One-cell and two-cell embryos from three random-bred strains of mice--CF1, Dub:(ICR), and CFW (Swiss-Webster)--were cultured to the blastocyst stage in Spindle's, Earle's, Ham's F10, Whittingham's T6, or Hoppe and Pitts' medium. CFW embryos were more successful than CF1 and Dub:(ICR) embryos in developing to the blastocyst stage in all five media. Dub:(ICR) and CFW two-cell embryos showed the best development in Spindle's, Whittingham's T6, and Hoppe and Pitts', whereas CF1 two-cell embryos were most successful in developing in Hoppe and Pitts' medium. Similar results were obtained with one-cell embryos, although fewer developed to the blastocyst stage, and T6 rather than Hoppe and Pitts' medium sustained the best development of CF1 one-cell embryos. For all strains, the least successful development was in Ham's F10, but CFW embryos did show good development in this medium. In addition to the effects of various media on mouse embryo development, our results indicate that the strain of mouse used for the bioassay of media is of critical importance. Random-bred CFW (Swiss-Webster) mice are as suitable as a hybrid strain for this purpose.
Conventional serodiagnosis of Pythium insidiosum infections involves the use of the immunodiffusion (ID) test. This test specifically diagnoses human and animal pythiosis. The test, however, has limited sensitivity and does not detect some culturally proven cases of the disease. Because of the increased recognition of pythiosis among humans and animals, we developed and evaluated an enzyme-linked immunosorbent assay (ELISA) using a soluble antigen from broken hyphae of P. insidiosum. Studies were carried out with sera from five humans and eight animals with culturally and/or histologically proven pythiosis. Some of these sera were negative in the ID test for pythiosis. Heterologous case sera from thirteen humans and two horses, plus 5 sera from healthy humans and 17 from healthy animals, were tested. Of the pythiosis case sera tested, the ID test detected only 8 of 13 (61.5%), whereas the ELISA detected all of them (100%). The ID and ELISA tests were entirely specific and gave negative results or low titers respectively, with sera from humans and animals with heterologous fungal infections or with no apparent illness. No correlation was found between the height of the ELISA titers and negative or positive sera in the ID test. Our results indicate that the ELISA is a reliable serodiagnostic test for pythiosis. It is as specific as the ID test but more sensitive.
Hatching, attachment, and trophoblast outgrowth of mouse embryos in vitro were examined as a model for implantation. Mouse embryos attached and grew out on glass cover slips that were partially covered with cultured mouse cells (L cells, liver cells, transformed JLS-V11 cells, and teratocarcinoma cells). Scanning electron microscopy showed that processes of these cells made contact with trophoblast, but there was no evidence of cell lysis or of phagocytosis of the cells by trophoblast. Time-lapse cinematography showed that after contact the cultured mouse cells retracted from the trophoblast, which then spread into the areas vacated by those cells. This suggests a means by which the trophoblast gains entry into the endometrium without destruction of maternal cells.Neuraminidase (100 or 250 unitdml) had no effect on attachment of mouse embryos to glass. However, attachment was inhibited by trypsin a t concentrations of 0.25%, 0.025%, and 0.0025%. Treatment of early blastocysts with diazo0x0-norleucine, a n inhibitor of glycoprotein synthesis, decreased the number of embryos hatching from the zona pellucida; treatment a t the late blastocyst stage decreased hatching to a lesser extent. Among the late blastocysts that did hatch, the number forming trophoblast outgrowths was lower than in controls. These results suggest that glycoproteins may be of importance for embryo hatching, attachment, and outgrowth.Adherence of the embryo to the uterine epithelium can be considered the initial stage of implantation, and it is a consistent feature in all mammalian species studied to date (Schlafke and Enders, '75). In the rodent, adhesion is followed by movement of trophoblast into the endometrium (Schlafke and Enders, '75). Despite the importance of these interactions, little is known about them beyond the information provided by morphological studies (Enders, '75).When allowed to develop in vitro the mouse blastocyst attaches to glass or plastic. After attachment the trophoblast grows out on the substratum and the inner cell mass develops into primary germ layers that are morphologically comparable to those seen with embryo development in vivo during the peri-implantation period. The cultured mouse embryo has consequently been used as a model to study embryo development and the interaction of
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