Worm Breeder's Gazette 14(1): 66 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232
The arrest of the oocyte cell cycle in meiosis is a nearly universal feature of reproduction. In C. elegans, oocytes are arrested in the diakinesis stage of meiotic prophase I. Previously, we found that oocytes in ceh-18 mutants variably undergo multiple rounds of DNA replication without cytokinesis or karyokinesis to become polyploid in the proximal arm of the gonad (Endomitotic Oocyte or Emo phenotype)(Genes & Dev. 8: 1935-1948). We also observed eggs that were round or had abnormal sizes and shapes. In many cases these abnormally sized embryos could undergo embryogenesis though there is a significant fraction of embryonic lethality (~15%). We suggested that ceh-18 affects signals from the sheath cells that maintain meiotic arrest and influence oocyte structure. To further analyze oogenesis in ceh-18 mutants, I have begun using time-lapse video recordings (Kirby et al., Dev. Biol. 142: 203-215; McCarter et al., 1995 Meeting, p. 367-368) to observe the behavior of oocytes as they become polyploid and to determine the origin of the round eggs. In wild type (N2) (n=6) nuclear envelope breakdown (NEBD) occurs, the oocyte rounds up, and contractions of the proximal sheath cells push the mature oocyte into the spermatheca (ovulation) after a delay of 2.8 ± 0.3 minutes following NEBD. Following fertilization the egg has an oval shape and the posterior end of the embryo is determined by the sperm entry point which corresponds to the region of the oocyte that first enters the spermatheca (Goldstein & Hird, 1995 Meeting). In contrast, in ceh-18(mg57) the oocyte was often misshapen and the nucleus was mispositioned (7/12 cases) such that it was off center dorsally or ventrally prior to NEBD. NEBD occured (11/12 cases), the oocyte rounded up, and sheath contractions pushed the oocyte into the spermatheca after a delay of 3.1 ± 0.9 minutes following NEBD, similar to N2. This contrasts with let-23(sy10) which results in a delay of ~ 20 minutes following NEBD and also results in an Emo phenotype (McCarter et al., 1995 Worm Meeting, p. 367). Thus, ceh-18 and let-23 are likely to function at different steps of oocyte development. Possibly, let-23(sy10) affects the coupling between NEBD and sheath cell contractions which drive ovulation. In ceh-18(mg57), oocyte rounding occurred, and cytoplasts were extruded from the distal portion of the oocyte as the oocyte entered the spermatheca (4/12 cases). Cytoplast extrusion was not observed in N2. The size of the cytoplasts varied considerably from a small amount to ~30% of the oocyte volume in one case. In all cases, the oocyte was fertilized. It is unclear whether cytoplast extrusion occurs because of a defect in the oocyte cytoskeleton or because of abnormal force exerted on the oocyte by the sheath cells. Following fertilization the egg had a round or irregular shape in 6/12 cases (includes the 4 cases with cytoplast extrusion). In these 6 cases the sperm entry point was in an abnormal position as determined by the position at which the sperm pronucleus first appeared. In 5/6 of these round egg cases, the AP axis was determined by the sperm entry point in agreement with the results of Goldstein and Hird (see Figure). In one case, the sperm pronucleus was first seen at an abnormal position, but translated to the normal position which correlated with the posterior end. Three lines of evidence suggest that these observations are not an artefact of mounting and illumination: 1) round eggs are observed in unrecorded animals; 2) first ovulations were recorded to minimize deleterious effects of prolonged recording; 3) defects were not observed in N2. So far, I have not yet observed the behavior of an oocyte as it becomes polyploid. This is not surprising because the Emo phenotype in ceh-18 has a low penetrance with respect to an individual oocyte. A major issue to be addressed by further experiments is whether the Emo phenotype in ceh-18 requires ovulation. One line of evidence suggests that it does not. McCarter et al. have found that germline feminization suppresses the sheath activity that is necessary for ovulation (1995 Worm Meeting p. 368). Therefore, I constructed a ceh-18 fem-2 double mutant to observe whether germline feminization reduced the penetrance of the ceh-18 mutant phenotype. Surprisingly, I found that fem-2 enhances the Emo phenotype. In ceh-18(mg57) grown at 25 oC, 24% of the gonad arms (79/325) have an Emo phenotype when examined 24 h after the L4 molt. By contrast, in ceh-18(mg57) fem-2(b245ts), 83% of gonad arms (298/361) exhibit an Emo phenotype when grown at 25 oC and analyzed in a similar manner. This result suggests that the Emo phenotype does not depend on ovulation and is consistent with the hypothesis that ceh-18 affects oocyte arrest. Possibly, oocytes remain in the gonad longer in the double mutant. In ceh-18 mutants, ovulation and fertilization could be thought of as partially rescuing the Emo phenotype by changing the cell cycle state of the oocyte. My current working model is that the sheath cells influence both oocyte cell cycle arrest and the structure of the oocyte cytoskeleton prior to NEBD. In ceh-18 mutants a defect in the sheath cells leads to an oocyte defect prior to NEBD (nuclear mispositioning), results in an Emo phenotype, and affects the behavior of the oocyte during ovulation and fertilization (cytoplast extrusion, round eggs, and abnormal sperm entry point). Figure. Altered sperm entry point in ceh-18(mg57) round eggs. The left drawing shows the 6 cases in N2 and the 6 cases in ceh-18(mg57) in which the sperm pronucleus was observed in the normal position (indicated by an arrow). The right drawing shows the 6 round egg cases in ceh-18(mg57) in which the sperm pronucleus was seen at an abnormal position. In 5/6 cases this corresponded to the posterior end of the egg. In the exceptional case (indicated by *) the pronucleus moved to the normal position.