetus. We have used monolayer cultures of human trophoblastic cells for an investigation of the role of cathepsin D in the di1) Present adress: University of Connecticut Health Centre, Dept. of Medicine, Farmington, Conn., U.s.A.
446 . S. F. Contractor and K. Krakauer
gestion of endocytosed protein. In this paper we present immunofluorescent evidence for the presence of cathepsin D in lysosome-like organelles within these cells. The antibodies to both human and rabbit cathepsin D were those of Poole et al. (1972) who have investigated the specificity of these reagents in considerable detail. The method for culturing human trophoblast was modified from that of Fox and Kharkongor (1970). Human placentae were obtained within fifteen minutes of a normal term delivery. Cotyledons were sterilized by singeing with a plasterer's hot knife. Wedge-shaped sections of tissue from within the margins of these areas were removed and placed in sterile balanced salt solution Dulbecco, A, calcium and magnesium-free (BSS) (Oxoid Ltd., London). The burnt surfaces were removed and discarded while remalmng tissue was fragmented with forceps. Following several washes by decantation of BSS, the tissue fragments were suspended in 0,5% Bactotrypsin in BSS and trypsinized at 37° C for 40-60 minutes. This reaction was stopped by addition of an equal volume of Hank's solution and a final concentration of 1% newborn calf serum. Following filtration through two layers of surgical cotton, the cells were centrifuged (1,000 rpm, 10 minutes), washed once with Hank's solution and finally resuspended in 10% newborn calf serum in Medium 199 to a final concentration of I X 106 cells/ml. 1.0 ml of cells were grown in Leighton tubes, containing flying coverslips, at 37° C in a atmosphere of 5% CO2 in air. The medium was changed following overnight incubation and twice a week thereafter. For staining of cathepsin D, cells were fixed on day 7 of growth in 4% formaldehyde for 5 minutes. This short period of fixation was necessary in order to keep the non-specific autofluorescence to a suitably low level (Compare Fig. I a and I b with I c). Cathepsin D was localised in human trophoblastic cells in tissue culture by the method of Poole et al. (1972). Following fixation the cells were washed with three changes of 10 minutes each in 20 mM phosphate buffer
Fig. 1. Black and white reproductions of colour photographs showing immunofluorescent staining of cathepsin D in trophoblast cells fixed in 4% formaldehyde for 5 minutes. (a) and (b) Test. Stained with sheep anti-(human cathepsin D) Fab' followed by pig anti(sheep Fab') Fab' labelled with fluorescein isothiocynate. c) Control. Stained with sheep anti-(rabbit cathepsin D) Fab' followed by pig anti-(sheep Fab') Fab' labeled with fluorescein isothiocyanate. The enzyme activity can be seen localised in discrete cytoplasmic droplets (a) a multinucleated cell and a small epithelioid cell directly above it, and (b) a spindle-shaped fibroblast and a large ovoid cell. (c) Control shows autofluorescence.
Localisation of Cathepsin D . 447
Fig. I a
Fig. I b
Fig.
r C
44 8 . S. F. Contractor and K. Krakauer
at pH 7.2 in 0.15 M sodium chloride containing 5 mM cysteine (PBS-C). The cells were reacted with PBS-C containing reduced Fab' fragments from sheep anti-(human cathepsin D) antiserum; sheep anti-(rabbit cathepsin D) antiserum was used as control. The reaction time was 30 minutes at room temperature. The preparation was then washed by immersing in PBSC for 30 minutes and finally stained by incubation with fluorescein isothiocyanate conjugate of pig anti-(sheep Fab') Fab' fragments for 30 minutes at room temperature. The reaction was stopped by washing three times for 30 minutes with PBS-C and once with distilled water. It was mounted in 1 part of 0.2 M Tris buffer pH 8.5 + 9 parts of glycerol and looked at immediately in a Reichert fluorescence microscope. GAF colour film was exposed with Olsen filters BG 12 at excitation source and matched barrier filter (525) and BG 38. Cultures of trophoblastic cells were composed of four cell types which Fox and Kharkongor (1970) have histochemically related to counterparts in the intact tissue: (1) multinucleate cells, derived from syncytium; (2) epithelioid or medium-sized round cells, derived from cytotrophoblast; (3) large ovoid cells, derived from Hofbauer cells; and (4) fibroblasts, either spindle or stellate-shaped, of stromal or endothelial origin. Figures 1 a and 1 b illustrate the immunofluorescent staining pattern of cathepsin D in these cells. All cell types present in the cultures showed an intense yellow green fluorescence in discrete cytoplasmic droplets, together with some general diffuse staining. The control (Fig. 1 c) showed a dim green autofluorescence characteristic of these cells under these conditions. These droplets are characteristic of the appearance of lysosomes in other cells. These studies revealed that lysosomal cathepsin D was present in cultured trophoblastic cells which could be used as a model to investigate pinocytosis and intracellular digestion of proteins by trophoblast, with specific reference to cathepsin D.
Zusammenfassung Kathepsin D wurde durch Immunofluoreszenz innerhalb vier verschiedener Zellarten festgestellt, die von humanem Trophoblast in Monoschichtkulmr gewonnen waren. Die Zellen wurden mit frisch reduzierten Fab'-Fragmenten von Schaf-(anti-humanem Cathepsin D-)Antiserum, danach mit FITC, konjugiert mit Schweine-Anti-(Schaf Fab')Fragmen ten, gefarbt. Die Orte der griinen Fluoreszenz in den zytoplasmischen Tropfchen entsprechen den Lyosomen in anderen Zellen. Acknowledgements: We would like to thank Dr. Robin Poole of Strangeways Laboratories, Cambridge, for providing us with the necessary materials for immunostaining and also for showing us the technique.
Localisation of Cathepsin D . 449
References Fox, H., and Khargongor, F. N.: Morphology and Enzyme Histochemistry of Cells derived from Placental Villi in Tissue Culture. J. Path. 101, 267-276 (1970) Poole, A. H., Dingle, J. T., and Barrett, A. J.: The Immunocytochemical Demonstration of Cathepsin D. Journal of Histochemistry and Cytochemistry 20,261-265 (1972) Dr. S. F. Contractor, Charing Cross Hospital Medical School, Dept. of Obstetrics and Gynaecology, West London Hospital, Hammersmith Road, London W6, Great Britain.
Short Communications
Charing Cross Hospital Medical School, Department of Obstetrics and Gynaecology, West London Hospital, London W6, Great Britain
Immunofluorescent Localisation of Cathepsin D in Trophoblastic Cells in Tissue Culture Immunofluoreszenzlokalisation von Kathepsin D in Trophoblastenzellen in der Gewebekultur S. F. CONTRACTOR and K. KRAKAUER1) With 1 Figure· Received Januar
22,
1976. Accepted in revised form March 15, 1976
Key words: Immunofluorescent localisation - Cathepsin D - Trophoblastic Cells - Tissue culture - Lyosomes
Summary Cathepsin D was localised by immunofluorescence within four different types of cells derived from human trophoblast in monolayer culture. Cells were stained with freshly reduced Fab' fragments of s,heep anti-(human cathepsin D) antiserum followed by FITC conjugate of pig anti-(sheep Fab') Fab' fragments. The green fluorescence seen in cytoplasmic droplets were characteristic of the appearance of lysosomes in other cells.
Abundant microvilli and pinocytic vesicles at the maternal surface of the syncytiotrophoblast indicate that human placenta is well adapted for endocytic transport. Lysosomal digestion of pinocytosed molecules is an important adjunct to the endocytic-vacuolar apparatus in a wide variety of cells. It was, therefore, thought possible that placental lysosomes may have an important role in digestion of endocytosed molecules for their utilisation by the placenta or foetus. We have used monolayer cultures of human trophoblastic cells for an investigation of the role of cathepsin D in the di1) Present adress: University of Connecticut Health Centre, Dept. of Medicine, Farmington, Conn., U.s.A.
446 . S. F. Contractor and K. Krakauer
gestion of endocytosed protein. In this paper we present immunofluorescent evidence for the presence of cathepsin D in lysosome-like organelles within these cells. The antibodies to both human and rabbit cathepsin D were those of Poole et al. (1972) who have investigated the specificity of these reagents in considerable detail. The method for culturing human trophoblast was modified from that of Fox and Kharkongor (1970). Human placentae were obtained within fifteen minutes of a normal term delivery. Cotyledons were sterilized by singeing with a plasterer's hot knife. Wedge-shaped sections of tissue from within the margins of these areas were removed and placed in sterile balanced salt solution Dulbecco, A, calcium and magnesium-free (BSS) (Oxoid Ltd., London). The burnt surfaces were removed and discarded while remalmng tissue was fragmented with forceps. Following several washes by decantation of BSS, the tissue fragments were suspended in 0,5% Bactotrypsin in BSS and trypsinized at 37° C for 40-60 minutes. This reaction was stopped by addition of an equal volume of Hank's solution and a final concentration of 1% newborn calf serum. Following filtration through two layers of surgical cotton, the cells were centrifuged (1,000 rpm, 10 minutes), washed once with Hank's solution and finally resuspended in 10% newborn calf serum in Medium 199 to a final concentration of I X 106 cells/ml. 1.0 ml of cells were grown in Leighton tubes, containing flying coverslips, at 37° C in a atmosphere of 5% CO2 in air. The medium was changed following overnight incubation and twice a week thereafter. For staining of cathepsin D, cells were fixed on day 7 of growth in 4% formaldehyde for 5 minutes. This short period of fixation was necessary in order to keep the non-specific autofluorescence to a suitably low level (Compare Fig. I a and I b with I c). Cathepsin D was localised in human trophoblastic cells in tissue culture by the method of Poole et al. (1972). Following fixation the cells were washed with three changes of 10 minutes each in 20 mM phosphate buffer
Fig. 1. Black and white reproductions of colour photographs showing immunofluorescent staining of cathepsin D in trophoblast cells fixed in 4% formaldehyde for 5 minutes. (a) and (b) Test. Stained with sheep anti-(human cathepsin D) Fab' followed by pig anti(sheep Fab') Fab' labelled with fluorescein isothiocynate. c) Control. Stained with sheep anti-(rabbit cathepsin D) Fab' followed by pig anti-(sheep Fab') Fab' labeled with fluorescein isothiocyanate. The enzyme activity can be seen localised in discrete cytoplasmic droplets (a) a multinucleated cell and a small epithelioid cell directly above it, and (b) a spindle-shaped fibroblast and a large ovoid cell. (c) Control shows autofluorescence.
Localisation of Cathepsin D . 447
Fig. I a
Fig. I b
Fig.
r C
44 8 . S. F. Contractor and K. Krakauer
at pH 7.2 in 0.15 M sodium chloride containing 5 mM cysteine (PBS-C). The cells were reacted with PBS-C containing reduced Fab' fragments from sheep anti-(human cathepsin D) antiserum; sheep anti-(rabbit cathepsin D) antiserum was used as control. The reaction time was 30 minutes at room temperature. The preparation was then washed by immersing in PBSC for 30 minutes and finally stained by incubation with fluorescein isothiocyanate conjugate of pig anti-(sheep Fab') Fab' fragments for 30 minutes at room temperature. The reaction was stopped by washing three times for 30 minutes with PBS-C and once with distilled water. It was mounted in 1 part of 0.2 M Tris buffer pH 8.5 + 9 parts of glycerol and looked at immediately in a Reichert fluorescence microscope. GAF colour film was exposed with Olsen filters BG 12 at excitation source and matched barrier filter (525) and BG 38. Cultures of trophoblastic cells were composed of four cell types which Fox and Kharkongor (1970) have histochemically related to counterparts in the intact tissue: (1) multinucleate cells, derived from syncytium; (2) epithelioid or medium-sized round cells, derived from cytotrophoblast; (3) large ovoid cells, derived from Hofbauer cells; and (4) fibroblasts, either spindle or stellate-shaped, of stromal or endothelial origin. Figures 1 a and 1 b illustrate the immunofluorescent staining pattern of cathepsin D in these cells. All cell types present in the cultures showed an intense yellow green fluorescence in discrete cytoplasmic droplets, together with some general diffuse staining. The control (Fig. 1 c) showed a dim green autofluorescence characteristic of these cells under these conditions. These droplets are characteristic of the appearance of lysosomes in other cells. These studies revealed that lysosomal cathepsin D was present in cultured trophoblastic cells which could be used as a model to investigate pinocytosis and intracellular digestion of proteins by trophoblast, with specific reference to cathepsin D.
Zusammenfassung Kathepsin D wurde durch Immunofluoreszenz innerhalb vier verschiedener Zellarten festgestellt, die von humanem Trophoblast in Monoschichtkulmr gewonnen waren. Die Zellen wurden mit frisch reduzierten Fab'-Fragmenten von Schaf-(anti-humanem Cathepsin D-)Antiserum, danach mit FITC, konjugiert mit Schweine-Anti-(Schaf Fab')Fragmen ten, gefarbt. Die Orte der griinen Fluoreszenz in den zytoplasmischen Tropfchen entsprechen den Lyosomen in anderen Zellen. Acknowledgements: We would like to thank Dr. Robin Poole of Strangeways Laboratories, Cambridge, for providing us with the necessary materials for immunostaining and also for showing us the technique.
Localisation of Cathepsin D . 449
References Fox, H., and Khargongor, F. N.: Morphology and Enzyme Histochemistry of Cells derived from Placental Villi in Tissue Culture. J. Path. 101, 267-276 (1970) Poole, A. H., Dingle, J. T., and Barrett, A. J.: The Immunocytochemical Demonstration of Cathepsin D. Journal of Histochemistry and Cytochemistry 20,261-265 (1972) Dr. S. F. Contractor, Charing Cross Hospital Medical School, Dept. of Obstetrics and Gynaecology, West London Hospital, Hammersmith Road, London W6, Great Britain.