FREEZE-FRACTURE STUDY OF THE DEVELOPING
MORPHOLOGY OF HALIOTIS RUFESCENS OOCYTES TO NINE
HOURS POST-FERTILIZATION
By W. Howard Hess
Hopkins Mar ine Station
Pacific Grove, CA, 93950
ABSTRACT
The technique of freeze-fracture in conjunction with scanning
electron microscopy (FFSEM) was developed and succesfully used for
viewing the developing morphology of the abalone embryo, Haliotis
rufescens The nucleus and intracellular granules/vesicles could be
visualized and changes in the distribution of granules followed during
embryogenesis. The FFSEM procedure could be further developed as a
research tool, and 1 present some possible improvements in the
procedure.
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INTRODUCTION
The development of marine gastropods has been studied since the
late 1800's, and excellent accounts have been published, e.g., on
Crepidula (Conklin 1897), Trochus (Robert 1902), Patel/a (Patten
1886), and Haliotis (Crofts 1937). Yet, very little is known about
the ultrastructure, aside from EM studies on processes of
fertilization and maturation (Longo and Anderson 1969, 1970). Here
report on the Haliotis embryo, using a novel procedure, FFSEM.
report on both surface views but especially internal structures. The
most interesting aspect of FFSEM is the ability to view intracellular
granules/vesicles, most probably yolk granules, and their change in
distribution during their development.
MATERIALS AND METHODS
Aquisition and Cuturing of Embryos
The reported studies were all done with one batch of oocytes and
embryos, obtained from the Granite Canyon Laboratory of the
California Department of Fish and Game (Carmel, CA). The embryos
were maintained at 15 C° within a 1000 ml beaker containing
microfiltered sea water (MSW) and were suspended 1.5 cm off the
beaker bottom via nylon mesh (Nytex, 120 u) at a concentration below
5/ml, and aerated with an air-stone.
Light Microscopy
Whole oocytes and embryos were viewed at 0.0 and 30 min
post-fertilization (PF), and then viewed and photographed at 2, 3, 4.5,
6.5, and 9 h PF with a phase-contrast microsope using dark-field or
bright-field settings. Photographs were made on 100 ASA color slide
film with a fully-automatic 35 mm camera conected via a T-mount to
the light microscope.
Freeze-Fracture Preparation for Scanning Flectron Microscopy
Oocytes and cultured embryos were concentrated with nylon mesh
(Nytex, 120 u) and samples (0.1-0.25 ml) were pipeted into 25m1
beakers for fixation at 0.0, 10, 20, and 30 min PF, and then at 1, 2, 3,
4.5, and 6.5 h PF. Specimens were fixed in 22 glutaraldahyde in 802
MSW containing 1.22 HEPES (pH 7.3) for a minimum of 2 hrs at 4° (at
min ratio Iml Sample : 50ml Fix), washed twice for 5 min in distilled
water (DW), and post-fixed for 5-1 hr at 23 C° at a 1:1 ratio in 12
osmium tetroxide prepared in MSW, and rewashed twice for 5 min in
DW. Fixed samples were then mixed (1:1) with a warm suspension of
3% agar, pipeted onto paraf ilm sheets, air cooled for 1-2 min,
dehydrated by immersion for at least 10 minutes each in 20, 30, 40,
50, 60, 70, 80, 90, and 100% ethanol, frozen by immersion in liquid
nitrogen (1-2 min), fractured with a chilled razor and then
reimmersed in 100% ethanol. Fractured samples were then either
critical-point dried or immersed in hexamethyldisilazane for final
drying, "glued" onto metal stubs via colloidial graphite with the
fractured suface facing upwards, and sputter-coated with gold.
Coated samples were then viewed under the SEM or stored in a
dessicator until viewing.
RESULTS
Development of Embryos
In the batch of embryos used, 95% of the embryos developed
normally during the first nine hours, 3% did not undergo cleavage, and
28 developed abnormally (fig. 1). At twenty-four hrs PF 67% of the
embryos showed normal but slow growth, 30% of the embryos
appeared abnormal, and 3% had not undergone cleavage.
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Light Microscope Observations
For purposes of interpreting the images obtained by FFSEM, it is
necessary to describe development as seen under the light
microscope.
Examination of Haliotis oocytes and embryos via the light
microscope provided a general view of its development which seems
to follow that of the Crepidula as described by E. G. Conklin in 1897
(see Appendix I for dev. of Crepidula). The 150 u oocytes were
loosely surrounded by a translucent chorion and exhibited no
polarity/asymmetry in shape or color (fig. 1). When viewed
macroscopically, the eggs were green; however, under the microscope
the eggs were green-brown and there was no appearent pigment
localization (as opposed to later). By 2 hrs PF, the embryos had
developed into two seemingly identical cells, equivalent in size and
color (fig. 2. For comparision to 2-cell Crepidula see Appendix! fig.
5). By 3 hrs PF, the embryos were in a four cell stage with
blastomeres similar in size and color (fig. 3. For comparision to
4-cell Crepidula see AppendixI fig. 11). By 4.5 hrs PF, the embryos
were at the eight-cell stage and asymmetry was now apparant in
blastomere size and yolk distribution. These embryos consisted of a
micromere quartet of four translucent, green cells resting on a
macromere quartet of four larger opaque, brown cells (figs. 4-5. For
comparision to 8-cell Crepidula see Appendix 1 fig. 13). By 6.5 hrs
PF, the embryos were in the 25-cell stage consisting of twenty-one
micromeres and four macromeres. The mass of the micromere region
had increased and the mass of the macromere region had
corespondingly decreased (fig. 6. For comparision to 25-cell
Crepidula see Appendix I fig. 24). By 9 hrs PF, the embryos were
approximately sixty cells, of which four were macromeres, and
approximately 56 were micromeres. The micromeres were in the
process of enveloping the macromeres (No photographed image. For
60-cell Crepidula see Appendix 1 fig. 36)
SEM
Extracellular lmages
Extracellular items of interest included the chorion, the cell
surface, polar bodies and sperm. The chorion was seen at all stages of
development; its general charcteristics did not differ from stage to
stage but its relation to the oocyte or embryo varied significantly
because of differences in the fracture plane (compare figs. 7, 8, &11).
In contrast, veiws of the cell surface differed according to
developmental stage (compare figs. 7,1 1,14, &15 ). The cell surfaces
of oocytes, but not embryos, were wrinkled (perhaps the oocytes are
more suceptible to osmotic changes than are the embryos) (fig. 7). By
comparison, the cell surfaces of the embryos were relatively smooth,
but at high magnification (x 2.5 K) they showed irregularities which
may have been caused by the underlying yolk granules (fig. 12). The
polar bodies, seen only on 2-cell and 8-cell specimens, were located
at the animal pole of these embryos (figs. 11,12, &14). The sperm
cells, also seen primarily on 2-cell and 8-cell specimens, were found
on the chorion and on the cell's exterior (figs. 11,12, &14).
Intracellular lmages
The intracellular structures visualized were granules/vesicles and
nuclei. Most of the fractured vesicles were empty, but some
contained what appeared to be fractured or intact yolk granules (figs.
7-10, 13, 16, 17). The granule distribution in oocytes to 2-cell-stage
was in mosrt cases uniform (figs. 7-10, 13 but in a few cases some
localization was seen (figs. 7-10, 13. A polar distribution of
granules could be seen by the eight-cell stage (4.5 hrs PF) as shown in
fig. 13 lcompare with sectioned Crepidula (4-cell and 12-cell).
Appendix I figs. 83 & 841. Further asymmetry in the distribution of
the granules/vesicles could be seen in the 6.5 hr samples (figs. 16, &
17). Also, micromeres with associated nuclei could be seen
surrounding the granule/vesicle filled cells, macromeres as shown in
figs. 15 & 16 Icompare with sectioned Crepidula (29-cell), Appendix
fig. 851.
DISCUSSION
The FFSEM images of this study are most interesting when
compared with Conklin's images of the developing Crepidula and
with the light microscope images. One set of structures seen in both
the FFSEM images and Conklin's images is polar bodies. These
structures were first found on the Haliotis at the 2-cell stage (figs.
11 & 12), but are shown to emerge in the Crepidula prior to first
cleavage (Appendix 1 fig. 2). Also, a comparison should be made
between the nuclei seen in the FFSEM images and Conklin's sectioned
images. In both cases, the nuclei could only be seen in micromere
cells (figs. 16 &17 and Appendix I figs. 84 & 85) (either the cells
containing the granules had no nucleus, or the granules were shielding
the nuclei from view).
The most interesting comparison involves the FFSEM images, LM
images, and Conklin's images. First, the distribution of
granules/vesicles in the FFSEM images seems to correspond to the
distribution of pigment in the light microscope images (seen most
easily by comparing fig. 13 to fig. 4). Second, these pigment and
granule/vesicle distributions seem to correspond to the patterns of
yolk distribution shown in Conklin's images of the Crepidula (this
relation can be visualized most easily be comparing figs. 16 & 17 to
Appendix I fig. 85). Therefore, it is likely that the granules/vesicles
seen in the FFSEM images and the brown pigment seen in the LM
images are yolk granules which are being distributed in the
developing Haliotis embryos in a manner similar to that of the
Crepidula.
The technique of FFSEM used in this investigation is a relatively
simple, but highly effective, method for visualization of intracellular
structures: FFSEM provided a unique three-dimensional view of the
internal as well as external aspects of the embryo. With a few
modifications, FFSEM could become particularly useful for viewing
intracellular vesicles. Such improvements might include, but not
AKNOWLEDGMENTS
wish to thank the California Department of Fish and Game and
the members of the Granite Canyon Laboratory (especially Michael
Harris) for Haliotis specimens and use of their facilities, Greg Baxter
for his assistance and advice, Chris Patton for his technical support
on SEM, and Dr. David Epel for his assistance, advice and use the of his
facilities.
LITERATURE CITED
Conklin, E. G. (1897) J. Morph., 13, 1-226.
Crofts, D. R. (1937) Philos. Trans. Roy. Soc. Lond., 228B,
219-268.
Longo, F. J. and Anderson, E. (1969a) Cytological aspects of
fertilization in the lamellibranch, Mytilitus edulis 1. Polar
body formation and development of the female pronucleus. J.
Esp. 200l, 172, 69-96
Longo, F. J. and Anderson, E. (1970a) An ultrastructural analysis of
fertilization in the surf clam, Spisula solidissimal. Polar
body formation and development of the female pronucleus. J
Ultrastruct. Res, 33, 495-514.
Longo, F. J. and Anderson, E. (1970b) An ultrastructural analysis of
fertilization in the surf clam, Spisula solidissimall.
Development of the male pronucleus and the association of the
maternally and paternally derived chromosomes. J.
Ultrastruct. Res, 33, 515-527.
Patten W. (1886) Arb. zool. Inst. Univ. Wien., 6, 149-174
Robert A. (1902) Arch. Zool. exp. gén. (3), 10, 270-538
FIGURE LEGEND
Fig. 1. Dark-field image (6.5 h stage) showing an abnormal embryo
(upper-left), and an unfertilized oocyte (lower-right).
Fig. 2. Dark-field image (2 h stage) showing 2-cell embryos with
sperm attatched to chorion.
Fig. 3. Bright-field image (3 h stage) showing a 4-cell embryo
Fig. 4. Dark-field image (4.5 h stage) showing 8-cell embryos with
sperm attatched to chorion and asymmetrical distribution of
pigments: green is present mostly in the micromeres and brown is
present mostly in the macromeres.
Fig. 5. Same as fig. 4.
Fig. 6. Dark-field image (6.5 h stage) showing 25-cell embryos
with sperm attatched to chorion and asymmetrical distribution of
pigments
Fig. 7. FFSEM image of an oocyte showing the cell surface and
internal structures (vesicles,fractured granules and intact
granules). (x660)
Fig. 8. FFSEM image of oocytes showing variations in the fractue
plane, the chorion, the cell surface and internal structures
(vesicles, fractured granules and intact granules). (x200)
Fig. 9. FFSEM image (20 min stage) showing the internal structure
of a L-cell embryo (vesicles, fractured granules and intact
granules). (x1000)
Fig. 10. FFSEM image (1 h stage) showing the cell surface, chorion,
and internal structure of a Lcell embryo (vesicles, fractured
granules and intact granules are asymmetrically distributed).
13
(x880)
Fig. 11. FFSEM image (2 h stage) of 2-cell embryos showing the
cell surface, chorion, sperm, polar bodies, and granules. (x360)
Fig. 12. FFSEM image (2 h stage) of a 2-cell embryo showing the
cell surface, chorion, sperm, and a polar body. (x2.5 k)
Fig. 13. FFSEM image (4.5 h stage) showing the intertnal
characteristics of an 8-cell embryo: asymmetrical distribution of
granules and vesicles (most of the granules and vesicles are
present in the macromeres, while few are present at the perimeter
of the micromeres). (x650)
Fig. 14. FFSEM image (4.5 h stage) showing the extertnal
characteristics of an 8-cell embryo: two polar bodies resting in
the cleavage furrow of the micromere quartet which, in turn, is
resting on the macromere quartet. In addition, a bit of chorion and
some sperm can be seen on the cell surface. (x650)
Fig. 15. FFSEM image (6.5 h stage) showing the extertnal
characteristics of a fractured 25-cell embryo: the cell surface and
the cleavage pattern of the micromeres (foreground) as well as
macromeres (background) can be seen. (x650)
Fig. 16. FFSEM image (6.5 h stage) showing the intertnal and
external characteristics of a 25-cell embryo: asymmetrical
distribution of granules and vesicles is a key feature of the
internal morphology. Note that nuclei can be seen in some of the
granule-free micromeres. On the exterior, the cell surface and the
chorion can be seen. (x650)
Fig. 17. FFSEM image (6.5 h stage) showing the intertnal
characteristics of a 25-cell embryo: asymmetrical distribution of
granules and vesicles is a key feature of the internal morphology
Note that nuclei can be clearly be seen in some of the granule-free
micromeres. (x810)
Fig. 2.
Fig. 4.
Fig. 3.
Fig. 6.
Fig. 5.
ig. 11
Fig. 12.
Fig. 14.
19
83.










oga
808
8o



oog
alo
FIG. 84. Section of egg
Eight cells.


so

8888
Sog
Logggdegs
FIG. 83. Section of egg
Four cells.
84.

8
o
9
G
B
888888
FIG. 85. Section of egg
Sixty cells.
24
necessarily be limited to reduced lipid extraction, reduced osmotic
changes, elimination of "ice" crystal formation, and most importantly
improved membrane resolution. Lipid extraction might be reduced
simply by the shortening of the duration of glutaraldehyde fixation,
but if this proves ineffective or other-wise undesireable, then
simultaneous fixation by gluteraldehyde and osmium tetroxide
mixtures might be used. In addition, intracellular membranes might
be better resolved by metallic impregnation of the the phospholipids
from which they are comprised. Then, to prevent osmotic disruption,
sucrose (or other compounds) might be added (probably prior to
osmium tetroxide fixation), or changes in drying media might be
tried. Also, "ice" crystal formation, if occuring, might be remedied by
a change in the drying medium, or by the addition of an "antifreeze" to
the medium. Finally, selective alignment of embryos (if possible) and
or serial-fracture of embryos might produce images which are more
easily interpretable than those obtained by the present method of
FFSEM.
10
Fig. 8.
Fig. 9.
Fig. 10.
Fig. 16.
ig. 17
21
22
APPENDIX
Fiqures from E. G. Conklin's The Embryology of the Crepidula.
FIG. 5.
Completion of first cleavage furrow.







FIG. 11.
Four cells.

1


P..



FIG. 13.
Eight cells.

Pr
23. jaii. 3a ja
ta
La'.
2a
sa
id

ed' MEsed)
FIG. 23.
Twenty-nine cells.
25“
36. Ja

Lale
Lee

34
3d“

Jeu Jc
FIG. 36.
Sixty cells.
1b“
15"
e1 G  1
1c
1c
2b“..

10
gu
c
23
94.
d  a
1c“
la




ME(4d)
àd'
FIG. 24. Side view of egg
of about the same stage as Fig. 23.