A M . ZOOLOCIST, 3:193-207(1963).
HETEROSYNTHESIS AND AUTOSYNTHESIS IN THE
EARLY STAGES OF ANURAN DEVELOPMENT
GEORGE W. NACE AND LORA HARRER LAVIN
Department of Zoology, University of Michigan
Ann Arbor, Michigan
Any attempt to explain the formation
of independent metazoan organisms from
dependent unicellular "germs" must consider several preconditions. First, the originating cell must possess a potential for
great lability, and have available an appropriate stimulus to initiate the expression of this potential. This is true for the
oogonium, for the spermatogonium or
other multipotential cell which makes an
abortive attempt to form an embryoid
body (Pierce, 1961; Stevens, 1962), for regeneration blastema cells, and for whichever cells give rise to vegetatively-produced
invertebrates and plants (see reviews in
Balinsky, 1960, and Rudnick, 1962). Other
preconditions for these cells and their
derivatives are: (1) the mechanisms required for the maintenance of life; (2)
the materials required for maintenance
and for the elaboration of new structural
and functional systems; (3) labile systems
capable of utilizing the raw materials for
the formation of structural and functional
systems characteristic of any given developmental phase; (4) labile patterns which
order the successive developmental events;
and (5) stable patterns which assure the
continuity of form.
Among these preconditions, our knowledge of the mechanisms for maintenance
and of the genetic mechanisms which comprise the stable patterns is reasonably complete. But! Our definition of "potential
for lability" remains empirical. We find
little in common among the stimuli which
can release this potential in the various
cells where it exists. Our knowledge of
the necessary raw materials is limited (see
papers in this symposium). We find it
difficult to distinguish between the labile
Supported in part by research grant GM-05409
fiom the National Institutes of Health, United
States Public Health Service and by grant IX-40C
from the American Cancer Society.
functional systems and the maintenance
systems. The former characterize specific
developmental phases and drive the developing complex to new levels of structural-functional organization; the latter
maintain life and intrude upon the work
of the investigator who attempts experimental manipulation of the former. We
recognize the labile patterns by the manipulation of developmental geometry.
This reveals patterned interactions between components at each level of organization—interactions which result in epigenetic development. Our analysis at each
level of this epigenesis leads us to the preceding level in the hope of unraveling the
skein of events from its origin. This intellectual process has led to the concept
of formative centers in the egg (Dalcq,
I960), which we visualize as centers for all
subsequent organization.
The bibliography of Dr. A. M. Schechtman, to whom this symposium is dedicated,
reveals his long interest in this question
of ultimate origins. His search began with
the analysis of inductive interactions
(Schechtman, 1934), continued into consecutively earlier phases of development
(Schechtman, 1935), and finally (Schechtman, 1955, 1956) was directed toward the
question: How does the egg acquire its
original organization? This question
places formative centers in perspective as
products of a prior organization resulting
from the origin, character, and geometry
of deposition of the macromolecules acquired by the egg during its growth phase
or during early in utcro life. Many of
these molecules possess antigenic properties. Thus, Schechtman turned to the
use of immunochemical tools to explore
the sequence of developmental events.
Various considerations which arose in
connection with these studies led Schecht-
(193)
194
G. W. NACE AND LORA LAVIN
man to the recognition of autosynthetic and
heterosynthetic components in the egg. He
defined the autosynthetic substances as
macromolecules elaborated by the egg and
embryo, and the heterosynthetic substances
as molecules produced by the adult organism and transferred to the embryo (Schechtman, 1955) .
Autosynthetic and heterosynthetic processes in mammals, birds, and insects are
treated elsewhere in this symposium. All
the evidence favors the view that the developing system is comprised of components
resulting from the action of both processes.
Flickinger (1960) has indicated evidence
of functional significance for the constituents o£ frog yolk (see also Nace and Inoue,
1957; Nace, 1963) ; however, in no instance
does clear evidence exist for the possible
autosynthetic or heterosynthetic origins of
specific macromolecules which have been
assigned specific roles as "raw materials",
as participants in functional systems of
maintenance or development, or as components of significant organizational patterns.
In considering some of these questions in
the frog, I will first review the major descriptive facts which, on one hand, place
limits on our concepts and, on the other
hand, show the existence of relationships
which are prerequisites to our concepts.
Some, but not all, of these facts are applicable to other amphibians. I will then consider several aspects of the growth of the
egg as it relates to both autosynthesis and
heterosynthesis. Finally, I will present two
sets of data. The first, collected by Dr. L. B.
Slobodkin and Miss Jill Clarridge (1963),
demonstrate a developmental significance
for heterosynthetic components in the egg
jelly. The second, collected in this laboratory (Nace et al., 1960) and extended by
one of us (Lavin), were gathered to explore
the relationship between a possible heterosynthetic molecule, its localization in the
egg, and specific developmental functions.
Although the newest data add to the complexities which must be considered in concepts of fertilization, the antigen under
study seems to be of autosynthetic origin,
and the data illustrate the difficulty of re-
lating developmental phenomena to the
origin and function of specific molecules.
OOGENESIS IN ANURANS
Recalling the general features of oogenesis in the frog, we note that the germinal
epithelium of the theca externa which encloses the saccular ovary of the frog gives
rise to the oogonium. As it grows and expands between the theca externa and the
theca interna, which provides the circulatory bed, the primary oocyte becomes surrounded by follicle cells which also arise
from the germinal epithelium. These cells
invest the oocyte and are in intimate but
not direct contact with it through the interdigitation of microvilli from the egg and
of processes from the follicle cells (Kemp,
1956; Wartenberg and Schmidt, 1961).
Soon, the originally central nucleus enlarges as the germinal vesicle, and becomes
eccentric (Brachet, 1947; Rugh, 1951);
yolk platelets appear in the cortical layer
and gradually fill the cytoplasm (Wittek,
1952; Kemp, 1953, 1956); pigment granules
appear in the cortex of the hemisphere
which contains the germinal vesicle and
the symmetry of the egg has become established (Wittek, 1952; Devillers, 1960).
The first meiotic division occurs in association with ovulation. The secondary
oocyte is then carried from the coelomic
cavity to the oviduct which deposits jelly
about the egg as it passes to the uterus.
During this period meiosis is blocked at
the metaphase of the second meiotic division. After some hours in the uterus the
egg leaves the female, is fertilized, the second polar body is extruded, the jelly swells
and the zygote passes through the familiar
developmental stages.
Several features relative to possible
sources of materials are evident from this
sequence of events. In particular, note
that growth of the oocyte occurs in the
presence of a diploid germinal vesicle and
in close contact with the diploid follicle
cells, while any contributions to the egg
after ovulation, e.g., from the oviduct, occur
in the presence of a haploid set of chromosomes.
EARLY ANURAN DEVELOPMENT
AUTOSYNTHESIS AND SYMMETRIZAT1ON
The existence of important quantities of
materials produced by autosynthetic mechanisms in the egg is amply demonstrated
by studies in many laboratories, and the
involvement of the germinal vesicle and
its genetic information in this synthetic
activity is undeniable (Brachet, 1947, 1957,
I960, 1962; Grant, 1953; Kemp, 1953; Ficq,
1960). However, we are still largely ignorant of the identity and function of the
specific macromolecules which are produced as a result of this activity, especially
during early oogenesis when normal egg
symmetry is established. As Devillers
(1960, 1961) has so rightly pointed out
in his lucid analyses of the problem, it
is at this level that the early organization
is established, and it is the precise nature
of these macromolecules that determines
the degree of lability, stability and, indeed, the very character of this organization.
Regardless of the nature of these materials, the geometry of their deposition in
the egg and, consequently, the organization of the egg, is related to the location
of the nucleus in the cytoplasmic mass
(Wittek, 1952). During the early portion
of the previtellogenesis period, the centrally-located germinal vesicle distributes
its products radially in all directions.
These nuclear products form centers of
synthetic activity, e.g., the so-called "yolk
nuclei," whose products cause further radial expansion of the cell mass, with the
nucleus retaining a central position (cf
Wittek, 1952, Fig. 1). During the next
phase of previtellogenesis, the germinal
vesicle takes an eccentric position and provides the first grossly visible evidence of
polarity in the egg (cf Wittek, 1952, Fig.
2). At this time the products of synthetic
activity accumulate in a particular sector
of the egg (Devillers, I960).
Wittek (1952) and Devillers (1960, 1961)
are the most recent investigators to attempt
explanations of the causative factors which
lead to this polarity in amphibian eggs (cf
Raven, 1963). Wittek notes that a basophilic "cape animale" exists at the time
195
the germinal vesicle is found in the eccentric position, and concludes that the monaxial polarity is already complex and that
its origin cannot be attributed to the position of the germinal vesicle, which is not
always in exact accord with the axis of egg
polarity. She attributes the polarity to a
symmetry which exists from the start of
oogenesis. Devillers suggests, more concretely, that the original organization of
the egg results from a tridimensional system formed in response to the intrinsic
properties of molecules present in the
oocyte at this time. He excludes external
factors as the cause of this original polarity.
This is in contradiction to Fankhauser
(1948), who, while noting the absence of
satisfactory evidence for an effective external factor, feels that the axiation of the
egg must be initiated by some factor in
the ovarian environment.
It is surprising that, in this connection,
no one seems to have noted that cellular
polarity is the rule rather than the exception, and that, while it is always difficult
to explain in specific instances, this cellular polarity is usually attributed to extracellular forces such as crowding, as in
columnar epithelia; directed motion or
outgrowth, as in nerve cells; cellular affinities, as in the alignment of muscle cells,
etc. In the case of the egg it is noteworthy
that the suppression of cell division in the
growing oocyte leads to conditions which
do not exist in other cells. For example,
the products of synthesis in the oocyte are
not secreted but accumulate within this
non-dividing cell which is thus forced to
acquire an enormous volume. With synthesis occurring in or near the nucleus
(Ficq, 1960; cf Wischnitzer, 1958) the fresh
products must accumulate near the nucleus
while previously synthesized products are
displaced radially as diagrammed by Wittek (1952, Fig. 5; cf Weiss and Hiscoe,
1948). As this process continues, the products which accumulate in the far reaches
of the cell must, by virtue of their mass,
tend to insulate these regions from further
direct nuclear control (see Fig. 1). It is
perhaps under these conditions that mito-
196
G. W.
NACE AND LORA LAVIN
of intrinsic and extrinsic factors, and the
randomness of oocyte orientation within
the ovary (Child, 1941; Wittek, 1952;
Bronsted and Meyer, 1950), lends support
to this idea that the polarity of the egg
may be the result of random pressure applied to the egg at a critical time during
its early growth phase.
The autosynthetic activities described
above are referred to the activities of the
germinal vesicle. However, the possibility
of a second type of autosynthesis seems to
have received little attention. This might
best be described as synthesis within the
oocyte but utilizing synthetic machinery,
e.g., messenger RNA, passed into the egg
from outside sources. Such machinery,
formed under the control of follicle-cell
nuclei, might well pass into the egg by
way of the microvilli or through pinocyAs growth processes continue, crowding tosis at the crypts between microvilli
in the saccular ovary distorts the oocytes. (Kemp, 1956; Press, 1959; Schjeide et al.,
Such crowding may be seen in several pub- 1963; but see Ward, 1962 b). The foci of
lished figures — for the amphibian, Rugh, synthesis established in this way would
1961, p. 44; Kemp, 1953, Fig. 1; for the simulate autosynthesis, but would mainmammal, Anderson and Beams, 1960, Fig. tain a peripheral localization far removed
1. These figures illustrate the extent to from the influence of the egg nucleus bewhich the shape of oocytes at the pre- cause of the accumulation of materials in
vitellogenesis stage is distorted from the the intervening; space. This could be the
spherical. If distortion such as this occurs synthetic activity in the cortex of the egg
at a critical period the machinery for auto- found by a number of investigators
synthesis could be displaced from a sym- (Kemp, 1953, 1955; Ficq, 1960). This is,
metrical to an asymmetrical distribution also, consistent with the evidence for inwithin the egg. Thus, the appearance of tense uptake of nucleic acid and protein
the "yolk nucleus" 1 as a condensed juxta- precursors in the follicle cells (Ficq, 1960)
nuclear body or bodies (Brachet, 1947; which, as evidenced by their squamous
Kemp, 1953; Anderson and Beams, 1960) character, do not seem to accumulate the
and the newly acquired eccentric position products of the synthesis implied by this
of the nucleus may well result from purely uptake. Direct evidence for the transfer
physical distortion. Further autosynthetic of such macromolecular systems comes
activity with concomitant organization of from observations that insect and bird eggs
the products at the sub-cellular level would engulf follicle cells or their fragments
(Brambell, 1926; Painter, 1940; Schultz,
accentuate the development of this asymmetry, and could well be the origin of the 1952; Press, 1959; Brachet, 1960). Perhaps
heterogeneity which results in the final the cortical accumulation of yolk platelets
establishment of the axial polarity of the represents such simulated autosynthetic
egg. The failure to find positive correla- activity. The ready availability of raw
tions between egg polarity and a variety materials to these peripheral centers of
synthesis, and the less favorable position
1
According to Ward C1962 b) this term may not of the juxtanuclear autosynthetic centers
be strictly applicable to the bodies seen in amphibcould accentuate the developing asymme-
chondria shift from their normal functions
and become converted into sites of protein deposition in the production of yolk
platelets (Lanzavecchia, 1960; Ward, 1962
a,b; Karasaki, 1963; Schjeide et al., 1963).
Note that this view differs from that which
suggests that the mitochondria are the
sites of yolk synthesis (Ward, 1962 b).
Since it is suggested that these events
result from a failure to secrete cell products, one is constrained to ask what special
conditions exist to prevent this very active
cell from secreting its products. Perhaps
the sequestering of yolk within mitochondria or other membrane systems (Karasaki,
1959), or activity of the complex cortical
structures of the egg (see references in
Wartenberg and Schmidt, 1961) are significant in preventing secretion of these
materials.
ian oocvtes.
EARLY ANURAN DEVELOPMENT
try of the egg, and result in the differential formation and shifts of the yolk mass
as described by Wittek (1952).
HETEROSYNTHESIS AND SYMAI ETRIZATION
In addition to the two sources of synthesis just described, heterosynthesis forms
a third possible source of macromolecules
for the egg. Evidence for the transfer of
macromolecules to the eggs of mammals,
birds, and insects is presented or cited in
many of the papers in this symposium
(Glass, 1963; Schjeide et al., 1963; Telfer
and Melius, 1963). In the case of the frog,
Cooper (1950), Flickinger and Nace
(1952), Flickinger and Rounds (1956),
Nace (unpublished), and others have evidence for the existence within the egg and
developing embryo of molecules with the
immunochemical specificity of several of
the adult serum proteins. Glass (1959) has
further contributed the application of
fluorescent-antibody techniques which reveal a pattern of distribution for serumlike antigens in the frog oocyte during the
course of its growth that suggests serum
transfer to the egg from the maternal circulation. Although Raster and Schechtman (1957) could not demonstrate a transfer of human serum antigens to the frog
egg, they note that the failure of this
heterologous system may not be pertinent
to a consideration of the transfer of homologous proteins.
In none of these cases has clear evidence
been presented to demonstrate that the
serum-like antigens in the oocyte of the
frog are identical to those found in the
serum. However, several sources of heterosynthesized macromolecules can be imagined. First, molecules synthesized in the
follicle cells could be passed into the egg
through mechanisms suggested above (cf
Nace et al., I960). Second, materials synthesized in any organ of the adult body
and passed into the circulation could filter
through the interstitial spaces between follicle cells and be accumulated in the egg
by pinocytosis (Schjeide et al., 1963).
Third, follicle cells could accumulate molecules from the circulatory system and pass
197
them with or without modification, into
the egg.
These three mechanisms could operate
at any time during the growth phase of the
oocyte, and would lead to the accumulation of materials in the cortical regions of
the egg. These processes could occur over
the entire surface of the egg, or they could
be differentially localized as a result of
any of several factors operating either
within or around the egg. Some evidence
for such differential uptake is to be seen
in the figures published by Glass (1959) ;
however, her sections through individual
eggs were not sufficiently extensive to establish this point.
In considering the role in egg symmetrization of molecules present in the cortex
of the egg, be they of heterosynthetic or
autosynthetic origin, it is frequently suggested that they move in a centripetal
direction (Wittek, 1952; Kemp, 1953; Devillers, 1960). However, it is equally possible that only raw materials for autosynthesis move in this manner and that macromolecules and formed structures such as
yolk platelets accumulate at the periphery
of the egg. It is difficult to collect evidence to choose between these alternatives,
but the details of the elaboration of symmetry must depend upon which phenomenon occurs. Fig. 1 shows oocyte growth
accurately to aid in clarifying this question. The diagram was constructed on
the basis of measurements and figures by
Kemp (1953). The diagram indicates that
germinal vesicle diameters are approximately the same for Yo, Y1; Y2, and Y3
oocytes, but that the germinal vesicles of
Y4 and Y5 eggs are as large as the entire
Yo oocyte. The proximal margin of the
yolk ring of the Y] oocyte, which is the
stage when this ring is first visible, is more
distal than the surface of the Yo oocyte. At
stages Yo and Y3 this margin appears somewhat closer to the center but this may be
the result of either centripetal yolk movement or of perinuclear synthesis. More
striking at these stages is the distal accumulation of the growth ring. Finally, at stages
Y4 and Y5 the margin of the germinal ves-
198
N :
G. W.
NACE AND LORA LAVIN
Nucleus
Y . Stag*
Yolk
of
Deposition
FIG. 1. Scale diagram of the growth of Rana
pipiens oocytes. The egg surface at each stage is
represented by the heavy line; the germinal vesicle
surface by the light line. Stage designations and
measurements fiom Kemp (1953). Note that normal adult cell si/.e is smaller than the figure "N"
which designates the germinal vesicle.
icle is beyond the inner margin of the
yolk ring at Y3. Taken together, these observations suggest that, although they may
occur, neither perinuclear synthesis nor
centripetal migration of yolk need be
invoked to explain the organization of the
oocyte.
been shown by the studies of Humphries
(1956, 1960), Subtelny and Bradt (1961),
Shaver and coworkers (1960, 1962), and
Shivers and Metz (1962), who have implicated substances of oviducal origin in the
phenomena of meiosis, fertilization, and
cleavage. Nace et al. (1960), moreover,
have presented evidence that materials of
oviducal origin may be found within the
egg itself. It is of particular interest, especially in view of the supposed cleidoic
character of the amphibian egg, that Slobodkin and Clarridge (1963) have obtained data which indicate that egg jelly
contributes important, if not essential,
sources of eneigy utilized by the embiyo
prior to hatching.
A fourth source of heterosynthetic macromolecules is the oviduct. This is evident from the absence of egg jelly on
coelomic eggs, and its presence on uterine
eggs. It is less evident that materials from
the oviduct contribute to the organization
of the egg or are significant to development
(for other aspects of this question «,ee Savage, 1961). Such significance has recently
EARLY ANURAN DEVELOPMENT
in jelly
60
1 0 0 140
200
hrs. of d e v e l o p m e n t
300
FIG. 2. Graph of caloric measurements made on
Rana pipiens embryos from fertilization through
post-hatching stages. Vertical lines represent 95%
confidence intervals; open circles represent data on
which confidence intervals were not determined.
From Slobodkin and Clarridge (1963).
EGG JELLY AS A HETEROSYNTHETIC
SOURCE OF ENERGY
The data of Slobodkin and Clarridge
(1963) have not been made available in
the embryological literature. They have
been kind enough to permit me to summarize it here.
A micro-calorimeter (McEwan and Anderson, 1955) sensitive to differences of
1% for samples weighing 5-10 mg was
used to measure the caloric content of
Rana pipiens embryos. Some embryos were
raised within their jelly and vitelline membrane, others were raised free of jelly but
in their vitelline membrane. Jelly, but not
the vitelline membrane, was always removed before making the calorimeter
measurements. Figure 2 illustrates representative and comparable sets of data from
among their results. The first four points
of the curve for Batch I represent embryos
raised free of jelly; the points at 100 hours
and at 140 hours for Batch I and all points
199
for Batch III represent embryos raised in
their jelly. 'H' designates the time of
hatching, while points 'a' and 'b' were
obtained from embryos of the same age
which had not yet hatched ('a') and which
had hatched ('b'). 'MO' designates the
time when the mouth is opened. 'FM' designates the time when the embryos were
placed in fresh medium, free of the jelly
and vitelline membranes remaining in the
culture dish after hatching.
Attention is drawn to the similarity of
the caloric content of these two batches of
eggs at the initiation of development. The
surprising observations were made on the
subsequent stages of development. Whereas, as might be expected, the caloric content of eggs free of jelly dropped between
fertilization and hatching, the caloric content of eggs raised in jelly continued to
rise until after hatching. It is of particular
interest that those embryos which hatched
precociously contained a lower caloric content ('b') than did those with slightly
retarded hatching ('a'). Note that the latter were measured within their vitelline
membranes and that the processes of jelly
dissolution are maximally active at this
time. Evidently the products of jelly degradation enrich the caloric content of the
perivitellin space and are available for
transfer to the embryo. The mechanisms
for uptake of these products from the
medium seem to be lost after hatching,
so that the caloric content of the embryos
dropped until they were able to feed; it
then rose as long as the embryos were in
the presence of jelly, but rapidly declined
in the absence of jelly or other nutrient
sources.
Based on these direct calorimetric measurements, Slobodkin and Clarridge (1963)
estimated that an amount of energy equal
to approximately 5-8% of the initial energy content of the egg was utilized in the
first 100 hours of development, and that
the amount of energy entering the egg
from the jelly was more than the energetic cost of development until that time.
This is evident in Fig. 2, Batch III, which
shows a higher caloric content in post
200
G. W.
NACE AND LORA LAVIN
hatching embryos (100 hrs) than in freshly the antisera show common, though comfertilized eggs. It is also of interest that plex, characteristics in agar diffusion analythe drop in caloric content for jelly-free sis. When made fluorescent, these antisera
eggs (Fig. 2, Batch I) and the rise in cal- localized on the follicle cells surrounding
oric content for eggs in jelly (Fig. 2, Batch immature primary oocytes; did not localize
III) was greatest prior to the neurula stage. on the follicle cells surrounding mature
This corresponds to a period during which primary oocytes; localized heavily on the
histological evidence suggests there is little cortex and cytoplasm of immature primor no utilization of yolk platelets (Kara- ary oocytes but less heavily on the cortex
saki, 1963).
and not on the cytoplasm of mature primFurther analysis of their data suggested ary oocytes.
Only slight localization was seen on the
to Slobodkin and Clarridge (1963) that
cortex
of coelomic oocytes just before or
carbohydrate was being used as the major
energy source during the first 60 hours of after the first meiotic division. Extremely
development. This corresponds with the heavy localization was seen on the epitheknown fact that carbohydrates are a major lium and in the lumen of pre-ovulation
component of frog egg jelly (Folkes et al., oviducts and on the oviducal epithelium
1950). Calculating from oxygen consump- during the passage of secondary oocytes,
tion data (Moog, 1944), and assuming a but this localization was much reduced
carbohydrate source, Slobodkin and Clar- after the passage of eggs through the oviridee (1963) found that 0.2 calories per ducts was complete. Jelly glands in the
embryo were consumed from fertilization oviduct were negative at all stages. Heavy
through hatching. This compared with localization was again observed on the cor0.33 calories per embryo consumed during; tex of eggs passing through the oviducts.
the same period as determined calorimetri- There was some evidence of an animalcallv (Fief. 2, Batch I), and with 2.3 calories vegetal gradient in the distribution of this
available in the jelly around each effg but localization on the cortex of uterine eggs.
excluding the caloric content of the fertili- Finally, all localization disappeared from
zation membrane and the contents of the the cortex of eggs about the time of the
first cleavage division; instead, it appeared
perivitelline space.
adjacent
to the egg in the perivitelline
This evidence reveals that the eggs of
in
a
region which may correspond
space
R. pipiens are not closed systems, and
the
surface
coat. With the exception of
to
that energy is transferred from the heterothe
epithelium
of the male "vestigial" ovisynthesized macromolecules of the jelly to
duct,
an
extensive
series of adult tissues
the egg, presumably as the products of jelly
for
this antigen proved
and
serum
tested
degradation.
to be negative.
MACROMOI.F.CULAR CONTRIBUTIONS OF
THE OVIDUCT TO THE EGG
In the course of examining antigenic
changes which occur in R. pipiens eggs
during their growth and passage through
the female reproductive tract, Nace et al.
(1960) made a series of observations which
suggested the transfer of at least one antigen from ovarian follicle cells and from
the oviducal epithelium to the egg. Replicate sets of observations were only produced by antisera directed against soluble
extracts of ovaries. In addition, it was
necessary for the replication of data that
These observations suggested that an
antigen(s) with high specificity for the
reproductive system was synthesized in the
follicle cells and oviducal epithelium, and
transferred to the egg where it has a developmental role, perhaps as a regulator
of meiosis and mitosis or as a participant
in fertilization reactions. This interpretation was consistent with the observations
of Humphries (1960) and others (see discussion in Nace et al., 1960) which indicate that contact between the oocyte and
the oviduct inhibits the second meiotic
division, at least in some amphibian eggs.
EARLY ANURAN DEVELOPMENT
Other evidence for the transfer of developmentally significant molecules from the
oviduct to the egg in R. pipiens comes
from the studies of Shaver and his coworkers (Shaver et al., 1960, 1962; Shivers
and Metz, 1962). Usinsr, primarily, antisera directed against jelly, these workers
have demonstrated the antigenically complex character of jelly. Drawing parallels
between their studies and those of Tyler
(1959), Perlmann (1959) and others, they
have further shown that these anti-jelly
sera can inhibit fertilization as measured
by the suppression of cleavage.
When made fluorescent, the antisera
used by Shaver and his co-workers localized
on both the jelly secreting glands and the
epithelium of the oviduct (Nace et al.,
I960, Fig. 12). Thus, it differed from the
anti-ovarian sera used to demonstrate the
localization specific to oviducal epithelium.
In addition, when subjected to agar diffusion analysis, the reaction spectra of the
antisera used by Shaver and his co-workers showed many features which distinguished them from the antisera used by
Nace et al. (1960) .
2
AN ANTIGEN OF THE EGG SURFACE
The observations of Nace et al. (1960)
remained incomplete without first demonstrating that the localizations detected in
the follicle cells, eggs, and oviduct were
all attributable to a single antigenic entity and second, experimentally demonstrating the developmental role of the
antigen (s) following principles previously
discussed and applied (Nace, 1955; Nace
and Inoue, 1957; Nace, 1963). Unfortunately, the anti-ovarian sera used to obtain the observations described above were
exhausted. Tn their absence, similar,
though not identical, antisera were tested
biologically to determine their ability to
modify development.
The biologicnl test system. Several possible biological responses to the antisera
- Illustrations and further details supporting the
observations and data recorded here will be included in the thesis of one of us (Lavin) and will
be published elsewhere.
201
were anticipated on the basis of previous
suggestions, i.e., that the antigen(s) concerned was involved in meiosis and mitosis or the fertilization reaction (Nace et
al., 1960). One possibility was that exposure to the antisera might cause premature second meiotic division by coelomic eggs and activation of uterine eggs.
Another possibility was that no effect
would be observed on coelomic or uterine
eggs, but that fertilization or early development might be rendered abnormal.
In either case it was assumed that the significant immunochemical reactions would
occur at the surface of the egg (Tyler,
1963). In addition, it was known that frog
egg membranes are not readily permeable
to antibodies (Flickinger and Nace, 1952;
Nace, unpublished); also, that it would be
undesirable to have any appreciable quantity of free antibody in solution at the time
of fertilization, if responses of the eggs to
fertilization were to be assessed (Shivers
and Metz, 1962). For these reasons we
wished to coat the surface of the egg with
antibodies while microvilli still protuded
through the vitelline membrane (Kemp,
personal communication), and prior to
jelly deposition and elevation of the fertilization membrane. Thus it was deemed
necessary to expose coelomic eggs to the
test antisera prior to their passage through
the oviduct.
To attain this objective, ovulation was
stimulated in several gravid females; one
to serve as egg donor, the others as hosts.
Preliminary tests revealed the time when,
at each season and with appropriate progesterone and pituitary dosage3 (Wright
and Flathers, 1961), maximal numbers of
eggs would be present in the coelom of
females which had undergone no prior
surgical treatment (cf Arnold and Shaver,
1962; Humphries, 1956). Coelomic eggs
were collected from the donor female,
washed, and held at 18-22°C in full
strength Holtfreter's solution or in the
3
About 5 nig of progesterone (Sigma Chemical
Co.) in 0.2 ml corn oil administered via the dorsal
lymph sac and one pituitary in saline via the coelomic cavity.
202
G.
W.
NACE AND LORA LAVIN
TABLE 1. Activation* and cleavage of inseminated uterine eggs exposed to various treatments prior to passage through the oviduct.
Treatment
Standard Hoitfreter's solution
Normal rabbit serum
Rabbit antiserum to:
Adult frog serum
Oviduct supernatant
Large oocyte supernatant
Neurula supernatant
Tailbud supernatant
Oocyte lysozyme
50% (NH,)2SO4 precipitate
from large oocyte supernatant (did not contain A.F)
Fraction obtained from
large oocyte supernatant (A.F)
No. of
eggs
No.
activ'd
%
activ'd
8
12
457
758
350
531
70
263
366
75
69
21
15
7
4
7
362
174
258
119
147
171
118
174
91
62
23
25
119
4
48
52
24
19
17
69
3
71
51
10
0
3
102
0
41
56
16
0
12
87
0
34
8
18
12
6
12
0
32
14
64
100
67
10
0
453
78
17
3
4
2
2
2
2
77
Normal Cleav., as
cleav. % activ'd
Abn. cleav.
Abn. as % total
cleav. cleavage
No. of
tests
75
* Rotation was taken to be indicative of activation.
test antisera or globulin fraction of the
test antisera which had been dialyzed
against full strength Holtfreter's solution.
Previous tests showed that the eggs could
be maintained in this way for as long as
12 hours at 18°C with only minor loss in
viability. Under experimental conditions,
however, they were seldom held longer than
one hour. After treatment, the eggs were
washed, lightly stained with 0.1% neutral
red, passed through several additional
washes with Holtfreter's solution, introduced into a 5 ml syringe fitted with a
shortened #13 needle, and about 100 were
injected into the coelomic cavities of each
of the ovulating host females.
After the appropriate lapse of time, host
and donor eggs were stripped onto dry
Petri dishes, and were artificially inseminated. Rotation data, selected as indicative
of activation, were collected on the black
and white eggs of the host and the
black and red donor eggs. The intensity of the staining on the experimental
eggs did not change between injection and
recovery; never were more stained eggs recovered than were injected. Thus, there
seems little likelihood that host eggs were
mistaken for donor eggs. After rotation,
the jelly masses were freed and host and
donor eggs were isolated in separate dishes.
Data on evidence of cleavage attempts and
on actual cleavages were then recorded (cf
Shaver and Barch, 1960).
Biological tests with complex antisera.
None of a variety of antisera (see Table 1)
administered to coelomic eggs stimulated
premature second meiotic division or activation of eggs, either before or after passage through the oviduct. In fact, no effects
were observed on these stages although
fluorescent-antibody data indicated that
the antisera used in the biological test system did coat the surfaces of the eggs.
These observations suggested either that
none of the antigens involved inhibit meiosis or activation, or that their combination
with antisera at the egg surface does not
influence their participation in these phenomena.
Table 1 contains a tabulation of data
collected after insemination of the eggs.
Information on the techniques for preparing and assaying the antisera may be found
in previous publications (see references in
Nace et al, 1960; Nace and Alley, 1961).
The figures for activation, normal cleavage, and abnormal cleavage obtained for
control eggs treated with standard Holtfreter's solution before being passed
through the host oviducts differ somewhat
from those usually obtained from normal
laboratory fertilization of R. pipiens eggs.
We believe this loss of viability may be
EARLY ANURAN DEVELOPMENT
attributed to damage produced in the
course of manipulating the eggs, but have
not submitted this to a rigorous test. The
response of control eggs exposed to normal rabbit serum differed slightly from
the response of controls exposed to standard Holtfreter's solution, but we have not
yet been able to attach significance to this
difference (cf Fedoroff and Webb, 1962).
Among the experimental results, activation
and cleavage data obtained with antisera
directed against the adult derivatives, i.e.,
adult frog serum and oviduct supernatant
(Table 1), differed significantly from the
control data, but not as markedly as did
the data obtained with antisera directed
against the embryonic derivatives, i.e., large
oocyte, neurula, and tailbud supernatants.
Evidently all these complex extracts contained antigens whose antibodies were
capable of producing effects upon fertilization and early cleavage.
Agar diffusion analyses of all these reagents revealed that the only reciprocal
patterns which always contained at least
one identifiable line in common were those
produced with the systems of embryonic
origin. We arbitrarily designated this common line as line F. T o determine that line
F indeed represented an antigen whose
antibody inhibited activation and cleavage,
it was necessary to isolate the antigen, prepare antisera to it, and test these antisera
in the biological test system.
The preparation of antigen F. The
preparation of antigen F was accomplished
by following the protocol diagrammed in
Figure 3. The products obtained at each
step of the protocol were tested by immunodiffusion to determine the presence or
absence of antigen F. The product containing the preponderant portion of antigen F was then subjected to the next step
in the protocol, until the final preparation
appeared as a single peak when eluted
from DEAE cellulose by 0.3 M Tris buffer.
Antisera to this product were prepared
by a single subscapular injection of the
rabbits using 1.0 mg of the product appropriately immulsified in Freund's complete
203
adjuvant (Nace and co-workers, op. cit.).
High titer antisera were obtained in bleedings made between the fourth and sixteenth week following such injection.
These antisera (A.F) were compared with
the previous antisera (prepared against
large oocyte supernatant) in agar diffusion
and produced one major line that coalesced
with line F of the other systems. A.F also
produced one to two minor lines in some
tests.
Biological tests of A.F. A.F produced
marked reduction in activation when used
in the biological test system. In addition,
only 10% of the activated eggs showed any
sign of cleavage and 75% of these were
abnormal (Table 1 — last line).
It is well known that activation and
cleavage are highly susceptible to adverse
environmental conditions of many kinds
(Brachet, I960). Therefore it was necessary to demonstrate that our results were
truly in response to the activity of the
antibody, and not to a non-specific toxic
component of the antisera. The specificity
was suggested by the preliminary control
tests, but it was most clearly demonstrated
by tests using antisera directed against two
other fractions obtained from oocytes.
Thus, high titer antisera directed against
a basic protein obtained from mature
frog ovaries and identified as lysozyme
(Nace, 1962; Nace, in preparation) did
not differ from normal rabbit sera with
regard to their effect on activation or
cleavage. This was in contrast to antisera
directed against the product obtained by
precipitation with 50% saturated ammonium sulfate in the course of preparing
antigen F. These antisera did not contain
antibodies to antigen F; yet they were more
effective as inhibitors of activation and
cleavage than were the antisera directed
against antigen F (Table 1).
Thus, of antisera directed against three
products obtained from the same starting
material, i.e., mature ovaries, one showed
no effect on activation and cleavage, while
the other two showed marked effects on
these same phenomena. We can only con-
204
G. W.
NACE AND LORA LAVIN
Mature Rana pipiens ovaries
homogenized in equal volume
phosphate buffered saline (pH 7.4, 4°C)
10,000 X g, 30 min
precipitate
supernatant
100,000 X g, 60 min
precipitate
supernatant
(NH4)2SO4 to 50% saturation
precipitate
P50
supernatant
to 70% saturation
supernatant
S70
precipitate
P70
I
dissolved
and dialyzed
10'3 M TRIS-HC1 buffer (pH 7.4)
precipitate
supernatant
Adsorbed on DEAE cellulose column
equilibrated with 10"3 M TRIS-HC1
(pH 7.4)
|
Eluted with stepwise gradient 10"3 to
1 M TRIS-HC1 (pH 7.4)
|
Antigen F eluted at 3 X H H M TRTSHC1
FIG. 3. Flow diagram of steps in preparation of
antigen F.
elude that the observed biological reactions
to these antisera were in response to their
specific activity and not to non-specific
toxic factors.
Three hypotheses were compatible with
these observations. Each assumed that
binding with antibody inhibits the normal
function of antigen F, that for 100% effectiveness the antibody must bind a minimal
proportion of the antigenic reactive sites
available on the egg surface, and that this
condition was not met in the case of eggs
which partially or fully escaped the action
of the antisera.
The first hypothesis was that antigen F
carries the reactive sites for sperm attachment and penetration (Tyler, 1963), and
that binding of these sites on the egg by
antibody prevented activation by inhibiting syngamy. The possibility that direct
action of the antibody on the sperm prevented syngamy was eliminated by insuring the absence of free antibody in the
medium at the time of insemination; by
the failure of sperm agglutination tests
using A.F; and by the ability of washed
sperm, which had been exposed to A.F, to
fertilize eggs. The second hypothesis was
that antigen F is a participant in the activation response following sperm attachment. The third hypothesis was that antigen F participates in both activation and
cleavage mechanisms.
The possibility of the participation of
antigen F in syngamy was eliminated by
two lines of evidence. First, abnormal effects
(i.e., abnormal cleavage) occurred even
when snygamy, as revealed by activation,
had taken place. Second, in one experimental series parthenogenetic stimulation
led to activation of 83% of the control eggs
but of only 32% of the eggs exposed to
A.F. Thus, the observed inhibition of activation was independent of the occurrence
of snygamy, and must be attributed to
direct participation of antigen F in the
activation response. It is more difficult to
assess the possible role of antigen F in
cleavage mechanisms, since we have not yet
exposed eggs to antibody in the period between activation and cleavage — a manipulation which is necessary to distinguish between the action of the antibody on activation and cleavage. The existence of abnormal cleavages is not definitive since it may
be a measure of "incomplete activation"
(Allen, 1958). Thus we conclude that antigen F is a participant in the activation
response and may or may not be a participant in cleavage mechanisms.
Antigen F and the antigen of the oviducal epithelium. Preliminary immunohistological analyses of the distribution of
antigen F using fluorescent A.F under conditions comparable to those used by Nace
et al. (1960) have failed to reveal the presence of this antigen in follicle cells of immature or mature oocytes or in the ovi-
205
EARLY ANURAN DEVELOPMENT
duct. It was present in the cytoplasm of
immature oocytes but was more sharply
limited to the cortex of mature oocytes.
Following fertilization and as late as the
mid-gastrula stage, the localization was
limited to a region of the perivitelline
space which corresponds to the position of
the surface coat (cf Wartenberg and
Schmidt, 1961).
This immunohistological evidence, together with the agar diffusion analyses, suggest that antigen F does not correspond
to the antigen detected by Nace et al.
(1960) in the follicle cell, oocyte, and oviducal epithelium. However, tests using
antisera directed against the precipitate obtained at 50% saturation with ammonium
sulfate (Fig. 3) do show intense localization on oviducal epithelium. This precipitate may contain the antigen previously
described.
SUMMARY AND CONCLUSIONS
Consideration of the preconditions necessary for development lead the investigator to the examination of increasingly
earlier stages of development. Following
this path, Schechtman was led to the examination of the macromolecular composition of the egg and was forced to define
concepts of autosynthesis and heterosynthesis in order to explain the components
he found in the hen's egg, and which others
(cf other papers in this symposium) have
found in eggs of other animals.
We have reviewed evidence indicating
that autosynthesis and heterosynthesis occur in the anuran, and have also suggested
the possible existence of products synthesized in the egg under the control of genetic information transmitted to the egg
from the follicle cells. In considering the
role of these mechanisms in the establishment of the original polarity of the egg,
we drew attention to the possibility that
asymmetry of the egg may result from random pressure applied to the egg early in
its growth phase as a consequence of its
expansion within the limited space available in the ovary. This implicates external
forces as critical to symmetrization, and
constitutes a return to earlier ideas in contrast to recent hypotheses which ascribe
symmetrization to the intrinsic properties
of the molecules comprising the egg.
New evidence demonstrating the participation of autosynthetic and heterosynthetic
mechanisms in anuran embryogenesis is
also presented. Slobodkin and Clarridge
(1963) have revealed the non-cleidoic character of frog eggs with respect to energetic
reserves by showing the transfer of energy
from egg jelly to embryo, and thus have
implicated the oviduct as a site of heterosynthesis of macromolecules utilized by the
embrjo. Immunochemical evidence of a
follicle cell and oviducal epithelium origin
for an antigen of the egg cortex was recalled (Nace et al., I960), and efforts to
isolate this component were described. It
was shown that the isolated component
differs from the component previously described; instead, the isolated component
seems to be of autosynthetic origin. Immunohistological and biological evidence,
however, suggest that the heterosynthesized
component may be in the ovarian fraction
obtained at 50% saturation with ammonium sulfate.
The isolated component was designated
antigen F. Biological tests using antisera
directed against this antigen suggest that
it is a participant in the activation response, as well as a possible participant in
cleavage mechanisms. Chemical analyses
of this entity have not yet been made; thus
it is impossible to compare it with the sea
urchin egg antigens described by Tyler
(1963) or by Perlmann (1959).
ACKNOWLEDGEMENTS
We wish to thank Dr. Isadore A. Bernstein, Department of Dermatology, the
University of Michigan, for advice and
equipment used for the protein fractionations; and Mrs. Marie Coon and Mrs.
Diane Ar for technical assistance.
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