[
International Worm Meeting,
2003]
Oocyte meiotic maturation is essential to prepare the oocyte for fertilization and embryonic development. During meiotic maturation, C. elegans oocytes undergo nuclear envelope breakdown, cortical rearrangement, and meiotic spindle assembly in a spatially restricted manner such that the most proximal (-1) oocyte matures, is ovulated, and fertilized. Sperm stimulate oocyte meiotic maturation and gonadal sheath cell contraction using the major sperm protein (MSP) as a signaling molecule. The discovery of MSPs signaling role raised the intriguing question of how sperm release MSP to signal oocytes and sheath cells at a distance. The release of MSP from sperm likely occurs through a non-canonical mechanism since spermatozoa do not possess cellular components required in standard models of protein secretion, such as ribosomes, ER, or Golgi. Moreover, MSP does not have a N-terminal leader sequence, and in vitro release does not involve protein processing. Non-canonical or leaderless secretory pathways are widespread, and in general, poorly understood, despite figuring prominently in cell signaling, disease, and host defense. Using specific antibodies, we detect MSP both inside and outside of spermatids and spermatozoa in vitro. MSP release does not require motility or a pseudopod, and does not involve sperm lysis. We observe extracellular MSP up to approximately 90 m away from spermatozoa in the spermatheca, frequently reaching the -1 oocyte. Examination of extracellular MSP reveals it is localized in a graded distribution with the highest levels of MSP adjacent to spermatozoa and lower levels distally. MSP receptors on the -1 oocyte appear to bind and concentrate MSP resulting in a sharp boundary of staining, which is eliminated when trafficking of oocyte membrane proteins is blocked. Confocal microscopy shows extracellular MSP to be punctate. We observe large (~100-200 nM) MSP-staining puncta near spermatids or spermatozoa in the uterus. We speculate that these puncta are MSP-containing vesicles. Using immuno-EM we examined MSP localization in spermatozoa to pinpoint its localization in the cell. In addition to localization of MSP within the pseudopod, we also found MSP located in the cell body. Interestingly, we found a fraction of MSP associated with the plasma membrane of the spermatozoa. Based on these results we propose a model in which spermatids and spermatozoa shed vesicles containing MSP. In this model, MSP-containing vesicles released by spermatozoa in the spermatheca are unstable, forming a diffusible MSP signal that correlates with meiotic maturation rates and MAP kinase activation. Biochemical tests of this model are underway.
[
International Worm Meeting,
2019]
Oxidative stress and inflammation are critical components in the development of obesity. Culinary herbs and spices, abundant in anti-oxidant and anti-inflammatory phytochemicals, may be useful in the prevention of obesity. In the present study, we report strong anti-obesity properties of culinary spices. Wild-type Bristol N2 strain (Caenorhabditis Genetics Centre) were grown on NGM plates seeded with E. coli (OP50). Worms were exposed to extract (1mg of dry powder per 1ml of NGM) of red chilli, black pepper, ginger, and turmeric. Fat content was determined on day 5 (adult stage) by staining with Oil-Red O. Compared to control group, chilli, ginger and pepper reduced the fat content of the worms by 92, 89 and 57% respectively. Turmeric had no effect on the fat content of C elegans. In conclusion, culinary herbs and spices may be useful in the prevention of obesity and C. elegans is a useful model for screening the anti-obesity properties of culinary spices.
[
International C. elegans Meeting,
1999]
We have tested a variety of antibodies in post-embedding protocols to localize proteins in thin sections of C. elegans. Despite recent improvements, about 50% of tested antibodies fail to work satisfactorily. Those that fail apparently involve antigens that are denatured during alcohol dehydration prior to LR Gold embedment. Fixation of chilled whole worms within a microwave oven provides more uniform, rapid fixation than worms fixed by immersion or cut open in fixative by a blade (1,2). Just as for immunofluorescence, it is still necessary to compare fixative strengths to obtain optimal preservation while retaining antigenicity. Here we compare the merits of several monoclonal and polyclonal antibodies against GFP to localize yolk protein (YP170::GFP strain) in thin sections, using a gold-linked secondary Ab. Commercial anti-GFP antibodies were obtained from Clontech, Quantum and RDI. Until now, it has been difficult to prove which organelles contain yolk, or to trace the exact path by which yolk travels from intestine to gonad and embryos (3). The YP170::GFP label has also been used by light microscopy to monitor endocytosis of yolk in wild type and mutant tissues (4). By immunoEM, GFP label in YP170::GFP animals is concentrated in two classes of dark-staining organelles in the intestine. Labeled yolk particles are found in the pseudocoelom. Labeled granules and free label above background levels are seen in the cytoplasm of oocytes in the proximal gonad and egg chamber. We will present TEM evidence for yolk passage through sheath pores and endocytosis into oocytes separately (5). We thank Barth Grant (Columbia U.) for providing the YP170::GFP strain used in this study. 1. Li and Kimble, International C. elegans Meetings, 1995, 1997 2. Miller and Hall, Worm Breeder's Gazette 15: 15, 1998 3. Bossinger and Schierenberg, Int. J. Dev. Biol. 40: 431-9, 1996. 4. Grant, Zhang, Pedraza, Hall and Hirsh, this meeting. 5. Hall, Winfrey, Blauer, Hoffman, Furuta, Rose, Hobert and Greenstein, this meeting.