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Sequential Changes Occurring on the Surface of Entamoeba invadens During Encystation Observed by Scanning Electron Microscopy
Archives of Medical Research 31 (2000) S200–S201
Sequential Changes Occurring on the Surface of Entamoeba invadens During Encystation Observed by Scanning Electron Microscopy Pilar Carranza-Rosales,*,*** Salvador Said-Fernández,* José Ruiz-Ordoñez,** Licet Villarreal-Treviño,*** Jorge Verduzco-Martínez,*** Delia E. Cruz-Vega* and Mario Morales-Vallarta*** *División de Biología Celular y Molecular, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social (IMSS), Monterrey, Nuevo León, Mexico **Departamento de Histología, Facultad de Medicina, Universidad Autónoma de Nuevo León (UANL), Monterrey, Nuevo León, Mexico ***Departamento de Biología Celular, Facultad de Ciencias Biológicas, UANL, San Nicolás de los Garza, Nuevo León, Mexico
Key Words: Entamoeba invadens, Encystation, Cyst surface, Scanning electron microscopy.
Introduction Cervantes-Mamoa and Martínez-Palomo (1) described the entire life cycle of Entamoeba invadens under axenic conditions. Nevertheless, the sequential changes occurring on the surface of amebas during cyst formation and maturation had not been described. In the present work we describe those changes, observed with the scanning electron microscope.
Materials and Methods Entamoeba invadens strain IP-1 was used in the present study. Twenty-seven 16 ⫻ 125-mm glass screwcap culture tubes containing 11 mL TPS-1 medium (2) were inoculated with 1 ⫻ 103 trophozoites/mL and incubated at room temperature for 5 days. The tubes were chilled in ice water for 10 min and centrifuged at 600 ⫻ g for 10 min at 4⬚C. The trophozoites were washed twice with two volumes of phosphate-buffered saline, pH 7.4 (PBS) and 5 ⫻ 105 amebas/ mL were inoculated in 13 ⫻ 100-mm screwcap tubes containing 5 mL encystation medium AEM (3) and incubated at room temperature for variable periods of time (0–48 h). Afterward, the amebas were harvested, washed twice with 3 mL PBS, fixed with 2.5% glutaraldehyde in PBS, and dehydrated with 50–100% ethanol. Amebas maintained in AEM for 48 h were incubated in 0.1 N HCl (5 min) and 0.1% Nonidet P-40 (30 min) before fixing and dehydrating. Then,
Address reprint requests to: Salvador Said-Fernández, Centro de Investigación Biomédica del Noreste, Av. 2 de Abril y San Luis Potosí, Colonia Independencia, 64720 Monterrey, Nuevo León, México. Telefax: (⫹52) (8) 190-4035; E-mail: [email protected] Presenting author: Mario Morales-Vallarta.
a drop from each preparation was placed on a 5 mm2 coverglass, dried with a Jeol JCPD-5 critical-point drier (JEOL Co., Tokyo, Japan), and covered with a 70-nm thick ionized gold film. The latter was performed with a JEOL JFC1100E ion sputtering device operated at 15–20 kV (3 min/ sample). The preparations were observed and photographed with a JEOL JSM-35 C scanning electron microscope operated at 1–15 kV.
Results and Discussion Trophozoites growing in TPS-1 medium showed typical pseudopodia and a smooth surface (Figure 1A). After 6 h incubation in AEM, the amebas clustered, adopted a rounded shape, and reduced their cellular volume from an average of 22 to ca. 8 m, maintaining their smooth surface (Figure 1B). At this stage, several typical pinocytic vesicles were observed on the encysting amebic surface. Within the next 6 h, numerous rounded protrusions appeared, as well as thick and long cord-like structures on the encysting amebic surface (Figure 1C). Each cord-like structure had numerous branches joining several others, forming a web. While the encystation progressed, more similar structures appeared. With time, the ameba was completely covered with them (Figure 1D). The entire encystation process lasted 48 h (Figure 1D). During encystation, the amebas clustered in large aggregates (Figures 1B–D), and many showed a cavity on their surface, sized approximately 2.0–2.5 m in diameter (data not shown). The cord-like structures that appeared during E. invadens encystation could be made of chitin (4) and the buttons observed in the earliest encystation stages could correspond to centers of chitin synthesis. Nevertheless, studies to confirm this hypothesis must be performed.
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 2 0 7 - 1
XIV Seminar on Amebiasis, Mexico City / Archives of Medical Research 31 (2000) S200–S201
S201
identified on the cyst surface. This material could grow on the surface of contiguous cells, covering all of them. Metacystic amebas from several species emerge through a specific cyst wall structure called operculum, which is a cavity located on the cyst wall. Thus, the cavity we observed in many cysts could be their operculum.
Acknowledgments We thank A. Luna de la Rosa and F. Treviño-González for their photographic assistance. We are further grateful to VITROTEC, Monterrey, Nuevo León, Mexico, for use of their scanning electron microscope facilities. This study was partially supported by Conacyt (Mexico) Grant No. PCSABNA-021534.
Figure 1. Sequential changes occurring on the Entamoeba invadens surface during encystation. (A) Trophozoites grown in TPS-1 medium. Bar ⫽ 10 m. (B) Two amebas after 6 h incubation in AEM medium, showing several pinocytic mouths (arrowheads). Bar ⫽ 1 m. (C) A cluster of encysting amebas showing different stages of differentiation. Arrowheads point out the buttons often observed on the surface of amebas that are in the early phases of encystation. Bar ⫽ 10 m. (D) Mature cysts after 48 h incubation in AEM medium. Bar ⫽ 1 m.
Whenever E. invadens was induced to encyst, several clusters of variable numbers of cysts (2 to ⬎50) were formed. The cysts were bound together by the same material
References 1. Cervantes Mamoa A, Martínez-Palomo A. Estudio del ciclo vital de Entamoeba invadens mediante cinematografía espaciada. Arch Invest Med (Mex) 1980;11(Suppl 1):31. 2. Diamond LS. Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and Entamoeba histolytica like amebae. J Parasitol 1968;54:1047. 3. Rengpien S, Bailey GB. Differentiation of Entamoeba: a new medium and optimal conditions for axenic encystation of Entamoeba invadens. J Parasitol 1975;61:24. 4. Arroyo Begovich A, Carabez Trejo A, Ruíz Herrera J. Identification of the structural component in the cyst wall of Entamoeba invadens. J Parasitol 1980;66:735.
Sequential Changes Occurring on the Surface of Entamoeba invadens During Encystation Observed by Scanning Electron Microscopy Pilar Carranza-Rosales,*,*** Salvador Said-Fernández,* José Ruiz-Ordoñez,** Licet Villarreal-Treviño,*** Jorge Verduzco-Martínez,*** Delia E. Cruz-Vega* and Mario Morales-Vallarta*** *División de Biología Celular y Molecular, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social (IMSS), Monterrey, Nuevo León, Mexico **Departamento de Histología, Facultad de Medicina, Universidad Autónoma de Nuevo León (UANL), Monterrey, Nuevo León, Mexico ***Departamento de Biología Celular, Facultad de Ciencias Biológicas, UANL, San Nicolás de los Garza, Nuevo León, Mexico
Key Words: Entamoeba invadens, Encystation, Cyst surface, Scanning electron microscopy.
Introduction Cervantes-Mamoa and Martínez-Palomo (1) described the entire life cycle of Entamoeba invadens under axenic conditions. Nevertheless, the sequential changes occurring on the surface of amebas during cyst formation and maturation had not been described. In the present work we describe those changes, observed with the scanning electron microscope.
Materials and Methods Entamoeba invadens strain IP-1 was used in the present study. Twenty-seven 16 ⫻ 125-mm glass screwcap culture tubes containing 11 mL TPS-1 medium (2) were inoculated with 1 ⫻ 103 trophozoites/mL and incubated at room temperature for 5 days. The tubes were chilled in ice water for 10 min and centrifuged at 600 ⫻ g for 10 min at 4⬚C. The trophozoites were washed twice with two volumes of phosphate-buffered saline, pH 7.4 (PBS) and 5 ⫻ 105 amebas/ mL were inoculated in 13 ⫻ 100-mm screwcap tubes containing 5 mL encystation medium AEM (3) and incubated at room temperature for variable periods of time (0–48 h). Afterward, the amebas were harvested, washed twice with 3 mL PBS, fixed with 2.5% glutaraldehyde in PBS, and dehydrated with 50–100% ethanol. Amebas maintained in AEM for 48 h were incubated in 0.1 N HCl (5 min) and 0.1% Nonidet P-40 (30 min) before fixing and dehydrating. Then,
Address reprint requests to: Salvador Said-Fernández, Centro de Investigación Biomédica del Noreste, Av. 2 de Abril y San Luis Potosí, Colonia Independencia, 64720 Monterrey, Nuevo León, México. Telefax: (⫹52) (8) 190-4035; E-mail: [email protected] Presenting author: Mario Morales-Vallarta.
a drop from each preparation was placed on a 5 mm2 coverglass, dried with a Jeol JCPD-5 critical-point drier (JEOL Co., Tokyo, Japan), and covered with a 70-nm thick ionized gold film. The latter was performed with a JEOL JFC1100E ion sputtering device operated at 15–20 kV (3 min/ sample). The preparations were observed and photographed with a JEOL JSM-35 C scanning electron microscope operated at 1–15 kV.
Results and Discussion Trophozoites growing in TPS-1 medium showed typical pseudopodia and a smooth surface (Figure 1A). After 6 h incubation in AEM, the amebas clustered, adopted a rounded shape, and reduced their cellular volume from an average of 22 to ca. 8 m, maintaining their smooth surface (Figure 1B). At this stage, several typical pinocytic vesicles were observed on the encysting amebic surface. Within the next 6 h, numerous rounded protrusions appeared, as well as thick and long cord-like structures on the encysting amebic surface (Figure 1C). Each cord-like structure had numerous branches joining several others, forming a web. While the encystation progressed, more similar structures appeared. With time, the ameba was completely covered with them (Figure 1D). The entire encystation process lasted 48 h (Figure 1D). During encystation, the amebas clustered in large aggregates (Figures 1B–D), and many showed a cavity on their surface, sized approximately 2.0–2.5 m in diameter (data not shown). The cord-like structures that appeared during E. invadens encystation could be made of chitin (4) and the buttons observed in the earliest encystation stages could correspond to centers of chitin synthesis. Nevertheless, studies to confirm this hypothesis must be performed.
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 2 0 7 - 1
XIV Seminar on Amebiasis, Mexico City / Archives of Medical Research 31 (2000) S200–S201
S201
identified on the cyst surface. This material could grow on the surface of contiguous cells, covering all of them. Metacystic amebas from several species emerge through a specific cyst wall structure called operculum, which is a cavity located on the cyst wall. Thus, the cavity we observed in many cysts could be their operculum.
Acknowledgments We thank A. Luna de la Rosa and F. Treviño-González for their photographic assistance. We are further grateful to VITROTEC, Monterrey, Nuevo León, Mexico, for use of their scanning electron microscope facilities. This study was partially supported by Conacyt (Mexico) Grant No. PCSABNA-021534.
Figure 1. Sequential changes occurring on the Entamoeba invadens surface during encystation. (A) Trophozoites grown in TPS-1 medium. Bar ⫽ 10 m. (B) Two amebas after 6 h incubation in AEM medium, showing several pinocytic mouths (arrowheads). Bar ⫽ 1 m. (C) A cluster of encysting amebas showing different stages of differentiation. Arrowheads point out the buttons often observed on the surface of amebas that are in the early phases of encystation. Bar ⫽ 10 m. (D) Mature cysts after 48 h incubation in AEM medium. Bar ⫽ 1 m.
Whenever E. invadens was induced to encyst, several clusters of variable numbers of cysts (2 to ⬎50) were formed. The cysts were bound together by the same material
References 1. Cervantes Mamoa A, Martínez-Palomo A. Estudio del ciclo vital de Entamoeba invadens mediante cinematografía espaciada. Arch Invest Med (Mex) 1980;11(Suppl 1):31. 2. Diamond LS. Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and Entamoeba histolytica like amebae. J Parasitol 1968;54:1047. 3. Rengpien S, Bailey GB. Differentiation of Entamoeba: a new medium and optimal conditions for axenic encystation of Entamoeba invadens. J Parasitol 1975;61:24. 4. Arroyo Begovich A, Carabez Trejo A, Ruíz Herrera J. Identification of the structural component in the cyst wall of Entamoeba invadens. J Parasitol 1980;66:735.