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University

 

This is to certify that the

dissertation entitled

(3H)Serotonin binding to cell fractions and
dissociated cells of blastula, gastrula,
prism and pluteus sea urchin (Arbacia

unctul ta embr as
P a ) ‘ypresented by

 

Kenneth Michael Brown

has been accepted towards fulfillment
of the requirements for i

Ph . D. degree in ZOO] 09y

 

 

a,
/

”722/ LQ/ZWL

Major professor

 

Date 5/20/82

 

MS U L! an Affirmative Action/Equal Opportunity Institution 0. 12771

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u.n.. I it a t i=1 .i I I it {.9. ii...- sto::L‘-..-.s»0...m»tnn.»‘. :0...‘l-b‘.::.$:.72.... .cuuu:

[3H]SEROTONIN BINDING TO CELL FRACTIONS AND DISSOCIATED CELLS OF

BLASTULA, GASTRULA, PRISM AND PLUTEUS SEA URCHIN (ARBACIA PUNCTULATA)

 

EMBRYOS

by

Kenneth Michael Brown

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology

1982

ABSTRACT

[3HJSEROTONIN BINDING TO CELL FRACTIONS AND DISSOCIATED CELLS OF

BLASTULA, GASTRULA, PRISM AND PLUTEUS SEA URCHIN (ARBACIA PUNCTULATA)

 

EMBRYOS

BY

Kenneth Michael Brown

The presence and location of serotonin binding sites in early

sea urchin (Arbacia punctulata) embryos was investigated by the

 

binding of radiolabelled serotonin to cell fractions of blastulae,
gastrulae, prism larvae, early pluteus larvae and later pluteus
larvae cell fractions and to dissociated cells of these stages.
Specific [3HJserotonin binding was demonstrated in some cell
fractions from each developmental stage studied. Cell fractions were
partially characterized by enzymatic and microscopical analyses. The
localization of serotonin binding sites to particular subcellular
components was suggested from comparisons between the binding
activity and organelle composition of the various cell fractions. In
the older plutei serotonin binding sites were tentatively determined

to be located on cell surface membranes. In earlier embryos the

correlation between serotonin binding activity and the quantity of
cell surface membranes in cell fractions was less distinct, and the
possibility of serotonin binding sites on other cell organelles was
examined. [3H]Serotonin bound to nuclei of late blastula, gastrula
and prism embryos while no binding to nuclear fractions of early and
advanced plutei was detected. Specific [3H]serotonin binding to
dissociated cells of prism, early pluteus and older pluteus embryos
was demonstrated, but no binding to late blastula cells was detected.
Results of binding studies suggest that serotonin functions in early
sea urchin embryos as it does in adult animal tissues by first
binding to a receptor molecule. Possible serotonin functions and

mechanisms of action are discussed.

©1982

KENNETH MICHAEL BROWN

All Rights Reserved

DEDICATION

I would like to dedicate this work to my parents, Ray and Beverly

Brown.

11

ACKNOWLEDGEMENTS

I would like to thank my thesis advisor, Dr. John R. Shaver, for
everything. Dr. Shaver is both an excellent scientist and a fine
person. I was very fortunate to have been his graduate student.

For their help and advice I thank all of the members of my
committee, Dr. Neal Band, Dr. Evelyn Rivera and Dr. Frank Welsch.

Special thanks to my wife, Amy, for her help, support and

fortitude.

iii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . . .
MATERIALS AND METHODS . . . . . . . . . . . .

Embryo CUlture o o o o o o o o o o o o o

 

Cell Dissociation . . . . . . . . . . . .

 

Cell Fractionation . . . . . . . . . . .

 

[3H]Serotonin Binding Assays

 

Binding to Cell Fractions . . . . .

 

Binding to Whole Cells . . . . . .

 

Transmission Electron Microscopy . . . .

 

Enzyme Analysis and Protein Determination

 

5'-NUCIeOtidase o o o o o o o o o o

 

NADH oxidase . . . . . . . . . . .

 

Cytochrome c Oxidase . . . . . . .

 

Protein Determination . . . . . . .

 

RESULTS 0 O O O O O O O O O O O O O O O O O O

Embryo CUIture O O O O O O O O O O O O 0

 

Cell Fractionation . . . . . . . . . .

 

Explanation of Methods . . . . . .

 

Enzymatic Analysis . . . . . . . .

 

5' Nucleotidase . . . . . . .

 

iv

Page
vi

viii

14
14
15
15
21
21
22
23
24
24
25
25
25
27
27
34
34
36

37

Cytochrome c Oxidase . . . . . . . . .

 

NADH Oxidase . . . . . . . . . . . . .

 

Microscopy . . . . . . . . . . . . . . . .

 

[3H]Serotonin Binding Assays . . . . . . . . . .

 

Binding to Cell Fractions . . . . . . . . .

 

Binding to Whole Cells . . . . . . . . . .

 

DISCUSSION 0 O O O O O O O O C O O O O O O O O O O 0

Cell Fractionation . . . . . . . . . . . . . . .

 

[3H]Serotonin Binding to Embryo Cell Fractions .

 

[3H]Serotonin Binding to Dissociated Embryo Cells

 

Serotonin in Embryonic and Nonembgyonichystems

 

Mechanisms of Serotonin Action in Early Sea Urchin Embryos

 

LIST OF REFERENCES . . . . . . . . . . . . . . . . .

Page
48
48
49
6O
61
67
70
70
73
77

80

86

Table

i

ii

iii

iv

vi

LIST OF TABLES

Distribution of [3HJserotonin binding activity in
subcellular fractions isolated from the homogenate of

24-11011: Sea urChin embryos. o o o o o o o o o o o o 0

Distribution of [3H]serotonin binding activity in
subcellular fractions isolated from the homogenate of

36-hour Bea “rehin embryos. o o o o o o o o o o o o 0

Distribution of [3Hlserotonin binding activity in
subcellular fractions isolated from the homogenate of

48-hour sea urchin embryos. . . . . . . . . . . . . .

Distribution of [3H]serotonin binding activity in
subcellular fractions isolated from the homogenate of

60-hour sea urchin embryos. . . . . . . . . . . . . .
Distribution of [3ulserotonin binding activity in
subcellular fractions isolated from the homogenate of

72-hour embryos O O O O O O O O O O O O O O O O O O 0

Comparison between [3H]serotonin binding and

5'-nucleotidase activity in subcellular fractions isolated

from 72-hour sea urchin embryos . . . . . . . . . . . .

vi

Page

37

41

43

45

47

63

Table Page

vii [3H]Serotonin specific binding recovery from

72-hour sea urchin embryo cell fractions. . . . . . . . 65

vii

LIST OF FIGURES

Figure

1 Procedure for the separation of sea urchin embryo cell

fractions. 0 O O O O O O O O O O O O O O O O O O O O 0

ii Procedure for dissociating sea urchin embryo cells . .

iii Unstained light micrograph of Arbacia punctulata

 

swimming blastula embryos (24 h). x 1580 . . . . . . .

iv Phase contrast micrograph of an Arbacia punctulata

 

gastrula embryo (36 h). x 630. . . . . . . . . . . . .

v Phase contrast micrograph of Arbacia ppnctulata prism

 

embryos (48 h). x 810. . . . . . . . . . . . . . . . .

vi Phase contrast micrograph of an Arbacia punctulata

 

early pluteus larva (60 h). x 1010 . . . . . . . . . .

vii Unstained light micrograph of Arbacia punctulata

 

plutCUSIErvae (7211).)‘3900 o o o o o o o o o o o 0

viii Phase contrast micrograph of an Arbacia punctulata

 

pluteus larva (72 h). x 1010 . . . . . . . . . . . . .

viii

Page

l7

19

29

29

31

31

33

33

Figure

ix

X

xi

xii

xiii

xiv

XV

xvi

xxvii

Phase contrast micrograph of the 24 hour embryo

fraction. x 2520. . . . . . . . . . . . . . . .

Phase contrast micrograph of the 36 hour embryo

fraCtiono x 1580. O O O O O O O O I O O O O O 0

Phase contrast micrograph of the 48 hour embryo

fraction. x 2020. . . . . . . . . . . . . . . .

Phase contrast micrograph of the 60 hour embryo

fraction. x 1010 O O O O O O O O O O O O O O 0

Phase contrast micrograph of the 72 hour embryo

fraction. x 2520. O O O O O O O O O O 0 O O O 0

Phase contrast micrograph of the 48 hour embryo

interface fraction. x 2520. . . . . . . . . . .

Phase contrast micrograph of the 48 hour embryo

interface fraction. x 2520. . . . . . . . . . .

Electron micrograph of the 24 hour embryo 34/40

interface fraction. x 38,200. . . . . . . . . .

Electron micrograph of the 72 hour embryo 34/40

interface fraction. x 53,800 . . . . . . . . .

ix

nuclear

40/55

Page

51

51

53

53

55

57

57

59

59

Figure Page

xviii Comparison between serotonin content in Paracentrotus

 

lividus embryos and [3H]serotonin binding to dissociated

Arbacia ppnctulata embryo cells. . . . . . . . . . . . . . 69

 

INTRODUCTION

Several chemical compounds which function as neurotransmitters
or hormones in tissues of adult vertebrate and invertebrate animals
have also been detected in early embryos that have not yet developed
nervous or endocrine systems. These substances include serotonin,
acetylcholine, dopamine, epinephrine and norepinephrine. One or more
of these compounds have been identified in embryos from the
unfertilized egg through gastrula and postgastrula stages in animals
of almost every animal phylum studied including echinoderms (Buznikov
e£_213, 1964, 1968, 1972a), mollusks and nemerteans (Buznikov,

1973), polychetes (Emanuelsson, 1974), insects (Furneaux and
McFarlane, 1965a, 1965b), amphibians (Baker, 1965), birds
(Emanuelsson, 1976; Wallace, 1979) and mammals (Renson, 1971; Burden
and Lawrence, 1973). The presence of neurotransmitters and hormones
in embryos which have not developed neural or endocrine structures
raises questions as to the function of these molecules in early
embryos. The concentrations of these compounds fluctuate throughout
development, but in general they are of the same order as in tissues
of adult animals (Buznikov, 1971). This observation, along with
studies which have demonstrated inhibition of early development in
the presence of inhibitors of these compounds, suggest that they

perform a regulatory role in early development.

2
The best case for a regulatory role of a neurohormone in early
embryogenesis can be made for serotonin (5-hydroxytryptamine). In

the nemertean, Tineus desori, and the polychete, Anaitides maculata,

 

 

serotonin levels dropped twentyfold in the period between
fertilization and the first cleavage divisions. This lower serotonin
level was maintained through gastrulation except for a fivefold
increase just prior to gastrulation (Buznikov, 1973). Serotonin has
been detected in early embryos of several mollusc species. In the

gastropod mollusc, Acanthodoris pilosa, serotonin levels increased up

 

to the midblastula stage and then dropped to undetectable levels
during gastrulation (Buznikov, 1973). Serotonin antagonists have
been shown to inhibit cleavage in several nudibranch (Mollusca)
species, and this inhibition could be reversed by the addition of
serotonin into the embryo culture medium (Buznikov SE 31., 1970). In

the loach, Misgurnus fossilis, drastic changes in serotonin content

 

have been monitored during cleavage divisions. At later stages the
serotonin level was barely detectable except for two short term
increases at early and late gastrula stages (Buznikov egflgl., 1964).
Baker (1965) has shown that serotonin levels in the frog, Xenopus
laevis, were higher during blastulation and neurulation than during
gastrulation. When neurula embryos of Xenopus laevis were exposed to
the serotonin antagonist lysergic acid diethylamide (LSD), monoamine
oxidase and 5-hydroxytryptophan decarboxylase activities (Baker,
1972) and serotonin levels (Baker, 1971) were altered compared with
untreated controls. Exogenous serotonin stimulated morphogenesis in

explanted neurula cells of the frog, Rana temporia; this effect was

 

blocked by serotonin antagonists (Martynova and Balousov, 1978).

3

In the chick embryo serotonin was detected throughout gastrulation
and neurulation (Emanuelsson, 1976). Injection of
S-hydroxytryptophan into chick gastrulae induced several
malformations (Schowing ES 31., 1977). Serotonin antagonists induced
disturbances in blastoderm expansion, primitive streak formation,
neurulation, somitogenesis and brain formation in the chick embryo
(Palen e£_§l,, 1979). Serotonin is present in rat ova, fertilized
eggs, two-cell and four-cell embryos (Burden and Lawrence, 1973),
and can reverse the depolarizing effect of the serotonin antagonist
chlorpromazine on the membrane potential of mouse two-cell embryo
blastomeres (Leonov SE 31., 1975).

Most of the investigations concerning the role of serotonin in
early embryogenesis have been made with sea urchin embryos. Using a
very sensitive biological assay based on the ability of serotonin to
stimulate motility in larvae of different nudibranch molluscs,
Buznikov SE 21. (1964) determined the serotonin levels in Strongylo-

centrotus drobachiensus sea urchin embryos from the fertilized egg

 

through advanced pluteus larval stages. Serotonin levels peaked at
each cleavage division until blastomeres started dividing
asynchronously. Levels also peaked at hatching, when primary
mesenchyme cells were separating from vegetal plate cells in the late
blastula, during the initial invagination of the vegetal plate in the
early gastrula, and at midgastrulation. Peaks were sharp, and embryos
at stages inbetween peaks contained two to five times less serotonin.
At the end of gastrulation, serotonin levels slowly rose and kept

rising through advanced pluteus stages. In embryos of the sea

4

urchins, Strongylocentrotus drobachiensis and Strongylocentrotus

 

 

intermedius, serotonin levels, measured by a fluorometric assay,

 

remained unchanged from the unfertilized egg through the first three
cleavages except for a four- to fivefold decrease at prometaphase of
each division (Buznikov e£_gl., 1972a). Using a histochemical
fluorescence technique, Buznikov e£_gl, (1972a) demonstrated the
presence of serotonin in every cell of embryos of various stages up
to the end of gastrulation, and the stage specific fluctuations in
serotonin levels from this study agreed fairly well with levels
determined by the biological assay discussed earlier. Using a
different histochemical fluorescent technique, this study failed to
detect serotonin until the early gastrula stage when it was shown to
be specifically localized in the archenteron and ventral ectoderm
region during gastrulation. Both histochemical fluorescence
techniques demonstrated the presence of serotonin in pluteus stages
only in the digestive tube, cells of the ventral ciliary band and
possibly in pigment cells. Histochemical studies of serotonin

localization in Psammechinus miliaris plutei confirm these

 

observations (Ryberg, 1974). Tbneby (1973, 1977a, 1977b), used
fluorometric and thin layer chromatographic techniques and could not
detect serotonin during cleavage, formation of primary mesenchyme

cells and gastrulation in embryos of the sea urchins, Paracentrotus

 

lividus and Strongylocentrotus fransiscanus. These results

 

contradict those of Buznikov SE 31. (1964, 1972a). From the end of
gastrulation until formation of a pluteus larva, serotonin was
detected by Toneby (1973, 1977a, 1977b), and serotonin levels were

shown to continually rise during this period. The inability of

5
Toneby to detect serotonin during cleavage and gastrulation may have
been due to the lower sensitivity of the assays he used as compared
with the biological assay of Buznikov, or it may reflect the greater
specificity of the assays used by Toneby. Renaud ep 31. (1979)
demonstrated the presence of S—methoxytryptamine in cleavage stages

of Paracentrotus and Sphaerechinus embryos when assayed by

 

 

dansylation and thin layer chromatography but not of serotonin.

The amount of 5-methoxytryptamine seemed to increase at the end of
the cell cycle, and this compound was postulated to be the important
molecule in the regulation of cell division. No spot corresponding
to 5~methoxytryptamine was detected in the thin layer chromatograms
of cleavage stage embryos as described by Toneby (1977a).

Several studies have examined the effect on early sea urchin
development of serotonin and various antisertonin compounds by
incubating embryos in sea water containing these substances.
Molecules which have been shown to block serotonin function in adult
animals have been shown to lower the membrane potential of

Strongylocentrotus intermedius fertilized eggs (Korobtsov and

 

Sorokin, 1974) and to inhibit both protein synthesis and cleavage in

Strongylocentrotus drobachiensus, Strongylocentrotus nudus,

 

Strongylocentrotus intermedius, Paracentrotus lividus, Scephechinus

 

mirabilis, Sphaerechinus granularis, Echinus melo and Arbacia lixula

 

 

embryos (Buznikov e£_gl., 1970, 1972b, 1974, 1976; Landau SE 31.,
1981). Lower concentrations of these inhibitors blocked cytoplasmic
divisions but allowed nuclear division to continue (Buznikov £5 31.,
1970). Buznikov (1971) and Buznikov fig El’ (1971a) have demonstrated

that various serotonin blocking agents in small concentrations

6
stimulate and in higher concentrations inhibit protein synthesis in

Arbacia lixula and Strongylocentrotus nudus oocytes, fertilized eggs

 

 

and early cleavage embryos. This action was not connected with the
effect of antagonists on cell division, as indicated by a completely
different shape of the dose-response curve. Early gastrulation

events in Psammechinus miliaris embryos, including the migration of

 

primary mesenchyme cells and the initial invagination of the vegetal
plate, were blocked by some antiserotonins and serotonin synthesis
inhibitors while other inhibitors delayed the onset of later
gastrulation events by preventing the formation of secondary
mesenchymal pseud0pods (Gustafson and Toneby, 1970, 1971; Gustafson,
1973). In all inhibitor studies, the effects of inhibitors could be
reversed by the addition of serotonin to the incubation medium.

Serotonin stimulated ciliary movements in Psammechinus miliaris

 

hatched blastulae (Gustafson, 1969). In low concentrations serotonin
has been shown to inhibit hatching by blocking synthesis of hatching
enzyme, and to increase the rate of protein synthesis in 2-16 cell

stage Paracentrotus lividus embryos, and in higher concentrations

 

serotonin completely inhibited cleavage (Deeb, 1972). Serotonin

stimulated Psammechinus miliaris pseudopodal activity in primary

 

mesenchyme cells, and these cells eventually formed a skeleton
containing a spicule arrangement closer to that of another sea urchin

genus Echinocardium (Gustafson, 1969).

 

Gustafson and Toneby (1970, 1971), Gustafson (1973), Buznikov
(1971) and Buznikov e£_313 (1972a) have proposed the following
functions for serotonin in sea urchin embryos from late blastula

through pluteus larva stages:

1.

5.

6.

7

Serotonin is involved in ciliary movement at hatching.

Serotonin produced by vegetal plate cells of late blastulae
elicits their pulsatory and other movements that play a role
in the release of primary mesenchyme cells and in the
initial invagination of the vegetal plate during
gastrulation. Serotonin may still be required for the
formation and exploratory movements of secondary mesenchymal

pseud0pods, necessary for the continuation of gastrulation.

Serotonin, probably released from pigment cells (derived
from secondary mesenchyme cells), induces the bubbling
activity of the coelomic rudiment, an important activity in
the morphogenesis of the coelom which begins in prism stage

embryos.

Serotonin, probably released from pigment cells, stimulates
cilia of the circumventral ciliary band which in turn

stimulates swimming activity in the pluteus larva.

Serotonin, probably released from pigment cells of the
pluteus larva, induces some ciliated ectodermal cells to
enter the body cavity and send out pseud0pods. These cells
eventually become nerve cells, and the pseud0pods become

axons o

Serotonin functions as a neurotransmitter in serotonergic

neurons of the advanced pluteus larva.

8
The functions proposed for serotonin in blastula and gastrula stages
were determined from studies already discussed; those in
postgastrular, preneural development (functions 3-5) were postulated
from detailed time-lapse light microscopy and electron microscopical

studies (Gustafson, 1969; Gustafson and Toneby, 1971) of Psammechinus

 

miliaris embryos. From these studies it was determined that pigment
cells were derived from secondary mesenchyme cells which are released
from the archenteron tip at the completion of gastrulation. The
cells accumulate pigment and show a chromaffin reaction, indicating
the presence of a monoamine which is postulated to be serotonin.
Pigment cells are initially located adjacent to the coelomic rudiment
during its formation and are later localized adjacent to ciliated
ectodermal cells. Agents which degranulate mast cells in adult
mammals stimulate pigment cells to release their contents, and this
results in a stimulation of ciliary activity in adjacent ectodermal
cells. Addition of serotonin to the embryo culture mimicked the
effect of degranulating agents. From these observations they
postulated that serotonin, released from pigment cells, provided the
stimulus for ciliary and coelomic activities in adjacent cells. In
early plutei the ciliated ectodermal cells that are adjacent to
pigment cells eventually break away from other ectodermal cells and
form neurons. Cell processes of developing neurons accumulate a
greenish substance which was presumably obtained from pigment cells.
This transfer of serotonin was postulated to be the initiator for
separation of future neurons from other ectodermal cells and for
elaboration of neuronal processes. Ryberg (1977) used histochemical

stains to identify neurons and confirmed the presence of nerve nets,

9

nerve strands and clustered cell bodies in Psammechinus miliaris

 

pluteus larvae. Pharmacological studies with this species have
suggested the presence of serotonergic and cholinergic neuronal
components in advanced plutei (Gustafson e£_§l,, 1972a, 1972b).

In summary, the roles of serotonin in early sea urchin embryos
cannot be totally determined from the available data. Consequently,
the analysis of the mechanism by which serotonin functions is still
at a theoretical level. Numerous pharmacological investigations, as
well as biological and histochemical serotonin assays, suggest a role
in cleavage and protein synthesis in cleavage stage embryos. Fewer
studies of the same type, as well as microscopical analyses, have
implicated a role of serotonin in ciliary activity in blastula
through pluteus stages, in cell morphogenesis in the late blastula,
during gastrulation and during postgastrular development, and as a
neurotransmitter in the advanced pluteus larval nervous system. In
postgastrula, preneural embryos the functions of serotonin released
from pigment cells have been formulated principally from
microscopical studies which need biochemical support. Although all
investigations confirm a continual increase in serotonin levels
between the end of gastrulation and formation of the pluteus larva,
the chemical serotonin assays of Toneby, in contrast to the
biological and histochemical assays, have not detected the compound
in gastrula and pregastrula stages of deve10pment. It is possible
that newly synthesized serotonin does not accumulate during or prior
to gastrulation but is rapidly degraded after carrying out its
function and that at these low levels serotonin could not be detected

by Toneby's chemical methods. It is also possible that a different

10
monoamine, such as methoxytryptamine, may be the important regulatory
agent in the earlier embryos.

The functional activity of neurotransmitter and hormone mediators
in adult animals is realized by their reaction with corresponding re-
ceptor sites. If serotonin is directly involved in the regulation of
early sea urchin embryogenesis, then embryos should contain reactive
sites which could recognize and bind this substance. Determination of
the subcellular location of serotonin binding sites could help answer
the question as to how serotonin functions. For example, serotonin
may function in cleavage and cell morphogenesis by interacting with
the microfilaments and microtubules, cell organelles directly respon-
sible for these events. Alternatively, serotonin may interact with
other subcellular structures such as nuclei, yolk granules, mitochon-
dria or endoplasmic reticulum. As a probable neurotransmitter in
advanced pluteus larvae, serotonin would most likely function, as in
adult animals, by interacting with cell surface binding sites. Cell
surface binding sites, however, may also mediate serotonin activity
in preneural embryos. The presence of serotonin binding sites in
pregastrula and gastrula stages is supported by the experiments
already discussed which have demonstrated developmental alterations
in embryos exposed to serotonin binding site blockers accompanied by
a reversal of the effects after addition of serotonin to the embryo
culture. Results of several studies with early cleavage stage
embryos suggest the presence of intracellular serotonin binding
sites. All sea urchin species studied displayed the same sensitivity
to certain antiserotonins, as determined by the ability of these

compounds to block cleavage. Other antiserotonins elicited this

11

"normal" sensitivity with many species (Strongylocentrotus

 

drobachiensus, Strongylocentrotus nudus, Strongylocentrotus

 

intermedius, Paracentrotus lividus, Sphaerechinus granularis,

 

Scaphechinus mirabilis) but were hyperactive against other

 

"supersensitive” sea urchin species (Echinocardium cordatum, Arbacia

 

lixula, Echinus melo, Echinus esculentus), inhibiting cleavage at

 

concentrations 10-800 times less than those required to inhibit
cleavage in the other species (Buznikov SE 31., l974,l976,l979). In
supersensitive species, neuroactive drugs of normal activity
inhibited both protein synthesis and early cleavage in approximately
the same concentrations, whereas hyperactive substances inhibited
protein synthesis in concentrations much higher than those needed to
block cleavage (Buznikov e£_§13, 1972b). From these studies it was
suggested that two types of intracellular serotonin receptor sites,
supersensitive (S) and nonsupersensitive (N), were present in
supersensitive embryos; hyperactive inhibitors could bind to either
site, while normally active inhibitors bind only to N-receptors. It
was also assumed that normally sensitive embryos contain superactive
binding sites which are important for some cellular activities but
which are uncoupled with the cleavage process. Buznikov e£_gl,
(1979) fragmented sea urchin fertilized eggs into halves and the
halves into quarters and tested the sensitivity of various egg
fragments to different antiserotonins which was measured by the
ability of these compounds to block cleavage. All fragments from
normally sensitive eggs exhibited normal sensitivity to all drugs.
In supersensitive embryos, however, the centripetal pole egg halves

displayed normal sensitivity to hyperactive drugs while centrifugal

12
pole halves displayed supersensitivity to the same drugs. If the
centripetal pole half was split again by centrifugation,
supersensitivity to hyperactive substances was restored in the
centrifugal quarter while the other quarter displayed normal
sensitivity to these substances. The investigators concluded that
S-receptors were coupled to cell division by a sensitizer factor that
moved centrifugally, and that normally sensitive embryos do not have
the factor. This factor was postulated to be associated with yolk
granules, the only organelle known to move during centrifugation in a
manner similar to the postulated sensitizer factor. Landau _e_§ a_l_.
(1981) determined that hyperactive substances were in general more
lipophilic than normally active antiserotonins and concluded that to
reach supersensitive receptors the antiserotonins must first
penetrate a lipid barrier and that the sensitizer factor may function
by enhancing penetrability. Based on the effect of antiseritonins on
early cleavage stage embryos treated with puromycin (Buznikov SE 31.
1971a; Buznikov, 1971) and antimycin A (Buznikov e£_213 1972b;
Buznikov and Markova, l974b) N- and S-receptors were postulated to be
localized on endoplasmic reticulum and mitochondria, respectively.
Buznikov ES 31. (1975, 1977) and Manukhim‘e£_gl, (1973, 1976) allowed
serotonin and antiserotonins to attach to whole embryos and
determined the amount bound by chemically assaying for the compounds
in embryo pellet extracts, by spectrophotometrically measuring a
decrease in the concentration of free ligands in the culture medium,
or by lysing the embryo cells and determining the sensitivity of

Arbacia lixula embryos to the lysate. In these experiments serotonin

 

and antiserotonin binding occurred in two phases. They concluded

13
that the second phase apparently coincided with the uptake of
serotonin and antiserotonins into embryo cells since dead or
osmotically lysed embryos only showed the first phase
of binding, and bound chemicals could not be released by washing
embryos, but could be released by cell lysis.

From the experimental observations with cleavage stage embryos,
serotonin receptive structures were postulated to be on mitochondria,
endoplasmic reticulum and possibly on yolk granules. Localization of
receptor sites with such methods is indirect. These studies have not
determined that receptive structures are on these organelles, nor
have they ruled out the possibility of receptive structures on other
intracellular organelles or on the cell surface membrane. For this
reason it is necessary to create cell free systems suitable for the
direct study of structures that can specifically recognize and bind
serotonin. The nature of serotonin receptive structures in embryos
older than the early cleavage stages is almost totally unexplored.

In the research described in this thesis, cell fractionation was
performed on five developmental stages of the sea urchin Arbacia

ppnctulata ranging from late blastulae to pluteus larvae. The

 

presence and location of serotonin recognition sites was investigated
by the binding of radiolabelled serotonin to various cell fractions

and to whole dissociated embryo cells.

MATERIALS AND METHODS

Embryo Culture

 

Gametes of the sea urchin, Arbacia punctulata (Florida Marine

 

Biological Specimen Co., Panama City, Fl.), were obtained by
application of 15 volts AC to the oral surface of the animals. Eggs
were collected in Herbst's (1904) artificial sea water as modified by
Motomura (1954). Sperm were collected dry. Eggs were washed once in
sea water by settling and fertilized with a dilute sperm suspension.
Embryos were subdivided among two or three one-gallon jars to which
sea water was added to a total volume of approximately 3500 ml in
each jar such that the final embryo concentration was 2000 embryos/m1
(7 ml washed, settled eggs per jar). Embryos were cultured at 15°C
in a constant temperature chamber. In each jar embryos were
continuously agitated by a teflon paddle rotated at 60 cycles per
minute. In the case of 72 hour embryos, paddles were rotated at 30
cycles per minute after 60 hours of rotation at 60 cycles per minute.
At the desired times - 24, 36, 48, 60 or 72 hours after
fertilization, embryos were allowed to settle for one hour at 0°C.
Excess sea water was poured off and embryos in the remaining sea
water were collected by centrifugation in a Sorvall SS-34 rotor at
480xg for 3 min. Embryo samples were fixed in 32 formalin for later

observation.

l4

15

Cell Dissociation

 

Dissociation of embryo cells was performed according to the
method of Cuidice and Mutolo (1970) and is summarized in Figure 2.
Embryos were washed three times in 20 vol calcium-free sea water
(Okazaki, 1956), suspended in 5 vol 0.5M sucrose, 0.01M Tris-MCI
buffer, pH 8.0, and homogenized in a 20 m1 Dounce homogenizer with a
loose-fitting glass pestle until dissociation was complete. The
cells were collected by centrifugation at 100xg for 3 min in a
Sorvall HB-4 rotor. Cell pellets were suspended in sea water for
binding assays and cell counts. Cell counts were made in triplicate
using a hemocytometer (Brightline, Americal Optical).

Cell Fractionation

 

The procedure for the separation of embryo cell fractions is
summarized in Figure l. Embryos were washed three times by
suspension in 20 vol calcium, magnesium-free sea water (Marine
Biological Laboratory, Woods Hole, Mass.) followed by centrifugation
at lOOOxg for 5 min in a Sorvall HB—4 rotor. Embryo pellets were
then dispersed in 6 vol 0.25 M sucrose, ZmM MgSO4, SmM TriseHCl, pH
8.0 (S-TM) (Chlapowski and Band, 1971), and homogenized in a 40 m1
Potter-Elvehjem type teflon grinder with 15-20 strokes of the pestle
rotated at 2500 rpm. The extent of homogenization was monitored by
phase contrast microscopy and was continued until at least 95 percent
of the cells had been disrupted. The homogenate was centrifuged at
2000xg for 15 min in a Sorvall HBP4 rotor. The supernatant was
removed and saved, and the pellet was suspended in 20 vol 0.25M
sucrose, 25mM KCl, SmM Tris-HCl, pH 8.0 (S-TK), and centrifuged at

2000xg for 15 min in a Sorvall HB-4 rotor. The final pellet was

16

Figure 1. Procedure for the separation of sea urchin

embryo cell fractions.

17

SEA URCHIN EMBRYOS
l
Wash 3X in Ca,lMg-free sea water

Homogenlzation in 5-6 vol S-TM, pH 8.0

HOMOGENATE
, I
20009. 15 min
1
\V \l'

Pellet Supernatant

l
Suspended in S-TK. pH 8.0
I
20009, 15 min

WL Supernatant

NUCLEAR PELLET

 

 

15.0009, 20 min

 

 

 

 

 

l
Suspended in 55% (w/w) S-TKM. pH 8.0 \p l \L
MITOCHONDRIAL Supernatant
PELLET l
7m| 34% (w/w) S-TKM, pH 3.0 100.0009. 60 mm
20ml 40% (w/w) S-TKM, pH 8.0
. MICROSOMAL
12ml PELLET

 

 

 

.|..
100.0009. 5 hr

1

- 34/40 INTERFACE

40/55 INTERFACE
' NUCLEI

18

Figure 2. Procedure for dissociating sea urchin embryo cells as in

Giudice and Mutolo (1970).

19

SEA URCHIN EMBRYO PELLET

 

Washed 3X in 20 vol Ca, Mg-free sea water,
0.01M Tris, pH 8.0

Suspension in 5 vol 0.5M sucrose.
0.01M Tris, pH 8.0

Homogenization in a glass homogenizer,
loose fitting glass pestle

Cell collection by centrifugation at 10009, 3 min

 

EMBRYO CELL PELLET

 

Suspension in sea water for binding assays

20
the previous supernatant and centrifuged 15,000xg for 20 min in a
Sorvall SS-34 rotor. The resulting pellet was referred to as the
mitochondrial pellet. A microsomal pellet was obtained by
centrifuging the post-mitochondrial supernatant at 100,000xg for 90
min in a Spinco Type 30 rotor. The post-microsomal supernatant was
discarded.

The nuclear pellet was further fractionated by discontinuous
sucrose gradient centrifugation. The pellet was dispersed in 20 ml
55% (w/w) sucrose, 25mM KCl, 2mM MgSO4, SmM Tris-301, pH 8.0,
(55%(w/w)S-TKM) by homogenization in a 40 ml Potter-Elvehjem type
teflon grinder with 4 strokes of a pestle rotated at 2500 rpm.
Additional 55% (w/w) S-TKM, to a total volume of 74 ml, was slowly
mixed in with a glass stir rod; 12.0 ml were delivered to each of six
38.5 ml cellulose nitrate centrifuge tubes, and 19.5 ml 40% (w/w)
S-TKM and 7.0 ml 34% (w/w) S-TKM, respectively, were layered over
the suspended nuclear pellet. The tubes were centrifuged in a Spinco
SW-27 swinging bucket rotor at 100,000xg for 4 hours. The two
interfaces were collected by pipetting from the top of the gradients
with a straight-tipped Pasteur pipet. Each interface was mixed with
an equal volume of 25mM KCl, 2mM MgSO4, SmM Tris-HCl, pH 8.0, (TKM)
and sedimented by centrifugation at 100,000xg for 2 hours in a Spinco
Type 30 rotor. The fractionation of the nuclear pellet resulted in
three additional fractions - the two interfaces, designated as the
34/40 interface and the 40/55 interface and a pellet contianing
nuclei at the bottom of the gradient tubes.

Cell fractions were washed one time in sea water prior to

serotonin binding assays and transmission electron microscopy or one

21
in 0.05M Tris-MCI, pH 8.0, prior to enzyme assays. Nuclear and
nuclear pellet fractions were subsequently collected by
centrifugation at 5000xg for 20 min in a Sorvall HB—4 rotor while
mitochondrial, microsomal, 34/40 interface and 40/55 interface
fractions were sedimented by centrifugation at 100,000xg for 90 min
in a Spinco Type 30 rotor. An aliquot of homogenate destined for use
in enzyme assays was first dialysed against 1000 vol 0.05M Tris-HC1,
pH 8.0, for 36 hours with three changes of dialysis solution.

All centrifugation Operations required in embryo collection,
cell dissociation, cell fractionation and transmission electron
microscopical procedures were performed at 2°C-4°C. Sorvall 88-34
and HB-4 rotors were used in a Sorvall RC-S centrifuge. Spinco Type
30, SW-27 and SW-50 rotors were used in Beckman L2-50 and L3-50
ultracentrifuges. Cell fractions were stored overnight at 2°C for
use in serotonin binding assays and at -40°C for no longer than 14

days for microscopic and enzymatic studies.

[3H]Serotonin Binding Assays

 

Binding 52 Cell Fractions. Cell fraction pellets were dispersed

 

in sea water by homogenization in a 10 ml Potter-Elvehjem type teflon
grinder. Cell fraction protein (0.1 mg-0.5 mg) was incubated with
SnM (30,000 dpm) radiolabelled serotonin (S-hydroxy [G-3H1trypta-
mine creatine sulfate, New England Nuclear, 16.6 Ci/mmole) in a total
volume of 0.2 ml containing 10 mM pargyline, a monoamine oxidase
inhibitor. Control samples also contained 10 mM unlabelled
serotonin. All chemicals were made up in sea water before addition

to assay tubes. Pargyline was preincubated for 15 min with the cell

22
fractions before addition of other chemicals. Samples were incubated
in triplicate in 15mm x 75mm disposable glass tubes on a shaker table
for one hour at 23°C.

After incubation, fractions were quickly filtered over 2.4 cm
Whatman GF/B glass fiber filters. Sample tubes and filter wells were
rinsed once with 1.0 m1 of sea water followed by two additional 1.0
m1 rinses of the filter wells. Samples were prepared for
scintillation counting according to Kobayaski (1980). Filters were
dried overnight, placed in scintillation vials and incubated with 0.5
ml Protosol tissue solubilizer (New England Nuclear) at 60°C for 1.5
hours. Vials were cooled and 20 l glacial acetic acid followed by
10 m1 Econofluor (New England Nuclear) were added to each vial.
Radioactivity content in [3H]serotonin stock solutions was
determined by counting aliquots of these solutions in 10 m1 Biofluor
(New England Nuclear).

Binding £2_Whole Cells. In the whole cell binding experiment

 

the assay volume and chemical composition and the incubation method
were the same as used in cell fraction binding studies. After
incubation, 1.0 m1 sea water was added to each of the assay tubes
which were then centrifuged lOOOxg for 2.5 min. Supernatants were
removed and cell pellets were partially dispersed in 1.0 ml sea water
and collected by centrifugation at 1000xg for 2.5 min. Supernatants
were again removed and the sides of the tubes wiped clean. Centrifu-
gations were performed at 2°C in a table-top centrifuge (Damon/IEC).
Samples were digested and prepared for scintillation counting
according to Kobayaski and Mandalay (1974). Cell pellets were each

suspended in 0.5 m1 Protosol tissue solubilizer (New England Nuclear)

23

and transferred to glass scintillation vials. An additional 1.0 ml
Protosol was used to rinse each assay tube before being transferred
to the vials. After incubation at 55°C for 48 hours, 0.1 ml 30%
H202 was added to each vial and vials were incubated at 55°C for
an additional 30 min. After cooling, 10 m1 Econofluor (New England
Nuclear) was added to each vial before being counted.

Scintillation vials from serotonin binding experiments were
counted for 100 min each in a Beckman LS-lOOC liquid scintillation
counter. Vials from cell fraction and whole cell binding experiments

were counted with average efficiencies of 442 and 402, respectively.

Transmission Electron Microscqpy

 

Cell fraction samples were suspended in fixative for 1.5 hours
at room temperature. The fixative solution was composed of 2.5%
glutaraldehyde, sea water (pH 8.0) diluted in half and 0.09M NaCl.
The osmolality of the fixative was 935 milliosmoles which closely
approximates the osmolality of sea water (Maser SE 31,, 1967). Fixed
samples were collected by centrifugation at 85,000xg for 1.0 hour in
a Spinco SW-SOL rotor. The fixative was poured off and cell fraction
pellets were partially dispersed in a few drops of 22 (w/v)agar
(Noble). After cooling, the agar was sliced into small pieces which
were washed in sea water and post-fixed in a solution containing 1%
osmium tetroxide, seawater (pH 8.0) diluted in half, and 0.225M NaCl
for 2.0 hours at room temperature. The osmolality of this fixative
was 940 milliosmoles (Maser EEHEEI’ 1967).

Post-fixed samples were rinsed briefly in 50% ethanol and

dehydrated in an ethanol series - 50%, 70%, 95% and 3X in 100%, 15

24
min each. Samples were cleared by two changes of propylene oxide, 15
min each, placed in 2:1 followed by 1:2 mixtures of prOpylene
oxide:p1astic, 30 min each, and then into plastic for 30 min. The
samples, in a final change of plastic, were placed in a desiccator
jar for 17 hr followed by 20 hr in a 40° oven and 24 hr in a 60°
oven. Sections were cut on a MT-ZB Sorvall microtome using glass
knives made with a LKB knife breaker, picked up on uncoated grids,
and stained with 2% aqueous uranyl acetate followed by SmM lead
citrate, 0.05M NaOH. Sections were examined and micrographed in a

Philips 300 electron microsc0pe operated at 60 kv.

Enzyme Analysis and Protein Determination

 

5'-nucleotidase. The assay procedure was according to Cestelli

 

eE-gl, (1975). 5'-nuc1eotidase was assayed at 37°C in a total volume
of 0.5 ml containing SmM AMP (Sigma), mM EDTA and 0.05M Tris-HCl, pH
8.0. The reaction was stopped after 30 min by the addition of 1.0 ml
12% (w/w) cold trichloroacetic acid. Proteins were removed by
centrifugation, and inorganic phosphate was determined by the method
of Bonting egnel. (1961). A 0.5 m1 aliquot of phosphate solution was
mixed with 0.5 ml color reagent made by adding 730 mg

FeSO4'7H20, just prior to use, to 10 m1 of 1% (w/v) ammonium
molybdate, 1.15N 32504. After 10 minutes at room temperature,

the optical density at 700 nm was measured and compared with standard
phosphate to determine the amount of substrate hydrolysed. Enzyme
activity was expressed as nanomoles of substrate hydrolysed per

minute per milligram protein.

25

NADH Oxidase, This enzyme was assayed according to Avruch and

 

Wallach (1971) as modified by Cestelli e£_§l, (1975). NADH oxidase
was assayed in a 1.0 ml volume containing 0.35 moles NADH (Sigma),
16 moles Tris-HC1, pH 7.4, 0.66 moles potassium ferricyanide, and
enzyme suspension. The oxidation of NADH was followed by monitoring
the decrease in optical density at 340 nm. Enzyme activity was
expressed as nanomoles of substrate oxidized per minute per milligram

protein.

Cytochrome g_0xidase. The assay procedure was according to

 

Wharton and Tzagaloff (1967). One percent (w/v) ferrocytochrome c
was produced by dissolving cytochrome c (Sigma) in 0.01M potassium
phosphate buffer, pH 7.0, and reducing with a few milligrams of
potassium ascorbate. Excess ascorbate was removed by dialysis
against 1000 vol 0.01M potassium phosphate buffer, pH 7.0, for 24
hours with three changes of buffer. To each of two 1.0 m1 cuvettes
was added 0.1 ml of 0.1M potassium phosphate buffer, pH 7.0, 0.07 ml
12 ferrocytochrome c and 0.83 ml water. The blank cuvette was
oxidized with 0.01 ml 0.1M potassium ferricyanide and the cuvettes
were incubated at 37°C for 3 min. The reaction was initiated by the
addition of 25 ul enzyme, and the decrease in Optical density at 55
nm was measured. Enzyme activity was expressed as nanomoles of

substrate oxidized per minute per milligram protein.

Protein Determination. Protein determination was performed by

 

the methods of Lowry SE 31. (1951) and Bradford (1976) using bovine

serum albumin (Sigma) as a standard.

26
All enzyme and protein assays were done in duplicate. In
5'-nucleotidase and NADH oxidase assays 0.05 mg - 0.8 mg protein were
added per assay tube. In the cytochrome c oxidase assay 0.02 mg -
0.2 mg protein were added to each assay tube. All measurements were
done with a Gilford 2400-2 automatic recording spectrophotometer.
NADH oxidase and cytochrome c oxidase activities were monitored using

a Gilford Electronik 196 chart recorder.

RESULTS

Embryo Culture

 

Embryos were cultured at a concentration of 2000 embryos/m1 sea
water, or approximately 7x106 embryos per culture jar containing
3500 ml sea water. Denser cultures resulted in some embryo death and
greater variation in extent of developement between embryos in the
same culture vessel. Deve10pmental variation due to temperature
fluctuation was minimized by culturing embryos in a constant
temperature chamber. In cell fraction serotonin binding experiments
it was necessary to culture at least 2 x 107 embryos to the desired
developmental stage. This made it possible to perform serotonin
binding, microscopic, and enzymatic studies on the same batch of
embryos and allowed for a better comparison between binding capacity
and organelle composition of a particular fraction than would have
been possible if these studies were performed on different embryo
batches. In whole cell serotonin binding studies, approximately 4 x
106 embryos of each developmental stage were used.

Embryos were cultured for 24 (Figure 3), 36 (Figure 4), 48
(Figure 5), 60 (Figure 6) or 72 (Figures 7 and 8) hours after
fertilization, corresponding to swimming blastula (SB), gastrula
(G)(gut complete), late gastrula or prism (LGP)(change of larval axis
from ovoid to polygonal), early pluteus larva (P2) and pluteus larva
(P3), respectively. Staging and stage abbreviations are designated
according to Harvey, 1956. Morphogenetic processes characteristic of

27

28

Figure 3. Unstained light micrograph of Arbacia punctulata swimming

 

blastula embryos (24h). x 1580.

Figure 4. Phase contrast micrograph of an Arbacia punctulata

 

gastrula embryo (36 h). x 630.

29

 

30

Figure 5. Phase contrast micrograph of Arbacia punctulata prism

 

embryos (48h). x 810.

Figure 6. Phase contrast micrograph of an Arbacia punctulata

 

early pluteus larva (60 h). x 1010.

31

 

32

Figure 7. Unstained light micrograph of Arbacia punctulata pluteus

 

larvae (72h). x 390.

Figure 8. Phase contrast micrograph of an Arbacia punctulata

 

pluteus larva (72 h). x 1010.

33

 

34

these embryonic stages and significant in this study, as discussed in
the introduction, are: formation of primary mesenchyme cells in 24
hour embryos, completion of gastrulation and release of secondary
mesenchyme cells in 36 hour embryos, bending of the gut tube toward
the oral surface and the initiation of coelom formation in 48 hour
embryos, continuation of coelom formation and initiation of nerve
cell formation in 60 hour embryos, and elaboration of neuronal cell

processes in 72 hour embryos.

Cell Fractionation

 

Explanation 2£_Methods. A cell fractionation technique was

 

developed that resulted in the separation of six cell fractions from
an embryo homogenate (Figure 1). The entire cell fractionation
procedure was monitored by phase contrast microscopy. Most critical
in obtaining the best separation and integrity of cell organelles was
the homogenization procedure. The magnesium ion concentration in the
homogenization medium was varied between zero and 5mM. Without
magnesium or any other divalent cation in the homogenization medium
cells were easily lysed, but most cell surface membranes disintegrated
to microsomal size and nuclei clumped. With 5mM magnesium ions only
70% homogenization could be obtained. A compromise concentration of
2mM magnesium sulfate allowed for 95% homogenization, little nuclear
clumping, and cell surface membrane fragments large enough to pellet
with the nuclei and unbroken cells at low centrifugal force in the
nuclear pellet fraction. Homogenization was performed immediately

after suspension of embryos in the homogenization medium. Delayed

35
homogenization resulted in less complete cell lysis such that embryos
only 75% of the cells of dispersed in homogenization medium for 5 min
prior to homogenization could be broken.

The low speed pellet of the homogenate was quickly washed in a
divalent cation free medium, which removed much of the mitochondrial
and microsomal size particles adhering to cell surface membrane
pieces and nuclei. While standard mitochondrial and microsomal
fractions were being prepared, the nuclear pellet fraction was
quickly dispersed in 55% (w/w) S-TKM. The time delay between
homogenization and dispersal of the nuclear pellet fraction in 55%
sucrose had to be made as short as possible since longer time periods
resulted in some nuclear swelling and lysis, and loss of cell-surface
membrane to the post nuclear supernatant. Sucrose concentrations
used in the discontinuous gradient centrifugation were chosen to
obtain the best separation from other organelles of nuclei and cell
surface membranes in the nuclear and 34/40 interface fractions,
respectively. Gradients were centrifuged for 4 hours at 100,000xg.
Longer centrifugation times did not alter the distribution of
particles in the gradient. Dispersal of the nuclear pellet fraction
in sucrose concentrations greater than 55%, or centrifugation of the
gradients for longer than 4 hours resulted in distorted nuclei due to
nuclear shrinkage.

After sucrose gradient centrifugation, extensive variation in
the patterns of gradients prepared from different stage embryos was
observed. The 34/40 interface of 24, 48 and 72 hour embryos
contained a grainy component as compared with the homogeneous

appearance of this interface prepared from 37 hour and 60 hour

36
embryos. Approximately 40 times as much material was collected at
the 34/40 interface of 37 hour and 48 hour embryos than at the same
interface of 24, 60 and 72 hour embryos. This interface was
bilayered only in 24 hour embryos. The 40/55 interface in 24, 37, 48
and 72 hour embryos contained three layers which could not be
separated from each other during collection of this interface. The
same interface in 60 hour embryos was composed of four layers. The
top layer could be separated from the bottom three, and these two
fractions were designated as 40/55 interface-top and 40/55
interface-bottom. Gradient profiles were reproducible; the same cell
fractionation procedure, used in experiments not covered in this
text, produced the same stage specific gradient profiles as those

discussed above.

Enzymatic Analysis

 

The enzymes 5'nucleotidase, NADH oxidase and cytrochrome c
oxidase were assayed as markers for cell surface membranes (Touster,
1970; Cestelli Efipfllf’ 1975; McClay e£_gl,, 1977), endoplasmic
reticulum and outer mitochondrial membranes (Sottocasa e£_§1,, 1967;
Huber and Morrison, 1973) and inner mitochondrial membranes
(Sottocasa eghgl., 1967), respectively. Enzyme activities, expressed
as nanomoles of substrate hydrolyzed or oxidized per minute per
milligram protein, were calculated, and the results of enzymatic
analyses are summarized in Tables 1-5. Standard errors for 5'nucleo-
tidase, cytochrome c oxidase and NADH oxidase assays averaged 3.8%,
5.9% and 5.4%, respectively. Differences in appearance of the same

cell fractions isolated from different embryonic stages, as judged by

37
gross inspection and microscopy, were reflected in differences in
organelle distribution within the cell fractions of various embryonic
stages as determined by enzymatic analysis. Enrichment in the
concentration of various subcellular particles in cell fractions
over their concentration in the homogenate is given in parentheses in
Tables 1-5.

5'-nuc1eotidase. Densities of cell surface membranes in the

 

34/40 interface fraction, as calculated from 5'-nucleotidase
activities, were 1.6, 2.6, 2.6 and 5.6 times that of the homogenate
in 36, 48, 72 and 24 hour embryos, respectively. At these
developmental stages this fraction contained the greatest enrichment
in cell surface membranes over the homogenate except for the
microsomal fraction of 72 hour embryos with a 5'-nucleotidase
specific activity of 3.4 times that of the homogenate (Table 5). In
60 hour embryos (Table 4) the highest 5'-nucleotidase activity was
demontrated in the top layer of the 40/55 interface; the 34/40
interface fraction activity was only 0.8 times that of the
homogenate. Cell surface membranes were distributed fairly evenly
between microsomal, mitochondrial and crude nuclear pellet fractions
of 24 (Table l), 36 (Table 2) and 48 (Table 3) hour embryos. Of
these fractions, the highest 5'-nuc1eotidase specific activity was
located in the mitochondrial fraction of 24 hour embryos (4 times the
homogenate), the microsomal fraction of 36 hour embryos (1.3 times
the homogenate) as well as the microsomal and nuclear pellet
fractions of 48 hour embryos (1.3 times the homogenate). In 60 and
72 hour embryos cell surface membrane distribution between these

three fractions was not as uniform, and highest 5'-nuc1eotidase

38

Table 1

1Binding and enzymatic data were obtained from the same batch of
embryos and are averages of triplicate and duplicate measurements,

respectively, from one experiment.

2Each number in parentheses is a ratio of the specific activity of

a fraction over that of the homogenate.

39

 

3.3 ‘wa 3.3 0.0 3.3 a; GOV .+. was. 3.032
3.8 odor 8.3 0...: 3.3 flop omv a 0 00023:. mmxow
3.3 0.3.“ 3.3 0.0 3.3 «4; how « a9: 000:3... Diva
6.3 53. 3.: «.3 8.8 0.0 new a 96 3.3 3282
3.8 0.2. 8.3 5.x. 3.8 v.5 emu a. mac 3:63.25.
8.9 fivow 223 ode 8.3 a.» an? a new gaucocoozi
.. «.3 ad od mm a" map 22.80on
0002.8 0002.8 0000:00_0:z...m £0.05 0:. con 22.50 5:00: :00
:92 0 05020030 5:0..030mim

 

.0 @533 2:00am
«oexsgososcv >=>=0( 03.025 0.535

 

09:28.“. $505 new Sozlvm *0 30:39:01 05 So: 00.0.00.
30:09.“. .m_:__00n:m E >=>=0< mc_0c_mlc_c20._om Tm— eo 5:39:55

40

h)

Table

1... .. . .
‘aiacifig and enzyzatic data were obtained from the sane batch of
enbryos and are averages of triplicate and duplicate aeasureaents,

respectively, fro: one experiaent.

7 . . . . . . - . . . .
‘0 ca gunner in parentheses is a ratio or the specific actiVity of

a fraction over that of the honogenate.

41

 

8.3 0.000 8.3 «.3 00.00.00 .00 0. u 000. .2002
3.3 0.0.0 8.3 0....0 8.3 v... 0.0 a o 00020.0. 0080
“0.5 «.90. Cd. 0.00 8.3 0.0 00. a 0.0 00020.0. 0300
«0.3 0.0.0 8.3 0.00 8.5 0.0 ~00 fl 0...... .0..00 000.002
8.0. 0.000 3.3 0.0. 8.3 0.0 0v. 0 0 30.000000...
3.0. v.00... 8.3 0.0.0. 3.: 0.0 000 a 00: 320000000!

0.00 0.00 .0 00 M 00 0.0000200...
00000.0 00000.0 0000..00.0:2J0 0.0.00. 00. .00 2203 00:00.. =00

:92 0 0.0050030 0.0280090..—

 

.0E\0.0=00.0E$ >..>..0< 0.00000 0£>~0m

.0 00.003 09:00.5

 

003005 0.00.... 000 0:01:00 .0 0.0000080... 00. E00. 00.0.00.
002.00.“. 5.2.0095 0. >..>..0< 00.00.010.00850 Tm .0. 00:00:55

42

Table 3

1Binding and enzymatic data were obtained from the same batch of
embryos and are averages of triplicate and duplicate measurements,

respectively, from one experiment.

2Each number in parentheses is a ratio of the specific activity of

a fraction over that of the homogenate.

43

 

3.0. 0.50 2.0. 0.00 8.0. .0 000 u :0. .2002
2.0. 0.0. 8.... 0.3. .0... .0. 000 u 000 8002:. 00.0..
.00. 0.00 2... 0.0. 8.0. 0.0 0... u 000 002.20. 0200
.00. 0.0... 2.0. 0.00 .0... 0... 000 0 0 .230 .0282
8.0.. 0.00.. 3.0. c... .0... 0.0. 000 u 0 32820.2.
8.0. v.00 8.0. 3.0 8.8 «.0 t. u 0 0:05.33:
.00 0... 0.0 000 u 0 0.0000050...
00000.0 000008 000000200210 0.0.0.0 0E .00 2209 00:00.. =00
19.2 0 0E0.000.>0 0.030.003

 

.00..0.E.00.0.00. >..>..0< 00.0000 000.000

.0 00.00... 2:0000

 

02.5.00 0.00.: 000 50:00 .0 0.0000050... 00. E0.. 00.0.00.
0.00000... 0.20300 0. >..>..0< 00.00.010.022002 .0 000000.05

44

Table 4

1Binding and enzymatic data were obtained from the same batch of
embryos and are averages of triplicate and duplicate measurements,

respectively, from one experiment.

2Each number in parentheses is a ratio of the specific activity of

a fraction over that of the homogenate.

45

 

...0. 2.0 .0... 0.0 .Nd. ... 00 u o .2002
8.0. .00 .0... 0.0 .00. 0.0 000 a. 0 .000. 0.2.00
.06. .00. .0... 0... .00. 0.0. 000 u... 00.0 .000. 00...
002.20. 00.00
3.5 ohm 00.00.00 .00 8.0. 0.0 000 « 0N0. 00.3.0.0. 0300
.00. 000 .00. 0.0. 2...... 0.0 00. u 00. .0..00 00.002
.00. 000 ..... ..0 .0... 0.0 000 0 000. 35820....
.00. :0 3.“. TN. .0... .0 .mm a 0mm. 3.502.000!
0.. 0.0 0.0 00 u 000 0.0000200...
00000.0 00000.0 0000..00.00z...0. 0.0.0.0 00. .00 .20... 00:00.. =00
+322 0 00.00.00.00 0.00.0.00 Himm—

 

.00..0.E.00.0E0. >0>..0< 00.0000 000.000

.0 00.00... 00.0000

 

0030.00 0.00.: 000 001.60 .0 0.00000501 0.... 0.0.. 00.0.00.
000000.“. ...0_0_.00000 0. >..>..0< 00.00.010.00.0.00m...m_ .0 00000....05

46

Table 5

1Binding and enzymatic data were obtained from the same batch of
embryos and are averages of triplicate and duplicate measurements,

respectively, from one experiment.

2Each number in parentheses is a ratio of the specific activity of

a fraction over that of the homogenate.

47

 

.60. 0.00 .06. 0.00 .06. 6.. .v u o .2002
.06. 06... .N... 0.00 .0... 0.0 06. .o. 000 000.00.... 00.60.
.0... 0.0 .06. 6.0. .00. 0.0 00. H 600 000.00....- 6:00
.0... 0.00 .06. 0.0 .06. 0.0 00. w 000 .0300 30.002
.0.0. 6.00. .06. 0.00 .00. 0.0. 0.. M 000 35000.0...
.0... 0.00 .0... 0.0V .0... 0.0 06. H 006. 32000302.:

0.10 6.00 0.0 00. N 050 0.0000900...
000008 000008 0000..00.00ZJ0 0.0.0.0 00. .00 .200. 00:00.. =00

1.22 0 00.0.0000... 0.00.0.00HI0H

 

.0....0.E\00.00.0. >..>..0< 0.00000 0.0.300

.0 00.00... 03.0000

 

09.505 0.00.... 000 501-0,. .0 0.0000008: 00. .00.. 00.0.00.
002.00.“. .0.....00000 0. >._>..0< 00.050-500.200 firm— .0 02.09.65

48
specific activities were located in the mitochondrial and microsomal
fractions, respectively. 5'-Nuc1eotidase specific activities in the
nuclear fraction ranged from undetectable in 36 hour embryos to 0.6

times that of the homogenate in 24 hour and 48 hour embryos.

Cytochrome g_oxidase. Mitochondrial densities, as determined

 

from cytochrome c oxidase activities, was greatest in the
mitochondrial and 40/55 interface fractions. Specific activity
values ranged from 1.6 to 8.7 times the homogenate in 24 to 72 hour
embryo mitochondrial fractions. Mitochondrial densities in
microsomal, 34/40 and nuclei fractions were, in general, less than
the homogenate. Exceptions were the 34/40 interface fraction of 48
hour embryos (1.7-fold over the homogenate), the microsomal fraction
of 60 hour embryos (1.1-fold over the homogenate) and the nuclei
fractions of 48 hour (2.7-fold over the homogenate) and 60 hour

(1.7-fold over the homogenate) embryos.

NADH oxidase. A quantitative determination of the endoplasmic

 

reticulum purity in a particular cell fraction cannot be made from
NADH oxidase specific activity values when it is assumed that this
enzyme is localized in both endoplasmic reticulum and mitochondria.
Relative purity over the homogenate of endoplasmic reticulum in
various cell fractions can be estimated by subtracting the cytochrome
c oxidase enrichment ratio value from the NADH oxidase enrichment
ratio value. Using this calculation it can be demonstrated that the
endoplasmic reticulum density in mitochondrial and 34/40 interface

fractions was less than that of the homogenate except in the 36 hour

49
mitochondrial and the 60 hour 34/40 interface fractions. Endoplasmic
reticulum densities were, on the average, highest in nuclear pellet,
40/55 interface and nuclear fractions, and of these three fractions
highest endOplasmic reticulum densities were demonstrated in the

nuclei fraction of 24, 36 and 48 hour embryos.

Microscopy

 

Phase contrast and electron micrographs were taken of some
representative sucrose gradient fractions. Judging from
phase contrast observations, the gradient did achieve an effective
separation of nuclei and cell surface membranes in nuclear and 34/40
interface (40/55 interface-top in 60 hour embryos) fractions,
respectively. Nuclear fractions of 24, 36, 48, 60 and 72 hour
embryos are given in Figures 9-l3, respectively. Nuclei appear
intact and relatively free of cell surface membrane pieces or whole
cells. The shiny rod shaped structures in 48 and 60 hour embryo
nuclear fractions are skeletal spicules. The 72 hour embryo nuclear
fraction was centrifuged 480xg for 2 min in an HB-4 rotor, and the
nuclei were pipetted off from the heavier spicule layer before being
micrographed. The 34/40 interface fractions (Figure 14) contained
cell surface membrane fragments and few nuclei, while the 40/55
interface fractions (Fig. 15) contained a considerable amount of cell
surface membrane as well as whole cells and nuclei. Electron
micrographs of the 34/40 interface of 24 hour and 72 hour embryos are
given in Figures 16 and 17, respectively, and confirm 5'-nucleotidase
assays which indicated a greater purity of cell surface membranes in

this fraction in 24 hour embryos than in 72 hour embryos.

50

Figure 9. Phase contrast micrograph of the 24 hour embryo nuclear

fraction. x 2520.

Figure 10. Phase contrast micrograph of the 36 hour embryo nuclear

fraction. x 1580.

51

 

52

Figure 11. Phase contrast micrograph of the 48 hour embryo nuclear

fraction. x 2020.

Figure 12. Phase contrast micrograph of the 60 hour embryo nuclear

fraction. x 1010.

53

 

54

Figure 13. Phase contrast micrograph of the 72 hour embryo nuclear

fraction. x 2520.

55

 

56

Figure 14. Phase contrast micrograph of the 48 hour embryo 34/40

interface fraction. x 2520.

Figure 15. Phase contrast micrograph of the 48 hour embryo 40/55

interface fraction. x 2520.

57

 

58

Figure 16. Electron micrograph of the 24 hour embryo 34/40

interface fraction. x 38,200.

Figure 17. Electron micrograph of the 72 hour embryo 34/40

interface fraction. x 53,800.

59

 

6O

[3H] Serotonin Binding Assays

 

Specific or saturable serotonin binding, defined as total
binding (the amount of radioligand bound in the absence of excess
unlabelled ligand)minus nonspecific binding (control) (the amount of
radioligand bound in the presence of excess unlabelled ligand),
(Bennett, 1978). In a pilot cell fractionation serotonin binding
experiments 3.4 ug, 8.5 ug and 34 ug gastrula cell surface membrane
fraction protein were each incubated with 10'9M, 7xlO'9M and
10'7M radiolabelled serotonin in a total incubation volume of 1.0
ml. In this experiment, specific serotonin binding could not be
detected at 10’9M or 10'7M radioligand. At lO'7M [3H]
serotonin nonspecific binding was too high to obtain a significant
difference from binding in controls. Specific binding could only be
detected using 34 ug protein and 7 x 10‘9M radioligand, the
dissociation constant for serotonin and rat brain serotonin receptors
as estimated by Bennett and Snyder (1976); however, at this
concentration of radioligand a considerable amount of specific
binding to filter material was also detected. Several examples of
saturable or specific binding to nonbiological materials are
documented in the literature (Cuatrecasas and Hollenberg, 1975;
Snyder, 1975). Based on results of the pilot experiment, the total
incubation volume and radioligand concentration used in this study
were set at 0.2 ml and 5 x 10’9M, respectively. With this
radioligand concentration and smaller incubation volume the amount of
cell fraction protein or whole cells needed to perform binding
experiments was minimized and specific serotonin binding to the

filter was negligible.

61

Binding £2_Cell Fractions. Tables 1-5 list the specific binding

 

of [3H] serotonin, in disintegrations per minute (dpm) per
milligram protein, to different cell fractions from 24, 36, 48, 60
and 72 hour embryos. Of the 30,000 dpm added to the incubation
medium containing 0.1 mg-O.5 mg protein, a total of 500 dpm-1700 DPM
were bound (100 dpm - 1 pg - 2.7 fmol) and specific binding
approximated 20% of the total. Specific binding was detectable in
various cell fractions from all embryonic stages studied.

In 72 hour embryos (Table 5)[3H]serotonin bound specifically
to all cell fractions except the nuclear fraction. The amount of
radioligand bound to cell fractions from this stage correlated
positively with 5'-nuc1eotidase activities of the fractions. Table 6
shows the specific [3H]serotonin binding to 72 hour embryo cell
fractions per milliunit of 5'-nucleotidase activity. Except for the
mitochondrial fraction, these values were similar, suggesting that
binding was to cell surface membranes. Binding to the mitochondrial
fraction per milliunit of 5'-nucleotidase activity was approximately
double that of the other cell fractions. This suggests that in
addition to cell surface membrane sites other subcellular binding
sites may be present in the mitochondrial fraction of 72 hour
embryos. The total 72 hour embryo [3H]serotonin binding content in
cell fractions was calculated as the product of the specific binding
per milligram protein and the total milligrams of protein in each
fraction. Binding content data are given in Table 7. Approximately
60% of the total homogenate binding content was recovered in the
mitochondrial (332), microsomal (10%), and nuclear pellet (17%)

fractions. Of the total nuclear pellet binding, 89%, 7% and 0% were

62

Table 6

. 2355 con n=2< E9: r .o 205: w 3.9.3.. 3....
53:3 9: ma amazon m. 33:8 9535 .o :c: 95F

 

63

 

I 6832

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:_co.ocom_m..mm co 9.559 05025

 

wo>BEm 5205 wow Sozlmn So: ooaflom.
95:08... cm_:__mon:w E >:>:o< 03286.0:sz
new @535 5:20.5me coozzon. cowtmano

TTTTTT

 

 

 

 

 

65

 

o o c .5 .2032

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w... mm 0.9 m 005.3... on?»

92 won #6 m— .. 3:2. 30.032

m6 N0? 069 h N 35009.22

«do can. mdw mm .m..u:o:oo:2

0.8. 83 vs I o 226852.
“22.8052. .0 I. 320:5 2.30:. 9:20.95: 3.5 5:02. .30

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25:09.... ._oo 9.58m 55.5 new caozlwn Eo:
>co>ooom 9.65m 0...".on Ecouocom—HIm—

66

recovered in the 40/55 interface, 34/40 interface and nuclear
fractions, respectively.

In 60 hour embryos (Table 4) specific [3H]serotonin binding
was demonstrated in all fractions except 40/55 interface-bottom and
nuclei. Embryo cell fraction serotonin binding and
5'-nucleotidase activities showed less correlation than in 72 hour
embryos. For example, binding to the 34/40 interface fraction was
4.8 fold over the homogenate, while the S'-nucleotidase activity of
this fraction was only 0.8 times that of the homogenate. In 48 hour
embryos (Table 3) specific serotonin binding was demonstrated only in
the 34/40 interface, 40/55 interface and nuclear fractions. Specific
binding to the 48 hour embryo nuclear fraction per milliunit
5'-nuc1eotidase activity was approximately 5 times the values for
34/40 interface and 40/55 interface fractions. [3H]serotonin bound
specifically to 36 hour embryo mitochondrial, nuclear pellet, 34/40
interface and nuclear fractions, and the binding did not correlate
with 5'-nucleotidase activities (Table 2). Binding was highest in
mitochondrial and nuclear fractions. In 24 hour embryos (Table 1)
specific serotonin binding was demonstrated in all fractions except
the 40/55 interface. The amount of serotonin bound per milliunit
5'-nucleotidase activity was very similar for homogenate,
mitochondrial, microsomal and nuclear pellet fractions. The 34/40
interface and nuclear fractions bound two and eight times as much
serotonin, respectively, per milliunit 5'-nuc1eotidase activity than
did the other fractions. [3H]Serotonin binding did not correlate
with mitochondrial densities in cell fractions from any of the

embryonic stages studied.

67

Binding £2_Whole Cells. In this study 24, 48, 60 and 72 hour

 

embryos were used. Total [3H]serotonin binding to 2-9 x 106

whole, dissociated embryo cells was 1500 dpm - 4000 dpm, and specific
serotonin binding, amounting to 14%-38% of the total, was only
demonstrated for stages older than 24 hours. Figure 18 shows the

binding capacity of Arbacia punctulata embryos, as determined from

 

whole cell binding data, and serotonin content of Paracentrotus

 

lividus embryos of similar developmental stages. Serotonin content
data, determined by fluorometric and thin layer chromatographic
techniques, were taken from Toneby (1977a). Both serotonin binding
and serotonin content increased between 48 and 72 hours. Toneby

could not detect serotonin in Paracentrotus lividus blastula and, in

 

contrast to cell fraction binding studies, specific serotonin binding

to Arbacia punctulata blastula cells could not be demonstrated even

 

though more cell protein of this stage than other stages was added to

assay tubes.

Figure 18.

68

Comparison between serotonin content (---) in

Paracentrotus lividus embryos and [3H]serotonin binding

 

to dissociated cells of Arbacia punctulata embryos.

 

Serotonin content data and embryo figures were taken from

Toneby, M. (1977a).

Numbers of cells from 24, 48, 60 and 72 hour embryos used
in incubation media were 9.3x106, 2.1x106, 5.5x106
and 6.0x106 corresponding to 0.70, 0.07, 0.16 7 and

0.13 milligrams of protein, respectively.

 

69

 

80393 0935 hzwezoo 2.2055».

 

 

 

 

 

 

 

 

 

 

 

 

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A”. .n. .HV
3 2 1.

mo. x 8955 metacm ozaom z_zohommmm:m

DEVELOPMENT
(HOURS AFTER FERTILIZATION)

Discussion

Cell Fractionation

 

The relative ease with which sea urchin eggs can be obtained in
large quantities, fertilized, and cultured has resulted in the
widespread use of the sea urchin embryonic system in developmental
research, especially in biochemical studies. Isolation of
subcellular organelles, however, has proven difficult for embryonic
systems in general, and despite the large volume of research on the
sea urchin embryonic system, few cell fractionation techniques have
been reported. Cell surface membranes have been isolated from

fertilized and unfertilized Lytechinus pictus and Strongylocentrotus

 

 

purpuratus eggs (Barber and Foy, 1973) as well as Paracentrotus

 

 

lividus (Cestelli £5 31., 1975) and Lytechinus variegatus and

 

Tripneustes eschulentes (McClay et a1., 1977) gastrula. Cell

 

cortices have been obtained from unfertilized eggs of Hemicentrotus

 

pulcherrimus, Anthocidaris crassispina (Ohnishi, 1963),

 

Pseudocentrotus depressus (Sakai, 1960), Strongylocentrotus

 

 

purpuratus and Strongylocentrotus franscisianus (Vacquier, 1975; Begg

 

and Rubhun, 1979); Detering E£.2£' (1977) have obtained a cortical
granule fraction from Strongylocentrotus purpuratus and Arbacia

punctulata unfertilized eggs. Yolk granules from Strongylocentrotus

 

purpuratus, Strongylocentrotus drobachiensus and Bytechinus pictus

 

oocytes (Harrington and Easton, 1980) and nuclei from

70

71

from Strongylocentrotus purpuratus unfertilized egg through gastrula

 

stages (Hinegardner, 1962) have been isolated. Several of these
techniques can be applied only to specific species and/or embryonic
stages. For example, the nuclear isolation technique of Hinegardner
cannot be successfully applied to postgastrula stages (Hinegardner,
1962), and it has been difficult to apply the cell surface membrane

isolation techniques of McClay and Cestelli to Arbacia punctulata

 

gastrulae (author's results and personal communication with William
Eckberg, Howard University, Washington, D. C.). In most studies,
just as in the present one, it would be advantageous to obtain
several cell fractions from the same embryo homogenate. 0f the few
organelle isolation procedures reported, many exclude the possibility
of obtaining other cell fractions. The cell fractionation of

unfertilized Strongylocentrotus interme taph nvvh thelomin and

 

Svetashev, 1978) is one of a very few studies which have succeeded in
the separation and partial characterization of several crude
subcellular fractions from a common sea urchin egg or embryo
homogenate.

During the course of experiments described in this thesis, a
subcellular fractionation procedure was developed, and cell fractions

from Arbacia punctulata embryos were partially characterized by

 

enzymatic and phase contrast as well as electron microscopical
analyses. The same cell fractionation procedures were applied to
five stages of embryonic development. This method resulted in the
enrichment of various organelles in particular cell fractions,
although the distribution of organelles in some cell fractions was

stage specific. In general, the mitochondrial and microsomal

72
fractions were enriched in these organelles by the procedures used,
and the discontinuous gradient centrifugation of the nuclear pellet
fraction successfully separated nuclei from cell surface membranes in
the nuclear and 34/40 interface fractions, respectively. In 60 hour
embryos cell surface membranes were enriched in the top layer of the
40/55 interface. Although the cell fractions produced by this method
are relatively crude, complete purification of cellular organelles is
often not an absolute prerequisite for a variety of studies if the
integrity of various organelles is maintained by the fractionation
procedure and if their distribution and concentration are properly
characterized. In this study, homogenization, washing and gradient
media were carefully chosen to best preserve the integrity of various
cell particles as judged by phase contrast and electron microscOpy.
Furthermore, marker enzymes were present in sufficient quantities to
establish the usefulness of the cell fractionation methods used.

The merit of this fractionation technique as an initial step to
the final purification of cell organelles must be considered for
particular organelles of specific embryonic stages. In all stages
studied a fairly clean nuclear fraction was obtained whiCh could be
further purified. The advantage over the Hinegardner (1962) nuclei
isolation technique discussed previously is the ability to fractionate
nuclei from postgastrula as well as gastrula and pregastrula stages.
Only in 24 hour embryos are cell surface membranes sufficiently
concentrated in a cell fraction (34/40 interface) and relatively free
of other organelles to consider the use of this fractionation
procedure as an initial step in the purification of cell surface

membranes. The fractionation results provide a possible explanation

73
for the problems that have been encountered by several workers in
obtaining a pure cell surface membrane fraction from sea urchin
embryos, and from embryos in general, by differential and sucrose
density gradient centrifugation methods. With the exception of 24
hour embryos, the concentrations of cell surface membranes in nuclear
pellet fractions and corresponding homogenates were similar. In all
stages cell surface membranes were fairly evenly distributed between
the nuclear pellet, mitochondrial and microsomal fraction. This
indicated that despite efforts to produce predominantly large
membrane fragments which could subsequently be concentrated in the
nuclear pellet fraction, approximately equal quantities of
mitochondrial and microsomal size cell surface membrane fragments

were also produced by the homogenization procedure.

[3H1Serotonin Binding to Embryo Cell Fractions

 

[3H]Serotonin bound specifically to some cell fractions from each
of the five embryonic stages studied which ranged from late blastula to
pluteus larva. The identification of serotonin binding sites in
pregastrula and gastrula embryonic stages (24 and 36 hour embryos)
and the detection of serotonin reported previously at these stages by
bioassay (Buznikov 25.213, 1964) and histochemical fluorescence
(Buznikov 35 21" 1972a) methods correlate well with studies which have
demonstrated an inhibition of primary mesenchyme cell movement and
gastrulation by mammalian serotonin receptor blocking agents (Gustafson
and Toneby, 1970, 1971; Gustafson, 1973). Taken together, these studies
strongly suggest that serotonin has morphogenetic function(s) in

these embryos and that the inability of Toneby (l973,l977a,l977b) to

74
detect serotonin in these early embryos by chemical methods was
probably due to assay sensitivity limitations. Alternatively, the
present observations and all of the other studies discussed,
including the serotonin assays of Toneby, would support the concept
of the existence of a functional serotonin analog in 24 and 36 hour
embryos if the analog and serotonin were similar enough chemically to
compete for the same binding sites. In postgastrula stages all
serotonin content studies have demonstrated a continuous increase in
serotonin levels from the end of gastrulation through formation of the
pluteus larva, and in the present investigation [3H]serotonin
binding sites were demonstraed during this developmental period in
48, 60 and 72 hour embryo cell fractions. Results of this cell
fraction binding study, along with serotonin content and inhibitor
studies, suggest that serotonin, or an extremely closely related
serotonin analog, functions as in adult tissues by first binding to a
distinct biological recognition site (receptor molecule).

Comparisons between [3H]serotonin binding capacity and
organelle distribution in cell fractions were made in an attempt to
determine the subcellular location(s) of binding sites, knowledge of
which would contribute to a better understanding of the role of
serotonin in early sea urchin development. In 72 hour embryos a
positive correlation between serotonin binding and S'-nucleotidase
activities in various cell fractions (except the mitochondrial
fraction) was demonstrated. This suggests that [3B]serotonin bound
to cell surface membranes, although the possibility that binding
sites were localized on some other cell component that distributed

similarly to cell surface membranes among the various cell fractions

75
cannot be unequivocally ruled out. Along with studies which have
demonstrated, in embryos of this stage, the beginnings of a nervous
system (Ryberg, 1977; Ryberg and Lundgren, 1979) which probably
eventually contains a serotonergic neuronal component as suggested
from pharmacological studies on more advanced plutei (Gustafson £5
31,, 1972a), the results of this study suggest that the binding sites
detected in 72 hour embryos are specific cell surface serotonin
receptors which may mediate or eventually mediate serotonergic
neurotransmission. The correlation between [3H]serotonin binding
and its associations with the presence of cell surface membranes
is less distinct in embryos younger than 72 hours.

At several developmental stages at which serotonin binding and
5'-nucleotidase specific activities seemed to parallel one another in
some cell fractions, binding to 34/40 interface, mitochondrial or
nuclei fractions per milliunit 5'-nucleotidase specific activity was
greater than in other fractions. This suggests that binding sites
other than or in addition to cell surface sites were present in these
fractions. Several serotonin inhibitor studies discussed in the
Introduction led the investigators to propose that mitochondrial
serotonin receptors were present in early cleavage state embryos. In
the present experimental observations no correlation between
serotonin binding and the presence of mitochondria, as determined
from cytochrome c oxidase content, was observed in any cell fraction
from any of the developmental stages examined. While the possibility
that serotonin recognition sites are located on mitochondria and also
on other organelles which are distributed among cell fractions

differently than mitochondria cannot be ruled out, the results

76

suggest that mitochondrial binding sites are not present in these
embryos.

Ten-Cate (1952) pointed to an incongruity between the proteins
synthesized in the early sea urchin embryo and the composition of
the yolk granules expended, leaving a surplus of aromatic amino acids
not accounted for by protein synthesis. Gustafson and Toneby (1971)
postulated that these aromatic amino acids were precursors for
serotonin production in yolk granules of sea urchin embryos.
Although the presence of yolk granules in mitochondrial fractions was
not examined in the present investigation, the same fraction,
prepared from both unfertilized and fertilized sea urchin eggs by
experimental procedures similar to those used in this study (Chemomin
and Svetashev, 1978), was shown to contain a greater yolk granule
density than nuclear pellet or microsomal fractions. Yolk granule
serotonin storage sites may account for a significant amount of
serotonin binding to mitochondrial fractions and possibly to other
fractions as well.

Most interesting is the relatively large amount of
[3H]serotonin binding to 24, 36 and 48 hour embryo nuclear
fractions when contrasted with the total absence of binding to this
fraction in 60 and 72 hour embryos. In younger embryo nuclear
fractions, the density of endoplasmic reticulum was greater than in
60 and 72 hour nuclei. The possibility that binding sites on
endoplasmic reticulum accounted for the binding to nuclear fractions
of the younger embryos is not supported since no binding to 48 hour
and 36 hour embryo microsomal fractions was detected. Furthermore,

the microsomal fraction from 36 hour embryos was more enriched in

77

endoplasmic reticulum than any other fraction from any developmental
stage. The likelihood of the existence of nuclear serotonin binding
sites in 24, 36 and 48 hour embryos which disappear at later stages
is supported by these data. This may indicate the involvement of
serotonin in nuclear processes in these earlier stages. Buznikov
(1971) postulated that in early cleavage stage embryos serotonin may
affect protein synthesis by reducing nuclear membrane permeability to
mRNA and tRNA. Emanuelsson (1974,1976) demonstrated the presence of

serotonin in nuclei of early cleavage stage Ophryotrocha polychaete

 

embryos as well as gastrula and postgastrula chick embryos and
suggested a nuclear function for serotonin in these embryos. These
reports also localized serotonin in lipid droplets, plasma membranes
and microfilaments and demonstrated the greatest serotonin

concentration in yolk granules.

_LEB]Serotonin Binding to Dissociated Embryo Cells

 

The increase in the amount of serotonin bound to dissociated
cells of embryos between postgastrula and pluteus stages correlated
with an increase in sertonin content between these stages in other
sea urchin species as measured by the chemical assays of Toneby
(l973,l977a, 1977b) and the biological assays of Buznikov EEHEl'
(1976). This agreement strengthens the conclusions drawn from the
cell fractionation binding studies which demonstrated specific
serotonin binding to several cell fractions from 48, 60 and 72 hour
embryos. Surprisingly, no binding to late blastula (24 hour) embryo

cells was detected even though significant binding was demonstrated

78

in most cell fractions from this stage. Judged on the basis of cell
fractionation binding studies, the [3H]serotonin binding capacity
of 24 hour embryos was roughly one-half that of 72 hour embryos as
estimated from homogenate binding activities. In the 24 hour embryo
intact cell serotonin binding assay, 50% more cells, corresponding to
approximately 3 times the number of embryos and 5.5 times the amount
of protein, were used than in the 72 hour whole cell binding assay.
If the binding sites labelled in the 24 hour cell fraction binding
study were equally accessible to [3H]serotonin in the whole cell
binding assay, they should have been detected in this study.

Among the possible explanations for the discrepancy between 24
hour embryo cell fraction and whole cell [3H]serotonin binding
data, the most obvious one would be to assume that 24 hour embryos
contain only intracellular binding sites which, due to poor cell
surface permeability to [3H]serotonin, remained inaccessible to the
label after a 60 minute incubation period. This explanation cannot
be supported or ruled out since information concerning the
permeability of blastula sea urchin embryos to the radiolabelled
creatine sulfate-serotonin complex used in this thesis work is not
available; however, studies have shown that serotonin is readily
permeable to cleavage stage embryos. In early cleavage stage embryos
exogenously added serotonin at 50 g/ml has been shown to rapidly

accumulate into Arbacia lixula, Strongylocentrotus intermedius and

 

Strongylocentrotus drobachiensus embryo cells, reaching a maximum

 

concentration of 500 ng/lO6 embryos in 10 min (Manukhin £5 31,,
1973). Some antiserotonins could arrest cleavage after a 50 min

incubation with embryos, and this effect was diminished or eliminated

79
if serotonin was also added to the embryo culture (Buznikov g£_§13
1970). This observation, if it is assumed that the antidotal effect
of serotonin is due to its interaction with intracellular receptors
as discussed in the Introduction, also suggests that serotonin can
readily penetrate into cells of early cleavage stage embryos.
It is also possible that the cell dissociation procedure either
removed or altered the accessible serotonin binding sites in 24 hour
embryo cells.

One other explanation for the lack of [3H]serotonin binding to
dissociated 24 hour embryo cells deserves attention. Buznikov fit
21,, (l972b,l975,l976) described a sensitivity phenomenon related to
embryo density in which an increase in the number of cleavage stage
embryos in the culture reduced their sensitivity to antiserotonins.
The inhibitory effects of antiserotonins on cell division and protein
synthesis could be completely reversed by raising the embryo
concentration, and further increases in embryo density often resulted
in a stimulatory effect. Density sensitivity was demonstrated in
embryos up to the late blastula stage and was a result of nonspecies
specific protective factor(s) (AN-factor(s)) which were secreted into
the culture medium by embryo cells (Buznikov st 21" 1971b). Embryos
that had been previously lysed by osmotic shock could not release
AN-factor(s) as judged by their inability to protect intact embryos
from the cytotoxic actions of various antiserotonins (Buznikov st
213, 1977). It was demonstrated that AN-factor(s) were normally
released into the medium by untreated embryos, but this release was
accelerated if antiserotonins were also present in the culture.

Although serotonin was released by antiserotonin stimulated embryos,

80
its concentration was too low to account for the protective effects
observed. An endogenous ganglioside fraction was isolated from

cleavage stage Arbacia punctulata, Strongylocentrotus intermedius,

 

 

Stronglylocentrotus nudus and Paracentrotus lividus embryos that

 

 

mimicked the effects of the protective factor(s) (Buznikov £5 31.,
1973a, 1973b). Buznikov £5 31. (l973c) have postulated that the
AN-factor(s) participate in the regulation of the functional activity
of intracellular serotonin as its endogenous antagonist which
decreases or st0ps the effect(s) of serotonin on corresponding
receptive structures. In the light of these studies, the lack of
serotonin binding to late blastula dissociated cells could be due to
the formation of endogenous serotonin binding site competitors (AN
factor(s)) which would be heavily concentrated in the dense cell

suspensions used in this cell binding study.

Serotonin in Embryonic and Nonembryonic Systems

 

The function(s) of serotonin in embryonic systems may be better
understood by first examining the information available concerning
its roles in nonembryonic systems. The proposed roles for serotonin
in sea urchin embryos between the blastula and pluteus stages as
reviewed in the Introduction involved the stimulation of ciliary
activity, cell movements and cell shape changes, and neurotransmission.
All of these functions are also mediated by serotonin in many
nonembryonic systems. In adult vertebrate and invertebrate animals
serotonin is a neurotransmitter in leech giant Retzius cells (Erwin
and Thies, 1975), snail central neurons (Gerschenfeld and

Paupardin-Tretsch, 1974), mollusk (Cedar and Schwartz, 1972) and

81
cockroach (Nathanson and Greengard, 1974) ganglia, mammalian
gastrointestinal tract (Gershon, 1979; Burnstock, 1981) and mammalian
brain (Bennet and Snyder, 1976; Enjalbert stflal., 1978a, 1978b;
Quayle st 31, 1978; MacDermot 35 31., 1979). In the green alga,

Euglena gracilis (Keirns et a1., 1973), and the flagellated protozoan,

 

Tetrahymena pyriformis (Jankidevi s£_al,, 1966), serotonin is a

 

probable trigger for cell motility. Serotonin stimulates motility of
mammalian glial cells (Hertz st 31., 1979) and chemotaxis of fetal
mouse palatal cells (Clark et a1., 1980). In mollusks serotonin
increases the beat frequency of gill cilia. Gosselin (1966) gave
evidence that serotonin functions as a local hormone in the mollusk
gill, not a neurotransmitter, and acts as a pacemaker through
rhythmic synthesis and degradation. Serotonin is actively taken up
by platelets and is involved in platelet cell shape changes and
aggregation (Mester and Mester, 1978; Mester st 21., 1979; Chuang s5
.31., 1976). As a local hormone in mammalian tissues serotonin is
important in the constriction of small vessels following vascular
injury (Little, 1977), and blood borne serotonin acts directly on
microfilament bundles of blood vessel endothelium (Buonassisi and
Venter, 1976). Interestingly, the postulated functions of serotonin
in preneural sea urchin embryos are often also performed in
nonembryonic systems as a result of the local production of serotonin
within the cell or group of cells affected and are not related to
serotonergic neurotransmission.

As previously discussed, serotonin released from pigment cells
was prOposed to be the stimulus for ciliary activity, for cell

movements that result in coelom formation and separation of certain

82
ciliated cells from the rest of the ectoderm, as well as for
subsequent cytoplasmic movements leading to the outgrowth of nerve
processes from these ciliated ectodermal cells. Ryberg, 1979,
suggested that light activation of pigment granules may induce
serotonin release from pigment cells. This process may be comparable
with serotonin mediated photoreception in the chick retina, which
involves the activation of adenyl cyclase (Hauschild and Laties,
1973). The cytoplasmic movements observed during neuron elongation
in sea urchin embryos (Gustafson, 1969) were similar to serotonin
induced cytoplasmic flow in nerve processes of mammalian brain cell

cultures (Geiger, 1960,1963).

Mechanism of Serotonin Action in Early Sea Urchin Embryos

 

Conservation of the same functions for serotonin in embryonic
and adult systems may also imply that the chemical mechanism of
serotonin actions in early embryos and adult tissues are similar. In
the adult tissues in which the mechanism of serotonin action
has been examined, it has been demonstrated that serotonin must bind
to a cell surface receptor molecule to exert its effect. Formation
of a serotonin receptor complex triggers either a direct alteration
in membrane permeability or the activation of adenyl cyclase and
production of cyclic AMP. In some cases serotonin stimulated cyclic
GMP production in tissue cells. Little is known about the
mechanism(s) by which cyclic AMP mediates serotonin functions. It is
generally assumed, however, that the mediation process is similar to
that of cyclic AMP induced in several cell types by a variety of

hormones. In these studies two mechanisms of cAMP action have been

83

uncovered. In Escherichia coli cyclic AMP regulates the expression

 

of some genes by activating a cyclic AMP binding protein that
stimulates the transciption of a defined set of genes by directly
interacting with the trancriptional process (Emmer 35 31., 1970). In
both procaryotes and eucaryotes, cyclic AMP dependent protein
kinases, when activated by cyclic AMP, transfer a phosphate group
from ATP to proteins, including histones and enzymes. Enzymes may
be activated or inactivated (Sutherland, 1972). There is evidence
that some of the effects of serotonin in brain may be modulated by
cyclic AMP-stimulated protein kinase activity which phosphorylates
synaptic membrane proteins (Routtenberg and Ehrlich, 1975; Bloom,
1975). Cyclic AMP modifies the permeability of a cell to inorganic
ions, and inorganic ions are necessary for the generation and
destruction of cyclic AMP and for the action of cyclic AMP-dependent
protein kinases and phosphatases (for a review of cyclic
AMP-inorganic ion interaction see McMahon, 1974).

The processes which cyclic nucleotides regulate in adult systems
are numerous and include among others, RNA, protein and DNA
synthesis, cell growth, ion permeability, contractility, membrane
potential and metabolism (Sutherland, 1970; Butcher g£_gl,, 1968).
These processes completely overlap the characteristic processes of
development and differentiation. Inorganic ions have been
demonstrated to be important in several developmental processes.
Barth and Barth (1972) have shown that the cations Li+, Na+ or
Ca++ induce neuron differentiation from prospective amphibian
gastrula epidermal ectoderm cells. In echinoderm gastrulae calcium

is necessary for secondary mesenchyme cell pseudopodal activity (Dan

84
and Okazaki, 1956), an event in which serotonin has been proposed to
be involved. In early embryos of sea urchin and other animals
serotonin may, as in adults, exert its effects by directly modifying
cell surface or intracellular membrane permeability to various
inorganic ions, or by stimulating the synthesis of cyclic nucleotides
which then mediate deve10pmental processes by chemical mechanisms
similar to those used in adult animals. As previously discussed, a
wealth of indirect data support the intracellular location of
serotonin receptors in early cleavage sea urchin embryos. Although
the serotonin receptor and adenyl cyclase molecules are usually cell
surface components of adult tissues, intracellular adenyl cyclase
activity has been demonstrtated on intracellular membranes of rabbit
skeletal (Rabinowitz g£_21., 1965) and dog cardiac (Entman Efihfllf’

1969) muscle and in the cytoplasm of the bacterium, Brevebacterium

 

liquifascien (Hirata and Hayaishi, 1965). A few experiments have

 

suggested that the effects of serotonin on early cleavage sea urchin

embryos involve cyclic AMP. In the sea urchin Strongylocentrotus

 

intermedius, cyclic AMP (Persianinov ££_gl,, 1975) and prostaglandin

 

F2 (Persianinov 32 El“, 1973) were shown to have a protective
action against the cytotoxic effects of serotonin antagonists on
early cleavage. Prostaglandin F2 stimulates adenyl cyclase in
several mammalian tissues (Hinman, 1972). Cholerotoxin stimulates
adenyl cyclase in a variety of biological systems after binding to
cell membrane gangliosides (vanHeynigen, 1977). The response of rat
stomach to serotonin is mediated by ganglioside serotonin receptors
(vanHeynigen, 1974). As previously discussed, in early cleavage

embryos a protective action of gangliosides against cytotoxic

85

antiserotonin effects was demonstrated. McMahon (1974) has proposed
that the differentiation path of embryonic cells of most, if not all,
organisms is determined by their content of inorganic ions and cyclic
nucleotides which is regulated by inducer molecules. He postulated
that early embryonic inducers were the same molecules which modulate
adult tissue activities, hormones and neurotransmitters.

The first data obtained on the binding of [3H]serotonin to
late blastula, gastrula, prism and pluteus sea urchin embryo cell
fractions and dissociated cells are presented in this thesis. A
serotonin binding assay procedure was developed that allowed the
detection of serotonin binding sites in cell fractions and
dissociated cells. Additional binding studies should help determine
if the binding sites described are the receptors which directly
mediate serotonin effects, or if they represent uptake and/or storage
sites or biologically nonfunctional sites. The cell fractionation
procedure developed during the course of the experiments described
may provide an initial step to the final purification of many cell
organelles so that binding studies can be performed on pure cell
fractions. Since chemicals generally thought of as neurohormones
have been identified in preneural embryos of several animal phyla,
including mammals, the sea urchin embryonic system may serve as an
exceptionally convenient model for the study of universal mechanisms
of neurohormone-modulated developmental processes in early animal

embryos.

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