RADIOIMMUNOASSAY
ASSIGNMENT: 2007
The primary purpose of
this exercise was for you to gain some exposure to the Nobel Prize-winning
procedure known as radioimmunoassay.
What we did was take ovaries from rats at 5 different intervals during
the ovulatory process. These intervals were 0, 4, 8, 12, and 24
hours after the animals received hCG. (In the rat, ovarian follicles begin
rupturing approximately 12 hours after hCG. Thus, you want to keep in mind when
considering the questions in this assignment that the 24-hour group of ovaries
contained postovulatory luteal tissue, rather than ovulatory follicles.
Therefore, by extracting and measuring ovarian progesterone and
prostaglandin E2, we could monitor any changes in this steroid and prostanoid during ovulation. Also, at 3 hours after hCG we treated one group of animals with epostane (EPO), which is a potent inhibitor of the enzyme 3b-hydroxysteroid dehydrogenase (3b-HSD). (What would
you expect this enzyme inhibitor to do to ovarian progesterone synthesis?) In addition, since it is well known that prostanoids (i.e., prostaglandins) increase in inflamed
tissues, and since it has been hypothesized that ovulation is like an
inflammatory reaction, we also tested the effect of the potent
anti-inflammatory agent known as indomethacin (INDO),
which is a cyclooxygenase-2 (COX-2) inhibitor.
In particular, we wanted to determine the effect of this agent on the
normal rise in ovarian prostaglandins, and we wanted to see if the agent could
inhibit ovulation in this experimental model.
Thirdly, we wanted to determine the relative amount of blood in the
ovary in order to detect any ovarian hyperemia that would be expected during an
inflammatory reaction. To make this
latter determination, we measured the amount of absorbance (at ~410
nm) by the ovarian extracts. This wavelength is near the peak absorbance for
hemoglobin, and therefore it represents an estimate of the relative amount of
blood in the ovary at the different stages of the ovulatory
process.
Use the numbers below
to perform the assigned calculations and to answer the questions.
I.
Concentration of Progesterone (ng P4/ml)
during Ovulation:
TUBE 0-hour 4-hour 8-hour EPO* INDO** 12-hour 24-hour
#1
4.5 22.4 40.0 1.7 40.8 20.7 12.4
#2
1.8 32.5 37.6 2.0 35.7 26.4 13.1
#3
2.4 34.6 39.1 2.2 40.2 25.0
3.7
#4
3.9 26.9 39.5 1.8 38.4 20.8 18.9
#5
3.0 26.2 30.1 1.9 22.7 48.2 14.9
#6
1.1 21.6 50.1 2.4 41.1 23.8 18.8
*Note that these tubes contained extracts from rats that
received 5 mg of epostane at 3 hours after hCG, and their ovaries were taken
at 8 hours after hCG.
Epostane is a potent inhibitor of steroid
synthesis, and therefore it blocks the usual increase in progesterone synthesis
that occurs in the ovary when the ovulatory process
is stimulated by hCG. The principal action of this drug is to
inhibit 3b-HSD, which converts pregnenolone into progesterone. (You should be able to see whether it inhibits
progesterone synthesis by comparing this column of data with the 8-hour
controls.)
**Note that these tubes
contained extracts from rats that received 1 mg of indomethacin
at 3 hours after hCG, and
their ovaries were taken at 8 hours after hCG. Indomethacin is a
potent non-steroidal anti-inflammatory agent (NSAID) that is commonly
prescribed for patients with rheumatoid arthritis and other connective tissue
diseases. A principal action of this
drug is to inhibit cyclooxygenase, the enzyme that
converts arachadonic acid into prostaglandins.
II. Concentration of Prostaglandin E2
(pg PGE/ml) during Ovulation:
TUBE 0-hour 4-hour 8-hour EPO* INDO** 12-hour 24-hour
#1 177 2392 2431 2343 52 1476 565
#2
59 2003 2544 2431 63 1388 407
#3
68 1387 2071 2041 47 1382
70
#4
57 1818 2212 2837 62 1483 403
#5
62 338 2718 2502 39 2138 432
#6 107 2795 2781 2219 41 1750 692
III.
Absorbance (at 415 nm) for Hemoglobin in Each of the Extracts
TUBE 0-hour. 4-hour 8-hour EPO* INDO** 12-hour 24-hour
#1 0.32 0.29 0.34 0.36 0.23 0.93 0.44
#2 0.18 0.28 0.41 0.46 0.19 0.61 0.55
#3 0.22 0.26 0.59 0.31 0.23 0.99 0.15
#4 0.20 0.37 0.45 0.39 0.28 0.67 0.57
#5 0.08 0.31 0.46 0.50 0.24 0.77 0.24
#6 0.08 0.43 0.42 0.58 0.25 0.85 0.61
IV. Ovulation Rates
Without and With EPO, or INDO Treatment
While you were making
your injections and surgically removing the rats' ovaries, your professor was
working with several other groups of rats.
One of these groups served as a control in which hCG was administered, but the
ovaries were not taken until 24 hours later. In this group taken at 24 hours,
your professor also cut out the oviducts and placed them under a dissecting
microscope in order to count the number of eggs that ovulated. The numbers of eggs found in each of 6 rats
were: 57, 68, 0, 69, 63, and 55.
Thus, it is obvious that the hormone treatment successfully induced "super-ovulation"
in all but one of the animals. (NOTE: The 24-hour group
of rats were used to count the ovulation rate, i.e., to determine the number of
ova in the oviducts of the animals.)
Parallel groups of rats received either 5 mg of EPO or 1 mg of INDO at 3
hours after hCG. The ovulation
rates in these animals were: 2, 0, 0, 0, 0, and 0 ova/rat in the EPO group and 2, 5, 11, 1, 3,
and 3 ova/rat in the INDO group.
PROBLEMS/QUESTIONS (Due Tuesday, December 6, 2006)
NOTE: As per the Academic Integrity Rules of Trinity
University, you are on your honor to respond to the following
Problems/Questions without any consultation and/or assistance from anyone else.
NOTE: Be sure to
show the “numbers” you used to arrive at your conclusions.
NOTE: Within the given groups of
numbers above, several of the numerical values stand out as being considerably
different from the others in the group and there is the temptation to reject
that value in establishing the mean value.
A common statistical rule for omitting the doubtful value is as follows: Omit the doubtful value from the group and
determine in the usual way the mean and the average deviation (a.d.) of
the retained values. The rejection can
be considered as mathematically justified if the deviation (d) of the suspected value from the mean
is at least four times the average deviation of the retained values—that is, if
d ³ 4 a.d.
1. Obtain the means and standard errors of the
means (SEMs) for all of the groups of numbers for P4
values, PGE2 values, for absorbance values, and
for ovulation rates. (Use any
method you wish to obtain SEMs, but show the basic
mathematical formula you used.
Specifically, did you use the formula S.E.M. = Standard Deviation
divided by the square root of “n-1”,
or divided by the square root of “n”?) (20 points)
2. In the PGE2 RIA data, it is
obvious that the value for tube #5 at 4 hours after hCG is completely out of line with the other five
values. Can you give three
possible explanations for this discrepancy?
What is the most likely explanation?
(6 points)
3. In the P4 RIA data, it is obvious
that the value for tube #3 at 24 hours after hCG is completely out of line with the other five
values. What is the most likely
explanation for this discrepancy? (4
points)
4. By "rule of thumb," if SEM
values overlap when one compares any two groups, the means of those two
groups are not statistically different from one another. Using this simplified method for estimating
differences in means, did INDO cause a "statistically significant"
change in the ovarian P4 level at 8 hours after the administration
of hCG? (6 points)
5. Did EPO "significantly" affect PGE2
synthesis at 8 hours after hCG? (6 points)
6. What can you conclude about the speed of
action of EPO and INDO as inhibitors of ovarian P4 and PGE2
synthesis? (6 points)
7. Did INDO "significantly" reduce the
ovarian 8-hour PGE2 level below the 0-hour PGE2
level? (6 points)
8. Is an elevated level of ovarian PGE2
associated with (i.e., important to) corpus luteum
function in the ovary? How did you arrive
at your conclusion? (5 points)
9. Did EPO "significantly" inhibit P4
synthesis in the ovary at 8 hours after hCG? (5 points)
10. When comparing ovarian P4 levels
at 12 hours and at 24 hours after hCG,
was there a "significant" change in the level of this steroid at 24
hours after hCG?
(5 points)
11. Based on the limited data that is available,
what was the percentage increase in ovarian blood at 12 hours after hCG over the ovarian blood volume
at 0 hours after hCG?
(5 points)
12. Can you conclude whether EPO and/or INDO
inhibited the normal increase in ovarian blood volume? (Be sure to indicate which values you used to
arrive at your conclusions.) (6 points)
13. Obviously, both EPO and INDO inhibited
ovulation. Is there a statistically significant
difference in the anti-ovulatory actions (i.e., the
effectiveness) of these two agents? (5
points)
14. Is there any way for you to determine whether
3b-HSD (inhibited by EPO) or COX-2
(inhibited by INDO) is more important as an enzyme in the biochemical events of
ovulation? (5 points)
15. If you were the CEO of a pharmaceutical
company and wanted to invest in one of these agents (i.e., EPO or INDO) as a
potential birth control pill, which one would you choose? Explain your answer. (5 points)
16. Approximately 10 years ago, CELEBREX was a
highly touted anti-inflammatory agent that, for the most part, took the place
of INDOMETHACIN as a drug to treat patients suffering from rheumatoid
arthritis, osteoarthritis, and other such inflammatory diseases. In view of the inflammatory nature of the ovulatory process, do you think CELEBREX might be useful as
a potential anti-fertility agent?
Describe the experiment you would conduct to test the effectiveness of
CELEBREX as an anti-fertility agent. (5
points)
