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.

Oocyte Defects Prior to Nuclear Envelope Breakdown in ceh-18(mg57)

David Greenstein

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.