0
Introduction:
Although much is known about RNA activity in
developing oocytes, (2,3,4) little is known about the
activity of enzymes. In most organisms the oocytes are
embedded in tissue and cannot be obtained free from
contaminating cells. One of the advantages of marine
annelids is that oocytes from most stages of development
can be found free in the coelom. Nevertheless it is
difficult to obtain enough oocytes of the immature
stages for standard enzymatic determinations. In order
to overcome this problem, a simple photoelectric system
was developed to measure the rate of reduction of
tetrazolium salts resulting from enzyme activity in
single oocytes of Cirriformia spirabrancha. The results
indicate that glucose-6-phosphate dehydrogenase and
succinic dehydrogenase are both being synthesized at
a steady rate during oogenesis.
Instrumentation:
A block diagram of the microphotometer used is
shown in figure 1. The basic unit was a trinocular Nikon
microscope, Model STR, and an American Optical spot lamp
with voltage selector, Model 350. A Sola constant voltage
transformer, Cat. 30807, was used to reduce electrical
noise. Light passed through a Klett +66 filter with a
transmission band from 640 to 700 mu. A pinhole diaphragm
33
-2-
which reduced the field to 40 u at 400X was placed
over the filter. Incubation chamber slides filled
with several eggs could then be placed on the
microscope stage and positioned so that the center
of the egg completely covered the field.
The light sensing device, an Internation Rectifier
silicon photovoltaic cell, SiM, was attached to one
end of a stiff rubber tube with inner diameter of 1.0
inches. The end of this tube with the photocell attached
was fitted to a light tight metal box. The tube and
box were then affixed over the trinocular attachment.
The output of the photocell was amplified and recorded
by a Sargent Recorder, Model SRL.
With the trinocular prism closed the output voltage
was zero. With the prism open open field readings were
in the range of 1.7 millivolts + 0.2 mv. depending on
the room lighting. Measurements were made with red
light since the color produced by the formazan product
of the tetrazolium dye is blue. The combination of
red light and silicon cell was ideal since the maximum
spectral responce of the photocell was between 600
and 900 mu.
Materials and Methods:
Oocytes:
All oocytes used were taken from the coelom of
C. spirabrancha collected in the yatch harbor of
Monterey Bay, Monterey, California. Diameters of the
33
-3-
three developmental stages used were 70, 90, and 110
microns, the last of which represents maturity.
Incubation Proceedure:
Freshly collected oocytes were placed in a depression
slide and quick frozen on dry ice in a solution of sea
water containing 5-7% PVP. The eggs were thawed after
five minutes, and samples of 10 to 20 eggs were placed
in a chamber whose walls were made from 2 coverslips
sealed with vaseline. This chamber had a depth of greater
dimensions than the largest diameter egg used. To these
chambers were added two drops of either G-6-P dehydrogenase
or succinic dehydrogenase incubation solutions prepared
according to Barka and Anderson.(1) The Sargent recordings
were started at the exact time the incubation solutions
were added. Eggs and solution were mixed, covered, then
sealed with vaseline. The chamber was placed on the
microscope, an egg chosen, focussed, and centered over
the diaphragm, and last,the trinocular was opened. The
time interval between addition of solution and the
opening of the trinocular averaged 2.0 minutes. Photocell
outputwas recorded until the total elapsed time was
15.0 minutes. All runs were made at 24.0 + 0.5°0.
Results:
Qualitative Observations:
Qualitative observations of dye formation showed
that in the smallest oocytes, 50u, succinic dehydrogenase
activity only reduced a small amount of tetrazolim,
338
-4-
yielding little color even after 40 minutes of incubation.
Larger 80u oocytes present in the same dish under the
same conditions showed very obvious color production.
High G-6-P dehydrogenase activity was observed in both
large and small eggs, with intense color production
clearly visible in the first 20 minutes.
Quantitative Observations:
Three sets of control recordings were made. An open
field, a sample of incubation solution alone, and eggs
incubated in sea water, all showed consistent behavior
with no voltage change. This indicated that any drop
in recorded voltage during the test runs would be due
only to color production in the egg.
Ten runs were made for each size egg for each enzyme.
Figure 2 shows a typical recorder tracing picturing the
voltage drop resulting from the enzymatic activity. These
records were analysed by two different methods to determine
the rate of reduction of the tetrazolium salts. In the
first method the rate in microvolts per minute was
measured from the voltage drop during the interval
between five and ten minutes. In the second the rate
was determined from the line which best approximated
the slope of the tracing, generally straightest in the
interval from 4 to 12 minutes. The averages and standard
deviations were determined for the ten runs, and the
results are listed in the uncorrected rates columns of
Tables 1 and 2.
336
5
Beer's Law prompted consideration of the optical
path length of the absorbing medium. The optical path
length in this case is the oocyte diameter while the
absorbing medium is the formazan impregnated cytoplasm.
Although it is questionable whether the oocyte test
situation is a pure application of Beer's Law, the rates
and their standard deviations have also been computed
with this correction factor considered, and the results
of this appear in the "corrected rates" columns of
Tables 1 and 2.
Taking standard deviations into account the data
shows that the activity of G-6-P dehydrogenase remains
constant throughout the period of development studied.
Succinic dehydrogenase apparently follows the same
pattern, although the results are a little less conclusive.
Discussion:
Tetrazolium Methods:
Tetrazolium salts are excellent dyes for histochemical
studies of enzyme activity. Methods have even been
developed to extract the tetrazolium from incubated
tissues in order to determine spectrophotometrically
enzyme activity. (5) Thus it is evident that tetrazolium
reduction can be used for enzyme rate studies in oocytes
provided substrate, temperature, and pH all remain
constant.
Scattering Effect:
Since the amount of scattering due to oocyte
3
334
-6-
cytoplasm changes little if at all during a 15 minute
run, any change in readings is primarily the result
of absorbtion by the blue formazan. A minor scattering
effect may be caused by the deposition of the formazan,
but this is likely to be consistent. Thus the relative
relationships in the results would still hold. Visual
observations indicated that scattering by the cells was
not very extensive.
Linearity of the Rate Curve:
Although the slope was determined for each trial,
this slope generally had to be extrapolated from the
section of tracing from 4 to 12 minutes. Within this
time period the tracing was straight, ignoring minor
noise, approximately 70% of the time. At the beginning
of each run when the trinocular was opened, the recorder
needle would move to its highest value. The slope from
this point would decrease rapidly for about two minutes
and then remain constant in the interval from 4 to 12
minutes. After this the line would begin to level off,
indicating reduced rate. This may result from deposition
of formazan around the sites of enzyme activity. It is
possible that the initial steep drop could be an equilibration
effect or actually the best measurement of enzyme rate.
Since this high rate was not consistently present, and
when present had a variable slope, the slope lines were all
extrapolated from the 4 to 12 minute segment, and therefore
still hold their relative relationship.
Interpretation of Data:
-7-
The rate of tetrazolium reduction due to G-6-P
dehydrogenase remains constant in all stages studied.
This implies that there is a continuing synthesis of this
enzyme during oogenesis. If therewere no synthesis, the
concentration of enzyme would be diluted with yoke and
cytoplasm during the growth process. Thus the rates
from a decreasing concentration system corrected for
optical pathllength would decrease. Conversely, constant
readings imply that there would have to be increasingly
more enzyme present to prevent dilution and result in
the production of an equal amount of color per unit volumn.
The data for succinic dehydrogenase is not as obvious.
When the standard deviations are carefully considered
it is seen that the 70 and 110 micron stages are very
similar, and that the upper limits of the 9Ou stage could
correspond to the lower limits of the other two stages.
It is therefore possible to state with some reservation
that the succinic rate remains almost constant during
the period of development studied. By the same argument
used for G-6-P dehydrogenase, the results imply that
succinic dehydrogenase, and therefore mitochondria, are
also being produced at a steady rate during development
from 70 to 110 microns in diameter.
Summary:
1. A simple new microphotometric method was developed
for studying the rate of tetrazolium reduction in single
oocytes.
33.
-8-
2. When corrections were made for the different
optical path lengths of these eggs, the results showed
that the tetrazolium reduction by the activity of
G-6-P dehydrogenase and succinic dehydrogenase were
very nearly constant in all stages. Constant enzyme
activity in different stages of development implies
constant synthesis of G-6-P dehydrogenase and succinic
dehydrogenase over the course of oogenesis.
3. Future application of this method may be very
helpful in studying enzyme activity under a variety
of conditions, especially for material not available
in large amounts.
ACKNOWLEDGEMENTS
The author wishes to acknowledge the valuable
guidance given by Dr. David Epel.
This work was supported in part by the Undergraduate
Research Participation Program of the National Science
Foundation: Grant GY - 4369.
REFERENCES
Barka, T., and Anderson, P. J., Histochemistry:
Theory, Practice, and Bibliography, p.313-315,
Harper and Row, Publishers, Inc., New York, (1963)
Brown, D. D., and Dawid, I. B., Science, 160,
272 (1968)
3. Brown, D. D., and Littna, E., J. Mol. Biol.
8,688 (1964)
4. Davidson, E. H., Allfrey, V.G., and Mirsky, A. E.,
Proc. Nat. Acad. Sci. U. S., 52,501 (1964)
5. Jardetzky, C. D., and Glick, D., J. Biol. Chem.
218, 283 (1956)
12
VOLTAGE
SOURCE
CONSTANT
VOLTAGE
FIGURE
METAL BOX
RECORDER
PHOTOCELL
RUBBER TUBE¬
TRINOCULAR
OBJECTIVE
INCUBATION CHAMBER¬
COVER SLIP.

(SL1DE
CONDENSER-
FIELD
DIAPHRAGM¬

FILTER
VOLTAGE
LIGHT
SELECTOR
SOURCE
C
E
A

2
2 —
2


2
e

A
V
S

1
A

9
FIGURE LEGEND
Figure 1. Block diagram of the microphotometer system.
Figure 2. Sample tracing showing curve analysis. of
a glucose-6-phosphate dehydrogenase recorder
tracing.
DIAMETER
microns
70
70
90
110
110
METHOD
5-10
slope
5-10
slope
5-10
slope
TABLE 1
UNCORRECTED RATE S.D. CORRECTED RATE S.D.
uV./min.
uV./min.
6.30
2.77
9.60
4.22
6.12
9.33
1.45
2.22
8.38
1.91
10.24
2.34
7.26
8.88
1.76
2.15
10.42
3.38
10.42
3.38
9.94
3.28
9.94
3.28
DIAMETER
microns
70
70
90
110
110
TABLE 2
METHOD UNCORRECTED RATE S.D.
uV./min.
5-10
4.34
1.59
3.98
slope
1.51
5-10
4.08
1.47
slope
4.06
1.09
5-10
7.40
2.71
6.93
1.39
slope
CORRECTED RATE S.D.
uV./min.
6.94
2.54
6.36
2.42
5.07
1.82
5.04
1.36
7.40
2.71
6.93
1.39
6
TABLE LEGEND
Table 1. Glucose-6-phosphate dehydrogenase activity
of different diameter oocytes.
Table 2. Succinic dehydorgenase activity of different
diameter oocytes.