ABSTRACT
The marine worm Urechis caupo exhibits a multixenobiotic
resistance (MXR) similar to multidrug resistance (MDR) in
mammalian tumor cells. Urechis oocytes from their developing
stages in the coelomic fluid to at least the two-day larval stage
possess a membranous protein of 160 kD that is immunologically
related to the mammalian MDR P-glycoprotein of 170 kD. However,
activity of the 160 kD protein is absent in developing coelomic
oocytes but is detected once the oocytes reach the storage organs as
immature oocytes. Further studies are necessary to investigate the
causes for the activity increase of the MXR protein at this stage.
INTRODUCTION
A major focus of current cancer research is on the multidrug
resistance phenomenon mediated by the multidrug transport protein
or P-glycoprotein. Different chemotherapeutic drugs similar only in
their moderate hydrophobicity are transported out by this protein,
rendering clinical chemotherapy virtually ineffective (West, 1990).
P-glycoprotein is a transmembrane protein with two ATP binding
sites (Chen et al., 1986; Gros et al., 1986). Hydrophobic compounds
including natural toxins and anti-cancer agents diffuse naturally into
the tumor cell, and P-glycoprotein then utilizes ATP to transport the
hydrophobic compounds out of the cell (Harper, 1993). This
multidrug resistance to a variety of drugs is also accompanied by a
decrease in the drug accumulation (Juliano, 1976) (Figure 1).
However, the MDR extrusion pump can be saturated and thus
inhibited by other MDR substrates such as verapamil. In this case,
the MDR transport pump becomes non-functional and
chemotherapeutic drugs and other hydrophobic compounds enter the
tumor cell and effectively destroy it.
A similar phenomenon to MDR is seen in some aquatic animals
including mussels, sponges, fishes, and worms. In these animals, a
multixenobiotic transport protein is as a possible defense mechanism
against natural and man-made xenobiotics. Studies on the echiuroid
worm Urechis caupo show that a protein immunologically and
functionally related to the mammalian MDR protein is present in its
oocytes and embryos.
Urechis caupo live in polluted mudflats where they inhabit U¬
shaped burrows. They circulate water and oxygen through the
tunnel by rhythmic peristalsis of their bodies, and feed by secreting
a mucous net to trap small food particles. Gametes continuously
develop in the coelomic fluid (Morris, Abbott, Haderlie, 1980). After
reaching a certain stage of development, they are then sequestered
by collecting organs and stored in three pairs of storage organs near
the anterior region (Figure 2).
A multixenobiotic resistance mechanism has been discovered in
Urechis caupo using fluorescence assays and immunoassays
(Toomey, 1993). The fluorescence assay measures the accumulation
of the dye rhodamine B, which is a substrate for the transport
protein (Neyfakh, 1988). Embryos or oocytes are incubated in
rhodamine B with and without verapamil, a potent competitive
substrate of the MDR protein. Oocytes in rhodamine have a lower
fluorescence than oocytes in rhodamine plus verapamil. Since
verapamil competes with rhodamine for export; less rhodamine
efflux occurs and the cells fluoresce more. MXR activity is thus
assayed by comparing the fluorescence of oocytes incubated in both
rhodamine and verapamil to those incubated in rhodamine alone
with a ratio of 2 to 8 usually indicating MXR activity. However, the
ratio range is not absolute due to variance among oocytes from
different female Urechis.
Immunoassays used to detect a protein immunologically
related to the mammalian P-glycoprotein include gel electrophoresis
and Western blotting, which probe protein samples with an antibody
to the mammalian MDR protein. R7 cells or other cell lines containing
the mammalian protein are used to insure validity of the antibody
probe and for comparison with the protein samples.
Recent studies have investigated the MXR mechanism in
mature oocytes and embryos of Urechis caupo without any specific
focus on the different stages of development. In my study, I
focused on investigating the presence and activity level of MXR from
the coelomic oocyte to the two-day larval stage using Western blots
and the rhodamine fluorescence assay. My results showed the
presence of a a protein immunologically related to the mammalian P¬
glycoprotein in Urechis from coelomic oocytes through the two-day
larvae. However, MXR activity was absent in coelomic oocytes,
present only in the immature oocytes in the storage organs, in the
fertilized oocytes, and in the embryos. For clarification, developing
oocytes reside in the coelomic fluid, and immature oocytes are stored
in storage sacs. Oocytes reach maturity only when they have become
fertilized.
MATERIALS AND METHODS
COLLECTION AND MAINTENANCE
Adult Urechis caupo were collected at Elkhorn Slough near
Moss Landing, California. They were maintained in tanks with
running seawater and a layer of mud from the slough.
SPAWNING
Immature gametes were obtained by inserting a rounded
smooth-tipped glass pipette into one of six gonadopores and gently
rotating the pipette back and forth (Figure 3). Either sperm (white)
or oocytes (pink, yellow, or olive) gushed from the gonadopore. The
sperm was pipetted in its concentrated form into an eppendorf tube
and stored in a refrigerator, able to retain its viability for 3-5 days.
The oocytes were rinsed off the animal into a beaker with filtered
seawater. They were stored at 17°C until use, and fresh oocytes
were obtained every day.
FERTILIZATION
Urechis oocytes were fertilized by adding a drop of dilute
sperm into a beaker of oocytes in filtered seawater. After a few
minutes, the excess sperm were washed out by hand centrifugation
and suctioning off the supernatant with an aspirator. The fertilized
oocytes were then resuspended in fresh, filtered seawater.
EXTRACTIONOE COELOMICOOCYIES
Oocytes are continuously developing in the coelomic fluid of
female Urechis (Miller, 1973). The coelomic fluid was extracted by
puncturing the body wall near the posterior end with a needle and
squeezing the fluid out into a small beaker. The fluid was then
centrifuged in a swinging-bucket rotor for two minutes in an
international clinical centrifuge at 1500 g (Gould, 1967). The
developing oocytes form a light brown layer above the coelomocytes
and were selectively removed with a pipette. This centrifugation
step was repeated several more times to further purify the oocytes.
RHODAMINE ELUORESCENCE ASSAY
For each rhodamine assay,
a 10 ml egg suspension was first
obtained and then divided evenly into two 5 ml egg suspensions of
approximately equal concentrations. To each egg suspension, 5 ul of
either 1 mM or 5 mM rhodamine B were added for a final
To
rhodamine B concentration of either 1 uM or 5 uM, respectively.
one of each pair of egg suspensions 25 ul of either 2 mg/ml or .2
mg/ml verapamil were added, depending on the desired final
concentration of either 22 uM or 2.2uM verapamil. After the
samples were incubated for, one hour at 17°C, they were spun in a
hand centrifuge, the rhodamine supernatant was suctioned off with
an aspirator, and the eggs were resuspended in 1 ml of filtered
seawater. The egg cells were immediately positioned under a l6X
lens of an epifluorescence microscope with filters for rhodamine and
the aperture was closed to a fixed point.
At the later stages of development (e.g. gastrulation and 2-day
larvae), 37% formaldehyde was used to immobilize the moving
embryos. Fluorescence measurements were taken on 10-20 healthy
oocytes or zygotes by a photosensor attached to the microscope. The
light emitted by each embryo was picked up by the photosensor and
converted to a numerical value by a voltmeter. In immature and
fertilized oocytes, mean values were calculated for each sample, and
ratios of the mean values of cells with and those without verapamil
were calculated as an index of MXR activity.
The second set of experiments involved performing rhodamine
fluorescence assays on coelomic and immature oocytes from the
same female Urechis. Oocytes of different sizes develop in the
coelomic fluid; the diameter of each coelomic oocyte was measured
with a reticule and the fluorescence was measured as above. One
unit on the reticule equaled 66.7 microns.
WESTERN BLOTS
Protein Sample Assays
Oocytes at various stages of development were washed, hand
centrifuged, and concentrated into about 100 ul in an eppendorf
tube. A minimum amount of lysis buffer (approximately 170 ul) was
added to the egg concentrate. The eggs were then homogenized into
a milky solution and were put on ice for a while. Approximate 100
ul of 5% SDS was added to the homogenate which was then sonicated
ten times for 3 seconds each. Protein concentrations of each sample
were obtained using the BCA protein assay(Pierce). A standard
curve using bovine serum albumin (BSA) diluted with 5% SDS was
constructed using the following concentrations of BSA: 0 mg/ml, 0.1
mg/ml, 0.25 mg/ml, 0.50 mg/ml, and 1.00 mg/ml. 5 ul samples of
each sample and dilutions of the egg samples were placed in
triplicate in a 96 well micro-titer. 100 ul of BCA working reagent
was then added to each well, and the samples were incubated at
60°C for 30 minutes. The protein concentrations of the unknown
samples were determined using a Molecular Devices VMax plate
reader at 550 nm. Each egg sample was mixed with an equal amount
of sample buffer and boiled for five minutes in a water bath. The
samples were then stored in a freezer at -200C.
Gel Electrophoresis and Western Blotting
Samples were loaded onto a 7.5% polyacrylamide gel at
approximately 40 ug of protein per lane. The proteins were
separated on a BioRad minigel apparatus using a tris/glycine running
buffer and transferred to nitrocellulose membranes using a BioRad
electrophoretic transfer system. The proteins were transferred at 13
V for 1 hour, 24 V for 2 hours, and 36 V overnight. The blots were
blocked with PBS-Tween 20 (.05%)-BSA(lOmM) for 30 minutes and
then incubated for 2 hours with C219 monoclonal antibody
(Centocor) (1:250 in PBS-Tween 20-BSA,). The nitrocellulose
membrane was washed for 10 minutes with 10 mls of the PBS-
Tween20-PBS blocking solution three times and then incubated for 1
hour with goat anti-mouse IgG conjugated to alkaline phosphatase
(Sigma) (1:1000 in PBS-Tween 20-BSA). It was then washed three
times for ten minutes each with 10 ml of PBS-Tween 20 (.05%) and
incubated in alkaline phosphatase developing buffer (100 mM Tris,
100 mM Nacl, 5 mM MgCl2, pH 9.5 with nitro-blue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate).
RESULTS
Rhodamine Fluorescence Assays
In the later developmental stages of Urechis caupo, the
embryos develop cilia and become motile. To stop motility, I found
that 37% formaldehyde immobilized the embryos, enabling a more
accurate fluorescence reading. A control was performed to test the
effects of formaldehyde on rhodamine fluorescence since
formaldehyde kills the cells. The control showed that formaldehyde
decreases rhodamine fluorescence by approximately 44% (Figure 4).
Rhodamine fluorescence assays showed that an MXR
mechanism is active in unfertilized eggs, two-cell and four-cell
embryos, gastrulae, and two-day larvaes since the + verapamil
ratio at each stage was 2.3 or greater. The average + verapamil
ratio was 2.3 at the unfertilized stage, 3.7 at the 2-cell stage, 2.7 at
the 4-cell stage, 3.3 at gastrulation, and 2.6 at the 2-day stage. The
+ verapamil ratios corrected for formaldehyde at gastrulation and
the 2-day stage were 6.3 and 4.9 (Figure 5).
Rhodamine fluorescence assays were performed on the
coelomic and immature oocytes of three different Urechis females at
two rhodamine concentrations, 5 uM and 1.0 uM and at 2.2 uM
verapamil. At .5 uM rhodamine B, the first trial had coelomic oocyte
diameters ranging from 33 to 127 microns. No apparent trend was
observed between the +verapamil and -verapamil graphs, which
overlapped at two points (Figure 6). The second trial at 5 uM
rhodamine involved coelomic oocytes with diameters ranging from
47 to 140 microns. The +verapamil graph was slightly higher in
fluorescence than the -verapamil graph at all diameter sizes (Figure
7). The third trial at 5 uM rhodamine included coelomic oocyte
diameters ranging from 47 to 120 microns. This trial also showed no
apparent pattern between the +verapamil and -verapamil graphs,
which overlapped at four places (Figure 8). The immature oocytes
incubated at 5 uM rhodamine from the second and third female
exhibit MDR activity with + verapamil ratios of 1.7 and 1.9,
respectively (Figure 9).
At 1 uM rhodamine concentration, the coelomic oocytes from
the first trial ranged in diameter size from 60 to 134 microns. No
trend was observed between the +verapamil and -verapamil graphs,
which overlapped in three places (Figure 10). For trial 2 at 1 uM
rhodamine, the coelomic oocyte diameters ranged from 40 to 134
microns. The -verapamil graph is slightly higher in rhodamine
fluorescence than the +verapamil graph for all oocyte diameters
(Figure 11). Finally, the coelomic oocytes in the third trial at 1 uM
rhodamine have diameters ranging from 33 to 127 microns. No
apparent trend was seen between the +verapamil and - verapamil
graphs, which overlapped in five places (Figure 12). The immature
oocytes from the three female Urechis incubated in 1 uM rhodamine
showed the following + verapamil ratios, respectively: 2.1, .87, and
1.9, indicating MDR activity in the first and third trials (Figure 13).
Western blots were performed at the following stages of
Urechis caupo: coelomic, 2-day, and gastrulation. All three stages
showed a band at around 160 KD (Figure 14).
DISCUSSION
The + verapamil ratios of immature oocytes and also
the embryos at the 2-cell, 4-cell, gastrula, and 2-day stages suggest
that these cells possess MXR activity. Furthermore, the Western blot
analysis shows that gastrulae and 2-day old embryos possess a
protein of MW 160 kD that is labeled by a monoclonal antibody to
the conserved ATP-binding region of the mammalian P-glycoprotein.
These results indicate a multixenobiotic resistance mechanism
similar to the MDR resistance in mammals is present in Urechis early
embryos.
The control performed on immature oocytes with and without
formaldehyde indicates that formaldehyde does decrease rhodamine
considerably. Another way of immobilizing the later ciliated stages
of Urechis embryos should be found and used, preferably a way that
does not damage the embryo. Mireles has found methyl cellulose to
be an effective but harmless immobilizer for Urechis embryos
(Mireles, personal communication).
The rhodamine assays performed on the coelomic and
immature oocytes from the same female show the absence of MXR
activity in the coelomic oocytes but the presence of MXR activity in
immature oocytes. In the first and third trials, assays with both .5
uM and 1.0 uM rhodamine B show no significant differences between
the +verapamil and -verapamil graphs of the coelomic oocytes.
These results indicate that verapamil does not inhibit the transport
function of the MXR protein because the protein in coelomic oocytes
is not yet active or simply not present. The rhodamine assays of the
immature oocytes in the first and third trials at both rhodamine
concentrations suggest MXR activity with + verapamil ratios of 1.7
or greater.
The second trial of coelomic oocytes did not give as clear
results as the first and third trials. At 5 uM rhodamine
concentration, the +verapamil graph was always above the
-verapamil graph. The + verapamil ratios between the two graphs
never exceeded 1.3. These results suggest some MXR activity in the
coelomic oocytes. One possible explanation for this is that this
particular worm may have been stressed, having some MXR activity
in its coelomic oocytes. In addition, + verapamil ratios from the
coelomic oocytes were not as valid as those from the immature and
fertilized oocytes because the +verapamil and -verapamil graphs
involved coelomic oocytes of different sizes. During each rhodamine
assay, finding coelomic oocytes of one particular size to measure
their fluorescence was difficult so the + verapamil and -verapamil
graphs did not always consist of the same-sized oocytes. Both
coelomic and immature oocytes were saturated at 1 uM rhodamine
but not at .5 uM rhodamine as indicated by the + verapamil ratio of
87 in the immature oocytes at 1 uM rhodamine and the +¬
verapamil ratio of 1.7 at 5 uM rhodamine. The former ratio is less
than one, indicating that adding verapamil to 1 uM rhodamine made
no difference in fluorescence, although verapamil enhanced
fluorescence at 5 uM rhodamine. Therefore, the data from the
second trial at 1 uM rhodamine should be disregarded due to
oversaturation of the oocytes by rhodamine.
Western blot analysis also shows that a 160 kD protein is
present in coelomic oocytes. This result demonstrates the presence
of a protein immunologically related to the mammalian P¬
glycoprotein. However, rhodamine fluorescence assays indicate a
lack of MXR activity in coelomic oocytes, suggesting that the MXR
protein is present but inactive in the coelomic oocytes. However, this
protein is present and active in immature and fertilized oocytes.
What then activates MXR in Urechis coelomic oocytes? One
possible explanation is that the oocytes in the coelomic fluid undergo
some kind of post-translational modification, specifically
phosphorylation, before becoming immature oocytes in the storage
organs. A past study has indicated approximately a 50% increase in
MXR activity between unfertilized and fertilized Urechis oocytes
2(Toomey, personal communication). This increase in MXR activity at
fertilization and the increase between coelomic and immature
oocytes may be due to phosphorylation. Center showed that the
mammalian P-glycoprotein transporting function can be modified by
phosphorylation. An increase in P-glycoprotein phosphorylation
produces a decrease in drug accumulation and an increased
resistance to the drugs (Center, 1985). Both protein kinase C and
cAMP-dependent protein kinase catalyze phosphorylation in MDR
cells (Chambers et al., 1990; Ma et al., 1991; Miyamoto et al., 1990).
Inhibitors to the two kinases such as staurosporine and H-7,
respectively, decrease the drug efflux from the cell, whereas protein
kinase C activators such as phorbol esters increase drug efflux (Ma et
al., 1991; Miyamoto et al., 1990). To test if either kinase is involved
in the MXR activity increases between coelomic and immature
oocytes and/or immature and fertilized oocytes, further rhodamine
assays should be conducted using staurosporine and H-7 as inhibitors
at the more active stage.
Environmental conditions of the oocytes may also influence
their MXR activity. Coelomic, immature, and fertilized oocytes live in
different media in nature. Coelomic oocytes develop in the coelomic
fluid surrounded by erythrocytes and proteins. Immature oocytes
are collected from the coelomic fluid and stored in storing organs.
Finally, immature oocytes become fertilized in seawater where
sperm can swim to them. These environments are different in many
ways, one of which may be their pH. The coelomic oocytes may be
activated by a change in pH. Furthermore, the different
environmental conditions may serve as a trigger to phosphorylation.
Coelomic oocytes reside within their mother, sheltered from
any external hazards. Thus, the coelomic oocytes may not need yet
to exert a self-defense mechanism. They can use the mother's
defense mechanisms. In addition, the MXR protein binds to many
hydrophobic substrates and transports them out of the cells. While
still developing, the coelomic oocytes may need for certain
hydrophobic compounds to be retained in the cell. Thus, MXR
inactivity may be beneficial to the oocytes at their developing stages.
Discovering why MXR activity is turned on as Urechis caupo
oocytes mature can prove helpful in further understanding both MXR
activity against pollutants in aquatic animals and MDR activity
against anti-cancer drugs in tumor cells. If MDR activity can be
turned off in tumor cells as it is in Urechis coelomic oocytes, then
chemotherapy would become much more effective.
In addition, further research on MXR may provide insight on
its natural role in the cell. The transport function of the MXR protein
is inherent in many marine organism, not requiring induction by
pollutants or drugs(Kurelec, 1992). Also, the MXR protein exists and
is taxonomically highly conserved across many species (Kurelec,
1992). This information implies that this mechanism has a role(s)
that is important in the functioning of certain cells. One possible role
may be to excrete natural toxic products in the diet or endogenous
metabolites (Gottesman, 1988).
Another role of P-glycoprotein in
mammals may be to secrete progesterone and other steroids in
mammalian adrenal cortex (Qian, 1990). Regardless of other possible
roles, the MXR mechanism plays an important role in detoxifying
cells in marine and terrestrial life.
ACKNOWLEDGEMENTS
I would like to thank the Epel lab for all the time, patience, and
support given to me during my stay here at Hopkins Marine Station
during this past quarter. Thank-you, Dave, for all the time and
advice you gave me over the quarter. I really had fun and learned a
lot during lab meeting and lunches. Thank-you, Barbara, for sharing
with me your knowledge on Urechis, rhodamine assays, and Western
blots. Thank-you, Beth, for sharing your lab bench with me, and also
for your smiles in times of frustration. Thank-you, Paul, for sharing
your insights on how to do things easier such as skating in the mud.
Thank-you, Robin, for your help in the lab including showing me
where things were hiding. Thank-you, Francois, for your interests
and sense of humor in my project. Thank-you, Chris, for making us
laugh in frustrated times, developing wonderful pictures, and making
corny jokes about pre-meds. Thank-you, Molly, for being a great
T.A., developing great slides, and for being a all-around wonderful
person. Thank-you, Maria, for being my Urechis buddy and helping
to dig me out of the mud. Finally, thank-you, Annie and Jenny, for
being wonderful labmates as well as housemates and Esther, for
being a great housemate. Without you all, my quarter would have
been not the same.
REFERENCES
Center, M. S., Mechanisms regulating cell resistance to adriamycin:
evidence that drug accumulation in resistant cells is modulated
by phosphorylation of a plasma membrane glycoprotein.
Biochemical Pharmacology, 34, 1471, 1985.
Chambers, T. C., Chalikonda, I., and Eilon, G., Correlation of protein
kinase C translocation , P-glycoprotein phosphorylation and
reduced drug accumulation in in multidrug resistant human KB
cells. Biochemical Biophysical Research Communications, 169,
253, 1990.
Chen, C.-J., Chin, J. E., Ueda, K., Clark, D. P., Pastane, I., Gottesman, M.
M., and Roninson, I. B., Internal duplication and homology with
bacterial transport proteins in the mdrl (p-glycoprotein) gene
from multidrug-resistant human cells. Cell, 47, 381, 1986.
Gottesman, M. M., Pastan, I., The Multidrug Transporter, a Double¬
aged Sword. The Journal of Biological Chemistry. 263, 12163,
1988.
Gould, M. C., RNA and Protein Synthesis in Eggs and Embryos of
Urechis caupo. Stanford University, 1967. (dissertation)
Gros, P., Croop, J., and Housman, D. E., Mammalian multidrug
resistance gene: complete cDNA sequence indicates strong
homology to bacterial transport proteins. Cell, 47, 371, 1986.
Harper, K., The Relationship of MDR P-Glycoprotein and the Swelling
Activated Chloride Channel. Stanford University, 1993.
(Honors Thesis)
Juliano, R. L. and Ling, V., A surface glycoprotein modulating drug
permeability in Chinese hamster ovary cell mutants,
Biochemical Biophysical Acta, 455, 152, 1976.
Kurelec, B., The Multixenobiotic Resistance Mechanism in Aquatic
Organisms. Critical Reviews in Toxicology. 22, 23, 1992.
Ma, L., Marquardt, D., Takemoto, L., and Center, M. S., Analysis of P¬
glycoprotein phosphorylation in HL6O cells isolated for
resistance to vincristine, Journal of Biological Chemistry, 266,
5593, 1991.
Miller, J. H., Studies of Oogenesis in Urechis caupo: Method for
Separating Different Size Classes of Oocytes. Developmental
Biology, 32, 219, 1973.
Mireles, M., Hopkins Marine Station, Stanford University, Pacific
Grove, CA, personal communication.
Miyamoto, K. I., Wakusawa, S., Nakamura, S., Koshiura, R., Otsuka, K.,
Naito, K., Hagiwara, M., and Hidaka, H., Circumvention of
multidrug resistance in P388 murine leukemia cells by a novel
inhibitor of cyclic AMP-dependent protein kinase, H-87, Cancer
Lett., 51, 37, 1990.
Morris R. H., Abbot, D. P., Haderlie, E. C., Intertidal Invertebrates of
California. Stanford University Press., Stanford, 1980.
Newby, W. W., The Embryology of the Echiuroid Worm Urechis caupo.
The American Philosophical Society. Philadelphia, 1940.
Neyfakh, A., Use of fluorescent dyes as molecular probes for the
study of multidrug resistance, Experimental Cell Research, 174,
168, 1988.
Qian, X., and Beck, W. T., Progesterone photoaffinity labels P-
glycoprotein in multidrug-resistant human leukemic
lymphoblasts, Journal of Biological Chemistry., 165, 18753,
1990.
TToomey, B. H., Multixenobiotic transport in Urechis embryos:
Protection from environmental toxins. Hopkins Marine Station,
Stanford University, 1993. (unpublished)
2Toomey, B. H., Hopkins Marine Station, Stanford University, Pacific
Grove, CA, personal communication.
West, I.C., What determines the substrate specificity of the
multidrug-resistance pump?, TIBS, 5, 42, 1990.
FIGURE LEGENDS
Figure 1. Mechanism of P-glycoprotein transport function.
Moderately hydrophobic compounds diffuse into the cell. P¬
glycoprotein binds to the compounds and transports them out using
ATP as its energy source.
Figure 2. Anatomy of Urechis caupo. Immature gametes are stored
in the storage organs ready to be spawned, and developing gametes
are in the coelomic fluid. Oocytes do not reach full maturity until
they are fertilized when they finish meiosis II.
Figure 3. Spawning of Urechis caupo. A glass probe is inserted and
gently rotated into one of six gonadopores. Extrusion of sperm
(white) and eggs (pink, yellow, olive) follows.
Figure 4. Effects of formaldehyde on rhodamine B fluorescence in
U. caupo oocytes. Formaldehyde decreases rhodamine fluorescence
by 44%.
Figure 5. + Verapamil ratios of U. caupo oocytes at different
stages incubated in 1 uM rhodamine B and 22 uM verapamil. Ratios
corrected for formaldehyde are also shown.
Figure 6. Trial 1: Coelomic oocytes in 5 uM rhodamine B and 2.2
uM verapamil. No apparent trend between +verapamil and
-verapamil graphs.
Figure 7. Trial 2: Coelomic Oocytes in 5 uM rhodamine B and 2.2
uM verapamil. +Verapamil graph is higher than -verapamil graph at
all diameter sizes of oocytes.
Figure 8. Trial 3: Coelomic oocytes in 5 uM rhodamine B and 2.2
uM verapamil. No apparent trend between the +verapamil and
-verapamil graphs.
Figure 9. + Verapamil ratio of immature oocytes incubated in .5
uM rhodamine B and 2.2 uM verapamil. Both trial 2 and trial 3
indicate MXR activity with + verapamil ratios of greater than 1.7
Figure 10. Trial 1: Coelomic oocytes in 1 uM rhodamine B and 2.2
uM verapamil. No apparent trend between the +verapamil and
-verapamil graphs.
Figure 11. Trial 2: Coelomic oocytes in 1 uM rhodamine B and 2.2
uM verapamil. -Verapamil graph is higher than the +verapamil
graph at all diameter sizes of oocytes.
Figure 12. Trial 3: Coelomic oocytes in 1 uM rhodamine B and 2.2
uM verapamil. No apparent trend between the +verapamil and
-verapamil graphs.
Figure 13. + Verapamil ratios of immature oocytes Incubated in 1
uM rhodamine B and 2.2 uM verapamil. Trial 1 and trial 3 indicate
MDR activity with + verapamil ratios greater than 1.7.
Figure 14. Western blot of the different development stages of
Urechis caupo. A band at 160 kD is seen in coelomic oocytes,
gastrula, and 2-day larvae.
FIGURE 1
HYDROPHOBIC COMPOUND
A
AT
6
S
P-GLYCOPROTEIN
STORAGE ORGANS
Immature Gametes

4
FIGURE 2
ANTERIOR
COELOM
Developing Gametes
u
POSTERIOR
GONADOPORES
FIGURE 3
MOUTH

—
—
—
—


MV SETAE
ANUS
naehe
lien
Figure 4
0-
mature
2-ce
Figure 5
4-cell
—
gastrul.
2-day
E CORRECTED
□ OBSERVED
Figure 6

10—


2 -
kvv-
—
0-
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
Size (1 un. = 66.7 microns)
—— Ver
—— 4Ver
Figure 7
18 +
14
12 -


k vkva-
oaaa
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0
Size (1 un. = 66.7 microns)
—
— -Ver
—— 4Ver
30 -
Figure 8




O
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
Size (1 un. = 66.7 microns)
——Ver
—— 4Ver
2.5
2.0
1.0
0.5-
0.0 +

Trial 2
Trial 3
Figure 9
Figure 10


60
50
A

30-
20
10
0 +
—
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
Size (1 un. = 66.7 microns)
——Ver
—— +Ver
Figure 11
100
90 -
80
70
60
40
30
20 -
10 -

O+
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0
Size (1 un. = 66.7 microns)
——Ver
—— 4Ver
Figure 12
p


70—
60 -
50 -
40 -
30
20

10 -
O +
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
Size (1 un. = 66.7 microns)
—Ver
—— 4Ver
2.5
2.0
1.0
0.5
0.0 +
Trial
Figure 13
2
Trial 2
2
Trial 3
200 KD
97.4 KD
69 KD
46 KD
30 KD
FIGURE 14
WESTERN BLOT: Different Development
Stages of Urechis caupo