Impaired secretion and fluid-phase endocytosis in the End4 mutant of Chinese hamster ovary cells

Mutant V.24.1 defines the End4 complementation group of temperature-sensitive Chinese hamster ovary cell mutants selected for resistance to protein toxins. We investigated the secretory pathway in the mutant cells and found: 1) The hemagglutinin of influenza virus failed to reach the plasma membrane and was retained in a form sensitive to endoglycosidase H at the restrictive temperature. 2) Transferrin receptors synthesized at the restrictive temperature remained sensitive to endoglycosidase H. 3) Secretion of total soluble protein into the medium was strongly reduced at high temperature. These data indicate that V.24.1 cells are defective in secretion at the restrictive temperature. To see what effect the lesion had on the endocytic pathway, we measured the accumulation and recycling of the fluid-phase marker horseradish peroxidase. Accumulation was inhibited by 50% while recycling was barely affected, suggesting that the rate of fluid-phase endocytosis was reduced. We previously showed that the clathrin-coated pit pathway of endocytosis was not affected in the mutant, indicated by a normal transferrin cycle (Colbaugh, P. A., Stookey, M., and Draper, R. K. (1989) J. Cell Biol. 108, 2211-2219). Thus, the secretory lesion correlates with reduced fluid-phase endocytosis without impairing the clathrin-dependent pathway of receptor-mediated endocytosis. We also investigated the delivery of endocytosed material to lysosomes and found that delivery was partially, but not completely, impaired in the mutant. This suggests that endocytosed material can enter lysosomes, although slowly, in the absence of a functional secretory pathway.     

Plasmodium spp.: dispersion of malarial oocyst populations in anopheline and culicine mosquitoes

The numbers of malarial oocysts developing in individual, like mosquitoes fed concurrently on a single vertebrate malarial host were found to be distributed according to the negative binomial distribution in 169 experiments utilizing 6 species of Plasmodium, 6 species of mosquitoes and 3 species of vertebrate hosts. Dispersion constants ranged upward to 8.0, and mean clump sizes ranged upward to 298.4. The dispersion constant was demonstrated to be contingent on the species, strain and identity of the mosquito, the parasite and the vertebrate host; on the genetic state of the mosquito; and on the state of the infection in the vertebrate host. It was concluded that the concentration of oocyst production in particular mosquitoes was produced by varying levels and combinations of numerous factors associated with the parasite, the mosquito and the vertebrate host and that the pattern of oocyst distribution favors parasite survival and the maintenance of malaria in the field.     

Effects of epidermal growth factor and follicle-stimulating hormone during in vitro maturation on cytoplasmic maturation of porcine oocytes

The present study was undertaken to investigate the influence of epidermal growth factor (EGF) and follicle-stimulating hormone (FSH) during in vitro maturation on cytoplasmic maturation of porcine oocytes as revealed by the success of fertilization and by the changes in the pattern of protein synthesis in oocytes and cumulus cells. For fertilization studies, oocyte-cumulus cell complexes (OCC) were cultured in media containing human recombinant EGF (1 ng/ml) or FSH (1.5 μg/ml) or both for 44 hr prior to fertilization with fresh sperm for 6–8 hr. The oocytes were then fixed, stained, and examined as whole mounts following an additional 14 hr of culture. Addition of EGF, FSH, and EGF + FSH significantly increased the proportion of oocytes reaching MII stage. The addition of EGF alone significantly decreased the percentage of polyspermic oocytes and increased the proportion of monospermic oocytes forming 2 normal pronuclei. FSH abolished these effects of EGF and significantly increased the percentage of polyspermic oocytes forming more than 2 pronuclei when added alone or with EGF. For protein analysis, OCC were cultured in media containing the above hormones for 6, 24, and 44 hr and exposed to 0.5 mCi/ml L-[³⁵S]methionine during the last 3 hr of cultures. The oocytes and cumulus cells were separated prior to lysis in SDS sample buffer, and denatured polypeptides were separated by 1-dimensional SDS-PAGE. In the oocyte, addition of EGF and FSH alone stimulated the synthesis of 34, 45, and 97 kDa proteins after 6 hr of culture; however, the addition of EGF and FSH together was without any effect. After 24 hr, EGF alone inhibited the synthesis of these peptides, whereas FSH alone and with EGF maintained the stimulation of synthesis of 34 and 45 kDa proteins. Two additional peptides corresponding to 66 and 200 kDa appeared at this time as a result of exposure to FSH alone or with EGF. After 44 hr of culture, these 2 new peptides were observed in all groups and the stimulatory effect of FSH and FSH + EGF was still evident. An additional peptide of 26 kDa appeared at this time as a result of FSH and EGF + FSH treatments. In the cumulus cells, EGF and FSH each alone induced the synthesis of a new peptide of 26 kDa after 6 hr of culture. FSH when added alone or with EGF induced the synthesis of an additional peptide of 29 kDa, the synthesis of which remained unchanged at 24 and 44 hr. After 24 hr, FSH alone and in combination with EGF induced the synthesis of an additional 38 kDa peptide and its synthesis was still maintained at 44 hr. EGF alone had no effect on protein synthesis in cumulus cells at 24 and 44 hr. These studies indicate that EGF may have a physiological role in the regulation of cytoplasmic maturation of porcine oocytes. Mol. Reprod. Dev. 46:401–407, 1997. © 1997 Wiley-Liss, Inc.