Amy Levenson
Self-fertilization in Botryllus
INTRODUCTION
The phenomenon of self-sterility in Botryllus is maintained
by a dual system. In the synchronous life cycle of the colony,
ovulation precedes the release of sperm by one or two days
(Milkman, 1967) preventing self-fertilization in most cases,
In their natural environment, Botryllus colonies are exposed
to large amounts of sperm released by neighboring colonies.
Cross fertilization occurs rapidly between organisms at
different sexual stages and it is rare for a colony containing
unfertilized eggs to be collected directly from the field. A
second block to self-fertilization, presumably residing in the
diploid maternally derived egg envelope, is also present (Oka
1970, Scofield 1981). This barrier is not absolute, and
gradually breaks down in an isolated colony (approximately one-
half or two days after ovulation, Scofield). With this
breakdown, asynchronous embryonic development is accompanied
by a high frequency of maldevelopment of embryos and failure
of the larvae to complete metamorphosis (Sabbadin 1971),
Ascidian eggs are surrounded by a three-layered envelope.
Within the perivitelline space test cells rest on the ovum itself.
The acellular chorion surrounds the egg and test cells, and is overlai
by flattened follicle cells (figure 1). In previous
experiments, the precise location of the internal barrier to
self-fertilization was not determined because enzymatic and
mechanical techniques did not differentially affect the
different egg layers (Morgan 1923, Rosati 1978). In Botryllus,
the extremely close opposition of the three layers further
complicates any detailed investigation of the exact location
Self-fertilization in Botryllus
Amy Levenson
of the barrier in this species.
The present work presents a successful new technique
allowing selective dechorionation and removal of follicle cells
of the Botryllus egg. With the aid of this technique the
phenomenon of colonial self-sterility was examined. The
localization of the barrier to self-fertilization in the
chorion or follicle cells is documented with an increased
degree of certainty and detail.
MATERIALS AND METHODS
Colonies ofBotryllus were gathered from beneath the docks
of the Monterey Marina during the months of April and May.
Individual colonies were separated and placed in 700 ml.
glass beakers with approximately 600 ml. filtered sea water
at room temperature (20° C) with a constant source of aeration.
The containers were washed with a 1% solution of 7x detergent
(Linbro) and refilled daily. Eggs were removed from the
adult by means of a slit between the oral and atrial siphons
and gentle pressure on the tunic. Colonies were categorized
according to their degree of embryonic development and monitored
for hatching and ovulation.
Dechorionation
Eggs were pipetted from the atrial cavity of Botryllus
and placed in polypropylene dishes (PolyCons, Cole-Parmer
Plastics), in which they were washed three times in filtered
sea water. One drop/three milliliters of penicillin-streptomycin
Amy Levenson
Self-fertilization in Botryllus
solution (5000U/m1/5000 g/ml) was added to the culture if not
used immediately. The filtered sea water was aspirated off
and a 1% solution of cellulase (Sigma no. C-7377) added for
three hours. (All solutions were made in filtered sea water
with o.5 M TRIS buffer (pH 9.1) to pH 6.8 and sterilized with
a millipore filter.) The cellulase solution was then aspirated
off and replaced in successive five minute intervals by a 0.5%
solution of protease (Sigma no. P-5130), a 2% solution of
bovine serum albumin (Sigma no. A-4503) and a 0.5% solution
of bovine serum albumin. The eggs were then washed three
times in filtered sea water with a triple concentration of
penicillin-streptomycin (figure 2).
Fertilization
Approximately ten testes were removed from the atrial cavities
of Botryllus colonies and collected in a PolyCon dish. The
testes were washed three times in filtered sea water (with
one drop/three milliliters of penicillin streptomycin added
to the culture) and refrigerated at approximately 4° C. An
appropriate number of eggs were also removed and washed. In
a sterile depression dish, one drop of penicillin-streptomycin
solution was added to 25-50 microliters of sterile filtered
sea water. The testes were then transferred to the depression
dish and were gently teased apart. The resulting suspension
was triturated several seconds in the pipette to evenly
distribute the sperm. Eggs were next gathered in a pasteur
pipette and allowed to sink to the tip. The eggs were carefully
trans ferred onto the top of the sperm solution in two to three
drops (total volume approximately 100 ml.) The depression dish
Self-fertilization in Botryllus
Amy Levenson
was then loosely covered with aluminum foil and placed in a
moisture chamber. After approximately four hours first
cleavages could be observed.
Self-fertilization
Immediately upon release of larvae, colonies were divided
in half. At this time, eggs are maturing in the new adult
zooids from the second generation buds. One half of each
colony was stored at 15° C to slow the breakdown of the
egg's internal barrier to self-fertilization. The other half
was left undisturbed at room temperature to develop mature
testes. When the testes of the colony held at room temperature
contained mature sperm, the fertilization protocol was followed.
RESULTS
Dechorionation of Eggs with Cellulase
In the first series of experiments, unfertilized Botryllus
eggs were dechorionated enzymatically with a combination of
cellulase and protease. The chorion and overlaying follicle
cells were selectively removed, leaving the test cells and
the egg plasma membrane intact (figure 3). To verify the
success of this unique treatment, fertilization of the
dechorionated eggs and an untreated control were simultaneously
undertaken. Experimental results indicate that the percentage
fertilization was not significantly different between treated
and untreated eggs (table 1). Dechorionated eggs, however,
were much more susceptible to contamination, necessitating
the modification of a normal fertilization protocol (Scofield)
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Self-fertilization in Botryllus
with the use of sterile solutions, sterile glassware and a triple
concentration of penicillin -streptomycin in the wash solution.
Examination of the Barrier to Self-fertility
In the second series of experiments, Botryllus colonies
were divided and their development manipulated until
simultaneous maturation of the gametes was obtained. Four
sets of crosses were run simultaneously: normal (egg) x self
(sperm), dechorionated (egg) x self (sperm), normal (egg) x
cross (sperm), dechorionated (egg)x cross (sperm). Results
for two experiments (A and B) appear in table 2. In experiment
A, there was a higher percentage of fertilization in the
dechorionated (egg) x self (sperm) relative to that seen in
the normal (egg) x self (sperm). The fertilization frequency
for the dechorionated (egg) x self (sperm), on the other hand,
closely parallels that for normal (egg) x cross (sperm) and
dechorionated (egg) x cross (sperm). The low percentage of
normal (egg) x cross (sperm) in experiment B is anomalous and
was not repeated in the dechorionated (egg) x cross (sperm).
Possible reasons for this result are discussed below. However,
these preliminary results suggest that the barrier to self-
fertilization is located in the chorion.
DISCUSSION
This report describes the development of an enzymatic
treatment which selectively removes the chorion and follicle
cells of Botryllus eggs, leaving the test cells and egg plasma
membrane intact. This technique has permitted the investigation
of the barrier to self-fertility in greater detail than was
Self-fertilization in Botryllus
Amy Levenson
possible in earlier studies (Morgan 1923, Rosati 1978), and
has allowed its tentative assignment to the chorion.
Dechorionation of the ascidian egg is useful for many
experimental procedures concerning the embryo. Difficulties
encountered in separating and removing the closely associated
layers of the egg envelope have limited experimental work
on colonial ascidian development. The dechorionation procedure
presented here creates a highly efficient means of attaining
a large number of dechorionated eggs for experimentation. It
is also unique in that it allows selective removal of the chorion.
The finding that cellulase removes the Botryllus chorion
also provides an unexpected insight into the structural nature
of the egg envelopes; the egg apparently resides in its own
tunic, even before development begins. The increased susceptibility
of dechorionated eggs to contamination also opens speculation
concerning the function of this extra-embryonic layer.
Seemingly, the chorion occupies an important role in the creation
of a highly protected structure which will allow the embryc
to successfully grow and develop freed from the hazards of a
non-sterile, and often hostile, environment. Since removal
of the chorion and attached follicle cells by this technique
increases fertilization frequency for eggs fertilized by self
sperm (table 2- A), it appears that one of these layers contains
the structural elements which block self-fertilization. The
finding by Rosati (1978) that follicle cell removal does not
affect self-fertilization in Ciona intestinalis eliminates
their participation in the block to selfing. The location of
Self-fertilization in Botryllus
Amy Levenson
this elusive barrier to self-fertility in colonial tunicates
can now be placed with greater certainty onto the chorion
itself.
The protocol for this investigation was difficult to maintain
successfully to completion. In the above experiments, egg¬
bearing colony pieces were isolated in the cold after ovulation.
For experiments in progress, the timing of temperature
separation of the colony halves has been altered to insure
simultaneous development of the gametes. If mature testes are
placed in the cold environment, the possibility that the barrrier
to self-fertilization has broken down (in the colony kept at
room temperature) at the time of ovulation will be greatly
reduced. Fertilization in vitro will be performed immediately
upon ovulation. Because of the variability of the timing of
the barrier's breakdown the quickest possible removal and
utilization of newly ovulated eggs would prevent self-fertilization
from becoming a source of technical failure.
The phenomenon of self-sterility in Botryllus is of special
immunologic interest because of its close genetic linkage with
the genes controlling the fusion-rejection reaction between
Botryllus colonies. The ampullae of these oozoids actively
fuse or reject with neighboring ampullae indicating some
type of allogeneic recognition (Nagashima, Scofield).
Each individual colony is heterozygotic at one locus determining
fusibility at which there are approximately forty different
alleles. Two colonies must share one allele to fuse. Eggs
of a colony of fusibility genotype AB cannot be fertilized by
A or B sperm, whether they come from the same individual, or
Self-sterility in Botryllus
Amy Levenson
from a neighboring parent or sibling. This suggests that
the fusibility gene locus,or link to it, controls self-sterility
in Botryllus through the diploid, maternally derived test cells
or their products in the chorion. This organization of
genetic locii controlling both histocompatibility and fertilization
is reminiscent of the linkage between the mouse T-complex affecting
spermatogenesis and embryonic development and the H-2 region
which is responsible for producing both the histocompatibility
and immune response (Scofield and Weissman 1980).
ACKNOWLEDGMENTS
I wish to thank Dr. David Epel for his advice concerning
this project. I would especially like to thank Dr. Virginia
Scofield for her incredible enthusiasm, patience and editing
capabilities. The use of her laboratory facilities and
suggestion for this investigation were critical to its
completion. Keith Kohatsu and Jay Schlumpberger donated
both time and concernand egg pulling aid. Finally,
I would like to acknowledge the support given to me by the
other members of the Hopkins spring class.
REFERENCES
MILKMAN, R. (1967). Genetic and developmental studies on
Botryllus schlosseri. Bio. Bull. 132, 229-24:
MORGAN, T. H. (1923). Removal of the block to self-fertility
in the ascidian Ciona. National Academy of Sciences 9
(no. 5), 170-171.
NAGASHIMA, L. and V, SCOFIELD, in preparation.
OKA, H. (1970). "Profiles ofJapanese Science and Scientists.'
H. Yukawa, (ed.) Kodansha, Tokyo, pp. 195-206.
ROSATI, F. and R. DE SANTIS. (1978). Studies on fertilization
in the ascidians I. Exp. Cell Research 112, 111-119.
(1971). Self- and cross-fertilization in the
SABBADIN, A.
ascidian Botryllus schlosseri. Dev.Bio. 24, 379-391.
SCOFIELD, V. (no date given), personal communication.
p
SCOFIELD, V. and I.L. WEISSMAN. (1980). Protochordate
allorecognition is controlled by an MHC-like system.
awaiting publication.
FIGURE LEGEND
FIGURE 1-
The flatened follicle
Botryllus egg with its envelopes.
cells (fc) lie outermost, with the chorion (ch) beneath.
The test cells (tc) have been sheared away but depressions
in the egg surface show where test cells were formerly
present.
FIGURE 2-
Schematic diagram of dechorionation procedure.
FIGURE 3-
Comparison of normal and dechorionated Botryllus eg
stained with methylene blue. a) normal egg with darkly
stained surrounding chorion and test cells b) dechorionated
eggs with darkly stained test cells and absence of outer
chorion.
TABLE 1-
Percentage fertilization of normal and dechorionated eggs
with cross sperm.
TABLE 2-
Self-sterility data comparing fertilization results of
normal and dechorionated eggs with self and crossed sperm.
FIGUF
99995. 15KU
DECHORIONATION PROCEDURE
EGGS — 1% cellulase 3 hours
0.5% protease five minutes
2% bsa five minutes
0.5% bsa five minutes
DECHORIONATED
wash 3X
EGGS
in sterile fsw
I
L
L

TABLE 1
CROSS FERTILIZATION OF NORMAL
AND DECHORIONATED EGGS
normal dechorionated
54
58
A
58
B
55
A
TABLE 2
SELF AND CROSS FERTILIZATION OF
NORMAL AND DECHORIONATED EGGS
normal x self dechor. x self normal x cross dechor. x cross
60
68
2
72
48
33
10