Worm Breeder's Gazette 14(2): 51 (February 1, 1996)

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.

Formation of Endomitotic Oocytes and Haploid Embryos in ceh-18 Mutants

David Greenstein

Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232

Several critical events in the late stages of oocyte development including maintenance of
meiotic prophase I arrest, oocyte maturation, ovulation, and fertilization must be correctly
executed in order for viable progeny to be produced. These crucial changes in the oocyte must
be orchestrated in concert with changes in the somatic gonad so that ovulation and fertilization
occur at the appropriate moment. I have been using time-lapse video recordings [1, 2] to
determine the origin of the endomitotic oocytes and the maternal effect lethal embryos in ceh-18
mutants. This analysis has revealed multiple incompletely penetrant defects in the late stages of
oocyte development including: formation of endomitotic oocytes; delays in oocyte maturation;
abnormalities in oocyte shape and nuclear positioning; abnormal accumulation of oocyte
granules and visible abnormalities in the oocyte cytoplasm; cytoplast extrusion; abnormal sperm
entry point (altered A/P axis); and formation of haploid embryos. In addition, ceh-18 mutant
sheath cells are less active than those in wild-type. I have also noticed that there is sometimes a
defect in the way the spemathecal opening is pulled over the oocyte in ceh-18 mutants such that
the oocyte is deformed during ovulation. This deformation of the oocyte may affect the kinetics
of fertilization and might explain the cases of complete reversal of A/P polarity I have observed
(see Figure G-J-Electronic Gazette only). Every ceh-18 mutant animal exhibits sheath cell and
oocyte defects, though the particular defect exhibited varies. The mutant animals are usually
fertile because the defects don't always interfere with fertilization.

Recent results suggest that endomitotic oocytes in ceh-18 mutants can arise in at least two
ways. In the first way, oocytes mature in the gonad arm but are not ovulated. Generally this
correlates with a situation in which the oocytes no longer form a single row in the proximal half
of the gonad arm. Possibly a defect in the sheath allows the oocytes to be pushed into an
abnormal arrangement. In the wild-type, oocyte maturation is spatially restricted such that only
the most proximal oocyte matures. In these misshapen gonad arms this rule is broken and
oocytes can mature out of order before they reach the most proximal position. I have also
observed one case in which the most proximal oocyte matured, sheath contractions occurred,
but ovulation never took place. The gonad was not misshapen in this animal. In the second
way, endomitotic oocytes form when the oocyte breaks in two during ovulation but the nucleus
remains in the oocyte fragment that is retained in the gonad arm (see Figure K-O). In these
instances, the gonadal oocyte fragment underwent multiple rounds of nuclear envelope
breakdown. DAPI staining of the recorded animal showed that the endomitotic nucleus was
highly polyploid. In contrast, the anucleate fragment was fertilized, an eggshell formed, and
only a single nucleus, presumably the sperm pronucleus, was seen in the egg. In these
embryos, pseudocleavage occurred, the single nucleus migrated to the center of the egg, and the
first cleavage was asymmetric as in wild-type with the position of the sperm pronucleus
corresponding to the posterior pole. The extent of embryonic development correlated with the
size of the oocyte fragment fertilized. These embryos exhibited a reduced intensity of DAPI
staining per nucleus compared to wild-type embryos, further suggesting that they were haploid.
Previously, Schierenberg and Wood observed the early development of haploid embryos
formed by extruding the female pronucleus from the fertilized egg with a laser: early cleavages
were normal, but embryogenesis arrested prior to morphogenesis [3]. These results from
ceh-18 mutants suggest that fertilization, pseudocleavage, sperm pronuclear migration, and
determination of A/P polarity do not require that the female pronucleus be present in the egg.
Taken together, these observations suggest that proper functioning of the gonadal sheath cells
is critical for multiple aspects of oogenesis and that ceh-18 is a crucial determinant of sheath cell

[1]. C. Kirby et al. (1990). Dev. Biol. 142: 203-215. [2]. J. McCarter et al. (1995). WBG 14 (1): 68.
[3]. E. Schierenberg and W. B. Wood. (1985). Dev. Biol. 107: 337-354.

Figure. Multiple defects in the late stages of oocyte development in ceh-18 mutants. A.
Representation of one of the two gonad arms of the wild-type adult hermaphrodite. Syncytial
germ-line stem cells proliferate in the distal half of the gonad arm, enter meiosis, and
differentiate as cellularized oocytes in the proximal half of the gonad arm. The position of
representative sheath nuclei is indicated (1-5). B. Endomitotic oocytes (arrows) in
ceh-18(mg57) detected by DAPI staining. C. CEH-18 expression in wild-type sheath cell
nuclei (orange) was detected with specific antibodies. DNA was also visualized by DAPI
staining. D-F. Wild-type ovulation and fertilization. Nomarski micrographs showing
ovulation (D), pronuclear meeting (E) in the proximal half of the egg, and first cleavage
(F) generating the anterior blastomere AB, and the posterior blastomere P1 (a different animal
was photographed in D vs. E-F). G-J. An example of polarity reversal in ceh-18(mg57).
G. Ovulation, note the oocyte is slightly distorted (compare D and G). H. Pronuclear meeting
occuring in the distal half of the egg. I. First cleavage, the position of AB (shown dividing)
and P1 are switched (compare F and I). J. The late stage embryo (arrow) from G-I hatched
and grew into a fertile adult. K-O. Formation of a haploid embryo and an endomitotic oocyte
in ceh-18(mg57). The oocyte in K broke in two during ovulation leaving a smaller oocyte
containing the nucleus in the gonad arm (small arrow) and a cytoplast which was fertilized in
the spermatheca (large arrow). M. The embryo contains a single nucleus, presumably the
sperm nucleus, migrating to the center after pseudocleavage. N. First cleavage of the haploid
embryo. O. DAPI staining of the recorded animal showing the haploid embryo and the Emo
oocyte. Compare the intensity of the DAPI staining of the haploid embryo to the diploid embryo
on the right.