Endocytosis of EGF-receptor complexes at various cell cycle stages

Parameters of EGF-receptor complex endocytosis have been studied in the early and late G1 phase and in mitosis. As a model, mouse mammary epithelial cells HC11 were used, whose growth depends on EGF presence in the medium. The Scatchard analysis has demonstrated that the surface receptors are represented by two receptor populations: 4800 high affinity (KD = 10(-11) M) receptors, and 73,000 low affinity (KD = 4.10(-9) M) receptors. Incubation of cells with the growth factor (5 ng/ml) resulted in a decrease in 125I-EGF binding, with its level being low until entering the S-phase. Under these conditions, receptors disposed on the plasma membrane presented a homogeneous population (KD = 8.10(-11) M, 14,000 receptors per cell). No reliable difference was revealed between the EGF-receptor complexes, internalized in early and late G1 phases, in respect to the internalization rate, level of recycling, degradation, and dynamics of compartmentalization. However, endocytosis of EGF-receptor complexes was found to be completely blocked in mitosis at the stage of internalization.     

Delayed incorporation of H3-thymidine by primordial cells

Roots of Vicia faba were treated with H³-thymidine (1 c/ml) for 1 h and incorporation of H³-TdR into nuclei of primordia was studied. Large primordia (>1,500 cells) did not incorporate H³-TdR. Cells of small primordia did not incorporate the labeled precursor immediately but did so after a delay of several hours. The frequency of labeled nuclei became similar to that of lateral meristems only after a delay of 12–14 h. The gradual increase in labeling index also occurs in colchicine treated cells, which do not divide; this shows that the rise in labeling index is not due solely to the division of labeled cells. It has been estimated from H³-TdR and colchicine labeled cells that small primordia have a population of cells in which intermitotic time is about 12 h. The delay in incorporation appears to indicate that pools of H³-TdR can be maintained in small primordia for several hrs and that the precursor is not used until the cells synthesize thymidine kinase.This research has been supported by the U.S.A.E.C. [Grant AT (11-1) 1625-12].     

Method for selecting cells during various phases of the mitotic cycle

Exponentially growing L5178Y cells in suspension culture were separated according to their position in the cell cycle on the basis of their volume with a velocity sedimentation method in which a linear and continuous ficoll gradient was used. Highly purified populations of G1 and S cells were obtained, containing about 90% G1 phase cells and 80% S phase cells. The method is rapid and a larger number of cells can be easily processed with no loss of viability.     

Lethal effects of fluorodeoxyuridine on cultured mammalian cells at various stages of the cell cycle

The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10^?5 M FUdR was very low (0.0001–0.0003) whether the drug was administered during early G₁, late G₁, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10^?7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.     

In vitro developmental competence of buffalo oocytes collected at various stages of the estrous cycle

The objective was to determine the in vitro developmental competence of buffalo oocytes collected from abattoir-derived ovaries at various stages of the estrous cycle and follicular status. In Experiment 1, ovaries (n=476 pairs) were collected and divided into the following five groups: (a) ovaries with a corpus hemorragicum and no dominant follicle (CH-NO-DF); (b) ovaries with a mature functional corpus luteum (CL) and a dominant follicle (CL-DF); (c) ovaries with a mature functional CL and no dominant follicle (CL-NO-DF); (d) ovaries with a regressing CL and a dominant follicle (RCL-DF); and (e) ovaries without any luteal structures and only small follicles (ANEST). In Experiment 2, 144 pairs of ovaries with a CL (or regressing CL) and a dominant follicle were collected and follicles were classified as dominant, largest subordinate, and subordinate. In both experiments, the dominant follicle was defined as any follicle >10mm in diameter that exceeded the diameter of all other (subordinate) follicles. Although oocytes were collected from each group of ovaries, only Grades A or B oocytes were used for in vitro embryo production. Cleavage rates were higher (P<0.05) from oocytes collected from ovaries in the CH-NO-DF (59.6%) and CL-NO-DF (59.2%) groups than those collected from CL-DF (52.2%) and ANEST (43.6%) groups. The yield of transferable embryos was higher (P<0.05) from oocytes collected from CH-NO-DF (27.4%) and CL-NO-DF (24.0%) ovaries than from CL-DF (16.2%), RCL-DF (15.4%), and lowest (P<0.05) from ANEST (8.8%). In Experiment 2, oocytes from the dominant follicle had a higher (P<0.05) cleavage rate (65.2 %) and transferable embryo yield (30.2%) than those collected from the largest subordinate and subordinate follicles. In conclusion, oocyte competence depended on the morphofunctional state of ovaries. Oocyte development was maximal in pairs of ovaries with a corpus hemorragicum or CL and no dominant follicle; in paired ovaries with a CL and a dominant follicle, development was maximal in oocytes derived from the dominant follicle.