CALIFORNIA STATE UI-HIJERSITY, NORTHRIDGE
FREEZE FRACTURE ANALYSIS
OF THE
EARLY SEA URCHIN EMBRYO
A thesis submitted in par·tial sati·::.faction of the
requirements for the degree of Master of Science in
Biology
by
Samuel Louis Shultz
May, 1982
The Thesis of Samuel Louis Shultz is approved:
Dr. Phillip Sheeler·
Date
Dr·. Mar·y Lee Barber
Date
Date
California State University, Northridge
ii
TABLE
OF
CONTENTS
LIST OF PLATES------------------------------ iv
ACKN()AILEDGEMENT - - - - - - - - - - - - - - - - - - - - - - - - - - - - vi
ABSTRACT - - - - - - - - - - - - - - - - - - - - - - - - - - - vii
INTRODUCT I Cf~ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1
MATERIALS AND METHODS--------------------Urchin collection and storage-------------------Ferti I ization - - - - - - - - - - - - - - - - - - - - Embryo culture and fixation-------------------Freeze-fracture methodology----------------Membrane particle assessment -------------------Covers! ip ferti 1 ization - - - - - - - - - - - - - - - - - Scanning electron microscopy----------------
6
6
6
7
8
9
9
10
RESULTS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Two cell e m b r y o - - - - - - - - - - - - - - - - - - - - - - - - Four eel 1 e m b r y o - - - - - - - - - - - - - - - - - - - - Eight cell embr-yo - - - - - - - - - - - - - - - - - - - - - - - - - Particle c l u s t e r s - - - - - - - - - - - - - - - - - - - Cover-s 1i p deve I opmen t - - - - - - - - - - - - - - - - - - - - -
11
11
12
12
13
14
DISCUSSION - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 15
BIBLIOGRAPHY - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 19
iii
LIST
OF
PLATES
Scanning electron micrographs of development
of the sea urchin embryo to the 16-cell
stage.
Figures 1-6 - - - - - - - - - - - - - - - - - - - 23
2 Freeze-fracture electron micrographs of
the 2-cell embryo PH-PF.
Figures 7-9-------------- 25
3 Histogam of particle size frequency of
the 2-cell embryo PH-PF
- - - - - - - - - - - - 27
4 Freeze-fracture electron micrographs of
the 4-cell embryo PM-PF.
Figures 10-14 - - - - - - · - - - 29
5 Histogram of particle size frequency of
the 4-ce II embryo PM-PF - - - - - - - - - - - - - - - - - - - 31
6 Freeze-fracture electron micrographs of
the 8-cell embryo PM-PF.
Figures 15-17--------- 33
7 Histogram of particle size frequency of
the 8-cell embryo PM-PF - - - - - - - - - - - - - - - - - 35
iv
a Freeze-fracture electron micrographs of
the 8-cetl PM-PF and EF.
Figures 1a-20
--------37
9 Particle frequencies of the
2, 4, and a-celt embryo PM-PF ------------------- 39
19 Freeze-fracture electron micrographs of
four a-cell particle clusters. Figures 21-24 - - - - - - 41
11 Histogram of particle size frequency of
the a-cell PM-PF particle clusters--------------------- 43
12 Table summarizing particle frequencies and
densities of the 2, 4, 8-cell embryo-------------- 45
v
ACKNOWLEDGEMENT
I wish to thank. my major professor, Dr. Edward Pollock., whose patience, guidance
and fr-iendship made this wor-k possible. I especially appr-eciate the impar-ting of his
philosophy of science and scientific investigation. I also thank. Dr. Mary Lee Barber
for her· friendship and inter-estirtg discussions on many facets of developmerttal biology.
I also thank. Dr. Phillip Sheeler for his friendship, assistance, and many interesting
and pertirtar•t discussior.s. Lastly, I wish to thank my family for all their· suppor-t
through the years.
vi
ABSTRACT
Plasma membrane molecular topography in the early development of the sea urchin
embr·yo, Stror.gylocentr-otus pur·puratus was analyzed using freeze-fracture
electron microscopy. Membrane topography was analyzed with respect to intramembranous
particle densities and size class fr·equencies in the 2, 4, and 8-cell embryonic
stages. Results show similarities in membrane topography between cells of the 2-cell
stage, and between cells of the 4-cell stage. Overall membrane particle size class
frequencies between the 2 and 4-cell stages are similar, yet the 8-cell embryo is
comprised of cells that differ· fr·om each other· and the pr-evious embr·yordc stages
topographically in membrane particle densities and size class frequencies. Both the 2
and 4-cell embryonic stages cor.tain cells with homogeneous particle distr·ibutions,
while some cells of the 8-cell stage show membrane particle heterogeneities. Membrane
par·ticle cluster·s appear· in some cells of 8-cell embryos, yet no clusters were
apparent in membranes of the 2 and 4-cell stages. Results indicate that membrane
particle homoger.eity is apparent in blastomeres of the 2 and 4-cell stages, yet
massive membrane particle insertion and reorganization occurs during division of the
4-cell embr·yo. The membrane structural changes observed correlate well with the
overall pattern of determination in the early development of the sea urchin embryo.
vii
INTRODUC:TION
The study of development in the sea urchin embryo has been an area of intense
inter·est for almost a centUI'y (for r·eviews, see C:zihak., 1975>. Although many facets
of cellular interaction and behavior in development have been discovered, the basis of
zygote animal-vegetal polarity remains obscure. The pr-ocess by which individual cells
in an embryo undergoes change or differentiation and subsequently forms .the adult
or·ganism is deperrdent or• mar.y deter·mir.ing factors. Cellular cor.stituents such as
enzymes, morphogenetic substances, quantities of ribosomes, RNAs, mitochondria, other
or·ganelles, the distribution of membrane receptor· molecules and structur·al
constituents all unite in a concerted fashion during the elaboration of the adult
orgardsm <Berr·il ar.d Karp, 197 6>.
The problem of determir.ation of polar·ity in an organism such as the sea urchin
can best be understood by examining the early development of the embryo. In
Str·ongylocentr·otus purpuratus, the zygote undergoes three equal divisions. The
first and second cleavage planes are meridional, and the third is equatorial,
r·esultir•g ir• an embr·yo that contains eight blastomeres of equal size. The fourth
division is unequal and gives rise to a 16 cell embryo, containing three different
cell types: the micromeres, mesomeres, and macromeres. Plate I shows the sequence of
early development of the sea urchinS. purpuratus up to the 16 cell stage
embr·yo <figure 6). It is at this stage that the fir-st overt manifestations of a polar
axis become evident. The micromeres comprise the vegetal pole and are the smallest of
the blastomeres; they give rise to the primary mesenchyme cells of the blastula.
These cells ultimately secrete the spicules of the larval skeleton. The macromeres
1
2
are the largest size blastomtre; they lie in the plane of the tquatOI' arad give rise to
some of the ectodermal and all of the endodermal structures. The mesomeres are the
intermediate size blastomeres comprising the animal pole of the embryo and give rise
to the remaining ectodermal structures of the adult organism <Okazaki, 1975;
Horstadius, 1973; HDI'stadius, 1939>.
Quantities of pigment, yolk. and mitochondria are different in the three cell
types <Schroeder, 1980; Aunchman, 1979; Laming, 1971, Lanning and Hagstrom, 1965>.
There are no differences in the amount or classes of soluble proteins present in the
blastomeres o-f tht 16-cell stage <Tufaro and Brandhorst, 1979; Brandhorst, 1976), and
the proteins present in the cytoplasm are synthesized from maternally supplied mRNAs
!Hough-Evans, et al., 1977>. Czihak. and HDI'stadius C1970> have shown no appreciable
RNA syn-thesis occurring prior to ihe formation of the micromeres at ihe 16-cell
stage. The stage of developmerat ai which the preparatory events are completed for
faie determination of the blastomeres is unclear. However, for unequal polar
divisions to result in the three cell types of the 16-cell stage, partial
differentiation must have occured before the 8-cell embryo began to divide. lt is not
clear whether these events that lead to embryo differentiation occur during or pr-ior
to the 8-c:ell stage.
Ear·ly exper·iments by Driesch (1906) showed that isolated 2 and 4-cell stage
blastomeres develop into normal plutei, though not all 8-cell stage blastomeres
develop raor·mally whera isolated !Horstadius, 1975>. furthermore, 1/8 fr-agments o-f
whole eggs can develop into normal plutei !Horstadius and Wolsky, 1936>. These facts
support the totipoteratcy of fr·agmerats of the fertilized egg and blastomeres of the 2
and 4-cell stages, yet it seems that some blastomeres of the 8-cell embryo have been
par-tially dttermintd.
Evidence for the raature of the morphogenttic substance arad/or- action that is
3
involved in sea ur-chin embryo differen-tiation has been sought for· i.lmos't a century,
'though cellular processes which bring abou11hese changes remain obscure. It has been
confirmed by Schroeder <1986) 'that the pigment band <in some species>, the jelly car.al
and polar bodies are the markers for 'the animal pole in unfer-tilized Paracentrotus
lividus eggs. Loeb (1899) found that ar-tificially ac'tivated sta urchir• eggs
differentiate into plu'tei, suggesting the existence of a pre-fer-tilization polar
axis. Shroeder <1980> summarizes the evidence of a differtntial distribution of
pigmen-t granules in the "banded" eggs of P.lividus, and a clearing of thtse
granules from the vegetal region in unbanded P.lividus, Arbacia
lixula, and A. punc'tula'ta during 'the 'time of the fourth division. Though
thest events occur, it is unlikely thai 'they car. be implicated as the pr·imary cause of
embryo differentiation, as cytoplasmic s'tra'tifica'tion experments using centrifuga-tion
have shDINn r10rmal development 'to the plutei s'tage ir• centrifugtd eggs <Harvey, 1933;
Morgan and Spooner, 1909).
Horsiadius <1975> summarized decades of cellular· tr·art5plan1a1ion experimen-ts
involving isolated animal and vegeial blastomeres. Results indica-te the presence of
an animal-vegetal gr·adien't along the A-V axis of the embryo. Blasiomeres thai
comprise the animal or vegetal pole of 16-cell and larger embryos can exert 'their
respec-tive tffec'ts when 'transplanted to isola'ted por'tions of embryos; the resul't is
varying degrees of animaliza'tion or vegetaliza.tion during further developmen-t.
For these effec'ts 'to occur, 'the cell periphery is implicated as a jur.c'ture
'through which cellular communica-tion is accomplished. Indeed, it has been shown thai
micr·omeres form cy-toplasmic connec-tions with adjacent blas'tomeres <Lanning and
Hagstrom, 1971; Hags'trom and Lanning, 1969). Though resul'ts shown by Horstadius <1975>
iriCiicate s'trong vege1ali2ir.g influtnces on tht embr·yo brought about through contact
with the micromeres, no differences in the ra'te of cleavage of macromeres and
4
mesomer-es wer·e noticed when these cells wer-e sepera'ted fr-om 'the micr-ometes.
Moreover, micromeres seem dependent upon the effects of neighbor-ing macr-omeres and
mesomer-es; isolated micr·omer·es slow their- r-ate of cleavage when isolated <Hagstr-om
and Lonning, 1965, 1969; Lenning and Hagstrom, 1969) and do not develop into normal
plutei <Hor-stadius, 1975).
!liHer-ences in cell sur-face pr·operties of the differ-ent classes of blas'tomeres
have suggested a possible role of the cell periphery in embryo morphogenesis. The
elector-phor-etic mobilities of micr-omer-e derived cells have been showr• to differ- fr-om
cells derived from macromeres or mesomeres <Sane, 1977>, suggesting differences in
over-all cell surface char-ge. !lifferential changes in plasma membrane permeability to
specific ions occur during early development of Fucus embryos <Nuccitelli and
Jaffe, 1976>. These observations suggest that widespread plasma membrane structural
changes occur- dur-ing embryo morphogenesis.
The plasma membr-ane has also beer. shown to be comprised of highly specific
elements that mediate many important cellular properties. Cell adhesion studies
involving isolation ar•d r-eaggregation of blastomer-es of the early sea urchin embr·yo
show a species-specific response upon reaggregation. In addition, the three cell
types terld to reform the or-iginal spatial configuration that the cells were in before
blastomere separation. These results suggest the presence of unique antigenic sites
or• the membr·anes of different species of sea urchir•s <Spiegel and Spiegel, 1975>.
A recent approach to the study of membrane architectural changes in dissociated
cells and tissues is the application of -freeu-fracture electron microscopy. This
technique allows visualization of intramembranous particles <IMPs> within the lipid
bilayer· of cell membranes. These integral membrane constituents have been shown to
participate in many cellular functions. Studies on erythrocyte plasma membranes have
shown IMPs to represent proteinacious macr-omolecules participating in the fONnation
5
O'f anionic, enzymatic, and antiger•ic sites on the cell membrane <Nicolsor., 1975; Ila
Silva et al., 1973; DaSilva et al., 1970>. Furthermore, IMP differences are
appar·er.t between contact-inhibi-ted and transformed cells, indica-ting a possible role
of IMPs and the cell surface in 'the refledion of the func-tional properties of 'the
cells <Furch't and Scott, 1975>. Recent evidence on membrane molecular ar-chitecture
of the 16-cell sea urchin embryo has shown significant topographical differences in
the three cell types found in the 16-cell embryo <Kasparian, 1980). Differences in
both IMP densities and size distributions among plasma membranes of the three cell
types is indicative of basic char.ges that could lead to cell speciali:zation artd
determination of eventual cellullar function. These findings suggest a possible role
for the differentiation of the cell periphery in embryo morphogenesis.
The present work attempts to characterize the cell surfaces of the blastomeres
O'f the sea urchin embryo, S. purpuratus, using free:ze-fracture electron
microscopy. An assessment is made of differences in the plasma membrane molecular
architecture between the blastomeres of 'the 2, 4, and 8-cell sea urchin embr·yos by
examining the hydrophobic region of lipid bilayers in the plasma membranes of
individual blastomeres. Statistical data are a.naly:zed in terms of si:ze class
frequencies and plasma membrane distributions of intercalated membrane particles and
their· possible implications with respect to development.
MATERIALS AND METHODS
URCHIN COLLECTION AND STORAGE
Sea urchins were purchased through PacHic Bio-Marine of Venice, California, ar.d
collected from November to April at intertidal rock. foNnations of the Palos Verdes
Mar·ine Biological Preserve in Palos Verdes, California. The animals wer-e stored in
filtered and aerated natural seawater of specific gravity 1.023t a temperature of
18oc, and a pH of 8.2 • If storage of the live urchins exceeded one week. the
animals were fed a variety of local algae (Leahy et al.t 1978).
FERTILIZATI~
Gravid males and females were induced to spawn with a single 0.5 ml
intr-acoelomic injection of
e.55 M KCl (Leahy et al •• 1978; Tyler· and Tyler-. 1966).
Eggs were collected in filtered sea water (FS'W) of pH 8.2t washed three times and
stor·ed in FSW at 40C. Sper-m was collected on a cooled Petr-i dish at 40C and
kept undiluted until just before fertilization. Eggs were examined for normal
mor-phology and sperm wer-e examined with respect to mobility when diluted.
Ten ml of a 1:100 sperm:FSW suspension were added to 5-20 ml of packed eggs
suspended ir• se.e ml of FSW at r·oom temperature. Fertili2ation was assesed by per·cent
populational elevation of the vitelline layer. Zygote cultures that showed less than
95% vitelline layer· elevation were discarded. Only those cultures which were above
95% and showed normal egg-zygote morphology were used in exper-iments.
6
7
EMBRYO CULTURE AND FIXATION
Ten to 39.0 ml of packed zygotes were diluted with FSW to 3999 ml in a 4999 ml
beaker, placed on a magnetic stirrer·, stir-ted slowly and aer-ated in an incubator- set
at 15 oc. Development was followed using phase contrast and dark field micr-oscopy
employing either- a Wild binocular- micr-oscope and magnifications of 100-400
x, or an
Amer-ican Optical dissection microscope at magnifications of 10-40 X.
Modified Katnofsky's fixative <Pollock, 1970; Kamovsky, 1965) was used to fix
the embryos and consisted of a 9.25 M glutaraldehyde, 0.66 M pataformaldehyde, and
0.2 M sucr-ose in 0.2 M sodium cacodylate buffet at pH 7.4. The embr-yos wer-e fixed
using the schedules below fot two hours at r-oom temperatur-e then placed in the
r·efr·iger·ator·. Other- embryos wer-e fixed in 2.5~ glutaraldehyde in sea water- using the
schedule below.
Collection of embryos for fixation was begur. at fertilizatior. and ever·y five
minutes thereafter until 99Y. of the embryos were at the 16-cell stage. In anotherfixation schedule embr-yos wer-e collected at each of the following stages: early,
middle, and late substages of the 2, 4, and 8-cell embr-yos. Both methods seem to
pr-ovide excellerl't sepatatior. of substages at each stage of development. Or.ly embryos
fixed between 8-12 minutes before or- after- division <late or early> were used in the
subsequent exper-iments. This ensur-ed that membr-ane topogr-aphy was not affected by
upheavels in membrane or-ganization during cytokinesis. In addition, only those
cultur-es which showed
95~
synchrony Cno more than 5% embr-yos of a differ-ent
developmental stage> were used.
8
FREEZE-FRACTURE METHODOLOGY
Glycerinated embryos, equilibrated with 25% glycerol in fixative for one hour,
and unglycer·inated contr·ols were pipetted onto gold car-r-ier- gr-ids. The carr-ier-s wer·e
then dropped into a well of rapidly freezing freon 22 at -1470C cooled by, and
ther• transferr-ed to, liquid nitrogen. Froun embryos wer-e then mounted on the
precooled stage of a Babers model 360M freeze fracture apparatus.
Razor- blade micr·otome and double r-eplica fractur-e techniques were utilized using
the standard Babers double replica recovery device. With both techniques specimens
were fr·actur·ed at a stage temper·ature of -11eoc, microtome knife (if used) at
-tseoc, and a bell pressur-e less than or equal to 1x10-7Torr.
If the knife
arm fr·acture method was used, specimer•s were etched for 60 seconds. A carbon
electrode charged with 7.0 em of a 0.1 mm diameter platinum wire was evaporated onto
the fr·actur-ed specimens at an angle of 450 for- 7 seconds and r-einfor-ced with
carbon at an angle of '900 for 14 seconds <Hudson et al., 1979; Bullivant, 1973;
Muhlethaler-, 1971; Moor, 1969>.
Replicas wer-e then recovered by flotatior• onto the ir.cubatior• medium <25%
glycerol in fixative for cryoprotected specimens, fixative only for unglycerinated
specimens). The r·eplicas wer-e then slowly equilibrated with distilled water-, washed
three times and passed with a platinum loop through increasing concentrations to 40%
chr-omic acid. Replicas wer·e cleaned in chromic acid for three hour-s, trar.sfer·r·ed to
distilled water, washed, and slowly equilibrated with 109% bleach. After 1-2 hours
in bleach, the r·eplicas wer-e gradually tak.er• back to distilled water and rinsed six
times. Replicas were mounted on Pelco no. 180 grids and examined with a Zeiss EM 9
S2 electron microscope using a driving voltage of 50 Kev.
9
MEMBRANE PARTICLE ASSESMENT
Freeze fracture electron microscopy was used to assess plasma membrane
molecular· topography through measurements of intramembranous particle size ar.d
density in the 2, 4, and 8-cell sea urchin embryos. Similar measurements were
obtair.ed for membrar.e particle clusters, which wer·e observed only in plasma membranes
of the 8-cell embryo. IMPs were measured using an illuminated Edmund 6X
comparator/reticle with divisions of e.1 mm. Ilistr·ibutior•s of IMPs were analyzed on
an Apple II microcomputer, using the program, Statistics with Daisy. The
fr·eeze-fractur·e nomer.clatur·e of Br·anton et al. <196i) was adopted in this work..
COVERSLIP FERTILIZATION
In an attempt to or·ient the 8-cell embryos for freeze-fracture, separate
experiments were conducted to examine the relationship between site of sperm entry
and subsequer.t A-V axis polarity. Three types of experiments were performed. The
first method was the fertilization of eggs in normal sequence, and adhering these
eggs to a glass coverslip coated with 8.1% polylysir.e <M.W. se,eee, Sigma Chemical
Co.) and observing subsequent development <Sanders et al., 1975; Mazia et al.,
1975). The second method employed dithiothr·eotol <DTT> digestior. of the vitelline
envelope <Epel et al., 1978), fertilization of eggs in the normal sequence, and
subsequent attachment to a polylysine-coated coverslip. In the third method, sper·m
were layered onto a polylysine coated coverslip, and treated eggs were deposited onto
the attached sperm.
10
[Jevelopment was allowed to proceed until 90% oi the cells were in the 16-cell
stage. All embryos on coverslips were monitored periodically for normal cellular
mor·phology and development. The cells were then fixed on the coverslips in various
developmental stages and subsequently examined for possible correlation of sperm
entr·y site to the development of the A-V axis.
SCANNING ELECTRON MICROSCOPY
Embryos were mounted on 0.1% poly lysine coated coverslips, dehydrated with
ethanol and critical point dried using liquid carbon dioxide CMazia et al., 1975;
Sander·s, 1975), Coverslips were subsequently mounted on SEM stubs, shadowed with 200
~ of gold, and examined in an lSI mini SEM II, using a driving voltage of 25 ICev.
RESULTS
The majority of fracture planes revealed the protoplasmic faces <PF> of the
blastomeres. The followirtg data on the three embryonic stages all reflect analysis of
the plasma membrane PF of the cells.
Ther·e were few blebbing artifacts produced by the use of glycerine as a
cryoprotectant. Membranes which showed blebbing were not used for data acquisition,
though they appeared to have particle topographies similar· to other, rton-artifactual
membranes of that stage of development. Controls incubated in fixative and
cryopr·ott:tcted material t:txhibited similar membrar•e topopgraphies for a given embryortic
stage. Plate I, figures 1-6 are scanning electron micrographs of the early
developmt:tnt of the sea urchin, S. pur-puratus, to the 16-cell stage.
TWO CELL EMBRYO
The dertsity of IMPs was homogeneous on the Pf of both blasiomeres of the 2-cell
embryo. IMP size range dis-tributions were similar in both blasiomeres. No membrane
specializations, such as particle clusters or particle aggregates, were observed.
Plate II, figure 7 shows a portion of a fracture through an intact 2-cell embryo.
figures 8 and 9 are high magrtificaiions of represen-tative membrane fractures thr·ough
each cell in a 2-cell embryo. Plate III tabulates the data presented below.
Measur·ements were obiairted using iwo differ·t:tnt 2-cell t:tmbryos.
The mean IMP diameter on the plasma membrane PF was 88
R, and the IMP size range
was 50-180 R. Mean population dt:tnsities of IMPs were 170 pa.r·ticles/J.lm2, The
11
12
population density r·ange obser·ved was 130-217 par·ticles/ll m2. A 'total of 1303
shadow bases were measut'ed. These measurements apply to both blastomer-es of the
2-cell embr·yo. Data taker• separ·ately for· both blastomeres r·evealed str·iking
$imilarities in IMP sile and density distributions.
FOUR CELL EMBRYO
Plate IV, figure 10 shows a fracture through an intact 4-cell embryo. Figures
j
1, 12, 13, ar1d 14 are higher· magrlifications of ar·eas pr·esent in each blastomer·e of
one whole 4-cell embryo. IMP densities for four· blastomeres from one embryo were
similar·. No membr·ar1e speciali:zations were evident.
Data taken from two separate 4-cell embryos shows the IMP popula:tion density
r·ar.g~:
was 53-104 par·ticles/P.m2, with a mean density of
901~'m2,
The
IMP size range was 50-170 A, with a mean of 86 A. Shadow bases of 1402 particles
wer·e measur·ed. Data taker. separ·ately for· each blastomer·e analyzed <S altogether·>
showed similar IMP size and density distributions.
EIGHT CELL EMBRYO
Membrane particle densities in approximately
sero of the 8-cell stage blastomeres
examined wer·e distinctly heter·ogeneous. Other blastomeres ir1 which IMP density was
homogeneous displayed a high particle density relative tu the 2 and 4-cell stage
blastomer·es. lr• those blastomer·es that displayed heter·oger.ous particle
distributionst membrane particle clusters were apparent.
13
Platt VI shows r-epresentative topographies from an 8-cell blastomere, and figure
15 a portion of the plasma membrane of one cell that exhibited many particle clusters
and a heterogeneous IMP density distr·ibu'tion. Figl.ll'ts 16 and 17 ar·e
high-magnification views of areas in figure 15. Plate VIII shows an 8-cell
blastomer-e <PF> that did not exhibit an heter-ogeneous IMP distribution ot membtarre
specializations, though the relative density of membrane particles is high. Plate X
shows four configurations of particle clusters which wett apparent in about half of
the 8-cell blastomeres analyzed.
The mearr IMP population density for thtt 8-cell embryo was 548/J.l m2, with
a range of 370-760/J.lm2. The IMP size range was 50-220
X.
X, with a mean of 97
A total of 1915 par-ticles were measur-ed.
PARTICLE CLUSTERS
Membtanes of 8-cell blastometts that showed IMP density heter-ogeneities irr all
cases exhibited particle clusters or particle aggregates. Plate X shows
repr·eserrtative cluster- configur·ations from four separ-ate membrarre fractures.
Generally, IMPs contained within the clusters exhibited longer shadows, indicating
that the particles protrude further above the membrane than other-, disper-sed
particles.
The mean IMP population density was 229 /J.l m2, ranging fr·om
122-395/J.lm2. The mean IMP diameter within the cluster was 108 .i, with a
r·ange of 55-207 .X. A total of 314 particles on six sepata'te membtanes wer-e
measured.
Var·ious cluster- configur·ations showed the IMPs in depr-essions, elevations, and
14
flush with the plane of the membrane. About
2er. of the clusters showed particles
that were arranged in small circles Cas in Plate
x, figure 21>.
COVERSLIP ORIENTATION EXPERIMENTS
There were no indications of animal-vegetal polarity with respect to embryo
or·ientation in any of the configlll"ations of embryo coverslip development. Corrtrol
experiments (fertilized eggs deposited on coverslips> displayed 16-cell embryos that
wer·e
oriented with their micromeres downward against the coverslip, upward, and in
gradients of hor-izontal placement.
The development of eggs that were DTT treated, fertilized, and deposited onto
coverslips was similar to the controls, though the vitelline layer was absent, and
fewer· embryos r·emained attached to the coverslip, leaving plasma membrane remnarrts.
The DTT treated eggs that were deposited onto coverslips with the attached sperm
showed non-unifor·m orientation as in the other· control expermimerrts.
DISCUSSION
This study shows that a change in plasma membrane topography is apparent prior
to the formation of the blastomeres of the 8-cell embryo of the sea urchin, S.
purpuratus. In addition, coverslip fertilization experiments confirm earlier
reports that the initial site of sperm errtry cannoi be cOffelated with subsequerrt
development of animal-vegetal polarity (Schroeder, 1986; Endo, 1966>. This is
contr·ary to the findings of Schatten and Schatten <1979) and Runnstrom <1925).
The 2 and 4-cell stage blastomeres show homogeneous membrane particle
distr·ibutions and no evidence of membr·ane speciali:zaiions. Further-more, particle
densities in the 2-cell embryo are almost twice those of the 4-cell embryo,
indicatir.g that IMPs pr-estmt on blastomeres of the 2-cell embryo art par-titioned
equally between the blastomeres of the 4-cell embryo. The mean IMP size and size
rarrge in the 2 and 4-cell embr·yos ar·e virtually identical, suggesting that no majornew membrane particle insertions have occurred between the 2 and 4-cell stages.
The 8-cell stage IMP densities and siu distr·ibutions clear·ly show a massive
reorganization of membrane topography during division of the 4-cell embryo. In
pr·eliminar-y exper-iments, it was found that membrane topographies wer·e similar- ir•
embryos examined at the early, middle and late substages of each major developmental
stage. Collection of embr·yos for· fixation in the ear·ly and late substages was begun
at least 10 minutes prior to or after division, thus placing the timetable of
membrane reor·garli:zation -from about i6 minutes befor-e division of the 4-cell embryo to
16 minutes after establishment of the 8-cell embryo.
A shift of 10 .X in mean particle si:ze and the increase in IMP density suggest
new membrane particles have been added to the PM-PF of the 8-cell stage blastomeres.
15
16
The particle der,sities found in membranes of the 8-cell embryo increase three to
seven times those found in the 4-cell embryo. These changes seem to be indicative of
overall embryonic diHerentiation, sirJCe blastomeres of the 2 ar,d 4-cell stages
evidence totipotentcy when seperated and cultured, whereas 8-cell stage blastomerP.s
do not. These findings shO'H a consequential, or· possibly causal, relatior•ship
between plasma membrane differentiation in the 8-cell stage and subsequent narrD'Hing
of developmental potential o-f the individual blastomeres.
Recent data on the plasma. membrane topography of blastomeres of the 16-cell
embryo reveal str·iking differences in IMP sin and membrane densities between the
three cell types !Kasparian, 1980>. These data also shD'H that membrane particle
cluster·s appear· on the macr·omer·e PM-PF; these heterogeneities were not apparer•t in
membranes of the micromeres or mesomeres. In Kaspar-ian's study, the IMP diameter
found within cluster·s on the macromer·e PM-PF was 137%, with a membrane density r-ange
of 334-385 IMPs/Jlm2. In the present study, particle clusters were observed
ir, the 8-cell blastomer·es with a mean IMP diameter of 108 K, and a cluster IMP
density range of 122-395 IMPs/Jlm2. Differences in IMPs found in clusters in
thee and 16-cell embl"yos shD'H a gradual predominance of lar-ger diameter IMPs and
greater densities in clusters found on macromeres than those found on blastomeres of
the 8-cell embr·yo. The trend toward larger particle sin is also evident in IMPs
during transition from the 4 to the 8-cell stage.
Membr·ane par-ticle cluster·s occurred in about 50% of the random fr-actures of the
PM-PF of 8-cell stage blastomeres. In the study conducted by Kasparian <1980), only
the macromeres shO'Hed evidence of membrar.e par·ticle clusters. Four cells of the
8-cell stage are destined to give rise to four- micromeres and four ma.cromeres, while
the other· tier of four cells gives rise to eight mesomeres. Although on a
statistical basis, half of the cells at the 8-cell stage shD'H clusters, clusters are
17
seen later- or. membr-ar.es of only fol.ll' of the macr-omer-es in 'the 16-cell embr-yo.
Micromer-es do not display pal'ticle clusters, though the distr-ibution of IMPs in the
membr·anes is heter-ogeneous. Macr·omel'es contain PAr-ticle cluster·s, yet show a
homogeneous pa1'1:icle distribution. In marked contrast, 8-cell stage blastomeres show
both clusters and membrane par·ticle density heterogeneities. These observAtior.s
suggest that the non-random distribution of membl'ane particles and the particle
clusters ar-e distinct in their functional relatior.ships dur·ing embryoger•tsis, and
become segregated into specific cell types.
The change of membr-ane topography observed in S. purpur-atus is one among
a number of phenomena known to occur in sea urchin development. Rodgers and Gross
(1978> have found a large asymmetr-y in maternal single copy RNA transcr·ipts in the
three cell types of the 16-cell sea. urchin, Lytechinus pictus. This
informational asymmetr·y is irJCiicative of mor·phogenetic factO!' segr-egation, such as
those processes which take place in mosaic ol'ganismslike Ilyanassa
obsoleia. Horstadius and Josefsson <1972> have isolated animaliling substances
from lyophilized sea urchin embryos and identified two of the strongly animalizing
substances as nucleoiides. Stratification of cytoplasmic subs'ti'tuents has showr,
little effect on sea urchin development (Harvey, 1933, Morgan and Spooner, 1909),
suggesting 'thai whatever· is being segr-egated within blastomer-es of the embryo are r10t
displaced during centl'ifuga'tion. Molecules l'esponsible for embryo differentation may
be per·ipher-al 'to the membr-ane but anchor-ed to por-tiorJS of the transmembrane integr-al
pl'otein system, rendering these molecules fixed in place at the cell periphery.
Since 'the cleavage plar.e of the fourth division has been set by oocyte syrnmetr·y
(Schroeder, 198&>, the possibility exists that morphogenetic factOI's are being
segregated by membrane reor·ganization during embr-yo cluvage. Since blastomer-es of
the 8-cell embryo seem to lose the totipotentcy pr-esent in blastomeres of the 4-cell
18
embr·yo <Hors'tadius, 1975> and exhibit massive membrane component reorganizaton, it is
possible that the change in membrane topography is accompanied by the segregation of
informational molecules at the 16-cell stage. Scant evidem:e fDI" this mechar.ism
exist, and is discussed only in vague terms (Schroeder, 1988; Freeman, 1977>.
The r·esults of this study show that a change in membrane or-ganization takes
place from 18 minutes before to 11 minutes after division of the 4-cell sea urchin
embr·yo. The subsequent changes in membrane character- of cells in the 8-cell embr·yo
accompany other changes that lead 1:o determination of these cells by the 16-cell
stage, and possible earlier·. Future work. in this area should include studies of
in situ localization of 1:he morphogenetic factors. Additionally, it would be
interestir.g to character·ize the plasma membrane topography of animalized vs.
vegetalized blastomeres of the sea urchin embryo.
This
wo~k
was funded in
pa~t
by a
g~ant
f~om
the
California State University, Northridge Foundation
No. 3234.30.317
B I BL I CtGRAPH·y·
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Czihak, G. Ced.>, 1975. The Sea Urchin Embryo. Springer-Verlag,
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Da Silva, P. F. and Br-anton,
Cell Bioi. 45: 598-605.
[1,,
1970. Membrar.e splitting irt freeze-etching.
~.
Dr-iesch, H., 1906. Studien zur Entwicklungsphysiologie der Bilateralitat. Ar·ch.
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Ertdo, Y., 1960. The first cleavage furr-ow in sea urchin eggs does not pass through
the sperm entrance point. Exp. Cell Res • .ll: 432-434.
Epel D., Weaver, A.M. and Mazia, D., 1970. Methods for removal of the vitelline
membrane of sea urchin eggs. Exp. Cell Res. 61: 64-68.
Fr·eemar., G., 1977. The establishmertt of the or·al-aboral axis ir• the Ctenophore
embryo. ~· Embryo!. Exp. Morph. 42: 237-260.
Furcht, L. T. and Scott, R. E., 1975. Modulatiort of the distribution of plasma
membrane intramembranous particles in contact-inhibited and transformed cells.
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sea ur·chin blastomer·es. Sarsia 18: 1-9.
19
20
Har-vey, E. B., 1933. The development of half and quar-ter- eggs of Ar-bacia
punctulata and of strongly centrifuged whole eggs. Biol. Bull.
62-63: 155-167.
Hor-stadius, s., 1975. Isolation and tr-ansplantation exper-iments. In The Sea
Ur-chin Embryo, <G. Czihak, ed.) pp. 364-406. Springer-Verlag, Berlin.
Hor-stadius, s., 1973. Experimental Embr-xology of Echinoder-ms.
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Hor-stadius, s., 1939. The mechanics of sea ur·chin development, studied by oper-ative
methods. Bioi. Rev. 14: 132-179.
Hor-stadius, S. and Josefsson, L., 1972. Mor-phogenetic substances from sea urchin
eggs. Isolation of animalizing substances from developing eggs of Paracentrotus
lividus. Acta. Embr-yol. Exp. pp. 7-23.
Horstadius, S. and H'olsk.y, A., 1936. Studien uber- die Determination der
Bilater·alsymmetrie des .iungen Seeigelk.eimes. Wilhelm Roux' Ar-ch.
Entwicklungsmech. Organismen 135: 69-113.
Hough-Evans, B. R., Wold, B. J., Er-r.st, S. G., Br-itter., R. J. artd Ilavidson, E. H.,
1977. Appear-ance and persistence of maternal RNA sequences in sea urchin
developmertt. Ilev. Bioi. 60: 258-277.
Hudson, S.C., Rash, J. E and Graham, H'. F., 1979. In Freeze-Fracture:
Methods, Ar·tifacts, and Interpretations <Rash arid Hudson, eds.>.
Raven Press, New Yor-k..
Kar-novsk.y, M. J ., 1965. A for-maldehyde-glutaraldehyde fixative of high osmolarity
for use in electron micr-oscopy • .Jl.. Cell Biol. 27: 137a.
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Sea Urchin Embryo. Masters Thesis, California State University,
Nor-thridge.
Leahy, P. s., Tutschulte, T. c., Br·itter., R. J. and Ilavidson, E. H., 1978. A
large-scale laborator-y maintenance system for gravid purple sea ur-chins
<Str·ongylocer.tr·otus pur·pur·atus>. ~. Exp. Zool. 204:
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Amer-.~. Physiol. ~: 135-138.
Lenning, S. artd Hagstr-om, B. E., 1971. Cleavage and differ-entiation in the sea
urchin embr-yo transplantation studies of micromeres. Protoplasma 73:
303-322.
Mazia, II., Schatten, G. and Sale, H'., 1975. Adhesiort of cells to surfaces coated
with polylysine. J.. Cell Biol. 66: 198-200.
21
Moor·, H., 1969. Freeze-etching. Int. Rev. Cy'tol. 25: 391-412.
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Muhlethaler, IC., 1971. Studies on freeze-etching of cell membranes. Int.
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Wiley and Sons, New Yor·k.
22
PLATE
I
Figure 1
Scanning electronrnicrograph of an
unfertilized sea urchin egg.-------- 999 X
Figure 2
Scanning electronrnicrograph of a
OTT-treated fertilized egg.---------- 1,099 X
Figure 3
Scanning electronmicrograph of
a OTT-treated 2-cell embryo.--------- 1,175 X
Figure 4
Scanning electronmicrograph of
a OTT-treated 4-cell embryo.------- 1,109 X
Figure 5
Scanning electronrnicrograph of
a OTT-treated 8-cell embryo. _ _ _ 1,225 X
Figure 6
Scanning electronrnicrograph of
a 16-cell embryo.
Notice the
intact vitelline envelope.-------- .1,959 X
24
PLATE
II
Figure 7
Freeze-fracture micrograph
of an intact 2-cell embryo,
showing the PF of both blastomeres. -- 6,650 X
Figures 8 - 9
High magnification freeze-fracture
micrographs of a portion of the PF
of each blastomere of a 2-cell
embryo.
Arrows indicate direction
of shadowing. ------------------- 98,000 X
-
-~
-
__... __
26
PLATE
III
Histogram shoVJing the IHP
size frequency distribution of the
plasma membrane PF of the 2-cell embryo.
40T
2 - C E L L E MB
A 36 PLASMA MEMBRANE
R
MEAN PARTICLE SIZE:
T 32 PARTICLE SIZE RANGE:
I
# PARTICLES MEASURED:
C 28
L
p
P
E
R
C
E
N
R Y 0
PF
88 A
50-180 A
1303
T E 24
0
s
20
F M
E 16
T A
0 s
T U
A R
8
D
4
L E
0
40
70
100
130
160
PARTICLE SIZE <ANGSTROMS)
190
28
PLATE
IV
Figure 19
Freeze-fracture micrograph of an
intact 4-cell embryo showing the
PF of the four blastomeres. ------- 5,935 X
Figures 11 - 14
High magnification freeze-fracture
micrographs of the PF of each
blastomere of a 4-cell embryo.---- 74,200 X
30
PLATE
V
Histogram showing the IMP size
distribution of the PH-PF of the four
eel I embryo.
4 - C E L L E MB
PLASMA MEMBRANE
MEAN PARTICLE SIZE:
PARTICLE SIZE RANGE:
# PARTICLES MEASURED:
48
P A 36
p
E R
R T 32
C I
E C 28
R Y0
PF
86 A
50-170 A
1482
N L
T E 24
s
20
F M
0
E 16
T A
0 s 12
T U
A R
8
D
4
L E
8
38
68
90
120
150
PARTICLE SIZE <ANGSTROMS)
180
32
PLATE
1..)
I
Figure 15
Freeze-fracture micrograph
of the PH-PF of a blastomere
from an 8-cell embryo. Notice
the numerous particle clusters
and heterogeneous distribution
of I HPs. - - - - - - - - - - - - - - - - - 31 , 850 X
Figures 16 - 17
High magnification freeze-fracture
micrographs showing two portions
of the membrane from figure 15.
Notice the steep gradient of IMP
density between portions of the
same membrane. -------------------- 89,635 X
34
PLATE
a......•I I
Histogram showing IMP size
frequency distribution on the PH-PF
of the 8-cell embryo.
30T
A 27
R
T 24
I
C '21
L
E 18+
s
15+
M
E 12+
A
s 9+
U
R 6+
E
p
P
E
R
C
E
N
T
0
F
T
0
T
A
L
D
8 - C E L L
E MB R Y0
PLASMA MEMBRANE
MEAN PARTICLE SIZE:
PARTICLE SIZE RANGE:
# PARTICLES MEASURED:
PF
97 A
50-220 A
1915
~
3+
0
40
I
I
80
120
160
200
PARTICLE SIZE <ANGSTROMS)
+
240
36
PLATE
VIII
Figure 18
Freeze-fracture electron
micrograph of two adjacent
blastomeres from an 8-cell
embryo.
Cell on left reveals
the EF; cell on right shows the
PF.
Notice the paucity of
IHPs on the exoplasmic face. ----- 55,519 X
Figure 19
Higher magnification of a
portion of the EF shown
in figure 18. --------------------- 112,eee X
Figure 20
Higher magnification of a
portion of the PF shown in
figure 18. ------------------------ 112,899 X
38
PLATE
I><
Particle size frequency distributions
of the PH-PF of the 2, 4, and 8-cell embryos.
Notice the 10 A shift in the predominant particle
species as developme-nt proceeds from the 2-cell
to the 8-cell embryo.
..
30
p
P A 27
E R
R T 24
C I
E C 21
T E 18
N L
0
s
F M
C 0 MB I N E D
2~4~8
C E L L
T 0 T A L S
S T A G E S
- 2-CELL EMBRYO
4-CELL EMBRYO
= 8-CELL EMBRYO
15
E 12
T A
0 s
9
T U
A R
6
D
3
L E
al40
_,-~~"+
I
I
80
120
160
......200
.t..;.
PARTICLE SIZE <ANGSTROMS)
I
240
40
PLATE
X
Figure 21 - 24
Freeze-fracture micrographs
showing various configurations of
PH-PF particle clusters.
Figures 21
and 23 show clusters flush
with the membrane surface.
Figure 22
shows an elevation, while figure 24
shows a cluster in a depression. ------------ 196,999 X
42
PLATE
><I
Histogram showing the size
frequency distribution of the
particle clusters on the PH-PF of an
8-cell embryo.
40
P A 36
p
E R
R T 32
C I
E C 28
N L
T E 24
s
0
20
F M
E 16
T A
0 s 12
A R
8
D
4
L E
T
u
0
P A R T I C L E C L U S T E R S
PLASMA MEMBRANE
PF
MEAN PARTICLE SIZE:
108 A
PARTICLE SIZE RANGE: 55-207 A
# PARTICLES MEASURED: 314
l_j~iiiiiiii~IL~~~~~--+--
40
80
120
160
200
PARTICLE SIZE <ANGSTROMS)
240
44
PLATE
XI I
Table of particle PH-PF IMP
densities and size frequency
distributions of the 2, 4 and 8-cell
sea urchin embryo, Strongylocentrotus
purpuratus.
Data also show
distributions for the 8-cell particle
clusters.
MEMBRANE
FRACTURE
FACES
2- CELL EM~RYO
PLASMA PF
MEMBRANE
PARTICLES
POPULATION DENSITIES
NUMBER
MEAN
SIZE
STD
STD
MEAN
RANGE
MEASURED
DIAMETER
RANGE
DEY
ERROR
[ #/tp2]
[#jtp2]
A
1
88
50-180
24
0.66
1303
170
130-217
i
4-CELL EMBRYO
PLASMA PF
1402
86
l
50-170
23
0. 61
90
53-104
I
I
8- CELL
EMBRYO
PLASMA PF
8- CELL EMBRYO
PLASMA PF
PARTICLE CLUSTERS
1915
97
50-220
28
0.64
541
370-760
314
108
55-207
31
1.6
229
122-395
I